Plant material

Seeds of Adenanthera pavonina were collected during March 2009 from the Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra, India. The plant was authenticated by Dr. P.G. Diwakar, Joint Director, Botanical Survey of India, Pune as Adenanthera pavonina Linn. (Mimosaceae) with a voucher specimen (BSI/WRC/Tech/2010/463) kept in herbarium, BSI, Pune.

Preparation of extract

The seeds were washed with distilled water, shed dried and latter powdered. This powder was then defatted with petroleum ether which was further macerated with distilled water for 72 h with occasional shaking. It was then filtered and evaporated. The yield of APSAE was 2.5 % w/w.

Preliminary phytochemical screening

The preliminary phytochemical screening of APSAE was carried out for qualitative identification of type of phytoconstituents present (Latha and Pari 2004; Kokate 1994).

Animals

Healthy adult male wistar rats weighing 150-200 g and Swiss albino mice weighing 25–30 g were obtained from in house breed at the animal house of M.E.S. College of Pharmacy, Sonai and were housed in polypropylene cages lined with husk in standard environmental conditions (Temperature 25 ± 2°C; relative humidity 55 ± 10 %; and 12:12 light: dark cycle,). The animals were fed on a standard pellet diet (Amrut rat and mice feed, Sangli, India) and water ad libitum.

Animals were acclimatized to the laboratory condition for at least 8 days prior to the experiment and were maintained in a well ventilated animal house. The experimental protocol was approved by the Institutional animal Ethical Committee (MESCOP/IAEC/07/2010) and the care of the laboratory animals was taken as per the current CPCSEA regulation.

Experimental designAcute toxicity study (OECD 425, 2001)

Acute toxicity of APSAE was done using Swiss albino mice (25–30 g) according to the procedure of Organization for Economic Co-operation and Development (OECD) guideline no. 425 (OECD, 2001). The animals were fasted overnight prior to the experiment and maintained under standard conditions. Animals were observed for general behavioral change and mortality for a period of 14 days post treatment.

Effect of APSAE on normoglycaemic rats

The rats were divided into four groups of 6 animals (n = 6) each. Group I served as control and received distilled water. Group II, III and IV received APSAE orally at doses 50,100 and 200 mg/kg/day b.wt. Blood glucose levels were determined at 0,1,2,3 and 4 h following treatment by retro-orbital plexus of the eye under mild ether anesthesia.

Oral glucose tolerance test in normal rats (OGTT)

The rats were divided into four groups of 6 animals (n = 6) each. Group I served as control and received distilled water. Group II, III and IV received APSAE orally at doses 50,100 and 200 mg/kg/day b.wt. All the animals were given glucose (2 g/kg) 30 min after dosing. Blood samples were collected from the retro-orbital plexus of the eye just prior (0 h) and 1, 2, 3 and 4 h. after the glucose loading and blood glucose levels were estimated.

Induction of diabetes

Diabetes was induced in rats by single intraperitoneal injection of STZ (55 mg/kg b.wt.) dissolved in freshly prepared 0.01 M citrate buffer, pH 4.5. (Gupta et al. 2004) after 72 h rats with marked hyperglycemia (fasting blood glucose ≥250 mg/dl) were selected and used for the study.

Treatment schedule

Total of 36 Wistar rats were used (30 Diabetic surviving and 06 nondiabetics). The rats were divided into six groups of 6 animals (n = 6) each as follows- The solution of APSAE was prepared with 1 % gum acacia, an emulsifying agent. Glibenclamide was served as a reference standard. Group-I (Nondiabetic Control) animals were received only 1 % gum acacia (1 ml/kg/day, p.o.). Group-II (Diabetic Control) animals were diabetic and received 1 % gum acacia (1 ml/kg/day, p.o.). Group-III (Diabetic + Glibenclamide) animals were diabetic and received glibenclamide (0.25 mg/kg/day, p.o.) (Sun Pharmaceuticals Ltd, India). Groups IV, V, VI animals were diabetic and received three different doses of APSAE 50, 100 and 200 mg/kg, p.o. respectively. All the animals received above treatment for 30 days.

Evaluation of antihyperglycaemic activity

Antihyperglycaemic activity of APSAE was evaluated by estimation of blood glucose levels and body weight measurement on 1st, 10th, 20th and 30th day of the study by the glucose oxidase/peroxidase (GOD/POD) (Trinder 1969) method using a standard kit obtained from Span Diagnostics, India.

Evaluation of antihyperlipidaemic activity

At the end of the experiment, the animals from each group were sacrificed by cervical dislocation and blood and organs were collected to estimate various biochemical and histological studies (Chakrabarti et al. 2005). Blood was collected from the heart and allowed to clot and the serum was separated by centrifuged at 3,500 rpm for 10 min. Serum was assayed either immediately or stored at -20°C. The tissue like pancreas was collected and used for histological studies. Serum samples were analyzed spectrophotometrically for triglycerides, total cholesterol, high density lipoprotein (HDL-C), using their respective kits UV- visible spectrophotometer (Jasco V-630, Japan), VLDL-C and LDL-C were calculated as per Friedwald’s equation (Richterich and Colmbo 1981).\( \matrix} = }/}; \\ } = } - \left( } + }} \right)} \\ }<!end array> \).

(Alayash et al. 1988; Burstein et al. 1970; Friedwald et al. 1972).

Estimation of glycated hemoglobin

After 30 days of treatment, the 12 h fasted rats were sacrificed by cervical decapitation, blood was withdrawn by retro orbital puncture under light ether anesthesia and the glycated hemoglobin was estimated (Sadasivam and Manickam 1996).

Statistical analysis

The results were expressed as mean ± S.E.M., statistical difference was doone by using one-way analysis of varience (ANOVA) followed by Dunnette’s multiple comparison test. A difference in the mean P value <0.05 was considered as statistically significant.