Gen spectrophotometer

Toxicological effect of both plant extracts were evaluated on human Chang liver cell lines using microculture CellTiter-Blue viability (Promega, USA) assay. Briefly, 96-well microplates were seeded accurately, with 100 μl DMEM + high glucose, L-glutamine and sodium pyruvate (Thermo Scientific, South Logan, Utah, USA) containing 3.0 × 10³ cells in suspension and incubated in a CO₂ incubator regulated at 37 °C and 5% CO₂. After 24 h incubation and attachment, the cells were treated with 1000, 500, 250, 125, 75, 25 and 5 μg/ml concentration of each extracts. Exactly 60 μm of curcumin (Sigma-Aldrich, South Africa) was used as positive control and 0.1% DMSO as negative control. After 24, 48, and 72 h of incubation, cell viability was determined by adding CellTiter-Blue as an indicator and further incubated for 4 h. Fluorescence was read at 570/620 nm using Analytical & Dignostic product Gen spectrophotometer (Bio Tek, USA).

The cytotoxicity assays were conducted in 96-well microplates (Cat no. 6905A11, Thermo Scientific™ Nunc™, Thermo Fisher Scientific, AEC-Amersham SOC Ltd., South Africa) by seeding at a density of 2000 cells/well with 100 μl of suspension. Cells were treated with 100 μl of 1, 0.5, 0.25, 0.125, 0.062, 0.031, and 0.015 mg/ml plant extract in DMEM working solutions, respectively. The following control combinations were included: (i) negative controls containing DMEM (100 μl) and (ii) positive control containing 0.1 mg/ml curcumin (100 μl). The microplates were incubated for 24, 48, and 72 h as described above. Following incubation, the medium was removed by aspiration and replaced with DMEM (100 μl) and MTT (5 mg/ml in phosphate buffered saline, pH 7.4; 20 μl) and further incubated for 4 h. Finally, the medium was aspirated and replaced with fresh DMSO (100 μl) to solubilize the formazan crystals formed in the cells. The absorbance was measured at 570 nm with a fluorescence kinetic synergy MX analytical diagnostic reader (Gen spectrophotometer, Bio Tek, Vermont, USA).