Immunomodulatory and Anticancer Activity of Terminalia Bellirica Extract in Mice

Immunomodulatory and Anticancer activity of Terminalia bellirica extract in mice

M.Pharm Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka Bangalore– 560 041

Mr. Hiral P. Bhalodiya B.Pharm.

Under the Guidance of

Mr. T. Prakash M.Pharm. Asst. Professor

Department of Pharmacology, Acharya & B.M.Reddy College of Pharmacy, Chikkabanavara Post, Soladevanahalli, Hesaraghatta Main Road, Bangalore – 560 090

2009-10

1 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE

ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. Name of the candidate & Mr. Hiral P. Bhalodiya Address. 304,Golden point, Golden park, Nana mava main road, Rajkot-360005 (GUJARAT) Phone No:09036188277 E-mail:[email protected]

2. Name of the Institution. Acharya & B.M. Reddy College of Pharmacy Chikkabanavara Post, Soladevanahalli, Hesaraghatta Road, Bangalore-560090. Phone No: 080 65650815 Fax No: 080 28393541

3. Course of the study M.Pharm. & subject. Pharmacology

4. Date of admission. 13/05/09

Immunomodulatory and Anticancer activity of 5. Title of the Topic Terminalia bellirica extract in mice

6. Brief resume of intended work

6.1 Introduction and need of the work Enclosure I

6.2 Review of Literature Enclosure II

6.3 Aim and Objective of the study Enclosure III

2 7. Materials & Methods

7.1 Source of data Enclosure IV

7.2 Methods of collection of data Enclosure V

7.3 Does the study require investigation on animals? Enclosure VI If yes give details

7.4 Has ethical clearance been obtained from your institution in case Yes (Applied) of 7.3

8. List of references Enclosure VII

9. Signature of the candidate

10. Remarks of the guide

11. Name & Designation of 11.1 Guide Mr. T. Prakash M.Pharm. Asst Professor Department of Pharmacology, Acharya & B.M.Reddy College of Pharmacy, Bangalore – 560 090

11.2 Signature of Guide

11.3 Head of the Department Dr. Kalyani Divakar M.Pharm. Ph.D Professor & Head Department of Pharmacology, Acharya & B.M.Reddy College of Pharmacy, Bangalore– 560 090

11.4 Signature of HOD

3 12.1Remarks of Principal 12.

Principal Prof. Divakar Goli M.Pharm. Ph.D Professor Acharya & B.M.Reddy College of Pharmacy, Bangalore– 560 090

12.2 Signature

4 ENCLOSURE – I

6. BRIEF RESUME OF INTENDED WORK

6.1 Introduction and need of work: Malignancy is one of the most serious diseases that damage human health in the modern world. There exists close relationship between the occurrence, growth and decline of tumor and immune states. The low immune function of an organism may not only result in the generation and development of a tumor, but also be one of the most important factors that prevent the tumor patients’ recovery. Immunomodulation through natural or synthetic substances may be considered an alternative for the prevention and cure of neoplastic diseases. The enhancement of host immune response has been recognized as a possible means of inhibiting tumor growth without harming the host. Therefore, it is very important to investigate novel antitumor substances with improving immunity potential.1 Terminalia belerica is one of the ingredients of Ayurvedic purgative medicament of ‘triphala’ available in the Indian market for the treatment of digestion and liver disorders. It is the centuries old healing system and is a main stay of Ayurvedic Pharmacy. Terminalia belerica is also known as Vibhitaki in Sanskrit, meaning fearless. It is so safe to use and so highly valued for its efficacy. The fruit of the plant has been used in various ailments in the indigenous system of medicine. Among the array of medicinal properties attributed to it, a significant one is its therapeutic value in the treatment of liver disorders. The fruit is also reported to have purgative, cardiac depressant, hypotensive and choleretic effects. It reduces lipid level from the liver and heart, which can lower the disease risk associated with those organs2. However, The literature survey offered no scientific claim on Immunomodulatory activity of Terminalia bellirica in mice and anticancer activity of Terminalia bellirica extract against Ehrlich ascites carcinoma in mice. Ehrlich ascites carcinoma was developed by Loewenthal and Jahn from one of the several lines of Ehrlich carcinoma that arise from spontaneous epithelial tumors, probably of mammary gland origin.3

5 ENCLOSURE – II

6.2 REVIEW OF LITERATURE:

“Terminalia bellirica” is belonging to family: Combretaceae. It is locally called by different names in different languages: English- Belliric myrobalan, Sanskrit- Vibhitaki4.

Distribution: It is found in abundance in Madhya Pradesh, Uttar Pradesh, Punjab, Maharashtra, and also in Ceylon and Malaya5.

Chemical Constituents: Principal Constituents of Terminalia bellirica are Sitosterol, Gallic acid, Ellagic acid, Chebulagic acid, Galloyl glucose5.

Medicinal uses:

The fruits of Terminalia bellirica are used for the medicinal purpose.It is used both, internally as well as externally.The seed oil or the paste of its fruit is applied externally on the swollen and painful parts.The paste of its fruits is applied on the eyelids in conjunctivitis5. The Terminalia belerica fruits extract have demonstrated significant anti-tumorous, anti-cancerous and selective toxicity against various types of cancer cells. It can also be used with confidence to treat Blood Cancer along with other conventional treatments with chemotherapeutic agents6.

6 Scientific publication: I. The Preventive actions of Terminalia bellirica in experimentally induced atherosclerosis has been studied in rabbits.Results of the study have reported that Terminalia bellirica reduced the levels of lipids in hypercholesterolemic animals7. II. The Anti-salmonella activity of Terminalia bellirica has been found. Pretreatment of mice with aqueous extract of T.bellirica conferred protection against experimental Salmonellosis and 100% survival of animals have been reported when challenged with lethal dose of S.typhimurium8. III. The antimicrobial activity of crude and methanol extract of Terminalia bellirica dry fruit has been tested by using disc diffusion method against 9 human

microbial pathogens. Results of the study indicated that T. bellirica dry fruit possesses potential broad spectrum antimicrobial activity9. IV. Evaluation of growth inhibitory effects of Phyllanthus emblica and Terminalia bellirica extracts on human hepatocellular carcinoma (HepG2) and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin10. V. The antispasmodic and bronchodilatory properties of Terminalia bellirica fruit has been studied. Results indicated that Terminalia bellirica fruit possess a combination of anticholinergic and Ca+2 antagonist effects, which explain its folkloric use in the colic, diarrhea and asthma11. VI. In vitro growth and shoot multiplication in Terminalia bellirica roxB. under controlled carbon dioxide environment12. VII. Evaluation of antimutagenic potency of water, acetone, and chloroform extracts of Terminalia bellirica by using the Ames Salmonella or microsome assay13.

7 ENCLOSURE – III

6.3 AIM AND OBJECTIVE OF THE STUDY: The present study is intended to investigate the Immunomodulatory activity in mice and also to investigate anticancer activity of Terminalia bellirica extract against Ehrlich ascites carcinoma in mice. Objective: 1) Collection of fruits of plant of Terminalia bellirica. 2) Preparation of ethanolic extract of Terminalia bellirica by using Soxhlet extractor. 3) Preliminary phytochemical analysis of ethanolic extract of Terminalia bellirica.

4) Evaluation of acute toxicity study (LD50) of plant Terminalia bellirica. 5) Immunomodulatory activity of Terminalia bellirica extract in swiss albino mice. 6) Anticancer activity of Terminalia bellirica extracts against Ehrlich ascites carcinoma in swiss albino mice.

8 ENCLOSURE – IV

7. MATERIALS AND METHODS 7.1 Source of Data: Data will be obtained from-  Laboratory based studies  Literature survey, CD ROM, Chemical abstracts  National & International Journal  Text books.  Internet.  RGUHS-website-Helinet.

9 ENCLOSURE – V

7.2 METHOD OF COLLECTION OF DATA

Plant: The fruits of Terminalia bellirica will be collected and dried. They will be then coarsely powdered & stored in an air tight container. Animals: Adult Swiss male albino mice (20-25 g) will be selected for evaluation of toxicity study, Immunomodulatory and Anticancer activity of Terminalia bellirica. Cells: EAC cells will be obtained from the Manipal Cancer Research Center, Manipal. Mice will be maintained by weekly intraperitoneal inoculation of 106 cells/mouse.

Methodology:

1. Preparation of Extraction: - Powdered fruits of Terminalia bellirica will be extracted by using ethanol with Soxhlet extractor. The dried extract will be used for determination of the Immunomodulatory and Anticancer activity of Terminalia bellirica in swiss albino mice. 2. Preliminary phytochemical analysis of the ethanolic extract. 3. Acute toxicity study will be conducted according to OECD guidelines. 4. Pharmacological studies: Evaluation of the Immunomodulatory activity of ethanolic extract of Terminalia bellirica by using following Parameters: a) Determination of the effect of Terminalia bellirica on the Organ weight. Mice will be divided into 5 groups, each group consisting of 6 animals. Group I: Animals will receive normal saline. Group II: Animals will receive Cyclophosphamide (25mg/kg). Group III: Animals will receive high dose of extract. Group IV: Animals will receive Cyclophosphamide (25mg/kg) + low dose of extract.

10 Group V: Animals will receive Cyclophosphamide (25mg/kg) + high dose of extract 14,15.

b) Determination of haematological parameter. Mice will be divided into 5 groups, each group consisting of 6 animals. Group I: Animals will receive normal saline. Group II: Animals will receive Cyclophosphamide (25mg/kg). Group III: Animals will receive high dose of extract. Group IV: Animals will receive Cyclophosphamide (25mg/kg) + low dose of extract. Group V: Animals will receive Cyclophosphamide (25mg/kg) + high dose of extract 14,15.

c) Determination of Phagocytic index by using Carbon clearance test. Mice will be divided into 3 groups, each group consisting of 6 animals. Group I: Control. Group II: Animals will receive low dose of extract. Group III: Animals will receive low dose of extract.16,17.

5. Evaluation of the Anti cancer activity of ethanolic extract of Terminalia bellirica by using following screening method: a. Effect of Terminalia bellirica on survival time (MST): Mice will be divided into 4 groups, each group consisting of 6 animals. Group I: Tumor bearing control mice. Group II: Tumor bearing mice + Low dose of extract. Group III: Tumor bearing mice + high dose of extract. Group IV: Tumor bearing mice+5-FU18.

b. Effect of Terminalia bellirica on normal peritoneal cells: Mice will be divided into 3 groups, each group consisting of 6 animals. Group I: Normal mice.

11 Group II: Normal mice will receive extract for 1 day. Group III: Normal mice will receive extract for 2 day 18.

c. Effect of Terminalia bellirica on heamatological parameter: Mice will be divided into 4 groups, each group consisting of 6 animals. (1) Tumor-bearing mice. (2) Tumor bearing mice will be treated with low dose of Terminalia bellirica. (3) Tumor bearing mice will be treated with high dose of Terminalia bellirica. (4) Control group of mice (normal) 18.

d. Effect of Terminalia bellirica on solid tumor: Mice will be divided into three groups, each group consisting of 6 animals. Group I: Tumor control. Group II: tumor control+ Terminalia bellirica (low dose). Group III: tumor control+ Terminalia bellirica (high dose) 18.

Data analysis The data obtained from the above study will be subjected to Statistical analysis and data will be evaluated by analysis of variance (ANOVA) followed by Dunnets test.

Duration of completion of project:  Total duration for the completion of whole project may be 9 months I. Duration of experimentation Six months. II. Literature survey one & half months. III. Thesis writing one & half months.

12 ENCLOSURE – VI

7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly.

The above study requires investigation on 162 Swiss albino mice for evaluation of Immunomodulatory and anticancer activity of Terminalia bellirica extract and also for acute toxicity study.

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

Applied for IAEC clearence

13 ENCLOSURE – VII

8. REFERENCES:

1. Hai-Shun Xu, Yuan-WenWu, Shi-Fang Xu, Hong-Xiang Sun, Feng-Yang Chen, Li Yao. Antitumor and Immunomodulatory activity of polysaccharides from the roots of Actinidia eriantha. J Ethnopharmacol 2009;125:310–7. 2. Anjana Jadon, Monika Bhadauria, Sangeeta Shukla. Protective effect of Terminalia belerica Roxb. and gallic acid against carbon tetrachloride induced

damage in albino rats. J Ethnopharmacol 2007;109:214–8. 3. Gupta M, Mazumder UK, Rath N and Mukhopadhyay DK. Antitumor activity of methanolic extract of Cassia fistula L. seed against Ehrlich ascites Carcinoma. J Ethnopharmacol 2000 Sep 1;72(1-2):151-6. 4. Terminalia bellirica synonyms [On line] [2009 Nov 14] Available from: URL: http://www.herbsnspicesinfo.com/medicinal-herbs/myrobalan-belliric.aspx 5. Bibhitaka (Terminalia belerica) [On line] [2009 Oct 30] Available from: URL: http://www.herbalcureindia.com/herbs/bibhitaka.htm 6. Blood Cancer Home Remedy Using Terminalia Belerica [On line] [2009 Nov 17] Availble from :URL:http://www.mamaherb.com/blood-cancer-home-remedy- using-terminalia-belerica 7. Shaila HP, Udupab AL, Udupa SL.Preventive actions of Terminalia belerica in experimentally induced atherosclerosis. Int J Cardiol 1995;49:101-6. 8. Madani A and Jain SK. Anti-salmonella activity of Terminalia bellirica: in vitro and in vivo studies. Indian J Exp Biol 2008 Dec;46:817-21.

9. Elizabeth KM. Anti-microbial activity of Terminalia bellirica. Indian J Clin

Biochem 2005;20(2):150-3. 10. Pinmai K, Chunlaratthanabhorn S, Ngamkitidechakul C, Soonthornchareon N, Hahnvajanawong C. Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents:

14 Doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells. World J Gastroenterol 2008 March 14;14(10):1491-7. 11. Gilani AH, Khan AU, Ali T, Ajmal S. Mechanisms underlying the antispasmodic and bronchodilatory properties of Terminalia bellerica fruit .J Ethnopharmacol 2008 Mar 28;116(3):528-38. 12. Suthar RK, Rathor P, Purohit SD. In vitro growth and shoot multiplication in Terminalia bellerica RoxB. under controlled carbon dioxide environment. Indian J Exp Biol 2009 Mar;47(3):204-9. 13. Kaur S, Arora S, Kaur S, Kumar S. Bioassay-guided isolation of anti mutagenic factors from fruits of Terminalia bellerica. J Environ Pathol Toxicol Oncol 2003;22(1):69-76. 14. Azmanthulla S, Hule A, Naik SR. Evaluation of Adaptogenic activity profile of herbal preparation. Indian J Exp Pharmacol 2006;44:574-9. 15. Nayak S and Dixit VK. Immunomodulatory activities of the seeds of Celastrus paniculata linn. Int J Pharmacol Biol Sci 2008;2(1):1-12. 16. Kale KR, Kale KK. Practical human anatomy and physiology.11th ed. Pune:Nirali prakashan;2004. 17. Sengupta J. Synopsis of clinical pathology and microbiology. 2nd ed. Culcutta:Roy and Sen publisher;1985. 18. Mgbemene CN, Ohiri FC. Anti-sickling potential of Terminalia catappa leaf extract. Pharmaceutical Biology (Formerly International Journal of Pharmacognosy) 1999 Apr;37(2):152-54.