Human catechol-O-methyltransferase (hCOMT) is considered a therapeutic target due to its crucial roles in the metabolic inactivation of endogenous neurotransmitters and xenobiotic drugs. There are nevertheless few safe and effective COMT inhibitors and there lacks a diversity in structure. To discover novel safe and effective hCOMT inhibitors from herbal products, in this study, 53 herbal products were collected and their inhibitory effects against hCOMT were investigated. Among them, Scutellariae radix (SR) displayed the most potent inhibitory effect on hCOMT with an IC50 value of 0.75 μg mL-1. To further determine specific chemicals as COMT inhibitors, an affinity ultrafiltration coupled with liquid chromatography-mass spectrometry method was developed and successfully applied to identify COMT inhibitors from SR extract. The results demonstrated that scutellarein 2, baicalein 9 and oroxylin A 12 were potent COMT inhibitors, showing a high binding index (>3) and very low IC50 values (32.9 ± 3.43 nM, 37.3 ± 4.32 nM and 18.3 ± 2.96 nM). The results of inhibition kinetics assays and docking simulations showed that compounds 2, 9 and 12 were potent competitive inhibitors against COMT-mediated 3-BTD methylation, and they could stably bind to the active site of COMT. These findings suggested that affinity ultrafiltration allows a rapid identification of natural COMT inhibitors from a complex plant extract matrix. Furthermore, scutellarein 2, baicalein 9 and oroxylin A 12 are potent inhibitors of hCOMT in SR, which could be used as promising lead compounds to develop more efficacious non-nitrocatechol COMT inhibitors for biomedical applications.
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