2.13. Anti-Acetylcholinesterase Assays

In vitro acetylcholinesterase (AChE) activity was assessed exactly as in Ka et al. (2020) [12] following Ellman’s colorimetric protocol [43] with the Acetylcholinesterase Assay Kit (Abcam Inc., Boston, MA, USA). Briefly, 50 µL serial dilutions (3.9–500 µM) of compounds 1–5 were prepared in Tris-HCl pH = 7.9 buffer into designated wells of a clear 96 well-plate. A total of 5 µL of DTNB was added in each well, then 50 µL of diluted acetycholinesterase was added. The plate was incubated for 10 min in the dark. Matched concentrations of DMSO were used as a negative control. Kinetic of reaction was measured in a multiple plate reader at 410 nm in kinetic mode for 40 min at room temperature. The percentage of anti-AChE inhibition was calculated according to the following formula: 100 − [((E − S)/E) × 100], where E is the activity of the enzyme with matched concentrations of DMSO and S is the activity of the enzyme with the test sample.