Mpr a4i

The effects of the three plant extracts on cell viability were evaluated on human epithelial colorectal adenocarcinoma cells (Caco-2 cells). Caco-2 cells are a valid system for evaluating the biological effects of plant extracts for human use [57 (link),58 (link),59 (link)]. Caco-2 cells were obtained from the ATCC cell bank (Rockville, MD, USA) and were propagated in DMEM (Gibco BRL, Life Technologies, Mumbai, India) supplemented with 10% fetal calf serum, 1% sodium pyruvate, 1% L-glutamine solution, and 1% streptomycin/penicillin in a humidified atmosphere of 5% CO₂ at 37 °C.
The effect of Artocarpus lakoocha Roxb. and Artocarpus heterophyllus Lam. flower extracts on cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to obtain the range of toxic and nontoxic concentrations. Caco-2 cells were seeded in 96-well plates at a density of 5 × 10⁴ cells per well for 24 h in complete medium. Cells were then treated with flower extracts at various concentrations (1–50 μg/mL) or with vehicle (DMSO at 0.001–0.05%) for 48 h; then, the cells were exposed to the MTT reagent (0.4 mg/mL in PBS) for 30 min at 37 °C, 5% CO₂. The absorbance at 570 nm was measured using the microplate reader MPR A4i (Tosoh, Tokyo, Japan).

The AR42J cell viability was analyzed using the Cell Counting Kit-8 assay. The cells were seeded into 96-well plates and incubated overnight in a humidified CO₂ incubator (95% air and 5% CO₂) to allow the cells to adhere and recover. The culture medium was then replaced with either H₂-rich or normal medium containing cerulein at a concentration of 10⁻⁸ M for 24 h as described above. The cells were then incubated with a WST-8 solution at 37°C for 1 h, and the absorbance at 450 nm was measured with a microplate reader (MPR-A4i, Tosoh Corporation, Tokyo, Japan). Cells cultured in normal medium served as controls. The cell viability index was calculated according to the following formula: experimental OD value/control OD value×100% [16 (link)]. The cells were viewed under a laser-scanning confocal microscope (LSM-510, Carl Zeiss Jena GmbH, Jena, Germany) to visualize those that had been injured and undergone necrosis. Annexin V was excited at 488 nm and emitted at 530 nm, and PI was excited at 536 nm and emitted at 617 nm. Five randomly selected microscopic fields were examined to determine the percentage of positive cells. All of the experiments were performed in triplicate and repeated three times.