Collection of plant material:
Fresh rhizomes of S. Hyacinthoides were collected from Gazipur in October 2009 and identified by the taxonomist of Bangladesh National Herbarium, Dhaka, where a voucher specimen (No. 31563) has been deposited.
Preparation of the solvent extracts (cold extraction):
Freshly collected rhizomes of S. Hyacinthoides were dried in an oven at 380C and crushed in pieces. The crushed powder (390g) was extracted with methanol for 5 days. The extract was concentrated to gummy mass (45.9 g) using Buchi Rotary Evaporator, USA. The methanol extract (13.8 g) was then partitioned by separatory funnel by using n-hexane, followed by ethyl acetate and n-butanol. These extracts were then concentrated by using rotary vacuum evaporator to provide n-hexane (5.0 g), ethyl acetate (3.2 g), n-butanol (3.4 g) and water (5.8 g).
General experimental procedure:
The UV absorbance was performed with a PerkinElmer Shelton, CT 06484 USA, Lambda 25 UV/VIS spectrometer. Vacuum rotary evaporator (BUCHI, Rotavapor R-210 Switzerland) was used for evaporating solvents. All solvents were of analytical grade and obtained from commercial sources (Sigma-Aldrich, St. Louis, MO, USA).
Cytotoxicity bioassays:
The cytotoxic activity was performed by brine shrimp lethality bioassay method (Meyer et. al., 1983). The test samples for crude MeOH extracts as well as n-hexane (4.0 g), ethyl acetate (2.2 g), n-butanol (2.4 g) and water (3.8 g) extract were dissolved in DMSO and serial dilution were made as 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 and 0.3095 μg/mL. Vincristine sulphate (positive control) was dissolved in DMSO and serial dilution were made as 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.0781 μg/mL. Then each of these test solutions was added to test tubes containing 12 shrimps in simulated brine water (5 mL) and incubated at room temperature for 24 h. After 24 h, the median lethal concentration (LC50) of the test samples was determined by a plot of percentage the shrimps against the logarithm of the sample concentrations (Finney method; Finney, 1952). Vincristine sulphate (LC50= 0.52) was used as a positive control in this assay to compare the cytotoxicity of the test samples. Results are presented.
Antibacterial screening:
The test samples were dissolved separately in a specific volumes of chloroform or methanol depending their solubility. The antibacterial screening was then carried out by the disc diffusion method (Barry, 1980; Bauer, 1996). The diluted samples were applied on to sterile blank discs (Oxoid, UK) at a concentration of 100 (g/disc for this test where Streptomycin 10 g/disc, Oxoid, UK) used as a standard. Results are presented.
Free radical scavenging activity:
The free radical scavenging activity was assayed spectrophotometrically by DPPH method (Rieser et al., 1996). The DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical has a deep violet color due to its unpaired electron and radical scavenging activity can be followed spectrophotometrically by a loss of absorbance at 525 nm. Sample stock solutions (1 mg/mL) were diluted to final concentrations of 100, 50, 10, 5 and 1 (g/mL in 70% ethanol or DMSO. DPPH ethanol solution (0.2 mM, 0.5 mL) was added to 1 mL of sample solutions of different concentrations, shaken well by vortex and allowed to react at room temperature. The absorbance values were measured after 10 min at 525 nm by UV/Vis spectrophotometer. The free radical scavenging activity of samples was calculated according to the formula:
DPPH radical scavenging activity (%) = [1-(Abs sample-Abs blank)/Abs control] × 100.
Where, Abs sample is the absorbance of the experimental sample, Abs blank is the absorbance of the blank, Abs control is the absorbance of the control. As a blank, 70% EtOH or DMSO solvent (0.5 mL) and sample solution (1.0 mL) were used. DPPH solution (0.5 mL, 0.2 mM) and 70% EtOH or DMSO solvent (1.0 ml) was used as a negative control. The ascorbic acid (vitamin C) was used as a positive control. Each treatment was replicated thrice.