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.

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).

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.

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.

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).

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 gastroin-testinal 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 (21).

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.

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.

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.

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.

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.

The total phenolic content of extract was determined using Folin-Ciocalteu reagent (27). M.sapientum seed extracts (100 µl) were mixed with the Folin-Ciocalteu reagent (500 µl) and 20% sodium carbonate (1.5 ml). The mixture was shaken thoroughly and made up to 10 ml with distilled water. The mixture was allowed to stand for 2 hr. Then the absorbance at 765 nm was determined with a Shimadzu UV-160A spectrophotometer (Kyoto, Japan). These data were used to estimate the phenolic contents using a standard curve obtained from various concentration of gallic acid.

The flavonoids content was determined by aluminium chloride colorimetric method (28). Quercetin was used to make the calibration curve. The different concentration of extract (0.5 ml) were separately mixed with 95% ethanol (1.5 ml), 10% aluminum chloride (0.1 ml), 1 M potassium acetate (0.1 ml) and distilled water (2.8 ml). After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in blank. All the determinations were carried out in duplicates. These data were used to estimate the flavonoid contents using a standard curve obtained from various concentration of quercetin.

Sterile 6.0 mm diameter blank discs (BBL, Cocksville, USA) were impregnated with test substances at the dose of 500 µg/disc. This disc, along with standard discs (Ciprofloxacin, Oxoid Ltd, UK) and control discs were placed in petri dishes containing a suitable agar medium seeded with the test organisms using sterile transfer loop and kept at 4°C to facilitate maximum diffusion. The plates then kept in an incubator (37°C) to allow the growth of the bacteria. The antibacterial activities of the test agents were determined by measuring the diameter of the zone of inhibition in terms of millimeter. Antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, Salmonella typhi, Shigella boydii, Shigella flexneri and Shigella dysenteriae were obtained from International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B) (29).

All values were expressed as the mean ± standard error of the mean (SEM) of three replicate experiments and were analyzed using the GraphPad program (GraphPad, San Diego, CA, USA). The analysis was performed using student's t-test. P<0.001 was considered to be statistically significant.

In the castor oil induced diarrheal mice, the methanolic extract of the seeds of M.sapientum, at the dose of 100 and 200 mg/kg, significantly (p<0.001) lessened the total number of faeces as well as delayed the onset of diarrhea in a dose dependent manner (Table 1).

M.sapientum seed extract exhibited significant antidiarrheal activity against magnesium sulfate-induced diarrhea (Table 2). The extract at both dose levels significantly (p<0.001) reduced the extent of diarrhea and also notably delayed the onset of diarrhea in a dose dependent manner.

With the gastrointestinal transit experiment, the methanolic extract, at the dose of 100 and 200 mg/kg, retarded (p<0.001) the intestinal transit of charcoal meal in mice when compared to the control (Table 3).

Methanolic seed extract of M.sapientum (500-5000 mg/kg, body weight) given p.o did not cause any death in the different dose groups. The LD₅₀ value for oral administration of the plant extract was found to be greater than 5000 mg/kg.

The total phenols and flavonoids content was found to be 15.94±0.12 mg/g plant extract (in GAE) and 29.98±0.32 mg/g plant extract (in quercetin equivalent), respectively, in crude extract of M.sapientum seeds (Table 4).

Percentage yield of methanol extract of M. sapientum seeds and its total antioxidant capacity are given in Table 4. Total antioxidant capacity of the extract is expressed as the number of equivalents of ascorbic acid and was found to be 197.24±0.69 mg/g equivalent of ascorbic acid.

The percentage (%) scavenging of DPPH radical was found to be concentration dependent i.e. concentration of the extract between 5-80 µg/ml greatly increasing the inhibition activity (Figure 1). Crude extract of M.sapientum seed (IC₅₀ value 12.32±0.33 µg/ml) showed similar activity than the standard ascorbic acid (IC₅₀ value 12.30±0.15 µg/ml).

The percentage inhibition of nitric oxide production was illustrated in Figure 2. It is observed that scavenging of nitric oxide by the extract is also concentration dependent and statistically significant (p<0.001). The IC₅₀ value of the extract of M.sapientum seed was 18.96±1.01 µg/ml, while ascorbic acid showed the value of 8.22±0.22 g/ml.

For the measurement of the reductive ability, we investigated the Fe^{3 +} to Fe^{2 +} transformation in the presence of crude extract of M.sapientum. Like the antioxidant activity, the reducing power of M.sapientum seed extract increased with increasing concentration of the sample and effect was statistically significant (p<0.001). Figure 3 shows the reductive capabilities of the M.sapientum compared with quercetin, gallic acid and ascorbic acid.

Table 5 expressed the antibacterial activity (zone of inhibitions) of the seed extract of the M. sapientum. The extract showed significant activity against the entire tested bacterial flora except Shigella flexneri and Shigella boydii. The highest zone of inhibition was found against Escherichia coli (zone of inhibition 18.59±0.22 mm), followed by Shigella dysenteriae (zone of inhibition 16.92±0.62 mm) and the moderate activity was shown against Pseudomonas aeruginosa (zone of inhibition 12.21±0.14 mm). The weakest activity was shown against Staphylococcus aureus.