AChE activity was measured by spectrophotometer based on Ellman’s method (Ellman et al.,1961). In the enzyme hydrolysis the substrate acetylthiocholine results in the product of thiocholine which reacts with Ellman’s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2- nitrobenzoate which can be detected at 412 nm. In test tube 1710 μL of 50 mMTris–HCl buffer pH 8.0 and 250 μL of plant extracts at the concentrations of 25 – 400 μg/ mL, 10 μL 6.67 UmL-1 AChE and 20 μL of 10 mM of DTNB (5,5’-dithio-bis[2- nitrobenzoic acid]) in buffer were added. Positive control namely galanthamine were prepared in serial concentration as same as test extract by dissolving in 50 mMTris–HCl buffer pH 8.0. The mixture was incubated for 15 min at 37oC.Then,10 μL of acetylthiocholine iodide (200 mM) in buffer were added to the mixture and the absorbance was measured at 412 nm every 10 sec for 3 mins, for a blank with buffer instead of enzyme solution was used.
The enzyme inhibition (%) was calculated from the rate of absorbance change with time (V= Abs/Δt) the calculation as follows.
The enzyme inhibition (%) was calculated from the rate of absorbance change with time (V= Abs/Δt) the calculation as follows.