4.5. AChE Inhibition Assay

The AChE inhibitory property was evaluated using slightly modified Ellman’s assay.³⁹ Electric eel AChE (type-VI-S, EC 3.1.1.7, Sigma) was used as the enzyme source, while acetylthiocholine iodide (ATCI, Sigma, St. Louis, MO, USA) was used as the substrate of the reaction. 5,5-Dithiobis-(2-nitrobenzoic acid) (Sigma, St. Louis, MO, USA) was used for the measurement of the cholinesterase activity. Test samples were reconstituted with 50% DMSO. About 140 μL of 0.1 mM sodium phosphate buffer (pH 8.0), 20 μL of 3 mM DTNB, and 20 μL of 0.36 U/mL of AChE were added in the reaction, followed by incubation for 15 min at 37 °C and addition of 10 μL of 15 mM ATCI. The microplates were then read at 412 nm using a Glomax microplate reader (Promega, Madison, Wisconsin). Galantamine was used as the reference drug. The IC₅₀ value is defined as the concentration of the plant material required to inhibit AChE activity by 50%.