4.8. Antiviral Activity Assessment

MTT [59] was used to study cytotoxicity of the compounds. Briefly, series of three-fold dilutions of each compound (300–4 µg/mL) in MEM were prepared. MDCK cells (ATCC CCL-34) were incubated for 72 h at 36 °C in 5% CO₂ in the presence of the dissolved substances. The degree of destruction of the cell monolayer was then evaluated in the MTT. The cells were washed twice with saline, and a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/mL) in cell culture medium was added to the wells. After 1h incubation, the wells were washed and the formazan residue dissolved in DMSO (0.1 mL per well). The optical density of wells was then measured on a Multiscan FC photometer at wavelength of 540 nm and plotted against concentration of the compounds. Each concentration was tested in three parallels. The 50% cytotoxic dose (CC₅₀) of each compound (i.e., the compound concentration that causes the death of 50% cells in a culture, or decreasing the optical density twice as compared to the control wells) was calculated from the data obtained. Values of CC₅₀ obtained in µg/mL were then calculated into micromoles.

The compounds in appropriate concentrations were added to MDCK cells (0.1 mL per well). Cells were further infected with A/Puerto Rico/8/34 (H1N1) influenza virus (m.o.i 0.01). Plates were incubated for 72 h at 36 °C at 5% CO₂. After that, cell viability was assessed by MTT test as described above. The cytoprotective activity of compounds was considered as their ability to increase the values of IC₅₀ compared to control wells (with virus only, no drugs). Based on the results obtained, the values of IC₅₀, i.e., concentration of compounds that results in 50% cells protection, were calculated using GraphPad Prism software. Values of IC₅₀ obtained in μg/mL were then calculated into μM. For each compound, the value of selectivity index (SI) was calculated as ratio of CC₅₀ to IC₅₀. The compounds with SI of 10 and higher were considered as active [39].