“EVALUATION OF ENDOPHYTIC FUNGAL FRACTIONS OF OCIMUM SANCTUM Linn. FOR ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY IN RODENTS”
M. Pharm. Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore – 560041
By
Mrs. Mary Rosalin, B. Pharm.
Under the Guidance of
Dr. P V Habbu, M. Pharm Ph.D
Professor and Head
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy,
S. R. Nagar, Dharwad,
Karnataka-580002.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE –II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1. / NAME OF THE CANDIDATEAND ADDRESS / Mrs. Mary Rosalin
DEPARTMENT 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-580002
3. / COURSE OF STUDY AND
SUBJECT / MASTER OF PHARMACY IN
PHARMACOGNOSY
4. / DATE OF ADMISSION TO
COURSE / JULY-2011
5. / TITLE OF THE TOPIC “EVALUATION OF ENDOPHYTIC FUNGAL FRACTIONS OF OCIMUM SANCTUM Linn. FOR ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY IN RODENTS.”
6.
7.0 / BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY:
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. 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 discovery.
A variety of microorganisms, mainly bacteria, fungi and actinomycetes inhibit plants and are, therefore, known as endophytic. Since the discovery of endophytes in Darnel, Germany, in 1904, various investigators have defined endophytes in different ways, which are usually dependent on perspective from which those organisms were being isolated and subsequently examined4. Bacon and White (2000) give an inclusive and widely accepted definition of endophytes. “Microbes that colonize living, internal tissues of plants, without causing any immediate negative effect”. The endophytic microorganisms penetrate plants tissue mainly by the root. However aerial parts such as stomata, flowers cotyledons also can serve as entrance5.
Pain is an unpleasant sensation no doubt, but on the whole it is usually beneficial. It is mainly a protective mechanism for the body, occurs whenever any tissue is being damaged and it causes the individual to react to the pain stimulus. It is a response to an untoward event associated with tissue damage, such as injury, inflammation or cancer, but severe pain can arise independently of any obvious predisposing cause (e.g. trigeminal neuralgia), or persistent long after the precipitating injury has healed (e.g. phantom limb pain)6.
Pain is associated with electrical activity in afferent fibers of peripheral nerves. These nerves have sensory endings in peripheral tissues which are activated by various stimuli (mechanical, thermal and chemical). These stimuli sufficient to excite afferent C-fibers to evoke painful sensation7. With many pathological conditions, tissue injury is the immediate cause of the pain and this result in the local release of a variety of chemical agents, which are assumed to act on the nerve terminals, either activating them directly or enhancing their sensitivity to other forms of stimulation6.
Inflammation is a normal response to any noxious stimulus that threatens the host and may vary from localized response to a generalized one8. The inflammatory process protects our bodys from diseases by releasing cells and mediators that combat foreign substances and prevent infection9. Inflammation is a complex process involving various mediators, such as prostaglandins, leukotrienes and platelet activating factors. Inflammation is caused by release of chemicals from tissues and migrating cells10.
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. In the Indigenous system of medicine like Ayurveda, many herbal medicines have been recommended for the treatment of diabetes or madhumeha and some of them experimentally evaluated11
6.2 REVIEW OF LITERATURE
Management of acute pain in chemotherapy is accomplished by employing non-steroidal anti-inflammatory drugs (NSAIDs) which target the cyclooxygenase (COX) enzyme. Narcotic analgesics are also used in the management of acute pain.
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. Endophytes can promote plant growth, 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.
Endophytes 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 antimicrobial15,16 Further many more examples in which endophytes producing various secondary metabolites such as taxol17 asperagenase18 campothecin19 as anticancer compounds and artimisinin20 as antimalarial etc.
Ocimum sanctum Linn. (Tulsi) has been used for thousands of years in Ayurveda for its diverse healing properties. Marked by its strong aroma and astringent taste, it is regarded in Ayurveda as a kind of “elixir of life” and believed to promote longevity21. Tulsi has been claimed in classical texts to possess antioxidant, antiasthmatic and antitussive antimalarial, antipyretic, anti-inflammatory, antidiabetic, antiasthmatic, nematicidal effect and is a good immunomodulatory agent with other numerous properties22. Recent studies proved the efficacy of the plant for antidiabetic23, hypoglycemic and antioxidant24,25, hepatoprotective26, cardioprotective27, antistress28 ,wound healing29, antianxiety30 activities. Some of the main secondary metabolites reported from Tulsi are Oleanolic acid, Urosolic acid, Rosmarinic acid, Eugenol, Carvacrol, Linalol and β-caryophyllene, Ocimumosides A and B and Ocimarin31,32. Hence based on the previous literature that endophytes of medicinal plants are the potential source for secondary metabolites and possess the same biological activity, it was thought worthwhile to isolate fungal endophytes from Ocimum sanctum Linn. and assess the crude fractions scientifically for analgesic and antiinflammatory activities in animal models.
In our earlier studies on endophytes in our laboratory, we have isolated four fungi (TRF1, TRF2, TRF3 and TRF6) from the roots of Ocimum sanctum Linn. and screened these fractions for hepatoprotective and immunomodulatory activities. The results of these studies were encouraging. Further we have characterized TRF1 by PCR sequential analysis. Hence in this investigation an attempt will be made to evaluate endophytic crude fungal fractions of Ocimum sanctum Linn. for analgesic and anti-inflammatory activity in rodents.
6.3 OBJECTIVES OF STUDY.
Ø Isolation of endophytes (fungi) from the roots of Ocimum sanctum Linn.
Ø Characterization of endophytes by qualitative and quantitative analysis.
Ø To evaluate the analgesic and anti-inflammatory activity of potential endophytic crude fractions from Ocimum sanctum Linn. using animal models.
MATERIALS AND METHODS
7.1 SOURCE OF DATA:
§ Indian Journal of Physiology and Pharmacology
§ Indian Journal of Pharmacology
§ Indian Journal of Pharmaceutical Sciences
§ The FASEB
§ FEMS Microbiology Letters
§ Journal of Natural Products
§ Journal of General and Applied Microbiology
§ Applied Biochemistry and Microbiology
§ Planta Medica
§ Pubmed
§ J-Gate@Helinet(RGUHS)
7.2 METHOD OF COLLECTION OF DATA:
The data is generated using laboratory experimental techniques. Isolated pure fungal cultures of Ocimum sanctum root endophytes are available in our laboratory.
7.3 PHARMACOLOGICAL EVALUATION:
Animals:
Albino wistar male rats weighing 150-200 g 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 studies:
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.
Analgesic activity
a. Writhing test33:
In this method, animals will be administered orally with standard drug Aspirin (20 mg/kg , p.o), test drug and vehicle. Thirty minutes after treatment, the mice will be given intraperitonial (i.p.) injection of 0.6% v/v acetic acid in a volume of 10ml/kg to induce the characteristic writhings. The number of writhings occurring between 5 and 15 min after the injection will be recorded. The response of treated animals will be compared with that of control. The data will be expressed in mean + SEM values by using one way ANOVA test (Tukey’s test ).
b. Hot plate method34:
In this method, the animals (mice) will be grouped as per the above table. The animals will be treated with drugs 60 minutes before experimentation. They will be placed on hot plate maintained at a temperature of 55 ± 0.50C. The latency to flick the hind paw or lick or jump from the hot plate is the reaction time (noted at 0, 15, 30, 45, 60, 90, and 120 min). The cut of time will be considered as 15 seconds. The data will be expressed in mean + SEM values by using one way ANOVA test ( Tukey’s test ).
Anti-inflammatory activity
a. Carrageenan induced rat paw oedema method35:
In this method, the animals (rat) will be grouped as per the above table, treated with drugs 60 minutes before injection of carrageenan (0.1ml of 1%w/v). Carrageenan will be injected into sub plantar tissue of left hind paw of each rat. Swelling of carrageenan injected paw will be measured at different time interval such as 0, 30min, 1h, 2h, 3h and 4h using Plethysmometer. The right hind paw will be injected with 0.1 ml of vehicle. Indomethacin (10mg/kg p.o.) serves as reference standard. The data will be expressed in mean + SEM values by using one way ANOVA test ( Tukey’s test ).
b. Cotton pellet granuloma in rats36:
In this method, the autoclaved cotton pellets 50±1 mg will be implanted subcutaneously by incision on the lumbar region of rats, under ether anaesthesia. Drugs will be administered orally for 7 days. Animals will be sacrificed on day 7 and the granuloma will be separated out, dried in an oven at 600C and weighed to determine the percent inhibition of granuloma and compared with standard drug (Indomethacin 10 mg/kg p.o.). The data will be expressed in mean + SEM values by using one way ANOVA test (Tukey’s test ).
7.4 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTION TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? IF SO, PLEASE MENTION BRIEFLY.
The above study requires investigations to be done on the albino rats, qswiss mice of Wister strain for the determination of analgesic and anti-inflammatory 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.
8. / REFERENCES:
1. Bary DA. Morphologie und physiologie der Pilze, Flechten, und Myxomyceten. Hofmeister’s handbook of physiological botony. Leipzig, Germany 1866;2.
2. Stone JK, Bacon CW, White JF. An overview of endophytic microbes: endopytism defined. In: Bacon CW, White JF, editors. Microbial edophytes. Marcel Dekker Inc., New York, USA 2000;01:03-29.
3. Freeman EM. The seed fungus of Loliiium temulentum L. Phil Trans R Soc Lond (Biol) 1904;196:01-27.
4. Strobel G, Daisy B. Bioprospecting for microbial endophytes and their natural products. Microbial Mol Biol Rev 2003;67:491-502.
5. Kobayashi DY, Palumbo JD. Bacterial endophytes and their effects on plants and uses in agriculture. In: Bacon CW, White JF, editors, Microbial endophytes. Marcel Deckker, New York ISBN 2000;0-8247-8831-1.
6. Paschapur MS, Patil S, Patil SR, Kumar R, Patil MB. Evaluation of analgesic and antipyretic activities of male flowers of Borassus flabellifer L. Int J Pharm Pharmaceut Sci 2009;1(2):98-106.
7. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. New Delhi: Elsvier 2005;562-4.
8. Williams DA, Lemke TL. Foye's principles of medicinal chemistry. 5th ed. Philadelphia (PA): Lippincott Williams & Wilkins 2002;751-93.
9. Frank MM, Fries LF. The role of complement in inflammation and phagocytosis. Immunol Today 1991;12:322.
10. Vane J, Botting R. Inflammation and the mechanism of action of anti-inflammatory drugs. The Faseb J 1987;11:89-96.
11. Roa K, Kirishna MB, Srinivas N. Effect of chronic administration of Boerhavia diffusa Linn. Leaf extract on experimentally induced diabetes in rats Trop J Pharm Res 2004;3(1):305-59.
12. Ryan RP, Germaine K, Franks A. Bacterial endophytes: Recent developments and application. FEMS Microbiol Lett 2008;278(1):1-9.
13. 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.
14. 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):1-6.
15. 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.
16. 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.
17. Li JY,Strobel G,Sidhu R,Hess WM. Endophytic taxol-producing fungi from bald cypress, Taxodium distichum. Microbiol 1996;142( 8):2223-26.
18. Theantann T, Hyde KD, Lumyong S. Asparaginse production by endophytic fungi isolated from some Thai medicinal plants. KMITL Sci Tech J 2007 ;7(1):13-8.
19. 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.
20. Tiwari R, Saini RK, Singh AK, Gupta MM. Effect of endophytes on the yield enhancing capabilities in Artemisia annua. NIM 2008;56.
21. Puri SC, Singh H . Rasayana: Ayurvedic Herbs for Longevity and Rejuvenation. CRC Press .2002;272-80.
22. The wealth of India. A dictionary of Indian raw materials & industrial products. CSIR: National institute of science communication & information resource. Vol:4 ;2006;4; 219-221
23. Mani UV, Ravi V, Lyer U.Effect of Tulsi (Ocimum sanctum) leaf powder supplementation on blood sugar levels, serum lipids and tissue lipids in diabetic rats. Plant Food Hum Natr 1997;50:9-16.
24. Sethi J, Sood S, Seth S, Talwar A. Evaluation of Hypoglycemic and Antioxidant affect of Ocimum Sanctum Linn. Ind J Clin Biochem 2004;19:152-155.
25. Devi P, Ganasoundari U.A modulation of glutathione and antioxidant enzymes by Ocimum Sanctum and its role in protection against radiation injury. Ind J Exp Biol 1999;37:262-268.
26. Razvi SU. Effect of Ocimum sanctum Linn. leaf extract on hepatotoxicity induced by antitubercular drugs in rats. Ind J Physiol Pharmacol 2003; 47:465-470.
27. Panda VS. Evaluation of cardioprotective activity of Ginkgo biloba and Ocimum sanctum Linn. in rodents. Altern Med Rev 2009;14(2):161-171.
28. Jyoti S,satendra S,sushma,S anjana T, shashi S; anti stress activity of Ocimum sanctum (Tulsi) against experimentally induced oxidative stress in rabbits. Methods and findings in experimental and clinical pharmacology, 2007; 29(6): 411-6.
29. Goel A, sandeep k,dilipkumar s ,ashokkumar b,wound healing potential of Ocimun sanctum Linn with induction of tumor necrosis factor alpha,ind j. of expt biology;48(2):402-6.
30. Manavi Chaterjee, Pinki Verma, Rakesh Maurya, Gautam Palit. Evaluation of ethanol leaf extract of Ocimum sanctum in experimental model of anxiety of depression. Pharmabiol 2011;49(5):477-483.
31. Kuhan M, Winston D. Winston and Kunn’s herbal therapy and supplements: A scientific and traditional approach. IInd Edn.Lippincott Williams & Wilkins.2007; 260.
32. Gupta P, Yadav DK, Siripurapu KB, Palit G, Maurya R.Constituents of Ocimum sanctum with antistress activity. J Nat Prod 2007;70:1410-1416.
33. Shastri LA, Ghate MD, Kulkarni MV. Dual fluorescence and biological evaluation of paracetamol ethers from 4-bromomethylcoumarins. Ind J Chem 2004;43(B):2416-22.
34. Turner RA. Screening Methods in Pharmacology. New York (NY): Academic press; 1971;100-13.
35. Bachhav RS, Gulecha VS, Upasani CD. Analgesic and anti-inflammatory activity of Argyreia speciosa root. Ind J Pharmacol 2009;41(4):158-61.
36. Agarwal RB, Rangari VD. Antiinflammatory and antiarthritic activities of lupeol and 19a-h LUPEOL isolated from Strobilanthus callosus and Strobilanthus ixiocephala roots. Ind J Pharmacol 2003;35:384-7.
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/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 and Head
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S. R. Nagar,
Dharwad. Karnataka – 580002.
Mobile No.: +91-9449143440
E-mail:
12
/NAME AND DESIGNATION OF CO – GUIDE
SIGNATURE / --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 – 580 002.
Mobile No.: +91 – 9448357804
E-mail:
10