International Journal of Pharmaceutical Sciences

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1 International Journal of Pharmaceutical Sciences INT.J.PH.SCI.,JAN-APRIL 2010;2(1): ISSN Original Research Manuscript ANTIDIABETIC EFFECT OF JATROPHA CURCAS L. LEAVES EXTRACT IN NORMAL AND ALLOXAN-INDUCED DIABETIC RATS 1 Shanti Bhushan Mishra*, M. Vijayakumar 2, Sanjeev Kumar Ojha 2, Amita Verma 3 1 Roorkee College of Pharmacy, Roorkee (UA), India. 2 Ethnopharmacology Division, National Botanical Research Institute, Lucknow (UP), India 3 Department of Pharmaceutical Sciences, College of Health Sciences, AAI-DU, Allahabad (UP) shanti321181@yahoo.co.in ABSTRACT Jatropha curcas (Euphorbiaceae) is a multipurpose plant with many attributes and considerable potential. Different parts of the jatropha curcas plant are employed in Indian traditional medicine for the treatment of several disorders. Our aim was to investigate the antihyperglycemic effect of 50% ethanolic extract of leaves of jatropha curcas (JCE) in alloxan induced diabetic rats. Oral administration of JCE at a dose of 250 and 500 mg kg -1 bw respectively showed potent antihyperglycemic activity in alloxan induced diabetic rats. The LD 50 determination was done in mice by OECD guidelines 423. The LD 50 of the JCE was found to be 2500 mg/kg. At the end of treatment, reduction in blood glucose level in treated rats with dose 250 mg/kg was , (p< 0.001). and oral administration of 500 mg/kg of JCE on blood glucose level was found to be , (p< 0.001). While in case of Glibenclamide 600µg/kg, the results was found to be mg/dl, (p< 0.001). Our results indicate that JCE have prominent antidiabetic effect in experimental diabetes and can therefore be used as an alternative remedy for the treatment of diabetes mellitus and its complications. Key Words: Jatropha curcas, antidiabetic, ethanolic extract, Glibenclamide, Alloxan INTRODUCTION: The incidence of each type of diabetes varies widely throughout the world. The vast majority of diabetes patients have type II DM. About 90% of all diabetic patients have type II DM. There are more than 125 million persons with diabetic in the world today. There is increase in current interest and demand for herbs as worldwide phenomenon, WHO currently encourages, recommends and promotes traditional/herbal remedies in national health care programmes because such drugs are easily available at low cost, and comparatively safe with the people s faith in such remedies. [1] A plenty of traditional herbal medicinal practices have been adopted for the diagnosis, prevention and treatment of diabetes. Jatropha curcas L. (physic nut or purging nut) is a drought resistant shrub or tree belonging to the family Euphorbiaceae, which is cultivated in Central and South America, South-East Asia, India and Africa [2]. Some of the ethnomedical uses of the extracts of Jatropha curcas leaves and roots include use as a remedy for cancer, as an abortifacient, 482

2 antiseptic, diuretic, purgative and haemostatic [3] The nut of the plant has also been used traditionally for the treatment of many ailments including burns, convulsions, fever and inflammation. In spite of the myriad of ethnomedical uses to which various parts of the Jatropha curcas plant have been put, it is important to note that toxic properties have also been adduced to parts of the plant, especially the seeds. [4-5] For example, the seed contains curcin, a toxalbumin which is highly irritant and produces deleterious effects on blood. The latex is acrid and irritable to the skin. [4] Curcain, a protease has been isolated from the latex of Jatropha curcas [6] Some of the ethnomedical uses of Jatropha curcas have received support from the results of scientific investigations in recent times. For example, some compounds with antitumor activities were reportedly found in this plant [7] Furthermore various solvent extracts of Jatropha curcas have an abortive effect. [8] Traditionally It is taken internally with ripe banana to treat dysentery in adults. The sap from twigs is considered styptic and is used for dressing wounds and ulcer. The bark rubbed with asafoetida and buttermilk is reportedly taken internally in Konkan to relieve dyspepsia and diarrhoea. A decoction of bark is used externally for treating rheumatism and leprosy. The decoction of root bark is used to rinse the mouth to relieve toothache and sore throat among the tribals inhabitants of southern Andhra Pradesh [9] Some anecdotal reports are there, which reveals plant has been used in the indigenous system of medicine and literature in the treatment of various ailments. Its leaves have acrid taste, which is considered as an excretory product or secondary metabolites, may be of therapeutic use and have action on metabolism. Since diabetes is considered as metabolic disorder so action on liver or pancreas can be postulated hence an attempt has been focused for its anti-diabetic activity. MATERIALS AND METHODS Plant Material The medicinal properties of plants have been investigated in the light of recent scientific developments throughout the world, due to their potent pharmacological activities and economic viability. As per the informations collected from local vaidhyas and other traditional medicine practitioners, the plant Jatropha curcas was selected for our study. The leaves of plant Jatropha curcas Linn (Family- Euphorbiaceae) were collected in the month of July 2008 from botanical garden of National Botanical Research Institute, Lucknow and the plant material was authenticated by a Taxonomist and the voucher specimen No was deposited in the departmental herbarium of N.B.R.I. Lucknow, India for future reference. Extract preparation The freshly collected leaves (4 kg) of J. curcas were first airdried and then dried in tray drier under control conditions and powdered. The powdered leaves (1000g) were macerated with petroleum ether to remove fatty substances; the marc was further exhaustively extracted with of 50% ethanol for 3 days (3 X 3L) by cold percolation method and centrifugation at 10,000 rev/min. The extract was separated by filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco, USA) under reduced pressure and thus 95.0 g of solid residue (yield 9.5 % w/w) was obtained. The extract was stored in a desiccator for use in subsequent experiment. Phytochemical analysis Preliminary phytochemical screening of 50% ethanolic extract of leaves revealed the presence of alkaloids, tannin, flavonoids, saponins, and triterpenoids. [10] Animals Sprague-Dawley rats ( g) and Swiss albino mice (20-25g) of either sex and of approximately the same age were procured from the animal house of the Central Drug Research Institute, Lucknow. They were kept in the departmental animal house at 26 ± 2 C and relative humidity 44-56% in polypropylene cages. The animals were exposed to alternate 12 hrs of darkness and light each. Animals were provided with standard rodent pellet diet (Dayal, India) and the food was withdrawn18-24 h before the experiment though water was allowed ad libitum. All experiments were performed in the morning according to current 483

3 guidelines for investigation of experimental pain in conscious animals. [11] The experimental protocol has been approved by the Institutional Animal Ethics Committee and by the regulatory body of the government. Chemicals Alloxan was purchased from Sigma Aldrich Chemicals Pvt, Ltd, Bangalore. All other chemicals and reagents used were of analytical grade. Evaluation of Toxicological studies of J.curcas Oral acute toxicity studies Acute toxicity study is generally carried out for the determination of LD 50 value in experimental animals. The LD 50 determination was done in mice by OECD guidelines 423. The aim of performing acute toxicity study is for establishing the therapeutic index of a particular drug and to ensure the safety in vivo. The LD 50 of the 50% ethanolic extract of J. curcas was found to be 2500 mg/kg. Table 1: LD 50 value of the 50% ethanolic extract of J. curcas leaves. Animal used - Swiss albino mice Weight of animals g No. of Animals - 3 Route - oral Sl.No. No. of animals Dose No. of death of animals mg/kg mg/kg mg/kg mg/kg 1 LD 50 value = 2500 mg/kg ED 50 value = 250 mg/kg Antihyperglycemic Studies Experimental induction of diabetes All animals were allowed to adapt to cages for 3 days, after which they were fasted overnight and 150 mg/kg of alloxan monohydrate freshly dissolved in normal saline was injected intra-peritoneally. After alloxan treatment, all animals were given free access to food and water. Blood glucose levels were measured 2 days after alloxan injection and used as parameters to obtain matching pairs of rats with diabetes of similar level of severity. Only rats with fasting blood glucose levels greater than 200 mg/dl were considered to be diabetic and were used in the experiment. The mean blood concentration of glucose in normoglycemic rats was 95 mg/dl. The animals were randomly assigned to four different groups i.e. group II to V. Group I served as normal control containing 6 normal rats. All treatments started 2 days after alloxan injection [12]. Experimental Design Five groups of rats were used to study the effect of 50% ethanolic extract of J. curcas. Each group consisting of six rats. Group I - Control rats received vehicle solution (1% CMC) Group II - Diabetic control rats received vehicle solution (1% CMC) Group III and IV - Diabetic rats treated with extract 250 & 500 mg/kg body weight in (1% CMC), respectively. Group V - Diabetic rats treated with standard drug Glibenclamide 600 µg/kg body weight in aqueous solution The vehicles and the drugs were administered orally using oral gavage tube daily for three weeks. Blood samples were collected for the measurement of blood glucose level from the tail vein on 0 day, 7 th, 14 th and 21 st day. The blood glucose level was determined by glucometer (one touch). The values of sample treated were compared with that of the standard group which was treated with Glibenclamide. Then the animals were sacrificed by cervical dislocation. The liver, kidney and pancreas were exposed and perfused with cold saline phosphate buffer of ph 7.4 for histopathological examination. Blood free liver and kidney were taken out and homogenized in a glass Teflon homogenizer separately (10% w/v). Incubation was done at 37 C under controlled conditions for biochemical estimation. The collected blood samples were immediately centrifuged at 2500 rpm for 15 min. The serum separated was collected in fresh 484

4 serum tubes and stored in refrigerator (2-4 C) after tightly capped [13] using computerized graph pad in stat version 3.05 graph pad software USA. Statistical Analysis Data were statistically calculated by utilizing one way ANOVA and expressed as mean ± SEM followed by Donnett s t test RESULTS The effect of 50% ethanolic extract of leaves of Jatropha curcas on alloxan-induced animals is indicated in table no. 2. (Short term study) and table no. 3 (long term study). Table 2: Effects of 50% ethanolic extract of leaves of J. curcas (JCE) on blood glucose level on normal experimental animals (Short term study) Treatment/Dose Blood glucose at different hours after the treatment in mg/dl 0 hr 1 hr 2 hr 4 hr Control 1% CMC (10 ml/kg) 83± ± ± ±1.18 JCE (250 mg/kg) 81.6± ± ±2.62 a 69.5±3.15 b JCE (500 mg/kg) 82.5±2.31 a 76.3±3.05 b 70± ±1.01 Standard (600µg/kg) 84.3±2.45 a 85.3± ±2.57 a 65.83±1.68 Values are expressed as Mean ± SEM of 6 rats in each group a P<0.05, b P<0.01 as compared to control Table 3: Anti hyperglycemic activity: Effects of 50% ethanolic extract of leaves of J. curcas (JCE) on blood glucose levels in alloxan induced diabetic rats. Groups Treatment/Dose 0 day (mg/dl) After 7 days (mg/dl) After 14 days (mg/dl) After 21 days (mg/dl) I Normal control 1% CMC (10 ml/kg) ± ± ± ± 4.61 II Diabetic control 1% CMC (10 ml/kg) ± ± z ± z ± z III JCE (250 mg/kg) ± ± ± ± c IV JCE ( 500 mg/kg) ± ± ± b ± c V Glibenclamide (600µg/kg) ± ± ± b 94.5 ± 5.46 c Values are expressed as Mean ± SEM of 6 rats in each group z P<0.001 in comparison to normal group a P < 0.05, b P<0.01 and c P<0.001 in comparison to diabetic group Table 4: Effects of 50% ethanolic extract of leaves of J. curcas (JCE) on the total cholesterol and triglyceride level in Blood serum after 21 days Triglyceride Total cholesterol Groups Treatment/ Dose (mg/dl) (mg/dl) I Normal control 1% CMC (10 ml/kg) ± ± 5.46 II Diabetic control 1% CMC (10 ml/kg) ± 6.36 z ± 7.54 z III JCE (250 mg/kg) ± 5.25 c ± 5.38 c IV JCE (500 mg/kg) ± 4.37 c ± 6.56 c V Glibenclamide (600µg/kg) ± 4.23 c ± 6.46 c Values are expressed as Mean ± SEM of 6 rats in each group z P<0.001 in comparison to normal group a P < 0.05, b P<0.01 and c P<0.001 in comparison to diabetic group 485

5 The results in table 2 showed that after a single dose of extract on alloxan diabetic rats there was a significant reduction (p<0.01 at 4 hour) of fasting blood glucose level with in the period of study (4h) as compared with control group. The antidiabetic effect was found comparable to that of standard drug Glibnclamide (p<0.01 at 4 h). Table 3 shows the effect of oral administration of 250 mg/kg/day and 500 mg/kg/day of JCE on blood glucose level in 21 days, at the end of treatment reduction in blood glucose level in treated rats with dose 250 mg/kg was , (p< 0.001). and oral administration of 500 mg/kg of JCE on blood glucose level in 21 days, the result was found to be , (p< 0.001). while in case of Glibenclamide 600µg/kg, the results was found to be mg/dl, (p< 0.001). Table 4 shows that the alloxan treated group, the level of cholesterol ( , p<0.001), triglyceride ( , p <0.001). In contrast, the groups treated was 50% ethanolic extract of leaves of J. curcas at dose 250 and 500 mg/kg once daily for 21 days prevented the diabetic condition in a dose related manner. The results were found to be significant viz. cholesterol ( , p<0.001), triglyceride ( , p<0.001), while in case of standard the results was found to be, cholesterol ( , p<0.001) and triglyceride ( p<0.001). DISCUSSION Diabetes mellitus arises from the irreversible destruction of the pancreatic beta cells causing degranulation and reduction of insulin secretion [14]. The present study demonstrated that the 50% ethanolic extract of Jatropha curcas had an antihyperglycemic effect in the alloxan induced diabetic rats when administered orally. It has been demonstrated that insulin deficiency in diabetes mellitus leads to a variety of derangements in metabolic and regulatory process, which in turns leads to accumulation of lipids such as cholesterol and triglyceride in diabetic patients. The abnormal high concentration of serum lipids in the diabetic subject is mainly due to increase in the mobilization of free fatty acids from the peripheral fat depots. In present study, the 50% ethanolic extract of leaves of J. curcas decrease the cholesterol and triglyceride levels in the significant manner. 486 CONCLUSION The current study provides some useful insight into the antihyperglycemic potency of Jatropha curcas leaves in alloxan induced diabetes. However, we suggest that further work should be carried out at molecular level to find out the absolute mechanism of action of plant Jatropha curcas in experimental diabetes. ACKNOWLEDGEMENT The authors are thankful to Dr. Sayeeda Khatoon, National Botanical Research Institute for their valuable guidance and support for the identification of medicinal plants. REFERENCES: 1. Handa SS. Herbal raw materials and traditional remedies. Eastern Pharmacist 1995; 38: Martinez-Herrera J, Siddhuraju, P, Francis G, Davila-Ortiz G, Becker K. Chemical composition, toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico. Food Chem. 2006; 96: Dalziel JM, The Useful Plants of West-Tropical Africa. Crown Agents for Oversea Governments and Administration, London, pp Watt JM, Breyer-Brandwijk MG. The Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd ed. Livingstone, Edinburgh, pp el Badwi SM, Adam SE, Hapke HJ. Comparative toxicity of Ricinus communis and Jatropha curcas in Brown Hisex chicks. Deutsche Tierarztliche Wochenschrifte, 1995; 102: Nath LK, Dutta SK. Extraction and purification of curcain a protease from the latex of Jatropha curcas Linn. J. Pharm. and Pharmacol. 1991; 43: Van den Berg AJ, Horsten SF, Kettenes van den Bosch JJ, Kroes BH, Beukelman CJ, Loeflang BR, Labadie RP. Curcacycline A: a novel cyclic octapeptide isolated from the latex of Jatropha curcas Linn. FEBS Letters 1995; 358: Goonasekera MM, Gunawardana VK, Jayasena K, Mohammed SG, Balasubramaniam S. Pregnancy terminating effect of Jatropha curcas in rats. J. Ethnopharmacol. 1995; 47: Parotta JA, Healing plants of peninsular india. CAB international, Wallingford U.K., pp Kokate CK, Practical Pharmacognosy, New Delhi, India, Vallabh Prakashan Zimmerman M. Ethical guidelines for investigation of experimental pain in conscious animals. Pain, 1983;16: Jothivel N, Ponnusamy SP, Appachi M, Singaravel S, Rasilingam D, Deivasigamani K, Thangavel S. Anti diabetic activity of methanol leaf extract of Castus pictus D. Don in alloxan induced diabetic rats. J. Health Sci. 2007; 53: Pushpangadan P, Vijayakumar M, Govindarajan R, Rao GMM, Rao ChV, Shirwaikar A, Mehrotra S. Action of Hygrophila auriculata against streptozotocin-induced oxidative stress. J. Ethnopharmacol., 2006; 104:

6 14. Junod A, Lambert AE, Atanffacher W, Renold AE. Diabetogenic action of streptozotocin relationship of dose to metabolic response. J. Clin. Invest, 1969; 48: Article History: Date of Submission: 12/01/2010 Date of Acceptance: 17/03/2010 Conflict of Interest: NIL Source of support: NONE 487