C3389

Cholinesterase (ChEs) inhibition of EO was determined for the enzymes (i) acetylcholinesterase (AChE) and (ii) butyrylcholinesterase (BuChE). The procedure was followed as described by Ellman et al. [62 (link)] and Calva et al. [57 (link)]. Phosphate buffered saline (pH = 7.4), DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) ion (1.5 mM) a reagent that reacts with thiocholine to give the yellow coloration and the EO sample in DMSO (1% v/v) were prepared. The reaction of DTNB is monitored by measuring its absorption at 412 nm. AChE, from Electrophorus electricus (Sigma-Aldrich, C3389, St. Louis, MO, USA) and BuChE, from horse serum, (Sigma-Aldrich, SRE020, St. Louis, MO, USA) are dissolved in PBS (pH = 7.4) at 24 mU/mL. Preincubation was carried out for 10 min and acetylcholine iodide (1.5 mM) is added to initiate the reaction. The reaction is monitored for 30 min at 30 °C in a PherastarFS detection system (BMG Labtech). Inhibitory concentration (IC₅₀) values were calculated in the online package GNUPLOT (www.ic50.tk, www.gnuplot.info) (accessed on 1 March 2022). Measurements were performed by triplicate. The reference drug inhibitor of ChEs was Donepezil, for AChE and BuChE with an IC₅₀ value of 100 nM and 8500 nM, respectively. False positives are not excluded for high concentrations (>100 ug/mL) of amine or aldehyde compounds [59 (link)].

AChE activity was measured within a 96-well microtitre plate based on the Ellman [58 (link)] method. Forty µL of plant extracts at concentrations of 200, 20, 2, 0.2 and 0.02 µg/mL were mixed with 50 μL of 3 mM DTNB, 50 µL of AChE (1 mg/mL, Sigma, C3389, Irvine, UK) or rat brain homogenate (prepared at 10% (w/v) according to references [55 (link),56 (link)], and 35 μL of 50 mM Tris-HCl (pH 8.0) containing 0.1% BSA, and samples incubated for 5 min at 37 °C. The reaction was initiated by addition of 25 μL of 15 mM ATCI resulting in the production of a 5-thio-2-nitrobenzoate anion read at 412 nm every 5 s for 10 min using a Spectramax microplate reader (ThermoFisher, Stafford, UK).
To establish suitable optical density changes and linearity of signal, a 1:10 dilution of rat brain homogenate (in 10 mM Tris-HCl pH 8.0) was used for AChE measurements. This rat brain positive control for AChE activity was inhibited in a dose-dependent manner by either eserine or organophosphorus pesticides [57 (link)].

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.