Selegiline

Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, hMAO-A, hMAO-B, H₂O₂, tyramine hydrochloride, selegiline and moclobemide were purchased from Sigma-Aldrich (Steinheim, Germany) and retained under the suggested conditions by supplier. All pipetting processes were performed using a Biotek Precision XS robotic system (BioTek Instruments, Winooski, VT, USA). Measurements were carried out by a BioTek-Synergy H1 microplate reader based on the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.
In the enzymatic assay, three different daily prepared solutions were used. (I) Inhibitor solutions: Synthesized compounds and reference agents were prepared in 2% DMSO in 10⁻³–10⁻⁹ M concentrations (10 mL for each concentration). (II) Enzyme solutions: Recombinant hMAO-A (0.5 U/mL) and recombinant hMAO-B (0.64 U/mL) enzymes were dissolved in the phosphate buffer and final volumes were adjusted to 10 mL. (III) Working solution: Horseradish peroxidase (200 U/mL, 100 μL), Ampliflu™ Red (20 mM, 200 μL) and tyramine (100 mM, 200 μL) were dissolved in the phosphate buffer and final volume was adjusted to 10 mL.
The solutions of inhibitor (20 μL/well) and hMAO-A (100 μL/well) or hMAO-B (100 μL/well) were added to the flat black bottom 96-well micro test plate, and incubated at 37 °C for 30 min. After this incubation period, the reaction was started by adding a working solution (100 μL/well). The mixture was incubated at 37 °C for 30 min and the fluorescence (Ex/Em = 535/587 nm) was measured at 5 min intervals. Control experiments were carried out simultaneously by replacing the inhibitor solution with 2% DMSO (20 μL). To check the probable inhibitory effect of inhibitors on horseradish peroxidase, a parallel reading was performed by replacing enzyme solutions with 3% H₂O₂ solution (20 mM 100 μL/well). In addition, the possible capacity of the inhibitors to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition was determined by mixing inhibitor and working solutions.
The specific fluorescence emission (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from vials containing all components except the hMAO isoforms, which were replaced by phosphate buffer (100 μL/well). Blank, control and all concentrations of inhibitors were analyzed in quadruplicate and inhibition percent was calculated by using following equation:
$\% \mathrm{Inhibition}=\frac{\left({\mathrm{FC}}_{\mathrm{t}2}-{\mathrm{FC}}_{\mathrm{t}1}\right)-\left({\mathrm{FI}}_{\mathrm{t}2}-{\mathrm{FI}}_{\mathrm{t}1}\right)}{{\mathrm{FC}}_{\mathrm{t}2}-{\mathrm{FC}}_{\mathrm{t}1}}\times 100$
where FCt₂: Fluorescence of a control well measured at t₂ time, FCt₁: Fluorescence of a control well measured at t₁ time, FIt₂: Fluorescence of an inhibitor well measured at t₂ time, FIt₁: Fluorescence of an inhibitor well measured at t₁ time. The IC₅₀ values were calculated from a dose-response curve obtained by plotting the percentage inhibition versus the log concentration with the use of GraphPad “PRISM” software (version 5.0, GraphPad Software Inc., La Jolla, CA, USA). The results were displayed as mean ± standard deviation (SD).

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.

All animal experiments were approved by the Institutional Animal Care and Use Committee (Approval No. KA2018-18) of the Korea Advanced Institute of Science and Technology (KAIST, Republic of Korea). Mice were maintained on a standard 12-h light-dark cycle in a specific-pathogen-free facility at KAIST. Wild-type male mice aged 8 to 10 weeks with a C57BL/6J background were fed a Lieber-DeCarli ethanol diet (#710260, Dyets Inc.) containing isocaloric maltose-dextrin (Pair) or 5% EtOH for 2 to 8 weeks. C57BL/6J background Adrb1/2 double knockout (DKO) and Alb-Cre mice were purchased from Jackson Laboratories (Stock Nos. 003810 and 003574, respectively). C57BL/6N background Gdf15^f/f and Cyp2e1^f/f mice were purchased from the EMMA mouse repository (Stock Nos. EM10460 and EM06364, respectively). Flippase mice were provided by the Korea Research Institute of Bioscience and Biotechnology Laboratory Animal Resource Center (Stock No. PX00003369). In all experiments with genetically modified mice, littermates were used as controls. Body weight changes and diet intake were measured daily. For pharmacological inhibition of monoamine oxidases, mice were treated with selegiline (Sigma‒Aldrich; 10 mg kg⁻¹ body weight, i.p., every other day) for the last 2 weeks of the experiments. All interventions were performed during the light cycle.

The ability of the extracts to inhibit hMAO was determined according to Košak and coworkers [108 (link)]. Each extract was prepared as a solution so that the final concentration in the reaction tube was 1 mg/mL. Briefly, 100 μL of 50 mM sodium phosphate buffer, pH 7.4, 0.05% (v/v) Triton X-114 containing either extracts or reference inhibitors (pargylin, clorgyline, isatin, and L-deprenyl) and hMAO-A or hMAO-B were incubated for 15 min at 37 °C in black 96-well flat-bottomed microplates placed. After 15 min of preincubation, the reaction was started by adding p-tyramine, Amplex Red, and horseradish peroxidase (HRP) (1 mM, 250 μM, and 2 U/mL, respectively, in a final volume of 200 μL) and different extract concentrations or reference inhibitors. Resorufin fluorescent emission was quantified (λex = 530 nm, λem = 590 nm) at 37 °C, for 30 min where fluorescence increased linearly. Ethanol:water (1:1) was used as a control. To determine the blank (b), the enzyme solution was replaced by buffer solution. Initial velocities were calculated from the trends obtained, with each measurement performed in duplicate. Extract ability to inhibit MAOs was expressed as the percentage of inhibition according to the following equation: $$Inhibition (\%)=100-\frac{V_i-b}{V_o-b}\times 100$$
V_i = velocity in the presence of the extracts or reference compounds.
V_o = control velocity in the presence of ethanol:water 1:1.
IC₅₀ was calculated by plotting percent inhibition against inhibitor concentrations, with experimental data fitted to a four-parameter Hill equation using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA).
Extracts showed no interference with the reagents in the medium of the assay.