Chendhooram " in In-Vitro Studies.: Dr. R.Abinaya

SCIENTIFIC VALIDATION OF ANTI-ORAL CANCER, ANTI-TUMOUR
AND ANTI-MICROBIAL ACTIVITIES OF SIDDHA METALLO-

MINERAL FORMULATION “ KAALAMEGA NARAYANA
CHENDHOORAM ” IN IN-VITRO STUDIES.
The dissertation Submitted by

Dr. R.ABINAYA
Reg. No: 321512101
Under the Guidance of

Dr. R. KAROLIN DAISY RANI, M.D.(S).,
Dissertation submitted to
THE TAMILNADU DR. M.G.R MEDICAL UNIVERSITY

CHENNAI-600032
In partial fulfilment of the requirements
For the award of the degree of
DOCTOR OF MEDICINE (SIDDHA)

BRANCH-II GUNAPADAM
POST GRADUATE DEPARTMENT OF GUNAPADAM
THE GOVERNMENT SIDDHA MEDICAL COLLEGE
CHENNAI -106
OCTOBER 2018
GOVT. SIDDHA MEDICAL COLLEGE,
CHENNAI-106
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation entitled Scientific Validation of Anti -Oral
Cancer, Anti-Tumour ,Antimicrobial Activities of Siddha Metallo-Mineral
Formulation “Kaalamega Narayana Chendhooram ” in Cell Line Studies is a
bonafide and genuine research work carried out by me under the guidance of
Dr.R.Karolin Daisy Rani M.D(S)., Lecturer, Post Graduate Department of
Gunapadam, Govt. Siddha Medical College, Arumbakkam, Chennai-106 and the
dissertation has not formed the basis for the award of any Degree, Diploma,
Fellowship or other similar title.
Date: Signature of Candidate
Place: Chennai Dr. R.Abinaya

CHENNAI-106
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled Scientific Validation of Anti-Oral cancer,
Anti tumour, and Anti-microbial activities of Siddha Metallo mineral Formulation
“Kaalamega Narayana Chendhooram ” in cell line studies is submitted to the Tamilnadu
Dr.M.G.R. Medical University in partial fulfillment of the requirements for the award of degree
of M.D (Siddha) is the Bonafide and genuine research work done by Dr.R.Abinaya Under my
supervision and guidance and the dissertation has not formed the basis for the award of any
Degree, Diploma, Fellowship or other similar title.
Date: Seal and Signature of the Guide
Place: Chennai Dr.R.Karolin Daisy Rani M.D(S)

CHENNAI-106
ENDORSEMENT BY THE HOD AND
PRINCIPAL OF THE INSTITUTION
This is to certify that the dissertation entitled Scientific Validation of Anti-Oral
cancer, Anti tumour, and Anti-microbial activities of Siddha Metallo mineral
Formulation “Kaalamega Narayana Chendhooram ” in cell line studies is a
Bonafide work carried out by Dr.R.Abinaya under the guidance of Dr.R.Karolin
Daisy Rani MD(s), Lecturer, Post Graduate Department of Gunapadam, Govt.
Siddha Medical College, Chennai - 106.
Seal and Signature of the HOD Seal and Signature of Principal
Dr. M.D.Saravanadevi M.D(S) Dr.K.Kanakavalli M.D(S).

ACKNOWLEDGEMENT
This work presented in this thesis wouldn’t have been possible without my close
association with many people who were always there when I needed them the most. I take this
opportunity to acknowledge them and extend my sincere gratitude for helping me make this
dissertation thesis a possibility.
Writing this dissertation has been fascinating and extremely rewarding. I would like to
thank a number people who have contributed to the final result in many different ways.
First and foremost I would like to thank the Almighty for his showers and grace and the
strength and caliber he gave in handling, understanding the difficulties during the tenure of this
work, enabled to complete this tough task, zeal and the light. In the name of Siddhars who has
given me power and courage to accomplish this dissertation work, I bow my head on thanks
and gratitude to Siddhars for their blessings.
This dissertation is the beginning of my foot step to the validation of Siddha drugs
through scientific way and end of my journey in obtaining my Post graduate in Gunapadam.
This dissertation has been kept on track and been seen through to completion with the support
and encouragement of numerous people including my well wishers, my friends and various
institutions. At the end of my dissertation I would like to thank all those people taken a part to
complete this dissertation possible and it’s an unforgettable experience in my life. So it is a
pleasant task to express my thanks to all my college people who contributed in many ways to
the success of this study and made it a memorable experience for me.
I wish to express my sincere thanks to Dr.K.Kanakavalli M.D(S).Principal, Govt.

Siddha Medical College, Chennai, for her permission to perform this study and also for her
valuable ideas and support throughout the course of the study.
I take this opportunity to express my profound gratitude and deep regards to my guide
Dr.R.Karolin Daisy Rani M.D(S), lecturer, PG Gunapadam Govt. Siddha Medical College,
Chennai, for her exemplary guidance, monitoring and constant encouragement throughout the
course of this dissertation. The blessing, help and guidance given by him time to time shall
carry me a long way in the journey of life on which I am about to embark
I feel intensely grateful to Dr. M.D.Saravanadevi M.D(S), proff, Head of Department,

PG Gunapadam Govt. Siddha Medical College, Chennai, for his valuable guidance,
suggestions for completion of my whole study.
I owe my special thanks and sincere gratitude to my advisor Dr.V.Velpandian
M.D(S).,Ph.D., for his support towards my dissertation topic discussions and selection. His
guidance helped me in all time of my research work.
I express my sincere thanks to Former Principal and Head of the PG Dept of
Gunapadam and presently Director of National Institute of Siddha, Chennai. Prof Dr. V.
Banumathi M.D(S), Director of National Institute of Siddha, Tambram Sanatorium, Chennai
for her guidance towards this study.
I wish to express my thanks to co-guide Dr.K.Rajamma Devi Sorubarani M.D(S),

Lecturer, Department of PG Gunapadam for his valuable ideas and suggestions to my study.
I acknowledge my thanks to Dr.A.Ganesan M.D(S)., Dr.S.Shankar M.D(S).,

Dr.K.Nalina Saraswathi M.D(S)., for their support and guidance.
I extended my gratitude to the animal Ethical Committee Members for their approval
to do animal studies in pre clinical studies.
I cordially register my humble thanks to Dr. P. Muralidharan M.pharm., Ph.D.,

HOD, Department of Pharmacology, C.L Baid Metha College of Pharmacy, Dhuraipakkam,
Chennai, for helping in the preclinical study. It was under their direct supervision that the work
contained in the dissertation was performed.
I acknowledge my thanks to Prof Dr.R.Rajesh M.Phil, Ph.D., Biogenix Research

Centre, Trivandrum. Mr. Prabhu Shankar M.Sc chemistry, Tamilnadu test laboratory,
Vaanagaram, Chennai. Prof Mr. Selvaraj M.Sc,M.Phil, HOD, Department of Bio-Chemistry
and Lab Assistant Mrs. Rajalakshmi, for helping me to prepare the test sample for instrumental
analysis of the trial drug Govt. Siddha Medical College, Chennai. I would like to aknowledge
Dr.N.Kabilan BSMS.,MD(s), Ph.D, The Tamilnadu Dr.MGR Medical University for doing
physico chemical analysis.
I am also my thankful to our Librarian Mr.V.Dhandayuthapani, B.Com, M.Lib.Sc

and staffs for their kind co-operation for my study.
I am also thankful to Mrs.Kanniyammal, D.Pharm, Pharmacist, Post Graduate

Department of Gunapadam for her kind co-operation for the purification and preparation of the
trail drug for my study and successful completion of dissertation
I would like to thank Vice Chancellor, The Tamilnadu Dr.MGR Medical University
for giving permission to carry out my dissertation work and to approve my dissertation topic
for research and to the Additional Chief Secretary and Commissioner of Indian Medicine
and Homeopathy Department, Arumbakkam, Chennai-106, for giving consent to do the
dissertation.
I am also thankful to all my college staffs for their kind co-operation for my study
My acknowledge will never be complete without the special mention, to express my
gratefulness to All My Classmates and my Department juniors for lending their helping
hands whenever needed during the course of the study. Words are short to express my deep
sense of gratitude towards apart from my academic friends, who willingly and selflessly
donated theirs valuable time for my experiments during my research endeavor.
I thank the almighty for giving me the strength and patience to work through all these
years. So that I can stand proud with my head held high.
Finally, I would like to acknowledge the people who mean world to me, My Parents,
My Sisters. I don’t imagine a life without their love and blessings. Thank you mom for showing
faith in me and giving me liberty to choose what I desired. I consider myself the luckiest in the
world to have a such supportive family, standing behind me with their love support.
.
CONTENTS
S.No TITLE Page. No
1. INTRODUCTION 1
2. AIM AND OBJECTIVES 8
3. REVIEW OF LITERATURE 10
3.1.1. SIDDHA ASPECT 10

3. 1. DRUG REVIEW 3.1.2. MODERN ASPECT 30
3.2.1. SIDDHA ASPECT 53

3.2. DISEASE REVIEW 3.2.2. MODERN ASPECT 56
3.3. PHARMACOLOGICAL REVIEW 75
3.4. PHARMACEUTICAL REVIEW 96
3.5 LATERAL REVIEW 100
4. MATERIALS AND METHODS 103
4.1 PREPARATION OF THE DRUG 107
STANDARDIZATION OF THE DRUG 119
4.2.1 PHYSICO CHEMICAL INVESTIGATION 120

PRELIMINARY BASIC AND ACIDIC
4.2.2 122
RADICAL STUDIES
4. 2
4.2.3 ANTI-MICROBIAL ACTIVITY 124
4.2.4 INSTRUMENTAL STUDY 126
4.2.5 TOXICOLOGICAL STUDY 135
4.3 PHARMACOLOGICAL STUDY 144
4.3.1 ANTI-CANCER ACTIVITY 144
4.3.2 ANTI-TUMOUR ACTIVITY 147
4.3.3 ANTI-MICROBIAL ACTIVITY 147
5. RESULTS AND DISCUSSION 149

S.No TITLE Page. No
6. CONCLUSION 191
7. SUMMARY 192
8. BIBLIOGRAPHY 193
TABLE CONTENTS
S. NO TITLES PAGE NO.
1. TUMOUR MARKERS 61
2. CLASSIFICATION OF ANTI-CANCER DRUGS 80
3. ANTI-CANCER DRUGS 81
4. DIFFERENT ASSAYS TAKE DISADVANTAGE OF 85

VARIOUS PROPERTIES OF CELLS
5. CANCER CELL LINES 91
6. ANALYTICAL SPECIFICATIONS OF 99
CHENDOORAM
7. RESULTS OF SIDDHA STANDARDIZATION 151
8. PHYSICAL CHARACTERIZATION OF 153

KAALAMEGA NARAYANA CHENDHOORAM
9. RESULTS OF PHYSICAL PARAMETERS 153
10. RESULTS OF BASIC RADICALS 155
11. RESULTS OF ACID RADICALS 156
12. BACTERIAL AND FUNGAL LOAD 158
13. FTIR ANALYSIS RESULTS 159
14. ICP-MS INTERPRETATION OF KMNC 165
15. ACUTE ORAL TOXICITY 167
EFFECT OF KAALAMEGANARAYANA
16. 168
CHENDHOORAM ON BEHAVIOURAL SIGNS IN
RATS
17. EFFECT OF KAALAMEGANARAYANA 169
CHENDHOORAM ON BODY WEIGHT IN RATS
CHENDHOORAM ON WATER INTAKE IN RATS
S. NO TITLES PAGE NO.

CHENDHOORAM ON FOOD INTAKE IN RATS
28 DAYS OF REPEATED ORAL TOXICITY
20. 171
STUDY BODY WEIGHT OBSERVATION IN
RATS
21. 171
CHENDHOORAM ON WATER INTAKE OF 28
DAYS OF REPEATED ORAL TOXICITY IN RATS
22. 172
CHENDHOORAM ON FOOD INTAKE OF 28
DAYS OF REPEATED ORAL TOXICITY IN RATS
23. CHENDHOORAM ON HAEMATOLOGICAL 172
REPORTS OF 28 DAYS OF REPEATED ORAL
TOXICITY IN RATS
24. 173
CHENDHOORAM ON LFT OF 28 DAYS OF
REPEATED ORAL TOXICITY IN RATS
25. CHENDHOORAM ON BIOCHEMICAL 174
RESULTS OF 28 DAYS OF REPEATED ORAL
TOXICITY IN RATS
26. 174
CHENDHOORAM ON RFT OF 28 DAYS OF
REPEATED ORAL TOXICITY IN RATS
27. ANTI-CANCER ACTIVITY CELL LINE: KB CELL 178
LINE
28. 187
ANTIMICROBIAL ACTIVITY OF E.Coli
29. ANTIMICROBIAL ACTIVITY OF Pseudomonas 187

aeroginosa
30. ANTIMICROBIAL ACTIVITY OF Klebsiella 187
pneumoniae
31. ANTIMICROBIAL ACTIVITY OF Staphylococcus 188
aureus
32. ANTIMICROBIAL ACTIVITY OF Streptococcus 188
mutans
FIGURE CONTENTS
S.NO. TITLE OF FIGURES PAGE
1. IMAGE OF POTASSIUM NITRATE 30
2. IMAGE OF ALUMINUM SULPHATE 32
3. IMAGE OF COPPER SULPHATE 34
4. IMAGE OF SODIUM BORATE (BORAX) 35
5. IMAGE OF AMMONIUM CHLORIDE 37
6. IMAGE OF SODIUM CHLORIDE IMPURE 38
7. IMAGE OF IMPURE OF SODIUM CARBONATE 40
8. IMAGE OF MERCURY 42
9. IMAGE OF CINNABAR 45
10. IMAGE OF YELLOW ORPIMENT 47
11. IMAGE OF SULPHUR 49
12. IMAGE OF REALGAR ( RED ORPIMENT ) 51
13. INGREDIENTS OF KAALAMEGA NARAYANA 109

CHENDHOORAM
14. PREPARATION OF KAALAMEGA NARAYANA 113
CHENDHOORAM
15. FTIR INSTRUMENT 126
16. FTIR MECHANISM 127
17. SEM INSTRUMENT 128
18. SEM MECHANISM 129

S.NO. TITLE OF FIGURES PAGE
19. ICPMS- INSTRUMENT 131
20. ICP MS MECHANISM 132
21. XRD - X-RAY POWDER DIFFRACTION 133
22. XRD MECHANISM 133
23. MICROPLATE READER 146
24. CO2 INCUBATOR 146
25. COLOUR OF KMNC 151
26. FLOATING ON WATER OF KMNC 152
27. FINGER PRINT TEST OF KMNC 152
28. LUSTERLESS AND TASTELESS NATURE OF KMNC 152
29. IMAGES OF SEM 164
30. HISTOPATHOLOGY SLIDES 177
31. KB CELL LINES 183

CHART CONTENTS
S. NO CHART NAME PAGE NO.
1. FT-IR CHART
159
166
2. XRD CHART
3. KB CELL LINE CHART 179
4. TUMOUR CELL LINE CHART 184
5. TUMOUR CELL ANNEXIN V ASSAY 185
6. TUMOUR CELLS VARIATIONS CHART 185
7. CELL CYCLE ANALYSIS 185

ABBREVIATIONS
ALP Alkaline Phosphatase
ALT Alanine Transaminase
AST Aspartate Amino Transferase
ANOVA Analysis of Variation
BUN Blood Urea Nitrogen
CT Computed Tomography
COX Cyclooxygenase
CMC Carboxy Methyl Cellulose
CAMP Cyclic Adenosine Monophosphate
CPCSEA Committee for the Purpose of Control and Supervision of Experimental
Animals.
DMEM Dulbecco’s Modified Eagle’s Medium
DNA DeoxyRibo Nucleic acid
DC Differential Count
DSC Differential Scanning Calorimeter
EDX Energy Dispersive X-ray Spectrometry
FDG-PET F-18 Fluoro-2-deoxy-D-glucose
FAD-Assay Flavine Adenine Dinucleotide

FTIR Fourier Transform Infrared Spectrometry
GOT Glutamate Oxaloacetate Transaminase
GPT Glutamate Pyruvate Transaminase
HPV Human Papilloma Virus
HDL High Density Lipoprotien
ICPMS Inductively Coupled Plasma Mass Spectrometry
IAEC Institutional Animal Ethical Committee
ICMR Indian Council of Medical Research
LDL Low Density Lipoprotein
LD50 Lethal Dose
MCV Mean Corpuscular Volume
MRI Magnetic Resonance Imaging
MTT 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- Diphenyl Tetrazolium Bromide
MCH Mean Corpuscular Haemoglobin
MCHC Mean Corpuscular Haemoglobin Concentration
NCRP National Cancer Registry Programme
OECD Organisation for Economic Corporation and Development
PCR Polymerase Chain Reaction
PCV Packed Cell Volume
PET Positron Emission Tomography
RBC Red Blood Cells

SEM Scanning Electron Microscope
SEM Standard Error Mean
SGOT Serum Glutamate Oxaloacetate
SGPT Serum Glutamate Pyruvic Transaminase
VLDL Very Low density Lipoprotein
WDS Wavelength Dispersive Spectroscopy
WBC White Blood Corpuscles
WHO World Health Organization
XRD X-Ray Diffraction

INTRODUCTION
1. INTRODUCTION
Pharmacology is the science that deals with the study of drugs, which means a
drug is any substances or product that is used to modify or explore physiological
systems or pathological status for the benefit of the recipient and interactions with the
living systems.
The knowledge of drug usage often rested with the priest or holyman. Drugs
were thought to be magical in their actions, several cultures like the Chinese, Greek,
Indian, Roman, Persian, European and many others contributed to the development of
medicines in early times. Though the medicines developed simultaneously in several
countries. By the beginning of the 1st century, it was realised that there was a need to
standardize the method of obtaining uniform medical preparations. With the growth of
Science and development of scientific methods of research. Rigorous steps are followed
and care is exercised in the introduction of new drugs. Because of the advanced
scientific method of research and high incidence of cancer, I have preferred to select
Anti-cancer activity. The term anti-cancer describes natural methods of health care that
contributes in preventing the development of cancer or as a compliment to its
conventional approaches[1].
A bird view of oral cancer:
A cell is regarded as the true biological atom.

– George Henry Lewes
Cells are the building blocks of life. Cell is defined as the structural and
functional unit of living organisms. Our human body is made up of many tiny units
called cells. The organ system is defined as group of organs that work together to carry
out specific functions of the body. An organ is composed of two or more primary types
of tissues. Normally, cells grow and divide to form new cells as the body needs them.
When cells grow old, they die, and new cells take their place. Sometimes this orderly
process goes wrong. New cells form when the body doesn’t need them and old cells do
not die .The extra cells can form a mass of tissue called a growth or tumour. The kinds
of cells found in the tumour determine how the tumour behave. Likewise oral cavity is
ANTICANCER ACTIVITY OF KAALAMEGA NARAYANA CHENDHOORAM 1

INTRODUCTION
also made up of cells. Sometimes changes in the oral cavity can cause precancerous
conditions. These conditions may lead to a higher chance to become oral cancer[2].
The cells of your body are like a trillion light bulbs. When you relax, purify, and
connect to source, you are a powerhouse of light.
– Deane cooper
Oral health is the reflection of the physiological, social factors that are essential
to our quality of life. Peculiarities of the oral cavity are unique, it have a unique
anatomical structure, characteristics by the juxtaposition of soft and hard tissue. Oral
cavity begins at the border between the skin, lips, sides of the cheeks. The roof of the
mouth is formed by the hard palate. The lowest part of the oral cavity is the floor of the
mouth, which is covered by the tongue.
Poor Oral health care can result in poor overall health.

– George Jaylor
The first and foremost functions are the 1st part of digestion takes place in oral
cavity, source of respiration, sound manipulation, sensory organ for taste. Due to its
anatomical structure of oral cavity is continuously subject to challenge by the external
environment and foreign materials. In some cases, changes in cells of oral cavity can
cause cancer. Most often, Oral cavity cancer starts in flat, thin cells called squamous
epithelium, which is a layer of the mucous membrane. This type of cancer is called
squamous cell carcinoma of the oral cavity. [3].
Oral health is just as important as getting a regular physical. Its not just about
getting a cavity filled, its about the overall health of the individual.
– Jennifer Williams
Cancer is a disease characterized by uncontrolled proliferation of cells may form
a mass of tissue. Oral cancer is defined as an abnormal growth of cells in any part of
the oral cavity. Oral cavity is sometimes termed as head and neck cancer.Alcohol and
tobacco use including smokeless tobacco such as chewing tobacco or snuff, betel quid,
human papilloma virus (HPV), especially HPV-16, is a risk factor in the oropharyngeal
cancer are the most important risk factors in the cancers of head and neck. The signs
and symptoms include Lumps, nodules, White, smooth style scaly plaque appeared,

INTRODUCTION
Red patches, ulcers, difficult in swallowing, inflammation and other symptoms district
cannot be cured by a longer period, Oral repeated bleeding for no apparent reason,
Mouth for no apparent reason numbness, burning or dryness, Difficulty in speaking or
swallowing unusual.
Bitterness is like cancer. It eats upon the host, but anger is like fire, it burns it all
clean.
– Maya Angelo
Among the various types of cancer oral squamous cell carcinoma is the most
common type of cancer contributing to about 95% of all cancer and ranks at number
six globally. OSCC remains the major cause of mortality and morbidity in patients with
head and neck cancers. There are about 14.1 million of new cases estimated and 8.2
million people die out of cancer every year and 32.6 million people living with cancer
(within 5 years of prevalence) in 2012 worldwide.
Now-a-days there are so many therapies and treatments are validated includes
chemotherapy, radiation and surgery. Some of the most commonly used drugs for oral
cancer such as Cisplatin, 5-fluorouracil (5-fu) other drugs like carboplatin, Bleomycin,
Methotrexate. Prolonged use of the drugs produces certain side effects. Such as
Cisplatin which is used to treat different types of cancer such as cancers of head and
neck, cervical cancer, ototoxicity, neurotoxicity, electrolyte disturbances such as
hypokalemia, hypomagnesiumia, hypocalcemia [5].
Whereas 5- fluorouracil produces diarrhoea, soreness, redness and peeling on the

palm of the hand, sole of the feet. Bleomycin produces allergic reactions, fainting,
confusion, breathing difficulties. Methotrexate produces birth defects, weakness,
diarrhoea. Cetuximab produces pruritis, GIT abnormal pain, constipation, vomit.
However radiotherapy produces certain side effects such as mucositis, oral
candidiasis, loss of taste, xerostomia, osteo radio necrosis of bone within the radiation
field mostly in mandible, osteocytes[6].
Healing is a matter of time, but it is sometimes also a matter of opportunity.

– Hippocrates

INTRODUCTION
Siddha system of Medicine:

As the number of people, preferring natural health remedies and herbal health
remedies are increasing day by day and Indian medical systems are gaining popularity
all over the world. Now-a-days, this is the best time to introduce Siddha system to the
world.
Medicine is a science of uncertainly and an art of probability.
- Hippocrates
Siddha medical science is very ancient in origin, as old as the earliest
civilization. The exact time of its existence cannot be as certained as it is also
catagorically stated that it was before the spitting out of stand from the stone.
“கல்த ோன்றி மண் த ோன்றோக் கோலத்த
வோத ோடு முந்த ோன்றிய மூத் குடி”
- முதுமமோழி
The word Siddha comes from the word Siddhi which means an object to be
attained or perfection, or heavenly bliss. The Siddha medical system is the component
of Dravidian system of medicine belonging to Tamil Nadu. Age of the Siddha or Tamil
medical system can be dated back to 5000 B.C (History of Siddha Medicine). This
system is based upon the literary evidences and oral commentaries which were passed
from one generation to other. Siddhars who were eminent scholars and spiritual adepts
documented their experiences in palm leaf manuscripts, scriptures etc., for the welfare
of humanity. Of which, some were published and many remain unexplored.Generally
Siddhars were considered to be super human beings, who have defined age and other
laws of nature.
Siddha system of medicine is a part and parcel of the earliest Tamil medicine.
It provides a cheap and efficient service to the people. The aim of this system is to keep
the body and mind in a good condition. Siddhars had completely investigated the exact
cause, effect of diseases, all kinds of drugs and thereby came to realize what was
beneficial and what was not, to their existence in life.

INTRODUCTION
[7]
In Siddha system oral cancer is referred as Kanna putru or Vaai putru .
Cancer described in the modern medical system co-relates with that of the features
described in the Siddha literature under various names such as Vippurudhi, Putru noi,
Pilavai, Kazhalai etc.
The importance and fundamental principles of siddha system of medicine

were embedded in Thirukkural, and it clearly shows the longevity of the system not less
than two thousand years. Thiruvalluvar in his monumental work Thirukkural had
devoted a chapter for medicine. A deep study of those couplets threw more light on the
social and medical area of those ages in Tamilnadu which gave rise to these pithy
sayings,
“தநோய்நோடி தநோய்மு ல் நோடி அது ணிக்கும்
வோய்நோடி வோய்ப்பச் மெயல்”
- ிருக்குறள்
Disease is a state in which the body is not allowed to move about and the mind
to think about or a state which makes the mind static and fastens the body as if every
thing in a dominant state. It is pointed by Thollkappiyar, that the disease means
suffering and depression[8].
“பபயுளும் ெிறுபமயும் தநோயின் மபோருள்”
- ம ோல்.உோி. 34
Role of Metals in Cancer vs Siddha System:

Nature is the nature of diseases.
– Hippocrates
Nature has created innumerable plants, herbs, metals, poisonous substances,
minerals, salts, and other organic substances. Metals are essential cellular components
selected by nature to function in several indispensable biochemical process for living
organisms.
Nature itself is the best physician.
– Hippocrates

INTRODUCTION
Metals were endowed with unique characteristics that include redox activity,
Variable co-ordination modes and reactivity towards organic substrates. Due to their
reactivity metals were tightly regulated under normal conditions and aberrant metal ion
concentrations were associated with various pathological disorders including cancer.
The extremely diverse structural chemistry and interactions of metal complexes with
bio-molecules such as nucleic acids, proteins have resulted in the exploration of
anticancer activity[9]. However I would preffered to choose metal based Siddha trail
drug mettalo - mineralo based “KAALAMEGA NARAYANA CHENDHOORAM” as
my dissertation drug.
Whenever the art of medicine is loved, there is also a love of humanity.

– Hippocrates
Role of Nano Medicine :
The rich wealth of our country is selectively utilized for the welfare of mankind.
The unique nature of siddha medical system is its vast and complex pharmacology.
The raw materials used in siddha medicine are of plants, metals, minerals and
animal origin. The extensive use of metallo herbominsed formulations are considered
as higher order.
The unique preparations of siddha system of medicines like parpam,

chendhooram, kattu, and padhangam are like “life saving” and “miracle”. Which were
prepared by the siddhars on the basis of nano medicine.
Recent advances in science explored the nano particles, which find potential
usage in bio-medical field especially in cancer.
The aim of medicine is to prevent disease and prolong life, the ideal of medicine is
to eliminate the need of a physician.
- William James
Most of the medicines of the above category were found to contain nano
particles and it seems siddha has used special technique to prepare each and every
medicines. Coarse particles- 10000-2500nm, Fine particles-2500-100nm, Ultrafine
particles 1-100nm.

INTRODUCTION
Medicine is a science of uncertainly and an art of probability.

- Hippocrates
Many siddha nano medicines are already used in routine clinical practice and
are claimed to be the very effective and potent. Nano particles exhibits unique physico-
chemical properties such as ultra small size, greater surface area per weight than larger
particle and reactivity. They can deliver drugs at cellular level and nuclear level. They
are proved to be more effective since they improve the drug bio-availability. Nano
particles medicated delivery may provide a means of alternate route, circumvenating
the blood brain barrier.
With the effects of nano partical , chendhooram is a category of medicines

made from metals or minerals (arsenicals or mercurials or salts) by grinding them with
specified juices or distillates or extractives and subjecting them to a process of
sublimation or calcination or burning or frying or exposing to insolation till the
characteristic reddening of the product takes place. The chendhooram are said to retain
their potency for 75 years[9].
The good physician treats the disease, the great physician treats the patient who has
the disease.
– Hippocrates.
From this treasure, in-order to reduce the adverse effects of alternate

medicines, less cost effectiveness, due to increased rate of prevalence among the World
population, with the importance of Siddha medicine with its greater efficacy, nano
partical in nature of my trial drug. I hope to strengthen the fight against oral cancer.
“KAALAMEGA NARAYANA CHENDHOORAM” will be a valuable weapon of choice
for dealing with Oral Cancer.

AIM
2. AIM AND OBJECTIVES

AIM:
To justify the ancient Siddha drug for management of oral cancer with its
ultimate formulation and give good progress. The aim of this study is to establish the
scientific validation of the Anti-cancer, Anti-tumor and Anti-microbial activities of
“KAALAMEGA NARAYANA CHENDHOORAM” for obviously managing Oral
Cancer through pre-clinical aspects. In the present medical world, there is a need for
proper treatment for Oral Cancer. The aim of this study is to validate a new drug for the
management of oral cancer.
OBJECTIVES:
The main objective of the present study is to highlight the efficacy of
“KAALAMEGA NARAYANA CHENDHOORAM” on Oral Cancer, the following
methodology was adopted to validate the drugs and its standardization studies.
The key objectives of the study are:
 Collection of various Siddha and modern literature relevant to the study.
 Identification of drugs in this formulation.
 Preparation of “KAALAMEGA NARAYANA CHENDHOORAM” as per the
Siddha classical text.
 Physicochemical and phytochemical investigation of the test drug.
 To evaluate bio-chemical analysis of the test drug to derive acidic and basic
radicals.
 To estimate the presence of elements, functional groups and particle size
through instrumental analysis of the trial drug.
 Evaluation of the Acute and 28 days repeated oral Toxicity of the test drug
according to OECD guidelines.
 Validation of the pharmacological study of the drug through the following
activities
 Evaluation of Anti-Cancer activity
 Evaluation of Anti-tumor activity
 Evaluation of Anti-microbial activity of “KAALAMEGA NARAYANA
CHENDHOORAM”

REVIEW OF LITERATURE
3. REVIEW OF LITERATURE
3.1 DRUG REVIEW
3.1.1. SIDDHA ASPECT OF TRIAL DRUG
1. VEDIUPPU (Pottasium Nitrate)
Chemical name: Pottasium Nitrate.
Other names : Pottiluppu, Inangan, Padairasan, Boomikoormai, Navachara

Mithru.
Preparation:
The sand containing the crude salt is placed in a mud pot. Water is added to it
and mixed well and a straw is placed inside the pot and filtered. The filtered mixture is
heated to get the salt.
The Potassium nitrate salt is used for the preparation of explosives. It is also used
for cooling alcohol and to polish the gold ornaments.
General Properties:
“மல்லாரு மட்டகுன்ம மாதருத ரக்கட்டி

கல்லா மததப்புநீர்க் கட்டருக- லலல்லாமம
கம்பிகம்பி லென்றுங் கருவுண்டா மங்கிநின்ற
கம்பிகம்பி லென்றுதரக்குங் கால்”.
-குணபாடம் தாது சீவ வகுப்பு
It cures ulcers, tumours, Urinary disorders,Ascites, vatha diseases.
Purification:
1. Salt – 100gm
2. Water – 400gm
3. Fermented butter milk – 100gm
4. Lime juice – 100 gm
ANTICANCER ACTIVITY OF KAALAMEGA NARAYANA CHENDHOORAM

9
Water is added to the salt and boiled on a hearth with mild flames. The white of
eggs (4 nos ) is added to every 1400gm of salt and the bubbles appearing with impure
substances are removed with wooden spoon.
The ingredients are then transferred to another pot, sealed with mud pasted
cloth, filtered and transferred to another pot, sealed with mud pasted cloth, filtered and
kept in places without aeration. Next day the water is filtered and salt is dried under
sun shade. This process is repeated for seven times to get it purified.
Properties and uses:
 Potassium nitrate salt has got Demulcent, Diuretic And Diaphoretic

properties. This should be given by dissolving in large quantities of water.
 The salt is also useful in the treatment of eight types of Gunmam, Uterous
fibroids, Anorexia, Anaemia, Urinary Tract Infection, Dysuria, Strangulate,
Ascites, Menopause disorders, Abdominal Distention and Asthma. It
improves fertility in women.
 The salt is also effective in fever, swelling, rheumatic disorders,
haemorrhage, gonorrhoea, eye disease and sore throat.
Dosage: 650 mg – 1300mg.
This is described in the following Tamil verses:
 The Potassium nitrate salt is also used as one of the ingredients in tooth
powders.
 The Potassium nitrate salt is dissolved in water and given for the treatment
of silver nitrated poisoning[10]
2. PADIGARAM (Alum )
Chemical name: Aluminium potassium sulphate (Alum )
Other names : Cheena kaaram, cheenam, padigi.

10
Occurance:
This is available in nature and found in combination with special form of clay
in places such as Nepal, Kathiyawar, Punjab, Bihar. The alum is separated from the
clay. This appears like clusters and white in colour. It has sweet, sour and also astringent
in taste.
Purification and detoxification:
The alum is dissolved in water filtered, boiled and dried to get purified form.
Dosage and actions:
650mg to 1.3gm. It has astringent, antiseptic and antispasmodic in actions.
General properties:
“சீனலமனுங் காரமது சீறிவரு பல்லரதண

ஆதனக்கால் கண்மணாய் அனிலலமாடு - மாநிலத்தில்
துன்மாங் கிசம்வாயு மதாலாத உள்ளழதல
குன்மமிதவ மபாக்குலமனக் கூறு”.
It cures gingivitis, Eye Diseases, Ophthalmia, Elephantiasis, Tumours, Sense

of Heat, Gastric Ulcer, Pharyngitis, Gonorrhoea.
Uses:
 35 gm of alum dissolved in 10.4ml of water is used as mouth wash and for

washing the ulcers.
 In leucorrhoea with bleeding, alum is given with the juice of Adathoda
vasica juice daily.
 2.6gm of alum is given with sugar syrup for guinea worm infestations.
 For severe head injury 130mg of alum is administered along with sugar.
 Alum is used in hair dye preparations.

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Other preparations:
 Padigara parpam:
Absolute suppression of dysuria, stricture of urethra and padigara
chendooram.
 Padigara chendhuram:
Dysentery, blood stained dysentery, menorrhagia.
 Padikara patru:
Redness of eye, lacrimated eye. The medicine is spoiled if water is

added.
The alum is also one of the ingredient of Linga thuvar is used for control of
diarrhoea and poongavi chendhuram for menstrual bleeding[10a].
3. THURUSU (Copper sulphate)
Chemical name: Copper sulphate
Copper sulphate is available in nature and also synthesized chemically. It is

combined with sulphuric acid to form the copper sulphate salt which is blue in colour.
When powdered it is green in colour. This is soluble in water. Lead sulphate, Pottassium
nitratre, Thottiphasanam, Camphor, mica, alum, white phasanam, lead ore, toddy, rock
salt, zinc, lead, soap are considered antagonistic to Copper sulphate.
Yellow arsenic trisulphide, ammonium chloride, borax, per chloride of mercury,

sulphur, mercury, bismuth, cinnabar etc., are considered as agonistic to copper sulphate.
The copper available now a days in the market is not pure and it should be used
only after proper purification.
Methods of purification of Thurusu:
 The impure thurusu (copper sulphate) is dissolved in hot water and filtered.
It is then heated till the salt is formed.

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 Thurusu is triturated with honey and ghee and boiled in a crucible. Then
soaked in decanted milk / water for 3 days and dried. Thus purified thurusu
is free from toxicity and never induces vomiting.
 The thurusu is placed in cow’s urine and heated. It is then washed with
water and dried in sunshade to get purified.
 The thurusu is fried with ghee, till it turns to whitish.
General properties:
“புண்ணாற்றுங் காமிெத்தின் புண்ணாற்றுங் கண்மணாதெ

விண்மணற்று முத்மதாட வீறடக்குஞ் - சண்ணுகின்ற
வாந்திலொடு மபதிதரும் வாய்மநாய் சுரந்தணிக்குங்
காந்தி தருந்துாிசு காண்”.
Properties and uses of Thurusu:
It is a powerful astringent, emetic and antiseptic. It is an external stimulant,

styptic, and mild caustic. It acts as an astringent with the dose of is 1/8 th to 2 gm
weight and as an emetic with a dose of 5gm weight and it is also used in cases of
poisoning.
Thurusu cures ulcers, eye diseases, disorders of three humors, fever and mouth
diseases but causes vomiting, diarrhoea.
 Thurusu (325mg) mixed in 14 ml of honey is applied over the ulcers of the

mouth.
 It is used for the treatments like bleeding from nose, thurusu 260mg is mixed
in 28 ml of water and inhaled into the nose.
 Thurusu is powdered and dissolved in water, made it as a solution and
applied for hypertrophied ulcers.
Signs and symptoms of thurusu poisoning:
Since thurusu dissolves in rapidly water and absorbs in blood. It produces toxic
effect quickly. Over dosage of thurusu produces unpleasant taste, vomiting, nausea,
hematemesis, blue coloured vomitus, abdominal pain, dryness of throat, excessive

13
thirst, jaundice, paralysis and watering of eyes. Thurusu poisoning may also cause
death.
Antidote for poisoning:
 42ml of lemon juice is given 3times daily. Alternatively ginger juice, honey
and sugar may also be given together.
 Stomach wash has to be done for thurusu poisoning after which white of
egg or milk should be given.
Other preparations:
 Thurusu chenduram - Bilious heat, bilious, nausea, tetanus, ascites,

delirium.
 Pachai ennai -Carbuncles. ulcers[10b].
4. VENGARAM (Borax)
Chemical name - Sodium biborate,Borax
Other names - Porikaram, Karam, Urukkinam, Urukkumithran, Danganam,

Thoomathaiyadakki
Occurance:
Vengaram is obtained abundantly in California. It is also found in Tibet and

Nepal. Naturally it is obtained along with sand and dust.
Synthetic Preparations:
Vengaram is available in shops are not pure. Hence four parts of hot water and
a small amount of calcium carbonate (lime) are added to it, filtered, insolated and heated
till the water evaporates completely. The salt so obtained is pure and can be used. The
salt appears clean white and shiny which is soluble in water and insoluble in alcohol. If
it expands to air, white powder is deposited on the surface. If it is heated, the moisture
evaporates and seen with minute hole.

14
Actions:
 Demulcent
 Diuretic
 Sedative
 Tonic
 Alterative
 Antiseptic
 Astringent
General Properties:
“லசாறிபுதட லெண்குன்மநதம மசாாி ொசம்

பறிகிரகணி கல்லூனம் பன்மனாய் - லநறிதெத்
தடங்கணங்க பங்கிருமி சர்ப்பவிடஞ் சந்நி
ெிடங்கணங்க லக்கிற்மபா லமண்”.
 Toad skin
 Gastric ulcer
 Carbuncle
 Itching
 Haemorrhoids
Purification of Vengaram:
Vengaram is bundled and hanged in the buffalo’s dung solution and boiled.
The bundle is cleaned with fresh water and insolated to get it in purified form.
 It is washed in cow’s dung solution

 It is soaked in buffalo’s urine for 72 mins
Uses:
 35gm of Vengaram dissolved in water of 10.4 litres is used as a mouth

wash in case of oral ulcers and sore throat.
 It is also used in anal fissures and ulcers.

15
 Powdered Vengaram, sulphur, acacia catecheu ( kaichukatti) are taken

in equal quantities, mixed with ghee and applied as a ointment for
scabies, eczema, itching, and ulcer
 Vengaram powder is sprayed over silver fish to kill them.
 To destroy the worms which multiply in the drug, 42gm of Vengaram
is dissolved in 11.3 litre of water and sprayed
Other Preparations:
Venkkara parpam -Pitha diseases
Venkara maathirai - Gastric ulcers, throbbing pain, hernia, anaemia,

splenomegaly.
Venkkara kattu - It is used with pudam to get it as a parpam[10c].
5. NAVACHAARAM (Ammonium chloride)
Chemical name: Ammonium chloride
Other names : Istigai, salligai, sooligai, padu.
General properties:
“குன்மம் குடற்சூதல லகால்லும் மமகாதரத்தத

வன்தமயுறு கல்லதடப்தப மாற்றுங்காண் - சன்மக்
கவிச்சுமுத் மதாடங் கனவாத நீக்கும்
நவச்சார மாமத நவில்”.
General properties:
Abdominal pain, Distended abdomen, urinary calculus, bad odour in the skin,
sinusitis, amenorrhoea, whooping cough, intermittent fever, three humours, indigestion,
hepatomegaly, hepatitis, splenomegaly, rhinitis, tuberculosis, haematemesis and facial
palsy.
Dosage: 325mg to 975 mg. If given in high doses it may produce diarrhoea.

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This is available in small quantities in brick stone furnace. This is also obtained
by sublimation of coal, salt and dung ashes of camel. It has no smell, solid in state, fiber
in nature and so it is hard to powder. It is dissoluble in water and alcohol.
Colour: White or grey colour.
Taste : Bitter, sour, urine smell.
Synthetic preparation of Navachaaram:
The sand available at the places where animals and human beings defecate is
collected and placed in a pot. To one part of the sand, four parts of the urine is added;
the clear liquid obtained is taken out. Camphor, alum, and potassium nitrate (3500 gm
each) are powdered and burnt and added to 1300 liters of the liquid. This mixture is
poured in another pot and subjected to sublimation. Navachaaram settles as a
sublimate.
Purification and detoxification:
Navachaaram is dissolved in hot water and filtered. After it has cooled, it is

poured in a broad mouthed vessel and insolated; the salt is formed in a purified form.
It is preserved with small quantity of root of jequirity in a bottle.
Other uses:
 Navachaaram 4.2 g
 Alcohol 28 ml
 Rose water 560 ml
 Navachaaram is dissolved in alcohol and rose water mixture. A cloth is
soaked in this solution and applied over the mammary gland.
Indications:
 Suppresion of the secretion of breast milk, breast enlargement, abscess in the

breast and ulcer in the nipple.
 Navachaaram and potassium nitrate solution may be used for pain in the eye
and excessive lacrimation

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 It is used in the preparation of philosopher’s liquid.
Non-medicinal use of Navachaaram:
Navachaaram is used for welding the metals such as tin, iron, copper and
lead. It is also used in dying industry as a colouring agent.
Other preparations:
 Navachaara kuzhambu - Urinary retension, abdominal distention, anasarca,

ascites.
 Navachaara Ennai - ascites, anasarca, eight types of gastric ulcers, jaundice.
 Navachaara chendooram -ulcer, colic, paralysis of the limb, dropsy, eczema
 Navachaara kattu - Anaemia, jaundice, white discharge[10d].
6.KALLUPPU (Sodium chloride impure)
Chemical Name: Sodium chloride impura
Synonyms : Kadar kuruvi, Anna koormai, Arusuvai saathi, Uvaruppingunam.
General characters:
“ஐெமறுஞ் சூதல ெமராசிபித்தஞ் சத்திமொடு
லவய்ெபிணி ெட்டகுன்மம் விட்மடகும் - லபய்வதளமெ
வாதமதி தாகம் மலக்கட்டும் மபாமுலகிற்
மகாதறுகல் லுப்தபக் லகாடு.”
-குணபாடம் தாது சீவ வகுப்பு [10e]
It is effective in the treatment of Kapha, Pricking pain, Loss of appetite, Pitha

diseases, Eight types of Ulcers, Vatha diseases, Polydipsia and Constipation
7. POONEERU( Impure Sodium Carbonate)
Chemical name: Impure Sodium Carbonate
Other name: Poovazhalai
This is present in brackish soil. It is considered that the fuller’s earth should be
collected from the brackish soil during winter season and in the early morning before

18
the sunrise. It is collected from Sivaganga, Kalasthri, Mosur from the earth in the dew
season, before sunrise.
General properties:
“பார்த்திட்ட பூநீற்றின் பருவங்மகளு

பங்குனியுஞ் சித்திரதவ காசிக்குள்மள
பூர்த்திட்ட ரவிசுருக்கிற் லபாங்கிநீறும்
பூப்மபான்மம னிற்குமதத வாாிக்லகாள்ளு
Purification:
 Pooneeru 1.3 litre is soaked in dew’s water 5.2 litres and allow to settle.
Next morning it is churned well and the outer cream layer is removed. The
remaining mixture is kept in procelin plates and insolated to obtain purified
form. This process is repeated for ten times and stored in a bottle.
 According to Bogar fuller’s earth is dissolved in lemon juice and filtered.
The filterate is boiled till the water is completely evaporatesd to get purified
form.
Uses:
 Pooneeru and limestone are added in equal ratio and obtained clear water
solution. The solution is used to purify the tortoise shell. egg shell pearl
oyster, asbestos, forsil of crab, conch shell. The above materials are
individually kept with the above said solution and boiled to get purified
form. Arsenic compound may be purified with this solution.
 Pooneeru is mixed with the hot water. For curing, arthritis in the ankle joint
and the foot is kept in the above solution for sometimes [10f].
8. RASAM ( MERCURY)
Synonyms: Mercury or Quick Silver
Chemical name: Hydragyrum

19
Mercury is comes under the classification of ‘Pancha soothaam’. It has many

connotations such has Sootham, Punniyam, Bharatham, Inimai, Sivasathi, Kesari etc,
according to Dasangu nigandu.
Mercury is obtained from its ores from countries like Spain, California, Russia,
China and Japan. It is separated from its ore Cinnabar.
Types of Mercury:
Mercury was classified into five types.
1. Rasam
2. Rasendhiran
3. Sootham
4. Misaragam
5. Bharatham
Properties:
1. Vitalizer
2. Tonic
3. Laxative
4. Diuretic
5. Neutralising pitham
6. Silagogue
7. Anti-inflammatory
8. Medicine for venereal disease (Meganasini)
Taste : Six tastes dominated by sweet
Potency: Hot and cool (both -speciality)

20
Special properties of Mercury: Unlike other drugs Mercury is useful in the treatment
of diseases caused by both heat and cold.
Dhosam (Impurities) of Mercury: It is considered that there are two types of Dhosam
of Mercury. They are
1. Dhosam 2. Sattai (Kavasam)
In Dhosam there are 8 types of impurities in Mercury producing various

diseases as shown below
Impurities Disease caused by them
1. Undheenam Soolai (Throbbing pain)
2. Kowdilayam Kabhala noi (Diseases of the head)
3. Anavartham Biramai (Manic illness)
4. Sangaram Thathu nattam (Spermatorrhoea)
5. Sandathvam Sattium (distress)
6. Panguthvam Kuttam (Leprosy)
7. Samalathvam Moorchai (Syncope)
8. Savisthavam Sareera Elaippu (Loss of weight)
Sattai is an another type of classification, there are 7 types of impurities in

Mercury which produces various diseases as shown below
Impurities Diseases caused by them
1. Naagam Moolam (Haemorrhoids)
2. Vangam Tholnoikal (Skin disease)
3. Malam Arivinmai (Idiocy)
4. Vidam Maranam (Death)

21
5. Akkini Thaha moham (Polydypsia)
6. Giri Sattium (Distress)
7. Sabalam Thathu nattam (Spermatorrhoea)
General properties of Mercury:
“விழிமநாய் கிரந்திகுன்மம் லமய்ச்சூதல புண்குட்

டழிகாலில் விந்துவினால் அத்தத – வழிொய்
புாியு விதி ொது புாிெிமனா லெல்லாம்
இாியுவிதி ொது மில்தல”.
Proper use of Mercury as a medicine has the ability to cure the following
diseases they are disease in eyes, syphilis, eight types of ulcers (gunmam), throbbing
pain (soolai), chronic ulcers (perumpun), and leprosy (kuttam)
Purification and detoxification of Mercury:
 Mercury - 35gram
 Brick powder - 100gm
 Turmeric powder - 100gm
 Acalypha indica juice - 1.3 lit
Mercury was triturated with finely powdered brick and with turmeric powder for
one hour respectively and washed with water. Mercury is then boiled with the juice of
Acalypha indica, it is detoxified and then finally it is washed with water thus the
mercury is purified
Other preparations of Mercury:
 Sootha karuppu
 Rasa mezhugu
 Rasa thailam
 Megavirana kalimbu
 Rasa kuligai

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 Rasa parpam
 Mega virana kalimbu
 Rasa guru
 Rasagandhi mezhugu
 Rasa kattu
Signs and symptoms of mercurial poisoning:
Bleeding, dropsy, anaemia, excessive body heat, sweating, diarrheoa, thirst,

flatulence, blabbering, skin diseases, burning sensation of the limbs, head diseases,
fever, shivering, hiccough and finally death will occur.
Antidote for mercurial poisoning:
 For nephrotoxicity- Saya pattai (dye plant) root bark is powdered and given
along with jaggery.
 For loosening of teeth - the stem juice of ivy gourd (Coccinia indica) may be
poured on the tongue.
 If there is burning sensation in limbs, urticarial, dryness of throat and
unconsciousness- Arugankizhnagu (Cynodon dactylon) is triturated and mixed
in any one of the following milk such as goat’s milk, cow’s milk, or cotton seed
milk and administered[10g].
9. LINGAM (CINNABAR)
Synonyms: Natural Cinnabar, Vermilion.
Chemical name: Red Sulphide of Mercury.
Other names:
Inkuligam, Raasam, Kadai vanni, Karpam, Kalikkam, Kaanjanam, Kaaranam,

Sandagam, Samarasam, Saaniyam, Chendooram, Maniragam, Milechem, Vani and
Vanni.
Nowadays, The Lingam used by us is called as Jaathi linga paadanam, grouped

under Vaippu paadanam.

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Preparation of Vaippu paadanam:
 Rasam (Mercury) – 280 gm

 Gandhagam (Sulphur) – 70 gm
 Vediuppu (Pottassium nitrate) – 70 gm
Procedure:
Mercury is thoroughly mixed and triturated with Sulphur. Potassium nitrate is

then added, placed in a conical flask and burnt for 18 hours, after cooling with the red
Sulphide of Mercury is collected out.
Gunam (Properties):
It is hard, when it is put into fire it develops smoke; not soluble in water, has no
smell and taste.
General properties:
“மபதிசுரஞ் சந்நி லபருவிரண நீலராடுத

காதகடி காசங் கரப்பான்புண்-மணாத
வுருவிலிங்க சங்கதமா யூறுகட்டி யும்மபாங்
குருவிலிங்க சங்கமத்ததக் லகாள்
ஆதி ெிரதவுருக் காதலாற் சாதிலிங்க

மமாதி லிரதகுண முற்றுடலிற் – றீதுபுாி
குட்டங் கிரந்தி லகாடுஞ்சூதல வாதமுத
லுட்டங்கு மநாய்கதளமொட் டும்”.
It is effective in the treatment of diarrhoea, pyrexia, delirium,tuberculosis,

scabies, unknown insect bites, syphilis, leprosy, eczema, skin diseases, throbbing pain
and vadha diseases.
Method of purification:
Lime juice, cow’s milk and the Acalypha indica juice are mixed together in
equal proportion and allowed to fuse Cinnabar so as to get it in a purified potent form.

24
Other preparation:
Sanda Rasa Parpam - syphilis, arthritis, tremor, delirium and venereal diseases
Padigalinga Chendooram - dysentery, diarrhea, menorrhagia and fever
Saathi Sambera Kuzhambu - diarrhea, nausea, vomiting, syncope, fever and thirst
Linga chendooram - fever, syphilis.
Linga kattu - angina, syncope, anaemia, stomach ache.
Sign and symptoms of Cinnabar toxicity:
Dyspepsia, loss of taste, ulcers in the buccal cavity, uvula, inner portion of the
tongue, larynx and large intestine, foul odour from mouth, burning sensation are the
toxic symptoms of red Cinnabar.
Antidote:
Nutmeg (Myristica fragrans) - 4.2 gm
Cubeb pepper (Piper cubeba) - 4.2 gm
Root bark of red cotton tree (Gossypium arboreum) - 4.2 gm
Sugar candy - 4.2 gm,
These are mixed together and made into decoction and administered twice daily
for 48 days[10h].
10. THAALAGAM ( YELLOW ORPIMENT)
Synonyms: Yellow Orpiment
Chemical names: Yellow Arsenic Trisulphide, Trisulphuret of Arsenic
Other names:
Peethagi, Aalmbi, Paluppu, Kothantham, Maalam, Arithaaram, Kaalpuththi,

Ponvarni, Manjal varni, Maaldevi and Arithalam

25
Types:
Depending upon the colour, appearance and properties, Thaalagam has been
classified into four types.
1. Sivappu Aridharam (Red Orpiment)

2. Madal Aridharam
3. Pon Aridharam (Gold Orpiment)
4. Karattu Thalagam
General properties:
“தாளகத்தின் மபருதரக்கத் தாலுகவுள் மநாய்குஷ்டம்

நீளக் குளிர்காய்ச்சல் நீடுகபம்-நாளகங்லகாள்
துஷ்டப் பறங்கிப்புண் சூழழுகண் மண்தடமநாய்
கிட்டப் படுபமா கிளத்து”.
It is effective in the treatment of skin diseases, diseases of head and tongue,

kapha diseases, urinary tract diseases, and venereal focus ulcer in the urethra
Actions:
Expectorant, antipyretic, convalescent, tonic, emetic
Thaalagam is bundled and kept immersed in the mixture of lime stone, cow’s
urine, Aclypha indica juice and heated to get the purified form.
Other medicines:
Thalaga ennai (Virana sanjeevi thailam) - Heals chronic ulcers
Thalaga karuppu - Asthma, fever
Thalaga maaththirai - Fever, poisonous attack.

26
Signs and symptoms of Thaalagam poisoning:
Thaalagam if not prepared properly, it leads to be highly toxic. The following

symptoms such as burning pain of the stomach, gastritis, hoarseness of voice, nasal
bleeding, bleeding from the nail buds, itching over the head and redness in the tip of
the hairs, mental disorders, lower abdominal swelling and throbbing pain in the lumbar
region, bronchitis and sciatica are present.
Antidote:
Root Bark of Ceylon lead wort (Plumbago zeylanica) _ 8.75gm
Pepper (Piper nigrum) – 8.75gm
These are added together and made into decoction. Culinary salt (4.37gm) is
then added and the mixture is consumed twice daily for 21 to 42 days[10i]
11. GANDHAGAM (SULPHUR)
Chemical name: Sulphur, Sulphur
Other names:
Kaarizhain naatham, Parai veeriam, Atheetha prakaasam, Peejam, Sakthi,

Sakthi peesam, Selvi vindhu, Chendoorathaadhi, Naatham, Naatram, Deviuram,
Ponvaruni.
General properties:
“லநல்லிக்காய்க் கந்திக்கு நீள்பதிலனண் குட்டமந்தம்

வல்தல கவிதசகுன்ம வாயுகண்மணாய் - லபால்லா
விடக்கடிவன் மமகமநாய் வீறுசுரம் மபதி
திடக்கிரக ணீகபம்மபாந் மதர்”.
Gandhagam is bitter and astringent in taste. Its actions are laxative, tonic and
antiseptic. It increases the various secretions of the body including skin. When used in
high doses, it causes diarrhea.

27
Types:
Gandhagam is divided into four types depends upon their colour, appearance
and properties.
1. White coloured Sulphur
2. Red coloured Sulphur
3. Golden yellow coloured Sulphur
4. Black coloured Sulphur.
In addition, gooseberry Sulphur and stick Sulphur (Vaana gandhagam) have

been mentioned in most of the text books of ancient Siddha medicines. Gooseberry
Sulphur is one which is often used in medicinal preparations.
Gooseberry Sulphur (Nellikai gandhagam):
It is used in the treatment of 18 types of skin diseases, liver enlargement,

abdominal distension, eye diseases, chronic venereal diseases, chronic diarrhea, gastric
ulcer, poisonous bites, fever, and chronic dysentery.
Sulphur is placed in an Iron spoon. A small quantity of cow’s butter is added and
the spoon is heated till the butter melts, this mixture is immersed in inclined position in
cow’s milk. This procedure is repeated for 30 times to get purified Sulphur. Fresh milk
is to be used every time[10j].
12. MANOSILAI (REALGAR) RED ORPIMENT
Synonyms: Realgar
Chemical names:
Arsenic Disulphidum, Bisulphuret of Arsenic

28
Types:
Manosilai (Bisulphuret of Arsenic) is of two types
1. Piravi sarakku – It is naturally available.
2. Vaippu sarakku – It is obtained by adding 5 parts of Arsenic trioxide and 3 parts of

Sulphur.
General properties:
“லகாடிெ குஷ்டம் காய்ச்சல் நடுக்கலஜ கல்லிதரப்

புச்சிலந்திப் மபசறும மனாசிதலக்குப் மபசு”.
It has body strengthening and rejuvenating properties. Its potency is good. This is
effective in the treatment of leprosy, fever with chills, asthma, eye diseases, urinary
tract infections, kapha diseases and cervical adenitis.
Manosilai is triturated with any one of the following juices for 3 hours, Ginger
juice, lemon juice or butter milk, it is then dried to a get purified form.
Medicinal uses:
 It is not used alone but mostly in combination with other drugs, pills and oil.
 The oil is effective in the treatment of fistula.
Other medicines:
Kasthruri karuppu, Vishnu chakkara maaththirai, Gandhaga urundai,

Paashana maaththirai, Bhramanandha bhairavam, Sivanar amirtham, Sandhirodhaya
maaththirai[10j].

29
3.1.2 MODERN ASPECT OF TRIAL DRUG
1. POTASSIUM NITRATE
Potassium nitrate is a chemical compound with the chemical formula KNO3. It

is an ionic salt of potassium ions K+ and nitrate ions NO3−, and is therefore an alkali
metal nitrate. It occurs in nature as a mineral, niter. It is a source of nitrogen, from
which it derives its name. Potassium nitrate is one of the several nitrogen-containing
compounds collectively referred as salt peter or salt peter. Major uses of potassium
nitrate as a fertilizers, tree stump removal, rocket propellants and fireworks. It is one of
the major constituents of gun powder (black powder) and has been used since
the Middle Ages as a food preservative.
Fig no:1 Potassium nitrate
Etymology:
Potassium nitrate, because of its early and global use and production, has many
names. Hebrew and Egyptian words for it had the consonants n-t-r, indicating
likely cognation in the Greek nitron, which was Latinised to nitrum or nitrium. Then
Old French had niter and Middle English nitre. By the 15th century, Europeans referred
to it as salt peter and later as nitrate of potash, as the chemistry of the compound was
more fully understood.
Properties:
Chemical formula : KNO3
Molar mass : 101.1032 g/mol
Appearance : white solid

30
Odour : Odourless
Density : 2.109 g/cm3 (16 °C)
Melting point : 334 °C (633 °F; 607 K)
Boiling point : decomposes at 400 °C
Solubility in water : 133 g/L (0 °C), 242 g/L (20 °C), 2439 g/L (100
°C)[3]
Solubility :slightly soluble in ethanol, soluble

in glycerol, ammonia
Basicity (pKb) : 15.3[4]
Magnetic susceptibility (χ) : −33.7·10−6 cm3/mol
Refractive index (nD) : 1.335, 1.5056, 1.5604
Specific heat capacity (C) : 95.06 J/mol
Properties:
Potassium nitrate has an orthorhombic crystal structure at room temperature,

which transforms to a trigonal system at 129 °C (264 °F).Potassium nitrate is
moderately soluble in water, but its solubility increases with temperature. The aqueous
solution is almost neutral, exhibiting pH6.2 at 14 °C (57 °F) for a 10% solution of
commercial powder. It is not very hygroscopic, absorbing about 0.03% water in
80% relative humidity over 50 days. It is insoluble in alcohol and is not poisonous; it
can react explosively with reducing agents, but it is not explosive on its own.
Uses:
 It is used in Thailand as main ingredient for kidney tablets to relieve the

symptoms of cystitis, pyelitis and urethritis.
 Potassium nitrate is desensitise sore throat. It is used as a diuretic. It is used to

cure stomach ailments to arthritis.
 It also have aphrodisiac effects.

31
 It reduces blood pressure and vascular diseases.
 It improves the functioning of muscles and nerves when added with sodium,
creating the nervous systems electrical potential[11].
2. ALUMINUM SULPHATE
Aluminium sulphate, is a classical adjuvant most often used in vaccines in

humans, includes a range of salts of aluminum precipitated under basic conditions,
usually aluminum sulphate mixed with a sodium or potassium hydroxide plus a variable
amount of phosphate. The relative proportions will determine the size, charge, and
solubility of alum. The composition of alum used as an adjuvant varies in currently
available vaccines and may influence vaccine immunogenicity. Alum is utilized as an
adjuvant in many of the currently available vaccines composed of inactivated toxins or
recombinant proteins (live attenuated vaccines do not include alum or other adjuvants).
Alum, any of a group of hydrated double salts, usually consisting of aluminum
sulphate, water of hydration, and the sulphate of another element. A whole series of
hydrated double salts results from the hydration of the sulphate of a single charged
cation (e.g., K+) and the sulphate of any one of a number of triple charged cations
(e.g., Al3+).
Most alums have an astringent and acid taste. They are colourless, odourless, and exist
as a white crystalline powder. Alums are generally soluble in hot water, and they can
be readily precipitated from aqueous solutions to form large octahedral crystals.
Fig no:2 Aluminum sulphate

Properties:
Taste : Astringent
Colour : colourless
Odour : Odourless

32
Solubility : Generally soluble in hot water

Molecular weight : 258.192g/mol
Physical Description : Large transparent crystals or white crystalline powder.
Melting point : 92.5°C
Density : 1.725
PH : 3-4
Uses:
 Alums have many uses, but they have been partly supplanted by aluminum
sulphate itself, which is easily obtainable by treating bauxite ore with sulphuric
acid.
 The commercial uses of alums mainly stem from the hydrolysis of the aluminum
ions, which results in the precipitation of aluminum hydroxide. This chemical
has various industrial uses.
 Paper is sized, for example, by depositing aluminum hydroxide in the interstices

of the cellulose fibres. Aluminum hydroxide adsorbs suspended particles from
water and is thus a useful flocculating agent in water-purification plants. When
used as a mordant (binder) in dyeing, it fixes dye to cotton and other fabrics,
rendering the dye insoluble. Alums are also used in pickling, in baking powder,
in fire extinguishers, and as astringents in medicine[12].
3. COPPER SULPHATE
Copper sulphate, also known as cupric sulphate, or copper sulphate, is

the inorganic compound with the chemical formula CuSO4 (H2O)x, where x can range
from 0 to 5. The pentahydrate (x = 5) is the most common form. Older names for this
compound include blue vitriol, bluestone, vitriol of copper and Roman vitriol.
The pentahydrate (CuSO4·5H2O), the most commonly encountered salt, is

bright blue. It exothermically dissolves in water to give the aquo complex
[Cu(H2O)6]2+, which has octahedral molecular geometry.
The structure of the solid pentahydrate reveals a polymeric structure where in

copper is again octahedral but bound to four water ligands. The Cu (II) (H2O)4 centers

33
are interconnected by sulphate anions to form chains. Anhydrous copper sulphate is a

white powder.
Fig no:3 Copper sulphate
Properties:
Chemical formula :CuSO4 (anhydrous),CuSO4·5H2O

(pentahydrate)
Molar mass :159.609 g/mol (anhydrous), 249.685 g/mol

(pentahydrate)
Appearance : gray-white (anhydrous), blue (pentahydrate)
Solubility in water : 1.055 molal (10 °C), 1.26 molal (20 °C), 1.502
molal (30 °C)
Solubility : anhydrous insoluble in ethanol
Magnetic susceptibility (χ) : +1330·10−6 cm3/mol
Refractive index (nD) : 1.724–1.739 (anhydrous), 1.724–1.739

(anhydrous)
Uses:
 Copper sulphate pentahydrate is used as a fungicide. However, some fungi are

capable of adapting to elevated levels of copper ions. Copper sulphate is used
to test blood for aneamia. The blood is tested by dropping into a solution of
copper sulphate of known specific gravity –blood which contains
sufficient hemoglobin sinks rapidly due to its density, whereas blood which
does not sink or sinks slowly has insufficient amount of hemoglobin.

34
 Copper sulphate is used as an anti-fungal agent to protect seeds against fungus

and to protect horse hooves from infection. It inhibits growth of bacteria such
as Escherichia coli. Copper sulphate was used in the past as an emetic. It is
now considered too toxic for this use.
 It is a antiseptic agent. Antifungal agent for topical use. Treatment for copper
deficiency. Copper sulphate can be applied to your plants before disease starts
as a preventative as well as when you begin to notice the dark spots of infection
show.
4. SODIUM BORATE (BORAX)
Borax, also known as sodium borate, sodium tetraborate, or disodium

tetraborate, is an important boron compound, a mineral, and a salt of boric acid.
Powdered borax is white, consisting of soft colourless crystals that dissolve in water.
A number of closely related minerals or chemical compounds that differ in

their crystal water content are referred to as borax, but the word is usually used to refer
to the term dehydrate. Commercially sold borax is partially dehydrated.
Fig no:4 Sodium borate
Etymology:
The English word borax is Latinized: the Middle English form was boras,
from Old French boras, bourras. That may have been from medieval Latin baurach
(another English spelling), along with Spanish borrax and Italian borrace, in the 9th
century. Another name for borax is tincal, derived from Sanskrit.
History:
Borax was first discovered in dry lake beds in Tibet and was imported via
the Silk Road to the Arabian Peninsula in the 8th Century AD. Borax first came into

35
common use in the late 19th century when Francis Marion Smith's Pacific Coast Borax
Company began to market and popularize a large variety of applications under the 20
Mule Team Borax trademark, named for the method by which borax was originally
hauled out of the California and Nevada deserts in large enough quantities to make it
cheap and commonly available.
Properties:
Chemical formula : Na2B4O7·10H2O or Na2[B4O5(OH)4]·8H2O
Molar mass : 381.38 (decahydrate), 201.22 (anhydrate)
Appearance : white solid.
Density : 1.73 g/cm3 (solid)
Melting point : 743 °C (1,369 °F; 1,016 K) anhydrate
Boiling point : 1,575 °C (2,867 °F; 1,848 K)
Magnetic susceptibility (χ): −85.0·10−6 cm3/mol
Uses:
 It is used to make buffer solutions in biochemistry, as a fire retardant, as an anti-

fungal compound, in the manufacture of fiberglass, as a flux in metallurgy,
neutron-capture shields for radioactive sources, a texturing agent in cooking, as
a precursor for other boron compounds, and along with its inverse, boric acid,
is useful as an insecticide. In artisanal gold mining, the borax method is
sometimes used as a substitute for toxic mercury in the gold extraction process.
 Borax was reportedly and used it as gold miners in parts of the Philippines in
the 1900s. Borax has been in use as an insecticide in the United States with
various restrictions since 1946[14].
5. AMMONIUM CHLORIDE
Ammonium chloride is an inorganic compound with the formula NH4Cl and a
white crystalline salt that is highly soluble in water. Solutions of ammonium
chloride are mildly acidic. Sal ammoniac is a name of the natural, mineralogical form
of ammonium chloride. The mineral is commonly formed on burning coal dumps from
condensation of coal-derived gases. It is also found around some types of volcanic

36
vents. It is mainly used as fertilizer and a flavouring agent in some types of liquorice.
It is the product from the reaction of hydrochloric acid and ammonia. a white or
colourless, odorless, water-soluble, cubic crystalline salt with a biter taste.
Fig no:5 Ammonium chloride

History:
The earliest mention of ammonium chloride was in 554 A.D in China. At that
time, ammonium chloride came from two sources: (1) the vents of underground coal
fires in Central Asia, specifically, in the Tian Shan mountains (which extend
from Xinjiang province of north western China through Kyrgyzstan) as well as in
the Alay (or Alai) mountains of south western Kyrgyzstan, and (2) the fumaroles of the
volcano Mount Taftan in south eastern Iran. (Indeed, the word for ammonium chloride
in several Asian languages derives from the Iranian phrase anosh adur (immortal fire),
a reference to the underground fires.) Ammonium chloride was then transported along
the Silk Road eastwards to China and westwards to the Muslim lands and Europe.
Around 800 A.D. the Arabs of Egypt discovered ammonium chloride in the soot that
resulted from burning camel dung, and this source became an alternative to the sources
in Central Asia.
Properties:
Chemical formula : NH3
Appearance : Colourless
Odour : Strong pungent odour
Density : 0.86 kg / m3
Melting point : 77.73°C

37
Solubility : Soluble in chloroform, ether, ethanol, methanol
Refractive index : 1.3327
Viscosity : 0.276
Uses:
 Ammonium chloride is used as an expectorant in cough medicine. Its

expectorant activity is caused by irritative action on the bronchial mucosa,
which causes the production of excess respiratory tract fluid, which presumably
is easier to cough up.
 Ammonium salts as an irritant to the gastric mucosa and may induce nausea
and vomiting.
 Ammonium chloride is used as a systemic acidifying agent in treatment of

severe metabolic alkalosis, in oral acid loading test to diagnose distal renal
tubular acidosis, to maintain the urine at an acid pH in the treatment of some
urinary-tract disorders[15].
6. SODIUM CHLORIDE IMPURE ( KALLUPPU )
Chemical Name : Sodium chloride impura

Chemical Formula : NaCl (impure sodium chloride)
Fig no:6 Sodium chloride impura

38
Characters:
Black salt is type of rock salt. It is also known as Himalayas black salt. The
condiment is composed largely of sodium chloride with several other components
lending the salt its colour and smell. The smell is due to its sulphur content, due to the
presence of iron sulphide it forms brownish pink to dark violet translucent crystals when
whole and when ground into a powder it is light purple to pink in colour.
Composition of black salt:
It consist primary of sodium chloride and trace impurities of sodium sulphate,

sodium bisulfide, sodium sulfide, iron sulphide.
Unrefined sea-salt contains small amounts of magnesium and calcium halides

and sulphates and sulphates traces of algae products, salt resistance bacteria and
sediment particle[16].
7. IMPURE OF SODIUM CARBONATE
Fuller’s earth is a naturally occurring earthy substance that has a substantial

ability to adsorb impurities or colouring bodies from fats, grease, or oils. Its name
originated with the textile industry, in which textile workers (or fullers) cleaned raw
wool by kneading it in a mixture of water, and other contaminants from the fibres.
'Fuller's earth''' is a clay material that has the capability to decolourize oil or other
liquids without chemical treatment.
Modern uses of fuller's earth include absorbents for oil, grease, and animal
waste (cat litter) and as a carrier for pesticides and fertilizers.
Minor uses include filtering, clarifying, and decolourizing; active and inactive
ingredient in beauty products; and as a filler in paint, plaster, adhesives, and
pharmaceuticals.

39
Fig no:7 Impure of sodium carbonate
Etymology:
The English name reflects the historic use of the material for cleaning or
"fulling" wool by textile workers called "fullers". In past centuries, fullers kneaded
fuller's earth and water into woollen cloth to absorb lanolin, oils, and other greasy
impurities as part of the cloth finishing process.
Fuller's Earth is also known by the following other names:
 "Bleaching clay", probably because fulling whitened the cloth.

 "Whitening clay", particularly when used to treat facial pigmentation, such
as melasma.
 "Multani mitti", or "mud from Multan" in ancient India (current day Pakistan),
where it was used in cosmetics.
History:
Fulling is an important step in the production of woolen garments, and can be

traced back to ancient times. Cuneiform texts from Mesopotamia mention a raw
material, im-bab-bár (Akk. gaṣṣu ‘gypsum, plaster’), literally “white earth”, which was
delivered to fullers for the finishing of cloth. Pliny the Elder mentions several types of
fuller’s earth (creta fullonia in Latin) from a variety of locations, each with different
properties and therefore different uses.The first references to fulling mills are from
Persia, and by the time of the Crusades in the late eleventh century, fulling mills were
active throughout the medieval world, the use of Fuller's Earth across the Indian
subcontinent dates back to at least 1879. While its household use and transportation by

40
local carts in the Sindh region predates the 1800s, export by rail was first recorded in
1929 in British India.
Composition:
Fuller's earth consists primarily of hydrous aluminum silicates (clay

minerals). Common components are montmorillonite, kaolinite and attapulgite. Small
amounts of other minerals may be present in fuller's earth deposits,
including calcite, dolomite, and quartz. In some localities fuller's earth refers
to calcium bentonite, which is altered volcanic ash composed mostly of
montmorillonite.
Properties:
Density: 32- 37
Specific gravity: 32- 37
PH: 6.7
Uses:
In addition to its original use in the fulling of raw fibers, fuller's earth is now
utilized in a number of industries. Most important applications make use of the minerals
natural absorbent properties in products sold as absorbents or filters.
 Treatment for poisoning. Even given the risk of salmonella, the clay content of soil
could save the life of a person exposed to paraquat, for example, as paraquat is
intended to break down in soil.
 Decontamination: Fuller's earth is used by military and civil emergency service

personnel to decontaminate the clothing and equipment of servicemen and CBRN
(chemical, biological, radiological, nuclear) responders who have been
contaminated with chemical agents.
 Cleaning agent: In the Indian subcontinent, it has been used for centuries to clean
marble. As a good absorbent, it removes dust, dirt, impurities and stains from the
surface and replenishes the shine of the marble. It has been used numerous times to
clean the Taj Mahal, India with positive results.

41
 Litter box: Since the late 1940s, fuller's earth has been used in commercial cat litter.
 Cosmetology and dermatology:
The same properties that make fuller's earth effective at removing oils, dirt, and
impurities from wool are also effective on human hair and skin. Since ancient times
it has found extensive uses in the beauty industry, both as a cosmetic and as a
treatment for acne and other skin problems. Some clays have antiseptic properties,
which enhance their effectiveness as skin treatments, though not all forms of fullers
earth are truly antiseptic[17].
8. MERCURY
Mercury should not have less than 99.5 percent of Hg. It occurs naturally as a
sulphide ore called Cinnabar HgS. It also occurs in small globules disseminated through
rocks and as amalgam of Silver and Gold.
Symbol - Hg
Atomic number - 80
Atomic mass - 200.59g.mol-1
Isotopes - 12
It is the only metallic element that is liquid at standard conditions of temperature

and pressure[18].
Fig no:8 MERCURY

42
Preparation:
 It’s obtained by roasting Cinnabar in a current of air HgS+O2→Hg+SO2

 The free Mercury gets liberated it may be either purified by volatilization or
chemically by dropping Mercury into a column of dilute Nitric acid for
removing basic impurities.
Properties:
 It has shinning silvery white in nature. Heavy liquid easily divisible into
globules and extremely mobile it easy volatilizes on heating. It boils at
359.58°C.
 Almost insoluble in water, alcohol and HCl. it dissolves in cold and dilute Nitric
acid, giving mercurial nitrate and Nitric oxide.
6Hg+8HNO3→ 3Hg2 (NO3)2+2NO+4H2O
Density:
13.581ml at 25°C
Mercurial preparations:
 Mercury with Chalk (Gray powder)

 Yellow Mercuric Oxide (HgO)
 Mercuric Oxide
 Oleated Mercury
 Mercurous Chloride (HgCl-Calomel)
Tests for Purity:
It has been tested for weight per ml (at 25°C is about 13.5g).Non-volatile matter
residue at 300°C (not more than 0.02℅w/w).
Assay:
An accurately weighed quantity (0.49g) is dissolved in equal parts (20ml) of

water and Nitric acid. It is heated gently until the solution become colourless. The

43
solution is then diluted with water (150ml) and sufficienct quantity of Potassium
permanganate is added till a permanent pink colour is produced. A trace of Ferrous
sulphate to discharge pink colour is added. Then the solution is titrated with standard
0.1N Ammonium thiocyanate (1ml of 0.1N Ammonium thiocyanate =0.01003g), using
Ferric Ammonium sulphate as indicator. The temperature during the titration should
not exceed above 20°C.
3Hg+8HNO3→3Hg (NO8)2+4H20+2NO↑
Hg (NO3)2+2NH4SCN→2NH4NO3+Hg (SCN) 2
Uses:
It is a pharmaceutical aid for preparing Mercury with chalk .Formerly metallic

Mercury found use as such therapeutically as a cathartic and parasiticide. But which is
used as excess of has been extremely poisonous and prolonged inhalation of even very
minimal amounts of Mercury prove fatal. Almost all the salts of Mercury with the
exception of the Sulphide, has been poisonous.
1. Mercury with chalk (Grew powder)
 It is having 31 -35℅ w/w of Mercury and 62-70℅ w/w of CaCO3

 It is used as a purgative (Dose 60-300mg)
2. Yellow mercuric Oxide (HgO)
 It is having not less than 99.5℅ HgO. It is used as a mild anti-septic, as anti-
infective and antibacterial agents.
3. Mercuric Oxide:
 It contains not less than 95℅ but not more than105℅ w/w of the stated amount
of yellow Mercuric oxide
 It is used in ophthalmology, 1℅ ointment to treat mild inflammatory conditions
for the treatment of blepharitis and conjunctivitis.

44
4. Oleated Mercury:
 It has the equivalent of 20℅ of yellow Mercuric oxide

 It is used as an anti-infective.
5. Mercuric chloride (HgCl) (Calomel):
 It is being not less than 99.6℅ of HgCl

 It has been used for centuries as a cathartic but recently it is replaced by other
drugs.
Calomel has been insoluble in gastric juice and has been not absorbed from the
stomach. It gets absorbed in the intestine by the alkaline pancreatic juice where it slowly
gets dissociated into Mercury and irritant Mercuric compounds which have been
exerting a cathartic action [19].
9. CINNABAR
Cinnabar (red Mercury (II) Sulfide (HgS), vermilion) is the ordinary ore of
Hg. It is normally found in a substantial, granular form and is bright scarlet to brick-
red in colour. It is a chemical compound composed of the chemical elements Mercury
and sulphur (Mercury 86.22 % Sulphur 13.78 %).
Fig no:9 CINNABAR
Properties:
Formula - Mercury (II) sulfide
Symbol - HgS
Molecular formula - HgS
Number - 32

45
Colour - brownish red and lead-grey
Specific gravity - 8.176
Solubility - Soluble in water,
Molecular Weight - 232.66 gm
Melting point - 580 °C decomp.
Other anions - Mercuryoxide, Mercury selenide, Mercury

telluride
Other cations - Zinc sulphide, Cadmium sulphide
Fermion Index - 0.26
Boson Index - 0.74
Radioactivity - 0GRapi i.e not radioactive (Gamma Ray

American Petroleum Institute Units)
HgS which has long been used in combination with traditional Siddha and
Chinese medicine as a Sedative, Hypotonic, Ant inflammatory, Anti pyretic and
Analgesic for more than 2000 years and is still widely used in Asian countries[20].
An overdose of cinnabar in drugs such as Bapuslsan, which is used as a sedative

and in the management of external intoxication in the Chinese population[21].
It must be aware of its toxic effects due to high Mercury content. Previous
studies have shown that the insoluble form of HgS (or) cinnabar (10 g / 1water at about
20) can still be absorbed from GIT and liver[22].
Uses:
 Cinnabar is the only important ore of mercury.

 The bright red pigment ‘Vermilion’ and ‘Chinese red’ are made from
cinnabar[23].

46
Toxic symptoms of Cinnabar:
 Most of the soluble salts of Mercury are absorbed slowly from the intestinal
mucous membrane of the alimentary tract and produce their toxic effects.
 The long term use of cinnabar containing traditional medicines could result in
renal dysfunction due to accumulation of Mercury in kidney.
 Skin allergic reaction may occur when cinnabar is used in tattoo dyes.
 Blurred vision due to accumulation of Mercury in brain is possible.
10. YELLOW ORPIMENT
Arsenic compounds have been known since at least the days of Ancient Greece
and Rome (thousands of years ago). They were used by physicians. The compound
most often used for both purposes was arsenic sulphide (As 2 3)[24].
Orpiment is a deep orange-yellow coloured Arsenic Sulfide mineral. Its formula is

As2S3. It is formed by sublimation of Arsenic (60.90%) and Sulphur (39.10%). It takes
its name from the Latin Auripigmentum (Aurum − Gold + pigmentum − pigment)
because of its deep-yellow colour.
Fig no:10 YELLOW ORPIMENT
Synonyms:
Arrhenicum Operment, Orpiment, Yellow Arsenic, Yellow Ratsbane, Auripigment,

Arsenicum flavum
Physical Properties:
Colour - Lemon yellow, Orange yellow

47
Density - 3.49 - 3.56, Average = 3.52
Melting point - 300 °C to 325 °C
Diaphaneity - Transparent to translucent
Fracture - Sectile
Crystal Habit - Foliated
Hardness - 1.5-2 - Talc-Gypsum
Luminescence - Non-fluorescent
Luster - Pearly
Streak - Pale yellow
Electron Density - 3.23 gm / cc
Specific Gravity - 3.49 gm / cc
Boson Index - 0.9970676253
Photoelectric - 44.66 barns / electron
Radioactivity - 0 GRapi (Gamma Ray American Petroleum Institute

Units)
Chemical Properties:
Formula - As2S3
Elements - As, S
Common Impurities - Hg, Ge, Sb

48
Uses:
 Orpiment was traded in the Roman Empire and was used as a medicine in China.
 It has been used as a fly poison and to tip arrows with poison.
 Because of its Golden colour, it was used by alchemists, both in China and the
West, searching for a way to make Gold.
 It is used in the tanning industry to remove hair from hides[25].
11. SULPHUR:
Sulphur or Sulphur is a Greek word which means “to burn”. Sulphur is a

chemical element with the symbol S. It is a plentiful, multivalent non-metal. It occurs
in nature as the pure element and as Sulfide and Sulphate minerals. Sulphur is referred
in the Bible as brimstone (burn stone) in English. It is the sixth most abundant macro
mineral in the breast milk.
Fig no:11 SULPHUR
History:
It is discovered by Chinese Before 2000BC and is recognized as an element by Antoine

Lavoisier in 1777.
General properties:
Symbol - S
Atomic Number - 16
Element category - polyatomic nonmetal

49
Physical properties:
Phase - solid
Density - 1.96 g·cm−3
Liquid density at M.P - 1.819 g·cm−3
Heat of fusion - 1.727 kJ·mol−1
Heat of vaporization - 45 kJ·mol−1
Molar heat capacity - 22.75 J·mol−1·K−1
Electronegativity - 2.58 (Pauling scale)
Chemical properties:
Solubility - insoluble in water
Vanderwaals radius - 0.127 nm
Ionic radius - 0.184 (-2) nm; 0.029 (+6)
Isotopes - 5
Electronic shell - [Ne] 3s23p4
Standard potential - 0.51 V
Biological role:
Sulphur is a vital component of all living cells, it is the seventh most occurring
element in the human body by weight, and is used in biochemical processes. The
average person takes in around 900 mg of Sulphur per day, mainly in the form of
protein. In metabolic reactions, Sulphur compounds serve as both fuels and respiratory
materials. Its organic form is present in the vitamins biotin and thiamine. It is an
essential part of many enzymes and in antioxidant molecules.

50
Uses of sulphur:
 Organo sulphur compounds are used in pharmaceuticals and agrochemicals.

 Magnesium sulphate known as Epsom salts when in hydrated crystal form can
be used as a laxative.
 Elemental sulphur is one of the oldest fungicides and pesticides.
 Sulphur is the most important fungicide in organic production.
 Octa sulphur is used in pharmaceutical skin preparations for the treatment of
acne. It acts as a keratolytic agent and also kills bacteria, fungi, scabies mites
and other parasites[26].
Toxic effects of Sulphur:
Elemental Sulphur is nontoxic, but many simple Sulphur derivatives are, such
as Sulphur dioxide (SO2) and Hydrogen Sulfide are toxic which include neurological
effects, disturbance of blood circulation, heart damage, reproductive failure, stomach
and gastrointestinal disorder, dermatological effects[27].
12.REALGAR ( RED ORPIMENT)
Realger, α-As4S4 is an Arsenic Sulphide mineral, also known as “Ruby

Sulphur”. It is orange-red in colour, melt at 3200C, and burns with the bluish flame
releasing fumes of Arsenic and Sulphur. It is a photosensitive mineral and will alter to
Para Realger upon prolonged exposure to light. It has an Arabic name Rahj al ghar
which means "powder of the mine."
Other names: Ruby Sulphur, Ruby of Arsenic.
Fig no:12. REALGAR

51
Physical Properties:
Formula - As4S4 or AsS
Colour - Red to yellow-orange
Density - 3.56
Diaphaneity - Transparent
Specific gravity - 3.56
Melting point - 320 °C
Refractive index - 2.538
Luminescence - Non-fluorescent
Lustre - Sub Metallic
Streak - Orange
Electrical Properties:
Electron Density - 3.30 gm / cc
Boson Index - 0.9977521227
Radioactivity - 0 GRapi (Gamma Ray American Petroleum Institute

Units)[28].
Uses:
The Chinese name for Realgar is Xionghuang, literally 'masculine yellow'. It

was used to repel snakes, rats, weeds and insects, as well as being used in Chinese
medication. The ancient Greeks called it as “Sandaracha”. It is used in combination

52
with Potassium Chlorate to make a contact explosive known as "red explosive" for
some types of torpedoes[29].
3.2. LITERATURE REVIEW OF DISEASE
3.2.1. SIDDHA ASPECT OF THE DISEASE
Siddha system of medicine deals with cancer and its treatment widely. In ancient Siddha
literature, cancer is explained in the name of Putru which gives the direct meaning and
as Arpudham and Vanmeegam. This name comes from the appearance of the cut surface
of a solid malignant tumour, with "the veins stretched on all sides as the animal the crab
[30]
has its feet, whence it derives its name" . In Siddha system of medicine, Cancer is
referred to Vippuruthi or Putru.
For the Purpose of diagnose and treatment following reference books evaluates
great ideas about cancer.
1. Yugi Vaidhya Chintamani
2.AnubogaVaidhyaNavaneetham
3. Pulipani 500 [31]
4.Agathiya Vaidhya Vallathi[32]
In this medical system of life, the cancerous growth and tumours are headed as
Arputhaviranangal and Arputhakatttikal.
According to Yugimamuni varvaidhya sinthamani 800 I part, some kinds of cancer

clarified under different systemic diseases. Yugi classification of disease is compared
with Western system of medicine by means of symptoms for quick and easy approach.
For example, Ukkarasoolai is understand as prostatic cancer
Vilperuvayiru is known as Testicular cancer
Mamisamagotharam and Kalperuvayiru as cancerous growth within

abdomen.

53
Hippocrates (ca. 460 BC – ca. 370 BC) described several kinds of cancer,
referring to them with the Greek word Karkinos (Crab or Crayfish). This name comes
from the appearance of the cut surface of a solid malignant tumour, with "the veins
stretched on all sides as the animal the crab has its feet, whence it derives its name". In
Siddha system of medicine, Cancer is referred to Vippuruthi or Putru.
“புற்று மநாதெ லமளனப்பதகவன்

மதறந்திருந்து லகால்லூம் பதகவன்”.
-திருமந்திரம்
Generally, a chronic tumour or swelling or ulcer is first the identification of Putru in
Siddha system. Tumour grows gradually and finally look like a Putru or cauliflower.
Causes:
 Taking excessive amount of salt and pungent.

 Taking large quantity of fish and meat.
 Making sleep in day time.
 Doing frequent sex.
Symptoms are varying depending on the particular type of cancer. To handle cancer
effectively it is considered as Vippuruthi.
Types of Vippuruthi:
Vippuruthi is classified into seven types,
1. Karppa Vippuruthi
2. Kuvalai Vippuruthi
3. Vatha Vippuruthi
4. Pitha Vippuruthi
5. Seththuma Vippuruthi
6. Santhu Vippuruthi
7. Oodu Vippuruthi

54
Appearance
Causes of various classes look like one or more following appearance.
 Kazhalaikatti
 Spreading ulcer
 Initially like warts then grows and develops as turtle shell with
oozing
 Hyper pigmentation of skin, affects hair follicles and destroys
entire body.
1. Karppa Vippuruthi:
Gastric regurgitation, pain in side and lower abdomen, dryness of skin, lower
abdominal swelling like pregnancy and head ache.
2. Kuvalai Vippuruthi:
Pain in lower back, anal region and side of the chest, fever with shivering, cough
with expectoration, abdominal pain and swelling.
3. Vatha Vippuruthi:
Pain and swelling in the lower abdomen, this swelling look like a frog, fever,
wound in the abdomen, pus discharge from the wound and abdominal distension.
4. Pitha Vippuruthi:
Hematemesis, paleness of the skin, burning sensation all over the body,
shivering, mental disorder , hiccup, tastelessness of the tongue, fever, dehydration,
hematoma and abdominal pain.
5. Sethuma Vippuruthi:
Small tumour and abscess in the abdomen, abdominal pain, fever, cough and
swelling of the body.

55
6. Santhu Vippuruthi:
Swelling in the side of the abdomen, this swelling is characterized by shining,

hardness, cool and itching.
7. Oodu Vippuruthi:
Some time fever, blackish discolouration of skin, abdominal pain, giddiness,

vomiting, diarrhea and body pain.
Curable type of Vippuruthi:
1. Karpa Vippuruthi
2. Kuvalai Vippuruthi
3. Pitha Vippuruthi
4. Oodu Vippuruthi
Incurable type of Vippuruthi:
1. Santhu Vippuruthi
2. Sethuma Vippuruthi
3. Vatha Vippuruthi[33].
3.2.2. MODERN ASPECT OF THE DISEASE
Cancer:
Cancer known medically as malignant neoplasia, is a broad group of diseases

involving unregulated cell growth. In cancer, cells divide and grow uncontrollably,
forming malignant tumours, which may invade nearby parts of the body. The cancer
may also spread to more distant parts of the body through the lymphatic system or
bloodstream. Not all tumours are cancerous; benign tumours do not invade
neighbouring tissues and do not spread throughout the body. There are over 200
different known cancers that affect humans.

56
History:
Cancer has existed for all of human history. The earliest written record
regarding cancer is from circa 1600 BC in the Egyptian Edwin Smith Papyrus and
describes cancer of the breast. Hippocrates (ca. 460 BC – ca. 370 BC) described several
kinds of cancer, referring to them with the Greek word Karkinos (Crab or Crayfish).
This name comes from the appearance of the cut surface of a solid malignant tumour,
with "the veins stretched on all sides as the animal the crab has its feet, whence it derives
its name."Galen stated that "cancer of the breast is so called because of the fancied
resemblance to a crab given by the lateral prolongations of the tumour and the adjacent
distended veins".
Celsus (ca. 25 BC – 50 AD) translated Karkinos into the Latin cancer, also
meaning crab and recommended surgery as treatment. Galen (2nd century AD)
disagreed with the use of surgery and recommended purgatives instead. These
recommendations largely stood for 1000 years.
In the 15th, 16th and 17th centuries, it became acceptable for doctors to dissect
bodies to discover the cause of death. The German professor Wilhelm Fabry believed
that breast cancer was caused by a milk clot in a mammary duct.
The Dutch professor Francois de la Boe Sylvius, a follower of Descartes,

believed that all disease was the outcome of chemical processes, and that acidic lymph
fluid was the cause of cancer. His contemporary Nicolaes Tulp believed that cancer was
a poison that slowly spreads, and concluded that it was contagious.
The physician John Hill described tobacco snuff as the cause of nose cancer in
1761. This was followed by the report in 1775 by British surgeon Percivall Pott that
cancer of the scrotum was a common disease among chimney sweeps.
With the widespread use of the microscope in the 18th century, it was
discovered that the 'cancer poison' spread from the primary tumour through the lymph
nodes to other sites ("metastasis"). This view of the disease was first formulated by the
English surgeon Campbell De Morgan between 1871 and 1874.

57
Epidemiology of cancer:
Nearly seven lakh Indians die of cancer every year, while over 10 lakh are newly
diagnosed with some form of the disease. According to the latest World Cancer Report
from the World Health Organisation (WHO), more women in India are being newly
diagnosed with cancer annually. As against 4.77 lakh men, 5.37 lakh women were
diagnosed with cancer in India in 2012.
In terms of cancer deaths, the mortality rate among men and women in India is
almost the same. While 3.56 lakh men died of cancer in 2012 in India, the corresponding
number for women was 3.26 lakh.
One in every 10 Indians runs the risk of getting cancer before 75 years of age,
while seven in every 100 runs the risk of dying from cancer before their 75th birthday.
Cancer of lip and oral cavity has emerged as the deadliest among Indian men
while for women, is breast cancer. The top five cancers in men are lip/oral cavity, lung,
stomach, colourectum and pharynx, while among women they are breast, cervix,
colourectum, ovary and lip/oral cavity.
The global cancer burden jumped to 14.1 million new cases in 2012, with WHO
saying the marked increase in breast cancers must be addressed.
The International Agency for Research on Cancer (IARC) 2012 estimated 14.1
million new cancer cases and 8.2 million cancer-related deaths occurred in 2012,
compared with 12.7 million and 7.6 million, respectively, in 2008.
The most commonly diagnosed cancers worldwide were those of the lung (1.8
million, 13% of the total), breast (1.7 million, 11.9%), and colourectum (1.4 million,
9.7%). The most common causes of cancer death were cancers of the lung (1.6 million,
19.4% of the total), liver (0.8 million, 9.1%), and stomach (0.7 million, 8.8%).
Projections based on IARC 2012 estimates predict a substantive increase to 19.3

million new cancer cases per year by 2025, due to growth and ageing of the global
population. More than half of all cancers (56.8%) and cancer deaths (64.9%) in 2012

58
occurred in less developed regions of the world, and these percentage will increase
further by 2025[34].
Causes:
Cancers are primarily an environmental disease with 90–95% of cases attributed

to environmental factors and 5–10% due to genetics. Environmental, as used by cancer
researchers, means any cause that is not inherited genetically, such as lifestyle,
economic and behavioural factors, and not merely pollution.
Common environmental factors that contribute to cancer death include tobacco

(25–30%), diet and obesity (30–35%), infections (15–20%), radiation (both ionizing
and non-ionizing, up to 10%), stress, lack of physical activity, and environmental
pollutants.
It is nearly impossible to prove what causes a cancer in any individual, because

most cancers have multiple possible causes. For example, if a person who uses tobacco
heavily develops lung cancer, then it was probably caused by the tobacco use, but since
everyone has a small chance of developing lung cancer as a result of air pollution or
radiation, then there is a small chance that the cancer developed because of air pollution
or radiation[35].
SYMPTOMS:
 Persistant cough
 Change in bowel habits
 Blood in the stool
 Unexplained anaemia
 Breast lump or breast discharge
 Lumps in the testicles
 Change in urination
 Haematuria (blood in urine)
 Hoarseness
 Indigestion
 Unusual vaginal bleeding

59
 Unexpected weight loss

 Continued itching in anal or genital area
 Non healing sores
 Back pain, Pelvic pain[36].
Classification of cancer:
There are five broad groups that are used to classify cancer.
1. Carcinomas are characterized by cells that cover the internal and external parts
of the body such as lung, breast, and colon cancer.
2. Sarcomas are characterized by cells that are located in bone, cartilage, fat,
connective tissue, muscle, and other supportive tissues.
3. Lymphomas are cancers that begin in the lymph nodes and immune system
tissues.
4. Leukemias are cancers that begin in the bone marrow and often accumulate in
the bloodstream.
5. Adenomas are cancers that arise in the thyroid, the pituitary gland, the adrenal
gland, and other glandular tissues.
Viruses in Human Cancer:
Certain human malignancies are associated with viruses. Examples include

Burkitt’s lymphoma (Epstein-Barrvirus), Hepato cellular carcinoma (hepatitis virus),
cervical cancer [Human Papilloma Virus (HPV)], and T cell leukaemia (retroviruses).
The mechanisms of action of these viruses are varied but always involve activation of
growth-promoting pathways or inhibition of tumour suppressor products in the infected
cells.
For example, HPV proteins E6 and E7 bind and inactivate cellular tumour
suppressors’ p53 and pRB, respectively. Viruses are not sufficient for cancer
development but constitute one alteration in the multistep process of cancer[38].

60
Table no:1 TUMOUR MARKERS[38a]

NON-NEOPLASTIC
TUMOUR MARKERS CANCER
CONDITIONS
Hormones Gestational trophoblastic disease,
 Human chorionic gonadal germ cell tumour. Pregnancy
Gonadotropin
 Calcitonin Medullary cancer of the thyroid.
 Catechol amines Pheochromocytoma.
Oncofetal antigens Hepato cellular carcinoma, Cirrhosis, hepatitis
 Alpha fetoprotein gonodal germ cell tumour.
Adenocarcinoma of the colon,
 Carcino embryonic pancreas, lung, breast, ovary. Pancreatitis, hepatitis,
antigen inflammatory bowel
disease, smoking
Enzymes
Prostatic acid phosphates Prostatic cancer Prostatitis, prostatic
Neuron specific enolase, Small cell cancer of the lung, hypertrophy Hepatitis,
Lactate dehydrogenase neuroblastoma. hemolytic anemia
Lymphoma, Edwing’s sarcoma.
Metastasis:
Metastasis is the spread of cancer to other locations in the body. The new tumours
are called metastatic tumours, while the original is called the primary tumour. Almost
all cancers can metastasize. Most cancer deaths are due to cancer that has spread from
its primary site to other organs[39].

61
Head and neck cancer:
Tumours of the head and neck are the sixth most common malignancy in the
world, with a yearly incidence of more than 500,000 cases, and it comprises
approximately 4% to 5% of all new cancers and 2% of all cancer deaths (100,000 per
year). Most patients are older than 50 years, and incidence increases with age; the male-
to-female ratio is 2.5:1. Approximately 34% of oral and pharyngeal cancers present as
localized disease, 46% present as locoregional (i.e., locally advanced or involving
regional lymph nodes) disease, and 10% present as metastatic disease. Ninety percent
of these cancers involve squamous cell histology. The most common sites are the oral
cavity, pharynx, larynx, and hypopharynx. Nasal cavity and paranasal sinus cancers,
salivary gland malignancies, and various sarcomas, lymphomas, and melanoma are less
common.
Site-specific head and neck tumours:
Oral cavity:
The oral cavity includes the lip, anterior two thirds of the tongue, floor of the
mouth, buccal mucosa, gingiva, hard palate, and retro molar trigone. Squamous cell
carcinoma is the histologic type observed in most cases.
Oropharynx:
The oropharynx includes the base of the tongue, tonsils, posterior pharyngeal
wall, and the soft palate.
Larynx:
Risk factors are history of tobacco and/or alcohol intake. In addition, certain
dietary factors and exposure to wood dust, nitrogen mustard, asbestos, and nickel have
been implicated as etiologic factors. The male-to-female ratio for laryngeal cancer is
4.5:1, with a peak incidence in the sixth decade of life. More than 95% of laryngeal
cancers are squamous cell carcinomas.
Laryngeal cancers can be supraglottic, glottic, and/or subglottic. Early cancers not
requiring laryngectomy are usually treated with radiation. If lymph nodes are involved,

62
neck dissection and/or neck radiation is indicated. Locally advanced resectable tumors
may be treated with surgery and adjuvant radiation if locoregional risk factors are
present. An alternative is the use of combined radiation and chemotherapy.
Hypopharynx:
Early cancers not requiring laryngectomy can be treated with surgery or

radiation. Locally advanced respectable tumours may be treated with surgery followed
by radiation or sequential or concomitant chemoradiation. In these cases, surgery
involves total laryngectomy and partial or total pharyngectomy and neck dissection.
Nasal Cavity and Paranasal Sinuses:
Most tumours are squamous cell carcinomas and are usually slow growing with
low incidence of metastasis. Carcinomas of the nasal cavity and paranasal sinuses are
usually detected in patients in advanced stages because of the relatively silent tumour
location. Treatment follows the same general guidelines as those for oral cancer.
Nasopharynx:
It is extremely rare in most parts of the world, with an incidence of less than 1
case per 100,000 population. However, it is endemic in certain areas, including North
Africa, Southeast Asia, China, and the far northern hemisphere. EBV is strongly
associated with nasopharyngeal carcinoma. This association has been demonstrated by
serologic studies and by the detection of the viral genome in tumour samples. Diet (salt-
cured fish and meat) and genetic susceptibility are other probable risk factors; tobacco
and alcohol are not risk factors, except in a minority of cases.
Salivary Gland Cancer:
Salivary gland cancers make up about 3% of all head and neck cancers
diagnosed in the United States yearly. Tobacco and alcohol consumption are not risk
factors, except possibly in women. Ionizing radiation and certain occupational
exposures (e.g., in workers of rubber and automotive industries, wood workers, and
farm workers) have been associated with the development of salivary gland cancer.

63
The salivary glands are classified as major (parotid, submandibular, and

sublingual) and minor (distributed along upper aero digestive tract, predominantly in
the oral and nasal cavities and the paranasal sinuses). Most of the salivary gland cancers
arise from the parotid glands; sublingual and minor salivary gland cancers are rare.
Most salivary gland tumours are benign, and the most common histology is
pleomorphic adenoma, which is characterized by slow growth and few symptoms, and
is most frequently seen in the parotid gland. The most common presentation of benign
salivary gland tumours is asymptomatic swelling of the lip, the parotid, or the
submandibular or the sublingual glands. Persistent pain or neurologic involvement
(mucosal or tongue numbness and facial nerve weakness) suggests malignant disease.
Surgery is the main stay of treatment for all localized stages of salivary gland
tumours. Postoperative radiation is indicated for localized tumours of high-grade
histology that are large, with close or positive margins, and/or positive regional lymph
nodes. Radiation is the primary treatment for unresectable tumours. The role of
chemotherapy is limited to management of locally recurrent, unresectable disease or
distant metastatic disease. There is no established standard chemotherapy for salivary
gland cancer. Regimens employing cisplatin, carboplatin, anthracyclines, taxanes,
cyclophosphamide, and 5-FU result in transient responses in 14% to 30%[40].
Other head and neck tumours:
Sarcoma:
Soft tissue sarcomas of the head and neck are relatively rare. Of head and neck
sarcomas, 80% are seen in adults and 20% are in children. These tumours are
heterogeneous and can present in any head and neck site, commonly as a submucosal
or subcutaneous painless mass. In the hypopharynx and nasopharynx, the presenting
symptoms may be cranial nerve abnormalities or airway or swallowing difficulties. As
in sarcomas at other sites, grade is an important prognostic indicator. High-grade,
aggressive tumours such as malignant fibrous histocytoma, angio sarcoma, osteogenic
sarcoma, neuro fibrosarcoma, and soft part sarcomas tend to be locally aggressive and
spread along neurovascular structures, fascia, and bone. In addition to aggressive local
behaviour, there is a high risk for metastatic disease, particularly in lung, bone, central

64
nervous system, and liver. Metastatic disease may occur without local lymph node
involvement. Sarcomas may arise after radiation therapy, but this is very uncommon in
the head and neck region. The prognosis for these secondary sarcomas may be worse
than for primary sarcomas.
Treatment depends on stage, age of the patient, tumour type, location and size.
Wide margin resection is the goal, but may not be possible because of the proximity of
vital structures. Adjuvant postoperative radiation and/or brachytherapy can improve
local control in aggressive sarcomas. The major indications for adjuvant radiation are
high-grade sarcomas or positive margins, lesions greater than 5 cm, and recurrent
sarcoma. Elective neck radiation is not necessary because the incidence of occult
positive lymph nodes is low. Soft tissue and possibly osteogenic sarcomas may benefit
from adjuvant or neoadjuvant chemo radiation. Such patients should be referred to
clinical trials when possible. Overall survival rate approaches 60% for patients with
sarcomas of the head and neck.
Melanoma:
Mucosal melanomas represent less than 1.5% of all melanomas. About 50% of
mucosal melanomas occur in the head and neck, and more than 20% of melanomas that
occur in the head and neck region are mucosal. The age of diagnosis is 60 to 80 years.
The hard palate is the most common site. Nearly one-third of these tumours are
amelanotic. The proportion of mucosal melanomas is higher in African American and
Hispanic populations than in white populations. Although rare in the United States,
mucosal melanomas are more frequent in Japan and in some parts of Africa. Mucosal
melanomas may be multiple, may have satellite lesions, may invade angio lymphatics,
and can metastasize. They behave more aggressively than skin melanomas. Lymph
node metastasis is observed at presentation in up to 48% of patients. Surgery is the
mainstay of treatment for local or locoregional disease. Prophylactic lymph node
dissection is not recommended. Radiation, when used, is usually employed adjuvantly
for positive margins or used palliatively for local recurrence or unrespectability.
Adjuvant use of radiation has not been shown to improve survival. Prophylactic nodal
radiation is not recommended. Chemotherapy and Immunotherapy have been studied,
but the effect of these interventions on survival when used as palliation or as adjuvant

65
therapy has not been defined. Patients should be encouraged to enter clinical trials
where available. Mean overall 5-year survival is 17%[41].
Oral cancer:
Oral cancer is one of the more common in head and neck malignancies. Oral
cancer is a general term for oral cavity cancers. It occurs in the majority were squamous
cell carcinoma, which is called the mucosa mutate. In clinical practice, oral cancer,
including cancer gums, tongue, hard and soft palate cancer, carcinoma of the mandible,
floor of mouth cancer, oropharyngeal cancer, salivary gland cancer, lip cancer, and
maxillary sinus cancer occurs in the facial skin and mucous membranes of cancer and
so on.
Causes of Oral cancer:
Long-term habit of tobacco, alcohol:
Most oral cancer occurs in patients with long-term history of smoking and
drinking. Poor oral hygiene:
Poor oral hygiene, bacteria or fungi in the mouth breeding, breeding to create
the conditions, thus contributing to the formation of nitrosamines and their precursors.
Coupled stomatitis, proliferation of some cells in the state, more sensitive to
carcinogens, so a variety of reasons may contribute to oral cancer.
Long-term stimulation of foreign body:
Root or sharp cusp, inappropriate dentures long-term stimulation of oral

mucosa, resulting in chronic ulcers and even cancer.
Malnutrition:
Vitamin A deficiency can cause oral mucosal epithelial thickening,

hyperkeratosis with the occurrence of oral cancer. Demographic studies show that
countries with low intake of vitamin A have high incidence of oral cancer. There are
also inadequate intake of trace elements considered relevant, such as low zinc content
of foods. Zinc is indispensable for the growth of animal tissue elements, zinc deficiency

66
may lead to mucosal epithelial damage, and create favorable conditions for the
occurrence of oral cancer. Also total protein and animal protein intake may be
associated with inadequate oral cancer[42].
Leukoplakia and Erythema:
Oral Leukoplakia and hypertrophic Erythema often a precancerous lesion.
Associated lesions:
The relationship between oral cancer and precancerous lesions:
White ulcers or blisters are often occurs in the buccal mucosa occurs, often
occurs in pressure, poor sleep or eating habits (such as insufficient fruit) on the
occasion, the general will heal within two weeks; If more than two weeks is not cured,
must be examined to rule out the possibility of epithelial cell carcinoma.
Changes in the oral mucosa colour:
Normal epithelium pink, red or a white colour of polarization are not normal.
If its red with white, it is more serious situation, another example of the tongue appears
dark red with white dots occur like, highly suspicious of cancer.
Ulcer:
Over more than two weeks of oral mucosa has not yet healed.
Clinical manifestations:
1. Lumps, nodules;
2. White, smooth style scaly plaque appeared;
3. Red patches, ulcers, inflammation and other symptoms district cannot be cured by a
longer period.
4.Repeated Oral bleeding for no apparent reason.
5. Mouth for no apparent reason numbness, burning or dryness.

67
6. Difficulty in speaking or swallowing unusual[43].
Differential diagnosis:
Traumatic ulcers:
This ulcers often occur in the tongue side edge, the corresponding total at the fangs
and ulcers, or irregular teeth and root dental prosthesis, indicating that ulcers are caused
by the stimulus. Ulcers soft, soft substrates, no induration. Eliminate these irritants 1 to
2 weeks after the ulcer to heal.
Tubercular ulcers:
These are almost secondary, mostly open tuberculosis direct result of the spread,
often occurs in the soft palate, buccal mucosa and tongue back, shallow ulcers
compared with cancerous ulcer, ulcer base induration soft non-invasive, effective anti-
TB treatment. Imaging and biopsy can accurately help identify and diagnose.
Diagnosis:
When neck mass is the first presentated, the primary site can be located and
biopsied in approximately 80% of cases. If no primary site is obvious, tissue diagnosis
can be obtained by fine needle aspiration (FNA) biopsy of the node, with sensitivity
and specificity approaching 99%. A non - diagnostic FNA does not rule out the presence
of tumour.
Computerized tomography scan (CT scan) remains the primary imaging study
for evaluation of metastatic adenopathy. Magnetic resonance imaging (MRI) may
complement with CT scan. Positron emission tomography (PET) scans are being used
more frequently to detect tumours that are not obvious on other scans.
Laryngoscopy and naso pharyngoscopy should be performed. With occult

primary tumours, directed biopsy of the nasopharynx, tonsil, base of tongue, and
pyriform sinus should be performed Bilateral tonsillectomy will sometimes reveal the
source of an occult cancer[44].

68
Treatment:
The management of patients with head and neck cancer is complex. The choice
of treatment modality depends on the stage and site of disease. Patients with locally
advanced disease should be evaluated (prosthodontics, nutrition, speech, and
swallowing) by a multidisciplinary team including otolaryngologist or head and neck
surgical oncologist, radiation oncologist, medical oncologist, dentist, and personnel
involved in rehabilitation before treatment is initiated[46].
In general, either surgery or radiation is effective as single-modality therapy for

patients with early-stage disease (stage I or II) for most sites. The choice of modality
depends on local expertise, patient preference, and functional result. For the 60% of
patients with locally advanced disease (stage III, IV, and M0), combined-modality
therapy is indicated.
TREATMENT:
 Surgery
 Radiation therapy
 Chemotherapy
 Immunotherapy
 Targeted therapy
 Hormone therapy
 Stem cell transplant, Precision medicine.
Surgery:
The nature of the surgical procedure is determined primarily by the size of the
tumour and the structures involved. Extensive surgeries and those cancers involving
function of the tongue. Frequently require myo cutaneous flaps or microvascular free
flaps to achieve a more functional reconstruction.
Resectability depends on the experience of the surgeon and the rehabilitation

team. In general, a tumour is unresectable if the surgeon believes that all of the gross
tumour cannot be removed or that local and distant control will not be achieved after

69
surgery even with adjuvant radiation therapy. Generally, involvement of the skull base,
pterygoid, and deep neck musculature, and of the major vessels portends a poor
outcome with surgery as a primary modality.
Cervical lymph node dissections may be elective or therapeutic. Elective neck

dissections are done at the time of surgery in patients with necks that are clinically
negative when the risk of a positive lymph node is at least 30%. Therapeutic neck
dissections are done for clinically obvious masses. This surgery is now rarely performed
because of excessive morbidity, especially loss of shoulder function. The modified
radical dissection preserves one or more of the nonlymphatic structures. In selective
neck dissections, only certain levels of lymph nodes are removed on the basis of the
specific lymphatic drainage from the primary site.
Radiation Therapy:
The use of radiation as a single therapy in early-stage tumours (i.e., T1 and T2)
is as efficacious as surgery. The choice of therapy depends on expected quality of life,
functional outcome, sequelae of therapy, and options for treatment in case of
recurrence.
In locally advanced tumours (i.e., T3 and T4), radiation therapy is combined

with surgery. In general, postoperative radiation is preferred over preoperative radiation
according to the results of two randomized prospective studies that show superior local
control and minimally increased survival in the postoperative radiation arm in hypo
pharyngeal cancer patients. Postoperative radiotherapy is recommended for patients at
high risk for local recurrence [i.e., T4 tumour, close or positive margins (<5 mm),
perineural or perilymphatic or vascular invasion by the tumour, multiple or large
positive nodes, and/or extracapsular invasion].
The radiation type varies for specific sites and for definitive versus adjuvant
therapy. The standard fractionation regimen in the United States is 1.8 to 2.0 Gy once
daily, 5 days per week. The total dose of irradiation for definitive treatment is in the
range of 70 to 80 Gy depending on the treatment schedule given and on the ability to
shield normal tissue.

70
Common severe acute radiation toxicity includes epidermitis, mucositis, loss of

taste, xerostomia, dysphagia, and hair loss. Dental evaluation and necessary extractions
should be performed before radiation because dental extractions in a radiated mandible
can lead to osteonecrosis. Dentulous patients should be given prophylactic fluoride.
Patients receiving radiation are at high risk for tooth decay due to the xerostomia caused
by injury to the salivary glands as well as mucosal damage.
Brachytherapy can be used as a definitive treatment for early-stage tumours or

combined with external beam radiation in more advanced lesions in selected tumours
(e.g., tongue, floor of mouth, tonsil, and nasopharynx) with excellent results.
Brachytherapy is an option for recurrent cancers in the head and neck, particularly in
previously irradiated patients[46].
Chemotherapy:
Until relatively recently, chemotherapy was used mainly for palliation of patients
with locally recurrent or disseminated disease without proven survival advantage.
Combination chemotherapy yields higher response rates but has increased toxicity with
no proven survival advantage when compared with single agents. The choice of single-
agent or combination chemotherapy depends on the patient's preference and
performance status. Several Combination regimens have been developed to improve
response rates. The combination of cisplatin and infusional 5-fluorouracil (5-FU)
produces a 70% response rate and a 27% complete remission (CR) rate in
chemotherapy-naive patients.
Platinum-based chemotherapeutic regimens and the single agent methotrexate

are the most commonly used regimens for metastatic disease. Carboplatin may be
slightly less active than cisplatin for head and neck squamous cancer, but carboplatin
combinations with other chemotherapy agents are generally better tolerated than those
with cisplatin. Carboplatin is preferred in patients at high risk for cisplatin toxicity, that
is, those with renal dysfunction, neuropathy, or hearing loss.
Both docetaxel and paclitaxel have shown antitumor activity. Several dosing
schedules for paclitaxel have been investigated. Three-hour infusions are probably the

71
best balance between theoretically optimum exposure and tolerable toxicity. Docetaxel
is usually administered at doses of 60 to 100 mg per m2 every 3 to 4 weeks.
The role of chemotherapy has expanded significantly over the last decade because
of the results of clinical trials incorporating chemotherapy in multimodality regimens
for previously untreated disease.
Induction Chemotherapy:
Induction chemotherapy followed by definitive radiation therapy in patients

responding to chemotherapy has been studied for organ preservation in patients with
locally advanced cancers of the larynx and of the hypopharynx. No significant survival
difference has been demonstrated for chemotherapy followed by radiotherapy
compared to surgery followed by radiotherapy in these patients. For laryngeal cancer,
concomitant cisplatin and radiation therapy leads to better local control. Presently,
induction chemotherapy followed by radiation therapy can be considered standard only
for patients with previously untreated locally advanced squamous cancers in the
hypopharynx.
Concomitant chemoradiation:
The rationale for concomitant chemoradiation is based on experimental

evidence of synergism between chemotherapy and radiation that is theoretically
mediated by interference of chemotherapy with multiple intracellular radiation-induced
stress-response pathways involved in apoptosis, proliferation, and DNA repair. The
finding that certain chemotherapeutic agents (e.g., cisplatin, 5-FU, taxanes, and
hydroxyurea) can induce radiosensitivity and increase log cell kill for radiation supports
this treatment strategy. Cisplatin, the most extensively evaluated drug in recent large
randomized trials, has the advantage of not having mucositis as toxicity, although as a
radiation enhancer, it does increase radiation-induced mucositis.
Adjuvant Chemotherapy:
A large randomized study in resected patients with stage III or IV disease

compared adjuvant radiation therapy with adjuvant chemotherapy followed by

72
radiation. This trial showed improved local control and overall survival rates
approaching statistical
Significance for a subset of patients treated with chemotherapy who were at

high risk for local recurrence. Patients with low-risk disease did not benefit from
adjuvant chemotherapy.
Adjuvant concomitant cisplatin and radiation in patients at high risk for

recurrence after surgery has been studied both in Europe and in the United States. Both
studies found a possible benefit in disease-free or overall survival for patients receiving
concomitant cisplatin and radiation[47].
Prevention:
1. Avoid unnecessary prolonged illumination, prevent the emergence of lip

cancer
2. Avoid smoking and drinking.
3. Patients wearing dentures:
a. Found that tissue under dentures pain, inflammation, should seek

immediate medical attention. Strive to achieve early detection of cancer, early
diagnosis and early treatament, and insisted regularly checked.
4. Balanced diet
5. Do not drink and eat which will give irritation to oral tissues.
6. Unplug the residual tooth root and crown
7. Good wear dentures, does not stimulate tissue.
8. Develop good oral hygiene habits
9. Regular brushing. Pay attention to nutritional balance, timely treatment

residual root.

73
Four symptoms of oral cancer to be alert:
If the mouth turns white, brown or black, it means a change in mucosal epithelial
cells. Especially the oral mucosa becomes rough, thickened or showed induration,
appeared oral leukoplakia, erythema, is likely to have cancerous.
Unhealed ulcer:
Oral ulcers lasting more than two weeks with burning sensation, pain and other
symptoms should be altered
Obvious pain:
Early generally painless or only partial exception sense of friction, ulceration

obvious pain, with further violations of the nerve tumor, can cause ear and throat pain.
Lymph nodes:
Multiple oral cancer to nearby lymph node metastasis neck, and sometimes the
primary lesion is small, and even the symptoms are not obvious, but they are found in
lymph node metastasis of cancer cells. Therefore, such a sudden neck lymph nodes,
need to check the mouth.
Vaccines:
A number of infectious agents cause cancer. Hepatitis B and C are linked to liver
cancer, some human papilloma virus (HPV) strains are linked to cervical and head and
neck cancer, and Helicobacter pylori is associated with gastric cancer and lymphoma.
Vaccines to protect against these agents may reduce the risk of their associated cancers.
The hepatitis B vaccine is effective in preventing hepatitis and hepatomas due to
chronic hepatitis B infection. Public health officials are encouraging widespread
administration of the hepatitis B vaccine, especially in Asia, where the disease is
epidemic. A four-valent HPV vaccine (Gardasil) is 100% effective at preventing
infection. The vaccine is recommended for girls and women ages 9–26 years. Reduction
in these HPV types could prevent >70% of the cervical cancers worldwide.

74
PALLIATIVE TREATMENT IN TERMINAL STAGE
 Pain relief with Morpia and Tramadol.

 Vomiting: correct dehydration and electrolyte balance[48].
3.3.PHARMACOLOGICAL REVIEW
Our great Siddhars explained many medicinal preparations to cure the life
threatening cancer disease.
Herbal Origin:
Compound herbal preparations
Pills
 Asuvagandhathi vadagam
Chooranam
 Garudakodi Chooranam
 Karanthai Chooranam
 Kukilathy Chooranam
 Megaroga Chooranam
 Vallathy Chooranam
Mineral and Metal Origin:
Siddhars identified and worked on many metal and mineral preparations

which had anti-cancer activity.
Preparations:
Pills
 Mahakodasuzhi mathirai

75
Parpam
 Kariya parpam[49]
 Naga parpam[50]
 Rasa parpam
 Kandhaga poora parpam[51]
 Sootha parpam [52]
 Thalaga parpam
 Sanda rasa parpam
Chendooram
 Gowri chinthamani Chendooram[53]

 Gandhaga Chendooram[51a]
 Linga Chendooram [54]
 Kala mega narayana Chendooram[55]
 Navachara Chendooram[56]
 Karuvanga Chendooram [56b]
 Narayana Chendooram [57]
 Pavalavanga Chendooram[58]
 Sandamarutha Chendooram [53a]
 Astabairava Chendooram
 Naga Chendooram [59]
 Rasa Chendooram
 Thambira Chendooram [55a]
Pathangam
 Guru pathangam [60]

 Veera rasa pathangam
 Putrupathangam [61]

76
Thylam
 Singi thylam[62]
 Pupudhakkar mega thylam
 Pachai thylam[62a]
 Sengathari thylam [63]
 Vippuruthiennai [63a]
 Mega rasangaennai [63b]
 Chinthamaniennai
 Sengottai thylam
 Visharajanga thylam
 Meganathiennai
 Magasanthanathy thylam[64]
Nei
 Kukkilnei [65]
 Thengainei
 Vallarainei
Mezhugu
 Korosanai mezhugu
 Gandhaga mezhugu [51b]
 Kanagalinga mezhugu
 Guru sanjeevi mezhugu
 Valai rasa mezhugu
 Veera mezhugu [66]
Others
 Madhusmeegi rasayanam
 Kulirntha pachai
 Veelai seelai
 Rana pugai [62b]

77
 Chitravallathi lehiyam.
Siddha drugs for oral cancer:
Pills
 Chithramoola kuligai
Chooranam
 Karanthai chooranam
Rasayanam
 Parangichakkai rasayanam
Parpam
 Thambiraparpam
 Rasa parpam[67]
 Gandhagaparpam
 Karuvangaparpam
 Naga parpam [68]
Chendhooram
 Kalamega narayana chendhooram[69]

 Pancha padanachendhooram[70]
 Swarnapushpa rasa chendhooram
 Muthuchendhooram
 Sadakshara chendhooram
Nei
 Chitramoolanei [71]
Ennai
 Perungayaennai

78
 Gandhagathylam
Mezhugu
 Rasagandhimezhugu[72]
 Gandhagamezhugu [73]
 Korosanaimezhugu
 Markandeya mezhugu
 Vaan mezhugu
 Nandhi mai
Kattu
 Poorakattu
Padhangam
 Linga padhangam
 Bhramasthiram [74]
 Guru padhangam [75]
Anticancer Drugs- Modern Aspect:
The drugs which prevent neoplasm are known as anticancer drugs. They also
called antineoplastic drugs.They may be divided into two classes
 Cycle specific
Cycle specific drugs act only at specific points of the cell’s duplication cycle,
such as anaphase or metaphase
 Non- cycle specific
Drugs may act any point in the cell cycle, In order to gain maximum effect, anti-
neoplastic drugs are commonly used in combinations.

79
Table no:2 Classification of Anti -Cancer Drugs [76]:
1.Alkylating agents Mechlorethamine, cyclophosphamide, ifosfamide,

Nitrogen mustards chlorambucil, melphalan, bendamustine
Ethylenimines Thio-Tepa, Altretamine
Alkyl sulfonate Busulfan
Nitrosoureas Carmustine, streptozocin
Triazine Dacarbazine, temozolomide
Methyl hydrazine Procarbazine
Antimetabolites Methotrexate (amethopterin), pemetrexed
Folate antagonist 6-Mercaptopurine, thioguanine, pentostatin,
Purine analogues fludarabin, cladribine.
Pyrimidine analogues 5-Fluorouracil, floxuridine, capecitabine, cytarabine
(cytosine arabinoside) gemcitabine
Natural and semisynthetic products Actinomycin-D (Dactinomycin), daunorubicin,

Antibiotics doxorubicin, bleomycin, mitomycin-C,
Epipodophyllotoxins mithramycin
Camptothecins Etoposide, teniposide
Taxanes Topotecan, irinotecan
Vinca alkaloids Paclitaxel, docetaxel
Vincristine, vinblastine, vinorelbine
Miscellaneous Hydroxyurea, cisplatin, I-asparaginase, imatinib,
bortezomib, thalidomide, monoclonal antibodies.
Hormones and their antagonists Glucocorticoids, androgens, antiandrogens,
oestrogens, antioestrogens, progestins, aromatase,
inhibitors.
Biological response modifiers Interferon alpha, interleukin 2, amifostine,
haematopoietic growth factors

80
Interactions:
Anticancer drugs may interact with a number of other medicines. When this
happens, the effects of one or both of the drugs may change or the risk of side effects
may be greater
Table no:3 ANTI-CANCER DRUGS:
DRUGS MOA USES

Cyclophosphamide Forms reactive derivates, NHL CLL, breast, ovarian
alkylates DNA and cancer, soft tissues sarcoma,
important Wilms tumour,
groupscytotoxicity Rhabdomyosarcoma.
Busulfan Same as above CML

Methotrexate Folate antagonist-MTX, Choriocarcinoma, NHL, breast,
polyglumates decreases bladder, head, and neck cancer,
DHFR-inhibits protein osteogenic sarcoma.
synthesis.
Mercaptopurine Purine analog- AML
incorporated into DNA
and RNA- breaks into
DNA, inhibits DNA
synthesis.
5-Fluorouracil Pyramidine analog- Colourectal, anal,
incorporated into DNA hepatocellular, gastric, ovaries,
and RNA- inhibits DNA head, and neck cancers.
synthesis, inhibits TS.
Actinomycin-D Inhibits DNA dependent Wilmstumour, Ewings tumour
RNA synthesis. rhabdomyo sarcoma, chorio
carcinoma, kaposis and soft
tissue sarcoma, immuno
suppressant.

81
DRUGS MOA USES
Bleomyocin Bind iron, generates free Testicular tumours, Head and

radicals-breaks into neck cancer, HL and NHL
DNA
Daunorubicin & Bind DNA and inhibits Testicular tumours. Head and
Doxorubicin topoisomerase. neck cancer.
Etoposide Inhibits topoisomerase II Lung and Gastric cancer.
Topotecan Inhibits topoisomerase I Lung and Ovarian cancer.
Vinblastine Inhibits mitosis Breast cancer, Kaposi’s

sarcoma
Vincristine Inhibits mitosis Neuroblastoma

Cisplatin Active form inhibits Lung, breast, bladder, testis,
DNA synthesis. ovarian, head and neck cancers.
Common adverse effects of anti-cancer drugs:
Because antineoplastic agents do not target specific cell type, they have a number
of common adverse side effects.
 Hair loss is common due to the effects on hair follicles

 Anaemia
 Immune system impairment
 Clotting problem caused by destruction of the blood forming organs, leading to
reduction in the number of red cells, white cells and platelets.
 Bone marrow depression
 GIT-stomatitis, glossitis, esophagitis.
 Reduced spermatogenesis in men and amenorrhoea in women
 Nausea and vomiting are immediate side effects
 Hyperuricaemia (increased plasma uric acid levels) leads to renal failure
 Carcinogenicity (cause secondary cancer).

82
SCREENING METHODS:
The pharmacological screening of plants, minerals and animals is an essential

mean for the invention of new, harmless and effective drugs. Over 50,000 plants have
therapeutic virtues in the world, and around 80% of human use medicines based on
plants and salts at least once in their life, Medicinal plants and mineral have diversified
chemical constituents which are important for the discovery of new active molecules
against many types of cancer.
Active compounds from many medicinal plants and minerals with effective
cytotoxic properties were developed in to anti-cancer drugs.
Nowadays it has become mandatory to monitor the quality of life of patients

while in treatment of cancer. It is healthy aware that the quality of life of cancer patients
treated with chemotherapeutic drugs are very much affected even long time after
withdrawal of drugs.
Therefore, the challenging task at this moment is to identify the quick and novel
methods that can identify and develop molecules, which can be of therapeutic value in
human cancers.
Cancer is one of the thrust area for which effective drugs at comfortable prices
are not available as yet probably due to lack in understanding the cancer Patho
physiology. For such a dreadful disease anti-cancer drugs have been developed from a
variety of sources ranging from natural products (plants and microbes) to synthetic
molecules. One of the cause of treatment failure is the development of resistance to
anticancer agents.
The widely used drugs which are called as cancer chemotherapeutic agents have
many side effects such as bone marrow suppression, alopecia, nausea and vomiting.
This necessitates screening of a large number of compounds .For this purpose

both in-vitro and in-vivo models are employed for systematic screening of an anticancer
drugs[77].

83
INVITRO METHODS:
In studies in vitro cytotoxicity on cell line, various cell staining methods are
used in order to indirectly estimate the number of viable cells present after treatment.
An ideal test in assessing cell proliferation and cytotoxicity should have main feature
in vitro: be simple, fast, efficient, economical, reproducible, sensitive, safe, and
effective as far viable cell population and do not show interference with to evaluate the
compound. In-vitro testing is a potential chemotherapeutic agent.
ADVANTAGES[78]:
 Easier to manage
 Less time consuming
 Small quantities and large number of compounds can be tested.
 Reduce the usage of animals
 Testing the ability of the compound to kill the cells by taking the advantage of
various properties of cell
 Able to process the large number of compounds quickly with minimum
quantity.
 Range of concentrations used is comparable to that expected for in vivo studies.
DISADVANTAGES:
 Pharmacokinetics in determining drug effects cannot be evaluated.

 Growing Solid tumour is poor compared to in-vivo method.
 Difficulty in maintaining the culture
 Show negative results for the compounds which gets activated after metabolism
and vice versa
 Impossible to ascertain the pharmacokinetics.
How to culture cell line
 Tumor cell line derived from several cancer types.

 Adaptable to a suitable growth medium.
 Show reproducible profile for growth and drug sensitivity.

84
 The lines were prepared and preserved using regents such as DMSO during
freezing.
 Thawing- bringing the freezed ampoule to room temperature by slow agitation.
Ideal Characteristics of an In vitro Screening Method:
An ideal in vitro screening method should be simple, economical, reproducible, rapid

and sensitive. The assay should be applicable to large number of tumour types and test
compounds. The choice of the cell lines should be representative of clinical situation as
close as possible. The range of drug concentrations used in vitro should be comparable
to that expected for in vivo treatment. The assay should be able to process a large
number of samples quickly and in an automated fashion. Data acquisition should be
simple, easily interpreted and applied. The goal of a screening assay is to test the ability
of a compound to kill cells, at the same time, the assay should be able to discriminate
between replicating cells and non-replicating cells (quiescent cells that are dead or
dying apoptosis).
Table :4 Different assays take disadvantage of various properties of cells as

mentioned below:
S.NO CELL PROPERTIES ASSAYS
1 Enzymatic properties Tetrazolium salt assay(MTT)
2 Protein content/synthesis Sulphorhodamine B assay
3 DNA content/synthesis H-Thymidine uptake Newer fluorescent

analogues with flow cytometry
Clonogenic assay
4 Membrane integrity Dye exclusion tests
5 Clonogenic properties Clonogenic assay
6 Cell division Cell counting assaY

85
Tetrazolium Salt Assay (Microculture Tetrazolium Test or MTT):
MTT assay is an international accepted in vitro method for anticancer drug

screening. Though viable cells can be measured using several methods other staining
procedures also used these which suffer from drawbacks that the require washing steps
thereby increasing processing time and sample variation. The multi well plate scanning
spectrophotometers can quickly measures large number of samples with high degree
precision and accuracy. Ideally, a colourimetric assay for living cells utilize a colourless
substrate that is modified to a coloured product by any living cells, but not by non-
viable or dead cells or culture medium. However, MTT assay utilizes a colour reaction
as a measure of viable cells. The assay is dependent on the cellular reduction of 3-
(45dimethylthaizol-2-yl) -25-diphenylltetrazolium bromide, a tetrazolium salt to a blue
formazan product by the mitochondrial dehydrogenase of viable cells/ metabolically
active cells. The intensity of blue coloured formanzan produced is directly proportional
to cell viability.
The cells from a particular cell line when in log phase of growth are trypsinized,
counted in a hemocytometer and adjusted to appropriate density in a suitable medium
and then inoculated in different multi well plates (usually 96-well plates). The cells are
treated with various concentrations (in replicates) of drugs for specific duration (usually
1 to 4 days), after which MTT dye is added in each well and plates are incubated at 37°
for 4 h in a co2 incubator. The plates are taken out of incubator, dark blue coloured
formazan crystals are thourougly dissolved in isopropanol / DMSO at a room
temperature. The plates are then read on an ELISA reader at 570 nm. The percent cell
viability with respect to control is calculated using the formula:
OD of treated cells
% Cell viability = _______________________ × 100
OD of control cells
This assay has been successfully used by us. The DMSO as a solvent rapidly
solubilizes the serum as well as formazan and use of spectrophotometric grade DMSO
gives stable "background" absorbance levels. Other solvents like isopropanol, propanol,

86
hexane and dimethyl formaldehyde, though used, do not solubilize serum at

concentrations exceeding 0.0625 percent.
The advantage of this assay is that, it can be run on microtiter dishes on

hundreds of cell samples at one time so that the various drug concentrations can be used
to get an idea of the dose response relationship for each drug tested. As a result, this
assay can be adopted for the determination of IC50 of drugs (concentration of drug
required to inhabit 50% cell growth).
Further this assay is relatively simple and therefore easy to perform. It can be
used for both adherent and suspension cell lines. This method is cheap, requires low
number of cells, manageable and a large number of drugs can be quickly screened for
anti-proliferative activity. However, the assay suffers from a drawback of giving false
results due to the inclusion of cells that might metabolically be active due to cells are
not capable of dividing (non-replicating). In addition, drugs whose mechanism of
section might spare mitochondria may not yield positive results in this assay, especially
for short incubation times. Also, use of DMSO warrants safe handling by laboratory
personnel.
Sulphorhodamine B Assay:
The Sulphorhodamine B (SRB) assay measures whole-culture protein content,

it should be proportional to the number of cell. Cell cultures are stained with a protein
staining dye, Sulphorhodamine B. It is a bright pink dye, which binds to basic amino
acids of cells.Un bound dye is then removed by washing with acetic acid, and protein-
bound dye extracted using unbuffered. Trisbase for determination of optical density in
a computer-interfaced, 96-well microtiter plate reader. Since dead cells either lyse or
are lost during the procedure, the amount of SRB binding is propotional to the number
of live cells left in a culture after drug exposure. This assay can be used to measure the
cellular protein content of both adherent and suspension cultures. Screening capacity,
reproducibility and quality control all appear to be enhanced in this assay relative to the
tetrazolium salt assays. The assay is more cumbersome and time consuming compared
to the MTT assay. Non-replicating and dead cells might contribute to the total protein
and interfere with the results.

87
H-thymidine Update Assay:
In this assay tumor cell suspensions are exposed to the drug continuously for 5
days, after which a radio-labeled precursor (3H-thymidine) is added during the final 48
hours of the assay to label proliferating cells. The replicating cells will incorporate
[3H]- thymidine into their DNA, which can then be determined either by
autoradiography or by liquid scintillation counting, Auto radiographic determination of
the [3H]-thymidine, though, Is time- consuming but it provides information on tumor
growth kinetics. This can generate DNA histograms, which can provide information on
the ploidy status of the cells. This assay looks at cells, which have actively replicating
DNA and hence are viable, Non replicating or dead cells will not be counted in this
case. The assay can be used for both adherent and suspension cell lines. The assay is
rapid, relatively inexpensive, and feasible in the majority of tumor types. However, it
will not differentiate between malignant and non-malignant cells and might lead to
false-negative predictions if lethally damaged cells undergo a final division.
Fluorescence:
Fluorescent dyes may be used in conjunction with microscopic evaluation

methods as an in vitro chemo sensitivity assay. Cells are exposed to fluorescent-labeled
precursors after drug-exposure. The replicating cells will incorporate labeled precursor
into their DNA and the resulting fluorescence is then measured by flow cytometry. This
assay also looks at actively replicating cells and hence dead or non-replicating cells are
not counted. In addition, the assay does not involve the use of radioactivity and is useful
for adherent and suspension cell-lines. Also using flow cytometry it is possible to
determine that in what phase of the cell cycle the cells are. The quantitation of apoptotic
cells is also possible. However this, assay requires the data to be analyzed by an
expensive and sophisticated fluorescence activated cell-sorter (FACS) instrument.
Because of technical difficulties in applying flow cytometry to primary tumor
specimens, data on the predictive value for clinical response for this assay are too scarce
to permit definitive conclusions.

88
Dye Exclusion Tests:
Early attempts to use exclusion of vital dyes like trypan blue, eosin, or nigrosin
to predict chemo sensitivity were unsuccessful. These assays relied on the structural
integrity of the cells .Dead cells would have lost membrane integrity and hence would
take up vital dyes like trypan blue. This method was mainly used because of its
technical simplicity No prospective trials of these assays have yet been performed,
however, to demonstrate their ability to predict as lack response or lack of response.
This DISC assay is drug sensitive assay, which relies on structural integrity of the cells.
In this assay, cells are incubated with drugs for 4 days. Dead cells are stained in
suspension with fast green dye with or without nigrosin. The specimen is centrifuged
and disks of cells are collected in the microscopic slides. Live cells are then stained
with hematoxylin-eosin. As control duck erythrocytes are used. The end point of the
study is the morphologic identification of tumor-cell cytotoxicity compared with the
internal control standard of duck erythrocytes. The DISC assay measures cell kill in
both dividing and non dividing and tumor cell population.
Clonogenic Assays:
A concern in the use of anti-proliferative assays is that they measure growth

inhibition rather than cell killing. This is particularly important for drugs that act by
arresting cells at check points in the cells cycle. Checkpoint arrest is a survival response
of cells that allows repair of DNA damage and is therefore not directly related to the
induction of cell death. Thus, cells that act by arresting cells at checkpoints may show
lower IC50 but increased survival. Clonogenic survival assays on the other hand,
measure loss of tumor cell reproductive viability(the ability of a single cell to form
colonies).it is the most direct method of measuring cytotoxic activity of a drug, In
clonogenic assays single-cell suspension are prepared from tumor biopsies and exposed
to anticancer agents to be tested . Cells are then rinsed and plated in a semisolid medium
(agar or methy1 cellulose),a medium that precludes proliferation of nonmalignant cells
in the specimen.15 After 14 to 28 days, some cells will have undergone several
divisions and will have formed tumor colonies, which can be quantified in a visual or
semi-automated fashion. Non-replicating and dead cells are not coated in this case. The
number of colonies from the treated cells is compared with the number of colonies from

89
the untreated control cells and the fraction of control growth provides an index of drug
activity. Traditional clonogenic systems suffer from a number of significant technical
problems like long incubation time(at least 14 days)before results can be made available
to the clinician. The assay is labor-intensive, costly, and cannot be used for suspension
cell-lines.
Cell Counting Assay:
Cells are cultured in the presence of drug for 2-5 culture-doubling times, after
which the cell number is estimated using a hemocytometer or a cell counter. The assay
is easy to perform, rapid and can be used for both adherent and suspension cell lines.
However, dead and non-replicating cells can be counted in this assay by the cell
counter. The IC50 values can be calculated in all the above assays.
Assay for Energy metabolism and autophagy
 FAD assay
 ATP assay
 Lysosome detection
 Mitochondrial membrane potential assay
 Reactive oxygen species test
For nuclear signaling, DNA damage and cell proliferation
 P53assay
 Topoisomerase II assay
 P21assay
 Cell proliferation assay
 Mdm2 assay
For inflammation, angiogenesis and metastasis
 Cytokine and chemokine assay

 STAT 1,2,3,6 assay
 COX-2 activity assay
 LDL uptake assay

90
For apoptosis, pyroptosis and necrosis
 Caspase 1 assay
 Bax assay
 Cytolysis assay
For cancer signaling pathway and phenotype
 ERK assay
 c- AMP assay
 c- Jun test [79]
Cell lines for cancer
There are plenty of cell lines available for research purpose. Only very few are listed
[80]
.
Table no: 5 Cancer cell lines
S.no Cell name Tissue Species
1 UM-UC Bladder Human
2 FM3A Breast Mouse
3 C 170 Colon Human
4 SHP 77 Lung Human
5 RAG Kidney Mouse
6 HF 1 Liver Rat
7 MEWO Skin Human
8 TT Thyroid Human
9 OV Ovary Human
10 C6 Neural(Glialtumor) Rat

91
IN VIVO MODELS:
In vivo models are advantageous over in vitro models in the sense that they can
detect host-mediated activity, are relatively predictable and estimate therapeutic ratio.
However, as compared with in vitro systems, their sensitivity is low, are costly, time
consuming and large number of samples cannot be handled and are difficult to manage.
After all, animal models are used both toxicological studies and for detecting preclinical
anticancer efficacy. They are able to detect agents irrespective of their mechanism of
action. The drugs with high degree of efficacy and broad spectrum of activity in animal
models are usually expected to be effective in clinical cancer, however, there are
exceptions also which could be due to metabolic differences and heterogeneity of
cancer cells between human and rodents. Despite these differences animal models are
widely used to support the results obtained from in vitro studies. The most promising
candidate compound is tested in more than one animal model. Dose response
relationship, combined effect of drugs, modes of their anticancer action and organ
specificity are established. Varied drug dosage forms, doses and animal strains and
animals of a particular age group may be used. The selected animal models should be
representative of high incidence of human cancers. The in vivo anticancer drug
screening methods are described under the following headings:
A. Chemically induced tumor models
B. Models involving cell line/tumor pieces implantation.
Chemically Induced Tumor Models:
Chemical carcinogens are well-known to account for about 80% of all cancers
and are used to induce cancer in animal models. Carcinogens require metabolic
activation before inducing carcinogenesis. The epidemiological studies indicate that
human carcinogenesis occurs through multiple steps in the same way as in mouse skin.
The concept of multistep carcinogenesis was first of all developed in rodent skin models
in 1940s and applies to cancers to many species and cell types. Experimental
carcinogenesis involves following three steps:

92
1. Initiation is due to exposure to carcinogens transforming the normal cell to a

cancer cell. Many animal species develop cancers spontaneously and are valuable for
understanding the biology of sporadic cancer development in humans. The major use
of spontaneous cancer models is to compare the biology with human; these animals are
increasingly valuable for cross-comparison of response or resistance to the same
clinical agents used for patients.
2. Promotion is due to the triggering of uncontrolled growth of the transformed

cell.
3. Malignant conversion is caused due to unlodging of cancer cells from the

original site its transportated by circulation and the establishment of secondary tumors
in the body.
The exact sequence of cellular, biochemical and molecular genetic events may
differ between tissues and species, the overall concept seems to be directly applicable
to clinical cancer and thus in future multistage mouse skin carcinogenesis model will
be of immense utility for further understanding the mechanisms of epithelial
carcinomas in human beings.
The experiment is well designed; dose of the carcinogen as well as drug

treatment schedule is standardized by conducting pilot studies. This helps in accurate
evaluation of the test compound[81].
DMBA-induced Mouse Skin Papillomas:
This is a classical two-stage experimental carcinogenesis model. Mouse skin is

generally most sensitive to epidermal carcinogenesis, Rats, hamsters and rabbits are
less sensitive and guinea pig is very resistant 2. SENCAR mice are highly sensitive to
DMBA induced skin tumors .Swiss albino mice are relatively less susceptible to tumor
induction. DMBA acts as an initiator and 12-o-tetradecanyl-phorbol-13-acetate(TPA)
is used as a promotor to induce skin papillomas and squamous cell carcinomas. Mice
are topically applied a single dose of 2.5µg of TPA in 0.2 ml acetone twice weekly on
the same site starting one week after DMBA application. Papillomas begin to appear
after 6 to 7 weeks of application of TPA.

93
Weekly observations are made to monitor tumor development till the

experiment terminates after 18 weeks. Percent tumor incidence and multiplicity of
treatment group is compared with DMBA control group. Drug under test can be
administrated either topically or by oral route. The tumor incidence in this model is
usually about 100% in DMBA controls. In various laboratories, however, repeated
topical application of DMBA alone has also been shown to induce carcinogenesis
3. The development of papillomas in DMBA treated swiss albino mouse (24th

week).
MNU-induced Tracheal Squamous Cell Carcinoma in Hamster:
In this model, 5% solution of MNU in the normal saline is administrated once

a week for 15 weeks using specially designed catheter, which exposes a defined area of
the trachea of male Syrian golden hamsters to the carcinogen.
Fifteen weeks MNU administration produces tumors in 40-50% animals within

6 months. Test drug efficacy is measured as percentage reduction of tumor incidence
compared with carcinogen control.
DMBA-induced Oral Cancer in Hamster:
Oral cancer can be induced in a male Syrian hamsters painting right buccal
mucosa, 3 times/ week for 16 weeks with 0.5% solution of DMBA in liquid paraffin
(approximately 10 µl containing 100 µg). Tumour size, number and tumour burden of
drug treated animals can be compared with that of control animals at the termination of
experiment.
Animal models:
1.Mouse cancer models
a. GEM-Genetically Engineered mouse Models

b. Inbred mice (systematic sibling mating)
c. Transplantation models
Allograft models (syngeneic tumour tissues derived
from same genetic mouse)

94
Xenograft models (actual human cancer cells or solid

tumours are transplanted into host mouse)
d. Carcinogen induced and spontaneous models
1. Digestive system cancer induced by polycyclic
aromatic
2. Chemically cancer induced by Cadmium and Arsenic
3. Radiation-skin cancer by ultraviolet radiation,
leukemic changes by ionizing radiation.
2. Rat cancer models:
a.Genetically altered rats
1. Treat embryos with DNA damage causing chemical

mutagen, Frequently N-ethyl-N-nitrosurea (ENU) is
used.
2. Insertion of mutagenesis strategies (Retro viruses)
3. Transgenic strategies (pronuclear injection of DNA)-

quickly developed and more effective models.
b. Inbred rats.
3. Other laboratory animal models
a. Hamster
b. Rabbits
c. Zebrafish
4. Other animal models
a. Dogs
b. Cats
c. Goats

95
d. Horses
e. Pigs.
3.4. PHARMACEUTICAL REVIEW
Chendooram:
Definition:
Chendooram is a category of medicines made from metals or minerals

(arsenicals or mercurial’s or salts) by grinding them with specified juices or distillates
or extractives and subjecting them to a process of sublimation or calcinations or burning
or frying or exposing to insolation till the characteristic reddening of the product takes
place. The Chendooram are said to retain their potency for 75 years
Method of preparation:
Usually two method of preparation are adopted in their processing, with some
exceptions and variants. Such as:
1. Sublimation by the sand – bath process
2. Calcination.
1. Sublimation by the sand - bath process (Kuppi Erippu):
If the Chendooram has Sulphur and Mercury as its components, sulphur is

ground to a fine powder in the mortar and grinding should be continued with the
addition of the given quantity of Mercury, till a black impalpable mobile powder is
obtained. Only after this, ,other ingredients are to be added.
In the conventional set up of the sand –bath sublimation contrivance, a heat

resistant glass flask with a long neck is used as the container for the drug ingredients.
Ceramic ware is also been in used. Before being put to use, these container are wound
around with clay smeared cloth ribbons so as to give seven superimposed layers,
leaving open the mouth of the flask. The flask thus encased should be kept for perfect
drying of the covering.

96
It has been found in recent times that one could make use of the enameled iron
bowls instead of glass flasks. When using enameled iron bowls, two identical bowls of
appropriate dimensions and capacity should be selected and checked for neat contact of
rims when juxtaposed. Then small holes should be punched along the margins so that
the two bowls could be fastened with a bonding wire (metallic). Then a perforation is
made in the center of the bottom of one of the bowls. Having prepared the bowls thus,
they should be secured and bound by pasting the binding wire through the marginal
holes. This would produce a capsule with a top orifice. Clay smeared cloth tape is
wound around as would be done for the glass flask, leaving the central opening
uncovered. This opening is the one through which the reaction going on inside is
inspected by inserting a probe.
The sand – bath is set up by taking a wide earthen trough and spreading fine
gravel or coarse sand at the bottom to a depth of two centimeters.
The capsule into which the drug ingredients are put is placed on the gravel or
sand and is properly cantered. Then the sides packed with sands, leaving the top two
centimeters unpacked and exposing the capsule. When using glass flasks, the neck
should be just out of the sand. This setup is placed on the oven and heat is applied, by
burning fire wood.
In the application of heat, there gradations are recognized. These three stages,
mild, moderate and intense are best understood and mastered with some experience.
It is said that, if the flames are convergent and resemble a single tongue of flame
as in a lamp, it is mild fire (Deepakkini). If several such tongues of flame lick the vessel
and diverge like the flower of lotus, it is moderate (Kamalakkini). If the multiple
tongues of flame fill the oven and enrich the sand bath. It is the intense stage of fire
(Katakkini).
These stages of fire should be manipulated and followed as prescribed in the

method of preparation. In general, the heating is spread over three continuous days. In
such cases, mild, moderate and intense stages are maintained for 24 hours each, in that
order of succession.

97
According to the composition and amount of sulphur in the preparation, the

mixture of drugs placed in the capsule will start melting sooner or later. Sulphur starts
escaping first in the form of yellow vapour through the opening. Later it will start
burning sending out a jet of blue flame. Just when the blue flame goes out, it a long
probe of steel wire is inserted into the orifice and drawn out the portion that enters the
container will show a whitish coating. If the sulphur is still present and not totally burnt
out, the probe will have a black sticky coat, when there is no blackening of the probe
and when whitish coat indicating should be closed and heating continued for one or two
hours and then the heat withdrawn and the setup is allowed to cool by itself.
When the setup has cooled down, the capsule containing the medicine is taken
out and the clay tape winding cut out. The material that has sublimed in upper bowl is
gently tapped with suitable beater or lifted with a spatula. The sublimate collected
should be finely ground in a mortar.
If the glass flasks have been used, the flask is carefully broken, open to collect
the medicine that has sublimed in around the neck.
2. Calcination (Pudam):
The powder is ground in a Kalvam with specified fluids for a specified time.
The paste is made into small discs and dried. They are put in earthen saucers (mann
agal) covered with another and the edge well sealed with mud cloth. It is allowed to
dry. The cups are placed in the middle of cow – dung cakes and burnt. For Pudams,
generally pits of various depths and circumferences are made in the ground. Half of the
pit is covered with cow – dung cakes. The earthen cups are placed and it is covered
again with cow-dung cakes. The fire is put in the middle of the heap on all the four
sides so that there would be uniform heat from all the sides.
All the metals and other ingredients are taken after the usual purification. In
specified cases, specific purification (Suddhi) is mentioned; otherwise, it is to be taken
as general method of purification for the drug as mentioned in Materia- Medica books.

98
Other method of preparations:
1. Prepared without heating (Araippu Chendooram)
2. Prepared by open heating (Erippu or Varuppu Chendooram)
3. Prepared by applying heat in the range close to 100◦c (LaguPuda Chendooram).
Specifications for Chendooram
1. Chendooram is red in nature, well fine in particle size and tasteless.
2. With suitable adjuvant they possess therapeutic values.
3. They are said to retain their potency for 75years[82].
Table no:6 ANALYTICAL SPECIFICATIONS OF CHENDHOORAM [83]
S.no Test
1. Description-colour, odour
2. Identification-chemical
3. Particle size-200 to 300
4. Loss on drying at 105ºc
5. Total ash
6. Acid-insoluble ash
7. Water soluble ash
8. Assay of element (s)
9. Siddha specifications
10. Lusterless
11. Fine enough to enter the cervices of finger
12. Floats on water
13. Smokeless
14. Tasteless
15. Irreversible

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3.5 LATERAL REVIEW

Potassium nitrate (Vediuppu)
Medical Education Online reports that "potassium supplements, 40-80

mEq/day, lower blood pressure, an effect that is largely lost in patients who are also on
a low sodium diet." The scientific community continues to be uncertain about the way
in which potassium is related to high blood pressure and vascular diseases. Potassium
and blood pressure appear to be inter-related through changes in sodium excretion[84].
Aluminum sulphate ( Padikaram (alum):
Alum with its concentrations of (5 and 10) gm/100 ml sterilized by a Millipore
filter and gauze was affected against most bacterial isolates while alum in concentration
2.5 gm/ 100 ml not showed any inhibited affect against all selective bacterial isolates[85].
Copper sulphate (Thurusu):
In the Microbial analysis, The drug, ‘Thurusu chendooram’ in shows sensitive

to all the organisms tested. This drug has which copper sulphate as a main drug
moderate sensitivity to E.Coli, Klebsiella, Styphylococcus aureus, Pseudomonas
aeruginosa, Candida albicans and highly sensitive to Proteus and Streptococcus
pneumoniae. This drug has been indicated to Gunmam (A.P.D), Peruvayiru (Ascities)
and diseases due to the derangement of Vatha, Pitha and Kapha[86].
Sodium borate (Vengaram):
Borax shows a potent anti-inflammatory and healing properties. Hence, it has

been used as a treatment in chronic tonsillitis in the form of a gargle. To ensure
scientific validity of the efficacy, a comparative study is conducted between Aspirin
tablet and borax which was statistically analyzed. In the study borax showed a significant relief
from symptoms that were statistically significant [87].
Ammonium chloride (Navachaaram) :
 Ammonium chloride shows a diuretic effect, diuretics are able to reduce the
blood pressure which plays important role in treatment of angina. Diuretic drugs
influences the reabsorption of ionic sodium at tubular levels. Thus it reduces the
blood pressure, cardiac filling, and ventricular stroke volume etc.

100
 Ammonium chloride which is a ingredients of Veera Mezhugu possess both an

antioxidant and anticancer potentials justifying scientifically its administration
in cancer patients by Siddha practioners[88].
Mercury (Rasam):
 The drug Arkashara Rasa showed potent activity against pancreatic cancer cells
(MIA-PaCa-2). LDH activity confirmed that AR was active against Pancreatic
Cancer cells.
 The incineration process of the Mercury and Sulphur macro particles are
became very smaller and this may possible for devoid of toxicity and more
potent in Anticancer therapeutic.
 The results acquired from the in-vitro studies achieved via the HeLa cell lines
reveals the unique Siddha medicine Gowri Chinthamani Chendhooram
(contains Mercury and Sulphur) have a potent Anticancer activity[89].
Cinnabar (red Mercury (II) Sulfide (HgS) (Lingam):
The study shows a Mupoora chendurum in which cinnabar is one of the
ingredients was prepared as per the Siddha textual references, with Sophisticated tests
reveals the absence of heavy metals like Arsenic, Lead, Cadmium and Copper. Mercury
was within the permissible limit. The Particle size of the drug was 83.3 nm. In spectra
by visual gratitude, there are no significant difference in the characteristic absorption
bands but the intensity of certain wavelength do differ from each other especially at the
fingerprint region (3584–1035 cm-1). This report is a fingerprint for future references
in analysis of Mupoora chendurum, the drug has antibacterial activity against Bacillus,
Streptococcus and Vibrio[90].
Yellow Orpiment (Thaalagam):

 In the Microbial analysis, the ‘Thalaga parpam’ was sensitive for only one
organism, in which Orpiment is one of the ingredients shows a Streptococcus
pneumoniae with 18 mm of inhibition.
 Low doses of arsenic trioxide can induce complete remissions in patients with
APL who have relapsed. The clinical response is associated with incomplete
cyto differentiation and the induction of apoptosis with caspase activation in
leukemic cells[91].

101
Sulphur (Gandhagam):
 The growth inhibitory and apoptosis-related effects of a newly developed highly

purified sulphur (HPS) on immortalized human oral keratinocytes (IHOKs) and
on oral cancer cells representing two stages of oral cancer (HN4, HN12) based
on a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, Western blotting, cell cycle analysis, and nuclear staining.
 The both diol-containing compounds, 2a and 3, were the most cytotoxic of the
sulfide series against V-79 cells in vitro (IC(90) = 2.1 micro M and 1.9 micro
M, respectively). A preliminary anticancer screening against P388 leukemia
showed that 2a is highly active in vivo as well.
 Ally sulphur compounds from garlic are reported to reduce the incidence of
Breast, Colon, Skin, Uterine and Lung cancers and to depress proliferation of
tumour cells[92].
Realger (α-As4S4) Arsenic Sulphide (Manosilai):

Arsenic disulfide, a major effective component of realgar, has been investigated
for its anti-cancer potential and shown to have therapeutic efficacies in hematological
and some solid tumors. However, its effect against breast cancer is rarely reported. The
study shows a anti-cancer effects of As2S2 in human breast cancer cell lines MCF-7 and
MDA-MB-231. As2S2 significantly inhibited cell viabilities, induced apoptosis, and led
to cell cycle arrest in both cell lines with a dose- and time-dependent manner.
As2S2 upregulated pro-apoptotic proteins like p53 and PARP in MCF-7 cells. Besides,
As2S2 downregulated anti-apoptotic proteins like Bcl-2 and Mcl-1, as well as cell cycle-
related proteins cyclin A2 and cyclin D1 in both cell lines. Of note, the expression level
of cyclin B1 was downregulated in MCF-7 cells, whereas, upregulated in MDA-MB-
231 cells. Moreover, As2S2 significantly inhibited the pro-survival signals in PI3K/Akt
pathway in both cell lines. These results provide fundamental insights into the clinical
application of As2S2 for treatment of patients with breast cancer[93].

102
MATERIALS AND METHODS
4. MATERIALS AND METHODS:
SELECTION OF THE DRUG:
For this present study, the metello-mineral formulation “KAALAMEGA

NARAYANA CHENDHOORAM” was taken as the compound drug preparation for
oral cancer mentioned in the classical Siddha literature “Athmarakshamirtham Ennum
Vaithiya Saara Sangeraham” written by Kandhasamy Mudhaliyaar, pg no:493,First
Edition 1931[94].
Ingredients of the drug:
1. Purified Vediuppu [Potassium nitrate ] – 840 gm

2. Purified Thurusu [ Copper sulphate ] – 210 gm
3. Purified Padikaaram [Aluminium potassium sulphate ( Alum )] – 840 gm
4. Purified Vengaram [ Sodium bicarbonate ( Borax) ] – 210 gm
5. Purified Navacharam [Ammonium Chloride ]-210gm
6. Purified Pooneeru [Impure Sodium Carbonate (Fullers Earth) ] – 105 gm
7. Purified Jaathilingam [ Red sulphate of mercury ]-525gm
8. Purified Gandhagam [Sulphur ] – 420 gm
9. Purified Kalluppu [Sodium chloride ]- 210 gm
10. Purified Rasam [ Hydragyrum ] – 1050 gm
11. Purified Aritharam [Tri sulphate of Arsenic (Yellow Orpiment) ]- 350 gm
12. Purified Manosilai [Di suphate of Mercury (Red Orpiment) ]- 140gm
Collection of the raw materials:
All the raw materials were purchased from R.N. Rajan country drug store,
Parrys corner, Chennai.
Identification and Authentication of the drug:
The raw materials were identified and authenticated by the experts of

Gunapadam, Government Siddha Medical College, Arumbakkam, Chennai- 106.
The specimen sample of each raw material has been kept in the PG Gunapadam
department individually for future reference.
103
ANTI CANCER ACTIVITY OF KAALAMEGA CHENDHOORAM
Purification of the drugs:
Purification process was done as per the classical Siddha literature [10j].
1. Purification of Pottasium Nitrate (Vediuppu) :
Materials Required:
1. Salt – 100gm
2. Water – 400gm
3. Fermented butter milk – 100gm
4. Lime juice – 100 gm
Procedure:
Water was added to the pottasium nitrate and boiled on a hearth with mild
flames. The white yolk of eggs (4 nos ) were added to every 1400gm of salt and the
bubbles thus appeared with impure substances were removed with wooden spoon.
The ingredients were then transferred to another pot, sealed with mud pasted
cloth, filtered and transferred to another pot, sealed with mud pasted cloth, filtered and
kept in places without aeration. Next day the water was filtered and salt was sun shade.
This process was repeated for seven times to get it purified.
2. Purification of Padikaaram (Aluminium potassium sulphate ( Alum )
The alum was dissolved in water and it was filtered, boiled. Then it was
cooled to get purified form.
3. Purification of Thurusu (Copper sulphate):
The copper sulphate was fried, till it turns to whitish.
4. Purification of Vengaram (Sodium biborate):
Borax was bundled and hanged in the buffalo’s dung solution and boiled. The
bundle was cleaned with fresh water and insolated to get it in purified form.
5. Purification of Navacharam (Ammonium chloride):
Navacharam (Ammonium chloride) was dissolved in hot water and filtered.

After it was cooled, it was poured in a broad mouthed vessel and insolated; the salt
104
was formed in a purified form. It was preserved with small quantity of the root of
jequirity in a bottle.
6. Purification of Kalluppu (Sodium chloride):
Kalluppu was dissolved in vinegar and clean with a cloth, dried in a sunshade.
7. Purification of Pooneeru (Impure Sodium Carbonate) :
Fuller’s earth 1.3 litre was soaked in dew’s water 5.2 litres and allowed to settle.
Next morning it was churned well and the outer cream layer was removed. The
remaining mixture was in procelin plates and insolated to obtain purified form. This
process was repeated for ten times and stored in a bottle.
8. Purification of Rasam (Mercury)
Materials Required:
 Mercury - 35 gm
 Brick powder - 100 gm
 Turmeric powder - 100 gm
 Acalypha juice (Acalypha indica) - 1.3 litre
Procedure:
Mercury was triturated with brick powder and turmeric powder for one hour
respectively and washed with water. Then the Mercury was boiled with the juice of
Indian Acalypha till the juice completely evaporates. And thus mercury was purified.
9. Purification of Lingam (Cinnabar):
Lime juice, cow’s milk and the Acalypha indica juice were mixed together in
equal proportion and allowed to fuse Cinnabar so as to get it in a purified potent form.
10. Purification of Thaalagam ( Yelow Orpiment):
Materials required:
 Arsenic trisulphide – 35 gm
 Cow’s urine – 1 litre
 Indian acalypha juice – 300 ml
105
 Lime stone – 300 gm
Procedure:
Arsenic trisulphide was bundled and kept immersed in the mixture of limestone,
Acalypha indica juice and cow’s urine and heated to get purified.
11. Purification of Gandhagam (sulfur):
Materials Required:
Sulphur – 35 gm
Butter – 35 gm
Cow’s milk – 150 ml
Procedure:
Sulphur was placed in an iron spoon. Butter was added and the spoon was heated
till the butter melts, this mixture was immersed in inclined position in cow’s milk. The
procedure was repeated for about 7 times and thus sulphur was purified. Fresh milk was
used each time.
12. Purification of Manosilai (Red orpiment)
Materials required:
Red orpiment – 35 gm
Cow’s butter milk – 125 ml
Procedure:
Red orpiment was triturated with cow’s butter milk for 3 hours. It was dried to get
purified form[94a].
106
4.1. Preparation of the trial drug – “KAALAMEGA NARAYANA

CHENDHOORAM”
1. Purified Vediuppu [Potassium nitrate ] – 840 gm

2. Purified Thurusu [ Copper sulphate ] – 210 gm
3. Purified Padigaram [Aluminium potassium sulphate ( Alum )] – 840 gm
4. Purified Vengaram [ Sodium bicarbonate ( Borax) ] – 210 gm
5. Purified Navacharam [Ammonium Chloride ]-210gm
6. Purified Pooneeru [Impure Sodium Carbonate (Fullers Earth) ] – 105 gm
7. Purified Jaathilingam [ Red sulphate of mercury ]-525gm
8. Purified Gandhagam [Sulphur ] – 420 gm
9. Purified Kalluppu [Sodium chloride ]- 210 gm
10. Purified Rasam [ Hydragyrum( Mercury) ] – 1050 gm
11. Purified Aritharam [Tri sulphate of Arsenic (Yellow Orpiment) ]- 350 gm
12. Purified Manosilai [Di suphate of Mercury (Red Orpiment) ]- 140gm
Procedure:
 840 gm of 8th solution of Vediuppu [Potassium nitrate ] and Padigaram

[Aluminium potassium sulphate (Alum) ] were taken
 Along with that, 210 gm of Thurusu [ Copper sulphate ], Vengaram [
Sodium bicarbonate (Borax)], Navacharam [Ammonium Chloride ],
Kalluppu [Sodium chloride Impura ] were taken and then mixed with 105
gm of Pooneeru [[Impure Sodium Carbonate (Fullers Earth) ].
 Above ingredients were ground into fine powder and divided into 3 parts
 First part of the powder was underwent distillation process, the end product
was mixed with 2nd part of powder and dried.
 Second part of the powder was underwent distillation process, the end
product was mixed with 3rd part of powder and dried.
 Third part of the powder was undergoes distillation process, the final end
product was taken and kept in a sealed bottle.
107
 The Jaathilingam [ Red sulphate of mercury ]-525 gm, Aritharam [Tri

sulphate of Arsenic (Yellow orpiment)]-350 gm, Gandhagam [Sulphur ]
420 gm , Manosilai [ Di sulphate of mercury (Red Orpiment) ] 140 gm
were ground ,along with the end product of distillation for 12 hours (4
saamam) and made into fine powder and dried.
 Dried powder was kept in a mud pot which was sealed with 7 mud pasted
plaster.
 Another mud pot with small quantity of sand was taken and above
preparation was kept into it and sealed the lid with mud pasted plaster.
 The mud pot was ignited by using Aavarai stick for 30 hours ( 10 saamam),
after 30 hours “Chendhooram” was obtained.
Drug profile:
 Drug name : Kaalamega Narayana Chendhooram
 Dosage : 244 mg of Chendhooram [1/2 Panavedai ]
 Route : Enteral (oral)

 Adjuvant : Thipili chooranam with honey (bd for 48 days – 1 mandalam)
 Indications : Kannaputru [ ORAL CANCER ], Elaippu [Tuberculosis],
Kuttam 18 [ Hansen´s Disease]
 Reference : “AthmarakshaMirutham Ennum Vaithiya Saara Sangeeraham”.
108
Fig no: 13. Ingredients of Kaalamega Narayana Chendhooram :
Purified Vediuppu [Potassium nitrate ]
Purified Thurusu [ Copper sulphate ]
Purified Padigaram [Aluminium potassium sulphate ( Alum )]
109
Purified Vengaram [ Sodium bicarbonate(Borax)]
Purified Navacharam [Ammonium Chloride ]
Purified Kalluppu [Sodium chloride Impura ]
110
Purified Pooneeru [Impure Sodium Carbonate (Fullers Earth)]
Purified Rasam [ Hydragyrum ]
Purified Jaathilingam [ Red sulphate of mercury]
111
Purified Aritharam [Tri sulphate of Arsenic (Yellow orpiment)]
Purified Gandhagam [Sulphur]
Purified Manosilai [ Red Orphiment ]
112
Fig no: 14. Preparation of Kaalamega Narayana Chendhooram:
Process 1:
Preparing for Thravagam
Process 2.
Divided into 3 parts
113
Process 3.
1st part undergoes distillation process Collection of Thravagam
Process 4.
The obtained Thravagam is used Again the second part

underwent distillation to grind the second part process
Process 5.
The obtained Thravagam is used Again the third part underwent

distillation to grind the third part process
114
Process 6.
The end product of distillation of distillation was sealed in a bottle.
Process 7.
Grinding of prepared medicine
115
Process 7.
Final product was sealed with mud pasted cloth
116
Process 8.
Ignition of final Chendhooram
Final end product of Chendhooram Chendhooram
117
Analysis as per AYUSH guidelines[95]

1. Floating on Water:
A pinch of Chendhooram gently placed on the still surface of water in a vessel,
did not sink immediately. It was found that the Kaalamega Narayana Chendhooram
particles floated over the surface of water indicated lightness of the trial drug.
2. Lines on fingers:
Chendhooram in well prepared form should be as fine powder. When taken
between thumb and index finger, the fine powder will fill up the lines of the finger print.
A pinch of Kaalamega Narayana Chendhooram was taken in between the thumb and
index finger and rubbed. It was found that the Kaalamega Narayana Chendhooram
entered into the lines of the finger and was not easily washed out from the lines,
confirmed its fineness.
3. Irreversible reaction:
The well prepared Chendhooram does not get reversible to its metallic state
when heated with a mixture of cane jaggery, hemp powder, ghee and honey. A pinch
of Kaalamega Narayana Chendhooram was taken and mixed with cane jaggery, ghee
and honey. It was observed that Kaalamega Narayana Chendhooram did not reverse to
its metallic state.
4. Tasteless:
The well prepared Chendhooram should be completely tasteless. Presence of
any taste like sweet or bitter indicate incomplete preparation which needed another
Calcination process. When a small amount of Kaalamega Narayana Chendhooram was
kept on the tip of the tongue, no specific taste was found.
5. Lusterless:
If any shining particle is present in Chendhooram, it indicates that the
Chendhooram is not manufactured properly and contains unchanged substances like
minerals, metals and other toxic substances. There should be no shining particles
present in the well manufactured Chendhooram. Kaalamega Narayana Chendhooram
was taken in a petri bowl and observed for any lustre in daylight through magnifying
glass. No lustre was observed in the Chendhooram.
118
DRUG STANDARDISATION:
Standardisation of drug means confirmation of its identity, determination of its
quality, purity and detection of nature of adulterant by various parameters like
morphological, microscopical, physical, chemical and biological evaluations [95].
STANDARDIZATION OF THE DRUG KMNC:
Standardization of drugs helps to prove its identity and determination of its

quality and potency. Standardization of the Metallo-mineral formulation is based on the
qualitative and quantitative analysis through Physico-chemical investigations and
instrumental analysis. The Physico-chemical analysis of the prepared Metalo--mineral
drug have been done at Central Research Institute, Arumbakkam, Chennai and
elemental analysis have been done at IIT, Chennai. (FTIR, SEM, ICP-MS, XRD)[96]
Method of standardization:
Techniques Involved In Standardization of Compound Drugs:
 Macroscopic Methods
 Microscopic Methods
 Physical Methods
 Chemical Methods
 Biological Methods
Organoleptic character of the Chendhooram :
 The organoleptic characters of the sample were evaluated which include

evaluation of the formulation by its colour, odour, taste, texture etc.
Colour:
 A sample of Chendhooram were taken in watch glasses and placed against white
back ground in white tube light. The Chendhooram were observed for its color
by naked eye.
Odour:
 Chendhooram were smelled, the time intermission between two smelling was
kept 2 minutes to nullify the effect of previous smelling.
119
Taste:
 A sample of about Chendhooram was tasted and the taste was reported.
Size:
 The chendhooram was completely sieved through mesh size 120.
4.2.1. Physico‐Chemical Investigations[96]:
Physico‐chemical investigations like pH value, Loss on drying at 105°C, Ash

test have been done at The Tamilnadu Dr M.G.R Medical University, Anna salai,
Guindy, as per the guide lines of WHO.
Solubility Test:
A pinch of sample (KMNC) was taken in a dry test tube and to it 2 ml of the
solvent was added and shaken well for about a minute and the results are observed. The
test was done for solvents like distilled water, Ethanol, Petroleum ether, Propylene
glycol, Toluene, Benzene, Chloroform, Ethyl alcohol, Xylene, Carbon tetra chloride
and the results are observed individually.
pH value:
Potentio metrically, pH value is determined by a glass electrode and a suitable pH

meter. The pH of the KMNC was written in results column.
Loss on Drying:
An accurately weighed 2gm of Kaalamega Narayana Chendhooram

formulation was taken in a tarred glass bottle. The crude drug was heated 105 0 c for 6
hours in an oven till a constant weight. The percentage moisture content of the sample
was calculated with reference to the shade dried material.
Determination of total Ash:
Weighed accurately 2g of Kaalamega Narayana Chendhooram formulation

was added in crucible at a temperature 6000c in a muffle furnace till carbon free ash
was obtained. It was calculated with reference to the air dried drug.
120
Determination of acid insoluble ash:
Ash above obtained was boiled 5min with 25ml of 1M hydrochloric acid and
filtered using an ash less filter paper. Insoluble matter retained on filter paper was
washed with hot water and filter paper was burnt to a constant weight in a muffle
furnace. The percentage of acid insoluble as was calculated with reference to the air
dried drug.
Determination of water soluble ash:
Total Ash 1g was boiled for 5min with 25ml water and insoluble matter
collected on an ash less filter paper was washed with water and ignited for 15 min at a
temperature not exceeding 4500c in a muffle furnace. The amount of soluble ash is
determined by drying the filtrate.
Determination of water soluble extractive:
5gm of air dried drug. Coarsely powered Kaalamega Narayana Chendhooram

was macerated with 100ml of distilled water in a closed flask for twenty-four hours,
shaking frequently. The solution was filtered and 25 ml of filtered was evaporated in a
tarred flat bottom shallow dish, further dried at 1000c and weighted. The percentage of
water soluble extractive was calculated with reference to the air dried drugs.
Determination of alcohol soluble extractive:
2.5gm of air dried drugs coarsely powdered Kaalamega Narayana

Chendhooram was macerated with 50ml. alcohol in closed flask for 24 hours. With
frequent shaking, it was filtered rapidly talking precaution against loss of alcohol .10ml
of filtrate was the evaporated in a tarred flat bottom shallow dish, dried at 1000 c and
weighed. The percentage of alcohol soluble extractive was calculated with reference to
air dried drug.
BIO-CHEMICAL ANALYSIS[97]
The bio-chemical analysis was done to identify the acid and basic radicals
present in the KMNC.
121
Preparation of extract
5g of KMNC was taken in a 250 ml clean beaker and 50 ml of distilled water

was added, boiled well and allowed to cool and filtered in a 100 ml volumetric flask
and made up to 100 ml with distilled water.
4.2.2. PRELIMINARY BASIC AND ACIDIC RADICALS
Test for basic radicals

1. Test for Potassium
To a pinch of the KMNC 2 ml of sodium nitrate and 2 ml of cobalt nitrate

solution in 30% glacial acetic acid was added and observed for the presence of yellow
precipitate.
2. Test for Calcium
To 2 ml of KMNC extract, 2 ml of 4% ammonium oxide solution was added and

observed for the formation of white precipitate.
3. Test for Magnesium:
To 2ml of KMNC extract, drops of sodium hydroxide solution was added and
watched for the appearance of white precipitate.
4. Test for Ammonium:
To 2ml of KMNC extract few ml of Nessler's reagent and excess of sodium

hydroxide solution are added for the appearance of brown colour.
5. Test for Sodium
Hydrochloric acid was added with a pinch of the KMNC, made as paste and
introduced into the blue flame of Bunsen burner and observed for the appearance of
intense yellow colour.
6. Test for Iron (Ferrous)
The KMNC extract was treated with Conc. HNO3 and ammonium thiocyanate
and waited for the appearance of blood red colour.
122
7. Test for Zinc
To 2 ml of the KMNC extract drops of sodium hydroxide solution was added

and observed for white precipitate formation.
8. Test for Aluminium
To the 2m1 of the KMNC extract sodium hydroxide was added in drops and
changes are noted.
9. Test for Lead
To 2 ml of KMNC extract 2ml of potassium iodide solution was added and noted
for yellow coloured precipitate.
10. Test for Copper
a. A pinch of KMNC was made into a paste with conc. Hcl in a watch glass and
introduced into the non-luminous part of the flame and noted for blue colour
appearance.
b. To 2 ml of KMNC extract excess of ammonia solution was added and

observed for the appearance of blue coloured precipitate.
11. Test for Mercury
To 2m1 of the KMNC extract sodium hydroxide solution was added and noted
for yellow precipitate formation.
12. Test for Arsenic
To 2 ml of the KMNC extract 2ml of sodium hydroxide solution was added and
brown or red precipitate formation was noted.
Test for acid radicals
1. Test for Sulphate
To 2 ml of the KMNC extract 5% of barium chloride solution was added and

observed for the appearance of white precipitate.
2. Test for Chloride
The KMNC extract was treated with silver nitrate solution and observed for the
appearance of white precipitate.
123
3. Test for Phosphate
The KMNC extract was treated with ammonium molybdate and conc. HNO3 and
observed for the appearance of yellow precipitate.
4. Test for Carbonate
The KMNC extract was treated with conc.HCl and observed forth appearance of
effervescence.
5. Test for Fluoride & Oxalate:
To 2ml of KMNC extract 2ml of dilute acetic acid and 2ml calcium chloride
solution was added and heated and watched for cloudy appearance.
6. Test for Nitrate:
To 1 gm of the KMNC, copper turnings was added and again conc.H2SO4 was
added, heated and the test tube was tilted vertically down and observed for any changes.
4.2.3. MICROBIAL LOAD[98]
AVAILABILITY OF MICROBIAL LOAD:
Enumeration of bacteria by plate count – agar plating technique
The plate count technique was one of the most routinely used procedures
because of the enumeration of viable cells by this method.
Principle:
This method is based on the principle that when material containing bacteria are
cultured, every viable bacterium develops into a visible colony on a nutrient agar
medium. The number of colonies therefore is the same as the number of organisms
contained in the sample.
Dilution:
A small measured volume is mixed with a large volume of sterile water or saline
called the diluent or dilution blank. Dilution is usually made in multiples of ten. A single
dilution was calculated as follows:
124
Volume of the sample

Dilution =
Total volume of the sample and the diluent
Requirements:
 Sample or Bacterial suspension
 9 ml dilution blanks (7)
 Sterile petri dishes (12)
 Sterile 1 ml pipettes (7)
 Nutrient agar medium (200 ml)
 Colony counter.
Procedure:
1. Label the dilution blanks as 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7.
2. Prepare the initial dilution by adding 1 ml of the sample into a 9 ml dilution
blank labeled 10-1 thus diluting the original sample 10 times.
3. Mix the contents by rolling the tube back and forth between hands to obtain
uniform distribution of organisms.
4. From the first dilution transfer 1 ml of the suspension while in motion, to
the dilution blank 10-2 with a sterile and fresh 1 ml pipette diluting the
original specimen to 100 times.
5. From the 10-2 suspension, transfer 1 ml of suspension to 10-3 dilution blank
with a fresh sterile pipette, thus diluting the original sample to 1000 times.
6. Repeat this procedure till the original sample has been diluted 10,000,000
times using every time a fresh sterile pipette.
7. From the appropriate dilutions transfer 1ml of suspension while in motion,
with the respective pipettes, to sterile petri dishes.
8. Three petri dishes are to used for each dilution.
9. Add approximately 15 ml of the nutrient medium, melted and cooled to
450c, to each petri dish containing the diluted sample.
Mix the contents of each dish by rotating gently to distribute the cells
throughout the medium.
10. Allow the plates to solidity.
11. Incubate these plates in an inverted position for 24-48 hours at 370c.
125
Observation:
Observe all the plates for the appearance of bacterial colonies. Count the number
of colonies in the plates.
Calculate the number of bacteria per ml of the original suspension as follows:
Number of colonies (average of 3 replates)

Organisms per millimetre =
Amount of plated × dilution
4.2.4. SOPHISTICATED INSTRUMENTAL ANALYSIS
FT IR - Fourier Transform Infra-red Spectroscopy[99]
FTIR (Fourier Transform Infra-red Spectroscopy) is a sensitive technique

particularly for identifying organic chemicals in a whole range of applications although
it can also characterise some inorganics. Examples include paints, adhesives, resins,
polymers, coatings and drugs. FTIR is an effective analytical instrument for detecting
functional groups.
Fig no:15 FTIR INSTRUMENT
126
Fig no:16 FTIR MECHANISM
APPLICATIONS:
Quantative scans
Qualitative scan solids, liquids, gasess
Organic samples, inorganic samples
Unknown identification
Impurities screening
Formulation
Pharmaceuticals
Principle:
Spectrophotometric tests are commonly used in the identification of chemical

substances and quantification of polymorphic forms. The test procedures are applicable
to substances that absorb IR radiation. The IR absorption spectrum of a substance
compared with that obtained concomitantly for the corresponding reference standard /
reference substance provide conclusive evidence of the identity of the substance being
tested.
127
Recording Infrared spectrum of a solid as a disc (as per USP ‹197K›):
 Triturate about 1 to 2 mg of the substance to be examined with 300 to 400 mg,

unless otherwise specified, of finely powdered and dried potassium bromide. If
the substance is a hydrochloride it is preferable to use potassium chloride.
 Carefully grind the mixture and spread it uniformly in a suitable die.
 Submit it to the pressure of about 800 mPa (8 tons/cm2).
 Examine the disc visually and if any lack of uniform transparency is observed,
reject the disc and prepare again.
 Record the spectrum between 4000 to 650 cm-1 unless otherwise specified in
individual standard test procedure.
 When sample and standard are measured for concordance, the transmittance
obtained at the start of the scan range, should not deviate by more than 10%
between them ( For eg. If the standard shows a transmittance of 75%, the sample
transmittance can be between 65% and 85%).
FT-IR was the most advanced and the major advantage was its
 Speed
 Sensitivity
 Mechanical Simplicity
 Internally Calibrated
SEM (SCANNING ELECTRON MICROSCOPE)[100]
Fig no:17 SEM INSTRUMENT
128
DEFINITION
Scanning Electron Microscopy (SEM), also known as SEM analysis or SEM

microscopy, is used very effectively in microanalysis and failure analysis of solid
inorganic materials. Scanning electron microscopy is performed at high magnifications,
generates high-resolution images and precisely measures very small features and
objects [83].
Fig no:18 SEM MECHANISM
SEM ANALYSIS APPLICATIONS
The signals generated during SEM analysis produce a two-dimensional image

and reveal information about the sample including:
External morphology (texture)
Chemical composition (when used with EDS)

Orientation of materials making up the sample
The EDS component of the system is applied in conjunction with SEM analysis
to:
Determine elements in or on the surface of the sample for qualitative

information.Measure elemental composition for semi-quantitative results.Identify
foreign substances that are not organic in nature and coatings on metal.
SEM Analysis with EDS – qualitative and semi-quantitative results

Magnification – from 5x to 300,000x.Sample Size – up to 200 mm (7.87 in.) in diameter
129
and 80 mm (3.14 in.) in height.Materials analysed – solid inorganic materials including

metals and minerals.
THE SEM ANALYSIS PROCESS
Scanning Electron Microscopy uses a focused beam of high-energy electrons to

generate a variety of signals at the surface of solid specimens. In most SEM microscopy
applications, data is collected over a selected area of the surface of the sample and a
two-dimensional image is generated that displays spatial variations in properties
including chemical characterization, texture and orientation of materials. The SEM is
also capable of performing analyses of selected point locations on the sample. This
approach is especially useful in qualitatively or semi-quantitatively determining
chemical compositions, crystalline structure and crystal orientations.
The EDS detector separates the characteristic X-rays of different elements into an
energy spectrum and EDS system software is used to analyse the energy spectrum in
order to determine the abundance of specific elements. A typical EDS spectrum is
portrayed as a plot of X-ray counts vs. energy (in keV). Energy peaks correspond to the
various elements in the sample. Energy Dispersive X-ray Spectroscopy can be used to
find the chemical composition of materials down to a spot size of a few microns and to
create element composition maps over a much broader raster area. Together, these
capabilities provide fundamental compositional information for a wide variety of
materials, including polymers. In scanning electron microscope high-energy electron
beam was focused through a probe towards PP. Variety of signals was produced on
interaction with the surface of the sample. This results in the emission of electrons or
photons and it was collected by an appropriate detector [84].
The types of signal produced by a scanning electron microscope include
 Secondary electrons
 back scattered electrons
 characteristic x-rays light
 specimen current
 Transmitted electrons.
130
This gives the information about the sample and it includes external morphology,
texture, its crystalline structure, chemical composition and it displays the shape of the
sample.
ICP-MS - Inductively Coupled Plasma Mass Spectrometry[101]

Analysis of Trace Metal and Inorganic Materials
Inductively Coupled Plasma Mass Spectrometry is a technique routinely used
to analyze trace levels of a wide range of inorganic elements. The ICP-MS allows for
the detection and quantification of elements with atomic mass ranges 7 to 250. This
covers Lithium to Uranium.
The typical detection limits are in the parts per billion (ppb) range and even
parts per trillion (ppt) in some cases. The ICP-MS analysis methods available at LPD
Lab Services allow the detection, identification and quantification of a wide array of
elements using a Perkin Elmer ELAN 6000 ICP-MS
Fig no:19 ICPMS- INSTRUMENT
131
Fig no:20 ICP MS MECHANISM
Analysis: Analyze according to the manufacturer's suggestions for program and m/z.
Calculate and report results based on the original sample size.
Applications of ICP-MS
 Monitoring of trace metals in drinking water, ground water, rainwater,

wastewater or industrial effluent streams.
 Trace elements in product / raw materials or from washed or rinsed surfaces.
 Analysis of additives and purity in metal alloys.
 Analysis of low level contaminants in chemical products, beverages, foods,
cosmetics, pharmaceuticals.
 Analysis of soluble / leachable material from solid samples such as medical
devices, polymers, PCB`s.
 Analysis can be performed on a diverse range of sample
XRD - X-ray Powder Diffraction (XRD)[102]
X-ray powder diffraction (XRD) is a rapid analytical technique primarily used for phase
identification of a crystalline material and can provide information on unit cell
dimensions. The analyzed material is finely ground, homogenized, and average bulk
composition is determined.
DEFINITION
X-ray powder diffraction is most widely used for the identification of unknown
132
crystalline materials (e.g. minerals, inorganic compounds). Determination of

unknown solids is important to studies in geology, environmental science, material
science and biology.
Fig no:21 XRD - X-ray Powder Diffraction
Fig no:22 XRD Mechanism

APPLICATIONS:
 Characterization of crystalline materials
 Identification of fine-grained minerals such as clays and mixed layer clays that
are difficult to determine optically
 Determination of unit cell dimensions.
With specialized techniques, XRD can be used to:
 Determine crystal structures using Rietveld refinement

 Determine of modal amounts of minerals (quantitative analysis)
133
Characterize thin films samples by:
 Determining lattice mismatch between film and substrate and to inferring stress
and strain
 Determining dislocation density and quality of the film by rocking curve
measurements
 Measuring super lattices in multilayered epitaxial structures
 Determining the thickness, roughness and density of the film using glancing
incidence X-ray reflectivity measurements
 Make textural measurements, such as the orientation of grains, in a
polycrystalline sample.
Strengths and Limitations of X-ray Powder Diffraction:
Strengths:
 Powerful and rapid (< 20 min) technique for identification of an unknown

mineral
 In most cases, it provides an unambiguous mineral determination
 Minimal sample preparation is required
 XRD units are widely available
 Data interpretation is relatively straight forward.
Limitations:
 Homogeneous and single phase material is best for identification of unknown

 Must have access to a standard reference file of inorganic compounds
 Requires tenths of a gram of material which must be ground into a powder
 For mixed materials, detection limit is ~ 2% of sample
 For unit cell determinations, indexing of patterns for non-isometric crystal
systems is complicated.
Sample Collection and Preparation:
Determination of an unknown requires: the material, an instrument for grinding, and a

sample holder.
 Obtain a few tenths of a gram (or more) of the material, as pure as possible
134
 Grind the sample to a fine powder, typically in a fluid to minimize inducing

extra strain (surface energy) that can offset peak positions, and to randomize
orientation. Powder less than ~10 µm(or 200-mesh) in size is preferred
 Place into a sample holder or onto the sample surface.
4.2.5. TOXICOLOGICAL STUDIES[103]
ACUTE ORAL TOXICITY STUDY OF KAALAMEGA NARAYANA

CHENDHOORAM
(OECD GUIDELINE – 423)
Introduction:
 The acute toxic class method is a stepwise procedure with the use of 3 animals
of a single sex per step.
 Depending on the mortality and/or the moribund status of the animals, on
average 2-4 steps may be necessary to allow judgement on the acute toxicity of
the test substance.
 This procedure is reproducible, uses very few animals and is able to rank
substances in a similar manner to the other acute toxicity testing methods.
 The acute toxic class method is based on biometric evaluations with fixed doses,
adequately separated to enable a substance to be ranked for classification
purposes and hazard assessment.
 In principle, the method is not intended to allow the calculation of a precise
LD50, but does allow for the determination of defined exposure ranges where
lethality is expected since death of a proportion of the animals is still the major
endpoint of this test.
 The method allows for the determination of an LD50 value only when at least
two doses result in mortality higher than 0% and lower than 100%.
 The use of a selection of pre-defined doses, regardless of test substance, with
classification explicitly tied to number of animals observed in different states
improves the opportunity for laboratory to laboratory reporting consistency and
repeatability.
Principle of the Test:
It is the principle of the test that based on a stepwise procedure with the use of
a minimum number of animals per step, sufficient information is obtained on the acute
135
toxicity of the test substance to enable its classification. The substance is administered
orally to a group of experimental animals at one of the defined doses. The substance is
tested using a stepwise procedure, each step using three animals of a single sex.
Absence or presence of compound-related mortality of the animals dosed at one step
will determine the next step, i.e.
− no further testing is needed
− dosing of three additional animals, with the same dose
− dosing of three additional animals at the next higher or the next lower dose
level. The method will enable a judgment with respect to classifying the test substance
to one of a series of toxicity classes.
Methodology:
Selection of Animal Species
The preferred rodent species is the wistar albino rat, although other rodent
species may be used. Healthy young adult animals are commonly used laboratory
strains should be employed. Females should be nulliparous and non-pregnant. Each
animal, at the commencement of its dosing, should be between 6 to 8 weeks old and the
weight (150-200gm) should fall in an interval within±20 % of the mean weight of any
previously dosed animals.
Housing and Feeding Conditions
The temperature in the experimental animal room should be 22ºC + 3ºC.
Although the relative humidity should be at least 30% and preferably not exceed 70%
other than during room cleaning the aim should be 50-60%. Lighting should be
artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional
laboratory diets may be used with an unlimited supply of drinking water. Animals may
be group-caged by dose, but the number of animals per cage must not interfere with
clear observations of each animal.
Preparation of animals:
The animals are randomly selected, marked to permit individual identification,
and kept in their cages for at least 7 days prior to dosing to allow for acclimatization to
the laboratory conditions
Test Animals and Test Conditions:
Sexually mature Female Wistar albino rats (150-200gm) were obtained
136
from TANUVAS, Madhavaram, Chennai. All the animals were kept under standard
environmental condition (22±3°C). The animals had free access to water and standard
pellet diet (Sai meera foods, Bangalore).
Preparation for Acute Toxicity Studies

Rats were deprived of food overnight (but not water 16-18 h) prior to
administration of the, Kaalamega Narayana Chendhooram (Kmnc)
The principles of laboratory animal care were followed and the Institutional
Animal Ethical Committee approved the use of the animals and the study design
IAEC approved Number: IAEC/XL VIII/13CLBMCP/2016

Test Substance :Kaalamega Narayana Chendhooram (Kmnc)
Animal Source : TANUVAS,Madhavaram, Chennai.
Animals : Wister Albino Rats (Female-3+3)
Age : 6-8 weeks
Body Weight on Day 0 : 150-200gm.
Acclimatization : Seven days prior to dosing.
Veterinary examination : Prior and at the end of the acclimatization period.
Identification of animals : By cage number, animal number and individual
marking by using Picric acid.
Number of animals : 3 Female/group,
Route of administration : Oral
Diet : Pellet feed supplied by Sai meera foods Pvt Ltd,
Bangalore
Water : Aqua guard portable water in polypropylene bottles.
Housing & Environment : The animals were housed in Polypropylene cages
provided with bedding of husk.
Housing temperature : between 22ºC + 3ºC.
Relative humidity : between 30% and 70%,
Air changes : 10 to 15 per hour and
Dark and light cycle : 12:12 hours.
Duration of the study : 14 Days
137
Administration of Doses:
Kaalamega Narayana Chendhooram (KMNC) was suspended in water and
administered to the groups of wistar albino rats in a single oral dose by gavage using a
feeding needle. The control group received an equal volume of the vehicle. Animals
were fasted 12 hours prior to dosing. Following the period of fasting, the animals were
weighed and then the test substance was administered. Three Female animals are used
for each group. The dose level of 5, 50, 300 and 2000 mg/kg body weight was
administered stepwise. After the substance has been administered, food was withheld
for a further 3-4 hours. The principle of laboratory animal care was followed.
Observations were made and recorded systematically and continuously as per the
guideline after substance administration.
The visual observations included skin changes, mobility, aggressively,
sensitivity to sound and pain, as well as respiratory movements. Finally, the number of
survivors was noted after 24 hrs and these animals were then monitered for a further 14
days and observations made daily. The toxicological effect was assessed on the basis of
mortality.
Limit test
The limit test is primarily used in situations where the experimenter has
information indicating that the test material is likely to be nontoxic, i.e., having toxicity
only above regulatory limit doses. A limit test at one dose level of 2000 mg/kg body
weight was carried out with three animals per step. The test substance-related mortality
was not produced in animals, so further testing at the next lower level need not be
carried out.
Observations:
Animals are observed individually after dosing at least once during the first 30
minutes, periodically during the first 24 hours, with special attention given during the
first 4 hours, and daily thereafter, for a total of 14 days, except where they need to be
removed from the study and humanely killed for animal welfare reasons or are found
dead. It should be determined by the toxic reactions, time of onset and length of
recovery period, and may thus be extended when considered necessary. The times at
which signs of toxicity appear and disappear are important, especially if there is a
tendency for toxic signs to be delayed. All observations are systematically recorded
with individual records being maintained for each animal.
138
a. Mortality
Animals will be observed intensively at 0.5, 2.0, 4.0, 6.0, 12.0, 24.0 and 48.0
hours following drug administration on day 1 of the experiment and daily twice
thereafter for 14 days.
b. Body weight
Individual weight of animals was determined before the test substance was
administered and weights will be recorded at day 1, 7, and 14 of the study. Weight
changes were calculated and recorded. At the end of the test, surviving animals were
weighed and humanly killed.
C. Cage-side observation
These include changes in skin and fur, eyes and mucous membranes and also
respiratory, circulatory, autonomic and central nervous systems, somatomotor activity
and behavioral patterns. Attention should be directed to observations of tremors,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.
d. Gross necropsy
All animals (including those which die during the test period are
removed from the study) will be subjected to gross necropsy. Gross necropsy
includes examination of the external surface of the body, all orifices, cranial,
thoracic and abdominal cavities and their contents, brain, eye, thymus, lungs, heart,
spleen, liver, kidneys, adrenals, testes and uterus of all animals.
Histopathology
Microscopic examination will be carried out in organs to show the evidence of

any toxicity in gross pathology.
Data and reporting

All data were summarized in tabular form, (Table-1-4) showing for each test
group the number of animals used, the number of animals displaying signs of toxicity,
the number of animals found dead during the test, description of toxic symptoms,
weight changes, food and water intake.
139
Test substance and Vehicle

In order to ensure the uniformity in drug distribution in the medium the
suspension was made by mixing Kaalamega Narayana Chendhooram (KMNC)
with 2% CMC solution and it was found suitable for dose accuracy.
Justification for choice of vehicle
The vehicle selected as per the standard guideline was pharmacologically inert and
easy to employ for new drug development and evaluation technique [104].
REPEATED DOSE 28-DAY ORAL TOXICITY (407) STUDY OF

KAALAMEGA NARAYANA CHENDHOORAM (KMNC)[105]
Test Substance : Kaalamega Narayana Chendhooram (KMNC)
Animal Source : TANUVAS, Madhavaram, Chennai.
Animals : Wister Albino Rats (Male -24, and Female-24)
Age : 6-8 weeks
Body Weight : 150-200gm.
Acclimatization : Seven days prior to dose.
Veterinary examination : Prior and at the end of the acclimatization period.
Identification of animals : By cage number, animal number and individual
marking by using Picric acid
Diet : Pellet feed supplied by Sai Meera Foods Pvt Ltd,
Bangalore.
Water : Aqua guard portable water in polypropylene bottles.
Housing & Environment : The animals were housed in Polypropylene cages
provided with bedding of husk.
Housing temperature : between 22ºC±3ºC.
Relative humidity : between 30% and 70%,
Air changes : 10 to 15 per hour
Dark and light cycle : 12:12 hours.
Duration of the study : 28 Days.
140
Justification for Dose Selection:
The results of acute toxicity studies in Wistar albino rats indicated that
Kaalamega Narayana Chendhooram (KMNC) was non-toxic and no behavioural
changes was observed up to the dose level of 2000 mg/kg body weight. On the
basis of body surface area ratio between rat and human, the doses selected As per
OECD guideline three dose levels were selected for the study. They are low dose
(5X), high dose (10X). X is calculated by multiplying the acute toxicity dose
(2000mg) and the body surface area of the rat (0.018), 5X dose is (10mg/kg), 10X
dose is (20mg/kg) The oral route was selected for use because oral route is
considered to be a proposed therapeutic route.
Preparation and Administration of Dose:

Kaalamega Narayana Chendhooram (KMNC) suspended in with water, It was
administered to animals at the dose levels of 5X, 10X. The test substance suspensions
were freshly prepared every two days once for 28 days. The control animals were
administered vehicle only. The drug was administered orally by using oral gavage once
daily for 28 consecutive days.
Methodology
Groups No of Rats
1. Group I Vehicle control 20(10male, 10female)
2. Group II KMNC - low dose X (20mg) 20(10male, 10female)
3. Group III KMNC - Mid dose 5X (100mg) 20(10male, 10female)
4. Group IV KMNC – High dose 10X (200mg ) 20(10male, 10female)
Randomization, Numbering and Grouping of Animals:

80 Wistar Albino Rats (40M + 40F) were selected and divided into 4 groups.
Each group consist of 20 animals (Male -10 and Female-10). First group treated as a
control and other three group were treated with test drug (low, high) for 28 days.
Animals were allowed acclimatization period of 7 days to laboratory conditions prior
to the initiation of treatment. Each animal was marked with picric acid. The females
were nulliparous and non-pregnant.
141
Observations:
Experimental animals were kept under observation throughout the course of study
for the following:
Body Weight:
Weight of each rat was recorded on day 0, at weekly intervals throughout the
course of study. From the data, group mean body weights and percent body weight
gain were calculated.
Food and water Consumption:

Food and water consumed per animal was calculated for control and the
treated dose groups.
Clinical signs:
All animals were observed daily for clinical signs. The time of onset, intensity
and duration of these symptoms, if any, were recorded.
Mortality:
All animals were observed twice daily for mortality during entire course of
study.
Functional Observations:
At the end of the 4th week exposure, ‘sensory reactivity’ to graded stimuli of
different types (auditory, visual and proprioceptive stimuli), ‘motor reactivity’ and ‘grip
strength’ were assessed.
Laboratory Investigations:
Following laboratory investigations were carried out on day 29 in animals
fasted over-night. Blood samples were collected from orbital sinus using sodium
heparin (200IU/ml) for Biochemistry and potassium EDTA (1.5 mg/ml) for
Hematology as anticoagulant. Blood samples were centrifuged at 3000 r.p.m. for
10 minutes.
142
Hematological Investigations
Blood samples of control and experimental rats was analyzed for hemoglobin
content, total red blood corpuscles (RBC), white blood corpuscles (WBC) count and
packed cell volume (PCV).
Biochemical Investigations
Serum was used for the estimation of biochemical parameters. Samples of

control and experimental rats were analyzed for protein, bilirubin, urea, BUN,
creatinine, triglyceride, cholesterol and glucose levels was carried using standard
methods.
Activities of glutamate oxaloacetate transaminase/Aspartate aminotransferase
(GOT/AST), glutamate pyruvate transaminase/ Alanine amino transferase (GPT/ALT)
and alkaline phosphatase were estimated as per the colorimetric procedure.
Necropsy:
All the animals were sacrificed by excessive anesthesia on day 29. Necropsy of
all animals was carried out.
Histopathology:
Histopathological investigation of the vital organs was done. The organ pieces (5-
6µm thick) of the highest dose level of 300 mg/kg were preserved and were fixed in
10% formalin for 24 hours and washed in running water for 24 hours. Samples were
dehydrated in an auto technique and then cleared in benzene to remove absolute alcohol.
Embedding was done by passing the cleared samples through three cups containing
molten paraffin at 50°C and then in a cubical block of paraffin made by the “L” moulds.
It was followed by microtome and the slides were stained with Hematoxylin-eosin.
The organs included heart, kidneys, liver, ovary, pancreas, brain, spleen and stomach,
of the animals were preserved they were subjected to Histopathological examination.
Statistical analysis:
Findings such as body weight changes, water and food consumption,
hematology and blood chemistry were subjected to One-way ANOVA followed
143
by dunnet’S multi comparison test using a computer software programme –

GRAPH PAD VERSION-7 version.
4.3. PHARMACOLOGICAL ACTIVITY:
4.3.1. ANTICANCER ACTIVITY:
INVITRO ANTICANCER EFFECT DETERMINATION BY MTT ASSAY[106]

KB (Oral Carcinoma) cells was initially procured from National Centre for Cell
Sciences (NCCS), Pune, India and maintained in Dulbecco’s modified Eagles medium,
DMEM ( Sigma aldrich, USA).
The cell line was cultured in 25 cm2 tissue culture flask with DMEM
supplemented with 10% FBS, L-glutamine, sodium bicarbonate (Merck, Germany) and
antibiotic solution containing: Penicillin (100U/ml), Streptomycin (100µg/ml), and
Amphoteracin B (2.5µg/ml). Cultured cell lines were kept at 37ºC in a humidified 5%
CO2 incubator (NBS Eppendorf, Germany).
The viability of cells were evaluated by direct observation of cells by Inverted
phase contrast microscope and followed by MTT assay method.
Cells seeding in 96 well plate:
Two days old confluent monolayer of cells were trypsinized and the cells were
suspended in 10% growth medium, 100µl cell suspension (5x104 cells/well) was seeded
in 96 well tissue culture plate and incubated at 37ºC in a humidified 5% CO2 incubator.
Preparation of compound stock:
1mg of sample was weighed and dissolved in 1mL DMEM using a cyclomixer.
The sample solution was filtered through 0.22 µm Millipore syringe filter to ensure the
sterility.
Anticancer Evaluation:
After 24 hours the growth medium was removed, freshly prepared each
compounds in 5% DMEM were five times serially diluted by two fold dilution (100µg,
50µg, 25µg, 12.5µg, 6.25µg in 500µl of 5% DMEM) and each concentration of 100µl
were added in triplicates to the respective wells and incubated at 37ºC in a humidified
5% CO2 incubator. Non treated control cells were also maintained.
144
Anticancer Assay by Direct Microscopic observation:

Entire plate was observed after 24 hours of treatment in an inverted phase
contrast tissue culture microscope (Olympus CKX41 with Optika Pro5 CCD camera)
and microscopic observation were recorded as images. Any detectable changes in the
morphology of the cells, such as rounding or shrinking of cells, granulation and
vacuolization in the cytoplasm of the cells were considered as indicators of cytotoxicity.
Anticancer Assay by MTT Method:
Fifteen mg of MTT (Sigma, M-5655) was reconstituted in 3 ml PBS until
completely dissolved and sterilized by filter sterilization..
After 24 hours of incubation period, the sample content in wells were removed
and 30µl of reconstituted MTT solution was added to all test and cell control wells, the
plate was gently shaken well, then incubated at 37ºC in a humidified 5% CO2 incubator
for 4 hours. After the incubation period, the supernatant was removed and 100µl of
MTT Solubilization Solution (Dimethyl sulphoxide,DMSO, Sigma Aldrich, USA) was
added and the wells were mixed gently by pipetting up and down in order to solubilize
the formazan crystals. The absorbance values were measured by using microplate
reader at a wavelength of 540 nm (Laura B. Talarico et al., 2004).
The percentage of growth inhibition was calculated using the formula:
Mean OD Samples x 100

% of viability =
Mean OD of control group
APPENDIX
Instruments and reagents used:
DMEM media -Sigma Aldrich, USA D5648
Fetal Bovine Serum -Gibco, US orgin-
0.25% Trypsin - Invitrogen, USA 25200-056
Micropipettes - F1 Thermoscientific USA
CO2 Incubator - Eppendorf, GERMANY
Phase Contrast Microscope - Olympus, JAPAN with Optika Pro 5 Camera
MTT - Sigma Aldrich M5655
145
ELISA Reader - ERBA, GERMANY

Culture Plates and Flasks - NUNC, Thermoscientific USA
Image Magnification - 10X
Fig no:23 Microplate Reader
Fig no:24 CO2 Incubator
146
4.3.2. IN VITRO ANTI- TUMOUR ACTIVITY
CELL CULTURE MATERIAL
OSCC cell lines were purchased from American Tissue Collection Centre
(ATCC) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) and
Ham’s F12 (DMEM/F12) (Sigma-Aldrich, USA).HOS cell lines were purchased from
ATCC and were maintained in DMEM high glucose. Culture media was supplemented
with 10% Foetal Bovine Serum (FBS) and 1% penicillin/streptomycin.
Cell apoptosis assay by flow cytometry
Cellular apoptosis was determined using the AnnexinV-FITC Apoptosis
Detection Kit I (Clontech Laboratories Inc, USA) according to the manufacturer’s
protocol. OSCC and HOS cell lines were cultured at 6 × 10cells/ml and seeded in 60
mm dish. The cells were treated with free medium containing various concentrations of
ABC for 6, 12 and 24 hour. Cells were harvested by trypsinization, then washed twice
with cold PBS and centrifuged at 1000 rpm. About 1 × 105 -1×106cells were then re
suspended in 400 µl1×binding buffer, centrifuged again at 1000 rpm for 5 minutes and
then supernatant was removed. Cells were re-suspended in 200 µl 1× binding buffer
and transferred to a sterile flow cytometry glass tube. Five µl Annexin V-FITC and 10
µl propidium iodide were added and then incubated in the dark at room temperature.
Cells were analyzed by flow cytometer at 488 nm. The distribution of cells was
analyzed using Cell Quest software (Becton-Dickinson) in the flow cytometer within 1
hour of staining. Data from 10,000 cells was collected for each data file. Apoptotic cells
were identified as Annexin V-FITC-positive and P-negative cells
4.3.3.ANTIMICROBIAL ACTIVITY[107]:
AGAR- WELL DIFFUSION METHOD:
PRINCIPLE:
The antimicrobials present in the samples are allowed to diffuse out into the
medium and interact in a plate freshly seeded with the test organisms. The resulting
zones of inhibition will be uniformly circular as there will be a confluent lawn of
growth. The diameter of zone of inhibition can be measured in millimeters.
147
MATERIALS REQUIRED:
1. Muller Hinton Agar Medium (1 L)
The medium was prepared by dissolving 33.8 g of the commercially available

Muller Hinton Agar Medium (MHI Agar Media) in 1000ml of distilled water. The
dissolved medium was autoclaved at 15 lbs pressure at 121°C for 15 minutes. The
autoclaved medium was mixed well and poured onto 100mm petriplates (25-
30ml/plate) while still molten.
2. Nutrient broth (1L)
One litre of nutrient broth was prepared by dissolving 13 g of commercially

available nutrient medium (HI Media) in 1000ml distilled water and boiled to dissolve
the medium completely. The medium was dispensed as desired and sterilized by
autoclaving at 15 lbs pressure (121ºC) for 15 minutes.
3. Streptomycin (standard antibacterial agent, concentration: 10mg / ml)
4. Culture of test organisms; growth of culture adjusted according to McFards Standard,

0.5%
1. E.coli (ATCC 25922)

2. Staphylococcus aureus (ATCC 25923)
3. Pseudomonas aeroginosa (ATCC 27853)
4. Streptococcus mutans (MTCC 890)
5. Klebsiella pneumonia (ATCC 13883)
PROCEDURE
Petriplates containing 20ml Muller Hinton Agar Medium were seeded with
bacterial culture of E.coli, Streptococcus mutans, Pseudomons aeroginosa, Klebsiella
pneumoniae and Staphylococcus aureus (growth of culture adjusted according to
McFards Standard, 0.5%). Wells of approximately 10mm was bored using a well cutter
and different concentrations of sample such as 250μg/mL, 500μg/mL, 1000μg/mL were
added. The plates were then incubated at 37°C for 24 hours. The antibacterial activity
was assayed by measuring the diameter of the inhibition zone formed around the well
(NCCLS, 1993). Streptomycin was used as a positive control.
148
RESULTS AND DISCUSSION
5. RESULTS AND DISCUSSION

One of the Siddha metalo-mineral formulation, Kaalamega Narayana
Chendhooram had been exposed to several modern scientific studies to establish its
efficacy to scientific people and public. Literary collection, Physicochemical and
elemental analysis, toxicological studies and pharmacological studies were done to
justify the anticancer activity of KMNC against Oral cancer. Siddha literatures related
to the drug brings the evidence and importance of its utility in treating the cancer. The
desirable consequences are displayed and discussed for its anticancer nature.
 Gunapadam review brings the effectiveness of the drug in treating cancer.

 Siddha and Modern aspects of the disease were also reviewed. Disease review
brings about the knowledge of the disease.
 The pharmacological review explains about the methodology of Anti-cancer
activity and the drugs used.
 Pharmaceutical review describes about the Chendhooram, its properties and its
efficacy in treating cancer.
 Lateral research gives the effectiveness of the drug in treating cancer.
Discussion on Gunapadam review
 The poem for general properties of processed quicksilver directly

indicates its anticancer nature.
 Padigaram had a property of healing chronic ulcers.
 Vengaram was used in the treatment of ulcers.
 Thurusu was used for its healing deep chronic ulcers.
 Navachaaram is an ingredient of veera mezhugu shows a potent
anticancer effects.
 Lingam had a property of healing deep ulcer.
Discussion on modern drug review
 Alum had an anti - microbial activity, in which microbes also plays an

important role in causing cancer[85a]
 Mercury helps to destroy the cancer cells and reduces the tumour
growth [89a].
 Copper sulphate, Cinnabar, Orpiment have antimicrobial activity[86a]
ANTI CANCER ACTIVITY OF KAALAMEGA NAARAYANA CHENDHOORAM

149
 Borax have anti-inflammatory and healing properties[87a]

 Orpiment plays an important role in treating blood cancers[91a]
 Sulphur have an anticancer effect[92a]
 Arsenic exhibits anti -cancer activity [93a].
Discussion of pharmacological review
The cell lines for this anticancer activity evaluation was carried out using KB
cell line (oral cancer cell lines). They are the genomes of HPV 16 and HPV 18
respectively. These HPV 16 and HPV 18 are the responsible for 93% of Oral Cancer
[3a]
.
So, the analysis of pharmacological activity through KB cell lines is the novel
methods for validation. They explained effective anticancer character of KMNC.
Discussion on pharmaceutical review
 Chendooram.
 75 years of shelf life denotes its long time efficacy.
 Being very fine particles it increases the therapeutic effect.
Discussion on materials and methods

The selection of trial drug was taken from the book Athmaraksha Mirtham
Ennum Vaithiya Saara Sangeraham written by Kandhasamy Mudhaliyaar, was
approved by the Department of AYUSH as Per the Classical Siddha literature.
The ingredients were bought from the authenticated vender and they were
identified and authenticated by the experts in Post Graduate Department Gunapadam,
GSMC, Chennai. So the ingredients were perfect and original.
The preparation of medicine was done at the well-equipped lab of the Post
Graduate Department of Gunapadam.
The analytical parameters were conducted at registered and licensed

laboratories only. Thus the result of Kaalamega Narayana Chendhooram under
various analytical procedures showed the accuracy of it.

150
The Siddha metalo-mineral formulation Kaalamega Narayana Chendhooram

had been subjected to various studies for its scientific validation and safety
assessment.Literary collections, physicochemical and Elemental analysis,
Toxicological study, Pharmacological studies are done to prove its efficacy.
Results of Siddha Standardization

Table:7
S.No Parameters Results for ideal Results of Interpretation
Chendhooram KMNC
1 Colour Reddish Reddish Chendooram
brown colour.
2 Floating of Floats on water Floats on Lightness of drug.
water water
3 Finger Print Impinged in the Impinged in Indicates fine particles
test furrows of finger the furrows of powder.
of finger
4 Luster Lusterless Lusterless Change of specific
metallic character of
raw material after
incineration
5 Taste No specific taste No specific Change of specific
taste metallic character of
raw material after
incineration
Colour:
It is reddish brown in colour. The absence of shining indicates there is no free
form of metals.
Fig no: 25

151
Floating on water:
Kaalamega Narayana Chendhooram floats on water. It is due to its less
specific gravity. So, it possesses the property of Chendhooram.
Fig no: 26
Finger print test:
Kaalamega Narayana Chendhooram impinged on the cervices of finger. This

indicates the particles are fine and it is in micro size.
Fig no: 27.

Lusterless& tasteless:
It is lusterless and tasteless
Fig no: 28.

152
Physical characterization of Kaalamega Narayana Chendhooram

Table-8
S.no. Parameter Result
1. Colour Reddish brown in colour
2. State of the drug Powder
3. Consistency Fine powder
4. Solubility Sparingly soluble in water, DMSO.
Well soluble in acids (Hcl and H2So4)
5. Sense on touch Fine
6. Sense on taste Tasteless
7. Sense of smell No significant smell is observed
Results of Physical Parameters

Table-9
S.NO Parameter Result
1. Specific gravity 0.956
2. pH value 4.24
3. Particle size Completely passes through sieve no.120
Loss on drying at 105
4. 0.61%
degree Celsius
97.78%
5. Total ash value
6. Acid insoluble ash 0.23%

7. Water soluble ash 4.76%
Discussion on Physico - Chemical parameters:

Solubility
 Solubility is the major factor for the bioavailability of a drug substance.
 It is useful to determine the form of drug and processing of its dosage form.
 The most frequent causes of low oral bioavailability are attributed to poor
solubility and low permeability [108].

153
KMNC is soluble in major solvents (H2SO4, HCl) and sparingly soluble in water
proves that its efficiency of solubility in the stomach indirectly, increasing the bio
availability.
Specific gravity
The trial drug “Kaalamega Narayana Chendhooram” shows (0.956) low
specific gravity compared to water. Thus it flows in water and indicates lightness of
the medicine. This lightness of the medicine indicates its nature of absorption.
pHvalue
 Kaalamega Narayana Chendhooram shows acidic pH.
 The PH level plays a role in enzyme activity by maintaining the internal
environment, thus it exhibits an important role in regulating homeostasis.
 It is also an important factor for drug absorption [109]
. Because of the acidic
nature, the drug is more readily absorbed in an acidic medium like stomach
which enhances the bioavailability of the drug.
Loss on drying
 Loss on drying (LOD) of KMNC gives the total amount of volatile content and
moisture (water) present in the drug.
 The stability of a drug and its shelf-life are depends on moisture content.
 Moisture increase can adversely affect the active ingredient.
 Low moisture content- drug could get maximum stability and better shelf life.
 The low moisture content of KMNC indicates that it has long shelf life. Since
the drug has low loss on drying (0.61%), the moisture content is less which is
suitable for medicine.
Ash values
Total Ash value
High level of total Ash value of the trial drug KMNC contains (97.78%)
indicates the richness of organic substances. These organic compounds are
responsible for mineral supplements and therapeutic effect of KMNC and also it
indicates it was under the process of incinerations.

154
Acid insoluble ash

Lower acid insoluble ash value (0.23%) better will be the drug quality [110].
The drug ensures a low value of acid insoluble ash indicating that the preparation did
not contain any sand, dust and stones.
Water soluble ash
Water soluble ash value (4.76%) indicates the easy facilitation of diffusion and
the osmosis mechanisms.
Bio Chemical analysis:
Table No.10. Results of Basic radicals
Parameters Result
Test for Potassium Negative
Test for Calcium Positive
Test For Magnesium Negative
Test For Ammonium Positive
Test For Sodium Positive
Test for Iron (Ferrous) Negative
Test For Zinc Positive
Test For Aluminium Negative
Test For Lead Negative
Test for Copper Negative
Test For Mercury Positive
Test for Arsenic Positive

155
Table No.11. Results of Acid Radicals
Parameters Result
Test for Sulphate Positive
Test for Chloride Positive
Test for Phosphate Negative
Test for Carbonate Negative
Test for fluoride &oxalate Negative
Test For Nitrate Negative
DISCUSSION:
 The Biochemical analysis for basic radicals of KMNC shows the presence of
Calcium, Mercury, Arsenic, Sodium, Ammonium and Zinc .
 The Biochemical analysis for acidic radicals of KMNC shows the presence of
Sulphate and Chloride.
 The Presence of these radicals helps KMNC for its therapeutic effect.
Calcium:
A randomized controlled trial found that 1400–1500 mg supplemental calcium

and 1100 IU vitamin D3 reduced aggregated cancers with a relative risk. [111]
Ammonium:
Ammonia generated from the amino acid catabolism following glucose

deprivation can also stimulate autophagy. This ammonia induced autophagy also
promotes cell survival and thus represents a promising therapeutic target in cancer
treatments.[112]
Sodium:
Sodium has a cytotoxic effect on cancer cells.[113]

156
Zinc:
Zinc is needed for its immune function, wound healing and blood clotting.
Some experiments shows, that the Zinc slows the growth of cancer cells in the
laboratory.[114]
Mercury:
Miles (1926) introduced perchloride of mercury as an antiseptic agent in rectal

surgery. Goligher et al. (1951), Morgan (1955) and Keynes (1961) introduced the
technique of flushing the colon and rectum in restorative cancer surgery. Perchloride
of mercury solution was used as a anti-cancer agent in renal surgery. It is therefore
concluded that mercury perchloride is a safe anti-cancer agent when it is used in a
large bowel surgery. H Brendan Devlin et al.
Arsenic:
During the 18th and 19th centuries, a number of arsenic compounds were used
as medicines. In that Arsenic trioxide has been used in a variety of method of
treatment over the past 500 years, but most commonly used in the treatment of cancer.
In 2000, the FDA approved this compound for the treatment of acute promyelocytic
leukemia that is resistant to ATRA.
Sulphate:
Hydrazine sulphate is a chemical compound that has been studied as a

treatment for cancer. Sulphate contains anti-cancer property.
Chloride:
Chloride has cyto toxic effects on cancer cells.[115]

157
Availability of bacterial and fungal load in Kaalamega Narayana Chendhooram
Table No.12. Bacterial and Fungal dilution:
Microbes Dilution Result
Bacteria 10-4 1
Bacteria 10-6 Nil
Fungi 10-3 Nil
Fungi 10-2 2
DISCUSSION:
 The availabity of bacterial load in the KMNC has been performed by plate
count –Agar plate technique
 The microbial load have been analyzed for the micro-organisms
contamination. The contamination of a trail drug by micro-organisms not only
cause bio deterioration but also reduces the efficacy of drugs.
 The toxic effect produced by microbes makes the drugs to give no response
for human consumption because the contaminated drug may develop several
diseases instead of disease being cured.
 Here, the contamination of KMNC has been examined by bacterial and fungal
load.
 Total bacterial load in 10-4 dilution is 1 and in 10-6 dilution is
nil.
 Total fungal load in 10-2 dilution is nil and in 10-3 dilution is 2.
Here, the contamination of KMNC is within the WHO norms. Hence, the drug
is collected, prepared, stored and packed and decontaminated prior to
formulation.

158
INSTRUMENTAL ANALYSIS:
FTIR (FOURIER TRANSFORM INFRARED SPECTROSCOPY)
Kaaamega Narayana Chendhooram
Graph : 1 Peak values by FTIR
S.no Absorption peak (cm-1) Stretch Functional group
1 3846 O-H,H Alcohols, phenols
7 3516 O-H,H bonded Alcohols, phenols

159
8 3491 O-H,H bonded Alcohols, phenols
9 3470 N-H 1º,2ºamines, amides.
12 2287 H-C=O, C-H Aldehydes
13 1820 C=O α –β unsaturated aldehydes,

Ketones.
14 1862 -C=C Alkenes
15 1820 C=O Carbonyl ( General )
28 1710 C-O α –β unsaturated aldehydes,

Ketones
29 1691 C-O α –β unsaturated aldehydes,

Ketones
30 1678 -C=C Alkene
31 1658 -C=C Alkene
32 1630 N-H 1ºamine
33 1611 N-H 1ºamine
34 1547 N-O Asymmetric Nitro-compounds

stretch

stretch

160

stretch

stretch
38 1468 C-H Alkanes
39 1332 C-N Aromatics amines
Table-13
Interpretation
The wave numbers from 4000cm-1 to 1500cm-1 gives details for identification of
functional group.
The wave number from 1500cm -1 to 400 cm-1 provides particulars about a
molecular fingerprint.
The above result shows the presence of functional group like alcohols, Alkanes,
amides in Kaalamega Narayana Chendhooram.
They may be responsible for the presence of anticancer action of Kaalamega

Narayana Chendhooram in oral cancer.
Amides
Amide derivatives of Benzene- sulfonanilide, a Pharmaceutical composition is
used in cancer treatment [111].The lead molecule of these compound was methane
sulfonamide, a cyclo oxgenase (COX) inhibitor. They act as a efficient anti tumour
agents[116].
OH
OH group of KMNC has higher potential towards inhibitory activity against

microorganisms[117].

161
Ketones:
Ketones plays an important role in treating cancer cells. The eliminating

carbohydrates can quickly lower calorie intake, reducing the energy available to the
cells in the body. In turn, this may slow down the tumour growth and the cancer’s
progression[118].
Aldehydes:
Aldehydes plays an important role in maintain and differentiation of stem cells

as well as normal development. Aldehydes are the potential therapeutic target for
treatment of prostate cancer and also plays a key role in resistance to radiation therapy
and tumour recurrence in prostate cancer[119].
Nitro compounds:
Nitro and nitroso compounds are potent, selective and nontoxic inhibitors,
suppressants of cancer growth and viral infections. These compounds are particularly
useful for the treatment and suppression of tumours and viruses.
Phenols
 The effect of phenols is currently of great awareness due to their anti

carcinogenic activities.
 Phenolic acid components play an important role in the control of cancer and
other human diseases.
 Phenols and flavanoids possess diverse biological activities, for example,

antiulcer, anti-inflammatory, cytotoxic and antitumour, antispasmodic and
antidepressant activities.
Alkanes:
Alkane derivative like bis (4-amino-5-mercapto-1, 2,4-triazol-3-yl) possess

anticancer activity [120].

162
Alkanes:
Alkenes are the molecules containing a C=C double bond and is claimed to
reduce the risk of heart disease and cancer.
Carboxylic acid
 Benzene-poly-carboxylic Acid Complex (BP-CI) is a novel anticancer
complex against human cancer cells.
 Docosahexaenoic acid (DHA) is an omega-3 fatty acid. Its structure is a
carboxylic acid (-oic acid) with a 22- carbon chain (docosa-is Greek for 22)
and six (hexa-) cis double bounds [121].
 DHA was revealed to increase the efficacy of chemotherapy in prostate cancer
cells and a chemo protective effect in a mouse model was reported.
 It may also be used as a nontoxic adjuvant to increase the efficacy of
chemotherapy.
 In mice, DHA was found to reduce growth of human colon carcinoma cells
 The cytotoxic effect of DHA was caused by decrease in cell growth regulators.
Ether:
Certain ether lipids such as 1-0-octadecyl-2-0 methyl-rec-glycero-3-
phosphocholine represent a new class of anti -neoplastic agents. These ether lipids
have been shown to be cytotoxic for a wide variety of tumours.
SEM
(SCANNING ELECTRON MICROSCOPE)
The particle size and chemical elements were assessed by SEM is one of the
most widely used instruments in research areas.
The following image is done by 10 K X magnification via 1µm aperture shows

maximum depth focused.

163
96nm
120nm
134 nm
Fig no: 29 Showing nano particles in SEM image of 1µm

160nm
Discussion on SEM reports m
 The above SEM study shows of microscopic resolution gives the ranges from
1, 20µm.
 The difference in morphology as evident from the micrograph is due to the
342nm
presence of various substances in the sample.
 The presence of nanoparticles in a drug which are in size of 100nm has to be
considered as a nanomedicine. 105nm
Advantages of nano particles:
 Enhancing solubility of hydrophobic drugs.

 Prolonging circulation time.
 Minimizing nonspecific uptake.
 Increasing therapeutic effect for the drug.
 Decreasing toxicity, side effects.
 Preventing undesirable side effects.
 Improving intracellular penetration.
 Improving stability and increased biavailability
 Specific cancer targeting [122].
 The test drug Kaalamega Narayana Chendhooram contains Nano particles.
 Nano particles present in the drug results in a better bioavailability and
facilitates absorption. Thus they act on a cellular level and possess anticancer
activity.

164
 Nanotechnology is a promising way from the cancer management towards

cancer elimination.
 The particles of nano size shows that the drug may easily enter the cells at the
molecular level to treat the disease rapidly and increase its therapeutic effect.
The SEM images of KMNC shows the presence of particles are spherical in
shapes, aggregated morphology.
The observed size of the particles are 96nm, 120nm, 134nm.The size of the
particles are in and around nano range. This trial drug KMNC can be considered as
nanomedicine. So, the bioavailability of the drug will be high. In addition to that the
drug will be highly potent even in lower dose. Nowadays the research studies reveals
that the current trends of cancer medications not only depends on chemotherapy,
brachiotherapy, additionally it also needs a immunotherapy and especially
nanotherapy. This nano medicine will helps to achieve magical remedy in the
treatment of cancer in this modern World.
ICP-MS RESULTS AND DISCUSSION
ICP-MS interpretation of KMNC

Table-14
S. no Elements Detected levels
1. Arsenic BDL 2.465(below 3PPM)
2. Mercury BDL 0.992(below 1PPM)
3. Lead BDL (below 10PPM)
4. Cadmium BDL (below 1PPM)
Discussion on ICP-MS:
From the above results, that the presence of heavy metals Arsenic, Mercury
are observed within the WHO permissible limits. Cadmium and lead are under the
permissible limit. This ensures the safety of the drug for its therapeutic use and it will

165
give much more effectiveness in the treatment of diseases without causing any
damages to the body cells. Safety of this drug apart from toxicological study, it is
validated by heavy metal analysis through ICPMS as per WHO guidelines report.
XRD (XRAY DIFFRACTION)

Graph :2 XRD Interpretation
Strongest 3 peaks of XRD analysis.

No. Peak 2Theta
1. 7 31.2824
2. 3 26.5708
3. 5 28.2502

166
Discussion
The crystalline structure, the size and shape of the particles are highly
dependent on the route of synthesis and high lights the efficacy of the drug. The nano
particles may enhance the bio absorption of the drug.
XRD pattern of Kaalamega Narayana Chendhooram shows the good

crystalline after incineration process. The major diffraction peaks are identified after
XRD analysis KMNC concluded that in nano crystalline range (26-31nm) is
association with organic molecules probably plays an important role in making its
biocompatible and non toxic at its therapeutic doses. Other elements present in KMNC
act as a additional supplement and possibly helps in increase the efficacy of the
formulation [123]
ACUTE ORAL TOXICITY
Dose finding experiment and its behavioral Signs of Toxicity for Kaalamega
Narayana Chendooram:
Observation done:
Table-15
SL Group Observation SL Group Observation

CONTROL
TEST
1 Body weight Normal 1 GROUP
Body weight Normal
2 Assessments of posture Normal 2 Assessments Normal

of posture
3 Signs of Absence 3 Signs of Absence of sign (-)
Convulsion,Limb Convulsion
paralysis
Limb
4 Body tone Normal 4 Body tone Normal
paralysis
5 Lacrimation Normal 5 Lacrimation Absent
6 Salivation Normal 6 Salivation Normal
7 Change in skin color No significant 7 Change in No significant color

color change skin color change

167
8 Piloerection Normal 8 Piloerection Normal
9 Defecation Normal 9 Defecation Normal

10 Sensitivity response Normal 10 Sensitivity Normal
response
11 Locomotion Normal 11 Locomotion Normal
12 Muscle gripness Normal 12 Muscle Normal

gripness
13 Rearing Mild 13 Rearing
gripness Mild
gripness
14 Urination Normal 14 Urination Normal
Table-16
Dose
No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
mg/kg
1. Control + - - + - + - - - - - - - - - - - - + -
2000mg
2. + - - + - + - - - - - - - - - - - - + -
Behavioural Signs of Toxicity for KMNC
1. Alertness 2.Aggressiveness 3. Pile erection 4. Grooming 5.Gripping 6. Touch

Response 7.Decreased Motor Activity 8.Tremors 9 Convulsions 10. Muscle Spasm
11. Catatonia 12.Musclerelaxant 13.Hypnosis 14.Analgesia15.Lacrimation
16. Exophthalmos 17. Diarrhoea 18. Writhing 19 Respiration 20. Mortality

168
Table 17 ( Body weight Observation)
DAYS
DOSE
1 7 14
CONTROL 280.2±42.30 281.4 ± 64.12 282.6 ±26.18
HIGH DOSE 279.4± 21.24 279 ± 3.64 279.4 ± 2
NS NS NS
P value (p)*
NS- Significant, **(p < 0.01), *(p <0.05), n = 10 values are mean ± S.D (One
way ANOVA followed by Dunnett’s test
Table: 18 (Water intake (ml/day) of Wistar albino rats group exposed to

Kaalamega Narayana Chendooram
DOSE DAYS
1 7 14
CONTROL 61 ± 1.12 62±2.22 62.9±1.14
HIGH DOSE 61.2±1.1 61±1.14 61.20±24
P value (p)* NS NS NS
N.S- Not Significant, **(p < 0.01), *(p <0.05), n = 10 values are mean ± S.D (One
way ANOVA followed by Dunnett’s test)

169
Table 19: Food intake (gm/day) of Wistar albino rats group exposed to
DOSE DAYS
1 7 14
CONTROL 56.24±2.22 56.2±7.42 57.4±3.46
High DOSE 56.6±1.63 55.6±2.62 55.1±5.38
N.S- Not Significant, **(p < 0.01), *(p <0.05), n = 10 values are mean ± S.D (One
Acute toxicity Discussion:
In the acute toxicity study, the rats were treated with different concentration of
Kaalamega Narayana Chendooram from the range of 5mg/kg to 2000mg/kg.
 This dose level did not produce the signs of toxicity, behavioral changes, and
mortality in the test groups as compared to the controls when observed during
14 days of the acute toxicity experimental period.
 However the behavior changes, Body weight, Water intake, food intake does
not produce much significant, Thus the results are in non-significant.
 These results showed that a single oral dose of the extract showed no mortality
of these rats even under higher dosage levels indicating the high margin of
safety of this drug.
 In acute toxicity test the Kaalamega Narayana Chendooram was found to be
nontoxic at the dose level of 2000mg/ kg body weight.

170
28 DAYS OF REPEATED ORAL TOXICITY STUDY IN RATS
Table 20: Body weight of wistar albino rats group exposed to Kaalamega
Narayana Chendooram
DAYS
DOSE
1 15 28
CONTROL 280.2±10.03 281.2 ± 10.24 281.6 ± 24.61
LOW DOSE 279.2 ± 40.20 279.5 ± 30.14 279.8± 52.40
MID DOSE 281.3±20.04 282.3±9.04 280±42.03
HIGH DOSE 279± 01.10 278.89 ± 10.30 279±04.32
NS- Not Significant, **(p < 0.01), *(p <0.05), n = 20 values are mean ± S.D (One
way ANOVA followed by Dunnett’s test.
Table 21: Water intake (ml/day) of Wistar albino rats group exposed to
DOSE DAYS
1 15 28
CONTROL 98.4 ± 1.25 98±1.02 98.8±2.30
LOW DOSE 97.2±6.40 97.4±8.50 97.6±1.14
MID DOSE 97.3±5.4 97.55±7.9 97.5±6
HIGH DOSE 97.8.1±1.30 97.20±2.20 97.7±4.52
NS- Not Significant, **(p > 0.01),*(p >0.05), n = 20 values are mean ± S.D (One way
ANOVA followed by Dunnett’s test.

171
Table 22: Food intake (gm/day) of Wistar albino rats group exposed to
DOSE DAYS
1 15 28
CONTROL 190.4 ± 2.25 191±7.14 191.4±4.20
LOW DOSE 190.2±1.12 189.2±1.02 189.4±4.14
MID DOSE 189.4±2.22 188.1±2.25 189±4.2
HIGH DOSE 188.1±1.20 188.2±2.21 188.6±2.40
NS- Not Significant, **(p > 0.01),*(p >0.05), n = 20 values are mean ± S.D (One way
ANOVA followed by Dunnett’s test.
Table 23: Haematological parameters of Wistar albino rats group exposed to

Category Control Low dose Mid Dose High dose P value

(p)*
Haemoglobin(g/dl) 10.2±0.24 10.10±0.36 10.1±0.11 9.28±0.26 N.S
Total WBC (×103 l) 14.52±0.05 14.42±0.13 14.44±012 14.40±6.16 N.S
Neutrophils(%) 26.15±0.01 25.11±0.22 25.23±1.13 26.20±2.30 N.S
lymphocyte (%) 79.10±1.06 79.23±1.02 78.24±0.23 78.26±4.46 N.S
Monocyte (%) 0.9±0.03 0.8±0.05 0.7±0.004 0.7±0.07 N.S
Eosinohil(%) 3.2±0.04 3.2±0.06 4.24±0.05 4.53±0.02 N.S
Platelets cells103/µl 604.16±2.66 600±4.26 599±3.32 599.06±4.54 N.S
Total RBC 106/ µl 8.49±0.01 7.09±0.50 7.07±0.44 7.64±0.32 N.S
PCV% 37.65±0.6 37.30±1.32 37.44±1.44 37.66±2.24 N.S
MCHC g/dL 38.4±1.42 38.06±0.47 38.06 ±0.22 38.30±2.34 N.S
MCV fL(µm3) 54.04±4.60 54.06±3.43 54.05±3.23 54.34±2.14 N.S
N.S- Not Significant, **(p < 0.01), *(p <0.05),n = 20 values are mean ± S.D (One
way ANOVA followed by Dunnett’s test).

172
Table 24: Liver Function Test of Wistar albino rats group exposed to Kaalamega
Narayana Chendooram
Treatment Control Low dose Mid dose High dose
T BILIRUBIN
0.50±0.07 0.52±0.16 0.54±0.44 0.60±0.15
(mg/dl).
Triglycerides
66.24±7.74 66.14±6.22 65.12±5.33 65.14±09.32
(mg/dL)
Cholesterol
66.61±4.45 66.10±6.46 65.02±0.44 62.14±2.43
(mg/dL)
T.Protein 7.14±0.12 6.84±0.24 6.87±0.22 5.36±0.42
SGOT/AST
119.12±0.43 120.23±1.22 120.4±0.44 122±0.32
(U/L)
SGPT/ALT
82.64±6.57 82.11±3.39 84.45±5.44 87.11±1.24
(U/L)
ALP
215.08±10.48 228.40±11.68 232.6±0.99 236.44±10.94
(U/L)

173
Table 25 : Biochemical Parameters of of Wistar albino rats group exposed to

P
BIOCHEMICAL LOW MID HIGH
CONTROL Value
PARAMETERS DOSE DOSE DOSE
(p)*
108.63
GLUCOSE (R) (mg/dl) 111.08±0.9 110.04±0.3 108.3±0.47 N.S
±0.81
T.CHOLESTEROL(mg/dl) 93.21± 1.16 93.8±1.50 93.3±1.65 93.03±1.15 NS
TRIGLY(mg/dl) 54.16±1.52 53.12±1.42 54.12±1.22 54.16±1.23 N.S
LDL 72.4±2.14 72.12±2.54 71.22±2.22 71.24±10.20 NS
VLDL 11.2±1.30 11.20±2.21 11.20±2.22 11.14±12.14 NS
HDL 27.14±6.12 27.42±2.30 27.33±2.34 28.17±2.14 NS
Ratio 1(T.CHO/HDL) 3.41±1.16 3.42±1.40 3.45±1.44 3.64±2.03 NS
Ratio 2(LDL/HDL) 1.92±1.14 1.91±1.12 1.91±1.23 1.96±08.02 NS

Albumin (g/dL) 5.43±0.16 5.50±0.52 5.23±0.56 5.42±9.48 NS
way ANOVA followed by Dunnett’s test
Table 25: Renal function test of of Wistar albino rats group exposed to
LOW MID HIGH P Value
PARAMETERS CONTROL
DOSE DOSE DOSE (p)*
UREA (mg/dl) 26.70±0.19 26.50±0.26 26.6±0.22 27.68±1.24 N.S
CREATININE(mg/dl) 0.22±0.02 0.52±0.04 0.56±0.04 0.98±0.07 N.S
BUN(mg/dL) 21.10±0.20 25.16±0.90 22.3±0.33 29.14±1.22 NS

URIC ACID(mg/dl) 6.04±0.34 7.06±0.51 7.09±0.22 8.42±0.20 N.S
way ANOVA followed by Dunnett’s test).

174
28 days of Repeated oral toxicity Discussion:

 The dose selected for the 28 days of repeated oral toxicity study was
20mg,100mg ,200mg/kg of Kaalamega Narayana Chendooram
 All the animals were free of intoxicating signs throughout the dosing period of
28 days.
Observations:
Overall observations were similar in both sex rats.These studies were done
with certain doses like low dose X ( 20mg), 5X (100mg), 10X ( 200mg).The values
are non significant.
Clinical signs of toxicity
No clinical signs of toxicity were observed.There is a slight variations in the

values but they are within the non significat ranges.
Mortality
No mortality was observed after 28 days repeated dose administration of

KMNC. All animals were survived up to study termination period.
Body weight
There is a slight variations in the body weights when compared to their initial
weight. No significant alterations were observed in body weight.
Food and water consumption
No effect of treatment was noted, they are within the non-significant ranges.
Physiological activities
There is no changes in their general behavior.
Blood analysis
a. Hematology
No treatment related effects were observed. However there is a slight
variations in the result but they were within the permissible limit.

175
b. Biological parameters
There is a slight difference has been noted but it is normal within the normal
limit. No treatment related effects were observed.
c. Histological examination
Histological examination of organs did not show as much pathological
variations.
Discussion
 The acute and repeated 28 days oral toxicity studies of KMNC did not produce
any toxicity signs in wistar albino rats. Daily administration of KMNC at
different doses 50mg/kg, 100mg/kg for 28 days was tolerated by the rats
without any mortality and morbidity, indicates the drug tolerance.
 There was a slight changes were observed in hematological report
 Hence the metalo - mineral formulation of KMNC can be considered to be safe
drug for prolonged use as revealed by toxicological studies.
HISTOPATHOLOGY EXAMINATION:
 Histopathology studies were carried out on liver, kidney and spleen and
recorded. Blood samples for hematological and blood chemical analyses were
taken from common carotid artery.
 All rats were sacrificed after the blood collection. The internal organs and
some tissues were observed for gross lesions. All tissues were preserved in
10% neutral buffered formaldehyde solution for histopathological
examination.

176
Fig no : 30 HISTOPATHOLOGY SLIDES:
Control: High dose of KMNC :
Kidney
Liver
Spleen

177
Discussion on Histopathology:
 From the histopathological examination, the slides of animal’s organ didn’t
reveal abnormalities.
 From the acute and 28 days of repeated oral toxicity studies shows some
significant changes were observed. But the values were found within the
normal limits. So the drug KMNC was non-toxic and safe. So the drug KMNC
is considered to be no observed adverse effect level ( NOAEL ) drug.
 Thus the safety of the drug is revealed so that it can be administered for long
time without side effects.
PHARMACOLOGICAL STUDY
Anti-cancer activity CELL LINE: KB Cell line
Table-26
Sample OD OD value OD value Average Percentage

Concentration value I II III OD Viability
(µg/mL)
Control 0.3859 0.3922 0.3642 0.3808 100

\
Sample code: KMNC
6.25 0.3647 0.3666 0.3703 0.3672 96.43
12.5 0.3606 0.3102 0.3311 0.3340 87.70
25 0.2808 0.2986 0.2824 0.2873 75.44
50 0.2277 0.1891 0.1969 0.2046 53.72
100 0.1626 0.1685 0.1659 0.1657 43.50
IC50 Value : KMNC shows the 50 % inhibitory concentration at 50µg/mL

178
Concentration
Graph: 3 KB cell lines.
Graph -3 shows the drug dose and % of Inhibition of KB Cell line after treating with
KMNC. It can be observed by the result of MTT assay that the IC dose of KMNC is
50µg/ml. As the dose increases the KB cell viability decreases. It was found that the
% of growth inhibition increasing with increasing concentration of KMNC steadily up
to 6.25 µg/ml on KB line (Table-26, graph -3) and that IC value on KB cell line was
50 and R value was 0.3808.
Cytotoxicity Assay by MTT

MTT colorimetric method, is a method for detecting cell survival and growth
methods. This assay is based on the metabolic reduction of 3- (4,5- dimethylthiazol-2-
yl) -2,5-difeniltetrazol (MTT) by mitochondrial enzyme succinate dehydrogenase in a
colored compound blue (formazan), allowing to determine the functionality of the
mitochondrial treated cells. This method has been widely used to measure survival
and cell proliferation. The amount of living cells is proportional to the amount of
formazan produced. Cell lines derived from NCCS, Pune were free from any kind of
bacterial and fungal contamination.
KMNC at different doses (6.25-100 µg in100 µl of 5% MEM) was administered for
24 hrs. It was found that the number of cells decreases as the dose increases and at
approximately 50 µg/ml dose of extract, 50% of the cells (KB cells) were less as

179
compared to normal control as shown in graph no (3). The percentage of cells

viability was determined by calculating the O.D of treated against the control.
Reading optical density (OD) is performed in a spectrophotometer at a wavelength of
540 nm. Comparison values are made on a basis of 50% inhibition of growth (IC50) in
treated cells with specific agents. Results are tabulated in Table (25) and graphically
represented in Graph(3).
Analysis of Membrane Morphological Characteristics by Haematoxylin /Eosin

(H/E) Staining
Morphological changes such as changes to the cell membrane, loss of
membrane asymmetry and cell shrinkage, are the early stage of apoptosis was
analyzed by H/E staining. The IC dose (50µg/ml) treated cancer cells show features of
apoptosis whereas treated with same amount of dose, to normal treated cells appeared
without any significant changes.
 Since the discovery of the Cisplatin anti-tumour activity, great efforts have
focused on the rational design of metal-based anticancer agents that can be
potentially used in cancer chemotherapy.
 Over the last four decades, a large number of metal complexes have been
extensively investigated and evaluated in vitro and in vivo [124].
 The key focuses of these studies lie in finding novel metal complexes which
could potentially overcome the hurdles of current clinical drugs including
toxicity, resistance and other pharmacological deficiencies.
 Metalo-mineral and Minerals compounds have been used in medicine for
several thousands of years.
 The medicinal uses and applications of metals and metal complexes are of
increasing clinical and commercial importance. Monographs and major
reviews, as well as dedicated volumes, testify to the growing importance of the
discipline [125].
 The field of inorganic chemistry in medicine may usefully divided into two
main categories: firstly, ligands as drugs which target metal ions in some form,
whether free or protein-bound; and secondly, metal-based drugs and imaging
agents where the central metal ion is usually the key feature of the mechanism
of action.

180
 Arsenic trioxide, As2O3 (Trisenox, Cell Therapeutics Inc, Seattle,USA) which

was approved by the FDA in September 2000 for the treatment of Acute
Promyelocytic Leukemia (APL) in patients who have relapsed or are
refractory to retinoid and anthraycline chemotherapy.
 An estimated 1,500 new cases of APL are diagnosed yearly in the US, of
which an estimated 400 patients will not respond to, or will relapse from, first-
line therapy .
 The approval of arsenic trioxide as a chemotherapeutic agent invokes the
pioneering work of Ehrlich and the development of Salvarsan for use in
syphilis- the foundation stone for the science of chemotherapy [126].
 The use of chelating agents in medicine may even be traced to a collaboration
between Werner (the father of coordination chemistry) and Ehrlich (the father
of chemotherapy) to find less toxic arsenic compounds for the treatment of
syphilis [119].
 Arsenic has been used therapeutically for more than 2,000 years and was used
in the 1930s for treatment of chronic myelogenous leukemia until by the
development of newer chemotherapies.
 The past, present and future of medicinal arsenic has been described as a story
of ‘‘use, dishonor, and redemption’’.
 Recent interest in arsenic trioxide initially arose through Chinese reports of its
efficacy and use. Side effects are cardiotoxicity, skin rashes and
hyperglycemia [127].
 Arsenic trioxide apparently affects numerous intracellular signal transduction
pathways and causes many alterations in cellular function.
 Thus, the mechanisms of cell death induced by arsenic trioxide are multiple;
inductions of apoptosis, inhibition of proliferation and even inhibition of
angiogenesis have all been reported.
 In cellular studies, arsenic trioxide inhibits glutathione peroxidase, possibly
through generation of arsenic–GSH conjugates, and increases cellular
hydrogen peroxide content [128].
 The incineration process of the Mercury and Sulphur macro particles are
became very smaller and this may be possible for devoid of toxicity and more
potent in Anticancer therapeutic.

181
 Arsenic disulfide, a major effective component of realgar, has been

investigated for its anti-cancer potential and shown to have therapeutic
efficacies in hematological and some solid tumours.
 Several pieces of evidence indicate that iron deprivation could be an excellent

therapeutic approach:
(i) dietary iron restriction markedly decreases tumour growth in rodents
(ii) The antibodies which block transferrin-binding to cellular receptors
inhibit cancer cell growth in vitro and in vivo [129]
 Sulphur is commonly used in Asia as a medicine to treat inflammation and
cancer.
 Sulphur has both the diol-containing compounds, 2a and 3, were the most
cytotoxic of the sulfide series against V-79 cells in vitro (IC (90) = 2.1 micro
M and 1.9 micro M, respectively). A preliminary anticancer screening against
P388 leukemia showed that 2a is highly active in vivo as well
 Organic sulphur has been studied on oral and other cancers and has been
found to have remarkable benefit in anti-cancer therapy .
 Oncologists and scientists engaged in the research of cancer treatments should
conduct a comprehensive study on the efficacy of mercury which is being used
as an anti-cancer drug in the age old Siddha system [130].
 Three years of research has shown that metal (Mercury, Arsenic and Copper)
based Siddha drug is a safe alternative for Cisplatin therapy or arsenic trioxide
in selected cases of cancer treatments wherein the patients cannot bear the
adverse effects. The mice treated with Siddha drugs showed better health,what
did in cisplatin therapy in terms of appetite, haemoglobin, red blood cells and
white blood cells.
 Thus from the above study, it is evident that the cytotoxic property of
Kaalamega Narayana Chendhooram may be due to the synergistic
interactions between the metal complex.

182
KB Cell lines:
Fig no: 30. Cancer cells in treated with various concentrations of KMNC:
Control KMNC treated with 6.25 µg/mL
KMNC treated with 12.5 KMNC treated with 25

µg/mL µg/mL
KMNC treated with 50 µg/mL KMNC treated with 100µg/mL

183
RESULT AND DISCUSSION:
Anti-Tumour Activity:
Apoptosis Assay
To verify the results of cell viability assays, Annexin V and propidium iodide
(PI) double staining was used to quantify apoptosis. AMOS III cells were either
treated with Kaalamega Naarayana chendhooram at 500 nm for 48 hours. Cells were
labeled with Annexin V–FITC conjugate and PI using the Annexin V assay kit
following the manufacturer's instructions (Sigma, St Louis, MO) and analyzed using
the BD Cell Quest Pro software. These results were further verified using Western
blot analysis for specific caspases and Poly (ADP-ribose) polymerase (PARP) assay.
UNTREATED CELL VEHICLE CELL KMNC (500nm)
Graph no:4

184
UTC GraphVC
No:5 KMNC(500nm)
Graph : 5
UNTREATED CELSS VEHICLE CONTROL KMNC (500nm)
Graph no :6
UTC
VC
KMNC
Graph no:7 . Annexin V assay and Cell Cycle analysis.

185
(A) Kaalamega Naarayana Chendhooram treated AMOS III cells showed

significant increase in early and late apoptosis (38%) as compared to the
untreated (5%) and vehicle control cells (8%) by annexin V assay.
(B) Kaalamega Naarayana Chendhooram treated AMOS III cells show
significantly higher number of cells in the Sub Go phase and G2M phases as
compared to the untreated control AMOS III cells.
DISCUSSION
In this study, we investigated the anti-proliferative and apoptotic activities of

KMNC on human OSCC cell lines. The present study showed that KMNC has
potential time and dose dependent anti-proliferative effect on OSCC cancer cell lines.
We found that IC for KMNC was 38% for OSCC cell lines. As shown by the flow
cytometry results, when the concentration of KMNC was increased, the percentage of
early apoptotic cells also increased. For this reason, the mode of cell death appears to
be due to early apoptosis cell death pathway.
RESULT AND DISCUSSION:
Antimicrobial activity:
Antimicrobial activity of Kaalamega Narayana Chendooram
GRAM NEGATIVE BACTERIA.
1. Table: 28 E.coli
Sample Concentration Zone of inhibition

(μg/mL) (mm)
KMNC Streptomycin (100µg) 20
250 15
500 16
1000 19
14 mm – Low sensitive, 15 mm – Moderate, above 16 mm – Highly sensitive

186
2. Table: 29 Pseudomonas aeroginosa

(μg/mL) (mm)
250 19
500 20
1000 23
3. Table: 30 Klebsiella pneumoniae

(μg/mL) (mm)
250 17
500 19
1000 23
GRAM POSITIVE BACTERIA
4. Table: 31 Staphylococcus aureus

(μg/mL) (mm)
250 20
500 27
1000 29

187
5. Table: 32 Streptococcus mutans

(μg/mL) (mm)
250 20
500 22
1000 24
Note: Concentration of stock 10mg/mL DMSO
Inference:
1. Streptococcus mutans - Highly sensitive in 500 (μg/mL)
2. Staphylococcus aureus - Highly sensitive in 250 (μg/mL)
3. Escherchia.coli - Highly sensitive in 250(μg/mL)
4. Klebsiella pneumoniae - Highly sensitive in 250 (μg/mL)
5. Pseudomonas aeruginosa - Highly sensitive in 250(μg/mL)
Discussion:
The development of resistance against the presently available antibiotics arises the
necessity of rediscovery of new anti-bacterial agents in traditional systems of
medicine. Different dosages of test drug against the microbes in antimicrobial activity
of KMNC was compared with Standard drug Streptomycin (100µg)/ml disc for the
following pathogens, they are Escherchia.coli, Pseudomonas aeruginosa, Klebsiella
pneumoniae, Staphylococcus aureus, Streptococcus mutans. The results represents
KMNC potencially inhibit the growth of all above organism in 250µl, 500µl and
1000µl / disc. 14 mm – Low sensitive, 15 mm – Moderate, above 16 mm – Highly
sensitive. The findings reveal that the Siddha drug KMNC have anti microbial potency
against bacterial pathogens which is used in the treatment of diseases. The results of a
trail drug will build a challenging weapon to achieve its antimicrobial activity in
malignant tumours.

188
Conclusion:
Cancer is one of the most dreadful disease and have an increasing incidence in
recent years. There is a huge need in finding a permanent cure for oral cancer. Hence I
derived a new formulation based on the basic principles of Siddha system of
medicine. Rather, the destructive process can be reversed by adapting the time tested
higher order Siddha medicine loaded with positive. Hence I have preferred to choose
the higher order medicine “KMNC” in treating cancer as per Siddha classical
literature. Siddha formulation KMNC has proved its efficacy in preliminary physico-
chemical analysis, instrumental analysis and in vitro anticancer assay. . Kaalamega
Naarayana Chendhooram showed promising anticancer activity in oral cancer cell
lines killing cancer cells by apoptosis. Further, Kaalamega Naarayana Chendhooram
demonstrated promising anti-tumour activity in oral cancer tumour xenografts without
significant toxicity to normal tissues underscoring the pre-clinical efficacy of
Kaalamega Naarayana Chendhooram, as a potential anti-cancer therapeutic for oral
cancer management. Kaalamega Naarayana Chendhooram also shows a resistance
aginst the microbes which causing cancer. From the bird view, I would like to explore
that the trail drug KMNC plays a major role in treating cancer cells in cell lines, anti
tumour effect and anti-microbial effect.

189
CONCLUSION
6. CONCLUSION
Oral cancer is the sixth most common malignancy found worldwide and some
are highly resistant to radiotherapy. The chemotherapy drugs also deliver intolerable
side effects which are worse than the disease. This paved way for a novel anticancer
drug which cures Oral cancer in a non-invasive way.
The intention of this study is to provide a solution for the above need. For a non-
violent anticancer drug to Oral cancer, as a trial drug “Kaalamega Narayana
Chendhooram” was taken from the classic Siddha Literature Athmaraksha mirtham
Ennum Vaithiya Saara Sangeraham written by Kandhasamy Mudhaliyaar which
was categorized by the department of AYUSH as a classical text.
Throughout the study, the safety and efficacy were tested thoroughly. The
procedure for drug preparation and its techniques for standardization revealed GMP.
The trial drug KMNC has satisfied all parameters of testing protocol for
Chendhooram which was assigned by AYUSH. It showed the accurate production and
potency of “Kaalamega Narayana Chendhooram”
Physico-chemical analysis revealed better bio-availability and richness of its

mineral content. Favouring this study were the presence of inorganic matters which
were found through experiments for analyzing acid and basic radicals.
Various instrumental analysis of “Kaalamega Narayana Chendhooram”such as
FT-IR spectroscopy, X-ray diffraction and scanning electron microscope demonstrated
its chemical constituents, functional groups and particle size to support its indication to
counter oral cancer
Under OECD guidelines, the acute and 28 days repeated oral toxicity studies proved
the safety of “Kaalamega Narayana Chendhooram”at particular dose level. It is very
useful in therapeutic dose determination.
The pharmacological activities are justified by anticancer effect on KB
cell lines, anti-tumour effect on OSCC cell lines and anti-microbial activity. The anti-
microbial activity of trial drug was also considered for its potential.

190
CONCLUSION
Factors like safety, efficacy, long self like, bio-availability, presence of

significant elements, anions and cations and minerals favouring the activity justifies the
main perspective of this study.
KMNC’s anti-cancer effect could be validated scientifically. Due to its Non-
toxic anti-cancer effect, it would benefit the health community and the world.

191
SUMMARY
7. SUMMARY
The validation of therapeutic efficacy of the trial drug “Kaalamega Narayana

Chendhooram” was taken from the classic Siddha Literature Athmarakshamirtham
Ennum Vaithiya Saara Sangeraham written by Kandhasamy Mudhaliyaar for oral
Carcinoma (Kanna Putru), anti-tumour, anti-microbial activities were dealt with in the
entire study.
 Introduction of the study comprises about the Siddha concept, Modern concept,
prevalence of Oral Carcinoma worldwide, Complications of modern treatments
which is found to be the major cause of the death in human and the role of the
test drug in treating Oral cancer.
 Review of literature under various categories was carried out. It was elaborated
under Gunapadam and Modern aspect of ingredients, Siddha and modern aspect
of the disease, pharmaceutical aspect and pharmacological aspect in both
Siddha and modern.
 All the ingredients were identified and authenticated by experts.
 The drug was subjected to analysis such as physiochemical, biochemical
and instrumental analysis which provided the key ingredients present in the
drug thus it accounts the efficacy of the drug.
 The sample was also analyzed for anti-microbial activity to ensure its accuracy.
 For the study protocol, required animals were approved by the IAEC under
CPCSEA.
 Toxicological study was made according to OECD guidelines comprising both
acute and repeated oral dose 28days toxicity studies in wistar albino rats. It
showed the safety of the drug which attributes its utility in long time
administration.
 Pharmacological studies were completed. It revealed the anti-cancer, anti-tumor
and anti-microbial activities of “Kaalamega Narayana Chendhooram”.
 Results and discussion gives the essential validations to prove the potency of
the drug.
 Conclusion gives a Compiled form of the study and explains the synergistic
effect of all the key ingredients and activities that supports the study.

192
FUTURE SCOPE
8. FUTURE SCOPE
Trial drug for the study “KAALAMEGA NARAYANA CHENDHOORAM ”was

taken from the classic Siddha Literature Athmaraksha mirtham Ennum Vaithiya Saara
Sangeraham written by Kandhasamy Mudhaliyaar. Its validation for its Anti-cancer
nature was completed at preliminary level. The result enhanced and assured its Anti-
cancer property against oral cancer. More specific experiments on animal models and
also clinical trials are required to understand the exact molecular mechanisms of action.
So it could be used worldwide in treatment of oral cancer and satisfy the safe and painless
anti-neo plastic treatment.

193
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SCIENTIFIC VALIDATION OF ANTI-ORAL CANCER, ANTI-TUMOUR

AND ANTI-MICROBIAL ACTIVITIES OF SIDDHA METALLO-

The dissertation Submitted by

Under the Guidance of

THE TAMILNADU DR. M.G.R MEDICAL UNIVERSITY

In partial fulfilment of the requirements

For the award of the degree of

DOCTOR OF MEDICINE (SIDDHA)

POST GRADUATE DEPARTMENT OF GUNAPADAM

THE GOVERNMENT SIDDHA MEDICAL COLLEGE

DECLARATION BY THE CANDIDATE

Date: Signature of Candidate

Place: Chennai Dr. R.Abinaya

CERTIFICATE BY THE GUIDE

Date: Seal and Signature of the Guide

Place: Chennai Dr.R.Karolin Daisy Rani M.D(S)

ENDORSEMENT BY THE HOD AND

PRINCIPAL OF THE INSTITUTION

This is to certify that the dissertation entitled Scientific Validation of Anti-Oral

cancer, Anti tumour, and Anti-microbial activities of Siddha Metallo mineral

Formulation “Kaalamega Narayana Chendhooram ” in cell line studies is a

Bonafide work carried out by Dr.R.Abinaya under the guidance of Dr.R.Karolin

Daisy Rani MD(s), Lecturer, Post Graduate Department of Gunapadam, Govt.

Siddha Medical College, Chennai - 106.

Seal and Signature of the HOD Seal and Signature of Principal

Dr. M.D.Saravanadevi M.D(S) Dr.K.Kanakavalli M.D(S).

I wish to express my sincere thanks to Dr.K.Kanakavalli M.D(S).Principal, Govt.

I feel intensely grateful to Dr. M.D.Saravanadevi M.D(S), proff, Head of Department,

I wish to express my thanks to co-guide Dr.K.Rajamma Devi Sorubarani M.D(S),

I acknowledge my thanks to Dr.A.Ganesan M.D(S)., Dr.S.Shankar M.D(S).,

I cordially register my humble thanks to Dr. P. Muralidharan M.pharm., Ph.D.,

I acknowledge my thanks to Prof Dr.R.Rajesh M.Phil, Ph.D., Biogenix Research

I am also my thankful to our Librarian Mr.V.Dhandayuthapani, B.Com, M.Lib.Sc

I am also thankful to Mrs.Kanniyammal, D.Pharm, Pharmacist, Post Graduate

S.No TITLE Page. No

2. AIM AND OBJECTIVES 8

3.1.1. SIDDHA ASPECT 10

3.2.1. SIDDHA ASPECT 53

3.3. PHARMACOLOGICAL REVIEW 75

3.4. PHARMACEUTICAL REVIEW 96

3.5 LATERAL REVIEW 100

4. MATERIALS AND METHODS 103

4.1 PREPARATION OF THE DRUG 107

STANDARDIZATION OF THE DRUG 119

4.2.1 PHYSICO CHEMICAL INVESTIGATION 120

4.2.4 INSTRUMENTAL STUDY 126

4.2.5 TOXICOLOGICAL STUDY 135

4.3 PHARMACOLOGICAL STUDY 144

4.3.1 ANTI-CANCER ACTIVITY 144

4.3.2 ANTI-TUMOUR ACTIVITY 147

4.3.3 ANTI-MICROBIAL ACTIVITY 147

5. RESULTS AND DISCUSSION 149

S. NO TITLES PAGE NO.

2. CLASSIFICATION OF ANTI-CANCER DRUGS 80

4. DIFFERENT ASSAYS TAKE DISADVANTAGE OF 85

8. PHYSICAL CHARACTERIZATION OF 153

10. RESULTS OF BASIC RADICALS 155

11. RESULTS OF ACID RADICALS 156

12. BACTERIAL AND FUNGAL LOAD 158