The inhibitory activity of 1–3 for AChE and BChE was determined spectrophotometrically using modified Ellman’s method [25 (link)] to quantify them in terms of IC50 values.
The enzyme activity in the final reaction mixture (2000 µL) was 0.2 U/mL, the concentration of ATCh (or BTCh) was 40 µM, and the concentration of DTNB was 100 µM for all reactions. The investigated derivatives were dissolved in DMSO and then diluted with demineralized water (conductivity 3 μS, equipment supplier BKG Water Treatment, Hradec Králové, Czech Republic) to a concentration of 1000 µM. Five different inhibitor concentrations were used for all tested compounds in the final reaction mixture. The final concentration of DMSO was 0.2%. All experiments were performed in triplicate. The average values of the reaction rate (v0-uninhibited reaction, vi-inhibited reaction) were used to construct the dependence of v0/vi on the concentration of inhibitors. IC50 values were calculated from obtained regression curve equations.
Acetylcholinesterase originating from electric eels (Electrophorus electricus L.; EeAChE) and butyrylcholinesterase obtained from equine serum (EqBChE) were used. The clinically used drug rivastigmine was involved as a reference dual AChE and BChE inhibitor. All enzymes, substrates and rivastigmine were purchased from Merck (Prague, Czech Republic).
The enzyme activity in the final reaction mixture (2000 µL) was 0.2 U/mL, the concentration of ATCh (or BTCh) was 40 µM, and the concentration of DTNB was 100 µM for all reactions. The investigated derivatives were dissolved in DMSO and then diluted with demineralized water (conductivity 3 μS, equipment supplier BKG Water Treatment, Hradec Králové, Czech Republic) to a concentration of 1000 µM. Five different inhibitor concentrations were used for all tested compounds in the final reaction mixture. The final concentration of DMSO was 0.2%. All experiments were performed in triplicate. The average values of the reaction rate (v0-uninhibited reaction, vi-inhibited reaction) were used to construct the dependence of v0/vi on the concentration of inhibitors. IC50 values were calculated from obtained regression curve equations.
Acetylcholinesterase originating from electric eels (Electrophorus electricus L.; EeAChE) and butyrylcholinesterase obtained from equine serum (EqBChE) were used. The clinically used drug rivastigmine was involved as a reference dual AChE and BChE inhibitor. All enzymes, substrates and rivastigmine were purchased from Merck (Prague, Czech Republic).
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