HERBO-D, comprising of dried and powdered Agaricus bisporus mushrooms, dried and powdered fresh leaves of Catharanthus roseus, dried and powdered seed pulp of Syzygium cumini, and dried and powdered seeds of Trigonella foenum-graecum was obtained from Shahi Laboratories (Unani), Rajshahi, Bangladesh. The formulation was obtained as a dry powder form.
For preparation of methanol extract, 50g powder was extracted with 250 ml methanol over 48 hours at room temperature. Following extraction, the suspension was filtered, and the filtrate evaporated at 50oC to obtain the methanol extract. Final weight of the extract was 4g. Prior to administration by gavaging, 0.4g of the extract was suspended in 2 ml Tween - 20.
For preparation of aqueous extract, 5g of the powder was suspended in 30 ml distilled water and kept overnight at ambient temperature with stirring. The suspension was next filtered and the filtrate administered at doses of 300, 600, 1200, and 2400 l per kg body weight. For direct administration of powder, 0.4g powder was suspended in 3 ml distilled water. Administration was done by gavaging at doses of 50, 100, 200, and 400 mg powder per kg body weight.
Chemicals: Glibenclamide and glucose were obtained from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals and reagents were of analytical grade. Animals : In the present study, Swiss albino mice (male), which weighed between 13-19 g were used. The animals were obtained from International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B). All animals were kept under ambient temperature with 12h light followed by a 12h dark cycle. The animals were acclimatized for three days prior to actual experiments. The study was conducted following approval by th e Institutional Animal Ethical Committee of University of Development Alternative, Dhaka, Bangladesh.
Antihyperglycemic activity: Glucose tolerance property of various extracts or suspension of HERBO-D was determined as per the procedure previously described by Joy and Kuttan (1999) with minor modifications. In brief, fasted mice were grouped into fourteen groups of five mice each. The various groups received different treatments like Group 1 received vehicle (1% Tween 20 in water, 10 ml/kg body weight) and served as control, group 2 received standard drug (glibenclamide, 10 mg/kg body weight). Groups 3-6 received methanol extract of HERBO-D at doses of 50, 100, 200 and 400 mg per kg body weight. Groups 7-10 received aqueous extract of HERBO-D at doses of 300, 600, 1200, and 2400 l per kg body weight. Groups 11-14 received HERBO-D suspension in water at HERBO-D powder doses of 50, 100, 200, and 400 mg powder per kg body weight. Each mouse was weighed and doses adjusted ac cordingly prior to administration of vehicle, standard drug, and test samples. All substances were orally administered. Following a period of one hour, all mice were orally administered 2 g glucose/kg of body weight. Blood samples were collected 120 minute s after the glucose administration through puncturing heart. Blood glucose levels were measured by glucose oxidase method (Venkatesh et al ., 2004).
The percent lowering of blood glucose level was calculated as follows. Percent lowering of blood glucose level = (1 – We/Wc) X 100, where We and Wc represents the blood glucose concentration in glibenclamide or extract administered mice (Groups 2 - 6), and control mice (Group 1), respectively.
Statistical analysis: 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.