3.5. Colorimetric Determination of Acetylcholinesterase Activities

Ellman’s assay was used to measure acetylcholinesterase activity in 96-well microtiter plates in a final reaction volume of 200 μL. First, 50 μL of a 0.05 M sodium phosphate buffer (pH = 7.0) and 20 μL of 5 mg/mL compounds dissolved in 25% ethanol were added in each well. Then, 10 μL of 1 μg/mL EelAchE (Sigma-Aldrich, Inc., product number C2888) dissolved in a 0.02 M phosphate buffer (pH = 7.0) containing BSA (Beijing Dingguo Changsheng Biotechnology Company, FA016-5G, Beijing, China) was added in each well and put at 4 °C for 20 min. Secondly, 20 μL of 1.05 mM acetylthiocholine (Sigma-Aldrich, Inc., product number BCBR6567V) and 100 μL of 1.5 mM 5,5′-dithio-bis-nitrobenzoicacid (Shanghai Aladdin Bio-Chem Technology Company, J1530009, Shanghai, China) were added to each well before being mixed and reacted at 37 °C for 20 min. Thirdly, each well was subjected to colorimetric determination at 412 nm by a microtiter plate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). 20 μL of 0.11 mg/mL huperzine A (Aladdin, F1517037) was set as the positive control group. 20 μL of 25% ethanol in water was set the negative control. Percentage inhibition was calculated using the following formula:

where A₀ was the absorbance of the negative control and A₁ was the absorbance of the samples. Tests were carried out in triplicate.