A STUDY OF PHYTOCHEMICAL AND PHARMACOLOGICAL EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF MERREMIA EMARGINATA WHOLE PLANT M.

PHARM DISSERTATION PROTOCOL SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

BY

TATINENI TEJASWINI
B. Pharm

UNDER THE GUIDANCE OF

RAJSHEKAR BHANDE
M. Pharm., (Ph.D.)

P. G. DEPARTMENT OF PHARMACOLOGY R.R.KS COLLEGE OF PHARMACY, NAUBAD,BIDAR-585402 2010-11

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.

Annexure II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01

Name and Address of the Candidate

TATINENI TEJASWINI,D/O T.GANDHI,H.NO.,3-10-250, GUNTI JANGIAH NAGAR COLONY,L.B.NAGAR,RING ROAD, HYD-500074, ANDHRA PRADESH. R.R.KS COLLEGE OF PHARMACY, NAUBAD,BIDAR-585402, KARNATAKA. M. Pharm (Pharmacology) 22.11.2010

02

Name of the Institution

03 04

Course of the Study Branch Date of Admission to course

05

Title of the Topic

A STUDY OF PHYTOCHEMICAL AND PHARMACOLOGICAL EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF MERREMIA EMARGINATA WHOLE PLANT

Brief resume of the intended work 06

Enclosure I Enclosure II Enclosure III Enclosure IV Enclosure V Enclosure VI

Yes, Registration No: 361/01/c/CPCSEA (Copy enclosed) Enclosure VII

6.1. Need for the Study 6.2. Review of the Literature

6.3. Objective of the Study Materials and Methods

7.1. Source of data

7.2. Methods of collection of data 07

7.3. Does the study require any Investigations on animals?

If yes give details

7.4. Has ethical clearance been obtained form your institution in case of 7.3.
08 List of References

09

Signature of the Candidate (TATINENI TEJASWINI)

10

Remarks of the Guide

The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by the above said student under my supervision.

Name and Designation of

(in Block Letters) 11.1. Guide

Mr. RAJSHEKAR BHANDE

M. Pharm., ( Ph.D) Professor & H.O.D. P.G. Department of Pharmacology, R.R.KS College Of Pharmacy, Naubad, Karnataka.

11.2.Signature
-----------------------------

11

11.3.Co-Guide (if any) 11.4.Signature 11.5. Head of the Department

-No-

Mr. RAJSHEKAR BHANDE

M.Pharm., ( Ph.D.) Professor & H.O.D. P.G. Department of Pharmacology, R.R.KS College Of Pharmacy, Naubad, Karnataka.

11.6.Signature
Remarks of the Principal

The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.

12

12.1. Signature
(Dr. K.SRINIVAS RAO) M.Pharm., Ph.D. Principal, R.R.KS College Of Pharmacy, Naubad,Bidar, Karnataka.

ENCLOSURE- I O6. Breif Resume Of Intended Work:6.1. Need for the study:
Liver is the main organ system physiologically involved in the metabolism of various xenobiotics, environmental pollutants etc. consequently they are exposed to oxidative stress and free radicals. This results in the tissue necrosis and damage of the organ system. Therefore several attempts are been made to protect these organs from free radical challenges. The organ system like hepatic are highly prone to attack by generation of excessive concentration of free radicals. Many anti-oxidants have been used to protect the organs from thefree radical challenges. Several researches are attempting to explore the possibility of using herbs containing anti-oxidants as organ protective agents. In one of our field survey awidely grown, creeping, smooth and hairy herb by name Merremia emerginata1 to convolvulaceae found through out in India, in plains and hills2. The phytochemical constituents of Merremia emerginata is not clearly known but it is mentioned that plants of convulvulaceae family proven to show anti-oxidant property because of the presence of phenolic compounds3. However, the pharmacological and phytochemical profile of this plant is incomplete, but traditionally it has been useful in nepropathy, uropathy, cardiac diseases, gastropathy, metropathy, fever, anaemia, leucoderma, strangury, otolgia, and rat bite2. Keeping in review and hypothesis of anti-oxidant principles and organ protection in view the whole plant of Merremia emarginata is selected for assessing the anti oxidant and organ protective potential.

ENCLOSURE- II 6.2. Review of Literature:

Merremia emerginata (convolvulaceae) also commonly called as kupit-kupit and in english kidney-leaf morning glory1.

Synonyms:
Musikakarni, bhudari, bhava, musikahvaya, unduru-karnika4.

Description:
It is a procumbent herb spreading upto1m and contains yellow coloured flowers4. It is a creeping, perennial herb rooting at the nodes; leaves simple, long stalked, reniform or ovate-cordate; axillary on 1-3 flowered very short peduncles which are shorter than the petioles; fruits subglobulose, capsules wid 2-4 glabrous light brown seeds2.

Phytochemical constituents:
Merremia seeds contains biologically active chemicals which are p-coumaric acid,ferulic,caffeic,sinapic acid esters4.But,it is also proven that the plant contains tropane alkaloids5. So, yet exact phytochemical evaluation is not exactly known.

Medicinal uses of different parts of plants:

Root -laxative, diuretic, eye diseases. Whole plant -bronchitis, inflammation, fever, paralysis6. Review of literature till date regarding Merremia emerginata is as follows:1. A.V.Babu, R.S.C Rao, K.G.Kumar, B.H.Babu and P.V.V Satyanarayana(2009)3, Hexane(IA), Ethyl acetate(IB), Methanol(IC), Aqueos methanol(25%) extrats of Merremia

emerginata were tested for anti-oxidant and anti-obese activities. The extracts showed encouraging results. 2. Sudhavani V, Chinnikrishnaiah V, Raghu Moorty V, Raghavendra H.G, Ranganayakulu D(2010)5, The ethanolic extract of the Merremia emerginata has offered significant nephroprotector activity against cisplatin induced nephrotoxic rats. 3. Babu, A.V.; Sujatha,K.; Valli, A.S; Narayana, K.J.P.; Babu,B.H.; Satyanarayana,P.V.V.(2009)7, Hexane, Ethyl acetate, Methanol, extrats of Merremia emerginata were studied by testing the inhibitory activity against cancer cell proliferation and pro-inflammatory cytokine TNFalpha. It had shown significant anticancer and anti-inflammatory activities. 4. Kristina Jenett-Siems,Robert weigi ,Anke Bohm,Petra Mann,Britta Tofern-Reblin ,Sonja C.Ott,Azar Ghomian ,Maki Kaloga,Karsten Siems,Ludger Witte,Monika Hilker,Frank Muller,Eckart Eich(2005)8, Their GC-MS study mentioned that members of almost all sections af 18 Merremia species showed the occurance of tropane and pyrollidine alkaloids.

ENCLOSURE- III 6.3 Objective of study:

1. To asses acute toxicity of absolute alcohol(99%) extract of Merremia emerginata whole plant. 2. Extraction of whole plant of Merremia emerginata with 99% absolute alcohol

and phytochemical screening. 3. To asses anti-oxidant property(in-vitro). 4. To evaluate hepatoprotective activity of absolute alcohol(99%) extract of Merremia emerginata whole plant.

ENCLOSURE-IV 7. Materials and Methods 7.1 Source of data

Review of literature till date regarding Merremia emerginata was carried out by referring 1. Chemical abstracts, 2. Medicinal and aromatic plant abstracts 3. Other scientific journals.

ENCLOSURE-V 7.2 Method of collection of data

By animal experiments and laboratory investigations from the pharmacology laboratory of our institution. Albino rats and mice will be used for the screening purpose.

a. collection of plant material & extraction & phytochemical screening.

For this study whole plant of Merremia emerginata will be collected from the fields of surrounding Bidar district. Whole parts of the plant will be collected, dried in shade and powdered. The drug will be subjected to Soxhlet extraction under reduced pressure and controlled temperature. Crude extracts obtained will be used for phytochemical and pharmacological investigations .

b. Invitro antioxidant activity9.

Invitro antioxidant models are, 1. Reduing power 2. Superoxide anion scavenging activity 3. Hydroxyl radical scavenging activity 4. Nitric oxide radical scavenging activity

c. Determiantion of acute toxicity.

Fixed dose method(OECD guide line no.420) of CPCSEA will be adapted to perform acute toxicity of the extracts. The Albino mice (swiss strain) weighing between 20-25gms will be utilized for this purpose

d. Screening of hepatoprotective activity.

Hepatoprotective activity of alcohol extract of whole plant of Merremia emerginata will be evaluated for the possible preventive and curative effects on..

1. Ccl4 induced hepatotoxicity 10.

A. Inclusion criteria in the present study only healthy albino rats of either sex(wister strains), 150-200gms body are selected. Any animal not satisfying above criteria will be excluded. B. Five groups of rats containing six animals each. Group 1- negative control Group 2- positive control Group 3- standard Group 4- test I Group 5- test II

2. Paracetamol induced hepatotoxicity 11.

A. Inclusion criteria in the present study only healthy albino rats of either sex(wiater strains), 150-200gms body are selected. Any animal not satisfying above criteria will be excluded. B.Five groups of rats containing six animals each. Group 1- negative control Group 2- positive control Group 3- standard Group 4- test I Group 5- test II

Parameters assess for the liver function.

1. Serum glutamate pyruvate transaminase(SGPT) 2. Serum glutamate oxaloacetate transaminase(SGOT) 3. Serum alkaline phosphatase(ALP) 4. Serum bilurubin(total and direct)

e. Stastical analysis
The data obtained from the above findings will be subjected to Stastical analysis using one-way ANOVA followed by Student-t-test.

f. Work plan details

Total duration for the completion of proposed research work may be ten months 1 .Authentication and collection of plant material 2. Duration of experimentation on animals including preparation of crude extracts. 3. Literature review 4. Dissertion writing and communication of research -One month. -Five months.

-Two months. -Two months.

ENCLOSURE-VI
7. 4 Does the study require any investigations to be conducted on patients or other humans and animals? If so please describe briefly. The proposed study requires the investigations on albino rats of either sex (Wister strain) weighing 150200g for the hepatoprotective activity. Whereas albino mice of Swiss Strain weighing 20-25g will be utilized for the acute toxicity study. 7. 5 Has ethical clearance been obtained from your institution in case of 7.3? The present study clearance been obtained from Institutional Animal Ethics Committee(IAEC certificate is enclosed)

ENCLOSURE-VII 8. List of references.

1. Kupit-kupit, Merremi emarginata-Phillippine medicinal plants.

2. Indian Medicinal Plants, oriental legmen 1994, vol 4, coll.no. AVS 2136, pg.no.18. 3. Babu, A.V., R.S.C.Rao, K.G. Kumar, B.H. Babu and P.V.V. Satynarayana. Biological activity of Merremia emarginata crude exacts in different solvents. Res. J. Med. Plant. 3:2009, pg.no.134-140. 4. Illustrated Dravya Guna Vijnana by Dr. J. L. N. Sastry, farworded by Prof. K. L. Chunekar. A. M. G, Ph. d, F. N. A. A., IInd edition 2005, vol-II; pg.no-861-862.

5. Sudhavani V, Chinnikrishnaiah V, Raghu Moorthy V, Raghavendra H.G, Ranganayakulu D; Journal of advances in Drug Research, Vol 1, Issue 1, Dec.2010; Original article; ISSN:2230-7761.

6. Dr.K.Madhava Chetty, K.Sivaji, K.Tulsi Rao, Flowering plants of Chittoor district Andhra Pradesh, India, first edition-2008, pg.no.224. 7. Babu,A.V; Sujatha, K,; Valli,A.S,; Narayana,K.J.P,; Babu,B.H.; Satyanarayana, P.V.V.;Biosciences, Biotechnology Research Article 2009 Vol.6 No.2,pg.no. 835-838, ISSN 0973-1245,pg.no. 835-838.

8. Kristina Jenett-Siems, Robert Weigi, Anke Bohm , Petra Mann, Britta Tofern-Reblin, Sonja C.Ott, Azar Ghomian, Maki Kaloga, Karsten Siems, Ludger Witte, Monika Hilker, Frank Muller and Eckart Eich, Science Direct Phytochemistry, Volume 66, Issue 12, June 2005, pg.no.1448-1464.

9. In-vitro models for anti-oxidant activity evaluation and some medicinal plants possessing antioxidant properties:An overview, S.Chanda and R.Dave, African Journal of Microbiological Research Vol.3,Issue 13, December 2009, pg.no.981-996.

10. Hepatoprotective effect of the methanolic extract of whole plant of

Borreria articularis on carbon

tetrachloride induced hepatotoxicity in albino rats, H. Parameshwar, B.Ravi kumar, G.Krishna Mohan, Raju Vastavya, Y. Narsimha Reddy, JPRHC, Vol 2, Issue 4, pg.no.285-292.

11. Hepatoprotective activity of Hepax- A polyherbal formulation, VC Devaraj, B Gopala Krishna, GL Viswanatha, Jagadish V Kamath, Sanjay Kumar, Asian Pacific Journal of Tropical Biomedicine 2011 , pg.no .142-146.