Rajiv Gandhi University of Health Sciences Karnataka s54

PRELIMINARY PHYTOCHEMICAL INVESTIGATION, ANTIMICROBIAL AND ANTICANCER ACTIVITIES OF STEM AND LEAVES OF RAUWOLFIA TETRAPHYLLA LINN

M. PHARM. DISSERTATION PROTOCOL

Submitted to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BENGALURU

Mr. AMIT N. PANASKAR B. Pharm.

Under the guidance of

Dr. BHARATESH S. KITTUR M. Pharm., Ph. D.

DEPARTMENT OF PHARMACHEMISTRY H.S.K COLLEGE PHARMACY BAGALKOT-587 101

Annexure-II

Mr. AMIT N. PANASKAR Name and Address of the H.S.K. COLLEGE OF PHARMACY 01 Candidate BAGALKOT-587 101 KARNATAKA

B. V. V. SANGH’S 02 Name of the Institution H.S.K. COLLEGE OF PHARMACY BAGALKOT – 587 101

Course of the Study M. PHARMACY 03 Branch PHARMACEUTICAL CHEMISTRY

04 Date of Admission to course OCTOBER 2010

PRELIMINARY PHYTOCHEMICAL INVESTIGATION, ANTIMICROBIAL AND 05 Title of the Topic ANTICANCER ACTIVITIES OF STEM AND LEAVES OF RAUWOLFIA TETRAPHYLLA LINN

2 3 06 Brief resume of the intended work:

6.1) Introduction:

India is endowed with a rich wealth of Medicinal plants, microbes are closely associated with the health and welfare of human being, some are beneficial and some are detrimental. The increasing failure, chemotherapeutics and antibiotic resistance exhibited by pathogenic microbial infections agents have lead to the screening of several medicinal plants for their potential antimicrobial activity 1. Antibacterial properties of various plant parts like leaves, seeds and fruits have been well documented for some of the medicinal plants for the past two decades2. A special feature of higher angiospermic plants is their capacity to produce a large number of organic chemicals of high structural diversity. The so- called secondary metabolites3, which divided in to different categories based on their mechanism of function like chemotherapeutic, bacteriostatic, bactericidal and antimicrobial4. A medicinal plant represents a rich source of antimicrobial agents. A wide range of medicinal plant parts is used for extract as a raw drug and their posses varied medicinal properties. The different parts are including bark, root, stem and fruits and modified plant organ. With some of these raw drug are collected in smaller quantities by the local communities and folk healers for local used many other raw drugs are collected in large quantities and traded in the market as the raw material for many herbal industries5. Cancer and Tuberculosis (TB) are considered to be dreadful diseases which have created havoc for the global populations, despite lot of advances made in the field of chemotherapy, cancer and TB still remains one of the major health problem today particularly in developing countries. As we know very well, now a days the chemotherapeutic agents available in the market from which most of them either not effective up to the mark or has to develop resistance resulting in reoccurrence again.

Cancer is a disease in which there is uncontrolled multiplication and spread within the body of abnormal form of the body’s own cells. Term cancer, malignant neoplasm and malignant tumor are synonymous; they are distinguished from being tumors by the properties of dedifferentiation, invasiveness and the ability to metastasis. Cancer continues to represents the largest cause of mortality in the world and claims over six million lives every4 year. There are three main approaches for treating established cancer-surgical excision, irradiation and chemotherapy-and the role of each of these depends on 5 6.2) Need for the Study:

Indian traditional system of medicines like Siddha and Ayurveda have suggested to increase the body’s natural resistance to diseases9. Recent screening with plant has reveled many compounds like alkaloids, flavonides, terpenoids etc with pronounced antioxidant, antineoplastic, antiulcer, anti-inflammatory, antimicrobial and immunostimulating potential10. There are about 86 Rauwolfia species. Rauwolfia tetraphylla L is widely distributed in tropical area of South Africa, Caribbean, India and Australia. Rauwolfia tetraphylla L. belongs to the family Apocyanaceae, herbaceous plant. It is used in cholera, fever, eye disease and diarrhoea. It is also used for remedy of antihypertensive as well as in dysentery and intestinal disorders11. R.tertaphylla-an introduced and naturalized species are economically important for their alkaloids. It is a native of West Indies. Five species are recorded in India including one, which is introduced and naturalized. The government of India restricted the export of crud drug to conserve the natural growth and thus reduce, its exploitations, resulting in the shortage of these alkaloids in the world marker. This led of an active search of these alkaloid in other related species like Rauwolfia micrantha, a rare species endemic to the Western Ghats12 and R.tetraphylla and introduced and neutralized species, which is very common. However both are economical important13. They contain alkaloids such as reserpine, serpentine, reserpiline, ajamalicine and sarpagine14. Most of the Rauwolfia species contains serpentine, having activity against mammary cancer MS 301 in mice15. Hence the present study is undertaken for the phytochemical investigation of petroleum ether, ethanol and aqueous extract of stem and leaves of R.tetraphylla for antimicrobial and anti-cancer activity on different human cancer cell lines to evaluate its traditionally clamed activities.

6 6.3) Review of Literature:

Extensive literature survey was carried out in our college library, visiting various websites through internet and helinet of RGUHS , Karnataka. Bengaluru .

6.4) Object of the Study: The object of the present study is: 1. Collection and identification of plant 2. Collection and processing of stem and leaves of R.tetraphylla. 3. Extraction of stem and leaves of R.tetraphylla with petroleum ether, ethanol and water. 4. Preliminary phytochemical investigation of different extracts. 5. Screening for the antibacterial, antifungal, anti TB and anticancer activities of potent extract.

07 Material and Method: Stem and leaves of R.tetraphylla, microorganism strains will be taken for studies. 7.1) Source of data: The source of data for this study is based on laboratory experiments on microorganisms and cancer cell lines, also the data is obtained from the literature. 7.2) Method of Collection of data: (Including sampling procedure if any) The stem and leaves of R.tetraphylla selected for the study will be collected from in and around Bagalkot region and authenticated. Preliminary Phytochemical Investigation: The stem and leaves of R.tetraphylla will be collected, shade dried, powdered and were extracted with different solvents and studied for various chemical constituents.

7 7.3) Anti-microbial activity: The potent extracts will be evaluated for anti-microbial property by turbidimetric method. Turbidimetric method: This technique is based on Inhibition of the microbial growth by measurement of the turbidity (transmittance) of suspension of a suitable microorganism in a fluid media. Change in transmittance produced by the test compound is compared with those produced by known concentration of reference material16. 7.4) Anti-tubercular activity: The potent extract will be screened for their anti-tubercular activity against Mycobacterium tuberculosis by Alamar blue assay method for the determination of MIC17. 7.5) Anti-cancer activity: The anti-cancer activity is carried for the potent extract by MTT Assay Method18 or Trypan blue exclusion method19.

7.6) Does the study require any investigation or invention to be conducted on patients or other human or animals? If so please mention briefly.

-No- 7.7) Has ethical clearance been obtained from your in institution in case of 7.6.

- No- Not required for the studies.

8 08 References: 1. Ritch-Kro Em., Turner NJ and Towers GH., carrier herbal medicine and evaluation of the antimicrobial and anticancer activity in some frequently used remedies. J.ethnopharmacology, 1996; 5:151-156.

2. Leven M., Vannan Bergha DA and Mertens F. Medicinal Plants and its importance in antimicrobial activity. J Planta Med, 1979; 36: 311-321.

3. Evans JS., Pattison E., Morris P. Antimicrobial agents from plant cell culture, in-secondary metabolities in plant cell culture. Morris P., Seraggs A., Stanfford A, Fowler M. Cambridge University, London.1986; P.12.

4. Purohit SS., Mathur SK. Drug in Biothchnology fundamentals and applications. Purohit SS. Maximillan Publishers. India.1999; P.576.

5. Ahsan Rajib., Islam Montrul., Haque E., Massaddik A. Invitro Antibacterial; Screening and toxicity study of some different medicinal plants. World J. Agri.Sci. 2009; 5:617-621.

6. Rang HP., Dale MM., Pharmacology, 5th ed. New Delhi: Elservier; 2005:693.

7. Gupta M., Mazumdher UK., Sambath RK., Antitumar effect of Bauhinia racemosa against Ehrlich ascites carcinoma with reference to lipid peroxidation and antioxidant system in Swiss albino mice. Acta Pharmacol Sin. 2004; 25(8):1070-1076.

8. Harman JG., Limbird LE., Goodman and Gilman’s The Pharmacology Basis of therapeutics. 10th ed. New York: Mc Graw-Hill medicinal publishing division; 2001: 1381.

9. Sagrawat H., Khan K. Immunomodulator plants, A Phytopharmacology and Pharmacognosy Review, 2007; 1:2; 248-60.

10. Wagner H. Search for plant derived natural products with immunostimulatory activity (recent advances), pure and applied chemistry.1990; 62:7:1217-22.

11. Ananymous. The Wealth of India. Raw materials, Publications and Information, Directorate, CSIR New Delhi,1969; Vol.VIII.

12. Gamble JS., Flora of Madras Presidency. 1921; Vol.2: p.808.

13. Henry AN., Kumar Grand Chithra V. Flora of Tamilnadu, India. Botanical of India. Calcutta.1987; Vol.2, p.79.

9 14. Roja PC., Simpathimalani AT., Heble MR and Chandha M. J.Nat.Products.1987; 50: 872-875.

15. Pharmacognosy (Pharmacognosy and Phytochemistry) Mohammed Ali. CBS Publishers and Distributors. New Delhi.2008; Vol.1, p:616.

16. Gannaro R., Alfonso.Remington. The science and Practice of Pharmacy. Indian. Delhi. BI Publication, 20th ed: 2005, 1:548.

17. Gundurao K., Vinayak H., Khazi I.,Pramodh G.Synthesis and evaluation of anti TB activity of imidazo [2,1,-b (1,3,4-thaidiazole derivatives)].Bioorganic and Med. Chemistry. 2006; 14: 3069-80.

18. Mosmann T., Rapid Colorimetric assay for culture growth and survival; Application to proliferation and cytotoxicity assay. J.Immunological Methods. 1983; 16, 55-63.

19. Devi PU., Solomon FE and Sharad AC. In vivo tumor inhibitory and radio sensitizing effects of an Indian medicinal plant, Pulmago rosea on experimental mouse tumors. Indian Jornal of expt. Bio. 1994;Vol.32,no.8, p.523-528.

10 09 Signature of the Candidate (AMIT N. PANASKAR)

10 Remarks of the Guide The above information and literature has been extensively investigation ,verified and was found to be correct .The present study will be carried under my supervision and guidance .

Name and Designation of Dr. BHARATESH S. KITTUR 11 (in Block Letters) M.PHARM,Ph.D 11.1. GUIDE: PROFESSOR, Guide ship Reference No. of P.G. DEPT. OF PHARMACEUTICAL RGUHS:ACA/CDC/PGT- CHEMISTRY, M.PH/HCP/48/2005-06 H.S.K. COLLEGE OF PHARMACY, BAGALKOT-587 101. Date:17.11.2008

11.2.Signature

Dr. SHREENIVAS R. DESHPANDE 11.3. HEAD OF THE M.PHARM,Ph.D DEPARTMENT PROFESSOR, P.G. DEPT. OF PHARMACEUTICAL CHEMISTRY, H.S.K. COLLEGE OF PHARMACY, BAGALKOT-587 101. 11.4. Signature

The above mentioned information is correct I recommend the same for the approval. 12 12.1Remarks of the Principal

11 (Dr. I. S. MUCHCHANDI) 12.2. Signature