3.4.1. Acetylcholinesterase Inhibition

The acetylcholinesterase inhibitory activity was assessed by an adaptation of the Ellman’s method [38,39]. The assay solution contained 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (50 mM, pH 8.0), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, 3 mM), the stock solution of each compound in methanol (1 mg/mL), AChE (type VI-S, from electric eel) and the required quantity of methanol to reach the desired volume of sample mixture in a 1 mL cuvette. Samples were incubated for 15 min and then 75 μL of acetylthiocholine iodide (AThChI) solution (16 mM) was added. The reaction was monitored for 5 min at 405 nm. Assays were run with a blank containing all the reagents but AChE. Reaction rates were calculated as well as the enzyme activity. A positive control reaction was performed using methanol as a sample solution. The fraction of enzyme inhibition due to the existence of rising concentration of the test compound was calculated using Equation (1), where vi = starting reaction rate in the presence of inhibitor; vo = starting reaction rate of the control reaction.

The inhibition curves were achieved by plotting the percentage of enzymatic inhibition against inhibitor concentration and a calibration curve was got from which the linear regression parameters were obtained.