Anethole Cytotoxicity on Gingival Cells

Normal and cancer gingival cells (3 × 10⁵ cells/well) were seeded in 6-well plates and cultured for 24 h. Cells were then exposed or not to various concentrations of anethole for 24 h, then sued to assess the cell growth by means of MTT colorimetric assay (Sigma-Aldrich) as described by Semlali et al.^{20 (link)} Briefly, each culture was supplemented with 10% of MTT (5 mg/mL) and incubated for 3 h at 37 °C. Stained cells were then lysed using 1 mL of isopropanol-HCl 0.05 M solution with agitation for 15 min. Then, 4 × 200 µL of lysis solution were transferred to a 96-well plate and the absorbance was measured at the wavelength of 550 nm using an iMark microplate reader (Bio-Rad, Mississauga, ON, Canada). Cell proliferation levels were determined by means of the following formula: % of cell viability = [(OD _550 nm (treated cells) − OD (Blank))/(OD (control cell) − OD (Blank))] × 100, n = 7. With another set of experiments, anethole treated and non-treated gingival cells were detached by means of 0.05% trypsin: 0.1% EDTA solution (Sigma-Aldrich, Oakville, ON, Canada) and resuspended in fresh culture medium. The level of living cells in each culture was determined by means of the trypan blue exclusion assay, as described previously^{21 (link)}.