Modified TLC bioautographic method for screening acetylcholinesterase inhibitors from plant extracts

Abstract

The present work describes modifications to an existing TLC bioautographic method for detecting acetylcholinesterase inhibitors from plant extracts. The basic principle of the method is that the enzyme converts 1-naphthyl acetate into naphthol which reacts with Fast Blue B salt to make a purple-colored background on the TLC plates. Inhibitors of acetylcholinesterases produced white spots on the background. Our modifications involve changes in the concentration of the enzyme, the reagents, and the time of the reaction. With these changes, the consumption of the enzyme was reduced by 85% and the detection limits were decreased remarkably.

Skimmianine, a furoquinoline alkaloid from Zanthoxylum nitidum as a potential acetylcholinesterase inhibitor

“…The TLC bioautographic assay for AChE inhibitory activity was modified by our group (Yang et al, 2009). Firstly, 0.1 ll of 10 mg/ml compound (1) was spotted on a silica gel TLC plate and migrated by 35:1 chloroform:methanol solution.…”

“…False-positive results due to inhibition of 1-naphthol reaction with Fast Blue B salt were eliminated by the method of Yang et al, (2009). Another TLC plate identical to the one in the TLC assay was prepared.…”

Thin‐layer Chromatography of Plants, with Chemical and Biological Detection Methods

“…Furthermore, attempts to establish the LOD estimated a minimum inhibitory amount of 1 ng for physostigmine. Thus, this method is 100-fold less sensitive than the modified Marston's method previously published by Yang et al (2009) and does not appear to offer any advantage compared to existing bioautographic assays. Nevertheless, further assays should be attempted by spraying or dipping the layer, instead of dabbing it with soaked paper: a proper and uniform application of the chromogenic solution might improve the resolution of this bioassay.…”

“…In Marston's protocol, the acetylcholinesterase concentration is 6.7 U mL −1 . Yang et al (2009) coupling of 1-naphthol with the stain is incomplete and produces an unstable azo-product still containing one free diazoamino group, which gives a pale and transient coloration. Therefore, the modified method enables the enzyme consumption to be reduced by 85%, while dramatically improving the sensitivity, as the lowest amount of physostigmine required to observe a white inhibition spot is 0.01 ng, that is, 100-fold less than with Marston's method and 1000-fold less than with the Ellman's method modified by Rhee.…”

Essential oil of parsley and fractions to in vitro control of cattle ticks and dengue mosquitoes

“…The method described by Yang et al (2009) was used to determine the anticholinesterase activity through an autobiographic assay. The EO and FRs obtained by CC were solubilized in methanol indifferent concentrations from 100,000 to 0.012 µg/ml.…”