Plant materials The fruits of M.sapientum were collected from the local market in Mirpur, Dhaka, Bangladesh in the month of April, 2008 and identified by experts in Bangladesh National Herbarium, Mirpur, Dhaka where the Voucher specimen no: 38765 has been retained for future reference.
Preparation of plant extract The seeds of M.sapientum were manually separated from the whole fruits, dried in hot air woven, pulverized into coarse powder using mechanical grinder, seiving through sieve #40, and stored in an air tight container. The dried powder material (500 g) was refluxed with methanol for three hr. The total filtrate was concentrated to dryness, in vacuum at 40°C to render the methanolic extract (80 g).
Chemicals Ammonium molybdate, Folin-chiocaltu phenol reagent, sodium nitroprusside, were purchased from E. Merck (Germany). 1,1- diphenyl- 2-picryl-hydrazyl (DPPH), sodium nitroprusside, ascorbic acid, quercetin, and potassium ferric cyanide were purchased from Sigma Chemical Co. Ltd, (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade. In vivo antidiarrheal activity Castor oil-induced diarrhea: The experiment was performed according to the method described by Shoba & Thomas (19). Briefly, mice fasted for 24 hr were randomly allocated to four groups of five animals each. The animals were all screened initially by giving 0.5 ml of castor oil. Only those showing diarrhea were selected for the final experiment. Group I received 1% carboxy-methyl cellulose (CMC) (10 ml/kg, p.o), groups III and IV received p.o the drug extract (100 and 200 mg/kg), respectively. Group II was given antidiarrheal drug loperamide (3 mg/kg, p.o) in suspension. After 60 min, each animal was given 0.5 ml of castor oil, each animal was placed in an individual cage, the floor of which was lined with blotting paper which was changed every hour, observed for 4 hr and the characteristic diarrheal droppings were recorded.
Magnesium sulfate-induced diarrhea Diarrhea was induced by oral administration of magnesium sulfate at the dose of 2 g/kg to the animals 30 min after pre-treatment with vehicle (1% Tween 80 in water, 10 ml/kg, p.o) to the control group, loperamide (3 mg/kg) to the positive control group, and the methanol extract at the doses of 100 and 200 mg/kg to the test groups (20). Effect on gastrointestinal motility Animals were divided into four groups of five mice each and each animal was given p.o 1 ml of charcoal meal (5% activated charcoal suspended in 1% CMC) 60 min after an oral dose of drugs or vehicle. Group I was administered 1% CMC (10 ml/kg) and animals in groups III and IV received extract at the dose of 100 mg/kg and 200 mg/kg body weight, respectively. Group II received atropine sulfate (0.1 mg/kg), which decreased gastrointestinal tract motility and was used as the standard drug. After 30 min, animals were killed by light ether anaesthesia and the intestine was removed without stretching and placed lengthwise on moist filter paper. The intestinal transit was calculated as a percentage of the distance travelled by the charcoal meal compared to the length of the small intestine.
Acute toxicity study Animals were divided into groups of five mice each. The test was performed using increasing doses of test extract, given p.o, in a 10 ml/kg volume to different groups serving as test groups (22). Another group of mice was administered saline (10 ml/kg, p.o) as negative control. The mice were allowed food ad libitum during the 24 hr test and kept under regular observation for mortality.
In vitro antioxidant activity Determination of total antioxidant capacity: The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al (23). The assay is based on the reduction of Mo (VI)–Mo(V) by the extract and subsequent formation of a green phosphate/ Mo(V) complex at acid pH. Extract (0.3 ml) was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. Then the absorbance of the solution was measured at 695 nm using a spectrophotometer (Shimadzu, UV-150-02) against blank after cooling to room temperature. Methanol (0.3 ml) was used as the blank experiment. The antioxidant activity is expressed as the number of equivalents of ascorbic acid using the following formula: C = (c ×V)/m where: C-total antioxidant activity, mg/g plant extract, in Ascorbic acid; c-the concentration of ascorbic acid established from the calibration curve, mg/ml; V-the volume of extract, ml; m-the weight of pure plant extract, g.
Free radical scavenging activity measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) The free radical scavenging activity of extract, based on the scavenging activity of the stable 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical, was determined by the method described by Braca et al (24). Plant extract (0.1ml) was added to 3 ml of a 0.004% MeOH solution of DPPH. Absorbance at 517 nm was determined after 30 min, and the percentage inhibition activity was calculated from [(A0– A1)/A0] x 100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/ standard. IC50 value was calculated from the equation of line obtained by plotting a graph of concentration (μg/ml) versus % inhibition.
Nitric oxide radical scavenging assay The procedure is based on the method, where sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered solution (pH=7.4) was mixed with different concentrations of extract dissolved in 10% DMSO and incubated at room temperature for 150 min. The same reaction mixture without the extract but the equivalent amount of the solvent used served as the control. After incubation, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H3PO4 and 0.1% N- (1- naphthyl) ethylenediamine dihydrochloride was added. The absorbance was measured at 546 nm and the percentage inhibition activity was calculated from [(A0–A1)/A0] × 100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/ standard (25). IC50 value was calculated from the equation of line obtained by plotting a graph of concentration (μg/ml) versus % inhibition.
Reducing power activity The reducing power of M.sapientum seed extract was determined according to the method previously described (26). Extract at different concentrations in 1 ml of 10% DMSO were mixed with 2.5 ml of phosphate buffer (0.2 M, pH=6.6) and 2.5 ml potassium ferricyanide [K3Fe (CN) 6] (1%), and then the mixture was incubated at 50 °C for 30 min. Afterwards, 2.5 ml of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of upper layer solution was mixed with 2.5 ml distilled water and 0.5 ml FeCl3 (0.1%), and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.