Bioluminescent Assay for MAO-B Activity

MAO-B activity was tested using a two-step bioluminescent MAO-Glo assay (Promega, Milan, Italy) as previously reported [88 (link)]. Briefly, 250 µL of flower, leaf or pseudobulb methanol extract was placed in a speedvac (Heto-Holten, Frederiksborg, Denmark) to remove the solvent, and the dry pellets were solubilized in 250 µL of MAO reaction buffer, comprising 100 mM HEPES (pH 7.5), 5% (v/v) glycerol and 10% (v/v) dimethyl sulfoxide (DMSO). Samples were used undiluted or diluted to 1:5 and 1:10 (v/v) with the MAO reaction buffer. MAO-B inhibition was tested by adding 12.5 µL of MAO buffer containing 4 µM MAO-B substrate and 12.5 µL of the candidate inhibiting solution in 96-well flat-bottom white opaque plates (Thermo Fisher Scientific, Rodano, Italy). The reaction was initiated by adding 25 µL MAO buffer containing 20 µg/mL of MAO-B. The 50-µL reaction was incubated for 1 h at room temperature, then mixed with 50 µL of luciferin detection reagent and incubated for 20 min at room temperature. Luminescence was recorded on an Infinite 200 Pro microplate reader. Three technical replicates were analyzed for each sample. We used 12.5 µL of the irreversible MAO-B inhibitor l-deprenyl/selegiline (Sigma-Aldrich, St Louis, MO, USA) at a concentration of 2.5 µM in MAO reaction buffer as a positive control and 12.5 µL of the MAO reaction buffer as a negative control. Blank samples were also included in the experiment and contained only the MAO reaction buffer without the enzyme.