Advances-in-pharmaceutical-biotechnology-Patra 2020

Jayanta Kumar Patra
Amritesh C. Shukla
Gitishree Das Editors
Advances in
Pharmaceutical
Biotechnology
Recent Progress and Future Applications
Advances in Pharmaceutical Biotechnology
Jayanta Kumar Patra • Amritesh C. Shukla
Gitishree Das
Editors
Advances in Pharmaceutical
Biotechnology
Recent Progress and Future Applications
Editors
Jayanta Kumar Patra Amritesh C. Shukla
Dongguk University Department of Botany
Goyang-si, Gyeonggi-do University of Lucknow
South Korea Lucknow, Uttar Pradesh
India
Gitishree Das
Dongguk University
Goyang-si, Gyeonggi-do
South Korea
ISBN 978-981-15-2194-2 ISBN 978-981-15-2195-9 (eBook)

https://doi.org/10.1007/978-981-15-2195-9
© Springer Nature Singapore Pte Ltd. 2020

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Preface
Advances in Pharmaceutical Biotechnology: Recent Progress and Future

Applications is basically an edited book comprising of 30 chapters that provides a
unique, single source of valuable information on the biopharmaceuticals available,
from conventional to modern scientific researches, pertaining the information on
plant-mediated green synthesis of nanoparticles, nanomaterials in drug delivery, tis-
sue engineering and regenerative medicines, clinical trials, and IPR. The book also
explores ethnopharmacology, traditional and folk medicines, phyto-pharmaceuticals,
and natural products as targets for drug discovery.
In a nutshell, the book contains a series of chapters on new trends covering devel-
opments in traditional medicines and natural products as leads for pharmaceutical
drug discovery, which will be helpful for the professionals, researchers, and schol-
ars working in the field of natural products, in general, and botany, biotechnology,
and pharmaceutical sciences, in particular.
We are grateful to the authors of this book who helped to bring this volume to
light. We wish to thank Dr. Emmy Lee, Associate Editor, Springer Nature Korea
Limited, for her generous assistance and persistence in finalizing the edited volume.
Special thanks are to our esteemed fellow colleagues and university authorities for
their kind support and constant encouragement throughout the task. Finally, specific
thanks goes to our families and friends for their support and cooperation in putting
everything together.
Goyang-si, Gyeonggi-do, South Korea Jayanta Kumar Patra

Lucknow, Uttar Pradesh, India Amritesh C. Shukla
Goyang-si, Gyeonggi-do, South Korea Gitishree Das
v
Contents
Part I Traditional and Ethnomedicine

1 Traditional Folk Medicine and Drug Discovery:
Prospects and Outcome �� 3
Prakash Roy Choudhury, Anupam Das Talukdar, Deepa Nath,
Priyanka Saha, and Rajat Nath
2 Ethnomedicine for Drug Discovery�� 15
Yengkhom Disco Singh, Manasa Kumar Panda,
and Kunja Bihari Satapathy
3 Medicinal and Aromatic Plants: Store House
to Herbal Antimicrobials �� 29
Ravikant Singh, Anand Pandey, Rohit Kumar Mishra,
Amritesh C. Shukla, and Anupam Dikshit
4 Ethnomedicinal Plants of North-East India
as a Potential Target for Drug Discovery
Against Type 2 Diabetes Mellitus �� 39
Jijnasa Bordoloi, Anjum Dihingia, Jatin Kalita,
and Prasenjit Manna
5 Application of Phytochemicals in Pharmaceuticals�� 55
Sankhadip Bose, Jai Malik, and Subhash C. Mandal
6 The Herbal Drugs�� 69
Amritesh C. Shukla
7 Role of Natural Products as Alternative
of Synthetic Steroidal Drugs �� 77
Priyanka Tiwari, Rashmi Pandey, Reetika Singh,
and Bechan Sharma
vii
viii Contents
8 Phytochemicals as Therapeutics in Heavy Metal Toxicity �� 91

Nitika Singh and Bechan Sharma
9 Phytotherapies for Thyroidism: An Overview�� 101
Taniya Sengupta Rathore, Shekhar Jain, and Sonu Chouhan
10 Phytochemicals as Antidepressants�� 115
Khushboo, Abhishek Kumar, and Bechan Sharma
11 Phytochemistry and Medicinal Value
of Putranjiva roxburghii Wall�� 133
Narendra Kumar
12 Traditional Herbal Practices of Eastern Ghats, Odisha,
India, for Treatment of Bone Fracture�� 145
Subhashree Pattnayak, Devasish Murmu, Manasa Kumar Panda,
Rojali Maharana, Kalicharan Mandal, and Nabin Kumar Dhal
13 Screening of Certain Medicinal Plants of Manipur
for Their Antifungal Activity against Candida Species�� 155
Wangkheirakpam Radhapiyari Devi
14 Phytochemicals and Pharmaceutical: Overview�� 163
Jayakumari
15 Millettia Pachycarpa Benth: A Herbal Medicinal
Plant of Southeast Asia�� 175
Bishnupada Roy and Ravi Rao Bharti
16 Phytochemicals and Their Role in Pharmaceuticals�� 193
Anushree Suresh and Jayanthi Abraham
Part II Drug Discovery, Pharmaceutical Sciences

and Future Medicine
17 Plant-Mediated Green Synthesis of Nanoparticles �� 221
Balaprasad Ankamwar, Saili Kirtiwar, and Amritesh C. Shukla
18 Biomedical Applications of Green Synthesized Nanoparticles�� 235
Reetika Singh, Priyanka Tiwari, Nishi Kumari,
and Bechan Sharma
19 Role of Nanoparticles and Nanomaterials
in Drug Delivery: An Overview�� 247
Manasa Kumar Panda, Sujogya Kumar Panda,
Yengkhom Disco Singh, Bimal Prasad Jit,
Rajendra Kumar Behara, and Nabin Kumar Dhal
Contents ix
20 Micelleplexes: A Promising Nanocarrier for the Transport

of Genetic Material and Drugs �� 267
Jorge Faria, Mariana Magalhães, Francisco Veiga,
Ana Cláudia Santos, and Ana Figueiras
21 Phytomedicines and Their Prospects in Treatment
of Common Skin Diseases �� 289
Humaira Rani and Abhai K. Srivastava
22 Microbes as Natural Products for Drug Discovery�� 317
Sagarika Devi
23 Natural Products from Actinobacteria for Drug Discovery�� 333
Soumya Nair and Jayanthi Abraham
24 Anti-Leprosy Vaccine (Hansen’s Disease Vaccine)�� 365
Jerusha Santa Packyanathan, Ira Christabel Packyanathan,
and A. Indra Balini
25 Exploitation of Fibrinolytic Enzymes in Combating
Blood Clotting Disorders – Recent Advances and Strategies:
A Comprehensive Review�� 383
Sheela Kumari Sahoo and Sabuj Sahoo
26 Recent Development in Chronic Inflammation
Research and Mangroves as Potential Source
of Anti-inflammatory Agents�� 397
Dibyajyoti Samantaray and Swagat Kumar Das
Part III Clinical Trials and IPR in Pharmaceuticals

27 Tissue Engineering and Regenerative Medicines:
An Interdisciplinary Understanding�� 409
Benu George, Nidhi Lal, Jeyaram R. Damodaran,
and T. V. Suchithra
28 IPR: An Overview�� 439
Amritesh C. Shukla
29 Intellectual Property Rights and Its Role
in Natural Product Research�� 449
Rajat Nath, Anupam Das Talukdar, Priyanka Saha,
Subrata Das, Prakash Roy Choudhury, Deepa Nath,
and Manabendra Dutta Choudhury
30 Nanotechnology in Preclinical Pharmacokinetics�� 461
Santosh Malik, Ananya Ghosh, Rout George Kerry,
and Jyoti Ranjan Rout
Contributors
Jayanthi Abraham Microbial Biotechnology Laboratory, School of Biosciences

and Technology, VIT University, Vellore, Tamil Nadu, India
Balaprasad Ankamwar Bio-Inspired Materials Research Laboratory, Department
of Chemistry, Savitribai Phule Pune University (Formerly University of Pune),
Pune, Maharashtra, India
A. Indra Balini Guru Nanak College, Chennai, Tamil Nadu, India
Rajendra Kumar Behara School of Life Sciences, Sambalpur University, Burla,
Odisha, India
Ravi Rao Bharti Parasitology & Toxicology Laboratory, Department of Zoology,
North-Eastern Hill University, Shillong, Meghalaya, India
Jijnasa Bordoloi Academy of Scientific and Innovative Research, Chennai, India
Biological Science and Technology Division, CSIR-North East Institute of Science
and Technology, Jorhat, Assam, India
Sankhadip Bose NSHM Knowledge Campus, Kolkata – Group of Institutions,
Kolkata, India
Manabendra Dutta Choudhury Department of Life Science and Bioinformatics,
Assam University, Silchar, Assam, India
Prakash Roy Choudhury Department of Life Science and Bioinformatics, Assam
University, Silchar, Assam, India
Sonu Chouhan Faculty of Life Sciences, Mandsaur University, Mandsaur, Madhya
Pradesh, India
Jeyaram R. Damodaran School of Biotechnology, National Institute of
Technology Calicut, NITC Campus, REC, Calicut, India
Subrata Das Department of Life Science and Bioinformatics, Assam University,
Silchar, Assam, India
xi
xii Contributors
Swagat Kumar Das Department of Biotechnology, College of Engineering and

Technology, Biju Patnaik University of Technology, Bhubaneswar, Odisha, India
Sagarika Devi National Institute for Research in Tuberculosis, Chennai, Tamil
Nadu, India
Wangkheirakpam Radhapiyari Devi DM College of Teacher Education, Imphal,
Manipur, India
Nabin Kumar Dhal Environment and Sustainability Department, CSIR-Institute
of Minerals and Materials Technology, Bhubaneswar, Odisha, India
Anjum Dihingia Academy of Scientific and Innovative Research, Chennai, India
Anupam Dikshit Centre of Science and Society, IIDS, University of Allahabad,
Prayagraj, Uttar Pradesh, India
Jorge Faria Department of Pharmaceutical Technology, Faculty of Pharmacy,
University of Coimbra, Coimbra, Portugal
Ana Figueiras Department of Pharmaceutical Technology, Faculty of Pharmacy,
REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy,
Benu George School of Biotechnology, National Institute of Technology Calicut,
NITC Campus, REC, Calicut, India
Ananya Ghosh Post Graduate Department of Biotechnology, Utkal University,
Bhubaneswar, Odisha, India
Shekhar Jain Faculty of Life Sciences, Mandsaur University, Mandsaur, Madhya
Pradesh, India
Jayakumari Department of Pharmacognosy, School of Pharmaceutical Sciences,
Vels Institute of Science, Technology and Advanced Studies [VISTAS], Chennai,
Tamil Nadu, India
Bimal Prasad Jit School of Life Sciences, Sambalpur University, Burla, Odisha,
India
Jatin Kalita Academy of Scientific and Innovative Research, Chennai, India
Rout George Kerry Post Graduate Department of Biotechnology, Utkal University,
Khushboo Department of Biochemistry, University of Allahabad, Allahabad, Uttar
Pradesh, India
Contributors xiii
Saili Kirtiwar Bio-Inspired Materials Research Laboratory, Department of

Chemistry, Savitribai Phule Pune University (Formerly University of Pune), Pune,
Maharashtra, India
Abhishek Kumar Department of Biochemistry, University of Allahabad,
Allahabad, Uttar Pradesh, India
Nishi Kumari Botany Section, Mahila Mahavidyalaya, Banaras Hindu University,
Varanasi, Uttar Pradesh, India
Narendra Kumar Amity Institute of Biotechnology, Amity University Haryana,
Manesar, Gurgaon, India
Nidhi Lal School of Biotechnology, National Institute of Technology Calicut,
Mariana Magalhães Department of Pharmaceutical Technology, Faculty of
Pharmacy, University of Coimbra, Coimbra, Portugal
Rojali Maharana Environment and Sustainability Department, CSIR-Institute of
Minerals and Materials Technology, Bhubaneswar, Odisha, India
Jai Malik University Institute of Pharmaceutical Sciences – Centre of Advanced
Study, Panjab University, Chandigarh, India
Santosh Malik Departmentof Life Science, National Institute of Technology,
Rourkela, Odisha, India
Kalicharan Mandal Environment and Sustainability Department, CSIR-Institute
of Minerals and Materials Technology, Bhubaneswar, Odisha, India
Subhash C. Mandal Division of Pharmacognosy, Department of Pharmaceutical
Technology, Jadavpur University, Kolkata, India
Prasenjit Manna Academy of Scientific and Innovative Research, Chennai, India
Rohit Kumar Mishra Centre of Science and Society, IIDS, University of
Allahabad, Prayagraj, Uttar Pradesh, India
Devasish Murmu Environment and Sustainability Department, CSIR-Institute of
Minerals and Materials Technology, Bhubaneswar, Odisha, India
Soumya Nair Microbial Biotechnology Laboratory, School of Biosciences and
Technology, VIT University, Vellore, Tamil Nadu, India
Deepa Nath Department of Botany, Gurucharan College, Silchar, Assam, India
xiv Contributors
Rajat Nath Department of Life Science and Bioinformatics, Assam University,

Ira Christabel Packyanathan The Leprosy Mission Hospital, Chennai, Tamil
Nadu, India
Jerusha Santa Packyanathan Saveetha Dental College and Hospitals, Chennai,
Tamil Nadu, India
Manasa Kumar Panda Environment and Sustainability Department, CSIR-
Institute of Minerals and Materials Technology, Bhubaneswar, Odisha, India
School of Life Sciences, Sambalpur University, Burla, Odisha, India
Sujogya Kumar Panda Animal Physiology and Neurobiology, KU Leuven,
Belgium
Anand Pandey PG Department of Botany, Avvaiyar Government College for
Women, Karaikal, Puducherry, India
Rashmi Pandey Department of Pulmonary and Critical Medicine, King George
Medical University, Lucknow, Uttar Pradesh, India
Subhashree Pattnayak Environment and Sustainability Department, CSIR-
Institute of Minerals and Materials Technology, Bhubaneswar, Odisha, India
Humaira Rani Amity Institute of Biotechnology, Amity University Uttar Pradesh,
Lucknow Campus, Lucknow, India
Taniya Sengupta Rathore Faculty of Life Sciences, Mandsaur University,
Mandsaur, Madhya Pradesh, India
Jyoti Ranjan Rout School of Biological Sciences, AIPH University, Bhubaneswar,
Odisha, India
Bishnupada Roy Parasitology & Toxicology Laboratory, Department of Zoology,
North-Eastern Hill University, Shillong, Meghalaya, India
Priyanka Saha Department of Life Science and Bioinformatics, Assam University,
Sabuj Sahoo Post Graduate Department of Biotechnology, Utkal University,
Sheela Kumari Sahoo Post Graduate Department of Biotechnology, Utkal
University, Bhubaneswar, Odisha, India
Dibyajyoti Samantaray Department of Biotechnology, College of Engineering
and Technology, Biju Patnaik University of Technology, Bhubaneswar, Odisha,
India
Contributors xv
Ana Cláudia Santos Department of Pharmaceutical Technology, Faculty of

Pharmacy, University of Coimbra, Coimbra, Portugal
Kunja Bihari Satapathy Post Graduate Department of Botany, Utkal University,
Bechan Sharma Department of Biochemistry, University of Allahabad, Allahabad,
Uttar Pradesh, India
Amritesh C. Shukla Department of Botany, University of Lucknow, Lucknow,
Nitika Singh Department of Biochemistry, University of Allahabad, Allahabad,
Ravikant Singh Department of Biotechnology, Swami Vivekanand University,
Sagar, Madhya Pradesh, India
Reetika Singh Department of Biochemistry, University of Allahabad, Allahabad,
Yengkhom Disco Singh Department of Post Harvest Technology, College of
Horticulture and Forestry, Central Agricultural University, Pasighat, Arunachal
Pradesh, India
Abhai K. Srivastava Plant Diversity, Systematics and Herbarium Division,
National Botanical Research Institute Rana Pratap Marg, Lucknow, India
T. V. Suchithra School of Biotechnology, National Institute of Technology Calicut,
Anushree Suresh Microbial Biotechnology Laboratory, School of Biosciences
and Technology, VIT University, Vellore, Tamil Nadu, India
Anupam Das Talukdar Department of Life Science and Bioinformatics, Assam
University, Silchar, Assam, India
Priyanka Tiwari Department of Biochemistry, University of Allahabad, Allahabad,
Francisco Veiga Department of Pharmaceutical Technology, Faculty of Pharmacy,
About the Editors
Dr. Jayanta Kumar Patra, M.Sc., Ph.D. , is Assistant

Professor at Dongguk University, South Korea. He has

about 14 years of research and teaching experience in
the field of food, pharmacology, and nano-
biotechnology. To his credit, he has published more
than 140 papers in various national and international
peer-reviewed journals and around 30 book chapters in
different edited books and has also authored/edited 11
books in various publications like Studium Press,
India; Studium Press LLC, USA; Springer Nature;
Apple Academic Press, Inc., Canada; and CRC Press, a
member of Taylor & Francis Group.
Prof. Amritesh C. Shukla, M.Sc., Ph.D. , is Professor

in the Department of Botany, University of Lucknow,

India. He obtained his D.Phil. (1998) and D.Sc. (2010)
degrees in Botany from the University of Allahabad,
India, and served as Reader/Professor and Head in the
Department of Horticulture, Aromatic and Medicinal
Plants, Mizoram University, Aizawl, India (2007–2016).
He has published around 100 research papers/articles,
authored/edited 10 books, and handled many externally
funded research projects and has US, UK, Japan, and
Indian patents to his credit. He is in the panel of many
internationally reputed journals as an Editor, Associate
Editor, and Editorial Board Member and is Fellow of
various scientific societies. He has also been Invited
Scientist for Germany, China, Switzerland, the USA,
Australia, and Korea and was Visiting Professor at the
University of British Columbia, Canada, and University
of Mauritius.
xvii
xviii About the Editors
Dr. Gitishree Das, M.Sc., Ph.D. , is Assistant Professor

at Dongguk University, South Korea. She has 10 years

of research experience in the field of rice molecular
biology, plant breeding, endophytic bacteria, and green
nanotechnology and 3 years of teaching experience.
Her current research is focused on the biosynthesis of
nanoparticles using food wastes and plant materials
and their applications in the biomedical and agricul-
tural fields. To her credit, she has published around 70
research articles in international and national reputed
journals and 16 book chapters. She has also authored/
edited 6 books for Springer Nature, Apple Academic
Press, Inc., Canada and Lambert Academic publishers.
She is an Editorial Board Member of some national and
international journals.
Part I
Traditional and Ethnomedicine
Chapter 1
Traditional Folk Medicine and Drug
Discovery: Prospects and Outcome
Prakash Roy Choudhury, Anupam Das Talukdar, Deepa Nath,

Priyanka Saha, and Rajat Nath
Introduction
Traditional folk medicine has been a widely accepted phenomenon over the ages
and continues to be the most effective therapeutic entity around the world especially
in the underdeveloped countries; however, recently its acceptance in developed
nations can also be traced (Yuan et al. 2016). The surge in the popularity might be
due to its low risk of adverse side effects and positive attitude of people toward
using traditional medicine. In a broader context, this fact can be elaborated citing
examples of increased demand in herbal drugs and products, health products, cos-
metics, food supplements, etc. Moreover, herbal medicine has a profound influence
on the “health for all” slogan of WHO (Kesavan and Swaminathan 2017).
Before the advent of the modern medical system, there was exclusively one sys-
tem of therapeutic approach to combat the different diseases which are traditional
folk medicine. Members and healers of ethnic groups of different countries how-
ever, besides medicinal plant, used fungus, animal products, etc., to produce useful
herbal formulations in the past. Some of the public medicine systems of the globe
are Ayurveda, Siddha, Unani, traditional Chinese medicine, Iranian medicine, tradi-
tional Korean medicine, and traditional African medicine (Dias et al. 2012). Many
of the new age medications were developed based on these traditional medicine
system formulations. Therefore, traditional folk medicine system is the significant
and productive cradle of discovery and development of potent drug leads. In India,
Vedic scriptures were the oldest records which provide a reference to most of the
plant’s curative properties which were later transcribed to form the Ayurveda.
P. R. Choudhury · A. D. Talukdar (*) · P. Saha · R. Nath

Department of Life Science and Bioinformatics, Assam University, Silchar, Assam, India
e-mail: anupam@bioinfoaus.ac.in
D. Nath
Department of Botany, Gurucharan College, Silchar, Assam, India
© Springer Nature Singapore Pte Ltd. 2020 3

J. K. Patra et al. (eds.), Advances in Pharmaceutical Biotechnology,
https://doi.org/10.1007/978-981-15-2195-9_1
4 P. R. Choudhury et al.
Essential works on the aspect of Indian medicinal plant are the Charaka Samhita
and Sushruta Samhita. Knowledge, practice, and skills are the pillars of traditional
folk medicine attributed depending on the indigenous beliefs, theories, and experi-
ences of different cultures.
The World Health Organization (WHO) acknowledged the dependency of the
majority of the world population on traditional folk medicine. However, to avoid
undesirous consequences, WHO also stressed out the explicable use of herbal thera-
peutics (WHO 2013). Many researchers proposed different definitions of traditional
folk medicine. Some of them described that traditional medicines and folk medi-
cines are two separate entities. Others described traditional medicine as formalized
content of the long-standing herbal therapy communicated and practiced by human
beings from one to another generation, whereas folk medicine comprises of the
knowledge of healing and body physiology confined to experienced people, known
as healers, in a particular culture. This knowledge will later be inherited to the fam-
ily or members of the community (Wangkheirakpam 2018). Both traditional medi-
cine and folk medicine are supposed to be the equivalent form of one another.
However, adoption of these traditional and folk therapeutic knowledge by the cul-
ture of different origins can be called alternative medicine (Acharya and
Shrivastava 2008).
At present, a large number of traditional medicinal plants are under scientific
evaluation for their bioactive efficacy and safety, and as mentioned earlier, WHO is
actively participating on incorporating the information on medicinal plants in tradi-
tional folk medicine system. The successive sophistication in the field of phyto-
chemistry, pharmacology, computer-aided drug designing, etc., has supported to
decrypt these medicinal plants for getting a clear idea of their ethnomedicinal uses.
Noted examples of phytochemicals include Colchicine (Colchicum autumnale),
Paclitaxel (Taxus brevifolia), Artemisinin (Artemisia annua), Silymarin (Silybum
marianum), Quinine (Cinchona sp.), Salicin (Salix alba), Morphine (Papaver som-
niferum), Atropine (Atropa belladonna), Forskolin (Coleus forskohlii), Vincristine
(Catharanthus roseus), Capsaicin (Capsicum annuum), Vinblastine (Catharanthus
roseus), etc. In 2016, the value of the global herbal medicine market was USD 71.19
billion which was USD 63.05 billion in 2014. The worldwide trade market of herbal
medicine is annually growing at the rate of 10–15% (Joshi et al. 2015).
Lots of modern synthetic drugs today have their root in traditional medicinal
products discovered in the course of learning the use of traditional folk medicinal
plants. The urge for the synthesis and development of a new entity of drug from
herbal origin, which has no or negligible side effects, is in demand for many dis-
eases. Traditional folk medicine formulations are administrable in the form of raw
extract, decoction, or in the form of pellet or tablets. These are the standardized
forms in traditional folk medicine system. Any standardized extract or decoction
used for the therapeutic purpose must be screened for its efficacy to act consider-
ably. Investigations shall be conducted to identify the significant chemical active
moiety from the available or new natural products. Analytical methods developed
for identification, isolation, and characterization of natural bioactive components
1 Traditional Folk Medicine and Drug Discovery: Prospects and Outcome 5
Fig. 1.1 Discovery prospect of new drug entity from traditional medicine
are helping to accelerate the drug discovery process. However, it seems quite inef-
ficient and often soporific when it comes to product delivery. A sophisticated
approach for separation and fractionation hyphenated to UV and evaporative light-
scattering detector (ELSD)-based spectroscopic analysis along with spectrometries
like LC-MS, GC-MS, and NMR, X-ray crystallography, bioinformatics, network
modeling, etc. enable wide-range identification and assessment of plant-based natu-
ral product multiplicity and functionality (Fig. 1.1).
History of Traditional Medicine System
Natural products with unmatched diversity and functionality continue to inspire the
novel discoveries in physiology and medicine. Natural origin products are evolu-
tionarily enhanced as “drug-like molecules” and remain the paramount architect of
new leads of drug (Newman and Cragg 2016). Historically, medicinal discoveries
have extraordinary stories of drug development from natural products. The earliest
cuneiform records on clay plates, depicted from Mesopotamia (2600BC), acknowl-
edged the usage of cypress oil (Cupressus sempervirens) and myrrh (a gum or resin
extracted from Commiphora sp. for cough, colds, and inflammation) (Cragg and
Newman 2005). In Egyptian pharmaceutical record (2900BC), particularly the
Ebers Papyrus (1500BC), 700 plant-based drugs with use against various ailments
were documented (Borchardt 2002; Cragg and Newman 2013). Drug formulations
based on plant origin are the records documented in subscripts of the Chinese
Materia Medica (1100BC) namely Wu Shi Er Bing Fang (52 formulations),
Shennong Herbal (365 formulations), and Tang Herbal (850 formulations) (Cragg
and Newman 2005). Similarly, recorded documentation of Indian medicinal herbs
in Ayurvedic monographs date back from before 1000BC with approximately 857
formulations (Dev 2001). Dioscorides (100CE), a Greek philosopher, while travel-
ing throughout with the Roman armies to the then-known world, recorded the use,
collection methods, and storage of medicinally important herbs (Atanasov et al.
2015). Theophrastus (300BC), also a Greek naturalist and philosopher, contributed
to the documentation of medicinal herb (Sneader 2005). Galen (130–200CE), a
Roman teacher and practitioner of medicinal herbs, was well known for the com-
plex formulation of prescribing drugs (Sneader 2005). The Arabs, on the other hand,
preserved much of the Western world and Greco-Roman medicinal knowledge dur-
ing the Dark and Middle ages (fifth to twelfth centuries) (Cragg and Newman 2013).
Arabs, however, expanded their medicinal expertise by amalgamating their resources
with Indian Ayurvedic and Chinese herbs unknown to Greco-Romans. Further, in
the eighth century, Arabs were the pioneers to own a private pharmacy with Persian
pharmacist Avicenna who with his work The Canon of Medicine contributed a lot to
pharmacy and medicine (Cragg and Newman 2005).
Traditional Medicine System of India
In India, modeling archaeological expeditions followed by excavations showcased

information and evidence regarding the use of medicinal herbs and trees in the
middle Gangetic region during or in between the Middle and late Bronze Age (Sen
and Chakraborty 2017). India, a land of diverse culture, tradition, religion, belief,
and language, has varied medicine systems. Therefore, Indian society, more or
less, depends on traditional medicine systems since ancient times. A large part of
Indian rural population still receives plant-based therapeutics which stand on the
philosophies of three codified systems, namely, Ayurveda, Siddha, and Unani.
Among the three medicine systems in India, Ayurveda is possibly the most ancient
of the traditional medicine systems and practiced since the beginning of India’s
medicinal civilization (Sen and Chakraborty 2015). The Siddha system of medi-
cine originated in the southern part of India (Sen and Chakraborty 2015) mirrors
Ayurveda in many instances and has a concept, philosophy, and lifestyle-oriented
approach of its own. Unani, on the other hand, originated in Greece, developed in
Arabia and introduced in India by Arab and Persian settlers in the eleventh century.
Apart from these three codified medicine systems, the folk medicine system of
India has been playing a crucial role in maintaining health care in rural as well as
urban population. It is estimated that nearly 8000 plant species and approximately
25,000 active plant-based preparations are used by the rural folklore of India (Sen
and Chakraborty 2017).
The Necessity of Traditional Folk Medicine
Traditional medicines, particularly plant-based formulations, have been catering a

large mass of population for millennia. Several lifesaving drugs have been synthe-
sized from plant sources. It is assessed that approximately 75% of herb-based rem-
edy or therapeutics which has worldwide use were from traditional folk medicine
origin and a bunch of different synthetic equivalent of natural drugs is prepared
from plant-derived isolates (Saad et al. 2017). Reports also claimed that 60% of
available cancer drugs in the market or in phase II of clinical trials have a plant-
based origin (Cragg and Pezzuto 2016). Plant-based or derivative drugs of immuno-
suppressive, anticancer, antimicrobial, and cardiovascular are reported to be 80% of
the current drug share available for these diseases (Pan et al. 2013). More than or
approximately 70% active or mimetic units among 117 approved anticancer drugs
are produced and centered on natural products (Wachtel-Galor and Benzie 2011).
Globally, approximately 25% of prescription-based drugs are derived from herbal
origin, and it is estimated that not less than 121 such drugs are in use currently
(Wachtel-Galor and Benzie 2011). Between 2005 and 2007, 13 drugs approved in
America were of herbal origin, and 100 or more plant-based drugs are under clinical
trials (Katiyar et al. 2012). Among 252 drugs enlisted in the essential drug list of
WHO, 11% is particularly of plant origin (Pan et al. 2014; Sen et al. 2009). In India,
it is estimated that Ayurveda has 1200–1800 medicinal plants, Unnai has 400–700
medicinal plants, and Siddha compromises of 500–900 plants, while approximately
7500 medicinal plants are included in folk medicine (Sen and Chakraborty 2015).
Historically Important Natural Products
Traditional medicine has a profound contribution in developing most of the modern

age medicine. Some of the important medicines are listed in Table 1.1. Acetylsalicylic
acid or aspirin, an anti-inflammatory agent derived from salicin, a chemical found
in willow tree (Salix alba) bark, is probably the prominent example of natural
product-derived drugs. Morphine, an alkaloid isolated in 1803 from Papaver som-
niferum or opium poppy, is another example of a widely known natural product.
Morphine is very significant as a commercial drug and has been a source of parent
compound for several other famous molecules. After boiling in anhydrous acetic
acid, the crude extract of morphine is converted to heroine (diacetylmorphine)
which is then readily converted to codeine, a pain killer. Sumerians and ancient
Greeks used the poppy extract for the medicinal purpose, while Arabs used it as
addictive (Der Marderosian and Beutler 2002; Tarver 2014). In the seventeenth cen-
tury, digitoxin, isolated from Digitalis purpurea L. (foxglove), was found to be a
glycosidic cardiotonic which can improve cardiac conduction and cardiac strength
in respect to contractibility. Digitoxin and its derivative analogs have a long-standing
use in the management of inefficient blood pumping around the heart, a condition
Table 1.1 Some of the historically important natural product-based drugs

Parent compound Isolated from Used as/used against
Salicin Salix alba Anti-inflammatory agent
Digitoxin Digitalis purpurea Enhance cardiac conduction
Quinine Cinchona succirubra Antimalarial agent
Pilocarpine Pilocarpus jaborandi Chronic open-angle glaucoma, acute angle-closure
glaucoma and xerostomia
Paclitaxel Taxus brevifolia Anticancer agent
Artemisinin Artemisia annua Antimalarial agent
Silymarin Silybum marianum Hepatoprotective agent
Leptospermone Callistemon citrinus Anti-tyrosinemia
Galantamine Galanthus nivalis Alzheimer’s disease
Morphine Papaver somniferum Parkinson’s disease
Atropine Atropa belladonna Chronic obstructive pulmonary disease
Dronabinol, Cannabis sativa Pain relievers
cannabidiol
Capsaicin Capsicum annuum Pain relievers
Reserpine Rauwolfia serpentina Antipsychotic and antihypertensive agent
Galegine Galega officinalis Antidiabetic
Khellin Ammi visnaga Bronchodilator
Vincristine Catharanthus roseus Acute lymphocytic leukemia, acute myeloid
leukemia, Hodgkin’s disease, neuroblastoma, lung
cancer
Vinblastine Catharanthus roseus Hodgkin’s lymphoma, lung cancer, bladder cancer,
brain cancer, melanoma, testicular cancer
Penicillin Penicillium notatum Antifungal
Vancomycin Amycolatopsis Antibacterial and antifungal
orientalis
Erythromycin Saccharopolyspora Antibacterial
erythraea
Betulinic acid Betula pubescens Inhibitor of HIV replication
generally known as congestive heart failure (CHF), but has side effects (Der
Marderosian and Beutler 2002). The “US Department of Agriculture” (USDA)
under the scheme “exploratory plant screening program” in the 1960s at the
“National Cancer Institute” (NCI) extracted Taxus brevifolia (Pacific yew) which
led to the isolation of paclitaxel, a bioactive anticancer agent of plant origin (Cragg
1998). It was approved and marketed with the brand name Taxol in 1992. However,
due to the high–low ratio of demand–yield, it is now developed synthetically. Many
derived natural origin drugs are currently used to treat AIDS. After the epidemic in
the 1980s, the NCI and other alike organizations started to follow up researches to
explore the traditional medicine oriented for isolating a suitable drug entrant for the
effective control of HIV-I-infected lymphoblastic cells. This exploration results in
the isolation of Prostrain, from Samoan mamala tree (Homalanthus nutans) bark,
which outshined in human clinical trial (phase I) carried out by AIDS Research
Alliance in LA, California, in 2010 (Gustafson et al. 1992).
Natural organisms other than medicinal plants also provided a potent bioactive
entity with curative activity. Penicillin is the first example of such development in
this context. It was discovered in 1992 by Flemming from Penicillium notatum (a
fungus) (Abraham et al. 1941), a milestone discovery in the natural product history.
After the discovery of penicillin, which revolutionized the research in drug discov-
ery, the common interest in the development of antibiotics from microorganism has
paced up. New screening methods in the 1970s allowed the researcher to isolate new
antibiotics, namely, norcardicins, imipenem, and aztreonam. Seventy percent of the
surface of the Earth covered by ocean harbors marine organisms with diverse biodi-
versity (Der Marderosian and Beutler 2002). Therefore, marine organism acts as a
unique source for a potential drug candidate. A notable example of natural product
derived from the marine body is plitidepsin (Aplidin®), a depsipeptide isolated from
Aplidium albicans (Mediterranean tunicate) effective against various kinds of cancer
like melanoma, non-Hodgkin lymphoma, lymphoblastic leukemia, etc. (Urdiales
et al. 1996). Another potent drug isolated was Ecteinascidin 743 (ET743; Yondelis™)
from ascidian Ecteinascidia turbinata (Rinehart et al. 1990). In 2007, Ecteinascidin
743 (Trabectedin) has become the first anticancer drug of marine origin approved in
Europe, later then approved by the European Medicines Evaluation Agency (EMEA)
and is concluding phase III of the clinical trial for approval in USA (Mayer et al. 2010).
Challenges in Drug Development
The usage of traditional medicine sources as an initial step in the drug development
approach has specific advantages. Typically, the criteria for selection of a particular
species could be possible by following its ethnomedicinal use. The speculation
behind this approach is that the isolated active compounds from such types of plants
are supposed to be less harmful than those derived from plant species with no previ-
ous history of human use. The development of drug from Rauwolfia serpentina
(reserpine) and D. purpurea (digitoxin) is under this style of approach. Contrarily,
the development of the drug from natural source also has certain disadvantages.
Occasionally, development and subsequent commercialization of the drug would
noticeably coerce the natural resource and might trigger adverse ecological alarms.
However, synthesis of the active compound might be a route in this regard, but every
molecule is not compliant for comprehensive synthesis. Hence, a strong level of
dependency on the lead natural supply would continue. Chief challenges in the
development of new drug leads are primarily coming from the two categories: the
prevalent paradigm of drug discovery in pharmaceutical productions and limited
technical resource and possibility in the identification and isolation of new com-
pounds with the desired activity. In general, compounds isolated from natural origin
have incomparable characteristics like high steric complexity, higher number of chi-
ral centers and oxygen atoms, lower aromatic ring atoms, higher number of hydro-
gen bond donors and acceptors, greater molecular rigidity and broader distribution
of molecular mass, etc. These mentioned chemical properties of natural product
attribute to the complexity in drug development analogs and posed a significant

challenge to the researcher as they initiate the process either absorption improve-
ment or try to lower the toxicity by focusing upon addition or removal of the unde-
sired functional group for improved efficacy. It can be presumed that most of the
natural origin products having biologically active properties and ADMET (absorp-
tion, distribution, metabolism, excretion, and toxicity) profile does not match the
criteria of drug-likeness. Thus, the challenge is of developing a physicochemically
tuned library of natural products in addition to the lead molecules generation.
Christopher A. Lipinski in 1997 formulated a set of four characteristic properties
called as “Lipinski’s rule of five” to determine the drug-likeness of a compound or
to evaluate if a natural product-based, pharmacologically, and biologically active
lead molecule could make up to orally active drug in humans (Lipinski et al.
1997). To be a drug-like molecule, a candidate must have the following:
1 . <5 H bonds donors
2. <10 H bonds acceptors
3. <500 Da molecular weight
4. <5 partition coefficient log P
The “rule of five” is postulated to ascertain the bioactivity problems if a drug has
been found to violate two or more dug-likeness properties.
Prospects and Outcome
Among 450,000 higher plant species on Earth (Corlett 2016), more than 80,000
species are estimated to have at least 1 or more medicinal value (Chen et al. 2016).
There are about 400 flowering plants families in the world, of which approximately
315 species are found in India (Karunamoorthi et al. 2013). According to WHO,
around 21,000 plant taxa have the prospects for being used as medicinal plants
(Bose et al. 2001). Although some of the healing properties ascribed to medicinal
plants were proven to be inaccurate, however, the usage of medicinal plants in the
treatment of various ailments is a well-known fact and preserved since ancient times.
With herbal “renaissance” happening all over the globe, herbs are retaliating.
Herbal therapeutics today represent safety in contrast to their synthetic counterparts
that are supposed to have ill effects to humans and the habitat. The emphasis is now
on switching chemical entities with natural product-derived ones. People prefer tra-
ditional treatment for several reasons. They might be from a remote area where
modern medication is unavailable during an emergency condition. They may belong
to societies whose habits and behavior direct them to traditional medicine (TM) as
the first line of therapy. They may prefer TM by believing that traditional remedies
have fewer side effects and more curable accuracy. They may perceive modern-age
health conveniences as expensive and unfriendly. Over the decades, the scientific
community worldwide is interestingly inclined toward the ancient traditional medi-
cine systems to reconnoiter the opportunities in formulating novel phytotherapeutic
leads (Karunamoorthi et al. 2013). Recently there was a boom in the traditional drug
discovery approach with 10 of the 44 approved drugs (Newman and Cragg 2016)
based on TM and their derivatives, which accounts for 25% of the 44 approved new
chemical entities.
At present, there are still few diseases like antihistamines, diuretics, and hypnot-
ics for which only synthetically sourced drugs are available (Newman and Cragg
2016). Two plant-derived drug leads, omacetaxine mepesuccinate or homohar-
ringtonine (used in of chronic myeloid leukemia, first reported from Cephalotaxus
harringtonii) and ingenol mebutate (found in Euphorbia peplus), which act against
actinic keratosis (a precancerous condition, which usually leads to melanoma if
untreated), were approved in 2012 by the FDA (Newman and Cragg 2016).
The natural product-based small-molecule entities approved from 2011 to 2014
have pointed out that there were significant numbers of antitumor, antibacterial, and
antifungal drug leads approved so far (Newman and Cragg 2016). Recently, Nobel
Prize in Physiology or Medicine (2015) was shared by William C. Campbell and
Satoshi Omura for their groundbreaking discovery and development of the aver-
mectin/ivermectin complexes, fermented product by Streptomyces avermitilis, and
Prof. Youyou Tu for her discovery of artemisinin (Shen 2015), is convincingly an
excellent update in the particular field. These discoveries sourced from the natural
product are the living examples that traditional medicine has a lot to provide us in
the coming days. It is often assumed that the major challenge in producing a natural
product-based molecule and introducing the same in the market are not the isolation
and synthesis of the drug but the huge supply-related problems faced by scientists
in translating laboratory discoveries to the commercial product (Cragg and
Newman 2013).
Natural product and its derivative have a predominant role in the discovery of
lead molecule for the drug development associated with the wellness of human
beings. A multidisciplinary attitude to drug discovery will involve the generation of
novel diversity of molecules from traditional medicine sources, associated with
total and combinatorial synthetic procedures and with modified biosynthetic path-
ways, which in turn continue to provide the paramount solution to the existing pro-
ductivity crisis of natural product-based leads.
Conclusion
Traditional medicine system is an age-old practice that humans have mastered, in

particular, the usage of ethnomedicinal plants through minute observation followed
by hit-and-miss experiment. Even in the age of modern computational and pharma-
cological approach, ethnomedicinal plants aid as an important source and tool for
the treatment of several ailments in the developing nations as well as in some parts
of the developed countries. The world’s most effective antimalarial drugs such as
chloroquine and artemisinin are the gifts of valuable traditional medicine knowl-
edge and culture of ethnomedicinal plants. The recent accomplishment in drug
development from natural product motivates and inspires many process chemists to
scrutinize and authenticate the uses of ethnomedicinal plants. However, there are a
lot of burning issues and daunting encounters that need to be satisfied and addressed
efficiently and immediately for the elevation of traditional medicinal plants. The
associative efforts of researchers in the field of medicine and physiology, more par-
ticularly ethnobotanists, anthropologists, pharmacists, and physicians, could be an
attainable strategy to evaluate and validate the culture and custom of traditional
medicinal plants with the existing modern scientific procedures and advanced
techniques.
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Chapter 2
Ethnomedicine for Drug Discovery
Yengkhom Disco Singh, Manasa Kumar Panda, and Kunja Bihari Satapathy
Introduction
Ethnomedicine is a study about the traditional system of medicine practiced by vari-

ous ethnic groups or communities around the world to cure a particular disease,
especially by indigenous peoples of a particular locality. It comprises studies of the
traditional medicine and its methods of using ethnomedicine and anthropological
aspect of the system. However, a scientific method of ethnomedicine studies consti-
tutes drug discovery and identification of a potential drug with a marketable phar-
maceutical product. Folk and traditional medicines were the soul therapy that was
carried out in older time to treat many types of diseases before the modern concept
of medicine has evolved. A part of plant product along with fungus or animal mixed
with other minerals has been used to form the drug to heal a particular disease. The
traditional system of medicine is still prevalent in many areas around the globe, and
it is suggested to contribute 80% of the world population still depends on the herbal
product to cure their primary healthcare needs. The belief on traditional system of
medicine is still a boost to modern science due to the adverse side effect of using of
modern drugs. Perhaps, this is one of the main reasons why the folklore claim is
increasing all over the world (British Medical Association 1993).
Y. D. Singh (*)
Department of Post Harvest Technology, College of Horticulture and Forestry, Central
Agricultural University, Pasighat, Arunachal Pradesh, India
M. K. Panda
Environment and Sustainability Department, CSIR-Institute of Minerals and Materials
Technology, Bhubaneswar, Odisha, India
K. B. Satapathy
Post Graduate Department of Botany, Utkal University, Bhubaneswar, Odisha, India

https://doi.org/10.1007/978-981-15-2195-9_2
16 Y. D. Singh et al.
Traditional systems of medicine totally depend on the type of plants and resources
available in a locality, and those practices have been inherited from their ancestor to a
new generation. The medical system is confined to different tribal and ethnic groups
of the world. However, some known traditional medicines are Ayurveda, Siddha med-
icine, Irani, Unani, Islamic medicine, traditional Chinese medicine (TCM), traditional
Tibetan medicine (TTM), Greek medicine, Korean medicine, and African medicine.
The presence of natural compounds in plants in the form of secondary metabo-
lites, which are derived biosynthetically from plant primary metabolites such as
amino acids, and carbohydrate leads to drug discovery and development. The pres-
ence of secondary metabolites in the plants can be broadly divided into three groups
based on the chemical nature of the compound. Firstly, terpenes consist of a chemi-
cal compound made from mevalonic acid composed from carbon and hydrogen;
secondly, phenolics made from simple sugars are composed from benzene rings,
hydrogen, and oxygen; and thirdly, alkaloids are extremely diverse and usually con-
tain sulfur (Harborne 1984). Some of the metabolites may contain rings and sugars,
and some may contain nitrogen or not, or some may be soluble in various solvents.
In India, the earliest records on herbal care system were found in the Veda culture
that later formed the Ayurveda which is a roughly 5000-year-old system of natural
healing. It has been said that Tibetan medicine, traditional Chinese medicines, and
Greek medicine have their roots or concepts originally described in classical
Ayurveda. In the case of Chinese traditional medicine, it is well organized, and the
medical system is well accepted by the people. Perhaps, the reasons may be due to
poverty, unavailability of modern medical facilities, or strong affinity toward healing
suggested by the traditional practitioner (Sujata and Warjeet 2016a, b). Traditional
Tibetan medicine (TTM) is similar to other Asian medicine systems. The system is
greatly influenced by the Indian Ayurvedic system of medicine and is based on the
belief that the human body is balanced by three kinds of internal energies. One
major part of this system of medicine is that Buddhist theology and its concept of
“three poison” have been greatly accepted. The Siddha system of medicine is also an
Indian traditional system of medicine in which the entire literature is written in
Tamil and is of Dravidian origin. The basic concepts of Siddha medicine are the
same as those of Ayurveda except the root which is mostly influenced by ancient
Dravidian culture. Iranian traditional medicine (ITM) is an old system of medicine
with 1000 years of history rooted in the boundless land of knowledge (Najmabadi
1987). Islamic medicine was fully inspired by the Holy Quran and Ahlul-Bayt doc-
trine. Later on, it was integrated with ITM (Najmabadi 1987). The Yunani or Unani
system of medicine originated from the Perso-Arabic culture which was practiced in
the Mughal Empire in India and South Asian continents. It is a comprehensive medi-
cal system, which deals with health and disease. It provides curative and rehabilita-
tive healthcare. The Greek system of medicine is one of the most dominating systems
of holistic healing in Western civilization. It provides inspiration for many other
natural products and holistic and alternative traditional medicine system in devel-
oped countries in the United States and many other European countries.
Plant as a main source of ingredients with other natural products of a particular
region is being used for the treatment of various ailments. The common plants such
2 Ethnomedicine for Drug Discovery 17
as tulsi, turmeric, ginger, neem, marigold, etc., are used for treating different
diseases. Some of the lower plants such as mushrooms and bryophytes are also used
in healing the ailments. In recent years, the use of traditional medicine and their
products for drug discovery has been increasing all over the world, especially in the
areas like allergies, menstrual-related problems, high blood pressure, and general
well-being (Sujata and Joshi 2017, WHO 2000, Deepak and Anshu 2008).
Traditional knowledge and folk medicines are the knowledge of using different
plants and other natural products based on the beliefs, theories, and culture for the
maintenance of healthy life and freedom from mental illness. As par, the World
Health Organization (WHO) reports 80% of the world population is still dependent
on the traditional system of medicines. It is high time to carry forward research
activities on such traditional and folk medicine to ascertain the efficacy and safety
used of several practices of plants and other natural products. The system is not only
confined to Asia and Africa but also to other parts of the world as primary healthcare
needs. The traditional system of medicine may have a different outlook when the
terms are concerned. However, it can be referred to as alternative medicine, indige-
nous medicine, folk medicine, or natural healers. At present, a large number of plants
and other natural products are used for scientific investigation so as to protect from
implication which occurs due to improper utilization of the medicines. Further, with
the new advancement of tools and technologies, there is a gradual change in better
understanding of the plants for ethnomedical uses. The phytochemistry of such
potential plants used as traditional medicine can be exploited. In most of the plants,
the phytochemical composition for traditional medicine includes Taxol, vincristine,
vinblastine, colchicine, artemisinin, etc. The global rate of the medicinal plant is at
the rate of 10–15% with global trade of USD 100 million/year (Joshi et al. 2015).
Natural products and plants are the sources for the discovery of modern drugs.
The bioactive molecules present in the traditionally used medicinal plants took a
great role in curing and treating the different ailments, for example, paclitaxel (com-
mon name Taxol) which is a drug derived from traditional medicinal plants Taxus
baccata used in inhibiting the growth of cancer cells. The new drugs developed
from traditional medicine will have fewer or negligible side effects and are the
demand of the current research for many diseases. Some plant extracts such as sily-
marin are used to treat liver disorders. Extracts of plants such as Psidium guajava,
Azadirachta indica (neem), Ocimum sanctum, Momordica charantia, and Carica
papaya have been extensively used for the therapeutic use of dengue. A suitable
analytical method for extraction of single or standardized extraction and its identifi-
cation and quantification of molecules in herbal medicine should be established in
order to ascertain the traditional knowledge. Various analytical techniques such as
open column chromatography, gas chromatography mass spectrometry (GCMS),
liquid chromatography mass spectrometry (LCMS), ultraviolet (UV), infrared (IR),
nuclear magnetic resonance (NMR), and X-ray diffraction have been used to iden-
tify certain bioactive molecules present in the traditional system of medicine (Sujata
and Warjeet 2016a, b). Synergetic effects of bioactive molecules in the form of
extract works better than a single molecule.
Traditional Healing Practices
Traditional medicine system (TMS) is sometimes referred to as complementary and

alternative medicine (CAM). CAM is the modified form of TMS. TMS has become
an indispensable part of our healthcare system. It is well known that TMS covers a
wide array of therapies and practices around the world and different medicine sys-
tems were developed with available natural resources, minerals, and cultural beliefs
of a particular region. Some of the practices could be named as Ayurveda, Egyptian,
Mongolian, Tibetan, ancient African (Iboga, Muti, Nganga, Hausa, South Africa,
Yoruba), ancient Greek, ancient Iranian, Byzantine, Irani, Roman, Islamic, Unani,
Siddha, Sri Lankan, Thai, Vietnamese, Chinese, Japanese, Korean, American
(Aztec, Brazilian, Curandero, Kallawaya, Mapuche, Maya), Australian Bush, and
Western European. However, few are well-established traditional medicines such as
Ayurveda, Siddha, Unani, ancient Iranian, Irani, Islamic medicine, TCM, traditional
Korean medicine, etc. About 80% of the world populations rely on TMS; thus, a
phytochemical study and identification of a particular bioactive compound are an
essential part. Nowadays, every TMS needs to be well verified to ascertain the
proper medication, and this leads to drug discovery.
Ayurveda
Ayurveda is a well-known Indian system of traditional medicine. It originated in

India way back in the pre-Vedic period. The famous Ayurveda texts like Charaka
Samhita and Sushruta Samhita were documented about 1000 years BC. The term
Ayurveda means “life knowledge or science of life.” It deals with knowledge of
healthful living during its various phases and a wide range of therapeutic measures
against different diseases. Some have written that Ayurveda was well developed
during the time of Gautama Buddha (563–483 BC). The Charaka Samhita is more
related to Atreya Punarvasu and that of Sushruta Samhita with Dhanvantri. The
origin of Ayurveda was also found in Atharvaveda. It means knowledge storehouse
of Atharvanas, the procedures for everyday life. In fact, Atharvaveda consists of
around 730 hymns and 6599 mantras. Every prose line relates to the human body,
disorders, and possible cures. Atharvaveda is considered as the forebear of Ayurveda.
Perhaps, Ayurveda is one of the oldest forms of the healthcare system which has
close similarity with the concept of the World Health Organization (WHO) for mod-
ern drug therapy. Some of the important texts which consist the Ayurveda are Bhela
Samhita, Sushruta Samhita, Charaka Samhita, Astanga Samgraha, Ashtanga
Hridaya, Sharngadhara Samhita, and Bhavaprakasa. The Ayurveda also described
the five element ideas (Pancha Mahabhutas). These five elements originated from
Pancha Tanmatra. Tanmatra means subtle essence. In Ayurveda, the body functions
and the component are similar to nature’s component and functions. Pancha
Mahabhuta also states that everything is composed of five elements, i.e., Akash
(Ether), Vayu (air), Agni (fire), Jala (water), and Prithvi (earth). Ayurveda is not only
about medicine, but it is all about a way of living with healthful life, prevention
from disease, and personal hygiene (Robert 2004; Robert et al. 2005; Narayanaswamy
1981). Ayurveda is a science that describes various phases of life, including happi-
ness and unhappiness. This ancient science deals with not only medication but also
with maintaining a good, healthy body and mind. Ayurveda is the beacon of light for
those who believed in TMS. Hence it is a complete science that helps both the
healthy and the sick person.
It offers a body of wisdom with a sound mind. Ayurveda is the state of balanced
health and dynamic integration of body, mind, and spirit. It also describes three
fundamental energies that govern our environment: movement, transformation, and
structure. The three primary forces such as Vata (wind), Pitta (fire), and Kapha
(earth) (Fig. 2.1) are responsible for the characteristics of our mind and body.
Sometimes, these three energies are also termed as three doshas (also called as
humor). It can be described as balanced doshas are for mind–body harmony, whereas
imbalanced doshas are for disease or any stress in lifestyle.
Different ingredients of natural products are used in Ayurvedic medicine. A natu-
ral product from plants (roots, leaves, fruits, bark, or seeds of plants such as carda-
mom, ginger, turmeric, and cinnamon are used for therapy) and animals (milk,
bones, fats, and gallstones), toxic metals, and minerals are used in formulation of
the drugs by the traditional practitioner. It also mentioned that some other products
such as oil and tar can be used to stop the bleeding of blood. The addition of metals,
minerals, or gems in the plant extract not only helps in enhancing the potential of
drugs but also curing a particular ailment. Sometimes this addition of the different
Earth
Space Water
Kapha
Vata
Pitta
Air Fire
Fig. 2.1 Panchamahabhutha and tridosa relationship

products in the herbal drug is called Rasa Shastra. The minerals such as sulfur, arse-
nic, lead, copper sulfate, gold, lead, mercury, etc. are used in Ayurveda medicine
preparation. Ayurveda also uses alcoholic beverages and purified opium to balance
the three doshas. While making the ayurvedic formulation, the practitioner should
have in-depth knowledge on qualitative and quantitative properties of the plants,
otherwise, adverse side effects will hamper the balance in the body. In the sense, the
practitioner should have correct knowledge on dosage required for a particular for-
mulation. There is a huge communication gap between the traditional practitioner
and modern medicine. The Indian Government has taken some initiative by setting
up some of the research institutions on Ayurveda to seal the gap between these two.
The Traditional Knowledge Digital Library (TKDL) was set up in 2001 as one of
the networks for the repository of various formulations such as Ayurveda, Unani,
and Siddha. TKDL is a joint collaborative project between the Ministry of Science
and Technology and the Ministry of Health and Family Welfare (CCRAS and TKDL).
Traditional Korean Medicine
Traditional Korean medicine (TKM) is one of the most primitive systems of medi-
cal practice in Korea and was originally started around 5000 years ago. During this
period, many therapeutic methods and unique theories were developed (Kwon 2000,
Chae et al. 2003, Kim 1993, Eisenberg et al. 1998). TKM has a unique history with
a collection of different medical formulas that provide the most extensive databases
(Lim et al. 2016, Kee 1999). TKM is also focused on the careful observation of
natural bodily functions rather than dissection or experimentation. It never confined
to a particular cause of a minor ailment but examines the entire body to find out the
exact cause of the disease (Chae et al. 2003). It encompasses different types of prac-
tices such as acupuncture, moxibustion (burning of herbal parts above the skin),
aromatherapy (treating the ailments with aromatic products of the plant extract such
as essential oil from lemon grass), meditation (relaxing and calming the mind for
mental peace), and herbal medicine. Traditional remedies supplemented with acu-
puncture have found that a patient’s mental state is more stable and relaxed. TKM is
well recognized at the international level and has sufficiently reasonable solutions
as compared with Western medicine. The Korean book, Dong-eui Bo-Gam, was
written based on traditional medicine during the seventeenth century and got regis-
tered in UNESCO (Kim et al. 2013).
Traditional Chinese Medicine (TCM)
Traditional Chinese medicine (TCM) is a healing system of Eastern medicine origi-

nated in ancient China more than 2000 years ago. TCM involves herbal medicines
and various mind and body practices to prevent diseases. Chinese immigrants to the
United States began practicing TCM in the early 1820s; however, Americans
became interested about TCM through the practice of acupuncture, and now they
started using TCM as a complementary healthcare system even if they use their
conventional traditional therapies as well. In 1997, it was estimated that approxi-
mately 10,000 practitioners were serving more than 1 million patients each year in
the United States. According to the National Health Interview Survey (NHIS) 2007,
3.1 million US adults had already used acupuncture as a therapy for curing different
diseases. The number of patients who visit the acupuncture center had raised triple
in between 1997 and 2007. NHIS report also suggested that about 2.3 million
Americans practiced Tai chi and around 600,000 practiced qi gong as alternative
medicine (National Center for Complementary and Integrative Health (NCCIH)). In
addition to treating illness, TCM also focuses on keeping the mental relax by
strengthening the body’s defense mechanism. TCM encompasses many practices
including acupuncture (very fine needle placed gently in the skin); moxibustion
(burning an herb above the skin to apply heat to acupuncture points); Chinese herbal
medicine (teas, powders, and capsules made mostly from plants); tui na (Chinese
therapeutic massage); dietary therapy, tai chi, and qi gong (practices that combine
specific movements or postures, coordinated breathing, and mental focus); cupping
(heated cups that create suction on your skin); inspection of the tongue; and medita-
tion (a way to sit quietly and calm your mind) (Kam and Liew 2002, Asaf 2008).
The traditional systems of medicine which exist in other East and South Asian coun-
tries, including Japan and Korea, are greatly influenced by TCM, but each has
developed distinctive features of its own. TCM is also quite similar to Ayurveda in
many forms. Actually, it was guided by a holistic concept. The five-element theory
which makes the development and maintenance of the human body also applies in
TCM. However, the five substances are different from Ayurveda’s five elements.
Here, the five substances are Qi, blood (xue), essence (jing), spirit (shen), and fluid
(jin ye). According to this five-element theory, it is said that everything is main-
tained in kinetic equilibrium under the movement of five elements in the body. TCM
uses more than 365 different types of medicinal substances prepared from plant
extracts, animal products, minerals, and other natural products for the treatment of
diseases. The prescription of the medicine may comprise of a single product or for-
mulation of different mixtures of herbs in different dosages. There are four catego-
ries of prescribed medicines based on the properties: Zhing, Chen, Zhou, and Shi.
In most of the cases, complex herbs are prepared using different herbs. For example,
the formulation of Danggui-Nian-Tong-Tang for treating acute gouty arthritis needs
15 different herbs. Another example of drugs that were found to contain antimalarial
agent is artemisinin which was usually given against fever. Now, the artemisinin and
its synthetic derivatives are developed from Qing hao (Artemisia annua). Tu Youyou
got the Nobel Prize in 2015 for her works on artemisinin (Youyou 2011).
Iranian Traditional Medicine (ITM)
Iran (formerly called Persia) is located in Southwest Asia. It covers a territory of

1,648,195 square kilometers with a population of about 70 million people. The
unique features of Iran’s geographical position make it more diverse in climatic
conditions of 12 different geographical environments. Due to this variety of climate,
there are more than 7500 plant species available in the region, and about 1800 spe-
cies are used in medicine. However, many Indian medicinal plants were brought to
Persia during the Sassanid dynasty in the sixth century CE, Burzuya (Perzoes in
Latin). The ITM is greatly influenced by the Indian traditional medicine. The ITM
is an amalgamation of the prehistoric beliefs and practices of the early ancient civi-
lization such as Mesopotamian, Babylonians, Assyrians, and Elamites (Elgood
1951). Avesta (the holy book of the Zoroastrians, aka Zarathustrans) is one of the
main sources of herbal knowledge used by ancient inhabitants of Iran. According to
this book, Thrita (Serita) was the first mythological physician who gave thousands
of healing plants (urvaro baesada in the Avestan language) to the Ahura-Mazda (the
wise god of light in the theology of Zoroastrianism). Probably, the word drug was
derived from the ancient Iranian word darav, meaning the stem of a plant. This word
later changed into droga in Latin, drogue in French, daru in Persian, and drug in
English. Many herbal medicines are introduced in this book for various ailments. It
also described herbal extractions, infusion, decoction, abortifacients, and disinfec-
tants in section 2 of Avesta (Doustkhah 1991, Bahrami and Joneydi 2000,
Abulghassemi 2000, Faravashi 2002). In addition to this, two medical scholars who
contributed to the Persian medical system were Rhazes and Avicenna. They had
written two medical books called Liber Continens and The Canon of Medicine.
These books are the collection of Greek, Indian, and Persian ancient healing system.
African Traditional Medicine (ATM)
ATM is one of the oldest and assorted therapeutic systems. Africa is a land of diver-
sity with various regional differences in healing practices (Gurib-Fakim 2006). Like
other traditional medicine, ATM also involves both body and mind approaches. The
traditional practitioners typically diagnose and treat the patient based on the psy-
chological thinking before prescribing the medicine. The prevalent practice of ATM
in the African healthcare system is due to two major reasons. The first one is inad-
equate access to modern medical care as the people of Africa could not afford the
costly allopathy medicines. Second, there is a lack of awareness among the people
for modern medicine and also lack effective modern medical treatment for some of
the prone disease such as malaria or HIV/AIDS. Across Africa, medicinal plant
extracts are the most common medicine given to patients as they are easily acces-
sible. Most of the traditional practitioners in Africa are very close to their patients
and provide information, counseling, and treatment.
Africa is a land with various fauna and flora. Forty-five thousand plant species
are found in Africa, and out of these, 5000 are being used in traditional medicine
preparation. This land enjoys the beauty of diversity as it has a tropical and subtropi-
cal climate which is quite important for the plants in such a hostile environment to
produce secondary metabolites (Manach et al. 2004). Because of her geographical
tropical location, the region receives a large amount of radiation of ultraviolet rays
from sunlight, and this helps the numerous pathogenic microbes, plants, and other
animals to accumulate chemopreventive substances more than the plants from other
parts of the world. Research has found that some plant species like Dorstenia man-
nii (Moraceae) contained more biologically active molecules than other related spe-
cies found outside of Africa (Abegaz et al. 2004; Farnsworth et al. 1985). It is high
time to safeguard and document the African traditional medicine system as it is
becoming rapidly lost due to anthropogenic activities and also due to an erosion of
valuable traditional knowledge. Forest areas cover 216 million hectares in Africa, of
which 1% per annum is vanishing due to deforestation. Some of the common
medicinal plants practiced by the traditional healers in Africa are Acacia senegal,
Aloe ferox, Artemisia herba-alba, Aspalathus linearis, Centella asiatica,
Catharanthus roseus, Cyclopia genistoides, Harpagophytum procumbens,
Momordica charantia, and Pelargonium sidoides.
Ancient Greek Medicine
The ancient Greek medicine system embraced the concept of a healthy mind in a
healthy body which links the physical and mental well-being of a person. The sys-
tem was a compilation of theories and practices. The great physicians like
Hippocrates and Asclepius were some of the pioneer workers on ancient Greek
medicine. Hippocrates, the great physician and philosopher of ancient Greece, is
known today as the father of medicine. Asclepius was considered as the son of
Apollo. Asclepieia was constructed in healer’s god name. Asclepieia provided heal-
ing requirements to the patients. The Temple of Asclepius in Pergamum is famous
for its healing by drinking the spring water that flowed down into the underground
room. Medicine such as hot teas (chamomile) was used to calm and treat against
headaches. Ancient Greek medicine also began to revolve around the theory of
humors. According to this theory, good health comes from equilibrium of four
things: blood, phlegm, yellow bile, and black bile. The ancient Greeks believed that
illness was divine punishment and healing was a gift from the gods (Cartwright
2013). Nonetheless, by the fifth century BCE, led by Hippocrates, this concept of
the gods’ influence over health was erased, and attempts were made to identify the
material causes for illness rather than spiritual ones. They began to take a greater
interest in the body itself for exploring reasons for causing the disease. Hippocrates
and his followers documented numerous illnesses in the Hippocratic Corpus.
Actually, Greek medicine had a great influence on Islamic medicine and medieval
European medicine.
Drug Discovery
Drug discovery is the process of discovering new drugs from natural products. It
involves a wide range of scientific disciplines, including physiology, biochemistry,
and pharmacology. The discovery of drugs is a multistep process starting from iden-
tification of screening hits, the chemistry of the medicine, optimization of those hits,
to oral bioavailability. After the fulfilment of the required criteria, further, the drug
is processed for drug development prior to clinical trials. However, some computer-
aided design can also be done, but it has to confirm the properties of the drugs by
doing real experiments. The modern drug discovery is thus usually a capital-
intensive process which takes place huge investments by the pharmaceutical com-
panies as well as government organizations. The final product of the drug or
compound is patented before the companies buy the product. Some of the examples
of the drug discovered from traditional-based medicine are described as given below.
Jasmonates
Jasmonate is one of the lipid-based plant hormones that regulate a wide range of
functions including photosynthesis and reproductive development. Methyl jasmo-
nate, a derivative of jasmonate, is isolated from Jasminum grandiflorum oil.
Jasmonates are useful in response to injury and intracellular signals. They also
induced program cell death and protein cascade, have defense function, and regulate
response against the biotic and abiotic stresses. It can directly act on the mitochon-
drial membrane through the depolarization and release the metabolites (Rotem et al.
2005). They also help in responding to wound and tissue regeneration in plant cells.
Some researcher cited it as an antiaging effect on human epidermal cells (Michelet
et al. 2012). This discovery has introduced new interest in the skin repair using this
plant hormone.
Salicylic Acid (SA)
Salicylic acid is one of the phytohormones found in the plants and initially derived
from the willow bark. This chemical is used by plants as their defense response
against the pathogens. Structurally, it has an OH group at the ortho position as a
functional group with formula C6H4 (OH) COOH. It is called as 2-hydroxybenzoic
acid. It is used in removing the outer layer of the skin, warts, psoriasis, acne, ring-
worm, dandruff, and ichthyosis. In the animal, there are salicylic binding proteins
(SABPs) which affect the animal tissues (Klessig et al. 2016). Perhaps this is the
reason why this compound is used as antiseptic, antibacterial, and sometimes in
food preservation. This molecule can induce the suppression of cell proliferation
and death in lymphoblastic leukemia and other human cancer cells (Fingrut and
Flescher 2002). Aspirin is structurally a salicylate (acetylsalicylic acid) compound
used as anti-inflammatory and antipyretic (Klessig et al. 2016; Pierpoint 1994).
Artemisinin
It is a drug obtained from sweet wormwood called Artemisia annua. The plant has
been used in Chinese medicine against the malarial disease. However, the com-
pound was firstly isolated in 1972 by Tu Youyou, a Chinese scientist, and she got the
Nobel Prize in 2015. Chemically it contained peroxide bridge (a sesquiterpene lac-
tone) which is responsible for the drug’s action and its mechanism. Monotherapy
treatment is explicitly discouraged by the WHO due to its sign of developing resis-
tance to the drug. Hence, combined artemisinin or its derivatives with some other
antimalarial drug are usually used in treating malaria disease.
Paclitaxel
It is a compound isolated firstly from the bark of Pacific yew tree Taxus brevifolia.
This compound showed a different type of anticancerous activities such as lung,
breast, ovarian, etc. It is sold under the brand name of Taxol. This compound is also
found in plant T. baccata. Taxol and its derivatives are synthesized industrially to
meet the requirements for anticancer drugs. This compound as a single or combined
with other drugs is usually given to the patient. However, the synergistic effect
action shows better results than monotherapy. Taxol was discovered through tradi-
tional medicine and T. baccata was documented in Canon of Medicine for cardiac
treatment in 1021 (Tekol 2007). The plant was also used as traditional medicine in
the Central Himalayas for treating breast and ovarian cancer. Later, the compound
was isolated from the bark of the yew tree. The extract was tested in animal models
after a series of experiments. This was the first linking discovery in developing anti-
cancer drugs from traditional medicines. The drug may have a number of shortcom-
ings like insolubility or undesired side effects. So, further research was carried to
synthesize the derivatives of fewer side effects and more efficient Taxol. One major
drawback of paclitaxel was insolubility in water, and this was overcome by a
research team making water-soluble derivatives, e.g., smart taxanes. In an overall
mechanism of paclitaxel action, it targets the tubulin. Unlike other tubulin-targeting
drugs, such as colchicine, that inhibit microtubule assembly, paclitaxel stabilized
the microtubule polymer from disassociation. Thus, the chromosome will not
undergo metaphase spindle configuration and hence no further cell proliferation.
Though the Taxol was a big discovery for cancer therapy, there have been many
other drugs that use traditional knowledge as a basic source of information for iso-
lating a particular compound from the plants. The discovery of artemisinin from
Chinese medicine was also a breakthrough. Many other therapeutic compounds

have been isolated from Chinese medicine. Another such example is arsenic, which
is found to be an effective drug against acute promyelocytic leukemia (APL).
Huperzine A is another drug developed from Chinese medicine for treating memory
dysfunction.
Conclusions
In spite of the advanced medical technology and modern medicines, traditional

knowledge and folk medicine system are still prevalent for therapy of different ail-
ments. As allopathy medicines cause many side effects, people are referring to the
traditional system of medicines. Medicinal plants and other natural products pro-
vide the traditional medical system with great success in human history starting
from the Hippocrates to the Ayurveda. In almost all the discussed traditional sys-
tems of medicine, they used natural products with or without minerals as a prescrip-
tion. We have been using traditional knowledge for finding new drugs along with
important guidelines for curing the ailments. Suitable recognition of new drug from
traditional knowledge is an important element for the national health policies.
Unexplored natural resources, traditional knowledge of folk and ethnomedicine,
combined with modern technologies in screening, separation, and synthesis have to
be integrated into discovering a new drug.
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Chapter 3
Medicinal and Aromatic Plants: Store
House to Herbal Antimicrobials
Ravikant Singh, Anand Pandey, Rohit Kumar Mishra, Amritesh C. Shukla,

and Anupam Dikshit
Introduction
Aromatic plants own malodorous substances that are present as volatile oils, gum
exudates, balsam, and natural resins. These are found in one or other plant parts,
namely, root, wood, bark, stem, foliage, flower, and fruit. Complicated organic
chemical components are responsible for the characteristic aroma of these plants
(Gupta 1980). Those chemical entities which are oily in nature and can easily be
converted in the gaseous state by distillation are termed as volatile oil and represent
the active constituents of the plants (Fuehrer 1970). At standard temperatures, vola-
tile oils can evaporate when exposed to air. Essential oils are present in the volume
but highly targeted by pharmaceuticals, because of their high-value products. With
the advancement in oil extraction technologies, they have found extensive applica-
tion in the fields of pharmaceuticals, flavors, disinfectants, and oral hygiene. EOs
constitute a major portion in the flavor and fragrance market. Nature has a wonder-
ful collection of aromatic plants. Human has narrow knowledge about phytochem-
istry of all known aromatic plants (Clair 1961). EO contains some commercially
important product. Nowadays, EOs are widely used in different spheres of human
activity, such as incense cones, perfumery, medicine, etc. Herbal products are now
grooming in the world due to their significant aroma. Extracts and EOs are used as
R. Singh
Department of Biotechnology, Swami Vivekanand University, Sagar, Madhya Pradesh, India
A. Pandey (*)
PG Department of Botany, Avvaiyar Government College for Women,
Karaikal, Puducherry, India
R. K. Mishra (*) · A. Dikshit (*)
Centre of Science and Society, IIDS, University of Allahabad, Prayagraj, Uttar Pradesh, India
A. C. Shukla
Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India

https://doi.org/10.1007/978-981-15-2195-9_3
30 R. Singh et al.
functional ingredients in the pharmaceutical, food, and feed industries (Sacchetti

et al. 2005). Now industries are moving to provide more effective, safe, and cost-
efficient products (Singh et al. 2018a). Such demands can be fulfilled by plant-derived
components. Accurate and precise in vivo trials of EOs and aromatic compounds are
necessary before applying them as new-generation compounds for human health
and nutrition. This is important to understand the exact mechanism (Giannenas
2008). Aromatic plants are used as preservative and medicine in the Middle East
since approximately 5000 BC (Chang 2000; Li 2006; Piccaglia et al. 1993). From
immemorial times, plant products are the main source of medicine for the health-
care of people of many developed and developing countries. According to the World
Health Organization (WHO), nearly 80% of the present population can use this
plant and their products (Collin 2006; Gurib-Fakim 2006). Sometimes, plants or
plant products can be added to improve the productivity of animals and animal
products (Windisch et al. 2009). Restricted use of antibiotics by EU countries in
2006 made a path for EOs and herbs to replace them (Greathead 2003; Barton
1999). EOs consist of chemical substances such as polyphenols, quinines, flavonols/
flavonoids, alkaloids, polypeptides, or their oxygen-substituted derivatives
(Perumalla and Hettiarachchy 2011; Negi 2012; Cowan 1999). Some of these sub-
stances can act in clutches and hence enhancing their bioactivity (Tiwari 2008).
Some of the bioactive compounds exhibit antioxidant and antiseptic activities (Li
2006; Madsen and Bertelsen 1995). Thus, they may act as a risk reduction factor for
cancer or cardiovascular diseases (Duthie 1994; Milner 1994). EOs are also effec-
tive in case of respiratory diseases and stomach or inflammatory disorders (Kadri
et al. 2011). EOs of aromatic plants have antioxidant properties (Li 2006; Couladis
et al. 2003). Different strains of pathogens became resistant strains by enhancing
their capacity against drugs, and hence people try to switch to alternate methods
(Singh et al. 2018b).
Botanic Classification of Aromatic Plants Modified classification of aromatic
plants is based on classification done by Guenther (1952) (Fig. 3.1).
Abstraction and Deployment of Aroma Principles
Plants owe their fragrance to traces of EOs. Aerial parts, as well as underground
parts, are an important source of many fragrance materials. Gums, balsams, and
oleoresins are valuable raw materials for perfumes by virtue of their tenacious, how-
ever, soft odor. Extractions to obtain EOs can be done either singly or in combina-
tion depending on the nature of EOs. Roots, stems, barks, leaves, flowers, fruits, and
heartwoods are more important for many fragrance materials. For perfumes, some
materials are important such as gums, balsams, and oleoresins. Extraction of aro-
matic and essential oils can be done using any of the following – hydro-distillation,
steam distillation, hydro-diffusion, effleurage, maceration, expression, and solvent
extraction (Varshney 1992; Rajendra and Gupta 1964). Aforementioned processes
3 Medicinal and Aromatic Plants: Store House to Herbal Antimicrobials 31
Fig. 3.1 Families of aromatic plants
can be used either singly or in combination. The nature of EOs or material is con-
sidered to be the most important aspect during extraction.
Distillation
Most of the EOs are obtained by distillation. Hydro-distillation, hydro-steam distil-

lation, and steam distillation are the main distillation methods (Varshney 1992). Of
these, hydro-distillation system is the oldest known method which is widely used
for oil extraction. The material is kept inside the extraction chamber and is in direct
contact with boiling water in an exceedingly crude gold-bearing distillation outfit.
For perfumery materials, hydro-steam distillation is commonly used. In this distil-
lation method, the material is supported on a pricked grid. Steam distillation system
is one of the efficient and higher yield methods. In this method, stressed steam is
injected via haze tubes beneath the charge. The pressure inside the distillation vessel
is controlled according to the nature of the material. For delicate flowers, this
method is not used as such. Steam is extensively used in all these distillation proce-
dures to embrace the volatile odorous oil. Depending upon the thermostability of
the volatile materials, different intensities of steam is required to extract them.
32 R. Singh et al.
Rectification of liquid is settled by fragmental hydro-distillation at reduced pressure

for raising their color and odor. For the distillation of lemongrass, palmarosa, veti-
ver, linaloe berries, geranium, patchouli, cedars, etc., improved versions are utilized
that enhance the extraction occasionally (Ciaramello et al. 1972). Oils contain free
water and can be removed by decantation. By adding anhydrous sodium sulfate, the
remaining traces of water can be removed. For 4–5 h, anhydrous sodium sulfate will
be kept with the oil, and during this period, oil is filtered to get rid of the sodium
sulfate and different sediments. Foreruns and hind runs can be used to remove impu-
rities. Aerated compounds such as aldehydes, alcohols, ethers, esters, ketones, lac-
tones, phenols, etc., are major odoriferous constituents of most of the EOs. Vacuum
fractionation is typically used to separate terpenes and sesquiterpenes. Solvent
extraction may also be used for the same purpose. Diluted alcohol or similar sol-
vents are used to extract terpene-free oil. This method is also very useful to get rid
of waxes and sesquiterpenes.
Absolute Flower Oils
EOs of the entire flower can be obtained and purification with unpredictable sol-
vents, enfleurage, and expression using maceration method (Rajendra and
Gupta 1964).
Maceration Maceration stands for the softening or breakdown of substances.
According to this procedure, flowers containing EOs are ruptured by immersion in
a sizzling oil at 60–70 °C which is the opportunity to engross the EOs. Oil is unglued
from consumed flowers and recycled for interesting fragrance from the ensuing
batch of recent flowers. The oil conserved by flowers is mended by hydraulic
method. The resulting odorous pomade is often marketed intrinsically; however, it
is commonly extracted with robust alcohol to yield abstracts. Double maceration or
repeated maceration is used for concentrated infusions which contain volatile oil.
This technique is currently operated frequently for the abstraction of fragrant ingre-
dients from florets, leaves, and mosses (Varshney 1992).
(A) Extraction is settled by three sequential cleanings, purifying the total scent-rich
solvent for their particular reclamations (Guenther 1952). This is also known as
steady-state extraction.
(B) Rotating system pretends from a conserved metallic drum spinning on a hori-
zontal axis. Four perforated gold-bearing partitions create four sections to
carry charges of recent flowers. The lowest part of the metallic drum covers the
solvent. A liquid can be dripped through the perforation of the compartment to
the bottom. Each consignment of flowers takes three washes. Solvent regaining
is 88–93%. The distillate is chilled, cleaned, and additionally concentrated in
vacuum.
The distillate is bushed with hot alcohol for the extrication the wax and making
ready absolutely (Lawrence 1985). The processed alcoholic distillate is cool at
20 °C to −30 °C facilitating extra valediction of wax. The cleaned resolution is
employed intrinsically; however, it is additionally more targeted beneath vacuum to
a gelatinous constancy. The solvent ought to be hand-picked to fully and swiftly
dissolve the odoriferous principles of the plant resources and may not melt the tor-
pid principles of plant materials like waxes, colorants, and simple protein com-
pounds and even be inert to flower oil ingredients. The boiling process shouldn’t be
extremely high or too low; it necessarily should be unvarying. The solvent must be
inexpensive, noninflammable, and incompatible with water (Varshney 1992;
Chaudhuri 1991, 1992).
Enfleurage
The technique of abstraction of cologne by engrossing it from flowers in interaction

through icy fats is called as enfleurage. This process is assumed for musky flowers
of shrub still obvious with their distinctive fragrance even in a grasped situation.
Solvent deficiency to this method may cause the change in the property and smell of
the extracted EOs. The fats ought to be drenched and odorless to anticipate the
advent of fat odors. Settled fat or beef edible fat is most well liked. The fat is finely
bedded on either side of a glass plate maintained on an oblong picket border. Recent
musky flowers are gently bedded on the fat-coated chases. Many chases are located
one on top of the cramming the flowers between two layers fat. Expended flowers
are detached (defleurage), and a recent charge is created. Retreating of the glass
block is called as patage. Patage is completed many times to get the most fragrant
absorption. Furrows formed with combs to extend the absorption surface (Chaudhuri,
1991, 1992). The techniques of defleurage, current charging, and patage are sus-
tained to get fat off the specified scent asset.
Physical–Chemical Properties of Essential Oils
EOs possesses many communal physical characters like specific and characteristic
odors and high refractive indices. They are commonly optically dynamic and insol-
uble with water molecules but sufficiently soluble to convey their characteristic
odors to water. Preparations of perfumed waters are determined by on this miscibil-
ity. EOs are mix with ether, alcohol, and organic solvents very easily. In the labora-
tory, essential oils are distilled out using Clevenger’s apparatus (Fig. 3.2) (Singh
et al. 2018a), and the physical characters are deliberate in the case of definite gravity
by definite gravity bottle, refractive index by refractometer, and optical rotation by
polarimeter.
34 R. Singh et al.
Fig. 3.2 Distillation process
Generally, fragrance investigations are carried out by the following techniques:

I. By straight solvent extraction
II. By steam distillation through solvent purification
III. By simultaneous purification
IV. By extreme vacuum
V. By extremely critical fluid purification
VI. By separation of the odorous complex into specific components using chro-
matography (column chromatography, gas liquid chromatography (GLC),
thin-layer chromatography (TLC), high-pressure liquid chromatography
(HPLC), ion-exchange chromatography, permeation chromatography)
techniques
VII. Identification of the elements through UV, infrared, NMR spectroscopy, and
mass spectroscopy
VIII. Chemiluminescence or perceiving whether the test material has been derived
from species besides food aromas
For this purpose, the most popular procedure is the different chromatography
techniques along with mass spectroscopy (MS). Samples that have squat vapor pres-
sure and extreme molecular mass can be examined through liquid chromatography
with mass spectrometry (LCMS) because gas chromatography cum mass spectrom-
etry (GCMS) can refuse analysis. The quick analysis of samples (either tracer or
huge) is possible only due to the availability of liquid chromatography cum mass
spectrometry (LCMS) at the commercial level (Adams 2001).
The former can be applied to leaves, cones, needles, branchlets, etc. (Singh et al.
2018a). Liaison to ignis and breeze, the oils get oxidized; they are mainly composed
of aldehydes, ketones, esters, hydrocarbons ethers, and alcohols; various techniques
will be used for separation of an organic compound:
I. Freeze for crystallizing the stearoptenes.
II. Fractional condensation.
III. Fractional illustration from polar solvents.
IV. Deduction by chemical action: sodium carbonate is used to take out the free
acid group from the oil; hydrochloric acid can be used for elementary com-
pounds, phenols with sodium hydroxide.
Storage of Essential Oils
Freshly extracted EOs are pale yellow colored to colorless depending on the nature
of EOs (Fig. 3.3) (Singh et al. 2018a) and flows freely. Citrus, pine needle, and tur-
pentine contain high hydrocarbon and are significantly susceptible to spoilage by
chemical reaction and resinification. In order to avoid their resinification and oxida-
tion, they must be stored at 4 °C (Singh et al. 2018a) on a dry place in the tightly
stoppered amber glass bottles. Essential oils with high ester percentage turn acidic
by hydrolysis of esters when storage is improper. Antioxidants are added to preserve
fatty acid for a longer duration. EOs that contain alcoholic content are quite stable
and endure prolonged storage like sandal and geranium. Therefore, proper storage
must be done in order to avoid changes in the properties of EOs.
Mother Medicine of Nature – Tulsi Mata
Ocimum sanctum (L.) (Lamiaceae)
Tulsi (Ocimum sanctum) is a Sanskrit word that means “matchless one.” Ocimum
sanctum belongs to the family Lamiaceae. It is highly aromatic with mediational
value and a native of India and found throughout southeast Asia. It is briefly
described in ancient Indian Ayurveda as “Mother Medicine of Nature” (Bast et al.
Fig. 3.3 Extracted essential oils

36 R. Singh et al.
2014; Singh et al. 2010). Holy basil (Tulsi) is an Indian aromatic sub-shrub of
30–60 cm in length with the hairy stem. Leaves of the plant are simple, petiolated,
ovate, slightly toothed margin with decussate phyllotaxy and inflorescence is raceme
with the purplish flower. Two main morphotypes of Tulsi (green-leaved and purple-
leaved) are cultivated mostly in southeast Asia. Medicinal and therapeutic proper-
ties of Ocimum are well known from the long ethnic history of humans and well
described in Ayurveda, Greek, Roman, and Unani medicinal literature. Each and
every part of Ocimum plant has medicinal values. Eugenol, oleanolic acid, ursolic
acid, rosmarinic acid, carvacrol, beta-caryophyllene, linalool, etc., are different
phytochemicals and different essential oils present in this plant (Reuveni et al.
1984). Ocimum extracts showed a broad range of antimicrobial activity (antibacte-
rial, antiviral, antifungal, antimalarial, antiprotozoal, etc.) against many animal and
human pathogens. It is utilized in wound healing, animal rearing, as sanitizer, and
also utilized in cold–cough and chronic disease treatments (Singh et al. 2007). Their
different combinations were greatly utilized indifferently in 11 pharmacological
fields such as neuroprotective, cardioprotective, antihypertensive, anticarcinogenic,
analgesic, antidiarrheal, antioxidant, memory enhancement, anti-asthmatic, anti-
leukodermal, and anticoagulant activities. Modern researches utilize its pharmaco-
logical actions to address physical, chemical, metabolic, and psychological stress
(Mahajan et al. 2013; Mohan et al. 2011).
Conclusion
Trees are a boon to us, especially aromatic trees. Many aromatic plants exhibit
medicinal properties. Proper knowledge and continuous research provide a basic
platform to develop herbal antimicrobials. Right from the pre-Vedic period and
from the beginning of civilizations, herbal medicine was used to cure several dis-
eases. It is important to explore more and more about the antimicrobial efficacy of
plants due to accelerated population and the tendency of pathogens to become resis-
tant against several drugs. It is a better idea to develop herbals as they have no side
effects. There is a need to collect and concentrate the knowledge of traditional
medicinal herbs and their practices. With the combination of traditional knowledge
and advances in medical sciences, we became able to serve humanity as better as
possible.
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Chapter 4
Ethnomedicinal Plants of North-East India
as a Potential Target for Drug Discovery
Against Type 2 Diabetes Mellitus
Jijnasa Bordoloi, Anjum Dihingia, Jatin Kalita, and Prasenjit Manna
Introduction
Plants have been considered as an essential part of traditional healing practices

since prehistoric times and are well-recognized as an excellent source for therapeu-
tic agents for treating of different ailments. Ethnomedicine-based healing systems
are a vital part in the culture of native people around many parts of the world due to
their easy accessibility, reliability, and inexpensiveness compared to their modern
counterparts. In recent years, global interest is increasing in the ethnomedicine-
based health-care system due to the successful application of various plant-based
drugs in the treatment of several health disorders. Moreover, herbal medicines have
attracted scientific attention and have become one of the fastest developing sectors
in the global market because of the increased awareness regarding the health risks
associated with contemporary synthetic medicines. According to one report by the
World Health Organization (WHO), the international market for plant-based medi-
cines has a yearly profit of around US $43 billion, and the market is reportedly
expanding at a fast pace (Aschwanden 2001). Another estimate made by the WHO
shows that globally more than 80% of the population depends on traditional medici-
nal practices for basic health-care needs (Khan and Yadava 2010). Uncovering and
documenting the unknown and unpublished information, regarding the medicinal
herbs scattered among different ethnic tribes around the globe, is highly needed for
future advancement of the health-care system. Moreover, the scientific validation of
traditional medicines is of utmost importance for the production of efficient drug
candidates and therapeutic strategies.
Authors Jijnasa Bordoloi and Anjum Dihingia contributed equally in the study.
J. Bordoloi · A. Dihingia · J. Kalita · P. Manna (*)

Academy of Scientific and Innovative Research, Chennai, India
Biological Science and Technology Division, CSIR-North East Institute
of Science and Technology, Jorhat, Assam, India

https://doi.org/10.1007/978-981-15-2195-9_4
40 J. Bordoloi et al.
India has a renowned custom of using traditional medicines and is well known
for some highly established ethnomedicinal systems, namely, Ayurveda, yoga and
naturopathy, Unani, Siddha, and homeopathy (AYUSH) which have been an indis-
pensable part of Indian healing cultures. The North-east region of India comprises
eight sister states, namely, Assam, Arunachal Pradesh, Manipur, Meghalaya,
Mizoram, Nagaland, Sikkim, and Tripura, which are considered as the treasure
house of many medicinal plants with tremendous potential in the industrial sector.
Around 40% of the total geographical area of the North-east region is inhabited by
evergreen forests which harbor very rich and diverse natural resources including
various endemic species of flora and fauna. Moreover, the Indo-Burma hotspot
region, which is ranked second among the total 25 biodiversity hotspots identified
worldwide, falls in the North-east region. North-east states are inhabited by more
than 180 major ethnic tribal communities such as Abor, Garo, Dafla, Khasi, Kuki,
Mishi, Rabha, Naga, Apatani, etc., and each tribal group is known by their own
languages, rituals, cultures, ensembles, and housing patterns (Mondal et al. 2013).
The North-east people of different ethnic groups utilize various folk medicines as a
remedy for several ailments. These ethnic communities have been practicing a tra-
ditional healing system since time immemorial. Here in this article, we attempt to
present the existing information regarding the medicinal plants of North-east Indian
states which have been in use as the source of herbal medicines for the management
of diabetes and its associated complications since ages.
Diabetes and Its Burden in India
Diabetes is gaining acceptance as a leading health disaster of the twenty-first cen-

tury as it is considered among the top ten causes of death globally. Diabetes is
characterized by an increase in circulating glucose concentration due to complica-
tions in either insulin secretion, insulin action, or both. The prevalence of diabetes
is rising at an alarming rate causing serious health and economic burden to the
patients and the society at large. According to the 2017 reports of the International
Diabetes Federation (IDF), approximately 425 million people around the world
were found to be diabetic and is predicted to increase to 629 million by 2045 (IDF
Diabetes Atlas 8th Edition). It has been reported that more than 47% of the world’s
population is still undiagnosed for diabetes. Moreover, 352.1 million people world-
wide are estimated to have impaired glucose tolerance (IGT) and are at high risk for
developing diabetes. The worldwide scenario of the frequency of diabetes is increas-
ing in a significant rate, and more than 80% incidence has been observed in low- and
middle-income countries like India. In the IDF 2017 reports, South-east Asia has
been found as the epicenter of diabetes crisis. Among the top 10 countries of South-
east Asia, India stands second with 72.9 million populations aged 20–79 years as
diabetic and is expected to raise its population to 134.3 million by 2045 (IDF
Diabetes Atlas 8th Edition). In India, around 11 million of people older than 65 years
4 Ethnomedicinal Plants of North-East India as a Potential Target for Drug Discovery… 41
were found to be diabetic in 2017, and that is expected to rise to 28.2 million by

2045. Similarly, the number of people aged 20–79 years with IGT is estimated to be
24 million in 2017 and is predicted to rise to 41 million by 2045. In addition, around
42.2 million of people in India are undiagnosed for diabetes, and it stands in the
second position after China. It is important to mention that diabetes is not only a
health burden to a country but also a societal catastrophe. Besides serious health
sufferings, chronic diabetes also drives the families to poverty. Globally, India ranks
fourth in health expenditure on diabetes with a total expenditure of 31 billion as per
the IDF 2017 annual report. Due to an abrupt rise in the occurrence of diabetes, the
government of India is struggling to satisfy the expenditure on diabetes. Premature
disability and mortality due to diabetes have been found to be the two major draw-
backs in the economic development of a country. According to the IDF 2017 reports,
about 4 million people aged 20–79 years worldwide are estimated to die from dia-
betes in the year 2017. In addition, a recent report by the Indian Council of Medical
Research (ICMR) showed that according to the Global Burden of Disease Report
2015, the number of mortality due to diabetes increased from 2.24 to 3.46 lakhs
between the year 2005 and 2015 in India (India: Health of the Nation’s States,
ICMR). Unhealthy diet, lavish sedentary lifestyle, and rapid urbanization are caus-
ing an increase in diabetes and its associated complications; thus, governments are
struggling to satisfy the heavy economic burden on the health-care section. Hence,
a combined approach of the government, civil societies, NGOs, and international
health organizations is necessary for its primary diagnosis and management to
reduce the influence of diabetes on the individual and society.
thnomedicinal Plants of North-east India Utilized

E
for the Management of Type 2 Diabetes
Aegle marmelos
Commonly known as bael, a fruit-bearing plant species under the family of Rutaceae
(Rahman and Parvin 2014). This only species of genus Aegle is native of India,
Bangladesh, and spread throughout South-east Asia (Brijesh et al. 2009). This plant
species grows up to 15 m tall and bears thorns and fragrant flowers (Fig. 4.1a).
Almost all parts of A. marmelos (i.e., root, leaf, trunk, fruit, and seed) bear tremen-
dous health benefits and are widely used to cure tuberculosis, hepatitis, dysentery,
constipation, peptic ulcer, piles, diabetes, and other diseases (Rahman and
Parvin 2014).
Ethnical use: The decoction prepared from the leaves of this plant is a popular
remedy for curing diabetes among the Deori community of Assam. The leaf decoc-
tion made from about 100 g of leaves prepared in water is advised to be taken orally
once daily for 3 months for treating diabetes.
Fig. 4.1 Photographs of (a) the leaves and fruit of Aegle marmelos; (b) the plant of Amomum
linguiforme; (c) the leaves and fruit of Annona squamosal; (d) the leaves of Azadirachta indica; (e)
the leaves and seedpods of Cajanus cajan; (f) the leaves of Cinnamomum impressinervium; (g) the
whole plant of Colocasia esculenta; (h) the plants of Coriandrum sativum. (Source:
Self-photography)
Amomum linguiforme
Belonging to the family of Zingiberaceae, a medicinal herb mostly found in

Myanmar and North-east Indian region including Darjeeling (Fig. 4.1b). A. lingui-
forme is commonly known as Kalphul.
Ethnical use: The people of Tai-Ahom community use the rhizome decoction of
this herb as a treatment for diabetes. About 25 ml decoction prepared by boiling
50 g rhizome of A. linguiforme by removing the epidermis in water is recommended
to drink once a day until the symptoms of the disease show improvement.
Annona squamosa
A semi-deciduous, fruit-bearing, well-branched shrub or small tree under the family

Annonaceae, which is commonly known as sugar apple or Atla-kothal (India)
(Fig. 4.1c). This plant species is widely grown in tropical and subtropical countries
around the world for the edible fruit and its numerous health benefits such as antitu-
mor, anticonvulsant, antidiabetic, antioxidant, antidiarrheal, and anti-inflammatory
activities and provides protection against cold, cough, intestinal infections, and
acidity conditions (Chakraverty and Bhattacharya 2016).
Ethnical use: Among the Moran community of North-east India, it is commonly
used for management of diabetes. About 10 ml decoction of the bark is recom-
mended to be taken once daily on an empty stomach.
Azadirachta indica
Commonly known as neem or Maha-neem of the Meliaceae family, this plant spe-
cies is abundantly found in tropical and semitropical regions, like India, Bangladesh,
Pakistan, and Nepal (Alzohairy 2016). A. indica is a fast-growing and long-lived
evergreen tree species (Alzohairy 2016) (Fig. 4.1d). Besides being an excellent
medicinal herb, this plant provides timber and acts as efficient repellent and
insecticide.
Ethnical use: Among the Deori community of Assam, neem leaves are widely
used as an antidiabetic medication. An infusion prepared from about 100 g of leaves
of A. indica is prescribed to be taken orally once per day for 2 months for the man-
agement of blood sugar.
Cajanus cajan
Commonly known as Pigeon pea, one of the most common pulse crop species cul-
tivated in tropical and subtropical countries for its highly nutritious edible seeds
(Pal et al. 2011). This perennial member of the family Fabaceae is a fast-growing,
branched, and hairy shrub species with 1–2 m of height and known for its wide
adaptability and drought resistance (Fig. 4.1e). This plant species is considered as
the most important grain legume crop of semiarid tropical regions. It has been
widely used for treating diabetes, inflammation, skin irritations, measles, jaundice,
dysentery, and other illnesses (Pal et al. 2011).
Ethnical use: The leaf decoction is prescribed orally on an empty stomach as an
effective remedy for diabetes among the Tai-Ahom community of Assam.
Cinnamomum impressinervium
Commonly known as Tezpata, a small- to medium-sized evergreen tree under the

family of Lauraceae (Fig. 4.1f). The leaves of C. impressinervium are aromatic in
nature and possess a strong spicy odor due to which they are used as spice. Moreover,
the essential oil collected from the leaves is used as an effective fungicide. The plant
is harvested in the evergreen forests of East Asiatic regions especially in North-east
India due to their medicinal values.
Ethnical use: This plant is very popular among the Zemi-Naga tribe of North-east
India as an effective medication for treating diabetes, and a mixture of one to two
teaspoons of dry leaf powder of C. impressinervium in water is prescribed orally
once or twice per day.
Colocasia esculenta
A fast-growing perennial herbaceous plant of Araceae family which is more com-

monly known as Kola-Kachu in North-east India. C. esculenta originates from a
large fleshy corm, can grow up to 1.5 m in height, and has lateral thick runners
(Fig. 4.1g). It is widely grown in tropical and subtropical regions primarily for its
edible corms, leaves, and petioles because it is an important staple food crop in
many tropical countries with a long history of cultivation (Prajapati et al. 2011).
This plant has numerous medicinal benefits such as wound healing, antibacterial,
hypotensive, antidiabetic, and treatment of conjunctivitis and menstrual problems.
Ethnical use: The decoction obtained by boiling 50 g of roots in goat milk is pre-
scribed orally once per day for effective management of diabetes among the Tai-
Ahom community of Assam.
Coriandrum sativum
More commonly known as coriander or Dhania (India), an annual herb belonging to

the Apiaceae family that is commonly grown in herb gardens for its lacy, strong-
scented foliage and its aromatic seeds (Laribi et al. 2015) (Fig. 4.1h). Coriander is
grown throughout the world primarily as a spice crop for culinary use. It typically
grows in a clump of 25–60 cm height and has small, pinkish-white flowers, alternate
leaves, an erect stalk, and slender, spindle-shaped roots. In addition to its use as a
seasoning in food preparation, the leaves and seeds of this plant are widely used in
folk medicine.
Ethnical use: Among the Tai-Ahom community of Assam, it is popular as an anti-
diabetic remedy, and an infusion of 100 g of leaves is prescribed orally once or
twice per day for improvement of this disease.
Dillenia indica
Commonly known as elephant apple and locally known as Ou-tenga in Assam, an

evergreen large shrub or medium-sized tree growing up to 15 m height belonging to
the family Dilleniaceae (Talukdar et al. 2018) (Fig. 4.2a). This plant species widely
grows in the rainforest regions of many Asian countries and is cultivated for its edible
fruit. Different parts of this plant have various medicinal properties against several ail-
ments, such as wounds, cut and burns, diabetes, cancer, diarrhea, abdominal pains, etc.
Ethnical use: About 25 ml infusion of 100 g fleshy perianth of D. indica flower is
prescribed orally once per day for effective management of diabetes among the
Tai-Ahom community of Assam.
Fig. 4.2 Photographs of (a) the leaves and fruit of Dillenia indica; (b) the plants of Enhydra fluc-
tuans; (c) the plant of Bombax ceiba; (d) the leaves and fruits of Piper griffithii; (e) the rhizome of
Zingiber officinale; (f) the plant of Euphorbia ligularia; (g) the leaves of Ficus hispida; (h) a young
plant of Garcinia pedunculata. (Source: Self-photography)
Enhydra fluctuans
Commonly known as Helencha, a tropical semiaquatic herbaceous plant belonging

to family Asteraceae (Kuri et al. 2014) (Fig. 4.2b). This plant species is native to
India, Bangladesh, Burma, and Sri Lanka. Helencha is well known for its numerous
therapeutic properties, such as antimicrobial, hepatoprotective, hypotensive, antide-
pressant, antidiarrheal, and anti-inflammatory activities and acts as an effective
remedy against smallpox.
Ethnical use: The local people of North-east regions of India use Helencha as an
antidiabetic remedy. About 25 ml infusion prepared from 125 g of aerial parts or
leaves of E. fluctuans is recommended to be taken orally twice per day for treating
this disease.
Bombax ceiba
Commonly known as red silk cotton tree of the Bombacaceae family, a tall decidu-
ous tree with a spreading crown and grows to the height of 25 m or more (Fig. 4.2c).
This tree species is widely distributed in tropical and subtropical areas of Australia,
Africa, and Asiatic countries especially India (Kamble et al. 2017). Being a multi-
purpose tree, it is widely used for fibers, tannins, edible seed oil, and edible leaves
and flowers. The parts of B. ceiba bear numerous health benefits and are used for
treating various illnesses like cholera, fractures, toothache, coughs, urinary prob-
lems, influenza, and snake bites.
Erythrina stricta
Commonly known as corky coral tree, an evergreen thorny medium-sized tree

species of the family Fabaceae with a height of 7–35 m. This flowering plant species
is extensively seen in Asian countries like India, Nepal, Burma, Thailand, Vietnam,
and China. It is cultivated in India as a decorative plant and as a support for climbers
and pepper plants. In Thailand this tree species is widely used as a pioneer species
during reforestation projects. The leaves are used by the local people for various
therapeutic purposes including treatment of eye infections, toothache, ear pain, joint
pain, and wound healing (Umamaheswari et al. 2010).
Piper griffithii
A pepper plant species of the family Piperaceae. This herbaceous plant species is
native to the regions of the Eastern Himalayas to Peninsula Malaysia (Fig. 4.2d).
This herb is well recognized for its medicinal traits.
Zingiber officinale
Commonly known as ginger, a perennial herb species and member of the family
Zingiberaceae (Ali et al. 2008) (Fig. 4.2e). This plant species is widely cultivated in
the tropical regions and consumed worldwide for culinary purposes and its multiple
health benefits. The underground stem or rhizome of Z. officinale has been in use in
healing practices since thousands of years because of its various medicinal proper-
ties, such as anti-arthritis, anti-inflammatory, antidiabetic, antibacterial, antifungal,
anticancer, etc. (Ali et al. 2008).
Ethnical use of Bombax ceiba, Erythrina stricta, Piper griffithii, and Zingiber
officinale: These four plant species are used in combination by the members of the
Dimasa community of North-east India for curing diabetes. 20 g roots of each of
B. ceiba, E. stricta, and P. griffithii with a small amount of the rhizome of Z. officinale
are ground and mixed with about 50 ml of water and are advised to be taken orally
thrice a day for 7–10 days for effective management of the disease.
Euphorbia ligularia
Commonly known as Indian spurge tree or Siju (Assam), a large, glabrous, branched,
and xerophytic fleshy shrub with a height of 2–6 m belonging to the family
Euphorbiaceae (Fig. 4.2f). Although this shrub species is widely and primarily
distributed in North, Central, and South India, however, nowadays it is also culti-
vated in West Bengal, Sri Lanka, Myanmar, and Thailand. Apart from being an
ornamental plant, E. Ligularia has beneficial effects in treating tumors, bronchitis,
delirium, piles, loss of consciousness, spleen enlargement, inflammation, ulcers,
fevers and anemia (Mali and Panchal 2017).
Ethnical use: Among the Tai-Ahom community of Assam, this plant is a popular
remedy for management of diabetes. About 25 ml decoction obtained by boiling
125 g stem of E. ligularia removing the epidermis in water is mixed with little salt
and recommended to be taken orally once per day.
Ficus hispida
Commonly known as hairy fig or Dimaru in Assam, a moderate-sized tree of family

Moraceae (Fig. 4.2g). It is grown wild or cultivated for its edible fruits and medici-
nal values. F. hispida trees are distributed in evergreen to semievergreen forests of
India, Sri Lanka, Myanmar, South China, New Guinea, Australia, and the Andaman
Islands. Traditionally, various parts of this plant bear tremendous health benefits,
such as antidiarrheal, hepatoprotective, anti-inflammatory, antitussive, antipyretic,
astringent, anti-ulcer, etc. (Ali and Chaudhary 2011).
Ethnical use: This plant serves as an effective remedy for treatment of diabetes
among the people of the Tai-Khamti community of North-east India. About 2–3
teaspoons of root exudates of this plant is mixed with 50 ml of raw cow milk and is
prescribed to be taken orally one time per day on an empty stomach.
Garcinia pedunculata
Commonly known as Bor-thekera in Assam, an evergreen fruit-bearing tree species

of the Clusiaceae family (Fig. 4.2h). G. pedunculata is endemic to the South-east
regions of Asia and North-east India (Sarma et al. 2016). This plant species is very
much famous in North-east India especially in Assam due to its edible fruit and
diverse therapeutic properties. The edible fruit is used by the native people of Assam
to treat various stomach-related illnesses.
Ethnical use: Assamese people preserve the fleshy fruit pulp of G. pedunculata by
cutting into small slices and then subsequently drying the pieces. Traditionally, the
local people of the Tai-Ahom community of Assam utilize these dried slices as an
effective source for antidiabetic remedy. An infusion of 200 ml prepared by soaking
50 g of dried slices overnight in water is prescribed orally once per day.
Fig. 4.3 Photographs of (a) the plants of Justicia adhatoda; (b) the plants of Kalanchoe pinnata;
(c) a young plant of Litsea glutinosa; (d) the plant of Solanum indicum with fruits; (e) the plant of
Syzygium cumini; (f) the leaves and flowers of Tabernaemontana divaricate; (g) the leaves and
stems of Tinospora cordifolia; (h) seeds of Sesamum orientale; (i) seeds of Nigella sativa. (Source:
Self-photography)
Justicia adhatoda
Commonly known as Baikar or Vasaka, an erect, evergreen, gregarious shrub spe-

cies belonging to the family of Acanthaceae (Fig. 4.3a). This plant species is widely
distributed in Pakistan, India, Panama, Indonesia, Malaya, and South-east Asia.
Apart from being an ornamental plant, the leaves and roots of J. adhatoda are uti-
lized for curing cough, cold, asthma, rheumatism, pneumonia, snakebites, tubercu-
losis, and both eye and ear ailments (Khan et al. 2018). In India, Vasaka plant has
been a part of traditional healing practices since ages.
Ethnical use: Among the Tai-Ahom community of Assam, this plant is a popular
remedy for treatment of diabetes. A decoction prepared by boiling 50 g roots of
J. adhatoda in about 125 ml of cow milk is prescribed orally once per day.
Kalanchoe pinnata
Commonly known as air plant or life plant, this is a perennial, succulent medicinal
herb of 40–200 cm height belonging to the family of Crassulaceae (Fig. 4.3b). It is
commonly grown as an ornamental plant in tropical and subtropical parts of South
America, the Caribbean, Australia, and parts of Asia especially India (Menon et al.
2015). K. pinnata has a wide range of medicinal properties such as antinociceptive,
anti-inflammatory, and antidiabetic activities (Menon et al. 2015).
Ethnical use: Among the Tai-Khamti community of North-east India, this plant is
a popular remedy for treating diabetes. A 100 ml infusion prepared from 100 g of
K. pinnata leaves is prescribed orally twice per day.
Litsea glutinosa
Commonly known as Indian laurel or Naluka (Assam), an evergreen or deciduous

tree species of 3–15 m height belonging to the family of Lauraceae (Fig. 4.3c). This
aromatic medicinal tree species is distributed widely throughout tropical and sub-
tropical parts of Asia, North America, and subtropical South America (Wang et al.
2010). Ethnomedicinally, the bark of L. glutinosa has demulcent, emollient, antidi-
arrheal, anti-dysentery, and wound healing properties, and the leaves are known to
be valuable for treating respiratory ailments (Pradeepa et al. 2011).
Ethnical use: Traditionally, the local people of the Mishing community of Assam
utilize the leaves L. glutinosa as an effective source for antidiabetic medication. An
infusion prepared from about 200 g fresh leaves is administered orally twice a day
on an empty stomach.
Solanum indicum
Commonly known as Indian nightshade, a spiny, erect, and perennial herb species
belonging to the family of Solanaceae (Fig. 4.3d). It is geographically distributed in
Africa, Australia, and South-east Asian countries especially India. The parts of
S. indicum, such as the seeds, roots, and leaves, are useful in various ailments, like
bronchitis, asthma, dry cough, rhinitis, dysuria, leucoderma, sexual disorders,
insomnia, cardiac weakness, and pruritus (Sharma et al. 2017).
Ethnical use: Ethnically, the people of the Hmar community of North-east India
eat the raw fruits of S. indicum directly for preventing diabetes.
Sterculia villosa
Popularly known as hairy sterculia or Udal in Assam, a spreading medium-sized

deciduous tree with a height of 15–18 m belonging to the family of Sterculiaceae. It is
a fast-growing tree species broadly distributed in the North-east region of India (Ghosh
and Baruah 1997). The tree is harvested from the wild for firewood, fibers, gum, and
its edible roots. Traditionally the plant species is well known for its anti-inflammatory,
diuretic, aphrodisiac, and analgesic characters (Monir Hossain et al. 2013).
Ethnical use: Ethnically, the people of the Dimasa community of North-east India
use the roots of S. villosa as an effective remedy for curing diabetes. One teaspoon-
ful of root powder of S. villosa mixed with about 50 ml water is prescribed to be
taken orally thrice a day after meal for 15 days.
Syzygium cumini
Popularly known as Jamun in India, belonging to family of Myrtaceae, a fast-

growing, attractive, evergreen tree with a dense, frondose crown. S. cumini widely
grows in different tropical and subtropical areas in the world (Fig. 4.3e). Apart from
being an important medicinal tree, S. cumini is well known for its fruit and timber
and as a decorative plant. Although Jamun is well recognized primarily as an anti-
diabetic plant, it has numerous other medicinal traits such as astringent, anthelmin-
tic, blood purifier, diuretic, and stomachic and used for treating sore throat,
bronchitis, asthma, thirst, biliousness, dysentery, and ulcers (Ayyanar and Subash-
Babu 2012).
Ethnical use: Among the Tai-Ahom and Deori communities of North-east India,
this plant species is considered as a popular remedy for the treatment of diabetes.
An infusion prepared from about 20 g bark powder of S. cumini in 100 ml of cow
milk, either alone or in combination with small amount of sugar candy, is prescribed
to be taken orally once a day on an empty stomach for 15 days up to 3 months.
Tabernaemontana divaricata
Commonly known as crape jasmine, an evergreen shrub species of the family

Apocynaceae (Fig. 4.3f). Although this plant species is native to India, nowadays it
is widely cultivated as a garden plant in South-east Asia and other tropical countries
(Kam and Anuradha 1995). T. divaricata is well known for its various ethnomedicinal
values such as antiepileptic, antioxidant, and anti-maniac properties and is widely
used as a brain tonic (Chanchal et al. 2015).
Ethnical use: Among the Tai-Ahom community of Assam, T. divaricata leaves are
widely used as an antidiabetic remedy. About 125 ml decoction obtained by boiling
25 g of leaves in cow milk is prescribed orally once a day for management of the
disease.
Tinospora cordifolia
Commonly named as heart-leaved moonseed or Saguni-lota (Assam), a deciduous

and climbing shrub under the family Menispermaceae (Fig. 4.3g). This shrub spe-
cies is distributed all over India, especially in the tropical parts, and also in certain
parts of China (Mittal et al. 2014). T. cordifolia has multiple folkloric medicinal
values, such as antiperiodic, antispasmodic, antimicrobial, anti-osteoporotic, anti-
inflammatory, antiarthritic, anti-allergic, and antidiabetic (Saha and Ghosh 2012).
Ethnical use: T. cordifolia serves as a popular source of antidiabetic remedy
among the people of the Deori community of North-east India. An infusion of
100 ml prepared from about 100 g stem of T. cordifolia is recommended to be taken
orally once on every alternate day for 3 months.
Plumeria acuminata
Commonly known as white Champa in India, a small laticiferous tree species

belonging to the family of Apocynaceae. P. acuminata widely grows all throughout
the tropics, especially in India. Traditionally, this plant is used for treating diarrhea,
inflammation, hard tumor, rheumatic pain, and itching and as an effective purgative
(Gupta et al. 2006).
Sesamum orientale
Commonly known as sesame internationally, an herbaceous annual plant with white

bell-shaped flowers under Pedaliaceae family (Fig. 4.3h). It is mainly grown for its
valuable seeds that are extremely rich in protein and edible oil content. Sesame
plant is grown in the tropical regions throughout the world because of its nutritional,
medicinal, and industrial values. Sesame plant is well recognized for its various
medicinal benefits such as antihypertensive, lipid-lowering, and anticancer activi-
ties (Elleuch et al. 2011).
Nigella sativa
Commonly black cumin or Kalonji, an annual flowering plant of the family

Ranunculaceae (Fig. 4.3i). N. sativa is native to Southern Europe, North Africa, and
South-west Asia and is also broadly cultivated in the rest of the world because of
culinary purposes and therapeutic values. The seeds and their oil have shown to hold
numerous medicinal values, such as diuretic, antihypertensive, antidiabetic, anti-
cancer, analgesic, antimicrobial, anti-inflammatory, bronchodilator, gastroprotec-
tive, hepatoprotective, renal protective, and antioxidant activities (Ahmad
et al. 2013).
Ethnical use of Plumeria acuminata, Sesamum orientale, and Nigella
sativa: These three plant species are widely used in combination by the members
of the Shyam community of North-east India for curing diabetes. A decoction pre-
pared by boiling the bark of P. acuminata together with 5 g seeds of each of S. ori-
entale and N. sativa in water is advised to be taken orally thrice per day consecutively
for 7 days for effective treatment of this disease.
Conclusion
The North-east region of India is known for its richness in biodiversity; hence the
traditional healing practice based on diverse medicinal herbs is widely seen among
different ethnic communities. The present article summarizes the significance of the
ethnomedicinal plants that are being used in North-east India as a source of antidia-
betic medications. Although many plants having strong antidiabetic property have
already been documented, scientific exploration of several of these plants remains
unresolved. Therefore, the exploration of the indigenous knowledge, its scientific
validation, isolation, and characterization of the active antidiabetic constituents and
standardization and quality assessment of the folkloric medicines are of utmost
necessity for the production of efficient drugs. Natural indigenous herbal prepara-
tions would be relevant or commercialized only if these are tested scientifically and
subjected to the rigorous investigation for quality, safety, and efficiency. Moreover,
it is noteworthy to consider that most plants have a number of morphologically
similar indistinguishable related species, which may lead to utilization of incorrect
plant species with toxic activity or with less or no activity. Hence, it is extremely
crucial to assess the proper botanical identity of the medicinal plants used for the
preparation of herbal drugs. Because of their efficacy in management of various
diseases, these medicinal plants are harvested from the wild unsustainably, and this
overexploitation may result in the extinction of these important herbs with time.
Therefore, conservation of these medicinally important plants should be taken seri-
ously for the sake of mankind and future generations.
Acknowledgment The authors are thankful to the Director, CSIR-NEIST, Jorhat, for his support;
DBT, Government of India, for providing the Ramalingaswami Re-Entry Fellowship ((BT/RLF/
Re-Entry/34/2013); to Dr. Manna and the senior research fellowship to Ms. Jijnasa Bordoloi; and
CSIR, Government of India, for providing the senior research fellowship to Mr. Anjum Dihingia.
The authors thank Ms. Cassandra Warden (Vanderbilt University Medical Center, Department of
Ophthalmology, USA) for excellent editing of this manuscript.
Conflict of Interest None declared
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(2011). Antibacterial screening of the stem bark and leaf extracts of Litsea glutinosa (Lour.)
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74, 652–654.
Chapter 5
Application of Phytochemicals
in Pharmaceuticals
Sankhadip Bose, Jai Malik, and Subhash C. Mandal
Introduction
Phytoconstituents, the chemical compounds produced by plants, have a definite bio-

logical activity and are being thoroughly investigated in the recent era for their
potentiality to provide health benefits. Hippocrates literally stated, “let thy food be
thy medicine.” So it has been observed that regular consumption of nutritive food
(with potent phytochemicals) has various health benefits against both acute and
chronic metabolic or degenerative disorders like diabetes, neurodegenerative dis-
eases (Alzheimer’s, Parkinson’s, etc.), cardiovascular diseases, and cancer. This is
the preliminary but foremost matter to manifest the scientific sense and data to
enhance their use in foods and pharmaceuticals, as probable nutritional active ingre-
dients. Phytoconstituents exert their effects by acting as substrates, cofactors, and
inhibitors of biochemical and enzymatic reactions. As chelants they bind and
remove unwanted elements in the GIT, and as ligands they are used as agonist or
antagonist at intracellular receptors on the cell surface and as scavengers of toxic
chemicals. As necessary growth factors, they are also used as constituents that boost
the absorption and stability of different crucial nutrients. These phytochemicals
mainly comprise phenolic compounds, alkaloids, terpenoids, and fibers. These phy-
tochemicals are being investigated for their beneficial role to control various meta-
bolic and degenerative disorders by many in vitro and in vivo systems. Each
pharmacological action of a phytoconstituent and the results of its structure activity
S. Bose (*)
NSHM Knowledge Campus, Kolkata – Group of Institutions, Kolkata, India
J. Malik
University Institute of Pharmaceutical Sciences – Centre of Advanced Study,
Panjab University, Chandigarh, India
S. C. Mandal
Division of Pharmacognosy, Department of Pharmaceutical Technology,
Jadavpur University, Kolkata, India

https://doi.org/10.1007/978-981-15-2195-9_5
56 S. Bose et al.
relations must have a good in vitro–in vivo correlation (IVIVC). With the advent of
modern technology, there has been a significant increase in screening and use of
phytochemicals in therapeutics, nutritional components, and as foods. The main
resources of phytochemicals in foods are different herbs including grains, vegeta-
bles, fruits, and beans. This increased usage of plants has given birth to various
challenges, pertaining to the quality, safety, and efficacy of phytochemicals for
industries that are involved in the development of products for human health. These
phytochemicals, either alone or in combination, have extensive impact on human
health since they proffer defense and immunity against many deadly disorders.
Classification and Health Benefits of Phytoconstituents
Phytoconstituents have been classified into three important divisions – phenolic and
polyphenolic compounds, di- and triterpenoids, and alkaloids including additional
nitrogen-containing elements (Harborne and Baxter 1993). Among them the pheno-
lic moieties mainly comprise phenols and phenolic acids, flavonones, flavonols, iso-
flavonoids, coumarins, anthocyanins, chromones, benzofurans, anthochlors, and
lignans. This group also contains phenolic ketones, tannins, stilbenoids, xanthones,
phenylpropanoids, and quinonoids. On the other hand, the terpenoids contain mono-
terpenoids, iridoids, diterpenoids, sesquiterpenoids, and triterpenoids. Apart from
these, phytosterols, steroids, saponins, sesquiterpene lactones, cardenolides and
bufadienolides, other triterpenoids and carotenoids, nor-triterpenoids, and cucur-
bitacins are also considered as part of terpenoids (Harborne 1999). The alkaloids,
depending upon the basic nucleus, are of indole, pyrrolidine, quinolizidine, tropane,
piperidine, quinoline, isoquinoline, pyrrolizidine, steroidal, and betalain type. Apart
from this, nitrogen-containing constituents encompass purines and pyrimidines,
cyanogenic glycosides, nonprotein amino acids, glucosinolates, and amines.
Generally, all phytochemicals offer a specific health interest, and they play a great
role in pharmaceuticals. This chapter has been contemplated to encompass reports
of recently accepted health benefits of particular phytochemicals in different phar-
maceuticals (Table 5.1).
Group I: Terpenoids or Terpenes
The terpenoids and terpenes are also referred to as isoprenoids. These are the big-
gest group of phytochemicals present in vegetables and food grains. Using photo-
sensitizing pigments, the terpenoids maintains the carbon in the plants through the
photosynthesis reactions. Plants have a wide range of phytochemicals like terpenes
and terpenoids which can protect them from oxidative stress. The most deliberated
terpenes are tocotrienols and tocopherols, which are used as antioxidants. Animals
have expanded the utilization of these terpenes and terpenoids for the regulation of
5 Application of Phytochemicals in Pharmaceuticals 57
Table 5.1 Use of phytoconstituents in different pharmaceuticals

Class of
phytoconstituents Phytoconstituents Use in pharmaceuticals
Terpenoids
Monoterpene Pinene, citronellol, Plant–plant interaction
limonene
Sesquiterpene Abscisic acid Plant growth hormones
Ryanodine Insecticides
Diterpene Gibberellic acid Plant growth hormone
Triterpene Azadirachtin Antifeedant, disruption of oviposition, reduction in
fecundity
Saponins, glycosidic Antifungal
triterpenoids
Phenolics
Simple phenols Catechol Antifungal
Stilbenes Resveratrol Antifungal
Flavonoids Quercetin Pigmentation and defense
Polyphenols Lignins Blocking the pathogenic growth
Condensed tannins Disease resistance, feeding repellants
Furanocoumarins Umbelliferone Fungal defense
Isoflavonoids Naringenin Promoting the formation of nitrogen fixing nodules
by symbiotic rhizobia
Nitrogen- and sulfur-containing compounds
Alkaloids Pyrrolizidine Antimicrobial
Cyanogenic Amygdalin Broad spectrum defense
glucosides
Phytochelatins Glutathione Cellular antioxidant
Glucosinolates Allyl-cys-sulfoxide Antifungal
Dietary fiber High-fiber diets may help prevent colon cancer and
be used to treat constipation, diverticular disease,
irritable bowel syndrome and Crohn’s disease
their hormones and growth. Also, the existence of these moieties in animal tissues
contributes to protection from specific diseases, specifically those allied to chronic
and unregulated growth of the damaged cells. When terpenes react with free radi-
cals, it separates them into fatty membranes by breaking its elongated C side chain
(Harborne and Baxter 1993).
Group I (A): Tocopherols and Tocotrienols
These terpenes are normally available in different grains. The most effective apop-
totic human breast cancer inducers are tocotrienols and RRR-d-tocopherol (Yu et al.
1999). Tissue concentration of tocopherols and tocotrienols is not the salient feature
(Hayes et al. 1993) to designate their outcome on cancer cells in vivo. Mixed and
pure isoprenoids named ƴ-tocotrienol and β-ionone, respectively, vanquished the
58 S. Bose et al.
development of tumor cell via commencement of apoptosis and concomitant appre-

hension of cells in the G1 phase of the cell cycle (Mo and Elson 1999). A regular
diet containing grains, vegetables, and fruits reduces the cancer risk partly due to
the availability of these terpenes.
Group I (B): Carotenoid Terpenes
These carotenoid terpenes are globally distributed as antioxidants (Wolf 1982;

Burton and Ingold 1984; Liebler 1996). They are yellow, orange, and red pigmented,
available in vegetables, in fruits, and in egg yolk. Carotenoids are tissue specific in
their pharmacological properties as they contain two types of moieties, which are
carotenes and xanthophylls. Mostly, precursors of vitamin A, α-carotene, β-carotene,
and ƴ-carotene, have the same property as that of vitamin A. To alter health condi-
tion, these precursors of vitamin A are now being tested extensively. Other caro-
tenes like lycopene, ƴ-carotene, and lutein show protection against cancer in the
prostate, colorectal, lung, uterine, and breast (Bendich 1989) and also protect
against the cancer of the digestive tract (Francheschi et al. 1994). The absorption
and distribution of lycopene in the body tissue are diverse. It has been reported that,
by evaluating alterations in buccal mucosa cell content of the carotenoids, it’s uptake
through the oral cavity is very high (Paetau et al. 1999). The lycopene level in the
body is increased significantly after consumption of oleoresin (Paetau et al. 1999).
Zeaxanthin, cryptoxanthin, and astaxanthin containing xanthophyll groups provide
tissue-specific protection when compared with other antioxidants (Parker 1989;
Nair et al. 1984).
Group I (C): Limonoids Group
Limonoids, terpenes of citrus fruit, furnish chemotherapeutic activity by obstructing

Phase I enzymes and instigating Phase II detoxification enzymes in the liver (Hertog
et al. 1993). D-Limonene, the customary monocyclic monoterpene from orange
peel oil, impedes pancreatic cancer (Nakaizumi et al. 1997) which has been investi-
gated in the gastric carcinogenesis in Wistar rats induced by N-methyl-N′-nitro-N-
nitrosoguanidine (Uedo et al. 1999). They also defended the lung tissue effectively
(Dorow et al. 1987).
Group I (D): Phytosterol Group
Phytosterols constitute another important class of terpenes. All the sterols which are
produced in the plant body are β-sitosterol and its glycosidic derivatives. These
moieties reveal antineoplastic, antipyretic, anti-inflammatory, and immune-
modulating activity in vivo (Bouic and Lamprecht 1999). The mixture of β-sitosterol
and its glycosidic derivatives targets specific Th1 and Th2 cells (T-helper
lymphocytes) thereby developing natural killer cell and T-lymphocyte activity. The
prostaglandin pathways are modified by different phytosterols in such a way that it
shields platelets. It is also known to block inflammatory enzymes (Hertog et al.
1993). Cytostatic activity of steroidal saponins has also been observed against leu-
kemia HL60 cells (Mimaki et al. 1998). With regards to intestinal uptake, phytoster-
ols always compete with cholesterol. It also helps to remove the cholesterol from
the body. In lowering cholesterol concentrations, saturated phytosterols are more
potent than unsaturated compounds in the body (Ling and Jones 1995). These
actions lower plasma levels of low-density lipoprotein (LDL), cholesterol, and total
cholesterol (Jones et al. 1999). Due to the similar structure of plant sterols and cho-
lesterol, the competition of absorption of cholesterol at the intestine is expected
(Ling and Jones 1995). In humans the plasma phytosterol levels are low due to their
very little intestinal absorption and their speedy liver excretion (Ling and
Jones 1995).
Group II: Polyphenolic Compounds
Polyphenolic compounds constitute a significant part of phytochemicals due to the

presence of their phenolic groups. Polyphenolic compounds from the plant are one
of the important metabolites which are comprehensively dispersed in many plants.
Their discern properties are solubility in water, molecular weights (500 to
3000–4000D), phenolic groups (12–16), and aromatics rings (5–6). The intermo-
lecular complications and classification of phenolic compounds are described as
galloyl, proanthocyanidins (condensed), hexahydroxydiphenoyl esters, and phloro-
tannins (Haslam 1998). Polyphenols generally fight against cancer cells and inhibit
angiogenesis. They also fight against free radicals and maintain normal blood pres-
sure and blood sugar level. They have also been found to protect the skin against
ultraviolet radiations, cardiovascular diseases, and dementia (Salunkhe et al. 1983;
Bravo 1998).
Group II (A): Phenolic Components
Phenolic metabolites include phenolic acids like hydroxycinnamic and hydroxy-

benzoic acids and polyphenols like flavonoids and tannins (hydrolyzable and con-
densed) (King and Young 1999) which protect the plants from oxidative damage.
This is a challenge of the food industry to embrace antioxidative phytochemicals in
foods, especially with regard to cereals and whole grain products (Andlauer and
Furst 1998). It has been reported that whole grain intake shows an improvemental
role (Anderson and Hanna 1999) in lowering the possibility of coronary heart dis-
ease (Liu et al. 1999), hypertension (Ascherio et al. 1992), diabetes (Salmeron et al.
1997), and some types of cancer (Adlercreutz 1990). But, no long-term clinical tri-
als with specific foods have been done (Anderson and Hanna 1999). The universal
60 S. Bose et al.
interest in the probable benefits of phenolic antioxidants has showed appreciable

commercialization activities surrounding several commodity extracts.
Group II (B): Flavonoidal Components
The flavonoidal components include flavanones, dihydroflavonols, flavones, and fla-

vonols. Researches involving therapeutical potency of flavonoids from grapes
(Waterhouse and Walzem 1998) and wine (German 1988; Pieta 1998) have been
going on recently. Free radical scavenging and cellular signaling, defense against
allergies, inflammation, ulcers, platelet aggregation, hepatotoxins, and tumors are
the main properties of flavonoids (Kinsella et al. 1993). Flavonoids inhibit the
angiotensin-converting enzyme which increases blood pressure. It inhibits prosta-
glandins formed by cyclooxygenase. It also blocks estrogen-produced enzymes
thereby reducing heart disease and thrombosis, by preventing platelet aggregation
and inhibiting estrogen synthase. By binding with the estrogen receptors, flavonoid
decreases the risk of estrogen-mediated cancers. Kaempferol, apigenin, myricetin,
quercetin, and luteolin are present in various foods as flavonoids (Hertog et al.
1993). The main sources of these flavonoids are tea, onions, and apples. It has been
demonstrated that less intake of flavonoids is one of the main reasons of coronary
heart disease (Hertog et al. 1993). Some flavonoids like apigenin and luteolin have
also been reported as mutagenic (Hertog et al. 1993; Taj and Nagarajan 1996). It
also has anti-inflammatory and antibacterial activities (Harborne and Baxter 1993;
Lin et al. 1997). Apigenin in general has hypotensive, anti-inflammatory, diuretic,
and antibacterial properties. In addition, it encourages smooth muscle relaxation
(Harborne and Baxter 1993). Another hexahydroxyflavone, myricetin, shows anti-
bacterial and antigonadotropic activity though it is not a mutagen (Harborne and
Baxter 1993). Another important component of diet, kaempferol (directly muta-
genic), has antibacterial and anti-inflammatory properties (Harborne and Baxter
1993). One common flavonoid, quercetin, bestows kaempferol’s mutagenicity when
the microsomal metabolizing systems are present. Quercetin impedes many
enzymes that decrease smooth muscle contraction and lymphocytes proliferation
(Harborne and Baxter 1993; Silva et al. 1996).
Group II (C): Catechins and Gallic Acids
Grapes, blue and black berries, cocoa, and green tea are the main resources of cat-
echins, epigallocatechin gallate, epicatechin, and epicatechin gallate (major gallic
acid esters). Work done by earlier researchers have shown that these constituents
show protectiveness by their free radical scavenging property (Hanasaki et al. 1994)
and their reticence in synthesis of eicosanoid and platelet aggregation (Pace-Asciak
et al. 1995). For the circumvention of prostate cancer, possible use of tea, especially
green tea, should be increased (Gupta et al. 1999; Hollman et al. 1999). In wines,
mainly procyanidins and catechins have the astringency property (Haslam and
Lilley 1988). Many important phenolics (constituents of grapes and red wines)
impart the protective of red wine against atherosclerotic cardiovascular disease
(Pieta 1998; Frankel et al. 1993; Kanner et al. 1994). It has been reported that cat-
echin metabolites have been found in plasma for both alcoholized and dealcohol-
ized red wine (Donovan et al. 1999). These metabolites of catechin are abolished
from the blood very soon, which proves that catechin is well absorbed but rapidly
metabolized.
Group II (D): Isoflavonoids
Isoflavonoids are a separate but an important subclass of the phenolic constituents.

Genistein and daidzein are two major isoflavones obtained from soya beans.
Isoflavones bind to the estrogen receptors and are referred to as phytoestrogens.
Genistein retard the development of colonic carcinogenic cells which are both
dependent and independent to hormones (Messina 1999). It also inhibits prostate
cancer, breast cancer, and changed tumor biomarkers connected with angiogenesis
(Zhou et al. 1999; Messina and Bennink 1998). Many reports have revealed that the
isoflavonegenistein binds with the beta-estrogen receptor (Makela et al. 1999) and
shows cardioprotective effects, except adversely affecting cancer risk on reproduc-
tive tissues (Rohdewald 1998; Liu et al. 1998).
Group II (E): Anthocyanidins
Generally, anthocyanidins, aglycones of anthocyanins, are water-soluble flavonoids.

The anthocyanidins, obtained from nature, are cyaniding, delphinidin, pelargonidin,
malvidin, petunidin, and peonidin which have roles as pigments in fruits and flow-
ers. These dyes are affected by metal ion complexes and pH. Like the parent moiety,
these are antioxidants in vitro (Rice-Evans et al. 1996, Pool Zobel et al. 1999) and
antioxidative and antimutagenic in vivo.
roup III: Alkaloids and Other Nitrogen-Containing

G
Metabolites
Group III (A): Glucosinolates
In cruciferous vegetables, glucosinolates generally activate the liver detoxification

enzymes. To impart defense against some deadly disease like carcinogenesis, muta-
genesis, and toxicity of electrophiles, proper consumption of vegetables is essential
(Fahey et al. 1997). By using broccoli and cauliflower, 10–100 times more of gluco-
raphanin can be taken inside the body, which can decrease the risk of several can-
cers (Fahey et al. 1997).
62 S. Bose et al.
Generally, the transformed forms of glucosinolates are dithiolthiones, sulfora-

phane, and isothiocyanates. Glucosinolates are liberated from plant cells trans-
formed to isothiocyanates by the enzymes myosinase. Some of them show
antineoplastic effects on the human body. Mechanism of action involves the metab-
olism of carcinogen by the Phase II detoxification enzymes. Inhibition of Phase I
carcinogen-activating enzymes may also be involved in the process (Hecht 1999). A
decreasing outcome on tumor formation has been noticed in humans using indoles
and brassica vegetables. Ingested isothiocyanates and glucosinolates and their
human metabolites are excreted through the urine (Shapiro et al. 1998). In the
human body, significant portion of glucosinolates and isothiocyanates are converted
to dithiocarbamates in the urine and Phase II enzyme induction occurs in the body.
These Phase II metabolizing enzymes authorize the metabolism and excretion of
unfavorable substances in adults.
Group III (B): Indole Alkaloids
Indole alkaloids like Indole-3-carbinol (glucosinolate metabolite) have the potency

to inhibit specific carcinogenesis (Telang et al. 1997). These indole alkaloids reduce
many cancer cells like mammoplasty-derived 184B5 cells and mammary carcinoma-
derived MDA-MD231 cells. These alkaloids can determine the decreasing order of
aberrant proliferation in initiated and transformed cells. The reason of the efficacy
of these indole alkaloids on breast cancer is mainly due to its capacity to control cell
cycle progression. Other reasons include enhancement of the production of antipro-
liferative estradiol metabolite and induction of cellular apoptosis. Continuous appli-
cation of estrogen, without progestogen, may create endometrial cancer in
postmenopausal women (Weiss 1996; Kelloff et al. 1995). This estradiol expands
the human papilloma virus oncogenes in cervical cancer cells. But indole-3-carbinol
nullifies the high estrogen level in the oncogenes (Jin et al. 1999; Yuan et al. 1999).
Scientists observed that indole-3-carbinol enhance the level of cytochrome P450
enzymes which are accountable for 2-hydroxylation of estrogen. For obese pre-
menopausal women who are taking at least 400 mg of purified indole-3-carbinol
regularly, it helps to minimize estrogen-dependent cancer risk (Michnovicz 1998).
Group IV: Dietary Fiber
The dietary fibers play a direct role to prevent colon cancer. This may change the
GIT domain to protect against colorectal disorders. Increased amount of bile acids
and its metabolites may create colonic carcinogenesis, due to which the calcium
level, pH, ammonia, and fatty acid levels in the colon can also be altered. Generally,
it decreases the level of bile acid concentrations in the colon (Jensen et al. 1982).
Cholesterol synthesis also can be reduced by butyric acid (short chain fatty acids)
and dietary fibers (Hara et al. 1999). This may be due to the formation of butyric
acid through the bacterial fermentation of the fiber (Smith and German 1995; Smith
et al. 1998). The main activities of butyric acid are to control the growth and apop-
tosis of epithelial cells and tumor cells of the colon. So, rich intake of these dietary
fibers with the food gives protection against the abovementioned diseases and shows
health benefits. Short in vivo half-life is one of the main drawbacks of these
dietary fibers.
Regulatory Aspects of Phytochemicals as Nutraceuticals
In the pharmaceutical industry, the rapid growth of healthy foods is described as

nutraceuticals. The Nutrition Labeling and Education Act of 1990 and the Dietary
Supplement Health and Education Action of 1994 have given the guidelines to the
industries with regard to the nutraceuticals. These acts are delivering the required
particulars to the customers who are purchasing the natural drugs, foods, and sup-
plements regularly (Clydesdale 1997). Manufacturers and customers jointly need to
be involved in the development and use of the phytochemicals present in different
pharmaceuticals based on proper scientific information.
Future Prospects of Phytochemicals in Pharmaceuticals
In many countries, since a long time, vast use of medicinal plants can be observed.
But only in the recent era have some of them been scientifically proven for nutri-
tional and medicinal evidence. These are now being referred to as phytonutrients.
The sincere researches on “modern” pharmaceuticals of plant origin may unravel
new phytoconstituents that may be considered as a medicine for their potency to
prevent and cure various deadly diseases. The recent research interests focus on the
relationship between the structure and function of the phytoconstituents (Bidlack
et al. 1998). Nowadays, it has become important to recognize and purify the bioac-
tive markers from the plant sources. Recently, the pharmaceutical industries have
shown a significant interest to improve the medicinal property of natural foods by
modifying the food habits of the consumers. The useful ingredients containing food
should be manufactured in the food industries and make them most popular to the
people. The foods which are the choice for most people may not be filled with mark-
ers and active constituents to control diseases (Erickson-Otto 1998; Reid 1999;
Hathcock 1993). Consumer interest and its policies play a major part in the drug
development and in the trading of pharmaceuticals (Childs and Poryzees 1998).
Many scientific data have superscripted the subsequent markets for phytochemicals
in the use of pharmaceuticals (Brower 1998). Researchers have shown commercial
developments of use of phytochemicals in pharmaceuticals in terms of patents for
64 S. Bose et al.
anti-diabetogenic, anticarcinogenic, cerebro-active, prebiotic oligosaccharides,

Bifidobacterium probiotic foods, and things for prevention of rheumatoid arthritis
and osteoporosis (Shukla 1998). The usage of healthy ingredients which are not
traditionally considered as nutrients is increasing extensively (Aarts 1997).
Conclusions
The execution of the therapeutic potency of medicinal herbs against different deadly
diseases started long back. The use of secondary metabolites in this purpose is as
old as a bygone edification. Plant constituents deploy an important pharmacological
activity in the human body. There are two types of chemical compounds available in
the plant body known as primary and secondary metabolites. This has been decided
according to their chemical structure and the biosynthetic pathway of their synthe-
sis. These metabolites can be again categorized on the basis of different functional
groups attached to its structure. These functional groups are many times responsible
to treat many diseases. All the above discussed secondary metabolites are very
much important to treat and prevent various diseases and to be a part of modern
pharmaceuticals and medicines. Direct interaction with the specific targeted recep-
tors influences the therapeutic activity of these chemicals in a broad range. These
important secondary metabolites and their phytochemistry including applications in
recent pharmaceuticals may pave the way for information to recognize and purify
the markers in the modern discovery of drugs.
Acknowledgments The author (S C Mandal) is thankful to the Department of Biotechnology,

New Delhi, for financial assistance to Dr. Subhash C Mandal under DBT’s Twinning Program for
North-east (DBT-NER/AGRI/29/2015 (Group 2), Dated: 19/10/2016) India.
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Chapter 6
The Herbal Drugs
Amritesh C. Shukla
Introduction
The pharmaceutical companies play a vital role in the economic development glob-
ally. While development of synthetic drugs is attributed to serendipitous discovery,
more and more companies are focusing more on drug development from plants (Pan
et al. 2013). Natural/plant products have been the basis of management of various
diseases in human beings and will remain an important source of drug development
(Patwardhan et al. 2004). Since long back, plants have been the basis of many con-
ventional systems of medicine throughout the world and continue to provide man-
kind with novel remedies (Jachak and Saklani 2007). The use of herbal drugs can be
dated from the existence of human civilization and has been the pillar of modern
medicine (Patwardhan et al. 2004). It has been used since ages in various system of
medicines including Ayurvedic, homeopathic, naturopathic, and others for the pre-
vention and protection of several diseases and ailments and to support healing and
health (Pathak and Das 2013; Rout et al. 2009). Before the introduction of contem-
porary system of medicines, disease treatment was completely managed by plant-
based herbal remedies (Birdi et al. 2006).
There has been an increased interest in natural product research in recent times,
and medicinal plants account for a critical role in the development of potent thera-
peutic agent (Pathak and Das 2013; Rout et al. 2009). The diversity of natural prod-
ucts and development of new techniques for isolation, identification, purification,
and structure characterization of these active constituents/complex natural products
form the basis and interest of traditional medicine and therapies research and devel-
opment (Rout et al. 2009). The ancient and modern therapeutic skills can be wisely
used for the natural products drug discovery (Patwardhan et al. 2004).
A. C. Shukla (*)

https://doi.org/10.1007/978-981-15-2195-9_6
70 A. C. Shukla
Significance of Plants as Potential Source of Herbal Drugs
The use of plants in traditional system of medicine has been gradually increasing in
developed countries, because of the failure of modern medicine to provide efficient
treatment for persistent diseases and materialization of multidrug-resistant bacteria
and parasites. The drawbacks of synthetic drugs, questioning on the approaches and
assumptions of allopathic drugs, their rising costs, and greater public access to
information on conventional medicine have also led to an increasing interest in
alternative treatments. Since centuries back, plant secondary metabolites have been
the source of expectation as a wide group of medicinal plant formulations. Hence, it
has become essential to resume the importance of herbal medicines (Birdi
et al. 2006).
Ayurveda is one of the most traditionally using medical system, widely practiced
in the Indian subcontinent, and has a sound optimistic and experimental backup.
Charaka Samhita and Sushruta Samhita (100–500 BC) are main classics of
Ayurveda, which illustrates over 700 plants along with their taxonomy, pharmacol-
ogy, and therapeutic properties. Further, on the basis of Ayurvedic investigations,
various phyto-molecules, including alkaloids of Holarrhena sp. (Fabaceae) in
amoebiasis, Rauwolfia alkaloids for hypertension, psoralens for vitiligo, Mucuna
pruriens for Parkinson’s disease, guggulsterone as hypolipidemic agent, piperidines
as bio-enhancers, baccosides as mental retention, picrosides as hepatic protection,
Phyllanthis (also called stone breaker plant), as antivirals, curcumin as inflamma-
tory, withanolides and several other steroid lactones, and their glycosides as immu-
nomodulator have been developed (Patwardhan 2005).
Since time immemorial, terrestrial plants, especially the higher plants, are used
for the treatment of human diseases. Numerous well-known species, namely, lico-
rice (Glycyrrhiza glabra), poppy capsule latex (Papaver somniferum), and myrrh
(Commiphora sp.), were referred to by the first known written record on clay tablets
from Mesopotamia in 2600 BC, and these plants are still in use for the management
of various diseases, as composition of herbal drugs, in traditional systems of medi-
cine, etc. (Jachak and Saklani 2007). Besides, codeine, morphine, noscapine (narco-
tine), and papaverine isolated from Papaver somniferum were developed as single
synthetic drugs and are still clinically used. A semisynthetic derivative of glycyr-
rhetinic acid, hemisuccinate carbenoxolone sodium, found in licorice, is prescribed
for the healing of gastric and duodenal ulcers in various countries (Chin et al. 2006).
A significant segment of natural product-based pharmaceuticals are plant-derived
drugs. As per the World Health Organization (WHO), plants are used as medicinal
agents by almost 65% of the world’s population. Around 25% of the drug prescribed
are of herbal origin, for example, anticholinergics (atropine), analgesics (opium
alkaloids), antiparasitics (quinine), anticholinesterases (galantamine), antineoplas-
tics (vinblastine/vincristine), etc. (Pathak and Das 2013).
6 The Herbal Drugs 71
Drug Discovery/Herbal Drug
The research practice is complex, time-consuming, and expensive, and the end
products are also never guaranteed (Anon). Discovery and development of herbal
drugs have a historical record, and it long backs to the ancient days of human civi-
lization. In those prehistoric times, drugs were not simply used for physical reme-
dies but were also related with the spiritual remedies. Religious leaders were often
the administrators of herbal drugs. The early drugs or folk medicines were mainly
derived from plant products and supplemented by animal materials and minerals.
These drugs were perhaps discovered through the amalgamation of trial and error
experimentation, as well as observation of the results/reactions on human and ani-
mal, of ingesting such products.
Until the late 1800s, majority of the drugs were based on herbs or extraction of
constituents from plant sources. The chemical drugs, using synthetics were com-
menced at the beginning of the 1900s, and the pharmaceutical industries thus came
into existence. Many drugs were researched and manufactured, but mostly they
were used for therapeutic purposes rather than completely curing the diseases.
Since, the early 1930s, the drug discovery was determined on the basis of screening
of natural products and isolation of the active ingredients of the plants and for treat-
ing various diseases. The active ingredients/constituents were usually the synthetic
form of the natural products. These synthetics were known as new chemical entities
(NCEs) and have to go through several tests to ensure that they are safe, potent, and
effective.
Further, in the late 1970s, expansion of r-DNA process and utilization of knowl-
edge of cellular and molecular biology came into existence, and the biotechnology
industry came to the limelight. The pharmaceutical companies, together with the
advancement in gene therapy and unraveling the mechanisms of diseases infesta-
tion, and the research findings from the Human Genome Project, have opened up
plenty of opportunities and made possible the development and use of drugs specifi-
cally targeting the sites where diseases are caused (Rick 2004). The key factors that
necessitated change in biomedical knowledge and technology were the substantial
increase in the number of therapeutic targets and discovery of high levels of com-
plexity (Herrling 2005).
Generally, hundreds and hundreds of chemical compounds are tested in an effort
to find out a compound that can achieve the desirable result. There are no standard
routes through which drugs can be developed. A pharmaceutical company may
choose to develop a novel drug designed for a specific disease or medical stipula-
tion. Sometimes, scientists choose to track an interesting/promising line of research.
However, on the other cases, new findings from universities, government, or other
laboratories may show the path for pharmaceutical companies to follow with their
own research (Anon).
A typical herbal medicine drug development process includes differential aspects
such as isolation/synthesis of bioactive constituent(s) in herbal medicines; investi-
gation of safety measures, bioefficacy, using pharmacological methods; evaluation
72 A. C. Shukla
of safety and efficiency using conventional pharmacological methods; approval of

the therapeutic agent to be used by the regulatory authorities and post-marketing
monitoring (Pan et al. 2013; Pietersd and Vlietinck 2005).
Literature reveals that plenty of researches selecting plants/plant-based products
as candidates for drug discovery programs, including antibacterial, antidiabetic,
antifertility, antifungal, anti-hypercholesteremic, anti-inflammatory, antitumor, car-
diovascular, central nervous system depressant, diuretic, etc., have already been
published/patented; besides a number of such studies are in progress (Fabricant and
Farnsworth 2001; Shahi et al. 2001; Bahar et al. 2007; Itokawa et al. 2008; Osbourn
and Lanzotti 2009; Shukla and Dikshit 2016).
Herbal Drug Development
The complex process of drug development includes product development, nonclini-

cal studies, and clinical studies. A new chemical entity emerges as a candidate to
undergo safety test, toxicity tests, pharmacokinetics, and metabolism activity, under
in vivo and preclinical models, if it shows promising in vitro pharmacodynamic
activity on particular biological targets (Rundfeldt 2011). Pure active ingredients/
molecules, obtained from a bioassay-guided isolation process from extracts of
medicinally important plants, are investigated for structural activity relationship
studies (SAR). Efficacy, safety issues, as well as clinical tests are supposed to be
carried out; active ingredients have to be prepared/synthesized at the industrial
level; and a suitable pharmaceutical formulation has to be made before the com-
pound/ingredients can be permitted as a novel drug. In traditional system of medi-
cine, however, pharmacological assessment of extracts from medicinally important
plants may lead to the establishment of standardized extracts. In such case, com-
mercial production of these standardized extracts may start immediately after the
toxicity and safety studies. After developing the formulation of the standardized
extracts/compounds, clinical studies may be carried out, which may lead to get its
approval as novel drug(s) (Fabricant and Farnsworth 2001; Shahi et al. 2001;
Pietersd and Vlietinck 2005; Shukla 2014; Shukla and Dikshit 2016).
Clinical trials characterize the ultimate premarket testing ground for unapproved
drug formulations. An investigational ingredient/compound is administered to the
humans and is investigated for its safety and efficiency in treatment, prevention, or
diagnosing specific disease(s). The consequences of these investigations will be
considered as one of the most important factors in the approval or disapproval of a
novel drug (Anon). Further, a flowchart showing different stages of plant products
research in the drug development is depicted in Fig. 6.1 as follows.
PLANT (MEDICINAL/ AROMATIC)
Extract(s)
Screening of the Bioefficacy
Standardization & Purification of Extract/ Active Compound(s)
Detailed Bioefficacy Tests
In-silico Investigations/ SAR Studies
Pharmacological Investigations (Toxicity & Safety Studies)
Clinical Investigations
Industrial Production of Standardized Biomolecule/ Pure Compound(s)
Pharmaceutical Formulations
Clinical Trials/ Multi-locational Trial
APPROVAL AS DRUG(S)
Fig. 6.1 Flowchart of different stages of plant products research and drug development
Challenges
In spite of the achievement of plant-based drug discovery programs, in the past few
decades, future endeavors face many challenges. Scientists working on natural
products and pharmaceutical industries will need to continuously enhance the qual-
ity and quantity of compounds/molecules that enter the drug formulation stage to
keep lick with other efforts on drug discovery. The process of drug discovery
approximately requires an average duration of 10 years and costs more than 800
million dollars. Maximum time and expenses are supposed to be spent on several
leads that are redundant during the drug discovery processing (Jachak and Saklani
2007). Several pitfalls can emerge when deciding to use plants, through either ran-
dom selection or ethnomedical claims involving the targeted disease (Fabricant and
Farnsworth 2001). Since plant-based drug discoveries have traditionally been time-
consuming, faster and better methodologies for collection of plants, bioefficacy
investigations, isolation of potential compounds, and its development must be
employed. Raw materials/crude drugs from plants are mostly used to develop the
formulation as tablet and capsule as well as up to some extent as oral liquid formula-
tions. These forms of dosage are not successful due to the problems encountered in
their absorption, therapeutic efficacy, and poor compliance. A tablet or capsule form
74 A. C. Shukla
of dosage requires a powdered form of crude herbs, and the size of particles affects
the process of blending, firmness, and filling. Besides this, homogeneity is compli-
cated to achieve due to the handling of bulk quantities, high moisture content and
inherent nature of raw materials (crude drug) (Jachak and Saklani 2007).
There are both merits and demerits of using plants as the initial point in any drug
development program. If one elects to use information suggesting that specific
plants may be useful in drugs based on enduring use by humans (traditionally eth-
nomedicinal), one can rationalize that any isolated active ingredients/compounds
from the medicinal plants are probably safer than the active ingredients from plants
having no/any traditional use. Also, plants are a renewable source of starting mate-
rial in several cases but not in all the cases. It is unanimously believed that plants
provide an infinite source of novel and multifarious chemical compounds/mole-
cules, e.g., digoxin, d-tubocurarine, vinblastine, vincristine, and taxol. If the active
principles derived from plants have novel structures and useful biologic activity,
patent protection can be assured. Further, the modern trend, especially to establish
an industry, is to seek biologically active compounds from medicinal plants that will
serve as lead compounds for synthetic/semisynthetic drug formulation, to assure
patent protection. Thus, it reduces the need to isolate novel biological active com-
pound/agent from medicinal plants, since the final goal is to utilize the active ingre-
dients to generate synthetic derivatives with lesser toxicity and higher efficacy
(Fabricant and Farnsworth 2001).
Conclusion
So far, plant-based natural compounds/herbal products discovered from medicinally

important plants (and their analogues thereof) have provided several clinically use-
ful drugs. Despite the various challenges encountered in the plant-based drug dis-
coveries, natural products isolated from medicinal plants will still persist as an
essential constituent in the search for novel drugs/herbal medicines. Besides, it is
also the fact that only one-tenth (approx.) of the flowering plant species occurring
worldwide were investigated so for; hence their pharmaceutical potential, can be the
obvious advantage to begin with plant-based drug discovery program. Further, with
the fabulous increase in the universal use of medicinally important plant products,
several concerns regarding the bioassay and protection of the herbal medicines have
also been raised. Therefore, it has become essential to standardize the bioefficacy
and safety measures, to ensure the supply of medicinal plant materials/products
with good quality.
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Jachak, M. S., & Saklani, A. (2007). Challenges and opportunities in drug discovery from plants.
Current Science, 92, 1251–1257.
Osbourn, A. E., & Lanzotti, V. (Eds.). (2009). Plant-derived natural products. New York: Springer.
https://doi.org/10.1007/978-0-387-85498-4_1.
Pan, S. Y., Zhou, S. F., Gao, S. H., Yu, Z. L., Zhang, S. F., Tang, M. K., Sun, J. N., Ma, D. L.,
Han, Y. F., Fong, W. F., & Ko, K. M. (2013). New perspectives on how to discover drugs from
herbal medicines: CAM’s outstanding contribution to modern therapeutics. Evidence-Based
Complementary and Alternative Medicine, 2013, 1–25.
Pathak, K., & Das, R. J. (2013). Herbal medicine – A rational approach in health care system.
International Journal of Herbal Medicine, 1(3), 86–89.
Patwardhan, B. (2005). Ethnopharmacology and drug discovery. Journal of Ethnopharmacology,
100, 50–52.
Patwardhan, B. V., Ashok, D. B., & Chorghade, M. (2004). Ayurveda and natural products drug
discovery. Current Science, 86(6), 789–799.
Pietersd, L., & Vlietinck, A. J. (2005). Bioguided isolation of pharmacologically active plant
components, still a valuable strategy for the finding of new lead compounds. Journal of
Ethnopharmacology, 100, 57–60.
Rick, N. G. (2004). Drugs from discovery to approval (Ph.D. thesis). Weinheim: Wiley.
Rout, S. P., Choudary, K. A., Kar, D. M., Das, L., & Jain, A. (2009). Plants in traditional medicinal
system – Future source of new drugs. International Journal of Pharmacy and Pharmaceutical
Sciences, 1(1), 1–23.
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ment – A case study based insight into modern strategies. Rijeka: In Tech.
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New Delhi: Today and Tomorrow’s Publishers.
Chapter 7
Role of Natural Products as Alternative
of Synthetic Steroidal Drugs
Priyanka Tiwari, Rashmi Pandey, Reetika Singh, and Bechan Sharma
Introduction
Corticosteroid also called as glucocorticoids came into existence in the mid-1940s

and recognized as an anti-inflammatory agent, therefore popularly prescribed by
clinicians for the treatment of severe inflammatory and autoimmune diseases.
Glucocorticoid agonists were first time used by Philip Hench and his coworkers for
the treatment of rheumatoid arthritis (RA). Edward Kendall synthesized a synthetic
glucocorticoid and delivered it to patients who suffered from severe rheumatoid
arthritis. Few days later, the symptoms were interestingly improved (Glyn 1998). In
1950, a Nobel Prize was awarded in Physiology or Medicine jointly to Philip Hench,
Edward Kendall, and Tadeus Reichstein for their invention of glucocorticoids
(Baschant et al. 2012).
Corticosteroids can be considered as a class of chemical compounds which com-
prise synthetic molecules or naturally secreted hormones. In general, glucocorti-
coids are involved in regulation of metabolism and inflammation, while
mineralocorticoids are involved in regulating electrolyte balance. It is noted that
exogenous glucocorticoid administration is associated with alterations in carbohy-
drates, lipids, protein metabolism, and a large number of enzymatic secretions. So,
side effects due to treatment with corticosteroids are expected to occur in the users
(Becker 1964).
Nowadays, so many clinical aspects of steroids are known for their admirable
anti-inflammatory and immune-modulating properties. Numerous side effects of
steroidal drugs have been reported. However, the extent of side effects varies with
P. Tiwari · R. Singh · B. Sharma (*)

Department of Biochemistry, University of Allahabad, Allahabad, Uttar Pradesh, India
R. Pandey
Department of Pulmonary and Critical Medicine, King George Medical University,
Lucknow, Uttar Pradesh, India

https://doi.org/10.1007/978-981-15-2195-9_7
78 P. Tiwari et al.
respect to their route of administration and duration of intake (Kendall 1950). The
function of corticosteroids is concerned with protein binding, expression of
enzymes, molecular cascade, and endogenous cortisol. The general metabolism of
corticosteroid is governed by Cytochrome P450 (CYP) system (Xu et al. 2007).
Oral administration of glucocorticoids is popular in patients with chronic dis-
eases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory
bowel disease, and asthma. However, long course of treatment with the corticoste-
roids generates several side effects which include pathologic fracture of bones,
muscle weakness, sleep disturbances, weight gain, skin rashes, and cataracts (Curtis
et al. 2006). Some psychiatric side effects are also seen such as anxiety, mood dis-
orders, panic disorder, and delirium with the continuation of glucocorticoid therapy
for long term. However, these effects get reversed when doses of glucocorticoids are
discontinued or withdrawn. The adverse side effects mentioned above are found in
90% of patients who would have taken low dosages (≤7.5 mg/day) of glucocorti-
coids for >60 days (Curtis et al. 2006; Pereira et al. 2010). Plant-based principles/
natural products are reported to be cost-effective, easily available, and safe in use
because it causes minimal or no adverse effects on human health (Singh et al. 2016).
Therefore, there is an urgent need to explore the plant-based steroids to replace the
synthetic or semisynthetic steroids.
Steroidal drugs have been predominantly used for a long time in treatment of
chronic inflammatory diseases. However, its adverse physiological effects such as
immune suppression, hypertension, osteoporosis, and metabolic disturbances have
been reported to occur due to prolonged treatment with these medicines. The mech-
anism of action of these drugs relies on multiple signaling cascades by their binding
with steroidal receptors which in turn regulate the activation of inflammatory genes
(Turk and John 2005). Recently, it was found that a huge variety of medicinal plants
have some steroid-like constituents known as plant steroids, which share their struc-
tural resemblance with synthetic steroids. The clinical studies and observations
show that these components are having anti-inflammatory as well as some other
physiological actions (Hubert 2003).
Plants are found to synthesize biologically active secondary metabolites which
are not playing any prominent role in their own primary cellular functions, but they
are having extensive medicinal properties and hence used for mankind. However,
some secondary metabolites produced by some medicinal plants acting as steroids
offer defense system to the plants. The general structure of steroids comprises of
tetra cyclic 1, 2-cyclopentenoperhydrophenanthrene ring, with methyl substituent at
C-10 and C-13 and an alkyl group at C-17 positions. The great diversity found
among the steroids arises due to different range of oxidation states of the carbons
present in its tetra cyclic core and methyl groups and the side chain structure
(Hubert 2003).
In this chapter, an endeavor has been made to present updates on the wide appli-
cations of plant-based corticosteroids and their varied anti-inflammatory activity for
the safe and effective treatment of chronic inflammatory diseases. It is expected that
in the future, these compounds may act as better substitutes of synthetic corticoste-
roids as potential anti-inflammatory compounds without any systemic toxicity.
7 Role of Natural Products as Alternative of Synthetic Steroidal Drugs 79
Plant Steroids
Plant steroids have a diverse group of biologically essential secondary metabolites

which are produced by plants. Some vital functions of plants are controlled by the
plant steroids. They exhibit certain medicinal properties which are beneficial for
human beings. Plants having steroids are used as traditional medicines in different
countries. Hence, it is necessary that plant raw materials and the final products are
routinely analyzed for the recognition and quantification of specific plant steroids.
After the investigation of steroids in plants, and use of these plants as a traditional
medicine system, they are used as therapeutic agents or as herbal supplements in
various countries. The cyclization reaction of 2,3 epoxysqualene into cycloartenol
via acetate-mevalonate pathway leads to biosynthesis of plant steroids. Further,
enzymatic metabolism of cycloartenol provides biologically active steroids. Plant
steroids can be classified into several groups on the basis of their chemical structure,
pharmacological action, and taxonomic considerations (Patel and Savjani 2015).
Plant steroids exhibit various pharmacological properties such as anticancerous,
immunomodulatory, hepatoprotective, antimicrobial, mammalian steroidal hor-
mones, and plant growth hormone. They also possess antihelminthic, and cytotoxic
properties with abilities to increase the tonus of heart muscles. In a study of H. cau-
casicus by Martucciello et al. (2018), it was investigated that the steroidal com-
pound isolated from this plant species reduces cancer cell viability inducing
apoptosis and GRP78 downregulation. The summary of plant-based steroid group
of molecules and their properties is shown in Table 7.1.
atural Occurrence and Biological Importance of Plant

N
Steroids
The wide distribution of plant steroids is seen among different families of plants in
the plant kingdom. The list of some families of plants recognized as a source of dif-
ferent groups of phytohormones is shown in Table 7.2. Some specific plants and
their aired biological activities are described in the following text.
Withanolides from Plants
Withanolides, naturally occur in plants as steroidal lactones. They are derivative of

ergostane-type skeleton. The C-22 and C-26 of ergostane oxidized to form a six-
membered ring of lactones denoted as the “withanolide” (Lavie et al. 1965,
Habtemariam 1997). The Solananceae family comprises a large number of genera
containing withanolides such as Acnistus, Dunalis, Datura, Discopodium, Deprea,
Exodeconus, Hyoscyamus, Iochroma, Jaborosa, Larnax, Lycium, Nicandra,
80 P. Tiwari et al.
Table 7.1 Plant-based steroid group of molecules and their effects

Plant steroidal
groups and General Pharmacological Example of
sources chemistry activity molecules References
Brassinosteroids Cholestane ring Plant hormone Brassinolide Mandava
having two having growth- Castasterone (1988)
diols and C-24 enhancing properties
position having
different groups
Bufadienolides C-24 steroid Cardiotonic, Helleborine ouabain, Krenn and
with pyrone increase tonus of physodine, Kopp (1988)
ring at C-17 heart muscles scillaridine
Cardenolides C-24steroids,C- Cardiotonic, used as Digoxin, digitoxin, Concepcion
17position is arrow position, digitoxigenin et al. 2000;
occupied with antimicrobial Manuel et al.
lactone ring 2001
Cucurbitacins Oxygenated Antitumor, Arvenin 1, Dinan et al.
C-30 antimicrobial, cucurbitacin C, (1997);
triterpenoids hepatoprotective, cucurbitacin D. Dinan (2001)
with a methyl anti-inflammatory
group at C-4
Phytoecdysteroids C-27 steroids, Protect plants Ecdysone Oogonial, Dinan et al.
C-24 occupied against insects 20-Hydroxyecdyson, (1996);
with methyl (molting hormones) pterosterone, Barile et al.
group and anabolic, Turkesterone (2007)
7-en-6-one antiarrhythmic,
chromophore antioxidant
Phytosterols Steroidal hypocholesterolemic β-Sitosterol, Jones et al.
alcohol having activity, antitumor, campesterol, (1997);
double bond in anti-inflammatory stigmasterol Moghadasian
sterol ring (2000)
Sapogenins/ C-27 steroids, Fungicidal Spirostanol Quan et al.
steroid saponins C-3 Position antitussive medicine, (diosgenin, (2005);
occupied with sex hormone sarsasapogenin, Barile et al.
hydroxyl group glycyrrhetinic acid) 2007
Steroidal Steroidal Anti-inflammatory, Solasodine, Li et al.
alkaloids skeleton with sex hormone, solasonine, (2006)
nitrogen atom anti-asthmatic, solamargine,
integrated into α-glucosidase solanidine,
a ring or as a inhibitor guggulsterone,
substituent lycioside A,
ebeiensine
Withasteroids/ C-28 steroids Diarrhea, rheumatic Witharistatin, Kirson and
withanolides with a fever, anticancerous withaferin A, Glotter
δ-lactone, the immunomodulatory, iochromolide, (1981);
side chain cholinesterase withanolide D Dinan et al.
linked to the inhibitory, Bracteosin A (1996);
steroid nucleus hepatoprotective Muhammad
at 17α or 17ß et al. (1999)
Table 7.2 The list of some families of plants recognized as a source of different groups of
phytohormones
Steroidal groups Plant families References
Brassinosteroids Brassicaceae, Fabaceae, Asteraceae, Poaceae, Hartmann (2004);
Rutaceae, etc. Bajguz et al. (2011)
Bufadienolides Crassulaceae, Hyacinthaceae, Ranunculaceae, Steyn and Heerden
Iridaceae, Melianthaceae, Santalaceae (1998)
Cardenolides Apocynaceae, Plantaginaceae Wen et al. (2016);
Schaller (2010)
Cucurbitacins Cucurbitaceae, Cruciferae, Euphorbiaceae, Kaushik et al.
Rubiaceae, etc. (2015)
Phytoecdysteroids Lilialiaceae, Ranunculaceae, Malvaceae, etc. Chaubey (2017)
Phytosterols Lamiaceae Villaseñor et al.
(2002)
Sapogenins/steroid Amaryllidaceae, Asparagaceae, Liliaceae, Sobolewska et al.
saponins Melanthiaceae, Smilacaceae, Zygophyllaceae, (2016)
Solanaceae, Plantaginaceae, Fabaceae
Steroidal alkaloids Melanthiaceae, Solanaceae, Buxaceae Wiart (2013)
Withasteroids/ Solanaceae Chen et al. (2011)
withanolides
Physalis, Salpichroa, Trechonaetes, Tubocapsicum, Vassobia, and Withania (Chen

et al. 2011). Withanolides are reported to have amazing biological activities.
Withaferin A is an important compound among the withanolides, isolated from vari-
ous species of genus Wuhania. Withaferin A contains antimicrobial properties,
mainly against acid-fast bacilli and gram-positive microorganisms. Later on inves-
tigating on several withanolides, it was found that 27 deoxywithalerin A is biologi-
cally more active than others. Apart from this, Withanolide A having cholinesterase
inhibitory property (Chen et al. 2011), where as Withanolide D has been reported to
be antitumor and antimetastatic by Leyon and Kuttan (2004). Withangulatin A
exerts immunosuppressive effects (Sun et al. 2005), whereas Viscosalactone B dis-
plays anti-inflammatory property (Jayaprakasam and Nair 2003).
Phytosterols from Plants
Phytosterols are the group of steroidal alcohols having double bond in sterol ring
that are ubiquitously distributed among the plant kingdom. Phytosterols, which are
commonly found in plants, are stigmasterol, Δ5-avenasterol, campesterol, and
β-sitosterol. The hypocholesterolemic activity of phytosterols is recently detected,
and therefore scientists got attracted toward knowing the other properties of phytos-
terols. The plant sterols are interacted with other membrane lipid components and
actively involve maintaining stress response and their development. However, they
are not being structural components of plasma membrane. Plants adopt a variety of
climatic conditions; this kind of adaptations is found to be related with a great
diversity of steroidal alcohols of plants. The phenomenon behind it is found to be a
82 P. Tiwari et al.
characteristic ratio of different phytosterols such as stigma sterol and β sitosterol,

24ethyl, and 24methylsterols. The ratio of phytosterols with other membrane lipid
is also contributed such as the ratio of sphingolipids to phospholipids (Valitova et al.
2016). Some other important properties such as anti-inflammatory, antibacterial,
antifungal, and antitumor activities have also been reported (Othman and
Moghadasian 2011; Siddique and Saleem 2011).
Ecdysteroids from Plants
Ecdysteroids are defined as steroid hormones of all sections of arthropods and per-
haps other invertebrates (Dinan 2001). They are important in the developmental
processes of these insects. The ecdysteroids such as 20-hydroxyecdysone were
found in the fern and some other reported from Achyranthes fauriei (Amaranthaceae)
and Podocarpus nakaii (Podocarpaceae) (Dinan 2001). Since the ecdysteroids are
derived from plants, therefore they are considered as phytoecdysteroids.
Phytoecdysteroids are used as phytomedicines for a long time as a tonic. The herbal
formulations are also consumed by the athletes for their body building property
(Bathori and Pongracz 2005). Natural phytoecdysteroid, 20-hydroxyecdysone, has
been found to be anabolic, hepatoprotective, and hypoglycemic with immune mod-
ulator properties, but these effects are not correlated with any of the thymolytic,
androgenic, and antigonadotropic side effects (Bathori and Pongracz 2005,
Sarkar et al. 1999).
Brassinosteroids from Plants
Brassinosteroids (BRS) belong to the plant steroidal groups of endogenous steroidal

plant growth hormone that are present at very low concentrations in plants. They
consisted of over 70 members (Bajguz and Tretyn 2003; Kreis and Müller-Uri
2010). The classification of brassinosteroids is based on the number of carbon atoms
present as a side chain of C17 and therefore named as C27, C28, and C29 brassino-
steroids. Their structural diversity is observed, which may be due to the substitution
at ring A and ring B. Some important representatives of plant steroids among the
plant kingdoms are brassinolide (1st brassinosteroid) and castasterone (Kim et al.
2005). BRs are investigated to be structurally related to animal steroid hormones,
and they control the expression of a variety of genes. They modulate the activity of
certain metabolic mechanistic pathways of plants. Their effects have been also
observed on cell division and differentiation. The BRS help in the regulation of
overall developmental process which leads to morphogenesis, and their impact is
also seen flowering and cell expansion in the presence of a potentially growth-
limiting cell wall (Clouse 2011).
Steroidal Alkaloids from Plants
Steroidal alkaloid has great diversity among the plant steroids. These compounds are
obtained from the Solanaceae, Liliaceae, Buxaceae, Asclepiadaceae, and
Apocynaceae plant families (Roddick and Melchers 1985). The biosynthesis of ste-
roidal alkaloids is carried out by the inclusion of one or two nitrogen atoms into a
preformed steroid molecule. The diversity is thought to be due to the groups attached
on the positions of nitrogen atom and steroidal skeleton modifications. The solani-
dine and siprosolane are the steroidal glycoalkaloids. They have been reported to
have antitumor, antifungal, and fetal toxic properties (Torres et al. 2011; Wang et al.
2011). The antifungal, antibacterial, antimalarial, antiviral, as well as inhibitory
properties to the activities of glutathione-S-transferase and cholinesterase enzymes
have been observed with steroidal alkaloids consisting pregnene ring. The drugs used
to treat cancer patients are found to make addictions with glutathione-S-transferase
and emit from the body. Therefore, by blocking glutathione-S-transferase, these mol-
ecules can play a valuable role in cancer chemotherapy (Ata and Andersh 2008).
Plant Steroids Against Asthma
Recent reports have suggested that there are some plants which synthesize steroidal
compounds that have been found to function as potential antihymatics (Patel and
Savjani 2015). Solanum xanthocarpum, a plant from the family Solanaceae, is
reported to synthesize Solasodine, a steroidal glycoalkaloid, which is widely pre-
scribed as a drug against respiratory diseases such as bronchial asthma and chronic
obstructive pulmonary diseases (COPD) (Govindana et al. 1999, 2004). Plants from
this family are also reported to synthesize 16-dehydropregnenolone acetate, a pre-
cursor compound for preparation of anti-inflammatory steroidal drugs (Chitravanshi
et al. 1990). In an experiment using guinea pigs, it was observed that a compound,
Saponin, isolated from S. xanthocarpum was found to possess anti-allergic activity
(Gupta 1994). However, the precise mechanisms of action of these plant-based ste-
roidal compounds against respiratory diseases including asthma are not known.
Plant Hormones Like Mammalian Steroids
In the year 1926, the appearance of the first mammalian steroid hormone was
detected (Janeczko and Skoczowski 2005). Studies using radioimmunoassay (RIA)
technique of about 128 plants have shown that most plant species have androgen
and progesterone and some contain androgens and estrogens (Simons and Grinwich
1989; Janeczko and Skoczowski 2005).
84 P. Tiwari et al.
Identification and Analysis of Plant Steroids
The plant kingdom has a wide range of plants, which are used as traditional medi-
cines, contain steroids, and also have some physiological activities. Nowadays, it is
essentially required to detect and quantify a very little amount of these steroids in
plant preparations by means of convenient analytical techniques with high resolu-
tion. Advanced chromatographic techniques including thin-layer chromatography
(TLC), high-performance liquid chromatography (HPLC), ultrahigh-performance
liquid chromatography, and gas chromatography are being used for their identifica-
tion. Some hyphenated techniques such as mass spectrometry (MS) and tandem
mass spectrometry (MS/MS) are more sensitive and hence used for their structural
determination.
hin-Layer Chromatography (TLC) for Detection of Plant

T
Steroids
TLC has been found to be a commonly used technique for characterization and
analysis of plant steroids. TLC is one of the popular methods for characterization
purposes having many advantages with some disadvantages which are recovered by
the development of method. TLC has proved as a rapid and accurate method for
initial analysis of phytochemicals and for observation of plant steroids in column
fractions during the purification process. TLC application in analysis and identifica-
tion of steroids is already reviewed (Bhawani et al. 2010). A broad range of plant
steroids are being analyzed efficiently by the process of TLC such as withanolides,
steroidal alkaloids, phytosterols, and phytoecdysteroids. The qualitative and quanti-
tative estimations of these plant steroids are also carried out by TLC. TLC densi-
tometry is an optical density measurement technique used for the quantitative
measurements of light-absorbing substances present as a spot-on chromatogram of
TLC. The limitation of TLC is found in analysis of brassinosteroids and some
mammalian-like steroidal hormones in plants. Withanolides such as withaferin A
and withanolide A are separated and quantified from the root extract of W. som-
nifera L. using HPTLC (Devkar et al. 2012). Steroidal alkaloids are the promising
steroidal compounds in treatment of various complications but most of them are
polar compounds such as glycoalkaloids do not contain any chromophore. Therefore
their detection is so critical. However, by using HPTLC densitometry some of the
glycoalkaloids are quantified (Shanker et al. 2011). Steroidal glycoalkaloids are
separated by semi-purified fraction of Solanum xanthocarpum (Solanaceae) by
HPTLC and eluting with CHCl3/MeOH/H2O (70/30/10). Further, Dragendorff’s
reagent is used for detection and quantification purposes, followed by scanning by
TLC densitometry at 520 nm. The identification of compounds such as solasonine,
solamargine, and khasianine (β-2-solamargine) is also carried out by using
this method.
igh-Performance Liquid Chromatography (HPLC)

H
for Detection of Plant Steroids
Application of HPLC is also employed in plant steroidal analysis. The whole HPLC
analytical system consists of FL detectors, diode array detector, and refractive
index. Sometimes MS detectors are also attached to it for identification and deter-
mination of compounds present in samples. Further, quantification is carried out by
the observation and evaluation of the peaks by comparing with the standard ones.
The individual compounds are identified generally with the help of retention time
(tR). Compound’s identification is done by either molecular mass or molecular
structure. Steroids obtained from plants are polar in nature such as brassinosteroids,
phytoecdysteroids, withanolides, and steroidal alkaloids. Among them, the steroidal
glycoalkaloids are found to be highly polar in nature. HPLC becomes a popular
method of choice due to its high specificity toward separation, characterization, and
quantification of steroidal groups by using a specific range of column and having a
variety of modifiers in their mobile phase (Dinan et al. 2011).
Plant sterols are characterized by the application of a highly sensitive analytical
technique like reverse phase (RP)-HPLC, hyphenated APCI-MS/MS [Atmospheric
pressure chemical ionization tandem mass spectroscopy] (Igarashi et al. 2011).
This technique is employed for the identification of campesterol, desmosterol
ergosterol, 7-dehydrocholesterol, and β-sitosterol in femtomole quantity from
mulberry leaves, silkworm, and silkworm artificial diets. Another advanced tech-
nique utilized for the study of plant steroid is UHPLC. The steroidal compounds
which are present in trace amounts in plants such as mammalian steroidal hor-
mones require very sensitive method for their identification and characterization.
The leaf extracts of Digitalis purpurea L., N. tabacum L., and Inula helenium
L. contain mammalian-like steroidal hormones which are detected by using
UHPLC/ESI-MS/MS (Simersky et al. 2009). The columns used in UHPLC are
composed of fine particles with size of 1.7 μm. Therefore, the sensitivity, resolu-
tion, and number of sharp peaks per unit time in UHPLC are improved from HPLC
(Swartz 2005; Zhao et al. 2006).
GC/MS Analysis of Plant Steroids
The GC/MS is one of the highly evolving sensitive techniques used for the detection
of volatile components of plants. Volatile phytochemicals which are present in little
amounts in plants are separated with this technique. Brassinosteroids acquire from
the shoot of Camellia sinensis (L.) Kuntze (Tea Plant) by the application of GC/MS
technique (Gupta et al. 2004). In this process characterization of brassinosteroids is
done in fresh tea shoot methanolic (MeOH) extraction followed by solvent–solvent
partitioning and HPLC. The fraction having brassinosteroids treated with methana-
bolic acid in pyridine and converted into their methylboronates, which are volatile in
86 P. Tiwari et al.
nature, and then they are subjected to GC/MS analysis. Six brassinosteroids are rec-
ognized: 6-deoxotyphasterol, typhasterol, 3-dehydroteasterone, 6-deoxocastasterone,
24-epibrassinolide, and 28-homodolicholide.
andem Mass Spectrometry (MS/MS) for Analysis of Plant

T
Hormones
For the analysis and recognition of phytochemicals, HPLC hyphenated with

ESI-MS/MS is another method of choice among the scientists. It is highly sensitive
and less time-consuming with several advantages by comparing with other tradi-
tional techniques. This analytical technique assures the structural parameters avail-
able online for compound identification (Zhou et al. 2010).
Conclusion
The existing information illustrates the adverse effects of synthetic steroids when
taken to treat any chronic disease for long duration. In order to treat various health-
related problems, a relatively safer and more cost-effective option is required.
Therefore, the secondary metabolites separated from many plants with steroidal
properties could prove better substitutes to the synthetic drugs for human welfare.
The reports on application of plant-based steroids indicate that they have a wide
range of potential to cure the patients as they possess anti-inflammatory, antibacte-
rial, anticancer, and antihelmentic properties with no or less side effects. However,
an extensive research is required to identify such phytochemicals and also to opti-
mize the efficacy and therapeutic potential of such plant-based principles showing
great promises as viable alternatives of synthetic steroids to be used against varied
inflammatory and other chronic diseases.
Acknowledgments PT is thankful to UGC-New Delhi for providing financial support in the form
of a fellowship. RP is grateful to UPCST-UP Lucknow for financial support and KGMU-Lucknow
for providing research facilities. RS thanks DST-New Delhi for awarding NPDF to her.
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Chapter 8
Phytochemicals as Therapeutics in Heavy
Metal Toxicity
Nitika Singh and Bechan Sharma
Introduction
Phytochemicals are naturally occurring secondary metabolites present in all vegeta-

bles, fruits, and medicinal plants. These are consumed regularly or rarely may con-
tain a therapeutic potential for altering the organism metabolism and help in
prevention from some chronic and degenerative ailments. Nowadays, several
researches are being performed on pharmacological properties of the phytochemi-
cals that may help to discover their important physiological and biochemical impacts
on living system against heavy metal toxicity. Among phytochemicals, some of
compounds like anthocyanins, carotenoids, flavonoids, polyphenols, and stilbenes
are known to be effective for a number of health-promoting effects (Kamboh et al.
2015). Also, plant alkaloids have been reported to contain a number of pharmaco-
logical activities such as antimalarial, antiasthma, anticancer, cholinomimetic, anti-
arrhythmic, analgesic, antibacterial, and antihyperglycemic activities (Singh and
Sharma 2018). The main sources of antioxidants are phytochemicals that can be
present in many foods and remedial plants. They play a very crucial role in the treat-
ment of chronic diseases caused by oxidative stress and its prevention as well. In
general, the phytochemicals have strong antioxidant potential that help in scaveng-
ing free radicals, and also anti-inflammatory properties that serve as the basis for
other bioactivities and health benefits, including antiaging, anticancer, and antidia-
betes mellitus and help in the prevention of obesity and neurodegenerative and car-
diovascular diseases (Zhang et al. 2015). Polyphenols are abundantly present in
human diets from various sources, and the mechanism of free radical scavenging
activities involves the substitution of hydroxyl groups in the aromatic rings of phe-
nolics (Rokayya et al. 2013).
N. Singh · B. Sharma (*)


https://doi.org/10.1007/978-981-15-2195-9_8
92 N. Singh and B. Sharma
Reactive oxygen species (ROS) are produced as by-products during the normal
metabolic processes. Animal tissues are continuously exposed to these highly reac-
tive by-products which mainly include hydroxyl radical (•OH), superoxide anion
(O2−), and hydrogen peroxide (H2O2) (Scartezzini and Speroni 2000; Ganie et al.
2011). Normally these ROS are synthesized in small amounts in the body for vari-
ous physiological functions, but if they are produced in excessive amounts, they can
cause oxidative stress (Cabre et al. 2000). Excess production of free radicals in the
human body can cause a condition (imbalance between oxidants and antioxidants
defense system) that leads to oxidative damage to major biomolecules (lipids, DNA,
and proteins). Further, the biomolecule damage causes severe pathogenesis to the
human system including cardiovascular disease (CVD), certain types of cancers,
and aging (Poulose et al. 2014; Singh et al. 2014). Either directly or indirectly, the
oxidants mediate the inflammatory and oxidative signals that further disrupt the
equilibrium state of the cell, resulting into mitogenesis, mutagenesis, genotoxicity,
and cytotoxicity. It has been well reported that the free radical–mediated damage is
involved in the pathophysiology of several diseases including Alzheimer’s, athero-
sclerosis, cancer, diabetes, hypertension, and Parkinsonism (Abdel-Daim et al.
2017). Thus, the phytochemicals with antioxidant properties could play a significant
role in the amelioration of some chronic diseases (Soobrattee et al. 2005; Sung and
Lee 2010).
Keeping in view the growing incidences of heavy metals–induced adverse effects
in the living systems and challenges in its management, the present chapter has been
prepared to exemplify the salient features of heavy metals, their chemical and toxi-
cological properties, secondary metabolites of plants, and their biological activities
including their roles in the amelioration of heavy metals toxicity.
Heavy Metals Toxicity
The toxicity of heavy metal has been proved to be a cause of major threat, and it is
also associated with several health risks. The metals which do not have any biologi-
cal role induce lethal effects in the organism and also inhibit it from its proper
functioning. Arsenic, cadmium, chromium, lead, and mercury are considered sig-
nificantly toxic metals for public health among the metals due to their high degree
of toxicity. Recently, the concern of ecological and global public health associated
with environmental pollution has increased due to these metals.
Apart from this, because of the rapid increase in their use in many industrial,
agricultural, domestic, and technical applications, human risk has increased dra-
matically (Bradl 2002; Tchounwou et al. 2012; Sharma et al. 2014). In biological
systems, the heavy metals have been reported to alter cellular organelles and their
components including cell membrane, lysosome, mitochondria, endoplasmic retic-
ulum, nuclei, and some enzymes that are involved in the metabolism, detoxification,
and damage repair processes (Wang and Shi 2001; Singh et al. 2017).
8 Phytochemicals as Therapeutics in Heavy Metal Toxicity 93
Several public health measures have been taken to control, prevent, and cure
metal toxicity at various levels such as occupational hazard, accidents, and environ-
mental factors. The toxicity of metals depends upon various factors such as its
absorbed doses, the route, and duration of exposure, that is, acute or chronic. These
metals are also considered as systemic toxicants, and by using lower doses of expo-
sure, they induce multiple organ damage. The free radical formation can lead to
oxidative stress that damages the cells and can further result into numerous ailments
in the organism (Jaishankar et al. 2014). Metal ions have been found to interact with
cell components such as DNA and nuclear proteins, causing DNA damage and con-
formational changes that may further lead to cell cycle modulation, carcinogenesis,
or apoptosis (Chang et al. 1996; Wang and Shi 2001; Beyersmann and Hartwig
2008; Singh et al. 2018) (Fig. 8.1).
Studies have indicated that ROS production results in oxidative stress that plays
a significant role in the toxicity and carcinogenicity of metals including lead, arse-
nic, chromium, cadmium, and mercury (Tchounwou et al. 2012). Sometimes, the
metals act as a pseudo-element of the cell and interfere with the metabolic processes
of biological system. The accumulation of some metals in the plant and animals
exhibited a chronic nature, while few metals including aluminum were removed
through elimination activities (Jaishankar et al. 2014). It has been reported that met-
als such as chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg),
manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se), and zinc (Zn) are
essential nutrients and are required for many physiological and biochemical func-
tions. Insufficient supply of micronutrients results in varied deficiency diseases/
syndromes (WHO/FAO/IAEA 1996; Tchounwou et al. 2012).
Fig. 8.1 Cell death caused by toxic metal–mediated oxidative stress

Phytochemicals
The antioxidants like polyphenols and carotenoids are mostly contributing phyto-
chemicals to the foods and plants. For instance, cape gooseberry contains carotene,
myricetin, quercetin, and kaempferol antioxidant phytochemicals (Zhang et al.
2013), and the strawberry contains some major antioxidant compounds such as
anthocyanins and ellagitannins among its phytochemicals (Giampieri et al. 2012).
Polyphenols (dietary) could be categorized into the following five classes: flavo-
noids, coumarins, phenolic acids, stilbenes, and tannins. According to Yang (2009),
flavonoids can be further grouped in to flavonols, flavones, flavanols, flavanones,
anthocyanidins, and isoflavonoids. The total antioxidant activity and phenolic con-
tent of any phytochemical or plant extracts may have a direct relationship. The fruits
having higher level of total polyphenol show stronger antioxidant activity (Sun et al.
2002). Carotenoids, a group of phytochemicals, provide color like orange, yellow,
and red to the foods. Carotene, cryptoxanthin lycopene, and lutein are the major
carotenoids in the diet and the human body. In human diet, vegetables and fruits are
as the main sources of carotenoids. For example, lycopene is present in tomato
which provides its characteristic red color (Zhang et al. 2015; Kumar et al. 2018).
Flavonoids, a major coloring phenolic compound of flowering plants, is a com-
monly and broadly distributed group of phytochemicals, virtually occurring in all
plant parts, particularly the photosynthesizing plant cells. Recently, a large number
of researchers have investigated the therapeutic potential of medicinal plants.
Specifically, phenolic compounds (flavonoids and flavonoids) are responsible for
these properties of plants. They also constitute the integral part of human and ani-
mal diet (Pourmorad et al. 2006; Kumar and Pandey 2012). In addition, they have
more biological properties that help in promotion of human health and minimize the
risk of ailments (Kumar and Pandey 2013).
Phytochemicals as Therapeutics
Phytochemicals are a group of naturally occurring chemical compounds. On the

basis of the nature of chemical compounds, phytochemicals can be either beneficial
or toxic. The plant species which are rich in antioxidative potentials are also rich in
polyphenols, flavonoids, etc. However, these compounds also act as scavengers
against the heavy metal toxicity. A majority of workers reported the ameliorative
effects of phytochemicals in respect to heavy metal toxicity. The oral supplementa-
tion of Arthrospira maxima and soya bean minimizes the genotoxic and cardiovas-
cular effects of cadmium (Brochin et al. 2008; Argüelles et al. 2013). Some
phytochemicals such as caffeic acid, chlorogenic acid, vanillic acid, p-coumaric
acid, ferulic acid (Nambiar et al. 2010; Rajeshwari and Andallu 2011), and cur-
cumin isolated from coriander and turmeric, respectively, contain anti-inflammatory
and antioxidant properties (Barzegar and Moosavi-Movahedi 2011). These

phytochemicals also protect rats from cadmium-mediated nephrotoxicity (Singh
et al. 2010). Carotenes, phycocyanobilin, vitamin C, and vitamin E collected from
cyanobacterial species, including Chlorella and Spirulina, have been reported to
ameliorate against cadmium- and lead-induced toxicity (Amin et al. 2006; Shim and
Om 2008). Garlic extract with its strong potential of antioxidants (rich in allicin) has
shown to reduce mitochondrial injury and apoptosis caused by cadmium and lead
(Chowdhury et al. 2007; Sadeghi et al. 2013). Aslani et al. (2010) have reported that
the administration of garlic extracts along with dimercaptosuccinic acid (DMSA)
strongly mitigates the rats from lead poisoning. Also, the garlic extract reported to
be protective against sodium arsenite–induced adverse effects (Das et al. 1993;
RoyChoudhury et al. 1996). It is believed that the protective effect of garlic from
arsenic poisoning is provided through thiosulfate components present in the extracts,
which will make some stable chemical complexes after reacting with arsenic chemi-
cal species and inactivating arsenic-mediated toxicity (Flora et al. 2009).
Annona muricate (soursop) and Hippophae rhamnoides (seaberry) leaf extracts
are rich in carotenoids, vitamins, and organic acids and play beneficial role in
arsenic-mediated toxicity. (Gupta and Flora 2005b, 2006b; George et al. 2015).
Dietary supplementation of soybean reduces the risk of arterial and cardiac injuries
induced by cadmium (Brochin et al. 2008). Mohammed et al. (2014) have investi-
gated the therapeutic potential of Nigella sativa seed oil and revealed that it may
protect the brain and kidney due to its strong antioxidant properties against oxida-
tive damages. They have also shown the use of N. sativa seed oil and the virgin olive
oils by cement factory workers where CD is very frequently liberated into the envi-
ronment. Some researchers have not only shown the ameliorative properties of phy-
tochemicals against heavy metals toxicity, but also indicated that the phytochemicals
reduce the body burden of accumulated heavy metals. During lead- and cadmium-
mediated intoxication, tomato extracts have been used to reduce bioaccumulation of
heavy metals (Nwokocha et al. 2012).
The isoflavan glabridin, a major polyphenolic phytochemical present in
Glycyrrhiza glabra of the Fabaceae family, inhibits oxidation of low-density lipo-
proteins (LDL) through scavenging the free radicals (Fuhrman et al. 1997). More
than a few epidemiological studies have recommended that taking either black or
green tea may reduce the blood cholesterol level and blood pressure and provides
protection against some cardiovascular disease (Kumar and Pandey 2013). The fla-
vonoids obtained from berries may have a role in the improvement of memory in
elderly people and also may show a positive effect on the patient having Parkinson’s
disease. Antihypertensive activity has been reported in total flavonoid fraction of
Atragalus complanatus in hypertensive rats (Li et al. 2005). It has been reported by
some workers that Centella asiatica, Allium sativum, Hippophae rhamnoides, and
Aloe vera barbadensis extracts help in the reduction of arsenic-mediated toxicity in
the hematological, hepatic, and renal parameters in experimental animals (Gupta
and Flora 2005a, b; 2006a, b; Flora et al. 2009; Gupta et al. 2015) (Table 8.1).
Table 8.1 List of some plants effective against heavy metal–mediated toxicity
Effective
S. No. Plant against Effects References
1 Arthrospira Cadmium Minimizes the genotoxic and Argüelles et al.
maxima cardiovascular effects (2013)
2 Coriandrum Cadmium Possesses a potent organo- John et al. (2014)
sativum protective effect and also
reduces lipid peroxidation
3 Curcuma Cadmium Used as an effective antioxidant Singh et al. (2010),
longa for ROS protection and to Barzegar and
reduce nephrotoxicity Moosavi-Movahedi
(2011)
4 Allium sativum Cadmium, Reduces mitochondrial injury Chowdhury et al.
arsenic, and and apoptosis, acts as (2007), Flora et al.
lead antioxidants, and protects from (2009), Sadeghi et al.
neural damage (2013)
5 Annona Arsenic Antioxidative properties and George et al. (2015),
muricate reduces hepatic injuries Adegboyega (2018)
6 Hippophae Arsenic Antioxidant properties Gupta and Flora
rhamnoides (2005b, 2006b)
7 Nigella sativa Cadmium Protects the brain and kidney Mohammed et al.
from oxidative damages (2014)
8 Glycyrrhiza _ Protects low-density lipoprotein Fuhrman et al. (1997)
glabra against lipid peroxidation with a
decrease in atherosclerotic
lesion area
9 Centella Arsenic Reduces oxidative stress Gupta and Flora
Asiatica (2005a)
10 Aloe vera Arsenic Antioxidant properties Gupta and Flora
barbadensis (2006a)
11 Chlorella and Cadmium Antioxidant properties Amin et al. (2006),
Spirulina and lead Shim and Om (2008)
12 Solanum Lead and Reduces bioaccumulation of Nwokocha et al.
lycopersicum cadmium toxic metals (2012)
Conclusion
The underlying mechanisms of actions of many of the toxic metals rely on the
excessive free radical production as well as generation of oxidative imbalance. The
development of heavy metal–mediated side effects, including some diseases, might
be due to damage of biological cells and their key biomolecules by ROS. The infor-
mation available indicates that these metals have the ability to substitute the corre-
sponding metal ions in enzymatic catalysis. Further, these metals possess a tendency
to directly bind with the proteins containing thiol groups and/or enzymes so as to
negatively modulate their functions and hence the physiological cellular behavior. It
is therefore expected that the plant-based molecules carrying the antioxidants may
help quench the ROS and neutralize them, thereby protecting the living system from
their toxicity. It is also possible that the plant extracts containing many phytochemi-
cal species may form complexes with these metals thereby protecting the enzymes
or proteins or both allowing them to perform their normal functions. The mitigation
of metallotoxicity employing the herbal ingredients is of great significance as they
are safe and cost-effective. However, still extensive research is needed to delineate
several molecular mechanisms associated with interactions of these metals in living
systems and identifying suitable targets to develop efficient drugs in cases of heavy
metal toxicity.
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Chapter 9
Phytotherapies for Thyroidism:
An Overview
Taniya Sengupta Rathore, Shekhar Jain, and Sonu Chouhan
Introduction
The thyroid gland, one of the most important endocrine organs, regulates many
human body functions including metabolic, respiratory, cardiovascular, digestive,
nervous, and reproductive systems directly or indirectly. The thyroid dysfunction
causes many abnormalities in the human body.
Thyroid problems are found to be the most common endocrine disorders at pres-
ent throughout the world. Globally, millions of the adult population are suffering
from thyroid disorders (Delshad 2008). In India, nearly every third person manifests
with thyroid disorders, which most often causes excess weight gain and hormonal
imbalances and is generally found in women, as surveyed by The Economic
Times 2017.
Thyroid disorder patient suffers either from hyper- or hypo-functioning of the
thyroid gland, called hyperthyroidism and hypothyroidism, respectively.
Hyperthyroidism leads to increasing concentration of thyroid hormone in body
fluid, whereas in hypothyroidism it is reversed.
In Ayurveda, the ancient system of herbal medicine in India, the common thyroid
dysfunction is known as goiter, an enlarged thyroid which is a state of hypothyroid-
ism (Sengupta 1999). Some other diseases, described in the Ganong’s review of
medical physiology (Ganong 2010), are myxedema, cretinism, Grave’s diseases,
and nodular goiter, which have not been described in Ayurveda.
The main focus of this chapter mainly is to review the available literature on
thyroid disorder including the experimental studies so far undertaken to highlight
the therapeutic basis of Ayurvedic medicine for these endocrine gland-related
disorders.
T. S. Rathore (*) · S. Jain · S. Chouhan

Faculty of Life Sciences, Mandsaur University, Mandsaur, Madhya Pradesh, India
e-mail: taniya.sengupta@meu.edu.in

https://doi.org/10.1007/978-981-15-2195-9_9
102 T. S. Rathore et al.
Thyroid Gland: Hormones and Functions
The thyroid gland is an endocrine gland which lies in the front of the neck just below
the Adam’s apple. It is divided into two lobes, the right lobe and the left lobe, each
about the size of a half-cut plum. These two lobes are joined together by a small
tissue called “isthmus.” These two lobes are situated on either side of the windpipe.
The thyroid gland produces two major hormones: thyroxine (T4) and triiodothy-
ronine (T3). Thyroxine contains four atoms of iodine, whereas triiodothyronine con-
tains three. T4 is converted into T3 by the deiodinase enzyme system in multiple
tissues and organs but especially occurs in the liver, gut, skeletal muscle, brain, and
the thyroid gland itself. T3 derived either from T4 or directly secreted from the thy-
roid gland is biologically active and influences all the cells and tissues for their
normal activity. The physiological imbalance of these two thyroid hormones leads
to health issues.
Thyroid Abnormalities
The T3 form highly influences the metabolism of body cells which help to regulate
the body cells to work properly. The overproduction and secretion of thyroid hor-
mones lead to the faster body cell performance than the normal, called hyperthy-
roidism. Sometimes hyperthyroidism causes diarrhea by increasing the activity of
the intestine (Juckett and Trivedi 2011). On the other hand, hypothyroidism slows
down the heartbeat rate and intestinal activity which leads to constipation problem.
As thyroid hormones influence the metal and physical growth and development
of human, the hypothyroidic condition in children leads to the mental retardation
with low intelligence called “creatinism” .
Hyperthyroidism condition is considered to be related with several diseases in
humans such as thyrotoxicosis, exophthalmic goiter or Graves’ disease, thyroid car-
cinoma, and adenoma. These diseases are very serious, as these in patients cause
heart ailment and diabetes, the two most common health problems of the modern
world (Williams 1997).
Several symptoms are associated with both hypothyroidism and hyperthyroid-
ism. The most common of them are listed in Table 9.1.
Thyroid Abnormalities Etiology
Hypothyroidism
In Ayurveda though no disease condition is described similar to hypothyroidism,

several references with various texts have been mentioned in various literatures.
Eight types of Nindita Purushas (Agnivasha 2001) and Avarana (Agnivasha 2004)
9 Phytotherapies for Thyroidism: An Overview 103
Table 9.1 Common Hypothyroidism Hyperthyroidism

symptoms of hypo- and
Gain in weight Weight loss
hyperthyroidism
Husky voice Hair thinning
Tiredness Sweating/intolerance to heat
Intolerance to cold Nervousness
Dry skin and hair Mood swing
Puffiness of the body Tachycardia
Periorbital swelling Diarrhea
Memory loss Tremor
Constipation Muscle weakness/fatigue
Bradycardia Insomnia
Muscles stiffness Angina
Impotence Increased appetite
Anemia Eyelid pain
Depression Loss of libido
Brittle nails Exophthalmos
have been described through various hormonal disorders. In Ayurvedic literature,

imbalances of air (vayu), mucus (kapha), and fat (meda) cause the enlargement of
thyroid gland called as galaganda in Ayurveda and also known as bronchocele
(Sengupta 1999). The most common reason for hypothyroidism is deficiency of
iodine. As a preventive measure related to heart problem and high blood pressure,
people intake diet with low sodium, resulting in iodine deficiency which is consid-
ered as a major factor for this disease. Doctors’ recommendation is intake of about
150 μg iodine in the daily diet for normal functioning of thyroid gland; less than
50 μg/day for a long period can lead to goiter (Flynn et al. 2003). Insufficient con-
centration of the iodine impaires the production of the T3 and T4 in an appropriate
amount in the thyroid gland; as a result the TSH level increases in the body, which
leads to abnormal increasing of the thyroid gland.
Selenium deficiency (Kipp et al. 2015), heavy metals and pesticides consump-
tion (Tomer and Huber 2009), and excessive consumption of goitrogenic foods, for
example, soya bean, cabbage, cauliflower, peanut, cassava, sweet potatoes, etc.
(Canaris et al. 2000), enhance hypothyroidism. For the synthesis of thyroid hor-
mones, an enzyme called thyroid peroxidase (TPO) is the basic enzyme which not
only catalyzes iodination but also the coupling reaction. More than 90% of patients
of autoimmune hypothyroidism and Graves’ disease have anti-TPO autoantibodies.
Thyroglobulins (TG) are the predominant antibodies in autoimmune hypothyroid-
ism (AH). These antibodies are basically from IgG class mainly from excess IgG1
and IgG4 subclasses (Silva et al. 2003).
Hyperthyroidism
According to classical Ayurveda, hyperthyroidism developed due to the imbalance

in air (vayu) and fire (Agni), which govern the nervous as well as hormonal system.
Physicians correlate both psychological stress with excess secretion of thyrotoxico-
sis and cortisol. The main factor is genetical susceptibility; apart from this another
major factor for hyperthyroidism is an increase in IgG level immunologically, which
stimulates TSH receptors for hormone production (Hershman 2004).
Approximately in 70% of patients, overproduction of thyroid hormone from
entire thyroid gland is the main reason for hyperthyroidism, also called Graves’
disease. Graves’ disease is developed by antibodies present in the blood that cause
the thyroid to secrete a high amount of thyroid hormone. This type of hyperthyroid-
ism passes from generation to generation in families, and it generally occurs in
young women. Another type of hyperthyroidism occurs by the development of one
or more nodules in the thyroid which gradually increase in size and in their activity
resulting in an increase in thyroid output in blood than the normal level. This condi-
tion is called toxic nodular or multinodular goiter. Temporary symptoms of hyper-
thyroidism are also developed which are known as thyroiditis. This condition
develops due to an immune system disorder or any viral infection that causes the
gland to leak stored thyroid hormone. The same symptoms can also occur by con-
sumption of too much thyroid hormone in the form of drugs. In both types, excess
thyroid hormone is present, but the thyroid works normally (American Thyroid
Association, http://www.thyroid.org/hyperthyroidism/).
Diagnosis for Thyroid Dysfunction
Diagnosis for thyroid dysfunction is generally done on the basis of thyroid gland
inspection by both Ayurveda and Allopathy. For evaluating the thyroid-related man-
ifestation, medical history and physical examination of the patient are required;
particularly, medical examination of the eyes, skin, cardiology, and neurology is
most important.
A. Tests Related to Blood
• TSH (thyroid-stimulating hormones): The TSH level is negatively correlated
with thyroid-level concentration. In the presence of excess thyroid hormone,
the level of TSH is low; that is in case of hyperthyroidism. In the case of
hypothyroidism, when thyroid hormone level is negligible, the TSH level is
high. TSH level test is the only most useful diagnostic test for many thyroid
diseases.
• Free (T4): High level of T4 indicates hyperthyroidism, whereas low indicates
hypothyroidism.
• Triiodothyronine (T3): High level of T3 in blood indicates hyperthyroidism.

Low T3 indicates hypothyroidism.
• TSH receptor antibody: It is a Graves’ disease antibody.
• Antibody thyroid peroxidase: For Hashimoto’s and Graves’ disease, this anti-
body is produced.
B. Scan for nuclear thyroid: During this scanning technique, a small amount of
radioactive iodine or similar material is swallowed; thyroid imaging study
reveals the radioactivity localization after injecting 99 m-technetium in the
blood. Increased activity of radioactive material in the thyroid gland indicates
hyperthyroidism, while decreased activity shows hypothyroidism. This test is
prohibited for pregnant women.
C. Thyroid ultrasound: Through thyroid ultrasound the size, number, and types of
nodules in thyroid gland are determined. This scan also detects enlarged para-
thyroid glands or lymph nodes near the thyroid gland.
D. Fine-needle aspiration: Under this diagnosis technique, a thin needle is inserted
into the nodule of thyroid gland in order to collect thyroid tissue. It is recom-
mended to be carried out under ultrasound guidance. The tissue collected is
observed under a microscope for any sign of cancer.
E. CT scan: A CT scan is occasionally performed to detect the extent of a large
goiter or for displacement of the trachea from the goiter.
Ayurvedic Therapies
According to Ayurveda, to treat thyroid disorders, blocked channels should be

cleaned so as to balance out vata, pitta, and kapha. Different types of herbal prod-
ucts are available for treating thyroid dysfunction. Apart from supplements of food
for goiter, oils such as Vimbadi oil with sesame oil are used. Yograj guggul
(0.5–2.25 g/day) and Ashwagandha powder (1–6 g/day) are also used for treating
hypothyroidism. Liv52, an Ayurvedic tonic for the liver, has the ability to stimulate
thyroid hormone secretion, generally the T3 (Dhawan and Goel 1994).
harmacological Aspects of Herbal Products for Thyroid

P
Dysfunctions
For the treatment of thyroid dysfunction, different plant extracts have been identi-
fied. Some are summarized in Table 9.2. For several years people are using seaweed
for normal functioning of thyroid gland as an iodine supplement.
Table 9.2 Plants for treatment of thyroidism

Effective
Thyroidism Plant Tested on dose References
Hypothyroidism Bauhinia variegata Animal model 2 g/kg Panda and Kar (2007)
Echhorina crassipes Animal model 2 g/kg Veena et al. (1975)
Bauhinia purpurea Animal model 2.5 mg/kg Panda et al. (2009)
Commiphora mukul Animal model 0.2 g/kg Panda and Kar (2005)
Withania somnifera Animal model 1.4 g/kg Panda et al. (2009)
Achyranthes aspera Animal model 200 mg/kg Brinker (1997)
Saussurea lappa Animal model 400 mg/kg Choudhary (2015)
Inula racemosa Animal model 400 mg/kg Gholap and Kar (2003)
Hyperthyroidism Lycopus virginicus Human 2 g/kg Blumenthal (1998)
Azadirachta indica Animal model 100 mg/kg Panda and Kar (2000c)
Momordica charantia Animal model 500 mg/kg Panda and Kar (2000b)
Convolvulus Animal model 400 mg/kg Panda and Kar (2001)

pluricaulis
Emblica officinalis Animal model 30 mg/kg Panda et al. (2002)
Ocimum sanctum Animal model 0.5 g/kg Panda and Kar (1998a,
b)
Piper betle Animal model 2.5 mg/kg Tripathi et al. (2011)
Rauwolfia serpentina Animal model 0.11 g/kg Nagarathna and Deepa
(2013)
Trigonella foenum Animal model 175 mg/kg Toppo et al. (2009)
Lithospermum Animal model 23 mg/kg Yarnell and Abascal
ruderale (2006)
Moringa oleifera Animal model 175 mg/kg Tabassum et al. (2013)
Lithospermum Animal model 400 mg/kg Yarnell and Abascal
officinale (2006)
escription of Plants for the Treatment of Hypothyroidism

D
(Presented in Fig. 9.1)
Bauhinia variegata
Ayurvedic name: Kanchnara

Alcoholic extract of Bauhinia variegata Linn. was fractioned into two doses of
2 g/kg and was given to NeoMercazole (150 mg/kg)-induced hypothyroid rats
(n = 12 in each group) for 20 days. As a result, the experiment proved an increase in
thyroid weight (p < 0.001) for increasing thyroid function (Veena et al. 1975).
Fig. 9.1 Plants for the

treatment of
hypothyroidism
Echhorina crassipes
Ayurvedic name: Plavaka

Similar to B. variegata, its water-soluble ash at 2 g/kg/day for 20 days can stimu-
late the thyroid function (p < 0.001 in all) (Veena et al. 1975).
Bauhinia purpurea
Ayurvedic name: Kanchnara

Bark extract of Bauhinia purpurea Linn. 2.5 mg/kg when administered to seven
mice each in two groups and all are female (n = 7 in each group) for 20 days signifi-
cantly increased serum T3and T4 (p < 0.001 for both) (Veena et al. 1975).
Commiphora mukul
Ayurvedic name: Guggulu

Guggulu, the gum resin, has reported to increase the T3/T4 ratio in female mice
(Panda and Kar 2005), and it also reversed the effect of propylthiouracil by stimulat-
ing the thyroid function in hypothyroid mice (Panda and Kar 1999b).
Withania somnifera
Ayurvedic name: Ashwagandha

Research studies revealed Ashwagandha to work for balancing thyroid hormone
concentration. Serum T4 was found to increase significantly after administrating
Ashwagandha in mice for 20 days, which indicates this herb shows stimulatory
effect on slowdown of thyroid (Panda and Kar 1998a, b).
In another study, for 30 days, all three extracts (B. purpurea, C. mukul, and
W. somnifera) of doses mentioned above were administered simultaneously to mice
(n = 8). There was the increase in both T3 and T4 levels (p < 0.01 and p < 0.001,
respectively) which suggests that a combination of the three plant extracts proved to
be effective for hypothyroidism treatment (Panda and Kar 2000a).
Achyranthes aspera
Ayurvedic name: Apamerga

Leaf extract of Achyranthes aspera Linn. administered in rats (n = 7) for 7 days
at a dose of 200 mg/kg caused an increase of bothT3 and T4 (p < 0.001). It also
increases the blood glucose level (p < 0.05) which supported its thyroid stimulation
nature (Tahiliani and Kar 2000).
Saussurea lappa
Ayurvedic name: Kushta

For 14 days when rats (n = 7) were treated with 400 mg/kg of S. lappa root
extract, which also showed thyroid stimulation (Chaturvedi 1993).
Inula racemosa
Ayurvedic name: Pushkarmool

Thyroid stimulation was also reported in rats (n = 7) when treated with 400 mg/
kg of Inula racemosa after 20 days (Tripathi and Chaturvedi 1995).
escription of Plants for the Treatment of Hyperthyroidism

D
(Presented in Fig. 9.2)
Lycopus virginicus
Common name: Bugleweed

Aqueous as well as alcohol extracts of Bugleweed are commonly used. Tea made
of two to three teaspoons air-dried herb in one cup hot water three times daily could
be administered orally. Scientific studies on more than 300 patients showed statisti-
cally significant improvements from mild hyperthyroidism without any side effects
(Beer et al. 2008).
Fig. 9.2 Plants for the treatment of hyperthyroidism

Azadirachta indica
Ayurvedic name: Arishta

For 15 days 100 mg/kg of the leaf extract of A. indica was administered and
resulted in a decrease in serum concentration of T3 (p < 0.05). It has been concluded
that this inhibits conversion of T4 to T3 (Panda and Kar 2000c).
Momordica charantia
Ayurvedic name: Karavella

M. charantia fruit extract (500 mg/kg) orally administered for 15 days decreased
both T3 and T4 concentrations in the serum (p < 0.001 for both) in mice (n = 8 in
each group) (Panda and Kar 2000b).
Convolvulus pluricaulis
Ayurvedic name: Sankhpushpi

Convolvulus roots (400 mg/kg for 30 days) inhibit the 5′-deiodinase enzyme
required for T4 into T3 conversion (Panda and Kar 2001).
Emblica officinalis
Ayurvedic name: Amla

For 20 days uptake of Emblica fruit extract (30 mg/kg), it decreased both T3 and
T4 concentrations. The decrease in T3 was due to inhibition of peripheral conversion
of T4 to T3 in extra-thyroid tissues (Panda and Kar 2003).
Ocimum sanctum
Ayurvedic name: Tulsi

The leaf extract (0.5 g/kg) of O. sanctum when administered to male mice sig-
nificantly inhibited only T4 concentration (p < 0.001) after 15 days (Bharthi
et al. 2017).
Piper betel
Ayurvedic name: Pippali kul

P. betel leaf extract (0.1 g/kg) when administered in male mice (n = 7) signifi-
cantly decreased both T3 and T4 concentration in serum after 15 days (Panda and
Kar 1999a).
Rauwolfia serpentina
Ayurvedic name: Sarpagandha

R. serpentina root extract (2.5 mg/kg) administered on mice (n = 7) for 30 days
significantly decreased both T3 and T4 concentration in the serum. (p < 0.001)
(Panda and Kar 2000d).
Trigonella foenum
Ayurvedic name: Chandrika

The effects of Trigonella foenum seed extract administered for 15 days (0.11 g/
kg) in rats and mouse both (n = 7 for each) decreased T3 level (p < 0.01 mouse and
p < 0.001 rats) but increased the T4 level in serum (for both p < 0.01) (Panda
et al. 1999).
Nelumbo nucifera
Ayurvedic name: Padma

N. nucifera rhizome extract (400 mg/kg) for 15 days administered to rats (n = 8)
decreased T3 and T4 concentration in serum (p < 0.01 and 0.05, respectively).
Moringa oleifera
Ayurvedic name: Sobhanjana

Moringa leaf extract of 175 mg/kg concentration treated on female rats (n = 14)
for 10 days decreased T3 level and increased T4 in serum.
Lithospermum officinale
Common name: Paint Indian

A dose of 400 mg/kg of extract of powdered leaf, administered to rats (n = 42)
for 24 h, decreased theT3 concentration (p < 0.01).
Conclusion and Future Prospects
Several pieces of literature based on herbal and Ayurvedic therapies revealed that
herbal products have the capacity to treat and manage both types of thyroid dysfunc-
tions, that is, hypothyroidism and hyperthyroidism. As compared to Allopathic,
herbal therapies are also safe, because Ayurvedic herbs used for therapy not only
support many complex functions but also prepare the body to work more efficiently.
Moreover, herbal extracts are the natural antioxidants which not only work to cure
diseases but also improve the immune system strength of the body. On the other
side, drugs of Allopathic background interfere with the metabolic activities, and
other activities thus alter the natural process of the human body, the main cause of
side effects. Therefore, herbal therapies should be preferred for both long- and
short-term diseases. Thus, it should be considered as the most effective therapy for
treating thyroid dysfunction.
Many other disorders such as diabetes, gastrointestinal, neural, and cardiac are
indirectly related to thyroid problems. Studies on the plant with their long-term
effectiveness and specificity to their target should be carried out to control and opti-
mize thyroid disorders and other related problems.
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Chapter 10
Phytochemicals as Antidepressants
Khushboo, Abhishek Kumar, and Bechan Sharma
Introduction
Depression characterized as a case of despair or a disorder in brain functions devel-

ops symptoms of altered psychological functions and behavioral activities; prob-
lems associated to thinking, hunger, and sleep; lack of concentration; feelings of
disappointment; depression; and suicidal tendencies (Salmans 1995). It is a most
common and frequent disorder that causes significant deformities and mortality
across the world (Keller et al. 1986). Depression, a type of mental illness, involves
the stimulus of grief which can affect overall thinking process, behavior, and emo-
tions (Salmans 1995). Such people suffer from unbalanced sleep and sleep disorders
(Partonen and Lönnqvist 1998; Doghramji 2003; Leventhal and Rehm 2005). Many
workers (Pillemer et al. 2010; Lindert et al. 2014) have explained the factors respon-
sible for causing depression, including genetic, asymmetrical parental behavior
such as neglect, or sexual abuse of the siblings. Also, certain common conditions
such as unemployment and difficulties in searching for a job, uncomfortable rela-
tionships, natural calamities, lack of finance, childbirth, disorderly injury, death of
loved ones, as well as menopause might be playing crucial roles (Schmidt 2005;
Rashid and Heider 2008). According to some reports, different regions of the brain
can initiate depression in the beginning because they control emotions, nerve cir-
cuits, and moods. The reduced process of the brain’s hypothalamic region may be
linked to both very little sleep and excessive sleep, lack of sexual interest, or any
other activities of pleasure (Saper and Lowell 2014). However, meager information
is available regarding the underlying mechanism(s) of the role of hypothalamus in
depression.
Normally there are three main forms of depression such as (i) psychological
severe depression, (ii) postpartum depression especially due to the birth of a child
Khushboo · A. Kumar · B. Sharma (*)


https://doi.org/10.1007/978-981-15-2195-9_10
116 Khushboo et al.
and the levels of hormones and physical characteristics associated to the disease,
and (iii) seasonal influential disorder, especially with less sunshine or during winter
months (Gupta and Sharma 2016).
In women, depression also arises due to additional workload, household respon-
sibilities, stressful relationships, childcare, aging parents, and care toward poverty.
Apart from all these parameters, some other factors associated to the hormonal,
psychological, and biological indices also make significant contributions to depres-
sion. In women, the emergence of premenstrual dysphoric disorder, premenstrual
syndrome, osteoporosis, and depression has been observed (Reed 2017). Men with
depression may be prone to severe diseases, for example, cancer and heart disease,
extreme fatigue, burning sensation, one-time happy interest, balance loss, low sleep,
and aggressiveness. In elderly men, arterial depression also called “vascular depres-
sion” has been reported. Depression causing suicides in children might be associ-
ated to promising sexuality and the beginning of teenage stage (Khushboo and
Sharma 2017).
The antidepressants used to treat depression mainly act by affecting the levels of
the neurotransmitters such as serotonin, norepinephrine, and dopamine which are
responsible for different physiological functions of brain cells. For example, sero-
tonin regulates memory, mood, sleep, appetite, sexual desire, and social behavior;
norepinephrine is responsible for regulation of motor functions to control heartbeats
and blood pressure due to any stress; and dopamine plays key roles in motivation,
arousal, decision-making, and pleasure or reward signaling.
The different categories of antidepressant compounds being used these days in
therapy of depression include five different groups: (i) the antidepressants with tri-
cyclic chemical structure (tricyclic antidepressants); (ii) the compounds selectively
inhibiting serotonin reuptake (selective serotonin reuptake inhibitors); (iii) the mol-
ecules acting as inhibitors of the enzyme, monoamine oxidase inhibitors (MAOIs);
(iv) the chemical agents inhibiting the reuptake of serotonin norepinephrine inhibi-
tors (SNRIs); and (v) the non-TCA antidepressants. Many of these antidepressants
are synthetic in nature and pose harmful effects on the health of users through many
ways including generation of excess free radical species. These compounds possess
different modes of actions. Since these chemical agents are anthropogenic in nature,
their removal from a patient’s body is slow, and hence they tend to accumulate and
magnify in various tissues of body, especially in different brain regions. They are
reported to lead to adverse effects after crossing the threshold level. The toxicity of
the above therapeutics has been primarily reported to be initiated through enormous
production of several chemical species containing unpaired electrons and develop-
ment of an imbalance between the oxidative and antioxidative indices. Probably this
event is responsible to cause alterations in the functions of the brain. In this regard,
the plant-based principles are believed to offer the most viable options because they
contain plenty of antioxidants. These plant products are easy to obtain with low
cost, least toxicity, and high therapeutic potential. The herbal ingredients have
recently been utilized as a complementary therapeutic agent, which may help relieve
symptoms and signs of depression and prevent relapse of psychoneurotic disorders.
The chapter illustrates recent advances in research related to the phytochemicals
10 Phytochemicals as Antidepressants 117
acting as antidepressants. Their chemical structures, biological functions, mode of

actions, roles in pathophysiology, and side effects, if any, have been included.
Depression and Its Symptoms
The word depression is derived from a Latin word, deprimere, which means “press
down.” The most accepted hypothesis about depression relates functional reduction
in brain biogenic amines acting as neurotransmitters. In the North American regions,
mainly the lowering in the content of catecholamines (dopamine and noradrenaline)
is thought to be the root cause of depression (Schildkraut 1965; Bunney and Davis
1965). This notion received convincing evidences from the results of some experi-
ments which indicated that the drugs such as reserpine and α-methyldopa were able
to reduce brain amines and to increase the incidents of depression (Lemieux et al.
1956). The effective medicines in raising depressed mood were also found to
enhance the levels of brain’s amines by decreasing the metabolic rate of the inhibi-
tor of the activity of monoamine oxidase.
Some workers from other laboratories of National Institute of Mental Health
(NIMH), Bethesda, MD, USA, have reported that a huge dose of L-dopamine (daily
4–8 g, oral) may exert antidepressant special impact in a small number of slow patients
(Bunney and Davis 1969; Bunney et al. 1970). In such patients, the side effects par-
ticularly the gastrointestinal disorder were observed to be high. It was thought to be
associated to the production of catecholamine by decarboxylation of dopamine.
Serotonin is shown to act as a neurohumoral transmitter in some regions of the
CNS, and large stores are contained in synaptic vesicles of long descending nerve
fibers in the mesencephalon, spinal cord, and diencephalon. All the enzymes neces-
sary for synthesis as well as destruction of serotonin were contained in certain
nerves of brain called serotonergic nerves. They are termed as serotonergic or
5-hydroxytryptaminergic nerves. However, their functions are still not well studied
(Mcclure and June 1971).
Actions of Antidepressants During Depression Therapy
Antidepressants are considered as the medicines that help decrease the depression
symptoms by bringing back the levels of neurotransmitters to normal in the brain.
The neurotransmitters (primarily serotonin, dopamine, and norepinephrine) com-
municate between the brain’s neurons by establishing chemical linkages among
them. The neurotransmitters stay located in the small vesicles of the presynaptic
exonic end of the nerve cells. From there, these molecules are transported to the
postsynaptic exonic ends of neurons via synaptic cleft. The antidepressants arrest
the reuptake of neurotransmitters through specific receptors, which enhance the
content of concerned neurotransmitters in proximity of the brain’s nerves.
118 Khushboo et al.
Categories of Antidepressants
The antidepressants have been categorized into five different groups as mentioned
above in the first section. The first group of antidepressants comprises the com-
pounds known as tricyclic antidepressants (TCAs), which are involved in increasing
the levels of two neurotransmitters, that is, norepinephrine as well as serotonin,
whereas they arrest the function of another important neurotransmitter, that is, ace-
tylcholine. It is believed that the TCAs alleviate depression by establishing a bal-
ance among these neurotransmitters present in different regions of the brain
(Gillman 2007). However, the treatment of depression using TCAs lead to the
occurrence of severe drug-induced toxicity such as chronic pain (tension, migraine,
headache), nausea, sexual dysfunction, insomnia, tremors, nervousness, and itching
(Aronson 2009; Khushboo and Sharma 2017).
The second group of antidepressants called selective serotonin reuptake inhibi-
tors (SSRIs) are known to influence the concentration of serotonin mainly in the
brain’s cerebrum. Some of these compounds are Fluoxetine or Prozac, Citalopram
or Celexa, Fluvoxamine or Luvox, Escitalopram or Lexapro, Paroxetine or Paxil,
Vilazodone or Viibryd, and Sertraline or Zoloft. The SSRIs are also helpful in treat-
ment of anxiety and depressive disorders. These antidepressants are able to recover
from any sign of depression but may exert certain side effects, for example, the
allergic reactions, hyperhidrosis (excess sweating), pruritus (itching), etc. The
SSRIs ease depression by increasing the concentration of serotonin and block its
reabsorption in the brain (Aronson 2009; Khushboo and Sharma 2017).
The third group of antidepressants comprise the monoamine oxidase inhibitors
(MAOIs), a key enzyme in neurotransmitter metabolism in the brain. This enzyme
denoted as MAO is a flavin-containing mitochondrial enzyme, which catalyzes the
neurotransmitter metabolism. MAOIs play an important role in knowing clearly the
key functions of neurotransmitters such as serotonin (5-HT), dopamine (DA), and
norepinephrine (NE) during neurotransmission in CNS. MAOIs by inhibiting the
activity of MAO increase monoamine concentration in the peripheral tissues as well
as CNS. It is known that the classical MAOIs can nonselectively inhibit the activi-
ties of both types of MAO, that is, MAO-A and MAO-B. MAO-A occurs in the
neurons containing serotonin and norepinephrine. This enzyme catalyzes and regu-
lates the catabolism of catecholamines and serotonin. This is presumed to act as a
target of drug action against depression. The biochemical effects of MAOIs concern
with the elevation of the concentration of norepinephrine and serotonin. The
MAO-A inhibitors being selective and reversible in binding with the target are
highly effective in the treatment of the severe depression without much adverse
effects (Moussa and Youdim 2004). The SNRIs, a class of medications effectively
used in treating depression, are also sometimes used to treat other ailments such as
chronic nerve pains and anxiety disorders (Medford 2005).
The fourth group of antidepressants constitute the inhibitors of reuptake of sero-
tonin norepinephrine (SNRIs). SNRIs such as desvenlafaxine, duloxetine, levomil-
nacipran, and venlafaxine inhibit the reuptake of serotonin. These compounds affect
the communication of signals between nerve cells of the brain and regulate mood to
take the patient out of depression. The side effects associated to the intake of these
drugs include constipation, changes in sexual desire, drying of the mouth, insomnia,
headache, dizziness, excess sweating, loss of appetite, and tiredness. These mole-
cules exhibit the property of interacting with certain other drugs such as warfarin or
aspirin or herbal ingredients being taken by the patients during medication with
antidepressants (Parikh et al. 2017).
The fifth group of molecules used for the treatment of depression called non-
TCA antidepressants include mirtazapine, trazodone, bupropion, and venlafaxine.
The mirtazapine also known as Remeron acts as an antagonist to noradrenergic
molecules. It blocks the function of receptors of adrenaline hormone, that is., epi-
nephrine, and regulates alleviation of the stress and major depression. Bupropion or
Wellbutrin acts as an inhibitor of dopamine reuptake and reduces the level of depres-
sion in patients. Similar to Bupropion, Trazodone or Oleptro and Vortioxetine or
Brintellix act as reuptake inhibitors and as serotonin antagonist and block the func-
tions of adrenergic receptors. Another compound in this group, Symbyax, is also
used to treat depression. It works in association with the SSRI fluoxetine, an anti-
psychotic agent, which is highly useful in treating the depression of bipolar as well
as drug-resistant nature. The application of these molecules is associated with the
onset of toxicity in the users. Their side effects have been shown by Naples et al.
(2016). A list of antidepressants, their doses, and therapeutic indices has been pre-
sented in Table 10.1.
Mechanisms of Actions of Different Antidepressants
TCA blocks the reuptake of both the neurotransmitters, that is, norepinephrine (NE)
and serotonin (5 HT). This phenomenon causes changes in the physiological behav-
ior of the neuron receptors, which is the primary mechanism of actions of antide-
pressants. TCA has also been shown to block muscarinic, alpha1 adrenergic, and
histaminergic receptors. The SSRIs can block the reentry of 5-HT and increase syn-
aptic 5-HT concentration. SSRI has been found to exert little or no significant effect
on the resurgence of other neurotransmitters. It has been observed that SSRIs do not
show any activity in muscarinic and histaminergic receptors. It results in anticholin-
ergic (ACH) and sedative effects.
The mode of the actions for MAOIs (phenelzine or nardil and tranylcypromine
or parnate) concerns inhibition of monoamine oxidase activity. MAOIs are usually
prescribed in cases of inertia or drug-resistant depression. Moclobemide (Manerix),
in contrast, has been recognized as the first inverse inhibitor of MAO-A (RIMAs),
which is found to be relatively more effective and safe. In this context, Nefazodone
(Serzone) exhibits the properties of both: it acts similar to the SSRIs, which block
the production of 5-HT, and acts as the opponent of 5-HT2 receptor. Nefazodone
has structural and pharmacological similarities. The only difference is that nefazo-
done binds with α1 receptors with low affinity. A high dose of SNRIs such as
120 Khushboo et al.
Table 10.1 List of available antidepressant drugs

Sr. Class of antidepressants and Toxicity in overdose and
no. antidepressant substrate (common name) Doses (mg/day) therapeutic index
1 Amitriptyline Start with a dosage High Narrow
up to 100
2 Amoxapine 50–100; NA Narrow
Maximum dose:
600
3 Clomipramine 25,100, 250 NA Narrow
4 Desipramine 100–300 Moderate Narrow
5 Doxepin 25–300 NA Narrow
6 Imipramine hydrochloride 10–50 NA Narrow
7 Imipramine pamoate 10–50 High Narrow
8 Maprotiline NA High Narrow
9 Nortriptyline 1025 NA Narrow
10 Protriptyline NA High Narrow
11 Trimipramine NA NA Narrow
12 Imipramine NA High Narrow
13 Citalopram 20–40 Moderate Wide
14 Fluoxetine 10–20 and 4 mg/ Low Wide
ml
15 Fluvoxamine 50–100 Low Wide
16 Paroxetine 20–30 Low Wide
17 Sertraline 25–100 Low Wide
18 Nefazodone 100–200 NA Wide
19 Trazodone 50–100 NA Wide
20 Isocarboxazid 40–60 High Wide
21 Phenelzine 60 High Wide
22 Tranylcypromine 60 Low Wide
23 Moclobemide 300 High Wide
24 Agomelatine 25–50 Unclear Narrow
25 Bupropion 150 Moderate Narrow
26 Duloxetine 60 Moderate Wide
27 Mianserin 30–200 Low Narrow
28 Reboxetine 8 Low Narrow
29 Trazodone 150–400 Low Wide
30 Venlafaxine 37.5–75 with food Moderate Narrow
NA Not available
d uloxetine (Cymbalta), venlafaxine (Effexor), desvenlafaxine (Pristiq, Khedezla),

and levomilnacipran (Fetzima) has been reported to gradually increase the blood
pressure. The first line of antidepressants is non-TCAs (NTCAs). These compounds
are relatively safer with better tolerance. However, current reports indicate that sec-
ondary amine TCAs (desipramine and nortriptyline) exert more side effects than the
tertiary amine TCAs. The different categories of antidepressants, their doses, and
side effects are summarized in Table 10.2.
Table 10.2 Groups of antidepressant drugs and their toxicity

Reason for
Sr. Class and name of Considerations related rejection from
no. synthetic drugs Side effects to these drugs medication
1. TCAs Amitriptyline, Blurred vision, Increased NA
desipramine, doxepin, constipation, drying of anticholinergic effects
imipramine, mouth, nausea, and cardiotoxicity
nortriptyline sedation, tachycardia,
gain in weight
2. MAOIs Isocarboxazid, Fatigue, hypotension, Due to hypertensive NA
phenelzine, sexual dysfunction, crises and serotonin
tranylcypromine, gain in weight syndrome
selegiline
3. SSRIs Anxiety, fatigue, GI Relatively safer, some Drug–drug
Citalopram, distress, headache, side effects observed interactions,
escitalopram, insomnia, sexual inhibition of
fluoxetine, paroxetine, dysfunction, gain in hepatic enzymes
sertraline weight
4. SNRIs Desvenlafaxine, Nausea, insomnia, dry Side effects are NA
duloxetine, mouth, headache, similar to but may be
levomilnacipran, increased blood slightly more frequent
venlafaxine pressure, sexual than with SSRI
dysfunction, gain in
weight
5. Atypical or Non- Agitation, headache, Increase in seizure, Onset
tricyclic compounds insomnia, loss of eating disorder, ofsSeizures, but
Bupropion appetite and weight, helpful to quit lower doses were
sweating smoking safer
Mirtazapine Enhancement in Relatively lower level Lower level of
appetite, sedation, and of nausea, sexual nausea, sedation,
weight dysfunction, decrease and sexual
in leucocyte count dysfunction
Trazodone Nausea, sedation, Slight weight gain, NA
priapism (rare) sexual dysfunction,
induction in sleep
Vilazodone Diarrhea, insomnia, Relatively safer, slight NA
nausea, sexual dysfunction,
gain in weight
Vortioxetine Diarrhea, dizziness, Toxicity similar to NA
nausea, SSRI
NA Not available
Antidepressants’ Overdose and Consequences
The intake of overdose of antidepressants may be lethal. The deaths due to poison-
ing or toxicity from antidepressants either taken alone or when taken with other
substances are displayed in Table 10.3.
122 Khushboo et al.
Table 10.3 An estimate of deaths because of overdose of antidepressants

Total deaths
Sr. no. Class of antidepressants Single-drug overdose Combination
1. All antidepressants 302 448
2. SSRIs NA NA
3. Older TCAs 298 432
4. Newer TCAs and others 8 16
Source: The Office of Populations, Censuses and Surveys (OPCS) (1990) report, NA = data not
available
Role of Phytochemicals as Antidepressants
The chemical ingredients preset in many plants including some vegetables and
fruits have been found to act as potential antidepressants. In addition, they are
highly cost-effective, safer, and effective in their actions to treat several diseases
including cardiovascular, autoimmune, and neurodegenerative disorders. Mainly
being polyphenols in their chemical nature, some of them including curcumin, feru-
lic acid, fisetin, piceatannol, resveratrol, and smaller proanthocyanidin, have been
widely used as complementary and alternative medicines to treat various ailments
including depression. These phytochemicals possess strong antioxidative, antimi-
crobial, anticancerous, and anti-inflammatory properties. These phytochemicals
possess antidepressants like properties.
Curcumin
Curcuma longa, a key phytochemical, has been recognized as a chief constituent

of established Chinese medicines like Xiaoyao-san and has been broadly used in
most of the Asian countries to effectively administer mental disorders. Curcumin
is the chief active ingredient in C. longa and exerts impact like antidepressants. It
was first displayed in animal depression models by forced swimming test (FST)
(Xu et al. 2005) and in the old unpredictable light stress mode (Li et al. 2009). Its
effects, however, can be potentiated by different antidepressants, such as fluox-
etine, venlafaxine, and bupropion. After the administration of curcumin (Kulkarni
and Dhir 2008), the elevated serotonin concentration has been reported in rats.
Curcumin in c ombination with piperine may enhance bioavailability and boost
the notable concentration of serotonin (Bhutani et al. 2009). It has been observed
that in the FST, the antidepressant effect of curcumin may be at the levels of 5-HT
receptors, especially 5-HT1A/1B and 5-HT2C subtypes (Wang et al. 2008).
Curcumin may arrest the stress-induced decrease in the 5-HT1A mRNA concen-
tration in rat’s hippocampus (Xu et al. 2007). The molecular structure of curcumin
is presented in Fig. 10.1a.
a b OH
c
HO O O O
O O OH
OH HO OH
N OH OH
HO
H O
NH2 HO OH
O O
OH
OH
d e f g OH
O OH
O HO O
OH HO OH
HO HO OH
OH O
OH
Fig. 10.1 Chemical structures of some phytochemicals. (a) L-theanine; (b) proanthocyanidins; (c)
curcumin; (d) carvacrol; (e) ferulic acid; (f) resveratrol; (g) quercetin
Quercetin
Quercetin, a polyphenolic flavonoid (Fig. 10.1b), is present in vegetables, fruits, as

well as in the therapeutic herbs. This flavonol is known to arrest oxidation of other
molecules by functioning as a reductant in free radical–mediated oxidative chain
reactions (Murakami et al. 2008). This polyphenolic flavonoid is a nonspecific pro-
tein kinase enzyme inhibitor (Russo et al. 2014), but it activates estrogen receptors
(Moutsatso 2007). The dose of quercetin (20–40 mg/kg, PO) prohibited depression-
related behavior as a result of activation of axis of hypothalamic–pituitary–adrenal
(HPA) in rats in preclinical studies. The consequence was analogous with fluoxetine
(10–20 mg/kg, IP) (Bhutada et al. 2010).
Proanthocyanidins
An oligonol (catechin-type monomer, dimer, and trimer), known as proanthocyani-

dins and oligomers are usually present in grapeseeds (DalBo et al. 2006).
Proanthocyanidins (Fig. 10.1c) have a large variety of pharmacological influences,
including antioxidant effects and antinociceptive and cardioprotective properties
(Preuss et al. 2000; Sato et al. 2001; Uchida et al. 2008). Xu et al. (2010) have
shown that proanthocyanidins can reduce the duration of instability in both the tail
suspension test and FST. Apart from this, increased concentration of 5-HT in the
cortex, hippocampus, and hypothalamus in front of rats after the proanthocyanidin
administration was recorded. Conversely, the method underlying the antidepressant-
like activity of proanthocyanidin is still unclear (Fig. 10.1c).
124 Khushboo et al.
Resveratrol
The previous studies have shown the inhibitory effects of resveratrol on noradrena-
line and serotonin reuptake in rats (Yáñez et al. 2006). The application of resveratrol
has been found to significantly reduce time of immobility in mouse, an experimen-
tal model of despair tests. It also enhanced the concentrations of serotonin and nor-
adrenaline in different brain regions (Yáñez et al. 2006; Xu et al. 2010). In addition,
resveratrol caused significant reduction in MAO activity (Mazzio et al. 1998; Xu
et al. 2010). Thus, resveratrol reflects the activity similar to the antidepressants.
Resveratrol having a strong antioxidant property exists in red wine and grapes.
Trans-resveratrol, as an active constituent of Polygonum cuspidatum, is convention-
ally used to treat neuropsychiatric disorders in most of the Asian countries.
Resveratrol consists of anti-inflammation, amelioration of learning as well as mem-
ory destruction, and neuroprotection (Tredici et al. 1999; Chen et al. 2009; Kumar
et al. 2007; Ranney and Petro 2009). The molecular structure of resveratrol is dem-
onstrated in Fig. 10.1d.
Ferulic Acid
Ferulic acid (Fig. 10.1e) is a phytochemical, which is a highly powerful antioxidant

in nature (Srinivasan et al. 2007). This compound is derived from phenylalanine,
which is converted into 4-hydroxycinamic acid and then caffeic acid. It shows vari-
ous pharmacological functions with anti-inflammatory, antitumor, antidiabetic, and
neuroprotective properties (Kawabata et al. 2000; Balasubashini et al. 2004; Sultana
et al. 2005; Yogeeta et al. 2006). Yabe et al. (2010) have reported that the oral admin-
istration of ferulic acid (100 and 250 mg/kg, PO) could reduce the stressed abnor-
mal behavior in the rat depression model.
L-Theanine
This phytochemical present in Camellia sinensis (green tea) is derived from

L-threonine, an important amino acid (Fig. 10.1f). This amino acid has been utilized
for various therapeutic benefits including improvements in concentration and learn-
ing ability, enhancing antitumor activity, preventing vascular diseases, reducing
blood pressure, acting as antibiotics, improving immunity, and displaying neuropro-
tection (Mu et al. 2015). It binds with the glutamate receptors (Kakuda et al. 2002;
Nathan et al. 2006). L-theanine may influence CNS as it may cause inhibition of
reuptake of γ-aminobutyric acid, dopamine, and serotonin and glutamate (Lu et al.
2004). According to Gomez-Ramirez et al. (2009), L-theanine (1, 4, and 20 mg/kg,
PO) when administered for 10 days performed an antidepressant function in rats.
However, its overdose (250 mg/day up to 8 weeks, PO) may induce some serious
side effects such as hypothermia (Yin et al. 2011), anxiety, cognitive destruction,
sleep disturbance, and depression (Hidese et al. 2017).
Carvacrol
This compound belonging to the monoterpenic phenol group of phytochemicals

(Fig. 10.1g) exhibits anti-inflammatory, analgesic, antiarthritic, antiallergic, anticar-
cinogenic, antidiabetic, cardioprotective, gastroprotective, hepatoprotective, and
neuroprotective properties (Friedman 2014). It regulates human ion channel tran-
sient receptor potential (Xu et al. 2006). It can also suppress inflammation of COX-2
mediocre (Hotta et al. 2010). The carvacrol (12.5–50 mg/kg, PO), when taken
orally, acts as an antidepressant which is mediated by dopaminergic pathway in
brain of rat (Melo et al. 2011). Zotti et al. (2013) have demonstrated that carvacrol
(12.5 mg/kg, PO) when taken for 7 days can increase the concentration of dopamine
and 5-HT in two regions of the brain, that is, prefrontal cortex and hippocampus.
Other Phytochemicals Acting as Therapeutics
Several natural compounds derived from plants can be placed in three major classes,
phenylpropanoids, isoprenoids, and alkaloid, which are widely used as potential
antioxidants (Facchini 2001; Holstein and Hohl 2004; Iriti and Faoro 2004). The
polyphenolic phytochemicals (phenolic acids, flavonoids, stable bones, and lignin)
may regulate cell receptors and modulate the functions of various enzymes (Bravo
1998; Manach et al. 2004). Their antioxidative potential has been attributed to the
prevention of oxidative stress-mediated diseases, for example, the neurodegenera-
tive disorders and cancer (Scalbert et al. 2004). A summary of some phytochemicals
known for their functions as antidepressants is demonstrated in Table 10.4.
Future Directions The existing information has indicated the neuroprotective
potential of phytochemicals. These compounds also act as antidepressants through
monoamine neurotransmitters-based, HPA axis-based, and neurotropic factors or
neurogenesis-based mechanisms. Possibly phytochemicals affect via activation of
signaling cascade in the brain, which can trigger many reactions such as promoting
neuronal survival and differentiation and blocking neuronal apoptosis. Despite great
progress made in our understanding about depression, very little information is
available about the antidepressant action of many phytochemicals as their antide-
pressant property involves many complex mechanisms. Therefore, it is pertinent to
initiate extensive research to identify plant based biomolecules and signaling net-
works associated to the individual phytochemical in order to develop cost-effective,
safer, and more efficient drugs to treat depression and other concerning neurodegen-
erative diseases. In addition, creation of good database is needed to gather more
126
Table 10.4 Some phytochemicals, their sources, and functions as antidepressants

Sr. Phytochemical Scientific name of
no. group Antidepressant herbs Family Active constituents Mode of actions References
1 Flavonoids Apocynum venetum Apocynaceae NA Medicating central monoaminergic Kim et al. (2001)
systems
2 Alkaloids Areca catechu Arecaceae Arecaidine, arecoline, Inhibition of activity of MAO-A Dar and Khatoon
guvacine (1997)
3 Saponins Bacopa monnieri Scrophulariaceae Bacoside A Antioxidative function Singh and Dhawan
(1997)
4 – Cissampelos sympodialis Menispermaceae Warifteine Antioxidative function Thomas et al.
(1999)
5 Flavonoid Curcuma longa Liliaceae Curcumin MAO-A inhibition Park and Kim
(2002)
6 Flavonoids Hypericum perforatum Hypericaceae Hyperforin, rutin Inhibitor to 5-HT, DA, MAO-A, Cervo et al. (2002)
MAO-B, and NE reuptake
7 Oligosaccharides Morinda officinalis Rubiaceae NA Antioxidative function Cui et al. (1995)
8 Poly phenols Perilla frutescens Lamiaceae Rosmarinic acid, caffeic NA Takeda et al.
acid, apigenin (2002)
9 Alkaloids Withania somnifera Solanaceae Withanolides, withanols Stabilization of mood Bhattacharya et al.
(2000)
10 Polyphenols Angelica sinensis Apiaceae Ferulic acid Antioxidant action Yan et al. (2001)
Khushboo et al.
useful clues about the phytochemicals and their antidepressant properties through
in vivo as well as in vitro studies.
Acknowledgments Khushboo and AK are grateful to UGC–New Delhi and UGC–CSIR–New

Delhi, respectively, for providing financial support in the form of a research fellowship and to the
DST–FIST–New Delhi as well as UGC–SAP–New Delhi for providing research facilities at the
Department of Biochemistry of University of Allahabad, UP, India.
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Chapter 11
Phytochemistry and Medicinal Value
of Putranjiva roxburghii Wall
Narendra Kumar
Introduction
Jivanputra (Putranjiva roxburghii) belongs to family Euphorbiaceae and commonly

called putrajeevak. It is a native of the Indian Subcontinent, Southeast Asia, Japan,
southern China, and New Guinea. It is an evergreen dioecious tree which bears
pendent branches. It reaches a height of 18 m and girth of 2 m. The bark is gray and
shiny. The leaves are dark green which are 5–10 cm long. Male flowers looks yel-
lowish in color and found in clusters. The female flowers appear alone.
This has immense medicinal uses. It is used in rheumatism, cold, fever, and
inflammation (Chinmaya et al. 2009). It is Native to India called as Patravanti,
Kudrajuvi, Jivputrakand, and Nageia. In Sanskrit literature it is called as Putranjiva,
Jivanputra, Kumarajiva, Pavitra, Mava, and Putrajiva. “Pootranjeeva” is formed of
two words in which pootra indicates son and jeeva means life. It is applied by tribes
in treating various health problems. Fruits, stones, and leaves are used in coldness
and sore affections. Rosaries are prepared from firm gravels to place at children’s
neck to protect from sickness. Sometimes it became the topic of debate due to its
popularity for getting a male child. It has been widely used for the treatment of
catarrh, diuretic, azoospermia, constipation, and ophthalmopathy (Varma
et al. 2010).
It has been found effective for liver diseases, infertility, and fever. This contains
its potential as an anti-inflammatory, anthelmintic, anticancer, aphrodisiac, diuretic,
laxative, and antioxidant. The stones, leaves, and fruits are taken as a medicine dur-
ing colds, fevers, and rheumatic pains (Gangal et al. 2009).
Leaves are normally bitter, astringent, refrigerant, and procreant. It is useful in
treating skin aridity ailment along with curing rheumatism (Ashok 2005). The
seed’s oil are used in Unani medications Ayurveda and other herbals. The fruits and
N. Kumar (*)
Amity Institute of Biotechnology, Amity University Haryana, Manesar, Gurgaon, India

https://doi.org/10.1007/978-981-15-2195-9_11
134 N. Kumar
leaves are useful in removing arthralgia and muscle sprain in Thai therapy. For birth
of male child, women use nuts of Putranjiva orally (Khare 2007). Chemically bark
contains triterpenoids (Garg and Mitra 1968; Sengupta and Mukherjee 1968).
Leaves have bioflavonoid and triterpene acid (Garg and Mitra 1971).
This contains mustard oils which act as defense chemical against herbivores.
They produce glucosinolates (Soltis and Soltis 2004). It shows fluctuations in fatty
acids and oil composition in different ages. The seeds have major compounds, lin-
oleic and oleic acids (Gangal et al. 2009). It is utilized as potential healing plant and
harbors many chemicals. The aim of this chapter is to provide insights regarding its
phytochemistry and medicinal properties against various diseases.
Names in Different Tongues
Marathi name, Putajan; Hindi name, Putranjeev and Jiyapeeta; English name, Indian
amulet and child life tree; Gujarathi name, Putramjeeva, Putrajiva, and Putranjiv;
Kannada name, Putramjiva and Putrajiva; Malayalam name, Pongalam; Tamil
name, Irukolli; Telugu name, Putrajivika, Kuduru
Botany of Jivanputra Plant
It is an evergreen, moderate-sized tree which reaches up to 12 m and has pendent

branches. Its bark is dark gray in color and has horizontal lenticels. The leaves are
simple, distichous, alternate, stipule triangular, caduceus, acute. The petiole is
0.5–1-cm-long glabrous. In cross-section, it is plano-convex. The flowers bear uni-
sexual male yellow-colored flowers. It appears in the form of axillary clusters.
The fruits range 0.5–0.8-inch in diameter, and are ovoid or round in shape. Seeds
are slimy inside and solitary. Fruit formation occurs during months January to
March. The tree may be observed up to 3000 feet altitude in entire India.
Leaves are alternate, simple, and caduceus. Petiole is 5–7-mm-long and pubes-
cent and lamina is 3.4–11 × 1.4–4.4 cm elliptic oblong, with apex shortly acumi-
nate. The flowers are unisexual. The male flowers are sessile found in axillary
spikes, 2–2.5-mm across; 1.5–2-mm-long pedicels, glabrous; tepals are 3–5, ciliate
oblong, obtuse, and imbricate puberulous; stamens 1.5–2-mm-long and 2–4; fila-
ments connate toward the base; anthers hairy ovate; female flowers, 2 or 3 or soli-
tary, axillary; pedicel puberulous up to 15 mm long; lanceolate bracts; tepals 5–6,
2–2.5 × 1–1.5 mm, oblong, unequal, puberulous, acute ciliate; superior ovary, 3 ×
2.5 mm, tomentose, globose, ovules 2 in each cell, three-celled; style 3, tomentose,
spreading, connate below and dilated into broad fleshy stigma; stigma glandular,
crescent-shaped. Fruits drupe, ovoid–ellipsoid, 1.3–2 × 1.5 cm, white tomentose,
crustaceous, seed one, and 6–25-mm-long pedicels.
11 Phytochemistry and Medicinal Value of Putranjiva roxburghii Wall 135
Phytochemistry of P. roxburghii
The tropical regions of Asia are the central part where P. roxburghii are found abun-
dantly. The kernel projects a smell, having yield of 0.5%. The main constituents are
depicted in Fig. 11.1. The oil contains 2 butyl isothiocyanates having isopropyl as
prime components and 2-methyl-butyl isothiocyanates in little amount. The gluco-
cleomin and glucoside are also present in kernels. Glucoside is also present in
shoots and roots. The paste of fruitlet has sufficient amount of mannitol. This con-
tains little saponin, glucoside. This is also reported in stones of seed case (Supriya
et al. 2017). The seed contains oils which have isopropyl and 2-butylisothio-cyanates
as major constituents while 2-methyl-butyl isothiocyanate as minor component. The
formation of isothiocyanates takes place through hydrolysis of glycoside progeni-
tors enzymatically, namely glucocochlearin, glucojiaputin, and glucoputranjivin,
respectively. Thioglucoside glucocleomin, has also been found present in the seed
kernel. Seed coat contains putranoside, putranjivoside, beta-sitosterol, along with
beta-D-glucoside. The leaves contain amentoflavone. This has derivatives namely
isopropyl and2-butylisothio- 2-methyl-butyl

cyanates isothiocyanate
amentoflav
one
putric acid,
roxburgholone( 3-
friedelanol, friedelin,
alpha-hydro-
friedelanone, friedelan-
xyfriedelan- 7-one)
3,7-di-one
carboxylic
(putranjivadione)
acid,mannitol,putric
acid,
Phytochemistry of
Jivanputra(Putranjiva
roxburghii)
glucosides,saponin and
stigmasterol
alkaloids
putranjivoside,
polyphenols,palmite, putranoside
putranjiva saponin
beta- sitosterol
,tis beta-D-
glucoside
Fig. 11.1 Main chemicals present in P. roxburghii

136 N. Kumar
Table 11.1 Recent investigations on chemicals present in P. roxburghii

Plant part Chemical constituents References
Bark Ethyl acetate extract of the bark of Putranjiva reveals the Abhimanyu
presence of 15 phytoconstituents, from which friedelin, et al. (2018)
sitosterol, ergosterol are in higher quantity, and the bark part
of this plant is a rich source of steroids and terpenoids
Seed Glycosides, alkaloids, carbohydrates, phenols, flavonoids, Emasushan
fixed oils, tannins, saponins, coumarins, terpenoids, and John
and sterols (2018)
Seed Presence of lignins, starch, alkaloids, tannins, and calcium Minj and John
oxalate crystals Saponins in leaf and stem root except in seeds (2017)
Ethanol and Alkaloids, flavonoids, carbohydrates, glycosides, steroids, Emasushan
methanolic leaf phenols, tannins, saponins, terpenoids, coumarins, et al. (2015)
extracts and fixed oil
Seed Saponins, phenols, alkaloids, flavonoids, steroids, and Raghvendra
glycosides et al. (2010)
Seed Tannins, saponins, steroids, alkaloids, and flavonoids Sudharshan
et al. (2009)
Methanolic Saponins, steroids, alkaloids, and flavonoids Chinmaya
extract of fruits et al. (2009)
beta-amyrin, palmitate, polyphenols, stigmasterol, and putranjiva saponin. The bark

bears nitric acid, carboxylic acid, putranjivic acid, friedelin, friedelanone, friedela-
nol, 3-alpha-hydroxy-friedelan-7-one (roxburgholone), and riedelan- 3,7-di-one
(putranjivadione). Recently some investigators have also studied the chemistry of
P. roxburghii which is recorded in Table 11.1.
Medicinal Activity
This has many medicinal potentials, which are presented in Fig. 11.2.
Antibacterial Potential
The methanol solvent was used in extraction of dried and powdered seed material.
This extracted material exhibited marked antibacterial action against Gram-negative
and Gram-positive bacteria which cause food poisoning. It checked Gram-positive
more in comparison to Gram-negative bacteria (Chinmaya et al. 2009). Sudharshan
et al. (2009) found that the extract resulted more Gram-positive bacteria inhibition
than Gram-negative bacteria (Sudharshan et al. 2009). But the methanolic, acetone,
and ethanol leaf extracts were found more effective showing maximum inhibition
zone against bacterial strains (Kapoor and Pandita 2013) namely Proteus vulgaris,
Antibacterial and
Ethnobotanical
Antioxidant potential Antifungal activity
uses,Trypsin
inhibitor
Fumigant
potential,Harbours
Larvicidal activity
many fungi,useful in
biodiesel production
MEDICINAL VALUE OF
PUTRANJIVA
Cytotoxic
activity,natural remedy
Anthelmintic activity
for control of Fusarial
wilting
Analgesic, Antidiabetic activity

antioxidant,antiinfla Anti-
matory nociceptive,
antipyretic, and
anti-
inflammatory
activity
Fig. 11.2 Medicinal potential of P. roxburghii
Streptococcus pneumoniae,Bacillus cereus, Klebsiella pneumoniae, Proteus mira-

bilis, Vibrio cholera, Escherichia coli, and Enterobacter aerogenes in comparison
to aqueous leaf extract (Emasushan et al. 2015).
Antifungal Activity
Sudharshan et al. (2009) reported that A. flavus was more susceptible than A. niger
and A. nidulans when methanolic P. roxburghii extract was applied. The essential oil
of the seed kernel of P. roxburghii exhibited absolute toxicity at 500 ppm. It inhib-
ited growth of Fusarium solani and Aspergillus niger (Kumar 2014a, b). It exhibited
greatest toxicity (Kumar and Tripathi 2002, 2004) against Aspergillus flavus and
Aspergillus niger at 400 ppm (Kumar 2015a, b). The methanolic extracts showed
higher value for Trichophyton rubrum (Emasushan and John 2018).
138 N. Kumar
Larvicidal Activity
P. roxburghii exhibited dose-dependent larvicidal activity against Aedes aegypti

mosquito larvae. Its extract preparation caused larval mortality 100% at 2.5 and
5 mg/ml (Sudharshan et al. 2009).
Anthelmintic Activity
It caused paralysis with death of the worms when dried and powdered seed extract
was applied. The results recorded that its methanol extract contains active principles
which have anthelmintic activity (Chinmaya et al. 2009). A study revealed that
extract at concentration of 5 mg/ml resulted in paralysis (19 min) and death (38 min)
of worms in a shorter period of time in comparison to 1% piperazine citrate (29 and
44 min for paralysis and death, respectively) (Sudharshan et al. 2009). P. roxburghii
showed highest antibacterial activity under in vitro conditions through disk diffu-
sion method namely, Erwinia herbicola, Bacillus subtilis, Escherichia coli,
Pseudomonas putida, Salmonella typhi, Staphylococcus aureus, Vibrio cholera, and
Klebsiella pneumoniae. It is a potential source of good antibacteria (Kumar
2015a, b).
Antidiabetic Activity
Diabetes mellitus being a serious endocrine disorder is formed due to impairing in

glucose homeostasis resulting in severe diabetic problems producing nephropathy,
angiopathy, retinopathy, and neuropathy. This produces neurological problems due
to a check in the glucose utilization. Hypoglycemic activity of P. roxburghii leaves
was evaluated on animal models. It was found that this has potent antihyperglyce-
mic effect (Varma et al. 2011). The EAPR barks was administered in STZ-induced
diabetic rats at 250 and 500 mg/kg by oral route for 21 days. This revealed that
EAPR barks have antihyperglycemic, anti-cholesterolemic potential which
improved β-cells of islets of Langerhans cell density in diabetic rats (Abhimanyu
et al. 2018).
Antinociceptive, Anti-inflammatory, and Antipyretic Potential
The ether extract potential of P. roxburghii leaves was evaluated for nociceptive
responses. The antipyretic power was studied in yeast-induced fever in rats. Anti-
inflammatory activities were found out using croton oil-induced anus, ear edemas,
and carrageenin-induced paw edema of rats. P. roxburghii at 100, 200, and 400 mg/
kg, p.o. ether extract exhibited dose-related analgesic power. At 400 mg/kg, this
checked licking activity at late phase and decreased fever resulted by yeast in rats.
The extract has moderate inhibitory action against inflammation of carrageenin-
induced paw edema. The extract inhibited croton oil-induced ear edema at 1.25, 2.5,
and 5.0 mg/ear in mice, which indicates that the ether extract of P. roxburghii leaves
have antipyretic, analgesic, and anti-inflammatory powers (Reanmongkol et al.
2009; Hat and Songkhla 2009).
Analgesic Activity
Analgesic activity was studied through tail flick models and Eddy’s hot plate mod-
els. The results highlighted dose-dependent anti-inflammatory activity in ethanolic
extract at 250 and 500 mg/kg of P. roxburghii wall in comparison to indomethacin.
It produced analgesic power in acetic acid–induced writhing model which is dose
dependent but dose independently in hot plate and tail flick models (Khobragade
et al. 2013).
Cytotoxic Activity
The methanolic extract of P. roxburghii seeds produced cytotoxicity with at

427.74 μg/ml in brine lethality assay. The valuable cytotoxic power is mainly due to
active phytochemicals. Because of this, they have efficient treatment and protection
roles against cancer (Raghavendra et al. 2010).
Cytogenetic Toxicity
The young weaning Swiss albino mice were orally given leaf extract of P. rox-
burghii at 0.5, 1.0, or 2.0 g/kg body weight/day up to 7 consecutive days. This
resulted in the induction of mitosis disruptive chromosomal conversion of bone
marrow cells. There was no change of incidence in structural abnormalities noticed
in metaphase chromosomes. It can be proposed that extract interfered with the spin-
dle and other proteins resulting in polyploidy, aneuploidy, c-mitosis, etc. (Awasthy
et al. 2000).
Antioxidant Activity
The in vitro antioxidant activity in ethanolic leaf extract of P. roxburghii was
searched through the DPPH method in different concentrations. This showed anti-
oxidant power. It showed that it depends on concentration which may be because
high flavonoid content in leaves of P. roxburghii (Shahwar and Raza 2012; Rajagopal
et al. 2014).
140 N. Kumar
Natural Remedy for Control of Fusarial Wilting of Shisham
A survey of 20 places of Eastern UP districts revealed that Shisham wilting occurs

in all age groups which is due to Fusarium solani and Fusarium oxysporum. The oil
of Putranjiva roxburghii showed inhibition of both at 500 ppm. The oil is thermo-
stable at MIC of 500 ppm, has a wide range at 1000 ppm, and has a storage period
of 120 days. Field trial at nursery level done along with Putranjiva caused no wilt-
ing in comparison to control plants of sissoo (Kumar 2014a, b).
umigant Potential of Oil of P. roxburghii

F
Against Storage Pests
The Putranjiva oil protected sissoo seeds up to 6 months at 1000 ppm and 1500 ppm
in a container of 250 ml capacity holding 200 g seeds and has no adverse effect on
seed germination, seedling growth, and foliage of sissoo plants (Kumar 2014a, b).
Kumar (2016) found that seed oil of Putranjiva safely preserved matar seeds up to
6 in tin containers of 500 ml capacity having 400 g seeds and has no adverse reac-
tion on plants. Hence, it may be an alternate of synthetic pesticides.
Endophytic Fungi of P. roxburghii
In three different seasons, endophytic fungi from the inner bark of P. roxburghii
were investigated in years 2009–2010. Out of 30 endophytes recorded during rainy
season, fungal species namely, Aspergillus niger, A. flavus, Bispora punctata,
Curvularia lunata, Fusarium oxysporum, and Verticillium sp. were dominant in
occurrence followed by Nigrospora sp., Cladosporium sp., Rhizopus stolonifer, and
Drechslera sp. species (Nagaraja 2013).
Ethnobotanical Uses
Its seed powder is useful in improving sperm count if given at 1–3 g with milk in
males. Leaf pasties should be applied on the portion affected with burning. Fresh
juice at 10–15 ml is used in curing elephantiasis. A cold infusion of its leaves is
given of 20–30 ml to treat micturition. The paste prepared from seeds is applied
treat eye problems. Kottaimuthu (2008) highlighted his ethnobotanical survey taken
from southern and eastern ghats of Tamil Nadu that the application of stalk bark of
Putranjiva combined with leaves of Pterospermum suberifolium helps in broken
bones (Kottaimuthu 2008). Kernels are consumed orally for the birth of a boy
(Shailja 2011).
Ayurvedic Medicine
Lucap capsule is prepared. It is useful in iron deficiency anemia, dysmenorrhea,

menorrhagia, and vaginitis and corrects postpartum disorders.
Trypsin Inhibitor
The cation and anion exchange chromatography was done to find out a stable inhibi-
tor Putranjiva seeds. This inhibitor checked dense trypsin in 1:1 mole ratio. The
inhibiting nature of the stable inhibitor was lost on or above 90 °C. The structural
stability parameters of inhibitor were studied at high temperatures. Trypsin inhibitor
worked like protease inhibitors which have enzyme inhibitory action. The Putranjiva
seeds can express the same nature of protease inhibitors (Chaudhary et al. 2008).
Production of Biodiesel
It is may be an alternate for diesel engines for fuel. The diesel engines can be oper-
ated through 100% biodiesel extracted from Putranjiva without any modification in
the engine (Ghosha et al. 2008). Haldar et al. (2009) mentioned that oil blend of
Putranjiva may be replaced in place of fossil diesel. This can even force the diesel
engine through decreasing emissions which have beneficial impact on the humans
(Haldar et al. 2009).
Conclusion
The evolving new conditions and loss of natural resources necessitated new alterna-
tives. The plant of Putranjiva may be utilized for antioxidant, anti-inflammatory,
and febrifuge activities. It can be utilized as a biofuel. It has fumigant efficacy for
preservation of forestry and agriculture-related important seeds. It may be used as
trypsin inhibitor. This can inhibit growth of bacteria, worms, and even larvae at
in vitro conditions. This contains a wide spectrum of bioactive chemicals in which
many are selective and have no harmful effect on nontarget organisms and even on
the environment. Putranjiva may be utilized as antifungal, trypsin inhibitor, and
antipyretic and has antidiabetic attributes. Further researches may be done on this
plant on stem bark which may have extraordinary medicinal potential and high
commercial value.
142 N. Kumar
Acknowledgment Authors are thankful to the Amity University, Haryana, authorities for the
facilities and constant encouragement.
Conflict of Interest Statement We declare that we have no conflict of interest.
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Chapter 12
Traditional Herbal Practices of Eastern
Ghats, Odisha, India, for Treatment
of Bone Fracture
Subhashree Pattnayak, Devasish Murmu, Manasa Kumar Panda,

Rojali Maharana, Kalicharan Mandal, and Nabin Kumar Dhal
Introduction
According to the World Health Organization (WHO), about 80% of developing coun-
tries still depend on traditional medicines for their primary healthcare needs (Calixto
2005). The conventional knowledge concerning the medicinal uses of these native plant
materials has been passed from generation to generation through verbal communica-
tion (Hossan et al. 2010). For the treatment of many diseases, these ethnomedicinal
plants have been used for many centuries as a source of drugs (Alpuerto et al. 2012).
Due to constituents of many potentially valuable therapeutic agents, these medicinal
plants can be a source of raw material for the preparation of desired medicines. These
can be a better alternative for synthetic drugs due to its less toxic, more systemic, and
readily biodegradable properties. The data lead to the development of novel phyto-
chemicals for human use (Amazad Basha and Basha 2013). The purpose of the medici-
nal plant is increasing, but there is very little knowledge about its use patterns. It is
crucial to analyze the traditional awareness for the commercial importance and value,
as ethnomedicinal uses of plants are one of the most successful criteria in finding new
therapeutic agents used by the pharmaceutical industry (Cox and Balick 1994).
The floral diversity of India directly enhances the use of the traditional system of
medicine, especially the herbal products. India has 12 mega-diversity centers which
are responsible for the biodiversity zones (Sarvalingam et al. 2017) and which
enhance the ethnic diversity, where 537 tribal groups comprising 67.37 million
S. Pattnayak · D. Murmu · R. Maharana · K. Mandal · N. K. Dhal (*)

M. K. Panda

https://doi.org/10.1007/978-981-15-2195-9_12
146 S. Pattnayak et al.
tribal people reside in different geographical locations. Twenty five percent of all
medicines suggested by an expert today originate from plants, which means that
plant-derived drugs contribute to a significant portion of natural product for phar-
maceuticals. All over the world, including India, there exist several traditional
practices with herbal medicine which are used for human and animal treatment.
Depending upon prolonged experience, the researcher might identify a few of them
as globally necessary (Pan et al. 2013).
Tribal communities of Odisha are generally found in Eastern Ghat and all of
them have their own culture, customs, and practices (Rout and Panda 2010).
Previously many ethnobotanical studies have already been done in Eastern Ghats of
India for documentation of indigenous knowledge (Ratnam and Raju 2008) and also
the plants responsible for the treatment of rheumatism,v arthritis, gout, and lumbago by
the traditional and local people of the district (Singh et al. 2010). Traditional herbal
practices are widely documented for wound healing and known to cure eczema, piles,
constipation, and skin infection (Mallik et al. 2012; Kumar et al. 2012). Documentation
has also been done on ethnobotanical knowledge–selected plants that are used for pre-
vention of malaria (Nagendrappa et al. 2013) and for curing infectious diarrhea (Panda
et al. 2012). Fewer studies have been conducted (Lal 1988; Venkataratnam and
Venkataraju 2008) on understanding medicinal plants used in the treatment of bone
fracture, which is a complex physiological process (Vibha Singh 2017). Fracture heal-
ing not only needs surgery, slings, braces, or other devices, but also needs proper nutri-
tion and overall health; this is because broken bones can repair themselves naturally.
The treatment is just required to assist the natural healing process (Gupta and Kumar
2018). Therefore, the present work is an effort to document the traditional knowledge
regarding the practice and use of plants in the treatment of the bone fracture.
Experimental
(a) Study Area

Odisha is located between the parallels of 17.49° N and 22.34° N latitudes and
meridians of 81.27° E and 87.29° E longitudes. According to the report in 2015 by the
Forest Survey of India (FSI), Odisha (geographical area 155,707 km2) has 50,354 km2
of forests which cover 32.24% of the state’s total area. Periodical information been
collected from the tribes such as Bathudi, Bhumija, Birhar, Ganda, Munda, Mundari,
Santal, Saunti, and Kolh, which inhabit the Mayurbhanja, Malkagiri, and Koraput
districts of Odisha (Fig. 12.1). The field trips were planned to use topographical maps
to cover the different village pockets, namely Dudurchampa, Hatibarii, Bijatala,
Dongadiha, Joshipur, Kaptipada, Khunta, Lulung, Singtala, Papermelta, Alamanda,
Boipariguma, Mudulipada, and Kakamela. Some of the herbal medicine preparation
systems and the healing processes were also documented.
(b) Specimen Collection and Identification
Flowering twigs of plant specimens (minimum five samples) were collected in
polythene bags, and their habit and habitat were noted down. The specimens were
12 Traditional Herbal Practices of Eastern Ghats, Odisha, India, for Treatment… 147
Fig. 12.1 Site map of Eastern Ghats
identified through the references of flora of Odisha, Gamble flora, and finally from
Council of Scientific and Industrial Research–Institute of Minerals and Materials
Technology (CSIR-IMMT) herbarium.
(c) Preparation of Herbarium Specimens
Specimens were precisely arranged between the blotting paper and tightly pressed
with the help of wooden pressure and changed every 2–3 days for proper drying. After
drying, the specimens were dipped in a saturated solution of 1% HgCl2 in ethyl alco-
hol to prevent infection of fungi and insects and finally mounted on herbarium sheet.
(d) Collection of Data

A standard questionnaire method followed for data collection. Then proper
investigation and recheck were carried out with the help of village vaidyas.
Outcome
The floristic and ethnobotanical data were collected regarding the plant parts used,
different local uses, mode of preparation, and cure percentages of a particular dis-
ease. The village vaidyas mostly used the whole plants of herb species for healing
the pain (Fig. 12.2). However, tuber, bark, root, leaf, and fruits of some species are
applied by Santelli, Saunti, Bathudi, and Munda tribes of the Mayurbhanj district
for proper healing of the fracture. The recorded 39 plant species from 22 families
were Fabaceae > Euphorbiaceae > Orchidaceae > Rubiaceae > Acanthaceae
>Vitaceae (Fig. 12.3). Table 12.1 contains species names, their family, and the part
that remedies bone fractures. Most of the plant arts are used in paste form. Sometimes
combinations of various parts of the same plant as well different plant parts are used
for early recovery. The village vaidyas have provided authenticated information
relating easy fracture-healing methods (Table 12.2).
Conclusion
The current investigation could reveal some natural products as sources of medi-
cines with less side effect on bone fracture. These tribes have well adopted these
practices. However modern medicines are gradually replacing these practices. Both
the Ayurvedic concepts and scientific development of these folk medicines and
practices can establish a novel protocol for natural bone fracture healing.
Further studies should be conducted to find out the population of these plant species
and their conservation strategies. Screening of documented plants would be rather
beneficial for future drug developing programs.
Fig. 12.2 Plant species

used, based on their habitat
Vitaceae
Verbenaceae
Sterculiaceae
Rutaceae
Rubiaceae
Rhamnaceae
Poaceae
Orchidaceae
Malvaceae
Loranthaceae
Lamiaceae
Family
Gentianaceae
Fabaceae
Euphorbiaceae
Dipterocarpaceae
Dioscoreaceae
Cuscutaceae
Combretaceae
Asteraceae
Araceae
Anacardiaceae
Adiantaceae
Acanthaceae
0 1 2 3 4 5 6
Number
Fig. 12.3 Bar chat of the documented dominant family
Table 12.1 Enumerated plants and their traditional usages for bone fracture treatment
Plant details
1. Acacia arabica 21. Hoppea dichotoma (Griseb.) C.B. Clark.
Family: Fabaceae Family: Gentianaceae
Habit: Shrub Habit: Herb
Date of collection: 23.05.2015 Date of collection: 23.05.2015
Locality: Bagalata, Similipal Locality: Motu, Malkangiri
Parts used :Bark Parts used: Plant paste
2. Acanthus ilicifolius L. 22. Luisia trichorhiza (Hook.) Bl.
Family: Acanthaceae Family: Orchidaceae
Habit: Shrub Habit: Herb
Locality: Paparmetla, Malkangiri Locality: Boipariguda, Koraput
Parts used : Root Parts used: Plant paste
3. Adiantum philippense L.
Family: Adiantaceae
Habit: Herb
Date of collection: 21.05.2015
Locality: Kalakada, Similipal 23. Mitracarpus villosus (Sw.) DC.
Parts used: Root Family: Rubiaceae
Habit: Herb
Locality: Narayanpatnam, Koraput
Parts used: Plant paste
(continued)
Table 12.1 (continued)
Plant details
24. Murraya paniculata(L.) Jack

4. Bambusa bambos (L.) Voss Family: Rutaceae
Family: Poaceae Habit: Shrub
Habit: Herb Date of collection: 24.05.2015
Date of collection: 16.05.2015 Locality: Boipariguda, Koraput
Locality: Bisipur, Similipal Parts used: Leaf paste
Parts used: Stem
25. Mussaenda frondosa L.
Family: Rubiaceae
Habit: Herb
Locality: Narayanpatnam, Koraput
5. Belpharis maderaspatensis (L.) B. Heyne ex Parts used : Leaf
Roth.
Family: Acanthaceae
Habit: Herb
Locality: Karanjia, Similipal
Parts used: Leaf paste
6. Butea monosperma (Lam.) Taub. 26. Pelatantheria insectifera (Reichb. f.)
Family: Fabaceae Ridley
Habit: Large tree Family: Orchidaceae
Date of collection: 15.08.2016 Habit: Herb
Locality: Jashipur, Similipal Date of collection: 25.03.2016
Parts used: Root Locality: Podia, Malkangiri
Parts used : Plant paste
7. Butea superba Roxb. exWilld.
Family: Fabaceae
Habit: Climbing Shrub
Locality: Hatibari, Similipal
Parts used: Root
27. Pterocarpus marsupium Roxb.
Family: Fabaceae
Habit: Tree
Locality: Jadamba, Malkangiri
Parts used: Bark
8. Byttneria herbacea Roxb. 28. Pterospermum suberifolium (L.) Willd.
Family: Sterculiaceae Family: Malvaceae
Habit: Prostrate herb Habit: Tree
Locality: Paparmetla, Malkangiri Locality: Jadamba, Malkangiri
Parts used: Whole plant Parts used : Leaf
(continued)
Plant details
29. Pygmaeopremna herbacea (Roxb.)
Moldenke
Habit: Verbenaceae
9. Catunaregam spinosa (Thunb.)Tirven. Locality: Pottangi, Koraput
Family: Rubiaceae Parts used: Root
Habit: Shrub
Locality: Chitrakonda, Malkangiri
Parts used: Stem bark
30. Rhaphidophora glauca (Wall.) Schoot
Family: Araceae
10. Cissus quadrangularis L.
Locality: Narayanpatna, Koraput
Family: Vitaceae
Parts used: Plant paste
collection: 23.05.2016
Parts used: Whole plant
11. Cissus repens Lam. 31. Rubia cordifolia L.
Family: Vitaceae Family: Rubiaceae
Habit: Climbing Shrub Habit: Herb
Locality: Mirgimindi, Similipal Locality: Alamanda, Koraput
Parts used : Root Parts used: Stem and root
32. Semecarpus anacardium L.f.
Family: Anacardiaceae
Habit: Tree
Date of collection:
Locality: Pottangi, Koraput
12. Cuscuta reflexa Roxb.
Parts used : Fruit
Family: Cuscutaceae
Habit: Creeper
Locality: Whole Plant
Parts used : Whole plant
13. Dendrophthoe falcata (L.f.) Etting
Family: Loranthaceae
Habit: Hemiparasitic herb
Locality: Jadamba, Malkangiri 33. Shorea robusta Roth.
Parts used : Plant paste Family: Dipterocarpaceae
Habit: Tree
Locality: Sunabeda, Koraput
Parts used : Bark and leaf
(continued)
Plant details
14. Desmodium oojeinensis (Roxb.) Ohashi
Family: Fabaceae
Habit: Herb
Locality: Kendumundi, Similipal 34. Terminalia arjuna (Roxb.) Wight & Arn.
Parts used : Bark Family: Combretaceae
Habit: Tree
Locality: Nandapur, Koraput
Parts used: Bark
15. Dioscorea pentaphylla L.
Family: Dioscoreaceae
Habit: Shrub
Locality: Baliposi, Similipal
Parts used: Tuber 35. Terminalia elliptica Willd
Family: Combretaceae
Habit: Tree
Locality: Kalamela, Malkangiri
Parts used: Bark
16. Drypetes roxburghii (Wall.) Hurus. 36. Vanda tessellata (Roxb.) Hook.ex G. Don
Family: Euphorbiaceae Family: Orchidaceae
Habit: Tree Habit: Herb
Locality: Kalimela, Malkangiri Locality: Boipariguda, Koraput
Parts used: Stem Parts used : Plant Paste
37. Vanda testacea (Lindl.) Reichb. f.
Family: Orchidaceae
Habit: Herb
17. Euphorbia Hirta L. Locality: Mudulipada, Malkangiri
Family: Euphorbiaceae Parts used : Plant paste
Habit: Herb
Parts used : Whole plant paste
18. Euphorbia rosea Retz.

Family: Euphorbiaceae
Habit: Herb
38. Vitex negundo L.
Family: Lamiaceae
Locality: Mathili, Malkangiri
Habit: Shrub
Parts used: Stem
Locality: Narayanpatna, Koraput
Parts used : Leaf
(continued)
Plant details
19. Euphorbia tirucalli L. 39. Ziziphus rugosa Lam.
Family: Euphorbiaceae Family: Rhamnaceae
Habit: Herb Habit: Shrub
Locality: Potangi, koraput Locality: Lakshmipur, Koraput
Parts used: Plant paste Parts used : Root
20. Grangea maderaspatana (L.) Poir
Family: Asteraceae
Habit: Herb
Locality: Mathili, Malkangiri
Parts used: Leaf
Table 12.2 Details of the medicinal healers investigated through questionnaires

Sl. no. Name of the tribes Age Village
1 Kusha Badanayak 46 Malkarband
2 Rabi Kirshani 51 Thoria
3 Balaram Gutal 48 Karanjaguda
4 Ganga Sisa 52 Golikapadon
5 Alok Khore 55 Sindhiguda
6 Brajabanta Sounta 45 Lakateguda
7 Rabindra Killo 54 Padapali
8 Harihar Madhi 59 Naliguda
9 Narasingha Kalakura 53 Gundriguda
10 Gobra Majhi 44 Kapiguda
11 Minikaka Gadaba 49 Chitapari
12 Laxman Jani 47 Sripeta
13 Babula Nali 39 Panighata
14 Baruna Nayak 36 Bisipur
15 Ramsing Murmu 51 Baghalata
16 Anam charan Naike 44 Mirigimindi
17 Chambru Munda 49 Hatibari
18 Laxmidhar Naike 58 Jashipur
19 Thakura Tudu 43 Udla
20 Basudev Hansdah 49 Khalpada
21 Rabindra Mlurmu 55 Kendumundi
22 Bhagirathi Soren 41 Khadikudar
Acknowledgments The authors express their gratitude to the director of CSIR-IMMT for provid-
ing laboratory facilities to carry out the research work. We are also grateful to the villagers and the
traditional herbal healers for their cooperation in providing baseline data during the field tour.
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hills, a part of Eastern Ghats, Tamil Nadu. International Journal of Current Research, 5(12),
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use of medicinal plants in Bangladesh to treat urinary tract infections and sexually transmitted
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Kumar, S., Jena, P. K., Sabnam, S., Kumari, M., & Tripathy, P. K. (2012). Study of plants used
against the skin diseases with special reference to Cassia fistula L. among the king (Dongaria
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Development and Research, 4(2), 256–264.
Mallik, B. K., Panda, T., & Padhy, R. N. (2012). Traditional herbal practices by the ethnic people
of Kalahandi district of Odisha, India. Asian Pacific Journal of Tropical Biomedicine, 2(2),
S988–S994.
Nagendrappa, P. B., Naik, M. P., & Payyappallimana, U. (2013). Ethnobotanical survey of malaria
prophylactic remedies in Odisha, India. Journal of Ethnopharmacology, 146(3), 768–772.
Pan, S. Y., Zhou, S. F., Gao, S. H., Yu, Z. L., Zhang, S. F., Tang, M. K., Sun, J. N., Ma, D. L.,
Han, Y. F., Fong, W. F., & Ko, K. M. (2013). New perspectives on how to discover drugs from
herbal medicines: CAM’s outstanding contribution to modern therapeutics. Evidence-Based
Complementary and Alternative Medicine, 13, 1–25.
Panda, S. K., Patra, N., Sahoo, G., Bastia, A. K., & Dutta, S. K. (2012). Anti-diarrheal activities
of medicinal plants of Similipal Biosphere Reserve, Odisha, India. International Journal of
Medicinal and Aromatic Plants, 2(1), 123–134.
Ratnam, K. V., & Raju, R. R. (2008). Traditional medicine used by the Adivasis of Eastern Ghats,
Andhra Pradesh-for bone fractures. Ethnonbotanical Leaflets, 12, 19–22.
Rout, S. D., & Panda, S. K. (2010). Ethnomedicinal plant resources of Mayurbhanj district, Orissa.
Rout, S. D. (2004). Medicinal plants of Similipal Biosphere Reserve. Doctoral dissertation, Ph.
D. Thesis. Bhagalpur: TM Bhagalpur University.
Sarvalingam, A., Dhaarani, V., Pavithra, C., Sharmila, S., & Rajendran, A. (2017). Inventory and
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Chapter 13
Screening of Certain Medicinal Plants
of Manipur for Their Antifungal Activity
against Candida Species
Wangkheirakpam Radhapiyari Devi
Introduction
Plant has an antiquity of practice for healthcare from its parts and extracts. In gen-
eral, humans are prone to innumerable skin infections resulting from diverse cli-
matic conditions. These novel exotic plant varieties (Premanathan et al. 2000;
Krishnaraju et al. 2005) are leading to new scenarios projected toward innovative
drugs from the folklore medicine to modern healthcare (McNeil et al. 2001) to a
great extent. Many scholars are gradually shifting their devotion to folklore medi-
cine for developing healthier medications by creating new combinations after dis-
covering the Nobel active compounds found in the plants (Selitrennikoff 2001;
Srinivasan et al. 2001).
The state Manipur is situated in the northeastern corner of India at the Indo-
Myanmar border between the latitudes 23°50′N and 25°41′N and longitudes
93°01′E and 94°74′E. There are 35 different ethnic groups settled here wherein
various humanity and ironic customary awareness on the exercise of therapeutic
floras are being practiced. The key study is to figure out medicinal floras with dura-
ble antimicrobial activity for the progress of novel antimicrobial mediators and/or
consistent phytomedicines. In this broadsheet, we explore the outcomes for antimi-
crobial activity in the Manipuri customary medicine to cure contagious diseases
(Dhanapati 1995, 1998; Sinha 1996; Tombi 2009) and for their genuine documenta-
tion thereof (Hooker 1872–1897; Kanjilal et al. 1934–1940; Balakrishnan and
Sanjappa 1993; Sanjappa and Balakrishnan 1993; Sharma et al. 1993).
W. R. Devi (*)
DM College of Teacher Education, Imphal, Manipur, India

https://doi.org/10.1007/978-981-15-2195-9_13
156 W. R. Devi
Experimental
Plant Specimens
The fresh plant specimens were collected from various places within the state of
Manipur, India, which were scrubbed and then air-dried. For the taxonomy identifi-
cation, the specimens were identified by the Botanical Survey of India, Kolkata,
India, and the voucher specimen numbers were further conserved.
Extraction of Plant Specimens
The shade-dried, crushed specimens were extracted in the Soxhlet apparatus suc-
cessively with six times the amount of plant material (v/w) of each solvent (petro-
leum ether, chloroform and methanol, and water) each time until no more colored
matter was extracted. Each solvent extract was dried at 40 °C under reduced pres-
sure using a rotary evaporator and reserved in firmly lid bottles (Table 13.1).
Candida Strains Used
The crude extracts were experimented against American Type Culture Collection
(ATCC) Candida strains (C. albicans ATCC 90029, C. albicans ATCC 1162,
C. albicans ATCC Y-9-19, C. glabrata ATTC 91030, C. parapsilosis ATCC 20019,
C. krusei ATCC 6258, C. krusei ATCC 71061–1113, and C. kefyr ATCC 1110), sub-
cultured onto Sabouraud Dextrose Agar (Himedia) and created at 35 °C for 24 h.
Table 13.1 Medicinal plants

Voucher Common Part of Yield
Plant species specimens Family name plant Solvent (%)
Xylosma WR234 Flacourtiaceae Nongleisang Leaf Petroleum 1.39
longifolium Clos. ether
Bark Petroleum 0.61
ether
Ageratum WR10 Asteraceae Khongjainapi Whole Petroleum 0.36
houstonianum plant ether
Mill. Chloroform 0.64
Methanol 1.4
Ageratum WR9 Asteraceae Khongjainapi Whole Chloroform 1.88
conyzoides Linn. plant
Vitex trifolia WR231 Verbenaceae Urikshibi Leaf Water 0.1
Linn.
13 Screening of Certain Medicinal Plants of Manipur for Their Antifungal Activity… 157
Antimicrobial Susceptibility Testing
Supporting to the guidelines of the Clinical and Laboratory Standard Institute

(NCCLS 2002), the antimicrobial screenings were executed by agar diffusion and
micro broth dilution techniques.
Agar Diffusion Test
Agar diffusion was analyzed. Each crude extract and oil was readied in 100%
dimethylsulfoxide (DMSO) (Sigma) as stock solution 1 mg/ml and 1 ml/ml. Further,
each solution was diluted at the concentration 500 μg/ml and 250 μg/ml and 500 μl/
ml and 250 μl/ml to put a final volume of 50 μl in the bore wells on the microbial
suspension agar plates. These were cultivated at 35 °C for 24 h. The zone of inhibi-
tion was recited in diameter at which a sharp decline in growth occurred.
Amphotericin-B (Sigma), Fluconazole (Sigma), Itraconazole (Sigma), Voriconazole
(Sigma), and DMSO were used and compared the growth control against tested
Candida strains at a concentration 16 μg/ml to that of the crude extract concentra-
tions. The technique was repeated thrice.
Micro Dilution Test
The micro broth dilution technique was also evaluated wherein different stock solu-
tions (mg/ml and ml/ml in DMSO) of different crude extracts were prepared and
diluted nine times to get the concentration of 1000, 500, 250, 125, 62.5, 31.25,
15.625, 7.8125, and 3.90625 μg/ml or μl/ml, respectively. Thereafter 100 μl of each
diluted extract was dispensed into the bores of flat-bottomed 96-well micro titer
plates (Tarson) and inoculated with the continuous volume (100 μl) of each broth
Candida strain which was incubating at 35 °C for 48 h. Against tested Candida
strains, control growths were compared. The antifungal activities on different con-
centrations of crude extract were designed on Table 13.2. The analysis was
done thrice.
Table 13.2 MIC Conc. of crude extracts (μg/ml or μl/ml) Outcome of MIC

determination on different
Equal or less than 100 Strong
concentration of crude
extracts 100–500 Moderate
500–1000 Weak
Over 1000 Inactive
158 W. R. Devi
Outcome
The outcomes highlighted the antifungal activities using agar diffusion and micro
broth dilution assays of different crude extracts and standard drugs against different
Candida strains as given on Tables 13.3 and 13.4. The peak results were found on
C. kefyr ATCC 1110 and C. krusei ATCC 6258 (zone diameter 28.62 and 27.97 mm)
in leaves petroleum ether extract of X. longifolium at 500 μg and oil extract of V. tri-
folia at 500 μl (Table 13.3). Almost the Candida strains showed no inhibition prop-
erties at higher concentration of the crude extracts.
MIC’s results of the different crude extracts are highlighted on Table 13.4.
Petroleum ether leaves crude extracts of X. longifolium presented strong activity
against the C. kefyr ATCC 1110 with MIC of 62.25 μg/ml. Dark extracts in petro-
leum ether of X. longifolium showed moderate activity against the C. albicans
Table 13.3 Antifungal activities from the crude extracts of traditional medicinal plants (inhibition
zone)
Inhibition zone (mm) of different crude extracts on agar well (50 μl)
Crude extracts Conc. CA1 CA2 CA3 CG CP CKR1 CKR2 CKE
1 500 μg 11.2 – – – – 9.2 – 28.62
250 μg 10.87 – – – – 8.02 – –
2 500 μg – – – – – 13.02 – 20.32
250 μg – – – – – 11.63 – 15.91
3 500 μg – – – – – 8.48 – 15.56
250 μg – – – – – 7.38 – 11.62
4 500 μg – – – – – 7.92 – 9.47
250 μg – – – – – 7.87 – –
5 500 μg – 13.28 15.84 – 11.65 12.96 11.42 20.36
250 μg – – – – – 7.22 – 10.24
6 500 μg – – – – – 10.33 – 24.53
250 μg – – – – – 7.33 – 10.16
7 500 μl – 11.67 – – 11.57 27.97 12.17 23.88
250 μl – – – – 11 13.72 – 20.4
Fl 16 μg – – 20.92 – 27.16 11.35 20.2 –
Itr 16 μg – – 23.7 – 26.1 10.33 12.92 –
Vo 16 μg – – 22.36 – 20.52 8.11 20.56 21.34
Am 16 μg 26.35 22.15 25.18 29.15 23.15 27.93 19.19 22.6
DMSO 50 μl – – – – – – – –
CA1 C. albicans ATCC 90029; CA2 C. albicans ATCC 1162; CA3 C. albicans ATCC Y-9-19; CG
C. glabrata ATTC 91030; CP C. parapsilosis ATCC 20019; CKR1 C. krusei ATCC 6258; CKR2
C. krusei ATCC 71061–1113; CKE C. kefyr ATCC 1110
1 X. longifolium (leaf) from petroleum ether; 2 X. longifolium (bark) from petroleum ether; 3 A.
houstonianum (whole plant) from petroleum ether; 4 A. houstonianum (whole plant) from chloro-
form; 5 A. houstonianum (whole plant) from methanol; 6 A. conyzoides (whole plant) from chlo-
roform; 7 V. trifolia (leaf) its essential oil
Fl fluconazole; It Itraconazole; Vo Voriconazole; Am amphotericin-B
Table 13.4 Antifungal activity from the crude extracts of traditional medicinal plants (MIC)
MIC (μg/ml) or (μl/ml)
Crude extracts CA1 CA2 CA3 CG CP CKR1 CKR2 CKE
1 125 μg 125 μg 250 μg 500 μg 500 μg 250 μg 250 μg 62.25 μg
2 250 μg 250 μg 250 μg 500 μg 500 μg 125 μg 250 μg 125 μg
5 125 μg 250 μg 250 μg 250 μg 250 μg 62.25 μg 250 μg 125 μg
7 125 μl 62.25 μl 250 μl 250 μl 62.25 μl 15.625 μl 125 μl 62.25 μl
Fl 0.25 μg 0.125 μg 16 μg 16 μg 2 μg 32 μg >32 μg 0.5 μg
It 0.06 μg <0.06 μg 0.25 μg >32 μg <0.06 μg 0.5 μg 0.06 μg <0.06 μg
Vo 0.06 μg <0.06 μg 0.25 μg 0.25 μg <0.06 μg 1 μg 0.25 μg 0.06 μg
Am 0.25 μg <0.06 μg 0.25 μg 0.125 μg 0.25 μg <0.06 μg 0.25 μg 0.06 μg
ATCC 90029, C. albicans ATCC 1162, C. albicans ATCC Y-9-19, C. krusei ATCC
6258, C. krusei ATCC 71061–1113, and C. kefyr ATCC 1110 with MICs of 250,
250, 250, 125, 250, and 125 μg/ml, respectively. The extraction of whole plant parts
for A. houstonianum in petroleum ether resulted moderate activity against the
C. albicans ATCC 90029, C. albicans ATCC 1162, C. albicans ATCC Y-9-19,
C. glabrata ATTC 91030, C. krusei ATCC 6258, C. krusei ATCC 71061–1113, and
C. kefyr ATCC 1110 with MICs of 125, 250, 125, 250, 125, 250, and 125 μg/ml,
respectively. A. houstonianum (whole plant) from chloroform extract revealed mod-
erate activity against the C. albicans ATCC 1162, C. albicans ATCC Y-9-19, C. gla-
brata ATTC 91030, C. krusei ATCC 6258, C. krusei ATCC 71061–1113, and
C. kefyr ATCC 1110 with MICs of 250, 250, 250, 125, 250, and 250 μg/ml, respec-
tively. Methanol A. houstonianum (whole plant) extract opened a strong activity
against the C. krusei ATCC 6258 with MIC of 62.25 μg/ml. A. conyzoides (whole
plant) from chloroform showed moderate activity against the C. albicans ATCC
90029, C. albicans ATCC 1162, C. albicans ATCC Y-9-19, C. glabrata ATTC
91030, C. parapsilosis ATCC 20019, C. krusei ATCC 6258, C. krusei ATCC
71061–1113, and C. kefyr ATCC 1110 with MICs of 125, 250, 250, 250, 250, 125,
250, and 125 μg/ml, respectively. Essential oil of V. trifolia offered strong activity
against the C. albicans ATCC 1162, C. parapsilosis ATCC 20019, C. krusei ATCC
6258, and C. kefyr ATCC 1110 with MICs of 62.25, 62.25, 15.625, and 62.25 μl/ml,
respectively. Among the extracts, V. trifolia oil exposed the greatest antifungal activ-
ity (MIC = 15.625 μl/ml) which was compared with the standard antifungals –
Fluconazole, Itraconazole, Voriconazole, and Amphotericin-B at 16 μg/ml (Ahmad
et al. 2000; Rani and Murty 2006).
Screenings of antimicrobial activities from the plant extracts lead to a great
potential for discovering of new antimicrobial drugs (Alim et al. 2009).
160 W. R. Devi
Conclusion
Results suggest a legitimately noble relationship between traditional practice and

the in vitro antifungal activity. From the documents and experiments thus studied, it
has been found that a huge potential can be explored by way of ethnobotanical
investigations which in turn could be developed into bioactive compounds. Further,
bio-guided fractionation will be conducted for V. trifolia oil to identify the active
components to explore as antifungal agents and provide preliminary technical proof
for the traditional medicinal use. Innovation of phytoactive compounds will bring
forth further studies for beneficial and trade application.
Acknowledgments The author gratefully wishes to appreciate and acknowledge the remarkable
financial support of the Dept. of Biotechnology, Govt. of India which enabled to carry out the study
smoothly and successfully.
References
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ergistic activity of five traditionally used Indian medicinal plants. Journal of Medicinal and
Aromatic Plant Sciences, 22, 173–176.
Alim, A., Goze, I., Goze, H. M., & Tepe, B. (2009). In vitro antimicrobial and antiviral activities
of the essential oil and various extracts of Salvia cedronella Boiss. Journal of Medicinal Plants
Research, 3(5), 413–419.
Balakrishnan, N. P., & Sanjappa, M. (1993). Flora of India, Botanical Survey of India, Calcutta
(Vol. II). New Delhi: Deep Printers.
Dhanapati, D. L. (1995). Folklore on the use of Indigenous Plants & Animals in Manipur (Vol. I).
Imphal: Rajen Printers.
Dhanapati, D. L. (1998). Folklore on the use of Indigenous Plants & Animals in Manipur (Vol. II).
Imphal: Jagadanbee Printers.
Hooker, J. D. (1872–1897). The Flora of British India (Vol. 1–7).
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(2005). Assessment of bioactivity of Indian medicinal plants using Brine shrimp (Artemia
salina) lethality assay. International Journal of Applied Science and Engineering, 2, 125–134.
McNeil, M. M., Nash, S. L., Hajjeh, R. A., Phelan, M. A., Com, L. A., Plikaytis, B. D., & Warnock,
D. W. (2001). Trends in mortamycotic diseases in United States, 1980–1997. Clinical Infectious
Diseases, 33, 641–647.
National Committee for Clinical Laboratory Standards. (2002). Reference method for broth dilu-
tion antifungal susceptibility testing of yeasts: Approved standard, 2nd ed., M-27-A2, National
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N. (2000). A survey of some Indian medicinal plants for anti-human immunodeficiency virus
(HIV) activity. Indian Journal of Medical Research, 112, 73–77.
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Chapter 14
Phytochemicals and Pharmaceutical:
Overview
Jayakumari
Introduction
The Greek word “phyto” is the origin for the development of the scientific term
“phytochemical.” Phytochemicals are compounds that are produced by plants due to
metabolism. These are chemicals of host (plant) and exhibit the vital role in the
growth and development of plant and protect the plant against pathogens, predators,
and environmental factors. Since the phytochemicals are produced by secondary
metabolism and are not required by plants as their basic need, they are called sec-
ondary metabolites. Phytochemicals basically possess health benefits, but they are
not explored much scientifically; hence they are called research compounds. The
person who is involved in the discovery and development of these compounds are
known as phytochemists, who are involved in the research of the chemistry of these
plant constituents. The phytochemical research consists of isolation of the com-
pound from plant origin determination of its structure by spectral methods and eval-
uation of its biological property both through in vivo and in vitro studies. Based on
the complex structure of phytochemicals, they are broadly classified into steroids,
alkaloids, and polyphenolic compounds which include tannins, flavonoid, isoflavo-
noid, and coumarin. Some phytochemicals also possess nutritional value and energy
value, so they are known as nutraceuticals (Perez-Vizcaino and Fraga 2018). If they
produce toxic or undue side effects, they are grouped as poisonous compounds or
phytotoxins. Salicin was the first phytochemical isolated from the bark of the white
willow tree and was used as anti-inflammatory and analgesic property. Because of
its wide use and more demand, it was later synthesized in more quantity by chemi-
cal method. Till today the phytochemical salicin is used as a prodrug for the devel-
opment of many conventional NSAIDs. Now they are easily available as a very
common over-the-counter drug [OTC]. Even today, well-known anticancer
Jayakumari (*)
Department of Pharmacognosy, School of Pharmaceutical Sciences, Vels Institute of Science,
Technology and Advanced Studies [VISTAS], Chennai, Tamil Nadu, India

https://doi.org/10.1007/978-981-15-2195-9_14
164 Jayakumari
p hytochemical paclitaxel is obtained from English yew tree. In earlier days, the
various parts of this tree (leaves and berries) were found to be poisonous for animals
and infants who ate the fruits. Later the biological active anticancer phytochemical
paclitaxel was isolated from the same tree in 1971 and subsequently became an
important outstanding phytochemical for treatment of all types of cancers. The
widespread knowledge and increasing use of phytochemicals and their role in mod-
ern medicine and herbal medicine were estimated about 45–50% per annum.
Developed countries are now focusing in finding phytochemicals of the plant mate-
rials used as drug therapy in life-threatening diseases. The main purpose of phyto-
chemical is used to promote efficacy, to minimize toxicity, and to target disease at
molecular level. Plants are considered as biochemical lab as it can synthesize many
types of compounds as defense to fight against external factors. These phytochemi-
cals can remain quite beneficial when they are taken out of their plant system. It
means that these are potential goldmines of phytocompounds that could be used in
pharmaceuticals as herbal drugs. Phytochemicals are naturally occurring.
Nonnutritive compounds present in plants show biological significance and are used
as pharmaceuticals (Sravani et al. 2019). They play a vital role in biological system
for the treatment and to overcome the metabolic disorders at any time of improper
physiological function (Gupta and Kesarwani 2013). They are used either alone or
in combination. So the present chapter focuses to review the role of phytochemicals
in various fields in pharmaceutical industry as phyto-pharmaceuticals.
Role of Phytochemicals as Therapeuticals
Plant-based phytochemicals are broadly responsible for the protective health bene-
fits. Some are used as bioenhancers to improve the efficacy of active ingredients
(Randhava et al. 2011). Phytochemicals are naturally occurring plant constituents,
responsible for color and odor of the flower, fruits, leaves, roots, and stem of the
same. They also have defensive role for the plant. Since they have a physiological
role in biological systems, they are often called bioactive compounds (Taliraju et al.
2018). These may be bioactive compounds, phytonutrients, or natural coloring
agents. There are about 10,000 different types of phytochemicals; many are yet to
be identified.
Types of Phytochemicals
• Phytoestrogens
• Phytophenols
• Phytosteroids
• Bioflavonoids
• Terpenoids
• Isothiocyanate
• Carotenoids
14 Phytochemicals and Pharmaceutical: Overview 165
Phytoestrogens
Phytoestrogens are naturally occurring polyphenolic phytocompounds, produced

by Leguminosae plants such as beans, nuts, soybeans, and legumes by flax, sesame
seeds, and other plants which have estrogenic properties. Phytoestrogens are not
considered as essential nutrients. These heterogeneous types of photochemicals can
be grouped into four subdivisions based on their chemical structures:
1. Isoflavones
2. Lignans
3. Coumestans
4. Lignans
Most of the phytoestrogens have biological properties due to their free radical
scavenging properties, that is, their ability to scavenge the free radicals generated by
chelating complex with metal ion (Adlercreutz 2003). Most of the South Asian pop-
ulation consumes herbs rich in phytoestrogens and has reduced risk of breast cancer,
osteoporosis, and cardiovascular diseases (Adlercreutz 2002). Phytoestrogens are
found in about more than 300 different plant species and are consumed by humans
because of their potent health benefits (lowered neuropausal symptoms, lowered
risk of osteoporosis, and breast cancer).
Phytophenols
Phytophenols are the naturally occurring aromatic polyhydroxyl compounds

(Ramos-Hryb et al. 2018) mostly found in plants. They are synthesized as second-
ary metabolic product to protect the plants. In plants, they act as defensive sub-
stances against any environmental stress. They include well-known substances as
coumarins, lignans, flavonoids, tannins, etc. Polyphenolic compounds of plants may
act as antioxidants to scavenge the free radicals, and other oxidative species, prod-
uct due to pathogens, infection, environmental stress, and any deficiency of nutrients
Phenolic phytochemicals are more diverse in nature. Some are soluble in polar sol-
vents and are miscible with water, and others are insoluble polymers. The potential
therapeutic application of phytophenols are free radicals scavenging effect, bactericidal
effect, prevention of cancer, immunomodulating property, cardio tonic, and prevention
of skin damage due to ultraviolet (UV) rays (Lescano et al. 2019; Caban et al. 2019).
Phytosteroids
Phytosteroids are naturally occurring plants steroids that have biological properties
chemically. They are made up of cyclopentano per hydro phenanthrene ring arranged
in a specific molecular configuration. In plants, they are biosynthesized in an ace-
166 Jayakumari
tate–mevalonate pathway. Squalene is the important precursor of its production. In

plants, steroids occur either in free from in the form of steroidal glycoside alkaloids,
steroidal hormones, and sclerosis. Out of these, plant sterols are found more abun-
dantly present. Betasitosteroids is one of most important plant steroids of natural
origin. Phytosteroids have many therapeutic potentials used in pharmaceutical as
chemotherapy for cancer as immune-modifying agent, liver tonic, bactericidal, reg-
ulator of plant growth hormone, in worm infestation, cardiac disease, and cytotoxic-
ity, and also they may have antioxidant properties, which are needed for support of
each membrane, and it is also essential for the maintenance of healthy cholesterol.
Bioflavonoids
Bioflavonoids are naturally occurring yellow colored pigments, well distributed in

all parts of the plant. Chemically they are heterocylic pyran ring structures. The
attractive color of the fruits and brilliant shades of flower of plants are due to the
presence these polyphenolic compounds. Most of the bioflavonoids exhibit fluores-
cence when they are excited by UV rays. Nearly about 6000 different types of fla-
vonoids are found. Most of the bioflavonoids are recently used as bioenhancer to
enhance the absorption of active ingredients which facilitate the absorption of drug
(Bakoyiannis et al. 2019). FLAVO- means yellow. These are yellow-colored pig-
ments which occur in almost all parts of the plant.
Flavonoids include flavones, flavonols, flavones, isoflavones, flavan-3-ols, and
anthocyanidins (Table 14.1). Flavonoids possess many biological activities includ-
ing bactericidal and bacteriostatic, toxic against virus, free radical scavenger,
reduces the mutagenic activity of the cell and reduces the activity of several enzymes
(Perez-Vizcaino and Fraga 2018).
Terpenoids
The term “terpene” represents hydrocarbons (C5H8)n, while terpenoids are hydro-
carbons as well as their oxygenated derivatives (Ludwiczuk et al. 2017). Terpenes
and terpenoids are constituents of volatile oils, for example, eucalyptus oil (cine-
Table 14.1 List of S.no. Phytochemical Therapeutic uses

bioflavonoids and their
1. Carotene Free radical protection
therapeutic uses
2. Curcumin Block carcinogens and DNA damage
3. Quercetin Antioxidant
4. Isoflavonoid Increases blood dilation
5. Saponin Interfere with cell replication (cancer
cell)s
ole), peppermint oil (menthol), clove oil (eugenol), etc. Flavors and aromas of most
of the aromatic plants are due to the wide variety of terpenoids.
Terpenoids are grouped based on the carbon numbers and isoprene units present
(Ludwiczuk et al. 2017).
Monoterpenes: Contains 10-C atoms or 2 isoprene residues
Sesquiterpenes: Contains 15-C atoms or 3 isoprene residues
Diterpenes: Contains 20-C atoms or 4 isoprene residues
Triterpenes: Contains 30-C atoms or 6 isoprene residues
Tetraterpenes: Contains 40-C atoms or 8 isoprene residues
Polyterpenes: Contains of isoprene residues more than 40
Beyond their cancer-preventive effects, terpenoids also have analgesic, anti-
inflammatory, antibacterial, and antifungal properties (Gutierrez-del-Rio and
Fernandez 2018).
Isothiocyanate These phytochemicals are largely found in cruciferous plants. It
suppresses the proliferation of tumor cell. Plant foods are 64 times richer in antioxi-
dants than animal foods. Many evidences and research data showed that the food
containing rich amount of flavonoids can reduce the mortality of rat and attack of
myocardial infarction by 25%. Most of the phytochemicals (especially polyphe-
nols) protect us from cancer and heart diseases by inhibiting cell proliferation and
angiogenesis (Eggersdorfer and Wyss 2018), thereby regulating and increasing
blood flow production.
Carotenoids These are widespread in plants. There are about 600 naturally occur-
ring pigments produced in lower plant organisms such as algae and bacteria.
Carotenoids are mostly colored yellow, orange, and red as such seen in most of the
fruits and vegetables. The most common carotenoids are carotene, beta-carotene,
lutein, and lycopene which are widely used. Chlorophyll is a naturally occurring
green-colored plant pigment. It has antioxidant, anti-inflammatory, and wound-
healing properties (Huang et al. 2017).
Role of Phytochemicals as Nutraceuticals
The term “nutraceutical” refers to the combination of words nutrition and pharma-
ceuticals derived from the natural source, used for health benefits (Lealra 2003).
Most of the phytochemicals (derived from plants) have nutraceutical importance
consisting of bioactive constituents that promote health. These nutraceuticals are
available in food industry as dietary supplement, dietary fibers or beverages, health
drinks, herbal drinks, and so on. Nutraceuticals also have physiochemical benefits
against chronic disease. Nutraceuticals are nutritive plant chemicals that have either
cognitive or disease preventive properties. Nutraceuticals also known as dietary
phytochemicals may promote immunity and prepare the body cells to fight against
chronic diseases such as cancer, psoriasis, and neurodegenerative diseases (Jhanwar
168 Jayakumari
Table 14.2 Classification of nutraceuticals

S.no. Type of nutraceutical Examples
1. Inorganic mineral supplement (a) Calcium
(b) Potassium
(c) Zinc
2. Antioxidants (a) Vitamin C (ascorbic acid)
(b) Vitamin E (tocopherol)
(c) Fatty acid (omega 3-fatty acids)
(d) Polyphenols
(e) Carotenoids
3. Proteins/amino acids Glucasane
4. Dietary fibers Soluble fibers: gums, mucilages, pectin
Insoluble fiber: cellulose, hemicelluloses, and
lignin
and Gupta 2014). Majority of food used in day-to-day life such as cereals, fruits,
vegetables, green beans, and whole grains containing phytochemicals with nutritive
value and used as nutraceuticals. Nutraceuticals are bioactive constituents that pro-
mote health. They may be isolated as phytocompounds, dietary supplements, and
herbal products. Plants contain macronutrients such as fats, proteins, carbohydrates,
and vitamins and micronutrients such as minerals and essential elements which are
used as nutraceuticals (Table 14.2). In addition to that plants are also found to con-
tain many nonnutritive chemicals called phytochemicals (Telessy 2019). They may
play a vital role in pharmaceutical industry in various categories, so this chapter will
provide a brief outline of the role of phytochemicals as bioactive agents, bioenhanc-
ers, and nutraceuticals and pharmaceutical aids (Table 14.3).
Role of Phytochemicals as Bioenhancers
Bioenhancers are chemical compounds which improve or promote or increase the

bioavailability of the drug (Sharma et al. 2017). They do not exhibit synergistic
effect with the drug. Most of the phytochemicals are used as natural bioenhancer by
means of permeation enhancement of transdermally administered drug or to improve
the bioavailability of orally administered drug (Patil and Saraoge 2014) (Table 14.4).
Why Phytochemicals Are Used as Natural Bioenhancers
(Theide et al. 2018; Tatiraju et al. 2013)

• They should be nontoxic.
• They should be safe to use.
• They should be easily available from natural resource.
Table 14.3 List of phytochemicals and their roles as nutraceuticals

S.no. Phytochemicals Sources Nature of nutraceuticals
1. Ascorbic acid Citrus fruits, green pepper Boosts immunity for healthy
(vitamin C) (capsicum), broccoli and sweet gums.
potato, tomatoes Healthy skin and prevent
wrinkling, essential for the body
to make collagen.
Helps our body to absorb
calcium
2. Vitamin D Egg, cheese, fish, mushroom, Phosphorous reduces the cancer
(calciferol) salmon risk.
Support healthy bone.
Reduce the risk of rheumatoid
arthritis
3. Vitamin A Pumpkin, papaya, carrot, mango, Good for vision, maintenance of
apricot, broccoli, green leaf, immunity, antioxidant.
vegetables, dairy products, and Reduces hair loss
fish
4. Α-tocopherol Almond, green olives, avocado, Maintenance of healthy skin.
(vitamin E) cold-pressed plant oils, apricot Complete health
Antiaging, antioxidant
5. Omega-3 fatty Fish oil (cold fish) Regulate cell activity and CVS
acid function
6. Bioflavonoids Fruits, vegetables, green leaves, For utilization of vitamin, lower
cereals the chronic state of cancer, CVS
diseases.
Immune stimulator
7. Amino acid All protein-containing foods Maintenance of the muscle,
(meat, fish, chicken, nuts, and tissue repair, immunity, control
soya) on all body process (Girija 2018)
8. Glucasane Shellfish, crab, corn, fungal food Used as geriatrics.
Improve joint health, improves
digestion as supplement for
osteoarthritis
9. Lycopene Red tomatoes, watermelons, Antioxidant carotenoid.
strawberries, papaya Preventing cancer.
Health to the heart (Moosavi
et al. 2018)
10. Isoflavonoids Isoflavones, soybean, sprouts, Anticancer, CVS
peas
11. Carotenoids Fruits, carrots, tomatoes Natural antioxidant
• They should be non-compatible.

• They should be pharmacologically inert.
• They should be non-irritable.
• They should be nonallergic.
• They should be effective at low concentrations in a combination (Chavhan et al.
2018).
• They should be easy to formulate.
170 Jayakumari
Table 14.4 List of phytochemicals as bioenhancer

Phytochemicals Role as biochemicals
Curcumin Used as permeation enhancer to improve topical bioavailability of drug by
reducing skin barrier resistance. It modifies the physiological activity of GIT,
so better absorption of drugs (Jantarat et al. 2018)
Piperine It was the first phytochemical discovered in the world in the year 1979 as
bioavailability enhancer (Basu et al. 2013)
Piperine enhances antidiabetic drug to lower the blood glucose level
It enhances the immune boosting effect of anticancer drug (GINSENOSIDE)
by orally
It increases the bioavailability of rifampin to 60% in the treatment of TB in
humans and also reduces the dose not only by bioavailability but also by the
increase in absorption of the active site
Quercetin It is a flavonoid
It works by inhibiting CYP3A4 level so used as bioenhancer to anticancer and
antihypertensive drug to increase the bioavailability 1.25–2.25-fold (Randhava
et al. 2011)
Rutin It is loaded as bioenhancer in the development. Novel nano-formulation to
improve nano-drug delivery system with bioactive agent to improve
bioavailability of active drug
Gallic acid Suppressors of CYP-450 enzyme sits 180 enzyme dose 10 mg/kg significantly.
It increases the bioavailability of rosuvastatin (RSV) by 53.3% and nifedipine
(Wacher and Benetz 2001)
Ellagic acid It enhances the bioavailability and activity of poly (E-caprolactone) as
biodegradable polymeric nanoparticle
Ferulic acid It enhances the bioavailability of rice bran enzymatic extract (RBEE) and its
biological potential a nutraceutical ingredients
Allicin It increases and enhances the antifungal activity of fungicides for pathogenic
fungi. It enhances amphotericin B induced vacuole membrane. It enhances the
fungicidal activity of amphotericin B
Naringin This is a flavonoid glycoside. It makes the drugs, tamoxifen, more bioavailable
Glycyrrhizin Its combination with taxol is used against breast cancer. Cell growth inhibition
of taxol was reported to be more with glycyrrhizin
Lysergol It enhances the transport of broad-spectrum antibiotics across the intestinal gut
and cell membrane
Sinomenine It inhibits P-Gp efflux pump and other efflux pumps
Genistein It is phytoestrogen; isoflavone inhibits P-Gp, BCRP, and MRP-22. It increases
the bioenhancer potential of paclitaxel by more than 50%
Capsaicin It enhances the bioavailability of theophylline in a biological system and
regulates the GIT function to facilitate better absorption
Curcumin Curcumin suppresses drug-metabolizing enzyme (CYP 3A4) in the hepatic
system and facilitates the changes in the drug transporter
Ammaniol It increases the glucose uptake and shows the potent antihyperglycemic
activity
Niaziridin It enhances the bioavailability niazirin of common antibiotics rifampicin,
tetracycline, and ampicillin
Niazirin It is bioactive nitrite glycoside that has potent bioenchancer property by drug
and nutrients
Role of Phytochemicals in Cosmetics
Cosmetics are the chemical substance used to improve the elegance of the body.
They enhance the appearance of the face and texture of the body. Most of the cos-
metics are topically applied formulations. Chemically cosmetics are made up of
plant-derived chemicals – phytochemicals. Phytochemicals such as tannins and
vitamins play a vital role in the herbal dye and herbal hair oils. In the same way,
saponins are used in herbal shampoo. Flavonoids and coumarins are much used in
the herbal facial preparations [lipsticks, skin creams, and facial creams] (Table 14.5).
So the phytochemicals are more used in pharma industry in the form of herbal cos-
metics as aromatherapy and naturopathy.
Table 14.5 List of phytochemicals as cosmetics

Name of the Common
S.no. plant name Parts used Phytochemical Uses
1. Acacia Shikakai Pods Saponin, sugars Shampoo
concinna
2. Acorus Sweet flag Rhizome Beta-asarone Skin lotion
calamus
3. Lawsonia alba Henna Leaves Hanno-tannic acid, Shampoo
Lam. glucoside
4. Azadirachta Neem Leaves Limonoids Toothpaste,
indica soap, shampoo
5. Citrus medica Lemon Fruit Flavonoids Whitening
Linn.
6. Santalum Sandal Bark, wood Sesquiterpenoids Skin lotions
album Linn. wood
7. Malus pumila Apple Fruit Flavonoids, Antiaging,
Miller. saccharides moisturizing
8. Mentha Mint Whole plant Triterpenoids Antiperspirant
arvensis Linn.
9. Rheum Rhubard Root Flavonoids, Astringent
coreanum Saccharides lotions
Nakai.
10. Pterocarpus Red sandal Bark Santalins Skin creams
santalinus wood
Linn.
11. Foeniculum Fennel Fruit Saponins, flavonoids Deodorant
vulgare Mill.
12. Cucumis Cucumber Fruit Cucurbitacins Moisturizing
sativus Linn.
13. Cucumis Saffron Stigma Safranal Fairness creams
sativus Linn.
14. Olea europaea Olive Seeds Triolein Shampoo,
lotions
(continued)
172 Jayakumari
Name of the Common
S.no. plant name Parts used Phytochemical Uses
15. Aloe Aloe vera Inner part of the Amino acids, Moisturizing
barbadensis aloe plant leaf Saponin glycosides
Mill. (gel)
16. Curcuma longa Turmeric Underground Curcumol, Analgesic
stem Curcumenol
17. Emblica Amla Fruit Emblicanin A&B, Shampoo
officinalis L-ascorbic acid
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Chapter 15
Millettia Pachycarpa Benth: A Herbal
Medicinal Plant of Southeast Asia
Bishnupada Roy and Ravi Rao Bharti
Introduction
More than 250 species of Millettia comprising climbers, shrubs, and trees are visi-
ble throughout the tropics. Millettia pachycarpa Benth (Fabaceae) (Fig. 15.1) is a
widely used ethnomedicinal plant in different parts of India, China, Bangladesh,
Bhutan, and Myanmar. In India, the plant is found in West Bengal, Sikkim, Assam,
Arunachal Pradesh, Manipur, Tripura, Mizoram, Meghalaya, and Nagaland (Jainul
et al. 2013; Dominic and Ramanujam 2012).
In northeast India, the aqueous extract of the root peel, whole root, bark, leaf, and
seed of M. pachycarpa are used against antifertility, anthelmintic, and fish poison
(Roy et al. 2008; Lalthanzara and Lalthanpuii 2010; Shrivastava 2010; Rai and
Lalramnghinglova 2010; Yumnamcha et al. 2015). In different parts of China, peo-
ple use different parts of Millettia sp. to poison fish, cure tumor and antifertility, and
make strong ropes (Perry and Metzger 1980; Duke and Ayensu 1985; Dorsher and
Peng 2010). The whole plant is also used in China to induce the growth of RBC. In
Bangladesh, people use the plant as anti-inflammatory, blood tonic, and anti-cancer
and as cure to infertility (Chowdhury et al. 2013; Jainul et al. 2013). Bhutanese
people consume aqueous juice of the seed of M. pachycarpa against abdominal
disorder and tumor and also to cure kidney problem (Wangchuk et al. 2017). Nearly
40 phytochemicals were isolated and identified from the plant, of which promising
biological activities of many have already been established (Ito et al. 2006; Okamoto
et al. 2006; Ye et al. 2008, 2010; Xue-Hui et al. 2012; Wu et al. 2013).
Insecticidal property of the plant has been established by several scholars where
seed extract was recorded as contact poison against several agricultural insects
(Chiu et al. 1942; Gong et al. 2015). Seed extract at a concentration of 100 μg/ml of
water was spread for 7 days which showed reduction of cabbage aphid to the extent
B. Roy (*) · R. R. Bharti

Parasitology & Toxicology Laboratory, Department of Zoology, North-Eastern Hill
University, Shillong, Meghalaya, India

https://doi.org/10.1007/978-981-15-2195-9_15
176 B. Roy and R. R. Bharti
Fig. 15.1 Whole plant of

Millettia pachycarpa Scientific classification
Benth
Kingdom : Plantae
Order : Fabales
Family : Fabaceae
Genus : Millettia
Species : pachycarpa
of 92.2% (Lin et al. 2017). On the basis of antifeedant properties of leaf extract of
M. pachycarpa against Spodoptera litura (caterpillar), Ningombam et al. (2017)
proposed that the leaf can be used for controlling the caterpillar through organic
farming.
To establish the anthelmintic property of the plant, if any, as claimed by the
Lushai tribe of northeast India, different scientists observed that the alcoholic
extract of M. pachycarpa causes dose-dependent motility and mortality of the hel-
minth parasites (Lalchhandama et al. 2008; Roy et al. 2008). Giri et al. (2013)
observed the reduced viability of the helminth parasite to the extent of 89.33% when
treated in vitro with alcoholic root peel extract of M. pachycarpa. The author’s
recorded reduced viability and ultimately paralysis of M. pachycarpa-treated hel-
minths are caused through programmed cell death as confirmed by observing bro-
ken mitochondrial membrane, condensed nucleus, and fragmented DNA.
It is also recorded that the phytoproducts altered activities of several tegumental
enzymes like acid phosphatase (AcPase), alkaline phosphatase (AlkPase), level of
trace elements, and normal structure of syncytial tegument and cell organelles like
nucleus, nucleolus, nuclear membrane, mitochondria, etc. (Roy et al. 2008;
Lalchhandama et al. 2008).
Studies on cytotoxicity of M. pachycarpa leaves carried out on Brine shrimp
larvae revealed that LC50 value of the plant is 1.74 μg (Jainul et al. 2013). Aqueous
root extract of the plant causes developmental toxicity such as spinal curvature,
swim bladder deflation, and pericardial oedema in the embryos of zebra fish, where
LC50 was recorded to be 4.276 μg/ml of water. Apoptotic pathway was found to be
responsible for developmental defect in the experimental animal (Yumnamcha
et al. 2015).
Studies on acute toxicity of root tuber of the plant carried out by Bharti and Roy
(2017) showed that LD50 of the plant is 7800 mg/kg body weight of albino mice. At
LD50, the damage of the liver and kidney along with blood indices and reduced
weight of vital organs was recorded. It should be noted here that according to the
Organization for Economic Co-operation and Development, LD50 value above
5000 mg/kg body weight is not to be considered as toxic. Experiment on subacute
toxicity of the plant carried out by the authors revealed that the mice exposed to
200 mg, 500 mg, and 1000 mg root tubers/kg body weight for 28 days cause exten-
sive histological changes in the kidney, liver, intestine, and testes in a dose-dependent
15 Millettia Pachycarpa Benth: A Herbal Medicinal Plant of Southeast Asia 177
manner (Figs. 15.2 and 15.3). Stereoscan observations on surface features of the
liver, kidney, intestine, and testis showed deformed endothelial lining, and fenestrae
in the liver, fine surface of the kidney, microvilli of the intestine, and topography of
the testis in the mice exposed to highest concentration of alcoholic extract of the
plant were very clear (Fig. 15.4). Ultrastructural studies also showed concentration-
dependent damage of the nucleolus, nucleus, nuclear membrane, and mitochondria
in the hepatocytes and ruptured mitochondria, vacuolated proximal and distal con-
voluted tube in the kidney (Fig. 15.5). The extract-treated mice also showed
concentration-dependent disrupted microvilli in the intestine, condensed nucleus,
ruptured nuclear membrane, and vacuolated mitochondria in the intestinal and epi-
thelial cells and also in the testicular cells (Fig. 15.5).
Pharmacological properties of some major active compounds (Fig. 15.6) of
M. pachycarpa are as follows:
Millepachine (C22H22O4)
Millepachine is a polyphenolic flavonoid belonging to the group chalcone. The ben-

zopyran structure of the phytochemical having 2, 2–dimethyl benzopyran motif
made it an efficient penetrator into the cell through plasma membrane (Nicolaou
et al. 2000). Wu et al. (2013) showed anti-proliferative activity of millepachine
against different human carcinoma cells without affecting normal cells. The chemi-
cal arrests cell cycle (G2/M arrest) through inhibition of cyclin-dependent kinase1
Fig. 15.2 Photomicrographs of histological sections of control (a, e) and 200 mg (b, f), 500 mg
(c, g), and 1000 mg (d, h) methanolic extract of M. pachycarpa/kg body weight exposed albino
mice. (a) Section of liver showing normal central vein (CV), sinusoids (S), and portal vein (PV).
(b–d) Showing dose-dependent abnormality and damage of CV, S, and PV. (e) Section of kidney
showing normal Bowman’s capsule (BC), glomerulus (G), and compact arrangement of the cyto-
plasm. (f–h) Showing dose-dependent damage and alteration of BC, G, and nature of the cyto-
plasm. All scale bars = 200 μm
Fig. 15.3 Photomicrographs of histological sections of control (a, e) and 200 mg (b, f), 500 mg
(c, g), and 1000 mg (d, h) methanolic extract of M. pachycarpa/kg body weight exposed albino
mice. (a) Intestine showing normal intestinal gland (IG), muscularis mucosa (MM), and lamina
propria (LP). (b–d) Intestine showing dose-dependent damage of IG, MM, and LP. (e) Section of
testis showing normal spermatozoa (SZ), spermatocytes (ST), and basement membrane (BM). (f–
h) Testis showing dose-dependent damage of SZ, ST, and BM. All scale bars = 200 μm
activity. Millepachine also causes apoptotic cell death in hepatocarcinoma cell lines,
namely, HepG2 and SK-HEP-1 through ROS-mitochondria mediated pathway.
Wang et al. (2012, 2013) synthesized different derivatives of millepachine and
showed that the derivative compound (E)-8-(3-(4-(diemethylamino) phenyl)
acryloyl)-2,2-dimethyl-2H-chromen-5-yl propionate, with a propionyloxy group at
the fourth position of the left phenyl ring, can inhibit proliferation of HepG2 cancer
cell lines with IC50 value 0.15 μm. The compound also arrests cell cycle progress (at
G2/M phase) and restricts the polymerization of tubulin through binding with col-
chicine binding site of the protein and thus acts as an antimitotic agent for tumor
cells. The xenograft experiment in mice involving the derivative compound revealed
that it has more effective anticancer agent than the reference drug taxol, when
treated against lung cancer (Wang et al. 2013).
Alpinumisoflavone (C20H16O5)
Alpinumisoflavone, a flavonoid, acts as a molluscicide and larvicide for schisto-

somes and thus can directly take part in controlling schistosomiasis by killing larval
schistosomes as well as indirectly controlling helminth infections through killing
mollusks’ intermediate hosts (Perrett et al. 1995; Perrett and Whitfield 1995).
Antibacterial, antineoplastic, and atheroprotective properties of the isoflavone
have already been established by several workers (Namkoong et al. 2011; Mvondo
et al. 2015; Kuete et al. 2016; Gao et al. 2017). It is known that osteoporosis, an
age-related deformity of bone architecture where bone homeostasis is disturbed due
Fig. 15.4 Scanning electron micrographs of control (a, c, e, and g) and M. pachycarpa (1000 mg
crude extract/kg body weight) treated (b, d, f, and h) mice. (a) Liver showing intact parenchyma
cells and the fenestrae (arrows). (b) Liver surface showing severe damage and disintegration of
endothelial lining having increased number of deformed fenestrae. (c) Kidney showing normal
contour of proximal tubule. (d) M. pachycarpa treated kidney showing severe disruption in the
proximal tubules. (e) Lumen showing normal intestinal epithelium with regular arrangement of
microvilli (arrows). (f) Intestinal surface showing severe deformation and disruption of microvilli
(arrows). (g) Testis showing normal seminiferous tubule. (h) Testis showing abnormal seminifer-
ous tubules. All scale bars = 200 μm
to overactivity of osteoclasts, can be remodeled by alpinumisoflavone. Cong et al.

(2017) experimentally proved that the phytochemical can suppress differentiation of
osteoclast in ovariectomy-induced bone deformation and thus established its osteo-
protective potential.
Fig. 15.5 Transmission electron micrographs of control (a, c, e, and g) and M. pachycarpa
(1000 mg crude extract/kg body weight) treated (b, d, f, and h) mice. (a) Liver showing normal
nucleus (N), nucleolus (NL), nuclear membrane (NM), and mitochondria (MT). (b) Liver showing
distorted nucleus (N), nucleolus (NL), nuclear membrane (NM), and swollen mitochondria (MT),
with disintegrated cristae. (c) Kidney cell showing normal nucleus (N), nuclear membrane (NM),
and normal elongated mitochondria (MT). (d) Kidney showing shrunk and breakage nucleus (N),
nuclear membrane (NM), and vacuolated mitochondria (MT). (e) Intestine showing normal regular
lining of microvilli (MV), having uniform epithelial membrane (EM), compact cytoplasm, mito-
chondria (MT), well-defined nucleus, and other cell organelles. (f) Intestine showing abnormal
irregular lining of microvilli (MV), having discontinuous epithelial membrane (EM), condensed
nucleus and nucleolus, abnormal mitochondria (MT), and other cell organelles. (g) Testis showing
normal nucleus (N), mitochondria (MT), nuclear membrane (NM), and acrosomal vesicle (V). (h)
Testis showing damaged nucleus (N), mitochondria (MT), nuclear membrane (NM), and acroso-
mal vesicle (V). All scale bars = 1 μm
Fig. 15.6 Structures of biologically most active compounds recovered from M. pachycarpa. (The
software Chem Draw Ultra 12.0 was used for drawing the structures of the compounds)
Of the two types of esophageal cancers prevalent among humans, esophageal

squamous cell carcinoma (ESCC) is the most common (90%) occurrence and
responsible for millions of death every year (Ge et al. 2014; Rustgi and El-Serag
2014). Han et al. (2016) showed that alpinumisoflavone modulates miR-370/pIm1
signaling pathway in ESCC leading to programmed cell death. Radiotherapy
appears as the prime way of treatment for advanced ESCC; however, resistance
developed by ESCC against radiotherapy leads to inefficiency of the treatment.
Recently, Zhang et al. (2017) observed that the phytochemical increases radiosensi-
tivity of ESCC by stimulating ROS generation through downregulating expression
of nuclear transcription factor2, thus increasing the efficiency of the radiotherapy.
Namkoong et al. (2011) observed that alpinumisoflavone causes apoptosis in lung
carcinoma cells through downregulating mitogen-activated kinase and nuclear

factor-κB pathways.
Deguelin (C23H22O6)
Deguelin, a rotenoid known as a nontoxic (at low dose) anticancer agent, inhibits
growth of precancerous bronchial cells and also skin cancer. The compound inhibits
growth of malignant cells by inducing apoptosis through protein kinase B signaling
pathways. The therapeutic dose of deguelin has minimal adverse effects on the nor-
mal bronchial epithelial cells (Udeani et al. 1997; Lee et al. 2004). However, Caboni
et al. (2004) observed that deguelin causes neuropathological lesion in the brain of
rat leading to Parkinson’s disease like syndrome at a dose of 6 mg/kgbw/day, if
consumed continuously for 5–6 days. Anti-inflammatory activity of the compound
has also been established (Ye et al. 2014). The rotenoid revealed to reduce the
mRNA expression of vascular endothelial cell growth factor-D in Lewis lung cancer
cells (LL/2), inhibit multiplication and migration of LL/2 cells, and also act as an
inhibitor of lymphatic metastasis (Hu et al. 2010).
Genistein (C15H10O5)
Genistein is a polyphenolic compound having an aromatic A-ring which is known

for its estrogenic property. The compound is formed by hydrolysis of inactive form
of glucoconjugates and also by metabolism of biochanin A and formononetin.
Degradation of genistein occurs in the liver where it mainly conjugates with gluc-
uronin acid and excretes from the body through urine (Axelson et al. 1984; Setchell
et al. 2001). The chemical at a dose of 0.5 mg per ml of phosphate-buffered saline
(PBS) causes promising changes in the activity of acetylcholinesterase (AChE), an
enzyme associated with signal transmission in the central and peripheral nervous
system of giant intestinal fluke Fasciolopsis buski and the cestode Raillietina echi-
nobothrida (Pal and Tandon 1998a; Kar and Tandon 2000). An increase in nitric
oxide (NO) along with cGMP concentration due to increase in activities of nitric
oxide synthase (NOS) that ultimately results in paralysis of the giant fluke F. buski
indicates the putative vermifungal potential of the phytochemical (Das et al. 2009).
Quantitative increase in the amino acids citrullin and γ-aminobutyric acid in the
experimental fluke further supports the idea that an increase in these amino acids are
responsible for altered NO level and consequent paralysis of parasitic worms due to
nerve disorder (Tandon et al. 2001; Kar et al. 2002; Kar and Tandon 2004).
Tegumental enzymes like AcPase, AlkPase adenosinetriphosphatase (ATPase)
and 5- nucleotidase are very important as they take part in absorption, digestion, and
secretion in cestode and trematode parasites, who do not have functional digestive
tract. A decline in activities of these enzymes in the genistein which exposed
trematode and cestode as recorded by different workers indicates a probable trans-

tegumental mode of action of the phytochemical in the parasitic worms (Pal and
Tandon 1998b).
Helminth parasites, particularly flat worms (trematode and cestode), acquire par-
tially or fully digested carbohydrate from their host to meet the requirement of
energy so as to establish in their host and to propagate their progeny. Therefore, to
see the effect of genistein on the energy metabolism pathway in cestode, if any, Das
et al. (2004a) carried out experiments on R. echinobothrida involving genistein as
chemotherapeutic agent and observed that at a concentration of 0.2 mg/ml of PBS,
genistein reduces the glycogen concentration up to 44% compared to the control.
Similarly, quantitative decrease in glucose (19%) and increase in malate (134%)
and glycolytic enzymes, namely, hexokinase (39%), phosphofructokinase (49%),
phosphoenolpyruvate carboxykinase (45%), and lactate dehydrogenase (67%),
were recorded in the phytochemical-treated worms (Das 2003; Das et al. 2004b, c).
Das et al. (2006) also showed that genistein is responsible for quantitative decrease
(39–40%) in calcium ion (Ca++) concentration in the cestode and a simultaneous
increase (118–123%) in the Ca++ concentration in the media where worms were
incubated. Thus, genistein seems to alter the membrane homeostasis through regu-
lating Ca++ concentration.
Metacestodiasis caused by larval forms of Echinococcus granulosus and E. mul-
tilocularis in different vital organs of mammalian hosts causes different complica-
tions leading to organ failures. Naguleswaran et al. (2006) showed an effective
anti-metacestode activity of genistein; however, long-time treatment (which is nec-
essary to control metacestode infection) leads to adverse estrogenic effect on
the hosts.
Different workers established anticancer potential of genistein (Fotsis et al.
1995; Lamartiniere et al. 1995). Lamartiniere et al. (2000) showed that injection of
genistein (500 mg/kg body weight) during prepuberted period develops long-lasting
protection in rat against DMBA-stimulated cancer. This observation also supported
by the epidemiological data, where it is observed that people who consume diet hav-
ing high amount of genistein have reduced incidence of cancer in mammary gland
(Wu et al. 1996). Pagliacci et al. (1994) showed how genistein acts against hormone-
dependent breast cancer in mammals. The phytochemical at a concentration of
50 μg incubated for 72 h also inhibits proliferation of tongue cancer by 50% through
downregulating tumorigenesis proteins like survivin and Oct4 (Ardito et al. 2017).
Recently, Tandon and Das (2018) gave an account of different workers who stud-
ied and established antioxidant property of genistein. Ma et al. (2010) showed that
in vitro antioxidant property of the compound is responsible for reduction of mito-
chondrial membrane damage. On the basis of this observation, Ma et al. (2010,
2015) hypothesized its neuroprotective role through antioxidant and anti-
inflammatory properties.
Collagen is an important extracellular protein; alteration of its quantity and dis-
tribution adversely affect various cellular functions. The phytochemical genistein
showed to inhibit oxidative stress related to inhibition of collagen synthesis and thus
protect dermal fibroblast in humans (Sienkiewicz et al. 2008).
Pomiferin (C25H24O6)
Pomiferin, a prenylated isoflavone, is a strong antioxidant. The extent of its antioxi-

dant activity is found to be almost similar to that of dibutylhydroxytoluene, vitamin
C, and vitamin E (Tsao et al. 2003). Bartosikova et al. (2007) experimentally proved
that pomiferin also acts as antidiabetic agent, as it significantly reduces blood glu-
cose level compared to its control animals. The authors also observed an increased
level of glutathione peroxide and decreased in malondialdehyde, in the pomiferin-
treated group of animals, which indicates an antioxidant potential of the compound.
Pomiferin also acts as an anticancer agent as it induces DNA fragmentation and
apoptosis in human cholangiocarcinoma cells with IC50 value 0.9 μg/ml (Svasti
et al. 2005).
Daidzein (C15H10O4)
Daidzein, an isoflavone, naturally occurs in different leguminous plants including

M. pachycarpa which is produced in plants in response to pathogenic attacks
through the phenylpropanoid pathway. Lamartiniere et al. (2002) showed that the
compound when consumed (1000 mg/kg diet) by rats causes significant reduction
in body weight. High daidzein content in food of rat also causes reduced level of
progesterone in circulation. The authors observed that the phytoestrogen at a dose
as high as 1000 mg/kg body weight does not alter histomorphology of female repro-
ductive organs and also does not provide any protection against induced mammary
gland malignant tumours (Lamartiniere et al. 2002). Daidzein also found to have
beneficial potential on our circulatory system. Hermenegildo et al. (2005) showed
that the vasodilator prostacyclin which is synthesized by cyclooxygenase in human
is increased in production through oestrogen receptor-dependent mechanism by
daidzein. The compound also revealed to take part in immune regulation by sup-
pressing the maturation of dendritic cells (Wei et al. 2012).
Barbigerone (C23H22O6)
Barbigerone, an isoflavone, is known for its anti-malarial activity against

Plasmodium falciparum (Yenesew et al. 2003). Antioxidant and 15-lipoxygenase
inhibitory properties of the compound have also been established by Wangensteen
et al. (2006). Li et al. (2009) showed in vitro antitumor activity of the phytochemical
against lung cancer cell line (LL/2) in a dose-dependent manner. The phytochemical
arrests cancer cell proliferation through intrinsic pathway of mitochondria-mediated
apoptosis where an increased release of cytochrome C into the cytosol of LL/2 cells
followed by higher activities of proapoptotic proteins (caspase 3 and caspase 9) and
reduced activity of anti-apoptotic protein (Bcl-2) were observed in the lung carci-
noma cells (Li et al. 2009).
Auriculasin (C25H24O6)
The prenylated isoflavone auriculasin is known for its in vitro anticancer activity
(Oh et al. 1998). Cho et al. (2018) showed that the phytochemical causes apoptosis
only in prostate cancer cells (LN Cap) without affecting the normal cells of xeno-
graft mice in a time-dependent manner. Further, the mechanism involved in apopto-
sis recorded to be caspase-independent pathway, where the phytochemical induces
ROS accumulation in LNCap cells causing inhibition of PI3K, phospho-AKT, phos-
pho-in TOR, and cell cycle progress. Through two-stage carcinogenesis test carried
out by Ito et al. (2000) on skin papilomas of mice showed the anti-skin cancer prop-
erty of the phytochemical.
4-Hydroxylonchocarpin (C20H18O4)
4-Hydroxylonchocarpin is an isoflavone biosynthetically derived from phenylala-

nine. Mbaveng et al. (2008) established fungicidal property of the compound against
Microsporum audorium, Trichophyton rubrum, Candida albicans, and C. glabrata.
The authors also showed antibacterial activity of the chemical, particularly against
Streptococcus faecalis, Staphylococcus aureus, Bacillus cereus, B. subtilis, B. mega-
terium, and B. stearothermophilus. Kuete et al. (2010) reported anti-gonorrhoeal
activity of the isoflavone against ten strains of Neisseria gonorrhoeae with mini-
mum inhibitory concentration between 4.88 and 39.06 μg/mL. Several workers
demonstrated antibacterial activity of the compound including multidrug-resistant
strain of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and
Pseudomonas aeruginosa (Avila et al. 2008; Kuete et al. 2011).
Ye et al. (2014) showed a dose-dependent anti-inflammatory potential of
4-hydroxyloncholcarpin, carried out through suppression of nitric oxide synthase
expression to the extent of 66.5%. The compound also inhibits production of nitric
oxide and scavenge-free DPPH radical, thus indicating its probable use as a lead
compound to control neurodegenerative diseases (Lee et al. 2006; Shajarahtunnur
et al. 2008).
As per as cytotoxic potential of the phytochemical is concerned, it is revealed
that IC50 value is below 20 μg/ml for several cancer cell lines as well as normal
hepatocyte cells (Kuete et al. 2010). Clinical evaluation on toxicity of
4-hydroxylonchocarpin revealed that at high plasma concentration, the chemical is
not toxic (Batovska and Todorova 2010).
illewanin G (C26H26O7), Millewanin H (C26H26O7),

M
and Furowanin B (C55H70MgN4O6)
Antiestrogenic activity of the isoflavonoids millewanin G, millewanin H, and

furowanin B was established through observation on inhibition of β-galactosidase
activities exerted by 17β-estradial in the yeast two-hybrid assays. IC50 values of the
phytochemicals were found to be 29, 18, and 13 μM, respectively (Ito et al. 2006).
Acknowledgments The authors would like to thank the University Grant Commission (UGC) for
providing financial support (RGNF), Department of Zoology, North-Eastern Hill University,
Shillong for infrastructural facilities. Head SAIF NEHU and Head SAIF AIIMS, New Delhi, are
also being acknowledged for providing scanning and transmission electron microscopic facilities.
We wish to thank Mr. Deepjyoti and Ms. Keleni-i for helping in construction of the structure of the
chemicals.
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Chapter 16
Phytochemicals and Their Role
in Pharmaceuticals
Anushree Suresh and Jayanthi Abraham
Introduction
Phytochemicals are bioactive compounds found naturally in plants which provide

health benefits to humans (Hasler and Blumberg 1999). These are called secondary
metabolites as they do not have any direct action on plant growth, development or
metabolic action on plants. These play an important role in plant defence mecha-
nism. The word ‘phyton’ is derived from Greek meaning plant. These bioactive
compounds are mostly non-essential to plants. The common source of phytochemi-
cals are vegetables, fruits, whole grains, seeds, nuts and other medicinal plants
(Saxena et al. 2013). The majority of foods consisting of phytochemicals/phytonu-
trients has therapeutic or health beneficiary properties against diseases. They pro-
vide protection against inflammation, cancers, high blood pressure, viral illnesses,
coronary heart disease, diabetes, microbial and parasitic infections, osteoporosis,
ulcers, associated disorders, spasmodic conditions and psychotic diseases (Prakash
et al. 2013). Phytochemicals can be natural or synthetic composite produced by
plants to aid them to combat pathogenic diseases, harsh environmental conditions,
competitors or predators.
Previous study suggests that phytochemical compounds such as epigallocatechin
gallate (found in tea), capsaicin (commonly found in bell pepper), curcumin (found
in turmeric), resveratrol (found in peanuts and grapes), etc. have the properties to
cure neurodegenerative diseases, namely, Alzheimer’s and Parkinson’s (Wang
et al. 2012).
A. Suresh · J. Abraham (*)

Microbial Biotechnology Laboratory, School of Biosciences and Technology, VIT University,
Vellore, Tamil Nadu, India

https://doi.org/10.1007/978-981-15-2195-9_16
194 A. Suresh and J. Abraham
lassification of Phytochemicals and Their Physiological

C
Properties
Phytochemicals are classified depending on physical, chemical and protective char-

acteristics. So far 4000 phytochemicals have been catalogued (Meagher and
Thomson 1999). Phytochemicals are categorized according to different chemical
structural groups like curcumin, phenolics, flavonoids, alkaloids, plant steroids, ter-
penes, lignans, saponins and glucosides (Harborne 1998) as depicted in Fig. 16.1.
Phenolic Compounds
Phenolics are the abundantly found phytochemicals in plants. The derivative of

hydrocarbons having hydroxyl groups is called phenols and alcohols. A phenolic
group of phytochemicals is usually found in plants as listed in Table 16.1 (Walton
et al. 2003). The enzymes responsible for the production of phenolic compounds via
a metabolic pathway are polyphenol oxidase. Phenolic compounds obtained from
medicinal plants play an important role in the prevention and treatment of cancer;
they contain many other compounds such as curcuminoids, stilbenes, flavonoids,
quinones, tannins, lignans and phenolic acids (Huang et al. 2010). They impart a
good antioxidant property and have inhibitory action against carcinogenic com-
pounds present in food (Hughes et al. 1997). Previous reports also suggest a reduc-
tion in blood cholesterol, an increase in the secretion of bile, maintenance of the
blood lipid level and antimicrobial action against Gram-positive bacteria,
Staphylococcus aureus (Gryglewski et al. 2016).
Fig. 16.1 Picture depicting the different categories of phytochemicals

16 Phytochemicals and Their Role in Pharmaceuticals 195
Table 16.1 List of phenols from natural sources

Phenolic compounds Sources References
Flavonols, Apple peel Chen et al.
Quercetin-3-O-glycosides, Phloridzin, c (2012)
Cyanidin-3-O-galactoside,
Chlorogenic acid
Flavonoids Mandarin Zhang et al.
Flavanones (2014)
Flavones
Hydroxybenzoic acid, Hydroxycinnamic acid,
Chlorogenic acid
Salicylic acid Leafy vegetables Khanam et al.
Vanillic acid (2012)
Gallic acid
Caffeic acid
Chlorogenic acid
p-coumaric acid
Ferulic acid
m-coumaric acid
Isoquercetin
Rutin
Gallic, vanillic, caffeic, syringic, p-coumaric, Oat flour Kilci and
sinapic, ferulic, p-hydroxybenzoic acids Gocmen (2014)
Gallic acid, chlorogenic acid, coumaric acid, Tea Zielinski et al.
catechin, epicatechin, procyanidin, quercetrin and (2014)
caffeine
Flavones, flavonols, rutin and chlorogenic acid Medicinal plants from Chew et al.
Leguminosae family (2009)
Flavanols Beans Manach et al.
(2004)
Flavonoids
This phytochemical compound has been found ubiquitous in nature. They are cate-
gorized into subgroups such as flavonols (myricetin, quercetin, kaempferol, fisetin),
flavones (luteolin, flavone, apigenin) and flavanones (naringenin, flavanone, hesper-
etin) as listed in Table 16.2. These compounds show various biological properties
such as antimicrobial, cytotoxic, antiallergic, antitumour, oestrogenic, cytotoxicity,
antioxidant property, enzyme inhibition and vasodilative effect (Tapes et al. 2008).
These compounds are usually found inhabiting the photosynthesizing cells.
Flavonoid compounds obtained from medicinal plant source have physiological
activities such as antibacterial, antifungal and antiviral activity. Earlier reports sug-
gest the use of flavonoid compounds in the existing chemotherapeutic diagnosis
procedure to treat cancer. Quercetin is a flavonoid compound which can be used in
the inhibition of DNA gyrase in the process of DNA replication. Another flavonoid
compound, sophoraflavone G and epigallocatechin gallate, inhibits the cytoplasmic
membrane function (Tsuchiya et al. 2000).
Table 16.2 List of flavonoids found in plants

Flavanoid compounds Sources References
Kaempferol, quercetin, myricetin and Onion, red wine, olive oil, berries Stewart et al.
tamarixetin and grapefruit (2000)
Chrysin, apigenin rutin, luteolin and Fruit skins, red wine, buckwheat, red
luteolin glucosides pepper and tomato skin
Catechin, epicatechin Tea Lopez et al.
Epigallocatechin (2001)
Naringin, naringenin, taxifolin and Citrus fruits, grapefruits, lemons and Miyake et al.
hesperidin oranges (2000)
Genistin, daidzin Soybean Reinli and Block
(1996)
Apigenidin, cyanidin Cherry, raspberry and strawberry Stewart et al.
(2000)
Alkaloids
True alkaloids are natural compounds containing heterocyclic nitrogen atoms and
are basic in nature. These possess many pharmacological properties such as antima-
larial activity, antiarrhythmic effect, antihypertensive effects and anticancer activity
(Roberts and Wink 1998). These have significant activity against bacterial and fun-
gal infections. Many indole alkaloids found in the medicinal plants possess pharma-
cological activity such as muscle relaxant, antidepressant and antihypertensive,
wound healing, anticholinergic, sympathomimetic, antiulcer, anti-inflammatory and
other properties. Earlier reports demonstrated the mode of action of alkaloid com-
pounds against anti-inflammatory activity, as these inhibit or regulate the inflamma-
tion mediators like NF-κB, COX-2 and iNOS (Pradeep and Kuttan 2004; Yang et al.
2006; Matheus et al. 2007; Chen et al. 2011a, b; Zhao et al. 2015; Zhou et al. 2015;
Pacheco de Oliveira et al. 2015). Some of the alkaloids and their pharmacological
activities have been tabulated in Tables 16.3 and 16.4.
Terpenoids
Terpenoids are the essential oils present in the plants. These contain isoprene struc-
tures essential for the rich structural property of terpenoid compound. These have an
inhibitory action against all classes of bacteria, viruses, protozoa and fungi (Elbein
and Molyneux 1999). A recent study conducted reported the use of a triterpenoid
compound named betulinic acid against HIV (Tiwari et al. 2011). They disrupt the
cell membrane by the action of the lipophilic compounds (Tiwari et al. 2011). These
compounds have gained higher importance in the food and cosmetic industries due
to the fragrances and flavour the functional group present in their structure. Some of
the pharmacological properties of terpenoids are hepaticidal, antimalarial, anticar-
cinogenic, antiulcer, antimicrobial, antifungal, anticancer and diuretic (Dudareva
et al. 2004). The pharmacological activities of terpenoids are listed in Table 16.5.
Table 16.3 List of alkaloids found in plants

Alkaloid
compounds Sources References
Atropine Atropa belladonna Hamilton and Duffy
Datura stramonium (2014)
Mandragora officinarum
Members of genera Brugmansia and
Hyoscyamus
Caffeine Coffee, tea, cocoa, yerba maté and guarana Juliano and Griffiths
plants (2004)
Cocaine Leaves of coca plant Plowman (1979)
Codeine Poppy saps Prommer (2010)
Heroine Opium poppy (Papaver somniferum) Rang et al. 2014
Morphine Opium poppy (Papaver somniferum) Jonsson et al. (1988)
Nicotine Family of Solanaceae D’Souza and Markou
(2011)
Papaverine Opium poppy (Papaver somniferum) Liu and Couldwell (2005)
Solanine Leaves, fruit and tubers of family of Gao et al. (2006)
Solanaceae
Table 16.4 Alkaloid compounds and their pharmacological properties

Compounds Pharmacological properties References
Aconitine Activity against diarrhoea, colonic MPO activity, INF-γ, Liu et al. (2009)
against gram-positive and gram-negative bacteria
Berberine Activity against diarrhoea, colonic MPO activity, INF-γ,
against gram-positive and gram-negative bacteria
Sangrovit Used as appetizer supplement
Sanguinarine Against colon cancer
Chelerythrine Against colon cancer
Diterpenoid Decrease body weight, decrease piloerection and mobility Wangchuk et al.
score, decrease faecal consistency score, decrease INF-γ (2015)
secretion
Table 16.5 List of terpenoid compounds and their pharmacological activities

Compounds Pharmacological activities Reference
Perilla alcohol Anticarcinogenic property Dudareva et al. (2004)
Artemisinin Antimalarial
Glycyrrhizin Antiulcer, hepaticidal, antimicrobial,
diuretic
Taxol Anticancer
Saponin
It is a secondary metabolite found naturally in both medicinal and non-medicinal

plants. The aromatic ring present in the structure confers them as better detergents,
as sweeteners and as an emulsifying agent. Amphiphilic behaviour of these com-
pounds imparts the ability to form complexes with steroids, membrane phospholip-
ids and proteins present in the cell membranes. The binding property of saponin to
cholesterol can reduce the cholesterol serum level and anti-hypercholesteraemic
activity. The anti-inflammatory activity of saponin compounds is mainly due to the
complex structure called aglycone present in the saponin compound. Many sapo-
nins are known to have pharmacological properties such as antiparasitic, antimicro-
bial, antifungal, antitumour and antiviral and protect plants from insect attack
(Lasztity et al. 1998). The mode of action of saponins has not been fully understood
yet. Saponin compounds and their pharmacological activities are listed in Tables
16.6 and 16.7.
Quinones
Quinones are phytochemical compounds containing an aromatic ring. Apart from

producing stable free radicals, quinones are known to form complex irreversibly by
binding with a nucleophilic amino acid in proteins, thereby leading to proteins inac-
tivation. This makes the compound a potent antimicrobial agent (Shashidhara et al.
2014). Coenzyme Q7 plays an important role as a membrane stabilizer and antioxi-
dant, thereby preventing cellular damage leading to several chronic diseases such as
cardiovascular diseases, Parkinson’s disease and osteoporosis (Lanham-New 2008).
Some of the quinones obtained from plant species have been listed in Table 16.8.
Table 16.6 Saponins derived from different plant species

Saponins Sources References
Triterpenoid Alternanthera philoxeroides Fang et al. (2009)
Celosia cristata Wang et al. (2010)
Hydrocotyle bonariensis Tabopda et al. (2012)
Ilex kudingcha Zuo et al. (2012)
Barbarea vulgaris Nielsen et al. (2010)
Silene viscidula Xu et al. (2012)
Steroidal Digitalis ciliata Gvazava and Kikoladze (2010)
Cestrum diurnum Fouad et al. (2008)
Solanum lycocarpum Nakamura et al. (2008)
Yucca desmetiana Diab et al. (2012)
Asparagus filicinus Wu et al. (2010)
Agave utahensis Yokosuka and Mimaki (2009)
Table 16.7 Some of the saponin compounds and their pharmacological activities
Compounds Pharmacological activities Reference
Glycyrrhizin Antiviral activity Barbosa (2014)
Steroid saponin Synthesis of hormone
Tomadins Steroid hormones synthesis
Triterpenoid Acts against myocarditis
saponin
Ginsenosides Restore cardiomyocytes damage by producing
free radicals, action on blood clotting
Table 16.8 List of quinones

Quinones Sources References
Aloe-emodin Rheum palmatum Arosio et al. (2000)
Juglone Juglans mandshurica Aithal et al. (2011)
Β-lapachone Tabebuia avellanedae Medeiros et al. (2010)
Plumbagin Plumbago pearsonii Lai et al. (2012)
Shikonin Lithospermum erythrorhizon Chen et al. (2011)
Thymoquinone Nigella sativa Sayed-Ahmed et al. (2010)
Tannins
Tannins are secondary metabolite found in various plants and consist of complex
organic structures containing polyphenols. These phytochemical compounds are
usually found in many fruits. These act as a better antimicrobial agent against Gram-
positive bacteria and yeasts (Surya et al. 2011). They are commonly used against
diarrhoea, astringents, diuretics, antiseptic, anti-inflammatory and haemostatic
agent in pharmaceutical industries (Haslam 1989). Previous reports suggest the use
of tannins against AIDS and various other cancer (Karamali and Teunis 2001).
Tannin-containing compounds of pharmacological value are tabulated in Table 16.9.
Coumarin
This phytochemical compound consists of a benzene ring connected to a pyrone

ring (Lacey et al. 2015). Earlier reports suggest the use of coumarin-containing
plants as antimicrobial agent against pathogens. Some of the pharmacological prop-
erties are antithrombotic, anti-inflammatory, vasodilatory, antioxidant, antimicro-
bial, anti-HIV, anticoagulant, analgesic, etc. (Wu et al. 2014) as depicted in Fig. 16.2.
Warfarin is a well-known coumarin which is used as a rodenticide and as an oral
anticoagulant (Keating and O’Kennedy 1997). Refer Tables 16.10 and 16.11 for
coumarin derivatives with pharmacological properties.
Table 16.9 Tannin compounds with pharmacological properties

Compounds Pharmacological properties References
Chebulic acid Astringent, stomachic Ramasubramania Raja and Sreenivasulu
Gallic acid Astringent, antimicrobial (2016)
activity
Ellagic acid Cardiotonic, hypotensive
Catechutannic Astringent for treatment of
acid diarrhoea
Fig. 16.2 Use of medicinal plants for various health-related problems
Phytochemicals have the potential to induce the immune system to fight against
carcinogenic conditions, reduce inflammation, induce slow growth rate of cancer
cells, repair damaged DNA, proper functioning of programmed cell death (apopto-
sis), regulate gene expression, activate insulin receptor and cellular signalling
(Hanhineva et al. 2010). Table 16.12 provides a list of the medicinal plants and their
phytochemical constituents.
The Role of Endophytes and Production of Phytochemicals
Anton de Bary was the first scientist to introduce the term ‘epiphytes’ for fungi that
thrive on the surface of plant host and ‘endophytes’ for those living inside the plant
tissue (Dutta et al. 2014a). There are different types of endophytes known till date.
They are as follows:
Table 16.10 Different types of coumarins

Types Examples References
Simple coumarins Esculetin Witaicenis et al.
(2010)
Ammoresinol Hodak et al.
(1967)
Novobiocin Chain (1958)
Chartreusin Portugal (2003)
Fraxin Whang et al.
(2005)
Furanocoumarins Imperatorin Piller (1975)
Psoralen Bourgaud et al.
(2006)
Methoxsalen Kharasch et al.
(2000)
Dihydrofurano Anthogenol Chakthong
coumarin et al. (2012)
Felamidin Rosselli et al.
(2009)
Linear Grandivittin Basile et al.
Agasyllin (2009)
Angular Calanolide Kashman et al.
(1992)
Inophyllum Patil et al.
(1993)
Phenyl coumarin Isodispar B, mammea A/AB dioxalanocyclo F, Crombie et al.
dispardiol B, mammea A/AB cyclo E, disparinol D, (1966)
disparpropylinol B
Bicoumarins Dicoumarol Poole and
Poole (1994)
Table 16.11 Coumarin Compounds Pharmacological properties Reference

compounds and their
Ledebouviellol Anti-inflammatory activity Asif (2015)
pharmacological importance
Esculetin Treat psoriasis
Peuarenin Phototoxicity
Anomalin Antimicrobial property
Osthol Anti-inflammatory
• Competent endophytes: Organisms which colonize a plant host by directly enter-

ing into the plant tissue and thereby modulate plant physiology and selectively
aid in beneficial maintenance of the plant-microbe association.
• Facultative endophytes: Organisms that inhabit plants internally as well as on
another habitat.
Table 16.12 List of medicinal plants and their major phytochemicals produced
Botanical Major phytochemical Pharmacological
names Medicinal properties constituents properties
Capsicum Irritant, pungent, Capsaicin, fatty acid, Antimicrobial and
frutescens stimulant, stomachic and fixed oil, resin, solanine, antivenom activity
tonic volatile alkaloids, volatile
oil, oleoresin capsaicin
Carica papaya Anthelmintic, alternative Papain or papayotin, Antifertility activity,
digestive, diuretic, ecbolic, chymopapain, caprine, antioxidant activity
emmenagogue, laxative, caricin, caproic, citric and contraceptive
rubefacient acid, tartaric, malic,
palmitic, crystalline,
papyic and carica fat
acids, dextrin and resin
Centella Alternative, tonic, Vallerine, asiaticoside and Anticancer, antiulcer
asiatica adaptogen, central nervous oxy-asiaticoside, essential and antioxidant
system relaxant, peripheral oil, fatty oil, sitosterol, activity
vasodilator, sedative, tannin, pectic acid,
antibiotic, detoxifier, ascorbic acid, alkaline
blood∗ purifier, laxative, hydrocotyline and resins
diuretic and emmenagogue
Cissus Alternative, digestive and Carotene, calcium Analgesic, anti-
quadrangularis stomachic oxalate, ascorbic acid inflammatory,
venotonic and
antiulcer activity
Gymnema Astringent, diuretic, Gymnemic acid, resins, Antimicrobial and
sylvestre antiperiodic, emetic, tannins, pararapin, antidiabetic activity
refrigerant, stomachic and glucose, inositol,
tonic anthraquinone,
carbohydrates, tartaric
acid and calcium salts and
crystalline concretions
Hemidesmus Alternative, demulcent, Coumarin, volatile oil, Antinociceptive,
indicus diaphoretic, diuretic, hemidesmine, antioxidant,
sudorific and tonic smilasperic, essential oil, antithrombotic,
resin, tannins, 2-hydroxy- antiulcer and
4-methoxy benzaldehyde, antidiabetic activity
sterols
Hygrophila Diuretic, spasmolytic and Phytosterol, semi-drying Hepatoprotective and
schulli hypotensive oil, mucilage, diastase, antioxidant activity
lipase, protease and
alkaloids
Mangifera Antihelmintic, Tartaric, citric acid, gallic Antiulcer, antioxidant
indica antiscorbutic, astringent, malic acids, tannin, fat, and ant amoebic
diaphoretic, diuretic, sugar, gum, starch, activity
laxative, refrigerant, antiscorbutic vitamin C
stomachic and tonic
Mimosa pudica Alternative, aphrodisiac, Alkaline mimosine, Anticonvulsant,
antiseptic, blood purifier, colchicines, tubulin, hyperglycaemic and
carminative and resolvent 5-deoxyflavonol antivenom activity
(continued)
Morinda Astringent, cathartic, Morindin and crystalline Antimicrobial activity
pubescens emmenagogue and
febrifuge
Phyllanthus Astringent, carminative, Rich vitamin C, essential Anticancer activity
emblica diuretic, laxative, oil and fixed oil,
refrigerant and stomachic phosphatides and tannins
Strychnos Aphrodisiac, anodyne, Strychnine, Analgesic, anti-
nux-vomica pseudostrychnine,
cardiac, emetic, purgative, inflammatory and
stimulant, stomachic brucine, vomicine, anti-diarrhoeal
igasurine. Igasuric and activity
strychnic acid, loganine,
proteids, starch. Sugar
and phosphates
Tephrosia Anthelmintic, cholagogue, Tephrosin, isotephrosin, Wound healing and
purpurea deobstruent, diuretic, rotenone, osyritin, immunomodulatory
febrifuge, laxative and deguelin, quercetin, activity
tonic querritrin, glucoside rutin
Zingiber Antiviral, antimotion and Oil zingiberol, Exlipidemic,
officinale antinauseant, anti- zingiberene, phellandrene expectorant,
inflammatory, and linaloal, astringent,
anti-ulcerogenic diarylheptanoids hypolipidemic,
antiemetic
Dalbergia Antidiabetic, analgesic, Isoflavone-o-glycoside, Anti-inflammatory,
sissoo antioxidant biochemin A, tectorigenin antinociceptive
Buchanania Expectorant, purgative, Bark contains tannins, Anti-inflammatory,
lanzan blood purifier, digestive alkaloids and saponins antimicrobial,
disorders antidiabetic.
Antihyperlipidemic
Oroxylum Astringent, rheumatism, Phenols Anti-inflammatory,
indicum joint pains, urogenital anti-allergy,
disorder antimicrobial activity
Cymbopogon Reducing fever, stomach Tannins, saponins, Antimicrobial,
citratus cramps, flatulence and flavonoids, alkaloids, anti-inflammatory
colic phenols, anthraquinones,
citronellol
Ipomea digitata Vasoconstrictor and Beta-sitosterol, taraxerol Antimicrobial,
bronchoconstriction effect anti-inflammatory
Grevia tiliifolia Astringent, strengthens Flavonones, steroids, Antibacterial and
joints, treats ulcerative triterpenoids, phenols, antifungal activity
colitis tannins, saponins
Mitragyna Analgesic, antipyretic, Flavonoids, glycosides, Anti-inflammatory
parvifolia antiarthritic tannins, alkaloids and antioxidant
Garcinia indica Altering diarrhoea, Saponins Antioxidant,
abdominal colic pain antibacterial activity,
antifungal
(continued)
Schinus Urogenital disorder, Tannins, alkaloids, Antifungal,
terebinthifolius ulcers, urethritic, flavonoids, steroids, antibacterial
menstrual disorders saponins, sterols, terpenes
and a large amount of
essential oil
Caesalpinia Colic convulsions, uterine Peltogyenoides, Antifungal,
bonducella stimulant, astringent, pulcherrimin, antibacterial,
antihelmintic 6-methoxypulcheri, antimalarial,
alkaloids anti-filarial
Gmelina Antihelmintic, analgesic, Essential oils Anti-inflammatory,
arborea piles, anaemia antibacterial
Millingtonia Expectorant, purgative, Bark contains tannins, Anti-inflammatory,
hortensis blood purifier, digestive alkaloids and saponins antimicrobial,
disorders antidiabetic
Antihyperlipidemic
Tylofora Relief from asthma, pain, Alkaloids, tannins Antibacterial activity
asthmatica blood clotting, antipyretic
Pimenta dioica Digestion stimulant, Essential oil Antimalarial,
remove gas, anti-flatulent, antifungal activity
anti-inflammation (such as
for rheumatism and
arthritis), rubefacient,
stomachache, vomiting,
diarrhoea, fever, flu, colds
Embelia ribes Astringent, relieves worm Embelin, christembine, Antibacterial,
infestation, useful in homoembelin, vilangine, antifungal activity
diabetes, useful in quercitol, etc.
headache, improves
appetite, useful in
abdominal colic pain
Putranjiva Improves female fertility, Tannins, alkaloids Anti-inflammatory,
roxburghii treat allergic and red antibacterial
pimples, laxative,
astringent
Semecarpus Astringent, alterative, Amino acids, minerals, Antibacterial activity
anacardium antirheumatic, bioflavonoids and
carminative, phenolic compounds
counterirritant,
rubefacient, vesticant
Terminalia Laxative, hair growth, Sitosterol, gallic acid, Antibacterial and
bellirica good digestive property, ellagic acid, chebulagic anti-inflammatory
improves mental faculties acid, galloyl glucose, fatty activity
and enhances the body acid, protein, oxalic acid,
resistance to diseases tannin, palmitic acid,
oleic acid, linoleic acid,
galactose, glucose, ethyl
gallate
(continued)
Hevea For healing wounds, cuts Essential oil and fixed oil, Antibacterial activity
brasiliensis and sores. In the treatment phosphatides and tannins
of parasitic worms
Naravelia Astringent, bitter, Alkaloids, ethanolic Antibacterial activity
zeylanica antipruritic and anti- compounds, sterols
inflammatory,
helminthiasis,
dermatopathy, leprosy,
rheumatalgia, odontalgia,
cephalalgia, colic
inflammation, wound
healing & ulcer protection
Anacardium Good digestion Essential oils, alkaloids Anti-helminthic
occidentale activity, antimalarial
• Obligate endophytes: Organisms that are solely adhered to the inside organelle
of the plants. They do not possess life stages outside the plant host except for
plant-to-plant and plant-to-insect-to-plant transmission.
• Opportunistic endophytes: Organisms that enter the plant host and get benefited
from the internal environment of plants.
• Passenger endophytes: Endophytes that enter the plant by accident in the pres-
ence of selective external forces maintaining it in the internal tissue of the plant
(Hardoim et al. 2008).
Many different groups of organisms, namely, fungi, bacteria, actinomycetes and
mycoplasma, are referred to as endophytes. Endophytes usually habitat in the tem-
perate, aquatic environments and xerophytic. These interact with multiple hosts
other than plant host (Schulz and Boyle 2006).
The endophytes can be isolated from the endangered plants which are biologi-
cally and medicinally important as they produce secondary bioactive metabolite.
The plants that have ethnobotanical history, unique environmental settings, endemic
and unusual biology are considered as a specific host for the study of endophytes.
These are responsible for the production of secondary metabolites with pharmaco-
kinetic properties. Both fungi and bacteria help in the production of bioactive com-
pounds with therapeutic value like anticancer, immunomodulatory, antimicrobial,
antiviral, immunosuppressive and anti-inflammatory properties (Shukla et al. 2014).
Anti-Cancerous Drugs
Taxol, a million-dollar drug, is produced by the endophytic fungus named Taxomyces

andreanae which is used in effectively combating cancer and other tissue misfold-
ing diseases. The fungus was first discovered in the plant host, Taxus brevifolia.
Later on, various other species of the plant were known to produce taxol such as
Seimatoantlerium tepuiense, Tubercularia sp., Taxus wallichiana, Periconia sp.,
Corylus avellana, etc. (Strobel and Strobel 2007). The other most important alka-
loid used in cancer treatment is ‘camptothecin’ an effective antineoplastic agent first
isolated from the wood of the tree named Camptotheca acuminata Decaisne
(Nyssaceae) which destroys cancer cells via apoptosis. “Gliocladicillins A” and “B”
were notifiable antitumour agents in vitro and in vivo, since they accelerated tumour
cell apoptosis as well as showed inhibition on proliferating melanoma B16 cells
which were introduced into immunodeficient mice (Shukla et al. 2014).
Antimicrobial Drugs
The secondary metabolite isolated from some of the medicinal plants showed anti-
bacterial activity. Sixty-three endophytic fungi were isolated from the rhizome of
medicinal herb named Paris polyphylla var. yunnanensis, of which five showed
strong antibacterial activity against four Gram-negative bacteria (Xanthomonas
vesicatoria, Agrobacterium tumefaciens, Escherichia coli and Pseudomonas lach-
rymans) and two Gram-positive bacteria (Bacillus subtilis, Staphylococcus haemo-
lyticus) (Yu et al. 2010).
Antiviral Drugs
Many novel antiviral agents are obtained from endophytic fungi. Human cytomega-
lovirus (hCMV) protease inhibitors, cytonic acids A and B, were isolated from
solid-state fermentation of the endophytic fungus Cytonaema sp. Mellisol, and
1,8-dihydroxynaphthol 1-O-a-glucopyranoside was isolated from the endophytic
fungus named Xylaria mellisii, which acts against herpes simplex virus-type 1. The
aryl tetralin lignans such as podophyllotoxin and its analogues showed antiviral and
cytotoxicity activities and are used as the precursor for many drugs used for the
treatment of cancer and viral infections, like etoposide, ectolophs phosphate and
teniposide. Podophyllotoxin are produced by various fungal endophytes such as
Phialocephala fortinii, Trametes hirsute, Aspergillus fumigates and Fusarium oxy-
sporum (Behera et al. 2013).
Immunomodulatory Drugs
The immunomodulatory compounds are commonly classified into two groups such
as immunosuppressive and immune regulatory drugs. Due to the emergence of auto-
immune disorders, the need for new immunosuppressive drugs has risen consider-
ably. Immunosuppressive drugs are mainly used to prevent rejection of allograft in
transplant patients and also to treat autoimmune diseases like rheumatoid arthritis
and insulin-dependent diabetes. Two important immunosuppressive compounds
subglutinol-B and subglutinol-A are composed of non-cytotoxic diterpene pyrones.
These compounds were isolated from the fungal endophyte named Fusarium sub-
glutinans, inhabiting on the barks of tree, Tripterygium wilfordii. These compounds
are non-toxic and used as a main compound in drug formulation of thymocyte pro-
liferation (TP) assays and mixed lymphocyte reaction (MLR). Another important
immunosuppressive drug, cyclosporine-A (C62H111N11O12) produced by an
endophytic fungus, is 104 times more potent in the thymocyte proliferation assay
and roughly as potent in the mixed lymphocyte reaction (MLR) assay. Mycophenolic
acid (C17H20O6) is another reported potent immunosuppressive fungal metabolite
used for the treatment of organ transplantations and autoimmune diseases. This
compound has been reported to be produced by Penicillium sp., Aspergillus sp.,
Byssochlamys sp. and Septoria sps. (Dutta et al. 2014b). Endophytes and their phy-
tochemicals are listed in Table 16.13.
Molecular Targets and Mechanisms
Liu (2004) reported the use of phytochemicals, targeting the tumour cells which
promote differentiation of cells, immune system stimulation, hormone metabolism
regulation, antibacterial activity, DNA binding prevention, steroid hormone metab-
olism regulation and antiviral effects.
I nhibition of Oncogene Expression/Induction of Tumour

Suppress Gene Expression
There are, namely, two sets of genes responsible for controlling the action of can-
cerous cells – oncogenes and tumour suppressor genes. Noscapine, a phytochemical
produced by endophyte, is found to enhance the Bax expression and thereby signifi-
cantly decrease the production of bcl-2 in apoptosis-proficient HL60 cells, myeloid
cells and apoptosis-resistant K562 cells (Heidari et al. 2007).
Induction of Cell Cycle Arrest
The cell cycle consists of four different phases – G1, S, G2 and M phases – in most
of the living systems. All the phases of cell proliferation consist of specific check-
points to ensure the efficiency and accuracy of DNA replication. Flavonoids, a sec-
ondary metabolite produced by endophytes, have been reported to inhibit the
cancerous cell proliferation by inhibiting or arresting cell cycle progression at the
checkpoint, G1/S phase. Another report suggests the use of luteolin, a phytochemical
Table 16.13 List of various bioactive compounds from endophytic organisms and their use
against pathogenic microorganisms
Source of Bioactive compounds
endophytes from endophytes Against pathogen References
Boesenbergia Munumbicins Escherichia coli Golinska et al. (2015)
rotunda
Streptomyces Singh and Dubey (2015)
coelicolor
Cladosporium sp. Cardiac glycosides, Klebsiella Selvi and
phenolic compounds pneumoniae Balagengatharathilagam
(2014)
Cytonaema sp. Cytonic acids A and B Human Bhardwaj and Agrawal
cytomegalovirus, (2014)
Hepatitis virus
Diaporthe Fabatin, tyrosol Enterococcus hirae Godstime et al. (2014) and
helianthi Specian et al. (2012)
Fusarium Beauvericin Clostridium Meca et al. (2010)
proliferatum botulinum
Ganoderma Rapamycin, Bacillus subtilis Parthasarathi et al. (2012)
boninense cyclododecane, and Ismail et al. (2014)
petalostemumol
Hypericum Hypericin, emodin, Salmonella sp. Joseph and Priya (2011) and
perforatum, tyrosol Specian et al. (2012)
Diaporthe
helianthi
Nigrospora sp. Saadamycin Saadamycin El-Gendy and El-Bondkly
(2010) and Dutta et al.
(2014b)
Saccharothrix Capreomycin Mycoplasm (TB) Golinska et al. (2015)
mutabilis, Munumbicins
Streptomyces sp.
Streptomyces Coronamycin, Saccharomyces Ezra et al. (2004) and
hygroscopicus rapamycin cerevisiae Parthasarathi et al. (2012)
Streptomyces sp. Kakadumycin A, Shigella sp. Joseph and Priya (2011)
hypericin
Xylaria sp. Dihydroxynaphthol, Herpes virus Pittayakhajonwut et al.
glucopyranoside (2005)
compound produced by endophyte for the arrest of the cell cycle process during
prostate cancer cells and the human gastric process (Lin et al. 2008).
Induction of Apoptosis
Apoptosis is a natural phenomenon of cell programmed death for maintaining the

homeostasis. A proper cell programmed death can lead to the cancer therapeutic
pathway. There are two pathways for apoptosis to occur: the mitochondrial (intrin-
sic) pathway and the death receptor pathway (extrinsic) (Ferreira et al. 2002;
Li-Weber 2013). The phytochemical compound, berberine, plays an important role
in activating both intrinsic and extrinsic apoptosis pathways in the cell. It induces
apoptosis through the expression of caspases such as 8, 9 and 3 (apoptosis-inducing
factor).
Inhibition of Cell Adhesion/Invasion/Metastasis
Matrix metalloproteinases (MMPs) play a vital role in the metastasis and invasion
of cancer cells. They are required for the therapeutic process against diagnosing
cancer. The phytochemical compound, silibinin, also helps in regulating the expres-
sion of MMP-2 in lung and tongue cancer cells (Chu et al. 2004; Chen et al. 2006;
Ramasamy and Agarwal 2008).
Enzyme Induction/Inhibition
Phytochemical compounds have the ability to induce the production of phase I

enzymes (helps in blocking the activation of carcinogenesis) which also helps in
inhibiting phase II enzymes (usually overexpressed in malignant tumours).
Phytochemical compounds, apigenin and nobiletin help in blocking the COX-2
enzyme responsible for transcriptional activity in breast cancer cells (Murakami
et al. 2000; Yi Lau and Leung 2010).
ole of Phytochemical Compounds in Health

R
and Pharmaceutical Industries
Epidemiological reports suggest that phytochemical compounds play a pivotal role

in treating cardiovascular diseases and cancer. They also possess various pharmaco-
logical effect such as anti-inflammatory, analgesic, antiallergic, CNS stimulant,
neuroprotective, hypotensive agents, chemopreventive, antioxidant, antibacterial,
antifungal, antispasmodic, immunomodulator, hepatoprotective, hypolipidemic,
prevent osteoporosis, etc. (Prakash and Gupta 2009). Recent advancement in phar-
maceuticals includes the use of phytochemicals in nano-based drug delivery sys-
tems (Table 16.14).
Table 16.14 List of phytochemicals and their molecular target actions

Phytochemical
compounds Sources Molecular targets Reference
Camptothecin- alkaloid Nothapodytes foetida Affect DNA topoisomerase Wink
Gallic acid – Phenols Scrophulariaceae Induction of cell arrest (2003)
Triterpenes – Terpenoid Brassicaceae sp. Inhibition of cell invasion
Quinolizidine alkaloids Solanaceae Induction of apoptosis
rostratum
Pyrrolizidine – Quinone Adenocarpus Inhibition of enzyme
production
Canavanine – Saponin Phellinus robiniae Inhibition of metastasis stage
Types of Nanocarriers Commonly Used
Encapsulation of phytochemicals using block copolymer micelles, nanospheres or

nano-capsules, liposomes and solid lipid nanoparticles are the commonly used
types of nanocarriers.
Encapsulation of some of the phytochemical compounds and their role in the
diagnosis of chronic diseases are as follows:
Apigenin (Flavonoid)
These phytochemicals have shown a good therapeutic property in cancer treatment.

They are abundantly found in celery, parsley and chamomile tea. A recent report
suggests the use of apigenin to impart apoptosis in human breast, prostate and lung
cancer cells (Zhai et al. 2013). They have been classified as BCS class II drug.
Artemisinin and Dihydroartemisinin (Terpene)
These phytochemicals contain antimalarial drug and chemotherapeutic potential.

Neda et al. (2011) prepared a drug encapsulated with liposomes using cholesterol,
artemisinin and phosphatidylcholine. This nano-micelle (nano drugs) are generally
used to treat specific cancerous cells (Gabriels and Plaizier-Vercammen 2004).
Triptolide (Terpene)
Triptolide (TRP), a diterpenoid triepoxide, is a purified component of a shrub-like

vine named Tripterygium wilfordii Hook F (Mei et al. 2003). Triptolide inhibits the
proliferation and colon cancer cell migration, thereby inducing apoptosis of human
pancreatic cancerous cells and suppressing invasion of human thyroid carcinoma

cells and angiogenesis. Xu et al. (2014) constructed triptolide-loaded MPEG-PLA
copolymer micelles by the solvent evaporation method with an average size of
78.9 nm and encapsulation efficiency of 66.7%.
Challenges and Future Prospects
Phytochemicals have showed good biological property such as anticancer agents

and other therapeutic conditions. These compounds in combination with the exist-
ing drug can overcome the drawbacks and reach maximum efficacy. Recurrent
problems such as their low solubility, cytotoxicity, rapid release and side effects
have to be addressed. The use of nanocarriers provides a new pathway to valorize
these phytochemicals. A number of such systems have progressed to undergo clini-
cal trials, and others are still under supervision in in vitro studies. There are many
issues that still need to be verified such as using varying pharmacokinetics profile/
biodistribution/metabolism among different drugs, control of drug ratios, stability
of drugs inside the nanocarriers, control of drug leakage, etc.
Conclusion
Medicinal plants have gained importance in the field of medical research. Secondary
metabolites produced by endophytic microorganism’s aid in treating and preventing
several diseases. Some of the novel phytochemicals can pave a route towards the
production of drugs for cancer treatment and various other diseases. Bioactive com-
pounds produced by endophytic microorganisms play a pivotal role in research
institutes and pharmaceutical industries for the manufacture of new drugs. The
abovementioned phytochemicals are useful in binding to the particular region of the
virus or bacterial or fungal genetic material, thereby inhibiting the process of repli-
cation and other metabolic pathways. Recent advancements in pharmaceuticals
employ the use of phytochemical in nano-based drug delivery systems. However,
phytochemical nano-based drug delivery system is yet to be explored to meet the
future pharmaceutical challenges.
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Part II
Drug Discovery, Pharmaceutical Sciences
and Future Medicine
Chapter 17
Plant-Mediated Green Synthesis
of Nanoparticles
Balaprasad Ankamwar, Saili Kirtiwar, and Amritesh C. Shukla
Introduction
Human life has been influenced by the development of nanotechnology.

Nanotechnology is the branch of science which deals with the multiple aspects of
nanoparticles (Dauthal and Mukhopadhyay 2016a, b). Nanoparticles have been
known to have various physiochemical properties which have diverse from their
bulk counterparts. Nanoparticles have unique properties because of spatial confine-
ment, large fraction of surface atoms, reduced imperfections, high surface energy,
surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS),
plasmon light scattering, surface-enhanced Rayleigh scattering (Jain et al. 2007).
Because of these properties, nanoparticles have electronics, biological optoelec-
tronics, and chemical-sensing applications (Wong and Schwaneberg 2003;
Ramanavicius et al. 2005).
There are two approaches for synthesis of nanoparticles (Narayanan and
Sakthivel 2010):
1. Top-down approach
2. Bottom-up approach
In the top-down approach, the precursor bulk material is reduced to nano-size
with the help of biological, physical, or chemical methods (Mittal et al. 2013).
Controlled tools are used externally for milling, cutting, and shaping the materials
into the desired shape and size. This approach method includes pyrolysis (Tsai et al.
B. Ankamwar (*) · S. Kirtiwar

Bio-Inspired Materials Research Laboratory, Department of Chemistry, Savitribai Phule Pune
University (Formerly University of Pune), Pune, Maharashtra, India
e-mail: bgankamwar@chem.unipune.ac.in
A. C. Shukla

https://doi.org/10.1007/978-981-15-2195-9_17
222 B. Ankamwar et al.
Fig. 17.1 Approach for synthesis of nanoparticles
2004), thermolysis (Shubin et al. 2012), radiation-induced methods (Treguer et al.

1998), and lithography (Li et al. 1999). The major drawback of this approach is that
the surface structure of nanoparticles is an impact which alters their surface chem-
istry and physical properties of the metallic nanoparticles. Nevertheless, high tem-
perature and pressure maintenance are required for this synthesis process (Treguer
et al. 1998).
The bottom-up approach is also called as “self-assembly” approach. In this
method, the nanoparticles are formed due to the assembly of atoms and molecules.
These nanoparticles are then assembled into finished material using biological and
chemical methods. In this method, the surface impaction is less, and homogeneous
nanoparticles are synthesized at cheaper cost. This approach is used for wet synthe-
sis procedures like sonochemical (Nemamcha et al. 2006), electrochemical (Yang
et al. 2013), and polyol reduction (Xiong et al. 2005). This method uses toxic chemi-
cals, synthetic capping agents, and nonpolar solvents which make their applications
limited in clinical and biomedical field. Due to the use of these toxic chemicals in
synthesis procedure, it adversely affects the environmental and living cells (Gangula
et al. 2011; Devi et al. 2016). Due to these drawbacks, the researchers are now mov-
ing on to the green method for synthesis of nanoparticles. The use of plant materials
for synthesis of nanoparticles is receiving attention since recent years (Fig. 17.1).
Plants perform photosynthesis to convert light energy from the sun into chemical
energy. Thus, fabrication of nanoparticles uses plants, and plant materials as a
renewable resource plants are known to synthesize secondary metabolites. These
17 Plant-Mediated Green Synthesis of Nanoparticles 223
molecules have the potential to fabricate the nanoparticles. The use of plant
resources is beneficial than microbial system (Rai et al. 2008). Microbial system
requires maintenance and downstream processing (Narayanan and Sakthivel 2011).
Plant-Mediated Synthesis of Nanoparticles
Plant materials fabricate nanoparticles by uptaking, accumulating, utilizing, and

recycling different mineral species (Parajuli et al. 2007). This is fast method for
synthesis of nanoparticles, and nanoparticles can be produced in bulk amount with
high stability. Plant-based synthesis of nanoparticles uses aqueous medium for fab-
rication of nanoparticles and requires room temperature for its synthesis, which
saves huge amount of energy (Chen et al. 2003).
The advantages of plant-based synthesis of nanoparticles include that this method
uses aqueous solvents; plant material is easily available; extracts obtained from
plant species are biocompatible; it used simple procedure for synthesis of nanopar-
ticles; this method uses normal temperature and pressure; plant components act as
stabilizing as well as reducing agents; and it is cost-effective method, eco-friendly,
and safe method for therapeutic use; and suitable for large-scale production.
Fabrication of nanoparticles by biomolecules obtained from the plant is currently
under exploration. Nanoparticles are synthesized intracellular (in living plants),
extracellular (using plant extracts), and by using individual phytochemicals. This
method is gaining significant importance as compared to physical and chemical
methods (Iravani 2011). Plant-mediated approach for synthesis is shown in Fig. 17.2.
Plant-mediated method for synthesis of nanoparticles has produced nanoparticles
with various shapes and sizes. In this method, low-cost agricultural wastes materials
are utilized for synthesis of nanoparticles (Gan and Li 2012). To control the mor-
phology, size, and shape of nanoparticles, we need to concentrate on the individual
phyto-constituent (proteins, polyphenol, and organic acids) for the fabrication of
nanoparticles (Basha et al. 2010; Tamuly et al. 2014).
Approaches for Plant-Mediated Synthesis of Nanoparticles
iving Plant-Mediated Synthesis of Nanoparticles (Intracellular

L
Route)
Many plants species have shown to have potential for detoxification and heavy
metal accumulation (Yang et al. 2005; Milner and Kochian 2008).. The biomole-
cules present in plant extract show variation in stabilizing and reducing potential
due to which there can be variation in the size, shape, and properties of n anoparticles.
Nanoparticles are characterized based on the size, shape, and its composition. Even
Fig. 17.2 Methods for plant-mediated synthesis of nanoparticles
by using single plant species, polydispersed nanoparticles with diverse structures

have been synthesized through intracellular route. Diversity, stabilization and reduc-
ing agents contribute to the variation in morhology and polydispersity. Metal trans-
location also contributes to the variation of nanoparticles. The separation and
purification of nanoparticles synthesized by this route become a challenging task,
and this also contributes to the morphological diversity. The recovery of nanoparti-
cles synthesized by this route is tedious and time-consuming (Fig. 17.3). Table 17.1
summarizes the synthesis of different nanoparticles using intracellular route.
lant Extract–Mediated Synthesis of Nanoparticles

P
(Extracellular Route)
In this method, extracts from plants are extracted by using hot or cold extraction
methods or by using Soxhlet apparatus and are utilized for synthesis of nanoparti-
cles (Fig. 17.4). The crude phyto-constituent present in plant extract is responsible
for synthesis of nanoparticles. Since in this method the phyto-constituents are
extracted and used externally for synthesis of nanoparticles, it is known as
extracellular method for synthesis of nanoparticles. Nowadays, this method is
Fig. 17.3 Intracellular route for synthesis of nanoparticles
Table 17.1 Living plant-mediated synthesis of nanoparticles (intracellular route)

Size
Plant source Nanoparticles (nm) Shape
M. sativa (Gardea-Torresdey et al. Au 2–20 Icosahedron
2002)
Brassica juncea (Harris and Bali 2008) Ag 50 Roughly spherical
Chilopsis linearis (Gardea-Torresdey Au 1.1 Spherical
et al. 2005)
Avena sativa (Armendariz et al. 2004) Au 25–85 Tetrahedral, decahedral,
hexagonal
Medicago sativa (Gardea-Torresdey Ag 2–4 Icosahedron
et al. 2003)
mainly suitable for synthesis due to its easy downstream processing and scaling up.
Nanoparticles synthesized using this method have many potential commercial
applications. By using extracellular route, it is difficult to synthesize mono-
dispersive nanoparticles due to variation in phytochemical composition. This
method is renewable, nontoxic, biocompatible, and eco-friendly for fabrication of
nanoparticles. Due to its biocompatibility of these nanoparticles, it is known to have
various biological applications. Bio-fabrication method for synthesis works best
with metal ions that have high positive electrochemical potentials. Hence, this
method works best for synthesis of silver and gold nanoparticles (Haverkamp and
Marshall 2009). This method is known to synthesize spherical nanoparticles with
high reactivity and hence known to have various commercial applications.
Extracellular route for synthesis of nanoparticles is a controlled equilibrium pro-
Fig. 17.4 Plant extract mediated synthesis of nanoparticles
cess, which favors the synthesis of nanoparticles up to the specific structure and
shape. The synthesized nanoparticles have been known to have high negative poten-
tial and are highly stable in water. Table 17.2 summarizes the synthesis of nanopar-
ticles using different plant sources by extracellular route.
Phytochemical-Mediated Synthesis of Nanoparticles
In plant extract mediated synthesis of nanoparticles, it is extremely difficult to pre-

dict the exact mechanism involved in nanoparticle synthesis, due to the presence of
variation in phytochemical constituents (Ahmed et al. 2014). Hence, researchers are
trying to isolate the respective phytochemical and use them for synthesis of nanopar-
ticles to get control on size and shape of nanoparticles. It is predicted that flavonoids
and polyphenols are involved in synthesis of nanoparticles. Table 17.3 summarizes
the phytochemical-mediated synthesis of nanoparticles.
Table 17.2 Plant extract–mediated synthesis of nanoparticles (extracellular route)

Size
Neolamarckia cadamba (fruit) Ag 10 Spherical
(Ankamwar et al. 2015a, b; Kirtiwar et al.
2018)
Cordia myxa (Ankamwar et al. 2016a, b, Ag 50– Triangular and hexagonal
c) 150
Murraya koenigii (Ankamwar et al. Ag – –
2016a, b, c)
Emblica officinalis (Ankamwar et al. Ag 10–20 Spherical
2005)
Shikakai and Reetha (Ankamwar et al. Ag 50 Spherical
2018)
Averrhoa bilimbi/fruit (Isaac et al. 2013) Ag 75– Hexagonal and rhomboidal
150
Rosa rugosa/leaf (Dubey et al. 2010) Ag 12 Spherical, triangular, and
hexagonal
Chenopodium album/leaf (Dwivedi and Ag 10–30 Quasi-spherical
Gopal 2010)
Cassia roxburghii/leaf (Muthukumaran Ag 57–95 Spherical triangle, truncated
et al. 2015) triangles, and decahedral
Aloe vera/leaf (Medda et al. 2015) Ag 287– Cubical, rectangular,
293 triangular, and spherical
Neolamarckia cadamba (flower) Au 50 Triangular
(Ankamwar et al. 2015a, b)
Plumbago zeylanica (leaf) (Ankamwar Au 60–80 Triangular, spherical
et al. 2016a, b, c)
Piper betle (leaf) (Gaware et al. 2012) Au 660 Triangular
Saraca indica/bark (Das et al. 2011) Au 15–23 Triangular
Allium sativum/clove (Coman et al. 2014) Au 78 Spherical
Nyctanthes arbor-tristis/flower (Li et al. Au 19–20 Spherical
1999)
Rosa damascena/flower (Ghoreishi et al. Au 15–16 Quasi-spherical
2011)
Prunus armeniaca/fruit (Dauthal and Au 20 Spherical and irregular
Mukhopadhyay 2013a, b)
Terminalia chebula/fruit (Kumar et al. Au 6–60 Triangular, pentagons, and
2012) spherical
Ananas comosus/fruit (Basavegowda et al. Au 5–15 Spherical
2013)
Rosa rugosa/leaf (Dubey et al. 2010) Au 11 Spherical
Diospyros kaki/leaf (Song et al. 2010) Pt 2–12 Spherical
Cacumen platycladi/whole plant (Zheng Pt 2–3 Spherical
et al. 2013)
Punica granatum/peel (Dauthal and Pt 16–23 Spherical
Mukhopadhyay 2015)
(continued)
Size
Citrullus lanatus/rind (Lakshmipathy Pd 96–98 Spherical
et al. 2015)
Musa paradisiaca/peel (Bankar et al. Pd 50 Irregular
2010)
Curcuma longa/tuber (Sathishkumar et al. Pd 10–15 Spherical
2009)
Ulmus davidiana/bark (Mishra et al. Pd 5 Spherical
2015)
Delonix regia/leaf (Dauthal and Pd 2–4 Irregular
Mukhopadhyay 2013a, b)
Table 17.3 Phytochemical-mediated synthesis of nanoparticles

Size
Psidium guajava/guavanoic acid (Basha et al. Au 4–24 Spherical
2010)
Desmostachya bipinnata/β-sitosterol-D- Ag 8–60 Diverse shape
glucopyranoside (Ahmed et al. 2014)
Solanum torvum/β-glucosidase (Govindaraju et al. Au 8–22 Spherical
2011)
Vitex negundo/sodium parahydroxybenzoate Ag 26–39 Spherical
tetrahydrate (Durai et al. 2014)
Lawsonia inermis/apiin (Kasthuri et al. 2009) Au and Ag 21 and Spherical and
39 anisotrophic
Pelargonium graveolens/geraniol (Safaepour et al. Ag 1–10 Spherical
2009)
C. platyclad/leaf (Zhan et al. 2011) AuPd 6–8 Spherical
Euphorbia condylocarpa/root (Nasrollahzadeh AuPd 80 –
et al. 2014)
Delonix regia/leaf (Dauthal and Mukhopadhyay AuPd 3–31 Spherical
2016a, b)
Silybum marianum/seed (Gopalakrishnan et al. AuAg 3.5–8.1 Spherical
2015)
Table for Use of Plant as Source for Nanoparticles Synthesis
Table 17.1: Living plant-mediated synthesis of nanoparticles (intracellular route)

Table 17.2: Plant extract mediated synthesis of nanoparticles (extracellular route)
Table 17.3: Phytochemical-mediated synthesis of nanoparticles
robable Mechanism for Synthesis of Nanoparticles by Plant

P
Constituents
Synthesis and fabrication of nanoparticles require stabilizing agents, solvent

medium, and reducing agents (Vijayaraghavan and Nalini 2010). When plant extract
is used for synthesis of nanoparticles, they act as reducing as well as stabilizing
agents. Plant-mediated synthesis of nanoparticles is done mainly in aqueous medium
and therefore is termed as green process for synthesis of nanoparticles. Due to high
amount of phytochemicals present in plant extract, it becomes difficult to identify
the exact reducing and stabilizing agents required for fabrication of nanoparticles.
The possible phyto-constituent acting as reducing and stabilizing agents is described
as follows.
Flavonoids
It is a water-soluble secondary metabolite present in plants. Flavonoid consists of

flavanones, flavans, flavanonols, isoflavonoid, anthocyanidins, and anthoxanthins.
They are considered as important bio-reducing agents present in plant extract. The
scavenging capacity of molecular oxygen present in flavonoids is directly related to
their electron-donating ability, hence, acting as potential reducing and stabilizing
agents (Pietta 2000).
Phenolic Acid
They belong to the group of polyphenols and contain an organic carboxylic acid and
a phenolic ring. This phenolic acid has the capability to chelate different metal ions
due to its highly nucleophilic aromatic ring and hence can be used as bioreducing
agents for the fabrication and synthesis of nanoparticles. Phenolic acids such as caf-
feic acid (Aromal et al. 2012), protocatechuic acid (Kumar et al. 2012), and gallic
acid (Huang et al. 2010) are reported for synthesis of nanoparticles.
Terpenoids
Terpenoids are group of secondary metabolites produced by the plants. The hydroxyl
functional group in these molecules is known for bioreduction of metal ions to form
nanoparticles (Shankar et al. 2003).
Proteins
Proteins have free amino and carboxylic group which has the ability to bind to
nanoparticles and also bring about the reduction of metal ions to form nanoparticles
(Raghunandan et al. 2009).
Organic Acids and Alkaloids
Due to the presence of carboxylic group and hydroxylic group, these molecules can
bring about reduction of metal ions and bio-fabrication of nanoparticles.
Conclusion
Nanoparticles can be synthesized by using three approaches using plants: first is the
living plant or intracellular route-mediated synthesis of nanoparticles, second is the
plant extract–mediated synthesis of nanoparticles, and third is the phytochemical-
mediated synthesis of nanoparticles. The latter two approaches are extracellular
route for synthesis of nanoparticles. The plant contains primary and secondary
metabolites such as proteins, flavonoids, terpenoids, organic acids, alkaloids, etc.,
which act as bioreducing and fabricating agents for synthesis of nanoparticles.
These synthesized nanoparticles have activities such as antibacterial activity, anti-
fungal activity, anticancer activity, and so on. Due to these immense activities, such
nanoparticles are known to have various biomedical and electronic applications.
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Chapter 18
Biomedical Applications of Green
Synthesized Nanoparticles
Reetika Singh, Priyanka Tiwari, Nishi Kumari, and Bechan Sharma
Abbreviations
NPs Nanoparticles
AgNPs Silver nanoparticles
FTIR Fourier transform infrared spectroscopy
XRD X-ray diffraction
SEM Scanning electron microscope
TEM Transmission electron microscope
Introduction
The field of nanotechnology is the most vigorous and dynamic research area
(Sergeev and Shabatina 2008). Nanotechnology deals with the fabrication, charac-
terization, and management of materials at the nanoscale to synthesize the nanopar-
ticles of desired shape and size. Fabrication and practice of nanoparticles are
increasing exponentially due to its extensive applications range in the field of elec-
tronics, biosensors, healthcare, biotechnology including bio-nanotechnology, etc.
(Mehata 2015; Ratnesh and Mehata 2015).
Green synthesis method comprises NPs synthesis with plants (Makarov et al.
2014; Mittal et al. 2013), microorganisms (bacteria, fungus, yeasts (Narayanan and
Sakthivel 2010)), and DNA (Sohn et al. 2011). A number of bacterial species and
fungi have been scrutinized for the development of NPs of various composition and
R. Singh · P. Tiwari · B. Sharma (*)

N. Kumari
Botany Section, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India

https://doi.org/10.1007/978-981-15-2195-9_18
236 R. Singh et al.
size such as synthesis of gold by Verticillium sp, CdS quantum dots synthesis using
fungi, etc. (Ahmad et al. 2002; Narayanan and Sakthivel 2010). Different plant
materials such as stem, leaves, roots, etc. (Jha et al. 2009) for the NPs synthesis
exposed a new avenue that is comparatively unmapped. Green synthesis method of
NPs has more biocompatibility for medical use when compared to the various other
physical and chemical methods wherever toxic material may adsorb/accumulate on
the surface of NPs that may cause adverse effect during medical use (Verma and
Mehata 2016).
Synthesis of Nanoparticle
To obtain the NPs with desired shape and size, various physical and chemical meth-
ods have been established for the NPs synthesis, though these methods are costly
and environment unfriendly. Wide range of applications support the numerous
methods regarding the production of silver nanoparticles along with various silver-
based compounds containing ionic silver or metallic silver (Singh et al. 2014).
Green synthesis method has been measured as most favorable method for NPs syn-
thesis due to its high biocompatibility, little toxicity, and cost-effective nature
(Malik et al. 2014). The practice of plant extract for the synthesis of NPs (silver,
gold, chitosan) offers ample benefits of compatibility and cost-effectiveness for
pharmaceutical, biomedical, and other applications (Jain et al. 2009). Green synthe-
sis method of NPs has been used with a number of plants/plant extracts of Parthenium
hysterophorus, Pelargonium graveolens, Medicago sativa, Azadirachta indica, lem-
ongrass, Aloe vera, and Cinnamomum Camphora (Parashar et al. 2009; Jha et al.
2009; Mittal et al. 2013; Makarov et al. 2014). This method is alternative to conven-
tional methods and concentrated the attention of scientific workers. Plant extracts/
natural product-based NPs synthesis will be additional effective with lesser side
effects because natural products cause negligible side effects and more efficacy due
to its natural origin (Singh and Kumari 2015; Singh et al. 2015). Hence, there is an
urgency to opt the green synthesized method instead of conventional physical and
chemical methods.
Characterization of Nanoparticles
Characterization of nanoparticles is a major step to optimize the synthesis of

nanoparticles. Appropriate size and shape of nanoparticles enhance the efficacy of
activity. On the basis of different factors such as different functional groups, light
absorption and scattering, potentials, etc., various techniques have been used to
characterize the synthesized NPs.
18 Biomedical Applications of Green Synthesized Nanoparticles 237
UV-VIS Spectroscopy
UV-VIS spectrophotometer is a primary instrument to characterize the NPs. The

scanning range for the NPs is 300–700 nm. The double distilled water is used as a
blank reference. The UV-VIS absorption spectra showed the clear difference
between the control and NPs. A specific peak for the synthesis of nanoparticles
(silver) ranges from 420 to 430 nm.
Fourier Transform Infrared Spectroscopy (FTIR)
Fourier transform infrared spectroscopy works on the principle of that the most of
molecules have the ability to absorb the infrared waves of electromagnetic spec-
trum. This amount of absorption relates precisely to the bonds present in the mole-
cule. The frequency ranges are measured as wave numbers typically over the range
of 4000–600 cm−1. FTIR is a sensitive technique to identify the organic chemicals
in a whole range of applications although some inorganics can also be characterized
such as resins, polymers, coating drugs, etc. Isolation and characterization of
organic contamination are easy by using this technique. For the characterization of
NPs, dried powder of NPs has been used. FTIR spectrometer analysis detects the
different functional groups by showing peaks from the region of 4000 cm−1 to
600 cm−1. FTIR analysis has shown the presence of bands due to aldehydic C–H
stretching, carbonyl group, N–H bend, C–O stretch (dialkyl) and C–O stretching
(carboxylic acid), etc. (Singh et al. 2014).
X-Ray Diffraction (XRD)
X-ray diffraction is another important technique to confirm the nanoparticles syn-

thesis by the characteristic peaks. The control samples (lacking NPs) did not show
the characteristic peaks, while the samples that contain the NPs showed the charac-
teristic peaks and the presence of crystalline materials. The XRD pattern of these
peaks indicates the crystalline nature of NPs, and some of the nonspecific peaks
may also be present. These peaks may be due to the un-crystallization of few bio-
molecules/plant extracts. Sharp bands of Bragg peaks showed the NPs synthesis. It
was establish that the average size from XRD data using Debye-Scherrer equation
was 60.89 nm (Anuj and Ishnava 2013).
238 R. Singh et al.
canning Electron Microscopy (SEM) and Transmission

S
Electron Microscopy (TEM)
Scanning electron microscopy and transmission electron microscopy are one of the
best techniques to determine the shape and size of NPs. In SEM, the shape and size
of NPs are determined by the scanning of the NPs, while the TEM works on the
basis of electron transmission.
Dynamic Light Scattering (DLS) and Zeta Potential
Dynamic light scattering and zeta potential measurements can determine the size
distribution or average size of the NPs. For DLS analysis, much diluted sample was
used in appropriate molarity of phosphate buffer solution of pH 7.4. The measure-
ments have taken in the range from 0.1 to 10,000 nm (Singh et al. 2014).
Biomedical Applications of NPs
NPs are showing several pharmacological activities and frequently used in biomedi-
cal applications (Fig. 18.1). Among all biological activities, anti-oxidative, antibac-
terial, antifungal, and cytotoxic activities are the more common and frequently
showed by silver NPs. Although other nanoparticles have also shown these biologi-
cal activities, percentage is very low. Immunomodulation and wound healing are the
most recent aspects of application of green synthesized NPs. All these biological
activities are described in detail in the coming paragraphs.
Antioxidants Activity
The human body is continuously producing the free radicals from various anabolic
and metabolic pathways, and these free radicals initiate the lipid peroxidation and
cell damage and may activate the initiation factors of several diseases. Simultaneously
several anti-oxidative molecules and enzymes are also present in our body to deac-
tivate/neutralize these harmful free radicals. Plant-based antioxidants are highly
beneficial for human’s health and serve as nutraceuticals and pharmaceuticals
(Valko et al. 2007). Ascorbic acid, carotenoids, vitamins, flavonoids, and phenolic
compounds, etc. are potential natural antioxidants and are commonly present in
nature (Ghani 2003). Advances in plant biochemistry and herbal medicines carry
forward the insistence of scientific/experimental evidence to support the benefits
gained from antioxidants (Singh and Kumari 2015). Most of the medicinal plants
Anticancer
Wound
Antifungal
healing
Biomedical
applications
of
nanoparticles
Immuno-
Antibacterial
modulation
Antioxidant
Fig. 18.1 Biomedical applications of biosynthesized nanoparticles
such as Emblica officinale, Citrus sinensis, Sapindus mukorossi, etc. possess variety
of phytochemicals such as phenolic compounds, flavonoids, tannins, terpenes, vita-
mins, etc. which are showing antioxidant potential (Singh et al. 2016a, Singh and
Kumari 2020). Several metals such as silver, gold, copper, zinc etc. play an impor-
tant role in diagnosis well as in therapeutic field (Singh and Sharma 2018). The
synergistic effect of plant extracts and NPs proved as a promising implement for
their biomedical applications. The application of nanoparticles as therapeutic agents
has open new prospects for the upgrading of healthcare sector. Biomolecules oxida-
tion for the regulation of oxidative chain reaction and plant plays vigorous role in
synthesis of NPs as they are nontoxic in nature as well as provide natural capping
agents (Arya and Yadav 2011). Several workers have been reported that green syn-
thesized NPs having the antioxidant activity (Thilagavathi et al. 2016; Mitiku and
Yilma 2017). The plants/plant’s extracts which were used for NPs synthesis and
these NPs have shown the antioxidant activity that is summarized in Table 18.1.
Antimicrobial Activity
Presently, increasing antibiotic resistance in microbes is commanding severe threat

to the medical sciences. Antibiotic resistance is a significance of evolution, and this
happens through natural selection. The antibiotic resistance may also arise due to an
240 R. Singh et al.
environmental pressure, action of chemicals, drugs, etc. Among the all NPs, silver
nanoparticles (AgNPs) have shown more antimicrobial/antibacterial effects. Silver
is a nontoxic, safe inorganic antibacterial agent that has the capability to kill approx-
imate 650 types of different diseases causing microorganisms (Jeong et al. 2005);
therefore, scientists are showing their interest in AgNPs for antimicrobial potential
(Choi et al. 2008). The antibacterial activities of AgNPs are related to their size.
AgNPs with the smaller particles showed greater activities on the basis of equiva-
lent silver mass content. NPs have evidenced to be a potent antimicrobial mediator
since their big surface to volume ratio confirms a broad range of attack on bacterial
surface. The complete antimicrobial potential of AgNPs was summarized by Rai
et al. (2009). Gajbhiye et al. (2009) reported the significant antibacterial and anti-
fungal activity from AgNPs against many bacteria and fungus (Escherichia coli,
Staphylococcus aureus, Trichophyton, Trichosporon beigelii, Candida albicans).
Several plants/plant extract has been reported for their antibacterial activity (Singh
et al. 2016b). A number of plants named Azadirachta indica, Cassia roxburghii,
Cleome viscosa, Chenopodium murale, Olea europaea, etc. have been used for the
synthesis of AgNPs, and these NPs have shown the antibacterial potential (Verma
and Mehata 2016). The green synthesized plant-based NPs with the antimicrobial
potential are summarized in Table 18.1.
Table 18.1 Green synthesized NPs and their biomedical applications

Plant’s/fungus Plant parts/ Materials used
S.no. name compounds for NPs synthesis Biological activities References
1. Azadirachta indica Leaf Silver Antimicrobial Verma and
Mehata
(2016)
2. Bergenia ciliata Not Silver Antioxidant, Phull et al.
available cytotoxic, (2016)
antimicrobial
3. Bipolaris Culture Gold, silver Antimicrobial, Fatima et al.
tetramera anticancer, (2015)
immunomodulatory
4. Calliandra Leaf Silver Antioxidants, Raja et al.
haematocephala antibacterial (2017)
5. Cassia roxburghii Leaf Silver Antioxidants, Moteriya
antibacterial et al. (2017)
6. Cleome viscosa Fruit Silver Antibacterial, Lakshmanan
anticancer et al. (2018)
7. Chenopodium Leaf Silver Antioxidant,
murale antibacterial
8. Crocus sativus Wastages Silver Antibacterial Bagherzade
et al. (2017)
9. Curcuma longa Curcumin Poly lactic-co- Anticancer Azandeh
glycolic acid et al. 2017
(continued)
Plant’s/fungus Plant parts/ Materials used
S.no. name compounds for NPs synthesis Biological activities References
10. Lantana camara Leaf Silver Antioxidant, Patil and
antibacterial, and Kumbhar
cytotoxic (2017)
11. Limonia Leaf Silver Antioxidant Thilagavathi
acidissima et al. (2016)
12. Lippia nodiflora Aerial parts Silver Antioxidant, Sudha et al.
antibacterial, and (2017)
cytotoxic
13. Moringa Leaf Silver Antibacterial and Mitiku and
stenopetala antioxidant Yilma
(2017)
14. Musa species Peel Silver Antibacterial Ibrahim
(2015)
15. Nothapodytes Fruit Silver Antimicrobial Mahendran
nimmoniana and Kumari
(2016)
16. Phyllanthus Whole Silver Antimicrobial Singh et al.
amarus plants (2014)
17. Saccharomyces Cell culture Silver Wound healing Kaler et al.
boulardii, Pichia (2014)
anomala,
Rhodotorula
bogariensis,
Candida melibiosa
18. Tinospora Whole Silver Antimicrobial Singh et al.
cordifolia plants (2014)
Cytotoxic Activity
Cancer is most deadly disease and leading cause of deaths around the world.
Radiation therapy and chemotherapy are only the treatment for the cancer but unsat-
isfactory due to serious side effects on healthy and normal cell and the difficulty of
drug resistance (Shi et al. 2006; Azandeh et al. 2017).
Natural products have the capability to cure the cancer without causing any side
effects. These natural plant-based principles may inhibit the metastasis and prolif-
eration of cancerous cells and ultimately cause the apoptosis of cancerous cell.
Taxane, vinca alkaloids, camptothecin, curcumin, etc. are some highly active
anticancer natural compounds (Nobili et al. 2009). The green synthesized NPs hav-
ing these natural compounds showed enhanced anticancer activity.
242 R. Singh et al.
Wound Healing
Wound healing is a natural but multifarious process. The ultimate goal of wound
healing is fast recovery with utmost function and least scarring. Wound healing
takes through a coinciding pattern of actions such as coagulation, inflammation,
proliferation, and remodeling of matrix and tissue. A number of cell-signaling path-
ways are required for the efficient and highly controlled wound healing process.
Although cytokines are a major factor for initiation, maintenance, and regulation of
the post-injury response, these cytokines are also responsible for reduced wound
healing, anomalous scar creation, and unregulated inflammatory response (Tian
et al. 2007). Complete and rapid wound healing depends on the proper delivery of
medicine/ointment. A number of plants/plant’s extracts have been reported for their
wound healing properties. Several workers reported the wound healing property of
NPs. Topically applied AgNPs also showed the wound healing potential (Tian et al.
2007). Nanoparticle-based dressing materials are a new example of improvement in
the field of wound healing research (Berthet et al. 2017). Significant results were
observed on in vivo wound healing from biosynthesis of AgNPs using various bac-
teria (Kaler et al. 2014).
Immunomodulatory Activity
Immunomodulation is a process where an immune response is altered to a desired

level. Microorganisms have the ability to moderate the immune system response to
their presence according to the need. Fatima et al. (2015) have reported the immu-
nomodulatory effect of gold and silver NPs by calculating the reactive oxygen spe-
cies (ROS). Results indicated that both the metal synthesized NPs have the
immune-modulatory capability but AgNPs have more efficiency than gold nanopar-
ticles (Fatima et al. 2015).
Conclusions
Nanotechnology is an exponentially growing area of research with broad range of

implementation. Green synthesized methods of NPs are superior over the other con-
ventional methods. Green synthesized NPs have known for their extensive range of
biomedical applications. Because of the nano-size and large surface area of NPs
enhances the surface of contact, drugs delivery and effectiveness of natural products.
Few limitations like toxicity of metals, time of exposure of nanoparticles, and route
of administration are some factors that must be keep in mind before the extensive
use of NPs. Further research in the nanotechnology and nanoparticles will bring a
new possibility in the medical applications.
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Chapter 19
Role of Nanoparticles and Nanomaterials
in Drug Delivery: An Overview
Manasa Kumar Panda, Sujogya Kumar Panda, Yengkhom Disco Singh,

Bimal Prasad Jit, Rajendra Kumar Behara, and Nabin Kumar Dhal
Introduction
Nanotechnology is a branch of science, engineering, and technology and the study

and application of tiny things, i.e., 1–100 nanometers. In recent years, nanotechnol-
ogy has gained significant momentum worldwide. There are different biomedical
and therapeutic applications for nanoparticles of various sizes. The multidisci-
plinary branch of science includes knowledge from many different fields, including
physics, engineering, and material science; however, from the 1980s, the impact of
nanotechnology on industrial sectors began its application in the biomedical sector
is recent (Kim 2007). Specific size ranging from 1 to 100 nm is produced in such a
way that the properties must be more efficient than the non-nanoscale size of the
same composition (Auffan et al. 2009).
The nanotechnology industry has a wide range of advantages in the field of medi-
cal sector. Due to the small size and large surface area, the nanoparticles improve
M. K. Panda
S. K. Panda
Animal Physiology and Neurobiology, KU Leuven, Belgium
Y. D. Singh
Department of Post Harvest Technology, College of Horticulture and Forestry, Central
Agricultural University, Pasighat, Arunachal Pradesh, India
B. P. Jit · R. K. Behara
N. K. Dhal (*)

https://doi.org/10.1007/978-981-15-2195-9_19
248 M. K. Panda et al.
the biomedical effects of the nanoparticles (Zhang et al. 2008). Both nanobiomedi-
cine and nanomedicine are the advanced fields of nanotechnology to understand
materials and biosystems for the useful application in biomedical sectors such as
direct therapeutic effects, cancer treatment, the delivery of drugs to specific tissues,
and their diagnostic applications (Zhang et al. 2008). The recent developments in
science, medicine, agriculture, and other areas of science that provide new,
improved, and efficient medical procedures lead to the useful applications in bio-
medical sectors, before that knowledge of nanotechnology and its current use in
different aspects must be known first (Schiavone and Tan 2006).
In recent decades, the emerging research theme is on the most efficient and
advanced drug delivery medium using nanoparticles (NP) (Davoudi et al. 2018;
Zheng et al. 2016; Hu et al. 2017; Nguyen et al. 2016). Polymers are versatile chem-
ical compounds; hence polymer nanoparticles are capable of being used in nano-
medicine, drug delivery, and diagnostic methods based on advanced imaging
technology. In the last few decades, researchers focused on the synthesis of poly-
mers with well-defined characteristics and properties. The unique feature of poly-
mer is that it can include various domains that each has different functionality, but
they are arranged in the polymer structure in a well-organized manner (Gao and
Matyjaszewski 2009; Le Droumaguet and Nicolas 2010; Hadjichristidis et al. 2011;
Delplace and Nicolas 2015). This opens the door of new possibilities for exciting
innovations and developing multifunctional, dynamic, and improved nanomedi-
cines (Mura et al. 2013). However, it is essential to thoroughly identify each type of
functionality required for the predetermined application necessarily to define
whether the polymer will serve to enhance the efficacy or possibility of biological
response modulation (Mahon et al. 2012). The designed nanoparticles may be effi-
cient to answer the best use of the nanoparticles in clinical need and to face the
difficult challenge of its approval for clinical use (Merkle 2015).
Inorganic Nanoparticles in Biomedicine
Application of Iron Nanoparticles in Biomedicine
Despite the fact that iron nanoparticles (FeNPs) tend to participate in oxidation-
reduction reactions, the nanoparticles are to be used in various non-oxidative envi-
ronments by excluding oxygen and water. Moreover, under suitable condition,
FeNPs may act as potential catalysts, for the formation and breakdown of the
carbon-carbon bonds, which are difficult to break with conventional biocompatible
materials (Huber 2005).
The magnetic properties can be utilized in the pharmaceutical industry to target
a specific area, where a magnet could be used to attract FeNPs, leading to lowering
of the required dose of the drug (Huber 2005). FeNPs are also used to treat tumors
19 Role of Nanoparticles and Nanomaterials in Drug Delivery: An Overview 249
and cancerous target area. However, heat may be generated when the magnetic field
is introduced (Gilchrist et al. 1965). The generated heat that may be applied for
30 min at 42 °C is capable enough to destroy tumor cells and thus might serve as a
unique and novel way to treat cancer (Stevens et al. 2002). But due to the oxidative
nature of FeNPs, preventive steps need to be taken for the oxidizing environment
(Carpenter 2001).
pplication of Zinc Oxide Nanoparticles (ZnO NPs)

A
in Biomedicine
Zinc oxide nanoparticles (ZnO NPs) have some unique properties such as small
size, unique electric properties, and biologically active nanomaterial feature,
which allow the nanoparticles for biomedical applications such as cell labeling,
tumor targeting, and diagnostics (Loomba and Scarabelli 2013). Due to the speci-
ficity, it allows for more efficient imaging than traditional fluorescent dyes
(Tokumasu et al. 2005). Moreover, specifically, quantum dots (QDs) have the abil-
ity and potentiality to track the location and movement of various cells (Loomba
and Scarabelli 2013).
Apart from cellular imaging, ZnO NPs may also be used in tropical creams and
sunscreen lotions to prevent skin cancer and reduce skin damage by ultraviolet radi-
ations (Nohynek et al. 2007; Yamaki and Yoshino 2009). ZnO NPs can be targeted
to drug delivery systems and also with the attached hydroponics drugs that are not
soluble in water to provide more efficient targeted delivery (Rasmussen et al. 2010).
The effectiveness of the hydroponics drugs is applied in the case of an anticancer
drug as these drugs have serious side effects at high doses (Toda et al. 2010).
Moreover, it is reported that ZnO NPs can also be utilized to treat cancer directly
and to induce toxicity in cancer cells are reducing the effects of toxicity in normal
cells (Hanley et al. 2008).
Application of Cerium Oxide Nanoparticles in Biomedicine
The cerium oxide nanoparticles as biologically active materials and the potentiality
of the nanoparticles for use in the biomedical sector have attracted researchers from
all over the world, in recent years (Nelson et al. 2016). The nanoparticles have been
properly examined for properties including applications in engineering and medi-
cine (Reed et al. 2014). To use the nanoparticles broadly, the materials must be well
characterized for industrial polishing (Reed et al. 2014; Cook 1990) as well as for
applications in new biomedical avenues (Das et al. 2013).
Advances of Cerium Oxide Nanoparticles in Treating Cancer
Cerium oxide nanoparticles have their own unique properties and show anticancer
properties, so they can be used to treat melanoma and have the capability to affect
the growth and progression of different types of cancer cell, as well as the nanopar-
ticles, and can transport chemotherapeutics (Alili et al. 2013; Pérez-Herrero
Fernández-Medarde 2015). This prooxidant activity may be harmful to healthy
cells, but due to the selectivity prooxidant nature and antioxidant activities depend-
ing on the neighboring environment of cerium oxide nanoparticles, the prooxidant
activity is limited to cancer cells (Von Montfort et al. 2015). The nanoparticles
conjugated with anticancer therapeutics are shown to be more effective in most
cancers such as gastric cancer and melanoma. In contrast to the previous studies
describe, cerium oxide nanoparticles combined with folic acid can target one type
of cancer cells, i.e., ovarian cancer cells (Hijaz et al. 2016).
The cytoprotective nature of the cerium oxide nanoparticles is shown to have
effects on a wide variety of environments and the capability to localize the nanopar-
ticles to a specific tumor is critical and need to be established, for the development
of novel treatments of cancer (Hijaz et al. 2016). Moreover the cerium oxide
nanoparticles have the potentiality to reduce angiogenesis of ovarian cancer without
increasing the cytotoxicity, as well as the diverse application of nanoparticles as an
anticancer agent (Engelberth et al. 2014). The unique application of cerium oxide
nanoparticles is also used to deliver chemotherapeutic treatment including the drugs
used in cancer therapy such as anthracycline and/or doxorubicin on the surface of
nanoparticles which becomes the most effective treatment of cancer as a combina-
tion of drugs and the nanoparticles significantly reduced the spread of tumor cells
(Sack et al. 2014).
Some researchers conducted the experiments and exposed hydrogen peroxide on
healthy cells, which would typically cause significant damage to healthy cells, but
after cerium oxide nanoparticles treatment, protection against genotoxicity is
shown; this is usually not seen in traditional chemotherapy treatment (De Marzi
et al. 2013). The nanoparticles also protect against reactive oxygen species (ROS)
and radiation treatment, which is mainly used in the treatment of various types of
cancer (Wason and Zhao 2013). Moreover, cerium oxide nanoparticles were used
primarily to mitigate and defend against the stress response of healthy cells includ-
ing heat-shock protein pathways. The nanoparticles protect the healthy cells, at the
same time making the cancer cells more sensitive to treatment; therefore it is ideal
to use the particular nanoparticles for cancer treatment (Yuan et al. 2016). Due to
the unique potential role of the cerium oxide, nanoparticles emerged as a novel
treatment method used in different types of cancer, without the risks and side effects
that may happen due to the current therapies (Zhao et al. 2016). The use of cerium
oxide nanoparticles can improve the degenerative condition of the most dangerous
dreadful neurological disease such as Alzheimer’s disease (Li et al. 2013). Moreover,
the nanoparticles can act directly on amyloid proteins, protecting risk cells against
cytotoxicity that often leads to Alzheimer’s disease (Zhao et al. 2016).
Applications of Cerium Oxide Nanoparticles in Ophthalmology
The eye is the most sensitive organ of the human body which predominantly requires
an appropriate condition to maintain its fidelity, and any injury from ROS may
cause impairment in vision and other eye diseases (Wong and McGinnis 2014).
Unlike other eye diseases resulted by ROS, it has been observed that cerium oxide
nanoparticles play a crucial role in combating the ROS-induced damage, by slowing
retinal degeneration (Wong and McGinnis 2014). It is also reported that the nanopar-
ticles were also able to diminish angiogenesis (Wong and McGinnis 2014), which
shows a positive association with retinal dysfunction and blindness (Witmer et al.
2001). Some of the researchers experimented on knockout mice having low-density
lipoprotein receptor, and a reduction of abnormal blood vessel formation was
observed up to 6 weeks after injection of these nanoparticles (Cai et al. 2014). These
nanoparticles may serve as an efficient method for the treatment of these neovascu-
larizations, as it is the result of oxidative stress (Kong et al. 2011).
Cerium oxide nanoparticles can prevent neovascularization and also have shown
the potential to treat other causes of vascular disease. The eye is an immune-sensitive
portion of the body, and any inflammatory response acts as an annihilator leading to
permanent damage (Taylor 2016). During neurodegeneration of the retina due to
light-induced injury, the potential application of the nanoparticle is capable enough
to prevent the immune response activation, particularly during damage state
(Ramirez et al. 2017). These particular nanoparticles inhibit the application of the
immune response in the eye and can reduce the activation of the microglia in the
retina (Fiorani et al. 2015). Considering this approach, nanoparticles can be used as
a potential candidate to limit the inflammation mechanism within the eye which will
facilitate the restoration of normal vision by promoting the natural repair mechanism.
pplications of Cerium Oxide Nanoparticles in the Vascular

A
System
Application of cerium oxide nanoparticles systematically decreasing the angiogen-

esis has been previously reported (Lord et al. 2013). This approach enables the
nanoparticles to use for the treatment of endometriosis, an event characterized by
angiogenesis and oxidative stress-induced lesions of the uterus (Chaudhury et al.
2013). Angiogenesis and ROS neutralizing ability of cerium oxide nanoparticles
provide a potential therapeutic approach to treat endometriosis (Chaudhury et al.
2013). It has been observed that there is a beneficial effect of cerium-coated
nanoparticles on the cardiovascular system against angiogenesis, and the nanopar-
ticles have potentiality to be used as vasoconstrictors, by reducing endothelium-
dependent vasodilation (Farooq et al. 2014).
Avenues of Nanotechnology for Cancer Treatment
Nanoparticles
Encapsulation of therapeutic nanoparticles with polymeric matrix either in adsorbed

form or conjugation on to the surface must be helpful against a particular disease
(Sahoo and Labhasetwar 2003). Significa0nt surface modification of nanoparticles
might be ideal for precise biochemical interactions with the receptors localized in
the target cells of the targeted sites (Parveen and Sahoo 2008; Misra and Sahoo
2010). One of the most vital features of nanoparticles is controlled drug delivery in
the targeted site by crossing the blood-brain barrier. Further study by several authors
indicates intravenous injection of polysorbates-coated nanoparticles with drug
which can cross the blood-brain barrier and enable to target the brain (Wang et al.
2008; Malam et al. 2009; Owens and Peppas 2006; Rao et al. 2009).
Carbon Nanotubes
Carbon nanotubes (CNTs) are another nanodevice for biomarker detection (Nie
et al. 2007). CNTs are the carbon cylinders composed of benzene rings applied in
many aspects of the biological sector such as diagnostic devices and sensors to dis-
criminate the different type of proteins from serum samples and carriers to the deliv-
ery drug, vaccine, or protein (Grodzinski et al. 2006). Efficient structural,
mechanical, electrical, and optical properties of single-walled carbon nanotubes
(SWNTs) offer a breakthrough for their application in the emerging areas like bio-
sensors, drug delivery, and therapeutic agent (Ferrari 2005).
Nanoparticle Design
Before synthesis, designing of metallic nanoparticles (MNP) is the fundamental

prerequisite for the understanding of the nature of the nanostructure as (1) a phar-
maceutical structure that must navigate for its target, (2) must be biocompatible
with the host with limited toxicity (3) ability to use in an external biomedical sys-
tem. In this context, we will consider the ideal characteristics of nanoparticles (NP)
which can overcome the physical barrier and have greater accessibility (Veiseh
et al. 2010).
Drug Loading and Release
Conjugation of NP with a specific therapeutic agent makes it an efficient drug vehi-

cle system for controlled drug release with limited nonspecific cellular interaction
with flexible loading that can be tracked by NP imagery method. Design and devel-
opment of NP for diagnostic imaging needs a careful physiochemical and target
design where additional considerations are also required for loading, transport, and
release of drugs (Durán et al. 2008; Davis et al. 2010; Namdeo et al. 2008).
Nanoparticle Drug Delivery System
Loading of polymeric nanoparticles with therapeutic agents by encapsulation in the

core of the nanoparticles, where absorption of the particle surface or combination
with the polymer must be strictly monitored before the formation of nanoparticles
(Labhasetwar 1997). Loading and encapsulation efficiency of nanoparticles with
therapeutic agent depend on several confounding factors like property of drug and
polymer, nature of the solvent, and method of encapsulation. Combination of drug
and polymer complex is of tremendous importance for the formation of nanoparti-
cles for the drugs with reduced loading and encapsulation efficiency (Yoo et al.
1999; Yoo and Park 2004).
Polymer Chemistry
Polymer response toward external stimuli like temperature, pH, or IR offers a wide
range of applications in drug delivery (Peppas 2007; Schmaljohann 2006), for
example, poly (4-styrene sulfonate)/poly-(allyl hydrochloride) capsules whose per-
meability is strictly dependent on pH (De Geest et al. 2007). Nanoparticles which
are fabricated from a unique hydrophobic polymer like poly (1,4-phenyleneacetone
dimethyleneketal) readily undergo acid hydrolysis into low molecular weight,
hydrophilic components and can potentially deliver encapsulated therapeutics at a
more improved rate in acidic environments like tumors or endosomes (Heffernan
and Murthy 2005). Recently, it has been observed that thermosensitive and photo-
sensitive polymers are used extensively by the researchers (Sershen et al. 2000;
Cammas et al. 1997).
Multiple functionalizations of a single particle (Roh et al. 2006) require strategic
manufacture and development of biphasic particles (Roh et al. 2005, 2006) and tri-
phasic particles, which are synthesized by simultaneous electrohydrodynamic jet-
ting of parallel polymer solutions under the applied electric field. In addition to the
functionalization of the compartmental surface, different compartments can be
loaded with various biomolecules for specific applications.
Surface Chemistry
The role of surface chemistry is of tremendous importance in targeted delivery of

particles improving the systemic circulation. The hydrophilic polymer molecules
like polyethylene glycol (PEG) coated with nanoparticles can resist the serum pro-
tein adsorption and facilitate the prolonged systemic circulation of the particle
(Moghimi et al. 2001). After several modifications, PEG and other hydrophilic
polymers have improved the circulation (Alexis et al. 2008). Epidemiological evi-
dence indicates, polaxamers, polaxamines, PEO and PPO block copolymers have
been extensively studied (Cheng et al. 2007; Moghimi and Hunter 2000). PEG
(hydrophilic surface) block copolymers with PLGA (biodegradable) have high
capacity as delivery vehicles (Cheng et al. 2007). The target delivery is achieved by
the surface functionalization of particles (Brannon-Peppas and Blanchette 2012)
with biomolecules, such as peptides (Arap et al. 1998) and antibodies (Sudimack
and Lee 2000). For therapeutic applications, some tumor-specific antibodies are
being investigated. Many researchers have developed different strategies to stan-
dardize PEG surface concentrations and target ligands to balance prolonged circula-
tion with the sufficient accumulation of tissue. These include the optimization of
randomly distributed PEG and aptamers on the surface of nanoparticles (Gu et al.
2008) and compartmentalized particle manufacturing (Roh et al. 2005, 2006). The
particle load may also affect other functions, such as macrophage internalization.
Positive particles were able to show higher internalization of macrophages and den-
dritic cells compared to neutral or negatively charged particles (Thiele et al. 2003).
Size
Considering the concentration of polymer or surfactant, the agitation method (vor-

texing, sonication, and stirring), the speed of agitation, the diameter of the nozzle/
capillary, and the flow rate of the material (Soppimath et al. 2001), polymeric par-
ticles of different size ranging from few nanometers to hundreds of micrometers are
produced by different manufacturing parameters. Functional integrity of a particle
like degradation, vascular dynamics, targeting, clearance, and absorption mecha-
nisms depend upon its size (Champion et al. 2007). It is observed that the particles
have altered speeds, diffusion characteristics, and size-dependent adhesion proper-
ties (Patil et al. 2001). For intravascular applications, particle size is observed to be
less than 200 nm because of their half-life in circulation compared to larger particles
(Juliano and Stamp 1975). Transport across biological barriers such as the skin
(Ryman-Rasmussen et al. 2006) and mucosa (Jani et al. 1990; Vila et al. 2004) in
tissues and intracellular particle transport are dependent on size. Some researchers
experimentally proved that particles as large as 5 mm can be endocytosed by endo-
cytosis mediated by the receptor, which could open up new applications for targeted
vasculature delivery (Muro et al. 2008).
Mechanical Properties
Mechanical properties are always crucial in biological functions nowadays (Levental

et al. 2007). Recent studies have also observed that engulfment of rigid particles by
macrophages is significantly higher in comparison to the soft particles (Beningo and
Wang 2002). Synthetic materials having mechanical properties are used in implan-
tation, tissue engineering, and manufacture of particular drug carriers; while con-
version of a normal mature cell to a replicative, immortal, and motile tumor cell
involves a stiff and ordered cytoskeletal change to an irregular biopolymer network,
which can be used in cancer diagnostics (Suresh 2007). Further study revealed that
tissue cells could exhibit the matrix’s rigidity, which can influence their cytoskele-
ton, differentiation, and development (Discher et al. 2005).
Particle Shape
Polymer chemistry, size, and surface chemistry on particle functions have a substan-
tial impact. Recent findings indicate that along with particle size and surface chem-
istry, the shape of the particle plays a vital role on particle functions (Champion and
Mitragotri 2006; Geng et al. 2007; Gratton et al. 2008).
Nanomaterials
Till now, various types of nanomaterials like micelles, polymeric nanoparticles

(NPs), carbon nanotubes, quantum dots, and metallic NPs have been designed and
implicated to treat various neurological disorders (Srikanth and Kessler 2012).
However, copolymers of polyethylene glycol (PEG) are one of the most prominent
drug delivery systems. However, NPs can be functionalized with fluorescent mark-
ers, tumor marker antibodies, and gene delivery agents as well as cell-penetrating
peptides (CPP) to trigger rapid cell uptake (Bhaskar et al. 2010).
Application of Liposomes as a Nanomaterial
Liposomes are lipid bilayers having amphipathic phospholipids, uni- or multilamel-

lar, surrounded by a watery compartment that most often consists of phosphatidyl-
choline (lecithin). Sonication, extrusion, reverse evaporation, and injection of
solvents are the most common approved methods for the production of liposomes
by the Food and Drug Administration (FDA) (Johnston et al. 2008). By their overall
net load, liposomes can be categorized as cationic, anionic, or neutral (Sharma et al.
2012). The distinctive feature of this nanoparticle is that it can cross the blood-brain
barrier (BBB) more quickly than other compounds. One of the most peculiar fea-
tures of liposomes is that they can pass the blood-brain barrier (BBB) more quickly
than other compounds.
The main advantage of the liposomes is high tolerance and high degradability,
which makes the nanomaterial suitable for the biomedical application. The disad-
vantage of the liposome loaded medicine is that they are prone to degradation by
the reticuloendothelial system (RES). Thus, to overcome the problem, modification
in particle size (< 100 nm) or polyethylene glycol (PEG) can be done (Nair
et al. 2012).
Application of Micelles as a Nanomaterial
The natural aggregate of spherical vesicles is formed after amphiphiles are placed in
an aqueous medium because the nonpolar water environment pushes the nonpolar
portions of amphiphiles into the inner part of the water to form a hydrophobic core
that can sequester hydrophobic drugs, whereas the polar portion is reinforced to
form a hydrophilic shell on the exterior surface that can protect encapsulated drugs
and extend the blood circulation time (Bhujbal et al. 2014). Micelles can be easily
changed or modified in size, surface, and molecular composition which makes them
ideal to use in a universal application including the pharmaceutical industry. The
most prevalent material used for the micelle’s outer part is PEG, due to its hydrophi-
licity and the ability to prevent macrophage endocytosis. Due to their size and flex-
ibility, micelles may prevent glomerular filtration at the renal tissue level (Daum
et al. 2012).
Application of Polymer Nanoparticles as a Nanomaterial
Polymer NPs are developed from a variety of natural and synthetic polymers.
Dextrans are the glucose polymers with 1,6-glucopyranoside (95%) and 1,3-links
(5%) and are formed by the hydrolysis of other high molecular weight dextrans and
fractionation. Nowadays, 40–70KDa molecular weight dextrans are used for drug
delivery of pharmaceutical drugs (Bisht and Maitra 2009).
Biodegradable poly lactic-co-glycolic acid (PLGA) NPs have been compre-
hensively studied to provide several agents, like plasmid DNA, proteins, and pep-
tides and low molecular weight compounds sustained and targeted/localized
(Bharali et al., 2009). The stability of NP, their loading with other agents, and
facile surface modification help to reduce the activation of the reticuloendothelial
system (RES), platelets, and neutrophils, as well as control drug kinetics (Patel
et al. 2012).
Application of Carbon Nanotubes (CNTs) as a Nanomaterial
Single-walled or multi-walled CNTs are graphic carbon tubes with a high aspect
ratio > 100 with characteristic features of ease of modification, biocompatibility,
and fluidity (Nair et al. 2012). Technological advances make multifunctional CNTs
ideal for theranostics application (Xie et al. 2010). Due to their ability to absorb
near infrared light, it enables CNT to use in the field of image studies with high
resolution. Further studies have also shown that heat produced upon light absorption
by CNT has the tumoricidal property (Rossella et al. 2012). However, several stud-
ies have shown that CNTs have some effects on toxicity; therefore; the nanomaterial
size, shape, and density ratio must be carefully considered (Nanjwade et al. 2010).
As per the safety requirements of nanoparticles, not a single type of carbon-based
nanomaterials can meet the demand. So, although there is a significant advancement
to improve the compatibility of CNTs biologically, the carbon nanomaterials
couldn’t be used efficiently (Bianco et al. 2011).
Application of Quantum Dots (QDs) as a Nanomaterial
Quantum dots (QDs) are semiconductor detection with superfine having diameters
between 2 and 10 nm (Smith et al. 2008). Quantum dots (QDs) have unique advan-
tages over conventional fluorescent dyes due to absorption profiles, narrow emis-
sion spectra, and long-term photostability with reasonably long fluorescence life
(Wang et al. 2012). Another striking feature of QDs is using them beneficial donors
for the transfer of fluorescence resonance energy (FRET) technique used for cell
labeling, tissue imaging, and labeling of tests (Medintz et al. 2005). Notwithstanding,
QDs also exhibit harmful side effects, since their core component is cadmium sel-
enide, and cadmium telluride shows cytotoxicity (Ghasemi et al. 2009).
Application of Metallic Nanoparticles as a Nanomaterial
Metallic nanoparticles (NPs) are enclosed by shells constituting inorganic metal or

metal oxide cores, for example, gold NPs (AuNPs) having gold-coated dielectric
silica core, whose property depends on the gold thickness, which can determine the
light absorption and dispersion of NP. The gold NPs(AuNP) can be used as photoac-
tive ingredient in optical imagery, X-ray and computed tomography(CT). Magnetic
iron oxide nanoparticles are a type of magnetic NPs (MNPs) that have been widely
studied all over the globe for years and are approved by the US Food and Drug
Administration (FDA) for several clinical applications. The magnetic iron oxide
nanoparticles can also be classified as superparamagnetic and ultrasmall superpara-
magnetic NPs, named superparamagnetic iron oxide nanoparticles (SPIONs) and

ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs), by their hydro-
dynamic particle size (Wang et al. 2012).
Nanomaterial Applications in Neurodegenerative Diseases
Use of Nanomaterial in Parkinson’s Disease
The chronic neurodegenerative disease, i.e., in Parkinson’s disease (PD), and the
progressive loss of nigrostriatal dopaminergic neurons are common (Galvan and
Wichmann 2008). Loss of dopamine (DA) storage is the prominent feature in the
region of the striatal, cingulate, and frontal brain in the patients with PD (Brooks
2003). Dopamine is the primary source of dopaminergic neurons of the midbrain in
the mammalian central nervous system, and their loss is the main reason of the most
prominent and dreadful neurological disorders in Parkinson’s disease (PD). One of
the successful diagnosing technique for early-stage Parkinson’s disease (PD) is
available, where vertically aligned ZnO nanowire on 3D graphene foam designed in
such a way that it can detect lower uric acid (UA), dopamine (DA), and ascorbic
acid (AA) (Yue et al. 2014).
Application of Nanomaterial in Alzheimer’s Disease (AD)
The chronic neurodegenerative disorder, i.e., Alzheimer’s disease (AD), is charac-

terized by the tangles and extracellular amyloid protein deposits leading to senile
plaques (Jakob-Roetne and Jacobsen 2009). The abnormal self-assembly of the Aβ
peptide leads to the proliferation of the β-amyloid (Aβ) peptide plaques, and the
toxic aggregates rich in β is the crucial factor for the development of Alzheimer’s
disease (AD) (McKhann et al. 1984; Miners et al. 2008).
One of the dangerous neurodegenerative diseases such as Alzheimer’s disease,
metal nanoparticles like gold nanoparticles (AuNPs) combined with the specific
peptide sequence (CLPFFD) is used to improve the delivery into the brain and to
enhance the targeting efficiency. There could be an interaction between the sequence
and the transferrin receptor present in the microvascular endothelial cells of the
blood-brain barrier (BBB) (Prades et al. 2012). Polymeric NPs conjugated with bor-
neol used in vivo have shown the capability to enhance brain targeting (Zhang
et al. 2013).
Conclusion
Application of nanoparticles like iron, cerium oxide, zinc oxide, and nanomaterials
like micelles, polymeric nanoparticles (NPs), carbon nanotubes, quantum dots, and
metallic NPs in medicine, drug delivery in therapeutic applications such as chemo-
therapy or as diagnostic tool, tumor treatments, neurological disease treatment are
the essential features of the technology. Although considerable serendipitous
achievements have been achieved, many more challenges still present. Before the
application of the technology, more studies on some drugs and their toxicity for
clinical use need to be validated. The effect of nanoparticles (NP), molecular size,
composition, and the interaction with the skin are essential to understand and
improve the transportation of the nanoparticles across the cutaneous barrier (blood-
brain barrier). Various studies highlighted in the chapter show potentiality of the
nanoparticles as drug molecules and drug carriers, which can be adopted completely
shortly for the efficient pharmaceutical and biomedical application.
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Chapter 20
Micelleplexes: A Promising Nanocarrier
for the Transport of Genetic Material
and Drugs
Jorge Faria, Mariana Magalhães, Francisco Veiga, Ana Cláudia Santos,

and Ana Figueiras
Introduction
Generally, the success of gene therapy relies on the development of an efficient vec-
tor capable of delivering this material specifically into the target cells, reducing
off-target risks. The perception of these therapy advantages promoted a vast interest
within the scientific community, which consequently contributed to increasing the
research and development of new and promising vectors (viral and nonviral origin)
(Magalhães et al. 2014, 2018a).
Viral vectors are widely studied and firstly used in gene therapy clinical trials.
These vectors have a high transfection efficiency, although they present several
limitations and safety problems such as oncogenesis, mainly with lentiviruses and
retroviruses, toxicity, and immunogenicity verified with the use of herpes simplex
virus (Jayant et al. 2016; Magalhães et al. 2017). Thus, nonviral vectors are seen as
an attractive alternative to overcome some of the drawbacks associated with the use
of viral vectors. Moreover, nonviral vectors can be chemically designed to perform
a more efficient and directed delivery of the genetic material. Polymers and lipids
are, perhaps, the most studied constituents for the construction of nonviral vectors,
and those with more data and promising results.
J. Faria
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra,
Coimbra, Portugal
M. Magalhães · F. Veiga · A. C. Santos · A. Figueiras (*)
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra,
Coimbra, Portugal
REQUIMTE/LAQV, Group of Pharmaceutical Technology, Faculty of Pharmacy, University
of Coimbra, Coimbra, Portugal
e-mail: rfigueiras@ff.uc.pt

https://doi.org/10.1007/978-981-15-2195-9_20
268 J. Faria et al.
Therefore, this review focuses on the development of micellar nanosystems to

perform a concomitant transport of genetic material and/or a drug in anticancer
approaches. These nanocarriers are usually formed by an amphiphilic copolymer
with hydrophilic groups of ethylene oxide (EO) and hydrophobic groups of propyl-
ene oxide (PO) conjugated with a polymer with cationic properties. The micellar
nanocarrier has the ability, in aqueous medium, to self-assemble into a micellar
structure with a hydrophobic core capable of transporting hydrophobic drugs, such
as doxorubicin (Chen et al. 2018) or docetaxel (Liu et al. 2016), and a positively
charged hydrophilic shell which, through electrostatic interactions, has the ability to
bind the genetic material. These nanocomplexes have a small size avoiding renal
filtration and the recognition by the reticuloendothelial system (RES) organs, which
improves the permanence time in the bloodstream. Besides, they also have the capa-
bility to protect the genetic material against extracellular nucleases and endosomal
activity (Magalhães et al. 2017).
Thereby, it seems like micelleplexes appear as a “double nano-weapon” against
a wide variety of diseases, such as cancer.
Micelleplexes
Definition
The past few years have witnessed an increasing scientific interest in micelleplexes,
because of their stable and efficient nanotransport system to deliver genetic material
and/or drugs with therapeutic action (Pereira et al. 2016). They can be defined as
cationic polymeric nanoparticles linked to specific small molecules of deoxyribo-
nucleic acid (DNA) or ribonucleic acid (RNA), generally composed of amphiphilic
copolymers coupled to a cationic polymer (Magalhães et al. 2017) and a drug,
which spontaneously self-assemble to form polymeric micelles with cationic prop-
erties (Chitkara et al. 2016) and small sizes between 10 and 100 nm (Dong et al.
2004; Pereira et al. 2016).
Structure
As already mentioned in this work, micelleplexes (Fig. 20.1) are structurally consti-

tuted by amphiphilic copolymers, a cationic polymer, genetic material, such as
DNA and RNA (Chitkara et al. 2016; Magalhães et al. 2017; Almeida et al. 2018),
and/or a drug (Li et al. 2015; Liu et al. 2016).
A detailed description of micelleplexes constituents and their advantages will be
provided in the following sections.
20 Micelleplexes: A Promising Nanocarrier for the Transport of Genetic Material… 269
Fig. 20.1 Structure of the micelleplex
Amphiphilic Copolymers
Poloxamers and Poloxamines
Amphiphilic blocks of copolymer have the ability to spontaneously self-assemble in

a micellar structure in an aqueous environment. This micellar architecture is distin-
guished through a hydrophilic shell and a hydrophobic core that enable hydropho-
bic interactions with poorly water-soluble drugs (Alvarez-Lorenzo et al. 2012;
Pereira et al. 2016). Among the available block of copolymers, poloxamers and
poloxamines emerge as the most promising and required ones to be used in the
development of micelleplexes.
Poloxamers, or triblock copolymers, also known as Pluronics®, are the most
widely used copolymers for the development of micelleplexes. They are constituted
by hydrophilic groups of ethylene oxide (EO) and hydrophobic groups of propylene
oxide (PO), arranged in a triblock A-B-A structure, that is, EOx-POy-EOx, where
X and Y correspond to the number of units of each compound. The variability in the
number of EO and PO units assigns particularities to each Pluronic®, having influ-
ence in the size, molecular weight, melting point, and, consequently, the ability to
solubilize in an aqueous medium, also referred to as hydrophilic-lipophilic balance
(HLB) (Kabanov et al. 2002).
Poloxamines, also known as Tetronics®, are X-shaped amphiphilic block copoly-
mers formed by four arms of poly (ethylene oxide)-poly (propylene oxide) (PEO-
PPO) blocks bonded to a central ethylenediamine unit (Fernandez-Tarrio et al.
2008; Alvarez-Lorenzo et al. 2012). These polymers, compared to poloxamers, have
270 J. Faria et al.
a more versatile and unique structure which provides them with a multi-stimulus
response capability (Alvarez-Lorenzo et al. 2012).
The characteristics of poloxamines, namely, molecular weight, EO/PO ratio, and
HLB, determine their self-associative behavior and thermal sensitivity. In addition,
the physicochemical conditions of the medium, particularly pH and ionic strength,
alter the extent of protonation of the central ethylenediamine group, disturbing the
hydrophobic interactions leading to spontaneous self-assembly and consequent for-
mation of the micelle (Dong et al. 2004).
Therefore, in an aqueous medium, both Pluronics® and Tetronics® have the abil-
ity to spontaneously form polymeric micelles through a process known as micelliza-
tion. This phenomenon happens when the concentration of the block of copolymer
is higher than the critical micellar concentration (CMC), occurring a molecular dis-
persion of the individual copolymers known as monomers (Kabanov et al. 2002).
For example, Pluronics® with a higher number of propylene oxide (PO) units and
low values of HLB are more hydrophobic, presenting lower CMC values, which
leads to the formation of stabler micelles with smaller sizes and lower rates of dis-
sociation, consequently, improving their permanence time in the bloodstream
(Magalhães et al. 2017).
Cationic Polymers
Poloxamers and poloxamines are neutral particles, which preclude the existence of
a problem to encapsulate genetic material through electrostatic interactions.
Regarding this, these polymeric structures can be chemically modified, through the
conjugation with a cationic polymer, in order to be possible the establishment of an
electrostatic interaction between the amine groups (N) of the cationic polymer with
the phosphate groups (P) of the genetic material (Chitkara et al. 2016; Wang et al.
2016; Almeida et al. 2018).
Subsequently, the cationic properties of the nanosystem will allow the condensa-
tion and protection of the genetic material, against the action of intracellular and
extracellular nucleases and endosomal activity, until reaching the target site (Samal
et al. 2012). On the other hand, this positive charge may also improve the cell uptake
of micelleplexes by endocytosis, as micelleplexes have the ability to establish elec-
trostatic interactions with the negative charge of the cell membrane (Samal et al.
2012; Magalhães et al. 2017).
Cationic polymers can have a natural origin, such as poly-L-lysine polypeptide
(PLL), chitosan, and cyclodextrins, or a synthetic origin, such as polyethyleneimine
(PEI) and polyamidoamine (PAMAM) (Samal et al. 2012; Keles et al. 2016).
Natural Cationic Polymers

Natural cationic polymers (Table 20.1) have aroused great scientific interest, par-
ticularly regarding new therapeutic applications. These polymers come from natural
sources, such as polysaccharides or proteins, being recognized by their advantages,
Table 20.1 Main characteristics of natural cationic polymers used in the transport of genetic
material
Structure Advantage Disadvantage
Poly-L-lysine Homopolymers Biodegradable Poor transfection
polypeptide amino acid L-lysine efficiency
(PLL)
Chitosan N-acetyl Biodegradable Poor transfection

glucosamine and and Digestible efficiency
D-glucosamine Endosomal
elimination
Cyclodextrins Cyclic glucose Biocompatible Low solubility in
units linked by and water
α-1,4-linkage Biodegradable Difficulty in
processing
Adapted from Keles et al. (2016) and Samal et al. (2012)
Table 20.2 Main characteristics of synthetic cationic polymers used in the transport of genetic
material
Structure Advantage Disadvantage
Polyethyleneimine L-PEI: all High load High toxicity
(PEI) secondary amines capacity and
B-PEI: primary, transfection
secondary and efficiency
tertiary amines
Polyamidoamine Alkyl diamine Low toxicity Low
dendrimers nuclei and High biodegradability
(PAMAM) branching of transfection
tertiary amines efficiency
Ease of
manufacturing
Adapted from Keles et al. (2016) and Samal et al. (2012)
like a reduced cytotoxicity and immunogenicity, as well as a good biocompatibility

and biodegradability. In spite of these attractive properties, they also present some
disadvantages, such as lower transfection efficiency and reduced water solubility
(Samal et al. 2012; Keles et al. 2016; Almeida et al. 2018).
Synthetic Cationic Polymers

Synthetic cationic polymers (Table 20.2) can fulfill certain fragilities attributed to
natural polymers, like the control and modulation of their physicochemical proper-
ties. Specific functional groups may be incorporated in order to upgrade singulari-
ties; such example is the incorporation of a chemical group capable of preventing
polymer degradation. These polymers, such as polyethyleneimine (PEI) and poly-
amidoamine dendrimers (PAMAM), contrary to natural polymers, exhibit high
272 J. Faria et al.
transfection efficiency, a clear goal of gene therapy; however, they have some cyto-
toxicity associated and a low biodegradability (Samal et al. 2012; Keles et al. 2016;
Almeida et al. 2018).
Preparation Methods
The preparation of the micelleplexes involves two fundamental steps: firstly, the
nanosystem is developed based on the conjugation of polymeric micelles with a
cationic polymer [amphiphilic copolymer (s) + cationic polymer]. After this, the
incorporation of the genetic material and/or drug will be performed inside the nano-
system (Amjad et al. 2017).
reparation of the Polymeric Micelle Conjugated with the Cationic

P
Polymer
Amphiphilic Copolymer + Cationic Polymer
This preparation will depend on the polymers used in the study and, essentially,
consists of the activation of the amphiphilic copolymer with an “activating agent”
such as N, N-carbonyldiimidazole (CDI) (Nguyen et al. 2000; Zhang et al. 2011) or
p-toluenesulfonyl chloride (TsCl) (Liu et al. 2016) to allow the conjugation with the
cationic polymer. As example, Liu and colleagues developed a method to activate
Pluronic® F127 with TsCl and, consequently, link to PEI, resulting in the formation
of a polymeric system with cationic and amphiphilic properties, F127-PEI
(Scheme 20.1).
Incorporation of the Drug
For drug incorporation, several methods were developed. The main ones are men-
tioned below.
Direct Dissolution
Direct dissolution is a technique that is based on the dissolution of the drug and the
polymer in an aqueous environment (Gaucher et al. 2010; Tyrrell et al. 2010). In
other words, this methodology consists in the dissolution of an excess of the model
drug in a micellar solution. Moreover, this technique is applied to polymers and
drugs moderately hydrophobic to ensure the necessary amount of drug in the solu-
tion to induce saturation of the micellar nucleus (Fig. 20.2).
Scheme 20.1 Synthesis F127-PEI. (Adapted from Liu et al. 2016)
Fig. 20.2 Direct dissolution
Dialysis
Dialysis involves the use of organic solvents, such as acetonitrile, dimethylacet-

amide, and acetone (Gaucher et al. 2005), being usually employed with poorly water-
soluble polymers (Gaucher et al. 2010). This technique consists in the dissolution of
the polymer and the drug in a water-miscible organic solvent, followed by a slow
removal of the solvent through a dialysis membrane against deionized water (Tyrrell
et al. 2010; Shen et al. 2015; Almeida et al. 2018) (Fig. 20.3). According to the litera-
ture, the choice of the solvent may influence the particle size (Tyrrell et al. 2010).
274 J. Faria et al.
Fig. 20.3 Dialysis
Oil/Water Emulsion
The method for oil/water emulsion (O/W), unlike the technique described above,
uses water-immiscible organic solvents, such as chloroform (Almeida et al. 2018)
and ethyl acetate (Gaucher et al. 2005). O/W intends to form an emulsion of small
droplets of an organic solvent, which will serve as a model for the “self-assembly”
capability of micelles. In a first step, the dissolution of the drug and the polymer is
verified in an immiscible volatile organic solvent (internal phase), where water will
be added then (external phase). Through stirring or sonication, it is formed dis-
persed droplets from the complete external phase. The drug and the hydrophobic
portion of the polymer are confined in the core of the droplet, and the hydrophilic
portion of the polymer extends into the external phase, stabilizing the emulsion
(Tyrrell et al. 2010). In a second step, the organic solvent is evaporated, leaving the
micelles loaded with the drug. The main drawback of this method is the difficulty in
removing the drug that was not incorporated into the micelles, the “free drug,” and
the total evaporation of the organic solvent used (Almeida et al. 2018) (Fig. 20.4).
Salting-Out
This method consists in the dissolution of the drug and the polymer in a water-
miscible organic solvent, such as tetrahydrofuran (THF) and acetone (Zweers
et al. 2004; Tyrrell et al. 2010; Wang et al. 2016). After, a highly concentrated
electrolytic solution (aqueous solution), in which salts are not soluble in the
organic solvent, is added to a solution containing the drug and the polymer (oil
phase) (Wang et al. 2016). The mixture is emulsified by magnetic stirring or soni-
cation, although the high concentration of salt used (up to 60% by weight of salt)
to mix phases is not observed. Subsequently, distilled water is added in order to
decrease the ionic strength of the electrolyte while, at the same time, the organic
solvents migrate rapidly to the aqueous phase and induce the formation of the
nanoparticles (Zweers et al. 2004) (Fig. 20.5).
Fig. 20.4 Oil/water emulsion
Fig. 20.5 Salting-out
276 J. Faria et al.
Fig. 20.6 Dry-down
Dry-Down
The dry-down method is based on the dissolution of the copolymer with the drug in
an organic solvent, such as acetonitrile, which is posteriorly removed (Liu et al.
2006; Tyrrell et al. 2010), usually using nitrogen to create a film of copolymer
impregnated with drug. Thereafter, this film is hydrated with warm water or a buffer
under stirring to induce micellization. The extension of drug incorporation is depen-
dent on the solvent used for the initial dissolution of the copolymer and the drug,
preferably choosing a common solvent for solubilization, avoiding phase separation
during the evaporation process. Additionally, it is important to emphasize that simi-
lar solubilities of the copolymer and the drug will lead to interactions during the
drying period, enhancing the amount of drug incorporated in the hydrophobic core
of the micelle (Liu et al. 2006) (Fig. 20.6).
Incorporation of the Genetic Material
The most common protocol used to encapsulate the genetic material is the simple
method of complexation. This methodology involves electrostatic interactions
between the negative charges of the genetic material and the positive charges of the
nanoparticle used. Moreover, this complexation is performed taking into account the
stoichiometry of the reaction, directly related to the amount of amine groups (N) pres-
ent in the micelle and the amount of phosphate groups (P) present in the genetic mate-
rial, such as small interfering RNA (siRNA) (Kim and Song 2014; Oe et al. 2014) or
microRNA (miRNA) (Zhang et al. 2015; Magalhães et al. 2017). Thus, depending on
the different N/P ratios, the genetic material is added to the previously prepared
micellar solution (Kim and Song 2014; Oe et al. 2014; Li et al. 2017a), forming the
micelleplexes.
Scheme 20.2 Physicochemical characterization of the micelleplexes in the different stages of

synthesis
Physicochemical Characterization
The physicochemical characterization is a very important point to take into consid-

eration since the properties of micelleplexes have a direct influence in their effi-
ciency. This characterization should be assessed in different stages (Scheme 20.2),
after the development of the micellar nanosystem and after incorporation of the
genetic material (micelleplexes).
Particle Size, Polydispersity Index, and Morphology
The particle size, as previously mentioned, has a significant relevance since it is

directly related to the cellular uptake (Zheng and Yu 2016), toxicity, and dissolution
of the nanosystem (Bhattacharjee 2016). Micelleplexes usually have a small size,
between 10 and 100 nm, which can be determined by dynamic light scattering
(DLS) (Xu 2008; Bhattacharjee 2016).
The polydispersity index (PDI), also called the heterogeneity index, indicates the
“non-uniformity” of existing sizes in a given sample. It is calculated from the ratio
between the molar mass averages of the weight (Mw) and the molar mass averages
of the number (Mn), Mw/Mn. In this index, two extremes [0.0–1.0] are considered,
in which 0.0 is attributed to a sample highly monodisperse and 1.0 is assigned to a
sample with a very wide and dispersed particle size distribution and several polydis-
perse populations (Danaei et al. 2018).
Concerning the evaluation of micelleplexes morphology, microscopic techniques
are usually used, namely, transmission electron microscopy (TEM) (Chen et al.
2017) and scanning electron microscopy (SEM) (Cambón et al. 2018). These tech-
niques provide a two-dimensional and three-dimensional image of the sample,
respectively. In addition, the techniques referred above are also able to measure the
size and dispersion of the micelleplexes (Almeida et al. 2018).
278 J. Faria et al.
Zeta Potential
Zeta potential (PZ) or electrokinetic potential indirectly evaluates the surface charge
of the particle, since it provides only indirect evidence of the nature of the surface
charge (±), assuming that the predominant ions in the electric double layer are simi-
lar in comparison to the surface of the particle itself (Bhattacharjee 2016). As previ-
ously mentioned, a positive charge is relevant and an advantage to bind the genetic
material, besides the fact that allows the establishment of electrostatic interactions
with the negative charge of the cell membrane (Magalhães et al. 2017). This param-
eter can be determined, for example, by electrophoretic light scattering (ELS)
(Almeida et al. 2018).
Critical Micellar Concentration
CMC is defined as the minimum polymer concentration required for micellization,

in which concentrations below the CMC disassemble into rudimentary unimers.
Furthermore, when the concentration of the copolymer is above the CMC, there is a
dynamic equilibrium exchange between micelles and unimers (Kabanov et al.
2002). CMC should be taken into consideration, once determines the stability of
micelleplexes in an aqueous solution. In addition, it assesses the maximum possible
concentration of unimers in which cells will be exposed, as well as the modifying
effects on the biological response that polymer may exert on cells.
There are several processes that allow the determination of the CMC, namely,
DLS (Kabanov et al. 2002), tensiometry (Kabanov et al. 2002; Chakraborty et al.
2011), and the most usual fluorescence spectroscopy (Wang et al. 2008;
Chakraborty et al. 2011).
Composition
The analysis of the composition of nanosystems becomes relevant and indispens-

able to verify the efficiency of the sequential development of nanoparticles.
According to the literature, there are several techniques which are used to provide
specific and diversified information about composition, such as high-performance
liquid chromatography (HPLC) and gel electrophoresis. HPLC, a highly sensitive
and selective chromatographic method, is widely used for the quantification of
drugs and nucleic acids. Meanwhile, to analyze the efficient incorporation of genetic
material by micelleplexes, UV-Vis spectroscopy and gel retardation assay are still
the most used ones (Liang et al. 2011). Regarding stability, polymorphic transitions,
or chemical degradation, are often used the differential scanning calorimetry (DSC)
and the thermogravimetry (TGA). Furthermore, the chemical analysis of nanosys-
tem composition is performed by nuclear magnetic resonance (NMR) and Fourier
transform infrared spectroscopy (FT-IR), in which these techniques provide knowl-
edge about the functional groups in the sample and the ratio between the individual
compounds (Li et al. 2009).
Therapeutic Applications
Currently, there are several studies and trials involving micelleplexes as feasible
candidates for the treatment of cancer.
Recently, Magalhães and colleagues developed a micelleplex, Fig. 20.7, to apply
in a gene therapy strategy for osteosarcoma (OS). OS is characterized by genetic
and epigenetic changes, being an extremely aggressive bone tumor. Unfortunately,
the available treatment options are limited, which often leads to patient death. In this
sense, this group developed a micelleplex constituted by an amphiphilic copolymer,
Pluronic® L64, conjugated to a cationic polymer, PEI, to deliver a downregulated
therapeutic miRNA. The results obtained showed a micelleplex capable to effi-
ciently deliver miRNA-145, a tumor suppressor miRNA that is downregulated in
OS cells. miRNAs are a small group of noncoding RNAs that bind and recognize
the untranslated region of a target gene, 3’-UTR region, leading to repression or
degradation of the target messenger RNA (mRNA). In this study, miRNA-145 tar-
geted vascular endothelial growth factor (VEGF), repressing its expression and,
consequently, inhibiting cell migration in an efficient way (Magalhães et al. 2018b).
This research group activated Pluronic® L64 (Fig. 20.8a.1) with acryloyl chlo-
ride, resulting in an intermediate compound, Pluronic® L64 diacrylate (Fig. 20.8a.2).
Then, the activated Pluronic® was conjugated with PEI in order to form a cationic
amphiphilic copolymer, namely, Pluronic® L64-PEI (Fig. 20.8a.3). The developed
cationic micellar complex presented small sizes, between 130 and 160 nm, and a ZP
around 25 mV. The resulting product was characterized by FT-IR and NMR, both
indicating the successful synthesis of this nanosystem.
Fig. 20.7 TEM of the

L64-PEI/myR-145
micelleplex. (Magalhães
et al. 2018b)
280 J. Faria et al.
a
1 3
HN x
y
O O
HO O O
n O m n H N O
O N
CH3 x N O n Om n-1 O N
H H
NH CH3 y
2
O O
O O c
O n O m n-1 O
CH3
Fig. 20.8 (a1) Pluronic® L64 structure. (a2) Pluronic® L64 diacrylate structure. (a3) Pluronic®
L64-PEI structure. (b) FT-IR spectrum of activated Pluronic® L64 and non-activated Pluronic®
L64. (c) FT-IR spectra of the comparison between the different molar ratios of L64-PEI and
between L64-PEI and the corresponding polymers. (Magalhães et al. 2018b)
Figure 20.8b shows the spectra obtained by FT-IR, demonstrating the success of
Pluronic® L64 activation. As expected, in the activated Pluronic®, the band observed
between 3300 and 3600 cm−1 (1) disappeared and aroused a new band between
1700 and 1750 cm−1 (2), which corresponds to the presence of acrylate groups. In
Fig. 20.8c, the spectra of efficient conjugation with the cationic polymer are shown.
The band between 3200 and 3400 cm−1 (3) corresponds to the presence of second-
ary amine groups of PEI, -NH-, while the band between 1100 and 1150 cm−1 (4) is
referenced as the C-OH stretching from Pluronic® (Magalhães et al. 2018b).
After the development of the nanosystem, the authors encapsulate the small
RNA (sRNA) or the therapeutic miRNA-145 and performed an extensive physico-
chemical and structural characterization, as well as in vitro studies to assess the
antitumoral activity. From the results obtained by flow cytometry (Fig. 20.9a), they
verified that complexes containing 50 nM miR-145 promoted a significant decrease
in the viability of OS cells, as well as inhibited cell migration. Thus, this work sug-
gested that treatment with this miRNA induced an antitumoral response through the
activation of regulatory pathways involved in cell migration, proliferation, and
apoptosis. Hence, these micelleplexes, L64-PEI/miR-145, emerged as a promising
and viable nanosystem to efficiently deliver therapeutic miRNAs in alternative
approaches to treat cancer (Magalhães et al. 2018b).
The often failure of a chemotherapeutic therapy is also directly related to the
resistance of the cells to the drugs, either by the existence of efflux pumps, such as
P-glycoprotein (Gp), or by the activation of antiapoptotic pathways. It is known that
cellular autophagy is a process of evolutionary and conserved degradation of cel-
lular components, which under limited conditions of nutrients and toxic stimuli can
a
I II III
4
4
10
10
10
3
3
10
10
10
2
2
10
10
10
1
1
10
10
10
AV
AV
AV
0
0
10
10
10
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
IP IP IP
IV V
4
4
10
10
3
3
10
10
2
2
10
10
1
1
10
10
AV
AV
0
0
10
10
0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10
IP IP
Fig. 20.9 (a) Effects on cell death profile of MG-63 cells 48 h after transfection with the micellar
complexes containing miR-145. (b) Percentage of cell death after 48 h. Viable cells, AV−/PI;
apoptosis, AV+/PI−; necrosis, AV−/PI+; late apoptosis/necrosis, AV+/PI+. (Magalhães et al.
2018b)
282 J. Faria et al.
maintain the production of energy for cell survival. However, it can be inferred that
tumor cell death can be programmed by autophagy blockade, for example, with the
silencing of the gene that activates this process as Beclin1, and an antineoplastic
drug. Chen and collaborators started from this principle and developed a micelle-
plex capable of codelivery shBeclin1 and doxorubicin (DOX).
This micelleplex (FPDP-DOX-snBeclin1) consists of a bifunctional amphiphilic
copolymer based on pullulan (PDP), a cationic polymer, PEI, and folic acid. Folic
acid acts as a ligand to improve the target delivery by recognition of specialized
molecules present on the surface of the cell membrane. Through in vivo assays, the
authors injected female BALB/C nude mice with human cervical carcinoma cells
(HeLa), in which micelleplexes were administered at the tumor site. After adminis-
tration, it was verified a more effective suppression of the tumor growth with a
reduction of the tumor volume (Fig. 20.10b), when compared to the use of DOX or
shBeclin1 alone (Fig. 20.10a). These results demonstrated that the developed nano-
complexes were capable to perform an efficient and specific codelivery of both
DOX and shBeclin1, inducing a most pronounced anticancer effect of DOX by
blocking the process of autophagy (Chen et al. 2018).
Fig. 20.10 (a) Bioimaging of in vivo administration of DOX, monitored by NIR fluorescence
imaging system. (b) Tumor tissue volume images from different treatment groups. Female
BALB/C nude mice with HeLa. (Chen et al. 2018)
Liu and his collaborators have focused their studies on nasopharyngeal cancer, in
particular on the production of a nanosystem capable of delivering a gene that
encodes tissue factor-2 pathway inhibitor (TFPI-2), and an antineoplastic agent,
docetaxel (DOC). The aforementioned gene has been identified as a tumor suppres-
sor gene since it encodes a protein that can inhibit a variety of serine proteases and
the proteolytic activity of the matrix metalloproteinases (MMPs) family (Liu et al.
2016; Li et al. 2017b). DOC is a drug used in conventional therapies to induce apop-
tosis of tumor cells and decrease tumor growth, although it is associated with sev-
eral side effects. To surpass these problems, this group synthesized a micelleplex
containing a cationic polymer, PEI, and an amphiphilic copolymer, Pluronic® F127,
to encapsulate this anticancer drug. Their results demonstrated that this F127-PEI-
DOC-(TFPI-2) nanosystem performed an efficient drug delivery and high in vitro
transfection rate in human nasopharyngeal carcinoma cells (HNE-1), subsequently
promoting a significant increase of TFPI-2 expression levels and DOC presence.
This codelivery boosted a higher therapeutic effect by inducing cell apoptosis and
decreasing invasiveness of HNE-1 cells when compared with the observed for the
treatment with DOC or TFPI-2 alone (Liu et al. 2016).
The work of Shen and colleagues is another therapeutic example, in which the
authors intended to overcome the adverse reactions attributed to chemotherapeutic
agents based on platinum, such as cisplatin, in the treatment of hepatocellular car-
cinoma (HCC). In order to achieve their goal, the authors developed a nanosystem
capable to efficiently deliver siNotch I to suppress the expression of Notch I gene,
which is overexpressed in HCC and is related to the lack of efficacy of platinum
agents. Therefore, it was developed micelleplexes constituted of poly(ε-
caprolactone)-block-poly(2-aminoethylethylene phosphate) (PCL-b-PPEEA) and
platinum(IV)-conjugated poly(ε-caprolactone)-block-poly(ethylene glycol)
(Pt(IV)-PCL-b-PEG), associated with siNotch I: (IV) Pt MCP-siNotch I. This
delivery nanosystem was able to increase the drug concentration, platinum IV, in
the target tumor cells/tissues, suppressing tumor growth and reducing the inci-
dence of cancer stem cells (CSCs) in a synergistic manner, both in vitro and
in vivo. Through flow cytometry, it was found that the treatment with the micelle-
plex (IV) Pt MCP-siNotch I induced apoptosis in 36.1% of HCC cells, which indi-
cated an inhibition of CSCs, mediated by the negative regulation of Notch1 and a
concomitant increase in drug sensitivity to platinum. Concerning to Nocth 1, these
researchers have elucidated that Notch1 is probably an effective therapeutic target
in SMMC7721 xenograft cells. However, it should be performed more extensive
studies in other HCC cell lines, as well as it should be considered the application
of other therapeutic approaches based on RNA interference (RNAi) for a better
understanding of the relationship between Notch1 and CSCs. Despite this, these
researchers defend that this nanocarrier is a great tool to apply in several therapies
against cancer (Shen et al. 2015).
In short, all the studies detailed have shown the therapeutic potential of micelle-
plexes and reach the same conclusion: micelleplexes are effective nanocarriers to
transport genetic material and/or a drug in future alternative therapies for cancer.
284 J. Faria et al.
Conclusions and Future Perspectives
Technological evolution is happening now, and it will keep happening tomorrow

and afterward. This advance along with the breakthrough in other areas of knowl-
edge, such as pharmaceutical nanotechnology and human biology, may sustain an
improvement in the available treatments as well as an enhancement in the success
of treatment results.
The introduction of gene therapies in the market took place in 2017, with the
approval by the American Food and Drug Administration (FDA) of the first gene
therapy for the treatment of acute chronic leukemia. This approval led to the
endorsement of two more treatments based on gene therapy, namely, for retinal
dystrophy associated with the confirmed bi-allelic RPE65 mutation and for some
types of non-Hodgkin’s lymphoma.
The construction of smart and specific nanosystems will be the future of innova-
tive therapies. The cellular non-specificity of certain conventional chemotherapeu-
tic drugs or the lack of efficacy caused by acquired tumor defense mechanisms leads
to progressive wear and tear of patients’ quality of life. In this sense, the existence
of a “double weapon” capable of accurately target drug delivery and dethroning
some tumor mechanisms through genetic manipulation will surpass some of the
above problems.
Over this chapter, it was clearly established the importance and relevance of
micelleplexes, a promising nanosystem for the treatment of several pathologies,
including cancer.
In summary, the synthesis of new biocompatible polymers, both cationic and
amphiphilic, as well as the appearance of new drugs and the demystification of new
genetic targets found in tumor cells, will be the future with the development of new
nanosystems to deliver drugs and genetic material, such as micelleplexes.
Acknowledgments This work was supported by Portuguese National Funds (FCT/MEC,

Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through project UID/
QUI/50006/2013, co-financed by the European Union (FEDER under the Partnership Agreement
PT2020). It was also supported by the grants with the reference POCI-01-0145-FEDER-030255
(PTDC/BTM-MAT/30255/2017) and PTDC/CTM-BIO/1518/2014 from the Portuguese
Foundation for Science and Technology (FCT) and the European Community Fund (FEDER)
through the COMPETE2020 program.
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Chapter 21
Phytomedicines and Their Prospects
in Treatment of Common Skin Diseases
Humaira Rani and Abhai K. Srivastava
Introduction
Herbs have been reported for their healing properties since time immemorial. They
may “include herbs, herbal materials, herbal preparations, and herbal extract prod-
ucts” that are, indeed, a manifestation of traditional medicines. Traditional medi-
cines came into existence due to day-to-day practice and constant observation of the
nature in daily life, while sometimes, it comes from religious beliefs. Nevertheless,
traditional medicines are rooted very deep in culture and civilization of either the
country and have their validation through the generations without any side effect. In
the Indian context, where the cultures and rituals largely depend on scientific
approaches, traditional medicines have a greater impact on society. Nowadays, tra-
ditional medicines are gaining momentous attention in global healthcare consider-
ations and are regarded as the centerpiece of about 75–80% of the world population’s
healthcare, mainly in developing countries, because of better cultural acceptability,
better suitability to the human body, and lesser side effects. The potential of herbs
to cure a variety of diseases/disorders is attributed to the phytochemical compounds
present in plants. The ancient world’s literature of medicinal plants substantiates
from Sumerians, Akkadians, Babylonians, and Assyrians who already inscribed
several medical recipes on clay slabs or tablets (Hassan 2015). The ancient Chinese
text Shennong Bencaojing (Shennong’s Classic of Materia Medica) finds 365
entries on medicaments and their description dates back to around 2500 BC (Zhu
1998). The treatise dating to circa 1550 BC is a 110-page scroll, containing some
700 magical formulas and remedies which include herbal remedies, surgery, and
H. Rani
Amity Institute of Biotechnology, Amity University Uttar Pradesh,
Lucknow Campus, Lucknow, India
A. K. Srivastava (*)
Plant Diversity, Systematics and Herbarium Division, National Botanical Research
Institute Rana Pratap Marg, Lucknow, India

https://doi.org/10.1007/978-981-15-2195-9_21
290 H. Rani and A. K. Srivastava
magical spells (Ghalioungui 1987). The basis for modern Indian Ayurvedic medi-
cine can be found in ancient texts, recorded in Sushruta Samhita and Charaka
Samhita, which dates from about 1000 BC (Kapoor 1990; WHO 2010). The ubiqui-
tous use of traditional medicines could be accredited to simplicity, cultural suitabil-
ity or acceptability, adaptability, economic affordability, and efficacy in combating
or checking the certain type of diseases as compared to modern medicines. Despite
ample development, modern medicine is still a far-reaching goal for weaker section
of society. Thus, different local communities in countries across the world possess
an in-depth knowledge of the use of medicinal plants and their environment. Their
perceptions and aboriginal experiences have helped them to use plants and plant
parts in different ways to heal different health issues.
Among the human diseases treated with medicinal plants in the developing
world, skin diseases are very common in rural areas (Naidoo and Coopoosamy
2011). Even with the high rate of certain skin ailments in developing countries, they
have so far not been regarded as a significant health problem in the development of
public health strategies (WHO 2005). Being the most exposed organ of the body, the
skin forms an active barrier to the external environment and experiences direct con-
tact with various physical, chemical, and biological factors. Interfacing with the
external environment, the skin plays an important immunity role by protecting the
body against active pathogens and thereby constitutes the first line of defense.
Healthy and attractive skin contributes to one’s self-confidence levels, plays a major
role in most persons’ self-esteem, and is a key component of the image they present
to the outside world. Prevailing at all ages, permeating all cultures, and affecting
between 30% and 70% of individuals, skin diseases is one of the most detrimental
human illnesses that has deleterious effects on both physical and mental well-being
(Hay et al. 2014). Indeed, the diseases that unswervingly affect the skin is the fourth
most frequent root of all human diseases, affecting some 1.9 billion people at any
instance, almost one-third of the world’s population. It is, therefore, a leading rea-
son for seeking medical help in all societies (Hay et al. 2014, 2015).
A large number of medicinal plants have been investigated to treat skin diseases
due to their therapeutic activities, including antimicrobial and anti-inflammatory
and their ability to hasten blood clotting, wound healing, as treatments for burns,
and to relieve other skin disorders (Naidoo and Coopoosamy 2011; Pereira and
Bártolo 2016). Various prevailing skin conditions, such as eczema, psoriasis, acne
vulgaris, pruritus, alopecia areata, decubitus ulcer, urticaria, scabies, fungal skin
diseases, impetigo, abscess, and other bacterial skin diseases, cellulitis, viral warts,
and skin cancer can be treated using medicinal plants (Tabassum and Hamdani
2014). This chapter includes a database of some common but effective medicinal
plants used in traditional medicines against skin diseases (Table 21.1).
The changing environmental conditions and increased pollution level are making
the skin more susceptible for infections. On the other hand, people have become
more conscious about their self-esteem, but the healthcare systems are going to
become more expensive which poses a challenge to develop an integrated herbal
medicine system in our healthcare. It led the great demand for herbal medicine in
the developed as well as developing countries like India, because of their wide
21 Phytomedicines and Their Prospects in Treatment of Common Skin Diseases 291
Table 21.1 Database of some common but effective medicinal plants used in traditional medicines
against skin diseases
Plant name
[Family: habit/ Common names Mode of administration
SL. No. part(s) used] Hindi/English and uses References
1. Abrus precatorius Gungchi, Gunja/ Root paste is applied to Buragohain and
L. jequirity, Rati cure leucoderma Konwar (2007),
[Fabaceae: Shrub/ Seed oil along with Kumar et al.
root] crushed leaves of (2013, 2015),
Plumbago zeylanica is Anitha et al.
applied on leucoderma (2008), Gupta
et al. (2010),
Gupta et al. (2017)
and Gupta and
Gupta (2018)
2. Abutilon indicum Kanghi/country Fresh leaves made into Anitha et al.
(L.) sweet mellow a paste with water, and (2008),
[Malvaceae: the paste is applied Sivaperumal et al.
Climber/root] externally on the skin (2009) and Kumar
thrice a day to treat the et al. (2013)
ringworm infection
3. Acacia catechu Khair, Madan, Bark powder is applied Kumar et al.
(L. f.) Willd. Payor, Khayar, externally on wounds (2013) and Bhat
[Fabaceae: Tree/ Dant-dhavan/ et al. (2014)
bark] black cutch tree,
cutch tree, catechu
4. Acacia farnesiana Guh babool, The extracted gum is Saikia et al.
(L.) Willd. Gukikar/Mimosa mixed with egg, and (2006)
[Mimosaceae: bush then it is applied on the
Shrub/gum] skin burns
5., Acacia nilotica (L.) Babul/gum Arabic About two spoonful Kumar et al.
Delile tree powder of stem bark is (2013)
[Fabaceae: Tree/ given orally thrice daily
bark] in leprosy
6. Acalypha indica L. Kuppi/Indian Paste of leaves with Kumar et al.
[Euphorbiaceae: Acalypha water is applied (2015), Anitha
Herb/leaf] externally two times a et al. (2008),
day for a period of Sivaperumal et al.
1 week to treat skin (2009) and Kumar
diseases et al. (2013)
7. Achyranthes aspera Chirchita, Latjira/ Seed paste with little Gupta et al.
L. rough chaff, salt is applied on (2017), Kumar
[Amaranthaceae: prickly chaff carbuncles. Leaf paste et al. (2013),
Herb/leaf,seed] flower is applied on ringworm Policepatel and
affected area till it cures Manikrao (2013)
and Buragohain
and Konwar
(2007)
(continued)
Plant name
8. Adhatoda vasica Vasala/Malabar Decoction of leaf is Sharma et al.
Nees nut used to wash wounds (2012) and Kumar
[Acanthaceae: et al. (2013)
Shrub/leaf]
9. Justicia adhatoda Bahok tita Leaf paste with little Buragohain and
L. (Assamese) sugar is applied on Konwar (2007)
[Acanthaceae: abscesses and Bhat et al.
Shrub/leaf] (2014)
10. Aegle marmelos Bae/wood apple Bark paste is applied on Saikia et al.
(L.) Corr. the ringworm affected (2006), Kumar
[Rutaceae: Tree/ area till it cures, or leaf et al. (2015),
leaf] juice is applied on Anitha et al.
affected area daily (2008) and
twice for a week Policepatel and
Manikrao (2013)
11. Aerva lanata (L.) Chhaya Juice prepared from Sivaperumal et al.
Juss.Ex Schult. gorakhbuti, Khari, whole plant with water (2009) and Kumar
[Amaranthaceae: Gorakhganja/ is taken orally three et al. (2013)
Shrub/whole plant] Polpala times a day for a period
of 2 days to reduce
eczema
12. Ageratum Visadodi/ Leaf paste is applied Kumar et al.
conyzoides L. goatweed, externally on leprosy (2013)
[Asteraceae: Herb/ whiteweed, Appa wounds
leaf] grass
13. Albizia lebbeck (L.) Siris/Lebbek tree, Juice of the bark is Dashahre et al.
Benth. east Indian walnut, applied on the (2014)
[Caesalpiniaceae: woman’s tongue, ringworm, scabies, and
Tree/bark, fruit] black Siris septic ulcer. Fruit paste
is applied on sore eyes
14. Allium cepa L. Pyaz/onion Bulb paste mixed with Saikia et al.
[Liliaceae: Herb/ curcuma paste is (2006), Kumar
bulb] applied on itching et al. (2013),
affected area 1–2 days Policepatel and
Manikrao (2013)
and Gupta et al.
(2017)
15. Allium sativum L. Lassan, lahsan, Bulbs are crushed and Saikia et al.
[Liliaceae: Herb/ lahsun/garlic mixed with coconut oil (2006),
bulb] and applied on Buragohain and
ringworm and scabies Konwar (2007),
Kumar et al.
(2013), Policepatel
and Manikrao
(2013) and Gupta
and Gupta (2018)
(continued)
Plant name
16. Aloe vera (L.) Gheekumari/aloe Leaf juice is applied on Gupta et al.
Burm. f. [Liliaceae: vera, medicinal ringworm infected area (2010), Policepatel
Herb/leaf] aloe, burn plant for 3–4 days and Manikrao
(2013) and Gupta
and Gupta (2018)
17. Alpinia nigra Tora (Assamese)/ Tubers are eaten fresh Buragohain and
(Gaertn.) Burtt. black galangal, which is said to cure Konwar (2007)
[Zingiberaceae: wild ginger scabies and other skin
Herb/tuber] diseases
18. Alstonia scholaris Chitvan, Shaitan The bark is crushed Saikia et al.
(L.) R.Br. Ka Jhar/devil tree, together with (2006),
[Apocynaceae: dita bark Achyranthes aspera, Buragohain and
Tree/latex, bark] Piper longum, and Konwar (2007),
Piper nigrum, and the Kumar et al.
mixture formed is orally (2013) and Gupta
taken in leprosy. The et al. (2017)
gum extracted from the
bark is applied directly
on the toe to relive toe
cracks. The gum is
applied directly on the
skin to treat cellulites
19. Amaranthus Kanta Nutiya, Whole plant juice is Saikia et al.
spinosus L. Kanta Chaulai/ applied on allergic (2006),
[Amaranthaceae: thorny amaranth, infected area before Buragohain and
Herb/leaf] spiny pigweed bathing daily once Konwar (2007),
Anitha et al.
(2008), Sharma
et al. (2012) and
Policepatel and
Manikrao (2013)
20. Ammi majus Bishop’s weed, Leaf paste is applied to Sidi and
bullwort, lace cure vitiligo Bourgeious-
flower Gavardin (1952)
21. Andrographis Kalpanath, Leaf paste is applied on Buragohain and
paniculata Kirayat/ abscesses Konwar (2007),
(Burm.f.) Nees Andrographis, Anitha et al.
[Acanthaceae: Kalmegh (2008),
Herb/leaf] Sivaperumal et al.
(2009), Gupta
et al. (2010), Devi
and Das (2015)
and Gupta and
Gupta (2018)
(continued)
Plant name
22. Annona reticulata Ramphal/netted Paste of older leaves is Saikia et al.
L. custard apple, applied on ringworm (2006) and
[Annonaceae: Bullock’s heart affected area till it curesPolicepatel and
Shrub/leaf] Manikrao (2013)
23. Annona squamosa Sharifa, Dried leaf powder Saikia et al.
L. Seethaphal, soaked in Carthamus (2006), Kumar
[Annonaceae: Shareefa/custard tinctorius oil for 24 h et al. (2015),
Shrub/leaf] apple, sugar apple and applied on Gupta et al.
ringworm affected area (2010), Kumar
daily once for 5–6 days et al. (2013) and
Policepatel and
Manikrao (2013)
24. Areca catechu L. Udveg, pug, The nuts are dried in Buragohain and
[Arecaceae: Tree/ Supari, Guvak, the sun and crushed into Konwar (2007),
nuts] Chamarpushpa, powder, which is Kumar et al.
Khapur, Guwa/ applied on septic ulcer (2015) and Gupta
areca palm, betel for quick healing et al. (2010)
nut
25. Argemone Satyanashi/prickly Latex or whole plant Kumar et al.
mexicana L. poppy, Mexican paste is applied on (2015), Anitha
[Papaveraceae: poppy eczema affected area et al. (2008),
Herb/leaf, latex] for a week Sharma et al.
(2012), Kumar
et al. (2013),
Policepatel and
Manikrao (2013)
and Gupta and
Gupta (2018)
26. Aristolochia indica Hooka-bel/Indian Paste prepared from Anitha et al.
L. birthwort, duck vegetative parts of the (2008),
[Aristolochiaceae: flower plant in water is applied Sivaperumal et al.
Shrub/root] externally once in a day (2009), Gupta
for a week to treat et al. (2010) and
dandruff Bhat et al. (2014)
27. Artocarpus Katahal, Kathal/ Latex is applied Sharma et al.
heterophyllus lam. jackfruit, jackfruit externally as dressing (2012) and Devi
[Moraceae: Tree/ tree over septic wounds and Das (2015)
latex]
(continued)
Plant name
28. Azadirachta indica Neem/Margosa, Leaves are boiled, and Buragohain and
A. Juss Indian lilac, neem the boiled water is used Konwar (2007),
[Meliaceae: Tree/ tree, Margosa in bathing, which is said Kumar et al.
leaf, bark] to cure scabies and (2015), Anitha
other skin diseases such et al. (2008),
as ringworms, scabies, Sivaperumal et al.
and wounds. Crushed (2009), Gupta
leaves are applied on et al. (2010),
ringworm. Crushed Sharma et al.
bark is applied to cure (2012), Policepatel
abscess and Manikrao
(2013), Gupta
et al. (2017),
Gupta and Gupta
(2018) and Saikia
et al. (2006)
29. Basella alba L. Poi/Malabar The crushed leaves are Buragohain and
[Basellaceae: spinach, Indian mixed with cheese, and Konwar (2007),
Climber/leaf] spinach it is then applied on the Bhat et al. (2014)
skin burns. The and Saikia et al.
extracted juice is (2006)
applied directly on the
infected skin to cure
urticaria
30. Bauhinia variegata Kanchan, Leaf paste is applied on Buragohain and
L. Kachnar/ebony septic ulcer for quick Konwar (2007),
[Caesalpiniaceae: tree healing Kumar et al.
Tree/leaf] (2013) and Gupta
et al. (2010, 2017)
31. Murraya koenigii Kare bevu Leaf paste is applied
(L.) Spreng. (Kannada)/Mithi daily once on psoriasis
[Rutaceae: Shrub/ neem affected area till it cures
leaf]
32. Boerhavia diffusa Biskhapara, Leaf paste is applied on Saikia et al.
L. Shothagni, abscess (2006),
[Nyctaginaceae: Gadahpurna, Buragohain and
Herb/leaf] Gadhacand/ Konwar (2007),
common hogweed Anitha et al.
(2008) and
Sivaperumal et al.
(2009)
(continued)
Plant name
33. Bombax ceiba L. Simal, Semal, Thorns are crushed and Buragohain and
[Bombacaceae: Kaantisenbal, mixed with milk and Konwar (2007)
Tree/thorns] Semar Kanda/silk applied on pimples and Kumar et al.
cotton, kapok tree, (2013)
Malabar Semul,
Indian bombax
34. Brassica juncea Sarson/leaf The extracted oil mixed Saikia et al.
(L.) Czern. mustard, mustard with juices from (2006)
[Brassicaceae: cabbage, brown Cynodon dactylon and
Herb/seed] mustard, Chinese Curcuma longa and
mustard, mustard water. The mixture is
greens, Indian heated. The product so
mustard formed is applied on the
infected skin to cure
scabies. The crushed
seeds are applied on the
face for pimple
treatment
35. Bryophyllum Dupor tenga, pate Leaf paste is applied on Saikia et al.
pinnatum (lam.) gaja(Assamese)/−− abscesses and (2006),
Oken pediculosis Buragohain and
[Crassulaceae: Konwar (2007),
Herb/leaf] Kumar et al.
(2015) and Gupta
and Gupta (2018)
36. Butea monosperma Dhak, Palash, Latex is applied on Buragohain and
(Lam.) Taub. Palash, Tesu, ringworm Konwar (2007),
[Fabaceae: Tree/ Dhak/Forest Kumar et al.
latex] flame, Flame Of (2013, 2015),
The Forest, Policepatel and
Bengal kino tree, Manikrao (2013),
Bastard teak, Bhat et al. (2014)
Flame of Forest and Gupta and
Gupta (2018)
(continued)
Plant name
37. Calotropis gigantea Safed Aak, Ak, Latex is applied Kumar et al.
L. W. T. Aiton Arka, Mudar/ externally in skin (2015), Anitha
[Asclepiadaceae: bowstring hemp, diseases et al. (2008),
shrub/latex] crown flower, Sivaperumal et al.
giant calotrope, (2009), Gupta
giant milkweed et al. (2010),
Sharma et al.
(2012), Policepatel
and Manikrao
(2013), Devi and
Das (2015),
Kumar et al.
(2013), Bhat et al.
(2014) and Gupta
et al. (2017)
38. Calotropis procera Arka, Arki, Latex is applied on Buragohain and
(ait) R.Br. Mudar, Safed-ak/ carbuncles Konwar (2007),
[Asclepiadaceae: giant milkweed, Kumar et al.
Shrub/latex] calotrope, king’s (2015) and Gupta
crown, small and Gupta (2018)
crown flower,
rooster tree,
auricula tree,
apple of sodom
39. Cannabis sativa L. Ganja/marijuana One tea spoonful Kumar et al. (2013
[Cannabinaceae: powder of dried leaf is
Shrub/leaf] given orally twice daily
in leprosy and eczema
40. Carica papaya L. Papita/papaw tree, Fresh latex is applied Saikia et al.
[Caricaceae: Shrub/ papaya on ringworm, pimples (2006),
latex] and sore eyes Buragohain and
Konwar (2007),
Sharma et al.
(2012), Devi and
Das (2015),
Kumar et al.
(2013), Policepatel
and Manikrao
(2013), Bhat et al.
(2014) and Gupta
and Gupta (2018)
(continued)
Plant name
41. Cascabela thevetia Peeli kaner/yellow Leaf paste with latex is Saikia et al.
[Apocynaceae: oleander, cook applied on the (2006) and Gupta
Shrub/roots, leaf] tree, be-still tree, ringworm affected area, and Gupta (2018)
lucky nut, Dicky daily once for 2 weeks.
plant, Mexican The paste is layered on
oleander, Lucky the infected portion.The
Nut juice extracted from
leaves is mixed with
equal amount of water
and is orally taken to
treat leprosy
42. Cassia alata L. Ergaj, Prapunnad/ Leaf juice is applied on
Buragohain and
[Caesalpiniaceae: Ergaj, Prapunnad ringworm Konwar (2007),
Shrub/leaf] Anitha et al.
(2008),
Sivaperumal et al.
(2009), Sharma
et al. (2012),
Gupta et al. (2017)
and Gupta and
Gupta (2018)
43. Cassia fistula L. Swarn-pushpi, Leaf paste is applied on Saikia et al.
[Caesalpiniaceae: Amaltas, septic ulcer for quick (2006),
Tree/leaf] Amaltash/Indian healing. The leaves are Buragohain and
laburnum, the crushed and applied on Konwar (2007),
Indian laburnum, the ringworm infection Kumar et al.
pudding-pipe tree, (2013, 2015),
purging fistula Anitha et al.
(2008),
Sivaperumal et al.
(2009), Bhat et al.
(2014) and Gupta
and Gupta (2018)
44. Cassia tora L. Chakunda/pot Leaf paste is applied on Buragohain and
[Caesalpiniaceae: Cassia scabies and ringworm Konwar (2007),
Undershrub/leaf] Anitha et al.
(2008), Sharma
et al. (2012), Bhat
et al. (2014) and
Gupta and Gupta
(2018)
(continued)
Plant name
45. Caesalpinia bonduc Putikaranj, Putik, Seed cotyledon paste Policepatel and
(L.) Roxb. Gajga, Kat- mixed with castor oil is Manikrao (2013)
[Caesalpiniaceae: kaleji,Kat Karanj, applied on ringworm
Climber/seed] Kat-karanj, Pattil, infected areas till it
Panshul/Bonduc cures
nut, fever-nut,
fever nut,
physic-nut, physic
nut
46. Centalla asiatica Brahma Manduki/ Leaf paste is applied on Buragohain and
(L.) Urb. Pohekula, Gotu abscess and carbuncles Konwar (2007),
[Apiaceae: Herb/ kola, Spadeleaf, for quick healing Kumar et al.
leaf] Indian penny-wort (2015), Gupta and
Gupta (2018),
Kumar et al.
(2013), Sharma
et al. (2012) and
Gupta et al. (2010)
47. Cinnamomum Tejpatta/Indian Fresh leaf paste is Buragohain and
tamala (Buch.- bark, Indian applied on allergy for Konwar (2007)
ham) Nees & Cassia lignea quick relief
Aberm.
[Lauraceae: Tree/
leaf]
48. Citrus aurantifolia Nibu, Nimbu/key Fruit juice with little Buragohain and
(Cristm.) Swingle lime coconut oil is applied Konwar (2007)
[Rutaceae: Shrub/ on scabies
fruit]
49. Citrus limon (L.) Paharinimbu, Fruit juice is applied on Saikia et al.
Osbeck [Rutaceae: Paharikaghzi, the body to relieve from (2006) and
Shrub/fruit] Baranimbu, prickly heat Buragohain and
Gulgul/lemon Konwar (2007)
50. Citrus medica L. Bara Nimbu, bara The fruit juice is orally Saikia et al.
[Rutaceae: Shrub/ nimbu, Kutla, taken for skin softening (2006) and
fruit] Bijaura, Bijaura and making body color Policepatel and
Irula/citron fairer and wrinkled skin Manikrao (2013)
51. Clitoria ternatea L. Khagtu, Gokarni, The flowers are mixed Saikia et al.
[Fabaceae: Aparajitha, with roots of the Piper (2006) and Anitha
Climber/flower] Aparajita/Clitoria, longum plant, and the et al. (2008)
butterfly bean mixture is crushed.The
paste formed is layered
on the infected skin in
leprosy
(continued)
Plant name
52. Cocos nucifera L. Nariyal/coconut The coconut oil is Saikia et al.
[Arecaceae: Tree/ tree, coconut mixed with Citrus (2006)
frui] limon juice, and the
mixture is applied on
the infected place in
scabies
53. Corchorus Patta shaak/white Seed paste mixed with Policepatel and
capsularis L. jute castor oil is applied on Manikrao (2013)
[Tiliaceae: Shrub/ ringworm infected areas
seed] till it cures
54. Coriandrum Dhania/coriander Leaf paste is applied on Policepatel and
sativam L. allergic affected area Manikrao (2013)
[Apiaceae: Herb/ for a week
aerial part]
55. Crotalaria pallida San/striped Leaf paste is applied to Buragohain and
Aito. crotalaria, smooth cure scabies and Konwar (2007)
[Fabaceae: rattlebox, ringworm
Undershrub/leaf] Brusquillo,
smooth crotalaria,
salts rattlebox,
cascabelitos, ding
ding, streaked
rattlepod
56. Cryptolepis dubia Kala bel, Karanta/ Latex is applied on –
(Burm.f.) wax-leaved ringworm affected area
M.R.Almeida climber, Palvalli, on alternate days till it
[Asclepiadaceae: wax leaved cures
Climber/aerial part] climber, Indian
sarsaparilla
57. Curcuma aromatica Jangli Haldi/ Fresh rhizome paste is Buragohain and
Salisb. Cochin turmeric, applied on the Konwar (2007)
[Zingiberaceae: wild turmeric, ringworm and scabies and Gupta and
Herb/rhizome] wild turmeric/ Gupta (2018)
yellow zedoary,
yellow zedoary
58. Curcuma longa L. Haldi/turmeric The extracted juice is Sharma et al.
[Zingiberaceae: mixed with milk and (2012)
Herb/root] ghee, and the mixture is
orally taken in scabies
(continued)
Plant name
59. Cyanthillium Sahadevi/little Handful of leaves is Anitha et al.
cinereum (L.) ironweed, purple pound and gently (2008),
H.rob. feabane heated up in coconut Sivaperumal et al.
[Asteraceae: Herb/ oil; the extract is (2009), Kumar
leaf] applied externally to et al. (2013) and
treat leprosy and Gupta and Gupta
scabies. Leaf juice is (2018)
applied externally on
wounds
60. Cynodon dactylon Dubori-bon, Dhub Leaf juice is applied on Saikia et al.
(L.) Pers.[Poaceae: grass (Assamese)/ allergy and prickly heat (2006),
Grass/leaf] to get quick relief Buragohain and
Konwar (2007),
Anitha et al.
(2008), Sharma
et al. (2012),
Kumar et al.
(2013) and Bhat
et al. (2014)
61. Datura metel L. Dhatura/Datura, Leaves are gently Anitha et al.
[Solanaceae: Herb/ jimson weed, heated on flame and (2008),
fruit] thorn apple, devils applied on the face once Sivaperumal et al.
weed, purple in a day for a week to (2009), Kumar
horn-of-plenty treat pimples and other et al. (2013) and
skin infections Gupta and Gupta
(2018)
62. Datura stramonium Dhatura/Datura, Leaf paste is applied on Saikia et al.
L. mad apple, stink eczema (2006),
[Solanaceae: Shrub/ weed, jimson Buragohain and
leaf] weed, Jamestown Konwar (2007)
weed, common and Policepatel
thorn apple, thorn and Manikrao
apple, Devil’s (2013)
apple
63. Daucus carota L. Gajar/queen Root juice is applied on Saikia et al.
[Apiaceae: Herb/ Anne’s lace, wild scabies (2006),
root] carrot Buragohain and
Konwar (2007)
and Gupta et al.
(2010, 2017)
64. Dioscorea bulbifera Kadu Kanda, Paste of tuber is applied Kumar et al.
L. Ratalu, Gainth/ externally on boils to (2013)
[Dioscoreaceae: bulb bearing yam, discharge pus
Climber/tuber] air potato, potato
yam, potato yam/
air potato
(continued)
Plant name
65. Eclipta prostrata Bhringaraj, Fresh leaves are ground Anitha et al.
(L.) L. Kesharaj/false to paste; leaf paste is (2008),
[Asteraceae: Herb/ daisy, trailing used to treat the Sivaperumal et al.
leaf] eclipta ringworm infection. (2009) and Bhat
Leaves gently heated up et al. (2014)
in coconut oil, and the
extract is applied on the
head for a week to treat
dandruff
66. Elephantopus Samdudri, About half glass Kumar et al.
scaber L. ban-tambakhu/ decoction is given (2015), Anitha
[Asteraceae: Herb/ elephant foot, orally twice daily as et al. (2008),
whole plant] prickly-leaved blood purifier in skin Gupta et al.
elephant’s foot, diseases (2010), Sharma
Bull’s tongue, et al. (2012) and
ironweed Kumar et al.
(2013)
67. Embelia ribes Baba-rang, Embelia ribes, Emblica Saikia et al.
Burm. f. baberang, officinalis, Piper (2006)
[Myrsinaceae: bhabhiramg, longum, and Terminalia
Climber/fruit] karkannie, bellirica are mixed in
vayvidamg, equal amounts, and the
wawrung/Embelia, crushed form (powder)
Baoberang, is added to honey, and
Vidanga the resulting mixture is
then applied on the
carbuncle infection
68. Enhydra fluctuans Helecha,Harkuch/−− Leaves are taken as Buragohain and
lour. vegetable to cure Konwar (2007)
[Asteraceae: Herb/ leucoderma. Leaf juice
leaf] is applied on prickly
heat to get quick relief
69. Erythrina stricta Ronga modar/ Leaf paste is applied on Buragohain and
Roxb. Indian coral tree abscess Konwar (2007)
[Fabaceae: Tree/
leaf]
70. Euphorbia Danda thaur, The gum is mixed with Saikia et al.
neriifolia L. Danda-Thor, gum from Calotropis (2006) and Kumar
[Euphorbiaceae: Sehund/oleander gigantea and powder et al. (2015)
Shrub/branch] spurge, Indian from Berberis aristata
spurge tree, hedge in equal amounts, and
euphorbia the mixture is applied
on the infected skin to
treat carbuncle
(continued)
Plant name
71. Ficus benghalensis Barh, Bar, Bor, Latex is applied on Buragohain and
L. Bargad/east Indian abscess, septic ulcer, Konwar (2007)
[Moraceae: Tree/ fig tree, banyan and cracked heels for and Anitha et al.
latex] tree, banyan, quick healing (2008)
Indian fig tree,
Indian banyan
tree, east Indian
72. Ficus religiosa L. Pipal/Peepal, Barks are crushed and Buragohain and
[Moraceae: Tree/ Peepal tree, Pipul, mixed with little milk Konwar (2007),
bark, latex] sacred Bo tree, and applied on scabies. Anitha et al.
sacred fig Latex is applied on (2008),
cracked heels for quick Sivaperumal et al.
healing (2009), Bhat et al.
(2014) and Gupta
and Gupta (2018)
73. Flemingia Clipti, Kanphuta/ Pounded roots are Buragohain and
strobilifera (L.) wild hops applied on ringworm Konwar (2007)
W.T.Aiton
[Fabaceae: Shrub/
root]
74. Foeniculum vulgare Moti saunf/fennel, The Feniculum vulgare Saikia et al.
mill. sweet fennel, is crushed along with (2006)
[Apiaceae: Herb/ Florence fennel, Coriandrum sativum.
seed] Finocchio The mixture mixed with
ghee and sugar is orally
taken in scabies
75. Garuga pinnata Kharpat/Garuga The leaves are crushed Saikia et al.
Roxb. and directly applied on (2006)
[Burseraceae: Tree/ the skin to treat
leaf] ringworm
76. Gloriosa superba Bachnag, Rhizome paste is Sharma et al.
L. kadyanag, kari applied externally on (2012) and Kumar
[Liliaceae: hari, languli, ringworm et al. (2013)
Climber/rhizome] ulatchandal/glory
lily, gloriosa lily,
tiger claw, claw
77. Hemidesmus Anantamul, A handful of the dried Anitha et al.
indicus (L.) R.Br. Sugandi Pala/ roots are pounded and (2008) and Gupta
[Periplocaceae: Indian sarsaparilla boiled in 100 ml of and Gupta (2018)
Climber/root] coconut oil. Few drops
of the oil extract are
externally applied twice
a day to treat eczema,
scabies, and the
ringworm infection
(continued)
Plant name
78. Holarrhena Kura, Kora, The juice of the bark is Gupta et al. (2010)
pubescens wall. Ex Kureya, Kurchi/ taken to cure leprosy
G.Don Connessi bark,
[Apocynaceae: Coneru,
Tree/bark] Tellicherry bark
79. Holoptelea Papri, Chilbil, Leaf paste is applied Kumar et al.
integrifolia planch. Papri, Kanju/ over common as well as (2013)
[Ulmaceae: Tree/ Indian elm, jungle leprosy wounds bark
leaf, bark] cork tree, Kanju paste is applied over
ringworms
80. Indigofera tinctoria Neel, black henna, Juice prepared from Anitha et al.
L. common indigo, whole plant is mixed (2008),
[Fabaceae: Shrub/ commercial with goat’s milk and Sivaperumal et al.
whole plant] indigo,, Bengal taken orally three times (2009) and Gupta
indigo a day for 3 days to treat et al. (2017)
leukorrhea
81. Ipomoea aquatica Kalmi/water Leaf juice is applied on Buragohain and
Forsk. spinach, swamp prickly heat to get quick Konwar (2007)
[Convolvulaceae cabbage, water relief
trailer/leaf] morning glory
82. Jatropha curcas L. Ratanjot, Jangli The juice from leaves is Saikia et al.
[Euphorbiaceae: Arandi, Jamal mixed with egg yolk, (2006),
Shrub/leaf, branch] Ghota/physic nut, and the mixture is Sivaperumal et al.
purging nut applied on the skin (2009), Sharma
burns. et al. (2012), Bhat
Teeth are brushed with et al. (2014) and
the cut branches for Gupta et al. (2017)
protection from
microbes
83. Justicia adhatoda Arus, arusa, The juice extracted is Saikia et al.
L. pramadya, Rus, mixed with honey, and (2006), Anitha
[Acanthaceae: sinh-parni, vajini, the mixture is orally et al. (2008) and
Shrub/leaf] visauta/Adhatoda, taken in leprosy. The Sivaperumal et al.
Vasaka, Malabar juice extracted from the (2009)
nut, vasa leaves is also used for
taking bath in measles.
The leaves are crushed
along with Curcum
alonga and the
extracted juice is used
to wash the infection in
cellulitis. A bit of sugar
is added to the crushed
form, and the paste is
layered on the infection
to cure abscess
(continued)
Plant name
84. Lawsonia inermis Mehendi/henna, Leaf paste is applied on Saikia et al.
L. Egyptian privet, abscesses and septic (2006),
[Lythraceae: Shrub/ henna Egyptian ulcer for quick healing Buragohain and
leaf] privet Konwar (2007),
Sharma et al.
(2012), Kumar
et al. (2013),
Policepatel and
Manikrao (2013),
Gupta et al. (2017)
and Gupta and
Gupta (2018)
85. Leucas aspera Chhota Halkusa, Paste made from Anitha et al.
(Willd) link Gophaa, Gopha, macerating leaves is (2008) and
[Lamiaceae: Herb/ Chota Halkusa applied externally to Sivaperumal et al.
flower, leaf] Irula/common treat ringworm infection (2009)
Leucas, Thumba,
Thumbe
86. Linum Alsi/flax, common Seed paste is applied as Gupta and Gupta
usitatissimum L. flax, flaxseed, poultice over hard boils (2018)
[Linaceae: Herb/ linseed for easy discharge of
seed] pus
87. Mangifera indica Aam/mango tree, Latex is applied on sore Saikia et al.
L. mango, cuckoo’s eyes (2006),
[Anacardiaceae: joy Buragohain and
Tree/latex] Konwar (2007),
Policepatel and
Manikrao (2013)
and Bhat et al.
(2014)
(continued)
Plant name
88. Melia azedarach L. Bakain/bead tree, The leaves are boiled in Saikia et al.
[Meliaceae: Tree/ Persian neem water, and that water is (2006) and Kumar
leaf] used to wash the et al. (2013)
infected place. The
crushed form is mixed
with ghee, and the
mixture is applied on
the infected skin in
carbuncles. The leaves
are boiled in water, and
that water is used to
rinse scabies infected
place.
The paste(crushed
form)is layered on the
boils, and the extracted
juicei s used to wash the
infection for treating
abscesses
89. Mentha arvensis L. Podina/field mint, The juice from the Saikia et al.
[Lamiaceae: Herb/ corn mint leaves is applied on the (2006)
leaf] face for removal of
pimples
90. Mesua ferrea L. Nag-kesar, Leaf paste is applied to Buragohain and
[Clusiaceae: Tree/ nag-kesar-ka-aitr, cure pimples Konwar (2007)
leaf] Gajapushpam, nag
Champa,
Nagkesar/
ironwood tree
91. Meyna spinosa Bahu-vij, Ripe fruits are rubbed Buragohain and
Roxb. Ex link. dal-amal,main,mayan/−− on cracked heels for Konwar (2007)
[Rubiaceae: Shrub/ quick healing. Seed
fruit, seed] paste is applied on
pimples
92. Mimosa pudica L. Chui Mui, A handful of the entire Anitha et al.
[Mimosaceae: Lajvanti/humble plant is made to paste (2008),
Herb/root, leaf/ plant, sensitive and applied on cuts and Sivaperumal et al.
whole plant] plant, touch me wounds to promote (2009), Gupta
not healing. Fresh leaves et al. (2010), Bhat
are ground to paste et al. (2014),
which is applied Buragohain and
externally to treat Konwar (2007)
eczema. Root paste is and Gupta et al.
applied on septic ulcer (2010)
for quick healing
(continued)
Plant name
93. Mirabilis jalapa L. Gul abbas, Dried root tuber ground Anitha et al.
[Mimosaceae: Gulbakshi/4 O′ to paste with water; the(2008),
Herb/root, leaf] clock plant, paste applied externallySivaperumal et al.
marvel of Peru to treat the sebaceous (2009) and Kumar
cysts and polyps et al. (2013)
94. Moringa oleifera Senjana/the The gum is poured on Saikia et al.
lam. horseradish tree, the eyes to abscess on (2006), Kumar
[Moringaceae: drumstick tree the eyes et al. (2015),
Tree/root bark] Gupta et al.
(2010), Kumar
et al. (2013) and
Gupta and Gupta
(2018)
95. Mukia Madras pea Fresh leaves are ground Anitha et al.
maderaspatana (L.) pumpkin, rough to paste; leaf paste is (2008) and
M. Roem bryony/Aganaki, applied externally to Sivaperumal et al.
[Cucurbitaceae: Agumaki, Bilari treat scabies and (2009)
Climber/leaf] ringworm infection
96. Neolamarckia Kadam/Kadam, Leaf and bark paste is Kumar et al.
cadamba (Roxb.) Cadam applied externally on (2013)
Bosser wounds
[Rubiaceae: Tree/
leaf, bark]
97. Ocimum Kali tulasi/hoary Leaf crushed leaves are Buragohain and
americanum L. basil, wild basil, mixed with a pinch of Konwar (2007)
[Lamiaceae: lemon basil salt and applied on the
Undershrub] ringworm
98. Ocimum sanctum Tulosi Crushed leaves are Saikia et al.
L. (Assamese), mixed with a pinch of (2006),
[Lamiaceae: Tulasi(Kannada)/−− salt and applied on the Buragohain and
Undershrub/leaf] ringworm Konwar (2007),
Kumar et al.
(2015), Gupta
et al. (2010),
Sharma et al.
(2012) and Devi
and Das (2015),
Policepatel and
Manikrao (2013),
Gupta and Gupta
(2018)
(continued)
Plant name
99. Ocimum Tulsi/English holy Fresh leaves are ground Anitha et al.
tenuiflorum L. basil, sacred basil to paste; fine paste is (2008),
[Lamiaceae: Herb/ applied on the wound to Sivaperumal et al.
leaf] promote healing (2009), Kumar
et al. (2013) and
Bhat et al. (2014)
100. Osbeckia Bagafutkala The juice extracted Saikia et al.
nepalensis Hook. f. (Assamese)/Nepal from the leaves is used (2006)
[Melastomataceae: Osbeckia for treating carbuncle
Shrub/leaf, root] The juice from the roots
is mixed with the seed
extract of Croton
tiglium and ashes. The
resulted mixture is
applied in leprosy
101. Oxalis corniculata Amrit Sak/Indian Leaf juice is applied to Saikia et al.
L. sorrel, creeping cure scabies, and root (2006),
[Oxalidaceae: oxalis, Inda, paste is applied on Buragohain and
Creeper/leaf] creeping wood eczema Konwar (2007),
sorrel Kumar et al.
(2015) and Gupta
and Gupta (2018)
102. Paederia foetida Gandhali/Chinese Leaf juice is applied on Buragohain and
L. [Rubiaceae: fever vine, stink allergy. Leaf juice with Konwar (2007)
Climber/leaf] vine garlic is eaten to relieve
from allergy
103. Phyllanthus Brahma Vriksh, Dried fruits with little Buragohain and
emblica L. Aonla, Bahu-muli, amount of molasses are Konwar (2007),
[Euphorbiaceae: Amla, Anwla/ eaten to relieve from Anitha et al.
Tree/fruit] Indian gooseberry, allergy (2008) and
Emblic Sivaperumal et al.
myrobalan, (2009)
Gooseberry, Amla
104. Piper nigrum L. Jhaluk The crushed form of the Saikia et al.
[Piperaceae: (Assamese)/ fruit are mixed with (2006), Anitha
Climber/fruit] common pepper, ghee, and the product is et al. (2008),
pepper, black orally taken for scabies Sivaperumal et al.
pepper (2009), Sharma
et al. (2012),
Policepatel and
Manikrao (2013),
Bhat et al. (2014)
and Gupta and
Gupta (2018)
(continued)
Plant name
105. Pithecellobium Jungle Jalebi/ Decoction of the leaf Kumar et al.
dulce (Roxb.) Madras thorn and root bark is used to (2013)
Benth. wash leprosy
[Fabaceae: Tree/
leaf and root bark]
106. Plumbago Chitrak, Chita, Kumar et al.
Leaf juice is applied on
zeylanica L. Chitrak/white ringworm affected area (2015), Anitha
[Plumbaginaceae: Plumbago, wild daily thrice for et al. (2008),
Herb/leaf] Plumbago, white 3–4 days Sivaperumal et al.
leadwort, Ceylon (2009), Gupta
leadwort et al. (2010),
Kumar et al.
(2013), Policepatel
and Manikrao
(2013), Bhat et al.
(2014), Gupta
et al. (2017) and
Gupta and Gupta
(2018)
107. Polyalthia Ashok/false Crushed barks are made Buragohain and
longifolia (Sonn.) ashoka, mast tree, into paste and applied Konwar (2007)
Thw. cemetery tree, on scabies
[Annonaceae: Tree/ asoka
bark]
108. Pongamia pinnata Karanj/Indian Dried bark powder is Anitha et al.
(L) Pierre. beech, pongam oil gently fried in coconut (2008),
[Fabaceae: Tree/ tree, Hongay oil oil, and the extract is Sivaperumal et al.
bark] tree applied externally to (2009), Bhat et al.
treat eczema, psoriasis, (2014) and Gupta
rashes, scabies, and et al. (2017)
ringworm infection
109. Ricinus communis Arandi/ricin, Leaf paste is used to Saikia et al.
L. Castor, Castor oil cure carbuncles. Seed (2006),
[Euphorbiaceae: plant, castor bean, paste mixed with Buragohain and
Shrub/leaf, seed] Krapata turmeric powder is Konwar (2007),
applied on the itching Gupta et al.
affected area daily once (2010), Kumar
till it cures et al. (2013),
Policepatel and
Manikrao (2013)
and Gupta and
Gupta (2018)
(continued)
Plant name
110. Rubia cordifolia L. Majith, Lachkura/ Fresh roots or fresh Anitha et al.
[Rubiaceae: Indian madder tender shoot is made (2008) and
Climbing herb/root] into paste; paste is Sivaperumal et al.
applied externally on (2009)
iron weapon injured
part of the body
111. Santalum album L. Sandal, Chandan/ Leaf paste is applied on Policepatel and
[Santalaceae: Tree/ sandal, allergic affected area Manikrao (2013)
leaf] sandalwood, white daily once for 4–5 days and Gupta et al.
sandal (2017)
112. Sapindus mukorossi Henil, Risht, The seed covers are Saikia et al.
Gaertn. Ritha, Rishta/ rubbed and used for (2006) and Kumar
[Sapindaceae: Tree/ soapberry, soap washing the hairs for et al. (2013)
seed] nut, Reetha, controlling dandruff
Hlingsi, Dodan
113. Senna auriculata Tarwar, Awal/ Root decoction is Policepatel and
(L.) Roxb. Tanner’s senna, applied on eczema Manikrao (2013)
[Ceasalpiniaceae: styptic weed, infected area for
Tree/flower] Tanner’s cassia 1–2 weeks
114. Sesamum indicum Til, Gingli, Safed The seeds are orally Saikia et al.
L. Til/ginger oil plant taken along with water. (2006)
[Pedaliaceae: Herb/ The eyes become
seeds] brighter, and the hair
gets beautiful
115. Sesbania Hathya, Agasti, The crushed flowers are Saikia et al.
grandiflora (L.) Gaach-munga/ mixed with curd and (2006)
Pers. swamp pea cheese, and the mixture
[Fabaceae: Tree/ is applied on the dry
flower] skin
116. Sida acuta Burm.f. Kharenti, Kareta, The paste is layered on Saikia et al.
[Malvaceae: Herb/ Bariara, the boils for treating (2006) and Gupta
leaf] Paharibariara/ abscess et al. (2010)
common
wireweed,
morning mallow,
broomweed
117. Solanum Baigun/eggplant, Leaf paste is applied Sharma et al.
melongena L. Brinjal externally on leprosy on (2012) and Kumar
[Solanaceae: Shrub/ leprosy on cuts and et al. (2013)
leaf, fruit] wounds. Roasted fruit is
applied as poultice over
hard boils
118. Solanum surattense Bhatkataiya/bitter Decoction of plant is Kumar et al.
Burm. f., brinjal given orally twice daily (2013)
[Solanaceae: Herb/ as blood purifier in skin
whole plant] diseases
(continued)
Plant name
119. Solanum tuberosum Aloo, alu/potato It is peeled, and the Saikia et al.
L. segments are rubbed (2006), Kumar
[Solanaceae: Herb/ against the burnt places et al. (2013) and
root] Gupta and Gupta
(2018)
120. Stephania japonica Rajapatha/tape Leaf paste is applied on Buragohain and
(Thunb.) Miers. vine, Snake vine septic ulcer for quick Konwar (2007),
[Menispermaceae: healing Kumar et al.
Climber/leaf] (2015), Gupta
et al. (2010) and
Devi and Das
(2015)
121. Sterculia foetida L. Jangli Badam/ Seed paste mixed with Policepatel and
[Sterculiaceae: skunk tree, Indian leaf juice is applied on Manikrao (2013)
Tree/seed] almond, Java ringworm affected area
olive, hazel daily twice till it cures
sterculia
122. Streblus asper lour. Sihora, Dahia, Leaf decoction is used Saikia et al.
[Moraceae: Tree/ Sewra/Siamese to take bath for (2006)
leaf] rough bush, relieving itching and
toothbrush tree scabies
123. Swertia chirayita Chirayata, The dried form is orally Saikia et al.
(Roxb.) Buch.-ham. Charaita, Chirata/ taken in leprosy (2006) and Kumar
Ex C.B.Clarke Chirayita The juice is applied et al. (2013)
[Gentianaceae: directly on the infected
Herb/leaf] skin for treating scabies
124. Tephrosia purpurea Dhamasia, Fresh leaf paste is Kumar et al.
(L.) Pers. Sarphanka/wild applied on itching (2015), Anitha
[Fabaceae: Shrub/ indigo, common affected area daily once et al. (2008),
leaf] Tephrosia, purple till it cures Sivaperumal et al.
tephrosia (2009), Kumar
et al. (2013) and
Gupta and Gupta
(2018)
125. Terminalia bellirica Bahera, bahuvirya, Crushed fruit bark is Buragohain and
(Gaertn.) Roxb. bhutvaas, kalk, applied on septic ulcer. Konwar (2007)
[Combretaceae: karshphal/bastard The bark of the fruit is and Policepatel
Tree/fruit] myrobalan, beach removed, and the and Manikrao
almond, bedda nut remaining part is (2013)
tree crushed into a paste and
applied on sore eyes
(continued)
Plant name
126. Tinospora sinensis Gulbel, Gurch, The juice of the leaves Saikia et al.
(lour.) Merr. Gulancha, Giloy/ with little honey is (2006),
[Menispermaceae: moon creeper, taken to cure leprosy Buragohain and
Climber/leaf] Gulancha Konwar (2007),
Tinospora, Gupta et al. (2010)
heart-leaved and Policepatel
moonseed, Indian and Manikrao
Tinospora (2013)
127. Tribulus terrestris Gokhru/goat’s Whole plant juice is Anitha et al.
L. head, devil’s applied on psoriasis (2008), Gupta
[Zygophyllaceae: weed, bullhead, affected area daily et al. (2010) and
Herb/aerial part] land caltrops, cat’s twice till it cures and Policepatel and
head, caltrop, also applied on white Manikrao (2013)
puncture plant, patches of the skin
Tribulus, devil’s
thorn
128. Trichopodium Methi/fenugreek, Fresh leaves are ground Anitha et al.
zeylanicum Greek clover, to paste; the paste is (2008)
(Gaertn.) Thwaites Greek hay applied externally to
[Fabaceae: Herb/ treat scabies and
leaf] ringworm infection
129. Tridax procumbens Akal Kohadi, The paste is layered on Saikia et al.
L. Khal-muriya, the infected skin for (2006), Kumar
[Asteraceae: Herb/ Tal-muriya/ curing cellulites et al. (2015),
leaf] Mexican daisy, Anitha et al.
coat button (2008), Gupta
et al. (2010),
Kumar et al.
(2013) and
Policepatel and
Manikrao (2013)
130. Trigonella Methiguti The seeds are soaked in Saikia et al.
foenum-graecum L. (Assamese)/ water and then crushed. (2006), Kumar
[Fabaceae: Herb/ The pastes formed is et al. (2015) and
seed] layered on the burnt Gupta et al. (2010)
skin
131. Typhonium Chomahu, choma Latex is applied on Buragohain and
trilobatum (L.) Kachu abscesses and pimples Konwar (2007)
Schott (Assamese)/
[Araceae: Climber/ Bengal Arum,
latex] lobed leaf
Typhonium
132. Urena lobata L. Unga, Bachita, Leaf paste is applied on Buragohain and
[Malvaceae: Shrub/ Lapetua/Caesar’s septic ulcer for quick Konwar (2007)
leaf] weed, common healing
purple mallow
(continued)
Plant name
133. Vigna mungo (L.) Urd/black gram Seeds are crushed into aSaikia et al.
Hepper. paste and applied on (2006) and
[Fabaceae: Herb/ scabies and ringworm Buragohain and
seed] Konwar (2007)
134. Vitex negundo L. Nirgundi/ The crushed leaf is Saikia et al.
[Verbenaceae: Tree/ five-leaved chaste applied on the skin, and (2006) and
leaf, root] tree, common the extracted juice is Buragohain and
chaste tree, orally taken to cure Konwar (2007)
Negundo, urticaria. The juice
Negundo extracted from the
Chastetree, chaste crushed leaves and
tree roots is mixed with the
oil extracted from
Sesamum indicum and
applied on the skin for
treating carbuncles
135. Wrightia tinctoria Karayaja, Dudhi, Handful of leaves is Anitha et al.
R.Br. Mitha-indrajau, pound and gently (2008) and
[Apocynaceae: Indrajau, Dudhi, heated up in coconut oil Sivaperumal et al.
Tree/leaf] kala Kuda, Kuda/ and made to paste; the (2009)
Pala indigo, sweet paste is externally
indrajao, Dyer’s applied to treat eczema
oleander and scabies
136. Zanthoxylum --/prickly ash Young leaves are eaten Buragohain and
oxyphyllum Edgew. as vegetable to cure Konwar (2007)
[Rutaceae: Shrub/ leucoderma
leaf]
137. Zingiber officinale Adrak/spice The juice of the Policepatel and
roscoe ginger, garden rhizome with little Manikrao (2013)
[Zingiberaceae: ginger, ginger, amount of molasses is and Gupta and
Herb/rhizome] Canton ginger, eaten to get relief from Gupta (2018)
Halia urticaria
b iological activities, higher safety of margin than the synthetic drugs, and lesser
costs. Herbal industry in India uses about 8000 medicinal plants (Sharma et al.
2008), and about 25,000 licensed pharmacies of Indian herbal medicines are run-
ning including approximately 1000 single drugs and about 3000 registered com-
pound formulations.
This review includes a total of 137 plant species that have been documented for
their medicinal properties in reference to skin diseases on the basis of literature
survey. It includes information regarding biofunctional parts and their mode of
administration in various skin diseases which is regarded trifling health problem due
to their benign, not life-threatening minor botheration, and are at least priority when
it comes to individual’s health. But morbidity cannot be overlooked. Skin diseases
occur worldwide and amount to approximately 34–70% of all occupational diseases
encountered. Approximately 50% of plant species useful for treatment of skin dis-
eases appear to be restricted to forests, so activities, such as deforestation, habitat
destruction, urbanization, etc., may pose a serious threat to these species. The exten-
sive literature survey reveals an idea about more than 300 plant species which are
used in treatment of skin disease under various community practices. Till now, the
researchers have focused only up to the documentation of ethnobotanical knowl-
edge, but their proper validation of doses and mode of administration including
pharmacological screening of active principles are still a goal to achieve. Therefore,
the present chapter reviews several medicinal plants and mode of applications of
their processed formulation for enhancing complexion and other skin-related dis-
eases to make the herbal drugs more acceptable, valuable, lifesaving, and economy
reformer for the mankind.
References
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Chapter 22
Microbes as Natural Products for Drug
Discovery
Sagarika Devi
Natural Products in Historical Perspective
Natural products hold colossal structures and chemical assortment that remain
unmatched to any synthetic library of small molecules and endure to instigate
unconventional findings in chemistry, biology and medicine. They are progressively
modified as drug-like molecules and linger to be effective sources of medication and
leading drugs (Newman 2012). The depiction on clay tablets in cuneiform in
2600 B.C. from Mesopotamia on oils from Cupressus sempervirens (cypress) and
Commiphora species (myrrh) used till date for treatment of coughs, colds and
inflammation is the primitive annals of natural products (Cragg and Newman 2005).
In 2900 B.C., Ebers Papyrus is an Egyptian pharmaceutical chronicle that docu-
mented more than 700 herbal drugs ranging from gargles, tablets, potions to oint-
ments. In 100 A.D., Dioscorides, a physician from Greece, documented the
collection, storing and utilization of herbs with medicinal values, whilst around
300 B.C., Theophrastus, a philosopher and natural scientist from Greece, dealt with
curative herbs (Cragg and Newman 2005). During the Dark and Middle Ages, this
knowledge from the west was preserved by the English, French, Irish and German
monasteries whilst the Greco-Roman knowledge was preserved by Arabs intensify-
ing practices of their own unfamiliar resources, together with Chinese and Indian
curative herbs (Cragg and Newman 2005). The fungus Piptoporus betulinus was
steamed to yield charcoal which was valued for its antiseptic and disinfectant prop-
erties (Swanton 1915). Strips of the fungus P. betulinus were well-known to create
very soothing corn pads utilized for curtailing bleeding (Swanton 1932). Agaricus
campestris Linnaeus ex Fries (field mushroom) originating in north and south tem-
perate zones and the Caribbeans have been stewed together with milk to appease
throat cancer (Hatfield 2005). Marine red algae such as Chondrus crispus and
S. Devi (*)
National Institute for Research in Tuberculosis, Chennai, Tamil Nadu, India
e-mail: sagarika.d@nirt.res.in

https://doi.org/10.1007/978-981-15-2195-9_22
318 S. Devi
Mastocarpus stellatus were not only a source of beverage but also prevalent as tra-
ditional medication for colds, sore throats and in infections of chest including tuber-
culosis. It was stewed in water or milk and utilized for problems and burns in the
kidney (Vickery 1995; Moloney 1919). Medical history is thus full of notable sto-
ries of profound impact of natural product finding in biology and inspired discovery
of drugs and therapy. The Nobel Prizes in Physiology or Medicine to Ernst B. Chain
and Alexander Fleming for penicillin, to Howard Florey for finding streptomycin in
1945 and to Selman A. Waksman in 1952 presaged the beginning of golden age of
natural product drug discovery during 1950s–1960s.
Fundamental understanding in the biosynthesis of products from nature has
endorsed coherent manipulation of nature’s biosynthetic production of both natural
produces and their engineered variants. Mining of the genome for nature’s bounty
so-called “cryptic” natural biosynthetic pathways has started to promote these
“sleeping beauties.” Finally, evolving strain prioritization, analytical know-how and
bioinformatics have transformed dereplication of natural products, facilitating pre-
diction of the structure, rapid detection and isolation of promising natural products
at a prompt pace (Bouslimani et al. 2014; Rudolf et al. 2016). Combined with the
cutting-edge advances in biosynthesis of natural products, bioinformatics, metage-
nomics and high-throughput analytical tools, we are setting foot in the new golden
age of drug discovery from natural products. Though more than 20,000 secondary
metabolites from microbes have been defined, merely a fewer percentage of these
have been accepted as naturally derived drugs. Innovations in cultivation procedures
(Ling et al. 2015) and application of engineering principles to biology (Paddon et al.
2013) have radically put forth the chemical space for products from nature and vali-
dated the feasibility of engineering naturally derived products from uncultivated
species or endangered species in model heterologous hosts. Natural microbial prod-
ucts have resolved numerous other hitches of our world owing to their distinctive
activities. They include avermectins like anthelmintic agents (Campbell 2012;
Omura 2008), bioinsecticides, enzyme inhibitors, terpenoids, nutraceuticals, surfac-
tants, polysaccharides and the most imperative area of vaccines.
iscovery of Drugs from Natural Products and Development:

D
A Multidisciplinary Process
The progression of discovery of drugs for healing and precautionary medicines is

expedited by expanding understanding of the biotic mechanisms of every challenge
and requires factors such as cure competence, probable side effects and the increas-
ing emerging threat of drug resistance to be addressed. Strategies in drug discovery
for application in agrochemistry and pharmaceuticals are in a radical period of
change (Auerbach et al. 2002). The closing of dozens of microbial pathogenic
genomes and the end of Human Genome Project have delivered thousands of tech-
niques for screening of various disease-related targets. Since the “golden age of
antibiotics” and the global spur to find new antimicrobials, numerous contemporary
22 Microbes as Natural Products for Drug Discovery 319
pharmaceutical industries instigated programs to discover natural products (NPD)

that engross not only on targets such as antibacterial and antifungal but also on dis-
eases that are infectious. The search for lead compounds towards treatment of
microbial infections, tissue rejections during organ transplantations, hypercholes-
terolemia and cancer has been successful through programs (Baker et al. 2007;
Ojima 2008). The initiation of automated high-throughput screening (HTS) meth-
ods has augmented the biological testing drive and combinatorial chemistry is
endorsed as an enhanced method to produce “drug-like” compounds. Advancements
such as instrument systems with automation, robotic instruments, platforms for
high-throughput screening (HTS) and high-throughput chemical analysis have pro-
vided potent tools for cost-effective methods to screen large compound libraries.
Synthetic chemical, natural product and combinatorial chemical libraries offer
ample resources for screening methods which are target based. The triumph and the
failure in discovery of drugs is united to the originality and significance of applied
biotic test systems as well as to the quantity and chemical diversity of the available
test compounds (Fernandes 2001). Numerous protein targets that are pertinent to
various diseases are being identified and authenticated through system/computa-
tional biological approaches and diverse “-nomics.”
In the search of apt natural products for drug discovery, target selection, high-
throughput screening methods and assessment systems are aspiring for added pre-
cise, dependable and low-background searches (Cochran 2000). This highlights
discovering novel amalgams from prevailing natural product libraries. The “CE
assay” identifies effective ligands even in the existence of high concentrations of
frail viable ligands and added undesirable compounds. An integrated multidisci-
plinary process could be utilized for possible quick isolation and characterization of
potential drug leads in therapeutic targets (Pierceall et al. 2003). Rapid identifica-
tion and dereplication of potent compounds is now very much possible due to the
emergence of recent combined mass spectroscopy technologies such as HPLC-MS,
HPLC-NMR and HPLC-NMR-MS (Baker et al. 2007; Brkljaca and Urban 2011). A
combined strategy involving a blend of bioassays, separation techniques, mass
spectrometry and database search can be considered in chemical or biological
screening practices. The recent advancement in genomics over a couple of years has
led to the rampant growth of secondary metabolites with uncharacterized biosyn-
thetic gene clusters in public databases.
In the older eras, classical chemistry of naturally derived products has essentially
been substituted by molecular and targeted approaches of drug discovery. An inte-
grative approach by merging numerous discovery tools and new disciplines of inte-
grative biology will surely offer a solution in the discovery and development of
naturally derived drugs. Nevertheless developments in know-how and refined
instrumentation for prompt identification and structural elucidation of novel natural
bioactive products endure improvization in the discovery of natural products (Baker
et al. 2007). Natural products can be anticipated to remain an indispensable element
in the pursuit and expansion for first-hand, benign and inexpensive remedies
employing enormous combinatorial libraries to acquire competent lead molecules
(Ojima 2008). Pharmaceutical industry must thus arise in this instance to amend its
approach and reshuffle its assets.
320 S. Devi
Microorganisms in Drug Discovery
Microorganisms are an inexhaustible resource of bioactive metabolites with struc-

tural diversity and have borne several of the utmost substantial products of pharma-
ceutical industry. Secondary metabolites from microbes are currently being
employed in the production or catalysis of nutraceuticals, vitamins, enzymes,
proteins, organic acids, antibiotics and food ingredients other than antibacterial,
antifungals and antiviral infections. Microorganisms are distinguished as an inex-
haustible reserve of natural products numerous of which are therapeutically signifi-
cant products. Whilst researchers still have plentiful to comprehend from the nature,
the tremendous growth of technologies and tools has made it likely to isolate unique
diverse natural products. Through the perception of biosynthesis of natural prod-
ucts, DNA sequence data intensification, expansions in bioinformatics and analyti-
cal methodologies, discovery of natural products from microbes has continued as a
dynamic field providing compounds with distinctive biochemistry and mode of
action to propel the succeeding generation of drug development.
Fungi, both macro and micro, have been linked with human wellness since
decades. Apart from being utilized as food (mushrooms) and in production of alco-
holic beverages (yeasts), fungi have been used in conventional medicine and for
ethnic purposes as well. Presently with the progress in microbiology, utilization of
microbes and their metabolites has unfurled to enzymes, biological control, antimi-
crobials and other pharmacologically active products (Lorenzen and Anke 1996).
Undoubtedly discovery of penicillin by Fleming in 1929 from the fungus, Penicillium
notatum, remains one of the utmost illustrious natural product discoveries originat-
ing from a microbe (Mann 1994). Saving countless lives, in the early 1940s, this
discovery steered the re-isolation and experimental studies by Chain, Florey and
co-workers to promote commercialization of synthetic penicillins revolutionizing
the drug discovery research (Alder 1970; Lax 2004; Wainwright 1990; Mann 1999).
There was a global enterprise between 1942 and 1944 to find new antimicrobials
and bioactive natural products after the reports of the first scientific data on penicil-
lin G (Williams 1999; Buss and Waigh 1995). With the initiation of advanced
screening techniques in the 1970s and the production of bacterial strains susceptible
to β-lactams, inhibitory tests of β-lactamases and selectivity for sulphur comprising
metabolites ensued discovery of unique antibiotic structural categories (norcardi-
cins, carbapenems and monobactams) together with the isolation of antibiotics,
such as norcardicin, aztreonam and imipenem (Fig. 22.1a–c), respectively (Williams
1999; Fabbretti et al. 2011). Presently besides glycylcyclines, a novel class of broad-
spectrum antibiotics, there are nine β-lactams such as two cephalosporins, six car-
bapenems and one penem which are considered in clinical trials or undergoing drug
registration (Fabbretti et al. 2011). Following these accomplishments, new
compounds have continued to be discovered. In 1948, Lederle Laboratories of
American Cyanamid (now Pfizer) led by Benjamin Duggar’s group asserted discov-
ery of chlortetracycline (aureomycin, biomycin) produced by Streptomyces aureo-
faciens. It was consented for utilization against bacteria, both gram-positive and
Fig. 22.1 (a) Norcardicin, (b) Aztreonam and (c) Imipenem
gram- negative, and was shortly preceded by discovery of oxytetracycline

(Terramycin). Scientists at Parke-Davis (now Pfizer) together with workers at Yale
University and University of Illinois discovered chloramphenicol, an exclusively
diverse compound. Owing to its relatively simple structure, chloramphenicol pro-
duced by Streptomyces venezuelae has furthermore been synthesized chemically.
Additional aminoglycosides such as kanamycin and gentamicin with broad-spec-
trum efficacy were discovered which are bactericidal and chemically stable.
Polypores are largely wood-rotting macrofungi belonging to phylum Basidiomycota
(Basidiomycetes) and Ascomycota that form a chief source of pharmacologically
active substances. Approximately strong antimicrobial activity has been exhibited
by 75% of assessed polyporus fungi. Several of these compounds have demon-
strated cardiovascular, antineoplastic, anti-inflammatory, immune-stimulating,
cytotoxic, antiviral and anticancer activities (Zjawiony 2004; Stamets 2002).
Fungi or collectively known as endophytes are more common microorganisms
inhabiting trees, grasses, algae and herbaceous plants. They live inside the cellular
spaces of plant stems, petioles, roots and leaves and do not influence the host (Gwinn
et al. 1992; Tan and Zou 2001; Petrini 1986). Numerous of the promising natural
products relevant to pharmaceutical industry have been formulated from novel bio-
active metabolites derived from endophytes. Vancomycin, a glycopeptide antibiotic,
first isolated in 1953 by Edmund Kornfeld (Fig. 22.2) produced in cultures of
Amycolatopsis orientalis is an active inhibitor of gram-positive organisms such as
Staphylococci and Streptococci and gram-negative bacteria, fungi and mycobacte-
ria. It was approved by FDA in 1958 for treatment of severe infection and for inhib-
iting susceptible organisms in patients hypersensitive to penicillin (Butler 2004).
Erythromycin (Fig. 22.3), a macrolide isolated from Saccharopolyspora erythraea,
is an antibiotic consisting a 14-membered macrocycle composed exclusively of pro-
pionate units. Broadly active against gram-positive cocci and bacilli, erythromycin
is also used against mild to restrained upper and lower respiratory tract infections
(Butler 2004; Dewick 2002). Other uses of macrolides include immunosuppressants
such as sirolimus (rapamycin) and tacrolimus (FK506), antitumor agents such as
epothilones and antiparasitics such as avermectin. Presently, cethromycin (ABT-773,
Restanza™), EP-420 (by Enanta Pharmaceuticals) and BAL-19403 (by Basilea) are
the three semisynthetic ketolide derivatives of erythromycin which are being con-
sidered in clinical development (Mishra and Tiwari 2011). Fewer natural antiviral
322 S. Devi
Fig. 22.2 Vancomycin
Fig. 22.3 Erythromycin
products or synthetic analogues of fungal origin have been reported till date
(Newman and Cragg 2007). Significant anti-HIV-1 protease activity was demon-
strated by Ganoderma lucidum spores and ganoderic acid β derived from its fruiting
bodies with an IC50 value of 20 μM (Min et al. 1998). In 2002, from the fungus
Streptomyces peucetius two compounds related to anthracycline such as doxorubi-
cin (Fig. 22.4) (Adriamycin®) and amrubicin hydrochloride have been isolated.
Doxorubicin is used for the cure of critical leukaemia, soft tissue and bone
malignancies, cancer of the lungs, thyroid and both Hodgkin’s and non-Hodgkin’s
lymphomas (Butler 2004; Dewick 2002). Torreyanic acid (Fig. 22.5) evaluated in
numerous cancer cell lines has been extracted from an endophyte from Torreya taxi-
folia (Lee et al. 1996), the endangered tree, and is found to demonstrate enhanced
Fig. 22.4 Doxorubicin
Fig. 22.5 Torreyanic acid
potency/cytotoxicity around 5–10 times in cell lines sensitive to protein kinase C

instigating cell death by apoptosis (Li et al. 2003).
The introduction of immunosuppressants such as cyclosporin A, FK506 and
rapamycin (Fig. 22.6) in the recuperative utilization of metabolites from microbes
and their derivatives is another notable breakthrough. Several natural products
derived from microbes are used as anti-diabetic drugs, antagonists, anticancer drugs,
hormones (ion channel or receptor) and agricultural and pharmaceutical agents and
have been well established. The commercialization of antihyperlipidaemics such as
lovastatin followed by recent discovery of guggulsterone is amongst other exam-
ples. Natural enzyme inhibitors such as statins derived from microbial sources
inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting and
regulatory cholesterol biosynthesis enzyme in the liver. An antibiotic compactin
isolated from Penicillium brevicompactum and later from Penicillium citrinum is
the foremost member of the group. In 1970, lovastatin (monacolin K; mevinolin), an
ethylated form, was isolated from the broth cultures of Monascus ruber and
Aspergillus terreus. Other genera such as Doratomyces, Eupenicillium, Gymnoascus,
Hypomyces, Paecilomyces, Phoma, Trichoderma and Pleurotus are amongst few
which are involved in statin production.
The deep sea has a rich track record in offering unique entities with novel struc-
tural diversity. In the past 40 years, progressive technological advancements in the
exploration of marine milieu have given rise to the isolation of numerous unique
bioactive marine natural products with structural diversity. With the majority of
documented metabolites commencing from bacterial species such as Salinispora,
Pseudoalteromonas and Vibrio, marine microorganisms are a potential resource of
new bioactive compounds based on diverse chemical scaffolds (Mouton et al. 2012;
Richards et al. 2012; Debbab et al. 2010; Jensen et al. 2005; Holmstrom and
324 S. Devi
Fig. 22.6 Rapamycin
Kjelleberg 1999; Bowman 2007; Mansson et al. 2011). However, a plethora of bio-
logically active compounds have also been isolated from marine fungi inhabiting
mangrove and intertidal zones (Richards et al. 2012; Jones 2011a, b) with isolates
of Penicillium and Aspergillus as the foremost frequent sources compared to fungi
inhabiting true marine habitats (Debbab et al. 2010; Bugni et al. 2004). These com-
pounds primarily have not only been isolated from substrates such as driftwood
(Abdel-Wahab and Gareth Jones 2000) and macroalgae (Loque et al. 2010) but also
in deep sediments [42,54,55] (Mouton et al. 2012; Burgaud et al. 2009; Khudyakova
et al. 2000). It remains ambiguous however if these denote strains which are true
marine isolates or simply opportunistic strains which have accustomed to the marine
environment (Burgaud et al. 2013). Several Bacillus species produce secondary
metabolites which are structurally diverse such as lipopeptides, polypeptides, mac-
rolactones, fatty acids, polyketides, lipoamides and isocoumarins (Hamdache et al.
2011). An extensive array of biological activities such as antimicrobial, anticancer,
anti-algal and anti-peronosporomycetal (Baruzzi et al. 2011) is demonstrated by
these lipopeptides.
Amongst half of the known secondary metabolites such as antibiotics (Bull 2004;
Berdy 2005; Strohl 2004), antitumor agents (Olano et al. 2009), immunosuppres-
sive agents (Mann 2001) and enzymes (Pecznska-Czoch and Mordarski 1988;
Oldfield et al. 1998) are customarily produced by actinomycetes. Microbial meta-
bolic studies of aromatic amino acids demonstrate evolution of secondary metabolic
pathways with the capability of producing potent compounds unveiling an effective
array of pharmacological uses. A phenol terpenoid called rubrivivaxin is produced
by Rubrivivax benzoatilyticus JA2 bacterial species conferring broad-spectrum anti-
microbial activity and cytotoxicity against U937 (human leukaemic monocyte lym-
phoma) and Jurkat (T lymphocyte) cell lines (Ranjith et al. 2011). Rhodestrin,
isolated from anthranilate photo biotransformation by Rhodobacter sphaeroides
OU5, demonstrates antitumor and antimicrobial potency (Sunayana et al. 2005). A
group of marine-derived polyketides designated as daryamides have been isolated
from actinobacteria with potent antibiotic and antitumor activity (Ratnakar et al.
2006). Whilst almost 200 antibiotics are produced by strains of Streptomyces hygro-
scopicus, strains of Streptomyces griseus are well-known to produce over 40 diverse
antibiotics. Amongst other organisms, strains of Bacillus subtilis are proficient anti-
biotic makers producing over 60 such compounds.
In the context of global advent of multidrug-resistant (MDR) and extensively
drug-resistant (XDR) strains, antibiotic treatment remains a severe global health
threat. Glycolipids, lipopeptides, polysaccharides, proteins and lipoproteins or mix-
tures thereof are an extensive array of structurally different biosurfactants which
have been identified till date (Neu 1996; Banat et al. 2000; Ron and Rosenberg
2001; Maier 2003; Mulligan 2005; Muthusamy et al. 2008). Lipopeptide biosurfac-
tants (LPs) produced by fungi including Aspergillus and various bacterial genera
such as Streptomyces, Pseudomonas and Bacillus contain a lipid tail linked to a
small linear or cyclic oligopeptide. Lipopeptides have received substantial consid-
eration for demonstrating antimicrobial activity, cytotoxicity, antitumour, immuno-
suppressant and surfactant properties (Cameotra and Makkar 2004; Donadio et al.
2007; Gross and Loper 2009; Pirri et al. 2009). Myxobacteria are relatively large
gram-negative rods with diverse morphology forming fruiting bodies that glide or
creep and are evidently an uncommon resource producing varied secondary metab-
olites. Until 2005, more than 400 compounds had been reported from myxobacteria
but epothilones with potential antitumor activity were the first in clinical trials that
acted like taxol but demonstrated activity against taxol-resistant tumours. These
epothilones, originally developed as antifungal agents to inhibit rust fungi (Gerth
et al. 2000), are 16-member ring polyketide macrolide lactones isolated from the
myxobacterium Sorangium cellulosum, but have established their efficacy as antitu-
mor compounds (Goodin 2008).
Untapped Potential of Microbial Diversity
The outstanding intricacy and molecular diversity of products from nature has been
always emphasized but even more significant is the fact that the surface of these
exclusive natural products reservoir has scarcely been scrapped. Assuming the fact
that a handful of soil contains billions of microbial organisms (Pace 1997), the
microbial universe distinctly offers an enormous unexploited inventory for discov-
ery of drugs. Researchers have realized that the potency of natural products derived
from microorganisms is underestimated, as most of the biosynthetic gene clusters
are “silent” or “cryptic” in the host organism culture condition of the laboratories.
Stimulation of these silent gene clusters could spur much interest. To isolate
microbes with secondary metabolites of interest, samples from environment have
frequently been steered without well-defined approaches (Shrestha et al. 2001). The
biogeography of ecosystems, sample number with reference to frequency of sample
collection and procedures leading to isolation of desired microbial species is a
prerequisite in such programs. The potential of the deep sea as a cradle of unique
drugs remains virtually unexplored (Newman and Cragg 2004; Newman and Hill
2006), and until lately, the exploration of the marine had chiefly been constrained to
tropical and subtropical areas; however, the investigation is now being stretched out
326 S. Devi
to colder regions. The number and diversity of sampling sites needs to be increased
(Foissner 1999), and it is particularly vital to investigate untapped sites. Most identi-
fied compounds with pharmaceutical value are mainly from the offshore regions
owing to the ease in collecting samples and deep sea has an unusual environment
where not much investigation has been conducted due to its inaccessibility. But with
recent development in technology, scientists have been capable to acquire samples
and deduce the possible compounds with pharmaceutical potential. Lately there has
been a considerable shift toward research on microorganisms over macroorganisms
in deep sea as they can be cultured and manipulated in bioreactors and have a higher
possibility of producing bioactive compounds since they thrive in one of the utmost
extreme environments. Novel microorganisms which are potential providers of
novel compounds have mostly generated from varied expanses such as the profound
subsurface, the deep sea and locations with extreme temperature, salinity or pH
(Bull 2000). Organisms near the hydrothermal vents and the hydrocarbon seeps can
be stated as prospective candidates as there are large variations of temperature in
really short intervals of time. With the advent of novel isolation strategies, temper-
ate ecosystems ought not to be barred for but they too are potential providers of
several novel microbial species.
Potential Problems with Natural Product Drugs
Products from nature offer incomparable chemical diversity with structural intri-
cacy and biological efficacy (Verdine 1996). They serve as “pathfinders” in the
exploration of cellular functions (Hung et al. 1996) and could escort designing of
synthetic compounds (Breinbauer et al. 2002). However the dearth of methodical
utilization of ecosystems for discovery of novel compounds from microorganisms
has given rise to random sampling methods missing out the real potential of various
regions (Czaran et al. 2002).
The incongruity between the range of microorganisms distinguished by molecu-
lar methods and the count of strains in cultures validates that there still rests rela-
tively unexploited resources of novel strains in every ecosystem (Harvey 2000).
Isolation, purification and structure interpretation of target composites from com-
plex blends of crude extract are the key holdups in natural products chemistry.
Production processes may require extensive substrate optimization and scale-up,
aiming to isolate ample quantities of the active compound desired for drug profiling
(Strobel 2002). The spell, energy and cost to obtain novel chemical entities and lack
of definitive dereplication strategies have led to redundancy of strains and com-
pounds within several libraries for natural product (Handelsman et al. 1998). Crude
natural products encompass multifarious mixtures of possibly hundreds of u nrelated
compounds functioning in interaction when the concentrate is administered as a
whole. Strategies such as extraction of crude from the source, concentration, lyophi-
lization (in cases of polar solvents), fractionation and purification are included in
the process of discovering promising natural product hits and their advancement en
route for expansion to isolate a single bioactive compound of interest. During the
year 1990–2005 combinatorial chemistry was emphasized and departed by numer-
ous pharmaceutical companies from the area of natural products. Failing to create
sufficiently structurally diverse and pharmacologically active molecules this effort
proved futile to substitute natural products with synthetic molecules. Consequently,
the numbers of (a) FDA drug uses, (b) novel drug endorsements, (c) new and per-
mitted active constituents, (d) orphan drug applications and (e) new chemical enti-
ties of the therapeutic trade declined tremendously since the late 1990s. Much of the
discovery efforts have been left to small companies and the biotechnology industry
since the withdrawal of several of the major pharmaceutical companies. Thus the
prospect of natural product extract screening is probably in the utilization of a blend
of procedures that might comprise pre-fractionation, HTS expending “designer”
cell lines that have precise aspects of the cell’s metabolic machinery modified to
give a signal when repressed or stimulated, united to microliter capacities of extract.
Historically, nature has formed the basis for plentiful of available drugs in use
today. However products from nature are in feisty competition with vast chemical
libraries and with combinatorial chemistries. Many pharmaceuticals since the late
twentieth century however have considerably stepped back or forsaken their natural
product programs partly due to the outstanding developments in both high-
throughput screening (HTS) and combinatorial synthesis, thereby creating enor-
mous synthetic libraries for small molecules. In contrast, natural product libraries
have been perceived to be unsuitable for contemporary HTS platforms as they con-
sist of extracts, partially purified fractions in addition to pure compounds. Hence
from the beginning of environmental sample collection for strains selection, expres-
sion of metabolic pathways, genetic exploitation, sample preparation and chemical
dereplication, every stride in any natural product discovery program has to be more
proficient than ever.
Summary
Naturally derived products have been and will linger to be a bountiful resource of
novel drugs. With the advent of novel screening systems allied to the outburst of
genetic information accelerating, the necessity to identify potent novel lead struc-
tures rapidly is a vital necessity. Microbes are large mines of natural bioactive com-
ponents, namely, sugars, terpenoids, esters, acids, peptides, nucleopeptides, proteins
and ethers used immensely as antibacterial, antifungal, anti-inflammatory, antican-
cer and antiviral agents with diverse structural and functional forms. New natural
products, chemistry tools and new bioassay techniques with relevance to virulence
are existing and wait for keen employment by interdisciplinary investigation teams.
In prospect with the beginning of genetic methods that allow segregation and mani-
festation of biosynthetic cassettes, microbes and their marine invertebrate hosts may
well be the novel frontier for discovery of natural drug leads (Imhoff et al. 2011).
Coupling these novel sources to revamped phenotypic screening methods that uti-
328 S. Devi
lize high-content imaging systems and can be run in microliter volumes optimisti-
cally will empower analysts to swiftly evaluate the action of specific agents and
their prospective in future drug development. This will also necessitate the expertise
of talented synthetic chemists who have the capability to shape structures from
nature in order to augment their properties for forthcoming drug usage.
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Chapter 23
Natural Products from Actinobacteria
for Drug Discovery
Soumya Nair and Jayanthi Abraham
Introduction
The intense research in the field of translational drug discovery and synthesis has
been continuously developing from its early days. Due to the extensive research for
the past six decades, more than ten thousand natural compounds have been deriva-
tised from the different microbial sources. These products have been thoroughly
used because of its various applications in the field of pharmaceuticals, food and
health. Increasing need towards drug discovery has led to new technologies in clini-
cal trials. All the major antibiotic drugs were discovered towards the culmination of
the golden era revolution (late forties and early fifties). Examples of some major
antibiotic groups are tetracyclines, cephalosporins, aminoglycosides, macrolides,
etc. By the early sixties, major issue concerning the chemotherapy had been solved.
Reports suggest that most of the bioactive compounds were derivatised from rare
Streptomyces species of marine sources. This represents 70–80% of the total derived
bioactive compounds. These compounds were primarily active against most of the
microbial strain. This was when the discovery of antitumour, antiviral and enzyme-
inhibiting molecules had just begun. Actinomycetes group of organisms is mostly
distributed in environments like terrestrial, marine or in extreme conditions
(Abraham and Chauhan 2017; Raja et al. 2010).
Streptomyces is the largest genus of Actinobacteria, belonging to the family
Streptomycetaceae. Over the years, more than 600 rare Streptomyces strains have
been isolated and reported by Euzeby (2008). These are Gram-positive bacteria with
high GC content and predominantly inhabiting the soil biota. The characteristic
earthy odour is due to production of geosmin, a volatile secondary metabolite.
Streptomyces sp. is known for its production of over two-third of antibiotics (such
as neomycin and chloramphenicol). The antifungal metabolites belong to the group
S. Nair · J. Abraham (*)

Microbial Biotechnology Laboratory, School of Biosciences and Technology, VIT University,
Vellore, Tamil Nadu, India

https://doi.org/10.1007/978-981-15-2195-9_23
334 S. Nair and J. Abraham
macrolide polyenes. Other bioactive metabolites of the same class are nystatin
(Streptomyces noursei), amphotericin B (Streptomyces nodosus) and natamycin
(Streptomyces natalensis). There are numerous bioactive compounds which show
antibacterial activity, such as aminoglycosides, which include streptomycin
(Streptomyces griseus), neomycin (Streptomyces fradiae), kanamycin (Streptomyces
kanamyceticus), erythromycin (Streptomyces erythraea), tetracycline (Streptomyces
rimosus), chloramphenicol (Streptomyces venezuelae), vancomycin (Streptomyces
orientalis) and thienamycin (Streptomyces cattleya). Nevertheless, there are antibi-
otics produced by Streptomyces sp. which are too lethal for human consumption (for
any treatment purpose) due to its toxicity towards the cells. Nowadays, research is
focused on exploiting the degree of toxicity of certain antibiotics to be used as che-
motherapeutic drugs. Some examples include actinomycin-D, bleomycin
(Streptomyces verticillus), mitomycin (Streptomyces lavendulae) and plicamycin
(Streptomyces plicatus) (Birnbaum et al. 1985).
Approximately 60% of antibiotics isolated from the Streptomyces group is used
in the agriculture industry. Bioactive natural compounds from these organisms have
shown to possess different functions such as antibacterial (Ramesh and Mathivanan
2009), antifouling (Xu et al. 2010), antifungal (Ebrahimi Zarandi et al. 2009), anti-
inflammatory (Renner et al. 1999), anti-parasitic (Pimentel-Elardo et al. 2010), anti-
tumour (Hong et al. 2009), antiviral (Sacramento et al. 2004), insecticidal
(Pimentel-Elardo et al. 2010), growth-promoting metabolites (Sousa et al. 2008)
and enzyme inhibitors (Hong et al. 2009). This is one of the important reasons why
this group of organisms has been extensively documented as industrially important
organisms. Apart from the above-mentioned bioactive compounds, these microor-
ganisms are known to produce numerous extracellular hydrolytic enzymes
(Higginbotham and Murphy 2010; Ramesh and Mathivanan 2009), thereby making
it an important subject for research from academic and industrial point of view.
Marine habitat is still underexplored. It is an important source of lesser studied
organisms such as rare actinomycetes (Manivasagan et al. 2014; Tiwari and Gupta
2002). A recent study of the aquatic ecosystem and its prevailing microbial diversity
has shown that this environment may hold more than 1400 diverse actinobacterial
strains, a great proportion of which represents novel and rare species (Abraham and
Chauhan, 2017; Stach and Bull 2005). It is furthermore documented that these
strains can adapt and respond quickly to diverse conditions (pH, salinity, pressure,
dissolved oxygen, temperature, dissolved nutrients, etc., to name a few). They com-
pete for defence and their survival in such extreme environments. The marine-
derived secondary molecules play an important role in the pharmaceutical market
(Mayer et al. 2010). There is an urgent need to scrutinise the unexplored marine
habitats for novel microbial bioactive compounds.
The isolation of an obligate marine Streptomyces sp. of the genus Salinispora was
first reported in the year 2005. In the following years, genera such as Aeromicrobium,
Lamerjespora, Marinactinospora, Serinicoccus, Salinibacterium, Solwaraspora,
Sciscionella and Williamsia were discovered (Abraham and Chauhan 2017,
Manivasagan et al. 2014; Subramani and Aalbersberg 2012). Furthermore, certain
actinomycetes are an imperative natural source of bioactive products (Table 23.1).
23 Natural Products from Actinobacteria for Drug Discovery 335
Table 23.1 Bioactive products isolated from rare actinobacteria sp.

Genus Compounds Reference
Salinispora sp. Salinosporamide A Manivasagan et al. (2014)
Sporolides
Saliniquinone A–F
Salinosporamide K
Verrucosispora sp. Abyssomicins Kwon et al. (2009)
Micromonospora sp. Diazepinomicin
Marinispora sp. Marinomycins
Marinisporolides
Distribution of Bioactive Natural Products
Organisms produce bioactive metabolites as a result of their cellular metabolism.

However, the inert ability of producing secondary metabolites is not even amongst
the microorganisms. Unicellular bacteria, eukaryotic fungi and filamentous
Streptomyces sp. are the known producers of over 40,000 bioactive compounds
(Abraham and Chauhan 2017). This number constitutes almost 17% of all bioactive
metabolites known and isolated so far from microbes. Amongst this, the group of
filamentous Actinomycetales produces over 10,000 of the bioactive compounds, out
of which nearly 7500 compounds are derived from Streptomyces and approximately
2500 from the rare actinomycetes species. The rare group of Actinomycetes sp. rep-
resents around 46% of the bioactive metabolite producers.
arine Rare Actinobacteria: Novel Source of Bioactive

M
Compounds
Marine rare actinobacteria are economically and biotechnologically priceless pro-

karyotes. Representative genera of these rare actinobacteria include Actinomyces,
Arthrobacter, Corynebacterium, Frankia, Micrococcus, Micromonospora,
Streptomyces, etc. (Manivasagan et al. 2014; Solanki et al. 2008). It has numerous
metabolic potential which remains unchallenged due to the lack of a potential com-
petitor amongst the other microbial groups. A large number of rare Streptomyces sp.
has been isolated from the marine environment in the past several decades. The
chances of isolating a novel strain from its terrestrial counterparts have diminished.
It has been reported that above 500 spp. of Streptomyces account for approximately
70–80% of the present secondary metabolites. Amongst the 70–80% bioactive
compounds, genera of rare actinomycetes including Actinoplanes, Amycolatopsis,
Micromonospora and Saccharopolyspora are responsible for 20–25% contribution
(Abraham and Chauhan, 2017; Solanki et al. 2008).
The reason for exploring the marine habitat for the isolation of rare actinomycetes
is to overcome the delinquent issue of the resistant pathogens (cause of most of the
infection). The resistant pathogens are no longer vulnerable to the current available
treatment drugs (Ramabhai and Jayaprakashvel 2018; Manivasagan et al. 2014). The
rate of mortality due to such infection is on the rise. The extraction of the secondary
metabolites from rare actinobacteria genera may form the basis of novel therapeutic
drugs, which may be efficient to overcome or treat a wide range of multidrug-resis-
tant pathogens (Ramabhai and Jayaprakashvel 2018; Manivasagan et al. 2014).
The first report of the discovery of rare actinomycetes was in the year 1984 by
Helmke and Weyland. The scientists had reported the occurrence of Rhodococcus
marinonascens (rare genus) in the aquatic ecosystem. Certain actinobacterial strains
have the aptitude to form aggregates in the marine dregs and deposits. This consti-
tutes about 10% of the total marine rare actinobacteria. Most of the rare
Actinobacteria spp. metabolise using the non-ribosomal polyketide synthetase
(NRPS) or the polyketide synthetase (PKS) pathway to produce the secondary bio-
active compounds. Certain Actinobacteria spp. have also been isolated in free
swimming form from stationary and anchored marine organisms such as sponges,
marine algae and corals. Unusual species belonging to the class of Dermatophilaceae,
Gordoniaceae and Micrococcaceae have been isolated from the sessile marine
sponges (Ramabhai and Jayaprakashvel 2018; Manivasagan et al. 2014). Researchers
and scientists are racing to explore and discover new classes from marine ecosystem
in order to isolate new metabolite producers.
Rare actinobacterial genera from diverse environmental niches in the marine
ecosystem include Actinomadura, Actinosynnema, Amycolatopsis, Arthrobacter,
Blastococcus, Brachybacterium, Corynebacterium, Dietzia, Frankia,
Frigoribacterium, Geodermatophilus, Gordonia, Kitasatospora, Micromonospora,
Micrococcus, Microbacterium, Mycobacterium, Nocardioides, Nocardiopsis,
Nonomurea, Pseudonocardia, Rhodococcus, Saccharopolyspora, Salinispora,
Serinicoccus, Solwaraspora, Streptosporangium, Tsukamurella, Turicella,
Verrucosispora and Williamsia (Ramabhai and Jayaprakashvel 2018; Manivasagan
et al. 2014; Solanki et al. 2008). The above-mentioned groups have been character-
ised and identified using different cultural and molecular techniques. Metagenomic
methods are also used to characterise these rare actinobacterial strains.
Rare Actinobacteria: Source of Novel Antibiotics
Unusual actinobacterial spp. have been exploited significantly to isolate antibiotics

(25–30% of known antibiotics). In order to identify and characterise a novel com-
pound isolated from these rare species the following methods can be used: high-
throughput screening, selective sampling techniques or enrichment method to
cross-examine the different rare actinobacterial spp. (Berdy 2005). Some of the
antimicrobial agents produced by these rare groups of Actinobacteria have been
tabulated below (Table 23.2).
Table 23.2 Antibiotics isolated from marine actinobacteria

Rare actinobacteria Antibiotic isolated Reference
Amycolatopsis mediterranei Rifamycins Igarashi et al. (2008)
Saccharopolyspora erythraea Erythromycin Zhuge et al. (2008)
Actinoplanes teichomyceticus Teicoplanin Lazzarini et al. (2000)
Amycolatopsis orientalis Vancomycin Pfefferle et al. (2000)
Micromonospora purpurea Gentamicin
Species belonging to genera Actinoplanes, Amycolatopsis, Saccharopolyspora

and Micromonospora have been subjugated as a prolific source of novel secondary
metabolites (Ramabhai and Jayaprakashvel 2018; Manivasagan et al., 2014; Dasari
et al. 2012; Dharmendra et al. 2010; Beth et al. 2009; Zhang et al. 2009). Nevertheless,
certain rare genera like Actinomadura, Kitasatospora, Marinispora, Microbispora,
Nocardiopsis, Serinicoccus, Verrucosispora, etc., to name a few, are now drawing
attention (Berdnikova et al. 2009; Liras and Demain 2009; Solanki et al. 2008).
Antibacterial Activity
Infectious diseases caused by bacterial pathogens remain as the main cause of mor-
tality. Most of the bacterial infections remain untreated owing to the antibiotic resis-
tivity acquired by the pathogens. The rate or the frequency of resistivity remains to
expand at a distressing frequency around the world (Ravikumar et al. 2012a, b). Due
to the decrease in the effectiveness of the currently available marketed drug, research-
ers have started developing new alternatives for the treatment of infections caused by
pathogens. This has, therefore, led to the exploration of marine actinobacterial strains.
Antibiotics from marine Streptomyces have been applied in order to prevent or treat
diseases instigated by microorganisms. Nevertheless, due to the multidrug resistance
developed by the pathogens, these treatment drugs are going in vain. Several new
antibiotics derived from natural resources have decreased drastically over the years.
This is mostly due to the exhaustion of the available resources. Continuous research
is being conducted to create an effective therapeutic drug by the isolation of the bio-
active molecules from the rare actinobacterial sp. Marine actinobacteria are currently
being extensively studied for its antibacterial activity. The list of antibacterial com-
pounds isolated from the actinobacterial strains has been tabulated in Table 23.3.
Antifungal Activity
Much of extensive research work is underway to identify novel antifungal com-

pounds from unusual actinobacteria which are effective against pathogenic fungi
(Wanner 2009). For example, bioactive compounds such as scopafungin, candip-
lanecin, rapamycin, phthalates, etc., to name a few, have antifungal property. Marine
Table 23.3 Antibacterial bioactive compounds isolated from actinobacterial strains

Antibacterial compound Actinomycetes sp. Reference
Arenimycin Salinispora sp. Asolkar et al. (2010)
Atrop-abyssomicin C Verrucosisporamaris Roh et al. (2011)
Ayamycin Nocardia sp. El-Gendy et al. (2008)
Abyssomicins G, H Verrucosispora sp. Keller et al. (2007)
Asukamycin Streptomyces nodosus subsp. Hu and Floss (2004)
asukaensis
Apramycin Streptomyces tenebrarius UD2 Xu et al. (2006)
Arizonins A1 and B1 Actinoplanes arizonaensis sp. nov Karwowski et al. (1988)
Avilamycin A Streptomyces viridochromogenes Weitnauer et al. (2001)
Tu57
Benzanthrins A and B Nocardia lurida Theriault et al. (1986)
Biphenomycin A and B Streptomyces griseorubiginosus Ezaki et al. (1985)
Bisanthraquinone Streptomyces sp. Socha et al. (2006)
Bonactin Schumacher et al. (2003)
Chloramphenicol Streptomyces venezuelae Piraee et al. (2004)
Capuramycin Streptomyces griseus SANK 60196 Yamaquchi et al. (1986)
Coloradocin Actinoplanes coloradoensis sp. Jackson et al. (1987)
nov.
Cephamycin C Streptomyces lactamdurans Jacks et al. (1980)
Caboxamycin Streptomyces sp. Hohmann et al. (2009)
Cinerubin R Streptomyces eurythermus Nakata et al. (1992)
Chloro-dihydroquinones Streptomyces sp. Mercado et al. (2005)
Diazepinomicin Micromonospora sp. Charan et al. (2004)
Daptomycin Streptomyces roseosporus Wezel et al. (2006)
Enduracidin Streptomyces fungicidicus B5477 Hiquashide et al. (1968)
Essramycin Streptomyces sp. El-Gendy et al. (2008)
Fortimicin A (astromicin) Micromonospora olivasterospora Ohta and Haseqawa (1993)
Fosfomycin Streptomyces fradiae Rorers and Birnbaum (1974)
Frigocyclinone Streptomyces griseus strain NTK Bruntner et al. (2005)
97
Gentamicin Micromonospora purpurea var. Escalante et al. (1992)
violaceae
Granaticin Streptomyces thermoviolaceus James et al. (1991)
Glaciapyrroles A, B and C Streptomyces sp. Macherla et al. (2005)
Gutingimycin Maskey et al. (2004)
Helquinoline Janibacter limosus Asolkar et al. (2004)
Himalomycins Streptomyces sp. Maskey et al. (2003a)
Ipomicin Streptomyces ipomoeae Schully et al. (2006)
Kocurin Kocuria sp. Palomo et al. (2013)
Kitastatin Kitasatospora sp. Pettit et al.,(2007)
Kalafungin Streptomyces tanashiensis strain Johnson and Dietz (1969)
Kala UC5063
Kanchanamycins Streptomyces olivaceus Stephan et al. (1996)
Lipoxazolidinone Marinispora sp. Macherla et al. (2005)
(continued)
Lajollamycin Streptomyces nodosus Manam et al. (2005)
Lynamicins Marinispora NPS12745 McArthur et al. (2008)
Lincomycin Streptomyces sp. Peschke et al. (2006)
Lynamicins A–E Marinispora sp. McArthur et al.,(2008)
Lankacidin Streptomyces rochei Kinashi et al. (1994)
Lankamycin Kinashi et al. (1994)
Lomofungin Streptomyces lomodensis Johnson and Dietz (1969)
Lavendomycin Streptomyces lavendulae Komori et al. (1985)
Lipiarmycin Actinoplanes deccanensis nov. sp. Parenti et al. (1975)
Lynamicins Marinispora sp. McArthur et al. (2008)
Marinopyrroles Streptomyces sp. Hughes et al. (2009)
Maduramicins Actinomadura rubra Terekhova et al. (1991)
Mimosamycins Streptomyces lavendulae No. 314 Arai et al. (1976)
Methylsulfomycin I Streptomyces sp. RSP9 Gonzalez Holgado et al.
(2002)
Marthiapeptide A Marinactinospora sp. Zhou et al. (2012)
Midecamycin Streptomyces mycarofaciens Hara and Hutchinson (1992)
Monomycin Actinomyces circulatus var. Gauze et al. (1960)
monomycini
Midecamycin Streptomyces mycarofaciens Schlegel et al. (2001)
Marinomycins Marinispora sp. Kwon et al. (2006)
Novobiocin Streptomyces niveus Mitchell et al. (1990)
Nocardiamides Nocardiopsis sp. Wu et al. (2013)
Nocarimidazoles Leutou et al. (2015)
Nogalamycin Streptomyces nogalater Ylihonko et al. (1996)
Oxytetracycline Streptomyces rimosus Petkovic et al. (2006)
Pseudonocardians Pseudonocardia sp. SCSIO 01299 Li et al. (2005)
Phenalinolactones A–D Streptomyces sp. Durr et al. (2006)
Pyridinium Amycolatopsis sp. Dasari et al. (2012)
Pseudonocardians A–C Pseudonocardia sp. Li et al. (2011)
Phthalates Nocardia levis Kavitha et al. (2009)
Proximicins A–C Verrucosispora sp. Fiedler et al. (2008)
Pacificanones A and B Salinispora pacifica Oh et al. (2008)
Pristinamycin I Streptomyces pristinaespiralis Lagard et al. (1997)
Retimycin Salinispora sp. Duncan et al. (2015)
Rifamycin S Micromonospora rifamycinica Huang et al. (2009)
Ripromycin Streptomyces sp. Bertasso et al. (2003)
Shurimycins A and B Streptomyces hygroscopicus Kamazawa et al. (1994)
Streptomycin Streptomyces griseus Nomi (1963)
Scopafungin Streptomyces hygroscopicus var. Samain et al. (1982)
enhygrus var. nova UC-2397
Spiramycin Streptomyces ambofaciens Ikeda et al. (1982)
Streptocidins A–D Streptomyces sp. Tu6071 Gebhardt et al. (2001)
(continued)
Sohbumycin Streptomyces sp. 82-85 Umezawa et al. (1985)
Taromycin Saccharomonospora sp. CNQ-490 Yamanaka et al. (2014)
Thiolactomycin Salinispora pacifica CNS-863 Tang et al. (2015)
Thiopeptide Nocardiopsis TFS65-07 Engelhardt et al. (2010)
Tylosin Streptomyces fradiae Malanicheva et al. (1992)
Tubelactomicin A Nocardia sp. Iqarashi et al. (2000)
Tetracycline Streptomyces aureofaciens Ross and Schugerl (2005)
TP-1161 Nocardiopsis sp. Engelhardt et al. (2010)
Teichomycins Actinoplanes teichomyceticus nov. Parenti et al. (1978)
sp.
Tirandamycins Streptomyces lincolnensis Carlson et al. (2009)
Tirandamycin Streptomyces sp. Carlson et al. (2009)
Victomycin Streptosporangium Takasawa et al. (1975)
violaceochromogenes nov. sp.
Virginiamycin M Streptomyces virginiae Rezanka et al. (1992)
Vinylamycin Streptomyces sp. Igarashi et al. (1999)
Zelkovamycin Streptomyces sp. K96-0670 Zhang et al. (1999)
3-((6-Methylpyrazin-2-yl) Serinicoccus profundi Yang et al. (2015)
methyl)-1H-indole
1,4-Dihydroxy-2-(3- Streptomyces sp. Ravikumar et al. (2012a, b)
hydroxybutyl)-9,10-
anthraquinone 9,10-anthrac
unusual actinobacteria spp. have been found to be associated with marine inverte-
brates and vertebrates. Streptomyces sp. DA11 was found to be associated with the
sponge Craniella sp. This species produced the exoenzyme chitinase which showed
significant antifungal activity against Aspergillus sp. and Candida sp. (Han et al.
2009). Certain enzymes and its derivatives (e.g. chitinase) are known for its antifun-
gal activity and its highly biocompatible quality. These secondary metabolites have
been exploited for its application in the field of biomedicine, for example, cartilage
tissue engineering, drug transport and nerve regeneration. The following table
depicts the list of antifungal therapeutic compounds extracted from the unusual acti-
nobacterial strains (Table 23.4).
Anticancer Activity
Cancer is one of the most serious health-related problems (Ravikumar et al. 2012a, b).
Till date, the available therapeutic methods for treatment are as follows: chemother-
apy, immunotherapy, surgery and radiotherapy. These techniques offer an efficient
treatment for tumour. It has been reported that many of the antitumour compounds
have been extracted from marine Streptomyces (Ravikumar et al. 2012a, b). Table 23.5
depicts some of the therapeutic compounds isolated from marine actinobacteria.
Table 23.4 Secondary metabolites with antifungal activity isolated from actinobacteria sp.
(marine environment)
Antifungal compound Actinomycetes sp. Reference
Amphotericin B Streptomyces nodosus Caffrey et al. (2001)
Axenomycins Streptomyces lisandri nov. sp. Bruna et al. (1973)
Ascomycin FK520 Streptomyces hygroscopicus Wu et al. (2000)
Ayamycin Nocardia sp. El-Gendy et al. (2008)
Blasticidin S Streptomyces griseochromogenes Zhanq et al. (1998)
Chandrananimycin Actinomadura sp. Maskey et al. (2003b)
Candiplanecin Ampullariella reguralis subsp. Itoh et al. (1981)
mannitophila subsp. nov.
Dentigerumycin Pseudonocardia sp. Haeder et al. (2009)
Oh et al. (2008)
Forazoline A Actinomadura sp. WMMB-499 Hou et al. (2011)
Jinggangmycin Streptomyces hygroscopicus Jian et al. (2006)
Kitastatin Kitasatospora sp. Pettit et al. (2007)
Kalafungin Streptomyces tanashiensis strain Kala Johnson and Dietz (1969)
UC5063
Kanchanamycins Streptomyces olivaceus Stephan et al. (1996)
Lomofungin Streptomyces lomodensis Johnson and Dietz (1969)
Leptomycin Streptomyces lividans Hu et al. (2005)
Marinisporolides A and B Marinispora sp. Kwon et al. (2009)
Nikkomycins Streptomyces ansochromogenus Luo et al. (1998)
Neomaclafungin Actinoalloteichus sp. NPS702 Sato et al. (2012)
Pimaricin Streptomyces natalensis Recio et al. (2004)
Phthalates Nocardia levis Kavitha et al. (2009)
Rapamycin Streptomyces hygroscopicus Lomovskaya et al. (1997)
Resormycin Streptomyces platensis Iquarashi et al. (1997)
Rimocidin Streptomyces diastaticus var. 108 Seco et al. (2004)
Shurimycins A and B Streptomyces hygroscopicus Kamazawa et al. (1994)
Scopafungin Streptomyces hygroscopicus var. Samain et al. (1982)
enhygrus var. nova UC-2397
Saccharothrixmicines Saccharothrix espanaensis An 113 Kalinovskaya et al. (2010)
Antitumour Activity
Marine actinobacteria are known for rich source of antitumour compounds, for
example, actinomycin, anthracycline, aureolic, mitomycin, bleomycin, pentostatin,
resistomycin, etc. These bioactive compounds possess diverse chemical backbones
due to the presence of different gene clusters encoding for polyketide and non-
ibosomal peptide synthases. The different mechanisms by which these compounds
function as an antitumour drug are as follows: apoptosis, mitochondrial permeabili-
sation, hindrance in signal transduction pathway, morphological changes due to
irregular cellular differentiation, angiogenesis and intercalating DNA. Sometimes
antitumour compounds isolated from Streptomyces strains act by intercalating with
Table 23.5 Secondary metabolites with anticancer activity isolated from actinobacteria sp.
Anticancer activity Actinomycetes sp. Reference
Arenicolides Salinispora arenicola CNR-005 Williams et al. (2007)
Anthracyclinones Micromonospora sp. Sousa et al. (2012)
Albidopyrone Streptomyces sp. Hohmann et al. (2009)
Arenamides Salinispora arenicola Asolkar et al. (2009)
Actinofuranones A and B Streptomyces sp. Cho et al. (2006b)
Actinofuranones Streptomyces sp. Matsuo et al. (2007)
Bromosalinosporamide Salinispora sp. Lam et al. (2007)
Bendigoles Actinomadura sp. SBMs009 Simmons et al. (2011)
Caerulomycins Actinoalloteichus cyanogriseus Fu et al. (2011)
WH1-2216-6
Dermacozines Dermacoccus abyssi Abdel-Mageed et al. (2010)
Daryamides Streptomyces sp. Asolkar et al. (2006)
Fluorosalinosporamide Salinispora sp. Eustáquio and Moore (2008)
Isomethoxyneihumicin Nocardiopsis alba KM6-1 Fukuda et al. (2016)
Lucentamycins Nocardiopsis lucentensis (strain Cho et al. (2007)
CNR-712)
Lodopyridone Saccharomonospora CNQ490 Maloney et al. (2009)
Lucentamycins Nocardiopsis lucentensis Cho et al. (2007)
Levantilides Micromonospora M71-A77 Gärtner et al. (2011)
Lomaiviticin Salinispora sp. Kersten et al. (2013)
Marinomycins Marinispora strain CNQ-140 Kwon et al. (2006)
Marthiapeptide A Marinactinospora thermotolerans Zhou et al. (2012)
SCSIO 00652
Methylsalinosporamide Salinispora sp. Manam et al. (2008)
Neihumicin Micromonospora neihuensis Fukuda et al. (2016)
Retimycin S. arenicola strain CNT-005 Duncan et al. (2015)
Nocapyrones Nocardiopsis sp. HB383 Schneemann et al. (2010)
Piperazimycins Streptomyces sp. Williams et al. (2007)
Pseudonocardians Pseudonocardia sp. SCSIO 01299 Li et al. (2006)
Piericidins Streptomyces sp. Hayakawa et al. (2007)
Pacificanones Salinispora pacifica Williams et al. (2007)
Proximicins Verrucosispora sp. Schneider et al. (2008)
Piperazimycins Streptomyces sp. Miller et al. (2007)
Salinosporamides Salinispora tropica (strain Williams et al. (2005)
CNB-392)
Salinosporamide B and C Salinispora tropica
Salinipyrones Salinispora pacifica Oh et al. (2008)
Salinoquinones Salinispora arenicola CNS-325 Murphy et al. (2010)
Saccharothrixones Saccharothrix sp. 10-10 Gan et al. (2015)
Urukthapelstatin A Mechercharimyces asporophorigenes Matsuo et al. (2007)
YM11-542
Salinosporamide K Salinispora sp. Eustáquio et al. (2011)
Thiocoraline Micromonospora sp. Lombó et al. (2006)
Thiocoraline Verrucosispora sp. Hou et al. (2011)
Nonactin Streptomyces sp. Jeong et al. (2006)
Gorajana et al. (2005)
Hardt et al. (2000)
duplex DNA, which leads to detrimental effects on fast proliferating cells by inhib-
iting the DNA-dependent RNA polymerase activities (Demain and Sanchez 2009).
In such cases, rare marine actinobacteria have been exploited and have drawn spe-
cial attention due to their innate ability of producing bioactive metabolites (Williams
2009). Few of the secondary metabolites showing antitumour activity have been
enlisted below (Table 23.6).
Table 23.6 Bioactive compounds with antitumour activity isolated from actinobacteria sp.
Antitumour compound Actinomycetes sp. Reference
Aureoverticillactam Streptomyces aureoverticillatus Mitchell et al. (2004)
Aureoverticillactam Streptomyces aureoverticillatus
NPS001583
Actinofuranones Streptomyces sp. CNQ766 Cho et al. (2006b)
Arenicolides Salinispora arenicola Williams et al. (2005)
Arenimycin Salinispora arenicola Asolkar et al. (2006)
Ammosamides Streptomyces sp. CNR-698 Hughes et al. (2009)
Arcyriaflavin A Actinomycete sp. Z2039-2 Liu et al. (2007)
Arenamides Salinispora arenicola CNT-088 Asolkar et al. (2009)
Arenicolides Salinispora Arenicola CNR-005 Williams et al. (2007)
Altemicidin Streptomyces sioyaensis SA-1758 Takahashi et al. (1989a, b)
Butenolides Streptoverticillium Lee et al. (2005)
luteoverticillatum
Bohemamines Streptomyces sp. CNQ-583 Bugni et al. (2006)
Chandrananimycins Actinomadura sp. M048 Maskey et al. (2003b)
Chinikomycins Streptomyces sp. M045 Li et al. (2005)
Caboxamycin Streptomyces sp. NTK 937 Hohmann et al. (2009)
Chalcomycin Streptomyces sp. M491 Wu et al. (2007)
Chinikomycins Streptomyces sp. Li et al. (2005)
Chartreusin Streptomyces sp. QD518 Wu et al. (2006)
Chlorinated Actinomycete isolate CNQ-525 Soria-Mercado et al. (2005)
dihydroquinones
Cyanosporasides Salinispora pacifica CNS103 Oh et al. (2006)
Chalcomycin Streptomyces sp. Wu et al. (2007)
Daryamides Streptomyces sp. CNQ-085
Echinosporins Streptomyces albogriseolus A2002
Cui et al. (2007)
Fridamycin D Streptomyces sp. Maskey et al. (2003a)
Griseorhodin A Li and Piel (2002)
Gutingimycin Maskey et al. (2004)
Himalomycins Maskey et al. (2003a)
Iodinin Actinomadura sp. M048 Maskey et al. (2003b)
Lajollamycin Streptomyces nodosus NPS007994 Manam et al. (2005)
Lynamycins Marinispora sp. NPS12745 McArthur et al. (2008)
Manumycin A Streptomyces sp. M045 Li et al. (2005)
Marinomycins Marinispora sp. CNQ-140 Kwon et al. (2006)
(continued)
Antitumour compound Actinomycetes sp. Reference
Marineosins Streptomyces sp. CNQ-617 Boonlarppradab et al. (2008)
Marmycins Streptomyces sp. CNH990 Martin et al. (2007)
Mechercharmycin A Thermoactinomyces sp. Kanoh et al. (2005)
Pacificanones Salinispora pacifica CNS-237 Oh et al. (2008)
Streptokordin Streptomyces sp. Jeong et al. (2006)
4a,8a-Dimethyl-6-(2- Actinobacterium sp. MS1/7 Saha et al. (2006)
methyl-propenyloxy)-
3,4,4a,4b,5,6,8a,9-
octahydro-1H-phenanthren-
2-one
1,8-Dihydroxy-2-ethyl-3- Streptomyces sp. Huang et al. (2006)
methylanthraquinone
1-Hydroxy-1- Streptomyces chinaensis AUBN1/7 Gorajana et al. (2005)
norresistomycin Streptomyces sp. B8005 Kock et al. (2005)
1,6-Phenazinediol Actinomadura sp. M048 Lombó et al. (2006)
1,8-Dihydroxy-2-ethyl-3- Streptomyces sp. FX-58 Huang et al. (2006)
methylanthraquinone
3,6-Disubstituted indoles Streptomyces sp. BL-49-58-005 Sánchez López et al. (2003)
Anti-inflammatory Activity
Inflammation is considered as a primary non-specific immune response. In this

state, a body tends to react to any kind of infection, irritation or injury. The primary
cause of inflammation is a pure mechanical stress, including blunt trauma, foreign
bodies, vibrations and chronic pressure. Inflammatory responses occur in different
phases, each of which is mediated by different mechanisms. The secondary bioac-
tive metabolites from marine Streptomyces sp. have been proven to possess the anti-
inflammatory activity (Woolhouse 2008; Jones et al. 2008). Some of the bioactive
secondary metabolites showing anti-inflammatory activity have been enlisted below
(Table 23.7).
Antimalarial Activity
Malaria is caused by the protozoan, Plasmodium, which is parasitic in nature and

accountable for more than 200 million reported clinical cases and over 3 million
mortality annually. P. falciparum, the causative agent, is becoming increasingly
resistant to most of the drugs. New therapeutic strategies are urgently required to
battle this disease. Some of the compounds derived from the marine actinobacteria
Table 23.7 Secondary metabolites with anti-inflammatory activity isolated from actinobacteria
sp.
Anti-inflammatory compound Actinomycetes sp. Reference
Cyclomarine S. arenicola CNS-205 Schultz et al. (2008)
Cyclomarazine
Cyclomarin A Streptomyces sp. Schultz et al. (2008)
Diazepinomicin Micromonospora sp. Charan et al. (2004)
Komodoquinone A Streptomyces sp. KS3 Itoh et al. (2003)
Levantilides A and B Micromonospora sp. Gärtner et al. (2011)
Table 23.8 Secondary metabolites with antimalarial activity isolated from actinobacteria sp.
Antimalarial compound Actinomycetes sp. Reference
Axenomycins Streptomyces lisandri nov. sp. Bruna et al. (1973)
2-Allyloxyphenol Streptomyces sp. Arumugam et al. (2009)
Marinacarbolines Marinactinospora thermotolerans Huang et al. (2011)
Salinipostins Salinispora sp. Schulze et al. (2015)
Thiamycins Streptomyces michiganensis Cassinelli et al. (1970)
Trioxacarcin A, B and C Streptomyces ochraceus Maskey et al. (2004)
possess extremely high anti-plasmodial activity. Some of the secondary metabolites

showing antimalarial activity have been enlisted below (Table 23.8).
Antiviral Activity
Marine antiviral agents have been reported to have the following applications in the
treatment and prevention of a viral disease:
1. Biological control of human entero-pathogenic virus contamination and disease
transmission in sewage-polluted waters. This is mostly applied in coastal areas,
where the waters are used for recreational activities, or in food industries or in
regions where the loss of these marine bioactive resources would have a devas-
tating effect on the lifestyle and the economy of people.
2. Chemotherapy of viral diseases of humans and lower animals.
A list of antiviral compounds derived from the rare actinobacteria (marine eco-
system) has been enlisted in Table 23.9.
Table 23.9 Antiviral compounds derived from the actinobacteria sp. (marine ecosystem)
Antiviral compound Actinomycetes sp. Reference
Benzastatin C Streptomyces nitrosporeus Lee et al. (2007)
Cyclomarin Streptomyces sp. Renner et al. (1999)
Fattiviracin A1 Yokomizo et al. (1998)
Resistomycin Streptomyces corchorusii Shiono et al. (2002)
Table 23.10 Antioxidant compounds derived from actinobacteria sp. (marine ecosystem)
Antioxidant compound Actinomycetes sp. Reference
Dermacozines, phenazine Dermacoccus abyssi Abdel-Mageed et al.
derivatives (2010)
Dermacozines A–G Dermacoccus
JBIR-65 Actinomadura sp. Takagi et al. (2010)
Lipocarbazoles Tsukamurella pseudospumae Schneider et al. (2009)
Mumiamicin Mumia sp. YSP-2-79 Kimura et al. (2018)
Mangromicin A–I Lechevalieria Nakashima et al. (2015)
aerocolonigenes
Resistoflavin methyl ether Streptomyces sp. Kock et al. (2005)
Antioxidant Activity
An antioxidant is any substance or compound that averts deterioration, damage or

destruction by the process of oxidation. These bioactive compounds confer protec-
tion against oxidative stress in an organism by blocking or delaying the oxidative
damage caused by the reactive oxygen species (ROS). The antioxidants employ
multiple mechanisms to eliminate ROS such as inhibiting the formation of free radi-
cals, scavenging oxygen molecule and chelating metal prooxidants. A list of anti-
oxidant compounds extracted from the rare actinobacteria (marine ecosystem) has
been enlisted below (Table 23.10).
Secondary Bioactive Compounds from Marine Streptomyces
Secondary metabolites produced by marine actinomycetes can be classified on the

basis of their chemical structure as follows (Table 23.11):
Table 23.11 Different groups of secondary metabolites from actinobacteria sp.

Group Compound Organism Reference
Terpenes Azamerone Streptomyces sp. Cho et al. (2006a)
Glaciapyrroles A, B and C Macherla et al. (2005)
Amorphane sesquiterpenes Streptomyces sp. Wu et al. (2007)
M491
Neomarinone Streptomyces sp. Hardt et al. (2000)
Polyketides Saliniketal A and B William et al. (2007)
Jensen et al. (2007)
Abyssomicin C Verrucosispora sp. Bister et al. (2004)
Polyketide SBR-22 Streptomyces Sujatha et al. (2005)
psommoticus
Actinofuranones Cho et al. (2006b)
Peptides Mechercharmycins Thermoactinomyces Kanoh et al. (2005)
sp.
Thiocoraline Micromonospora sp. Romero et al. (1997)
Cyclomarins A–C Streptomyces sp. Renner et al. (1999)
Piperazimycins Miller et al. (2007)
Dehydroxynocardamine Lee et al. (2005)
Desmethylenylnocardamine
Urukthapelstatin A Mechercharimyces Matsuo et al. (2007)
asporophorigenes
Salinamides Streptomyces sp. Moore et al. (1999)
Caprolactones R-10-Methyl-6- Streptomyces sp. Stritzke et al. (2004)
undecanolide
(6R,10S)-10-Methyl-6-
dodeconolide
Polycyclic PX-IB-00208 Actinomadura. sp. Malet Cascon et al.
xanthones (2003)
Quinones Resistomycin Streptomyces Shiono et al. (2002)
Tetracenomycin D corchorusii Adinaryan et al. (2006)
Resistoflavine Streptomyces Kock et al. (2005);
chibaensis Gorajana et al. (2005)
Komodoquinone A Streptomyces sp. Itoh et al. (2003)
Himalomycins A and B Maskey et al. (2003a)
Helquinolines Janibacter limosus Asolkar et al. (2004)
Macrolides Chalcomycin A Streptomyces sp. Wu et al. (2007)
M491
Arenicolide Salinispora arenicola Jensen et al. (2007)
Marinomycins Marinispora sp. Kwon et al. (2006)
Terpenes and Terpenoids
Some of the secondary metabolites (terpenes and terpenoids) isolated from the rare
genus of marine actinobacteria have been mentioned in Table 23.11. Most com-
monly occurring terpenoids are the glycoside terpenoids. These are surfactants car-
rying the hydrophilic group linked with a carotenoid group and are very effective
against antibiotic-resistant bacterial species. Terpenoids are biosynthesised from
isoprenyl diphosphate which is formed by the condensation of the monomer isopen-
tenyl diphosphate (IPP) to its isomer dimethylallyl diphosphate (DMAPP). Two
distinct biosynthetic pathways are involved in the production of the primary metab-
olites IPP and DMAPP. They are as follows: the 2-C-methylerythritol 4-phosphate
pathway and the mevalonate pathway. Novobiocin was the first antibiotic with ter-
penoid side chain to be isolated from Streptomyces sp. in the year 1956. Examples
of terpene glycoside producing rare actinobacteria are as follows: Corynebacterium
sp., Arthrobacter sp., Rhodococcus sp. and Micrococcus sp.
Polyketides
These are a structurally varied group of secondary metabolites. They offer with a
wide range of medical and industrial applications. Polyketides are produced by the
enzyme group polyketide synthases. Several genes act together for the synthesis of
polyketides by the enzyme polyketide synthases. Examples of some of the
polyketides produced by unusual actinobacteria are as follows: rifamycins
(Salinispora sp.), daunorubicin and doxorubicin (Streptomyces peucetius sp.),
angucyclines (Saccharothrix espanaensis), jadomycin B (Micromonospora sp.) and
mayamycin (Streptomyces sp.), to name a few.
Phenazines
These are nitrogenous compounds with heterocyclic nature. Phenazines are impor-
tant secondary metabolites which have a vital role in the interaction between
Streptomyces sp. and its metabolic processes. These compounds are also known for
its wide array of bioactivities.
Peptides
These are group of non-ribosomal secondary metabolites synthesised using peptide

synthetases. These compounds show significant anticancer activity.
Indocarbazoles
These are group of secondary metabolites containing indole-pyrrole-carbazole

group. They are in great demand due to their varied therapeutic oriented applications.
Future Prospects and Strategies
Selective isolation and extraction of secondary bioactive compounds from unusual

actinobacteria sp. from the marine environment requires a sound understanding and
knowledge of the microbial physiology, metabolism and systematics (Hayakawa
2008; Goodfellow and Fiedler 2010; Kurtböke 2010). Protocols concerning the
detection and recovery of bioactive secondary metabolites from these marine spe-
cies should be derived from a thorough investigation and exploration of appropriate
cultural and ecological requirements of the targeted genus. The following points
must be borne in mind while isolating therapeutic secondary metabolites from the
marine sp.
1. Sound knowledge of the ecology and physiology of the marine rare actinobac-
terial strains (Goodfellow and Fiedler 2010).
2. The functional diversity and the physiological characteristics must be kept in
mind for performing the isolation and extraction of the novel bioactive com-
pounds (Ferrari et al. 2008; Gavrish et al. 2008; Vartoukian et al. 2010).
3. Study of the previously unexplored habitats such as the extreme biosphere
(Bull 2010) must be conducted (Alain and Querellou 2009).
4. Improvement in the mode of sampling, its estimation and evaluation procedures
must be designed chiefly for microorganisms inhabiting marine extreme habi-
tats (Jensen and Lauro 2008; Joint et al. 2010).
5. Provision must be allotted for an efficient identification system to determine its
novelty (Wink 2011).
6. Exploitation of new and advanced technology, such as the use of bioinformatics
to study the expression of certain antimicrobial secondary metabolite in gene
disruption or the heterologous expression of the drug encoded by Streptomyces
species (Bull 2010).
7. Cross-examination and assessment of databases pertaining to the taxon chem-
istry and its property revealed the capabilities of the rare actinomycetes group
producing species-specific secondary compounds. This thereby helps in build-
ing understandings and greater awareness into the processes that catalyses spe-
ciation in this particular genus (Goodfellow and Fiedler 2010; Wink 2011). For
example, one can procure information about the biodiversity of rare marine
Streptomyces producing a vast array of antibiotics.
8. Development of new robust and rapid protocol for the analysis of bioactive
compounds at a small scale must happen for an effective de-replication (Lang
et al. 2008; Genilloud et al. 2011).
9. Designing an effective screening approach must be brought about (Baltz 2008;

Asolkar et al. 2010).
10. Screening of known bioactive compounds with multifunctional applications for
novel targets must be performed (Vaishnav and Demain 2010). The mode of
action of the therapeutic drugs must be examined carefully in order to develop
new compounds which can be further exploited for its antibiotic action. The
success rate in recovering rare actinomycetes species from the marine environ-
ment solely depends on the knowledge and the understanding about its ecology,
physiology, cellular metabolism and the taxonomy. Success also depends on
the use of advanced molecular techniques and computer-aided tools for procur-
ing novel information. The use of polyphasic approach and computational clus-
ter analysis to monitor genomic information will provide a dominant and a
clear platform to answer key questions related to the organisms’ taxonomical
and chemical identities. It may also help in identifying previously undetected
bioactive marine actinobacterial strains.
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Chapter 24
Anti-Leprosy Vaccine (Hansen’s Disease
Vaccine)
Jerusha Santa Packyanathan, Ira Christabel Packyanathan,
and A. Indra Balini
Introduction
Leprosy exists in a bacteriologic, immunologic, clinical and pathologic spectrum.

The infection found in the peripheral nerves is the locus of diseases, from where
neuronal damage leads to functional impairment and phenotypic expressions of lep-
rosy. The precise pathogenetic mechanisms by which the underlying nerve is dam-
aged is not known (Joyce and Scollard 2004). The bacterium responsible for this
disease is Mycobacterium leprae, identified before 200 years, by Armauer Hansen,
a physician from Norway. Human beings are considered the sole reservoirs for the
infection and armadillos are a host for M. leprae (Talwar et al. 1978).
There are five categories according to Ridley-Jopling scale. This includes bor-
derline lepromatous (BL), lepromatous leprosy (LL), borderline-borderline (BB),
tuberculoid leprosy (TT) and borderline tuberculoid (BT) (Ridley and Jopling 1966;
Scollard 2004). Owing to limited resources and for ease of practice and treatment,
clinical criteria are preferred. The WHO classification, based on clinical criteria, is
based on the number of nerves involved and number of skin lesions and is classified
as multibacillary (MB) leprosy (presenting five or more lesions) and paucibacillary
(PB) leprosy (presenting with less than five lesions) (WHO global Leprosy situation
2010). An in-depth histological analyses showed that MB patients, possessing the
BB, BL and LL forms, revealed multiple skin lesions, characterized by highly dys-
functional lymphocytes. Extreme immune deficit and increased M. leprae devoid of
J. S. Packyanathan (*)
Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India
I. C. Packyanathan
The Leprosy Mission Hospital, Chennai, Tamil Nadu, India
A. I. Balini
Guru Nanak College, Chennai, Tamil Nadu, India

https://doi.org/10.1007/978-981-15-2195-9_24
366 J. S. Packyanathan et al.
specific cell-mediated immunity are seen in multibacillary lepromatous leprosy

(Ridley and Jopling 1966). Bacterial replication is not controlled in LL patients and
they have high bacterial indices (BI refers to a logarithmic scale that represents the
number of acid-fast bacilli that occur in the dermis). Patients with tuberculoid lep-
rosy (TT) have limited number of lesions with scanty bacilli showing low granulo-
matous dermatopathology and lower or negative BI (Skinsnes 1973).
On the diagnosis of leprosy, patients ought to be provided antibiotic therapy in
the form of multidrug therapy (MDT) indicated by WHO. Since 1995, WHO has
supplied MDT, free of cost, for all patients diagnosed with leprosy. It initially gave
the treatment through a drug fund funded by Nippon Foundation and currently
MDT therapy is sponsored by Novartis and the Novartis Foundation. A combination
of dapsone, rifampicin and clofazimine is provided for multibacillary patients for
12 months. On the other hand, dapsone and rifampicin is administered for pauci-
bacillary patients for 6 months (Joyce and Scollard 2004).
In India, the stigma towards those affected by the disease itself is a road block to
end the reign of leprosy. Another discouraging factor affecting the treatment is the
fear of the patients being termed ‘untouchable’ when people are aware they are
affected with the same. The war on leprosy has to be raged from various medical
and social aspects should this neglected complex disease be eradicated. MDT is just
one tool used by many and a vaccine may hold the future to end this ancient
suffering.
Leprosy: The Current Situation
Leprosy is not a disease that is native to India. However, research and surveys have
identified around 1, 27,326 cases in 2015 in India, contributing to 60% to the overall
number of new cases identified that year (accessed from WHO report, 2018).
Although in 2009, more than 1000 cases were newly reported in 16 countries, coun-
tries like Brazil, Mozambique and Democratic Republic of Congo were successful
in completely eliminating leprosy as it is prevalent at a rate of less than one case per
10,000 people (WHO global Leprosy situation 2010). When the spread of leprosy is
analysed it is found that it is not evenly spread across populations. However, the
close contacts with the leprosy patients have known to be at significantly greater
risk of contracting this disease. This leads to the occurrence of smaller pockets of
higher incidence rates (Bakker et al. 2002).
The introduction of the multidrug therapy (MDT) regime has created a fall in the
incidence of leprosy with prevalence rates dropping to presently 2,50,000 cases per
year internationally from figures as enormous as 12 million cases per year, approxi-
mately 20 years ago. The current emphasis is on early detection and timely admin-
istration of MDT (WHO global Leprosy situation 2009).
24 Anti-Leprosy Vaccine (Hansen’s Disease Vaccine) 367
Need for a Leprosy Vaccine
The advent of MDT was a saviour to all those crippled by the disease. It has made a
powerful change especially to the victims. But it does not end there; there are still
many who fall prey to this disease. In order to completely eradicate leprosy, a vigor-
ous effort has to be taken to bring down the incidence. This can be achieved with the
introduction of vaccines for the disease. This vaccine should be both prophylactic
and therapeutic in its action. It must be stated that some cases have been reported to
relapse in patients with raised BI noted at diagnosis, indicating that these patients
demand increased duration of treatment, particularly in the Indian population
(south) (Norman et al. 2004; Matsuoka et al. 2000).
There is strong reason to believe that lowered potential of MDT could be due to
drug resistance (Honrado et al. 2008; Roche et al. 2000). Resistance to dapsone has
been established which when coupled with no clofazimine compliance, the gross
therapy is rifampicin monotherapy (Ellard et al. 1988). It has been reported that
certain strains of M. leprae present with the disadvantage of multidrug resistance
(Honrado et al. 2008; Chen et al. 1999). In 2009, 213 MB relapse cases were studied
and the results showed that 12 cases were resistant to dapsone, 9 cases to rifampicin
and 2 cases to both the drugs (WHO Technical report series 2011). Recently, newer
drugs like ofloxacin and minocycline have been an addition to the drug arsenal for
the treatment of leprosy.
It is found that the relapse may be related to lower MDT compliance. Patients
often do not follow their treatments sincerely, as one of the drugs, clofazimine,
causes dark skin discolouration which could be stigmatising. Some patients were
stressed by the duration of treatment and, in consequence, commitment to the treat-
ment declined (Roche et al. 2000; Ellard et al. 1988). The largest reported study is
a 6-year follow-up of 47,276 patients conducted in China, which revealed an overall
relapse rate of 0.73/1000 person-years (Chen et al. 1999). A 10-year prospective
study in the Philippines observed an overall relapse rate equivalent to 2.8/1000
person-years (Cellona et al. 2003).
Attempts by educational campaigns to bring about awareness of the disease and
elimination of stigma continue globally; however, stigma continues to be a major
obstacle to reporting for diagnosis and prompt treatment. Research has shown that
the time period between the first noticeable symptom and clinical diagnosis of lep-
rosy ranges from 1 to 3 years among nearly 50% of patients (De Rojas et al. 1994).
The effect of the delay can be dramatic with crippling nerve damage leading to
functional impairment and disability (Ferreira et al. 2000; Van Veen et al. 2006).
Another challenge is misdiagnosis due to inadequate expertise, poor resources in
rural areas and poor patient compliance. This elevates anti-M. leprae antibodies
making existing untreated patients sources for further continued transmission in the
region (Duthie et al. 2011).
A decline in the incidence was reported with the introduction MDT but it was
premature. M. leprae still propagates and this is evident with new cases identified
ever year (WHO Global leprosy situation 2007). These concerns, along with the
current limitations in control and treatment strategies, suggest that the advancement
of additional tools and strategies is critically needed. The development of a subunit
vaccine that would both prevent and treat leprosy would herald a major step towards
eradication of this dreaded scourge.
Mechanism Behind the Vaccine
The French word ‘la vacche’ was the origin of the English word ‘vaccine’ which
literally means ‘the cow’, in reference to the cowpox extract that Dr. Jenner in 1812
to treat smallpox in humans. The concept of attenuated vaccines was introduced by
Louis Pasteur. The action of a vaccine is to enhance the immune system of the host
and is currently recognised as the most effective method of disease control
(Kartikeyan et al. 1991).
Ideally, vaccines are manufactured with killed or attenuated strains that are no
longer virulent but still possess ‘protective’ antigens. An alternate method uses non-
pathogenic attenuated or killed strains that are capable of cross-reacting antigeni-
cally with the respective pathogen. There is a third approach, which involves only
the immunogenic ‘subunit(s)’ in the preparation of a vaccine. The various leprosy
vaccines available thus far have employed the various methods available (Chirmule
et al. 1988a; Deo et al. 1981). The prospective vaccines for leprosy eradication are
BCG alone, Mycobacterium vaccae, BCG and killed M. leprae combination, Indian
Cancer Research Centre (ICRC) bacillus and Mycobacterium ‘w’(M. indicus pranii
(MIP) (Fine and Dockrell 1991; Gupte 1991).
Parameters for Determining Vaccine Efficacy
The utmost effect of a vaccine is decided by its capacity to reduce the incidence of
a disease. Hansen’s disease has an incubation period of 3–30 years, which hinders
studies regarding the efficacy of the vaccines. However, both clinical and experi-
mental research is essential prior to large trials being initiated, proving that the vac-
cine in study is efficient and provides ‘protective’ immunity. It has been found that
the cell-mediated immunity (CMI) which is the dominant host defence against
M. leprae and circulating anti-M. leprae antibodies have little role. Vaccines for
leprosy were developed in response to results from skin tests using the M. leprae
antigens (insoluble and soluble) (Lancet 1987). The Mitsuda test is a good indicator
of the vaccine capability to the bacteria, while the popular lymphocyte transforma-
tion test (LTT) is not as good as an indicator of protective immunity (Bjune et al.
1976). Persons with lower Mitsuda value, from endemic locations, possess a high
risk of acquiring multibacillary leprosy (Dharmendra and Chatterjee 1956). It is
found that Mitsuda test is indirectly related to the tissue bacillary load in persons
that are left untreated (Ridling and Jopling 1966).
As it is a spectral disease, the pathological and clinical features show the CMI
state of the host (Ridling and Jopling 1966). The CMI gradually improves as we
move from lepromatous (LL) side of the spectrum to the tuberculoid (TT) end
showing a fall in the tissue bacillary load. It is mandatory for the candidate vaccine
to show immune response in animal models also. However, it must be noted that
there is no concrete animal testing done for leprosy vaccines. Studies conducted in
the footpad of mice showed variable results in different laboratories.
Animal Studies
Every vaccine in study has to go through the phase of preclinical evaluation done on
animals to human clinical trials, a transition that can be challenging. A unique fea-
ture of armadillos is that, when they are naturally and experimentally inoculated
with M. leprae, they manifest histopathological and clinical features of the whole
spectrum of the disease identical to that seen in man. National Hansen’s Disease
Program (NHDP) has done immense groundwork in using armadillos to study
M. leprae-induced nerve damage, the immune system and armadillo genome (Job
et al. 1993; Adams et al. 2005). Despite limitations in using armadillo as a study
model, it is still well suited to evaluate vaccines. One of the challenges faced with
using armadillo is that it sustains M. leprae replication but does not reflect the nerve
damage which is manifested in humans (Shepard 1960). Nevertheless, the footpad
model is significantly used to analyse drug protocols and the occurrence of drug
resistance and for vaccine testing.
BCG
One of the earliest vaccines against leprosy was the introduction of BCG. It proved
efficient in battling against the replication of M. leprae in mice footpad (Fine 1982).
But human trials with the BCG vaccine conducted at four major locations showed
varied results (Kirchheimer and Storrs 1972). At Uganda, the average protection
rate was 80%, and that of Papua New Guinea showed 46%, whereas, in South India,
the studies showed 28% protection and in Burma 20%. It was therefore accepted
that the overall efficacy of the vaccine is modest, except in Uganda. But on its own,
BCG vaccination is no longer the standard for immunoprophylaxis of the disease. It
is still challenging to cultivate M. leprae in the laboratory but it propagates in arma-
dillos (Kirchheimer and Storrs 1972). A study by Convit et al. pointed out that the
immunogenicity is enhanced when the conventional BCG vaccines are administered
in combination with heat-killed armadillo-derived M. leprae [M. leprae-A’ + BCG]
(Convit et al. 1980).
The literature review of various studies approximately established the average
protective effect to be 61% with the BCG vaccine (95% CI 51–70%), and significant
heterogeneity was seen between the studies (p < 0·00001). It has been suggested by
Muliyil et al. (1991) that a change in immune state of the host was brought about by
vaccination by BCG responsible for differential protection of multibacillary form
and paucibacillary form of leprosy. The administration of BCG and its effect on the
immune system will reduce the severity of the attack and manifest only as milder
forms of the disease like paucibacillary leprosy and indeterminate leprosy. The effi-
cacy of the vaccine reduces with age, as a drop in the protection was observed. On
comparison, women were observed to have more protection than men with regard to
observational and experimental studies. BCG also offers higher protection among
contacts at home (Rodrigues et al. 1992).
The conventional BCG vaccine in leprosy patients revealed that it eventually led
to lepromin conversion, which is indicative of a type of delayed hypersensitivity in
man. Ganapati et al. (1989) believed that a combination of BCG and killed M. lep-
rae could enhance immune system. Studies have proved that several doses of BCG
given to the patient provided greater immunity against the same (Convit et al. 1993;
Bertolli et al. 1997).
The differing effect of the BCG vaccine could be explained based on the pres-
ence of environmental bacteria present in the study sites. Stanford et al. (1981) and
Rook et al. (1981) in their studies show environmental mycobacteria could alter the
cell-mediated response and thereby change the protective effect against tuberculosis
and leprosy. A study by Colditz et al. (1994) showed the effect of the vaccine (BCG)
was better at higher latitudes; i.e. the protection was increased as we moved towards
the poles away from the equator. But this finding was contradictory in the case of
leprosy, as another study (Fine 1985) argued that the protection of the vaccine was
greater at the equator highlighting that environmental and geographic factors play a
role in altering the effect of the vaccine.
The present BCG vaccine is a combination of different strains that vary pheno-
typically and genotypically (Behr and Small 1999). The different results obtained
from the various studies across the globe could be due the fact that different strains
were used in each study. The studies lacked uniformity. The genetic variation among
the different study populations is another contributing factor leading to changing
susceptibility to infection (Shields et al. 1987).
Mixed Vaccine
The combination vaccine has immunological effects in patients with leprosy and
therapeutic effects in healthy population. But when administered on their own the
vaccine proved inefficient. There is neither valid information on the stability of the
immune conversion nor details on the antigenic variation between M. leprae organ-
isms isolated from different armadillos. The combination vaccine demonstrated the
occurrence of lepromin in humans in a study conducted by Stanford (Stanford 1988)
The M. leprae bacillus has lipids in the cell wall which prevents the recognition of
the bacilli by the macrophages in our body and this is responsible for resistance in
LL patients. On the contrary, persons with TT leprosy show cell-mediated immune
response owing to increased levels of lipase, which eliminates the lipids from the
cell wall. Delipidified cell component (DCC) of the bacillus leads to the activation
of the macrophages and kills M. leprae (Robinson and Mahadevan 1989). However,
the toxicity and safety factor has not been studied to date.
M.W. Vaccine
According to another study, it is seen that the vaccine prepared from Mycobacterium
welchii (M.W.) brings about the conversion of lepromin in BL/LL patients
(Chaudhary et al. 1983). M.W. is a fast-growing mycobacterium and can be grown
in saprophytic soil (Dharmendra 1985). This vaccine was used against multibacil-
lary leprosy and the outcome was similar to the effect of ICRC vaccine (Deo et al.
1983; Zaheer et al. 1988). But this was an expected finding as ICRC and M.W. are
almost similar with respect to antigens (Girdhar and Desikan 1978; Mustafa and
Talwar 1978). Studies at Central Drug Research Institute at Lucknow established
CMI response in mice, langur and rhesus monkey (Dharmendra 1985) when treated
using M. Habana vaccine, which is a photochromogenic mycobacterium.
ICRC
In the hunt for an effective vaccine combination, antigenically similar cultivable

strains were traced and the Cancer Research Institute located in Mumbai introduced
the ICRC vaccine in 1979. ICRC is a cultivable mycobacteria strain belonging to the
complex M. avium-intracellulare (Deo et al. 1983). It is evident from the results
obtained from humans and animal studies that cross-reactivity of antigens is shown
by ICRC bacilli with M. leprae with respect to both T and B cell antigens (Girdhar
and Desikan 1978; Mustafa and Talwar 1978; Gangal and Khanolkar 1974).
T4 + clones obtained from tuberculoid leprosy patients have been tried against
M. leprae soluble protein, as they are produced in large numbers (Emmrich and
Kaufmann 1983). The interaction of many several clones with the ICRC bacilli
highlights its association with M. leprae with respect to T cell antigens. Studies
have revealed that as the antigens of the ICRC bacilli are easily accessible, it makes
the vaccine overall more immunogenic.
Research on ICRC vaccine has revealed that a single dose brings 53% lingering
immune conversion in LL patients who are on chemotherapy, associated conversion
in few patients with ‘increasing’ tissue reaction and faster rate of clearance of bacilli
from tissues (Fine 1985). Reversal reactions were observed in about 10% of the
participants immediately after vaccination (Bhatki et al. 1983). ENL was noted in
around one third of the participants with a high load of bacteria, 10–15 days after
vaccination (Zaheer et al. 1988). Newer lesions were not developed in the patients
after the dose of vaccination (Zaheer et al. 1988). Another study by Convit et al. also
showed similar results (Muliyil et al. 1991). Lepromin conversion was seen is 95%
of the cases when treated with ICRC vaccine (Chaturvedi et al. 1987) and this was
stable up to 5 years (Chirmule et al. 1986). Hypersensitivity to M. leprae antigens

was responsible for the pathogenesis of nerve damage observed in patients affected
by the disease (Godal 1978; Waters et al. 1971). No corroborating results have been
detected during the past 4 years in either the lepromin-positive or lepromin-negative
group of healthy household contacts (HHC) of MB leprosy patients, to whom the
vaccine was administered (Chaturvedi et al. 1987). The vaccine was reported to be
non-toxic and well accepted. It can be conveniently administered as a single dose.
The antibody levels remain unaltered (Scollard 2004). There is no defence provided
by the anti-M. leprae antibodies in the circulation but they are associated with
hypersensitivity reactions (Fine and Dockrell 1991). Since this vaccine is sourced
from cultivable organisms, it is cost effective and there is no chance of contamina-
tion. The immunity conferred is stable in contrast to the armadillo-derived M. leprae.
MIP
Also popularly known as Mycobacterium indicus pranii (MIP), this anti-leprosy

vaccine has an efficacy only below that of the BCG vaccine itself. However, studies
from this prospective vaccine were conducted as a field survey with a follow-up
period of 9 years (Sharma et al. 2005). The results showed the highest efficacy of
68% for a duration of 3 years, only next to BCG. But this protective efficacy fell to
60% after 6 years and 28% after the 9-year follow-up period. Currently it is still
under study in two districts in India, along with a combination of single dose of
rifampicin (SDR) (Kumar 2017). Another ongoing study has already been launched
in five high-risk areas, employing the autoclaved form of the vaccine. The associ-
ated family members are also being vaccinated at an interval of 6 months.
After intense research, it was found that the deficit in immunity was caused due
to the inability of the host to react against the microorganisms. In the presence of the
bacteria, the lymphocytes of the host remain unaltered and therefore do not produce
cytokines in response to the same (Talwar and Gupta 2017).
When used as an adjunct to multidrug treatment (MDT), it hastens bacterial
clearance and quickens the recovery period (Chaudhary et al. 1983). It changes
nearly 98% of normal negative lepromin persons who are healthy to persons with
lepromin positivity status. This gets more interesting when it is seen to clear all
granulomas. The patients heal completely and go on to lead normal lives without the
scars of the infection. The sensitivity in the peripheral nerves is restored to normal
sensitivity as well (Talwar 2014).
Additional Properties of MIP
(i) Tuberculosis
The antigens of MIP have commonalities with both M. leprae and M. tuberculo-
sis. This has made it an ideal candidate to be employed in treating ‘difficult to treat’
class II tuberculosis patients (Sharma et al. 2017).
(ii) Action Against Anogenital Warts and Other Lesions
When administered intralesionally, the vaccine is capable of clearing warts in the
anogenital region (WHO, Global leprosy situation 2009) and other lesions present
elsewhere (Singh et al. 2014).
(iii) Stimulates Humoral and Cellular Response
MIP may be used as an oral contraceptive adjuvant (Purswani and Talwar 2011).
(iv) Preventive of Myelomas and Cancers
MIP has both therapeutic and preventive measures that have been established in
SP2/O myelomas in mice, in a study conducted by the Indian Institute of Science
(Rakshit et al. 2012). This has been approved by the Drugs Controller General of
India (DCGI) for human use and consumption and is currently being employed in
treating cancers of various types.
Scope for Future Research
It is an established fact that the small peptides in a protein molecule are responsible
for its immunogenicity (Job et al. 1982). These small peptides are target specific in
nature and will lead to manufacturing newer vaccines both synthesized chemically
or biosynthesized (using recombinant DNA technology) which will be purer. One of
the limiting factors encountered is in cultivating the organism to produce sufficient
quantities of the subunit vaccine. But this has been overcome using the recombinant
technology where the entire genome of the organism is cloned (Young et al. 1985);
the coding of genes has established five immunogenic proteins from M. leprae
using monoclonal antibodies. Once the ‘protective’ antigen(s) are identified for the
immunogenic proteins, the non-cultivability of M. leprae will no longer challenge
the production of the pure vaccine and the supply of the antigens will not be a
constraint.
A technique that has been advocated to isolate the immunogenic antigens at the
Cancer Research Institute located at Mumbai is liquid chromatography (Chirmule
et al. 1988a, b). It uses columns of gel through which substances can permeate, and
a very high molecular weight fraction is obtained under sonicate (approx. 1 million
daltons) named PP-1, which is the immunogen with dominant T cell of the ICRC
bacilli (Chirmule et al. 1988a, b). On sonicating M. leprae, another similar fraction
can be isolated. The PP-I fractions obtained from the two different organisms dis-
play antigenic cross-reactivity. The glycoprotein PP-I is primarily a component of
the cell wall of the bacillus. A recent isolation of a high molecular weight cell wall
core (CWC) fraction of M. leprae has been achieved. CWC is a strong T cell
immunogen (Kaplan et al. 1988). When a vaccine contains PP-I of ICRC bacilli, it
promotes lepromin conversion in LL patients (Bhatki et al. 1988). As it is a glyco-
protein, PP-I has all essential components for the vaccine. These proteins can
thereby act as co antigens on their own or even act as carriers for sugars or fats
which play the role of an antigens. Phase I and II trials are ongoing, with regard to
this vaccine containing PP-I of ICRC bacilli being established as the ideal vaccine
for leprosy.
Subunit Vaccine for Leprosy
An ideal vaccine for leprosy is anticipated to not only protect from the disease but
also decrease the transmission rate by instigating a strong, lasting response in T cell
directed against M. leprae. Subunit vaccines would be more specific and targeted to
battle leprosy showing lasting effect against the bacterium. Since the completion of
the M. leprae genome fully in the year 2001, the production of production of recom-
binant antigens has been made easier. It is believed that a subunit lymph node (DLN)
cellularity can be used to determine the status of infection. Identifying the antigens
is important for efficient vaccination against the disease. The American Leprosy
Missions supports a project by Infectious Disease Research Institute (IDRI) where
they have discovered numerous antigens identified by PB leprosy patients and
which in turn stimulate secretion of IFN-γ (Duthie et al. 2008; Sampaio et al. 2011).
Increase in T cell concentration suggested a concomitant rise in DLN cellularity at
the site of infection. But these changes were not observed when killed M. leprae
was inoculated and the infection controlled by rifampicin therapy. One of the most
recent findings showed strong antigen-specific Th1 responses that reduce the inflam-
mation caused by the disease; however the bacterial load did not reduce (Raman
et al. 2009).
From the above studies, it is clear that although there is no effect on the bacterial
burden, it is effective in managing the local inflammation. Since a very common
problem encountered in leprosy is uncontrolled inflammation, this subunit vaccine
would be beneficial to individuals suffering from the disease which otherwise
causes significant discomfort, deformity and disability.
One Vaccine for All
It is interesting to note that most vaccines that have their effects in leprosy were
initially used to treat tuberculosis worldwide. It was only later that their potential
against leprosy was discovered (Wakhlu et al. 2001; Valdes et al. 2014). BCG is still
used against tuberculosis all over the world (Kaufmann et al. 1992) and was the first
vaccine given to newborns in over 172 countries worldwide (Zwerling et al. 2011).
Although it was recognised for its efficacy against the prevention of tuberculosis, it
was quickly identified to have effects against M. leprae too (Fine 1995). The use of
BCG vaccine, as a single vaccine, against both TB and leprosy has been analysed in
various clinical and observational studies. BCG’s therapeutic effects ranged from
2% to 83% and from 58% to 74% against pulmonary and extrapulmonary TB,
respectively (Mangtani et al. 2014); its effect in leprosy ranged from 26% to 41% in
experimental studies and 61% in observational studies, respectively. Mild variation
was seen in paucibacillary (62%) and multibacillary (76%) forms (Duppre et al.
2008). Aside from its efficacy in TB and leprosy, the vaccine is reported to have a
notable impact on diseases that are not associated, perhaps by inducing the innate
immune system to react more favourably to other assaults (Blok et al. 2015).
The efficacy of the vaccine against TB and leprosy, however, decreases over a
period of time, dropping to 14% after 10–20 years (Mangtani et al. 2017).
Revaccination is effective to a certain point in TB and in leprosy, the risk is reduced
to 50% (Ponnighaus et al. 1992). Revaccination with BCG was used in countries
like Brazil as early as 1970 in order to boost its efficacy (Merle et al. 2010). The
immunity conferred by the booster dose of BCG vaccine was 56%; this was inde-
pendent of its efficacy with previous vaccination (Duppre et al. 2008).
As mycobacterial diseases like tuberculosis and leprosy are diseases encountered
in Third World countries, a mixed single vaccine would greatly help in tackling the
dual threat. Now Mycobacterium avium (an intracellular group of organisms) is also
being frequently isolated from patients with HIV infection (Kichn et al. 1985). The
future could be in proposing a polyvalent mycobacterial vaccine offering immuni-
zation over a wide spectrum of mycobacterial diseases consisting of BCG and ICRC
(expand this) bacilli or its ‘subunits’. This would subsequently reduce the number
of vaccinations and this will be cost effective in mass campaigns.
Subunit Vaccines for Tuberculosis and Leprosy
As the leprosy bacteria M. leprae have been subjected to severe gene reduction
(Cole et al. 2001), it is evident that not every Mtb antigen will be a suitable target
for the TB vaccine as they possess corresponding homologs in the leprosy bacteria
M. leprae. A few instances are ID83/GLA-SE and ID93/GLA-SE, which are two
fusion proteins that are recombined, which are formulated with TLR4L having
adjuvant GLA-SE, possessing three Mtb proteins, namely: Rv1813, Rv2608 and
Rv3620, with the additional inclusion of Rv3619 in ID93. The sequence of amino
acid (aa) of Rv3620 and Rv3619 is 64% and 58% identical to the corresponding
M. leprae proteins, namely, ML1056 and ML1055, respectively. It is therefore pre-
dictable and has been proved that on account of these similarities, both the Mtb
hybrid recombinant proteins were identified from the blood in paucibacillary
patients. Moreover, when administered subcutaneously, the subunit vaccines
decreased inflammation induced by M. leprae and reduced the bacterial culture in
mice models of leprosy (Duthie et al. 2014), implying that TB subunit vaccines may
have beneficial therapeutic effects in leprosy.
With the same idea, a similar TB subunit vaccine candidate is also being tested,
possessing two majorly secreted proteins, Mtb ESAT6 and Mtb Ag85B, which are
both present short term in Mtb culture filtrates (Sorensen et al. 1995). On compari-
son, Ag85B-ESAT6 seems a suitable candidate for leprosy as well, as Mtb Ag85B
and ESAT6 share 89% and 68% amino acid overlap with M. leprae homologs,
namely, ML0049 and ML2028, respectively. It must be noted that they are widely
recognized by antibodies in multibacillary leprosy patients (Spencer et al. 2011)
and also by IFN-γ-secreting cells seen in paucibacillary leprosy patients (Duthie
et al. 2013). Cross-reactivity of T cells in both Mtb and M. leprae ESAT6 among
leprosy and TB patients has been established (Geluk et al. 2002). Furthermore,
another study showed overexpression of Ag85B in BCG significantly raising BCG’s
therapeutic efficacy against M. leprae too (Gillis et al. 2014).
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Chapter 25
Exploitation of Fibrinolytic Enzymes
in Combating Blood Clotting Disorders –
Recent Advances and Strategies:
A Comprehensive Review
Sheela Kumari Sahoo and Sabuj Sahoo
Introduction
Blood clots are regular fortification processes of our body against bleeding, formed
over a sequence of responses among special blood cells (platelets) and proteins
(clotting factors). Under customary condition, fibrin masses (final clotting factor)
are hydrolysed by means of plasmin to evade thrombosis in blood vessels. However,
under unhinged condition, it leads to form pathophysiological disorders, where
clots are not hydrolysed and fibrin is deposited in blood vessels initiating thrombo-
sis which obstructs the flow of blood through the circulatory system (Furie and
Furie 2008; Gopinath and Lingappa 2016). People with high immobility and genetic
disorders associated with clotting may result in formation of thrombus. Additionally,
damage of an artery, vein or surrounding tissue may also result the same. Such clot
formation inside the vessels may leads to innumerable disorder related to cardiovas-
cular system such as myocardial infarction, cerebral stroke, venous thromboembo-
lism and other diseases like deep vein thrombosis (DVT), pulmonary embolism,
antiphospholipid syndrome, thrombophlebitis and cerebral venous sinus thrombosis
(Tian et al. 2015).
In poor and developing countries, the situation is significantly alarming wherein
80% of deaths occurred in both women and men due to cardiovascular diseases
(WHO CVD 2017). As per the WHO by 2030, about 23.3 million deaths will be
from cardiovascular disease every year which may continue to get worse over time
(Vijayaraghavan and Vincent 2015). It is anticipated that globally, around 82% of
mortality is due to cardiovascular diseases at the age of 65 and older. The threat of
cardiovascular stroke doubles every decade after the age 55. Apart from CVD there
are approximately 10 million cases of DVT worldwide (13 October The Global
S. K. Sahoo · S. Sahoo (*)

Post Graduate Department of Biotechnology, Utkal University, Bhubaneswar, Odisha, India
e-mail: sabujbiotech@utkaluniversity.ac.in

https://doi.org/10.1007/978-981-15-2195-9_25
384 S. K. Sahoo and S. Sahoo
World Thrombosis Day Report). Considering the current scenario there is an urgent
need to develop a noble safe and potent therapeutic agent to confront such situation.
Blood Coagulation and Thrombus Formation
The steadiness of blood transmission in a fluid or coagulated form is known as hae-

mostasis that consists of two corresponding mechanisms: blood coagulation and
fibrinolysis. Coagulation constrains loss of blood from an impaired vessel by means
of clot formation and is recovered into circulating blood by means of fibrinolysis
system after refurbishment of the vessel endoepithelium (Takada et al. 1994, Lu and
Chen 2012). When the vessels are damaged it encourages the platelet activation and
amassing of clotting plasma proteins. Platelets adhere to the subendothelium and at
corresponding period activates blood coagulation cascade which consequence in
establishment of fibrin. This creates a meshlike structure over the platelet plug,
which ultimately sealed the injury site (Gentry 2004; Norris 2003; Wolberg 2007).
Under standard circumstances, fibrinolysis system quickly and safely removes
fibrin lumps formed in blood vessels. Plasmin which is a serine protease plays a
crucial role in the fibrinolytic system; it is triggered from a proenzyme plasminogen
through a tissue-type plasminogen activator (tPA) or a urinary plasminogen activa-
tor (u-PA). A number of coordinated interfaces between fibrin, specific inhibitors,
plasminogen and plasminogen activators are obligatory to degrade the thrombus by
the normal fibrinolytic system (Medved and Nieuwenhuizen 2003). Formation of
meshlike structure of fibrin activates the body’s fibrinolytic system and generates
the active plasmin and this plasmin disintegrates the fibrin structure followed by
dissolving of clot (Collen 1999) (Fig. 25.1a). But, when there is imbalance of the
any clotting cascade, it resulted into formation of thrombosis.
Thrombosis is the establishment of blood clot inside the blood vessel, hindering
the blood flow throughout the circulatory system. Virchow’s triad has been offered
three aspects which are obligatory for the formation of thrombosis: stasis of blood,
vessel wall injury and altered blood coagulation (Golan et al. 2011). Various other
factors which are the reason behind the formation of blood clot are smoking, ele-
vated cholesterol level, obesity, atherosclerosis, diabetes and stress.
Thrombolytic and Fibrinolytic Agents
Anticoagulants
Conventionally these thrombotic disorders are treated by IV/IM or an oral adminis-

tration of anticoagulants like heparin sodium, warfarin sodium, enoxaparin sodium,
etc. (National blood clot alliance report www.stop the clot.org), that act by either
25 Exploitation of Fibrinolytic Enzymes in Combating Blood Clotting Disorders … 385
Intrinsic pathway Extrinsic pathway
XII XIIa Native

VII VIIa condition
Denaturing condition
tPA, {Native lysis
(Denaturing buffer)
STREPTOKINASE, buffer)
UROKINASE
XI XIa WARFIRIN
Plasminogen
IX IXa Bind with Ni-
NTA coloumn
ENOXAPARIIN
VIII
DNasc Native Wash Wash Denaturing Wash buffer
buffer
X Xa
Plasmin HEPARIN XIII
Degrade V
Native Elution Elute
NATTOKINASE Prothrombin Thrombin buffer Denaturing Elution buffer
LUMBROKINASE XIIIa
Purified product
SERRATIO- Fibrinogen Fibrin Stable fibrin clot
PEPTIDASE Degrade
OTHER FIBRINOLYTIC
ENZYMES Western blotting
Fig. 25.1 Effect of various drugs on (a) blood coagulation cascade (red line shows inhibitory
effect and green line shows activation) and (b) Purification of the cloned gene using Ni NTA
overwhelming the synthesis or function of several clotting cascade factors that are
normally present in the blood. Heparin sodium binds with antithrombin III and acti-
vates it which inhibits activated thrombin and coagulation factor. Warfarin sodium
inhibits synthesis of hepatic vitamin K which blocks the activation of vitamin
K-dependent coagulation factors II, VII, IX and X. Enoxaparin sodium acts on fac-
tor Xa and inhibits the creation of thrombin from prothrombin (DeWald 2018).
Thrombolytic Therapies
Thrombolytic therapies such as tissue plasminogen activator (tPA), streptokinase

(SK) and urokinase (UK) are also used to treat thrombosis (Table 25.1). The avail-
able marketed drugs and mechanism for treatment of thrombosis are enlisted at
Table 25.1.
Tissue Plasminogen Activator
tPA is a serine protease found on endothelial cells that lines on blood vessels. Tissue
plasminogen activator cleaves clot by binding with fibrin on the superficial surface
of the clot and initiating attachment of plasminogen and plasminogen activators to
specific sites on fibrin molecule and to endothelial cell surface receptors (Tadayon
et al. 2015). Plasminogen activators convert plasminogen into plasmin which is a
proteolytic enzyme (Chandler 1996) and having capability of breaking down the
fibrin molecules, thereby dissolving the clot. Even though tPA is reasonably
Table 25.1 Available marketed agents for treatment of thrombosis

Drug Category Brand and dosage form Mechanism of action Reference
Heparin Anticoagulant Delcot (5000IU) Binds and activated DeWald
sodium Thinla (25000IU) antithrombin III; (2018)
Troparin (3000IU); inhibits activated
Injection thrombin and
coagulation factor
IV
Warfarin Anticoagulant Warf (3 mg) Inhibits hepatic DeWald
sodium Uniwarfin (2 mg) synthesis of vitamin (2018)
Sofarin (5 mg); tablet K-dependent
coagulation factors II,
VII, IX, X
Enoxaparin Anticoagulant Clexane 40 mg (0.4 ml) Acts on factor Xa; DeWald
sodium Dynalix 40 mg inhibits the synthesis of (2018)
Lupenox 40 mg thrombin from
prothrombin
Lovenox 40 mg
Injectable prefilled
syringes 30, 40, 60, 80,
100, 120 and 150 mg;
subcutaneous
tPA/alteplase Thrombolytic Cathflo activase, powder Binds with plasminogen Tadayon
for reconstitution, activator; converts et al.
Actilyse (10 mg); plasminogen into (2015)
injection IV plasmin degrade fibrin
Streptokinase Thrombolytic Myokinase 150000IU Form highly specific Albisetti
Stpase 150000IU enzymatic complex (2006)
Prokinase 0.75MIU with plasminogen;
convert inactive
fibrokinase150000IU;
plasminogen into active
injection
plasmin; break down
IV fibrin clots
Urokinase Thrombolytic Solokinase 500000IU/vial Acts on plasminogen; Albisetti
Uropase 250000 IU/vial generates active (2006)
Unikinase (10, 2.5, 5, 7.5 plasmin; degrades
lacs IU); injection fibrin; breaks down clot
IV
s elective for clot-bound plasminogen, it still activates circulating plasminogen and

released plasmin, which leads to breakdown of circulating fibrinogen and causes an
unnecessary systemic fibrinolytic state. Generally, plasmin is deactivated by circu-
lating α2-antiplasmin but salutary doses of tPA lead to adequate formation of plas-
min which overcome circulating concentrations of α2-antiplasmin resulting in
undesirable bleeding (Khursade et al. 2018).
Streptokinase
This drug was first derived from group C, β-haemolytic streptococci, in 1933 having
molecular Wt. of 47–50.2 kD. It is on the WHO Model List of Essential Medicines,
the most successful medicines needed in a health system. It forms a highly specific
enzymatic complex with plasminogen and converts inactive plasminogen molecules
into active plasmin which break down fibrin clots in addition to fibrinogen and other
plasma proteins. This in turn proceeds to the lysis of blood clots (Albisetti 2006).
Urokinase
Urokinase is obtained from human and animal cells having molecular weight 50–54
kD. Urokinase targets endogenous fibrinolysis system. It acts on plasminogen to
generate active plasmin which cleaves fibrin clots in addition to fibrinogen and other
plasma proteins (Albisetti 2006).
Limitations of Thrombolytic Drugs
Abnormalities caused by thrombosis can be treated with thrombolytic therapies that

are stated to be associated with adverse effects such as excessive bleeding, a short
half-life, recurrence at the site of the residual thrombosis, blood loss, induction of
gastrointestinal bleeding, immunogenic effects, vomiting, requirement of high ther-
apeutic dose, etc. These drugs also have low specificity towards the fibrin when
administrated intravenously and relatively expensive and develop resistance to
repercussion which limits its clinical use (Mahajan et al. 2012; Hua et al. 2008;
Cheng et al. 2015). Drawbacks of available drugs accentuate the need to develop
and exploit novel biochemical attributes for the development of safer, non-
immunogenic, nontoxic, direct-acting fibrinolytic enzymes with high specificity
towards fibrin.
Fibrinolytic Enzymes
Nattokinase
Nattokinase (NK) is a serine proteolytic enzyme extracted from natto, which is a

traditional Japanese food manufactured by the fermentation of soybeans with the
help of Bacillus subtilis having molecular mass 46.5kDA (Lin et al. 2015). NK
degrades blood clumps by directly cleaving fibrin and plasmin substrate, alters
endogenous prourokinase to urokinase (uPA), lyses PAI-1 (plasminogen activator

inhibitor-1) and elevates tissue plasminogen activator (t-PA) that aids in fibrinolytic
activity. This enzyme has an effect on injury mediated by oxidative stress such as
arterial thrombosis and inflammation-induced venal thrombosis (Weng et al. 2017).
Lumbrokinase
Lumbrokinases are isolated from the body cavity, intestine tissue, tissue fluid and
intestinal fluid of earthworms (Eisenia fetida). They are a group of proteases having
molecular mass within the range of 25–32 kDa. These kinases can directly degrade
fibrinogen and fibrin, convert plasminogen to plasmin and elevate endogenous
human tissue plasminogen activator (t-PA) activity to cleave fibrin clots. Studies
also recounted that lumbrokinases obstruct platelet activation and accretion and
restrict the intrinsic coagulation pathway (Li et al. 2012).
Serratiopeptidase
Serratiopeptidase (serratiapeptase/serralysin/serrapeptase) is a proteolytic enzyme

that is derived from the non-pathogenic enterobacterium Serratia sp. E-15 found in
silkworm. It is a sole chain protein having molecular mass of about 55 KDa. This
enzyme degrades insoluble products of proteins like fibrin and inflammatory media-
tors and eventually dissolves blood clot (Rajaram et al. 2016).
DNase
Deoxyribonuclease is an enzyme having molecular weight 31–60 kDA which catal-

yses the hydrolytic breakdown of phosphodiester linkages in the DNA backbone;
this combination with tPA could dissolve the blood (Rahman et al. 2011).
Other Fibrinolytic Enzymes
There are other fibrinolytic enzymes which are derived from several microbial
sources which also directly act on fibrin to degrade its meshlike structure and finally
dissolve the blood clot.
Sources of Thrombolytic and Fibrinolytic Enzymes
Fibrinolytic enzymes were reported to be isolated from diverse sources like plants,
bacteria, marine microorganisms, snake venom, insects, earthworm and mush-
rooms. Plants from which fibrinolytic enzymes were isolated are as follows:
Achyranthes Aspera, Eclipta alba, Hemidesmus indicus, Hibiscus rosa-sinensis,
Zingiber officinale (Aruna et al. 2016), Curcuma aromatica and Curcuma longa
(Shivalingu et al. 2015). There are several bacterial species capable of producing
fibrinolytic enzymes such as Bacillus sp. and β-haemolytic streptococci (Dubey
et al. 2011, Mukherjee et al. 2012, Majumdar et al. 2014, Yogesh and Halami 2015,
Kim et al. 1997, Han et al. 2009), Pseudomonas aeruginosa (Raj et al. 2012),
Candida guilliermondii, Streptomyces sp., Penicillium chrysogenum, Rhizomucor
miehei and Escherichia coli (Raju and Divakar 2013). Apart from these, some
marine bacteria are also present which are potent to produce the fibrinolytic enzymes
Serratia marcescens subsp. sakuensis (Krishnamurthy and Belur 2018), Shewanella
sp. (Vijayaraghavan and Vincent 2015) and Bacillus subtilis ICTF-1 (Mahajan et al.
2012). Venom obtained from the snake like Bothrops colombiensis (Girón et al.
2013), Agkistrodon saxatilis Emelianov (Koh et al. 2001), Crotalus atrox (Bajwa
et al. 1980), Protobothrops tokarensis (Tokara-habu) (Oyama et al. 2013) and
Agkistrodon contortrix (Chen et al. 1991) and insects such as Catharsius molossus
(Ahn et al. 2003) and Porcellio scaber Latreille (Tian et al. 2015) and earthworms
(Eisenia fetida) (Li et al. 2012) are also considered as a source of above said enzyme.
Instead of all these sources some species of mushrooms such as Cordyceps militaris
(lan Liu et al. 2016), Lyophyllum shimeji (Moon et al. 2014), Pleurotus ostreatus
(Choi and Shin 1998), Armillaria mellea (Kim and Kim 1999) and Tricholoma sap-
onaceum (Kim and Kim 2001) are also considered as a major source of fibrinolytic
enzymes.
creening and Isolation and Identification of a Fibrinolytic

S
Enzyme Producing Strain
Screening and isolation of fibrinolytic enzymes producing strain were reported to be

done by using sterilized skimmed milk agar and incubating the culture at 37 °C for
24 h. The hydrolytic zone formed by the isolate considered protease positive was
further screened by employing fibrin plate (Majumdar et al. 2014; Raj et al. 2012;
Yogesh and Halami 2015; Mahajan et al. 2012).
Production of Fibrinolytic Enzyme
Fibrinolytic enzymes are produced by liquid and solid state fermentation using suit-
able medium containing various nitrogen and carbon sources such as casein, yeast
extract, peptone, beef extract, gelatin, urea and sucrose, dextrose and maltose,
respectively (Khursade et al. 2018, Vijayaraghavan and Vincent 2015). For further
understanding of bioprocess parameters reported to enhance the fibrinolytic enzyme
production are summarised in Table 25.2.
Partial Purification of Fibrinolytic Enzyme
The partial purification of the fibrinolytic enzyme is reported to be done by employ-

ing ammonium sulphate precipitation followed by dialysis, size exclusion and ion
exchange chromatography. For size exclusion chromatographic separation Sephadex
G-25, G-50, G-75 or butyl sepharose FF hydrophobic column is preferred. While in
the case of ion exchange chromatographic separation sepharose DEAE (diethylami-
noethyl) grouped resin is basically used (Mahajan et al. 2012; Devi and Hema
Latha 2014).
Table 25.2 Bioprocess parameters required to enhance the fibrinolytic enzyme production
Parameters Optimum References
pH pH 5–pH 11 Liu et al. (2017), Dubey
et al. (2011), Sun et al.
(2016)
Temperature 30–70 °C Cheng et al. (2015),
Yoon et al. (2002), Park
et al. (2013), Gopinath
and Lingappa 2016
Carbon Sucrose, dextrose, maltose, fructose, galactose, ribose, Khursade et al. (2018),
source xylose, starch, dextran, cellulose and trehalose Vijayaraghavan and
Vincent (2015)
Nitrogen Casein, yeast extract, peptone, beef extract, gelatin, Khursade et al. (2018),
source urea, tryptone, meat extract, soya meal, casitone and Vijayaraghavan and
peptone (organic source). Ammonium chloride, sodium Vincent (2015),
dihydrogen phosphate (NaH2PO4), calcium chloride, Mahajan et al. (2012)
sodium nitrate, disodium hydrogen phosphate
(Na2HPO4), ammonium sulphate and ferrous sulphate
and potassium nitrate (inorganic salts)
Inhibitors Phenyl methyl sulfonyl fluoride (PMSF), ethylene Cheng et al. (2015),
diamine tetra acetic acid (EDTA), ethylene glycol- Yogesh and Halami
bis(β-aminoethyl ether) (EGTA) (2015), Liu et al. (2017)
Metal ions Cu2+, Fe3+, Fe2+, Zn2+, Co2+, K+, Na+, Ca2+, Liu et al. (2017), Tian
Mg2+ and Mn2+ et al. (2015).
olecular Cloning and Characterization of the Gene

M
Encoding Fibrinolytic Enzyme
Various cloning vectors like pMD18-T/ pGEM-T/pET26b, restriction enzymes such

as BamHI/XhoI, expression vector and competent cells like pET28a/pSUGV4 and
E. coli XL1 blue/E. coli DH5/E. coli DHα, respectively, were employed for molecu-
lar cloning and characterization of fibrinolytic enzyme (Jeong et al. 2004, 2015;
Peng et al. 2004; Jeong and Kwon 2007; Han et al. 2009; Leedagger et al. 2013).
Optimized reports and studies on molecular cloning and expression of fibrinolytic
genes are shown in Table 25.3.
Purification of Recombinant Fibrinolytic Enzyme
The purification of recombinant fibrinolytic enzyme was reported to be performed

by cultivation of the transformed cells with gene encoding for fibrinolysis, in LB
broth containing appropriate antibiotics and inducer such as IPTG to induce gene
expression. The supernatant and pellet of microbial cells were collected and the
recombinant proteins with 6xHistidine-tag were purified by Ni-NTA column (Jeong
et al. 2014). Schematic representation of purification of cloned fibrinolytic gene is
shown in Fig. 25.1b.
Table 25.3 Molecular cloning studies of fibrinolytic gene

Required
parameters Fibrinolytic agents References
Gene responsible Nattokinase: aprN Cook et al. (2012), Caballero et al. (1999),
for fibrinolysis Streptokinase: Ska, Skc Jeong et al. (2004), Sun et al. (2016), Peng
Other fibrinolytic enzymes: et al. (2004), Choi et al. (2004)
aprE2, afeE, subtilisin DJ-4,
subtilisin DFE
Cloning vector pMD18-T/pGEM-T/pET26b/ Jeong et al. (2004), Peng et al. (2004), Jeong
pBFA1/pTZ57R and Kwon (2007), Han et al. (2009),
Leedagger et al. (2013), Jeong et al. (2015)
Restriction BamHI/XhoI/EcoRI/SalI Jeong et al. (2004), Peng et al. (2004), Jeong
enzymes and Kwon (2007), Han et al. (2009),
Expression pET28a/pSUGV4/pET32a Jeong et al. (2004), Peng et al. (2004), Jeong
vector and Kwon (2007), Han et al. (2009),
Competent cells E. coli BL21 (DE3)/E. coli Jeong et al. (2004), Peng et al. (2004), Jeong
XL1 blue/E. coli DH5/ E. coli and Kwon (2007), Han et al. (2009),
DHα Leedagger et al. (2013), Jeong et al. (2015)
Conclusion
The case of thrombotic disorders including cerebral stroke, myocardial infarction,

venous thromboembolism and other disorders is rapidly increasing the mortality
rate worldwide. Though expensive but are an efficient plasminogen activators, like
streptokinase, urokinase and tissue plasminogen activators (t-PA) as an effective
means to combat these disorders. But these drugs are also reported to be associated
with a number of adverse effects such as excessive bleeding, re-occurrence at the
site of the residual thrombosis, haemorrhage and vomiting. They, further, have low
specificity towards fibrin upon intravenous administration. On the other hand, the
plasmin-like, direct-acting clot-bursting drugs such as nattokinase, lumbrokinase
and other fibrinolytic enzymes dissolve the thrombin rapidly and wholly without
enhancing the risk of bleeding or haemorrhage. Stability at a wide range of pH and
temperature that is pH 5–11 and 30–70 °C, respectively, is one of the physiological
advantages of these enzymes. The impact of various nitrogen, carbon sources and
metal ions elevates and ameliorates the enzyme production. An effort has been
made to illustrate the mechanism and adverse effects of present marketed drugs
used for treatment of thrombosis, their sources and bioprocess parameters. Moreover
molecular cloning, characterization of gene encoded for enzyme having fibrinolytic
property with minimal adverse effects and specific target to dissolve the clot have
also been described. The information documented in the review shall update
researchers to further derive a safer potent and stable fibrinolytic enzyme to warfare
blood clotting disorders.
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Chapter 26
Recent Development in Chronic
Inflammation Research and Mangroves
as Potential Source of Anti-inflammatory
Agents
Dibyajyoti Samantaray and Swagat Kumar Das
Introduction
According to the International Diabetes Federation, an estimated 425 million diabe-

tes were present in 2017 globally and the prevalence is expected to reach up to 629
million by 2045. Amongst these, the majority (80%) of diabetic patients come from
low- to middle-income countries. Such spatial distribution of patients is due to dif-
ferences in their genetic background, lifestyle and availability of healthcare facili-
ties. Diabetes is a chronic disease which progresses slowly and systematically.
Therefore there is requirement for treatments which have excellent and long-term
safety profiles. However, it is seen that some well-reported treatments could interact
with novel drugs in patients with type 2 diabetes. Hence, search for novel anti-
inflammatory agents in diabetic condition is the need for hour. These anti-
inflammatory agents also need further testing in long-term clinical studies involving
large and multi-ethnic pool of patient population to find the efficacy and variability
against different inflammatory factors (Kahn et al. 2006). Natural biomolecules iso-
lated from plant species such as mangroves may be an alternate treatment option
with lower side effects as they are reported to have anti-inflammatory activity.
Disease Pathogenesis
Type 2 diabetes mostly affect people as they grow by age, whereas it is more promi-
nent with those having genetic and epigenetic susceptibility. The disorder is more
heightened in those who do overeating or have lack of physical activity. For indi-
D. Samantaray · S. K. Das (*)

Department of Biotechnology, College of Engineering and Technology, Biju Patnaik
University of Technology, Bhubaneswar, Odisha, India

https://doi.org/10.1007/978-981-15-2195-9_26
398 D. Samantaray and S. K. Das
viduals with high rate of disease susceptibility, deformity in insulin secretion is high
and can be correlated with the decreased rate of insulin-mediated glucose uptake in
both liver and adipose tissues known as insulin resistance. During an individual’s
life journey, initially insulin resistance remains constant for a certain period of time
and increases gradually with age. However, deterioration of pancreatic β-cells may
occur at any age. This severely affects insulin secretion leading to development of
type 2 diabetes. Therefore, insulin secretion no longer replaces increased peripheral
insulin demand (Cerasi 1995; Kahn et al. 2006; Bonner-Weir 2000). The disease
progression from prediabetes to diabetes is mostly mediated by change in the secre-
tory potential of islets, which demand medications that could lead to indemnify
insulin replacement.
Multiple mechanisms have been reported that define and explain the underlining
defective secretion of insulin hormone and its role in onset of type 2 diabetes. These
factors can be discussed as glucotoxicity, oxidative stress, changes tin gut microbi-
ota, lipotoxicity, endoplasmic reticulum (ER) stress and amyloid deposits in islets.
However, contribution of the said factors in onset and progress of diabetes is still
unclear (Hotamisligil and Erbay 2008; Donath et al. 2003; Ehses et al. 2009). It is
more likely that they all participate in the disease pathology, with each differential
contribution that depends on genetics, physical activity, food and environmental fac-
tors. In pancreatic islets, it is reported that elevated glucose concentration leads to
increased level of metabolic activity, which increases production of reactive oxygen
species (ROS) along with activation of NLRP3, inflammasome and caspase that
leads to the production of interleukin-1β (IL-1β) (Hotamisligil and Erbay 2008). It
has also been found that rise in insulin demand and its increased production leads to
ER stress activating inflammatory inflammasome. Moreover, lipopolysaccharides
(LPS) from bacterial cell wall or fetuin-A-bound free fatty acids can also activate
Toll-like receptor 2 (TLR2) and TLR4, which eventually leads to translocation of
nuclear factor-κB (NF-κB) and induction of inflammation. Many of these compo-
nents are responsible for islet-derived IL-1β induction. This inflammatory process
could be beneficial as it promotes proliferation of β-cell and insulin production to
compensate insulin resistance. IL-1β is known to induce different cytokines and
chemokines, viz., IL-6, IL-8, tumour necrosis factor (TNF) and monocyte chemoat-
tractant protein 1 (MCP1) (Richardson et al. 2009; Butcher et al. 2014). Furthermore,
islets could produce polypeptide of amyloids, which later gets aggregate to form
amyloid fibrils in type 2 diabetes. Reports have suggested that both human islet
amyloid polypeptide and immune cells can interact with each other promoting IL-1β
synthesis. This causes resident islet macrophages to induce phenotype of a pro-
inflammation, which leads to islet dysfunction. It has been documented that islet
macrophage depletion facilitated glucose metabolism in experimental mice that
express human islet amyloid polypeptide, whereas improvement in glycaemic con-
dition was reported against IL-1 receptor antagonist (Butcher et al. 2014). Finally,
endocannabinoids, which get upregulated in the liver during obesity and mediate
satiety in the hypothalamus, may also promote activation of macrophages (Fig. 26.1).
Pathway imparting β-cell secretory function and its survival is correlated with
the cytokines, mediated by immune cells of type 1 diabetes which are associated
26 Recent Development in Chronic Inflammation Research and Mangroves… 399
Glucose,
Glucose, free fatty
TLR4
free fatty Feutin-A TLR4 acids,
Feutin-A
acids, endotoxins
endotox
ins TLR2
IL-1R1 IL-1R1
NF-κB
NF-κB
Macrophages
ROS
ß Cell Pro-IL-1ß IL-1ß Pro-IL-1ß ROS
NLRP3 CB1 Endocannabinoid

Caspase 1
Caspase 1
NLRP3
Amyloid
CB1 Plovpetides
Cytokines,
chemokines
Endocannabinoid
Fig. 26.1 Pathways showing interrelationship between diabetes and inflammation
with inflammatory response. It has also been reported that excessive lipid overload
may lead to death of adipocytes that can trigger inflammatory response. Formation
of localized hypoxia arising from the quick expansion of adipose tissues could also
trigger such responses. Obesity is also related with increased gut liking for bacterial
endotoxins that may trigger tissue inflammation (Freigang et al. 2013). Lipids,
endotoxins and free fatty acids recruit fetuin-A that activates TLR2 and TLR4 and
this leads to the NF-κB-mediated cytokine and chemokine release such as TNF,
IL-1β, IL-8 and MCP1. These cytokines are also known to promote various immune
cell accumulations. In macrophages, free lipids and hyperglycaemia condition facil-
itate formation of inflammasomes, which can lead to splicing of pro IL-1β to active
form of IL-1β (Koenen et al. 2011; Wen et al. 2011; Ramos-Zavala et al. 2011). This
high potent cytokine is seen to activate immune cells.
Unmet Needs in Type 2 Diabetes Therapy
Current type 2 diabetes treatments include insulin, metformin, thiazolidinediones,

sulphonylureas, α-glucosidase inhibitors, incretin and glucose co-transporter inhibi-
tors as these treatments can improve glycaemic condition and could even delay
diabetes onset. However, none of them have disease editing characteristics that
could show gradual decline in secretion of insulin. Diabetes is associated with com-
plex diseases like hypertension and dyslipidaemia and is also one of the dominant
causative factors for cardiovascular disease and other complications including neu-
ropathy, nephropathy and retinopathy. To treat and prevent such adverse conditions,
varied and multidrug therapy is being prescribed to diabetic patients along with
glucose-lowering medications. However, multidrug therapy has not been found
popular approach due to decreased patient satisfaction, as these drugs are having
various side effects. Some of the common symptoms include hypoglycaemia and
increased body weight (with sulphonylureas), gastrointestinal symptoms (metfor-
min), increased body weight and bone fractures (thiazolidinediones) and genital
infections (sodium-dependent glucose co-transporter inhibitors) (Sartipy and
Loskutoff 2003). It is therefore essential that an ideal treatment for diabetic patients
should not only be to lower glycaemia but also prevent the disease progression, with
long-lasting effects and lesser adverse effects.
Immuno-Metabolism as a Therapeutic Target
Earlier studies have suggested that immune system is closely associated to meta-
bolic changes. Such research studies have evolved a new field of study termed as
‘immunometabolism’ and it has contradicted to the earlier assumptions that metab-
olism and immunity have varied and distinct functions. It is very well-known that
metabolism regulates the transformation and disposal of nutrients, whereas the
immune system mediates host defence. Both these body functions aim at restoring
homeostasis in response to stress. The traditional and common factors that invoke
stress on immune system are chemical, mechanical and pathogenic insults. With
increasing consumption and intake of food, overnutrition has also become a stress
factor on the body. Such factors have led to draw the similarity between
microorganism-triggered stress response and the response due to metabolic stress.
Of lately, some clinical investigations have also reported existence of close interac-
tion between immunity and nutrient metabolism. Immune system’s chronic hyper-
activation by metabolic stress sometimes may be deleterious, possibly due to genetic
predispositions. It may lead to the development of one or more chronic inflamma-
tory diseases in which type 2 diabetes may fall into this category.
Anti-inflammatory Drugs with Unclear Mechanisms
Anti-inflammatory drugs used for type 2 diabetic patients have unclear mechanisms
of action. Diacerein, one of such promising drugs frequently used in the inflamma-
tory joint disease treatment, is known to decrease levels of IL-1β and TNF. In
patients with type 2 diabetes, 2-month treatment using diacerein had improved insu-
lin secretion and decreased placebo-corrected glycated haemoglobin by 1.6%
(Freigang et al. 2013). AC-2, another compound with anti-inflammatory effect, can
modulate IL-1β. However, its mechanism is unknown. Further, few clinical studies
have also reported lowering of glycaemic level in type 2 diabetic patients who were
treated with these drugs. It is possible that targeting different molecules may
improve various parameters associated with type 2 diabetes mellitus. Numerous
biologically active compounds have been identified or are being evaluated to be
used as putative drugs. One potential therapeutic approach which can be considered
includes inhibition of inflammasome that leads to prevention of the maturation of
IL-1β. This mechanism has been supported by clinically evaluated data that shows
IL-1β antagonism effectiveness and blockage of IL-1 receptor by anakinra.
Furthermore, anakinra also blocks both IL-1α and IL-1β. Of note, some reports have
suggested that IL-1α have a major role in development of atherosclerosis. As a
result, a human monoclonal antibody, MABp1 specific for IL-1α, has been trailed
out in diabetes patients (Ramos-Zavala et al. 2011). This approach is seen to improve
glycaemia and insulin production.
The recruitment of macrophages to adipose tissue and pancreatic islets can lead
to development of insulin resistance and impaired insulin secretion. MCP1, a
renowned chemokine, has a pivotal role in macrophage attraction and is able to
promote their infiltration into tissues of type 2 diabetic patients. Treatment orally
with available antagonist of the MCP1 receptor, CCX140-B (a C-C chemokine
receptor 2 (CCR2)), decreases placebo-corrected glycated haemoglobin by 0.14%
(Sartipy and Loskutoff 2003; Kanda et al. 2006). This effect is comparatively higher
in patients who were treated with a comparable standard pioglitazone. It is consid-
ered promising clinically after considering its short treatment. IL-6 is a cytokine
which releases into the blood circulation and also in abundance during inflamma-
tion. IL-6 stimulated glucose metabolism. However, these effects are dependent on
various tissue types. Under varied conditions, IL-6 can increase or decrease both
insulin resistance and enhance glucagon-like peptide 1 (GLP1)-mediated insulin
secretion. IL-6, a regulator of glucose disposal, is known to stimulate inflammation
and could precipitate insulin resistance in obesity and inflammation conditions.
(Hanefeld et al. 2012).
Anti-inflammatory Activity of Mangrove Plants
Mangrove forests comprised of a unique category of plants found in the intertidal

regions of the tropical and subtropical parts of the globe. The mangrove forest
accounts around 167,000 to 181,000 square kilometer area across the world and
majority of the mangrove forests is confined to Southeast Asia and Australia cover-
ing a span of 43% of the worldwide mangrove area (Spalding et al. 1997). The
mangrove plants are taxonomically grouped into 70 plant species and 27 genera.
However, mangrove forests also support the growth of non-mangrove plant species
as well. About 334 different plant species of 245 genera have been reported so far
from the Sundarbans mangrove forest, one of the largest mangrove forest areas of
the world (Alongi 2002). The mangrove forest flora is unique and different from
others. It is because of their unique habitat that extends along the intertidal region of
the sea. Unlike other terrestrial plants, the mangrove plants can withstand high salt
concentration and remain submerged in water for a longer time maintaining an effi-
cient nutrient retention. Although mangrove forests are limited in distribution across
the globe, still people inhabiting near mangrove forests area are heavily depended
for their livelihood including their healthcare. Along with the different therapeutic
potential of mangrove plants, the anti-inflammatory properties of mangrove plants
have also been reported in few mangrove species. Table 26.1 represents different
studies carried out to report the anti-inflammatory potential of mangrove plants.
The exact mechanism behind their anti-inflammatory activity in these mangrove
plants has not been elucidated yet. Only few reports are available which have high-
lighted anti-inflammatory mechanism of these mangrove extracts. Exploring meth-
ods to evaluate antinociceptive, anti-inflammatory and antipyretic activity of
mangrove plants could help to build and understand possible mechanisms of actions.
It is well understood that sensation of pain can be initiated either from peripheral
nervous system or else the central nervous system (CNS). The non-steroidal anti-
inflammatory drugs (NSAIDs) can block pathways responsible for pain resulting in
inhibition of peripherally mediated pain. However, opioid analgesics that inhibit
opioid receptors are proven to be useful in management of centrally acting pain.
Intraperitoneal administration of acetic acid or formalin can cause release of inflam-
matory mediators that may cause pain through the prostacyclin (PGI2). The other
tests like hot plate test, the tail flick test and the Randall-Selitto nociceptive test are
based on the principle of nociception through activating central mechanism.
Similarly, one of the popular anti-inflammatory models is rat paw oedema model in
which inflammation can be induced by carrageenan, formalin, kaolin, cotton pellet
granuloma and granuloma, pouch, etc. The inflammation in rat paw was stimulated
by administering inflammatory mediators like histamine or eicosanoids like
5-hydroxytryptamine and prostaglandin E-2. Other anti-inflammatory models being
used for assessment of inflammation include nitric oxide, TNF-α and IL-6 induction
(Shilpi et al. 2012).
Future Developments
The existing data preview supports the fact that inflammation is responsible for
pathogenesis of type 2 diabetes. Many anti-inflammatory drugs and those derived
from mangroves can improve glycaemic conditions without inducing hypoglycae-
mia. Treatments that could address inflammation could also be applied to prevent
insulin resistance. Type 2 diabetes can be manifested in various organs of the body
such as the brain, liver, etc., where there are also signs of inflammation. Therefore,
treatment should be beneficial to the organs and help in improvising multiple inter-
connected metabolic pathways. This correlation is also prominent in animal models,
where both genetic and pharmacological interventions could generate results which
are consistent and are in parallel with the observations in humans.
However, several questions remain unanswered. The optimal treatment dose and
its duration need to be carefully considered. Future genetic and biomarker studies
could be used to screen patient profiles with type 2 diabetes and identify those
having specific anti-inflammatory effect. Inflammation is now more evident and
considered as an important pathogenic pathway that helps in development of dia-
Table 26.1 Anti-inflammatory activity in mangrove plants

Sl. Mangrove Observed
no. plant Family Plant part tested activity Study method
1 Acanthus Acanthaceae Aqueous Antinociceptive Acetic acid induced in
hirsutus Aq extract of mice
Boiss MeOH fraction
of leaf extract
2 Acanthus Acanthaceae Aqueous Anti- Carrageenan-induced
ilicifolius Aq extract of inflammatoryrat paw oedema, COX
Linn. MeOH fraction (1 and 2) and 5-LOX
of leaf extract activity
3 Aegiceras Myrsinaceae n-Hexane, Antinociceptive, Acetic acid induced,
corniculatum EtOAc and anti- formalin-induced paw
(Linn.) MeOH extracts inflammatory licking and hot plate
Blanco. of stem test in mice
4 Aegiceras Myrsinaceae MeOH extract Anti- Rat paw oedema and
corniculatum of stem inflammatory peritonitis models
(Linn.) Human platelets and
Blanco. rat neutrophils were
stimulated with Ca2+
ionophore A23187
5 Avicennia Avicenniaceae MeOH extract Anti- Freund’s adjuvant-
officinalis of leaves inflammatory induced arthritis,
Linn. carrageenan and
formalin-induced rat
paw oedema
6 Barringtonia Lecythidaceae 98% n-Hexane, Anti- Inhibition of nitric
racemosa 98% CHCl3 inflammatory oxide formation in
Linn. and 95% EtOH RAW 264.7 cells by
extracts of leaf Griess assay amount
of lipid peroxidation
by ferric thiocyanate
method
7 Barringtonia Lecythidaceae Aqueous bark Antinociceptive Tail flick, hot plate
racemosa extract and formalin tests in
Linn. rat
8 Caesalpinia Leguminosae CH2Cl2 and Anti- Inhibition of
mimosoides acetone inflammatory lipopolysaccharide
Lamk. extracts, pure (LPS)-induced nitric
compounds oxide (NO)
production in RAW
264.7 cell line
9 Ceriops Rhizophoraceae EtOH extract of Antinociceptive Acetic acid induced in
decandra leaf and mice
(Griff.) pneumatophore
W. Theob.
(continued)
10 Calophyllum Clusiaceae EtOH extract of Anti- Carrageenan- and
inophyllum nut kernel inflammatory formalin-induced rat
Linn Pure paw oedemas, cotton
compounds pellet implantation,
tested cotton pellet
granuloma and
granuloma pouch
techniques, in normal
and adrenalectomized
rats
11 Excoecaria Euphorbiaceae Pure Anti- Suppression of the
agallocha compounds inflammatory expression of NF-κB
Linn tested and AP-1-targeted
genes including
TNF-alpha and IL-6
induced by
lipopolysaccharide
(LPS) in mouse
macrophages RAW
264.7 cell
12 Nypa Arecaceae MeOH extract Antinociceptive Acetic acid induced in
fruticans of leaf and stem mice
Wurmb.
13 Pongamia Fabaceae 70% EtOH Antinociceptive, Hot plate and tail
pinnata (L.) extract of leaf antipyretic, flick, acetic acid
Pierre PE, CHCl3, anti- writhing and
acetone and inflammatory Randall-Selitto
EtOH extracts nociceptive tests in
of seed mice and brewer’s
yeast-induced pyrexia
in rats.
Carrageenin,
histamine,
5-hydroxytryptamine
and prostaglandin
E-2-induced hind paw
oedema, kaolin-
carrageenan and
formaldehyde-induced
hind paw oedema,
cotton pellet
granuloma models of
inflammation
Bradykinin and
PGE-1-induced
inflammation
(continued)
14 Tamarix Tamaricaceae 80% MeOH Antinociceptive, Acetic acid induced in
indica Willd. extract of root anti- mice, using
inflammatory carrageenan-induced
rat paw oedema
15 Derris Fabaceae CHCl3 extracts Anti- Carrageenan-induced
scandens of leaf and root inflammatory paw oedema in rats;
(Roxb.) and pure eicosanoid inhibition
Benth. compounds;
aqueous extract
of stem and
pure
compounds
16 Ipomoea Convolvulaceae EtOH extract of Antinociceptive Acetic acid induced
imperati whole plant and hot plate test in
(Vahl) MeOH-water mice
Griseb. extract of leaf
17 Ipomoea Convolvulaceae MeOH extract Anti- Mouse ear oedema
imperati and two inflammatory, induced by croton oil,
(Vahl) fractions of antinociceptive arachidonic acid,
Griseb. aerial part cotton pellet-induced
Pure granulomas, inhibition
compounds of phospholipase A(2)
purified from Apis
mellifera bee venom
Acetic acid induced
and formalin test in
mice, carrageenan-
induced paw oedema
and ear oedema
induced in rats by
arachidonic acid or
ethyl
phenylpropiolate,
inhibition of
prostaglandin
synthesis in vitro
18 Heritiera Sterculiaceae Pure Anti- Nitric oxide (NO)
littoralis compounds inflammatory inhibitory effects
Aiton using RAW 264.7
macrophage cells
betic complications and other cardiovascular diseases. Finally, some anti-inflamma-

tory treatments from mangroves promise to be more effective for improvising
insulin secretion. Other medicaments (such as anti-TNF agents) may primarily act
on insulin-sensitive tissues and show up different pathways involved in the immune
response during progression of diabetes. Thus, by combining a variety of anti-
inflammatory drugs, effective therapeutic strategies could be developed for treating
inflammation in patients with type 2 diabetes.
References
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Conservation, 29(3), 331–349.
Bonner-Weir, S. (2000). Islet growth and development in the adult. Journal of Molecular
Endocrinology, 24(3), 297–302.
Butcher, M. J., Hallinger, D., Garcia, E., Machida, Y., Chakrabarti, S., Nadler, J., Galkina, E. V.,
& Imai, Y. (2014). Association of proinflammatory cytokines and islet resident leucocytes with
islet dysfunction in type 2 diabetes. Diabetologia, 57(3), 491–501.
Cerasi, E. (1995). Insulin deficiency and insulin resistance in the pathogenesis of NIDDM: Is a
divorce possible? Diabetologia, 38(8), 992–997.
Donath, M. Y., Størling, J., Maedler, K., & Mandrup-Poulsen, T. (2003). Inflammatory media-
tors and islet β-cell failure: A link between type 1 and type 2 diabetes. Journal of Molecular
Medicine, 81(8), 455–470.
Ehses, J. A., Ellingsgaard, H., Böni-Schnetzler, M., & Donath, M. Y. (2009). Pancreatic islet
inflammation in type 2 diabetes: From α and β cell compensation to dysfunction. Archives of
Physiology and Biochemistry, 115(4), 240–247.
Freigang, S., Ampenberger, F., Weiss, A., Kanneganti, T. D., Iwakura, Y., Hersberger, M., & Kopf,
M. (2013). Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent
IL-1α and sterile vascular inflammation in atherosclerosis. Nature Immunology, 14(10), 1045.
Hanefeld, M., Schell, E., Gouni-Berthold, I., Melichar, M., Vesela, I., Johnson, D., Miao, S.,
Sullivan, T. J., Jaen, J. C., Schall, T. J., & Bekker, P. (2012). Orally-administered chemokine
receptor CCR2 antagonist CCX140-B in type 2 diabetes: A pilot double-blind, randomized
clinical trial. Journal of Diabetes and Metabolism, 3(9), 1–8.
Hotamisligil, G. S., & Erbay, E. (2008). Nutrient sensing and inflammation in metabolic diseases.
Nature Reviews Immunology, 8(12), 923.
Kahn, S. E., Hull, R. L., & Utzschneider, K. M. (2006). Mechanisms linking obesity to insulin
resistance and type 2 diabetes. Nature, 444(7121), 840.
Kanda, H., Tateya, S., Tamori, Y., Kotani, K., Hiasa, K. I., Kitazawa, R., Kitazawa, S., Miyachi, H.,
Maeda, S., Egashira, K., & Kasuga, M. (2006). MCP-1 contributes to macrophage infiltration
into adipose tissue, insulin resistance, and hepatic steatosis in obesity. The Journal of Clinical
Investigation, 116(6), 1494–1505.
Koenen, T. B., Stienstra, R., Van Tits, L. J., De Graaf, J., Stalenhoef, A. F., Joosten, L. A., Tack,
C. J., & Netea, M. G. (2011). Hyperglycemia activates caspase-1 and TXNIP-mediated IL-1β
transcription in human adipose tissue. Diabetes, 60(2), 517–524.
Ramos-Zavala, M. G., González-Ortiz, M., Martínez-Abundis, E., Robles-Cervantes, J. A.,
González-López, R., & Santiago-Hernández, N. J. (2011). Effect of diacerein on insulin secre-
tion and metabolic control in drug-naive patients with type 2 diabetes: A randomized clinical
trial. Diabetes Care, 34(7), 1591–1594.
Richardson, S. J., Willcox, A., Bone, A. J., Foulis, A. K., & Morgan, N. G. (2009). Islet-associated
macrophages in type 2 diabetes. Diabetologia, 52(8), 1686–1688.
Sartipy, P., & Loskutoff, D. J. (2003). Monocyte chemoattractant protein 1 in obesity and insulin
resistance. Proceedings of the National Academy of Sciences, 100(12), 7265–7270.
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S. D. (2012). Antinociceptive, anti-inflammatory, and antipyretic activity of mangrove plants:
A mini review. Advances in Pharmacological Sciences, 2012, 1.
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International Society for Mangrove Ecosystems.
Wen, H., Gris, D., Lei, Y., Jha, S., Zhang, L., Huang, M. T. H., Brickey, W. J., & Ting, J. P. (2011).
Fatty acid–induced NLRP3-ASC inflammasome activation interferes with insulin signaling.
Nature Immunology, 12(5), 408.
Part III
Clinical Trials and IPR in Pharmaceuticals
Chapter 27
Tissue Engineering and Regenerative
Medicines: An Interdisciplinary
Understanding
Benu George, Nidhi Lal, Jeyaram R. Damodaran, and T. V. Suchithra
Introduction
The advanced method of changing the integrity of a cell with the capacity to regen-
erate induced by growth factors and supported by biological or biocompatible syn-
thetic polymeric network as bioimplants altogether account as tissue engineering
(Soler-Botija et al. 2012). It is an interdisciplinary area which draws from various
disciplines of life sciences as well as engineering to reconstruct, maintain, or alter
tissue function or generate an entire human organ for transplantation (Langer and
Vacanti 1999). This branch of engineering employs matrices as a scaffold which
serves as a substrate for the cell to adhere to and grow. Later, growth factors pro-
mote growth and proliferation in a controlled manner. The scaffold also acts as an
architectural support structure. Cell interaction with neighboring cells are achieved
with appropriate cellular signals and growth factors. As a promising tool for the near
future, tissue engineering offers a broad scope for tailored cells supporting adapt-
able immune response, vascularization, and automated modelling by a computer-
programmed system (Howard et al. 2008).
Tissue engineering and regenerative medicine aim to restore the potential of tis-
sues that have been damaged with a function of a regenerative characteristic by
replaceable native tissue (Atala 2008). These processes have achieved much success
in treatment with the replacement of healthy tissue/organs with minimal transplan-
tation problems, such as donor shortage, immune-rejection, and transplanted tissue
death (Lo et al. 2014). Strategically, a combination of scaffolds, stem cells, and
growth factors function together to revive the nonfunctional tissue and simulate the
native function with the transplanted tissue (Gao and Cui 2016). The sources of cell
range from stem cells, fibroblasts, chondrocytes, and keratinocytes of the same
B. George · N. Lal · J. R. Damodaran · T. V. Suchithra (*)

School of Biotechnology, National Institute of Technology Calicut, NITC Campus, REC,
Calicut, India
e-mail: drsuchithratv@nitc.ac.in

https://doi.org/10.1007/978-981-15-2195-9_27
410 B. George et al.
patient (autologous) or another individual (allogeneic). Besides, strategies for utiliz-

ing xenogenic cells originating from animals are also adopted in regenerative medi-
cine and tissue engineering (Kraeutler et al. 2018). Investigators have a
well-established basic concept of bottom-up and top-down methods (Fig. 27.1) to
develop tissues that can be altered as tissue equivalents. In older methods, various
immunosuppressants are used to neutralize an allogeneic cell’s immune response,
which depends on age, sex, and region to be transplanted. Regenerative medicine
facilitates the body’s own ability to heal (Guex et al. 2012).
Incorporation of synthetic or natural biological molecules, like growth factors,
has critical side effects. Reports suggest that rejection of non-human-origin stimu-
lants has adverse effects on cell proliferation. Subsequently, such purified molecules
are expensive; their half-life is too short, making them a costly option (Cai et al.
2017). Developing countries opt traditional practices that use medicinal plants and
herbs formulations for primary-level health care. But in the last 5 years there has
been an increase in their use for promoting good health as well as for the treatment
of various diseases in developed countries too. There is an exponential increase in
recent years in the use of medicinal plants and their health promotion in developed
countries, like the United Kingdom, Germany, and France (Banjari et al. 2017).
Various studies on the effect of plant-sourced extracts (Table 27.1) have been under-
taken to examine their effect on the growth and differentiation of stem cells. A
research revealed an increase in upregulation of collagen type II and the viability of
chondrocytes by resveratrol treatment. Thus, experiments with the usage of resvera-
trol in 3D bioprinting of cartilage constructs improved chondrocyte viability. Usage
of Pleurostylia capensis Turcz (Loes) ethanolic extracts has promisingly shown
antimicrobial, antioxidant, and anti-inflammatory activities too (Maepa et al. 2016).
Fig. 27.1 Traditional approaches to tissue engineering

27 Tissue Engineering and Regenerative Medicines: An Interdisciplinary Understanding 411
Table 27.1 Potential plant sources studied to augment tissue engineering

Source Function Refs.
Aconiti Lateralis Proliferation of mesenchymal stem cells (MSCs) Kim et al.
Preparata Radix (2013a)
Angelica and Chuan xiong Offers an angiogenic effect Meng et al.
Effective for myocardial infarction and (2008)
peripheral ischemia treatment
Curcumin longa Promotes adipose-derived endothelial Widowati et al.
differentiation of MSCs (2014)
Anti-inflammatory properties
Cyanococcus and catechin Proliferation of bone marrow stem cells Bickford et al.
from Malus domestica (2006)
Dipsaci Radix Enhances bone marrow–derived osteoblastic Liu et al. (2012)
differentiation MSCs
Herba epimedii Enhances bone marrow–derived osteogenic Peng-Zhang
differentiation of MSCs et al. (2009)
Hypericum perforatum Enhances keratinocytes differentiation Abakuks and
Deters (2012)
Mucuna gigantea Encourages bone marrow–derived MSCs Bramanti et al.
proliferation (2013)
Increases neural markers like nestin and β-III
tubulin expression
Supports formation of nerve in stem cell therapy
Olea europaea Promotes tubular endothelial differentiation of Calabriso et al.
MSCs an important component for formation of (2016)
blood vessel.
Pleurostylia capensis Antimicrobial, antioxidant, and Razwinani et al.
Turcz anti-inflammatory (2014)
Radix angelica sinesis Boosts cell differentiation Sgarbossa et al.
Decrement in neurotoxic formation of β-amyloid (2015)
peptide aggregation
Rhizoma drynariae Enhances bone marrow–derived osteogenic Peng-Zhang
differentiation of MSCs et al. (2009)
Salvia miltiorrhiza Neurogenic differentiation elicitation of Kim et al.
Wharton’s jelly-derived of MSCs (2013b)
Upregulates nestin, a glial fibrillary acidic
protein biomarkers
Similarly, Aconiti Lateralis Preparata Radix (ALR) enhanced the proliferation of

mouse bone marrow–derived mesenchymal stem cell (Kim et al. 2013a). Extracts
from medicinal plants are inexpensive and easily obtainable and have been used
since ancient times. A few drawbacks that queue along its usage are: variability,
purification, toxicity, complexity, lack of knowledge about the mode of action (Che
et al. 2016), and solvents used in extracts that can cause undesired effects in trans-
plantations and therapy (Amri et al. 2017). Use of medicinal plant extracts is usually
involves a combination of phytochemicals from various plant sources, which then
according to the pathological condition provide a synergistic effect.
Scaffold material or biomimic of extracellular matrix stimulates an architecture

and promotes an environment for the cell to grow and function as a tissue transplant
(Guan et al. 2017). An ideal tissue, engineered for transplantation, depends on cell
source, biomaterial, and the biomolecules that promote growth and differentiation.
At times organs exhibit some or no regeneration capability, like the cartilage and
cornea, whereas some have a very high potential for regeneration (Sadtler et al.
2016). In the last 10 years, advanced bioprinted 3D constructs and stem cell thera-
pies have been approved by the Food and Drug Administration (FDA) and the
European Medicines Agency (EMA). Appropriate growth-factor-like scaffold archi-
tectural bone morphogenetic proteins (BMP) and platelet-derived growth factors
can act as stimulants for bone and tissue regeneration (Mao and Mooney 2015).
Thus this chapter unfolds an interdisciplinary aspect of tissue engineering of novel
biomaterial, seed cells, and transplanted complications during the use of such
material.
Domains of Tissue Engineering Application
Blood Vessel
A scaffold, seed cells, and favorable environment comprise the underlying compo-
nents for building an artificial vessel (Hoerstrup et al. 2002). A structural support
and desired shape are achieved by extracellular matrix (ECM) since cells fail to
produce it for a transient period. The scaffold plays a significant role; it is com-
monly built from biodegradable polymer and collagen matrices. Alternatively, the
tubular mandrel acts as an anchoring device in vitro, which can be removed in the
later stages of implantation into the host (L’heureux et al. 1998). To mimic an
in vitro environment, bioreactors producing pulsatile flow with autologous or allo-
geneic smooth muscle cells (SMCs), fibroblasts, and endothelial cells (ECs) on
scaffolds are cultured until cells mature into vessels with optimum mechanical
properties (Hoerstrup et al. 2001).
Human umbilical veins graft, biopolymers, and endothelial cells in polymers are
better alternatives for biocompatible vascular blood vessels – these biocompatible
conduits act as 3D scaffolds for cell seeding (C. Wang et al. 2010). Mesenchymal
stem cells (MSCs), or differentiating cells, are cultured in an in vitro condition and
are subjected to a favorable environment to obtain the desired cell. MSCs are pre-
ferred over differentiated cells to achieve rapid increase in cell count (Williams and
Hare 2011). MSC-rich tissues are also preferred as harvesting sites. Eventually,
cells can be collected in a less tedious procedure with the aim of maintaining an
equivalent differential potential, e.g., adipose tissue has a denser concentration of
MSCs in the stromal-vascular region (Strem et al. 2005). These cells are subdued by
various growth factors to bring about the function of endothelial differentiation,
namely, platelet-derived growth factor and vascular endothelial growth factor (Sufen
et al. 2011). Advancements in tissue engineering helped distribute cells on heterolo-

gous decellularized scaffolds of biodegradable polymers or organic tissue procured
through in vitro tissue specimens (Jeon et al. 2006). This decreased the rate of tissue
rejection since it can mimic organ tissue and form high-quality equivalents (e.g.,
cardiovascular conduits like a native artery) (Elder et al. 2009). Three leading attri-
butes contribute for a good scaffold in tissue engineering. Firstly, the scaffold pore
size distribution should not render the mechanical property. Secondly, degradation
rate should be well balanced with ECM production speed by the seeded cells. This
shall ensure that ECM proteins are replaced in the scaffold (Bourget et al. 2012) and
finally, it should be biocompatible.
Bone
With inherited regenerative capacity, bone self-heals and there is limited tissue loss
during fracture. But in the case of trauma or tumor removal, tissue loss cannot be
retrieved by self-healing and requires surgical attention. In orthopedics, two options
for critical diaphyseal defect are limb salvage or amputation, which results in dis-
ability followed by recurring complications of infections and fibrosis. Additionally,
high-quality posttreatment and cost of care pose a significant concern (Bosse et al.
2002). Due to the intervention of tissue engineering and regenerative medicine,
reconstruction of bone with grafts and osteogenesis with Ilizarov devices has led the
treatment for bone defects to progress over the years from amputation (O’Malley
and Kates 2012). Cell sources include embryonic stem cells, the more recently
developed induced pluripotent stem cells (iPS), and postnatal and adult stem/pro-
genitor cells. One common approach is to isolate, modify, and reintroduce cells into
the host from tissue biopsies or aspirates (Netti 2014). Common cell sources for
bone regeneration include (Amini et al. 2012):
• Fresh bone marrow aspirates
• Osteoblasts and cells that have been modified genetically to express osteogenic
factors, such as rhBMP
• Purified, culture-expanded bone marrow mesenchymal stem/progenitor cells
• Umbilical cord blood cells
• Embryonic stem cells
• Adipose-derived stem/progenitor cells
These seedling cells require biomaterial scaffolding to form a 3D tissue which
will also act as a stimulant for tissue regeneration. The nominal necessities expected
from a biomaterial scaffold are as follows (Ning 2016):
• It should enable the cells to configure to a native state of appropriate size, induce
adhesion between cells, and minimize motion.
• It should bear a mechanical load.
• It should have sufficient size and distribution of porosity to achieve an active

mass transport, revascularization, osteoinduction, and osteoconduction.
• It should have biocompatible adaptability.
• Improved surface functionalization.
Bone consists of bone marrow, endosteum, periosteum, cartilage, nerves, and
blood vessels. Depending on age, sex, and diseases, it contributes to composition
and structural changes. It comprises 70% of a mineral phase, 9% of water; the
remaining 20% is organic matrix – collagen and other proteins; 1% is cellular ele-
ments, calcium phosphate – hydroxyapatite in the form of nanocrystals sized from
25 to 50 nm; and 90% of bone consist of collagen type I (Bosse et al. 2002). Scaffold
material selection (Table 27.2) is an essential factor, as it provides basement sub-
strate for the cells to adhere and promotes favorable environment to proliferate
(Ning 2016).
The advanced technology of solid freeform fabrication (SFF) functions for bone
defect by 3D modeling. It facilitates the internal monitoring of cell growth and its
architecture in the scaffold (Hutmacher and Cool 2007). Such 3D printing advance-
ments have contributed to low-temperature, high-resolution, multi-injector 3D
printing systems for rapid prototyping. Bone tissue engineering with biocompatible,
osteoinductive calcium phosphate powder and biocompatible binder for CT-guided
3D printing of personalized scaffolds are the outcome of advanced 3D printing sys-
tems (Igawa et al. 2006). Bone defects are usually treated by autologous osteoplas-
tic surgery. Alternatively, various osteoplastic materials are used for defect filling,
i.e., allogeneic and xenogeneic bone; synthetic and polymeric materials; and using
compression-distraction osteosynthesis. The location and size vary the selection of
defect filling. Instead of the conventional method of bone plastic surgery, a novel
approach of regenerative medicine for bone repair is developed. Bone regeneration
can be achieved by supervising the high doses of bone morphogenetic proteins or
recombinant human bone morphogenetic proteins to the defect zone. Regeneration
of bone tissue also uses immunomodulatory allogeneic cultured osteoprogenitor
bone marrow–derived multipotent mesenchymal stromal/stem cells (Jaiswal
et al. 1997).
Table 27.2 Common components used as scaffolds in bone tissue engineering

Components Example
Synthetic Polylactide (in the D and L chiral forms), polylactide-co-glycolide (with
biodegradable different contents of copolymers), polycaprolactone, polypropylene
polymers fumarate, polyethylene glycol, and polyethylene oxide
Natural polymers Chitosan, collagen, gelatin, elastin, silk, osteocalcin, and fibronectin
Inorganic Calcium phosphates, hydroxyapatite, (including nanohydroxyapatite),
compounds β-tricalcium phosphate, calcium carbonate, silica and boron, inorganic
compounds various metals: MgO, SrO, Au
Signaling molecules Morphogenetic proteins, platelet-derived growth factors, fibroblast growth
factors, proteins comprising a sequence arginine-glycine-aspartic acid,
insulin-like growth factors, etc.
Cartilage
Engineering cartilage appears to be simple since it consists of one cell type but a
functionally equivalent that has load-bearing potential is difficult (Huey et al. 2012).
The various stages of chondrogenesis are mesenchymal/pre-cartilaginous condensa-
tion, cavitation, interzone formation, and stabilization of articular cartilage. In the
lateral plate of the mesoderm, undifferentiated mesenchymal cells accumulate and
condense. In the initial stages, the prechondrocytic mesenchymal cells won’t prolifer-
ate, instead they increase by size and migrate away from the center to form cellular
packing (Janners and Searls 1970). The adhesive mechanism of the cell, gap junctions,
and cell-matrix interaction favor cell aggregation and cell contact (Zimmermann 1984).
Furthermore, maturation leads to alteration of matrix with the downregulation of
collagen type I (DeLise et al. 2000) and upregulation of tissue building components
like collagen type II, type IX, and type XI; Gla protein; aggrecan; and link protein
(Pitsillides and Ashhurst 2008), thus leading to pre-cartilaginous condensation.
Later, the chondrogenic differentiation of mesenchymal cells is entrapped in ECM
(Lefebvre and Bhattaram 2010). Thus they form a spherically shaped cartilage anla-
gen as they are transitioned by the ECM. In the fifth to sixth weeks of embryogen-
esis, nodes of prechondrocytes can be observed, and by the 11th to12th a
morphologically dense and minimal cartilaginous matrix architecture is visualized
(Wu et al. 2013).
Cell source for cartilage development is important. The cell source (seed) are
human articular chondrocytes, human embryonic stem cells, and mesenchymal
stem/stromal cells (MSCs) derived from three different sources: adipose tissue,
bone marrow, and embryonic stem cells (Tigli et al. 2009). To stimulate and regulate
the growth of cells in the scaffold (Table 27.3), the media is enriched with biochemi-
cal regulatory signals.
Pellet culture, or high-density micromass culture, has an inherent property of cell
adhesion and cellular aggregation, which leads to a scaffold-free approach for tissue
Table 27.3 Common types of scaffolds used for cartilage tissue engineering
Types Advantages Disadvantages Refs.
Alginate Homogeneous distribution of cell Absence of adhesion Kavalkovich
hydrogel Cell diffusion enhanced and motifs et al. (2002)
Cartilage ECM synthesis rapid Adhesion
Collagen Flexible matrix Physically weak Zhang et al.
hydrogels Enable superior cell migration and crosslinker (2012)
aggregation
Silk scaffolds Porous nature enable nutrient The signal events vary Wang et al.
diffusion mechanical property (2005)
Simplified chemical modification
and mechanical property
Silk-gelatin Quick post increase in Interference of Das et al. (2013)
scaffolds redifferentiation of articular crosslinking agent
chondrocytes
engineering (Ichinose et al. 2005). Micromass culture does mimic cellular conden-
sations during skeletogenesis. Favorably, MSCs undergo chondrogenesis under
such culture conditions (Centola et al. 2013). Still, these models fail by producing
heterogeneity in cell phenotype among the aggregates, even in the presence of cell
adhesion proteins, N-cadherin, and N-CAM (Wright et al. 1995). The micromass
forms three distinct layers: a peripheral layer of elongated cells and collagen type
I-rich matrix; a middle layer of round chondrocyte-like cells producing collagen
type I, II, and X and chondroitin sulfate; and the core region with the absence of
matrix, apoptotic, and/or dead cells (Johnstone et al. 1998).
Cartilage regeneration in a 3D system has limitations due to: (a) the existence of
dead and/or apoptotic core, (b) unoptimized culture conditions, (c) heterogenous
chemical gradient in the sample due to diffusion limitations, and (d) tight cell aggre-
gates since the initial unexpected condensation. Hanging drop culture and microflu-
idic system, microfluidic-based technological tools, and automated platforms have
overcome the above limitations. Through these techniques, the culture conditions
can be easily scaled down and chemically mimic the required environment. They
can also maintain the physical forces for a cell in tissue (Foty 2011). These recent
advancements have enabled robust chondrogenesis by (a) providing a continuous
laminar flow to the system, (b) handling cells and fluid in their native shape, and (c)
supplying sufficient quantity of nutrients and stimulating factors to the system
(Titmarsh et al. 2011). To synthesize cartilage, specific matrix scaffold-based sys-
tems are also adopted. They include hydrogels, porous scaffolds, and fibrous net-
works. Hydrogel provides sufficient water content similar to ECM, whereas porous
scaffolds and fibrous networks provide the best mechanical uniformity and mass
transport (Li et al. 2006). Traditional hydrogel materials include collagen, chitosan,
alginate, hyaluronic acid, poly(ethylene glycol), and poly(vinyl alcohol) (Lee et al.
2013), and the polymers/ biopolymers used are poly(esters), poly(vinyl alcohol),
poly(ethylene oxide), alginate, collagen, hyaluronic acid, chondroitin sulfate, and
silk protein (Coburn et al. 2012).
Heart
Injury to heart muscle, acute or chronic, has progressively led to heart failure. An
adult cell of heart, i.e., cardiac myocytes, fails to replace the injured cells even when
heart contains inherent cardiac stem cells at its disposal, and the cells cannot initiate
the repair process. Instead, over the damaged myocardium, a scar tissue evolves. It
can support the tissues by keeping it intact but cannot function to contract. Clinical
trials aim to replace scar tissue with functional scar tissue. But due to cell death,
such instigation had poor success rate (Murry et al. 2004). Tissue-engineered cells
require a favorable environment, which offers the right amount of nutrients, signal-
ing molecules, and oxygen flow.
Nevertheless, the basal requirement is a specific physical substrate that will con-
tain and organize engineered cells. Scaffolds are implanted in vivo which are seeded
by stem cells (Jawad et al. 2007). The engineered tissues are evaluated for anchor-
age, diffusion, integration, and vascularization.
Cell seed source for myocardial tissue are skeletal myoblasts, bone marrow–
derived stem cells: endothelial progenitor cells, hematopoietic stem cells, and mes-
enchymal stem cells, adipose tissue-derived stem cells, cardiac stem cells, embryonic
stem cells, iPSC, and fetal cardiomyocytes. An ideal choice depends on charterers
such as (a) in vitro scale up at faster rates, (b) quality of merging with injured tissue,
and (c) electromechanical coupling of new cardiomyocytes with the host tissue
(Gálvez-Montón et al. 2013). The scaffolding matrix (Table 27.4) is not obligated to
Table 27.4 Different matrix used in myocardial tissue engineering

Matrix Advantages Disadvantage Refs.
Artificial cardiac It had Enhanced drug delivery, The engineered tissue Eschenhagen
tissue survival and proliferation of fails to match et al. (2012)
seed cell extracellular cardiac
If natural materials used it matrix
enables high malleability Implanted cells colonize
Matrix pore size is alterable at peripheral or few
according to the requirement micrometers only
Hydrogel cell It can prevent anoikis A mechanostimulation Munoz et al.
tissue (ex vivo) Favour intercellular needed for the (2010)
communication immortality of
Seed cell can easily mature cardiomyocytes
and form extracellular matrix
Intramyocardial It facilitates the Injection pressure cause Kraehenbuehl
seeded in hydrogel cardiomyogenic differentiation a decrease in therapeutic et al. (2008)
of implanted cells effect
The system favours a control Least control on
on the elasticity, biophysical physiochemical
and biochemical growth properties and
environment degradation
Hybrid hydrogels proved to be Potentially cytotoxic to
much efficient than homo- cells
genus polymeric system
Monolayer cell The construct favours cell Failure to mimic Shimizu (2002)
constructs culture detachment without characteristics of human
any enzymatic interference heart functional tissue
Neonatal cardiomyocyte in
monolayer cell constructs have
superior intracellular
communication
Natural tissues Matrix efficiently contribute in Still under animal model Choi et al.
ECM cellular responses, clinical trials (2012)
proliferation, differentiation,
migration, and regeneration
Eliminates allogenic and
xenogenic antigens
Maintain proper mechanical
integrity and vascular structure
be the biological origin but must be biocompatible and anchor as a stable structural
support. It should facilitate streamlined migration and endurance within the isch-
emic tissue, which later should be biodegradable and nontoxic.
Muscles
Skeletal muscle loss due to injury, denervation, disease, or tumor excision that is
above the limit of regenerative capacity is known as volumetric muscle loss (VML).
It is impossible to repair by conventional methods and may cause chronic pain,
impaired function, and reduced mobility between the muscle process, and is often
rectified through surgery (Corona et al. 2015), which results in a functional and
structurally steady muscle (Mertens et al. 2014). Muscle flap surgery is usually done
to treat VML that involves an autologous tissue intact with blood. The affected
nerve is replaced in the patient from a donor (Mertens et al. 2014). This procedure
limits to a specific tissue and rarely results in a functional tissue. Healing of the
donor site is another concern with this method.
In recent years, transplantation of face, hand, larynx, and abdominal wall has
been successfully been carried out using composite tissue allografts (CTAs)
(Siemionow et al. 2009), which treats VML shortly. CTA has its negative effects.
This surgery may result in lifelong immunosuppression, tissue rejection, malignan-
cies, and infections. Thus, to overcome the constraints of surgical involvement in
VML treatment, skeletal muscle tissue engineering (SMTE) has an alternative
method, i.e. isolation of myogenic potential stem cells. The stem cells (Table 27.5)
(Pantellic and Larkin 2018) are seeded into a scaffold and allowed to proliferate and
differentiate in vitro. Later the engineered tissue is transferred to the patient’s
injured site (Machingal et al. 2011). There have been a few successful cases of the
use of biologic extracellular matrix (ECM) protein scaffolds as the decellularization
technique. Such method depends on ECM’s wound healing ability by minimizing
infection and enabling a moisture environment for the cell to promote self-repair of
tissue (Turner and Badylak 2015).
Thus the development of SMTE depends on cell source and cell potential. The
stem cell source should favor isolation, purification, and homogeneity. The isolates
should maintain myogenic potential throughout cell maturation, rapid growth in
vitro, and better in vivo performance, i.e., structurally and functionally. Satellite
cells are precursor skeletal muscle cells which yield a promising result. However,
the isolation and purification of an inadequate quantity of stem cell from a small
biopsy tissue retrieved pose a limitation in the usage of satellite cells (Rosenblatt
et al. 1995). Growth stimulants associated with skeletal muscle maturation are
dependent on multiple factors. Bone morphogenetic proteins (BMPs) as growth
stimulant facilitate bone and cartilage tissue engineering methods (Bessa et al.
2008). For a successful SMTE, cell source plays a major role. A variety of myogenic
stem cells including adipose-derived stem cells, bone marrow–derived mesenchy-
mal stem cells, perivascular stem cells, and induced pluripotent stem cells have
advantages over satellite cells for a safe and clinically attainable VML treatment.
Table 27.5 Different types of seed cells use in muscle tissue engineering
Source and
Isolation
Types of cell Function method Advantages Disadvantages
Satellite cells Allows Biopsy of Native cells of skeletal Isolation of cell
regeneration of skeletal muscle tissue are used as seed is painful and
skeletal muscle The procedure invasive
of single fiber Biopsy sample
explant culture to isolate cell
or enzymatic collection size is
digestion for small
isolation To achieve
significant yield
enzymatic
dissociation
which is a
difficult process
and produces
impure cells
Preservation
method not yet
explored
Adipose- These are the Biopsy of Yield of seed cells is Nominal
derived stem mesenchymal adipose tissue independent of donor contribution to
cells (ADSCs) stem cell (MSC) Procedure of site myofiber
which are lipoaspirate or Less chance of tissue development
multipotent and enzymatic rejection due to
have myogenic digestion for immune response since
potential isolation cells exhibit low
expression of major
histocompatibility
complex class I and do
not express major
histocompatibility
complex class II
Bone Heterogeneous Biopsy of bone Incapable to stimulate Low-yield
marrow– stem cell marrow allogeneic T cell process (>0.01%
derived population with Adult stem cell Better myogenic BM-MSCs
mesenchymal individual of bone marrow differentiation than Total BM cells)
stem cells carrying different A procedure of ADSCs
(BM-MSCs) markers enzymatic Successfully suppress
digestion for inflammatory
isolation similar cytokines promoting
to ADSC and anti-inflammatory
satellite cell cytokines, making
isolation structural and
functional recovery
easy
Contribute actively for
myogenesis and
angiogenesis
(continued)
Source and
Isolation
Types of cell Function method Advantages Disadvantages
Umbilical Capable Umbilical cord Rapid self-renewal Further research
cord–derived of self- renewal The procedure than adult MSCs necessary for
mesenchymal than adult MSCs of explant Painless process traumatic injury
stem cells and are similar to culture methods Can induce multiple treatment
(UCMSCs) embryonic stem or enzymatic lineages of mesoderm.
cells than adult digestion for They are multipotent
stem cells, and isolation progenitors with
they are capable self-renewing
of faster capability
self-renewal Suited for muscular
dystrophy therapies
Perivascular These cells are Adventitial cells Produce a myogenic Pericytes have
stem cells involved in are located at lineage not yet been
(PVSCs) growth of normal tunica adventitia Flexible with researched in
postnatal skeletal Pericytes are myogenic regards to VML
muscle by stem cell differentiation medium treatment
differentiating located at as well as co-culture
into muscle fibers capillary and with myoblasts
Invade satellite microvessel High differentiation
cell niche walls, basal potential
Myogenesis of lamina of Isolation from various
pericyte transpire various tissues convenient sources of
as a response to like skeletal tissue type biopsy
injury muscle and Co-stimulatory
adipose tissue molecules expression
Procedure of absence
explant culture
methods or
enzymatic
digestion for
isolation
Induced Culture able to Biopsy of skin Efficiently blend with Can prompt
pluripotent attain self- to isolate existing muscle fibers genetic
stem cells renewal, which epithelial cells Extracted from instability and
(iPSCs) Is an upper hand and fibroblasts myogenic progenitors abnormalities
satellite cells Laboratory these cells dystrophin Possibility of
procedure use and improve causing
upregulate contractility tumorigenic
transcription linage
factors that
induce
pluripotency
Embryonic Pluripotent stem Blastocyst inner Isolation is painless Can deviate to
stem cells cells cell mass Hurdle of ethical tumorigenic
(ESCs) challenges Isolated cells are
They are allogeneic generally
inefficient
Needs skilled
and expert
researchers
Nerves
Axons and specialized neural fibers in the nervous system form a complex network.
They are the fundamental base for the central nervous system (CNS) and partly
contribute to the peripheral nervous system (PNS). During embryonic development,
long-distance axon links are defined, a period where the source and target are in
adjacent vicinity (Varier and Kaiser 2011). Disease or injury to the nervous system
includes traumatic brain injury (TBI), stroke, spinal cord injury (SCI), peripheral
nerve injury (PNI), and neurodegenerative diseases like Alzheimer’s disease (AD)
and Parkinson’s disease (PD).
Commonly neuronal disorders relate to damage to long-distance axonal con-
nections. The CNS has no ability (Wang et al. 2005) for regrow compared to PNS
(Fitch and Silver 2008). Thus the result of loss or dysfunctional neural pathway
results in a severe or chronic condition in individuals. Recovery of a neuronal
network involves use biomaterial scaffolds, molecular signals, and cell seeds to
improve the loss of functional tissue. (Eberli and Atala 2006). A scaffold would be
a matrix of biomaterial seeded with neural cells that will enable the formation of
a 3D construct (Cullen et al. 2007). Replacing functionless tissue with scaffold-
generated tissue should also restore its neuronal activity in vivo. Tissue engineer-
ing also aims to resume the damage tissue neuroarchitecture. It helps to rectify
neuroregeneration, replaces neuron and/or axon tract to restore biological neuro-
modulation. Techniques that employ fabricated tissue engineering scaffolds –
nanofiber self-assembly, gas foaming, solvent casting and particulate leaching,
liquid-liquid phase separation, emulsification/freeze-drying, electrospinning, and
computer-aided design and manufacturing techniques – have attained a varied
level of success (Yang et al. 2005). Electrospun nanofibrous scaffold neuronal
repair substrate facilitates a safe and controlled delivery of the bioactive com-
pound. The substrate increases the contact guidance for seed cells to adhere and
regenerate in an uninterrupted way (Cao et al. 2009). Autologous grafts method to
treat neural defects has its limitations: insufficiency of nerves, donor-recipient
nerve size mismatch, formation of neuroma, and lack of functional recovery
(Bellamkonda 2006). Another method for acellular nerve grafting is by limiting
the viable cells deficiency and time lag in extracellular matrix formation and
regeneration. Matrix metalloproteinase (MMP) expression-induced regenerated
nerve grafts resulted in a potential for restoration and degraded inhibitory chon-
droitin sulfate and proteoglycans in the absence of cells (Krekoski et al. 2002).
Nerve cells have an inherent capacity to repair a gap of 6 mm (Haile et al. 2007).
Strategies have been explored to enhance the regeneration strength by growth fac-
tors cell therapy and tissue engineering. PNS damage can be repaired (Fig. 27.2)
to an extent, but CNS damage lacks the ability (Goldman 2016). The regrowth is
subdued by protein synthesis and degradation machinery in axons that favor the
process (Gumy et al. 2010).
Distinctive cells like astrocytes and oligodendrocytes of CNS fail the onset of the
regeneration process as they act as self-inhibitory molecules. Subsequently, myelin-
Larger gaps Regeneration of nerves

questionable
Injury to PNS
Forms fibrin network
Smaller gaps
across the gap
Encourage growth Formation of Establishment of

nutrients bungner bands Schwann cells
Gap repair
initiation
Fig. 27.2 Repair mechanism of nerve injury
associated inhibitors (chondroitin sulfate proteoglycans) favor glial scar formation

progressively (Schmidt and Leach 2003). Thus, CNS myelination varies to a greater
degree compared to PNS (Xiong et al. 2009). Moreover, a primary CNS injury
enlarges by secretion of free radicals via the blood-brain barrier, which begins the
formation of secondary injury that hurdles the regeneration efficiency of neural cells
(Spilker et al. 2001).
The tissue repair process is subdued by the involvement of growth factors
(Table 27.6). The growth factors helps in enhancing the regenerative property.
Similarly, bioactive components also help in the growth and differentiation of stem
cells. Scar formation is kept under control by nerve-guided channels that diffuse the
growth factors and avoid glial formation. Growth factors can reduce scar formation
if supplemented in the right quantity as nerve-steered channels distribute it to the
affected region. (Schmidt and Leach 2003).
Scaffold material for nerve tissue engineering (Table 27.7) (Subramanian et al.
2009) should be firm, flexible thin, porous, biodegradable, biocompatible, compli-
ant, neuroconductive, neuroinductive, and nontoxic (Haile et al. 2007). Surface-
erodible materials are preferred than bulk erosion. This property retains the
mechanical and structural stability of the blend in vivo (Sundback et al. 2005).
Various approaches, like the layer-by-layer (LbL) technique (He and Bellamkonda
2005) and photochemical methods employ growth factors on poly (hydroxyethyl
methacrylate) scaffolds to enhance the bioactivity (Kapur and Schoichet 2003).
This in turn modifies the biocompatibility and improves the operational challenges
of neural implants. Synthetic biodegradable polymer (e.g., cellulose, alginate,
poly(hydroxyethyl methacrylate), poly(hydroxybutyrate)) scaffold inherent hydro-
Table 27.6 Common growth factors in tissue engineering

Growth stimulant Function Refs.
Nerve growth factor (NGF) Promoting nerve regeneration Sofroniew et al.
(2001)
Lipoplexes Factors of neurotrophic repair the Whittlesey and Shea
nerve injury (2006)
Polyphosphoesters Delivery vehicle, controls acidic Xu et al. (2002)
products release
Collagen binding domain- nerve Restore the peripheral nerve Sun et al. (2009)
growth factor β function
Table 27.7 Common types of scaffolds used for nerve tissue engineering
Types of polymer Example
Natural polymers Chitosan, chitin, collagen, gelatin, and alginate
Synthetic PLGA, poly (ε- caprolactone), poly L-lactic acid, poly (glycerol sebacate),
biodegradable poly (hydroxyethyl methacrylate), and poly (hydroxybutyrate)
Conducting Polypyrrole, polyaniline
polymers
Polymer blending PLGA/polyurethane or poly (ethylene glycol), chitosan-gelatin, chitin/
chitosan, and chitosan/polyglycolic acid
phobicity, modification by ECM proteins (e.g., laminin, fibronectin, collagen), or

adhesive peptide sequences (e.g., RGD, IKVAV and YIGSR) are carried out to
induce hydrophilicity and adhesion property.
he Interdisciplinary Nature of Tissue Engineering

T
and Regenerative Medicine
It is no surprise that biology has far-reaching influence beyond the scientific and
technical domains, into arts, social sciences, and, most important of all, into our
daily lives. Biological and medical sciences have tremendously benefitted from the
amalgamation of various disciplines of science, such as engineering, nanotechnol-
ogy, chemistry, physics, and mathematics. Moreover, this close relationship has
been continuously evolving, blurring the lines that distinguish them, ultimately giv-
ing rise to entirely new branches of science, such as computational biology, nano-
biotechnology, molecular biophysics, tissue engineering, biomechanics, etc. The
terms “tissue engineering” and “regenerative medicine,” although used interchange-
ably, are not synonymous. Tissue engineering utilizes cells, biomaterials, and mol-
ecules that are necessary for tissue growth in an external environment, while
regenerative medicine comprises of these, or the use of genetically engineered cells

directly, without any 3D scaffolds or maybe even without the manipulation of devel-
opmental biochemical pathways of stem cells (Daar and Greenwood 2007).
ajor Disciplines Contributing to Tissue Engineering

M
and Regenerative Medicine
(A) Systems Biology

Systems biology is an interdisciplinary field that explains complex biological
phenomena and whole system interactions by computational and mathematical
modeling. A plethora of models exist that predict various cellular events like cell
differentiation, proliferation, adhesion, apoptosis, and migration (Mironov et al.
2004). It is almost impossible to predict system-wide effects and interactions asso-
ciated with transplanting tissues grown in vitro into the subject. Although it is indis-
pensable for the appreciation of the biological process at the molecular level, an
in vitro study can never yield a complete picture. Since all of the processes that
occur within a biological system are highly interconnected. The experimentation
involved is often costly and sometimes even impractical, due to ethical and legal
concerns as well as due to the lack of technological advancement. A systems bio-
logical approach becomes especially imperative in such a scenario.
Moreover, in silico studies can serve as a guide to the progression of therapy. The
following four attributes are central to obtaining a whole-system-level picture
(Kitano 2002):
1. System structure: A network consisting of the reaction mechanisms that result
from the various biochemical pathways, the genetic interactions to control them,
and physical manifestations that change the properties of the internal structures.
2. System dynamics: Slight variations in system response over time under various
experimental environments that can be examined through methods like dynamic
analysis, metabolic analysis, and sensitivity analysis.
3. Control method: Precise biological mechanisms that systematically control the
cellular state can be studied to manipulate the actual in vitro or in vivo process to
ensure effective therapy.
4. Design method: Biological systems can be modified or completely designed
from scratch to obtain desirable properties based on the understanding of the
cellular processes, which gives an immense advantage over traditional experi-
mental methods that rely heavily on trial and error.
The model predicted needs to be refined several times to improve its accuracy.
The knowledge derived from them can be used for the development of therapeutic
methods after this stage (Fig. 27.3).
Existing knowledge
about cellular
systems, organs and
physiological
processes
Formulation of
Wet lab experiments hypothesis
and data analysis
Preliminary wet lab

analysis (if
Predictions based on
necessary) and
the developed model
simulation
experiments
System level
Formulation of
analysis of the
theory; model
physiological
development
network
Fig. 27.3 Experimental cycle involved in the systems biology approach to regenerative medicine
(B) Material Sciences

Material science is an interdisciplinary science that deals with the study of the
properties of materials, and tries to design as well as discover new materials tailor-
made for specific usage. Its application in tissue engineering is primarily in the
fabrication of scaffolding material.
Characteristics of Scaffolding Material
For a scaffold material to be an excellent 3D scaffold it should meet following
requirements (O’Brien 2011):
1. Biodegradability: The scaffold material is intended to serve only as temporary
support for the growing tissue, after which it needs to be disposed of. Therefore,
it is designed so that as the implant grows, the scaffold is degraded naturally in
the body, usually by hydrolysis. It is essential that the scaffold not elicit severe
immune responses and that the material left after degradation is nontoxic.
2. Biocompatibility: The scaffold must allow the adherence and proliferation of
cells, while also facilitating their migration.
3. Mechanical properties: The implanted scaffold must be sturdy enough to resist

any damaged during the surgical implantation procedure. Moreover, the mechan-
ical properties must be close to that of the adjacent tissue at the site of
implantation.
4. Architecture of the scaffold: The material should have a highly porous intercon-
nected structure to allow sufficient cell migration, penetration, and nutrient sup-
ply. This is essential for the clearance of waste materials generated as a result of
the natural cellular processes.
(C) Nanotechnology
The primary goal of applying nanotechnology is to favor scaffold design. Most
commonly used scaffolds have surface features which are larger, compared to tis-
sues of biological origin. Therefore, it is hypothesized that adding nanoscale topo-
graphic features might augment tissue adhesion and growth. Changing the chemical
properties of the surface as well as alterations to the surface morphology, for exam-
ple, modification of roughness of the surface, may cause a dramatic change in the
results. It has also been observed that nanostructured implants can promote calcium
deposition and thereby improve bone cell integration (Webster et al. 1999). Some of
the approaches used for modifying the nanostructure of the scaffolding material are
given below (Engel et al. 2008):
1. Nanopattern fabrication: Two types of nanotopographies – regular controlled
patterns and unordered patterns – are being used to improve cell adhesion, rec-
ognition, and growth on the scaffolds. The consistent patterns can be obtained
using techniques such as chemical etching, polymer demixing and colloidal
lithography, whereas soft lithographic methods (using elastomeric stamps) and
radiation methods are used for unordered topographies.
2. Using nanophase materials and nanocoating: Nanophase materials are those with
grain surface structures on a nanometer scale. Such materials can be chosen for
the fabrication of scaffolds, or they can be made by depositing the grainy mate-
rial on the surface artificially.
(D) Immunology
A major challenge in using allograft cells is immune recognition and subsequent
responses elicited by the patient’s immune system, similar to those seen in organ
transplant patients as well as hematopoietic stem cell transplant patients. Engineered
autologous cells are likely to elicit immune response at a very minor scale. To
reduce immunologic complications after transplantation specific measures must be
taken to induce the dual tolerance, such as using drugs (regularly used to suppress
the host immune system in transplantation procedures), monoclonal antibodies, cell
therapies and hematopoietic chimerism (inducing tolerance by transplanting donor
HSC before the organ transplantation) (Zakrzewski et al. 2014). Some of the emerg-
ing strategies are described below (Zakrzewski et al. 2014):
1. Physical separation of donor and host cells using biomaterials, for example,
encapsulation of cells in hydrogels
2. Usage of a biomaterial that can send inward and outward signals (promoting cell
growth in transplanted tissue, by sending signals to adjacent tissues)
3. Appropriate transplant site selection
4. Inducing immune evasion by mimicking the mechanism used by tumor cells
5. Copying the peripheral tolerance mechanism
(E) Developmental Biology
Developmental biology and stem cell biology go hand in hand. They can be
thought of as the central core of regenerative medicine. Induced pluripotency or
nuclear reprogramming of differentiated somatic cells solves the ethical and legal
concerns regarding the usage of embryonic stem cells. It allows the use of a patient’s
cells to reprogram to become stem cells. Thus, it has an added advantage of being
nonimmunogenic, along with paving the way for a discovery-based approach,
regardless of the disease condition (Nelson et al. 2010).
(F) Biomechanics
Biomechanical factors profoundly affect the strength and endurance capabilities
of the regenerated tissue. For tissues that operate in high mechanical stress condi-
tions, such as cartilage, muscle, bone, intervertebral disc, ligament, and tendon, the
longevity of the implant is a major challenge. The stiffness of the matrix used has
been found to be an important factor in determining the lineage of the stem cell, for
example, soft matrices guide the differentiation of naive MSCs into neuronal cell
types, while stiffer and comparatively rigid matrices are myogenic and osteogenic,
respectively (Engel et al. 2008).
echanical and Biological Factors Influencing Cell-Scaffold

M
Interactions
There are three major constituents in tissue engineering – biomolecules, cells, and
scaffolds. Scaffolds are designed to mimic ECM in physiology conditions of tis-
sues. It aims to promote cell adhesion, proliferation, invasion, and migration. A 3D
scaffolds should have the strength and load-bearing capacity to withstand the
mechanical stress during tissue development as well as maintain the overall shape
of the construct over time. Mechanical properties of scaffolds play a crucial role to
bring out the desired actions involved in repair and regeneration of damaged tissues
and organs. Possessing a uniform topology similar to the natural ECM promotes
bioactivity (Chandrasekaran et al. 2011) Porosity, pore interconnectivity, and pore
size of scaffolds can alter the morphology and various biological events that occur
in tissue development. Porosity refers to the total void space or free space available
within a 3D structure. The microporosity of scaffolds promotes cell-cell interac-
tions, such as adhesion and direct signaling and facilitates capillary formation.
Macroporosity of scaffolds allows proper nutrient delivery and waste removal –
effective transportation of fluids through the construct as a whole. Porosity influ-

ences cell migration and vascularization. The pore size of scaffolds mediates
cell-cell adhesions and can affect cell migration. During migration, cells use neigh-
boring cells as a support to form a bridging mechanism to cross pores larger than
individual cells (Salem et al. 2002). In situations where the pores cannot be covered
by such a mechanism, they can only migrate through the joints in the scaffolds, this
can influence cell migration and migration speed (Reilly and Engler 2010). Optimum
porosity and pore size vary among cell types, the biomaterial used, and the applica-
tion of the construct. A large exposed surface area allows higher cell attachment and
anchorage. The rigidity of the scaffold impacts the formation of adhesion plaques
among cells mediated through the mechanics – sensitive receptors on the cell sur-
face (Chen et al. 2014). Degradability of the scaffold material influences the replace-
ment of the engineered tissue in the long term.
Cell-ECM adhesion is mediated through ECM proteins such as fibronectin, inte-
grin, laminin, and membrane proteins of cells. Functionalization of scaffolds with
cationized and surface-conjugated artificial membrane-binding proteins increases
cell adhesion (Burke et al. 2017). Formation of focal adhesion complex mediated by
proteins – vinculin, talin, and paxillin (Burridge and Connell 1983) – negatively
influences the migration of human gingival fibroblasts on three-dimensional nanofi-
brous gelatin scaffolds (Sachar et al. 2014). Regulatory biomolecules released by
cells attached to the scaffold and cells surrounding the construct modulate prolifera-
tion, migration, vascularization, and ECM synthesis. Activation of signaling path-
ways mediated by different tyrosine kinase receptors such as Estimated Glomerular
Filtration Rate (eGFR), FGFR (fibroblast growth factor receptors), PDGF (platelet-
derived growth factor), and serine threonine sulfate regulated pathways such as
TGF-Beta elicit various biological responses involved in development (Pritchard
2013). Scaffolds of composites combined with delivery vehicles for PDGF gene in
periodontal regeneration and controlled release of TGF-Beta for bone regeneration
have stimulatory effects on angiogenesis and proliferation, differentiation, and
matrix biosynthesis (Zhang et al. 2007).
The introduction of constructs into the body elicits host immune response against
the foreign material of the scaffold. Coating of scaffold materials with proteins or
other biomolecules reduces the chances of detection and triggering of the immune
system (Campoccia et al. 2013). Encapsulation of construct by selectively perme-
able hollow fibers and sheets (Hatziavramidis et al. 2013) and immunocloaking
using nanofilm to prevent antigen recognition by the immune system (Pareta et al.
2012) are the two commonly used methods to avoid immune responses.
Overexpression of anti-inflammatory factors in stem cells using transfection and
other techniques has increased the survival of cells in scaffolds (Holladay et al.
2011). Immunosuppression of the host positively influences tissue development.
Immunosuppression of mesenchymal stem cells by the modification of the hCTLA4
gene inhibits immune response and allows differentiation of cells in bone tissue
engineering (Dai et al. 2006).
Regenerative Medicine
Over the past years, there has been tremendous progress in tissue engineering, lead-
ing to the development of biocompatible scaffolds. Growth factor and other signal-
ing molecules stimulate the physiological condition which can replace the damaged
components of a system. The development of these scaffolds mainly focused on
compatible mechanical properties with little attention to the biological factors
involved, due to which the progress in the field of tissue engineering is now facing
certain limitations.
Regenerative medicine, on the other hand, focuses on utilizing cells, genes, and
other biological factors to promote self-healing to rejuvenate, repair, replace, or
regenerate damaged body tissues and organs. It relies solely on using the ability of
stem cells, especially embryonic stem cells, to restore and regenerate tissues dam-
aged by diseases, genetic or chromosomal disorders, or due to age. There are several
examples of organisms in nature that are capable of regenerating their damaged
body parts. In the human body, the only organ that can regenerate itself is the liver.
A healthy lobe of the human liver can proliferate and differentiate to develop a full-
sized liver.
According to the National Institutes of Health, the development of regenerative
medicine includes a blend of various interdisciplinary perspectives. It involves engi-
neering other quantitative sciences with biology and medicine to build functional
tissue and organs in vitro for implantation in vivo. It also partly contribute regenera-
tion of tissue in vivo to repair, replace, preserve, or enhance tissue or organ function
lost due to disease, injury, or aging. The field of regenerative medicine is a billion-
dollar global industry with unlimited potential that can be used to develop technolo-
gies to treat a wide array of new pervasive diseases like congestive heart failures,
Alzheimer’s disease, Parkinson’s disease, and diabetes mellitus. Progress in the
areas of stem cell biology, developmental biology, immunology, and other fields has
led to the improvement of existing techniques and the development of new therapies
(Mahla 2016).
Biological and Molecular Basis for Regenerative Medicine
Several events that occur during the development of multicellular organisms pro-
vide evidence of the ability of cells to repair or regenerate damaged tissues. One of
the most crucial evidence for tissue repair is shown by mesenchymal cells which are
formed during endothelial-mesenchymal transition of cells. Cells in tissues show
two types of organization – epithelial or mesenchymal (Kalluri and Weinberg 2009).
Epithelial cells are compactly arranged through interactions with each other and
adhere to the ECM through various proteins like E-cadherins. Mesenchymal cells
are present as individual cells and are loosely organized in a tissue held by a poorly
organized ECM.
The transition of epithelial cells to mesenchymal cells is brought about by cel-

lular mechanisms such as changes in cell-cell adhesion, cell-ECM matrix adhesion,
invasion of basal lamina, and an increase in the motility of cells. Molecular mecha-
nisms, such as secretion of several growth factors; activation of signaling pathways,
such as TGF-beta pathway, WNT Pathway; and the activation of several transcrip-
tion factors also bring about the necessary change. Epithelial mesenchymal transi-
tion (EMT) occurs during several developmental processes like implantation,
embryogenesis, and organ development, which leads to the formation of multipo-
tent mesenchymal cells that migrate, proliferate, and differentiate to generate vari-
ous tissues and organ systems (Kalluri and Weinberg 2009).
The ECM is primarily composed of collagen, proteins, glycoproteins, and pro-
teoglycans generated by different cellular components such as epithelial, fibroblast,
endothelial, and adipocyte cells (Frantz et al. 2010). In addition to the constituents
mentioned above, ECM also contains growth factors (GFs), cytokines, and inhibi-
tors that alter the cell morphology and modulate gene transcription through down-
stream signaling by binding to their receptors. The distribution of these components
and GFs vary from tissue to tissue and with the various stages of development.
Binding of the components of ECM with the receptors of cells regulates cell adhe-
sion (integrins, focal adhesion complex), proliferation – cell cycle progression
(EGF, FGF, PDGF), survival, and apoptosis (JNK Pathway). ECM-cell interactions
mediate fetal wound healing and liver regeneration (Meyer et al. 2009).
Regeneration of tissues is primarily carried out by the activation of adult stem
cells. Stem cells are a class of undifferentiated cells which can proliferate and dif-
ferentiate themselves to become specialized cells and sustain their population
through self-renewal. Activation of stemness is brought about by the activation of
transcription factors such as S0X2, OCT4, and NANOG as a result of various sig-
naling pathways elicited by growth factors such as BMPs, TGF-β, FGF, EGF, PDGF,
and IGF-1 and other small molecules (Biehl and Russell 2009).
Regenerative Therapies
Autologous or allogeneic transplantation of hematopoietic stem cells is being used

to treat or cure a range of genetic disorders such as thalassemia, sickle cell disease,
severe combined immunodeficiency, aplastic anemia, and acute and chronic myeloid
leukemia. Embryonic stem cell and induced pluripotent cell-based therapies are
currently undergoing experimental and preclinical testing for the treatment of neu-
rological disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s
disease, multiple sclerosis, and ischemic stroke (Tong et al. 2015). The use of regen-
erative therapy for recovery from trauma suffered by the central nervous system has
not yet been bridged from experimental to clinical phase. Bone marrow hematopoi-
etic progenitor/stem cells, endothelial progenitor cells, cardiac stem cells, and
embryonic stem cells are currently being studied for developments in stem cell-
based and cell-based therapy at the clinical level for coronary heart disease, chronic
ischemic heart disease, and chronic heart failure. Endothelial precursor cells have
been shown in rodent models to contribute to liver regeneration. Similarly, stem cell
therapies based on embryonic stem cells, tissue-specific adult stem cells, and mes-
enchymal stem cells are currently being proposed and tested for ocular regenera-
tion, vascular regeneration, renal and duct repair in kidney and for lung diseases
(Brody 2016).
Conclusion
It is essential to understand the concept behind tissue engineering. With a brief dis-
cussion about various theories and concepts supporting successful transplantation,
we understand the interdisciplinary nature of this field. Scaffold design, scaffold
material, selection of seed cell origin, and growth factor to stimulate healthy growth
of the cell all play a significant role in developing an engineered tissue. The future
is promising, yet the subject, “Tissue engineering and regenerative medicines,”
remains a little hazy because of complexity in the human body.
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Chapter 28
IPR: An Overview
Amritesh C. Shukla
Introduction
“If nature has made any one thing less susceptible than all others of exclusive property, it is
the action of the thinking power called an idea, which an individual may exclusively pos-
sess as long as he keeps it to himself…”
–Thomas Jefferson
In other words, patents can be defined as a lawful monopoly that is contracted to

the owner of the new invention that is capable of industrial utilization for a specific
phase of time. In other words, if an invention is novel, useful, and nonobvious, pat-
ent can be applied and granted after processing. The patents can be made of the
several things which have property, or mainly the intellectual property. Presently,
separate laws in the constitution of India as well as other countries of world com-
monly refer to it as “intellectual property rights.”
Hence, IP is a combination of both science and technology which mandates the
legally competent scientists or scientifically qualified lawyers to undertake the task
of developing and improving the intellectual property rights to suit the needs of the
modern digital and biotechnological world.
Patent: Some Important Milestones
1421: The first patent given to Filippo Brunelleschi by the United States of
Florence.
1474: The first notification related to patents was passed through a law of
Venice.
1520–98: Lord Barle, a minister of Queen Elizabeth (1533–1603), provided so
many patent to lots of foreign inventers, because they feel proud.
Later, they advertise their invention and/or products in England.
A. C. Shukla (*)

https://doi.org/10.1007/978-981-15-2195-9_28
440 A. C. Shukla
1856: In India, the main concept of IPR began with the act of conservation of
invention. This act was based on the law of patent, 1852, of Great Britain;
this was the time when law of patent was started in India.
1970: Indian Patents Act, 1970 (Amended in 1999, 2002, and 2005).
1995: India got the membership from the World Trade Organization (WTO).
2003: Patents Rules, 2003 (Amended in 2005 and 2006).
As per the WIPO rules and regulations (1979), at a behavioral point of view, the
“invention is a notion” which solves vital problems in the area of technology.
The word “invention” evolved from Latin word “invenire,” which means to
acquire something by chance.
Types of IPR
As per the Indian context, the intellectual property rights can be categorized as
follows:
Types of IPR
Corporeal Incorporeal
Movable Immovable Lease Securities Intellectual Patents

Geographical
indications
Trademark
Trade secret
Copy right
Neighboring rights
Integrated circuits
Industrial design
Why Make a Patent?
There are many reasons for inventing/filing a patent. It may be made to satisfy one’s
self, to resolve the problems in daily life, to become popular among colleagues,
desiring to be a part of the challenging profession, or to earn more and more capital.
It may be achieved alone or with a group of inventors (teamwork), and it may be in
the form of products or processes.
28 IPR: An Overview 441
Probabilities of Patenting
In India, depending upon the inventor’s inventions, the probability of getting a pat-
ent is either in the form of a product called as “product patent” or in any processes
called as “process patent.”
a. Product patent: it contains human body parts, human body, genetic material,
and unicellular and multi cellular organisms.
b. Process patent: it contains essentially biological and nonbiological processes,
genetical therapies, cloning, and medical treatment methods.
Patenting Criteria
Any invention which comes under the criteria of new, patentable matter, having util-
ity and uniqueness, or nonobvious can be patented.
What Does a Patent Cover?
It covers the composition of matter, methods of manufacture, methods of use, arti-

cles of manufacture, apparatus, machines, etc. On the other hand, computer soft-
ware, pharmaceuticals, processes, and microorganisms are not patentable, according
to Sections 3 and 4 of the Indian Patent Act.
When to File a Patent?
When any inventor searches and finds a new thing, usually he or she aims get it
patented, but there are certain limitations that must be considered before filing for
an application for a patent. It includes the following: the invention should be away
from public disclosure (written or oral), it should not be in public use, and it should
not have any offer of sale.
Further, if any one of these has publically been disclosed, then chances of patent
rights will be lost.
Earlier, a separate patent application has to be made for each country, but at pres-
ent, under PCT (Patent Cooperation Treaty) system, a single application can now be
made. It is an agreement for rationalizations and support with regard to the filing,
probing, and examining of patent application and the propagation of the technologi-
cal information it contains. The PCT is a system for filing a patent, not for granting
system of patent.
442 A. C. Shukla
Scope and Relevance of the Patent
The patent should be commercially viable, user-friendly, and beneficial to society.

The patent is also essential to protect our microbial biodiversity. A patent is directed
to the public purposes of nurturing technological advancement, research and devel-
opment investment, innovation, natural strength, formation of assets, and entrepre-
neurial and global competitiveness.
Fee for Getting a Patent in India (Approx.)
Patent office Fee (Rs) 1$ = ~ 60 INR

(e-filling)
Natural Other than natural person
person/ Small Excluding
S.N. Particular startup entity small entity Remarks
1 Patent application 1600 4000 8000 Compulsory
2 Fee for early publication 2500 6250 12,500 Non-
compulsory
3 Patent application, request 4000 10,000 20,000 Compulsory
for examination
4 Fee for each additional sheet, 160 per 400 per 800 per page Compulsory
above 30 sheets page page
5 Fee for each additional 320 per 800 per 1600 per Compulsory
claim, in excess of ten claims claim claim claim
Indian Patent Law: Characteristic Features

The Indian Patent Law includes some important features. It provides both product
and process patents, both for a tenure of 20 years; on request, an examination facil-
ity of the application of patent; creation of both pre-grant and post-grant opponent;
fast track system for clearance of appeals; provisions for safety of biodiversity and
conventional knowledge; application publication after 18 months with facility for
advance publication; obligatory license to ensure accessibility of drugs at realistic
prices; and condition to deal with civic health emergency. Besides this, the patent
can be revoked in public interest and also on security point of view.
However, with effect from 1.1.1995; Mail-Box for pharmaceutical and agro-
chemicals products have been made (TRIPS agreement requires for those countries,
who are not providing product patents, especially on chemical and pharmaceuticals
inventions have to put in a mechanism for accepting product patent applications
w.e.f. January 1, 1995. Such applications will only be entertained for granting pat-
ents, after proper amendments in their national patent law policy. This method of
a ccepting product patent application is known as the “mail box” system). Besides
this, provisions of exclusive marketing rights have also been made.
Further, w.e.f. 1.1.2000; Patent term increased up to 20 years; inclusion of inven-
tive step under the definition of the invention; Reversal of burden of proof – on the
infringer; provision of a compulsory license for food, drugs and chemicals have
been removed; as well as the right of patentee/ importation, also included.
Furthermore, w.e.f. 1.1.2005: Product patents for food, chemical, and pharma-
ceutical products have also been granted.
Hierarchy of Patent Offices in India
Head Quarter
KOLKATA
Controller general of Patents and designs
Nizam Palace
2nd MSO Building
5 – 7th Floors,
234/4 Acharya Jagadish Chandra Bose Rd
Kolkata – 700 020, WB
Ph- 033-22474401, 033- 22473851
Email: patentin@vsnl.com
Four Branch Offices
NEW DELHI MUMBAI CHENNAI NAGPUR
Assistant Controller of Assistant Controller of Assistant Controller of Assistant

Patents & Design Patents and design Patents and design Controller of
(ACP&D) (ACP&D) (ACP&D) Patents and design
W-5 Patel Nagar (W) Todi Estate Rajaji Bhawan (ACP&D)
N. Delhi- 110 008 3rd Floor, Sun Mill 3rd Floor, C wing 4th Floor
Ph- 011-25861255 Compound Besant Nagar C- Block
011- 25861258 Mumbai- 400013 Chennai- 600 090 CGO Complex
Email: Ph- 022-24925092 Ph-044-24901495 Seminar Hills
delhipatent@rediffmail.com 022- 24920622 044-24901492 Nagpur- 400006
Email: Email: Maharashtra
patmum@vsnl.net chpatent@tn.nic.in
444 A. C. Shukla
Patent Offices and Their Territorial Jurisdiction
(i) Patent branch office at N. Delhi: It includes Haryana, Himachal Pradesh, J&K,
Punjab, Rajasthan, U.P., and Delhi states.
(ii) Patent branch office at Mumbai: It includes Gujarat, Madhya-Pradesh, Goa,
Maharashtra, Daman and Diu, Dadar, and Nagar Haveli.
(iii) Patent branch office at Chennai: It includes Andhra-Pradesh, Kerala, Tamil-
nadu, Puducherry, and Lakshadweep.
(iv) Patent branch office at Kolkata (Head Office): It includes the rest India.
Stages from “Filing” to “Grant” of Patent
The Indian Patent Offices have three different types of provisions for filing patent
applications.
(i). Regular Application for Granting the Patent
(ii). Convention Application for Granting the Patent
India is a member of Paris Convention, since 1998, and so far, the Convention
has 177 member countries (till February 2017). The application filed in the Indian
Patent Office, claiming a precedence date based on the similar/same application
filed in one or more convention countries, is called as convention application. To get
convention status, an applicant has to file the application within a year, in the Indian
Patent Office, from the date of first filing of the similar application in the convention
countries.
(iii). Patent Cooperation Treaty (PCT) National Phase Application for Grant
of Patent
Since India is a PCT member country, therefore, a national phase application
may also be filed in this country. The World Intellectual Property Organization
(WIPO) has nominated the Indian Patent Office as one of the many International
Searching Authorities (ISA) and International Preliminary Examining Authorities
(IPEA), w.e.f. December 2007.
As per the Indian Patents Act and the rules, the following are the important steps
from “filing” to “grant” of a patent:
(i). To get the patent, file an application form
• The application form should be filed in one of the patent offices based on territo-
rial jurisdiction of the place of office or residence of the applicant /agent.
• Pay the requisite fee.

(ii). Formalities/screening of the papers

• An examiner checks the desired documents before accepting the application and
the fee, at the earliest.
• Allotment of application number and the fee receipt should be posted on the
same day.
• In case of application and fee receipt, received by post, the application number
and the fee receipt slip should be sent by post within 2–3 days.
(iii). Publications
• Application is kept confidentially for a period of 18 months from the date of
filing.
• The application is published in the official journal, in the 19th month; this journal
is made available on the website weekly.
• The candidate has a choice to get his application published before 18 months
also.
• In such case, the application is published within 01 month from the request.
(iv). Appeal for examination

• Application is examined, on demand.
• Examination request can be made either by the applicant or by a third party.
• From the date of filing, 48 months duration, is available with the party for mak-
ing examination request.
(v). Examinations
• From the date of examination request, an application is sent to an examiner
within 1 month.
• The examiner undertakes examination.
• Whether the claimed invention is not prohibited for grant of patent.
• Whether the invention meets the criteria of patentability.
(vi). Issue of first examination report (FER)

• 1 to 3 months period is available with the examiner to submit the report to the
controller.
446 A. C. Shukla
• A period of 1 month’s time is available in order for the controller to assess the
examiner’s report.
• FER containing substance of the objections is issued within 6 months from the
date of filing of request.
(vii). Reply from the applicant

• From the date of issue of FER, 12 months’ time is available to the applicant to
convene the objections.
• If objections are met, grant of patent may be approved by the controller – within
a period of 1 month.
(viii). Pre-grant opponent

• An opposition can be filed within a period of 6 months of publication.
• Opportunity of hearing the opponent is also available.
(ix). Examination of pre-grant opponent

• Opposition (documents) is sent to the applicant.
• 3 months’ time is allowed for receipt of the response.
(x). Consideration of pre-grant opponent

• After examining the opponent and the submissions made during the hearing, the
controller may:
• Either discard the opponent and grant the patent.
• Accept the opponent and revise/reject the patent application.
• It should be made within a period of 1 month from the date of completion of
opponent proceedings.
(xi). Patent granting

• A patent certificate is issued within 7 days.
• Grant of patent is published in the official journal.
To renew the patent, fees are to be paid within 3+6 months from the date of record-
ing in the register [sec 142 (4)], but there will be no fee for first and second year. The
renewal of fees is done on an annual basis, and renewal fee is required from 3rd to
20th years of patent, in order to keep the patent viable. Six months delay from the due
date is permissible on payment of fee, for extension of time. However, the patent will
be dropped down, if the renewal of fee is not paid within the stipulated time.
Note
• Information pertaining to application form and details of fees are available at
www.ipindia.nic.in.
• Guidelines for applicants are also available on this website: www.ipindia.nic.in.
Stages for Patent: A Schematic Representation
The details of the procedures, as mentioned above, are summarized in the form of
flow diagram:
Filing of Application *Note: if provisional specification (PS) is filed

Provisional / Complete then complete sp (CS) to be filed within
12months.
Publication of Application After 1.6 years from date of filing
Appeal for Examination Within 04 years from date of filing
Issue of FER (Examination) Third Party Representation
All Objections to be Complied within a year
Grant of Patent
Opponent
Decision of Controller
Appeal
Appellate Board Revocation/Amendment

Chapter 29
Intellectual Property Rights and Its Role
in Natural Product Research
Rajat Nath, Anupam Das Talukdar, Priyanka Saha, Subrata Das,

Prakash Roy Choudhury, Deepa Nath, and Manabendra Dutta Choudhury
Introduction
Natural products are best owed with immense medicinal properties that are used for
the treatment of almost majority of diseases. The endogenous people are rich in
knowledge of various core medicinal plants. Natural bioactive products are mainly
isolated from this knowledge. According to Stephen Brush, indigenous learning is
the composed data which covers the casual area and are normally unwritten or are
protected in oral custom as opposed to writings, culture explicit, while formal infor-
mation is decultured (Stabinsky and Brush 2007). From the last decades, pharma-
ceutical, human healthcare, and biotechnology industries have augmented their
curiosity in natural products as sources of new biochemical compounds for drug,
agro-products, and chemical development (Reid et al. 1993). The decade has also
perceived a revival of attentiveness in traditional knowledge and medicine. This
curiosity has been enthused by the significance of traditional knowledge as a prime
in new product development. In the world market, out of 119 drugs, 74% were dis-
covered from traditional herbal medicine (Laird 1994). In 1985, the yearly world
market had evaluated that medications from medicinal plants discovered from
indigenous people groups added up to US$ 43 billion (Posey 1990). It was found
that in most of the cases, indigenous people who created this knowledge did not get
recognition or compensation. On the other hand, pharmaceutical companies were
counting profits by developing drugs using their knowledge (Aguilar 2001).
Intellectual property in other words can be explained as a concept, knowledge infor-
mation, or other “matters of the mind” which, in the nonappearance of divulgence
R. Nath · A. D. Talukdar (*) · P. Saha · S. Das · P. R. Choudhury · M. D. Choudhury

Department of Life Science and Bioinformatics, Assam University, Silchar, Assam, India
e-mail: anupam@bioinfoaus.ac.in
D. Nath
Department of Botany, Gurucharan College, Silchar, Assam, India

https://doi.org/10.1007/978-981-15-2195-9_29
450 R. Nath et al.
or access to it, is neither known nor evident to other people. Specific intellectual
property is generally claimed only or is usually accompanied by an individual, or by
a clan of people, or by an association or firm, or by a legislature. A selective propri-
etor of a specifically protected innovation may do nothing with it themselves but
may give it away to other people, bargain or exchange it, or sell it to others (Bertha
1996; Kartal 2007).
Concept of IPR
Intellectual property may be plated in the form of some conserved information,

potential piece of knowledge, concepts, or other “matters of the mind” which, in the
absence of disclosure or access thereto, is neither known nor obvious to others.
Intellectual property may be considered as owned properties or physical ownership.
It certainly indicates that this property can be enjoyed or owned jointly and may
have detectable worth to the owner of such rights and, destitute of the passable
shield, issue to theft. By this concept, intellectual properties are something which
can be owned, and there should be an owner, and there should be rights bestowed to
the owner on his intellectual properties. So, there comes the intellectual property
rights (IPR). Just as with any physical stuff, an authentic proprietor of definite intel-
lectual property has the right to some different choices. For example, an exclusive
proprietor of specific intellectual property may choose to use or practice it oneself;
do nothing with it; exchange or trade it to others; sell it to others; or give it away to
others. Likewise, a multiparty owner of a given intellectual property may, unless
specified otherwise by prior contract, implement these same rights to part or all of
the together owned intellectual property (Boyd 1996).
Laws of IPR differ country to country in scope and nature. In spite of having
numerous international agreements for IPR protection, there are very controversial
issues arising at the time of acceptance where certified by a single country may be
not be feasible to the other country. As there exist many alterations in national laws,
for example, in the USA, genetically modified organisms can be patented by normal
requirements and other European Union countries, but so many other countries con-
flicted to such cases (Mugabe 2001).
Patents
A patent is regarded as any constitutional right protecting an invention that is

allowed for a restricted period to the patentee by the government, in return of full
revelation of his creation apart from others, from reusing, marketing, producing, or
trading during the patented period for producing that product for those tenacities
without his consent. The most authentic way of protecting one’s noble works and its
attributes is simply by patent itself. This is because patent is itself a total protected
29 Intellectual Property Rights and Its Role in Natural Product Research 451
package which bars the access without one’s consent. To meet the requirements for
a patent, a noble entity or simply an invention must follow some organized criteria’s
such as:
(i) It should be innovative in nature.
(ii) It must come up with some noble procedure with unique protocol so as to the
steps can be patented in that regard.
(iii) It must have some utility and industrially commercial object through which
one can enjoy a handful revenue annually or as designed to be.
(iv) It should not appeal the rations of sections “Patents” and “Patent on Natural
Products” of the Patents Act 1970 (Indian).
Also, the product to be patented should not be publicly known or used. Some
common techniques that inventors can attain patents for herbal medicines are if
they can:
(i) Find new uses for an existing herb.
(ii) Isolate new active ingredient.
(iii) Develop the technique for extraction of the active ingredient(s).
Claiming an old compound for new unknown uses may serve the purpose for
patenting herbal or natural medicine. For example, the composition of herbal medi-
cines of China is known for years resulting in no scope to patent those medicines.
But if any researcher gets any new information regarding those medicines, he might
be able to apply to get patent.
Procedures for Obtaining a Patent
Attaining patent comprises a process involving several steps:
Document Disclosure
The process for patent usually is initiated with “document disclosure.” It is a process
to get the legal recognition for an aspirant inventor. It protects the idea as evident
from being stolen by someone else. If it is the initial phase of applying for a patent,
it is termed as “idea generation” phase.
Patent Search
The main formality for processing for a patent started by the step “patent search,”
this determines the existence of applied invention/products, and this remains pro-
tected under the patent act. The patent search includes searching the existing patents
452 R. Nath et al.
which have already been documented. This step is accomplished by confirming that
there is no patent issued for that inventor’s invention.
Patent Application
An application should be sent by the applicant to the office of the patents and trade-
marks when the patent search process is completed. The application consists of
three parts:
(i) First of all, a written document should be submitted which contains a depiction
of the invention, along with its specification and claim.
(ii) After that, the second part of the application contains a set of diagram which
should be simple and precise.
(iii) The third part consists of a formal “declaration” or “oath” given by the
inventor.
All these items, convoyed with an application fee, are need to be submitted at the
patents and trademarks office. If the submission fulfills all the necessary criteria by
the patent office, then it will be recorded by conveying a file number to it, and the
inventor will also be notified.
Patent Examination
When the process of submission of application is over, the form will be examined
by the patent officials scrupulously; after that, they will draw a decision and specify
the claim, and/or if there is any problem found by the officials, it should be solved
by the applicant.
Patent Grant
“Patent grant” is the last and final step for processing and getting the patent. When
all the criteria get fulfilled, the official will grant a patent. The patent office will
documented new records for a new patent and will issue a letter to the inventor.
Consequently, finally the inventor will get a patent for his invention.
Patent on Natural Products
To get a reward for the significant cost spent during the years of natural product
research work, it is expected to get the right over the result you have got. Developing
a new chemical drug is so affluent that it may cost 80 million to several billion
dollars (Kartal 2007). To protect intellectual creations, some countries have the
legal endowment for providing economic benefit to the creators. Some of these
provisions are changed time to time depending upon the case of patent system
(Moreira et al. 2006).
A “patent” is a lawful instrument, these days for the most part including big busi-
nesses, organizations, and finance, to keep others from making, offering, bringing
in, or utilizing the ensured creation without a permit or approval, for a settled time-
frame (20 years in many nations). At the end of the day, it creates an imposing busi-
ness model that, by and by, may enable the patent proprietor to set up costs; however,
it may also restrain legislatures of creating nations from detailing advancement
strategies (Rights 2002).
The topic about natural products and plant protection in emerging nations is not
new. From one perspective, there is the benefit related with the patent, yet on the
other, there is much discussion of the ethics and morals of this sort of protection and
the advantages given by patents on natural products in developing countries.
To attain a patent for pharmaceutical products, there are only three main protect-
able issues in order in the conservative patent law:
1 . Patent for discovering new chemical components.
2. Patent for knowhow to producing the products.
3. Patent for trademark (Zhang 2000).
Eligibility of Patenting Natural Products
There are numerous misconceptions regarding the patent criteria for natural prod-
ucts patenting. Some key points to understanding whether the product is eligible or
not for patenting are discussed below. However there are many other conditions,
they have to pass for getting granted a patent.
The first step of patent eligibility is to check whether the product which is
claimed for patent falls under categories such as conformation of matter, machine,
process, and manufacture. To get a patent, the first criterion of patent application
must fall under any of these mentioned categories and in maximum cases comprise
natural products. Secondly, it must be considered whether patent application falls
under the legal exceptional cases or not like natural phenomena, abstract ideas, and
laws of nature. For patent, the claim should not fall under these exceptions.
Patent application for grant is eligible only if the claim doesn’t cover a big area so
that other persons can get a further chance to gain a patent on that same area. That is
why those exceptions, along with natural phenomenon or laws, abstract ideas, math-
ematical equations etc., do not become eligible for getting a patent. Medicines
derived from natural products that valuate patent eligibility are implemented by con-
flicting what is campaigned in the cases to their natural occurring counterparts. It
governs if they have prominently diverse properties than their natural counter-
part or not.
454 R. Nath et al.
Natural Products Are Not Patent Eligible if
(i) The component of the parent compound is highlighted, and it retains the natu-
ral properties of the mother (initial) compound.
(ii) The application is restricted to the constituents of the natural products. The
product of which is generally similar in their original state.
(iii) The claims which involve mere joining of two natural compounds or may be
synthetized ones keeping their primary properties unchanged with very little
differences in their bioactivities from that of their initial counterparts.
(iv) When the claims are generally restricted to the particular signaling pathways
or any metabolic pathways or suppose in extreme cases some natural interac-
tion like the glucose and insulin interaction.
Natural Products Are Patent Eligible If
(i) The claims that the newly presented molecule of interest will have the poten-
tialities of industrial applications and all total a new designed molecule upon
alteration of the parent molecule. In other words, the claims are coordinated to
the synthesis that has been altered.
(ii) The claims are guided toward how to synthesize them. Presently, the assur-
ance being looked for isn’t for the normally happening item itself yet for
another procedure that has been invented to integrate it.
(iii) The claim can be filed against some innovative methods for isolation of any
bioactive pure compounds for its indigenous habitat or from the whole plant
ingredients. The protocol should have something noble in its extraction pro-
cesses. Such new techniques which can yield a good amount of the pure com-
pounds that can be in high demand to the industrial sector in terms of its
productivity will procure high chances of being patented.
(iv) The claims are generally synchronized to the specificity of its usage.
(v) The claims that are synchronized to objects having details certainly having
notably extraordinary attributes from a usual occurrence from one with a like-
lihood to happen usually or to any other known set of plans. For illustration,
plans that adjoin appealing characteristics to the particular object item that
have to be patent qualified.
(vi) The claims in questions are coordinated to a product specifically produced
following a unique procedure, and the product is unique in accordance to its
natural counterpart.
(vii) This claims to patent the method of ways that is used to screen various natural
products for its uses in clinical applications. The methods or processes for
alteration of such compounds bringing about something innovative make it
patentable.
However there are numerous wrinkles to be rationally assessed, complete
a decent patent capable comprised right from the starting would be the best
process for enhancing the probabilities of gaining a patent for a natural prod-
uct-based innovation (Anand 2018).
Patent of New Chemical Components from Natural Products
One third of the total drugs used globally are mainly obtained from natural products
or from their derivatives (Strohl 2000). Isolation, purification, and illumination of
structure of unknown or new active lead and finding out the unraveled and depicting
unique useful biological properties and creation of valuable new analogues or deriv-
atives may contribute in the crucial phase of natural product discovery (Boyd 1996).
If the product discovered is abundantly available in nature, then it doesn’t undergo
patenting, but if the characterization is properly done or the isolation process or the
new parameters are found from the same product, then it may be considered for get-
ting the patent (Ganguli 2000).
In case of innovation, when a new compound comes into focus for the first time,
that doesn’t comply it to be patentable. This fact explains that a mere discovery hap-
pening doesn’t promise a compound to be patentable. Furthermore, we can say that
any originality in structural connectivity, biological activity, promising metabolic
studies with a slightest alteration, and expressing the properties of that same com-
pound can certainly be an object for patenting (Cordell 2000).
Patent for Known Natural Products Compounds
The circumstance is that protecting the compound, new or old, must be fixed to non-
evident biological action. It is possible to patent a known or freely existing natural
compound, if its new novel use is discovered. Authentic patents for traditional medi-
cine, if any, would most likely be “use patents” as well; apart from ‘new use’ from
patentability would therefore at the same time be an obstacle for genuine shield of
traditional medicine (Timmermans 2003). The possibilities of getting interesting
new potential utilizable biological activity of an existing known old compound are
very high. For example, betulinic acid was patented as it shows specific cytotoxicity
against melanoma cell, and this betulinic acid also exhibits in vivo antiproliferative
and cancer chemo-protective effects. Although known substances may be semi-
synthesized or modified to enhance its potency, plummeting its toxic effects, etc.,
purification and isolation procedure of known substances from nature and useful
derivatives or analogue synthesis could also be patentable (Cordell 2000).
Traditional Herbal Medicine and Products Patenting
Traditional medicines are the signpost for the screening of natural compounds for
therapeutic use and laboratory and scientific testing for safety and efficacy.
Pharmaceutical companies are focused on traditional medicine or natural products
456 R. Nath et al.
to market new drugs. Pharmaceutical products might be protected by existing con-

ventional patent law. As chemical drugs and herbal medicines differ from each
other, the available existing laws for protecting chemical drugs are unable to protect
the herbal medicine in the same way. Another big problem is to authenticate the
original founder of a particular herbal drug due to the deficiency of databases, and
the same medicinal plants are also cultivated and used to prepare herbal drugs
throughout the globe (Zhang 2000).
The “product patent” is more valuable than “process patent” in case of herbal
medicine. The new better methodologies may arrive to isolate the products from
medicinal plants so patenting the process is not so beneficial. The major herbal
therapeutic doses are simple and are directly derived from herbal preparations like
extracts, powder, fatty oils of herbal products, etc. The procedure or the methodolo-
gies of production of herbal drugs are also very simple, so it is also another matter
of concern to get the proper ideas on how to patent those processes (Weiss and
Eisner 1998).
Pharmaceutical companies also may claim the intellectual property rights by
somewhat altering the traditional knowledge and/or biological resources. The patent
is general if the technologies are used to produce useful products by using the tradi-
tional knowledge.
Process of Patenting Herbal Medicines
Some key steps of patenting any new methods of herbal medicine which have
inventing contributions are as follows:
I. To prepare herbal drugs.
II. To prepare pharmaceutical preparation using herbal drugs or herbal drug
preparations.
III. To get the active compounds by extracting and isolating from natural products
which are used in the production of pharmaceutical drugs.
IV. To prepare standardized fluid/solid extracts or powders or tinctures from
herbal drugs.
V. To dry and granulate the procedure of standardized extracts.
VI. To protect microbial infection and moisture of standardized extracts.
VII. To eradicate trace amount of impurity materials such as pesticides, toxins,
and surfactants from plant products.
VIII. To increase the production size of standardized extracts from natural

products.
IX. To reduce the preparation cost of standardized extracts from herbal drugs.
X. To surge the pureness of the extract isolated from herbal drugs.
XI. To reduce the side effects of herbal medicine (Kartal 2007).
Some Patented Drugs
From the isolation of secondary metabolites to drug discovery, the process should
pass multistep experimentation. A number of in vitro and in vivo experiments and
clinical trials should be passed mandatorily. To be an effective drug, this may be
approved for intellectual property right. Newman and Cragg (2016) listed numerous
natural compounds as well as some synthetic compound which received the patent
rights and now commercially trading in the market. In their review, they presented
different categories of marketed drugs of different sources starting from natural to
synthetic origin within the period from 1980 to 2014. During that time span, a total
of 326 (141, antibacterial; 32, antifungal; 138, antiviral; and 15, antiparasitic) anti-
infective drugs have come into existence and marketed. Some of these anti-infective
drugs are dalbavancin, oritavancin, finafloxacin hydrochloride, nemonoxacin, efina-
conazole, tavaborole, asunaprevir, and favipiravir, among others.
Considering the cases of anticancer drugs, huge breakthrough has been obtained
during the last few decades (1980–2014). A total of 202 anticancer drugs from natu-
ral and synthetic origin have been reported and marketed. Blinatumomab, nivolumab,
pembrolizumab, ramucirumab, apatinib mesylate, idelalisib, ponatinib, pembroli-
zumab, Rexin-G, PICN, and peplomycin are some of the latest inclusions in the last
decades.
A total of 28 antidiabetic drugs were also reported in various papers which were
introduced within last few decades (1981–2014). Some of them include albiglutide,
dulaglutide, liraglutide, lixisenatide, empagliflozin, ipragliflozin L-proline, tofogli-
flozin, luseogliflozin, and anagliptin, among others.
Conclusion
Techniques lined up with national laws and worldwide arrangements and practices
are the most important activity of the board of intellectual property and intellectual
property rights. Among the other multidimensional assignments, intellectual proper-
ties and its related rights are determined mainly according to their advertisement
reaction, market needs, cost involved in elucidation of IP in to the business adven-
ture, etc. It is not fully determined by the national viewpoint. There is diverse range
of subject requests for IPR regarding distinctive treatment, procedure, and taking
care of arranging, which will require a number of people having expertise in various
specialties such as medicine science, law, engineering, finance, marketing and
economics, etc. All the industries follow their own approaches regarding intellectual
properties, methodologies, and their management style and rely upon its zone of
specialty. In the field of pharmaceutical science-related industries, new procedures
for intellectual property rights are developing. The existence of some IPR laws
expanded the probability as invalid, antitrust law. That is why, it is not considerable
458 R. Nath et al.
that invalid rights are unlawfully declared to build up and look after ill-conceived but
guarded, daunting business models inside the pharmaceutical business. It also seems
that some countries, which are very negligible in number, do not realize the impor-
tance and necessity of patenting the traditional knowledge. Most of the countries
even don’t having the laws for protecting the traditional knowledge. The existing
IPR laws do not protect traditional knowledge adequately. Thus, it is of utmost
importance in the field of IPR to take the necessary steps to protect the traditional
knowledge with the shield of IPR. Meanwhile, various matters stay to be settled in
this context.
Acknowledgments The authors are thankful to DBT (Govt. of India) Sponsored Bioinformatics
Infrastructure Facility (BIF) of Assam University and DelCON’s e-Journal Access Facility.
Conflict of Interest The authors declare that there is no conflict of interest regarding the publica-
tion of this book chapter.
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Chapter 30
Nanotechnology in Preclinical
Pharmacokinetics
Santosh Malik, Ananya Ghosh, Rout George Kerry, and Jyoti Ranjan Rout
Introduction
In the era of modern science, the emergence of nearly unbeatable ailments and dis-
orders like diabetes, cancer, and Alzheimer’s demands the development of complex
drugs, and the engagement of orthodox machines in both clinical and preclinical
stage of pharmacological study has always been affected and effected by advanced
technologies. This evolving perception in the cure has also influenced the elemen-
tary knowledge of “what is called a disease” and has further included “the structural
motive of cure process.” Way back during the seventeenth and eighteenth century,
diagnosis and study of maladies were based on outward symptoms such as body
temperature, while treatment emphasizes on the solvent extract of different pharma-
cologically active plants. In the early seventeenth century, the bark of cinchona tree
was used for the healing of malaria, and later in 1820, Pierre J. Pelletier and Joseph
B. Caventou isolated quinine from cinchona (Roth and Streller 2013). However, in
recent times, the application of various diagnostic medical devices from micro-
scopes to DNA/RNA-based chips used in high-throughput screening assays and
effectively nanoparticles and nanorobot research has directly influenced the speed
and accuracy of diagnosis processes. Further, nanoparticles as therapeutics have
elevated the hope for effectiveness by elevating the drug efficacy through targeted
delivery, metal nanoparticles as antimicrobial agents against multidrug-resistant
(MDR) superbugs (they help in ROS production, a relatively unadoptable mechanism
S. Malik
Departmentof Life Science, National Institute of Technology, Rourkela, Odisha, India
A. Ghosh · R. G. Kerry
Post Graduate Department of Biotechnology, Utkal University, Bhubaneswar, Odisha, India
J. R. Rout (*)
School of Biological Sciences, AIPH University, Bhubaneswar, Odisha, India
e-mail: routjr@aiph.ac.in

https://doi.org/10.1007/978-981-15-2195-9_30
462 S. Malik et al.
to be outwitted by the microorganism), and finally green nanoparticles for deliver-

ing phyto-drugs with higher bioavailability (Kumari et al. 2012; Li et al. 2014; Fu
et al. 2014; Arakha et al. 2017; Baptista et al. 2018). The result of such high-end
diagnostic process and treatment methodologies with the integration of bioinfor-
matics databases for storing preclinical and clinical information about diseases
(Disease Database, MalaCards, etc.) and medicines (DrugBank) for further is help-
ing to understand the nature, etiology, and pathology of various acute diseases
(Wang and Liotta 2011; Gill et al. 2016).
Today’s advanced technology and interdisciplinary sciences have enhanced the
scientific understanding of the useful self-destructive mechanisms of our body such
as autophagy and immunological actions of healing. Although sometimes these
approaches fail to respond the clinical treatment, they have manifested many ave-
nues toward the understanding of evolutionary developmental modifications in the
host body. Due to progressive cognitive knowledge, it is feasible for us to under-
stand ourselves as a single system-social organism and furthermore as one of the
most manifold organisms in an evolutionary frame at cellular, tissue, organ, and
system level. These complex dynamic interconnected physiological systems repre-
sented by respiratory, neuron, digestive, circulatory, reproductive, excretory,
immune, hormonal, and cognitive systems have boosted the investigation and the
consequent discoveries of underlying molecular functioning. These understanding
would prove to be profitable in the detection of various abnormal conditions through
the exploitation of advanced omics procedure represented by proteomics, genomics,
epigenomics, metabolomics, systems biology, etc. (Dettmer and Hammock 2004;
Mischak et al. 2007; Mischak et al. 2012; Nicholson et al. 2012; Vucic et al. 2012;
Datta et al. 2013). In the last few years, these approaches have greatly contributed
for helping pharmaceutical science. Further integration of silico methods, real-time
analysis, and adaptive strategies, etc. have made possible the development of highly
promising personalized medicine (van der Greef et al. 2006). These heterogeneous
strategies have not only given a new hope for mankind against deadly diseases like
cancer (personalized medicine available are Herceptin and Iressa) and HIV/AIDS
(personalized medicine available is Ziagen) but also built a new path of advance-
ment in pharmaceutical science (Reynolds et al. 2014; Ann et al. 2017). Despite the
promising achievement in research and development, the high cost of such valuable
medicines/treatments has become a problem for economically backward people of
all countries.
With nanotechnology development, an advancement of clinical and preclinical
technologies such as laser therapy, thermotherapy (involving the use of magnetic
nanoparticles), and noninvasive imaging during in surgery (radiology-based gold
and iron oxide nanoparticles) (Stephen et al. 2011; Kiessling et al. 2014; Vegerhof
et al. 2016) is quite achievable. Imaging guided-drug delivery and disease progres-
sion detection have also improved the state of treatment in recent years (Lammers
et al. 2010; Kiessling et al. 2014; Ojha et al. 2015). Further, controlled drug release
(mesoporous silica nanovalves), real-time protein-ligand affinity, kinetic study (sil-
ica nanowire as biosensor), and real-time biomarker detection (nanoporous silica
nanochips) have immensely contributed in personalized medicine development (Hu
30 Nanotechnology in Preclinical Pharmacokinetics 463
et al. 2011; Duan et al. 2012; Yang et al. 2014). However, the nanorobot-mediated
artificial intelligence surgery is an advance technique in the medical field (Soto and
Chrostowski 2018).
Recent studies show the detrimental effect of some drugs on the host immune
system which may lead to an immune-compromising condition (Becattini et al.
2016; Benoun et al. 2016). This again may become compulsive for the host against
such drugs throughout the life instead of curing the diseases. Further, currently gen-
erated drugs are majorly focused on the development in our system such as high
functioning cognitive development, increasing athletic performance, etc., instead of
curing a particular disease or disorder. This has increased the momentum of psycho-
active and steroidal drug invention (Melia et al. 1996; Savulescu et al. 2004; Sjöqvist
et al. 2008; Christou et al. 2017). According to the World Drug Report (WDR) in
2017, opioid and cannabis drugs have been ruling in those sectors with inclusion of
739 new psychoactive (including synthetic opioids) drugs to worldwide use (WDR
2018). But a majority of them have been marked as illegal considering their multi-
ple side effects at the clinical or preclinical practice, which have been reported in
WDR 2018. Despite getting banned in a large number of the regions, they are still
being used illegally by certain social sector encouraging social imbalance and ter-
rorism and eliciting the financial flow (WDR 2018). Such fast revolutions in the
pharmacon sectors have also created an imbalance in the inner structure of our body.
Drugs used during pregnancy condition might increase the risk toward viability of
the fetus (Stover and Davis 2015). Adolescent young people (the major fraction of
human resource) are the predominant age group facing drug-related health conse-
quence during their most important phase of life (WDR 2018). About 4,50,000
death cases are reported in 2015 due to drug use, among which 167,765 of the inci-
dents were noticed to be due to overdose of opioids (WDR 2018). Further as con-
firmed by an FDA report, adverse drug reactions (ADRs) are also a major governing
reason of death, and around 106,000 deaths are reported per annum due to compul-
sive polypharmacy and drug-drug interactions (DDI). A study has revealed that
death from overdose involving opioid abuse in the USA has made a significant
contribution to the incidence of decreasing the period of life expectancy in 2015 and
2016 (Dowell et al. 2017; Hall and Farrell 2018). Henceforth, it might be speculated
that indisposition could be ameliorated without hampering the host.
The scientific research community has invested their faith with “ADME/Tox”
formula, which defines absorption, distribution, metabolism, excretion, and the
toxic effects of prescription medicines, respectively, which helps the host body to
maintain its pharmaco-response so that there is a possibility of control dose, absorp-
tion rate, bioavailability, volume of circulation, and elimination. These parameters
are commonly cited as PK/PD parameter. PK relates the prescribed measurement of
a drug and its concentration in the living system of an individual, while pharmaco-
dynamics relates concentration at the site of its activity and synthetic and physiolog-
ical repercussions on the body. This interplay between the body and drug relies on
physiochemical characters (size, polydispersity, surface ligand density, surface
charge, shape, stability, and purity) of the medication which are directly liable for
the reaction. Apart from that, the physiochemical characteristics of a living body
464 S. Malik et al.
directly affect the drug performance, invulnerability, and competence. These again
are comprehensively held accountable for functional properties exemplified by ther-
apeutic drug loading, stability, release, and targeting as conjugation, quantitation,
and activity (Yamashita and Hashida 2004; Ekins et al. 2006).
By that, a new understanding can be obtained which is the accessibility of “site
of action” whether it is specific to particular cell/tissue/organ/multiple organs that
can be directly proportional to the potency of drug molecules. In short, the PK/PD
parameter defines the efficacy and potency of drug molecules which are comprehen-
sively studied during preclinical and phase I clinical trial stage. Luckily, technologi-
cal advancements and interdisciplinary studies have made the evaluation of PK/PD
parameters a time cost in addition to that of the individual labor efficient via in silico
methods such as molecular docking, quantitative structure-activity relationship
(QSAR), three-dimensional QSAR, similarity searches, high-throughput solubility
assays (turbidometry, laser nephelometry), permeability assays (Caco-2 cells,
MDCK cells, PAMPA), and metabolism assays (hepatocytes, S9 fractions, recombi-
nant enzymes), among others (Yamashita et al. 2004). But still today, on an average
around 75–80% of 70 million to 1.4 billion financial supports is needed in drug
discovery which is mainly due to deficiency of the medication at phase I clinical
trial where tolerability, PK, and PD parameters are studied. The failures are mostly
due to the diverse physiological feedback to the similar drugs by individuals of dif-
ferent metabolic profiles, genotype, age group, lifestyle, and community (Couch
and Mott 2012).
Henceforth, the understandings of accessibility and PK/PD parameters of the
pharmacological study are extremely essential as it ensures an immense efficacy,
potency, and safety of drug administration. Further, the modern drugs based on
nanotechnology which are synthesized through biologically and/ or chemically
active small molecules are working with negligible side effects that introduced
through any mediums of transportation. The appliance of the drug in the system of
a living entity is a challenging job in the process of exploration in the area of nano-
technology and requires urgent precision in the preclinical study of drug discovery.
Therefore, keeping in views of significance in drug delivery, some of the relevant
physiochemical parameters involved in PK/PD are systematically represented here.
Pharmacokinetics Parameters
Pharmacokinetics deals with the kinetics of the drug with respect to its physiologi-
cal environment in time duration from intake to exclusion of the drug molecule
(Krauer and Krauer 1977). Hence, physicochemical trademarks of both drug and
body contribute toward the ideality of PK parameters that is absorption, circulation,
metabolism, and discharge which further along with PD parameters defines potency
and efficiency of the drug molecule. On the other hand, the measured efficacy is
defined as the therapeutic rate of success of the drugs in the body which solely relies
on the interaction of drug molecule with predefined target and consequences of
interaction at the cell, tissue, and organ or at the organism level. Whereas potency is
the revelation of the drug’s activity quantified by concentration, mandatory for pro-
ducing a defined effect. Considering the safety of drugs during clinical trial and the
fact that today discovering a drug takes on an average of 11–12 years with an out-
of-pocket cost of around US$1.4 billion, these PK/PD parameters are evaluated at
preclinical and phase I clinical phase of drug invention (Wein 2016). However, the
mode of absorption, distribution, metabolism, and excretion involved in PK are
reviewed as below, and different formulas defining the basic parameters of pharma-
cokinetics are schematically illustrated in Fig. 30.1.
Absorption
Absorption is defined as the relocation of the drug molecules from the site of deliv-
ery into the bloodstream so that it can be shifted to the location of the action.
Absorption of orally administrated drugs occurs through thee epithelial cells of the
stomach (Hamman et al. 2007; Gavhane and Yadav 2012). The bioavailability fac-
tors determine the rate and extent of retention subjecting to the solubility and per-
meability which further depends on surface chemical composition such as occlusive
dressings or additives, mode of intake and the environment it must pass, and poly-
dispersity index-dependent dose regimen (dose regimen defined based on the pre-
sumption that drug molecules are monodispersed in the medium) of the drugs
(Johnston et al. 2018).
Fig. 30.1 Schematic representation of different formulas defining the basic parameters of pharma-
cokinetics. (Adopted from Loftsson 2015)
466 S. Malik et al.
Distribution
It defines the transportation of drug from the blood to extravascular tissues. After
intake with an increase in absorption of the drug and creating a concentration gradi-
ent, the drug moves from the blood to the interstitial fluid followed by correspond-
ing cell or tissue by passive diffusion. However, sometimes, carrier-mediated active
transport and receptor-mediated endocytosis mechanism have also governed the
drug distribution (Atkinson 2012). The factors that affect drug distribution are solu-
bility, reversible interaction of the drug to macromolecules in blood, and tight junc-
tions which hinder the movement of the drug between cells. In a complex
heterogeneous biological medium, solubility and interaction of the drug are due to
the measurement of its surface charge, size, and polydispersity.
Solubility
Apart from the absorption of the drug, solubility also plays a pivotal function in the
distribution of a drug. Size and chemical and surface properties define the solubility
of a prescribed drug molecule in a medium which influences the movement of the
drug via the biological membrane, which further affects the volume of distribution
(VD) of a drug molecule. VD is a measurement of fluid volume indispensable to
maintain the same concentration of drug in the being as the concentration measured
in plasma. Or alternatively, it might be said that higher solubility in plasma indicates
its higher concentration in the blood, and hence a higher dosage of the drug is
required to retain the same concentration at different parts of the body for potential
therapeutic activity. This is the consequence of the time-dependent concentration
gradient established between the blood and perfused tissue which direct the redistri-
bution of drug molecule from perfused tissue to the bloodstream and try to maintain
the equilibrium between blood and tissue concentration (Alavijeh et al. 2005).
Distribution of drug relies upon the likeliness of drug to relocate away from hydro-
philic blood/interstitial region through lipophilic biomembrane again to hydrophilic
cytosol; hence the intensity of hydrophobicity and hydrophilicity plays a critical
role in biodistribution and potency of a drug. Highly hydrophilic drug molecule dif-
fuses slowly from the blood to extravascular tissue, and their distribution is condi-
tioned with the pace of drug diffusion. These drugs maintain an elevated
concentration in the blood and distribute to the liver and skeletal muscle through
loose-kink capillaries. On the other hand, lipid dissolvable drugs quickly move from
the blood to extravascular tissue because of the hydrophobic tendency of the lipid
bilayer which results in less hindrance of vessels. However, the high lipophilic drug
may have a surged plasma concentration owing to reluctance in moving from hydro-
phobic bilayer to hydrophilic cellular environment. Thus, it can be speculated that
the solubility of a drug controls its targeted distribution to the locus of activity
which further contributes to potency and adequacy of the drug molecule. For that,
medications with moderate solubility having a moderate value of partition coeffi-
cient are preferred these days (Alavijeh et al. 2005; Ishikawa and Hashimoto 2011).
Reversible Binding of Drugs to Macromolecules
In a blood medium, due to complementarity in surface chemical properties such as

charge and shape between the biological macromolecule and the drug, the interac-
tion between drug and macromolecules in the blood might be established which
further affects the distribution and efficacy of the drug as only unbound drugs show
pharmacological activity. Unbound fraction measures the scope of drug and plasma
protein binding. Further, prodrugs and soft drugs that metabolize in the liver to their
active form may bind within the liver to macromolecules in their active form which
disables them to follow enterohepatic cycling resulting in low dispersity to other
tissue. Also, high plasma protein interaction results in longer plasma half-life of the
drug and low hepatic and renal clearance.
Metabolism
It is designated as the biotransformation of the drug molecule into active (in the
event of a prodrug), inactive, or toxic products (reactive metabolites) through an
enzymatic system of the liver and in a few cases of the small intestine. Metabolism
occurs at two phases, facilitating the easy clearance of drug after their therapeutic
activity and refraining from the toxic effect due to drug accumulation for a pro-
longed period. Phase I metabolism involves the non-synthetic modifications of
drugs by the processes of oxidation, reduction, and hydrolysis which form certain
functional groups crucial for their chemical activity, mainly by the involvement of
Cytp450 family enzymes. While phase II metabolism involves the addition of
endogenous biomolecules represented as thione, sulfate, glycine, glucuronic acid,
etc., to result in accreting the water solubility of the product for easy clearance with-
out renal reabsorption (Jancova et al. 2010). Moreover, phase II modifications in the
liver are essential for drug elimination through the bile. The interaction between the
drug molecules and their corresponding enzymes is based on the structural and
more importantly chemical composition of the drug. The enzymes are sometimes
restricted for their specific substrate as per the nature of chemistry of drug during
the preclinical phase of drug discovery. However, the several methods or tools
which are involved in the prediction of metabolism of drug molecules are summa-
rized in Table 30.1.
468 S. Malik et al.
Table 30.1 Predicted metabolisms of drug molecules in relation to specific methods or tools
Sl. Methods/
No. procedure Metabolism Bibliography
1. SMARTCyp Predicts the sites of cytochrome P450-mediated metabolism Rydberg
of drug-like molecules et al. (2010)
2. XenoSite Predicts the atomic locations at which xenobiotics/drugs Matlock et al.
will experience metabolic modification by cytochrome P450 (2015)
enzymes
3. FAME Provides models which allow predicting the locality of Sicho et al.
metabolism. The software uses a slightly modified version (2017)
of the visualization implemented in SMARTCyp
4. StarDrop’s Helps quick identification of the regions of compounds that Zaretzki et al.
P450 are more vulnerable to be metabolized by the dominant drug (2015)
metabolizing isoforms of cytochrome P450 which can be
executed and predicted based on an expectation-
maximization algorithm
5. DEREK Knowledge-based software which gives predictions for a Greene et al.
diversity of toxicological endpoints (1999)
6. MetaDrug Functional and network analysis from a 2D compound Ekins et al.
structure (known or unknown), determining its primary and (2006)
secondary effects. Compares molecules for the purpose of a
comprehensive analysis of common, similar, and unique
targets and, in turn, understanding their functional,
interconnectivity, and system-level effects. Helps in the
classification and characterization of an unknown or poorly
understood metabolite, endogenous compound, and/or
nutraceutical
7. CypReact Software developed for in silico reactant prediction for Tian et al.
human cytochrome P450biocatalysts (2018)
Excretion/Elimination
Drug elimination is defined as the permanent removal of drug or toxic and nontoxic
drug metabolites from the living system. The major pathways of drug elimination
are renal and biliary excretion. In the former pathway, elimination of phase II
metabolized drugs occurs via passive diffusion due to hydrostatic and concentration
gradient across glomerulus-Bowman’s capsule junction. However, large-sized drugs
are not eliminated via renal excretion pathway. Similarly, liver enzyme metabolized
drug product excretion occurs through the active transportation from the liver and
afterward to the gut through hydrophilic bile medium (Kok-Yong and Lawrence
2015). As puissance of a drug is dependent upon its concentration, its activity and
toxicity may appear as a consequence to excess drug disposition at the certain cell
or tissue; hence, elimination of the medicinal drug is a necessary parameter to be
accurately reviewed during preclinical stage. However, the properties of drug mol-
ecules contribute toward its elimination and depend on the size, polydispersity and
aggregation propensity, chemical structure, and route of metabolism (Johnston
et al. 2018).
Problems with Conventional Drugs
The physicochemical traits of a drug as well as the physiological condition of the

organism during illness determine the ideality in PK parameters, and during that
point of time, a drug should be administrated in appropriate formulations so as to
achieve a better therapeutic effect. For example, class I biopharmaceutical classifi-
cation system (BSC) drugs having upsurged solubility and permeability can be for-
mulated as tablets, but class II BSC drugs having high solubility but low permeability
should be contributed in formulation after certain modifications like proportion
reduction or in the form of a liquid for higher bioavailability (Loftsson 2015).
Similarly, class III BSC drugs having high solubility and low permeability should be
formulated along with permeation enhancer such as sodium lauryl sulfate and pal-
mitoylcarnitine chloride for oral drug formulation and pyrrolidones, dimethyl sulf-
oxide, fatty acids, and esters for transdermal formulations (Whitehead et al. 2007;
Sinha and Kaur 2000). Further, it has been documented that highly soluble drugs
tend to metabolize faster and excreted through hepatic clearance, whereas low solu-
ble drugs are excreted via the renal clearance. Hence, if the drug does not stay for a
particular time course in the system due to rapid expulsion, its therapeutic activity
may decrease. This effect of rapid elimination can be compensated by increasing the
dose to maintain drug plasma concentration within the therapeutic window.
However, the tendency of a drug molecule inside a dynamic heterogeneous biologi-
cal system such as blood or targeted cell cannot be predicted accurately; hence, it is
a difficult task to sustain a constant therapeutically active concentration in the blood
or interstitial fluid or inside the targeted cells. This might be a consequence of the
reversible binding of the drug to plasma proteins, when in blood the other molecules
save for the target, when in a targeted cell and due to the fact that exclusively free
drug molecules are therapeutically active (Yang et al. 2014). Further, a high-dose
and rapid reversible drug-protein binding may culminate into a low or high concen-
tration, which further results in toxicity as a reaction to drug overdose. In addition
to a drug overdose, toxicity may result as a consequence of the production of reac-
tive metabolites during the first and second phases of metabolism by the enzymatic
action of Cytp450 family enzyme (Jancova et al. 2010). As active groups in conven-
tional drugs are significant for their therapeutic activity, which are more susceptive
to enzymatic modification, this may result in the reduction of potency of the drug.
Moreover, smaller-sized drug entities tend to diffuse rapidly resulting in uneven
dispersity of it and low bioavailability at the site of action. This again decreases the
competence of smaller molecule drugs if not fabricated for targeted delivery.
Another important hindrance with the conventional drug is its limited functional-
ity, as a result, inadequate figures of active sites (functional groups) participate for
the effective drug interaction that results a failure of target specific treatment. This
was conceived in a major worldwide crisis MDR or resistance of various pathogenic
microorganisms against a varying range of antibiotics (Nikaido 2009; Exner et al.
2017). Due to overuse of certain antibiotics, microorganisms have adopted various
mechanisms such as surface receptor modification, activation of the efflux pump,
470 S. Malik et al.
bypassing the targeted pathway, or by modification of the target molecules.

According to a report, over 58,000 babies died due to infection related to MDR that
was passed from their mother (Laxminarayan et al. 2013). The annual deaths due to
MDR-associated factors in the European Union and the USA are 25,000 and more
than 23,000, respectively (Nikaido 2009).
Nanotechnology and ADME Parameter
The therapeutic functionalities of the conventional drug maneuvered depending

upon the electrostatic communication with the target and its subsequent conse-
quences of the interaction. The enzyme-substrate alike interaction is based on the
shape, and the surface chemical tendency of the drug molecules has relied upon
charge distribution. Before a drug reaches the region of the target, it has to pass
through a series of diverse biological mediums like the blood, liver (hepatic first-
pass metabolism and hepatic second-pass metabolism), blood-brain barrier, etc.,
from the locus of administration. During this movement, it interacts with various
biomolecules due to outline and surface charge complementarily, which leads to its
structural change and hence deprivation of function and starts its therapeutic activ-
ity. Henceforth, to maintain the therapeutic ability of any kind of drug, it has to be
carefully transported in the biological medium to the location of action/target by
keeping its structure and surface properties intact during movement. This requires a
comprehensive examination of the host physiological system during clinical condi-
tion at a molecular level followed by the fabrication of drug molecule for safe trans-
port (Pisal et al. 2010; Zhang et al. 2012).
When drugs are conjugated with nanoparticles, either for imaging or enhancing
the therapeutic activity of the drug, they are considered as nanomedicines (Bobo
et al. 2016). High therapeutic functions of these nano-based formulations are a
result of their small size, prolonged circulation time, targeted delivery, and subsided
volume of distribution as well as drug localization at healthy non-targeted tissue
which increases the drug localization at target tissue or at the place of action (Sanna
et al. 2014). These characteristics enhance the drug’s ADME parameter, thereby
increasing efficacy and decreasing its toxic effects. Further, these characteristics,
apart from the host physiological system, are ascertained by the physicochemical
particularities of the drug. Those properties and fabrication of approach to achieve
its best effects are briefly described below.
Shape
The architecture of a nanomaterial influences its rate of diffusion from the blood to
the extravascular cells, later affecting its distribution. Small-sized spherical nanopar-
ticles are less likely to interact with the membrane of blood vessels and hence stay
in the blood without getting distributed to extravascular tissue or to the site of action.
However, discoidal and cylindrical nanoparticles due to their tumbling motion and
a high chance of communicating with the membrane of the blood vessel show better
biodistribution. Again, the inconsistent shape of nanoparticle shows higher antimi-
crobial activity than that of the regular shape. The outline of the nanoparticle can be
restrained by altering physical and chemical parameters as exemplified by tempera-
ture, the concentration of substrate used, and pH of the preparation medium in a
chemical as well as the biological procedure for the organization (Gatoo et al. 2014).
Size and Polydispersity
The magnitude of the nanomaterials shows a certain degree of impact on its absorp-
tion, biodistribution, and excretion. Due to smaller-sized nanomaterials, their ele-
vated surface to volume ratio give a better and more favorable circumstances for
surface modifications like ligand conjugation for targeted delivery as well as drug
loading for high efficacy. Similar to that of structure, the size of the nanomaterials
can also be controlled during the synthesis process. Polydispersity is characterized
as particles of varied sizes in the disperse phase of a dispersing medium and poly-
dispersity index (PDI) which indicates the size range of the drug. For safety and
stability, monodisperse nanocarrier formulations are required which will have simi-
lar physicochemical properties due to their narrow size range, and hence it is easy
to predict their behavior inside a biological medium (Johnston et al. 2018). A nano-
material having PDI values less than 0.05 is considered highly monodisperse, while
a value greater than 0.7 indicates a broad range of particle size (Danaei et al. 2018).
Further, PDI is used to enumerate the required deliver of prescribed dose of a for-
mulation for which monodispersity greatly contributes toward PK/PD parameter of
a drug (Johnston et al. 2018).
Surface Ligand and Ligand Density
Ligands used for nanomaterial fabrication are mostly the biomolecules such as minor
molecules, antibodies, and protein domains attached on to the periphery of nanomate-
rials. Further, ligand size and polarity determine the packed structure and orientation
of the matrix of the nanomaterials in their assembly contributing to their structural
properties (Feng et al. 2011). Surface modification of nanomaterials using biogenic
ligands increases their biocompatibility. Also, surface ligands provide a better speci-
ficity to nanomaterial to bind with a target which can be a diagnostic biomarker or
target to specific cell or tissue. Multiple ligands can be used at a time to increase the
functionality of nanomaterial-based drug that prompts the process of targeted delivery
system of drug along with further increases the efficacy (Friedman et al. 2013). Apart
from specificity, the efficacy of a drug also depends on its affinity toward the target
molecule, which can be increased by modifying the surface ligand density that will
enhance the number of probable interaction with the target (Verma and Stellacci 2010).
472 S. Malik et al.
Surface Charge
The surface charge of a nanomaterial will always define its possible interaction with
molecules present in its proximity. Hence, it plays a paramount role in defining
solubility, bioavailability, cellular internalization, and distribution during drug
administration. For example, nanomaterials having a positive surface charge are
internalized into cell irrespective of their size, while nanomaterials with neutral
surface charge do not (Verma and Stellacci 2010). For which, it is a necessary
parameter with respect to deciding the interaction profile and efficacy of a molecule
of pharmaceutical importance. However, the surface charge of the nanomaterial can
be manipulated by appropriate surface ligand modifications.
Porosity
It is defined as the quantitative measure of void volume to the total volume of a

nanomaterial; however, the fraction of void is an important factor in determining
therapeutic drug loading as well as control release of the drug. Porous nanoparticles
are also used for in vivo biomarker detection which helps in the preclinical pharma-
cokinetic study. Apart from these, the purity of the drug is also a vital factor that
may control toxicity level due to drug-drug interaction. Due to the promising results
of nanomedicines in terms of the efficacy, potency, and toxicity control, they have
been extensively used in the last two decades. According to a literature, more than
350 drug products containing nanomaterials have been submitted to FDA, and max-
imum data was submitted toward the treatment of various cancers (Bobo et al. 2016).
Alternative Medications
Allopathic medication has significantly progressed due to advancements in science

and technologies which launched a variety of medicines for the treatment of differ-
ent diseases. However, maybe due to their adverse effect and high cost, the public
prefers alternative medications which has no or less identified side effects (Kanungo
et al. 2013). Alternative medications are defined by their lack of scientific explana-
tion toward the pattern of disease cure, but they can be credited for the importance
of placebo effect in curing a disease. A survey between 1990 and 1997 in the USA
showed the implementation of either one out of sixteen alternative medicines
(Fig. 30.2) was surged by 8.3%, while the number of individuals visiting alternative
medicine practitioners had increased by 10% during these years (Eisenberg et al.
1998). During 2001 to 2012, the World Mental Health Survey in 25 different coun-
tries for 12 different mental disorders found that 3.6% of 138,801 individuals have
resorted to alternative medications. Further, the percentage of approach was two
Fig. 30.2 Different alternative medicines commonly implemented for the treatment of allied
diseases
times higher in high-income countries (de Jonge et al. 2018). Another study has
revealed that out of 453 studied cancer patients, 83.3% had used alternative medi-
cines at least once, and the most prefer medications are spiritual practices or intake
of vitamin supplements or consumption of herbal medicines (Richardson et al.
2000). The scientific understanding and more demand for alternative medications
showing a significant solution for curing different diseases, which indicate a better
and effective choice of treatment among the people of western countries.
Conclusion
Looking at the duration of drug discovery, a step for maintaining physical and men-
tal healthiness of mankind, the beneficence of science is noteworthy. Growing sci-
ence from the seventeenth century to the twenty-first century has immensely
contributed in the development of drugs from preparing medication extract of phar-
macologically useful herbal sources for identification and modification of func-
tional drug compounds for increasing efficacy and potency of the medication to
efficiently combat against continuously emerging deadly diseases. With this, the
duration and the certainty of therapeutics increased significantly, but the extent and
the finances involved in discovering a drug are still a threatening challenge to the
research and development sector of pharmaceutical science. It can’t be neglected
that discovery and illegal marketization of isolated drug compounds have been
adversely harming the living society, creating social imbalance and terrorism and
eliciting financial flow and death resulting from ADR being the fourth leading rea-
son of mortality. ADR being in question for safety clinical practices, evaluation,
competence, and safety of drug at the preclinical stage are primarily focused in the
field of therapeutic medicine development. In addition to the achievement due to an
474 S. Malik et al.
interdisciplinary approach, implementation of nanotechnology has fostered this

process not only by providing the opportunity to nanofabricate medicines for
achieving desired physicochemical properties to enhance the PK/PD parameters but
also helping to study the precisions of the physiological systems in real time at the
molecular level. This approach is not only focusing on the medicine but also on the
body at the preclinical stage, which is an evolutionary contribution of nanotechnol-
ogy to medicine. And again, the clinical application of laser therapy due to its ability
to selectively interact with certain photosensitive biomolecules in the living system
has further improved its utility. Hence, the application of laser nanotechnology in
union with nanodevices can conceive a mechanism for preclinical evaluation molec-
ular profile of a body at real time, considering it as a solitary physiological system.
A substantial prospective of this approach may reduce today’s drug failure which is
at 70–80%, at phase I clinical trial. Further, the integration of tissue engineering
with laser nanotechnology can be implemented for the treatment of tissue- and
organ-related abnormalities. From current achievements of nanotechnology such as
the use of nanodevices for real-time in vivo molecule detection, combination of
external radiotherapy for achieving desired function of nanoparticles in vivo, and
rapid progression toward nanorobot-based artificial surgery, it can be speculated
that nanotechnology invokes for curing disease by nano-based mediated regulation
of physiological organization similar to hormonal, immune, and cognitive system,
inducing the body to heal itself.
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What are some of the alternative medicines commonly used?

What are some of the alternative medicines commonly used?

How has nanotechnology contributed to preclinical drug development?

How has nanotechnology contributed to preclinical drug development?

Jayanta Kumar Patra

ISBN 978-981-15-2194-2 ISBN 978-981-15-2195-9 (eBook)

© Springer Nature Singapore Pte Ltd. 2020

Advances in Pharmaceutical Biotechnology: Recent Progress and Future

Goyang-si, Gyeonggi-do, South Korea Jayanta Kumar Patra

Part I Traditional and Ethnomedicine

8 Phytochemicals as Therapeutics in Heavy Metal Toxicity ������������������ 91

Part II Drug Discovery, Pharmaceutical Sciences

20 Micelleplexes: A Promising Nanocarrier for the Transport

Part III Clinical Trials and IPR in Pharmaceuticals

Jayanthi Abraham Microbial Biotechnology Laboratory, School of Biosciences

Swagat Kumar Das Department of Biotechnology, College of Engineering and

Saili Kirtiwar Bio-Inspired Materials Research Laboratory, Department of

Rajat Nath Department of Life Science and Bioinformatics, Assam University,

Ana Cláudia Santos Department of Pharmaceutical Technology, Faculty of

Dr. Jayanta Kumar Patra, M.Sc., Ph.D. , is Assistant

Professor at Dongguk University, South Korea. He has

Prof. Amritesh C. Shukla, M.Sc., Ph.D. , is Professor

in the Department of Botany, University of Lucknow,

Dr. Gitishree Das, M.Sc., Ph.D. , is Assistant Professor

at Dongguk University, South Korea. She has 10 years

Prakash Roy Choudhury, Anupam Das Talukdar, Deepa Nath,

P. R. Choudhury · A. D. Talukdar (*) · P. Saha · R. Nath

© Springer Nature Singapore Pte Ltd. 2020 3

History of Traditional Medicine System

Traditional Medicine System of India

In India, modeling archaeological expeditions followed by excavations showcased

The Necessity of Traditional Folk Medicine

Traditional medicines, particularly plant-based formulations, have been catering a

Historically Important Natural Products

Traditional medicine has a profound contribution in developing most of the modern

Table 1.1 Some of the historically important natural product-based drugs

Challenges in Drug Development

attribute to the complexity in drug development analogs and posed a significant

Traditional medicine system is an age-old practice that humans have mastered, in

Yengkhom Disco Singh, Manasa Kumar Panda, and Kunja Bihari Satapathy

Ethnomedicine is a study about the traditional system of medicine practiced by vari-

© Springer Nature Singapore Pte Ltd. 2020 15

Traditional Healing Practices

Traditional medicine system (TMS) is sometimes referred to as complementary and

Ayurveda is a well-known Indian system of traditional medicine. It originated in

Fig. 2.1 Panchamahabhutha and tridosa relationship

Traditional Korean Medicine

Traditional Chinese Medicine (TCM)

Traditional Chinese medicine (TCM) is a healing system of Eastern medicine origi-

Iranian Traditional Medicine (ITM)

Iran (formerly called Persia) is located in Southwest Asia. It covers a territory of

African Traditional Medicine (ATM)

Ancient Greek Medicine

Salicylic Acid (SA)

Chinese medicine was also a breakthrough. Many other therapeutic compounds

In spite of the advanced medical technology and modern medicines, traditional

Ravikant Singh, Anand Pandey, Rohit Kumar Mishra, Amritesh C. Shukla,

© Springer Nature Singapore Pte Ltd. 2020 29

functional ingredients in the pharmaceutical, food, and feed industries (Sacchetti

Abstraction and Deployment of Aroma Principles

Goyang-si, Gyeonggi-do, South Korea Jayanta Kumar Patra

Part I Traditional and Ethnomedicine

8 Phytochemicals as Therapeutics in Heavy Metal Toxicity �� 91

Part II Drug Discovery, Pharmaceutical Sciences

20 Micelleplexes: A Promising Nanocarrier for the Transport

Part III Clinical Trials and IPR in Pharmaceuticals

History of Traditional Medicine System

Traditional Medicine System of India

The Necessity of Traditional Folk Medicine

Historically Important Natural Products

Challenges in Drug Development

Traditional Healing Practices

Traditional Korean Medicine

Traditional Chinese Medicine (TCM)

Iranian Traditional Medicine (ITM)

African Traditional Medicine (ATM)

Ancient Greek Medicine

Salicylic Acid (SA)

Abstraction and Deployment of Aroma Principles

Absolute Flower Oils

Physical–Chemical Properties of Essential Oils

Storage of Essential Oils

Mother Medicine of Nature – Tulsi Mata

Ocimum sanctum (L.) (Lamiaceae)

Diabetes and Its Burden in India

thnomedicinal Plants of North-east India Utilized

Popularly known as Jamun in India, belonging to family of Myrtaceae, a fast-

Classification and Health Benefits of Phytoconstituents

Group I: Terpenoids or Terpenes

Group I (A): Tocopherols and Tocotrienols

Group I (B): Carotenoid Terpenes

Group I (C): Limonoids Group

Group I (D): Phytosterol Group

Group II: Polyphenolic Compounds

Group II (A): Phenolic Components

Group II (B): Flavonoidal Components

Group II (C): Catechins and Gallic Acids

Group II (D): Isoflavonoids

Group II (E): Anthocyanidins

roup III: Alkaloids and Other Nitrogen-Containing

Group III (B): Indole Alkaloids

Group IV: Dietary Fiber