AChE activity was assayed as described by Ellman et al. [64 (link)], with some modifications [65 (link)]. Fifteen μL of Electrophorus electricus AChE (Sigma-Aldrich, Darmstadt, Germany) in buffer phosphate (pH 7.6) and 15 μL of the tested compounds (1.4–4350 μM in methanol) were dissolved in 200 μL in the same buffer, and plated in 96-well plates. The mixtures were incubated for 30 min at room temperature before the addition of 30 μL of the substrate solution (15 μL 0.5 M DTNB, 15 μL 0.6 mM ATCI in buffer, pH 7.6). The absorbance was read on a Microplate Reader Biochrom EZ 800 at 403 nm, after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor, according to the following equation:
where Abscontrol is the absorbance of the control, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solvent (methanol/water for GAL); Abssample is the absorbance of the sample, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solution of the tested compound (in methanol/water for GAL).
where Abscontrol is the absorbance of the control, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solvent (methanol/water for GAL); Abssample is the absorbance of the sample, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solution of the tested compound (in methanol/water for GAL).