2.1. Plant Material. From March 2006 to May 2007, 16 plants were collected from tidal forests in the coastal Sundarbans (a swamp region in the Ganges delta), and other locations in the Khulna district of Bangladesh. The plant material was identified by the Bangladesh National Herbarium, Dhaka, Bangladesh and shade-dried. A specimen representing each collection was deposited in the Bangladesh National Herbarium, Dhaka, Bangladesh.
2.2. Chemicals. n-Hexane, dichloromethane and methanol were purchased from Merck, Germany. Advanced Dulbecco’s modified Eagle’s medium (DMEM) (Batch #497466 and ID: Gibco 12491), newborn calf serum (NBCS) (Batch #1280182 and ID: Gibco 2901), trypsin-EDTA (Batch #475919 and ID: Gibco 25200) and l-glutamine (Batch #371023 and ID: Gibco 25030) were all obtained from Invitrogen, Australia. Dimethylsulfoxide (DMSO) (Batch #038K07101 and ID: Sigma D8418-100 mL), [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] (MTT) (Batch # 02317KH and ID: Sigma M2128-1G) were supplied from Sigma Aldrich, Germany.
2.3. Preparation of Extracts. The dried plant material (50– 200 g) was ground into coarse powder and then successively extracted with solvents of decreasing lipophilicity (n-hexane, dichloromethane, methanol and milliQ-water) using a Soxhlet apparatus. The plant extracts were then filtered and the solvent was evaporated under reduced pressure followed by freeze-drying.
2.4. Cytotoxic Screening 2.4.1. Cell Culture. Normal mouse fibroblast cells (NIH/3T3, ATCC: CRL-1658) and three human cancer cell lines gastric adenocarcinoma cells (AGS, ATCC: CRL-1739), colorectal adenocarcinoma cells (HT-29, ATCC: HTB-38) and breast ductal carcinoma cells (MDA-MB-435S, ATCC: HTB-129) were used for cytotoxicity screening of the Bangladeshi medicinal plant extracts. All cell lines were purchased from ATCC, Manassas, VA 20108, USA. Cell lines were cultured in Advanced DMEM supplemented with 10% inactivated NBCS and 5 mM l-glutamine, and grown at 370C in a humidified atmosphere of 5% CO2 in air.
2.5. MTT Assay. The MTT [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide] colorimetric assay developed by Mosmann, and further modified by Popiolkiewicz and Kim, was used with minor modifications to screen for cytotoxic activity of Bangladeshi medicinal plant extracts. Briefly, the cells were seeded in 96-well plates at a density of 2.5 × 104 to 3.5 × 104 cells/well. Following 24-h incubation and attachment, the cells were treated with different concentrations of plant extract for 24 h. Following washing and incubation with MTT solution (0.5 mg mL−1 for cancer cell lines and 1 mg mL−1 for mouse fibroblasts) for 2 h, cells were lysed with DMSO. The absorbance was measured after 45 min using a microplate reader (Wallac 1420 Multilabel counter, PerkinElmer) at a wavelength of 560 nm. MilliQ-water and 0.75% DMSO served as the negative control for water and methanol extracts, respectively, while 25% DMSO served as the positive control. The MTT assay was validated using various concentrations of DMSO (0.25–25%). Extracts showing cytotoxic activity were further tested at additional concentrations to calculate the IC50 values. The results are generated from two independent experiments; each experiment was performed in triplicate. The IC50 values were calculated with probit analysis software (LdP Line software, USA).