Colorimetric Cholinesterase Activity Assay

The cholinesterase (ChE) activities were assayed following a colorimetric protocol adapted from Ellman et al. [43 (link),44 (link)]. ChEs efficiently catalyze the hydrolysis of acetylthiocholine (ATCh), the sulfur analog of the natural substrate of these enzymes. Upon hydrolysis, this substrate analog produces acetate ion and thiocholine. Thiocholine, in the presence of the highly reactive dithiobisnitrobenzoate (DTNB) ion, generates a yellow color, which can be quantitatively monitored by spectrophotometric absorption at 412 nm. All reagents were obtained from the Sigma-Aldrich trading house. A typical 200 μL inhibition assay volume contained phosphate buffered saline solution (pH 7.4), DTNB (1.5 mM), test sample in DMSO (1% v/v final). Both acetylcholinesterase from Electrophorus electricus (Type V-S, lyophilized powder, 744 U/mg solid, 1 272 U/mg protein) and butyrylcholinesterase from equine serum (lyophilized powder, ≥900 units/mg protein) were dissolved in PBS pH 7.4 and used at 25 mU/mL for the assay. After 10 min of pre-incubation, the substrate acetylthiocholine iodide (1.5 mM) was added to start the reaction. During 1 h of incubation at 30 °C, 96-well microtiter multiplates were read on a PherastarFS (BMG Labtech) detection system. All measurements were made in triplicate. When possible, the IC₅₀ values were calculated using the GNUPLOT package on line (www.ic50.tk, www.gnuplot.info). Donepezil was used as reference ChE inhibitor with an IC₅₀ = 100 nM for AChE and 8500 nM for BChE. In this assay, we did not exclude the possibility of false-positive inhibition results previously described for high concentrations (>100 μg/mL) of amine or aldehyde compounds [45 (link),46 (link)], but the lack of inhibition observed for the essential oil versus the AChE strongly minimized this possibility.