Rapid identifying of COX-2 inhibitors from turmeric (Curcuma longa) by bioaffinity ultrafiltration coupled with UPLC-Q Exactive-Orbitrap-MS and zebrafish-based in vivo validation

Turmeric (Curcuma longa), a typical source with recognized anti-inflammatory activity, is one such medicine-food homology source, yet its anti-inflammatory mechanisms and specific component combinations remain unclear. In this study, a net fishing method combining bio-affinity ultrafiltration and ultra-high performance liquid chromatography-mass spectrometry (AUF-LC/MS) was employed and 13 potential COX-2 inhibitors were screened out from C. longa. 5 of them (C1, 17, 20, 22, 25) were accurately isolated and identified. Initially, their IC50 values were measured (IC50 of C1, 17, 20, 22 and 25 is 55.08, 48.26, 29.13, 111.28 and 150.48 μM, respectively), and their downregulation of COX-2 under safe concentrations (400, 40, 120, 50 and 400 μM for C1, 17, 20, 22 and 25, respectively) was confirmed on RAW 264.7 cells. Further, in transgenic zebrafish (Danio rerio), significant anti-inflammatory activity at safe concentrations (15, 3, 1.5, 1.5 and 3 μg/mL for C1, 17, 20, 22 and 25, respectively) were observed in a dose-dependent manner. More importantly, molecular docking analysis further revealed the mode of interaction between them and the key active site residues of COX-2. This study screened out and verified unreported COX-2 ligands, potentially accelerating the discovery of new bioactive compounds in other functional foods.