Background: Cholinesterase inhibitors are routinely applied in the treatment of Alzheimer’s
disease, and seeking new cholinesterase inhibitors is a priority.
Objectives: Twenty seven compounds were compared, including ones not previously tested. An attempt
was undertaken to precisely describe the role of alcohol in the inhibitory activity. This paper underlines
the role of a “false positive” blank sample in the routine analysis.
Methods: The inhibition of cholinesterase was measured using Ellman’s colorimetric method with a few
modifications designed by the authors (including the “false-positive” effect). The inhibitory role of
ethanol and methanol was also carefully evaluated. The present and past results were compared taking
the source of enzyme and alcohol content into consideration.
Results: For the first time, new inhibitors were identified, namely: methyl jasmonate, 1R-(−)-nopol
((anti-acetyl-(AChE) and butyrylcholinesterase (BChE) activity)) and 1,4-cineole, allo-aromadendrene,
nerolidol, β-ionone, and (R)-(+)-pulegone (anti-BChE activity). Oleanolic acid and (+)-β-citronellene
(not previously studied) proved to be inefficient inhibitors. For a number of well-known inhibitors (such
as nerol, (−)-menthol, (+)-menthol, isoborneol, (−)-bornyl acetate, limonene, α-pinene, β-pinene, α-
ionone, and eugenol) some serious discrepancies were observed between our findings and the results of
previous studies. Ethanol and methanol showed no anti-AChE activity up to 0.29% (v/v) and 0.23%
(v/v), respectively. Similarly, ethanol up to 0.33% (v/v) and methanol up to 0.29% (v/v) did not inhibit
the activity of BChE.
Conclusion: It can be stated that the impact of alcohol should be precisely determined and that blank
“false-positive” samples should be processed together with test samples. Furthermore, the effect of the
enzyme origin on the result of this test must be taken into consideration.