3.5. Cholinesterase Inhibitory Assay

Acetylthiocholine iodide (ATCl), acetylcholinesterase (AChE, from electric eel), butyrylcholinesterase (BChE, from equine serum), S-butyrylthiocholine chloride, and 5, 50-dithiobis(2-nitrobenzoicacid) (Ellman’s reagent, DTNB) were purchased from Sigma–Aldrich (Kuala Lumpur, Malaysia).

Cholinesterase enzyme inhibitory potential of the test samples was determined following the method of Khaw et al. (2014) with slight modifications on the vehicle used. Briefly, test samples and galantamine were prepared in DMSO at the initial concentration of 0.5 mg/mL. The final concentration of DMSO in reaction mixture was 1%. At this concentration, DMSO has no inhibitory effect on both AChE and BChE enzymes. For AChE inhibitory assay, 140 µL of 0.1 M sodium phosphate buffer (pH 8) was added to a 96 wells microplate followed by 20 µL of test samples and 20 µL of 0.09 units/mL AChE enzyme. Then, 10 µL of 10 mM DTNB was added into each well followed by 10 µL of 14 mM of acetylthiocholine iodide. The absorbance of the colored end product was measured using Tecan Infinite 200 Pro Microplate Spectrophotometer at 412 nm for 30 min. Each test was conducted in triplicate. For BChE inhibitory assay, the same procedures were applied as AChE except for the use of enzyme and substrate, which were BChE from equine serum and S-butyrylthiocholine chloride. Absorbencies of the test samples were corrected by subtracting the absorbance of their respective blank (test samples in DMSO with substrate and DTNB, but without enzyme). A set of five concentrations was used to estimate the 50% inhibitory concentration (IC₅₀) for the compounds showing more than 50% inhibition at 5µg/mL concentration.