2.2. Monoamine Oxidase Assay, Determination of IC50 and Evaluation of Inhibition Kinetics

MAO inhibition assays were performed using a MAO-Glo™ Assay kit (Promega) according to the manufacturer’s instructions. Briefly, 1 µg of human recombinant MAO-A and MAO-B enzyme isoforms were incubated with 160 and 16 µM of MAO substrates, respectively. The enzyme reactions were performed in the presence or absence of EA or urolithin-A (UA), urolithin-B (UB) or urolithin-C (UC) in a 96-well white plate (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 1 h. The enzyme reaction was quenched by the addition of a luciferin detection reagent. The standard inhibitors of MAO-A (chlorgylline; 1 μM) and MAO-B (deprenyl; 5 μM) were used as positive controls. An equal volume of dimethyl sulfoxide (0.1%) was added to the enzyme assay as a control. After 1 h of incubation, the luminescence was measured with a multimode microplate reader (Synergy HT, BioTek, Winooski, VT, USA) to determine the MAO enzyme activities. Initially, the inhibition potential of all the test compounds was evaluated at a concentration of 100 μM. The control reaction (with no inhibitor) with vehicle (DMSO 0.1%) was set at 100% activity. To determine IC₅₀ of the test compounds, the dose dependent enzyme assays were performed. We used range of 0.1 to 50 μM concentrations for MAO-A and 0.1 to 200 μM concentrations of test compounds for the MAO-B enzyme activities. The type of enzyme inhibition was determined using variable concentrations of MAO-A substrate (10–360 µM) and MAO-B substrate (2–64 µM) for the respective enzyme assay. The enzyme assays were performed in the presence of UA (3, 12 μM), UB (0.4, 2 μM) or UC (15, 60 μM). A Lineweaver–Burk double reciprocal plot was generated, and the mode of inhibition was determined by visual examination of the plot. The slope of the reciprocal plots was plotted against the inhibitor concentrations and determined the x-axis intercept that represented the inhibitor constant, Ki value.