Rajiv Gandhi University of Health Sciences s10

ANTIDIABETIC ACTIVITY OF RHYNCHOSIA BEDDOMEI BAKER ON ALLOXAN INDUCED DIABETIC RATS

Protocol of Dissertation Submitted

Mr. BAHUBALI

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

Under the guidance of

Dr. NANU. R. RATHOD Asst. Professor

Department of Pharmacology, HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY, BAGALKOT- 587101, KARNATAKA. (2010-2011)

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. Name of the Candidate and Address Mr. BAHUBALI DEPARTMENT OF PHARMACOLOGY H.S.K.COLLEGE OF PHARMACY B.V.V.S. CAMPUS BAGALKOT-587101, KARNATAKA. 2. Name of the Institution H.S.K.COLLEGE OF PHARMACY B.V.V.S CAMPUS BAGALKOT-587101, KARNATAKA.

3. Course of Study and Subject MASTER OF PHARMACY IN PHARMACOLOGY

4. Date of admission to Course 06th July -2010

5. Title of the topic: “Antidiabetic activity of Rhynchosia beddomei baker on Alloxan

induced Diabetic rats’’

2 6. Brief resume of the intended work

6.1 Need for the study:

Diabetes is a condition in which the body either does not produce enough, or does not

properly respond to, insulin a hormone produced in the pancreas1. Diabetes is the world’s

largest endocrine disease with deranged carbohydrate, fats and protein metabolism. As per

WHO report, approximately 150 million people have diabetes mellitus world wide, and

this number may well double by the year 2025. Statistical projection suggests that the

number of diabetics will rise from 15 million in the year 1995 to 57 million in 2025,

making India apart the country with the highest number of diabetics in the world.

Although many drugs and interventions are available to manage diabetics, these are

expensive for a developing country like India apart from their inherent adverse effects.

Therefore, it is necessary to look for new avenues to manage this major health problem.

As part of the pathogenesis of Type II diabetes mellitus, skeletal muscle, liver and adipose

tissues become resistance to the hormonal effect of insulin, which in turn leads to

decreased insulin-mediated glucose disposal, hepatic glucose overproduction and a

marked increase in lipolysis. An addition to the above, hyperinsulinemia is a central

pathophysiological feature of Type II diabetes mellitus and has been shown to play a key

role in the disease evaluation and macrovascular complication. The plants kingdom has

become a target for the search by multinational drugs and biological active compound.

Ethnobotanical information indicates that more than 800 plants are used as traditional

remedies for the treatment of diabetes2.

Hence, the present study was under-taken to explore Antidiabetic activity of

Rhynchosia beddomei baker of different extracts on normal and alloxan induced diabetic

rats. The leaves are collected from widely growing plants in the region of Tirumala

3 Chittoor District, Andhra Pradesh, India3.

6.2 Review of the Literature:

Plant profile:

Title of plant : Rhynchosia beddomei baker.

Family : Fabaceae,

Synonyms : Vendiaku, vendu chettu.

Parts used : Leaves.

Habitat : India.

Chemical constituents: The plant is a rich source of Alkaloids, Anthracene glycosides,

Anthraquinones and Caratinoids, Coumarins, Flavonoids, Gallic tannins, Saponins

Triterpenoids, Volatile oils are reported in the Leaf extract4, 5.

Medicinal action and uses: The leaves of the plant are used for Diabetic mellitus

Hepatoprotective, Antibacterial, Abortifacient, Antifungal and Wounds, Cuts, Rheumatic

Pain by Adivasi Tribes inhabiting the forests of Eastern Ghats of Andhra Pradesh, India3,5.

Pharmacological actions: The Antimicrobial activity6.

6.3 Objective of the study:

Traditionally well known Rhynchosia beddomie baker, extract will be used for the

present study.

1. Evaluation of hypoglycemic activity in rats.

2. Antidiabetic activity in alloxan induced diabetic rats study.

Material and methods:

7. 7.1 Source of data:

a) Literature survey, books, Website- Pubmed, Medline, Scidirect, Scirus, etc.

4 b) Publications of Journal. c) Lab based studies.

7.2 Experimental animal groups:

Evaluation of hypoglycemic activity of extract in rat :

Group I - Serve as control receives 0.5% Tween 80

Group II - Low dose of chloroform extract

Group III - Medium dose of chloroform extract

Group IV - High dose of chloroform extract

Group V - Low dose of Ethyl acetate extract

Group VI - Medium dose of Ethyl acetate extract

Group VII - High dose of Ethyl acetate extract

Antidiabetic activity in alloxan induced diabetic rats:

Group I - Serve as control receives 0.5% Tween 80 ( Alloxan 140 mg/kg)

Group II - Glibenclamide (10 mg/kg)

Group III - Low dose of chloroform extract

Group IV - Medium dose of chloroform extract

Group V - High dose of chloroform extract

Group VI - Low dose of Ethyl acetate extract

Group VII - Medium dose of Ethyl acetate extract

Group VIII - High dose of Ethyl acetate extract

7.3 Materials:

Drug : Rhynchosia beddomei baker.

Parts used : Leaves.

5 Animals : Rats either sex.

Instruments : Tissue homogenizer (Dolphin), Refrigerated centrifuge (MPW), Enzyme

assay Kits (Erba Mannheim), Autoanalyzer (star 21 plus), Research

Centrifuge etc.

Chemicals : All the chemicals to be used in the present study will be AR graded.

7.4 Methods:

7.4.1 Preparation of Rhynchosia beddomei baker extract: The Plant is authenticated and collected in the ideal condition and air dried under the shade, powdered and passed through the sieve # 44 to get a uniform powder. The powders were extracted with petroleum ether (40-600 C) to defat, followed by Chloroform and Ethyl acetate solvent extraction for 24 hrs. The extract was concentrated under rotary evaporator and dried in lyophilizer. The extracts were formulated as suspension in distilled water using 5%

Tween 80 as, suspending agent. The % of yield of extract will be calculated and dose will be selected based on the toxicity studies7.

7.4.2 Evaluation of hypoglycemic activity: The acclimatized animals were fasted for 24 hrs with water ad libitum, fasted animals were divided in to seven groups of six rats each.

Initial blood sample were drawn at 0 hrs and on intervals 1½, 3, 5 hr after the extracts administration. Blood sample were collected from retro-orbital plexus under anaesthesia and were centrifuged at 3000 rpm for 10 min to obtained serum and used for estimation of glucose by using glucose kit2.

7.4.3 Antidiabetic activity in alloxan induced diabetic rats: Diabetes was induced by a single intraperitoneal injection of freshly prepared alloxan (140 mg/kg). The rats were maintained on 5 % glucose solution for 24 hr to prevent hypoglycemia. After three days the animals were checked for serum blood glucose level, and the animals with

6 hyperglycemic (glucose>200mg/dl) were used for the experiment. The treatments were continued daily for 28 days blood samples were collected from retro-orbital plexus under anesthesia. The obtained serum was used for estimation of glucose using glucose kit, after

1 hr of treatment on days 7, 14, 21 and 28 days of treatment8, 9.

7.4.4 Evaluation of biochemical parameters:

Serum blood glucose level, Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), Alkaline phosphate (ALP), Cholesterol

Triglycerides10. Superoxide dismutase (SOD) 11. Lipid peroxidations (LPO) 12. Catalase

(CAT) 13. And total protein in liver homogenate by using spectrophotometric method14.

7.4.5 Statistical analysis :

All the data were expressed in mean ± S.E.M. The significance of differences in mean between control and treated animals for different parameters were determined by using one way ANOVA and followed by Dunnet’s comparison test and significance difference between groups was evaluated by using student’s t-test.

7.5 Dose the study requires any investigations or interventions to be conducted on patients or other humans/animals? If so describe briefly:

Yes, for the study rats will be used. The effects of preparation on blood serum glucose liver, will be studied by considering physiological, pathological and biochemical parameters using animal models.

7.6 Has ethical clearance been obtained from your institution for performing various tests on animals?

Yes, the study is cleared Institutional Animal Ethics Committee (IAEC) and the copy is enclosed with the protocol.

7 8. REFERENCES:

1) http://en.wikipedia.org/wiki/Diabetes_mellitus.

2) Rathod N., Raghuveer I., Chitme H. R., Ramesh Chandra. Antidiabetic activity of

Nyctanthes Arbortistis. Phcog Mag 2008; 4: 335-339.

3) Chetty Madhava K., Sivaji K., Tulasi Rao K. Flowering plants of Chittoor district.

1st ed. Student offset printers 2008: pp 99.

4) Mankil Jung., Moonsoo Park., Hyun Chul Lee., Yoon-Ho Kang., Eun Seok Kang.,

Sang Ki Kim., et al. Antidiabetic agents from medicinal plants. Current Medicinal

Chemistry 2006; 13: 1203-1218.

5) Venkata Raju R. R., Bhakshu L. Antimicrobial activity Rhynchosia beddomei

baker. Fitoterapia 2001; 70: 579-582.

6) Jeevan Ram A., Bhakshu L., Venkata Raju R. R. In vitro antimicrobial activity of

certain medicinal plants from Eastern Ghats, India, used for skin disease J

Ethnopharmacol 2004; 90: 353-357.

7) OECD guidelines 425 acute oral toxicity-up and down procedure 2006.

8) Manonmani G., Bhavapriya V., Kalpana S., Govindasamy S., Apparanantham T.

Antioxidant activity of Cassia fistula flowers in alloxan induced diabetic rats.

J Ethnopharmacol 2005; 97: 39-42.

9) Pari L., Amaranath Satheesh. Antidiabetic activity of Boerhaavia diffusa L. effect

on hepatic key enzymes in experimental diabetes. J Ethnopharmacol l2004; 91:

109-113.

10) Achyut N. K., Kesari S., Gupta R., Watal G. Studies on the glycemic and

lipedimic Effect of Murraya Koenigii in experimental animals. J Ethnopharmacol

2007; 112: 305-311.

8 1) Beauchamp C., Fridovich I. Superoxide dismutase : Improved assays and an

assay Applicable to acrylamide gels. Anal biochem 1971; 44: 276-287.

9. SIGNATURE2) Ohkawa OF CANDIDATE H., Ohishi N., Yagi K. Assay for lipid peroxidase in animal tissues by ( Mr. BAHUBALI ) Thiobarbituric acid reaction. Anal Biochem 1979; 95: 351-358. The present study has been proposed based on 10. REMARKS3) Sinha OF THEA. Calometric GUIDE assay oftraditional catalase. claim. Anal Biochem The results 1972; obtained 47: 389-394. from the study may validate its traditional therapeutic claim. 4) Lowry O. H., Rosebrough N. J., Farr L., Randall R. J. Protein measurement

11. NAME ANDwith DESIGNATION theFolin- Phenol reagent. J Biol Chem Dr. NANU.1951; 193: R. RATHOD265-272. OF THE GUIDE Asst. Professor

12. SIGNATURE

13. CO-GUIDE ------

14. SIGNATURE ------

15. HEAD OF THE DEPARTMENT Dr. I.S.MUCHANDI H.O.D. Department of Pharmacology H.S.K.College of Pharmacy, B.V.V.S. Campus, Bagalkot-587101.

16. SIGNATURE

17. REMARKS OF THE The above mentioned information is correct and I PRINCIPAL recommended the same for approval.

18. NAME OF THE PRINCIPAL Dr. I.S.MUCHANDI H.O.D. Department of Pharmacology H.S.K.College of Pharmacy, B.V.V.S. Campus, Bagalkot-587101.

19. SIGNATURE

9 OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC) HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY, BAGALKOT-587101, KARNATAKA

REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE “BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision) RULES 1998”

Ref: HSKCP/IAEC, Clear /2010-11/1-12

CERTIFICATE

This is to certify that Mr. BAHUBALI a student of first M.Pharm is permitted to carry out experiments on animals for the dissertation /thesis work entitled as “Antidiabetic activity of Rhynchosia beddomei baker on alloxan induced diabetic rats’’ as per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.

Animal house in charge CHAIRMAN

Form B See rule [6 (a) and 8(a)] PART A

(1) Name and address of the HSK College Of Pharmacy, B.V.V.S Establishment Campus, Bagalkot. 587101, Karnataka (2) Date and Registration Number of the 821/01/a CPCSEA Establishment:

(3) Name, address and Registration No. OFFICE OF CPCSEA, of the breeder from whom acquired Ministry of environment and forest, and the date of acquisition: IIIrd seaward road, Valmiki nagar, Thiruvanmiyur, Chennai-600041. Tamil Nadu (4) Place where the animals are presently Animal House kept: HSK College Of Pharmacy, B.V.V.S Campus, Bagalkot.587101, Karnataka (5) Place where the experiment is to be PG Lab-II, Department of performed: Pharmacology, HSK College of Pharmacy, B.V.V.S Campus, Bagalkot. 587101, Karnataka (6) The date on which the experiment is to commence and the duration of the 5th April 2011 experiment:

The protocol form for the research proposal – PART B in the case of experiments using other than non-human primate animals for ongoing / new projects PART C for use of non-human primates for new projects and PART D for use of non-human primate for extension of ongoing projects – should be duly filled singed and annexed with this form.

Dated: Signature

Place: (Name and Designation) 11 PART – B

Protocol form for Research Proposal to be submitted to the Committee on use of small animals / Animals other than non human Primate in Biomedical Research for ONGOING / NEW PROJECTS

1. Project Title : Antidiabetic activity of Rhynchosia beddomei baker on Alloxan induced diabetic rats.

Investigation (s) : Dr. NANU. R. RATHOD 2. Designation Asst. Professor

Department (s) : Department of Pharmacology, 3. HSK College Of Pharmacy, BVVS Campus, Bagalkot. 587101, Karnataka

(a) Funding Source (s): if any 4. ------

(b) Are sufficient funds available for purchase and maintenance of the Yes animals

(c) Duration of present project :

(1) Number of months : 6 months

(2) Date of start of the Project : (Experiment) 5th April 2011

(3) Date of termination of the project : 16th November 2011

5. Date by which approval is needed in case the project is to be funded by outside ------agency (If less than six weeks from the date of admission, please justify below):

12 6. Summary of project briefly summarize in laymen’s term the background, the objective and the experiment approach.

(a) Background: Enclosed

(b) Objectives Enclosed

(c) Experimental procedure: Enclosed

7. (a) Name of species Albino rats

Age Sex Weight

4-8 Weeks Either 200-250 gms

(b) Rationale for selection

Approximate number of animals required during the 90 first 12 months.

Justification of number (define treatment group and Fifteen groups, Each group number per group) containing six animals.

Number of animals housed per weeks 20

8. List all invasive Non Surgical Animal Procedures and Invasive non-surgical Potentially Stressful Noninvasive procedures to be used procedure (Example IM injection, foot pad injection, venapunctures).

Procedure and Approximate Frequency: Enclosed

13 Anaesthetic and/or Analgesic and Dosage:

Anaesthetic ether

Test substance injected and/or applied:

Test substances administered post orally.

9. Does the protocol prohibit the use of anaesthetic and analgesic for the conduct of painful procedures?

10. With surgical procedure/Experimental procedure is performed?

(a) Will the animal be sacrificed after surgery?

INVESTIGATOR SIGNATURE

DATE: ______

14 1. Background Diabetes is a condition in which the body either does not produce enough, or does not properly respond to, insulin a hormone produced in the pancreas. Diabetes is the world’s largest endocrine disease with deranged carbohydrate, fats and protein metabolism. As per WHO report, approximately 150 million people have diabetes mellitus worldwide, and this number may well double by the year 2025. Statistical projection suggests that the number of diabetics will rise from 15 million in the year 1995 to 57 million in 2025, making India apart the country with the highest number of diabetics in the world. Although many drugs and interventions are available to manage diabetics, these are expensive for a developing country like India apart from their inherent adverse effects. Therefore, it is necessary to look for new avenues to manage this major health problem. As part of the pathogenesis of Type II diabetes mellitus, skeletal muscle, liver and adipose tissues become resistance to the hormonal effect of insulin, which in turn leads to decreased insulin-mediated glucose disposal, hepatic glucose overproduction and a marked increase in lipolysis. An addition to the above, hyperinsulinemia is a central pathophysiological feature of Type II diabetes mellitus and has been shown to play a key role in the disease evaluation and macrovascular complication. The plants kingdom has become a target for the search by multinational drugs and biological active compound Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes. Hence, the present study was under-taken to explore Antidiabetic activity of Rhynchosia beddomei baker of different extracts on normal and alloxan induced diabetic rats. The leaves are collected from widely growing plants in the region of Tirumala Chittoor District, Andhra Pradesh, India.

2. Objective of the study: Traditionally well known Rhynchosia beddomei baker extract will be used for the for the Present study. 1. Evaluation of hypoglycemic activity in rats. 2. Antidiabetic activity in alloxan induced diabetic rats study

15 3. Experimental Procedure

3.1 Antidiabetic activity in alloxan induced diabetic rats.

3.1.1 Evaluation of hypoglycemic activity: The acclimatized animals were fasted for 24 hrs with water ad libitum, fasted animals were divided in to seven groups of six rats each. Initial blood sample were drawn at 0 hrs and on intervals 1½, 3, 5 hrs after the extracts administration. Blood sample were collected from retro-orbital plexus under anaesthesia and were centrifuged at 3000 rpm for 10 min to obtained serum and used for estimation of glucose by using glucose kit.

3.1.2 Antidiabetic activity in alloxan induced diabetic rats: Diabetes was induced by a single intraperitoneal injection of freshly prepared alloxan (140 mg/kg). The rats were maintained on 5 % glucose solution for 24 hrs to prevent hypoglycemia. After three days the animals were checked for serum blood glucose level, and the animals with hyperglycemic (glucose>200mg/dl) were used for the experiment. The treatments were continued daily for 28 days blood samples were collected from retro-orbital plexus under anesthesia. The obtained serum was used for estimation of glucose using glucose kit, after 1 hrs of treatment on days 7, 14, 21 and 28 days of treatment8, 9.

3.1.3 Evaluation of biochemical parameters: Serum blood glucose level, Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), Alkaline phosphate (ALP), Cholesterol Triglycerides. Superoxide dismutase (SOD). Lipid peroxidations (LPO). Catalase (CAT). And Total protein in liver homogenate by using spectrophotometric method.