Determination of Maximum Non-toxic Dose (MNTD) and 50% Cytotoxic Concentration (CC50) Value

Cytotoxicity assay was carried out on the plant extracts to determine the MNTD and CC₅₀ on Vero cells. MNTD is defined as the highest concentration of extract that would not induce cell death (similar to negative control). CC₅₀ value was expressed as the concentration of extracts that would cause 50% cell death. The cells were seeded at a density of 2 × 10⁴ cells per well in 96 well plate and incubated for 24 h to allow cellular attachment. Two-fold serial dilutions of O. indicum extracts ranging from 10 000 to 39 μg/mL were freshly prepared in growth media supplemented with 2% FBS and 100 μL from each concentration were placed in triplicates. The cell viability was assessed using the CellTiter 96^® AQ_ueous one solution cell proliferation assay (Promega). After 48 h, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent were pipetted into each well and incubated for 37 °C for 3 h in 5% CO₂ incubator. The absorbance was read using ELISA plate reader at 490 nm. MNTD and CC₅₀ value were determined by plotting a graph of the concentration of extract (μg/mL) against percentage of viable cells (Fig. 1). The percentage of viable cells was calculated as: Survival (%) = (T/C) × 100 where C is the mean absorbance of healthy negative control without the extract and T is the mean of absorbance in treated wells with different extract concentration.

Cytotoxicity analysis of O. indicum a methanol and b aqueous extracts on Vero cells. The cells were incubated with increasing concentration of plant extract and the cell viability was measured after 72 h. Values were expressed as the mean ± SE for triplicate assays