Analysis of AChE and BChE activities were adapted from the method of Jung et al. (2009) (link), with modifications indicated in Kukreja et al. (2018) . The AChE assay mixture contained 20 ng AChE (100 μL), 0.8 mM acetylthiocholine (40 μL), 16 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) (10 μL), and the extract (50 μL). Enzyme and substrate were prepared in assay buffer (50 mM KPB, pH 7.0) (50 μL), while DTNB was prepared in methanol. Enzyme inhibitory activity was spectrophotometrically measured at a wavelength of 412 nm using the 96-well microplate reader. Inhibitory activities of pepper extracts were calculated as percentage of inhibition using the equation:
where A is an initial velocity of the reaction with enzyme, a is an initial velocity of the reaction without enzyme, B is an initial velocity of the enzyme reaction with extract, and b is an initial velocity of the reaction with extract but without enzyme.
The BChE assay mixture contained 100 ng BChE in 50 mM KPB, pH 7.0; 1 mM MgCl2 (100 μL); 1 mM butyrylthiocholine in 50 mM KPB, pH 7.0 (40 μL); 16 mM DTNB in 50 mM KPB, pH 7.0 (10 μL); and extract (50 μL). Enzyme inhibitory activity was spectrophotometrically measured at a wavelength of 412 nm using the 96-well microplate reader. Inhibitory activities of pepper extracts were calculated as percentage of inhibition as above.
Since inhibitory activities of AChE and BChE were determined utilizing enzyme kinetics, interferences from sample colors can be unconcerned. Inhibitory activities were determined using the rate of yellow color development during the enzyme assay (which becomes more yellow with time). Therefore, even though the samples had strong colors (yellow, green, red, and orange), the yellow color will only develop in the assay with enzyme. Thus, the yellow color measured in the assay only results from the reaction between the substrate and the enzyme. Eserine, a reversible anti-cholinesterase drug, was used as a control inhibitor for both AChE and BChE assays.
where A is an initial velocity of the reaction with enzyme, a is an initial velocity of the reaction without enzyme, B is an initial velocity of the enzyme reaction with extract, and b is an initial velocity of the reaction with extract but without enzyme.
The BChE assay mixture contained 100 ng BChE in 50 mM KPB, pH 7.0; 1 mM MgCl2 (100 μL); 1 mM butyrylthiocholine in 50 mM KPB, pH 7.0 (40 μL); 16 mM DTNB in 50 mM KPB, pH 7.0 (10 μL); and extract (50 μL). Enzyme inhibitory activity was spectrophotometrically measured at a wavelength of 412 nm using the 96-well microplate reader. Inhibitory activities of pepper extracts were calculated as percentage of inhibition as above.
Since inhibitory activities of AChE and BChE were determined utilizing enzyme kinetics, interferences from sample colors can be unconcerned. Inhibitory activities were determined using the rate of yellow color development during the enzyme assay (which becomes more yellow with time). Therefore, even though the samples had strong colors (yellow, green, red, and orange), the yellow color will only develop in the assay with enzyme. Thus, the yellow color measured in the assay only results from the reaction between the substrate and the enzyme. Eserine, a reversible anti-cholinesterase drug, was used as a control inhibitor for both AChE and BChE assays.
