2.7. Monoamine Oxidase B Activity Assay

The MAO-B activity was measured spectrophotometrically and is based on the formation of H₂O₂ from the conversion of benzylamine to benzaldehyde. The formation of H₂O₂ is detected by the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in its presence. The substantia nigra of the mice was homogenized in a 1 M monosodium phosphate buffer at pH 7.2 and centrifuged at 10,000 × g. To perform the kinetics, each well of a 96-well plate contained 20 μL of 1 M sodium phosphate buffer, 20 μL of 50 mM benzylamine, 20 μL of sample, and 40 μL of water. A control well with the sample without benzylamine and a control well without sample were also included. The samples were allowed to incubate for 3 min and were placed in a BioTek plate reader to take readings every 3 minutes for 30 minutes at a wavelength of 560 nm. At least seven animals per group were used for statistical analysis.