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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. NAME OF THE CANDIDATE

AND ADDRESS

SHUKLA SUNILKUMAR TRIVENIPRASAD

DEPT. OF PHARMACOLOGY

SETs COLLEGE OF PHARMACY

S.R.NAGAR,

DHARWAD-580002

2. NAME OF THE INSTITUTION SETs COLLEGE OF PHARMACY

S. R. NAGAR,

DHARWAD-580002

3. COURSE OF STUDY AND

SUBJECT

MASTER OF PHARMACY IN

PHARMACOLOGY

4. DATE OF ADMISSION TO

COURSE

JUNE-2009

5.TITLE OF THE TOPIC

EVALUATION OF HEPATOPROTECTIVE AND ANTIOXIDANT ACTIVITY OF

ENDOPHYTIC CRUDE FRACTIONS OF OCIMUM SANCTUMLINN. IN CCL4

INDUCED HEPATIC DAMAGE IN RATS

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6. BRIEF RESUME OF THE INTENDED WORK

6.1 NEED FOR THE STUDY:

Chronic liver diseases are common worldwide and are characterized by a progressive

evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis and hepatocellular

carcinoma.1Liver diseases are mainly caused by toxic chemicals (antibiotics, chemotherapeutics,

peroxidised oil, aflatoxin, CCl4, chlorinated hydro-carbons, etc.) excess consumption of alcohol,

infections and autoimmune disorder. Most of the hepatotoxic chemicals damage liver cells

mainly by inducing lipid peroxidation and other oxidative damages in liver2,3

.

Carbon tetra chloride (CCl4) is extensively used xenobiotics induces lipid peroxidation and

toxicity, also widely used as a model for screening hepatoprotectives4.Several studies have

previously demonstrated that antioxidant prevents CCl4 toxicity particularly hepatotoxicity by

inhibiting lipid peroxidation and increasing antioxidant enzyme activities3. Therefore protective

mechanism relevant to the liver is of particular interest.

Ocimum sanctum Linn. syn; O.tenuiflorum Family: Labiatae, a small branched herb,

mainly shows antimalarial, antipyretic, anti-inflammatory, antidiabetic, antiasthmatic,

nematicidal effect, and is a good immune-modulatory agent5. Ocimum sanctum leaf extract

shows hepatoprotective effect in hepatotoxic rats6. The plant has shown promise in alleviating

hepatic dysfunction in clinical trials on patients suffering from viral hepatitis5

.Endophytes, microorganisms that reside in the internal tissues of living plants without

causing any immediate overt negative effects, have been found in every plant species examined

to date and recognized as potential sources of novel compounds for exploitation in medicine

industry with more and more bioactive natural products isolated from the endophytes7.

Endophytic fungi isolated from Ocimum sanctum Linn. known asHyalopus sp. were screened to

determine their extracellular tannase producing ability8.

Therefore the present study is to evaluate antioxidant and hepatoprotective effect of

endophytic crude fractions ofOcimum sanctum Linn. on CCl4 induced hepatic damage in albino

wistar rats.

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6.2 REVIEW OF LITERATURE

CCl4 is a commonly used hepatotoxin used for production of experimental liver

toxicity. CCl4 is activated by cytochrome (CYP)2E1, CYP2B1 or CYP2B2, and possibly

CYP3A, to form the trichloromethyl radical, (CCl3-).This radical can also react with oxygen to

form the trichloromethylperoxy radical (CCl3OO+), a highly reactive species. Further initiates

the chain reaction of lipid peroxidation, which attacks and destroys polyunsaturated fatty acids.

This affects the permeabilities of mitochondrial, endoplasmic reticulum, and plasma

membranes, resulting in the loss of cellular calcium sequestration and homeostasis, which can

contribute heavily to subsequent cell damage. Among the degradation products of fatty acids are

reactive aldehydes, especially 4-hydroxynonenal, which bind easily to functional groups of

proteins and damage marker enzymes GOT(Glutamate Oxaloacetate Transaminase), GPT

(Glutamate Pyruvate Transaminase) and LDH(Lactate Dehydrogenase) are released in serum9.

Measurement of serum enzyme levels has provided a powerful tool for studies of

hepatotoxicity10

.

Ocimum sanctum Linn. contains major essential oils such as eugenol, carvacrol, nerol and

eugenolmethylether. Leaves have been reported to contain ursolic acid, apigenin, luteolin,

apigenin-7-O-glucuronide, luteolin-7-O-glucuronide, and molludistin. The ethanol extract (90%)

of the leaves showed hepatoprotective effect against paracetamol-induced liver damage

11,6

.Endophytes have been found virtually in every plant studied, where they colonize the

internal tissues of their host plant and can form a range of different relationships including

symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes appear to originate

from the rhizosphere or phyllosphere; however, some may be transmitted through the seed.

Endophytic bacteria can promote plant growth and yield and also can act as biocontrol agents.

Endophytes can also be beneficial to their host by producing a range of bioactive compound that

could be harnessed for potential use in medicine, agriculture or industry12

.

Endophytic microorganisms from medicinal plants are a potential source of a diverse array

of bioactive metabolites which can be used for the development of some potent drugs. Many

authors have isolated endophytic microbes from various medicinal plants with antioxidant13

,

antibacterial14

, antimicrobial7, anticancer, antidiabetic, immune suppressant activity

14. Further

many more examples in which endophytes producing various secondary metabolites such as

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taxol , asperagenase , campothecin , as anticancer compounds and artimisinin as

antimalarial etc.

Endophytes are also discovered with some novel potent molecules such as antibiotics7,

alkaloids19

and antidiabetics showing insulin mimetic effect by oral route20

.

Hence, in this investigation, an attempt will be made to evaluate endophytic crude fractions

from Ocimum sanctum Linn. for hepatoprotective activity on the basis of widely accepted

hypothesis and literatures that some endophytes from medicinal plants produce same secondary

metabolites as that of the parent plant20

.

6.3 OBJECTIVES OF STUDY.

To evaluate and establish the in-vitro antioxidant activity of different endophytic crudefractions from Ocimum sanctum Linn.

To carry out acute toxicity studies for potential endophytic fractions. To evaluate potential endophytic crude fractions of Ocimum sanctum Linn. for in-vivo

hepatoprotective activity in CCl4 induced liver damage in rats.

To establish histopathological and biochemical support for hepatoprotective activity.7. MATERIALS AND METHODS

7.1 SOURCE OF DATA: Indian Journal of Physiology and Pharmacology Indian Journal of Pharmacology Indian Journal of Pharmaceutical Sciences Indian Journal of Science and Technology Journal of General and Applied Microbiology Applied Biochemistry and Microbiology Planta Medica Pubmed J-Gate@Helinet(RGUHS)

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7.2 METHOD OF COLLECTION OF DATA:

The data is generated using laboratory experimental techniques. The isolated and

characterized endophytic crude fractions of Ocimum sanctum Linn. will be

procured from Department of Agricultural Microbiology, University of Agricultural Sciences,

Dharwad.

In-vivo pharmacological activities will be carried out on male wistar rats. The results and data

obtained from present study will be analyzed by statistical methods.

1. In-vitroantioxidant activity evaluation21,22

:

2,2-diphenyl-1-picrylhydrazyl [DPPH] Scavenging activity Hydroxyl radical scavenging activity Lipid peroxidation Glutathione Catalase

2. Animals :

Adult male Wistar albino rats weighing 150200 g will be used. The animals will be

maintained under controlled condition of temperature (23 2C), cycles. The animals are

randomized into experimental and control groups and housed each in sanitized polypropylene

cage containing sterile paddy husk. They will have free access to standard pellets as basal dietand waterad libitum.

3. Acute toxicity studies23

:

The guidelines described by OECD will be adopted for the determination of LD50 on Swiss

albino mice and 1/10th

of LD50 will be taken as dose for the study.

4. Pharmacological studies( Hepatoprotective activity)

Model: Carbon tetra chloride induced hepatic damage in rats.

(a) CCl4 induced peroxidation damage in liver24:

Albino wistar rats weighing 150-200 g, will be maintained in standard environmental

condition and fed with standard laboratory diet and water ad libitum will be used for the

experiment. CCl4 will used to induce hepatotoxicity in rats. Different endophytic crude

fractions will be administered orally at different dosage level to study the efficacy against

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CCl4 induced peroxidative damage in liver of rats.

(b) In-vivo hepatoprotective activity evaluation22

:

Animals will be sacrificed by cervical dislocation, livers excised and the

following parameters will be estimated.

Serum glutamate pyruvate transaminase (SGOT) Serum glutamate oxaloacetate transaminase (SGPT) Serum Alkaline phosphatase (SALP) Total Bilirubin Total Protein Total Triglycerides Total cholesterol

HISTOPATHOLOGICAL STUDIES:

The liver tissues will be dissected out and fixed in 10 % neutral buffer formalin and they

are subjected to histopathological examination.

7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTION TO

BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO,

PLEASE MENTION BRIEFLY.

Yes, the above study requires in-vivo screening techniques on Albino Wistar male rats.

7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN

CASE OF 7.3?

The Institutional Animal Ethical Committee (IAEC) has approved the proposed work.

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8 . REFERENCE :

1. Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free RadicBiol Med 2003;34:110.

2. Benzie IF. Lipid peroxidation: a review of causes, consequences, measurement anddietary influences. Int J Food Sci Nutr 1996;47:233.

3. Poly G, Albano E, Dianzani MU. The role of lipid peroxidation in liver damage. ChemPhysiol Lipids 1987;45:117-42.

4. Jeon TI, Hwang SG, Park NG, Jung YR, Shin SI, Choi SD, Park DK Antioxidativeeffect of chitosan on chronic carbon tetra chloride induced hepatic injury in rats.

Toxicol 2003 May 1;187(1):67-73.

5. The wealth of India, A dictionary of Indian raw materials & industrial products.Reprinted 2006(New Delhi). CSIR: National institute of science communication &

information resource. p. 219-21 (1st

supplement series. Vol 4 (J-Q),).

6. Razvi SU. Effect ofOcimum sanctum Linn. leaf extract on hepatotoxicity induced byantitubercular drugs in rats. Indian J Physiol Pharmacol 2003; 47(04):465-70.

7. Guo B, Wang Y, Sun X, Tang K. Bioactive natural products from endophytes: AReview. Appl Biochem Microbiol 2008;44(2):136-42.

8. Mahapatra S, Banerjee D. Extracellular tannase production by endophyticHyalopus sp.J Gen Appl Microbiol 2009;55:255-9.

9. Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action ofhaloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol

2003;33(2):105-36.

10.Achliya GS, Koyagale NR, Wadodkar GS, Dorle AK. Hepatoprotective activity ofPanchagavya Ghrita against carbon tetrachloride induced hepatotoxicity in rats.

Indian J Pharmacol 2003;35:308-11.

11.Khare CP. Indian medicinal plants, An illustrated dictionary. USA (NY):Springer; 2007. p.445-6.

12.Ryan RP, Germaine K, Franks A, Bacterial endophytes: Recent developments andapplication. FEMS Microbiol Lett 2008 Jan;278(1):1-9.

13.Huang WY, Cai YZ, Xing J, Cork H, Sun M. A potential antioxidant resource:

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Weber%20LW%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Boll%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Stampfl%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Stampfl%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Boll%20M%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Weber%20LW%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

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Endophytic fungi from medicinal plants. Econ botany 2007;61(1):14-30.

14.Gangadevi V, Sethumeenal S, Yogeswari S, Rani G. Screening endophytic fungiisolated from a medicinal plant, Acalypha indica L. for antibacterial activity.

Indian J Sci Tech 2008 Sep;1(5):1-6.

15.Li JY, Strobel G, Sidhu R, Hess WM, Endophytic taxol-producing fungi from baldcypress, Taxodium distichum.Microbiol 1996 Aug;142( 8):2223-6.

16.Theantann T, Hyde KD, Lumyong S. Asparaginse production by endophytic fungiisolated from some Thai medicinal plants.KMITL Sci Tech J 2007 Nov;7(1):13-8.

17.Touseef A, Khajuria RK, Puri SC, Verma V. Determination and quantification ofcampothecin in an endophytic fungus by liquid chromatography positive mode

electrospray ionization tandem mass spectrometry.Current sci 2006 July25;91(2):208-

12.

18.Tiwari R, Saini RK, Singh AK, Gupta MM; Effect of endophytes on the yieldenhancing capabilities inArtemisia annua . NIM 2008;56.

19.Sugawara K, Inoue T, Yamashita M, Ohkubo H. Distribution of the endophytic fungus,Neotyphodium occultans in naturalized Italian ryegrass in western Japan and its

production of bioactive alkaloids known to repel insect pests. Grassland Sci

2006;52:147-52.

20.Zhang B, Salituro G, Szalkowski D, Li Z. Discovery of small molecule insulin mimeticwith antidiabetic activity in mice. Sci 1999 May 7; 284:974-7.

21.Hayashi T, Maruyama H, Kasai R, Hattori K. Ellagitannins from Lagerstroemiaspeciosa as activators of glucose transport in fat cells. Planta Med 2002;68(2):1735.

22.Prabhakar KR, Veerapur VP, Parihar Vipan K, Priyadarsini KI. Evaluation andoptimization of radioprotective activity of Coronopus didymus Linn. In -Irradiated

Mice. Int J Radiat 2006;82(8):525-36.

23.Ghosh MN. Fundamentals of Experimental Pharmacology. 2nded. Culcutta: ScientificBook Agency; 1984. p.154-7.

24.Hukkeri VI, Akki KS, Sureban RR, Gopalakrishna B, Byahatti VV, Rajendra SV.Hepatoprotective activity of the leaves ofNyctanthes arbor-tristis linn. Indian J Pharm

Sci 2006;68:542-3.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Li%20JY%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Li%20JY%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Strobel%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sidhu%20R%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hess%20WM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Hess%20WM%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sidhu%20R%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Strobel%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstracthttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Li%20JY%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

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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 AND DESIGNATION OF

11.1 GUIDE

SINGNATURE

Dr. V. H. KULKARNI M. Pharm., Ph.D.

PROFESSOR & PRINCIPAL

S.E.TS COLLEGE OF PHARMACYDHARWAD-580002.

11.3 CO-GUIDE

SINGNATURE

Dr. K. S. JAGDEESH M.Sc(Micro)., Ph.D.PROFESSOR & HEADDEPT. OF AGRICULTURAL MICROBIOLOGY,

UNIVERSITY OF AGRICULTURAL SCIENCE

DHARWAD-580005.

11.5 HEAD OF THE

DEPARTMENT

SIGNATURE

Dr. V. H. KULKARNI M. Pharm., Ph.D.

PROFESSOR & PRINCIPALS.E.TS COLLEGE OF PHARMACY

DHARWAD-580002.

12. 12.1 REMARK OF THE PRINCIPAL

The above mentioned information is correct and I recommend the same for Approval.

12.2 SIGNATURE

Dr. V. H. KULKARNI M. Pharm., Ph.D.PRINCIPAL

S.E.TS COLLEGE OF PHARMACYDHARWAD-580002.