Introduction

Diarrhea is an alteration in the normal bowel movement, characterized by increased frequency of bowel sound and movement, wet stool, and abdominal pain (1). Clinically it is used to describe increased liquidity of stool, usually associated with increased stool weight and frequency (2). Regardless of the understanding causes, treatment and prevention of diarrheal diseases, an estimated 4.6 million people, with 2.5 million children, die from diarrhea every year, particularly in developing countries (3). Diarrhea, may be acute or chronic. Acute diarrhea being the most common, is usually caused by an infectious agent, even though drugs, poisons or acute inflammatory reactions can contribute a lot (4). Now a days, rotavirus is the major causative agent for infectious diarrhea, particularly in young children, however, other viral (adenovirus, enterovirus and norovirus), bacterial (Escherichia coli, Salmonella sp., Shigella sp., Camphylobacter and Vibrio cholerae) and parasitic (Cryptosporidium and Giardia) agents are important pathogens (5).
Oral Rehydration Therapy (ORT) has been identified as a key factor in the decline of child mortality rate due to diarrhea, although it does not reduce the volume or duration of diarrhea (6). Likely, antibiotics and gut motility suppressing agents bid the other treatment option, wherein reverse dehydration, shorten the length of illness and reduce the period of time when an individual is infected (7). Treatment with pharmacological agents that are pathogen specific or that suppress severe symptoms would be of benefit to patients suffering from prolonged diarrhea (8).
Despite the availability of a vast spectrum of approaches for diarrheal management, a vast majority of the people of Bangladesh have been known to treat diarrhea with a variety of medicinal plants one of which being M.sapientum (9). M.sapientum L. (family Musaceae) grows in humid lowland to upland tropical areas whose vernacular name is banana in Bengali. Agarwal et al (10) has studied the wound healing activity of plantain banana extracts. Plants parts like fruits, leaves, peels, root, and stalks from banana plants have been utilized orally or topically as a medicine for treating diarrhea and dysentery. It is also used in inflammation, pains and snakebite (11) as well as it has antilithic (12), antiulcerogenic (13), hypoglycemic (14), hypolipidemic and antioxidant actions (15). Houghton and Skari (16) have also reported the antivenom action of the stem juice from banana plant.
Literature reviews indicated that no studies combining the antidiarrheal, antioxidants as well as antibacterial of the seeds of M.sapientum have so far been undertaken. Taking this in view and as a part of our ongoing research (17,18) on Bangladeshi medicinal plants, the present study aimed to evaluate the antidiarrheal activity of seeds extracts of M.sapientum along with their antioxidant and antibacterial activities.

Materials and Methods

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 anti diarrheal 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), decreased gastrointestinal tract motility and 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).

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 (0.1 ml) 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] x 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.

The amount of phenolic compounds and flavonoids
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.

Antimicrobial activity
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).

Statistical analysis
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 by using student’s t-test. P0.001 was considered to be statistically significant.

Result

Effect on castor oil-induced diarrhea
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).

Effect on magnesium sulfate-induced diarrhea
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.

Effect on gastrointestinal motility
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).

Acute toxicity studies
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.

Total phenolic and flavonoid contents
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).

Total antioxidant capacity
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.

DPPH radical scavenging activity
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).

Nitric oxide (NO) scavenging activity
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 μg/ml.

Reducing power ability
For the measurement of the reductive ability, we investigated the Fe3+ to Fe2+ 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.


Antibacterial activity
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.

Discussion

Several mechanisms have been previously proposed to explain the diarrheal effect of castor oil including inhibition of intestinal Na+, K+-ATPase activity to reduce normal fluid absorption (30), activation of adenylate cyclase or mucosal cAMP mediated active secretion (31), stimulation of prostaglandin formation (32), platelet activating factor and recently nitric oxide has been claimed to contribute to the diarrheal effect of castor oil (33). However, it is well evident that castor oil produces diarrhea due to its most active component recinoleic acid which causes irritation and inflammation of the intestinal mucosa, leading to release of prostaglandins, which results in stimulation of secretion (34). Since the methanol extract of the seeds of M. sapientum successfully inhibited the castor oil-induced diarrhea, the extract might have exerted its antidiarrheal action via antisecretory mechanism which was also evident from the reduction of total number of wet faeces (not shown separately) in the test groups in the experiment. Again, flavonoids present in the plant extract (13) are reported to inhibit release of autacoids and prostaglandins, thereby inhibit motility and secretion induced by castor oil (35).

Conclusion

The MMSS showed antidiarrheal as well as antioxidant activities in a number of models of diarrheic condition in the test animals and in vitro antioxidant assay method, respectively. The obtained results thus give the experimental basis to understand the use of M.sapientum in traditional medicine, as an antidiarrheal agent. However, further bioassay guided phytochemical and pharmacological studies are required to identify the active principles and exact mechanism(s) of action.

Acknowledgement

We express our sincere thanks and gratitude to Professor Dr. Kabir Hossain Talukder, Dean, Faculty of Biological Science, Atish Dipankar University of Science & Technology, Banani, Dhaka, Bangladesh for providing laboratory facilities and necessary reagents support while doing the study.