Cholinesterase Inhibition Assay

Cholinesterase (ChE) inhibitory activity was measured using Ellman's method, as previously reported [25] with slight modification. Sample solution (50 µL) was mixed with DTNB ((5,5-dithio-bis(2-nitrobenzoic) acid) (125 µL) and AChE (acetylcholinesterase) or BChE (butyrylcholinesterase) solution (25 µL) in Tris-HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of acetyl thiocholine iodide (ATCI) or butyryl thiocholine chloride (BTCl) (25 µL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as equivalents of galanthamine (mgGALAEs/g extract).