**3.1 Evaluation of cytotoxicity using MTT assay**

The assay detects the living cells, and it be used to measure cytotoxicity, proliferation, or activation [18]. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay is based on the principle that mitochondrial dehydrogenase enzyme forms viable cells to cleave the tetrazolium rings of the pale yellow MTT and forms a dark blue formazan crystal which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The solubilization of the cells is by the addition of a detergent that cause in the liberation of the crystals, which are solubilized. The color can be quantified using a multi-well plate reader at 570 nm. The cells can be maintained in DMEM medium, supplement with 10% FCS. Briefly, cells in the log phase of growth can be harvested, counted, and seed (5 × 103 cells/well in 100 μl) in 96 well titer plates (Axygen), and add PBS to the outer wells (200 μl/well). After 24 h of incubation at 37°C in 5% CO2 to allow cell attachment, media will be removed; cultures will be treated with various concentration of compounds diluted with medium. Cells and media are used as the negative controls. The plates are further incubated for 24, 48, and 72 h. On completion of incubation, with the extract, media can be removed without disturbing the adherent cells. In the case of suspension cells lines, the media can be removed after the plates are centrifuge at 2000 rpm for 15 min. To each well, 100 μl of 5 mg/ml stock solution of MTT dye will be added, and plates can be incubated for 2 h in the dark at 37°C in a CO2 incubator. About 100 μl of lysis buffer can be added to each well, and the plates can be incubated for 4 h in the dark in a CO2 incubator and absorbance can be read using ELISA plate reader. Three replicates are set up for each concentration. The concentration required to reduce absorbance by 50% (IC50) in comparison to control cells is determined. In MTT assay colorless well indicates the cytotoxicity of KB human oral cancer cells (**Figure 2**)

1100 cens is uteerminneu. In M1 1 assasy conress wen mancaires une cyclouxent y  $\mathbf{B}$  human oral cancer celles (Figure 2)

1 % of Growth inhibition = 100 --  $\frac{\text{Absorbance of treated cells} \times 100}{\text{Absorbance of control cells}}$ 
