3.10. Acetylcholinesterase Inhibition Assay

Acetylcholinesterase activity was determined by the Ellman method [83] and Jin et al. [84] with slight modification. Briefly, the assay was carried out on a 96-well plate, with each well containing 140 µL 0.1 M phosphate buffer (pH 8.0), 10 µL of stock solution of extract and 10 µL AChE (1 U/mL). The plate was incubated for 10 min at 25 °C. After incubation, 10 μL of 10 mM DTNB was added to the reaction mixture. Next, the reaction was initiated by the addition of 10 μL of 14 mM acetylthiocholine iodide. The plate was shaken for 1 min and finally 20 µL of 5% SDS was added to stop the reaction. Control wells containing the same composition but without extract (10 µL 70% ethanol) were included. Absorbance at λ = 412 nm was recorded using plate reader (EPOCH, Bio Tech) after 10 min incubation. All the reactions were performed in five repetitions. The percent of AChE inhibition was calculated as follows:

where: A_E is the absorbance of the sample containing extract and A_C is the absorbance of the control sample.