*3.4. Cytotoxicity Assays*

The cytotoxic activity of the isolated and identified AEG671 as well as the activity of two other samples containing AEGs as the main components was tested. For the purpose the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assays with the application of a human breast adenocarcinoma cell line T47D (Merck KGaA, Darmstadt, Germany) were performed as described by Felczykowska et al. [48] and Szubert et al. [49]. T47D cells were plated at 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well of 96-well plate containing RPMI1640 (Carl Roth GmbH) medium supplemented with 10% fetal bovine serum (Merck KGaA) and penicillin-streptomycin solution (50 u and 0.05 mg per 1 mL of medium respectively; Merck KGaA) (24 h at 37 ◦C, 5% CO2). The cytotoxic effects of tested samples dissolved in 1% DMSO, at final concentrations 25, 50, 100 and 200 μg ml−<sup>1</sup> (in culture medium) were examined after 24 h incubation (37 ◦C, 5% CO2) using a microplate reader (Spectramax i3, Molecular Devices, LLC. San Jose, CA, USA). Cell viability was calculated as the ratio of the mean absorbance value, for the six replicates containing the samples, to the mean absorbance of the six replicates of the corresponding solvent control, and expressed as a percentage. The results were considered as significant when cell viability decreased below 50%.
