3.8.1. Acetylcholinesterase (AChE) Inhibition Assay

Acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 222 U/mg) inhibitory activity of extracts were determined according to previously described Ellman’s colorimetric method [59]. Acetylthiocholine iodide was employed as the substrate to assay the inhibition of AChE. The reaction mixture contained 130 μL of 100 mM sodium phosphate buffer (pH 8.0), 20 μL of test sample solution and 20 μL of AChE (0.36 U/mL), which were mixed and incubated for 15 min at 25 °C. The reaction was then initiated via the addition of 40 μL of the following mixture (freshly prepared): 20 μL 0.5 mM DTNB (5,5′-dithiobis (2-nitrobenzoic acid) and 20 μL acetylthiocholine iodide (0.71 mM). The hydrolysis of acetylthiocholine iodide was monitored by following the formation of yellow 5-thio-2-nitrobenzoate anion at 412 nm every 10 sec for 10 min using a 96-well microplate reader under a constant temperature of 25 °C, which resulted from the reaction of 5–50-thiobis-2-nitrobenzoic acid with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide. The percent inhibition of acetylcholinesterase enzyme was calculated using the equation:

where, Abs_control is the absorbance of the assay using the buffer instead of inhibitor (sample) and Abs_sample is the absorbance of the sample extracts. Neostigmine bromide was used as the positive control [49]. Phosphate buffer was used as the blank. Each standard or sample solution was analyzed in triplicate. The concentration of the extracts which caused 50% inhibition of the tyrosinase activity (IC50) was calculated by nonlinear regression analysis.