3.4. Evaluation of Acetyl- and Butyrylcholinesterase Inhibition

Human recombinant acetylcholinesterase (HuAChE), butyrylcholinesterase from equine serum (eqBChE), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were gained from Sigma-Aldrich. Assays of acetyl- and butyrylcholinesterase inhibitory activity were performed according to Ellman’s method described previously [34]. Briefly, these assays were performed as follows. The reaction mixture (250 µL), containing 50 µL of buffer (50 mM Tris-HCl buffer (pH 8.0), 0.1 M NaCl, 0.02 M MgCl₂.6H₂O), 25 µL of 1.5 mM of ATCI or BTCI, 25 µL of 100 µM of test compounds in EtOH and 125 µL of 3 mM DTNB were added. Then, 25 µL of HuAChE and equine serum of BChE in 50 mM Tris-HCl buffer containing 0.1% (w/v) BSA (pH 8.0). Reactions were initiated by the addition of the enzyme into the medium. This reaction resulted in the development of a yellow color, which was measured at 405 nm every 11s for 2 min in a Microplate Scanning Spectrophotometer. Each experiment was repeated in triplicate. In this study, tacrine and galanthamine were taken as reference drugs.

The percentage of enzyme inhibitory activity (%Inhibition) was calculated by the following expression: %Inhibition = ((Mean velocity of blank-Mean velocity of sample) × 100)/Mean velocity of blank. The IC₅₀ value (the concentration of the compounds required for a 50% reduction in cholinesterase activity) was calculated using GraphPad Priam 2.01 software. Eight difference concentrations of the inhibitor (50 µM–1.6 × 10⁻³ µM) were used. The results are expressed as the mean ± SD. The selectivity of acetylcholinesterase inhibitory activity of the compounds can be evaluated by the ratio between IC₅₀ of equine serum of BChE with IC₅₀ of HuAChE and shown as the Selectivity index (SI) [47].