Pharmacological Studies for Investigation Of

“PHARMACOLOGICAL STUDIES FOR INVESTIGATION OF

HEPATOPROTECTIVE ACTIVITY OF EXTRACT OF BERBERIS ARISTATA fruit IN RATS”.

M.Pharm Dissertation Protocol

Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, BangalorE

SYED HASHIM ALI

Dr. KONDURI PRASAD

M.Pharm., Ph.D.

Department of Pharmacology

H.K.E. Society’s College of Pharmacy, GULBARGA-585 105

2009-10

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 HASHIM ALI
H.NO-5-408/5C/4, MADINA COLONY, ROZA-(B), GULBARGA-585 104 (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 / 1/06/2008
5. / Title of the Research topic / PHARMACOLOGICAL STUDIES FOR INVESTIGATION OF HEPATOPROCTECTIVE ACTIVITY OF EXTRACT OF BERBERIS ARISTATA FRUIT 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 Hashim Ali)
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. KONDURI PRASAD, Professor
M.pharm. Ph.D
NO
------
Dr. Konduri Prasad
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 requiring homeostasis in the body.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 metabolites are always associated with the disturbance of hepatocytes results in generation of Reactive Oxygen Species (ROS) 2. Lots of 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 which proved to have hepatoprotective potential. Silymarin, a flavonoid ligan mixture extracted from silybum marianum (milk thistle) is a popular remedy for hepatic diseases. Today every herbal company is marketing formulations for liver disorders but the actual scene is that only selected medicinal herbs have been tested for hepatoprotective activity. Therefore, it is necessary to test the hepatoprotective and antioxidant activities of selected medicinal plants such as Berberis aristata. The fruit extract of Berberis aristata has been reported as liver protecting activity.

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 the indicates no similar reports.The following is a brief report

of literatures.

Literature surveys indicates Berberis aristata, Family - Berberidaceae, grows abundantly in Himalayas (India and Nepal). It is used in skin diseases, menorrhagia, diarrhoea, infections of eyes.

It is also used in painful micturation from bilius or acrid urine, is useful in application of aphthous ulcer, to patients of malarial fever7.

Literature survey reports following activities of Berberis aristata.

 Anti-diabetic activity8.

 Antihyperglycemic activity9.

 Anti-carcinogenic10.

 Immunomodulatory11.

 Normalises or changes in the rate of respiration12.

 antipyretic, antiprotozoal, antitumour and anti-inflammatory activities13.

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 the plant Berberis aristata fruit extract. Hepatoprotective 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 Berberis aristata fruit extract on 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 plant material will be procured from commercial reputed ayurvedic drug 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

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

Phase 2: To study the effect of Berberis aristata leaf extract in the above animals.

Phase 3: To study the antioxidant activity of Berberis aristata leaf 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 Sampling

1) 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

Ward FM, Daly MJ, Hepatc disease in Walker R, Ed Wards C. Editors, Clinical Pharmacy and therapeutics, churchil Livingstone, New York; 1999, Page no.195-212.
Fernendez – checa, Kaplowitz N. Hepatic mitochondrial glutathione: transport and role in disease and toxicity, Toxicol Appl Pharm. 2005; 204:263-273.
Vrbz J, Modriansky M. Oxidative burst of kupffer cells; Target for liver injury treatments. Biomed pap. 2002; 146:15-20.
Friedman, Scott E, Grendell, James H, Mc Quaid, Kenneth R. Current diagnosis and treatment in gastroentrology. New York. Lang Medical Books/Mc Graw-Hill, 2003;

664-679.

Shah RR. Drug induced hepatotoxicity: Pharmacokinetic perspectives and strategies for risk reduction. Adverse drug reactions and toxicological reviews 1999; 18(4):181-233.
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.
Kirtikar and Basu, Indian medicinal plants, Volume 1, Page no.102-03.
Kamal Shah, Nagendra Singh Chauhan, Bhupesh Chander Semwal, Rohit Badhe, Kalyani Divakar. Antidiabetic activity of stem bark of B.aristata D.C in alloxan diabetic rats, Internet Journal of Pharmacolgy 2008.
Jitendra Gupta1, Sonia Singh1, Yogesh Kumar2. Antihyperglycemic activity of root of B. aristata D.C. in alloxan-induced diabetic rats, International journal of Green pharmacy 2009,Vol-3, page no-259-62.
Anis KV et al, Evaluation of chemical carcinogenis by berberine in rats and mice, J Pharm Pharmacol 2001, May; 53(5):763-8.
Sohni YR and Bhatt RM. Activity of a crude extract formulation in experimental hepatic amoebiasis and in immunomodulation studies, J Ethnopharmacol 1996,

Nov; 54(2-3); 119-24.

Sebastiant1, Setty OH. Protective effect of B.aristata ethanol-induced mitochondrial damage in rats, Journal of clinical biochemistry and nutrition, ISSN 0912-2009.Vol-23.pp.1-13.
Sharma PC et al, Database on medicinal plants used in Ayurveda, Central council for Research in Ayurveda and Siddha, New Delhi, pp.291-92.
Anwar_Ul Hassan Gilani, Khalid Hussain Janbaz, Preventive and curative Of B.aristata fruit extract and CCl4 - induced Hepatotoxicity, Indian journal of Pharmacy 2006.

Application for Permission for animal experiments

Application 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) / cENTRAL ANIMAL HOUSE
M.R.MEDICAL College, sedam road
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. / 01.06.2010 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. KONDURI PRASAD

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 HASHIM ALI
H.NO-5-408/5C/4, MADINA COLONY, ROZA-(B), GULBARGA-585 105 (KARNATAKA)
2 / Area of Research / Pharmacology
3 / List of Experiments Carried out / PHARMACOLOGICAL STUDIES FOR INVESTIGATION OF HEPATOPROTECTIVE ACTIVITY OF EXTRACT BERBERIS ARISTATA FRUIT IN 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) / cENTRAL ANIMAL HOUSE
M.R.MEDICAL College, SEDAM road
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 HEPATOPROTECTIVE ACTIVITY OF EXTRACT BERBERIS ARISTATA FRUIT IN 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 HEPATOPROTECTIVE ACTIVITY OF EXTRACT BERBERIS ARISTATA FRUIT IN RATS
2 / Chief Investigator :

Name :

Designation :
Dept/Div/Lab :

Telephone number :

/ Dr. KONDURI PRASAD
Professor
Dept. Of Pharmacology
H.K.E.S’s College of Pharmacy, Sedam Road GULBARGA - 585 105 kARNATAKA
09844368785 (M)
3 / List of names of all individuals authorized to conduct procedures under this proposal. / Dr. Konduri Prasad
Dr. Nitin Mahurkar
Mr. Syed Hashim Ali
4 / Funding source / H.K.E.S’s. college of pharmacy,
GULBARGA - 585 105.
5 / Duration of the project :

Number of months :
Date of initiation :
Date of completion :

/ 10 months 01.06.2010 31.03.2011
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

Species
Age/weight/size
Gender
Number to be used
Number of days each animal will be housed

/ Albino Rats Adult Rats – 180-220 gm Either Sex
Rats 80 45 Days
9 / Rationale for animals usage :

Why is animal necessary for these studies?

Why are the particular species selected?

Why is the estimated number of animals essential?

Similar experiments conducted in the past. If so, number animals used and results obtained in brief.

If yes, why new experiment I required?

Have similar experiment (s) been made by any other organization/agency? If so, their result in your knowledge.

/ To find out a new safe and effective drug to treat liver disorders.
These species are suitable for screening hepatoprotective agents as they possess the complications that are comparable to disorders of human beings.
To meet minimal statistical requirements
- No. -
- No. -
- No. -
10 / Does the protocol prohibit use of anesthetic or analgesic for the conduct of painful procedures? / - No. -
11 / Will survival surgery be done? / - No. -
12 / Methods of disposal of post experimental Animals / Similar to Sl. No. 8 as in 1st page
13 / Animal transportation methods if extra institutional transport is envisaged. / - No. -
14 / Use of hazardous agents / - No. –

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:Signature

Place:

Name and designation of Chief Investigator

Dr. KONDURI PRASAD

M.pharm., PhD

Professor

H.K.E.S’s College of Pharmacy,

Sedam Road GULBARGA.

karnataka- 585 105

name / signature
Dr. KONDURI PRASAD, Professor (Guide)
Mr. Syed Hashim Ali, Student

(For iaec/cpcsea Usage)

Proposal Number: HKE COP / IAEC / 18 / 2009-10

Date first received: 25/11/2009

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

Approval Date: 1/12/2009

Expiry Date: 31/03/2011

Name of IAEC/CPCSEA Chair person: Dr. S. Appala Raju.

Principle,

H.K.E. Society’s College of Pharmacy,

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/18/2009-2010