Medicus No.1'2016

Hamdard Medicus Vol. 59, No.
1, 2016
MADINAT AL-HIKMAH
City of Education, Science and Culture
Shaheed Hakim Mohammed Said (1920-1998), a scion of the renowned South Asian
Hamdard family, decided in 1948 to make the newly created Pakistan his home. He
settled down in Karachi and by untiring, single minded devotion and commitment,
braving all handicaps, created Hamdard Pakistan. He developed it into the leading
pharmaceutical complex of Eastern Medicine in the country, run on the latest modern
lines and techniques, supplying drugs of high quality and purity, backed with free
clinical consultations to help ailing humanity. Hamdard Pakistan, under his leadership,
also emerged as the leading philanthropic organization, and also tried to motivate
people through dialogue, conferences, and journals like the Hamdard Medicus. In
addition, he provided help to various institutions and academic bodies.
He was restless to do more, and during one of his Hajj pilgrimages, he envisioned the
creation of a comprehensive City of Education, Science and Culture: the Madinat al-
Hikmah. Work on it was initiated and funded by the Hamdard Foundation Pakistan in
1981, in the picturesque surroundings of Bund Murad Khan, 35 kilometers away from
the city centre. Today it is a beehive of activity, with children and youth engaged in
academic, technical and sports activities, devoted to promoting learning and culture,
and through it, help achieve moral and physical welfare, peace and progress. The Bait
al-Hikmah Library, Hamdard Public School, free Hamdard Village School, Centre for
Horticulture and the Hamdard University are the major institutions found here. The
Hamdard University’s mission provides value-based education to all students in its
constituent institutions. Its prestigious institutions – some based at the main campus
and some in the city and some in Islamabad - include Hamdard Institute of Management
Sciences (HIMS), Hamdard College of Medicine and Dentistry (HCMD), Hamdard
Al-Majeed College of Eastern Medicine (HACEM), Shifa ul-Mulk Memorial Hospital,
Hamdard University Hospital – Naimat Begum Mother & Child Care Unit (part of the
Hamdard University Centre of Excellence), Hamdard Institute of Education and Social
Sciences (HIESS), Dr Hafiz Mohammad Ilyas Institute of Pharmacology and Herbal
Sciences (HMI-IPHS), Hamdard School of Law, Faculty of Pharmacy and Faculty of
Engineering Sciences and Technology (FEST). The Madinat al-Hikmah continues to
develop and grow.
Hamdard Medicus Vol. 59, No. 1, 2016
HAMDARD MEDICUS
Quarterly Journal of Science and Medicine
Regd. No. M-73. International Standard Serial Number (ISSN) 0250-7188
Vol. 59 January-March 2016 No. 1
Patron-in-Chief: Mrs. Sadia Rashid

Patron: Prof. Dr. Hakim Abdul Hannan
Editor: Prof. Dr. Ghazala Hafeez Rizwani
Managing Editor: Prof. Dr. Ahsana Dar Farooq
Advisory Board Members
Prof. Dr. Atta-ur-Rahman Prof. Dr. Hakim Zillur Rahman

Patron-in-Chief, ICCBS President, Ibn Sina Academy
International Center for Chemical and Aligarh, India.
Biological Sciences, University of Karachi
Karachi-75270, Pakistan. Prof. Dr. Stefan Reichmuth
Director, Der Geschaftfuhrende
Dr. Mahum Munir Ahmed Ruhr Univeristy Bochum, Germany.
Mutawallia, Hamdard Laboratories (Waqf) Pakistan
Vice President, Hamdard Foundation Pakistan Dr. Shigeru Suganami
Nazimabad, Karachi-74600, Pakistan. President, Association of Medical Doctors of
Asia (AMDA), Okayama City
Mrs. Lily Anne D’Silva Japan.
Vice President, Madinat al-Hikmah
Karachi-74700, Pakistan. Dr. Amos O. Abolaji
Drug Metabolism & Toxicology Research
Ms.Zahida Bano Laboratories, Department of Biochemistry
Former Director, English Publications University of Ibadan, Ibadan, Oyo State,
Hamdard Foundation Pakistan Nigeria.
Nazimabad, Karachi-74600, Pakistan.
Prof. Dr. Liu Xinmin
Prof. A.K. Azad Khan Institute of Medicinal Plant Development Chinese
President, Diabetic Association of Bangladesh Academy of Medical Science, Beijing China and
Chairman, Board of Trustees WHO Advisor on Traditional Medicine
Bangladesh University of Health Sciences (BUHS) Consultant & Advisor, State Administration of
Dhaka-1000, Bangladesh. Traditional Chinese Medicine, China.
Prof. Jurgen H. Hohnholz Prof. Dr. M. Iqbal Choudhary

Former Director, Institute for Scientific Cooperation Director, International Center for Chemical and
Tubingen, Germany. Biological Sciences
University of Karachi, Pakistan.
Mr. Salim Mehmud
Former Chairman SUPARCO Norazrina Azmi
Chief Scientist & Scientific Advisor Deputy Dean
MOD and Chief Scientific & (Undergraduate, Alumni & International Affairs)
Technical Advisor, MOC Faculty of Pharmacy, Universiti of Kebangsaan
91, St. 59, F-10/3, Islamabad-44000, Pakistan. Kuala Lumpur, Malaysia.
Editorial Board Members
Prof. Dr. Hakim Shahabuddin Dr. Shagufta Usmani

Dean, Faculty of Eastern Medicine Associate Prof., Department of Pharmaceutics
Hamdard University Faculty of Pharmacy
Karachi, Pakistan. Hamdard University
Karachi. Pakistan.
Prof. Dr. Azhar Hussain
Dean, Faculty of Pharmacy Dr. Tabiba Tasneem Qureshi
Hamdard University Associate Prof., Faculty of Eastern Medicine
Karachi, Pakistan. Hamdard University
Karachi Pakistan.
Prof. Dr. Muhammad Javed
Dean, Dr. Muhammad Faisal Khan
Faculty of Health & Medical Sciences Hamdard Institute of Engineering & Technology
Hamdard University Hamdard University
Karachi, Pakistan. Karachi, Pakistan.
Prof. Dr. Vali uddin Dr. Hina Zahid
Dean Assistant Professor, Faculty of Pharmacy
Faculty of Engineering Sciences & Technology Hamdard University, Karachi, Pakistan.
Hamdard University
Dr. Ghulam Abbas
Karachi, Pakistan.
Assistant Professor
Prof. Dr. Jawaid Siddiqui H.E.J. Research Institute of Chemistry
Acting Dean, Faculty of Humanities & International Center for Chemical and
Social Sciences Biological Sciences
Hamdard University, Karachi, Pakistan. University of Karachi, Karachi, Pakistan.
CONTENTS
1) Clinical Efficacy and Safety of Cupping Therapy

— Tasneem Qureshi and Abdul Hannan 5
2) Cuscuta reflexa L. & Roxb.: An Implication of Quercetin Mediated

Monoamine Oxidase Inhibition in Preclinical Models of Depression
Sara Zeeshan, Wahid Zada, Huma Aslam Bhatti and Ghulam Abbas 17
3) Anti-bacterial Activity and Qualitative Phytochemical Screening of

Fagonia cretica
— Kashaf Zia, Muhammad Qasim Hayat, Shaziad Anjum,
Muhammad Ashraf, Saima Hanif and Alia Sadiqa 24
4) Unani Treatment of Tinea Capitis: A Case Study

— Leena Hameed and Tasneem Qureshi 34
5) Phytochemical and Dermatological Investigations of Tribulus terrestris L.

— Khizar Abbas, M. Shehzad, Ayesha Asif, M. Imran Qadir and
M. Taufiq Ahmad 41
6) Inhibitory Effect of Aegiceras corniculatum Against Lung Injury in

Carrageenan-induced Mouse Pleurisy Model
— Sabahat Aziz, Talat Roome, Perbhat Ali and Anam Razzak 52
7) Mini Review: Herbal Medicinal Products Regulation in Pakistan

— Huma Shareef, Ali Akbar Sial, Farya Zafar and Huma Ali 60
8) Obituary 66
3
HAMDARD MEDICUS
Quarterly Journal of Science and Medicine
Vol. 59 January-March 2016 No. 1
INFORMATION FOR CONTRIBUTORS
The HAMDARD MEDICUS since 58 years has been publishing original articles, reviews, short
communications, history of traditional medicine and case reports on all aspects of complementary medicine
and pharmaceutical sciences in English. Manuscripts to Hamdard Medicus are accepted for consideration
with the understanding that the findings have not been published earlier.
Manuscript Preparation
Manuscripts should be typed double-spaced with at least one-inch margin on all sides.
Times New Roman 12-point is recommended for text, tables and figures. All text should be written in
the third person. The sections includes: Title page, abstract, text, references, tables, list of titles for all
figures (typed on one page), and figures. The page numbers of the entire manuscript should be at the
bottom right corner of each page.
Title Page Should Contain: Title of the manuscript, Short title, Authors names with superscripts
representing their affiliation clearly marked *corresponding author with Telephone, Fax and E-mail address.
Abstract: On a separate page (200-250 words) with Keywords (3-5).
The Text For Articles Should Contain: Main headings in caps and bold as:
1. INTRODUCTION, 2. MATERIALS AND METHODS (2.1 sub-headings [in bold italics] describing
animals, chemicals, preparation of extract, experiments and statistical analysis), 3. RESULTS AND
DISCUSSION, 4. CONCLUSION and ACKNOWLEDGEMENTS (if needed) before References,
5. REFERENCES..
Tables: On separate page, numbered as appearing in the text bearing title (bold) and legend underneath.
Figures: On separate page, numbered as appearing in the text Fig. 1 and so on.
References: Follow the pattern shown below: References should be in alphabetical order.
(Articles) 1. Quinton, R. (2012). The increase in the toxicity of yohimbine induced by imipramine and
other drugs in mice. Brit. J. Pharmacol. 21:51-66.
(Books) 1. Gaill, W., Jon, A.W. (1995). Manual of Clinical Microbiology. ASM Press, Washington, DC.
6th Edn., pp. 1327-1332.
Figure Title/Legend: On separate page.
Brief Reports
Should also follow same pattern as a full research paper with 1-2 short tables or figures.
Author’s Contribution
Conflict of interest: The authors declare that there is no conflict of interest.
Each contributor will receive two copies of the issue by airmail in which his or her paper has been published. Ten offprints
will be sent to the principal author by surface mail.
Copyright: Copyright of all material is held by the Hamdard Medicus. The Editor-in-Chief and the
Publisher are not responsible for the scientific contents and statements of the authors of accepted papers.
All ideas forwarded reflect the individual views of the authors.
Articles and photographs in this issue may not be reproduced unless previous permission has been
obtained from the Editor.
Publication Charge
There is no publication fee or charge for any submitted or accepted articles.
HAMDARD MEDICUS is indexed in:

Abstr. Hyg., Biol. Abstr., Curr. Adv. Ecol. Sci., Extra MED., Per. Islam., Trop. Dis. Bull., Index Medicus For
WHO Eastern Mediterranean Region, Medicinal and Aromatic Plants Abstracts, CAB Abstracts: Review of
Aromatic and Medicinal Plants.
Clinical Efficacy and Safety of Cupping Therapy
Tasneem Qureshi* and Abdul Hannan
Hamdard Al-Majeed College of Eastern Medicine,

Hamdard University, Sharae Madinat al-Hikmah, Muhammad Bin Qasim Avenue, Karachi-74600, Pakistan.
*Email: tas_qur@hotmail.com
Abstract depression. The relief from symptoms depended

In Unani System of Medicine, since o n temp er amental ev aluatio n an d its
centuries cupping therapy (Al Hijamah) has effectiveness appears to be: Sanguineous
been an integral part of regimental therapy to temperament harr (hot) and ratb (moist) >
treat various human diseases. The present study phlegmatic temperament barid (cold) and ratb
focuses on the therapeutic effect of wet cupping (moist) > bilious temperament harr (hot) and
therapy (combination of suction and controlled yabis (dry) > melancholic temperament , barid
medicinal bleeding) against various diseases. It (cold) and yabis (dry).
is the oldest form of alternative medicine in It is concluded that wet cupping therapy
which a suction is created locally on the skin, is effective against diseases of various origins
physicians consider that this act trigger blood and its efficacy is dependent on temperament
flow in order to support healing process. The and severity of the disease.
patients (n = 65) aged between 20-65 years,
enrolled in this study after obtaining their Keywords:
informed consents at Eastern Specialist Clinic, Unani system of medicine, Wet cupping therapy,
Karachi. They were clinically assessed and Temperament.
diagnosed on the basis of their past history, 1. INTRODUCTION
clinical ob ser vation and temp er amen tal The historical background of cupping
assessment grouped into: chronic constipation, therapy seems prominent to ancient Egyptian,
dyspepsia, knee joint pain/arthritis, migraine, Chinese, and Middle Eastern cultures as
shoulder pain/muscular spasm/cervical pain, indicated in the oldest medical textbooks in the
sciatica, stress/anxiety/depression and varicose world, the Ebers Papyrus, illustrates its practice
vein. The cupping therapy was applied on the during 1550 B.C by Egyptians to remove by
back and front of the body over different points bleeding the foreign matter from the body and
(5-6) for a period of 3-5 minutes comprising triggering blood flow thus encouraging the
3-10 sessions with an interval of 15 days. The curative process of various medical ailments.
wet cupping was effective in 72% of patients Hippocrates (Father of Modern Medicine) and
w hich sho w ed imp r ov emen t in th eir Galen were also enormous supporters of cupping
corresponding symptoms in order of chronic (Ahmad and Qadeer, 1998). Unani System of
constipation > migraine > sciatica > shoulder/ Medicine is based upon the Nazariyya-i-Akhlat
cervical/muscular pain > varicose veins > (theory of humors) as proposed by Hippocrates
dyspepsia > knee joints pain > stress/anxiety/ emphasizing the presence of four humours in
5
the body, namely dam (blood), balgham sufferer’s but to the disorder affecting or even
(phlegm), safra (yellow bile) and sawda (black to the type of prescription or activity suggested
bile) (Wasti, 1990). as therapy. It is stated in Unani-Tibb that
Each humor is associated with specific individuality of every patient should be considered
temperaments i.e. blood is harr (hot) and ratb during treatment (Bhikha and Haq, 2000).
(moist), phlegm, barid (cold) and ratb (moist); In order to release toxins and unwanted
yellow bile, harr (hot) and yabis (dry); black matter from the tissues and organs, cupping is
bile, barid (co ld) an d yabis (dr y). Any considered to be the oldest and most effective
imbalance in the humoral levels, if not controlled methods. Cupping can be classified as: fire
by innate system may lead to the development cupping, massage cupping and cupping by horn
of various diseases (Ahmed, 2009). method (Anjum et al., 2005). Other than that
The concept of temperament characterizes mainly there are two classes of cupping: 1) dry
the individuality of a human being (Rolfe, 2002) cupping and 2) wet cupping. In dry cupping
reflecting communal measure of an individual therapy the skin directly underneath the cup is
corporal structure and psychosomatic profile or sucked upwards by creating a vacuum within
personality. The above theory has laid a strong the cup and hold for 5-10 minutes while in wet
basis as its origin reflects back to centuries and cupping therapy superficial cuts are made on
is still applied with suitable adaptations the skin below the cup lightly lacerated enabling
concerning different medical and scientific the blood to be drawn from the skin within the
spheres (Azmi, 1995). As temperament is the cup (Li et al., 2006). A modified version of
amalgamation of physical, psychological, cupping therapy is massage cupping that has
emotional and spiritual features, depending on been used broadly in Chinese medicine. In this
these specific qualities there are four types of system suction and negative pressure is created
temperaments: Sanguinous (with qualities of Hot along with massage to bring blood flow to
and Moist) Phlegmatic (with qualities of Cold stagnant muscles and skin, loosen adhesions,
and Moist) Bilious (with qualities of Hot and connective tissue and stubborn knots in soft
Dry) Melancholic (with qualities of Cold and tissues, drain excessive fluids and toxins, to
Dry). However, each individual has trait from stimulate the peripheral nervous system. Before
all four temperaments with a dominance of one the application of the cup, preferably olive oil is
of the temperament types. Having a unique applied to the skin, after identifying the region
temperament a combination of qualities exists of tenderness and blockage in order to make
in an individual. That’s why there is a difference possible smooth movement. The vacuum is
in people experiencing either cold or hotness in created by placing the cup on to the affected
a unique way depending on the individual areas followed by gently gliding it on that area.
temperament. Predispositions to illnesses as well It is placed around 5 mins on persistent tender
as pathological and therapeutic processes may areas and on swollen joints as well as tissues.
result from the strong impact of temperament Strong massage cupping leads to reddened skin,
and associated qualities of a person. Qualities signifying blood movement towards the surface
associated with the temperament of the individual and ready for the application of liniments of
play an important role in the predisposition to natural origin (preferably sunflower, linseed,
specific illnesses, this concept in Unani-Tibb is energy producing castor oil and olive oil),
employed not only to the treated and to the analgesics (rose oil, babuna oil), plant hydrosols
6
( Ro se, Roman Ch amomile, Ner oli an d (60 ml) vacuum cupping cups of circumference
Lavender) or essential oils (Rose, Roman of 1 to 3 inches (25 mm-75 mm), vacuum pump
Chamomile, Jasmine and Lavender) (Anita J. with pistol grip (U.S Global, Karachi), sterilized
Shannon, A.C.E., Massage Cupping, www. and disposal items included: gloves, surgical
massagecupping.com) immediately facilitating blade, cotton and medical gauze and micro-pore
absorption deeper into the tissue for relieve of surgical tape (Medics shop at medicine market,
pain and obstructions. Local increase in blood Karachi), pyodine (Brookes Pharma, Pakistan)
flow nourishes the skin and muscles thereby, and honey (Al-Shifa) were purchased from the
allowing contaminated matter to be removed local market.
from the nearby tissues and organs towards the
surface to be eliminated. In case of wet cupping 2.2. Inclusion Criteria
therapy, incisions are made in order to remove The patients (65), both genders aged
blood which has been brought up just beneath between 20-65 years were enrolled in the trial
the surface of the skin The concept of cupping after obtaining their informed consent.
is to let out bad blood that is supposed to be
injurious to the body leading to improve in the Temperamental Evaluation:
flow of life energy, which travels throughout A questionnaire has been designed to
the body in channels called meridians (Arzani assess the temperament of the patients for
1940). analysis of the effect of cupping therapy on
The basic philosophy behind cupping is temperament.
that both inflammation(s) and pressure(s) are
diverted from the major organs including brain, 2.3. Exclusion Criteria
heart, liver, lungs, and kidneys to the skin. It Patients suffering from chronic serious
assists in the healing process by supporting the illnesses, dehydration, diarrhea, hypertension,
immune system, hence resulting in the optimal severe vomiting and uncontrolled diabetes,
performance of the body. It also aids the actions females during menstruation and pregnant
of physis, ward off toxins and other injurious women were excluded. Cupping was also
impurities from the major organs towards the avoided when the patient experiencing bleeding
skin, before expulsion, whereas, fresh blood disorders, inflammation, skin infection, ulcer, or
replenish the affected area (Ibn-ul-Kuf, 1935). who are prone to bruising.
2 MATERIALS AND METHODS 2.4. Instructions for the Patients

The study was conducted during November Prior to the cupping treatment regimen all
2012 to December 2014 at Eastern Specialist the patients were briefed as follow:
Clinic (PECHS), Karachi jointly by Brookes
Health and Education Foundation and Faculty i) To be mentally prepared and willing to
of Eastern Medicine, Hamdard University, adopt this therapy without any fear.
Karachi. ii) They should undergo 4h of fasting before
therapy.
2.1. Requirements and Equipments iii) It is an additional benefit, if the timings of
Cupping chair (self-fabricated), transparent the sessions are kept in mind such as
(polyvinyl) bell shaped small (30 ml) or larger weather conditions specially hot weather
7
Scarificat ion: Using surgical blades

(gauge 15-22) superficial incisions 3-4 mm long
and less than 0.1 mm deep were made on the
skin.
i) Blood letting: Immediately, the cup was

placed on the skin, as described above for
blood collection (3-5 mins) from the
capillary vessels.
ii) Removal of cup: Once the procedure was
completed it was removed and antiseptic
agent (Al-Shifa honey) was applied on the
affected area.
iii) Dressing o f affect ed a rea: A p re-
prepared medicated sterile fibrous material
or sterile muslin or cotton wool was placed
on it. This temporary dressing detached
Fig. 3a: Cupping points on the front side of the
itself within an hour. human body
The cupping therapy consisted for 3-7

sessions (duration: 1½ month-3½ months) with
an interval of 15 days. The cups of 4 and 5 size
n umbers ( Al- Ru baye, 2012) were u sed
throughout the procedure, while the position of
cup on the front (thorax) and back (trunk) of
the body was decided as described earlier (Cao,
Han, Li et al., 2010) with few modification and
were either common or different depending on
diseased conditions (Fig 3a and 3b).
3. RESULTS AND DISCUSSION

Patients (n = 65) underwent wet cupping
therapy against 8 d if ferent ailmen ts:
1) Constipation, 2) Dyspepsia, 3) Knee joint pain/
arthritis, 4) Migraine, 5) Shoulder pain/muscular
spasm/cervical pain, 6) Sciatica, 7) Stress/
anxiety/depression and 8) Varicose vein and
the results are presented in Table 1. The details Fig. 3b: Cupping points on the back side of the
of eight diseases are described individually human body
depending upon the their level of response.
Various parts of body mostly used for cupping therapy.
Constipation is generally defined as an The numbers within circles indicate specific locations to
inadeq uate defecatio n characterized b y place cups (5-7) as per disease condition.
9
Table 1: Responses of Cupping Therapy Against Different Disease Conditions in the Patients
Severity of Response to
Complaints and Dise ase Cupping Magnitude of Improvemnet
Respective parameters n Ratings in Cupping Points Y or N (%) and Percentage with
Patients and Temperament
Indications
Hamdard Medicus
Chronic Constipation 05 Mild = 2 1, 55, 121, 6,7, 8 Y = 90 Mild = 0

(Abdominal pain, hard or Moderate = 3 N= 0 Moderate = 10 (Melancholic)
scanty stool. trouble having Severe = 0 137, 138, 139, 140, Bowel Marked = 90 (Sanguineous)
a bowel movement/straining, 141, 142 movements
swollen tender abdomen, normalized with
vomiting) 28,29, 30, 31, 11, improvement in
12, 13 digestion.
Sciatica 03 Mild = 0 1, 55, 11, 12, 14, 16, Y = 100 Mild = 0

(Radiating pain from Moderate = 2 24, 26, 51, 132 N= 0 Moderate = 10 (Melancholic)
10
lumbar region to the Severe = 1 Flexion and Marked = 90 (Phlegmatic)
outer side of leg up 1, 55, 11, 13, 15, 17, extension with
till foot, difficulty in 25, 27, 52, 130 ease
walking without support)
Migraine 06 Mild = 3 1, 55, 20, 21, 23, Y = 90 Mild = 0

(Blurred vision, headache- Moderate = 2 41, 42, 45, 46, 47, 48 N = 0 Moderate = 10 (Bilious)
pulsating or throbbing pain Severe = 1 Pain episodes Marked = 90 (Sanguineous)
on one or both sides of and interval
head, irritability, nausea and prolonged up
vomiting, sensitivity to light, till a year
sounds and sometimes smells)
Knee Joint pain/arthritis 16 Mild = 5 1, 46, 48, 7, 8, 11, Y = 80 Mild = 10 (Melancholic)

(Pain, reduced mobility, Moderate = 9 25, 24 N = 20 Moderate = 30 (Bilious)
stiffness, swelling, tingling Severe = 2 Movement with Marked = 60 (Phlegmatic)
and numbness) ease
Vol. 59, No. 1, 2016
Severity of Response to
Complaints and Dise ase Cupping Magnitude of Improvemnet
Respective parameters n Ratings in Cupping Points Y or N (%) and and Percentage with
Patients Indications Temperament
Shoulder pain/muscular 17 Mild = 3 1, 55, 40, 41, 42, 11, Y = 90 Mild = 5 (Bilious)
Hamdard Medicus
spasm/cervical pain Moderate = 13 12, 13 N = 10 Moderate = 5 (Phlegmatic)

(Muscle spasm, neck pain, Severe = 1 49, 120, 4, 5, 6, 19 Decreased pain, Marked = 90 (Sanguineous)
and neck stiffness) movement improved
Varicose vein 03 Mild = 03 1, 55, 28,29, 30, 31, Y = 90 Mild = 0
(Enlarged and twisted veins, Moderate = 3 132 N = 10 Moderate =10 (Bilious)
painful, strain during walking Severe = 0 127, 128, 12, 26, 27 Able to stand Marked = 90 (Sanguineous)
or long standing posture) 51, 52 for longer durations
Stress/Anxiety/Depression 10 Mild = 3 1, 55, 11, 12, 13, 12 Y = 70 Mild = 10 (Melancholic)
(Irritability, mood swing, Moderate = 5 16, 46, 7, 8, 9, 10 N = 30 Moderate =20 (Bilious)
tension headaches, nausea, Severe = 2 Mental stress Marked = 70 (Phlegmatic)
negative thinking) lessened
11
Gastrointestinal 05 Mild = 1 1, 55, 121, 6, 7 Y = 90 Mild = 0
disturbance Moderate = 3 137, 138, 139, 140 N = 10 Moderate = 10 (Phlegmatic)
(Abdominal pain, abdominal Severe = 1 Symptoms Marked = 80 (Bilious)
bloating, abdominal distension, relieved with
belching, heart burn, loss of improvement
appetite, nausea, in digestive
regurgitation, vomiting) system.
Number of patients = n
Depending upon respective parameters for each disease within parenthesis, the rating scale for their severity is represented by a scale ranging from (0-10)
and classified into:
Mild = (1-3), Moderate (4-6) and Severe = (7-10).
Cupping points: The numbers indicate the trigger points on the front and back of human body for corresponding disease (8)
Response to cupping therapy: Yes (Y) or No (N). The numbers indicate the percentage of patients responded to the therapy.
Magnitude of improvement in patients upon clinical observation of the symptoms is classified arbitrarily as: Mild, Moderate and Marked.
Temperament is shown within parenthesis [Sanguineous temperament harr (hot) and ratb (moist), phlegmatic temperament barid (cold) and ratb (moist),
Vol. 59, No. 1, 2016
bilious temperament harr (hot) and yabis (dry), melancholic temperament , barid (cold) and yabis (dry)].
infrequent stools, difficult stool passage or both. sciatica from 3-12 months complaint of tolerable
It is well established that the pathophysiology pain with minimal disability, while patients
of constipation under primary conditions are due experiencing intense leg pain with high disability
to intrinsic problems of colon or anorectal scores and prolonged suffering were excluded.
functions, whereas, secondary causes are related After wet cupping therapy for 5-8 sessions the
to either organic (duodenal/jujenal perforations) flexion and extension of the legs particularly
and systemic diseases (hormonal, glandular) during walking showed marked improvement
or to medications (antibiotic) (Brandt et al., (90%) along with the reduction in inflammation
2005, Higgins and Johanson, 2004 and Pare of the lumbar region and enabled them to walk
et al., 2001). The patients suffering from without support.
constipation related to primary conditions, after Migraine is a disturbance of sub-cortical
6 sessions of wet cupping therapy showed aminergic sensory modulatory systems in the
mar ked ( 90%) and mo derate (10%) brain, or in brainstem, hypothalamic and thalamic
improvements as reflected by their normal bowel structures. The cortical spreading depression
movements. (CSD) is associated with the activation of
Sciatica is referred to a radiating pain or trigeminal nerve afferents which induces a
radicular pain arising from the hip joints radiating series of brainstem and meningeal events
towards the outer side of the leg and foot with appearing consistently at the time of migraine
the features of numbness and weakness. The episode. Prompt CSD enhancement leads to a
major cause refers to nerve compression long-lasting increase in blood flow within the
resulting from severe arthritis, disc herniation middle meningeal artery which is linked with
and spinal stenosis. Sciatica-like-distribution the activation of trigeminal and parasympathetic
called as pseudo-sciatica is resulted by the system. (Stovner et al., 2006). The patients in
trigger points in the gluteal minimus muscle the study complaint of at least 4 hours of migraine
hence creating referred pain similar to radicular pain or a day with which subsided after intake
pain from sciatica. Trigger points are usually of pain killers (panadol, aspirin). The intensity
muscle “knots” tender to touch and if active headache scale ranged from 0 (no headache)
result in pain that refers to other regions. It can to 10 (very severe headache). Efficacy of the
be caused by abrupt or frequent burden, treatment as a subjective evaluation by each
extended immobility, dislocation of the sacroiliac patient was recorded at the end of the study as
joint and nerve-root irritation. Trigger points marked, moderate or mild improvement. Pain
(localized areas of muscle spasm) are targeted intensity and duration was also compared before
until the muscle tension subsides. For sciatic and after treatment. In 6 patients after 3-10
nerve pain, the trigger points that are often sessions of wet cupping therapy a decrease in
responsible for the nerve irritation are located periodic pains and its duration was prolonged
in the gluteus medius and minimus muscles, as for about a year along indicating marked (90%)
well as the gluteus maximus, piriformis, and the while and moderate (10%) improvement.
lumbar paraspinal muscles. (Vroomen et al., Knee Joint pain/arthritis can be caused
1999). In the present study, a scale of 0-10 due to cartilage destruction and the deposition
(with 0 being no pain and 10 being the most of crystals in the joints from osteoarthritis
painful) was used to locate the trigger points (Levine et al., 1986). Primary sensory (afferent)
for sciatica. Patients (n = 3) suffering from fibers linking peripheral receptors with second
12
order neurons in the spinal cord result in pain and weakness in sensory symptoms (Eddy and
sensations arising from inflamed or damaged Loud, 2005).
joints. Afferent fibers are capable of enhancing Patient (n = 17) complaint about non-
and diminishing their capacity to detect and specific neck pain radiating into shoulder, upper
respond to various stimuli. Inflammatory pain is back, arm(s) and head leading to neck stiffness
associated with sensitization of sensory proteins and muscle spasm. Few patients also complained
at the nociceptive endings whereas pain that about unilateral neck, shoulder, or arm pain
results from damage to the nerve or neuropathic closer to dermatome. After 5-6 sessions of wet
pain has been linked to changes in axonal ion cupping therapy marked (90%), moderate (5%)
channels giving out ectopic discharge in and mild (5%) improvement was observed. It
nociceptors. was noted that the symptoms were aggravated
The patients complained about reduced by particular movements, posture and other
mobility of knee, warmth or swelling, pain physical activities in patients showing mild or
(stabbing, throbbing, burning, dull or sharp). Its moderate improvement who were also suffering
intensity varied fro m mild to severe or from cervical disc herniation and degenerative
tenderness, stiffness, tingling or other unusual changes.
sensations and numbness. It is well established Varicose veins are enlarged and twisted
that the joint pain mostly arises du e to most commonly observed on the legs. To prevent
inflammation, cartilage degeneration, crystal blood from flowing backwards that is retrograde
deposition, infection, and trauma (Fauci et al., flow or venous reflux, veins have pairs of leaflet
1998). valves leading to formation of cluster of veins
After treatment patients (n = 16) were that induces inflammation and pain (Mark, 2011).
relieved from pain, reflected as ease in joint 3 patients were treated through wet
movement and reduction in its stiffness. The cupping therapy applying 5-9 sessions. Results
improvement was evident in the patients after included easy movement, no inflammatory
treatment sh own w ithin par enthesis and conditions, patient can walk with ease, there
qualitatively classified as marked (60%), was mark ed imp rovemen t ( 90%) in the
moderate (30%) and mild (10%). The patients 2 patients and moderate (10%) in 1 patient was
displaying moderate and mild improvement seen because of severity of the case. The
could be due to unexplained fever, significant majority of cases of var icose veins are
joint-movement disability accompanied by considered to be benign although rigorous
history of medicatio n- ind uced immun e- varicosities can lead to major complications due
suppression as well use of steroidal injections to the insufficient circulation of the affected
prior to treatment. limb.
Shoulder pain/muscular spasm/cervical Stress/Anxiety/Depression, 10 patients
pain is frequently result from compression or after 5-6 sessions of wet cupping therapy
damage to the cervical spine nerve root reflected resulted in lessened mental stress indicating
as pain along with the motor dysfunction, sensory marked improvement (70%) in 6, moderate
deficits or adaptation in tendon reflexes. improvement (20%) in 3 and mild improvement
Cervical disc herniation and degenerative (10%) was seen in 1 patient. The terms stress
changes ar e its mo st co mmo n causes. or depression are linked with neuronal atrophy
Radiculopathy usually affects C5 to C7 levels characterized by loss of synaptic connections in
13
cortical and limbic brain regions implicated in ( ho t) an d ya bis ( dr y) > melancho lic
depression. The concept considered to occur temperament, barid (cold) and yabis (dry). The
through decreased expression and function of therapeutic effect of wet cupping therapy
growth factors like brain-derived neurotrophic followed a sequence of: chronic constipation >
factor in the prefrontal cortex and hippocampus. migraine > sciatica > shoulder/cervical/muscular
It’s difficult for typical antidepressants to reverse pain > varicose veins > dyspepsia > knee joints
str uctu ral alteration s. Accor ding to th e pain > stress/anxiety/depression.
Diagnostic and Statistical Manual of Mental It has been noticed that in case of
Disorders, 5th Edition (DSM-5), anxiety temperamental imbalance in the cold and dry
disorders include disorders that share features qualities, there was an increased stiffness that
of excessive fear and anxiety and related aggravates the natural cold and dry qualities of
behavioral disturbances. the connective tissues, however, other associated
Gastrointestinal disturbance including factors like overweight, hormonal variations,
dyspepsia, vomiting, hyperacidity, 5 patients after excessive intake of cold and dry foods.
5 sessions of wet cupping therapy resulted in Additionally, six essential factors of Unani
improvement of symptoms and better appetite medicine also play an important role in
indicating marked improvement (90%) in progression towards temperamental imbalance.
4 patien ts an d 1 h ad sh ow n mod er ate In patients having sanguineous and phlegmatic
improvement (70%). Dyspepsia is related to temperament a marked improvement in their ill-
the symptoms such as nausea, vomiting and health conditions, improved sleeping pattern as
postprandial fullness. Symptoms such as well as their emotional status.
postprandial pain, belching, and weight loss are The results presented above implies that
associated with hypersensitivity to gastric in cupping therapy a correlation between
distension. Psychosocial factors and altered temperament and improvement among patients
response to duodenal lipids or acid have also exists, however further studies on larger scale
been identified as pathophysiologic mechanisms. are r eq uir ed to co n firm o ur find in gs.
The present study supports the earlier Nevertheless, patients showing no response to
concept behind cupping therapy and the cupping were either affected by the prolonged
improvement in about 72% of patients observed medication or severity of the disease indicated
could be related to the transferring of discomfort marked disability or any other organic disorder.
through evacuation of morbid matters from I n so me patien ts temp or ary skin
the affected area referred to as Amalaa or discoloration was apparent that lasted for
Tanqiya-e-Mavad leading to required blood 3-5 days. Although, minor and temporary
circulation to the affected area thereby providing bruising in some patients were also prominent
appropriate nutrients. which were reversible but it needs to be taken
The marked improvement was linked with into account for those who are sensitive to
the temperament of the patients such as bruising. Nonetheless, most of patients were
sanguineous, phlegmatic, bilious, melancholic and satisfied with the cupping therapy and its
the o bserv atio n s were co nclu d ed as > outcome because of its efficacy, safety and
sanguineous temperament harr (hot) and ratb cost effectiveness. Considering the popularity
(moist) > phlegmatic temperament barid (cold) of cupping in Pakistan and to minimize its misuse
and ratb (moist) > bilious temperament harr guidelines for its safety needs to be prepared
14
and implemented. Moreover, in order to achieve 06. Bhikha, R.A.H. and Haq, M.A. (2000). Traditional
high level of medicinal significance along with Roots of Medicine in Modern Routes to Health,
Mountain of Light, South Africa.
complete saf ety an d eff icacy p ro per
07. Anjum, N., Jamil, S., Hannan, A., Akhtar, J. and
improvement in high techniques, scientifically Ahmad, B. (2005). Clinical efficacy of Hijamat
designed instrumentation and advanced or ultra (cupping) in Waja-ul-Mafasil (Arthritis). Indian J.
laboratory facilities for the selection of précised Traditional Knowledge. 4:412-415.
patients should also be needed to avail great 08. Li, CD., Fu, XY., Jiang, ZY., Yang, X., Huang,
benefits of cupping therapy. S.Q., Wang, Q.F., Liu, J. and Chen, Y. (2006).
Clinical study on combination of acupuncture,
It is concluded that if precautionary
cupping and medicine for treatment of fibromyalgia
measures are taken, wet cupping therapy is syndrome. Zhongguo Zhen Jiu. 26:765-767.
effective against diseases of various origins and 09. Arzani, M.A. (1940). Meezan-ut-Tib (Urdu),
appeared to be depended on the severity of the translated by Kabeeruddin, M. Daftarul Masih,
disease and temperament of the patients. Marol, Delhi, pp. 147-148.
However, further studies are still required for 10. Ibn-ul-Kuf. (1935). Kitab-ul-Umda Fil Jarahat.
Daeratul Moarif Usmania Usmania University,
other diseases as well as to prove or disapprove
Hyderabad, pp. 15-23.
claims of health benefits. 11. Xue, C.C. and O’Brien, K.A. (2003). Modalities
of Chinese Medicine. In: P.C. Leung, C.C. Xue,
Author’s Contribution and Declaration Y.C. Cheng (Eds.). A Comprehensive Guide to
Tasneem Qureshi: Conducted the therapy. Chinese Medicine. Singapore, World Scientific.
19-46.
Pro f. Dr . Hakim Abd ul Hann an :
12. Tait, P.L., Brooks, L. and Harstall, C. (2002).
Supervised the research study. Acupuncture: Evidence from Systematic Reviews
The author’s have no conflict of interest. and Meta-analyses. HTA 27: Series A – Health
Techno logy Ass es sment. Alberta Heritage
Acknowledgement Foundation for Medical Research, AB, Canada.
13. Al-Rubaye, K. (2012). The clinical and histological
Authors are thankful for the financial
skin changes after the cupping therapy (Al-
support provided by Hamdard University for Hijamah). J. Turk Acad. Dermatol. 6:1-7.
this research study. 14. Cao, H., Han, M., Li, X. et al. (2010). Clinical
research evidence of cupping therapy in China: A
systematic literature review. BMC Complement
4. REFERENCES Altern. Med. 10:1-10.
1. Ahmad, J. and Qadeer, A. (1998). Unani – The 15. Brandt, L.J., Prather, C.M., Quigley, E.M., Schiller,
Science of Greco-Arab Medicine. Lustre Press Pvt. L.R., Schoenfeld, P. and Talley, N.J. (2005).
Ltd., New Delhi, p. 53. Systematic review on the management of chronic
2. Wasti, N. (1990). Tibb-ul-Arab (Urdu) translation co ns tip ation in North Americ a. Am. J.
by Sir Edward Brown Book. Arabian Medicine, Gastroenterol. 100(Suppl 1):S5-S21.
Saqaf-e-Islamia, Lahore, pp. 444-446. 16. Higgins , P .D. and Johanson, J.F ., (2004).
3. Ahmed, S.I. (2009). Introduction to Al-Umur Epidemiology of constipation in North America:
al-Tabi’yah. Central Council for Research in Unani A systematic review. Am. J. Gastroenterol. 99:750-
Medicine, New Delhi, Ministry of Health & 759.
Family Welfare, Govt. of India, pp. 75-142. 17. Pare, P., Ferrazzi, S., Thompson, W.G., Irvine,
4. Rolfe R. (2002). The Four Temperaments, New E.J. and Rance, L. (2001). An epidemiological
York, USA, Marlow & Co. survey of constipation in Canada: Definitions,
5. Azmi A.H. (1995). Basic Concepts of Unani rates, demographics, and predictors of health care
Medicine, Jamia Hamdard, India. seeking. Am. J. Gastroenterol. 96:3130-3137.
15
18. Vroomen, P.C.A.J., Krom, M.C.T.F.M. de and of Neuroscience, the official journal of the Society
Knottnerus, J.A. (1999). Diagnostic value of history for Neuroscience. 6:3423-3429.
and physical examination in patients suspected of 21. Fauci, A., Braunwald, E., Isselbacher, K. et al.,
sciatica due to disc herniation: a systematic review. Editors. (1998). Harrison’s Principles of Internal
J. Neurol. 246:899-906. Medicine, 14th Edn., New York, McGraw-Hill
19. Stovner, L.J., Zwart, J.A., Hagen, K., Terwindt, Professional.
G.M. and Pascual, J. (2006). Epidemiology of 22. Eddy, D., Congeni, J. and Loud, K. (2005). A
headache in Europe. Eu ropean Journal of review of spine injuries and return to play. Clinical
Neurology. 13(4):333-345. Journal of Sports Medicine. 15(6):453-458.
20. Levine, J.D., Dardick, S.J., Roizen, M.F., Helms, 23. Mark, S. Whiteley. (2011). Understanding Venous
C. and Basbaum, A.I. (1986). Contribution of Reflux – The Cause of Varicose Veins and
sensory afferents and sympathetic efferents to Venous Leg Ulcers, 1st Edn., Whiteley Publishing
joint injury in experimental arthritis. The Journal Ltd.
16
Cuscuta reflexa L. & Roxb.: An Implication of Quercetin Mediated Monoamine

Oxidase Inhibition in Preclinical Models of Depression
Sara Zeeshan1, Wahid Zada1, Huma Aslam Bhatti2 and Ghulam Abbas2*
1
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad-22060, K.P.K., Pakistan.
2
H.E.J. Research Institute of Chemistry, International Center for Chemical & Biological Sciences,
University of Karachi, Karachi-75270, Pakistan.
*Email: ghulam.abbas@hotmail.com
Abstract antidepressant and this effect can be attributed

The current study was aimed at exploring to quercetin mediated rise in neuronal serotonin
the folkloric use of Cuscuta reflexa dodder a and noradrenaline levels possibly via MAO
parasitic plant (family convolulaceae) against inhibition.
depression. Briefly, the methanolic extract of
C. reflexa vine was assessed for antidepressant Keywords
action using various behavioral paradigms such Cuscuta reflexa L. & Roxb.; antidepressant;
as tail suspension test (TST), forced swim test phenelzine; serotonin; noradrenaline.
(FST) and lo co motor activity test. The
serotonergic and noradrenergic changes were
evaluated using 5-hydroxytryptophan (5-HTP) 1. INTRODUCTION
ind uced h ead twitch es and yo himb in e Depression is a complex mental disorder
potentiation tests, respectively. The fluoxetine that presents itself as sadness, fatigue, worthless-
and phenelzine were used as positive controls ness, disturbed weight/sleep, and loss of social
in the study. Our data demonstrated that the interactio ns. According to Wo rld Health
C. reflexa extract significantly declined the Or gan ization (WHO) estimatio n, arou nd
immobility time in TST (EC50 ~ 50 mg/kg) and 20% of the global population is affected from
FST without affecting the locomotor counts. depression. Furthermore, it is the 3rd foremost
The extract also significantly increased the cause of disability and would most likely be the
5-HTP induced head twitches and yohimbine first by 2030 (The Global Burden of Disease,
induced lethality. The aforesaid behavioral WHO, 2004).
outcomes were similar to that caused by standard The complexity of depression can be
drug phenelzine (monoamine oxidase inhibitor, reflected by numerous hypotheses that have
MAOI). It is known that quercetin is an inhibitor been proposed underlying its pathogenesis. The
of MAO and also the constituent of C. reflexa. most significant, among all, is the monoamine
Our HPLC data confirmed the presence of hypothesis which states that depression can be
quercetin (0.156 mg/25 mg of extract; retention attributed to low levels of monoamine neuro-
time = 2.1 minutes) in the extract and was used transmitters (serotonin and noradrenaline) in the
in present study. In conclusion, the present study brain. The significance of this hypothesis can
validate the folkloric use of C. reflexa as an be reflected by the fact that most of the
17
clinically used antidepressants (SSRIs) possess Care Co mmittee of th e CII T that is in

monoamine elevation as core mechanism of accordance with the guidelines by National
action (Elhwuegi, 2004). Institute of Health (NIH publication no. 85-23,
The treatment with medicinal plants is very revised 1985) for the care and use of laboratory
common practice around the globe. These have animals.
been used as medicine since antiquity and many
commercially used drugs are initially obtained 2.2. Chemicals
f ro m plan ts. The WHO estimates that The following chemicals are used: 5-
approximately 75-80% of the world population Hyd roxytryp toph an su pplemen t ( 100 mg
(mainly in developing countries) uses medicinal cap su les, Natr ol, USA) , eth ano l ( BDH
plants against several diseases (Cass, 2004). laboratory, England), fluoxetine phenelzine and
Cuscuta reflexa (English name: Giant dodder) yohimbine (Sigma Aldrich, USA). Distilled water
is a parasitic plant that grows widely in Pakistan. was used during entire course of experiments.
It belongs to the family Convolvulaceae and
genus cuscuta comprising ~170 species. It is 2.3. Preparation of Extract and
usually a very long, branched, twining vine that Standardization
is found on several plants species. It fully or Cuscuta reflexa L. & Roxb (Fig. 1)
partially covers the host plant by its twining parasite on Ficus religiosa L.was identified
stem. It usually lacks chlorophyll or has meager (Mr . Sh er Wali, Herbarium Department,
amounts thus unable to undergo photosynthesis University of Karachi) and assigned a specimen
to synthesize its own food. It is a bitter plant voucher No. 44269. It was chopped (1 Kg)
with pungent odor. Our survey also revealed into small pieces and repeated (4×) extraction
that it is traditionally used by ‘hakims’ for the was performed with methanol (70%) at room
treatment of depression and also reported to temperature. The combined extract was later
posses anxiolytic potential (Pal et al., 2003). concentrated under vacuum affording thick
Keeping this in mind, the current study was syrup which constituted the crude methanolic
designed to assess the antidepressant potential
of C. reflexa using various preclinical behavioral
paradigms.
2. MATERIALS AND METHODS

2.1. Animals
Sprague Dawley rats (150-250 g) and
Balb/c mice (25-30 g) were obtained from the
animal house facility of COMSATS Institute of
Information Technology (CIIT), Abbottabad.
The animals were placed in plastic cages, each
cage comprised of ten animals at 25±1°C and
light/dark cycle of 12 h each. Food and water
was freely and sufficiently available to all the
animals. All experiments were performed Fig. 1: Cuscuta reflexa L. & Roxb.
according to the protocols of Animal Use and Host Plant: Ficus religiosa
18
extract. The extract was standardized against water (25±1°C) up to 20 cm. The test was
quercetin using HPLC-DAD (Agilent 1200, performed in two sessions i.e. pre-test and test
USA). Briefly, the methanolic extract of session with a time lapse of 24 hours. In the
C. reflexa (25 mg/10ml) and quercetin (1 mg/ pre-test session (15 minutes) male rats were
5 ml) were injected (5 µl) into HPLC containing placed in the FST tank. The animals showing
C18 column (4.6 × 50 mm, 1.8 µ, Zorbax SB, nose bleeding during pre-test session were
Agilent Technologies, USA) set at 20°C. The excluded from the experiment. After 24 hours,
mobile phase (0.25% acetic acid, 72% water the healthy animals was treated (intraperitoneal)
and 28% acetonitrile) was used at flow rate of with C. reflexa extract (IC50 dose), fluoxetine
0.5 mL/min. (30 mg/kg), phenelzine (30 mg/kg) or vehicle
(5 ml/kg). The treated animals after 1hour were
2.4. Tail Suspension Test (TST) individually exposed for 6 minutes to FST
It was performed as described by Steru session and immobility time (animal is motionless
(Steru et al., 1985) . The animals were or making slight motions to keep the head above
distributed in four groups (n=5) into: C. reflexa water) was recorded for last 5 minutes (providing
extract i) (25, 50 and 75 mg/kg), ii) fluoxetine 1st minute for familiarization) and compared
(30 mg/kg), iii) phenelzine (30 mg/kg) and with control.
iv) vehicle (10 ml/kg). After 1 hr of intra-
peritoneal treatment, the animals were suspended 2.7. 5-hydroxytryptophan Induced Head
by the tail (using adhesive tape) almost 15 cm Twitches
above the ground for 6 minutes. The immobility Test animals (n=5/group) were treated
time (w hen the an imal was static) w as with 5-HTP (100 mg/kg, i.p.). After 30 minutes,
videotaped for 5 minutes (excluding the first these animals were again tr eated w ith
minutes) and compared with that of control. C. reflexa extract (IC50 dose) or fluoxetine
(30 mg/kg) and head twitches were counted
2.5. Locomotors Activity Test for 30 minutes (Sallinen et al., 1998).
Prior to performing this test, the animals
were placed in activity cage (1.5 by 1 ft. box 2.8. Yohimbine Potentiation Test
having 2 × 2 inch markings on the floor) for In this test, mice were divided into three
5 minutes for acclimatization. Later, these mice groups (n=5) i) Yohimbine (40mg/kg dissolved
were treated (intraperitoneal) with C. reflexa in ethanol) or in addition (30 minutes before
extract (IC50 dose), phenelzine (30 mg/kg), yohimbine) with ii) C. reflexa (IC50 dose) or
fluoxetine (30 mg/kg) or vehicle (10 ml/kg). iii) phenelzine (30 mg/kg). After 24 hours,
After 1 h, the animals were placed again in the percentage of lethality was calculated (Quinton,
activity cage for 6 minutes and the number of 2012).
boxes crossed by animals were recorded for
5 minutes and compared with the control. 2.9. Statistics
The data is presented as mean ± SEM
2.6. Forced Swim Test (FST) (n = 5-10 / dose). Differences between various
FST was performed as detailed by Porsolt means were computed by using one-way
(Porsolt et al., 1977). This test involved the use ANOVA followed by least significant difference
of a glass tank (17 × 17 × 50 cm) filled with (LSD). The level of significance is: *p<0.05,
19
**p<0.01 and ***p<0.005 as compared to their caused by stimulants. Our results clearly showed
respective control. that C. reflexa did not significantly increase
the locomotor activity (data not shown) thereby
3. RESULTS AND DISCUSSION certifying its antidepressant property. The
The present investigation was aimed to literature also supported its other neurological
explore the traditional use of C. reflexa in effects as it is shown to be neuro-protective
depression. The tail suspension test has been (Aruoma et al., 2003), anxiolytic (Pal et al.,
p op ular ly emplo yed fo r assessment o f 2003) and anticonvulsant (Mehrabani et al.,
antidepressant activity (Steru et al., 1985). In 2007) in nature.
similar ity with stand ard antidepr essan ts Another widely used tool to assess the
(p hen elzin e and flu oxetine) , the extract antidepressant activity is FST. Its major
significantly reduced the immobility time (EC50 drawback is failure to detect SSRIs i.e. selective
do se = 50 mg/k g) th er eb y sup po rting its serotonin reuptake inhibitors (Cryan et al.,
traditional use (Table 1). As per literature, the 2002). Likewise, fluoxetine failed to manifest
psychomotor stimulants may lead to false its action, while phenelzine (monoamine oxidase
positive results in test (Cryan et al., 2002). inhibitor, MAOI) reduced the immobility time
Therefore, the locomotor activity test was also (Table 1). Interestingly, the C. reflexa extract
performed in order to confirm the antidepressant also displayed activity similar to phenelzine.
action and rule out any possibility of false results Keeping in view the results, it can be deduced
Table 1: Effect of C. reflexa Extract on the Immobility Time of

Rodents in Tail Suspension Test and Forced Swim Test
TST FST
Treatment
Dose Mean immobility Do se Mean immobility
(mg/kg) time (mg/kg) time
25 144±15
C. reflexa extract 50 082±6***
75 131±9** 50 102±13***
Phenelzine 30 90±11*** 30 139±8*
Fluoxetine 30 56±8*** 30 201±11
The values represent the mean immobility time ± S.E.M (n = 5).

Mean immobility time of control animals: 155±7 (TST) and 189±12 seconds (FST).
Asterisk(s) indicate the significance levels i.e. *p<0.05, **p<0.01 and ***p<0.005 as compared to respective
control.
20
that the extract probably belongs to the MAOI potentiating test has been frequently used in
class of antidepressants but more studies in order to signify the effect of substances that
presence of MAOI inhibitors are required for cause noradrenaline modulation (Quinton, 2012).
its verification. Our study demonstrated that the lethality caused
The extract of C. reflexa was further by yohimbine (35%) alone was raised to 70 %
subjected to different behavioural tests (at EC50 and 90 % after treatment with either C. reflexa
dose i.e. 50 mg/kg) in order to incriminate (50 mg/kg) or phenelzine (30 mg/kg; Table 2),
th e mo noaminergic mo dulatio n. Th e 5- respectively. This led us to suggest that
hydroxytryptophan (5-HTP) is an amino acid C. reflexa most likely has the ability to elevate
that acts as p recu r so r of mo no amin e levels of noradrenaline at neuronal level.
neurotransmitter serotonin, whose high level The aforementioned behavioural effects
manifests serotonin syndrome characterized by caused by C. reflexa extract are similar to those
symptoms such as tremors. The 5-HTP triggered caused by standard drug phenelzine that is a
head twitch test is used to identify the effect monoamine oxidase inhibitor (MAOI). In search
of antidepressant on serotonergic function of MAOI in extract, it was found through
(Sallinen et al., 1998). Our data revealed that literature that that the main active constituents
in conformity with fluoxetine, the head twitches in C. reflexa are amerbelin, quercetin, cuscutine
triggered by 5-HTP were significantly (p<0.005) and cuscutamide (Patel et al., 2012). Among
enhanced by C. reflexa (50 mg/kg) (Table 2) all, the quercetin was reported to possess the
suggesting that it is probably acting via elevating monoamine oxidase inhibitory potential (Singh
the levels of serotonin in the brain. et al., 2003; Yoshino et al., 2011). Our HPLC
Yo himbin e is a 2 -antagon ist and its data confirmed that the extr act contains
Table 2: Effect of C. reflexa Extract on 5-HTP Induced Head Twitches and

Yohimbine Lethality Test
5-HTP induced head twitches Yohimbine induced lethality
Treatment Do se Number of Treatment Do se Mortality

(mg/kg) head twitches (mg/kg) (%)
5-HTP 200 29 ± 5 Yohimbine 40 35
Fluoxetine + 5-HTP 30+200 100 ± 11*** Phenelzine+

Yohimbine 30+40 90
C. reflexa + 5-HTP 50+200 79 ±6*** C. reflexa+

Yohimbine 50+40 70
The values represent the mean number of head twitches ± S.E.M (5-HTP induced head twitches) and percent
mortality in mice (yohimbine induced lethality) (n = 10).
Asterisk(s) indicate the significance levels ***p<0.005 as compared to respective control.
21
0.156 mg of quercetin (retentio n time = Author’s Contribution and Declartion

2.1 minutes) per 25 mg of methanolic extract Ms. Sara Zeeshan and Mr. Wahid Zada
of C. reflexa (Fig. 2). have performed the bench work.
In conclusion, the C. reflexa extract Dr. Huma Aslam Bhatti has prepared the
elicited antidepressant effect that justifies its extract and standardized it against quercetin.
folkloric use. This action can be attributed to Dr. Ghulam Abbas has supervised the
quercetin mediated increase of serotonin and entire work.
noradrenaline levels in the brain probably via The authors declare that there is no conflict
monoamine oxidase inhibition. of interest.
Fig. 2: Identification of quercetin in Cuscuta reflexa methanolic extract using HPLC-DAD

HPLC-DAD chromatograms of: (a) Quercetin (1 mg/5 ml, retention time = 2.1 min) and (b) C. reflexa extract
(25 mg/10 ml) showing the quercetin peak at 2.1 min which is equivalent to 0.156 mg.
22
4. REFERENCES of Cuscuta reflexa Roxb. J. Chin. Integr. Med.

1. Aruoma, O.I., Bahorun, T., Jen, L.-S. (2003). 10:249-255.
Neuroprotection by bioactive components in 08. Porsolt, R.D., Le Pichon, M., Jalfre, M. (1977).
medicinal and food plant extracts. Mutat Res-Rev. Depression: A new animal model sensitive to
Mutat. 544:203-215. antidep ressant treatments, Natu re. 266:730-
2. Cass, H. (2004). Herbs for the nervous system: 732.
Ginkgo, kava, valerian, passionflower. Semin Integr 09. Quinton, R. (2012). The increase in the toxicity of
Med. Elsevier, 82-88. yohimbine induced by imipramine and other drugs
3. Cryan, J.F., Markou, A., Lucki, I. (2002). Assessing in mice. Brit. J. Pharmacol. 21:51-66.
antidep ressant ac tivity in rod ents: recent 10. Sallinen, J., Haapalinna, A., Viitamaa, T., Kobilka,
developments and future needs. Trends Pharmacol. B., Scheinin, M. (1998). d-amphetamine and l-5-
Sci., 23:238-245. hydroxytryptophan-induced behaviours in mice
4. Elhwuegi, A.S. (2004). Central monoamines and with genetically-altered expression of the sub 2C
their role in major depression, Prog Neuro- sub-adrenergic receptor subtype. Neuroscience.
Psychopharmacol. Biol. Psychiatry. 28:435- 86:959-965.
451. 11. Singh, A., Naidu, P.S., Kulkarni, S.K. (2003).
5. Mehrabani, M., Modirian, E., Ebrahimabadi, A., Quercetin potentiates L-Dopa reversal of drug-
Vafazadeh, J., Shahnavaz, S., Heidari, M.R. (2007). induced catalepsy in rats: possible COMT/MAO
Study of the Effects of hydro-methanol extracts of inhibition Pharmacology. 68:81-88.
Lavandula vera DC. and Cuscuta epithymum Murr. 12. Steru, L., Chermat, R., Thierry, B., Simon, P.
on the seizure induced by pentylentetranzol in (1985). The tail suspension test: a new method for
Mice. JKMS. 14:25-32. sc reening antid epres sants in mice Psycho-
6. Pal, D., Panda, C., Sinhababu, S., Dutta, A., pharmacology. 85:367-370.
Bhattac harya, S. (2003). Evaluatio n of 13. Yoshino, S., Hara, A., Sakakibara, H., Kawabata, K.,
psychopharmacological effects of petroleum ether Tokumura, A., Ishisaka, A., Kawai, Y., Terao, J.
extract of Cuscuta reflexa Roxb. stem in mice Acta (2011). Effect of quercetin and glucuronide metabo-
Pol. Pharm. 60:481-486. lites on the monoamine oxidase-A reaction in
7. Patel, S., Sharma, V., Chauhan, N.S., Dixit, V.K. mouse brain mitochondria. Nutrition. 27:847-
(2012). An updated review on the parasitic herb 852.
23
Anti-bacterial Activity and Qualitative Phytochemical Screening of

Fagonia cretica L.
Kashaf Zia,1 Muhammad Qasim Hayat,2 Shaziad Anjum,1* Muhammad Ashraf,2

Saima Hanif2 and Alia Sadiqa2
1
Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur, Pakistan.
2
Medicinal Plant Research Laboratory, Department of Plant Biotechnology,
Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology,
Islamabad, Pakistan.
*Email: m.qasim@asab.nust.edu.pk; shazia.anjum@iub.edu.pk
Abstract be associated with antibacterial activity. On

Fagonia cretica L.is traditionally used the o th er hand leucoan th ocyan in s an d
against many diseases including gastrointestinal diterpenes were absent. Thus present study
and respiratory ailments. The present study provides a scientific rational for medicinal use
reports its minimal inhibitory concentration of F. cretica L. to treat gastrointestinal, urinary
against bacterial strains. Thus crude methanolic tract and respiratory tracts infections in humans.
extracts of F. cretica was evaluated against Further studies are required to isolate and
17 bacterial pathogenic strains and effective identify the active coumarins, diterpenes and
only against 17% of bacterial cultures as saponins-based lead molecules.
reflected by Proteus vulgaris, Micrococcus
luteus and Staphylococcus saprophyticus Keywords
which showed minimal inhibitory concentration Fag on ia cretica; b actericid al activ ity;
of 50 µg/ml, 50 µg/ml and 100 µg/ml, respectively S. saprophyticus; P. vulgaris; S. typhimurium;
with possibly due to synergistic actions of M. luteus; qualitative phytochemical screening.
chemical composition but selectivity towards
M. Luteus and Proteus vulgaris. Further 1. INTRODUCTION
fractionation of F. cretica crude methanolic Med icin al plan ts have b een u sed
extracts with hexane, ethyl acetate and water traditionally for the treatments of various
enhanced bactericidal activity (MIC 25 µg/ml) ailments since ancient time which laid the
of the resp ective plant fractio ns against foundation of currently available phytochemicals
P. vulgaris. Similar magnitude of response with w ith th erapeutic pr op erties and also
hexane and ethyl acetate fractions against co mp onent( s) of many synthetic dr ugs.
Salmonella typhimurium were also noted, Atropine, ephedrine, digoxin, morphine, quinine,
w hereas cr u de extr act w as in ef fective. aspirin and reserpine are few such examples of
A qualitative phytochemical screening revealed drugs, invented based on folk knowledge of
that F. cretica methanolic extract coumarins indigenous people (Gilani and Atta-ur- Rahman,
and diterpenes predominates while in water 2005). The importance of plants with curing
fraction it is saponins and hence are possibly properties has been realized by researcher as
24
their medicinal uses have been associated with hepatoprotective (Qureshi et al., 2016; Bagban
~95,000 wild plants out of which only a small et a l., 2012), antitu mor , cytoto xic with
fraction (~7,500 plants) are used as a source of dipeptidyl peptidase-4 (DPP-4) inhibitory
medicine, 3,900 as edibles, 400 as a source of properties (Hussain et al., 2007; Saleem et al.,
fodder, 500 as fibre producers and 300 being 2014).
used as bio-pesticides (Mishra et al., 2007). The 7th largest desert in the world also
Despite of a large number of plants only ~700 known as the ‘Great Indian Desert’ is located
plant species have been exploited as food, in the Sindh province of Pakistan as well as in
medicinal source, flavouring agents and by Rajasthan, India. It covers an area of about
cosmetic industry etc (Shinwari, 2010). In 2,00,000 sq. Kilometres and divided into three
developing coun tries including Pakistan, parts in Pakistan. The Northern part is in South
~80% of the people rely on folk medicines using end of Punjab it is called ‘Cholistan’, spreading
natural products from medicinally important over an area of 16,000 square kilometers (30 km
plants for primary health-care (Igbinosa et al., from Bahawalpur city). The locals are nomadic
2009). herders living in huts made of mud and straw.
I t is w ell estab lished th at hu man Cholistan desert of Pakistan is a rich land
pathogenic bacterial strains with the passage of bestowed with many medicinal plants. It is
time have d ev elop ed r esistance against hypothesized that the harsh weather conditions
antibiotics accompanied by various undesirable of this desert (winters with low temperature
side effects. Therefore, it is crucial to explore touching the freezing point with frost formation,
alternatives to combat bacterial infections and while the summers are hot and dry with
in this context natural products from other temperature rising to 51°C) plays a pivotal role
sources particularly medicinal plants with in inducing and modifying secondary metabolites
significant potential to provide drug candidate(s) in the plants possessing plethora of medicinal
because of their rich molecular diversity and properties. Although, its antibacterial and
likelihood of producing lesser side effects. antifungal potential were associated with flowers
Fagonia. cretica, bitter in taste is used (Thetwar et al., 2006; Vaibhav et al., 2014)
for the treatment of cancer, fever, thirst,
vomiting, dysentery asthma, urinary discharges,
liver and kidney ailments, typhoid, toothache,
stomach discomforts and skin diseases (Pareek
et al., 2012 and Gulshan et al., 2012). It is an
antioxidant, febrifuge, astringent, antimicrobial
and prophylactic against small pox The powder
of whole plant is dusted on boils and skin
eruptions (Qureshi et al., 2010) and decoction
given orally is also effective for curing skin
eruptions. Its herbal tea has been reported to
be effective against breast cancer (Lam et al.,
2012). The leaves and twigs showed anti-venom
potentials against Naja naja karachiensis
(Razi et al., 2011) . F.cret ica po ssesses Fig. 1: Fagonia cretica Auct-non Linn Parker
25
however, information regarding its minimal Staphylococcus aureus, S. aureus ATCC,

inhibitory concentration is lacking. Therefore, S . Sa prop h yt icus, and St rep to coccu s
based on ethnomedicine and ethnobotanical p yo gen es. The Gr am-n egative strain s
information available for F. cretica from comprised: Acinobacter junii, Bordetella
Cho listan Desert it w as evaluated fo r b ro nchisep tica , Escherich ia co li,
antibacterial activities against pathogenic and Enterobacter cloacae, Klebsiella pneumonia,
non-pathogenic bacterial strains. Proteus vulgaris, Pseudomonas aeruginosa,
Sa lmo nell a t yph i, Serra tia m arcescen s,
2. MATERIALS AND METHODS Shigella dysenteriae, Salmonella paratyphi,
2.1. Plant Material and S. typhimurium ATCC-14097.
Healthy F.cretica bearing leaves, stems,
roots and flowers were collected from Chak 2.4. Chemicals
Lehar a location of Cholistan desert. After The following chemicals are used: Agar ,
identification by the taxonomist Mr. M. Warris organic solvents, isoamyl alcohol, sodium
a voucher number was assigned (3430/CIDS/ hydroxide and copper sulphate were purchased
IUB) and the specimen deposited in the from Sigma (USA). All the other chemicals
herbarium. used were of analytical grade.
2.4. Antibacterial Screening

2.2. Extraction The sensitivity of different bacterial strains
The plant was shade dried and milled to to F. cretica extracts w as measu red by
obtain its powder (6.0 kg) that was soaked in popularly used agar diffusion assay (Bauer
methanol (10 L) for a week, followed by et al., 1966). For bacterial culturing, Luria
filtration and evaporation under high vacuum. Bertani agar (28 gm) in distilled water (1 L)
The extraction process was repeated twice with was autoclaved for 15 min at 121°C. The
methanol (7.5 L) for 7 days, yielding 64 g of sterilized media was was poured into petri dishes
methanol extract. and left at room temperature for 20-30 minutes.
The F. cretica crude methanolic extract Modified method of Kirby-Bauer was used for
(11 g) was dissolved in of distilled water (50 mL) inoculation of bacterial cultures in petri plates
and solvent-solvent successive extractions were using sterile streaking loop by streaking in
performed with petroleum ether (50 mL × 3) to three different angles keeping the same central
obtain hexane extracts (0.36 g). Thereafter, position to acquire uniform bacterial colonies.
extraction of aqueous extracts with ethyl acetate Appropriate precautionary measures were aslo
layer (50 mL × 3) affo rded ethyl acetate taken to avoid contamination with other cultures
extracts (0.45 g). and the streaking-loop was sterilized on red
hot flame, before using new bacterial culture.
2.3. Bacterial Cultures The organic extracts were screened for their
The bacterial strains were generously antibacterial activity using a established disc
provided by Microbiology Laboratory of Atta- diffusion method (Jamil et al., 2012). A 6 mm
ur-Rahman, School of Bio-Sciences (ASAB) filter paper dics were prepared, autoclaved and
Islamabad, Pakistan. The Gram-positive strains a 10 µl sample of organic extracts (100 mg/ml)
included: Micrococcus luteus ATCC-4617, were applied individually. While on control plate
26
three discs without any sample was placed. th e u pper layer s indicating presen ce of
Two standard drugs gentamicin and ciprofloxacin leucoanthocyanins.
served as positive controls and dimethylsulpoxide
(DMSO, 50%) were used as a negative control. Saponins
All the plates were incubated at 37oC overnight On the addition of distilled water (5 mL)
for about 18-20 hours and zone of inhibition to 10 mL of test substances followed by vigorous
(mm) were measured. The experiments were shaking caused stable foam that was considered
performed in triplicate. as an indicative of saponins.
2.5. Minimum Inhibitory Concentration Steriods

(MIC) Equal volume of concentrated sulphuric
In the disc diffusion assay, the average acid and chloroform (10 mL) introduced in the
size of the zone of inhibition (n = 3) is presented test substances. The appearance of red colour
for those extracts that showed a consistent and in the upper layer and yellowish green colour in
noticeable clear zone around the filter paper the lower layer confirmed the presence of
disc (6 mm). The inhibitory zones of crude steroids.
methanolic extract, hexane, ethyl acetate and
aqueous fractions were expressed as the 3. RESULTS AND DISCUSSION
mean ± error and compared with control. In different parts of Cholistan desert the
local dwellers depends on infusions and
decoctions of F. cretica that is prepared using
2.6. Phytochemical Screening
water as a solvent for curing multiple ailments.
The phytochemicals such as coumarins,
Thus based on ethnomedicine and ethnobotanical
diterpenes, leucoanthocyanins, saponins and
information F. cretica was evaluated for their
steroids were determined in methanolic extracts
antibacterial actions against 17 bacterial species
and aqueous fraction as described earlier (Brain
that vary with respect to their disease modality
and Turner, 1975).
and virulence. For example, E. Coli, human
enteric species is generally avirulent but
Coumarins sometimes causes weak virulent gastroenteritis
Yellow colour was evident after addition and urinary tract infections and S. typhimurium,
o f 3 mL NaO H ( 10%) in 2 mL of test causing enterocolitis are both Gram-negative
substance. pathogens. While, S. aureus a Gram-positive
pathogen is most common cause of abscess,
Diterpenes food poisoning, and toxic shock syndrome; both
The extract and fraction were dissolved are considered to be more virulent than S. lactis
in water and upon addition of copper sulphate and E. coli (Levinson, 2008).
emerald green colour appeared, indicating the Our results demonstrated that F. cretica
presence of diterpenes. crude methnolic extract and its hexane, ethyl
acetate and water fractions induced zones of
Leucoanthocyanins inhibition against P. vulgaris. The crude
Addition of isoamyl alcohol (1.0 mL) to meth anolic extracts at 50 µg and 100 µg
test substances (1 mL) formed red colour in concentrations killed the bacteria in dose
27
Table 1a: Effect of F. cretica Methanol Extract and Hexane, Ethyl Acetate and Water Fractions on
Gram Negative Bacterial Growth Using Disc Assay Hamdard Medicus
Test agents
µg/ml
A. junii
B. bronchiseptica
E. coli
E. cloacae
K. pneumonia
P. aeruginosa
P. vulgaris
S. typhi
S. marcescens
S. dysenteriae
S. paratyphi
S. typhimurium
ATCC-14079
Methanolic
extract 025 – – – – – – – – – – – –
050 – – – – – – 16.6±0.5 – – – – –
100 – – – – – – 24.8±0.3 – – – – –
n- hexane 010 – – – – – – – – – – – –
015 – – – – – – – – – – – –
28
025 – – – – – – 15±0.2 – – – – 6.7±0.3
Ethyl acetate 010 – – – – – – – – – – – –
015 – – – – – – – – – – – –
025 – – – – – – 7.9±0.2 – – – – 13.4±0.5
Water 010 – – – – – – – – – – – –
015 – – – – – – – – – – – –
025 – – – – – – 13.5±0.5 – – – – –
DMSO – – – – – – – – – – – –
Gentamicin 010 7±0.2 23.4±0.4 7.1±0.1 – 7.0±0.1 – 12.3±0.6 – 7.8±0.3 10.7±0.5 – 20.1±0.2
Ciprofloxacin 010 – 34.9±0.2 6.9±0.1 21.8±0.4 5.8±0.3 11.9±0.1 29.6±0.5 24.8±0.2 24.9±0.4 31.1±0.2 21.4±0.5 30.1±0.1
The values represent mean ± SEM as diameter (mm) zone of inhibition of 3 independent experiments
Dimethylsulphoxide (DMSO, negative) and gentamicin and ciprofloxacin (positive) served as respective controls
Vol. 59, No. 1, 2016
No effect (–).
dependent manner forming 16.6 mm and F. cretica did not show any inhibition against
24.8 mm of inhibitory zones, respectively. While S. typhimurium ATCC 14097 which is known
the hexane fraction was more effective as a to cause gastroenteritis and fever. However, at
15 mm zone was evident at lower concentration 25 µg both hexane and ethyl acetate fractions
(25 µg). The ethyl acetate and water fractions showed zones of inhibition of 6.7 mm and
at 25 µg also showed zone of inhibition of 7.9 mm 13.4 mm (Table 1a) suggesting that bioassay
and 13.5 mm, magnitude, respectively. Thus it guided fractionation will be the most suitable
can be inferred that chemical constituents of strategy to isolate active principles against the
F. cretica act synergistically to inhibit the afo remen tion ed diseases in d uced b y
growth of P. vulgaris which is reputed for S. typhimurium.
urinary tract and wound infections in humans. The cru d e methano lic extr acts o f
Interestingly, crude methanolic extracts of F. cretica (100 µg) also induced
Table 1b: Effect of F. cretica Methanol Extract and Hexane, Ethyl Acetate and
Water Fractions on Gram Positive Bacterial Growth Using Disc Assay
Test agents µg/ml M. S. S. aureus S. S.

luteus aureus aureus pyogenes saprophyticus
ATCC-4617 ATCC
Methanolic
extract 025 – – – – –
050 – – – – –
100 6.7±0.3 – – – 10.7±0.2
n-hexane 010 – – – – –
015 – – – – –
025 – – – – –
Ethyl acetate 010 – – – – –
015 – – – – –
025 – – – – –
Water 010 – – – – –
015 – – – – –
025 – – – – –
DMSO – – – – –
Gentamicin 010 23.5±0.5 34.1±0.1 26.5±0.5 12.7±0.2 14.6±0.5
Ciprofloxacin 010 30.3±0.5 23.2±0.4 39.6±0.5 30.9±0.1 29.9±0.1
The values represents mean ± SEM as diameter (mm) zone of inhibition of 3 independent experiments
Dimethylsulphoxide (DMSO, negative), gentamicin and ciprofloxacin (positive) served as respective controls
No effect (–).
29
S. saprophyticus growth inhibition (10.7 mm). (Acinobacter junii, Bordetella bronchiseptica,

Likewise, inhibitory zone of 6.7 mm at 50 µg Escherichia co li, Ent erob acter cl oaca e,
against M. Luteus was also observed (Table 1b). Klebsiell a p neum o nia, Pseu do mo na s
However, all the fractions of F. cretica (such aeru gin osa, Sa lmo nella typh i, Serrat ia
as hexane, ethyl acetate and water extracts) marcescens, Shigella dysenteriae, Salmonella
were ineffective for both bacterial cultures paratyphi), thereby suggesting that its selectivity
supporting synergism again in this particular tow ar d s S . sa prop h yt icus; P. vul ga ris;
case and also supporting the notion that S. t yphimurium; M. lu teu s emphasize to
combination of different chemical constituents conduct in-depth studies on the extract and
together are more effective. The minimal fractions in the diseases affiliated with them
inhibitory concentrations of the fractions against and also elu cidate th e mechanism o f
bacter ial cultu res is 2× low er th an the bacterialcidal activities residing in the plant.
corresponding extracts (Table 2) clearly supports The DMSO was used as a negative
that bioassay directed fractionation is required control as expected showed no effect on the
to see if the MIC values could be further bacterial cultures on the contrary, gentamicin
improved. and ciprofloxacin serving as positive controls
This is to be noticed that crude methanolic were effective against all the strains used
extracts of F. cretica and its fractions were (Tables 1a and b) as reflected by observable
ineffective against 76% of bacterial isolates zone of inhibition around bacterial colonies.
including both Gram-positive (Staphylococcus Ciprofloxacin, fluorinated quinolones structurally
aureus, S. aureus ATCC, and Streptococcus related to nalidixic acid acts by inhibiting bacterial
p yo gen es) and Gr am-n egative strain s DNA gyrase. It is a broad spectrum antibacterial
Table 2: Minimum Inhibitory Concentration of F. cretica Methanolic Extract and

Fractions Against Bacterial Strains
Bacterial strains Extract/Fractions µg/ml
S. saprophyticus Methanolic Extract 100
P. vulgaris Methanolic Extract 050

Hexane Fraction 025
Ethyl acetate Fraction 025
Water Fraction 025
S. typhimurium ATCC 14079 Hexane Fraction 025

Ethyl acetate Fraction 025
M. luteus ATCC 4617 Methanolic Extract 050
The MIC of ciprofloxacin and gentamicin = 10 µg/ml.
30
drug as most of Gram-negative bacteria are hence provides a appreciable source of alternate
highly susceptible in vitro and many Gram- to antibiotics which are either not accessible
positive bacteria are moderately susceptible to and if so are not affordable. Considering that in
it. Similar pattern was also observed in our study rural areas herbal treatments are favored over
as only A.junii was resistant to it. The other the allopathic ones for their low cost and the
standard antibiotic used was gentamicin that rich traditional knowledge about healing systems
acts by irreversibly binding to the 30S subunit as well as compatib ility to th e hu man
of the bacterial ribosome, interrupting protein physiological system. Our results favoured that
syn th esis wh ich is similar to other crude extracts of F. cretica possess bactericidal
aminoglycosides. In the present study only activities. The results affirmed the claim by the
Ent erob act er clo a ca e, Pseu do mo na s herbal practitioners of the area and this plant
aeruginosa, Salmonella typhi, Salmonella can be used to treat gastrointestinal and
pa ratyphi were resistant to gentamicin. respiratory tracts infections in humans.
Furthermore, small variations in the sensitivity The phytochemical analysis of the crude
of bacterial culture to plant extract and fractions methanolic extracts and aqueous fraction of
and to standard drugs could also be attributed F. cretica, coumarins and diterpenes were
to the differences in growth rate of the tested predominant while, saponins and steroids
organisms, nutritional requirements, temperature w er e pr esent in small amo u nts wh ile
and inoculum size (Gaill and Jon, 1995) but all leucoanthocyanins was absent. On the contrary,
the precautionary measures were undertaken in the water fraction saponins was predominant
as described above. These results advocate that followed by coumarins and steroids while
the methodology adopted for the anti-bacterial diterpenes and leucoanthocyanins were absent
screening was accurate and supports that (Table 3). Thus coumarins, diterpenes and
antimicrobial property residing in F. cretica saponins are possibly be associated with
Table 3: Qualitative Phytochemical Analysis of F. cretica Methanolic Extract and

Aqueous Fraction
Chemical Groups Methanolic Extract Aqueous Fraction
Coumarins +++ ++
Diterpenes +++ –
Leucoanthocyanins – –
Saponins ++ +++
Steroids ++ +
The presence of the chemical group is represented as:

Strong (+++), moderate (++), weak (+) and absent (–)
31
antibacterial activity as they were predominantly 05. Gilani, A. H., Atta-ur-Rahman. (2005). Trends
present in F. cretica methanolic extract and in ethnopharmacology. J. Ethanopharmacol.
100(1-2):43-49.
aqueous fraction, while leucoanthocyanins and
06. Gulshan, A.B., Dasti, A.A., Hussain, S., Atta,
diterpenes were absent. M.I., Amin-ud-Din, M. (2012). Indigenous uses
It is concluded that, this study provides a of medicinal plants in rural areas of Dera Ghazi
scientific rational for medicinal use of F. cretica Khan, Punjab, Pakistan. ARPN. J. Agri. Biol. Sci.
against gastrointestinal and respiratory tracts 7(9):750-762.
07. Hussain, A., Zia, M., Mirza, B. (2007). Cytotoxic
infections in humans. However, further studies
and Antitumor Potential of Fagonia cretica L.
are required to explore its mechanism(s) of Turk. J. Biol. 131(1):19-24.
action of plants extracts and its secondary 08. Igbinosa, O.O., Igbinosa. E.O., Aiyegoro, O.A.
metabolite following bioassay directed approach (2009). Antimicrobial activity and phytochemical
screening of stem bark extracts from Jatropha
with emphasis on coumarins and diterpenes and
curcas (Linn). Afrc. J. Parm. Pharmacol. 3(2):58-
saponins. In parallel the synergistic effects noted 62.
also demands in-depth study in this direction 09. Jamil, M., Ihsan ul Haq, Mirza, B., Qayyum, M.
along with standardization of plant that can also (2012). Isolation of antibacterial compounds from
Quercus dilatata L. through bioassay guided
prove a worthy asset of Pakistani herbal fractionation. Annals of Clinical Microbiology and
medicines. Antimicrobials. 11:1-11.
10. Lam, M., Amtul, R. C., Helen. R.G. (2012). An
Author’s Contribution and Declaration aqueous extract of Fagonia cretica induces DNA
Ms. Kashaf Zia, Ms. Saima Hanif, and damage, cell arrest and apoptosis in breast cancer
cells via FOXO3a and p53 Expression. PLOS.
Ms. Alia Sadiqa performed the bench work. ON E (http://www. plo so ne. org/article/info%
Dr. Muhammad Qasim Hayat and Prof. 3Adoi%2F10.1371%2Fjournal.pone.0040152).
Dr. Shazia Anjum have supervised the entire 11. Levinso n, W. (2008). Review o f med ic al
work. microbiology and immunology, 10th Edn. New
York, McGraw-Hill Medical.
The authors declare that there is no conflict
12. Mishra, S.N., Tomar, P.C., Lakra, N. (2007).
of interest. Medicinal and food value of Capparis decidua,
Ind. J. Tradn. Knowl., 6(1):230-238.
4. REFERENCES 13. Parneek, A., Nikhil, B., Manoj, G., Prakash, N.B.
1. Bagb an, I.M., Ro y, S. P. , Chaud hary, A., (2012). Phytochemicals and biological activities of
Das, S.K., Gohil, K.J., Bhandari, K.K. (2012). Fagonia indica. Internat. Res. J. Pharm. 3(6):56-
Hepatoprotective activity of the methanolic extract 59.
of Fagonia indica burn in carbon tetra chloride 14. Qureshi, H., Asif, S., Ahmed, H., Al-Kahtani, H.A.,
induced hepatotoxicity in albino rats. Asian Pac. Hayat, K. (2016). Chemical composition and
J. Tradn. Biomed. S1457-S1460. medicinal significance of Fagonia cretica: A review.
2. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, Nat. Prod. Res. 30(6):625-639.
M. (1966). Antibiotic susceptibility testing by a 15. Qureshi, R., Bhatti G.R., Memon, R.A. (2010).
standardized single disk method. Am. J. Clinic. Ethnomedicinal uses of herbs from northern part of
Pathol. 45:493-496. Nara desert, Pakistan. Pak. J. Bot. 42(2):839-851.
3. Brain, K.R. and T.O. Turner. (1975). The 16. Razi, M.T., Muhammad, H.H.B.A., Taous, K.,
Practical Evaluation of Phytophamtaceuticals. Muhammad, Z.C., Muhammad, T.A., Muhammad,
Wright-Scientecknica, Bristol. pp. 152-153. A. A. , Q azi, N. S. (2011). Antihaemorrhgic
04. Gaill, W., Jon, A.W. (1995). Manual of Clinical potentials of Fagonia cretica against Naja Naja
Microbiology, ASM Press, Washington, DC. Karachiensis (Black Pakistan Cobra) venom. Nat.
6th Edn. pp. 1327-1332. Prod. Res. 25:1902-1907.
32
17. Saleem, S., Jafri, L., Haq, I., Chang, L.C., 19. Thetwar, L.K., Thakur, A.S., Shrivastana, S.,
Calderwood, D., Green, D.B., Mirza, B. (2014). Augor, M.R., Tandon, R.C., Deshmukh, N.C.
Plants Fagonia cretica L. and Hedera nepalensis K. (2006). Antimicrobial efficacy of methanolic extracts
Koch contain natural compounds with potent of Fagonia cretica. Asian J. Chem. 18(1):743-744.
dipeptidyl peptidase-4 (DPP-4) inhibitory activity. 20. Vaibhav, C., Raman, C., Hiral, M., Subhash, D.
J. Ethnopharmacol. 156:26-32. (2014). Selective in vitro antimicrobial properties
18. Shinwari, Z.K. (2010). Medicinal plants research of Fagonia cretica Linn. crude extract. J. Adv.
in Pakistan. J. Med. Plant Res. 4(3):161-176. Drug Del. 1(2):71-81.
33
Unani Treatment of Tinea Capitis: A Case Study
Leena Hameed* and Tasneem Qureshi

Faculty of Eastern Medicine, Hamdard University,
Sharae Madinat al-Hikmah, Muhammad Bin Qasim Avenue,
Karachi-74600, Pakistan.
*Email: leena.hameed@hotmail.com
Abstract such as hair, nail and skin. They are further

Tinea capitis (TC) ( ) is a divided into 3 major groups: Anthropophilic
dermatophytic infection of the scalp this infection dermatophytes which are limited to the human
most commonly affect children usually under h ost on ly causin g o nly mild ch ro nic
the age of 12 years and frequently seen in inflammation. Zoophilic dermatophytes are
boys. This is a case study of an 11 year old boy transmitted from pets, livestock and horses to
naïve patient who presented to the OPD with humans resulting in marked inflammatory
a history of severe scaly, crusting, bald patches reactions in the host. The last group is the
on scalp from 5months. The clinical diagnosis geophilic and its source is soil and can infect
was confirmed to be TC and the causative both human and animals (Tan, 2005).
organism is Trichophyton violecum. After Dermatophytes invade and multiply within
treatment with polyherbal formulation (Nuskha keratinized tissues and classified into three main
No. 9 and 10) for a period of 8-weeks with groups: trichophyton (skin, hair and nails),
follow up the sign and symptoms of the patient epidermop h yton ( sk in an d n ails) an d
resolved completely with healthy normal hair microsporum (skin and hair). It is characterized
growth pattern. This case study confirmed that by erythema, scaling, pruritus and alopecia and
nuskha No. 9 and 10 are effective against TC its transmission occurs via infected persons and
and it is cost effective. However, more data is animal vectors through shedding of hair.
required to justify its efficacy, safety and its Depending upon the site of infection, these have
mechanism of action. been classified clinically into TC (head), tinea
faciei (face), tinea barbae (beard), tinea corporis
Keywords (body), tinea manus (hand), tinea cruris (groin),
Unani treatment, Tinea capitis, Case study. tin ea p ed is (f oo t) , and tin ea un gu iu m
(nail) (Bennassar and Grimalt, 2010). Many
1. INTRODUCTION f un gal sp ecies can cause TC su ch as
Tin ea capit is (TC) r epresents tinea Microsporum canis (common in Europe),
(fungal) and capitis (scalp) is a dermatophytic Epidermophyton floccosum, Trichophyton
infection of the scalp (Elewski, 2000). The mentagrophytes, Trichophyton tonsurans
dermatophytes comprise of three genera: (mostly in the United States) and Trichophyton
a) Trichophyton, b) Epidermophyton and rubrum (Elewski et al., 2008).
c) Microspora. (Gupta and Summerbell, 2000). The dermatophytes invade the hair shaft
All of them grow in keratinized environment in any of two ways i.e., endothrix and ectothrix.
34
In the former hair shaft is filled with hyphae patient had contact with household animals like
and spores and arthroconidia is inside the hair goats and cattle. Majority of the patients
shaft only and the cuticle of the hair remains belonged to low socio-economic class and only
intact. However, in later hyphae and spores 2% were from higher socio-economic class
cover the outside of the hair shaft causing (Farooqi et al., 2014).
destruction of the cuticle. How ev er in Eur op e T. t on su ran s
(Anthropophilic) is the most common cause of
1.1. Prevalence TC since mid-1990s in London (Tan, 2005) and
The epid emio lo gy of TC v ar ies its incidence is increasing in different parts of
geo gr ap h ically du e to so cioecon omic, the world including USA. (Foster et al., 2004)
environmental, lifestyle, and climatic conditions Another microorganism is M. canis, a zoophilic
(Table 1) shows that Germany has the lowest dermatophyte is transmitted from household
prevalence and Philadelphia (USA) shows the pets and infections resulting from it are usually
highest prevalence of TC. The prevalence of sever e with er ythema and pustules. The
TC in a data in tertiary care center of Karachi incidence of M. canis, a zoophilic dermatophyte
on 202 patients there age ranged from 1 to is also increasing in central and southern Europe.
14 years. 9.4% of patients have previous history The common microorganism causing infection
of TC which was treated and they were in East Africa and the Indian sub-continent is
symptom free for a period of 6 months to 1 year T. violaceum (Moriarty et al., 2012).
before presenting again with the disease. Children are particularly susceptible to
dermatophytic infections because of their poor
personal hygiene habits and poor environmental
Table 1: Global Prevalence of Tinea Capitis
sanitation and hence most frequent in them
Countries Prevalence (%) particularly in boys under 12 years of age. It
has also been linked with their frequent visit to
Germany 0.1
the hairdresser as shorter hair also facilitates
Barcelona (Spain) 0.23 easy access for circulating spores (Friedlander
Italy 0.3 et al., 2003). M. canis infection is prevalent in
boys, while boys and girls are equally infected
Palestine 1 by Trichophyton species (Aly, 1999). TC is more
London (United Kingdom) 2.5 common in younger children as compared to
ad ults this was d ue to the presen ce of
Tanzania 7.1
Pityrosporum orbiculare (Pityrosporum ovale),
Cleveland (USA) 13 which is part of normal flora, and also from the
Philadelphia (USA) 14 fungistatic properties of fatty acids of short and
medium chains in post-pubertal sebum due to
The prevalence of TC in African, Asian, European and which adults are rarely infected (Gorbach et al.,
North American countries (Balci et al., 2014)
1997).
TC may occur sporadically or epidemically
40.1% of the patient’s siblings were also and its prevalence varies throughout the world
suffering from TC and the most prevalent with a rise over the last few decades (Kundu
species were anthropophilic. 11.9% of the et al., 2012).
35
2. MATERIALS AND METHODS morning and half in the evening) in a cup of

boiling water as decoction. Additonally, Nuskha
2.1. Case History No. 10 (Table 2b) was also prescribed as 1
An 11 year old boy visited Shifa ul Mulk tablet per day before bed-time. The patient was
Memorial hospital, Hamdard University Karachi advised for follow-up after every 2-weeks and
on April 2013. Initially, he was interviewed treatment was continued for a period of
regarding demographic data such as age, 8-weeks.
duration of disease, socio-economic group and
living conditions particularly sharing of towels, 3. RESULT AND DISCUSSION
combs, having pets at home, fungal infections The patient, a 11 year old boy suffering
among siblings and any prior antifungal from TC which is a fungal infection caused by
treatment. On physical examination particularly T. violaceum. This appears to be a predominant
the scalp there were signs of severe scaly, pathogen in children and adults with multiple
crusting with bald patches, indicating severe clinical variations globally (Table 1) including
manifestation of TC. The patient was suffering Pakistan (Farooqi et al., 2014) and India
from this complain since 3 months that gradually (Moriarty et al., 2012).
aggravated and worsen on the 5th month when The major complaint by the patient was
he visited the OPD. He did not receive any itching along with hair loss accompanied by dull
prior treatment and hence was a naïve patient. grey patches. These were the most common
Furthermore, enlarged post auricular lymph clinical signs and symptoms of TC and are in
nodes were noticeable with body temperature accordance with the descriptions reported earlier
of 100°F. However, there was no other past (Kundu et a l., 2012). After 2-w eeks of
medical history with normal progression of treatment with polyherbal Unani medicine i.e.,
childhood development. Nuskha No. 9 and No. 10 a slight improvement
was noted and patient remarked that the itching,
2.2. Laboratory Investigation and Diagnosis secretions and crusting has been reduced. It
The p atien t was r ef er red to DO W was reflected by the reduction in the abrasions
Laboratory, Karachi for identification of the on scalp which are usually produced by
microbial infection. The laboratory highlighted continuous scratching and thus advised to
that hair samples were collected by sterile continue the treatment. Upon 2nd follow-up visit
hairbrush technique, while skin scrapings were after 4-weeks of treatment (Fig. 1b) a significant
cultu red o n Sab ou raud ’s agar fo r th e improvement was observed and the treatment
identification of microbes. The clinical report of was continued for another 4-weeks. A significant
skin scrapings showed fungal hyphae which was improvement was noted followed by normal hair
identified as T. violaceum and the case was growth in the affected area (Fig. 1c).
diagnosed as TC. Lymphadenopathy, was also noted which
has been reported in other cases of TC
2.3. Treatment appearing as painful nodes that enlarges with a
A photo was taken before treatment passage of time possibly due to immunological
(Fig. 1a), followed by the prescription of Unani events particularly occurrence of inflammatory
product as Nuskha No. 9 (Table 2a). He was processes in the area drained by the lymph
instructed to use one sachet per day (half in the nodes (Karpf, 1990).
36
Before treatment For example, in Nuskha No. 9, Fumaria

(shaahtara) contain fuyuziphine, an alkaloid
with antifungal activity (Sarma et al.,1999);
Sphaeranthus indicus enchance the process
of wound healing comparable to neomycin
(Sadaf et al., 2006); Terminalia chebula
(a)
exhibiting significant anti-oxidant activity along
with steroids and flavonoids (Aruna et al., 2012)
After treatment and hence used in treating various skin diseases
and Rosa damascena having anti-inflammatory
related to vitamin C which also has antioxidant
effects (Hajhashemi et al., 2010).
Likewise, in Nuskha no 10 the analgesic
property of A.leucophloea was due to the
blockade of prostaglandin release or synthesis
(b) (c)
or other endogenous substances that excite pain
Fig. 1: Effect of Nuskha No. 9 and tablet Nuskha No. 10 nerve endings (Juan, 1979). Cassia absus is
used against Tinea capitis also useful in fungal skin infection and other
skin disease (Ahmed et al., 2011).
a) A 11 years old boy with TC; b) After 1 month
treatment with Unani formulation s hows marked Other poly herbal antifungal formulation
improvement in the scalp condition; c) Follow up visit including Melicon V ointment (www.poulvet.
after 2 months from the start of the treatment shows com) and candigone capsules (www.renewlife.
complete cure and normal healthy hair growth.
com/candigone.html) contain S. chirayita extract
and berberry root extract which are also present
in Nuskha No. 9.and 10, respectively.
The dermatophytes usually invade non- The conventional medicine are also
living keratinized layer of skin, stratum corneum available for treating TC e.g griseofulvin, is
and use keratin as a nutrient source by producing f un gistatic an inh ib its the mito sis o f
keratinase enzyme that facilitates its invasion. dermatophytes by interacting with microtubules
At the site of infection inflammatory reaction and disrupting the mitotic spindle (Roberts et al.,
induces redness, swelling, heat or burning and 2005). It is effective against trichophyton,
finally alopecia (Sultana et al., 2011). microsporum and epidermophyton. Griseofulvin
Both the Unani formulations (Nuskha in school screening programmes and almost
No. 9 and No. 10) containing medicinal plants eradicated TC as an endemic condition in the
(9 herbs in Nuskha No. 9 and 4 herbs in Nuskha developed world in the 1950s, but it re-emerged
No. 10) ar e r ep or ted to po sses diverse as a public health concern in the United Kingdom
pharmacological properties particularly anti- in the 1990s in school children (Hay et al.,
bacterial, anti-fungal and anti-inflammatory. 1996). Increased mass tourism and mobile
These p lan ts ar e kn o wn f or diverse populations may also have contributed to the
pharmacological actions were used against many changing epidemiological trends (Havlickova
diseases including skin infections and for blood et al., 2008). The griseofulvin shows many
purification. adv er se eff ects such as head aches an d
37
Table 2a: Constituents of Nuskha No. 9 Used Against TC
Botanical name Family Local name Unani name Medicinal use Dosage/gm Part used
Swertia chirayita Gentianaceae Indian Gentian Chirayta Blood purifier (Skin diseases) 6 Bark
Fumaria indica Fumariaceae Indian Fumitory Shahtara Blood purifier 6 Leaves
Hamdard Medicus
Tephrosia
purpurea Leguminosae Wild indigo Sarphoka Blood purifier 6 Bark
Sphaeranthus Compositae East India Gul mundi Anti-inflammatory, 6 Fruit
indicus Globe Thistle Wound healing activity
Ziziphus jujuba Rhamnaceae Jujube Unab Blood purifier 6 Fruit
Santalum album Santalaceae White Sanda Sandal safaid Blood purifier 6 Wood
Terminalia Combretaceae Chebulic Halyela siyah Digestive Tonic 6 Fruit
chebula Myrobalan
38
Tamarix dioica Tamaricaceae Tamarisk Jhao Liver tonic 6 Leave
Rosa damascena Rosaceae Damask rose Gul e surkh General tonic 6 flower
Table 2b: Constituents of Tablet Nuskha No. 10 Used Against TC
Botanical name Family name Local name Unani name Activity Dosage/gm Part used
Berberis aristata Berberidaceae Berberry Rasoot Blood purifier 3 Resin

Curcuma zedoaria Zingiberaceae White turmeric Nar kachur Blood purifier 3 Rhizome
Cassia absus Fabaceae Jasmeejaz chaksu Blood purifier 3 Seed
Acacia leucophloea Fabaceae White Catechu Kath safaid Anti-pyretic 3 Resin
Vol. 59, No. 1, 2016
gastrointestinal disturbances and sometimes it Author’s Contribution and Declaration

needs to be discontinued. Severe allergic Leena Hameed Afridi: Conducted the trial.
reactions, hepatic toxicity and leucopenia rarely Tasneem Qureshi: Supervised the research
occur; therefore routine blood studies are not study
necessary unless treatment is to last for many The authors declare that there is no conflict
months or the dosage is exceptionally high. It is of interest.
contraindicated in children with porphyria, lupus
erythematosus, or severe liver disease (Higgins
et al., 2000) 4. REFERENCES
Terbinafine is also fungicidal and acts by 1. Ahmad, W., Hasan, A., Ahmad, I. and Zeenat, F.
inhibiting squalene epoxidase, a membrane- (2011). Ethnomedicinal Plants of Mansoora,
bound enzyme in the biosynthetic pathway of Malegaon. Hamdard Medicus. 54(2):29-40.
2. Aly, R. (1999). Ecology, epidemiology and diagnosis
sterol synthesis of the fungal cell membrane. It
of Tinea capitis. Pediatric Infectious Disease
is well absorbed and binds strongly and Journal. 18(2):180-185.
nonspecifically to plasma proteins. (Gupta et al., 3. Aruna P ., Ses hagiri, J.R ., Mad hu, R . and
2003) Sudhakar, M. (2012). Antimicrobial and anti
Also orally itraconazole is effective in TC oxidant activity of seeds of Terminalia chebula
infections against Trichophyton and Micro- plant extracts. Journal of Pharmaceutical Research
sporum. Depending on its concentration it exhibit and Opinion. 2:(12)188-190.
4. Balci, E., Gulgun, M., Babacan, O., Karaoglu, A.,
both fungistatic and fungicidal properties by
Kesik, V., Yesilkaya, S., Turker, T., Tok, D. and
inhibiting the cytochrome P-450-dependent
Koc, A.N. (2014). Prevalence and risk factors of
enzymes and synthesis of ergosterol (the Tinea capitis and tinea pedis in school children in
principal component of fungal cell membranes), Turkey, JPMA. 64(5):514-518.
respectively. (Gupta et al., 1998). 5. Bennassar, A. and Grimalt, R. (2010). Management
The Unani formulations are cost effective of Tinea capitis in childhood. Clinical, Cosmetic
as from Nuskha No. 9 with 5 sachets (10 gm and Investigational Dermatology. 3:89-98.
6. Elewski, B.E. (2000). Tinea capitis: A current
each) cost approx Rs.1/sachet and total cost perspective. Journal of American Academy of
for entire treatment (8 weeks) is around Rs.45. Dermatology. 42:1-20.
However, Nuskha no 10 (12 tablets of 500mg) 7. Elewski, B.E., Cáceres, H.W., DeLeon, L. et al.
approximately costs Rs.9.0. The cost of 8 weeks (2008). Terbinafine hydrochloride oral granules
versus griseofulvin suspension in children with
treatment is a combination of both used for
Tinea capitis : R esults of two randomized,
8weeks of treatment co st Rs.94 app rox. investigator-blinded, multicenter, international,
(0.8971$). On the other hand, Grisol 500 mg controlled trials. J. Am. Acad. Dermatol. 59:41-
(Lisko Pharma) Rs.10 per tablet used for a 54.
period of 8 weeks with total cost of treatment 8. Farooqi, M., Tabassum, S., Rizvi, D.A. et al.
is ar ou n d Rs.440 (4.20$) . Th u s Un an i (2014). Clinical types of Tinea capitis and species
identification in children: an experience from tertiary
formulation is 4.6 times less costly than
care centers of Karachi, Pakistan. J. Pak. Med.
conventional medicine. Assoc. 64(3):304-308.
It is concluded the nuskha No. 9 and 10 9. Foster, K.W., Ghannoum, M.A. and Elewski, B.E.
are effective against TC, it is cost effective, (2004). Epidemiologic surveillance of cutaneous
however more data is required to justify its fungal infection in the United States from 1999 to
efficacy, safety and its mechanism of action. 2002. J Am Dermatol 50(5):748-752.
39
10. Friedlander, S.F., Rueda, M. and Chen, B.K., British Association of Dermatologists. Br. J.
Caceros-Rios, H.W., (2003). Fungal, protozoal and Dermatol. 143(1):53-8.
helminthic infections. In: Schachner, L.A., Hansen, 19. Juan, H. (1979). The pain enchancing effect of
R.C., Eds. Pediatric Dermatology, 3rd Edn., Mosby PG12. Agents and Actions Suppl. 4:204-212.
pp. 1093-1140. 20. Karpf, M. (1990). Lymphadenopathy. In: Walker,
11. Gorbach, S.L., Bartlett, J.G., Zorab, R., Blacklow, H.K., Hall, W.D. and Hurst, J.W., Eds. Clinical
N.R. (1997). Dermatophyte infections of the hair, Methods: The History, Physical, and Laboratory
Tinea capitis in fungal infections of the skin. Examinations, 3rd Edn. Boston, Butterworths. 711-
Infectious Diseases, 2nd Edn. Philadelphia, WB 714.
Saunders Co. pp. 1276-1295. 21. Kundu, D., Mandal, L. and Sen, G. (2012).
12. Gupta, A.K., Solomon, R.S. and Adam, P. (1998). Prevalence of Tinea capitis in school going children
Itraconazole oral solution for the treatment of Tinea in Kolkata, West Bengal J. Nat. Sci. Biol. Med.
capitis. Br. J. Dermatol., 139(1):104-106. 3(2):152-155.
13. Gupta, A.K. and Summerbell, R.C. (2000). Tinea 22. Moriarty, B., Hay, R. and Jones, R.M. (2012).
capitis. Med. Mycol. 38:255-287. The diagnosis and management of tinea.
14. Gupta, A.K., Adamiak, A. and Cooper, E.A. BMJ, 345:e4380.
(2003). The efficacy and safety of terbinafine in 23. Roberts, B.J. and Friedlander, S.F. (2005). Pediatr
children. J. Eur. Acad. Dermatol. Venereol. Ann. 34(3):191-200.
17(6):627-640. 24. Sadaf, F., Saleem, R., Ahmed. M., Ahmed, S.I. and
15. Hajhashemi, V., Ghannadi, A. and Hajiloo, M. Zafar, N. (2006). Healing potential of cream
(2010). Analgesic and anti-inflammatory effects of containing extract of Sphaeranthus indicus on
Rosa damascena hydroalcoholic extract and its dermal wounds in Guinea pigs. J. Ethnopharmacol.
essential oil in animal models. Iran. J. Pharm. Res. 107:161-163.
Spring 9(2):163-168. 25. Sarma, B.K., Pandey, V.B., Mishra, G.D. and
16. Havlickova, B., Czaika, V.A. and Friedrich, M. Singh, U.P. (1999). Antifungal activity of berberine
(2008). Epidemiological trends in skin mycoses iodide, a constituent of Fumaria indica. Folia
worldwide. Mycoses. 51(4):2-15. Microbiologica. 44(2):164-166.
17. Hay, R.J., Clayton, Y.M., de Silva, N., Midgley, 26. Sultana, J., Abid, M., Abbas, Q. and Wahid, Z.A.
G. and Rosser, E. (1996). Tinea capitis in south (2011). Dermatophytes, the causal organisms of
east London a new pattern of infection with public dermatomycosis an overview. Fuuast. J. Biol. 1(1):
health implication. Br. J. Dermatol. 135:955- 57-62.
958. 27. Tan, H.H. (2005). Superficial fungal infections seen
18. Higgins, E.M., Fuller, L.C. and Smith, C.H. (2000). at the National Skin Centre, Singapore. Nippon
Guidelines for the management of Tinea capitis. Ishinkin Gakkai Zasshi. 46:77-80.
40
Phytochemical and Dermatological Investigations of Tribulus terrestris L.
Khizar Abbas*1, M. Shehzad2, Ayesha Asif2, M. Imran Qadir3 and M. Taufiq Ahmad4
1
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, GC University, Faisalabad, Pakistan.
2
Univeristy College of Pharmacy, University of Punjab, Lahore, Pakistan.
3
Department of Biotechnology and Molecular Biology, BZU, Multan, Pakistan.
4
Akson College of Health Sciences, MUST, Mirpur, AJK.
*Email: khizarabbas_pharmacist@yahoo.com
Abstract plants preparations often referred as “Herbal

Trib u l u s t errest ris extr ac ts w er e dru gs” or “Herb al r emed ies” hav e been
prepared using solvents of different polarities playing appreciable role in keeping human
an d their irr itant in ducing ef fects were health and improving the quality of human
assessed in vivo on rabbit’s ears. The result life for chiliads of years (Craig, 1999, Roberts,
indicates that the polar components were in 1988, Tripathi and Tripathi, 2003). Now-a-
higher yield than the others and amounts of days the application of herbal drugs in the
th e extr a cted mater ial w er e d ir ectly world especially Europe and in America is
proportional to the polarities of solvents. growing rapidly for adjusting the imbalances
Preliminary phytochemical screening shows induced by consumption of modern diets and
diversity of compounds and comparative TLC sedentary lifestyles (Balandrin et al., 1985,
analytical behaviour further indicated the Cu lture, 1996). Many p lan ts have been
resolution of mostly five compounds from the reported to contain phytochemicals which
non-polar, intermediate polar and highly polar causes skin irritation. These chemicals often
materials by different solvent systems. Irritant occur in leaves, stems, bark, flowers and fruit
potency of water extract was assessed on etc. of certain plants. Some of them are
rabbit’s ears using a known method that was discharged only when the plant parts are
proved to be the most potent irritant (with crushed, while the others exudates on their
+++ response, propagated in an area of surfaces which cause dermatitis by their
2.70 cm 2 diameter). direct action (Evans and Schmidt, 1980,
Basketter et al., 2004, Slodownik et al., 2008).
Keywords Many plants that give rise to irritant reactions
Phytochemical screening, Irritancy, Chromato- are ordinary house plants, garden plants or
graphy, Tribulus terrestris. edible fruits and seeds etc. (Wattanakrai
et al., 2007, Evans and Schmidt, 1979).
1. Introduction Tribulus terrestris L., a weed of tillable
Large numbers of plants have been used land, gardens widely distributed worldwide as
globally as sources of medicine and these well as in warm dried areas of Pakistan and
41
commonly known as calatrops, small-caltrops, 2. Materials and M ethod

land-caltrops, puncture-vine in English and
bhakhra, bhakhrou, gokhru and chota-gokhru 2.1. Chemica ls
in different local languages. The plant is a Silica gel (60 F 254 Merck, Germany),
prostrate branched annual or biennial herb, organic solvents (BDH (Germany) and all
silky hairy with dragging stems(Plucknett and other chemicals were of analytical grade and
Holm, 1977). Leaflets are sub-equal, oblong, were purchased from the local market.
mucronate very variable in size. Flowers are
yellow on peduncle shorter than the leaves.
2.2. Animals
Its phytochemistry is well established and
Healthy adult male/female rabbits (1-
contains: Furostanol and spirostanol saponins
1.5 k g) o f alb in o str ain o f sp ecies
o f tigo genin , n eo tigo gen in , gito gen in ,
Oryctolagus cuniculus were purchased from
neogito genin , hecogen in, n eoh ecogenin,
th e lo cal mar k et. Th ese an imals w er e
d io sgen in , ch lo r o g en in , r u sco gen in ,
acclimatized in the animal house o f the
sarsasapogenin (Kostova and Dinchev, 2005,
University College of Pharmacy, University
Alavia et al., 2008, Simeonov et al., 2011),
of the Punjab, Lahore, for a period of 3 days.
qu ercetin 3-O -glycoside, q uercetin 3- O-
They were provided with carrots, fresh green
r u tin o sid e, k aemp f er o l 3- O - glyco sid e,
fodder (clover) and tap water ad libitum.
quercetin and kaempferol(Alavia et al., 2008),
methylprotodioscin, protodioscin (Kostova
et al., 2002), arboline, tribulusterine (Bremner 2.3. Plant Collection and its Processing
et al., 2003) rhamno-galact-uronan (Chen T. terrestris L. (Fig. 1) was collected
et al., 2002) and tribufurosides G and H(Xu from the botanical garden of Government
et al., 2009). T. Terrestris is used traditionally College University, Lahore Pakistan and was
as diuretic, antiseptic, anti-inflammatory for authenticated by Prof. Dr. Zaheer-ud-din
the mu cus membr ane o f u r in ary tr act, khan, Department of Botany, with voucher
demulcent, tonic, emmenagogue, also used no GC 1970 and specimen was deposited in
for spermatorrhoea, phosphaturia, dysuria, the herbarium of Pharmacognosy section,
impo tence, in chro nic cystitis, calculus University College of Pharmacy, University
affections, gonorrhoea, painful micturition and of the Punjab, Lahore. The plant was dried
in chronic kidney inflammation, reduces the under the shade at room temperature for about
high blood pressure, cholesterol, against 15 days then pulverized to a fine powder and
erectile dysfunction, increases the testosterone sto r ed in amb er co lou r ed bo ttles. Th e
level, fertility, libido and possesses anti-oxidant pulverized dried plant (100 g) were separately
properties (Neychev et al., 2007, Adaikan soaked and extracted with 500 mL of hexane,
et al., 2000, Chu et al., 2003, Gauthaman light petroleum ether (40-60o), chloroform,
et al., 2002, Gauthaman et al., 2003). dichlorometh ane, ethyl acetate, aceton e,
Our study on T. terrestris L. presents ethanol, methanol and water. Maceration was
the separation and identification of the active carried out in each solvent for 7 days at room
irritant principles residing in it through solvent temp er atu re (27± 2.5oC) f ollo wed by its
extractions and its dermatological effects on removal under reduced pressure and the
rabbit’s ears. residues were weighed and stored.
42
for the identification of main constituents present

in the dried powdered materials (Purohit et al.,
2007).
2.6. Thin Layer Chromatography

It was performed using silica gel using
different solvent systems for petroleum ether,
ch lo r o f o rm an d meth ano l extracts an d
compounds were visualized using UV light and
iodine vapors on TLC chromatogram (Stahl,
1969).
2.7. Irritancy Assay

The biological assay for irritancy was
performed as described earlier (Evans and
Schmidt, 1979). Group of 6 rabbits for each
dilution was used and a 10µl sample of the
most diluted solution was applied to one of
the ear of a rabbit while untreated ear served
as a control. The other group of animals were
treated similarly by increasing concentration
Fig. 1: Tribulus terrestris L.
of irritants. Rabbits were examined after
30 minutes of its application with 30 minutes
intervals. The number of ears showing marked
inflammation of the major blood vessels was
2.4. Preparation of Dilutions recorded. The irritant dose (ID50) corresponds
Dried extracted material of each sample to the 50 % cumulative frequency and the
was constituted as 20 mg/ml (w/v) solution evaluation of irritant response was determined
with acetone. The dilution series was prepared using Hacker method (Busch, 1967).
according to the equation (Evans and Schmidt,
1979). 3. Results And Discussion
Cm=C o × a –m T. terrestris L. is a herb aceous or
somewhat low shrubby plant often found as
Where, C o= initial concentration, Cm= weed in the fields of economically important
Concentration after m dilution, a = Dilution crops (Plucknett and Holm, 1977, Reddi, 1981,
factor. The dilution factor in all the cases Boydston, 1990) causing significant damages.
was kept at 2 and six or seven dilutions were It was a matter of familiar observation that
prepared. during the removal, it causes irritation on the
hands of workers (Zhai and Maibach, 2004,
2.5. Phytochemical Screening Mitchell and Rook, 1977, Calnan, 1975).
The condensed water extract were used Moreover, during the collection of plant fine
for the preliminary screening of phytochemical trichomes, present on the lower surface of
43
the leaves and on its spiny fruits produced ether (5.24%) and hexane (3.26%) were also
irritation on the flexor side of collector’s obtained. On the other hand, the components
hands. with intermediate polarities were extracted in
O n e o f th e imp o r tan t d er matitis chlo r of o r m ( 10.37%) , dich lo r o meth an e
parameters is irritancy which is very common (11.16%), ethyl acetate (10.75%) and acetone
and could easily be appraised in animals by (15.90%) in intermediate quantities. Thus
observing a rapid reaction of rabbits against T. terrestris contains a larger proportion of
the solvent extracts. Previously, many authors high and intermediate polar compounds than
separated, characterized and evaluated the its non-polar components.
structure-activity relationship of some of the
allergenic/irritants including alkaloids, saponins,
Table 2: Preliminary Phytochemical
flavonoid glycosides, terpenoids and similar Analysis of Aerial Parts of
phytochemicals compounds from various Tribulus terrestris L.
members of different the families (Misra,
1962, Athar and Mahmood, 1985, Alavia Phyto- Test T. terrestris
et al., 2008, Italo et al., 2009, Ntalli et al., constituent
2010). In Table 1 percent yield of both non- Alkaloid Mayer’s reagent +++
polar and polar ingredients are presented. The Replace test with
p olar comp on ents extr acted in eth ano l reagent in all
(25.42%) , methanol (30.72%) and water
Wagner’s test +++
(63.52%) were in higher yield. However, low
yields of non-polar components in petroleum Hager’s test +++
Dragendroff’s
test +++
Table 1: Percent Yield of the
Extracted Materials Glycoside Fehling’s test +
Using Different Solvents
Tannin Ferric chloride
test ++
S.No. Solvent Percentage
yield Flavonoid Alkaline reagent
test +
1. Hexane 03.26 %
Steroid and
2. Petroleum ether 05.24 % triterpenoid Salkowski test +
3. Chloroform 10.37 %
Lignin Phloroglucinol
4. Dichloromethane 11.16 % test –
5. Ethyl acetate 10.75% Saffranine test +
6. Acetone 15.90 % Amino acid Ninhydrine test –
7. Ethanol 25.42 %
Carbohydrates Molisch’s test –
8. Methanol 30.72 %
Fats and Copper sulphate
9. Water 63.52 % fixed oil test –
44
Table 3: Comparative TLC Analysis of Petroleum Ether Extract of T. Terrestris L.
Comp.
Solvent Systems Detection
No. of
Solvents Ratio UV light Iodine hRf values
P.E./CHCl3 90:10 3 blu, blu, pur l-yel, yel, yel 21, 41, 52
P.E./CHCl3 80:20 4 gry, yel, pur, blu l-yel,yel,yel,l-yel 14, 21, 61, 70,
P.E./CHCl3 70:30 3 yel, gry, blu l-bro,yel,yel 29, 40, 81
P.E./CHCl3 /MeOH 90:10:2 5 red, blu, yel, pin, blu l-yel,l-el,yel,yel,yel 26, 35, 55, 68, 83
P.E./CHCl3 /MeOH 90:10:5 5 blu, pur, pur, yel, blu yel,yel,yel,l-yel,bro 8, 24, 36, 41, 66
P.E./CHCl3 /MeOH 80:20:2 4 blu, pur, blu, blu l-yel,yel,yel,l-bro 18, 47, 68, 85
P.E./CHCl3 /MeOH 80:20:5 5 blu, pur, blu, blu, pur l-yel,yel,yel,yel,d-bro 27, 58, 67, 72, 82
P.E./CHCl3 /MeOH 80:20:10 5 pin, pur, yel, pin, bro yel,l-yel,yel,yel,d-bro 11, 30, 65, 79, 83
P.E./CHCl3 /MeOH 70:30:2 5 yel, blu, gry, pur, gry yel,l-yel,yel,yel,d-bro 9, 28, 56, 77, 89
P.E./CHCl3 /MeOH 70:30:5 5 bro, yel, blu, pin, yel yel, l-yel, yel, yel, d-bro 20, 65, 75, 82, 89
P.E./CHCl3 /MeOH 70:30:10 4 blu, yel, blu, red yel, l-yel, yel, l-bro 24, 57, 68, 89
P.E./CHCl3 /MeOH 60:40:1 4 blu, yel, pin, blu l-yel, l-bro, yel, yel 20, 54, 62, 78
P.E./CHCl3 /MeOH 60:40:2 3 blu, yel, blu l-yel, l-yel, bro 28, 65, 76
P.E./CHCl3 /MeOH 60:40:5 1 blu l-yel 81
CHCl3/MeOH 10:20 2 gre, blu yel, yel 64, 76
CHCl3/MeOH 15:25 3 yel, blu, red yel, d-yel, l-yel 65, 74, 80
CHCl3/MeOH 20:30 3 yel, blu, red l-yel, yel, d-yel 65, 70, 82
CHCl3/MeOH 25:35 3 yel, blu, red l-yel, d-yel, yel 77, 83, 87
HCl3 /MeOH/HCl 30:40:1 2 yel, blu yel, bro 75, 73
HCl3 /MeOH/HCl 30:40:2 2 yel, blu l-yel, d-yel 76, 80
HCl3 /MeOH/HCl 40:50:3 2 pur, yel. l-yel, d-yel 56, 78
HCl3 /MeOH/HCl 35:55:5 2 pur, l-blu l-yel, d-yel 60, 72
45
Table 4: Comparative TLC Analysis of Chloroform Extract of T. Terrestris
Comp.
No. of
hRf values
Solvents Ratio UV light Iodine
P.E./CHCl3/MeOH 90:10:2 4 red, red, gre, pin d-yel, yel, bro, yel 35, 59, 65, 79
P.E./CHCl3/MeOH 90:10:5 3 yel, gre, gre bro, d-yel, d-bro 24, 37, 64
P.E./CHCl3/MeOH 80:20:2 4 org, gre, gre, pin l-yel, d-bro, bro, yel 35, 45, 53, 68
P.E./CHCl3/MeOH 80:20:5 3 pin, bro, gre bro, yel, d-bro 34, 57, 73
P.E./CHCl3/MeOH 80:20:15 4 pin, pin, gre, blu l-yel, yel, d-yel, yel 61, 71, 75, 87
P.E./CHCl3/MeOH 70:30:1 3 gre, pin, blu l-yel, yel, yel 10, 25, 67
P.E./CHCl3/MeOH 70:30:2 3 gre, pin, blu l-yel, yel, yel 9, 25, 67
P.E./CHCl3/MeOH 70:30:5 4 yel, gre, yel, blu d-yel, l-yel, yel, yel 8, 20, 47, 64
CHCl3/MeOH 95:5 4 yel, gre, gre, pin l-yel, yel, d-yel, yel 41, 56, 72, 79
CHCl3/MeOH 90:10 2 blu, pin l-yel, d-yel 74, 81
CHCl3/MeOH 85:15 2 blu, pin l-yel, d-yel 76, 84
CHCl3/MeOH 80:20 1 gre, bro 88
CHCl3/MeOH/HCl 80:20:1 3 pin, yel, blu l-yel, yel, yel 21, 46, 68
CHCl3/MeOH/HCl 75:25:2 4 gre, pin, yel, blu l-yel, yel, yel, yel 42, 65, 76, 81
CHCl3/MeOH.HCl 70:30:3 4 gre, gre, yel, blu l-yel, yel, yel, yel 14, 29, 73, 85
CHCl3/MeOH/HCl 65:35:5 4 org, gre, pin, blu yel, l-yel, yel, yel 9, 17, 32, 64
CHCl3/MeOH/AcA 90:15:3 4 org, gre, pin, pin yel, yel, d-yel, yel 20, 44, 58, 69, 80
CHCl3/MeOH/AcA 80:20:5 3 blu, yel, gre d-bro, d-yel, yel 21, 50, 68
CHCl3/MeOH/AcA 70:30:10 3 gre, yel, blu d-yel, yel, l-yel 33, 42, 54
CHCl3/MeOH/AcA 65:35:12 4 org, pin, gre, blu d-yel, l-yel, yel, l-yel 71, 56, 77, 26
46
Table 5: Comparative TLC Analysis of Methanol Extract of T. Terrestris L.
Comp.
No. of
hRf values
Solvents Ratio UV light Iodine
CHCl3/MeOH 95:5 2 pin, blu — 39, 40

CHCl3/MeOH 90:10 2 d-pin, pin — 25, 51
CHCl3/MeOH 85:15 2 d-pin, pin — 9, 22
CHCl3/MeOH 80:20 5 pin, org, pin, pin, blu l-yel, yel, yel, yel, l-yel 9, 16, 36, 65, 77
CHCl3/MeOH 75:25 4 org, pin, gre, pin d-bro, l-yel, l-yel, yel 10, 16, 49, 63
CHCl3/MeOH 70:30 5 gre, org, pin, pin, blu l-yel, yel, yel, l-yel, yel 19, 27, 37, 57, 87
CHCl3/MeOH 65:35 3 pin, d-pin, blu l-yel, d-yel, yel 9, 23, 54
CHCl3/MeOH 60:40 4 pin, d-pin, red, blu l-yel, d-yel, yel, yel 30, 34, 53, 70
MeOH/AcA 95:5 1 yel yel 72
MeOH/ AcA 90:7 1 yel yel 75
MeOH/ AcA 90:10 1 l-yel l-yel 78
MeOH/ AcA 85:7 1 l-yel l-yel 70
MeOH/ AcA 85:10 2 gry, gre bro, bro 53, 69
MeOH/ AcA 80:15 2 gry, gre bro, d-bro, 54, 69
MeOH/ AcA 75:20 2 gry, gre yel, d-bro 43, 57
MeOH/ AcA 70:30 1 gre d-bro 55
MeOH/HCl 95:10 2 gry, gre l-bro, d-bro 43, 65
MeOH/HCl 90:15 1 blu yel 18
MeOH/HCl 85:30 4 yel, gre, pin, d-pin d-bro, d-yel, yel, yel 22, 34, 57, 89
MeOH/HCl 80:30 2 pin, yel yel, l-yel 20, 47,
MeOH/HCl 80:35 2 d-pin, d-yel l-bro, l-yel 25, 56
MeOH/HCl 75:30 2 l-pin, l-yel l-bro, l-yel 20, 41
Where:P.E. = Petroleum ether, CHCl3 = Chloroform, MeOH = Methanol. EtAc = Ethyl acetate, AcA = Acetic acid,
blu = blue colour, yel = yellow colour, gre = green colour, pur = purple colour, pin = pink colour, gry = grey colour,
red = red colour, l-yel = light yellow, d-yel = dark yellow, l-bro = light brown, d-bro = dark brown, org. = orange
colour, d-pin = dark pink.
47
Table 6: Irritant Response of Different Doses of the Water Extract of
T. Terrestris on Rabbit’s Ears
Do se s Responses after – Acute time Chronic time

g/ml
Hamdard Medicus
30min 1h 2h 3h 4h 5h 6h 7h 8h 9h 11h 12h 24h 48h 72h
10 – – – – – – – – – – – – – – –
15 – – – – – – – ± ± ± ± ± – – –
20 – + + + + + + + ++ + + + – – –
25 – + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + ± ±
48
30 + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ±
35 + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ±
40 + ++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ + + +
45 + ++ ++ ++ ++ ++ +++ +++ +++ ++ ++ ++ + + ±
50 + ++ ++ ++ ++ +++ +++ +++ +++ +++ ++ ++ + ± –
55 + ++ ++ ++ +++ +++ +++ +++ +++ +++ ++ ++ + – –
60 + ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + +
Where: – = No reaction, ± = doubtful reaction, + = Slight reddening of the main vessels, ++ = Marked reddening of the main vessels, +++ = Intense reddening
Vol. 59, No. 1, 2016
of the entire ear.

Thr ee solvent extract samples w ere substance (s) most likely interacted with the
analysed by comparative TLC, usin g 22 cell membrane and cellular con ten ts of
different solvent systems. The main intention superficial and deeper layers of epidermis
of this analysis was to have an estimation of inducing release of inflammatory mediators
total number and chromatographic behaviour in superficial and deeper layers epidermal
of the ingredients residing in each extract. layers causing damage which was reflected
The most appropriate solvent system which as localized inflammation. Similar strong to
resolved the mixture of petro leum ether moderate irritant reaction have been noted
extract of the T. terrestris materials into five by various compounds (Anderson et al., 2009,
o r mo r e co mp o n en t s, ap p ear ed to b e Loffler et al., 2001). The isolation of irritant
petroleum ether/chloroform/methanol (90:10:2; principle(s) from T. terrestris exact and its
90:10:5; 80:20:5; 80:20:10 an d 70:30:2) mechanism of action needs to be explored
(Table 2). Chloroform extract was segregated further that will be helpful to design the
maximally into four components by eight precautionary measures against it.
or ten different solvent systems but was not
further resolved (Table 3). The mixture of Author’s Declaration
polar components, present in the methanol The authors declare that there is no conflict
extract of samples was resolved into five of interest.
major compounds by chloroform/methanol
(80:20; 70:30; 65:35 and 60:40) (Table 4).
4. REFERENCES
Water extract was used for the irritancy
1. Adaikan, P., Gauthaman, K., Prasad, R. and N.G.S.
assay because in preliminary irritancy assay (2000). Proerectile pharmacological effects of
it was most active as compared to other Tribulus terrestris extract on the rabbit corpus
extracts probably, the elements residing in cavernosum. Annals of the Academy of Medicine,
these mixtures were unable to penetrate the Singapore. 29:22-26.
skin to induce deep damage to the epidermal 2. Alavia, S.H.R., Matin Yekta, M., Hadjiaghaee, R.
tissues, but strong enough to produce dilation and Ajani, Y. (2008). Flavonoid Glycosides from
of blood vessels and superficial redness Tribulus terrestris L. orientalis. Iranian Journal of
Pharmaceutical Sciences, 4:231-236.
(Anderson et al., 2009, Basketter et al.,
3. Anderson, S.E., Brown, K.K., Butterworth, L.F.,
2004). The water extracts seems to contain Fedorowicz, A., Jackson, L.G., Frasch, H.F.,
the most active mixture of phytochemical Beezhold, D., Munson, A.E. and Meade, B. (2009).
compounds than the other solvent extracted Evaluation of irritancy and sensitization potential
materials. Eight out of eleven water extracts of metalworking fluid mixtures and components.
of T. terrestris were dermatologically active Journal of Immunotoxicology. 6:19-29.
on rabbit’s ears and their irritant responses 4. Athar, M. and Mahmood, A. (1985). Observations
was dose dependent (10 to 60 mg/ml) as on nitrogen fixation by Tribulus terrestris Linn.
under natural habitat. Geobios, 12:44-46.
reflected by its severity and duration on the
5. Balandrin, M.F., Klocke, J.A., Wurtele, E.S. and
an imal ear s ( Tab l e 5) . I t p r o d u ced Bollinger, W.H. (1985). Natural plant chemicals:
commemorated erythema on rabbit’s ear after sources of industrial and medicinal materials.
one hour of application and in most cases Science, 228:1154-1160.
this reaction persisted for about 48 hours 6. Basketter, D.A., York, M., Mcfadden, J.P. and
(Table 6). The polar nature of penetrated Robinson, M.K. (2004). Determination of skin
49
irritation potential in the human 4h patch test. 20. Kostova, I. and Dinchev, D. (2005). Saponins in
Contact Dermatitis, 51:1-4. Tribulus terrestris – chemistry and bioactivity.
07. Boydston, R.A. (1990). Time of emergence and Phytochemistry Reviews, 4:111-137.
seed production of longspine sandbur (Cenchrus 21. Kos tova, I., Dinchev, D., Rentsc h, G. H.,
longispinus) and puncturevine (Tribulus terrestris). Dimitrov, V. and Ivanova, A. (2002). Two new
Weed Science, 16-21. sulfated furostano l sapo nins from Tribulus
08. Bremner, J., Sengpracha, W., Southwell, I., terrestris. Zeitschrift für Naturforschung C, 57:33-
Bourke, C., Skelton, B. and White, A. (2003). The 38.
Alkaloids of Tribulus terrestris: A revised structure 22. Loffler, H., Pirker, C., Aramaki, J., Frosch, P.J.,
for the Alkaloid Tribulusterine. III WOCMAP Happle, R. and Effendy, I. (2001). Evaluation of
Congress on Medicinal and Aromatic Plants, Vol. 3, skin susceptibility to irritancy by routine patch
Perspectives in Natural Product Chemistry. 677:11- testing with sodium lauryl sulfate. European
17. Journal of Dermatology, 11:416-419.
09. Busch, H. (1967). Methods in Cancer Research. 23. Misra, D. (1962). Tribulus terrestris weed in arid
10. Calnan, C. (1975). Petty spurge (Euphorbia zone farming. Indian Journal of Agronomy, 7:136-
peplus L.). Contact Dermatitis. 1:128-128. 141.
11. Chen, H.S., Leung, W.N. and Xu, Y.X. (2002). An 24. Mitchell, J. and Rook, A. (1977). Diagnosis of
acidic polysaccharide from Tribulus terrestris. contact dermatitis from plants. International
Chinese Chemical Letters. 13:625-628. Journal of Dermatology, 16:257-266.
12. Chu, S., Qu, W., Pang, X., Sun, B. and Huang, X. 25. Neychev, V., Nikolova, E., Zhelev, N. and
(2003). [Effect of saponin from Tribulus terrestris Mitev, V. (2007). S apo nins from Trib ulus
on hyperlipidemia]. Zhong yao cai= Zhongyaocai= terrestris L. are less toxic for normal human
Journal of Chinese Medicinal Materials, 26:341- fibroblasts than for many cancer lines: Influence
344.
on apoptosis and proliferation. Experimental
13. Craig, W.J. (1999). Health-promoting properties
Biology and Medicine, 232:126-133.
of common herbs. The American Journal of
26. Ntalli, N.G., Cottiglia, F., Bueno, C.A., Alché, L.E.,
Clinical Nutrition. 70:491s-499s.
Leonti, M., Vargiu, S., Bifulco, E., Menkissoglu-
14. Culture, U. (1996). Health orientation texts-world
spiroudi, U. and Caboni, P. (2010). Cytotoxic
decade for cultural development 1988-1997.
tirucallane triterpenoids from Melia azedarach
DocumentCLT/DEC/PRO-1996, 129.
15. Evans, F.J. and Schmidt, R.J. (1979). An assay fruits. Molecules. 15:5866-5877.
procedure for the comparative irritancy testing of 27. Plucknett, D. and Holm, L. (1977). The World’s
es ters in the tigliane and dap hnane s eries. Worst Weeds, Distribution and Biology. East-West
Inflammation, 3:215-223. Centre, University of Hawaii, Honolulu, USA.
16. Evans, F.J. and Schmidt, R.J. (1980). Plants and 28. Purohit, A., Kokate, C. and Gokhle, S. (2007).
plant products that induce contact dermatitis. A Text Book of Pharmacognosy. Publication, Nirali
Planta Medica. 38:289-316. Prakashan. 357-358.
17. Gauthaman, K., Adaikan, P. and Prasad, R. (2002). 29. Reddi, C.S. (1981). Breeding Structure and
Aphrodisiac properties of Tribulus terrestris extract Pollination Ecology of Tribulus terrestris. Proc.
(Protodioscin) in normal and castrated rats. Life Indian Nam. Sci. Acad. B47 No. 2, 1854930981.
Sciences, 71:1385-1396. 30 Roberts, M. (1988). Medicinal products through
18. Gauthaman, K., Ganesan, A.P. and Prasad, R. plant biotechnology. Manipulating Secondary
(2003). Sexual effects of puncturevine (Tribulus Metabolism in Culture, 201-216.
terrestris) extract (protodioscin): An evaluation 31. Simeonov, E., Koleva, V. and Chilev, C. (2011).
using a rat model. The Journal of Alternative & Solid liquid extraction of furostanal saponins from
Complementary Medicine, 9:257-265. Tribulus terrestris. Journal of the University of
19. Italo, C.G., Amanda, H.F. and Diego, L.R. (2009). Chemical Technology and Metallurgy, 46:309-314.
Physical and chemical characterization of Melia 32. Slodownik, D., Lee, A. and Nixon, R. (2008).
azedarach L. fruit and leaf for use as botanical Irritant contact dermatitis: A review. Australasian
insecticide. Journal of Dermatology, 49:1-11.
50
33. Stahl, E. (1969). Thin-Layer Chromatography: and ingredients by human single closed patch
A Laboratory Handbook, Springer Verlag. Berlin, testing. Journal of the Medical Association of
Heidelberg, NY, 779. Thailand = Chotmaihet thangphaet. 90:1116-
34. Tripathi, L. and Tripathi, J.N. (2003). Role of 1122.
biotechnology in medicinal plants. Tropical Journal 36. Xu, T., Xu, Y., Liu, Y., Xie, S., Si, Y. and Xu, D.
of Pharmaceutical Research. 2:243-253. (2009). Two new furostano l saponins from
35. Wattanakrai, P., Suwanachote, S., Kulkollakarn, S. Tribulus terrestris L. Fitoterapia, 80:354-357.
and Rajatanavin, N. (2007). The study of human 37. Zhai, H. and Maibach, H.I. (2004). Dermato-
skin irritation of a novel herbal skin care product toxicology, CRC Press.
51
Inhibitory Effect of Aegiceras corniculatum Against Lung Injury in

Carrageenan-induced Mouse Pleurisy Model
Sabahat Aziz, Talat Roome*, Perbhat Ali and Anam Razzak

Molecular Pathology, Department of Pathology, Dow Diagnostic Reference and Research Laboratory,
Dow Research Institute of Biotechnology & Biomedicial Sciences, Dow International Medical College,
Dow University of Health Sciences, Karachi, Pakistan.
*Email: talatroome@hotmail.com talat.roome@duhs.edu.pk
Abstract morphonuclear cells (PMNs) that enhanced the

Aegiceras corniculatum is a mangrove production of interleukin-1b, IL-6, tumor necrosis
plant traditionally used against rheumatism, pain factor-5a and cyclooxygenase (COX-2) which
and inflammation. The present study was aimed were more likely to be attenuated by the
to explore the anti-inflammatory activity of A. corniculatum extracts revealing its protective
methanol and ethyl acetate extracts derived from effect in acute lung inflammatory conditions.
A. corniculat um in carrageen an-induced
pleurisy mouse model of acute inflammation. Keywords
Mice were treated with different doses (10, 50 Aeg icera s co rnicu la t um , an ti-
and 100 mg/kg, i.p.) of methanol and ethyl inflammatory, carrageenan-induced acute lung
acetate extracts followed by induction with intra- inflammation.
pleural injection of 1% carrageenan (0.1 ml).
After 4 hours animals were euthanized and 1. INTRODUCTION
pleural lavage was collected in sterile PBS and Inflammation is a complex phenomenon
myelo p er oxidase (MPO ), nitr ate/n itr ite which involving multiple biochemical, cellular and
concentration and leukocytes migration were molecular processes for activating the host
determined. The methanol and ethyl acetate def ense and regulating the u ncon trolled
extracts at higher do ses ( 100 mg/kg) inflammatory developments causing various
demonstrated remarkable reduction ~82% in inflammatory diseases (Vane et al., 1998; Jiang
leukocytes migration. Likewise, MPO and et al., 2003; Willerson et al., 2004). In
nitrate/nitrite concentrations also showed a dose inflammatory conditions, oxidative burst of
dependent decline in the presence of both polymorphonuclear neutrophils (PMNs) produce
methanol and ethyl acetate extracts reaching extra- and intra-cellular reactive oxygen species
maximum value (83%) at 100 mg/kg thus (ROS) or free radicals namely, hydroxyl radical
implying its protective effect in the management ( O H) , h yd r ogen p er o xide (H2 O 2 ) an d
l
of acute lung inflammation. These results led superoxide anion (O 2 – ) via activation of
l
us to suggest that carrageenan administration NADPH oxidase and myeloperoxidase (MPO)

into the pleural cavity caused accumulation of enzymatic pathways and protect the body from
fluid containing infiltrated PMNs from lung immune responses. In neutrophil degranulation,
tissues as well as a large number of poly- myeloperoxidase, a heme-containing peroxidase
52
which are stored in azurophilic granules of d-galactose extracted from certain red seaweeds
neutrophilic granulocytes are released from the of the Rhodophyceae class are also a common
cells and are associated with inflammation and food additive. Its unique chemical structure
oxidative stress (Goldblum et al., 1985). In activates body immune responses, which has
phagosome of neutrophils, MPO also plays a been identified as a dangerous invader and
vital role in killing of microbes and forming causing inflammation (Prajapati et al., 2014).
hypochlorous acid (HOCl) from the chlorination In most animal studies, it is used to induce
cycle. But under adverse circumstances its inflammation (paw or ear edema) for evaluation
excessive production oxidizes vital biomolecules of anti-inflammatory activities of compounds/
such as DNA, proteins, and lipids thereby drugs and also provide information about their
leading to the development of various diseases mechanism of action against different pro-
inclu d in g n eu ro d egen er ative diso r ders, inflammatory mediators such as leukotrienes,
cardiovascular diseases and cancer (Selvakumar prostaglandin, bradykinin, histamine, platelet-
et al., 2011). activating factors, interleukins (ILs) (Saldanha
Globally, it is a common practice to treat et al., 2016).
different diseases with medicinal plant(s). Considering anti-inflammatory and anti-
A. corniculatum, a mangrove plant growing in oxidant properties of A. corniculatum, the
the wetland of tropical and subtropical regions present study was designed to investigate its
of Indus Delta valley of Pakistan (Roome et al., effect in carrageenan-induced pleurisy mouse
2008). Its phytochemical and pharmacological model to determine the inflammatory response
evaluation revealed that it is enriched with including PMN infiltration, myeloperoxidase
different metabolites, mainly including flavonoids, activity and release of nitrate/nitrite.
polyphen ols ( Tanin and lign in), stero ls,
pentacyclic triterpines (Aegicerin, genin) and 2. MATERIALS AND METHODS
its d er iv atives as well as sap on in s 2.1. Animals
(Corniculatonin) (Zhang et al., 2005). It Swiss mice either sex (20 to 25 g) acquired
possesses anti-diabetic, anti-asthmatic, anti- from the animal house of Dow University of
rheumatic, anti-inflammatory, anti-allergic, Health Sciences, Karachi, Pakistan, were used
antioxidant, and hepatoprotective activities throughout the study. The animals were housed
(Roome et al., 2008). In our previous study, under controlled environment and were provided
we reported that A. corniculatum extracts with standard rodent chow and water. Animal
significantly inhibited inflammatory pathways via studies were performed in accordance with the
suppressing COX-1, 2 and 5-LOX metabolites declaration of European Community guidelines
as well as antagonized oxidative stress. The for the ethical handling and the use of laboratory
anti-oxidant properties of n-hexane, methanol animals and through the clearance of institutional
and ethyl acetate extracts were also identified animal use committee.
which were associated with a variety of
chemical constituents residing in the plant that 2.2. Chemicals
inhibited the respiratory burst in cells (Roome The following chemicals were used:
et al., 2008). dimethyl sulfoxide (DMSO), l-carrageenan
Car rageen ans are su lp hated lin ear (Type IV), indomethacin, dexamethasone and
polysaccharides of d-galactose and 3,6-anhydro- rutin Sigma Chemical Company, USA. Solvent
53
diethyl ether (RDH, Sigma-Aldrich Laboratory Professor Dr. Surriya Khatoon from the
Chemicals Germany). Other chemicals including Department of Botany, University of Karachi,
sodium azide and the reagents used in the Pakistan. A voucher specimen representing the
preparation of PBS and assay buffer were of collection labeled as KHU G.H.S. No. 68219
analytical grade. has been deposited at herbarium of this
All chemicals were dissolved in saline department. Its morphological characteristics
( NaCl, 0.9%,) with th e excep tion o f are presented in Fig. 1.
dexamethasone and plant extract that were
dissolved in DMSO (10%) while indomethacin 2.4. Preparation of A. corniculatum Extracts
were solubilized in 1% Na2CO3. The extracts were prepared with the same
procedure as described in (Roome et al., 2008).
2.3. Plant Material Air-dried powdered stem (9.0 g) were soaked
A. corniculatum were collected during in 50 L of hexane, ethyl acetate and methanol
June 2001 from Indus Delta, Sindh, Pakistan at extracts for 7 days and n-hexane extract (6.0 g
lowest tide and identied by a taxonomist, or 0.07%, w/w), ethyl acetate extract (8.5 g or
Fig. 1: Morphological characteristic of Aegiceras corniculatum

(a) A. corniculatum is a shrub or small tree, 1.5-7 m tall usually gregarious and glabrous. (b) Leaves are obovate-
oblong to obovate, 4-8 cm long, 2.5-4.0 cm broad, pale green, shining above, glaucous beneath with petiole 3-6 mm
long. Flowers are 1.5 cm across usually white and fragrant. (c) Fruit are cylindrical, 2.5-5.0 cm long, 4-6 mm broad
and reddish brown (Jafri et al., 1975).
54
0.09%, w/w) and methanol extract (9.8 g or 2.7. Determination of Nitrate/Nitrite

0.11%, w/w) were obtained. These extracts Concentration
were prepared in collaboration with Professor Nitrate and nitrite levels in pleural fluid
Muhammad Iqbal Choudhary’s research group which is an indicator of nitric oxide production
at International Center for Chemical and was determined by u sing Griess method
Biological Sciences, University of Karachi, (Sautebin et al., 1998). The pleural lavage and
Pakistan. equal volume of fresh Griess reagent were
mixed and incubated for 10 min at room
2.5. Carrageenan-induced Pleurisy temperature, followed by its quantification (µM)
Swiss mice weighing 20-25 g were divided at 540 nm spectrophotometrically (Dalmarco
into groups (n=6) pretreated intraperitoneally et al., 2002).
with methanol and ethyl acetate extract (10, 50
and 100 mg/kg) derived from A. corniculatum 2.8. Statistical Analysis
or indomethacin (1, 5 and 10 mg/kg) followed Statistical differences between groups were
by intra-pleural injection of 1% carrageenan determined by analysis of two-way ANOVA
(0.1 ml). After 4 hours mice were euthanized, with Dunnet’s tests. Less than 0.05 p-value
th or ax op en ed an d w ashed w ith sterile was considered as significant.
phosphate buffered saline (pH =7.27) and
samples of pleural lavage were collected for 3. RESULTS AND DISCUSSION
the d etermin ation o f leu ko cytes cou nt, The carrageenan induction into the pleural
myeloperoxidase and nitrate/nitrite concentra- cavity induces lung injury which is characterized
tions. Total infiltrated leukocytes count and by cellular infiltration, edema, alveolar thickness,
differential count in pleural fluid were determined myelin bodies, and appearance of large vacuoles
using a hemocytometer and observed under light along with enhanced pro- inflammatory cytokine
microscope. levels, COX-2 and NOS (Cuzzocrea et al.,
1999). The present investigation showed that
2.6. Determination of 1% carrageenan (0.1 ml) administered into the
Myeloperoxidase (MPO) Activity pleur al cavity o f mice caused an acu te
Different concentrations of MPO (from inflammatory response leading to accumulation
human neutrophils) used as a standard samples. of fluid containing a large amount of PMNs.
Pleur al aliq uo t (40 µl) an d stand ar d The methanol extract of A. corniculatum
(dexamethasone) or test agents were transferred elicited dose dependent (10, 50 and 100 mg/kg)
into reaction cuvettes followed by addition of decline by 35%, 65% and 80 %, respectively.
assay buffer (0.167 mg/ml of o-dianisidine Likewise, at the similar doses, ethyl acetate
2HCl and 0.0005% H2O2) for the initiation of extract also inhibited infiltration of cells into the
reaction and stopped by sodium azide (1%). pleural cavity by almost similar magnitude (40%,
Afterwards, the samples were centrifuged at 64% and 84 %). Indomethacin (1, 5 and 10 mg/
50 × g for 5min, the supernatants were separated kg) used as a positive control also reduced
and the rates of changes in absorbency were infiltration of PMNS as shown in Fig. 2 as
determined spectrophotometrically at 450 nm compared to the control group.
and expressed as mU/ml (Dalmarco et al., The effect of A. corniculatum on cell
2002). influxes were also determined by measuring
55
Fig. 2: Effect of A. corniculatum extracts on migration of leukocytes in carrageenan-induced pleurisy model
Methanol (MeOH, s), ethyl acetate (EtOAc, n) extracts (10, 50 and 100 mg/kg) and indomethacin (Indo l 1, 5
and 10 mg/kg) were administered in mouse pleurisy model induced by carrageenan (1 %, 0.1 ml) and leukocyte
migration at the site of inflammation was compared with the carrageenan alone (Cg u).
The leucocyte count in control (saline treated animals = 2.1±0.6) while (carrageenan treated animals= 17.6±2.0)
The values within parenthesis represents percent reduction in leukocyte migration after induction with carrageenan.
Data represents Mean ± S.E.M (n=6) at *p<0.05, **p<0.01, ***p<0.005 as compared to control.
MPO enzyme activity which was significantly arthritis, lupus, psoriasis, or breathing disorders
elevated at 4 hour of carrageenan administration. (Pithadia et al., 2011). Based on these result,
The methanol and ethyl acetate extracts (10- extracts of A. corniculatum inhibited the
100 mg/kg) dose dependently inhibited (40-80%) neutrophils influx which is also linked with the
the MPO levels in lung tissue as depicted in attenuation of adhesion molecules CD11b and
Table 1. Likewise, dexamethasone (0.5 mg/kg) CD18 (Frode et al., 2001).
also caused 58% reduction in the enzyme levels. Moreover, the nitrate/nitrite levels in the
It is well established that this corticosteroid is pleural exudate were also estimated by Griess
u sed clinically to treat many d iff er en t reaction (Dore et al., 2007) in which both
inflammatory conditions such as allergic methan ol an d eth yl acetate extracts o f
disorders, skin conditions, ulcerative colitis, A. corniculat um caused dose d epend ent
56
decline in exudate formation accompanied by the level of NO in lungs like expression of iNOs
the reduction in the levels of nitrate/nitrite and stimulated neutrophils (Cuzzocrea et al.,
~80% at 100 mg/kg. (Table 1). Similarly, rutin 2000). Oxidative stress also plays an important
(10 mg/kg) a quercetin-3-O-rutinoside also role in inflammation because it activates the
reduced the nitrate/nitrite levels by 77% because neutrophils due to which it activates ROS. NO
of its strong antioxidant properties. The NO is synthesized by iNOs it is a free radical and
levels significantly increased in the exudate of enhanced inflammation and interact with ROS
carrageenan-induced untreated group and plays to increase the activity of free radicals. As a
an important role in mediating inflammatory co nsequence d iff erent pro -in flammato ry
responses a variety of sources that increased cytokines IL-1b, TNF-a are released that
Table 1: Effect of A. corniculatum Extracts on Myeloperoxidase and

Nitrate/Nitrite Production
Treatment Dose Myeloperoxidase Nitrate/Nitrite
(mg/kg) (mU /ml) (µM)
Saline (0.95% NaCl) 43±12.8
Standard control 0.5 (Dexamethasone) 196±36.8 (58%) –
10 (Rutin) – 20±4.0 (77%)

Carrageenan (1%) 0.1 ml 464.5±98.8 86±21.6
010 220±21.6* (53%) 56±4.4 (35%)
Methanol extract 050 113±26.2** (76%) 34±4.3** (61%)
100 80±8.1*** (83%) 20±4.0*** (77%)

010 253±36.8* (47%) 28±2.1* (68%)
Ethyl acetate extract 050 200±40.8** (57%) 15±4.0** (83%)

100 80±8.16*** (83%) 7.3±2.0*** (92%)
Data represents mean ± S.E.M (n=6) of myeloperoxidase and nitrate/nitrite levels after treatment with carrageenan
(1% ,0.1 ml) alone or in the presence of different concentrations of standards (dexamethasone and rutin), methanol
and ethylacetate extracts.
The values within parenthesis represents percent reduction as compared to corresponding controls.
The significant values are shown with asterisks *p<0.05, **p<0.01, ***p<0.005.
57
activates the transcription factor NF-kB (Elena 02. Cuzzocrea, S., Sauteb in, L., De Sarro, G.,
et al., 2012). Considering, the aforementioned Costantino, G., Rombola, L., Mazzon, E., Ialenti,
A., De Sarro, A., Ciliberto, G., Di Rosa, M. (1999).
mechan ism of nitr ic o xide an d its pr o-
Role of IL-6 in the pleurisy and lung injury caused
inflammatory derivatives nitrates/nitrites in by carrageenan. J. Immunology. 163:5094-5104.
regulation of acute and chronic inflammatory 03. Dalmarco, E.M., Frode, T.S. and Medeiros, Y.S.
process, inhibitory effects of A. corniculatum (2002). Effects of methotrexate upon inflammatory
again st MPO and n itr ate/nitrite clearly parameters induced by carrageenan in the mouse
d emon str ates its po tential again st an ti- model of pleurisy. Med. Inflammation. 11:299-
306.
inflammatory response. 04. Dore, C.M.G., Azevedo, T.C., de Souza, M.C.,
The present investigation validated the Rego, L.A., de Dantas, J.C., Silva, F.R., Rocha,
traditional use of A. corniculatum against H. A. , Bas eia, I. G. , Leite, E.L. (2007).
inflammation and rheumatism. Our data Antiinflammatory, antioxidant and cyto toxic
actions of b-glucan-rich extract from Geastrum
confirms the inhibitory effect of A. corniculatum
saccatum mushroom. Int. Immunopharmacol.
upon bo th neutrophils and monon uclear 7:1160-1169.
infiltration in pleural cavity against carrageenan 05. Elena, T., Rosanna, D.P., Emanuela, M., Esposito,
challenge in association with a parallel decline E., Virginia, M., Salvatore, C. (2012). Anti-
of nitrate/nitrite concentrations and MPO inflammatory effects of adrenomedullin on acute
lung injury induced by Carrageenan in mice. Med.
enzyme levels. Thereby, revealing a new
Inflammation, 2012.
protective mechanism of A. coriculatum 06. Fröde, T.S., Medeiros, Y. (2001). Myeloperoxidase
extracts against acute lung inflammation. and adenosine-deaminase levels in the pleural fluid
leakage induced by carrageenan in the mouse model
Author’s Contribution of pleurisy. Med. Inflammation. 10:223-227.
07. Goldblum, S.E., Wu, K.-M., Jay, M. (1985). Lung
Sabahat Aziz has performed the bench
myeloperoxidase as a measure of pulmonary
work and manuscript writing. leukostasis in rabbits. J. Applied Physio. 59:1978-
*Talat Roome has supervised the entire 1985.
work and designed the experimental model. 08. Jafri, S.M.H., Qaiser, S. (1975). Myrsinaceae. In:
Perbhat Ali and Anam Razzak performed Nasir, (Eds.), Flora of West Pakistan, 89:1-8.
09. Jiang, Q., Ames, B.N. (2003). Gamma-tocopherol,
experimental work and participated in literature
but not alp ha-to cop herol, dec reas es pro-
surveys. inflammatory eicosanoids and inflammation damage
Muhammad Iqbal Choudhary supervised in rats. FASEB. 17:816-818.
the chemistry part of the project. 10. Prajapati, V.D., Maheriya, P.M., Jani, G.K.,
Solanki, H.K. (2014). Carrageenan: a natural
Conflict of Interest seaweed polysaccharide and its applications.
The authors affirm that there is no conflict Carbohydrate Polymers. 105:97-112.
11. Pithadia, A.B., Jain, S. (2011). Treatment of
of interest in this study and publication of this
inflammatory boweldisease (IBD). Pharmacological
manuscript. Reports. 63:629-642.
12. Roome, T., Dar, A., Ali, S., Naqvi, S., Choudhary,
5. REFERENCES M.I. (2008). A study on antioxidant, free radical
1. Cuzzocrea, S., Mazzon, E., Calabro, G., Dugo, L., scavenging, anti-inflammatory and hepatoprotective
De Sarro, A., Caputi, A.P. (2000). Inducible nitric actions of Aegiceras corniculatum (stem) extracts.
oxide synthase-knockout mice exhibit resistance to J. Ethnopharmacol. 118:514-521.
pleurisy and lung injury caused by carrageenan. 13. Roome, T., Dar, A., Naqvi, S., Ali, S., Choudhary,
Respiratory Medicine. 162:1859-1866. M.I. (2008). Aegiceras corniculatum extract
58
suppresses initial and late phases of inflammation Baskar, V. (2011). Antio xidant as says in
in rat paw and attenuates the production of pharmacological research. AJ Pharmacy. 1:99-103.
eicosanoids in rat neutrophils and human platelets. 16. Sautebin, L., Ialenti A., Ianaro A., Di Rosa M.
J. Ethno-pharmacol. 120:248-254. (1998). Relationship between nitric oxide and
14. Saldanha, A.A., de Siqueira, J.M., Castro, A.H.F., prostaglandins in carrageenan pleurisy. Biochem.
de Azambuja Ribeiro, R.I.M., de Oliveira, F.M., Pharmacol. 55:1113–1117.
de Oliveira Lopes, D., Pinto, F.C.H., Silva, D.B., 17. Vane, J.R., Botting, R.M. (1998). Anti-inflammatory
Soares, A.C. (2016). Anti-inflammatory effects of drugs and their mechanism of action. Inf. J. Eur.
the butanolic fraction of Byrsonima verbascifolia Hist. Res. Society. 47, Suppl. 2, 78-87.
leaves: Mechanisms involving inhibition of tumor 18. Willerson, J.T., Ridker, P.M. (2004). Inflammation
necrosis factor alpha, prostaglandin E 2 production as a cardiovascular risk factor. Circulation. 109:11-
and migration of polymorphonuclear leucocyte in 12.
vivo experimentation. Int. Immunopharma. 31:123- 19. Zhang, D., Wu, J., Zhang, S., Huang, J. (2005).
131. Oleanane triterpenes from Aegiceras corniculatum.
15. Selvakumar, K., Madhan, R., Srinivasan, G., Fitoterapia. 76:131-133.
59
Mini Review: Herbal Medicinal Products Regulation in Pakistan
Huma Shareef*, Ali Akbar Sial, Farya Zafar and Huma Ali
Faculty of Pharmacy, Ziauddin University, Karachi, Pakistan.
*Email: phr_huma@hotmail.com
Abstract 1. INTRODUCTION
Pakistan is gifted with a rich prosperity of Her bal medicines p ro du cts ar e th e
curative plants from All Mighty Allah. Herbs manufacture of remedial experiences, by the
have always been most important form of generations of local practitioners of native
medicine in Pakistani population and currently systems of medicine as a dietary supplements
plants have got popularity throughout the globe. derived from food plant, phytochemicals and
Standardization, quality control and import and pro-vitamins that now being described as
export of raw materials and herbal formulations f un ctio n al f oo d s, n utr iceu ticals an d
materialize the key dispute for herbal drug nutraceuticals or health foods etc. may support
man ufacturers w hich is due to the n on in maintaining good health and combating the
availability of sufficient regulatory strategy, different disease. Herbal medication are also in
principally for good manufacturing practice great insist in the developed world for primary
(GMP), no execution of good agricultural and health care due to their effectiveness, safety
co llection p ractices ( GACP), and w eak and fewer side effects, as well as providing
implementation of the Herbal product drug act treatment for th ose disorders which are
from the ministry of health of Pakistan. Now associated with age such as memory loss,
there ar e sev er al o p po rtun ities fo r th e osteoporosis, arthritis and immune disorders, etc.
development of quality of herbal products on due to the unavailability of modern medicine
the basis of data based studies which is essential and only palliative treatment is processed. This
for the establishment of the herbal products is lead to unexpected enlargement in the herbal
quality, safety and efficacy in the domestic and drug manufacturing and number of countries in
export market. Finally, strong Regulatory spite of its prosperous traditional knowledge,
implementation becomes now necessary to cu stoms of herb al medicines and lar ge
moderate the hindrance for the commercializa- biodiversity only has a large share of the world
tion of the herbal medicinal drug in Pakistan market due to export and import of crude
with the help of scientific research at Institutions, extracts and herbal drug products (Robinson
Universities and hi-tech advancement in the field and Zhang, 2011; WHO Geneva 2005 ). In
of herbal medicine and their related products. these circumstance consumers, professionals and
regulatory officials have struggled to change
Keywords: this scenario by gathering information about the
Herbal med icinal drugs, Quality control, quality, efficacy and safety of these products,
Standardization, Regulatory requirement, Global and dev eloped new guidelin es fo r th eir
scenario. registration in the regulatory framework as those
60
in the place of conventional pharmaceuticals product is “Herbal medicine product is containing

according to their habitat and their distribution the active ingredients from plant origin or/and
in the different localities. vegetative dru g pr eparations”. Th ey are
available in the many dosage forms as shown
1.1. World Paradigm of Herbal Medicine in Scheme 1. Dry extract available in pills/
Traditional medicines are categorized in capsules/lozenges/ lyophilized powders in the
many ways throughout the various jurisdictions market that was prepared by local Hakims or
around the world; a few of these are shown in well known companies and dawakhanas (Shaista
Fig. 1. World Health Organization (WHO) and Shahid, 2011) .
published a report with name of “Traditional
medicine strategy 2002-2005”, which support
the concept of a national policy on Traditional 1.4. Growth of
medicine/ Complementary and alternative Herbal Medicinal Product Market
medicine (TM/CAM). It has been exposed from The modern annual sales trend of herbal
that report, only 32% WHO member countries medicines, from nine countries including
had a national policy. Response of another Pakistan showed in Fig.3. The global market
survey, based on whether laws or regulations share or trade of traditional medicine was about
existed on TM/CAM, indicated that 65% 2500 species internationally and estimated at
countries have law or regulations on herbal US$83 billion annually in 2008, with an increase
medicine (WHO, 2002-2005). rate of sale per year and estimates suggest that
it will reach up to the US$ 5 trillion by 2050
1.2. Alternative Medicinal Trend in (Kamboj, 2000; Hoareau and DaSilva, 1999;
Pakistan Robinson and Zhang, 2011).
In Pakistan, 80% country’s population lives 12% of growing flora of Pakistan has been
in rural areas where they followed alternative used in medicines and more than 300 medicinal
system of medicines as a first line of treatment.
plants are traded. In 1990s, 2 million kg of 200
In 2009, a group of Government officials
medicinal plants were annually consumed by 10
conducted the survey regarding the trend of
principal Dawakhanas (Herbal manufacturers)
utilization of medicine in different health care
of Pakistan that was bigger in the last two
systems. Thus the assessment showed in Fig. 2
decades. According to calculated trade of
that revealed the trend of alternative therapies
medicinal plants approximately, 22 species of
in Pakistan.
From the graph we can easily revealed medicinal plants worth Rs.14.733 million while
that almost half of the population used TCAM in 2002, this value eight-and-a-half times
which is to be a sign of an increasing popularity increased to more than Rs.122 million. Whereas
of CAM in Pakistan and report also showed consumption of 95 species of medicinal plants
the common practice of combined use of in 1990, valued Rs. 36 million while in 2002,
biomedicine with TCAM (Shaikh et al., 2009). valued Rs. 218 million which indicates the 6-
fold increased in 12 years (Shinwari et al.,
1.3. Dosage Forms of 2002). The 2012 market value of herbal products
Herbal Medicinal Products in contrast with the conventional medicine is
Europe union definition of herbal medicinal 12% and now it is up to16%.
61
Fig. 1: Categories of regulation of herbal medicine in the world (WHO, 2002-2005)
TCAM: Traditional complementary alternative medicine

TCM: Traditional complementary medicine
Fig. 2: Trends of alternatives therapies in Pakistan (Shaikh et al., 2009)
Scheme 3: Different available dosage forms of herbal medicinal products
62
Fig. 3: Annual market sales of herbal medicines
1.5. Policy Concern in Relation to requirements. WHO published the various

Standardization and technical guide lines regarding the above
Regulation Globally mentioned issues such as:
From most recent decades there are
various policies available in the national and a) Good agricultural and collection practices
international level for the regulation of alternative
(GACP)
medicines. But the herbal drug and their products
b) Good manufacturing practice for the herbal
globally face the a variety of challenges like
medicine (GMP)
identification, recognition, consistent quality,
c) Guideline for the selection of the material
learning, confirmation based research, safety
for the quality control of herbal medicine
and efficacy, coherent use, herb-drug relations,
in 2004 and 2005
poor consideration of socio-cultural background
of their practice and handling, security of patent d) Monograph for various herbal drugs
rights of knowledge container, guarantee of e) Guidelines for the assessment of herbal
natural supply, guideline and facility of non- drug and quality control method of
formal practitioners, appropriate methodologies medicinal plant material
for evaluation and resolving conflicts. These all
factors can be erasing by the linkage of raw Through the utilization of above mentioned
material and finished herbal product in terms of guidelines we can meet the increased demand
quality control, standardization and regulatory of quality herbal medicinal products.
63
1.6. Herbal Drug Regulatory Status in 2. FUTURE PROSPECTIVE

Pakistan The importance of medicinal plants cannot
In Pakistan, Traditional/Complementary be diminished in any society of the world due
medicines are being practiced under folklore, to its ancient utilization. As we know that the
Unani, Ayurvedic and Homoeopathic (UAH) population of the world increases in both
Act of 1965. In the year 2003 Government developed and developing world through the
cabinet was approved amendments in the Unani, migration and birth rate and its gives the rising
Ayurvedic and Homeopathic Practitioners Act, interest in the industrialized nations to have
1965 as the Tibb-e-Unani/Eastern Medicine greatly expanded the claim of medicinal plants
Practitioners Act 1965 with a vision of officially themselves and their prepar ed produ cts.
registering Bachelors of Eastern Medicine and Therefore, in this scenario the regulatory
Surgery (BEMS) to practice Tibb. The Drugs authorities of the state have play a crucial role,
Control and Traditional Medicines Division of in terms of value of herbal medicines, financing
the National Institute of Health Islamabad serve and appreciating training and research in the
as the national institute on traditional medicine development of this field. For this instance,
and were established in 1991. Moreover, Ministry inclusion of some introductory modules of herbal
medicinal knowledge into the basic medical
of Health of Pakistan was established the expert
curriculum of allopathic medical schools
committee o n TM/CAM in 2001 for the
must be considered. Finally academicians,
development of national policy on Traditional
researchers, law & order regulators, herbal drug
medicines. The Drugs Act of 1962 only
producer, and prescribers must join their hands
considers the regulation of herbal medicines
together and could bring a new perspective of
advertising and prevention of its misuse. Later
outstanding revolution by reorganization and
the bill had been prepared in 2010 for the
redesign the herbal drugs. When all above
regulation of manufacturing, storage, imports
concerns are given their valuable inputs as
and export of Tibb-e-Unani, Ayurvedic,
equivalent, successful, economical and safe
Homoeopathic, Herbal and Non-allopathic
partners then it can be possible to face the
medicines (Shista and Shahid 2011). challenges of the 21st century for herbal
Currently Drug Regulatory Authority of drug.
Pakistan directives to regulate herbal medicines
according to the laws designed for allopathic
medicines and they provide for effective 3. REFERENCES
coordination and enforcement of the Drugs Act, 1. Hoareau L.and DaSilva E.J. (1999). Review,
Medicinal plants: a re-emerging health aid. EJB
1976 (XXXI o f 1976) that w ill br in g
Electronic Journal of Biotechnology. 2:2, Issue of
synchronization in inter-provincial buying and August 15.
selling commercialization of therapeutic goods 2. Kamboj V. P. (2000). Herbal medicine. Current
including herbal products as well (DRAP, Act, Science. 78(1):35-44.
2012). 3. Notification SRO No. (I) 2013 sec. 23 DRAP Act,
As an agricultural country Pakistan still 2012 (XXIof 2012) [homepage on the Internet]
4. National policy on traditio nal medicine and
need some strong policies to furnish this law at
regulation of herbal medicines Report of a WHO
the ground level that is from agricultural to the Global survey. WHO Geneva, 2005.
consumer hand. 5. Robinson M.M. and Zhang X. (2011). Traditional
64
medicines: global situation issues and challenges Medicine. May, 15(5):545-550.

the world medicines situation WHO, Geneva. 08. Shinwari, Z.K., Gilani, S.S. and Shoukat, M.
1-49. (2002). Ethnobotanical resources and implications
6. Shaista Kokab and Shahid Ahmad . (2011). for curriculum. In: Shinwari, Z.K., A. Hamilton
Op po rtunities and potentials for herb al and A.A. Khan (Eds.). Proceedings of Workshop
pharmaceutical products development in Pakistan. on C urriculu m Developm en t in App lied
Managing Natural Resources for Sustaining Future Ethnobotany. May, 2-4, Nathiagali, Abbotabad,
Agriculture, Natural Resources Division, Pakistan WWF-Pakistan. 21-34.
Agricultural Research Council, Islamabad, Pakistan. 09. World Health Organization Guidelines on good
Research Briefings. 19. agricultural and collection practices for medicinal
7. Shahzad H. Shaikh, Farnaz Malik, Henry James plants [homepage on the Internet]. Online
and Hamid Abdul. (2009). Trends in the use of document at:http://whqlibdoc.who.int/publications/
co mplementary and alternative med icine in 2003/9241546271.pdf .
Pakistan: a po pulatio n-b as ed survey. Th e 10. World Health Organization. Traditional Medicine
Journal of Alternative and Complementary Strategy 2002-2005. Geneva: WHO, 2001.
65
Obituary
Prof. Dr. M. Ataur Rahman

(1929-2015)
Inna lillahi wa inna ilayhi raji’un He also served as Professor of Bio-

chemistry and Dean of Faculty of Science,
“We surely belong to Allah and to Him we Hamdard University, Karachi.
shall return His contributions brought him many
Al-Baqarah, II:156”. national and international honours and awards,
including President of Pakistan’s Award
Hamdard Medicus is sad to report the
“Pride of Performance” and “Hilal-i-Imtiaz”
demise of Prof. Dr. Mohammad Ataur
by the Government of Pakistan. He received
Rahman (D. Sc) an eminent scientist and
one of the pioneer members of its Advisory Go ld Medal b y Pakistan Academy of
Board. He breathed his last in Karachi on Sciences for outstanding contributions in
Saturday, 26th September, 2015. May research.
Allah bless the departed soul with
eternal peace (Ameen). Hamdard Medicus
66
PUBLICATIONS
HAMDARD FOUNDATION PAKISTAN
Hamdard Foundation Pakistan also publishes two quarterly academic journals in

English.
Hamdard Islamicus, focusing on the problems faced by the Muslim world,

interaction and dialogue within and outside, historical studies and research.
Historicus: Journal of the Pakistan Historical Society, mostly dealing with South
Asian studies and history in general, especially of the SAARC countries.
These journals have an international academic clientele and circulation.
We have regular exchange programmes with most of the leading research journals
but wish to extend and improve these arrangements further to help in advancing academic
exchange and co-operation.
Both journals are abstracted and referred to in international indexes.
We invite learned bodies and organizations, having no exchange programmes with

us, to accept our offer to further the cause of research.
Please contact:
Hamdard Foundation Pakistan
Nazimabad, Karachi-74600, Pakistan
Telephone: 36616001-4 lines; Telefax: (92-21) 36611755
e-mails:
islamicus@hamdard.edu.pk
hfp@hamdardfoundation.org
phs@hamdard.edu.pk
Websites:
Hamdard Foundation Pakistan – www.hamdardfoundation.org
Hamdard Laboratories (Waqf) – www.hamdardlabswaqf.org
Idara-e-Said – www.hakimsaid.info
Published by Hamdard University, Madinat al-Hikmah, Shahrah-e-Madinat al-Hikmah,

Muhammad Bin Qasim Avenue, Karachi-74600, Pakistan.
Telephones: 92-21-36440035-40, Fax: 92-21-36440045
E-Mail: hamdardmedicus@hamdardfoundation.org; Websites: www.hamdard.edu.pk
P rice
Inland Pak.Rs. Foreign US$
Annual 750/- 150/-
Single Copy 200/- 040/-
(inclusive of airmail charges)
Printed by MAS Printers, Nazimabad, Karachi-74600, Pakistan.

Medicus No.1'2016

Uploaded by

Copyright:

Available Formats

Medicus No.1'2016

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Medicus No.1'2016

Uploaded by

Copyright:

Available Formats

Hamdard Medicus Vol. 59, No.

Patron-in-Chief: Mrs. Sadia Rashid

Editor: Prof. Dr. Ghazala Hafeez Rizwani

Managing Editor: Prof. Dr. Ahsana Dar Farooq

Advisory Board Members

Prof. Dr. Atta-ur-Rahman Prof. Dr. Hakim Zillur Rahman

Prof. Jurgen H. Hohnholz Prof. Dr. M. Iqbal Choudhary

Editorial Board Members

Prof. Dr. Hakim Shahabuddin Dr. Shagufta Usmani

1) Clinical Efficacy and Safety of Cupping Therapy

2) Cuscuta reflexa L. & Roxb.: An Implication of Quercetin Mediated

3) Anti-bacterial Activity and Qualitative Phytochemical Screening of

4) Unani Treatment of Tinea Capitis: A Case Study

5) Phytochemical and Dermatological Investigations of Tribulus terrestris L.

6) Inhibitory Effect of Aegiceras corniculatum Against Lung Injury in

7) Mini Review: Herbal Medicinal Products Regulation in Pakistan

HAMDARD MEDICUS is indexed in:

Clinical Efficacy and Safety of Cupping Therapy

Tasneem Qureshi* and Abdul Hannan

Hamdard Al-Majeed College of Eastern Medicine,

Abstract depression. The relief from symptoms depended

2 MATERIALS AND METHODS 2.4. Instructions for the Patients

Scarificat ion: Using surgical blades

i) Blood letting: Immediately, the cup was

The cupping therapy consisted for 3-7

3. RESULTS AND DISCUSSION

Chronic Constipation 05 Mild = 2 1, 55, 121, 6,7, 8 Y = 90 Mild = 0

Sciatica 03 Mild = 0 1, 55, 11, 12, 14, 16, Y = 100 Mild = 0

Migraine 06 Mild = 3 1, 55, 20, 21, 23, Y = 90 Mild = 0

Knee Joint pain/arthritis 16 Mild = 5 1, 46, 48, 7, 8, 11, Y = 80 Mild = 10 (Melancholic)

spasm/cervical pain Moderate = 13 12, 13 N = 10 Moderate = 5 (Phlegmatic)

Cuscuta reflexa L. & Roxb.: An Implication of Quercetin Mediated Monoamine

Abstract antidepressant and this effect can be attributed

clinically used antidepressants (SSRIs) possess Care Co mmittee of th e CII T that is in

2. MATERIALS AND METHODS

Table 1: Effect of C. reflexa Extract on the Immobility Time of

Phenelzine 30 90±11*** 30 139±8*

Fluoxetine 30 56±8*** 30 201±11

The values represent the mean immobility time ± S.E.M (n = 5).

Table 2: Effect of C. reflexa Extract on 5-HTP Induced Head Twitches and

5-HTP induced head twitches Yohimbine induced lethality

Treatment Do se Number of Treatment Do se Mortality

5-HTP 200 29 ± 5 Yohimbine 40 35

Fluoxetine + 5-HTP 30+200 100 ± 11*** Phenelzine+

C. reflexa + 5-HTP 50+200 79 ±6*** C. reflexa+

0.156 mg of quercetin (retentio n time = Author’s Contribution and Declartion

Fig. 2: Identification of quercetin in Cuscuta reflexa methanolic extract using HPLC-DAD

4. REFERENCES of Cuscuta reflexa Roxb. J. Chin. Integr. Med.

Anti-bacterial Activity and Qualitative Phytochemical Screening of

Kashaf Zia,1 Muhammad Qasim Hayat,2 Shaziad Anjum,1* Muhammad Ashraf,2

Abstract be associated with antibacterial activity. On

however, information regarding its minimal Staphylococcus aureus, S. aureus ATCC,

2.4. Antibacterial Screening

2.5. Minimum Inhibitory Concentration Steriods

Test agents µg/ml M. S. S. aureus S. S.

S. saprophyticus growth inhibition (10.7 mm). (Acinobacter junii, Bordetella bronchiseptica,

Table 2: Minimum Inhibitory Concentration of F. cretica Methanolic Extract and

Bacterial strains Extract/Fractions µg/ml

100 80±8.1* (83%) 20±4.0* (77%)

Ethyl acetate extract 050 200±40.8 (57%) 15±4.0 (83%)