2.3.2. Glutathione S-Transferase Inhibition Assay

The activities of the GSTs were determined according to the method described by Habig et al. (1974) [48] using a cell imaging multi-mode reader (Cytation 3; BioTek, Winooski, VT, USA). The stock solutions of inhibitors were prepared as described for the ChE inhibition assays. Then the stock solutions of the potential inhibitors and negative controls were added to the wells, and progressively diluted in 100 mM sodium phosphate buffer (pH 6.5) to the final volume of 50 μL. 1-Chloro-2,4-dinitrobenzene (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol to 50 mM, and then diluted with 100 mM sodium phosphate buffer (pH 6.5) to a final concentration of 4 mM. This solution (50 μL) and 2 mM reduced glutathione (100 μL) in the same buffer were added into the microtiter plate wells. Two GSTs were used as the enzyme sources: horse liver GST (hlGST) and human placenta GST (hGST) (Sigma-Aldrich, St. Louis, MO, USA). These were dissolved in 100 mM sodium phosphate buffer (pH 6.5), and 50 μL of these enzyme solutions were added into the wells to start the reaction. The final enzyme concentration was 0.044 U/mL. The blank reactions without the inhibitors were run with the appropriate dilutions of the solvents in which the tested compounds were initially diluted (100% MeOH or 5% aqueous DMSO), and the readings were corrected according to the appropriate blanks. The reactions were followed spectrophotometrically at 340 nm at 25 °C over 4 min. Each measurement was repeated at least three times. For determination of the inhibitory constants (K_i), the kinetics were monitored using three different final substrate concentrations (200, 400, 800 μM). The data were analysed using the OriginPro software (OriginPro 2020, OriginLab Corporation, Northampton, MA, USA).