Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [<sup>14</sup>C]AA or PGG<sub>2</sub> is used as substrate.

Abstract

<p>The incubation mixtures consisted of 20 µM [<sup>14</sup>C]AA (0.2 µCi) or 10 µM PGG<sub>2</sub> as substrate, COX I or COX II as enzyme (0.5 or 0.97 µg/mL, respectively), 10 mM EDTA, 1 mM reduced glutathione, 1 µM hematin, and myricetin in 200 µL Tris-HCl buffer (100 mM, pH 7.4). The reaction was incubated at 37°C for 5 min and terminated by adding 15 µL of 0.5 N HCl to each test tube. Ethyl acetate (600 µL) was added immediately for extraction. The dried extracts were re-dissolved in acetonitrile or EIA buffer (Cayman Co. Michigan, USA), and the metabolites were analyzed using HPLC (with radioactivity detection) when [<sup>14</sup>C]AA was used as substrate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a> or using an EIA kit when PGG<sub>2</sub> was used as substrate. Note that in this experiment, the COX I and II enzymes with or without aspirin pretreatment were both tested. For aspirin pretreatment, enzymes were pre-incubated with aspirin at 0.5 mM for COX I or 5 mM for COX II for 30 min at room temperature and then were immediately used as the enzyme source in the assay.</p

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