The cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of prostaglandins, its inhibitors are effective for the treatment of inflammation. In this study, an analytical method based on bio-affinity ultrafiltration coupled with ultra performance liquid chromatography and quadrupole-time-of-flight mass spectrometry (BAUF-UPLC-Q-TOF-MS) was established for rapidly screening and identifying COX-2 ligands. Meanwhile, the specificity of the method was verified by denatured COX-2 and inactive compound. Next, the biological activity of ligands screened was proved by enzyme inhibition assay. The preferred binding modes for these COX-2 inhibitors were then determined by molecular docking. Finally, network pharmacology was used to explore the pathways involved anti-inflammatory effects. As a result, five COX-2 inhibitors were selected in the extract of Andrographis Herba (AH), including andrographolide (1), 14-deoxy-11,12-didehydroandrographiside (2), andrographidine E (3), andrographidine D (4), and deoxyandrographolide (5). Among them, compounds 2, 3, 4 and 5 were reported to have COX-2 inhibitory activity for the first time. The result of COX-2 inhibition assay showed that compound 3 had an IC50 of 19 μM, compounds 2 and 5 had an IC50 of >200 μM. And each ligand could bind to multiple amino acid residues of COX-2 based on molecular docking. At last, combined with network pharmacology, these ligands could exert anti-inflammatory effects through three pathways related to COX-2, arachidonic acid metabolism, synthesis of prostaglandins, and prostanoid ligand receptors. The method established in the study could be used to rapidly screen other enzyme inhibitors in complex mixtures, and help to understand the mechanism of action.
Keywords: Andrographis paniculata; BAUF-UPLC-Q-TOF-MS; COX-2 inhibitor; Inflammation.
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