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“PHARMACOLOGICAL STUDIES FOR INVESTIGATION OF HEPATOPROTECTIVE ACTIVITY OF EXTRACT OF VIGNA

MUNGO SEEDS IN RATS”

M.PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

BY

SYED IFTHEKARUDDIN QUADRI

UNDER THE GUIDANCE OFDR. NITIN MAHURKAR

M.Pharm., Ph.D.

DEPARTMENT OF PHARMACOLOGYH.K.E. SOCIETY’S COLLEGE OF PHARMACY,

GULBARGA-585 1052010-11

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and

Address (in block letters)

SYED IFTHEKARUDDIN QUADRI

H.NO. 9-588/12/B1/1, SHAIK ROZA,

GULBARGA-585 101 (KARNATAKA)

2. Name of the Institution H.K.E. Society’s College of Pharmacy,

Sedam Road, GULBARGA - 585 105

3. Course of the study

Branch

M. Pharm.

Pharmacology

4. Date of Admission to Course 07/07/2010

5. Title of the Research topic PHARMACOLOGICAL STUDIES FOR

INVESTIGATION OF

HEPATOPROCTECTIVE ACTIVITY OF

EXTRACT OF VIGNA MUNGO SEEDS IN

RATS

6. Brief Resume Of Intended Work

6.1 Need for the study Enclosure-I

6.2 Review of Literature Enclosure-II

6.3 Objective of the study Enclosure-III

7. Materials and Methods

7.1 Source of data Enclosure-IV

7.2 Methods of collection of data Enclosure-V

7.3 Does the study require any

Investigation on animals?

If yes give details

Enclosure-VI

7.4 Has the ethical clearance been

obtained from your institution in

case of 7.3

Yes, Registration number: 142/1999

CPCSEA.5th July 1999.

8. List of References Enclosure-VII

9. Signature of the candidate

(Syed Ifthekaruddin Quadri)

10. Remarks of the guide Enclosure –VIII

11. Name and Designation of

(in Block Letters)

11.1 Guide

11.2 Signature

11.3 Co-Guide (if any)

11.4 Signature

11.5 Head of the department

11.6 Signature

Dr. Nitin Mahurkar

Professor

M.PHARM.,Ph.D

NO

---------

Dr. Nitin Mahurkar

Professor

M.Pharm.,Ph.D

12. Remarks of the Principal

12.1 Signature

The present study was permitted for

executing in the institution and IAEC

permission has been granted.

(Dr. S. Appala Raju)

Enclosure-I

6. Brief Resume on the Intended Work

6.1 Need of the Study:

The liver is the key organ of the body playing major role in maintaining homeostasis. It is involved

with almost all the biochemical pathways related to growth, fight against disease, nutrient supply, energy

provision and reproduction1. Hepatoprotection is the ability to prevent damage to the liver. Hepatotoxicity

implies chemical-drive liver damage.

Liver is the main organ involved in the metabolism of biological toxins and medicinal agents. Such

agents are always associated with the disturbance of hepatocytes resulting in generation of Reactive Oxygen

Species (ROS) 2. Liver damage ranging from subclinical icteric (Jaundice) hepatitis to necro-inflammatory

hepatitis, cirrhosis, and carcinoma has been proved to associate with the redox imbalance and Oxidative

Stress (OS) 3.

More than 900 drugs have been implicated in causing liver injury4, and it is the most common reason for the

drugs to be withdrawn from the market, such as troglitazone, bromfenac, rovafloxacin, ebrotibine,

nefazodone, and ximedagatran5.Injury to hepatocyte and bile duct cells lead to accumulation of bile acid

inside liver. This promotes further liver damage6.

Medicinal herbs are significant source of drugs. Latest trend has shown increasing demand of

phytodrugs and some medicinal herbs have hepatoprotective potential. Silymarin, a flavonoid ligand mixture

extracted from Silybum marianum (milk thistle) is a popular remedy for hepatic diseases. Therefore, it is

proposed to test the hepatoprotective and antioxidant activity of Vigna mungo.

Enclosure-II

6.2 Review of literature

An extensive literature review has been carried out to find whether similar work has been reported elsewhere and indicates no similar reports. The following is a brief reportof literatures. Literature surveys indicates, Vigna mungo Family - Fabaceae, grows 90% in South Asia and

adjacent regions (India, Pakistan, Afghanistan, Bangladesh and Myanmar)

It is used as excellent sex tonic, as nervine tonic, used in hair disorders, digestive system disorders like

dysentery and diarrhea, and also used in rheumatism.

Vigna mungo L.(Fabaceae) was reported to be used in a variety of disease conditions of liver in Indian

traditional system of medicine7.

Literature survey reports following activities of Vigna mungo.

Antidiabetic activity8.

Antifungal activity9.

Antioxidant10.

Immunostimulatory activity11.

Hepatoprotective activity12.

Enclosure-III

6.3 Objective of Study

Hepatic disorders pose a great difficulty in the treatment especially the hepatitis, jaundice, necrosis, cirrhosis

etc.

To evaluate the hepatoprotective activity of seeds of Vigna mungo. Hepatoprotective activity is the ability to

prevent damage to the liver. The liver plays a central role in transforming and cleaning chemicals and is

susceptible to the toxicity from these agents, which results in many liver diseases.

Therefore, in the present study, the hepatoprotective activity of extract of Vigna mungo seeds in

hepatotoxicity induced by the models like ethanol, rifampicin will be evaluated.

Enclosure-IV

7.1 Source and Data

Journals and publication of pharmacognosy and pharmacology.

Electronic data like CD-ROM and internet.

Helinet, Delnet.

Medicinal and aromatic plants Association.

National institute of science communication and information resources.

The normal adult rats (200-250 g) of either sex will be procured from central animal house M. R.

medical college, Gulbarga.

Rifampicin pure sample will be collected from manufactures.

Ethanol will be procured from bonded laboratory of H.K.E’S College of Pharmacy, Gulbarga.

The seeds will be procured from commercial suppliers.

Diagnostic kits will be purchased from local suppliers.

Standard drug silymarin would be procured from reputed manufacturer.

Enclosure –V

7.2 Method of Collection of Data

The study is planned on normal rats and work is designed in following phases.

Phase 1: In this phase it is proposed to induce liver damage by rifampicin and alcohol and

assess the same by measuring enzyme levels in normal adult albino rats of either sex.

Phase 2: To study the effect of Vigna mungo seeds extract in the above animals.

Phase 3: To study the antioxidant activity of Vigna mungo seeds extract in normal adult

Albino rats of either sex.

Inclusion Criteria

1) Normal Wistar albino rats of either sex weighing between 180-220 gms.

Exclusion Criteria

1) The albino rats which do not fall the above mentioned weight are excluded from study.

2) The animals where the enzyme levels (SGOT, SGPT, ALP and Total Bilirubin) are not in normal

range.

Study Sampling1) Blood sampling is done from retro orbital sinus.

2) Enzyme levels are measured at different interval of time.

3) Statistical significance is calculated by student t- test.

4) The total duration will be approximately 10 months.

Enclosure –VI

7.3 Does the study require any investigation on animals? If so, please prescribe briefly

Yes, the above study requires investigation on animals that is albino rats for hepatoprotective

activity.

7.4 Has the ethical clearance has been obtained from your institution in case of 7.3

Yes, the study is cleared from institutional animal ethics committee (IAEC Certificate enclosed).

Enclosure-VII

List of References

1. Ward FM, Daly MJ, Hepatic disease in Walker R, Ed Wards C. Editors, Clinical Pharmacy and

therapeutics, churchil Livingstone, New York.; 1999, 195-212.

2. Fernendez – checa, Kaplowitz N. Hepatic mitochondrial glutathione: transport and role in disease

and toxicity, Toxicol Appl Pharm.; 2005, 204:263-273.

3. Vrbz J, Modriansky M. Oxidative burst of kupffer cells; Target for liver injury treatments. Biomed

pap.; 2002, 146:15-20.

4. Friedman, Scott E, Grendell, James H, Quaid Mc, Kenneth R. Current diagnosis and treatment in

gastroentrology. New York. Lang Medical Books/Mc Graw-Hill.;

2003, 664-679.

5. Shah RR. Drug induced hepatotoxicity: Pharmacokinetic perspectives and strategies for risk

reduction. Adverse drug reactions and toxicological reviews.; 1999, 18(4):181-233.

6. Patel T, Roberts LR, Jones BA, Gores GJ. Dysregulation of apoptosis as a mechanism of liver

disease: an overview. Semin. Liver Dis.; 1998, 18(2):105-14.

7. Anonymous 2003. The Ayurvedic Formulary of India, Part-I. Government of India, Ministry Of

Health and Family Welfare. Department Of Indian Systems of Medicine and Homeopathy, New

Delhi.

8. Mushtak Ahmed, Rahmatullah Qureshi, Muhammad Arshad. Traditional Herbal Remedies Used for

treatment of Diabetes. Pak.J.Bot.; 2009, 41(6):2777-2782.

9. Hafidh Rand R, Abdulamir AS, Law Se Vern, Fatimah Abu Bakar. Inhibition of Human Pathogens.

American Journal of Biochemistry and Biotechnology.; 2010, 6 (1): 40-46.

10. Ashfaq M, Aslam Khan M, Javed N. Effect of UrdBean Leaf Crinkle Virus Infection on Total

Soluble Protein and Antioxidant Enzymes In Black gram Plants. Pak.J.Bot.; 2010, 42(1):447-454.

11. Solanki YB, and Jain SM. Immunostimulatory activities of Vigna mungo L. extract in male

Sprague-Dawley rats. J. Immunotoxicol, Doi.; 2010, 10.3109/15476911003792278.

12. Solanki YB, and Jain SM. Hepatoprotective Effects of Clitoria ternatea and Vigna mungo against

Acetaminophen and Carbon tetrachloride-induced Hepatotoxicity in Rats. Journal of Pharmacology

and Toxicology.; 2011, 6 (1):30-48.

Application for Permission for animal experimentsAPPLICATION TO BE SUMITTED TO SEND EITHER TO THE CPCSEA (ADDRESS IN FORM A

ABOVE) OR INSTITUTIONAL ANIMAL ETHIC COMMITTEE (IAEC)

part A

1 Name and Address of Establishment H.K.E.S’S COLLEGE OF PHARMACY,

SEDAM ROAD, GULBARGA.

KARNATAKA- 585 105

2 Registration Number and Date of

registration.

142/1999 CPCSEA. 5th JULY 1999

3 Name, address and Registration number of

breeder from whom animal acquired (or to

be acquired) for Experiments mentioned in

part B and C.

CENTRAL ANIMAL HOUSE

M.R.MEDICAL COLLEGE, SEDAM ROAD

GULBARGA-585 105 KARNATAKA

142/1999 CPCSEA. 5TH JULY 1999

4 Place where the animals are presently kept

(or proposed to be kept)



GULBARGA-585 105 KARNATAKA

5 Place where experiment is to be performed PHARMACOLOGY & TOXICOLOGY

LABORATORY. POST GRADUATE

DEPARTMENT. H.K.E.S’S COLLEGE OF

PHARMACY-GULBARGA

6 Date on which the experiments is to

commence and duration of Experiment.

01.06.2011

10 MONTHS

(THE APPROPRIATE PROTOCOL FORM FOR THE RESEARCH PROPOSAL PART-B IN THE CASE

EXPERIMENTS USING ANIMALS OTHER THAN ON HUMAN PRIMATES, PART-C FOR USE OF

NON-HUMAN PRIMATES TO BE DULY FILLED IN SIGNED AND ATTACHED TO THIS FORM).

Date:

SIGNATURE

Place:

Name and designation of Chief Investigator

Dr. Nitin Mahurkar

M.PHARM., PhD.

Professor.

H.K.E.S’S COLLEGE OF PHARMACY,

SEDAM ROAD, GULBARGA.

KARNATAKA- 585 105

ANIMAL EXPERIMENT FOR RESEARCH PURPOSE

1 Name and Address SYED IFTHEKARUDDIN QUADRI

H.NO. 9-588/12/B1/1, SHAIK ROZA,

GULBARGA-585 101 (KARNATAKA)

2 Area of Research PHARMACOLOGY

3 List of Experiments Carried out PHARMACOLOGICAL STUDIES FOR

INVESTIGATION OF

HEPATOPROTECTIVE ACTIVITY OF


RATS

4 No. of animals used annually (species-wise) 80 Rats

5 Duration of the Project 10 Months

6 Source of Experimental animals (In-house

Breeding/other Institution/import/others)



GULBARGA-585 105. KARNATAKA

7 No. of Animals to be sacrificed No sacrifice

8 Method of euthanasia and disposal The Euthanasia will be done under mild ether

anesthesia in rats when needed.

9 Whether a Veterinary doctor is employed YES

10 Housing details (Dimension/No. of animals) 254 Sq.m.

11 No. of animals rehabilitated ----

12 In vitro research ----

13 Scientific findings (update when project is

over)

PHARMACOLOGICAL STUDIES FOR

INVESTIGATION OF



RATS

Kindly fill in the appropriate from-research / Educational details to be precise.

Signature of the IAEC Chairman,

PART – B

Protocol forms for research proposals to be submitted to the committee / Institutional Animal Ethics

Committee, for new experiments or extensions of ongoing experiments using animals other non-

primates.

1 Project Title PHARMACOLOGICAL STUDIES FOR

INVESTIGATION OF


EXTRACT OF VIGNA MUNGO SEEDS

IN RATS

2 Chief Investigator :

a. Name :

b. Designation :

c. Dept/Div/Lab :

d. Telephone number :

Dr. NITIN MAHURKAR

Professor

Dept. Of Pharmacology


SEDAM ROAD GULBARGA - 585 105

KARNATAKA

08472-221392 (O)

08472-241926 (R)

9945835706 (M)

3 List of names of all individuals

authorized to conduct procedures under

this proposal.

Dr. Nitin Mahurkar

Mr. Syed Ifthekaruddin Quadri

4 Funding source H.K.E.S’S. college of pharmacy,

GULBARGA - 585 105.

5 Duration of the project :

a. Number of months :

b. Date of initiation :

c. Date of completion :

10 months

01.06.2011

30.04.2012

6 If day by which approval is needed is less

than six weeks from date of submission.

Justification for the same.

Doesn’t arise.

7 Study objectives (the aims of study and

why they are important) to be explained

non-technical terms as for as possible.

TO EVALUATE A SAFE AND NEW

HEPATOPROTECTIVE AGENT.

USEFUL IN TREATING CONDITIONS

LIKE JAUNDICE, HEPATITIS ETC.

8 Animals required

a. Species

b. Age/weight/size

Albino Rats

Adult Rats – 180-220 gm

Investigator’s declaration

1) I certify that I have determined that the research proposal here in is not unnecessarily duplicative of

previously reported research.

2) I certify that all individuals working on this proposal and experimenting on the animals have been

trained in animal handling procedures.

3) For procedure listed under item 11, have reviewed the pertinent scientific literature and have found

no valid alternative to any procedure described here in which may cause less pain or distress.

4) I will obtain approval from the IAEC/CPCSEA before initiating any significant changes in study.

5) Certified that performance of experiment will be initiated only upon review and approval of

scientific intent by appropriate expert body (to be named).

6) Institutional bio-safety committees (IBC) Certification of review and concurrence will be taken

(required for studies utilizing DNA Agent of human pathogens.

7) I shall maintain all the records as per format (form-D).

Date:

Place:

Signature

Name and designation of Chief Investigator

Dr.NITIN MAHURKAR

M.PHARM., Ph.D

Professor


SEDAM ROAD GULBARGA-585105

KARNATAKA -

.

(For IAEC/CPCSEA Usage)

Proposal Number: HKE COP / IAEC / 32 / 2010-2011.

Date first received: 25/11/2010.

Date received after modification (if any): ----

Approval Date: 6/12/2010.

Expiry Date: 30/04/2012.

Name of IAEC/CPCSEA Chair person: Dr. S. Appala Raju. Principal, H.K.E. Society’s College of Pharmacy,

NAME SIGNATURE

Dr. NITIN MAHURKAR, Professor

(Guide)

Mr. Syed Ifthekaruddin Quadri,

Student

Gulbarga.

Enclosure-VIII

Remark of the guide

The above work is genuine and original and is feasible in our laboratory, hence recommended

for favour of consideration.

HKE COP/IAEC/32/2010-2011.

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