Collection of plant material The leaves of A. carambola, F. hispida, and S. samarangense were collected, respectively, during May 2010 from Dhaka district, Bangladesh, December 2009 from Brahmanbaria district, Bangladesh and July 2010 from Dhaka district, Bangladesh. The leaves were identified by the Bangladesh National Herbarium, Mirpur, Dhaka (Accession Nos. 34,972, 34,490 and 35,070) and sample specimens have been kept over there.
Preparation of the test samples The leaves of the plants were separately air-dried in the shade and pulverized into a fine powder and were mixed with methanol at a ratio of 1:3 (w/v). After 24 hours, the mixtures were filtered; the filtrate was collected and the residue was again mixed with methanol at a ratio of 1:2 (w/v) for 24 hours. After filtration, filtrates were combined and evaporated to dryness (approximate yields 10.4, 2.2 and 5.8% for A. carambola, F. hispida, and S. samarangense, respectively) using rotary evaporator. Extracts were suspended in 1% Tween 80 in water prior to administration.
Animals Swiss albino mice (male), weighing 15-20 g bred in the animal house of ICDDR,B (International Centre for Diarrheal Disease and Research, Bangladesh) were used for the present experiments. All the animals were acclimatized one week prior to the experiments. The animals were housed under standard laboratory conditions (relative humidity 55-65%, room temperature 25.0 ± 20 C, and 12 hrs light-dark cycles). The animals were fed with standard diet from ICDDR, B and had free access to water. The study was approved by the Institutional Animal Ethical Committee of the University of Development Alternative, Dhaka, Bangladesh.
Anti-hyperglycemic activity test Antihyperglycemic activities of the extracts were studied through the glucose tolerance test method. Glucose tolerance test was performed following the procedure as described by Joy and Kuttan (1999) with slight modifications (Rahman et al., 2011; Ahmed et al., 2011). In brief, fasted mice were divided into fourteen groups. Each group received a particular treatment: group-I served as control and received vehicle (1% Tween 80 in water, 10 ml.kg-1 body weight), while group-II received standard drug (glibenclamide, 10 mg.kg-1 body weight). Groups III-VI received leaf extract of A. carambola at four different doses of 50, 100, 200 and 400 mg extract.kg-1 body weight, respectively. Groups VII-X was administered the leaf extract of F. hispida at doses of 50, 100, 200 and 400 mg extract.kg-1 body weight, respectively. Groups XI-XIV was administered the leaf extract of S. samarangense at doses of 50, 100, 200 and 400 mg extract.kg-1 body weight, respectively. Each mouse was weighed properly and the doses of the test samples, standard drug, and control materials were adjusted accordingly. Test samples, control, and glibenclamide were given orally. After one hour, all mice were orally treated with 2 g.kg-1 of glucose. Blood samples were collected two hours after glucose administration. Serum was separated and blood glucose levels were measured immediately by glucose oxidase method (Venkatesh et al., 2004).
Statistical analysis for anti-hyperglycemic activity Experimental values are expressed as mean ± SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.
Acute toxicity study The study was carried out as previously described (Ganapaty et al., 2002) with minor modifications. For each plant leaf extract, selected animals were divided into nine groups of six animals each. The control group received 1% Tween 80 in normal saline (2 ml.kg-1 body weight). The other groups received respectively, 100, 200, 300, 600, 800, 1500, and 3000 mg leaf methanolic extract.kg-1 body weight. Animals were monitored closely after dosing for the next 8 hrs for any behavioral changes and were kept under observation up to 14 days to find out if there is any mortality.