Mao glo kit

The hMAO-A and hMAO-B isozymes, produced in BTI-TN-5B1-4 insect cells containing human cDNA, and their analyzed active units (U), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isozymes stocks were diluted with 1% of 1 M HEPES in Hank’s Balanced Salt Solution (HBSS) (pH 7.4) and aliquots stored at −80°C for single use. Standards of pirlindole, a reversible inhibitor for MAO-A (RIMA), deprenyl (DEP), an irreversible MAO-BI, and cell culture media and supplements were also purchased from Sigma-Aldrich. Different plant dry parts (leaves, stems, roots, petals, barks, resins, or whole herbs) were purchased from and identified by their trades companies including, East Earth Trade Winds (Redding, CA, USA), Mountain Rose, Herbs (Eugene, OR, USA), Mayway Corp. (Oakland, CA, USA), Monterey Bay Spice Comp. (Watsonville, CA, USA). The plants used were not specific to one region. Western blotting equipment and reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA) and BCA Protein Assay Kit from Peirce (Rockford, IL, USA). Amplex™ Red MAO Assay Kit was purchased from Molecular Probes by Life technologies™ (Eugene, OR, USA), and tyramine HCl from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). MAO-Glo™ Kit was purchased from Promega Inc. (Madison, WI, USA).

Hippocampus was collected on ice plate and weighed; and mitochondrial fraction was prepared using the method previously described by Xia et al. [21 (link)]. Briefly, the hippocampus tissue was washed in cold sodium phosphate buffer (PBS; 10 mM, pH 7.4, containing 250 mM sucrose) at 9-fold volume and then resuspended at 100 mg/mL in ice-cold homogenization buffer and homogenized by hand using a small glass homogenizer. Tissue homogenates were then centrifuged at 800 ×g for 5 min at 4°C twice. The supernatant was transferred to a new tube and centrifuged at 12000 ×g for 10 min at 4°C. The pellets were resuspended in the same buffer. Protein concentration was estimated by the BCA method using BSA as the standard. MAO-A activities were measured using a MAO-Glo kit (Promega, USA) according to the manufacturer's instructions. Briefly, an opaque white 96-well plate was prepared and 25 μL of 80 μM MAO-A substrate solution was added to each well. Then the reaction was initiated by adding 25 μL of mitochondrial fraction solution, and the plate was incubated at room temperature for 1 h. The reaction was terminated by adding 50 μL of reconstituted luciferin detection reagent and mixed briefly. Finally, the plate was incubated at room temperature for 20 min, and the luminescence was measured by the luminescence detector (PE, USA).

The potential of the test compounds for human MAO inhibition was evaluated via a chemiluminescence technique using the MAO-Glo kit (Promega, Madison, WI, USA). Detailed experimental conditions and procedures were reported previously [64 (link),65 (link)]. The test compounds were evaluated at a concentration of 6, 30, and 120 µM. Selegiline was used as a positive control. The kinetic analysis of hMAO inhibition was analyzed at different concentrations of hMAO substrate depending on the isozyme (40, 80, and 160 µM for hMAO-A and 4, 8, and 16 µM for hMAO-B) following the same method of enzyme inhibition. The concentrations of the test compounds for the kinetic study are presented in Figure 2 and Figure 3. Kinetic parameters were analyzed using SigmaPlot (v12.0, SPP Inc., Chicago, IL, USA).