4.11. Human Monoamine Oxidase (MAO)-A and MAO-B Inhibitory Activity Assay

The recombinant human MAO-A and MAO-B (Sigma-Aldrich, St. Louis, MO, USA) inhibitory activity assays of the D. alatus leaf extract were performed according to the previously described procedure [41]. Briefly, 100 mg of the crude extract was dissolved in 400 µL DMSO to prepare 250 mg/mL as a stock solution. Serial dilution of test samples (20 µL) for each concentration was used to test the activity. Concentration of MAOs was 0.0075 mg/mL for MAO-A inhibitor test, 9 µL of kynuramine (Sigma-Aldrich, St. Louis, MO, USA) was mixed with 469.5 µL of potassium phosphate buffer (100 mM, pH 7.4, made isotonic with KCl, 20.2 mM). For MAO-B inhibitor test, 6 µL of kynuramine was mixed with 472.5 µL of potassium phosphate buffer (pH 7.4). Then 1.5 µL of the enzymes were added before incubation at 37 °C for 20 min. The reaction was stopped by adding 400 µL of 2N NaOH and 1000 µL of water. Measurement of 4-hydroxyquinoline (Sigma-Aldrich, St. Louis, MO, USA) was carried out by fluorescence spectrophotometry with the excitation wavelength at 310 nm and the emission wavelength at 400 nm. Sigmoidal dose-response curves were constructed by plotting the initial rates of MAO-catalyzed kynuramine oxidation versus the logarithm of the inhibitor concentrations and expressed as IC₅₀ values which were determined in duplicate and expressed as mean ± standard deviation (SD). Data were analyzed by Prism 5 software package (GraphPad Software, version 5, San Diego, CA, USA). The IC₅₀ values were converted to the corresponding Ki values according to the equation Ki = IC₅₀/(1 + [S]/Km). Clorgyline (Sigma-Aldrich, St. Louis, MO, USA) and deprenyl (Sigma-Aldrich, St. Louis, MO, USA) were used as control for selective MAO-A and MAO-B inhibitors, respectively.