Induction of diabetes and experimental design

Antidiabetic activity was carried out on selected healthy albino mice [16]. The experiments were carried out in accordance with the National Institute of Health guidelines of care and use of laboratory animals [17]. Diabetes was induced in mice using freshly prepared solution of alloxan monohydrate dissolved in normal saline (0.9% w/v of NaCl). For inducing diabetes, the mice were kept on fasting for 12 h and were given a single IP injection of alloxan monohydrate (150 mg/kg b. wt.). To prevent fatal hypoglycemia initially due to massive pancreatic insulin release, the mice were provided with 5% glucose solution after six hours supplied in water bottles in their cages for next 24 h. Animal were kept at room temperature (27 ± 2 °C) and humidity (55 ± 5%) and a 12 h’ cycle of light and dark. After 72 h, the glucose level of the fasting animals was measured. After acclimatization, the animals were separated into following groups (six mice in each group); Groups A, Normal control treated with saline; B, Diabetic control; C, Diabetic mice treated with 500 mg/kg body weight of fruit extract; D, Diabetic mice treated with 500 mg/kg body weight of bark extract; E, Diabetic mice treated with 500 mg/kg body weight of leaves extract F, Normal mice given 500 mg/kg of Gt-MeOH extract and G, reference control treated with glibenclamide (10 mg/ kg). An identification mark was given to the mice of each group on the tail with permanent marker. Each of mice was weighed and the doses were calculated accordingly. The extract was given orally. All the groups were given respective treatments daily for 15 days. To check the effect of the extracts on the weight of animals, weight of the mice was recorded prior to the administration of the extracts and at the end of the study as well i.e. on the 15th day.

The blood samples were collected (in glass tubes) and left for 1 h at 37 °C to allow to clot. The blood was collected using capillary tubes into Eppendorf Tubes® containing heparin for analysis of plasma profile. Using a glass Pasteur, carefully, the clot was loosened from the sides of the tube. The serum was centrifuged at 5000 rpm for 5 min at 4 °C. The serum was removed from the clot by gently pipetting off into a clean tube using a micropipette. The serum was labeled with the animal number and the estimations were made [18].