3.8. In Vitro Acetylcholinesterase Assay

The in vitro AChE inhibition assay was evaluated using a spectrophotometry method developed by Ellman with some modifications [41]. The assay was conducted in a 96-well plate with a total volume of 200 µL assay mixtures in comparison to galantamine as the positive control. Plant extracts were dissolved in dimethyl sulfoxide (DMSO) and tested at a final concentration of 0.2 mg/mL, while compounds were tested at 0.1 mg/mL. The final concentration of DMSO was fixed at 1% in the 96-well plate. In a 96-well plate, 178 µL of 50 mM phosphate buffer, 2 µL of 1 mg/mL compound and 20 mg/mL extract, and 10 µL of 0.5 U/mL AChE were added. The first incubation period was 15 min at 25 °C. Five microliters of 14 mM ATCI substrate and 10 mM of the color indicator, DTNB, were added equally into each well and the well was then incubated at 25 °C for 30 min to initiate the enzyme reaction. The absorbance was measured at 415 nm using Promega Glomax^® Multi Plus Reader (Promega, Madison, WI, USA). Each sample was done in triplicate and eight 2-fold serial dilutions were performed for each sample to determine the fifty-percentage inhibitory concentration (IC₅₀). The percentage of inhibition was calculated using formula (1) [42]: