Infinite 200 pro microplate spectrometer

Cholinesterase inhibitory potential of the samples was determined using a spectrophotometric method that was modified from that described by Ellman et al [23 (link)]. For the acetylcholinesterase (AChE) inhibition assay, 140 μL 0.1M Na₂PO₄ buffer (pH 8) was added to a 96-well microplate, followed by the addition of 20 μL test sample and 20 μL acetylcholinesterase enzyme (0.09 U/mL). Ten microliters 10mM 5,5′-dithiobis (2-nitrobenzoic acid) was then added to each well, followed by addition of 10 μL acetylthiocholine iodide (14mM). The absorbance of the colored end-product was measured at 412 nm at designated intervals for 30 minutes after initiation of the enzymatic reaction by a Infinite 200 ProMicroplate Spectrometer (Tecan, Männedorf, Switzerland).
For the butyrylcholinesterase (BChE) inhibition assay, the same procedure as described for AChE was followed; however, the enzymes and substrates were substituted with butyrylcholinesterase from equine serum and S-butyrylthiocholine chloride, respectively.
A set of five concentrations of each plant extract or isolated compound was used to estimate the 50% inhibitory concentration (IC₅₀). Physostigmine, a cholinesterase inhibitor, was used as the reference standard. Absorbance of the test samples was corrected by subtracting the absorbance of their respective blank. Percentage inhibition was calculated using the following formula: