Keywords: Mangiferaindica, hypoglycaemic, streptozotocin, diabetes. Sarkodie J A et al, SPJPBS.2013,(1), ISSN

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1 COMPARATIVE STUDY OF THE HYPOGLYCAEMIC PROPERTY OF AQUEOUS EXTRACT OF MANGIFERA INDICA LEAVESIN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND GENETIC DIABETIC MICE SarkodieJA a *, EdohDA c, SakyiamahM c, AppiahAA c,antwis d,nyarkoak b, OwusuPD a, Debrah P a, Jackson NAB a and AmoatengP b adepartment of Pharmacognosy and Herbal Medicine, bdepartment of Pharmacology, Faculty of Pharmacy, University of Ghana, Accra-Ghana cdepartment of Phytochemistry, ddepartment of Pharmacology, Centre for Scientific Research into Plant Medicine, Mampong-Akwapim, Ghana Abstract Mangiferaindica is locally called mango and grows in most African countries. It is an important medicinal plant used to treat dysentery, leucorrhoea, jaundice, pains, malaria,diabetes, and haemorrhoids. The present study aims to investigate the hypoglycaemic property of aqueous extract of the leaves of M. indicain streptozocin-induced diabetic rats and genetic diabetic mice. The leaves of M. indica (65 g) werepulverized and extracted using aqueous distilled water. The doses for the study were fixed based on the Irwin test. The hypoglycaemic effect of the extract (with doses 1 mg/kg, 5 mg/kg and 1 mg/kg) were studied for both streptozotocin-induced diabetic rats and genetic diabetic mice. The extracts showed reduction of glucose levels in the streptozotocin-induced diabetic rats and genetic diabetic mice to some extent after 6 hours and 28 days period. The findings demonstrate the potential of the leaves of M. indicain streptozocin-induced diabetic rats and genetic diabetic mice justifying the traditional use of the leaves of M. indicain the management of diabetes by traditional healers in Ghana. Keywords: Mangiferaindica, hypoglycaemic, streptozotocin, diabetes Introduction Diabetes, a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action (or both) is one of the most significant public health problems in the world (Dineshkumaret al., 21). The disease is classified into two main categories; type 1 and type 2. Whereas type 1diabetes is characterized by an inability of the body to produce insulin, type 2 diabetes reflects deficient insulin secretion and/ or insulin resistance. According to the WHO, there are over 15 million diabetic patients and it is expected that this population would appreciate to 3 million by the year 225; with the impact borne more by the developing countries (Desai et al., 212). The number of patients with diabetes (mainly Type 2) is increasing due to population growth and a growing prevalence of physical inactivity leading to obesity (Wild et al., 24). Type 2 diabetes represents approximately 9% of all cases of diabetes. However, the high cost and inaccessibility associated with the use of synthetic oral hypoglycaemic agents such as biguanides and sulfonylureas, or insulin to lower blood glucose levels to normal ranges has turned out to be a limitation in the treatment of diabetes (Teves et al., 24; Kennan et al., 25). On the contrary, the developing countries are endowed with over 12 plant species claimed to possess hypoglycaemic effects, and have been used empirically to alleviate symptoms of diabetes (Shim et al., 23). Mangiferaindicais a large evergreen tree belonging to the family Anacardiaceae. It is commonly known as mango and its fruits are eaten all over the world. In the tropics, mangos grow at elevations up to 12 m and trees may reach 4 m or more in height and live for several hundred years. The different parts have been used for treatment of various diseases. Unripe green mangoes cut into pieces and dried in the 37

2 sun, are believed to be valuable in the treatment of scurvy. Ripe mangoes are rich in Vitamin A and are used in the treatment of night blindness. The bark, roots and leaves of M. indicaare used to treat ailments such as asthma, cough,diarrhoea, dysentery, leucorrhoea, jaundice, pains and malaria (Madunaguet al., 199). Also, the fresh leaves of M. indicahave been used in Asian traditional medicine to treat diabetes mellitus (Muruganandanet al., 25; Bakhru, 2). In Ghana however, there is little or no scientific data on the hypoglycaemic property of M. indica leaves.the aim of the present study therefore, was to evaluate the hypoglycaemic property of M. indica leaves in both STZ induced diabetic rats and genetically diabetic mice. Materials and Methods Collection of plant materials Fresh leaves of M. indicawere obtained from Arboretum region of CPMR in the Eastern region, and authenticated by the Plant Development Division of the CPMR. The leaves were washed with distilled water, shade dried and pulverized. Extraction Pulverized dried leaves of M. indica (76. g) was boiled in 6. liters of water for 45 min and cooled. The resultant extract was filtered through cotton wool, pre-frozen and lyophilized into powder using a freeze dryer. The dried powdery extract was weighed to determine the yield (9.7 % w/w) and stored in a desiccator at room temperature. The powder was reconstituted in sterilized distilled water (to prevent contamination) before administered to the animals. Animals Adult Sprague Male Dowley rats weighing g, and db/db mice weighing 4-52 g were housed under standard conditions of room temperature. The rats were fed with standard pelleted feed with tap water ad libitum in the Animal House of the Department of Pharmacology (CPMR). All the animals were treated in accordance with the National Institute of Health Guidelines for the care and use of laboratory animals. The research protocol was approved by the Ethics Committee of CPMR. Toxicity Studies Aqueous extract of M. indica leaves suspended in distilled water was administered orally at doses of 25 and 5 mg/kg body weight to two groups of six animals (rats) respectively, after an overnight fast. The animals were observed over a 48 hour period under the profile; mortality and general behavior. Surviving animals were observed for a further period of 12 days for toxic symptoms such as pilo-erection and defects in lachrymatory, locomotory and respiratory activities. Induction of diabetes in rats using streptozotocin (STZ) Diabetes was induced in rats according to the description of Nagarajanet al. (25) with some modification. The animals were injected intraperitoneally with a single dose of STZ (6 mg/kg body weight) after an overnight fast, and allowed free access to feed and water. Blood glucose levels were recorded before (baseline) and after a resting period of 48 hours to confirm state of diabetes. Rats with blood glucose level higher than 1mmol/l were considered to be diabetic for the experiment. Hypoglycaemic Effect of the aqueous extract of M. indicaleaves on diabetic rats and mice The STZ induced diabetic rats and genetic diabetic mice (representing Type 1 and Type 2 diabetes models, respectively) were randomly divided into five groups of five animals each; Groups A, B, C, D and E. Groups A, B and C of both rats and mice were given oral doses of 1, 5 and 1 mg/kg body 38

3 weight of aqueous extract of M. indica leaves, respectively. Group D of the diabetic rats set up was given 2 units/kg body weight of insulin, a standard antihypergylcaemic agent to serve as positive control while Group D of the diabetic mice set up was given 1mg/kg body weight of glibenclamide, an anti-diabetic reference drug to serve as positive control. Group E of both set ups received 1 ml/kg body weight distilled water representing the negative controls. Blood glucose level measurement Blood glucose (BG) levels were initially measured hourly over a six hour period starting at hour after which the M. indica extract was administered. Blood samples were obtained from tail vein using Accu- Chek Active glucometer (Roche Diagnostics GmbH, Mannheim Germany). Both rats and mice received standard pelleted diet and water every morning throughout the study period. Statistical analysis All the data provided in this hypoglycaemic study represents means ± S.E.M. The results were analysed by one-way ANOVA followed by Bonferroni s multiple comparison test to establish significance (p.1) between the treated and the control groups. Results and Discussion Acute Toxicity Studies The effect of a single oral dose of aqueous extract of M. indica leaves (5 mg/kg) administered to six animals showed no deaths within 48 hours, suggesting that the oral LD 5 is greater than 5 mg/kg. There were no observations of any other signs of weakness as supported by normal respiratory, locomotor and lachrymatory activities and the absence of pilo-erection after 12 days monitoring. This finding suggests that aqueous extract of M. indicaleaves is safe or non-toxic to rat. Hypoglycaemic Effect of the aqueous extract of M. indicaleaves on diabetic rats and mice The aim of the study was to evaluate the hypoglycaemic property of M. indica leaves in both STZ induced diabetic rats and genetic diabetic mice representing. Administration of streptozotocin causes reduction in the number of β-cells, responsible for the production of insulin and induces hyperglycemia (Brenna et al., 23). In Type I diabetes, patients are administered with insulin due to their inability to produce sufficient insulin for the metabolism of glucose. Insulin enhances the uptake of glucose into the cells for metabolism (McGibbonet al., 213). The effect of M. indica on the blood glucose level in STZ-induced rats over a 6hr and 28 days, are shown in Fig 3a (i) and Fig 3b (ii) respectively. From Fig 3a (i) there was a dose-independent percentage reduction in blood glucose level among the treatment group over the 6 hour period (23.8%, 12.7%, 16.3% reduction for 1 mg/kg, 5 mg/kg and 1 mg/kg respectively); this reduction however, was not comparable to the positive control (Insulin-1 unit/kg, 85.4% reduction). Fig 3a(ii) shows that, whereas the lowest dose of M. indica(1 mg/kg) showed progressive percentage reduction in the blood glucose level over the 28 days period, the positive control (Insulin-1 unit/kg) showed an initial significant decrease in the blood glucose level but increased within 1 days period and decreased significantly again. This suggests that M. indica barely has any acute hypoglycaemic effect on Type 1 diabetes, but after a prolonged period at 1 mg/kg may present promising hypoglycaemic effect. This observation corroborates with the earlier findings by Aderibigbeetal., 1999.In that study, the effect of aqueous extract of leaves of M. indicaon blood glucose level was assessed in normoglycaemic and streptozotocin (STZ) induced diabetic rats. The aqueous extract given orally (1 gm/kg) of aqueous 39

4 extract of leaves M. indicadid not alter the blood glucose levels in either normoglycaemic or STZ induced diabetic rats. 1 INS-1unit/kg Time/hrs -1 Fig. 3a (i) Hypoglycaemic effect of aqueous extract of M. indica leaves on STZ-induced diabetic rats over a 6 hour period. All values are expressed as mean ± S.E.M. (N = 5); MGI = Aqueous extract of M. indicaleaves; INS = Insulin 1 INS-1unit/kg Days -1 Fig. 3a (ii) Hypoglycaemic effect of aqueous extract of M. indica leaves on STZ-induced diabetic rats over 28 days period. All values are expressed as mean ± S.E.M. (N = 5); MGI = Aqueous extract of M. indicaleaves; INS = Insulin Type 2 diabetes is a very complex metabolic disorder in which there is interplay between genetic background and environmental factors (such asobesity, age etc.) to contribute to the establishment of the disease. The disease is usually characterized by peripheral insulin resistance (approximately 9% of 4

5 all cases of diabetes), hyperinsulinemia, and/or β-cell dysfunction (with or without decrease of β-cell mass) and thus resulting in hyperglycaemia (Chatzigeorgiouet al., 29).Glibenclamide, an antidiabetic drug in the class of sulfonylureas is administered to stimulate insulin release to aid the metabolism of glucose (Harper et al., 213). Fig 3b (i) and Fig 3b (ii) show the effect of M. indica on the blood glucose level in genetically diabetic mice over a 6hr and 28 days, respectively. Over the 6hr observation, there was an apparent general dose-dependent percentage reduction in the blood glucose levels compared to the untreated group as shown in Fig 3b (i). The highest dose of M. indica extract (1mg/kg) was very comparable with the positive control (glibenclamide-1mg/kg); 32.2% and 28.4% reduction, respectively. Likewise, over the 28 days observation there was a general percentage reduction in the blood glucose level. This reduction was all comparable with the positive control (glibenclamide-1mg/kg) as shown in Fig 3b (ii). The results therefore suggest that M. indica has hypoglycaemic effect to Type 2 diabetes and this effect could be due to either or both an increase in the endogenous insulin release from pancreatic β-cells and the promotion and facilitation of peripheral glucose uptake and utilization, similar to glibenclamide (Moller, 21). However further studies should be performed to confirm this hypothesis Time/hrs GB-1 mg/kg Fig. 3b (i) Hypoglycaemic effect of aqueous extract of M. indica leaves on genetic diabetic mice over a 6 hour period. All values are expressed as mean ± S.E.M. (N = 5); MGI = Aqueous extract of M. indicaleaves; GBI = glibenclamide GB-1 mg/kg Days Fig. 3b (ii) Hypoglycaemic effect of aqueous extract of M. indica leaves on genetic diabetic mice over 28 days period 41

6 All values are expressed as mean ± S.E.M. (N = 5); MGI = Aqueous extract of M. indicaleaves; GBI = glibenclamide Conclusion The present study has shown that the aqueous extract of M. indica leaves possesses hypoglycaemic properties in genetic diabetic mice comparable to glibenclamide. The leaf extract of M. indica may therefore be useful in the management of diabetes especially, Type 2. Also, this study provides some scientific rationalization for the traditional use of M. indicain the management of diabetes in Ghana. Acknowledgement The authors are thankful to Department of Pharmacognosy and Herbal Medicine, University of Ghana, Legon and Centre for Scientific Research into Plant Medicine at Mampong-Akwapim, Ghana, for providing support for this study. References 1. Aderibigbe, A..,Emudianughe, T. S. and Lawal, B. A. (1999)Antihyperglycaemic effect of Mangiferaindica in rats. Phytother Res; 13(6): Bakhru, H. K. (2) Foods that heal (16 printing). Orient Paperback, a division of Vision Book Pvt. Ltd, India, pp Chatzigeorgiou, A., Halapas, A., Fatakis, K.K.andKamper, E.(29)The Use of Animal Models in the Study of Diabetes Mellitus: a Synopsis (Review),in vivo23: Desai, R.,Parul, V. and Dhruv, P. (212) Effect of Awareness of Diabetes on Clinical Outcomes of Diabetes: An Observational Study at a Private Hospital in Gujarat. Nat J. Med Res2(4): Dineshkumar, B., Mitra, A. and Manjunatha, M. (21)Antidiabetic and hypolipidaemic effects of few common plants extract in Type 2 diabetic patients at Bengal.Int J Diabetes &Metab18: Harper, W., Clement, M., Goldenberg, R., Hanna, A. Main, A., Retnakaran, R., Sherifali RN, D., Woo, V. and Yale, J. (213) Pharmacologic Management of Type 2 Diabetes. Can J Diabetes37: Kennan, R.P., Takahasi, K., Pan, C., Shammoon, H. and Pan, J.W. (25) Human cerebral blood flow and metabolism in acute insulin induced hypoglycaemia.j. Cereb. Blood Flow Metab Madunagu, B.E. Ebana, R. and Ekpe, E.D (199) Antibacterial and Antifungal Activity of some medicinal plants of AkwaIbom state. West Aft J BiolApplChemi.35: McGibbon,.A, Richardson, C., Hernandez RN, Cher and Dornan, J. (213) Pharmacotherapy in Type 1 Diabetes. Can J Diabetes37: Muruganandan, S., Srinivasan, K., Gupta, S., Gupta, P.K. and Lal, J (25) Effect of Mangeferin on hyperglycaemia and atherogenicity in streptozotocin diabetic rats. J Ethnopharmacol.93: Nagarajan, N.S, Murugesh,N., Thirupathy-Kumaresan, P., Radha, N. and Murali, A. (25). Antidiabetic and antihyperlipemic effects of Clemeo felina, Fitoterapia76: Shim, Y.J., Doo, H.K., Ahn, S.Y, Kim, Y.S., Seong, J.K., Park, I.S. and Min, B.H (23).Inhibitory effect of aqueous extract from the gall of RhusChinensis on alpha-glucosidase activity and post prandial blood glucose.j Ethnopharmacol85:

7 13. Teves, D., Videen, T.O., Creyer, P. E., Powers, W. J. (24). Activation of human medial refrontal cortex during autonomic response to hypoglycaemia.proc. Natl. Acad. Sci. 11: Wild, S., Roglic, G., and Green, A. (24). Global Prevalence of Diabetes: Estimates for the Year 2 and Projections for 23. Diabetes Care.27 (5):