Polyphenol Cytotoxicity Evaluation in Cells

The CellTiter-Glo^® Luminescent Cell Viability Assay Kit (Promega, Southampton, UK) was used as a homogeneous method to determine the number of viable cells in culture was based on a quantification of ATP levels. This assay was used to determine the effect of each polyphenols on cellular proliferation in each of the cell lines. Cells were seeded into white 96-well plates (Fisher Scientific, Loughborough, UK) at 2.5 x 10³ cells per well and treated with each polyphenol dissolved in ethanol: quercetin, apigenin, chrysin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene (Sigma, Poole, UK) at concentrations between 2 - 500 µM for 24, 48 and 72 h together with ethanol vehicle controls at 0.1 % (v/v) ethanol. All treatments were performed in triplicate, in three independent experiments. Following treatments, cellular proliferation was measured as per manufacturer’s instructions. The IC₅₀ was determined for each polyphenol in each cell line. This was defined as the treatment concentration at which 50% reduction in cellular proliferation was observed. This was calculated from a linear regression equation of each standard curve for each polyphenol with each cell line. The IC₂₅ was also determined in order to provide treatment ranges for apoptosis detection, and cell cycle treatments, but were not used to determine the effectiveness of treatments.