2.9. In vitro anti-cholinesterases inhibition assay
The evaluation of AChE and BChE inhibitory activities was based on Ellman’s
method (Ellman et al., 1961), using a 96-well microplate reader. Firstly, 3 mM
DTNB, 15 mM substrate (ATCI or BTCI), 100 mM phosphate buffer (pH 8.0) and extract (2.5 mg/ml), buffer or galanthamine (standard inhibitor) were mixed. Finally,
AChE or BChE (0.28 U/ml) were added and the absorbance was read at 405 nm for
5 min. The reaction enzyme activity was calculated as a percentage of the velocities
compared to that of the assay using buffer without any inhibitor. Inhibitory activity
was calculated from 100 subtracted by the percentage of enzyme activity.