Screening of dual targeted inhibitors of 5-lipoxygenase and cyclooxygenase-2 from Oroxylum indicum by off-line two-dimensional liquid chromatography coupled with mass spectrometry

Screening and developing dual-target inhibitors of natural 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) as anti-inflammatory drugs with higher efficacy and fewer side effects is becoming increasingly popular. In the current study, an approach by off-line two-dimensional liquid chromatography combined with mass spectrometry (2D-LC-MS) was built and verified, and was used to rapidly screen 5-LOX/COX-2 inhibitors in the extracts of Oroxylum indicum. First, the extracts were incubated with 5-LOX/COX-2 to optimize the incubation conditions. Then, off-line 2D-LC-MS screening was performed on the mixture, and the potential inhibitors were analyzed by comparing the chromatograms of the active group, blank group, and inactivated 5-LOX/COX-2 group. In addition, in vitro enzyme inhibition experiments, LPS-stimulated RAW 264.7 cells experiment and molecular docking technology were employed to confirm the findings. As a result, a total of nine compounds were confirmed as possible 5-LOX/COX-2 inhibitors, and in vitro activity assays showed that oroxin B, baicalein, oroxin A, and chrysin possessed strong inhibitory effects on 5-LOX/COX-2 (IC50 ≤154.6 μM). Meanwhile, the possible mechanism of interaction between ligands and enzyme active sites was further elucidated by molecular docking technology. The results demonstrated that off-line 2D-LC-MS was a fast, efficient, and accurate method for screening and analyzing bioactive compounds from natural products.