2.2. Human Monoamine Oxidase Inhibition Assay

The inhibitory activities on hMAO-A and hMAO-B by three tanshinones: 1, 2, and 3 were evaluated by using a MAO-Glo^TM chemiluminescent assay kit (Promega, Madison, WI, USA). The experimental procedures for this experiment were as described earlier [30,31]. Briefly, 12.5 µL of the test compound or l-deprenyl/clorgyline.HCl was added to a 12.5-µL aliquot of beetle luciferin derivative substrate (the initial concentrations of hMAO-A and hMAO-B were 160 µM and 16 µM, respectively) in each well of a 96-well plate. An enzyme solution (25 µL) was then added to the test samples to initiate the reaction. After an hour of incubation at 25 °C, a reconstituted luciferin detection reagent (50 µL) was added to each well to stop the reaction and produce a luminescent signal. The final mixture was incubated for an additional 20 min at 25 °C. Then, the luminescence was recorded on a FilterMax F5 Multi-Mode microplate reader (Molecular Devices LLC, San Jose, CA, USA). The amount of luminescence was directly proportional to the residual activity of the hMAO isoenzymes.