Glucose kit

To evaluate effects on fasting blood glucose, the B. monosperma extract (250 mg/kg, 500 mg/kg and 1000 mg/kg) was suspended in distilled water and orally administered to 12 h fasted type 2 diabetic rats. The control animals received an equal volume of distilled water.
Effects on glucose tolerance were similarly evaluated by administration of B. monosperma extracts together with glucose (2.5 g/10 mL per kg body weight) after a fasting period of 12 h. Control group received only glucose solution.
In either cases blood was collected from the tail vein, and serum separated by centrifugation and stored at −22°C until further analysis. Blood glucose was analysed by GOD-PAP method [23 ] (glucose kit, Randox, UK).
To evaluate chronic effects of B. monosperma, type 2 diabetic rats were given extract at 250, 500, and 1000 mg/kg doses by gavage, twice daily for 48 d. Control rats were similarly administered water alone (10 mL/kg body weight). Blood samples were collected from the cut tip of the tail at the times indicated in the figures (Figure 4). Serum was separated by centrifugation, stored, and analyzed as mentioned above.

Oral Glucose Tolerance Test (OGTT) was performed following 8weeks of B. vulgaris administration, as described previously[11 (link)]. Briefly, after a designated fasting period, animals were anesthetized with an intra-peritoneal injection (100 mg/kg) of pentobarbitone sodium (Therapon, Burwood, Victoria, Australia), and a silastic catheter filled with heparinized saline (20 U/ml) was inserted into the left carotid artery. The mice also underwent tracheotomy to facilitate breathing. A bolus of glucose was delivered into the stomach by a gavage needle (20-gauge, 38 mm long curved, with a 21/4 mm ball end; Able Scientific, Canning Vale, Western Australia, Australia), and 200 μl of blood was sampled at 0, 30, 60, 90, and 120mins for plasma glucose and insulin analyses. Blood was immediately centrifuged and the plasma was separated and stored at −20°C until assayed. The red blood cells were re-suspended in an equal volume of heparinized saline and re-infused into the animal via the carotid artery prior to the collection of the next blood sample to prevent anemic shock. Blood glucose levels were analyzed by GOD-PAP method[12 (link)] (glucose kit, Randox, UK) and plasma insulin levels were determined using Mice Insulin ELISA Kit (Crystal Chem, USA)

Venous blood samples (∼6 mL) were collected into EDTA tubes, stored on ice, and then centrifuged at 4°C and 1,000 g for 15 min. Aliquots of plasma were then stored at −70°C and later analyzed for glucose, lactate, nonesterified fatty acids (NEFA), and glycerol using commercially available kits (Glucose kit, Lactate kit, NEFA kit, Glycerol kit; Randox, London, UK) using an automated photometric clinical chemistry analyzer RX Daytona+ (Randox, London, UK). Plasma galactose concentration was determined using a colorimetric assay (Galactose Assay Kit, Sigma Aldrich, St. Louis, MO) and insulin using an enzyme-linked immunosorbent assay (Invitrogen, Life Technologies, California).

Plasma was analyzed for glucose, lactate, nonesterified fatty acids (NEFA), and glycerol with commercially available kits (Glucose kit, Lactate kit, NEFA kit, Glycerol kit; Randox, London, UK) using an automated photometric clinical chemistry analyzer RX Daytona+ (Randox, London, UK). Plasma galactose concentration was determined using a colorimetric assay (Galactose Assay Kit, Sigma Aldrich, St Louis. MO) and insulin using an enzyme-linked immunosorbent assay (Ultrasensitive Insulin, Mercodia, Uppsala Sweden). Breath samples were analyzed for ¹³C:¹²C ratio by gas chromatography isotope ratio mass spectrometry (IRMS), Hydra 20-20, Europa Scientific, Crewe, UK).

Venous blood samples (~6 mL) were collected into EDTA tubes, stored on ice and then centrifuged at 4°C and 1,006 × g for 15 min. Aliquots of plasma were then stored at −70°C and later analyzed for glucose (Glucose kit; Randox, London, UK) and lactate (Lactate kit; Randox, London, UK) using an automated photometric based clinical chemistry analyzer RX Daytona+ (Randox, London, UK).

Rats were weighed and blood samples collected from the tail vein for baseline plasma glucose (glucose oxidase method) estimation using Randox glucose kit (Randox Laboratories Ltd., BT29 QY, United Kingdom). Subsequently, the animals were divided equally into two (2) groups.
Group 1 (control group) were injected with freshly prepared sterile saline. Group 2 (test group) received 170 kgbwt⁻¹ alloxan preparation (2,4,5,6-tetraoxypyrimidine; 2,4,5,6-pyrimidinetetrone). All injections were done through single intraperitoneal administration using a total volume of 0.5 mL after estimating the effective dose and administered volume with respect to their weights [13 (link)].