1.0 INTRODUCTION:

Diabetes Mellitus is a clinical syndrome, characterized by hyperglycemia caused by a relative or absolute deficiency of insulin at the cellular level. It is the most common endocrine disorder, affecting mankind all over the world, prevalence of which is increasing, daily (Tong and Cockrum, 2003). Experimental diabetes in animals has provided considerable insight into the physiologic and biochemical derangement of the diabetic state. Many of the derangement have been characterized in hyperglycemic animals. Significant changes in lipid metabolism and structure also occur in diabetes (Sochar et al., 1985).In these cases the structural changes are clearly oxidative in nature and are associated with development of vascular disease in diabetes (Baynes and Thropes, 1999). In diabetic rats, increased lipid peroxidation was also associated with hyperlipidemia (Morel and Chisolm, 1989).

Diabetes mellitus (DM) is associated with an increased risk of thrombotic, atherosclerotic and cardiovascular disease. Hyperlipidemia is metabolic complication of both clinical and experimental diabetes (Gandh, 2001). Low-density lipoprotein in diabetic patients leads to abnormal metabolism and is associated with increase in very low-density lipoprotein (VLDL) secretion and impaired VLDL catabolism. Ultimately this leads to atherosclerotic plaque (Howard, 1987). A number of known factors for coronary artery disease such as hypertension, obesity and dyslipidemia are more common in diabetics than in the general population. The World Health Organization (WHO) predicts that the number of cases worldwide for diabetes, now as of 171 million, will touch 366 million or more by the year 2030 (Wild et al., 2004). Patients with DM are more likely to develop microvascular and macrovascular complications than the non diabetic population (Baynes, 1991). Dyslipidemia is a frequent complication of DM and is characterized by low levels of high density lipoprotein-cholesterol (HDL-C) and high levels of low density lipoprotein-cholesterol (LDLC) and triglyceride (TG).

Hyperlipidemia is the presence of high levels of cholesterol in the blood. It is not a disease but a derangement that can be secondary to many diseases and can contribute to many forms of diseases, most notably cardiovascular disease. The treatment of hyperlipidemia depends on the patients’ lipid profile. Many antihyperlipidemic agents like statin, fibrates, niacins, bile acids, ezitimibe etc., reduce cholesterol level with different conditions. Currently, available drugs have been associated with number of side effects (Brown, 1996).

Recent studies have shown that lipid associated disorders are not only attributed to the total serum cholesterol, but also to its distribution among different lipoproteins. The low density lipoprotein (LDL) are the major carriers of cholesterol towards tissues having atherogenic potential while the high density lipoprotein (HDL) carry cholesterol from peripheral tissues to the liver. HDL, thus gives protection against many cardiac problems and obesity.

The injection of streptozotocin and alloxan leads to the development of hyperlipidemia (O’mearaet al., 1991 and Agardhet al., 1999) and this is practically shown in this study with the injection of 160mg/kg of streptozotocin.

Treatment of hyperlipidemia in diabetes involves improving glycemic control, exercise and the use of lipid lowering diets, drugs and hypoglycemic agents (Betterridge, 1997; Miller et al., 2001).

Atherosclerosis is the most prevalent long-term diabetic complication. The increased mortality and morbidity in diabetics caused by atherosclerosis has been related to the frequent occurrence of hyperlipidemia in diabetes.

This study is designed to evaluate the effect of the combined therapy metformin and reducdyn on streptozotocin induced diabetic-hyperlipidemic male rats.

2.0 MATERIALS AND METHOD:

2.1 Drugs and Equipment.

Glucophage (metformin), and reducdyn were obtained from Dooka Pharmarcy, Ltd opposite the main gate of University of Port Harcourt teaching hospital, Alakahia, Port Harcourt while Streptozotocin was obtained from Glaxosmithline Ltd Ibadan. All other reagents were of analytical grade. Optical densities were measured using digital spectrophotometer (model 752S).

2.2. Animals

Adult male Wistar albino rats weighing 170-200g were obtained from the animal House of the Department of Biochemistry, University of Port Harcourt, Port Harcourt, Nigeria. The rats were randomly sorted into five groups of nine animals each (these nine animals were subdivided into three groups of three animals each for the 3, 6 and 9 weeks of treatment) so that the average difference was ± 2.0g. The animals were housed in plastic cages. The animals were allowed normal feedi. Eguinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria) and water ad libitum.

2.3 Experimental Design

The rats were divided into five groups of nine rats each after the induction of streptozotocin diabetes.

Group-I-- Normal control rats

Group-II -- Diabetic control rats

Group-III-- Diabetic rats received a standard drug, Glucophage^TMMetformin (1.4mg/kg)

Group-IV-- Diabetic rats received 1.4mg/kg of Glucophage^TM(metformin) and 0.25mg/kg of Reducdyn.

Group-V-- Diabetic rats received 1.4mg/kg of Glucophage^TM(metformin) and 0.5mg/kg of Reducdyn.

2.4. Anti-Diabetic Study/Drug Administration

The rats were randomly sorted into five groups of nine animals each (these nine animals were subdivided into three groups of three animals each for the 3,6 and 9 weeks of treatment) so that the average difference was ± 2.0g. The animals were housed in plastic cages. After a one week acclimatization period on guinea growers mash (Port Harcourt Flour Mills, Port Harcourt, Nigeria), the animals were fasted overnight and diabetes was induced by intraperitoneal injection of freshly prepared solution of streptozotocin (160mg/kg body weight) in distilled water, while the normal control rats (NCR) were injected with distilled water alone. Seven days after administration of streptozotocin, the animals were again fasted and blood collected via tail cutting (Burcelin et al., 1995), for the determination of their fasting glucose levels. Then the rats were kept for 3 days to stabilize the diabetic condition (Jyoty et al., 2002) before commencing treatment, which lasted for 9 weeks.

The first and second groups (normal control rats (NCR) and diabetic control rats (DCR) received appropriate volume of water using a gavage via intubation. The third group i.e the first treatment group received daily by intra-gastric gavages, 1.4mg/kg body weight of Glucophage^TM (metformin); the fourth group received 1.4mg/kg body weight of Glucophage^TM (metformin) and 0.25mg/kg body weight of Reducdyn^TMvia the same route and the fifth group received 1.4mg/kg body weight of Glucophage^TM (metformin) and 0.5mg/kg body weight of Reducdyn^TMvia the same route.

The animals were allowed normal feed and water ad libitum. At the end of each treatment period, i.e., 3 week, 6 week, and 9 week, the rats were weighed, fasted overnight and their fasting glucose level estimated by an automatic one touch glucometer. They were anaesthetized by exposure to chloroform. While under anesthesia, they were painlessly sacrificed and blood was collected from each rat into heparin sample bottles. The heparin anti-coagulated blood samples were centrifuged at 1000 x g for 10 min, after which their plasma was collected and stored for subsequent analysis.

Table 1: Effect of co-administration of metformin and reducdyn on plasma glucose level of normal andstreptozotocin induced diabetic male rats.

Group	Treatment	Serum glucose level (mmol/l)
Group	Treatment	3 weeks	6 weeks	9 weeks
I	Normal Control Rats (NCR)	4.2±0.26^a,b	3.9±0.85^a,b	4.8±0.91^a,b
II	Diabetic Control Rats (DCR)	8.1±0.32^a,b	7.0±0.40^a,b	6.3±0.43^a,b
III	DCR on Metformin (1.4mg/kg)	4.6±0.97^b	4.2±0.80^b	4.5±0.20^b
IV	DCR on Metformin (1.4mg/kg) + Reducdyn (0.25mg/kg)	5.6±0.41^a,b	3.8±0.20^b	4.2±0.05^b
V	DCR on Metformin (1.4mg) + Reducdyn (0.5mg/kg)	6.3±0.77^a,b	4.9±0.98^b	4.4±0.98^b

Values are expressed as Mean ±SD; n=3, per group/week. Values in the same column with common superscript letters (a,b,…) are significantly different at p<0.05.

Superscript A^(a) represents significant difference when group I (normal control rats) are compared with other groups at p<0.05.