Kynuramine

Quercetin, isorhamnetin (3′-O-methyl Quercetin), luteolin and diosmetin (4′-O-methyl luteolin) were purchased from Extrasynthese (Genay Cedex, France). Penicillin (5000 U/mL) and streptomycin (5000 μg/mL) were obtained from Invitrogen (Carlsbad, CA, USA). Serotonin was a product of Nacalai Tesque (Kyoto, Japan). Kynuramine and Dulbecco's modified Eagle's medium (DMEM)/Nutrient F-12 Ham (D 8437) were from Sigma-Aldrich (St. Louis, MO, USA). Cell Proliferation Reagent WST-1 was from Roche Diagnostics (Mannheim, Germany). Other reagents were of the highest grade available from commercial sources.

MAO-A
and MAO-B inhibitory
activities were assayed using the method in our previous report with
slight modification.^{16 (link)} Human recombinant
MAO-A solution (3 μL, M7316, Sigma-Aldrich, St. Louis, MO) or
7 μL of MAO-B solution (M7441, Sigma-Aldrich) was diluted with
1100 μL of potassium phosphate buffer (0.1 M, pH 7.4). Potassium
phosphate buffer (140 μL), 8 μL of kynuramine (final concentration
is 30 μM, Sigma-Aldrich) in potassium phosphate buffer, and
2 μL of a dimethyl sulfoxide (DMSO) inhibitor solution [final
DMSO concentration of 1% (v/v)] were mixed and preincubated at 37
°C for 10 min. Diluted MAO-A or MAO-B solution (50 μL)
was then added to each well. The reaction mixture was further incubated
at 37 °C, and the reaction was stopped after 20 min by the addition
of 75 μL of 2 M NaOH. The product generated by MAO-A or MAO-B,
4-quinolinol, is fluorescent and was measured at Ex 310 nm/Em 400
nm using a microplate reader (SPECTRA MAX M2, Molecular Devices, Tokyo,
Japan). DMSO without the test compound was used as the negative control,
and pargyline (Sigma-Aldrich) was used as a positive control.^{17 (link)} The IC₅₀ values were estimated using
Prism software (version 5.02; GraphPad, San Diego, CA).