Bche from equine serum

Recombinant MAO-A and MAO-B were used in MAO inhibitory activity assays using kynuramine (0.06 mM) and benzylamine (0.3 mM) as substrates, respectively [23 (link)]. The MAO-A and MAO-B reactions were carried out at 25 °C and monitored for 30 min at 316 and 250 nm, respectively, in 0.5 mL mixture of 50 mM sodium phosphate (pH 7.2). Clorgyline and toloxatone were the drug references used for MAO-A, and pargyline and lazabemide were used for MAO-B as reference compounds. The IC₅₀ values were determined by measuring the residual activity at different concentrations of the compounds and by using GraphPad Prism software 5.0. The K_m value of benzylamine against MAO-B was measured to be 0.40 mM [24 (link)]. On the other hand, multitarget analysis, ChE (AChE, BChE) inhibitory activity assays were carried out at 25 °C and monitored for 15 min at 412 nm in a 0.5 mL mixture of 100 mM sodium phosphate (pH 7.5). BACE1 inhibitory activities were tested by the BACE1 activity detection kit. The assay method was described previously [25 (link)]. Recombinant human MAO-A and MAO-B, AChE from Electrophorus electricus, BChE from equine serum, BACE1, and their substrates and reference inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The cholinesterase inhibitory activities of the extracts and fompounds 1–6 were performed as previously described [42 (link)]. AChE from Electrophorus electricus and BChE from equine serum were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). An amount of 0.1 M of sodium phosphate buffer (pH 8) was added to a 96-well microplate followed by the sample and 1U of the AChE or BChE. Then, 10 mM of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was added followed by 14 mM of acetylthiocholine iodide or S-butyrylthiocholine chloride. The final concentration of DMSO was 1%. Galanthamine was used as the standard for validation and result comparison. The absorbance was measured using a Tecan Infinite 200 Pro Microplate Spectrometer at 412 nm for 30 min.