Sample collection
The leaves of M. esculenta were collected from Katmandu, Nepal in May 2014. After collection, the undesirable parts and dusts were washed by water.
Preparation of extract
Shade dried leaves were ground into a coarse powder with the help of a suitable grinder. Powdered leaves (250 g) were soaked in 600 ml of ethanol in a glass container for fifteen days accompanying regular shaking and stirring. After fifteen days, the extract was separated from the plant debris by filtration using a piece of clean, white cotton cloth. The filtrate obtain is then evaporated by rotary evaporator.
Microorganism Both Gram positive and Gram negative bacteria were used for antibacterial activity test and these are Staphylococcus aureus, Escherichia coli, Salmonella typhi, Staphylococcus epidermidis, Bacillus megaterium and Vibrio cholerae. Microorganisms were collected from ICDDRB.
Parasites
Live parasites Paramphistomum cervi (Trematoda) and Haemonchus contortus (Nematode) were used for test for anthelmintic activity. Live parasites were collected from freshly slaughtered cattle at local abattoirs, Khulna.
Chemicals
Molisch's, Mayer's, Dragendroff's, Fehling's, Libermann-Burchard and Benedict's reagent, Sodium bicarbonate, sodium hydroxide, hydrochloric acid, potassium dichromate, sulphuric acid, nitric acid, chloroform, ferric chloride, DPPH, loperamide, albendazole and ascorbic acid.
Phytochemical tests
In each test, 5% (w/v) solution of extract in ethanol was taken unless otherwise mentioned in individual test such as aqueous extract for reducing sugar, chloroform extract for steroid, sodium hydroxide extract for flavonoids (Trease et al., 1989; Sofowara et al., 1982).
Libermann-Burchard test was performed to identify steroids. For alkaloid identification both Mayer's reagent and Dragendroff's reagent test were performed. On the other hand, ferric chloride and pottasium dichromate test were performed to identify tannins and Molisch's test, Fehling's test and Benedict's test were performed to investigate the presence of reducing sugars. For saponins, flavonoids (Agoha et al., 1981; Barakat et al., 1973), terpenoids, acidic compounds (Amer et al., 2004) and glycosides, standard identifying tests were performed.
In-vitro Antioxidant activity test
DPPH free radical scavenging assay (Qualitative assay) Thin-layer chromatography (TLC) was performed for the qualitative investigation of weather antioxidant compounds are present or not (Lewis et al., 1989).
Diluted stock solution was spotted on pre-coated silica gel TLC plates and the plates were developed with solvent systems of different polarities (polar, medium polar and non-polar) to resolve polar and non-polar components of the extract. The plates were dried at room temperature and were sprayed with 0.02% 1, 1-dipheny1-2-picrylhydrazyl (DPPH) in ethanol. Bleaching of DPPH by the resolved bands was observed for
10 minutes and the color changes (yellow on purple background) were noted (Sadhu et al., 2003). DPPH forms deep pink color when it is dissolved in ethanol. When it is sprayed on the chromatogram of the extract, it forms pale yellow or yellow color which indicates the presence of antioxidants. DPPH free radical scavenging assay (Quantitative assay)
The anti-oxidant potential of the extract of M. esculenta was determined on the basis of their scavenging of the stable 2,2-dipheny1-1-picryl hydrazyl (DPPH) free radical (Prakash et al., 2001). DPPH is a stable free radical containing an odd number of electrons in its structure and usually utilized for detection of radical scavenging activity in chemical analysis. Aliquots of different concentrations of the extracts were added to three ml of 0.004 % ethanol solution of DPPH. Absorbance at 517 nm was measured after 30 minutes and IC50 (Inhibitory concentration 50%) was calculated from these data. IC50 value denotes the concentration of sample require to scavenge 50% of the DPPH free radical (Gupta et al., 1971).
The formula used for % inhibition ratio is -% inhibition = {(Blank absorbance — Sample absorbance) / Sample absorbance} X 100
Anti-bacterial activity test
Antibacterial activity of the ethanolic extract of M. esculenta was determined by disc diffusion method (Bauer et al., 1966; Ahmed et al., 2003).
In this method, measured amount of test samples were dissolved in definite volumes of solvent to give solutions of known concentrations (µg/m1). Sterile filter paper discs were impregnated with known amount of test substances using micropipette and dried. Standard antibiotic discs and discs on which the solvent used to dissolve the samples was adsorbed and dried were used as positive control and sample, respectively. These discs were then placed in petridishes (85.88 mm in diameter) containing a suitable agar medium seeded with the test organisms. The plates were then incubated at 37° C for 12-18 rs. After this time, the zone of inhibition for standard, sample and blank were estimated by slide calipers.
Anti-diarrheal activity test
Anti-diarrheal activity of the extract of M esclulent was determined by slight modification of castor oil induced diarrhea in mice by Ahmadu et al., 2007.
The mice were fasted 12 hrs prior to the commencement of the experiments and were randomly divided into four groups of five mice in each. Mice in the first group received 10m1/kg distilled water containing 1% tween-80, the third and fourth group received 250 mg/kg, 500 mg/kg body weight of ethanol extract of M. esculenta, while the second group received loperamide (3 mg/kg body weight). After I hr of administration of extract, castor oil 0.7m1/mouse was administered intragastrically. The animals were placed on individual cages over clean filter paper. The presence of characteristic diarrhea droppings (number of stools or any fluid material that stained the filter paper) were counted at each successive hour during the 4 hrs period and were noted for each mouse. Their absence was recorded as a protection from diarrhea, and the percentage of inhibition was calculated (Diurno et al., 1996; Akah et al., 1996). The latent period of each mouse was also counted.