Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
International Journal of Applied Research in Natural Products Vol. 1(2), pp. 32-36, June/July 2008 Available online http://www.healthy-synergies.com ©2008 Healthy Synergies Publications Original Article Hypoglycemic Activity of Methanolic Stem Bark of Adansonnia digitata Extract on Blood Glucose Levels of Streptozocin-Induced Diabetic Wistar Rats Tanko Y1*, Yerima M2, Mahdi MA2, Yaro AH3, Musa KY4, Mohammed A1 1 Department of Human Physiology, Ahmadu Bello, University, Zaria, Nigeria. 2Department of Pharmacology and Clinical Pharmacy Ahmadu Bello, University, Zaria. Nigeria. 3Department of Pharmacology, Bayero University, Kano, Nigeria. 4Department of Pharmacognosy and Drug Development Ahmadu Bello, University, Zaria. Nigeria. Summary: Evaluation of hypoglycemic activity of methanolic stem bark extract of Adansonnia digitata in Wistar rats has been investigated. Antidiabetic potentials of the plant extract at the doses of 100,200 and 400 mg/kg intraperitoneally administered on Streptozocin diabetes Wistar rats. Treatment of streptozocin diabetic Wistar rats with the extract caused a significant (P<0.05) reduction in the blood glucose levels when compared with control. The dose of 100mg/kg shown a significant decreased (p<0.05) after 1, 3 5 and 7 hours of extract administration when compared to control normal saline. Also the dose of 200 mg/kg shown a significant decreased (p<0.05) after 3, 5 and 7 hours of extract administration. The dose of 400 mg/kg also shown a significant decreased( p<0.05) after 5 and 7 hours of extract administration when compared to control normal saline. The highest activity resides at the dose of 100 mg/kg with percentage glycemic change of 51 % after 7 hours of extract administration while the other two doses 200 and 400 mg/kg have glycemic change of 39% and 31% respectively after 7 hours of extract administration. The phytochemical screening revealed the presences of tannins, carbohydrate, terpenes, saponins, flavonoids and alkaloids. The median lethal dose ( LD50) in mice was calculated to be 1264.9 mg/kg bodyweight. This result suggests that the methanolic stem bark of Adansonnia digitata possess antidiabetic effect on streptozocin induced diabetic Wistar rats. Industrial relevance: The herbal medicines are getting more importance in the treatment of diabetes and others ailments because of the hazardous adverse effect of the current therapy used to treat those ailments using synthetic drugs. Herbal medicine is free from side effects and less costly when compared to the synthetic hypoglycemic agents. The present study will help the industry to produce herbal drug with less side effect, less costly affordable and more effective in the treatment of diabetes mellitus. In vivo models were utilized to test the antidiabetes activity and important to optimized the dose in the herbal formulation. Finally the phytochemical screening or elucidation of the bioactive compounds from the plant would be effective drug against diabetes mellitus. Keywords: Hypoglycemic activity, streptozocin Phytochemical Adansonnia digitata, Introduction Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia and defective metabolism of glucose and lipids. Diabetes was estimated to affect 177 million people world wide in 2000 and this figure is projected to increase to 300 million by 2025 (Porter and Barret, 2005). Diabetes is not a single disease rather it is a heterogeneous group of syndromes characterized by an elevation of blood glucose caused by relative or absolute deficiency of insulin. Diabetes can be divided into two main groups based on their requirements of insulin: insulin dependent diabetes mellitus (Type 1), and non-insulin dependent diabetes mellitus (Type 2). However, other types of diabetes have also been identified. Maturity Onset Diabetes of the Young (MODY) is now classified as Type 3 and gestational diabetes classified as Type 4. NIDDM type 2 diabetes account for about 90 percent of diabetic cases (WHO, 2002). Insulin resistance and β-Cell dysfunction are the metabolic abnormalities in the type 2 diabetes (Sa’ad et al., 1991). Glycemic control is one of the targets for managing diabetes mellitus. Studies have confirmed that for the type 2 diabetes, effective control of blood glucose substantially decrease the risk of developing diabetic complications (Ohkubo et al., 1995; UKPDS, 1997). Orthodox treatment of diabetes mellitus includes a modification of life style, such as diet and exercise and the use of insulin and/or oral hypoglycaemic drugs. These pharmacologic agents target increased insulin secretion, decreased hepatic glucose production and increased sensitivity to insulin (Kelly and Mandarino, 2000). Management of this disease with insulin and/or oral hypoglycaemic agents have certain drawbacks (University Group Diabetes Program, 1974; *Corresponding Author: Tel: +1-234-8037054274 Fax: +1-234-62248314 E-mail: [email protected] Accepted 27 March 2008 32 Hypoglycemic activity and Adansonia digitata Knatterud et al., 1978). For insulin such drawbacks include ineffectiveness on oral administration, short shelf life, requirement of constant refrigeration and in the event of excess dosage-fatal hypoglycaemia. The use of oral hypoglycaemic drugs like sulfonylureas and biguanides is also associated with side effects such as propensity to gain weight (Rang and Dale, 1991). Adansonnia digitata. L. ( Bombacaceae). An African plant known as baobab tree. Leaves, bark and fruits of this plant are traditionally employed in several African regions as food stuffs and for medicinal purposes, and for that reason baobab is also named “the small pharmacy or chemist tree” (Etkin and Ross 1982; Kerharo and Adam,1974).The native African population commonly used, baobab fruit as famine food to prepared, decoctions, sauces and natural refreshing drink due to it nutritional properties ( Obizoba and Anyika 1994; Lunven and Adrian, 1960). The pulp is therapeutically employed as analgesic anti-diarrhea and for treatment of smallpox and measles (Kerharo and Adam, 1974). The present study was designed to test the hypoglycemic effect of methanolic extract of Adansonnia digitata stem bark on streptozocin- induced diabetes. Materials and Methods Plant material: The stem bark of Adansonnia digitata was collected around area of Basawa, Sabongari L.G.A. Zaria of Kaduna State Nigeria in the Month of August 2007. The plant specimen was identified by Mal A.U Gallah of the Herbaruim Unit, Department of Biological Sciences, Ahmadu Bello University, Zaria- Nigeria, where a voucher specimen has been deposited. Extract preparation: The stem bark of Adansonnia digitata was dried under shade for one week and the pulverized into fine powder in a pestle and mortar and passed through a mesh sieve. The powdered was macerated with 70% methanol for 72 hours. Later the extract was filtered then concentrated using rotary evaporator and was kept in dessicator until use. Chemicals used: All chemicals and drugs were obtained commercially and were of analytical grade. Streptozocin (Sigma).The Biphasic Isophane insulin AS Mixtard 30 HM Pen fill (Novo Nordisk AIS 2880 Bagsvaerd, Denmark. NAFDAC Reg.no 04-1601). Acute toxicity study: The lethal dose (LD50) of the plant extract was determined by method of Lorke (1983) using 13 mice. The animals were handled in accordance with international principles guiding the use and handling of experimental animals (United State National Institute for Health, 1985). In the first phase mice were divided into 3 groups of 3 mice each and were treated with the extract at doses of 10, 100 and 1000mg/kg body weight intraperitoneal. They were observed for 24 hours for signs of toxicity. In the second phase 4 mice were divided into 4 groups of 1 rat in each group and were also treated with the extract at doses of 600, 1000, 1600 and 2900 mg/kg bodyweight (i.p).The median lethal dose ( LD50 ) was calculated using the second phase. Phytochemical screening: The preliminary phytochemical screening of the crude extract of Adansonnia digitata was carried out in order to ascertain the presence of its constituents utilizing standard conventional protocols (Trease and Evans, 1983). Induction of Diabetes Mellitus: Twenty five Wistar rats of both sexes weighing 150-180 grams were used for the study of the effects of Adansonnia digitata extract on the blood glucose levels of the animals. They were kept in standard cages at 250C and 12 hour light/dark condition in the animal room of the Department of Human Physiology, AB.U, Zaria. The animals were fed on commercial feeds and were given water ad libitum. The animals were fasted from feeds for 12 hours before the commencement of each experiment, but were allowed water ad libitum. The rats were injected with streptozocin dissolved in citrate buffer pH 4.5 in a dose of 60mg/kg body weight intraperitoneal. Since Streptozocin is capable of producing fatal hypoglycemia as a result of massive pancreatic release of insulin, the rats were treated with 20% glucose solution intraperitoneally after 6 hours (Stanley et al., 2001).The were kept for the next 24 hours on 5% glucose solution bottles in their cages to prevent hypoglycemia. After a period of three days the rats with a blood glucose levels greater than 180mg/dL were considered diabetic and used for this research work Experimental Design The Streptozocin-induced diabetic Wistar rats were randomly assigned into five groups (1-5) of five rats (n=5) each as follows, namely Group 1- Received normal saline i.p Group2- Received Biphasic Isophane Insulin 6 i.u/kg i.p(Stanley et al.,2001) Group3- Received 100 mg/kg body weight of the Adansonnia digitata extract i.p Group4- Received 200 mg/kg body weight of the Adansonnia digitata extract i.p Group5- Received 400 mg/kg body weight of the Adansonnia digitata extract i.p Determination of blood glucose levels Blood samples were collected by cutting the tail-tip of the rats, for blood glucose determination at intervals of 0, 1, 3, 5 and 7 hours. Determination of the blood glucose level was done by the glucose-oxidase principle (Beach and Turner 1958) using the ONE TOUCH Basic (Lifescan, Milpitas, CA) instrument and results were reported as mg/dl (Rheney and Kirk, 2000). Statistical analysis: Blood glucose levels were expressed in mg/dl as mean ± SEM. The data were statistically analyzed using ANOVA with multiple comparisons versus control group. The values of p<0.05 were considered as significant (Duncan et al., 1977). 33 Tanko et al Results Phytochemical analysis Freshly prepared extracts were subjected to preliminary phytochemical screening test for various constituents. This revealed the presence of tannins, carbohydrate, terpenes, saponins, flavonoids and alkaloids. Acute toxicity study (LD50) The sign of toxicity were first noticed after 10-12 hours of extract administration. There was decreased locomotor activity and decreased in sensitivity to touch. Also there was decreased feed intake, and prostration after 18 hours of extract administration. The median lethal dose (LD50) in rats was calculated to be 1264.9 mg/kg body weight. Anti diabetic study Table 1 showed the results of the effects of three doses (100mg/Kg, 200mg/Kg and 400 mg/Kg) of Adansonnia digitata extract, Insulin and control groups in streptozocin-induced diabetic Wistar rats. A drop of blood samples was collected by cutting the tail-tip of the rats, for the blood glucose determination at intervals of 0, 1, 3, 5 and 7 hours. The dose of Insulin and the dose of the extract 100mg/kg there was a significant decreased the blood glucose levels when compared to control after 1 hour of extract administration while the other two doses of the extract 200 and 400 mg/kg did not show any significantly change in the blood glucose levels after 1 hour of extract administration when compared to control group with percentage glycemic change of 19 and 4 % After 3 hours of extract administration there was a significant decrease in the blood glucose level in 100 and 200 mg/kg when compared to control group with percentage glycemic change of 38 and 29% while the dose of 400 mg/kg there was no any significant change in the blood glucose levels with percentage of glycemic change of 9% . However, after 5 and 7 hours of treatment there was a significant decrease (p<0.05) in the blood glucose levels when compared to control in all the three doses given with percentage glycemic change of 43, 29 and 20 respectively. And after 7 hours of extract administration the glycemic change of 51, 39 and 31 % respectively. Table 1. Effect of stem bark of aqueous methanolic Adansonnia digitata on streptozocin-induced diabetic Wistar rats Treatment 0 hour BLOOD GLUCOSE LEVELS(Mg/dl) 1 hours 3 hours 5 hours 7 hours Group 1 Control (N/Saline) 189±7.6 226±18.2 231±17.1 232±13.8 239±16.2 Group 2 (Insulin 6.i.u/kg) 193±11.9 170±10.2 a (25%) 143±8.66 a (38%) 115±6.42 a (50%) 103±4.27 a (56%) Group 3 (100 mg/kg) 192±9.13 ns 165±14.4 a ( 27%) 143±10.7 a (38%) 131± 11.8 a (43%) 115±3.31 a (51%) Group 4 (200 mg/kg) 189±14.0 ns 182±12.9 ns (19%) 162±10.8 a (29%) 165±12.1a (29%) 146±12.1 a (39%) Group 5 (400 mg/kg) 192±57.37 ns 216.±19.2ns (4%) 209±18.3 ns (9%) 185±14.9 a (20%) 166±13.9a (31%) Vales are given as mean ± SD for 5 rats in each group; experimental groups are compared with diabetic control. Values are statistically significant at a=P<0.05 ns=not significant % Glycemic change= Glucose concentration (2, 4, 8 or 24) – fasting blood glucose x 100 Fasting blood glucose Figure in parenthesis represent percentage glycemic change Discussion Medicinal plants are widely used by the populations of underdeveloped countries as alternative therapy. In Africa, hundreds of plants are used traditionally for the management and/or control of diabetes mellitus. Unfortunately only a few of such African medicinal plants have received scientific scrutiny. The present work was therefore designed to study the anti-diabetic property of Adansonnia digitata stem bark Streptozocin-diabetic rats. Streptozocin-induced hyperglycaemia has been described as a useful experimental model to study the activity of hypoglycemic agents ( Szkudelski, 2001). Streptozocin selectively destroyed the pancreatic insulin secreting β –cells, leaving less active cell resulting in a diabetic state (Kamtchouing et al., 1998; Szkudelski, 2001). 34 Hypoglycemic activity and Adansonia digitata Many secondary metabolites participate in a variety of anti-diabetic functions in vivo (Kako et al., 1997). The glycemic change in blood glucose levels of diabetic rat at different time intervals after intraperitoneal administration Adansonnia digitata extract of at the doses of 100, 200, and 400mg/kg as showed in Table 1. In relation to the diabetes rats that received 100, 200 and 400 mg/kg bodyweight of Adansonnia digitata extract there was a significant (p<0.05)reduction in the blood glucose levels when compared to the control group after 2 hours of extract administration as regard to the dose of 100 mg/kg and the reference drug . In relation to the dose of 200 and 400 mg/kg of the Adansonnia digitata there was no any significant change in the blood glucose levels after 1 hour of extract administration. Also after 3 hours of extract administration as regard to the doses of 100 and 200 mg/kg there was significant reduction in the blood glucose levels extract administration when compared to control group. After 5 and 7 hours of extract administration there was a significant change in the blood glucose level in all the three doses of the extract given when compared to control group. In relation to the reference drug biphasic insulin 6.i.u/kg there was a significant decrease in the blood glucose level when compared to control group. The dose of 100 mg/kg was found to be more effective in the glycaemic change after 7 hours of extract administration than the other two doses of the extract 20 and 400 mg/kg body weight. The extract might possess Insulin like effect on peripheral tissues either by promoting glucose uptake and metabolism or inhibiting hepatic Gluconeogenesis. The phytochemical studies of Adansonnia digitata extract of revealed the presence of tannins, carbohydrate, terpenes, saponins, flavonoids and alkaloids. flavonoids . Flavonoid and terpenes isolated from the other antidiabetic medicinal plants has been found to stimulate secretion or possess an insulin like-effect (Marles and Farnsworth 1995). Effect of the flavonoids quercetin and ferulic acid on pancreatic β-cells leading to their proliferation and secretion of more insulin have been proposed by Mahesh and Menon (2004) and Sri-Balasubashini, et al., (2004) as the mechanism by which they reduced hyperglycaemia caused by streptozocin in diabetic rats. The flavonoids present in Adansonnia digitata may also be acting similarly thereby decreasing the high blood glucose levels of streptozocin-diabetic rats. In conclusion, the experiment evidence obtained in the present laboratory animal study indicate that methanolic extract Adansonnia digitata stem bark possess anti-diabetic properties which suggest the presence of biologically active components which may be worth further investigation and elucidation. Acknowledgements The authors wish to thank Mallam Bala Mohammed, a technical staff of the Department of Human Physiology, ABU, Zaria for taking care of the experimental animals throughout the period of this research work. References Beach, E.F., and Turner, J.J. 1958. An enzymatic method for glucose determination uptake in body fluids. Clin Chem. 4:462-468. Brain, K. R and Turner, T. D.1975 The Practical Evaluation of Phytopharmaceuticals, Wright Scientechnica, Bristol, pp. 57 – 58. Dalziel, J.M. 1936. The useful plants of West Tropical African Watmonghs, Idle, London. Pp 354-355. Duncan, R.C., Knapp, R.G., Miller, M.C. 1977. Test of hypothesis in population. In: Introductory Biostatistics for the health sciences. John Wiley and Sons Inc. NY pp.71-96. Etkin,N.L.,Ross,P.J.1982: “Food as medicines and medicines as food” Soc.sci med,16,1559-1573. Kako, M., Miura, T., Nishiyama, Y., Ichimaru, M., Moriyasu, M., Kato,1997:Hypoglycemic Activity of Some Triterpenoid Glycosides. J. Nat. Prod. 60:604-605. Kamchouing, P.,Sokeng,D.S,Moundipa,F.P.,Watcho,P.,Jatsa,B.H and Lontsi, D. 1998:.Protective role of Anacardium Occidentale extract against streptozocin- induced in rats. J. Ethnopharmacol 62:55-99. Kameswara Rao, B. Kesavulu., M.M. Giri., and Appa Rao, CH.1999: Antidiabetic and hypolipidemic effects of Momordica cymbalaria Hook, fruit powder in alloxan diabetic rats. J.of Ethnopharmacol. 67:103-109. Kelly, D.E. and Mandarino, L.J. 2000. Fuel selection in human skeletal muscle in insulin resistance. Diabetes, 40: 677681. Kerharo, T., Adam,J.G 1974 “ la pharmacopia senegalaise traditionanelle” Plantes medicates et toxiques Editions Vigot Frerez, Paris Kokwaro, J.O .1976 Medicinal plants of East African. East African literature bureau, Kampala, Nairobi,Dar’salaam Pp 368. Krisna, B., Nammi, S., M.K. and Krshna Rao, R.V. 2004. Evaluation of hypoglycemic and antihyperglycemic effect of Datura metel Linn seeds in normal and alloxan-induced diabetic rats. J. of Ethnopharmacol. 9:95-95. Knatterud, G., Klimt, C.R., Levin, M.E., Jacobsen, M.E. and Goldner, M.G. 1978. Effect of hypoglycaemic agents on vascular complication in patients with adult onset diabetes. Section VIII. Mortality and selected non fatal events with insulin treatment. Journal of the American Medical Association, 240: 37-42. Lunven, P., Adrian, J 1960: Interer alimentaire dela Seville et de la pulped u fruit de baobab ( Adansoia digitata ) Am Nutr.14,263-276. Lork D 1983. A New Approach to Practical Acute Toxicity Testing Archieves of Toxicology: 275-287. Mahesh, T. and Menon, P.V. 2004. Quercetin alleviates oxidative stress in streptozotocin induced diabetic rats. Phytotherapy Research, 18: 123-127. Marles, J.R.and Farnsworth, N.R. 1995.Antidiabetic plants and their active constituents.Phytomedicine 2 (2 )123-89. Obizoba, I.C.,Anjika J.U 1994 “Nutritive value of baoba milk ( gubdi) and mixture of baoba( Adansonnia digitata L.) hungry rice, acha ( Digitaria exillis) flours” Plants food Hum Nutr, 46,157-165. 35 Tanko et al Porter, J.R. and Barrett, T.G. 2005. Monogenic syndromes of abnormal glucosehomeostasis: clinical review and relevance to the understanding of the pathology ofinsulin resistance and β cell failure. Journal of Medical Genetics, 42: 893-902. Rang, H.P. and Dale, M.M. 1991. The Endocrine System Pharmacology. Longman, Harlow. Pp. 504-508. Rheney, C.C., Kirk, K.K. 2000. Performance of three blood glucose meters. Ann Pharmacother. March, 34 (3) 317-21. Sa’ad, M.F., Knonwler, W.C., Pettitt, D.J., Nelson, R.G., Charles, M.A. and Bennett, P.H. 1991. A two step model for development of non-insulin dependent diabetes. American Journal of Medicine, 90: 229-235. Stanley,A mainzen P,Venugopal MP.2001.Anti-oxidant action of Tinosporacordifolia root extract in alloxan diabetic rats.Phytother Res.15:213-218. Sri Balasubashini, M., Rukkumani, R., Viswanathan, P. and Menon, P.V. 2004. Ferulic acid alleviates lipid peroxidation in diabetic rats. Phytotherapy Research, 18: 310-314. Szkudelski, T.2001. The mechanism of Alloxan and Streptozocin action in β cell of the rats pancreas.Physiol Res. 50:536-46. Trease, G.E. and Evans, M.C. 1983. Textbook of Pharmacognosy, 13th ed. Bailliere, Tindall, London, pp. 683-684. United State National Institute for Health.1985. publication no 85-23. University Group Diabetes Program 1974. A Study of the effects of hypoglycaemic agents on vascular complications in patients with adult onset diabetes: Section V. Evaluation of phenformin therapy. Diabetes, 24: 65-184. World Health Organisation 2002. Diabetes Mellitus fact sheet 138. Geneva. 36