2.7. Acetylcholinesterase Inhibition Assay

The assay for AChE inhibition was based upon the method of Ellman et al. [55], but modified for a 96-well microtiter plate format, as reported in Nwidu et al. [54]. In a microtiter plate, 40 μL of plant extract (at concentrations of 200, 20, 2, 0.2 and 0.02 µg/mL) was mixed with 35 µL of 50 mM Tris-HCl (pH 8.0) containing 0.1% BSA, 50 μL of 3 mM DTNB, and 50 µL of AChE. The AChE used was either from electric eel at 1 mg/mL (Sigma, Poole, UK) or that present within rat brain homogenate (prepared at 10% (w/v), according to the procedure of Carter et al. [56,57], which had been diluted 1:10 in 10 mM Tris-HCl pH 8.0 for assays. Plates were incubated at 37 °C for 5 min before the cholinesterase reaction initiated by the addition of 25 μL of 15 mM ATCI substrate, resulting in the production of 5-thio-2-nitrobenzoate anion that was read at 412 nm every 5 s for 10 min using a Spectramax microplate reader (Thermo Fisher, Stafford, UK). Eserine was employed at 0.02 µg/mL as a positive control for AChE inhibition. At this concentration (or above), eserine inhibits AChE to ≈100% [55]. All reactions were performed in triplicates, from which an average reading was generated.