Rajiv Gandhi University of Health Sciences s99

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the candidate and Mr. ABHISHEK KUMAR address DEPT.OF PHARMACOGNOSY AND PHYTOCHEMISTRY, K.L.E.SOCIETY’S COLLEGE OF PHARMACY, J.N.M.C.CAMPUS, NEHRU NAGAR, BELGAUM-10.

2. Name of the Institution K.L.E.SOCIETY’S COLLEGE OF PHARMACY, BELGAUM-10

3. Course of study and MASTER OF PHARMACY IN PHARMACOGNOSY subject AND PHYTOCHEMISTRY

4. Date of admission to course JUNE - 2008

5. Title of the topic: “ASSESSMENT OF ANTIDIABETIC POTENTIAL OF RHIZOME OF KYLLINGA MONOCEPHALA ROTTB. IN STREPTOZOTOCIN DIABETIC RATS.” 6. BRIEF RESUME OF THE INTENDED WORK:

INTRODUCTION: Diabetes mellitus (DM) is a chronic metabolic disorder affecting approximately 5% of the population1. There are an estimated 171 million people worldwide suffering from diabetes mellitus and will probably doubled to 221 million by 2010 and 366 million in 2030 2,3. The WHO reported that diabetes mellitus (DM) is fast becoming pandemic4. It is characterized by hyperglycemia in the postprandial and/or fasting state, and in its severe form is accompanied by ketosis and protein wasting. Beside hyperglycemia, several other symptoms, including hyperlipidemia, are involved in the development of microvascular and macrovascular complication of diabetes, which are the major causes of morbidity and death5. Currently available therapies for diabetes include insulin and various oral antidiabetic agents such as sulfonylureas, biguanides, α-glucosidase inhibitors and glinides.6 In most instances these are expensive for a developing country like India and they may also have adverse effects, including hypoglycemia, obesity haematological effects, coma and disturbances of the liver and kidney and are not suitable during pregnancy 7,8.

6.1 NEED FOR THE STUDY:

For a long time, diabetics have been treated with several medicinal plants or their extracts based on the folklore medicine. Synthetic hypoglycemic agents can produce serious side effects and in addition, they are not suitable for use during pregnancy. Therefore, the search for more effective and safer hypoglycemic agents has continued to be an important area of active research. Furthermore, after the recommendation made by WHO on diabetes mellitus, investigation on hypoglycemic agent from medicinal plants has been more important A number of investigations, of oral antihyperglycemic agents from plants used in traditional medicine, have been conducted and many of the plants were found with good activity9.

Over 1,200 organisms (representing 725 genera in 183 families) have been used or experimented with ethnopharmacologically in treating diabetes, or the obvious symptoms of the disease. The plants which have been used represent a wide range of families. Of these, 127 species are found in the Fabaceae (pea family), 98 in the Asteraceae (aster or daisy family), 36 in the Lamiaceae (mint family), 35 in the Liliaceae (lily family), 30 in the Poaceae (grass family), and 30 in the Euphorbiaceae (spurge family). Screening these plants for hypoglycemic (blood sugar lowering) activity has shown that 81% yield positive results. This figure reflects the "great variety of possible active constituents and mechanisms of action," and illustrates the excellent potential for discovering new sources of antidiabetic drugs. Promising leads reflecting this wide range of antidiabetic constituents and mechanisms of hypoglycemic activity are being studied. The ethnopharmacological use of herbal remedies for the treatment of diabetes mellitus is an area of study ripe with potential as a starting point in the development of alternative, inexpensive therapies for treating the disease10.

Kyllinga monocephala Rottb. (Family: Cyperaceae) commonly known as “Nirbisi” in India, is a weed and has been regarded to possess various medicinal properties. In indigenous system of Ayurvedic and Unani system of medicine Kyllinga monocephala (rhizomes) is used to treat diabetes11-14.

The Literature survey indicates that there that the antidiabetic activity of Kyllinga monocephala has not been scientifically investigated. Hence, the present study is undertaken for the phytochemical investigation of the whole plant of Kyllinga monocephala and to evaluate it’s traditionally claimed antidiabetic activity in Wistar albino rats. 6.2 REVIEW OF LITERATURE:

Plant - Kyllinga monocephala Rottb.

Family - Cyperaceae

Habitat - Kyllinga monocephala Rottb. is found throughout India, in shady and moist places. The plant is known by various names in different languages, as follows:-11-13

Sanskrit Svetnirvasi.

English Bitter gourd.

Hindi Nirbisi, Svet gottubi.

Tamil Velutta Nirbasi.

Telgu Gandala. Bengali Nirbishaghas. Sveta-gotubh.i

Malayalam Palnirvasi, Pimuttanna, Velutta Nirvasi.

Marathi Musta.

Kyllinga monocephala Rottb. is perennial creeping sedge which grows up to 30 cm height and spread by means of long- creeping rhizome, 7.5 -30 cm in height, culms tufted, three angled. Leaves simple, linear, as long as or shorter than stem, 1.5-3mm wide, acute with strong mid nerve, and leaves sheath brown to purple brown. 1Flowers minute in spikes, solitary or 2-3 together, rachis naked when spikelets fall off, spikelets one flower. Fruits obovoid or oblong compressed nuts, pale reddish brown11,13,14. The plant is considered diuretic, stomachic and anthelmintic, and is given for fistula, postules, tumors and stomach and intestinal complaints. In Malaya, it is used for diarrhoea and in Celebes, for measles. The spikes are applied as poultices for gathered nails 13 . The rhizome is fragrant, aromatic, sweet, astringent, bitter, diuretic, refrigerant febrifuge, sudorific, antidiarrhoeal, expectorant, and stomachic. They are also useful in vitiated conditions pitta and vata, hyperdipsia, fever, stranguray, verminosis, cough, bronchitis, hepatopathy, splenopathy, diabetes and dermatitis 11,12,13,14. The study indicated the presence traces of hydrocyanic acid in roots, stems and nutles13. Review of literature reveals that the methanolic extract of Bixa orellana, Kyllinga monocephala, and Luffa acutangula posse’s analgesic and hypoglycemic activity 15. The pharmacognostic studies on Kyllinga monocephala has been reported14. Indian plants which are most effective and the most commonly studied in relation to diabetes and their complications are: Allium cepa, Allium sativum, Aloe vera , Cajanus cajan , Coccinia indica , Caesalpinia bonducella , Ficus bengalenesis , Gymnema sylvestre , Momordica charantia , Ocimum sanctum, Pterocarpus marsupium, Swertia chirayita , Syzigium cumini, Tinospora cordifolia and Trigonella foenum graecum etc16. 6.3 OBJECTIVES OF THE STUDY:  Collection and identification of plant.  Collection and processing of rhizomes of Kyllinga monocephala.  Successive solvents extraction of rhizomes of Kyllinga monocephala.  Pharmacological screening of rhizomes of Kyllinga monocephala for antidiabetic activity.  Separation of major chemical constituents from potent extract by chromatographic techniques.  Characterization of separated chemical constituents from spectroscopic analysis. 7. MATERIALS AND METHODS: 7.1. SOURCE OF DATA:

The source of data for this study is based on the laboratory experiments on animals, also the data obtained from the literature will be the source of data.

7.2 METHOD OF COLLECTION OF DATA: (Including sampling procedure if any) The rhizomes of the Kyllinga monocephala selected for the study will be collected as per its availability and will be authenticated. Standardization of Kyllinga monocephala Rottb. rhizomes:

. Botanical Evaluation:

Colour, Odour, Taste, Size, Shape, Texture.

. Physical Evaluation:

Ash value, Extractive values, Loss on drying17.

. Phytochemical Evaluation:

The rhizomes of the Kyllinga monocephala will be collected, shade dried and powdered. Extract of Kyllinga monocephala rhizomes will be prepared by subjecting the powdered drug to successive solvent extraction with soxhlet apparatus and using the different non-polar and polar solvents like per-ether, chloroform and ethanol. The aqueous extraction will be carried out by cold maceration process. The extracts so obtained will be concentrated in vacuum using rotary flash evaporator to get a semisolid or solid mass. The extracts will be stored in a glass container in a refrigerator until further use.

. Phytochemical Investigation: The extracts will be subjected for preliminary phytochemical investigation to identify different phytoconstituents18. Then subjected for chromatographic techniques for the separation of phytoconstituents. Later for spectroscopic methods for the characterization of individual phytoconstituents. . Pharmacological Evaluation:

i) Animal selection:-

Wistar albino rats of either sex weighing between 180-220 gms will be selected for experiments. They will be employed for assessing antidiabetic activity.

ii) Acute toxicity studies:

The acute oral toxicity study will be carried out as per the guidelines set by the Organization for Economic Cooperation and Development (OECD) regulations. 19

iii) Screening of Antidiabetic activity in rats:

Methods of Santosh kumar 20 will be followed with necessary modification as a model for the present study.

1. Assessment of hypoglycemic activity in normal healthy rats -

Twenty-four normal healthy male rats will be fasted overnight. The fasted animals will be divided equally into four groups of six rats each. Pretreatment FBG levels of each group will be evaluated. Group I served as untreated control given vehicle (water only), whereas the other three groups II, III and IV will be given lyophilized aqueous extract suspended in distilled water orally in doses of 250, 500 and 1000 mg/kg bw, respectively. Blood samples will be collected from the tail vein after 2, 4 and 6 h and the percentage change in blood glucose will be calculated for each group.

2. Assessment of antidiabetic activity on glucose tolerance in mild-diabetic rats

The antidiabetic effect of aqueous extract will be assessed in mild-diabetic rats using a glucose tolerance test. The overnight fasted rats will be divided into five groups (V, VI, VII, VIII and IX) of six rats each. Pretreatment FBG levels of each group will be evaluated. Group V (diabetic control) received vehicle (water only) whereas doses of 250, 500 and 1000 mg/kg bw of aqueous extract and a dose of 250 mg/kg bw of tolbutamide will be given orally to groups VI, VII, VIII and IX, respectively. BGL of each group was further evaluated after 90 min of the treatment considered as 0 h value. Two grams per kilogram bw glucose solution will be given to all the groups and their BGL will be estimated after 1 and 2 h of glucose administration.

3. Assessment of antidiabetic and hypolipidemic potential in severely diabetic rats A study will be carried on three groups (X, XI and XII) of six rats each. Group X served as normal control, group XI as diabetic control and group XII as diabetic treated groups. Control rats (group X and XI) will be given vehicle (distilled water) only while group XII will be orally treated with a single dose of 500 mg/kg bw of aqueous extract suspended in distilled water daily for 2 weeks. Fasting blood glucose, total cholesterol, HDL cholesterol, urine sugar and triglycerides levels will be estimated, before and after the treatment, weekly up to 2 weeks and LDL cholesterol will be calculated by using Friedwald formula.

Statistical Analysis:

All the results of biochemical estimation will be analyzed using One-way ANOVA followed by Dunnett’s test. 7.3 Does the study require any investigation or invention to be conducted on patients or other humans or animals? If so please mention briefly.

Yes. The above study requires investigation on Wistar rats for determination of LD50 & antidiabetic activity. Albino rats will be used and strictly follow all the rules laid down the CPCSEA, the ethical committee and any other regulatory body. 7.4 Has ethical clearance been obtained from yours institution in case of 7.3

Yes. The ethical clearance for said work will be obtained from institute animal ethical committee of K.L.E.S’s College of Pharmacy, Belgaum. 8. REFERENCES:

1. Li-Qin Tang , Wei Wei, Li-Ming Chen, Sheng Liu,2006. Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats. Journal of Ethnopharmacology 108, 109–115.

2. Aslan M, Orhan DD, Orhan N, Sezik E, Yesilada E, 2007.In vivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp. plicatum capitulums in streptozotocin-induced-diabetic rats. Journal of Ethnopharmacology 109, 54–59.

3. Orhan N, Aslan M, Orhan DD, Ergun F, Yesilada E, 2006. In vivo assessment of antidiabetic and antioxidant activity of grapevine leaves (Vitis vinifera) in diabetic rats. Journal of Ethnopharmacology 108, 280–86.

4. World Health Organization: Diabetes mellitus: Report of a WHO Study Group WHO Technical Report Series 727. Geneva: WHO, 1985.

5. Jun-En Suna et al., 2008. Antihyperglycemic and antilipidperoxidative effects of dry matter of culture broth of Inonotus obliquus in submerged culture on normal and alloxan-diabetes mice. Journal of Ethnopharmacology.

6. Ndiaye M, Diatta W, Sy A.N., Dièye A.M., Faye B, Bassène E, 2008. Antidiabetic properties of aqueous barks extract of Parinari excelsa in alloxan-induced diabetic rats. Fitoterapia79, 267–70.

7. Bhavna S, Viswanath G, Rajani S, Partha R., 2008. Effects of flavonoid-rich extract from seeds of Eugenia jambolana (L.) on carbohydrate and lipid metabolism in diabetic mice. Food Chemistry 110, 697–705.

8. Aguilar FJA, Estrada MJ, Chipla RR, Ramos RR, 2000. Hypoglycaemic effect of extracts and factions from Psacalium decompositum in healthy and alloxan-diabetic mice. Journal of Ethnopharmacology 72, 21-7.

9. Kumar S, Kumar D , Deshmukh R, Lokhande P, More S., Rangari V., 2008 Antidiabetic potential of Phyllanthus reticulatus in alloxan-induced diabetic mice Fitoterapia 79, 21–23.

10. HerbalGram. 1997; The Journal of the American Botanical Council 40:21.

11. Arya VS, Indian Medicinal Plants – A Compendium of 500 Species. Madras: Orient Longman Ltd., 1994: 235-6. 12. Nadkarni KM. The Indian materia medica. Mumbai: Parakashan Pvt. Ltd.; 1976. 405.

13. The wealth of India, 1952. Vol. V (H-K), New Delhi: CSIR 331-332.

14. Raju H.V , Padmalatha K, Narayana T.V,Ramesh R,2007. Pharmacognostic study

of K.moncephala, Indian Nat. Prod. 23,36.

15. Jusal P. Quanico, Evangeline C. A, Grace C.P.,2008. Analgesic and Hypoglycemic

Activities of Bixa Orellana, Kyllinga monocephala, and Luffa acutangula

Phillippine journal of science, 137.

16. Grover J.K., Yadav S., Vats V., 2002. Medicinal plants of India with anti-diabetic

potential. Journal of Ethnopharmacology 81, 81-/100.

17. Khandelwal KR. Practical Pharmacognosy Techniques and Experiment. 4thed.

Pune: Nirali Prakashan 1994: 107-11

18. Kokate CK. Practical Pharmacognosy. 4th ed. Delhi: Vallabh Prakashan 1994:

107-11.

19. OECD Guidelines for Testing Of Chemicals: October, 2000.

20. Santosh Kumar Singh, Achyut Narayan Kesari, 2007. Assessment of antidiabetic

potential of Cynodon dactylon extract in streptozotocin diabetic rats. Journal of

Ethnopharmacology. 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 (in block letters)

11.1 Guide Dr. S.S.JALALPURE M.Pharm.Ph.D. ASSOCIATE PROFESSOR, Department of Pharmacognosy & Phytochemistry, K.L.E.Society’s College of Pharmacy, Belgaum.

11.2 Signature 11.3 Co-guide (if any) 11.4 Signature

11.5 Head of the Department Dr. S.S.JALALPURE M.Pharm.Ph.D. ASSOCIATE PROFESSOR & HEAD, Department of Pharmacognosy & Phytochemistry, K.L.E.Society’s College of Pharmacy, Belgaum.

11.6 Signature 12. 12.1 Remarks of the The above mentioned information is correct and I Chairman & Principal recommend the same for approval.

12.2 Signature Dr. F.V.MANVI M.Pharm.Ph.D. PRINCIPAL, K.L.E.Society’s College of Pharmacy, Belgaum-10