Rapid Discovery, Guided Isolation, and Mechanism Study of Anti-neuroinflammatory Constituents from Glycosmis craibii var. glabra Targeting 5-LOX

Objective: In this study, we aimed to screen and identify potent 5-lipoxygenase (5-LOX) inhibitors in Glycosmis craibii var. glabra extract and elucidate the mechanisms underlying their inhibitory effects. Materials and Methods: A 95% ethanol extract of G. craibii var. glabra was analyzed using an online ultra-performance liquid chromatography–photodiode array–5-LOX–fluorescence detector (UPLC–PDA–5-LOX–FLD) system. Guided by liquid chromatography–diode array detection/mass spectrometry (LC-DAD/MS) analysis, major peaks were isolated using various chromatographic techniques, and their structures were determined using high-resolution electrospray ionization mass spectrometry (HRESIMS), one-dimensional/two-dimensional nuclear magnetic resonance (1D/2D NMR), and electronic circular dichroism (ECD) data. Molecular docking, surface plasmon resonance (SPR) assays, and enzyme activity assays were performed to evaluate the binding capacity and inhibitory effects of the isolated compounds on 5-LOX. The anti-neuroinflammatory activities and mechanisms of action of these compounds were further examined in lipopolysaccharide (LPS)-stimulated BV-2 cells. Results: Eight main peaks were detected using the online UPLC–PDA–5-LOX–FLD system: one coumarin ( 1 ), one quinoline ( 2 ), three quinolones ( 3, 5, 8 ), and three flavonoids ( 4, 6, 7 ). Among these, compounds 4 and 5 were newly characterized, while compounds 5 and 6 exhibited the strongest binding affinity for 5-LOX. Mechanistic investigations revealed that compounds 5 and 6 suppressed TNF-α and IL-6 expression in LPS-stimulated BV-2 cells in a concentration-dependent manner by modulating the 5-LOX/CysLT 1 R pathway. Conclusions: This study successfully employed an online UPLC–PDA–5-LOX–FLD system to identify two major anti-inflammatory components of G. craibii var. glabra that target 5-LOX and exert their effects by regulating the 5-LOX/CysLT 1 R pathway.