4.3. In Vitro Assay for AChE Inhibitory Activity

To investigate the in vitro AChE inhibition activity, the manufacturer’s protocol for the Acetylcholinesterase Assay Kit (Abcam, Cambridge, UK) based on a modified Ellman’s colorimetric method [47] was used. The stock solutions of EHRT and five CHRTs in DMSO (100 mg/mL) were diluted with 0.1 M sodium phosphate buffer (pH 8.0) to 100 μg/mL to use as sample solutions. These sample solutions was then serially diluted with the same buffer to final concentrations of 100, 50, 25, and 12.5 μg/mL. AChE in 0.1% bovine serum albumin/H₂O (25 U/mL) was dissolved in assay buffer (0.1 M sodium phosphate buffer, pH 7.3) to 35.2 mU/mL final concentration. To prepare the reaction mixture, the substrates 5,5′-dithiobis-(2-nitrobenzoic acid) and acetylthiocholine iodide were dissolved in assay buffer and H₂O, respectively, to 10 mM, and then mixed in assay buffer to 0.5 mM final concentration. To assess the enzymatic reaction, the sample solution (50 µL) and the reaction mixture (50 µL) were mixed in 96-well plates, and pre-incubated (10 min) at room temperature. The AChE solution (10µL) was treated to each well to start the enzymatic reaction, which was conducted for 60 min at 10-min intervals and measured using an Epoch microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) at 412 nm. All experiments were conducted in triplicate, and berberine was used as a positive control [48]. The percentage inhibitory activity of AChE was calculated using the following equation:

where S is the sample volume (reaction mixture, sample solution, and enzyme), S’ is the sample volume without enzyme (reaction mixture, sample solution, without enzyme), C is the control volume (0.1 M sodium phosphate buffer (pH 8.0), reaction mixture, and enzyme, and C’ is the control volume without enzyme (0.1 M sodium phosphate buffer (pH 8.0), reaction mixture, without enzyme).