RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. NAME OF THE MR.WADKAR GANESH HINDURAO. CANDIDATE AND DEPT.OF PHARMACOGNOSY AND ADDRESS PHYTOCHEMISTRY, K.L.E.SOCIETY’S COLLEGE OF PHARMACY, J.N.M.C.CAMPUS, NEHRU NAGAR, BELGAUM-10.

2. NAME OF THE K.L.E.SOCIETY’S COLLEGE OF PHARMACY, INSTITUTION BELGAUM-10.

3. COURCE OF STUDY & MASTER OF PHARMACY IN PHARMACOGNOSY SUBJECT AND PHYTOCHEMISTRY. 4. DATE OF ADMISSION JUNE-2007. TO COURSE

5. TITLE OF THE TOPIC “PHYTOCHEMICAL STUDIES AND HEPATOPROTECTIVE ACTIVITY OF THE LEAVES OF CAESALPINIA BONDUCELLA (LINN.) ROXB.”

6. BRIEF RESUME OF THE INTENDED WORK:

1 INTRODUCTION:

Liver is the largest and most complex internal organ in the body. It plays an important role in the maintenance of internal environment through its multiple and diverse function. It is involved in the intermediary metabolism of proteins, fats, and carbohydrates and in the synthesis of a number of plasma proteins such as albumin, fibrinogen and fasting factors, in the production of various enzymes and formation and excretion of bile. It acts as a storage depot for proteins, glycogens, various vitamins and metals. It has an important role in regulation of blood volume and also detoxification and excretion of many endogenous and exogenous compounds.

The liver, which is the largest gland in the body, plays a central role in metabolic homeostasis, it serves as the primary regulatory site for energy metabolism taking up and processing ingested nutrients for controlled distribution to extra hepatic tissue. In addition liver synthesizes essential proteins, enzymes and co- factors for digestion and normal body function. Diverse homeostasis mechanism are affected if the liver function is impaired, with potentially serious consequences for the individual concerned .1

Drug induced liver injury is an unresolved problem and often limits drug therapy in clinical practice. Liver injury may follow the inhalation, ingestion or parentral administration of a number of chemical and pharmacological agents.2 Drug induced liver disease can mimic all forms of acute and chronic hepatobilary diseases. However, the predominated clinical presentation resembles acute icretic hepatitis or cholestic diseases.3 Chronic hepatitis can present itself under various forms, from the very active and progressive disease with multi organ involvement, rapid onset of cirrhosis and evolution liver insufficiency to an almost asymptomatic disorders. The disease will be discussed with regard to serological parameter, histological aspects and management.4 Development of liver injury takes place in three stages i.e. necrosis, fibrosis and cirrhosis. Cirrhosis of liver is one of major health problem in developed countries. Cirrhosis of liver is the fourth leading cause of death in American adults, about 70% is associated with alcohol.

2 6.1 NEED FOR THE STUDY:

Nature has been a boon to mankind, not only it nourishes us with food but also produces beneficial effect in the treatment of various diseases, thus referred as medicinal plants. Traditionally plants have been used to cure many ailments from tribe to tribe in various geographical locations.

The efficacy of the drug along with its safety factor has made it to be widely used by physicians now a day. The modern medicine doesn’t have suitable remedies for many conditions such as liver disorder, asthma, cardiovascular disorders etc.

The survey of literature reveals that the seeds, leaves and bark of medicinal plant Caesalpinia bonducella are used in fever, asthma, colic. Seeds are used in dispersive swelling, restraining hemorrhage and keeping off infectious diseases. Tender leaves are efficacious in liver disorders. Oil expressed from them is useful in convulsions and nervous complaints.5,6,7Caesalpinia bonducella has shown to have property as an anti oxidant.8

Flavonoids , triterpeniods, ascorbic acid shows hepatoprotective effects against paracetamol induced hepatic toxicity in rats.9

6.2 REVIEW OF LITERATURE:

Caesalpinia bonducella (Linn.) Roxb. (Family- Caesalpiniaceae).

It is a large thorny shrub distributed throughout the hotter parts of India, particularly in the coastal area. Common in West Bengal and southern India. The plant is known by various names in different languages as follows:

Sanskrit - Latakaranja.

Hindi - Kanthekaranja.

English - Fever nut.

Marathi - Sagargota.

Kannada - Gajkkayi.

Gujarat - Kakachia.

Telugu - Gachakaya. 10

3 Caesalpinia bonducella (Linn.) Roxb. is a large scandent prickly shrub, branches armed with hooks and straight hard yellow prickles; leaves binnate, large, stipule foliaceous, pinnae 7 pairs, leaflets 3-8 pairs with 1-2 small recurved prickles between them on the underside; flowers are yellow in colour, in dense long penducle supra-axillary racemes at the top; fruits- 6 cm. in length, flat and thorny, 1 to 2 seeds. Shell is tough and bluish grey. Seed pulp is white in colour.10

A bitter substance bonducin, phytosterinin, fatty acids, caesalpins (α, β, γ, δ and ψ), new diterpene caesalpins, a new homoisoflavone-bonducelline and citrulline. The seeds of Caesalpinia bonducella contain a bitter principle bonducin-2%, fatty oil-25%, proteins-20% and starch-35.5%. Four cassane furanoditerpenes, designated bonducellpins A, B, C & D were isolated;11 two new cassane diterpenes, named caesaldekarins F & G have been isolated & identified; α-amyrin, β-amyrin, lupeol and lupeol acetate have been identified and isolated. 12

The whole plant is used as emmenagogue, febrifuge, astringent, anthelmintic, digestive, stomachic, liver tonic, depurative, expectorant, antipyretic, aphrodisiac, thermogenic, splenomegaly, hepatomegaly, amenorrhoea, dysmenorrhoea, pharyngodynia & tonic.

The seeds are bitter, acrid, anodyne, anti-inflammatory, antifertility, cough, asthma, leucoderma, leprosy, skin diseases, dyspepsia, dysentery, colic, haemorrhoids, hepatopathy, diabetes and intermittent fevers. 13

The leaves are anthelmintic ,febrifuge, and are useful in elephantiasis, intestinal worms,splenomegaly and fevers .The young leaves are used in hepatic disorders

The fatty oils obtained from the nucleus of the seeds are useful in convulsions and paralysis.

Review of literature reveals that Caesalpinia bonducella possesses antipyretic and analgesic, hypoglycaemic, antihyperglycaemic and hypolipidemic, and antibacterial activities. 14,15

4 6.3 OBJECTIVE OF STUDY

The objectives of the present study are-

 Extensive Review of literature of plant Caesalpinia bonducella.

 Preparation of Herbarium of the plant Caesalpinia bonducella.

 Extraction of leaves of Caesalpinia bonducella by using polar and non- polar solvents.

 Phytochemical studies of the leaves of Caesalpinia bonducella.

 Identification of various extracts of Caesalpinia bonducella using chromatographic techniques (Thin layer chromatography, Column chromatography and High performance TLC).

 Acute toxicity studies for all the extracts of Caesalpinia bonducella.

 Activity of extracts on CCl4 and Alcohol Induced Hepatotoxicity.

 Histopathological studies of various extracts from the leaves of Caesalpinia bonducella.

 Formulation of extract and showing significant activity. 7. MATERIALS AND METHODS: 7.1. SOURCE OF DATA: The sources of data for this study is based on laboratory experiments on animals; also the data obtained from the literature will be source of data 1. Fresh leaves of Caesalpinia bonducella will be taken for the study. 2. Successive hot extraction with polar and non polar solvents. 3. Hepatoprotective activity on experimental animals (Albino rats).

7.2 METHOD OF COLLECTION OF DATA:

(Including sampling procedure if any)

The leaves of Caesalpinia Bonducella (Linn.) Roxb. will be collected from local areas of Pune, Jath (Sangli), Bijapur .

5 Standardization of Caesalpinia bonducella (Linn.) Roxb. leaves . Botanical Evaluation Colour, Odour, Taste, Size, Shape, Texture. . Physical Evaluation Moisture content, Total ash, Acid insoluble ash. . Phytochemical Evaluation16 The leaves of Caesalpinia bonducella will be collected, shade dried, & powdered. The powdered material will be extracted in soxhlet assembly with alcoholic solvent. Aqueous extract is prepared by maceration process. Each extract will be concentrated by distillation off the solvent and then evaporated to dryness on water bath. The extract obtained with each solvent will be weighed. Its percentage will be calculated in terms of air dried weight of plant material. The colour and consistency of extracts will be noted. . Phytochemical Investigation 17 The obtained extract will be subjected for: 1. Identification of phytoconstituents by using various qualitative tests and chromatographic techniques. 2. Separation and isolation of phytoconstituents. 3. Subjecting isolated compounds for spectral studies. 4. Animal activity. . Pharmacological Activity: *Animal selection:- Healthy adult albino rats weighing between 150-200 gms will be selected for the experiments. They will be then employed for assessing hepatoprotective activity by post treatment of drug extracts (1ml/kg.p.o.), 5% w/v gum acacia will serve as suspending agent for the extracts.

*Acute toxicity studies The acute oral toxicity study will be carried as per the guidelines set by Organization For Economic Co-operation and Development (OECD) regulation.20 The isolated phytoconstituents will be screened for hepatoprotective activity against CCl4 induced and Alcohol induced hepatotoxicity on group of six albino rats of either sex.The activity will be compared with standard Liv-52.,18,19,20

6 For hepatoprotective activity :- By enzyme estimation such as Serum glutamtic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT),21 Serum alkaline phosphate (SALP) using reagent kits and histopathological studies.

*Statistical analysis:- All the results of biochemical estimation will be analyzed using student’t’ test22.

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

Yes. The above study requires investigation on Wistar albino rats for determination

of LD 50 Hepatoprotective activity. 7.4 Has ethical clearance been obtained from yours institution in case of 7.3?

Yes. The study has been cleared by institutional animal ethical committee. (certificate enclosed)

7 8. REFERENCES:

1. Zakim and Boyer. Anatomy and Physiology of the Liver. A Text Book of Liver Diseases Hepatology. 4 th ed.USA: W B Saunders Company 1982; (1):3.

2 .Dienstag JL, Isselbacher KJ, Toxic and Drug Induced Hepatitis. Chapter 296 in Harrison’s Principle of Internal Medicine. 15 th ed. Braunwald E. et.al. USA: The McGraw-Hill Companies; 2001,(2), 1737-42.

3. Neil K, Laurie D. Text Book of Drug Induced Liver Disease. New York: Mareel-Dekker 2000; (2):2.

4. Sidney C, Roger D. Long Term Follow –up and Management of Asymptomatic Chronic Active Hepatitis. In : chap 3C Chronic Acute Liver Disease, London: Churchill Livingstone Edinburgh 1983;(2):332.

5. Chatterjee A, Prakashi SC. The Treatise on Indian Medicinal Plants. New Delhi: Publication and information Directorate, CSIR; 1994 ;(2): 27-29.

6. Vaidya Gogte VM.. Ayurvedic Pharmacology and Therapeutic Uses of Medicinal Plants .Swami Prakashananda Ayurveda Reasearch Centre (SPARC); Mumbai: Chaukhambha Publication 2000: 474-75.

7. Kirtikar KR, Basu BD. Indian Medicinal Plants.2nd ed. Dehradun:1999;(2):842- 45.

8. Gupta M, Mazumdar U K, Sambath Kumar R, SivakumarThangave, Gomathi P , Rajeshwar Y, Antioxidant defense system induced by methanol extract in rats liver. Pharmaceutical Biology .2005;43(5):411-19.

9.MazumderU K, Ramanathan S,Hepatoprotective Effect and Antioxidant Role of Caesalpinia bonducella on Paracetamol Induced Hepatotoxicity. Natural Product Science 2003;186-91.

10. Arya Vaidya Sala. Indian Medicinal Plants. Chennai: Orient Longman Ltd 1996; (1):320-22.

11. Kapoor LD. Handbook of Ayurvedic Medicinal Plants. 1st ed.Washington: CRC Press 2005:87-88.

12. Peter SR, Tinto WF, Mcleans S, Reynolds WF, Yu M. Cassane diterpenes from

8 C. bonducella. Phytochemistry; 1998; 47(6):1153-55.

13.Archana P, Tandan SK, Chandra S, Lal J. Antipyretic and Analgesic activity of C. bonducella seed. Phytotherapy Research 2005;19(5):376-81.

14. Sharma SR, Dwivedi SK, Swarup D. Hypoglycaemic, antihyperglycaemic and hypolipidemic activities of C. bonducella seeds in rats. Journal of Ethnopharmacology 1997;58(1):39-44.

15. Saeed MA, Sabir AW. Antibacterial Activity of C. bonducella seed. Fitoterapia 2001; 72(7):807-09.

16. Kokate CK. Practical Pharmacognocy. 4th ed. Delhi: Vallabh Prakashan 1994:107-11.

17. Khandelwal K R. Practical Pharmacognocy Techniques and Experiments 13th ed. Pune: Nirali Prakashan 2005:157-60.

18.Malaya Gupta, Mazumder UK, Thangavel SK, Gomathi P, Ramanathan SK “Antioxidant and Hepatoprotective Effects Of Bauhinia racemosa Against

Paracetamol and CCl 4 Induced Liver Damage in Rats. Iranian J of Pharmacol & Therapeutics 2004;3(1).

19.Jalalpure S, Patil M B, Prakash N S, Hemlata K, Manvi F V. Hepatoprotective activity of fruit of Piper longum Linn. Indian J Pharm Sci 2003; 65(4):363-66.

20.OECD/OCDE, OECD .Guidelines for the testing of Chemicals revised draft guidelines, Acute Oral Toxicity class methods, Revised Documents, October 2000:423.

21. Nobert Textbook of Fundamental Clinical Chemistry. USA: W B.Saunders Company 1970:747-70.

22. Kulkarni S K.Handbook of Experimental Pharmacology . 2 nd ed. New Delhi. Vallabh Prakashan 1993: 82-87.

9 9. Signature of Candidate 10. Remark of the guide The above information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance. 11. NAME & DESIGNATION OF (in block letters)

11.1 Guide Mr. PRAMOD H JM.Pharm. ASSISTANT PROFESSOR, DEPARTMENT OF PHARMACOGNOSY & PHYTOCHEMISTRY, K.L.E.SOCIETY’S COLLEGE OF PHARMACY, BELGAUM-10.

11.2 Signature 11.3 Co-guide (if any) - 11.4 Signature -

11.5 Head of the Department Dr S S JALALPURE M.Pharm.Ph.D PROFESSOR & HEAD, DEPARTMENT OF PHARMACOGNOSY & PHYTOCHEMISTRY, K.L.E.SOCIETY’S COLLEGE OF PHARMACY, BELGAUM.

11.6 Signature 12. 12.1 Remarks of the The above mentioned information is correct and I Chairman & Principal recommend the same for approval.

12.2 Signature

Dr. F.V.MANVI M.Pharm.Ph.D. PRINCIPAL, K.L.E.SOCIETY’S COLLEGE OF PHARMACY, BELGAUM-10

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