4.4. Monoamine Oxidase A and B (MAO-A and -B) Enzyme Inhibition Assay

A monoamine oxidase assay was carried out using a fluorimetric method as previously reported [40], in which the oxidation of kynuramine by monoamine oxidase led to the production of 8-hydroxychinoline, a compound which becomes fluorescent in alkaline conditions. The 250 µL reaction mixture was prepared in a 50 mM potassium phosphate buffer, pH 7.1, and contained 40 µM kynuramine in the absence or presence of the indicated concentration of the AFPE. The reaction was started by adding 3.75 µg monoamine oxidase A or B and allowed to proceed for 20 min. The enzymatic oxidation of the substrate was stopped by adding 150 µL of 2 M NaOH, and after 10 min incubation at room temperature, 240 µL of water. The resulting mixture was centrifuged for 10 min at 15,000 rpm and the fluorescence was read on 500 µL supernatant using a Cary Eclipse Spectrofluorimeter (Varian). The fluorescence signal was recorded at room temperature (20–25 °C) using an excitation and emission wavelength of 315 and 380 nm, respectively; slits were set to 10 nm, both for the excitation and the emission beam. The residual activity was referred to that measured in the absence of AFPE, and the data were collected in three different experiments. The concentration leading to 50% residual activity (IC₅₀) was derived from a semilogarithmic plot in which the logarithm of the residual activity was plotted against AFPE concentration.