Acetylcholinesterase Inhibition Assay Protocol

Acetylcholinesterase (AChE) activity was measured in a 96-well microtiter plate assay based on the method of Ellman et al. (29 (link)), as published previously (30 (link)). For each assay data point, 50 μL of 3 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 50 μL of AChE (1 mg/mL) (Sigma, C3389) or rat brain homogenate (prepared according to previous publications (31 (link), 32 (link))), 35 μL of 50 mM Tris/ HCl pH 8.0, and 40 μL of polyherbal extract were mixed and incubated at 37 °C. The assay was initiated by addition of 25 μL of 15 mM acetylthiocholineiodide (ATCI), with production of 5-thio-2-nitrobenzoate anion read at 412 nm every 30 sec for 10 min using a Spectramax microplate reader (ThermoFisher, UK). Assay reactions with polyherbal extracts were all performed in triplicate at concentrations of 200 μg/mL, 20 μg/mL, 2 μg/mL, 0.2 μg/mL and 0.02 μg/mL. A negative control assay performed in the absence of AChE provided a reagent blank. Eserine (Sigma, E8375) was used as a positive control to inhibit electric eel or rat brain AChE in a dose-dependent manner. The percentage inhibition of AChE by polyherbal extract was calculated relative to inhibition by Eserine, with the herbal extract concentration producing 50% inhibition (IC₅₀) of AChE calculated using GraphPad Prism (version 5.03, Inc., 2010, San Diego California USA) via non-linear regression analysis.