3.5. AChE Inhibition Assay

Inhibition potency of the test compounds against AChE was determined using Ellman’s method [53]. Enzyme solutions were prepared in gelatin solution (1%, w/v), at the concentration of 2.5 units/mL. The synthesized compounds, and donepezil, which was used for reference, were prepared at 10⁻³ M and 10⁻⁴ M concentrations using 2% DMSO solution for initial measurements. AChE solution (20 µL/well) and test solution (20 µL/well) were added to phosphate buffer solution (140 µL/well, pH 8.0 ± 0.1), and incubated at 25 °C for 5 min. The reaction was started by addition of the chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, 20 µL/well, 10 mM) and the substrates acetylthiocholine iodide (ATCI, 10 µL/well, 75 µM) to the enzyme-inhibitor mixture. The production of the yellow anion was recorded by multimode microplate reader for 10 min at 412 nm. As a control, an identical solution of the enzyme without the inhibitor was processed. The control and the inhibitor readings were corrected with blank-readings. All processes were assayed in four independent determinations.