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Tropical Journal of Pharmaceutical Research, Vol. 8, No. 1, February, 2009, pp. 19-26 Research Article Protective effect of methanol-methylene chloride extract of Terminalia glaucescens leaves on streptozotocin-induced diabetes in mice Guy BSN Njomen1, René Kamgang1*, Petit RN Soua1, Jean LE Oyono2, Njifutie Njikam1 1 General Endocrinology and Metabolism Systems (GEMS), Laboratory of Animal Physiology, Faculty of Sciences, Code Number: pr09004 Abstract
Purpose: Terminalia glaucescens (Combretaceae) is traditionally used in Cameroon in the treatment of diabetes. The anti-hyperglycemic effect of the methanol-methylene chloride extract of the leaves of this plant was investigated in streptozotocin (STZ)-induced diabetic mice. Keywords: Terminalia glaucescens, streptozotocin, diabetes mellitus, glycemia, lipemia. INTRODUCTION Diabetes mellitus is a metabolic disorder characterized by fasting hyperglycemia and alterations in carbohydrate, fat and protein metabolism, associated with absolute or relative deficiencies in insulin secretion and/or insulin action1 . As the number of people with diabetes multiplies worldwide, the disease has taken an ever-increasing share of national and international health budgets. Type 1 diabetes is an auto-immune disease characterized by a local inflammatory reaction in and around pancreatic islets that is followed by selective destruction of insulin-secreting � cells2-4 . Experimentally, streptozotocin (STZ) or alloxan are used to induce diabetes in rodents. STZ is effective in triggering islet cell death by acute oxidative stress. STZ-induced diabetic mice are one of the animal models of insulin-dependent diabetes mellitus characterized by high fasting blood glucose levels and drastic reduction in plasma insulin concentration5 . Though different types of oral hypoglycemic agents are available along with insulin for the management of diabetes mellitus, there is a growing interest in herbal remedies due to the side effects associated with orthodox therapeutic agents6,7 . Furthermore, plants have played a major role in the discovery of new therapeutic agents. A medicinal plant, Galega officinalis, led to the discovery and synthesis of metformin; however, there is still a need for new oral antidiabetic drugs with minimal side effects8,9 . The available literature shows that there are more than 400 plant species showing antidiabetic activities10-12 . The protective effect of some plants or natural substances against the death of � cells has been reported13 . Information provided by practitioners of traditional medicine in the Centre Province of Cameroon suggests that Terminalia glaucescens possesses useful anti-diabetic properties, although this was not cited in the national ethnobotanical survey of Cameroonian medicinal plants. Terminalia arjuna, Terminalia belerica, Terminalia chebula, Terminalia catappa and Terminalia pallida are other species of the same family (Combretaceae) known for their antidiabetic properties1 . The present study was undertaken to investigate the anti-hyperglycemia effect of the methanol/methylene chloride extract of Terminalia glaucescens leaf on the hyperglycemia induced by a multiple dose of STZ in diabetic mice. MATERIALS AND METHODS Plant extract Fresh leaves of Terminalia glaucescens were harvested in October 2005 in Mbalmayo in the Centre Province of Cameroon. T. glaucescens was identified by Dr Simeon Tchoulagueu of the Teachers' Training College of the University of Yaoundé I, who kept a voucher specimen (no. 4016/BS) in the laboratory. Two kilograms of the sun-dried powdered leaves were macerated in a mixture of methanol/methylene chloride (1:1) for 7 days (with occasional stirring) at room temperature. The mixture was filtered with Whatman No.1 filter paper and the filtrate concentrated in a rotary evaporator (Rotavapor) at 45°C to yield 125g of a dry dark extract. Doses of 20 mg mL-1 and 60 mg mL-1 were obtained by dissolving 2 g and 6 g, respectively, of the concentrated extract in 100 mL of 10% dimethyl sulfoxide (DMSO) solution. The experimental dose was 5 µL g-1 body weight. Experimental animals Male albino Wistar mice (26-30 g weight, 8-10 weeks old) were raised in the animal house of our laboratory under natural conditions with free access to water and regular rodent chow. For this experiment, the mice were fasted overnight prior to blood sugar determination and randomly divided into 6 groups of 8 animals each as follows:
To induce diabetes, the mice received 45 mg kg-1 (i.p.) of freshly prepared STZ (Sigma Aldrich No. SO 130) dissolved in citrate buffer 100 mM pH4.5 (Cb) daily for five consecutive days. The plant extract was administered to the fed mice and STZ injection was preceded by 4 h of fasting. Animals DE100 and DE300 orally received a dose of plant extract, daily for five consecutive days, followed 6 hours later by i.p. injection of STZ. T. glaucescens extract administration began one day (day 0) before the beginning of STZ injection and continued for up to day 14. Animal housing and in vivo experiments conformed to the guidelines of the European Union on Animal Care (CEE Council 86/609) and were ethically approved by the Institutional Committee of the Ministry of Scientific Research and Innovation of Cameroon. Measurement of body weight gain, food and water intake Food and water intake were monitored on days 1, 3, 6 and 12 while body weight was measured on days 0, 3, 6, 9, 12 and 15. Determination of blood parameters Blood samples for glucose determination were obtained from the tail tip of 4 h fasted mice on days 0, 6 and 12 of the experiment. Blood glucose level was estimated using a glucometer (Accu-Check, Roche). Mice with fasting blood glucose ≥ 14 mM were considered as diabetic14 . At the end of the treatment (day 15), mice were weighed and anesthetized with sodium pentobarbital (60 mg kg-1 i.p.). Blood was rapidly collected by cardiac puncture in syringes containing EDTA. Blood samples were centrifuged (1 min, 8000 g), from which plasma was collected, aliquoted and snapfrozen in liquid nitrogen. Plasma parameters were assayed using commercially available kits according to the manufacturers’ instructions: triglycerides (TG: Triglycerides PAP, bioMérieux, Marcy l’Etoile, France), non esterified fatty acids (NEFA, NEFA-C, Wako), and cholesterol (RTU, bioMérieux). Plasma insulin levels were measured by radioimmunoassay using porcine insulin kit (SB-insulin-CT, CIS Biointernational, Gif-sur-Yvette, France) with guinea pig anti-porcine insulin serum-coated tubes. Statistical analysis
The results are expressed as mean RESULTS Body weight, food and water intake There was no significant difference in body weight between the groups prior to STZ injection. The body weight progressively decreased in diabetic control group (DC) and diabetic group treated with 100 mg kg-1 body weight extract (DE100). The decrease was significant on days 6 (P< 0.05), and 9 and 15 (P< 0.001) compared to the normal control group (-28% DC and – 25% DE100) as shown in Fig 1. Body weight gain for normal control (NC), normal treated (NE100 and NE300) and 300 mg kg-1 treated diabetic (DE300) mice more marked on the 12th day, whereas other increased progressively and were groups (NC, E100; E300 and DE300) did not comparable. Furthermore, DC and DE100 exhibit significant variations in food and water presented significant (P < 0.01) increases in intake (Fig 2). food and water intake on day 3 which became more marked on the 12th day, whereas other groups (NC, E100; E300 and DE300) did not exhibit significant variations in food and water intake (Fig 2). Plasma metabolites and insulin levels STZ administration significantly (P < 0.001) reduced insulin plasma levels (Fig 3). T. glaucescens at 100 mg kg-1 did not prevent the decrease of the plasma insulin level. The plant extract also did not significantly change the insulin level in normal mice (NE100 and NE300). STZ did not modify insulin secretion in mice treated with 300 mg kg-1 extract. In DC mice, the observed reduction of insulin secretion was accompanied by a significant (P< 0.05) increase in all plasma lipids: + 45% TG, + 32% NEFA, + 29% cholesterol (Table 1). T. glaucescens extract reduced the STZinduced elevation of plasma lipids in a dosedependent manner. Blood glucose level in DC mice was very significantly (P < 0.001) increased on the 6th day and beyond (Fig 4). The dose of 100 mg kg-1 body weight of the extract did not show a marked protective effect in mice which had STZ-induced hyperglycemia, but there was a significant fall in blood glucose in DE300 mice to the extent that glycemia was comparable to NC mice. In normal mice, T. glaucescens treatment resulted in a decrease of blood glucose levels (-9 %) in a dose-dependent manner in NE300 (P < 0.05). DISCUSSION This study was carried out to assess the protective effect of the methanol/methylene chloride extract of Terminalia glaucescens leaf on impaired glucose level induced by STZ. Our results show that intraperitoneal administration of multiple doses of STZ (45 mg kg-1 for 5 consecutive days) effectively induced diabetes in normal fasted mice as reflected by hyperglycemia, polyphagia, polydipsia and body weight loss, compared with normal control (NC) mice. The T. glaucescens extract dose of 300 mg kgbody weight significantly inhibited the hyperglycemic action of STZ, while the blood glucose level of diabetic control was distinctly increased (+117%). Streptozotocin induces hyperglycemia by selective destruction of pancreatic �-cells that secrete insulin9,15 . This suggests that the extract (300 mg kg-1 ) may prevent decreased insulin secretion by protecting �-cells against destruction caused by STZ. In the extract-pretreated mice, diabetic manifestations after STZ administration were reduced, suggesting that the extract may involve direct inhibition of STZ. The extract may act by competing with STZ for glucose associated-receptors on �-cell membranes. A phytochemical analysis of T. glaucescens has revealed the presence of tannin, alkaloids, flavonoids, triterpenoids and glucose16. The anti-hyperglycemic activity of T. glaucescens may be due to any one or more of these constituents in the extract. STZ is a 2-desoxy-D-glucose derivative of N-methyl-N-nitrosourea17,18. Thus, it is possible that the glucose moiety of STZ, which can bind to glucose receptors on �-cells, may directly compete with the glucose contained in the extract19. T. glaucescens extract might also have acted by increasing the resistance of �-cells by a direct activation of superoxide dismutase, a widely distributed enzyme that scavenges superoxide anion radical which may be responsible for the toxic effects of STZ20,21 . The secretion of insulin correlated with the reduction of glycemia. Moreover, T. glaucescens extract in normal mice and 300 mg kg-1 extract in diabetic test mice did not increase insulinemia beyond the normal control value. This suggests that in diabetes, T. glaucescens sensitizes insulin peripheral action rather than stimulating insulin secretion.T. glaucescens probably lowers blood lipids (TG, NEFA, cholesterol) due to improvement of peripheral insulin action, while body weight loss as well as increase in food consumption and the related water intake result from the lack of insulin action. The present study showed high plasma NEFA in diabetic mice. Excess plasma NEFA can inhibit insulin-stimulated glucose utilization in muscle and promote hepatic production of glucose22,23 , whereas reduction of plasma NEFA concentration improves glucose utilization and the suppression of hepatic glucose production by insulin24,25 . The reduction of plasma NEFA may also explain the anti-hyperglycemia effect of T. glaucescens extract. CONCLUSION The results from this study indicate that T. glaucescens extract in STZ-induced diabetes could have protective effects. The antihyperglycemia effect of T. glaucescens extract in STZ-induced diabetes could explain the traditional use of this plant for diabetes treatment or management. Further studies are required to elucidate the site(s), as well as cellular and molecular mechanism of actions of T. glaucescens extract. REFERENCES
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. The following images related to this document are available:Photo images[pr09004t1.jpg] [pr09004f1.jpg] [pr09004f4.jpg] [pr09004f2.jpg] [pr09004f3.jpg] |
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± standard error of mean (S.E.M.). Mean values were obtained by one-way analysis of variance (ANOVA) using the computer software, StatView 4.5, followed by Dunnets multiple comparison test. The significance of difference between and within various groups was determined. Values of P < 0.05 were taken to imply statistical significance. 
