*3.8. Enzyme inhibitory Assay*

A modified Ellman's method [30] was used to evaluate AChE inhibitory activities of compounds **1**–**7** in 96-well microplates. Tacrine was used as the standard inhibitor, and control test was performed without the presence of AChE inhibitors. All the inhibitors, solubilized in MeOH, were diluted stepwise from initial concentration of 32 μM. Every experiment was performed in triplicate. 5 μL inhibitor was added to each well and dried, then 50 μL phosphate buffer (PBS, 10 × 0.01 M, pH 7.2–7.4) was dispensed followed by 10 μL AChE (2 U/mL) and 20 μL 5,5-dithiobis 2-nitrobenzoic acid (DTNB, 5 mM). After 10 min culturing at 37 ◦C, 20 μL acetylthiocholine iodide (ATCh, 10 mM) was added and then OD was read at 405 nm over another period of 10 min culturing at 37 ◦C. The enzymatic inhibitory activity was calculated according to the following equation: Inhibition % = ((C − Cbackgroud) − (A − Abackgroud))/(C − Cbackgroud) ×100%, where C is the OD value of the control and A is the OD value in the presence of the inhibitor. As for the background, ATCh was replaced by PBS in A and C and bovine albumin (BSA, 1mg/mL) took the place of AChE in C.
