SCREENING OF MICROBIAL ENDOPHYTIC FRACTIONS OF Murrayakoenigii(L.)Spreng.FOR ANTIDIABETIC ACTIVITY IN RATS

M. Pharm. Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560041

By

Mr. Manjunath. V. Salagare,B. Pharm.

Under the Guidance of

Shri. B.S. Patil,M. Pharm(Ph.d)

Asst. Professor

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 CANDIDATE
AND ADDRESS / MANJUNATH VASANT SALAGARE
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-580002
3. / COURSE OF STUDY AND
SUBJECT / MASTER OF PHARMACY IN
PHARMACOGNOSY
4. / DATE OF ADMISSION TO
COURSE / JULY-2010
5. / TITLE OF THE TOPIC “SCREENING OF MICROBIAL ENDOPHYTIC FRACTIONS OF Murrayakoenigii(L.) Spreng.FOR ANTIDIABETIC ACTIVITY IN RATS”
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 century.3 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 examined.4Bacon 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 entrance.5
Diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose concentration-hyperglycemia (fasting plasma glucose > 7.0 mmol/l or plasma glucose > 11.1 mmol/l 2 hours after a meal) – caused by insulin deficiency, often combined with insulin resistance. Hyperglycemia occurs because of uncontrolled hepatic glucose output and reduced uptake of glucose spills over into the urine (glycosurea) and causes an osmotic diuresis (polyurea), which in turn results in dehydration, thirst and increased drinking (polydipsia).6The IDF (International Diabetes federation) has subsequently released estimates of the numbers of people with diabetes for 2003 and forecasts for 2025 of 194 million and 334 million, respectively.7 International Diabetes Federation, (2006) published the number of people with diabetes in India currently is around 40.9 million and is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken.8
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 evaluated.9
6.2 REVIEW OF LITERATURE
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. Most endophytes appear to originate from the rhizosphere or phyllosphere; however, some may be transmitted through the seed. Endophytescan 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 industry.10
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 antioxidant,11 antibacterial,12 antimicrobial.13,14 Further many more examples in which endophytes producing various secondary metabolites such as taxol,15 asperagenase,16 campothecin,17 as anticancer compounds and artimisinin18 as antimalarial etc.
Murrayakoenigii (L.) Spreng. Is a well known plant in Indian system of medicine. Traditionally the leaves, bark and root of Murrayakoenigii(L.) Spreng. are used as tonic and stomachic. Pharmacological reviewreported the stimulant, antidysentery,19 antidiabetic,20 antioxidant, hypolipidaemic and antiartherosclerotic properties.21Phytochemical investigations revealed the presence of Carbazole Alkaloids like, Mahanimbine, Girinimbine, Murryanine, Murrayafoline-A, Bismurryafoline E, Mahanimbicine etc from the title plant.22, 23
Hence in this investigation an attempt will be made to isolate and evaluate endophytic crude fractions (bacteria, fungi or actinomycetes) from Murrayakoenigii (L.) Spreng. for antidiabetic activity in rats, on the basis of widely occupied hypothesis and literatures that some endophytes from medicinal plants produce same secondary metabolites as that of the parent plant.24
6.3 OBJECTIVES OF STUDY.
Isolation of endophytes (bacteria, fungi or actinomycetes) from Murrayakoenigii(L.) Spreng.
Characterization of endophytes by qualitative and quantitative analysis.
In-vitro antioxidant activity of endophyte crude fractions.
To evaluate the antidiabetic activity of potentialendophytic crude fractions from Murrayakoenigii(L.) Spreng. 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
  • Indian Journal of Science and Technology
  • Journal of General and Applied Microbiology
  • Applied Biochemistry and Microbiology
  • PlantaMedica
  • Pubmed
  • J-Gate@Helinet(RGUHS)
7.2 METHOD OF COLLECTION OF DATA:
The data is generated using laboratory experimental techniques. The isolated and characterized endophytic crude fractions of Murrayakoenigii(L.) Spreng. will be procured from Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad.
In-vitro free radical scavenging activity:25,26,27
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.
7.3 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 studies:28
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.
OGTT in normal rats:
A Single dose of endophyte fraction/glibenclamide (GLB) and vehicle will be given orally to normoglycemic rats fasted for 18 h. Thirty minutes later; glucose (2 g/kg body weight) will be administered orally. Serum glucose will be estimated at 0 min (i.e. immediately after glucose load), 30, 60, 90 and 120 mins after glucose administration. The results are expressed as integrated area under curve for glucose (AUCglucose) as calculated by trapezoid rule.
Standardization of STZ dose to induce diabetic condition in rats:
A single intraperitoneal injection of different doses of STZ (35,45 and 65mg/kg body weight) will be administered to 6 animals per group. After 7 day post STZ administration, serum glucose levels will be estimated and the extent of glucosurea will be estimated using Diastix (Bayer Diagnostics, India). Rats showing glucose level > 200 mg/dl will be considered as diabetic and included in the study. A single oral dose of 10 mg/kg of GLB will be administered to all groups of rats. Glucose levels will be estimated at 0, 2, 4, 6 and 10 h after drug administration (single-dose one-day study).
Effect of endophyte fractions on stz-induced diabetes in rats:
Optimized dose of STZ will be used to induce diabetes in rats. These diabetic rats will be randomizing into different groups based on their glucose levels. Single-dose one-day study will be carried out as mentioned earlier. Further these groups of rats will be treated with single dose of endophyte fractions for 15 consecutive days [Multiple-dose fifteen-day study]. Serum glucose levels will be estimated on days 0, 7, 10 and 15thday.
Oral glucose tolerance test (OGTT) in diabetic rats:
At the end of 15 days of treatment, glucose (2 g/kg body weight) will be administered to 12 h-fasted rats and blood samples will be collected at 0 (immediately after glucose load), 30, 60 and 120 min after glucose administration. Glucose and insulin will be estimated and results were expressed as integrated area under curve for glucose (AUCglucose) and insulin (AUC insulin)
Biochemical parameters:
Serum insulin
Serum triglyceride (TG)
Serum total cholesterol (TC)
HDL-c
VLDL-c, LDL-c calculated as described in literature29
TC/HDL-c & LDL-c/HDL-c.
7.4DOES 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 of Wister strain for the determination of antidiabetic 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.

/ REFERENCE :
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  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 LoliiiumtemulentumL. 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. [cited 2008 Nov 30th]
  7. Hayashi T, Maruyama H, Kasai R, Hattori K, Takasuga S, HazekiO, et.al. Ellagitannins from Lagerstroemia speciosa as activators of glucose
    transport in fat cells. Planta Med 2002;68(2):173–75.
  8. Anthony M. The Evaluation of Diabetes Knowledge in Relation to the
    Theory of Scientific Revolutions. DiabEdu 2002;28(5): 688-96.
  9. Roa K, Kirishna MB, Srinivas N. Effect of chronic administration ofBoerhavia diffusa Linn. Leafextract on experimentallyinduceddiabetes inrats Trop J Pharm Res 2004;3(1):305-59.
  10. Ryan RP, Germaine K, Franks A. Bacterial endophytes: Recent developments and application. FEMS MicrobiolLett 2008;278(1):1-9.
  11. 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.
  12. Gangadevi V, Sethumeenal S, Yogeswari S, Rani G. Screening endophytic fungi isolated from a medicinal plant,Acalyphaindica L. for antibacterial activity. Indian J Sci Tech 2008;1(5):1-6.
  13. SetteLD, Passarini MRZ, Delarmelina, Salati F, Durate MCT. Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants. World MicrobiolBiotecnol 2006;22:1185-95.
  14. 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.
  15. Li JY,Strobel G,Sidhu R,Hess WM.Endophytictaxol-producing fungi from bald cypress, Taxodiumdistichum.Microbiol 1996;142( 8):2223-26.
  16. Theantann T, Hyde KD, Lumyong S. Asparaginse production by endophytic fungi isolated 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 of campothecin in an endophytic fungusby liquid chromatography – positive mode electrospray ionization tandem mass spectrometry.Current sci2006;91(2):208-12.
  18. Tiwari R, Saini RK, Singh AK, Gupta MM. Effect of endophytes on the yield enhancing capabilities in Artemisia annua. NIM 2008;56.
  19. Adebajo CA, Gbola O, Eugen VJ, Ezekiel OI, Omisore NOA, Dieter B. et.al. Evaluation of the ethnomedical claims of MurrayaKoenigii.Pharm Biol 2004;42:610-20.
  20. Grover JK, Yadav SP, Vats V. Effect of feeding MurrayaKoenigiiandBrassicajunceadiet kidney functions and glucose levels in streptozotocindibetic mice. J Ethenopharmacol 2003;85:01-05.
  21. Vinuthan MK, Girish KV, Narayanaswamy M, Veena T. Lipid lowering effect of aqueous leaves extract of MurrayaKoenigii(curry leaf) on alloxan induced male diabetic rats. Phcog Mage. 2007;3:112-15.
  22. Noor HA, Modh AS, Mawardi R, Atan MS, Kaida K, Umi KY. Chemical constituents from stem barks and roots of Murrayakoenigii (Rutaceae). The Malaysian J of Analytical Sciences 2007;11:173-76.
  23. Yukari T, Hiroe K, Nordin HL, Nobuji N. J Agric. Food Chem.2001;49:5589-94.
  24. Zhang B, Salituro G,Szalkowski D, LiZ. Discovery of smallmolecule insulin mimetic with antidiabetic activity in mice. Sci 1999;284:974-77.
  25. Kunchandy E, Rao MNA. Effect of curcumin on hydroxyl radical generation through Fenton reaction. IntJ Pharm1989;57:173-76.
  26. Narla RS, Rao MNA. Scavenging of free radicals and inhibition of lipid
    peroxidation by 3-phenyl syndone. J Pharm Pharmacol 1995;47:623–25.
  27. Mahmood RM, Soheila M, Said A. Scavenging and Reducing power of Salvia Mirzayaniisubfractions. Molecules 2008;13:2804-13.
  28. GhoshMN. Fundamentals of Experimental Pharmacology. 2nded. Culcutta: Scientific Book Agency;1984:154-7
  29. Friedewald WT, Levy RI, Fredrickson DS. Estimation of low-density lipoprotein cholesterol in plasma without use of the preparative centrifuge. ClinChem1972;18:499.

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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

/ Shri.B.S.Patil,M. Pharm.,( Ph.D).,
Assistant Professor
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 / Dr.K.S.JagdeeshM.Sc(Micro).,Ph.D.
Professor and 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:

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