IN-VITRO AND IN-VIVO EVALUATION OF ENDOPHYTIC FRACTIONS OFTINOSPORA CORDIFOLIA(Willd.) MiersFOR HEPATOPROTECTIVE ACTIVITY IN RATS

M. Pharm Dissertation Protocol Submitted to

RajivGandhiUniversity of Health Sciences, Karnataka

Bangalore – 560041

By

Mr.Hanamant Koppad.

B.Pharm.

Under the Guidance of

Dr. P. V. Habbu.

M. Pharm., Ph. D.

Professor

Post Graduate Department of Pharmacognosy,

SET’S College of Pharmacy,

S.R.Nagar, Dharwad,

Karnataka-580002

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / NAME OF THE CANDIDATE AND ADDRESS / HANAMANT KOPPAD
DEPT. OF PHARMACOGNOSY
SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
2 / NAME OF THE INSTITUTION / SET’S COLLEGE OF PHARMACY
S.R.NAGAR, DHARWAD,
KARNATAKA – 580002.
3 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOGNOSY
4 / DATE OF THE ADMISSION / JUNE2010
5 /

TITLE OF THE TOPIC

“IN-VITRO AND IN-VIVO EVALUATION OF ENDOPHYTIC FRACTIONS OFTINOSPORACORDIFOLIA(Willd.) Miers FOR HEPATOPROTECTIVE ACTIVITY IN RATS”
6.0 / BRIEF RESUME OF THE INTENDED WORK
6.1 / Need for study
The need for new and useful compounds to provide assistance and relief in all aspects of the human condition is ever growing. Endophytes (Gr. Endon, within; phyton, plant) – the term was first coined by de Bary1 and has become deeply embedded in the literature ever since. At present, endophytic organisms are defined as “microbes that colonize living, internal tissues of plants without causing any immediate, over negative effects”2. First reports describing these microbes date back to the turn of 19th and 20th century3. The most frequently encountered endophytes are representatives of the fungi; however, the existence of many endophytic bacteria has been documented as well. A variety of microorganisms, mainly bacteria, fungi and actinomycetes inhibit plants and are, therefore, known as endophytic. Endophytes are relatively unstudied and these are potential sources of novel natural products for exploitation in medicine. The recent development and implementation of new technologies offers unique opportunities in the screening of natural products and will reestablish them as a major source for drug discovery4.
Free radical induced lipid peroxidation is believed to be one of the major causes of cell membrane damage leading to a number of pathological situations5-7. A number of recent reports clearly demonstrated that in addition to hepatic problems, CCl4 also causes disorders in kidney, lungs, testis and brain as well as in blood generating free radicals8-11. Lipid peroxides produced from unsaturated fatty acids via radicals, cause histotoxicity and promote the formation of additional free radicals in a chain reaction type. It is thought that, if the in-vivo activity of enzymes or scavengers is not high enough to inhibit the radicals, various diseases such as liver disease, diabetes and accelerated aging may result12. In the modern medicine, plants occupy significant birth as raw materials for some important drug preparations13-15.
In recent years, there has been renewed interest in the treatment against different diseases using herbal drugs as they are generally non-toxic and WHO has also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs. No scientific data regarding the in-vitro and in-vivo evaluation of endophytic fractions of Tinospora cordifolia for hepatoprotective activity is available. Hence, the present study is designed to evaluate the hepatoprotective activity of Endophytic fractions of Tinospora cordifolia on experimental rats.
6.2 / Review of literature
The traditional Indian medicinal plants act as antiradicals and DNA cleavage protectors.16Moringa oleifera,17Eclipt alba,18Phyllanthus niruri,19,21Picrorrhiza kurroa22 possess hepatoprotective property against toxins and drugs induced hepatotoxicity.23
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, commensalisticand trophobiotic. 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 industry.24Endophytes 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 antioxidant,25 antibacterial,26 antimicrobial.27,28 Further many more examples in which endophytes producing various secondary metabolites such as taxol,29 asperagenase,30 campothecin,31 as anticancer compounds and artimisinin32 as antimalarial etc.
Guduchi [Tinospora cordifolia (Willd) Miers] is a large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae33,34. It is distributed throughout tropical India subcontinent and China, ascending to an altitude of 300m. It is widely used in veterinary folk medicine/ayurvedic system of medicine for its general tonic, antiperiodic, antispasmodic, anti-inflammatory, antistress, antiarthritic, antiallergic, antidiabetic, antioxidant, antileprotic, antimalarial, hepatoprotective, immunomodulatory, and antineoplastic activities34-39.
Phytochemical review revealed the presence of tinosporafuranol, tinodporafurandiol, tinosporaclerodanol40, clerodane diterpene, tinoscorside and aporphine alkaloids41.
In view of the diversified contribution of endophytes in the discovery of novel secondary metabolites and on the basis of widely occupied hypothesis and literatures that some endophytes from medicinal plants produce same secondary metabolites as that of the parent plant42 an attempt will be made in this protocol to isolate and evaluate endophytic crude fractions (bacteria, fungi or actinomycetes) from Tinospora cordifolia for hepatoprotective activity.
6.3 / Objectives of the study
Isolation of endophytes (bacteria, fungi or actinomycetes) from Tinospora cordifolia.
Characterization of endophytes by qualitative and quantitative analysis.
In-vitro antioxidant activity of endophyte crude fractions.
To evaluate the hepatoprotective activity of potential endophytic crude fractions from Tinospora cordifoliausing animal models.
7 / MATERIALS AND METHODS
7.1 / Sources of data
The source of data are
  • Literature survey
  • Books and journals
  • Laboratory experiments on animals
  • Internet.

7.2 / Method of collection of Data
The data is generated using laboratory experimental techniques. The isolated and characterized endophytic crude fractions of Tinospora cordifoliawill be procured from Department of Agricultural Microbiology, University of Agricultural Science, Dharwad.
7.3 / Methodology
  1. In-Vitro Free Radical Scavenging Activity43-45:
As per reported studies,the following parameters will be studied.
A.Reducing power.
B. Hydroxyl radical scavenging activity.
C. 2,2-diphenyl-1-picrylhydrazyl [DPPH] Scavenging activity.
  1. Pharmacological Evaluation:
Animals:
Albino wistar male rats weighing 150-200g will be used. The animals will be maintained under controlled conditions of temperature (23 ± 2C), humidity (50 ± 5%) and 12-h light-dark cycles. The animals are randomized into experimental and control groups and housed each in two sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellets as basal diet and water ad libitum.
Acute toxicity studies46 :
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.
HEPATO PROTECTIVE ACTIVITY
  1. CCl4 induced hepatotoxicity47:
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 CCl4 induced peroxidative damage in liver of rats.
B. Biochemical parameters48 :
Animals will be sacrificed by higher dose of thiopental sodium, livers excised and the following parameters will be estimated.
 Serum glutamate pyruvate transaminase (SGPT)
 Serum glutamate oxaloacetate transaminase (SGOT)
 Serum Alkaline Phosphatase (SALP)
Direct Bilirubin
Total Bilirubin
Total Protein
C. Histopathological studies:
The liver tissues will be dissected out and fixed in 10% neutral buffer formalin and they are subjected to histopathological examination.
D.Measurement of antioxidant enzymes:
Five percent liver homogenate will be prepared with 0.15 M KCl and centrifuged at 1000 rpm for 10 min. The cell free supernatant will be used for the estimation of Super oxide dismutase (SOD)49, Catalase50 and Lipid Peroxidase51.
7.4 / Does the study require any investigation or interventions to be conducted on patients or other humans or animals?If so, please describe briefly.
The above study requires investigations to be done on the albino rats of Wister strain for the determination of hepatoprotective activity. The study is planned in accordance with the procedure reported in the literature.
7.5 / Has ethical clearance been obtained from your institution in case of 7.3?
The Institutional Animal Ethical Committee (IAEC) has approved the proposed work (copy enclosed).
8 / REFERENCES:
  1. Bary DA. Morphologie und Physiologie der Pilze, Flechten, und Myxomyceten. Hofmeister’s Handbook of Physiological Botony. Leipzig, Germany. 1866;II.
  2. Stone JK, Bacon CW, White JF. An overview of endophytic microbes: endopytism defined. In: Bacon CW, White JF, editors. Microbial edophytes. New York(NY): Marcel Dekker Inc; 2000. p. 13-29.
  3. Freeman EM. The seed fungus of Loliiium temulentum L. Phil Trans R Soc Lond, Biol 1904;196:1-27.
  4. Strobel G, Daisy B. Bioprospecting for microbial endophytes and their natural products. Microbial Mol Biol Rev 2003;67:491-502.
  5. Halliwell B. Oxygen species in pathology with special reference to the skin. In oxidative stress in dermatology. New York(NY): Marcell Dekker Inc; 1993. p. 3-11.
  1. Oberley LW. Free radicals and Diabetes. Free Rad Biol Med. 1988;5:113-24.A
  2. Slater TF. Free radical mechanism in tissue injury. Biochem J 1984;222:1-15.
  3. Chaurbonneua M, Brodeur J, Souich DP, Plaa GL. Correlation between acetone potentiated CCl4- induced liver injury and blood concentration after inhalation or oral administration. Tox appl Pharmacol 1986;84:286-94.
  4. Ahamad FF, Cowan DL, Suns AY. Detection of free radical formation in various tissues after acute carbon tetra chloride administration in gerbil. Life Sci 1987;41:2469-75.
  5. Ohata Y, Nikida K, Sasaki E, Kongo M, Ishiguro I. Atteneuation of disrupted hepatic active oxygen metabolism with the recovery of acute liver injury in rats intoxicated with carbon tetra chloride. Res Commun Mol Pathol Pharmacol 1997;95:197-201
  1. Ozturk F, Ucar M, Ozturk IC, Vardi N, Batcioglu K. Carbon tetra chloride induced nephrotoxicity and protective effects of betaine in Sprague-dawley rats. Urol 2003;62:353-56.
  2. Nikis E. Antioxidants, free radicals and biological defence. In: Niki E, Shimasaki SH, Mino M (Eds), Tokyo: Japan scientific societies Press; 1995. (vol 3).
  3. Mejia DDG, Mares RMV. Leaf extracts from Ardisia compressa protects against 1- nitropyrene induced cytotoxicity and its antioxidant defense disruption in cultured rat hepatocytes. Toxicol 2002;179:151-62.
  4. Iwu MM, Jackson JE, Schuster BG. Medicinal plants in the fight against leishmaniasis. Parasitol 1004;10:65-68.
  5. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants, Public and information directorate. New Delhi: CSIR; 1986. p. 44-46.
  6. Russo A, Izzo AA, Cardile V, Borelli F, Vanella A. Indian medicinal Plants as Antiradicals and DNA cleavage protectors. Phyt med 2001;8:125-32.
  7. Pari L, Kumar NA. Hepatoprotective activity of Moringa oleifera in antitubercular drug induced liver damage in rats. J Med Food 2002;5:171-77.
  8. Singh B, Saxena AK, Chandan BK, Agarwal SG, Anand KK. In vivo hepatoprotective activity of active fraction of ethanolic extract of Eclipt alba leaves. Ind J Physio Pharmacol 2001;45:435-41.
  9. Syamasundar KV, Singh B, Thakur RS, Husain A, Kiso Y, Hikino H. Antihepatotoxic principles of Phyllanthus niruri herbs. J.Ethnopharmacol 1985;14:41-44.
  10. Venkateswaran PS, Millman I, Blimberg B.S. Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies. Proc Natl Acad Sci 1987;84:274-78.
  11. Unander DW, Webster GL, Blumberg BS. Usage and bioassays in Phyllanthus (Euphorbiaceae).IV. Clustering of antiviral uses and other effects. J Ethnopharmacol 1995;45:01-18.
  1. Chauhan CK, Nanivadekar SA, Billimoria FR. Effect of a herbal hepatoprotective product on rug metabolism in patients of cirrhosis and hepatic enzyme function in experimental liver damage. Ind J Pharmacol 1972;24:107-10.
  2. Habbu PV, Shastry RA, Mahadevan KM, Hanumanthachar Joshi, Das SK Hepatoprotective and antioxidant effects of Argyreia Speciosa in rats. Afr J Trad Cam2008;5(2):158–64.
  3. Ryan RP, Germaine K, Franks A, Bacterial endophytes: Recent developments and application. FEMS Microbiol Lett 2008;278(1):01-09.
  4. Huang WY, Cai YZ, Xing J, Cork H, Sun M. A potential antioxidant resource: Endophytic fungi from medicinal plants. Econ botany 2007;61(1):14-30.
  5. Gangadevi V, Sethumeenal S, Yogeswari S, Rani G. Screening endophytic fungi isolated from a medicinal plant, Acalypha indica L. for antibacterial activity. Indian J Sci Tech 2008;1(5):01-06.
  6. Sette LD, Passarini MRZ, Delarmelina, Salati F, Durate MCT. Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants. World Microbiol Biotecnol 2006;22:1185-95.
  7. Souwalak P, Nattawut R, Vatcharin R, Jariya S. Antimicrobial activity in cultures of endophytic fungi isolated from Garcinia species. FEMS immunol Med Microbiol 2006;48:367-72.
  8. Li JY,Strobel G,Sidhu R,Hess WM. Endophytic taxol-producing fungi from bald cypress, Taxodium distichum.Microbiol 1996;142(8):2223-26.
  9. Theantann T, Hyde KD, Lumyong S. Asparaginse production by endophytic fungi isolated from some Thai medicinal plants.KMITL Sci Tech J 2007;7(1):13-18.
  10. Touseef A, Khajuria RK, Puri SC, Verma V.Determination and quantification of campothecin in an endophytic fungus by liquid chromatography – positive mode electrospray ionization tandem mass spectrometry.Current sci 2006;91(2):208-12.
  11. Tiwari R, Saini RK, Singh AK, Gupta MM. Effect of endophytes on the yield enhancing capabilities in Artemisia annua. NIM 2008;56.
  12. Wealth of India: Raw Materials. New Delhi: CSIR; 1976. (vol 10).
  13. Nadkami KM, Nadkami AK. Indian Meteria Medica. 3rd ed. Mumbai: M/ S Popular Prakasan Pvt. Ltd; 1976.
  14. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. New Delhi: CSIR; 1956.
  15. Kirtikar KR, Basu BD. Indian Medicinal plants. New Connaught Place. 2nd ed. Dehradun: M/s Bishen Singh, Mahendra Pal Singh; 1975. (vol 1)
  16. Chopra RN, Chopra LC, Handa KD, Kapur LD. Indigenous Drugs of India. 2nd ed. Kolkata: M/s Dhar VN & Sons; 1982.
  17. Nayampalli S, Ainapure SS, Nadkami PM. Study of antiallergic acid Bronchodilator effect of Tinospora cordifolia.Ind J Pharm 1982;14:64–66.
  18. Zhao TF, Wang X, Rimando AM, Che C. Folkloric medicinal plant: Tinospora sagittata var.cravaniana and Mohonia bealei. Planta Med 1991;57:505–13.
  19. Ahmad F, Ali M, Alam P. New phytoconstituents from the stem bark of Tinospora cordifolia Miers.Nat Prod Res 2010;24(10):926-34.
  20. Phan VK, Chau VM, Nguyen TD, La VK, Dan TH, Nguyen HN, et al. Aporphine alkaloids,clerodane diterpenes, and other constituents from Tinospora cordifolia. Fitoterapia 2010;81(6):485-89.
  21. Zhang B, Salituro G, Szalkowski D, Li Z. Discovery of small molecule insulin mimetic with antidiabetic activity in mice. Sci 1999;284:974-77.
  22. Kunchandy E, Rao MNA. Effect of curcumin on hydroxyl radical generation through Fenton reaction. Int J Pharm1989;57:173-76.
  23. Narla RS, Rao MNA. Scavenging of free radicals and inhibition of lipid
    peroxidation by 3-phenyl syndone. J Pharm Pharmacol 1995;47:623–25
  24. Mahmood RM, Soheila M, Said A. Scavenging and Reducing power of Salvia Mirzayanii subfractions. Molecules 2008;13:2804-13.
  25. Ghosh MN. Fundamentals of Experimental Pharmacology. 2nd ed. Culcutta: Scientific Book Agency; 1984. p. 154-57.
  26. Hukkeri VI, Akki KS, Sureban RR, Gopalakrishna B, Byahatti VV, Rajendra SV. Hepatoprotective activity of the leaves ofNyctanthes arbor-tristislinn. Indian J Pharm Sci 2006;68:542-43.
  1. Prabhakar KR, Veerapur VP, Parihar Vipan K, Priyadarsini KI. Evaluation and optimization of radioprotective activity of Coronopus didymusLinn. In -Irradiated Mice. Int J Radiat 2006;82(8):525-36
  2. Beauchamp CF. Super oxide dismutase: improved assay and an assay applicable to acrylamide gel. Anal Biochem 1979;10:276-79.
  3. Aobi H. Catalyse invitro. Methods Enzymol 1984;105:121-25.
  4. Nicholas MA. A Spectrophotometric assay for iodide oxidation by thyroid peroxidase. Anal Biochem 1969;4:311-15.

9

/

SIGNATURE OF THE CANDIDATE

10 / REMARKS OF THE GUIDE / The above mentioned 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 GUIDE

SIGNATURE

/ Dr. P. V. HABBU,M. Pharm., Ph.D.,
Professor & HOD
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580 002.
Mobile No.: +91 – 9448224894
E-mail:

12

/

NAME AND DESIGNATION OFCO – GUIDE

SIGNATURE / Dr. K. S. JAGDEESHM. Sc (micro)., Ph.D.
Professor & Head
Dept. Of Agricultural Microbiology,
University Of Agricultural science.
Dharwad-580005.
13 / NAME AND DESIGNATION OF HOD
SIGNATURE / Dr. P. V. HABBU,M. Pharm., Ph.D.
Professor & HOD
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580 002.
Mobile No.: +91 – 9448224894
E-mail:

14

/

REMARKS OF PRINCIPAL

/ The above mentioned information is correct and I recommend the same for approval.
15 / NAME OF THE PRINCIPAL
SIGNATURE / Dr. V. H. KULKARNI,M. Pharm., Ph.D.
Principal, SET’s College of Pharmacy,
S.R.Nagar, Dharwad. Karnataka – 580002.
Mobile No.: +91 – 9448357804
E-mail: