Orthogonal chemical-biological profiling of bioactive components from Olea europaea L. fruits for mitigating hypoxia-induced cellular injury.

Hypoxic stress, a pathological hallmark of cardiovascular, oncological, and neurodegenerative diseases, underscores the need for safer hypoxia-targeting therapeutics beyond conventional agents with dose-limiting toxicities. The protective effect of polyphenols from Olea europaea L. fruit (OEL-F) against hypoxic injury in PC12 cells was investigated in this study. Furthermore, the main bioactive components responsible for this protective effect were isolated and characterized. An integrated chemometrics approach facilitated the systematic identification of 23 characteristic markers through HPLC analysis of 17 OEL-F extracts. Coupling with CoCl₂-induced hypoxia modelling in PC12 cells (cell viability assessed via MTT assay) and orthogonal biological validation to identify five hypoxia-alleviating bioactive markers precisely. S6 extract demonstrated significantly superior biological activity in the repair of hypoxic injury compared to all other groups (p < 0.01). Bioactivity-directed fractionation led to the identification of luteolin-4'-O-β-D-glucoside and oleuropein as primary bioactive constituents. Molecular docking analysis indicated that these compounds exhibit structural motifs analogous to vadadustat (a clinical PHD2 inhibitor), engaging residues His313/Tyr310 in the substrate pocket. Unlike vadadustat's Fe²⁺-chelating mechanism, these constituents act as potential competitive PHD2 inhibitors via non-chelating interactions at the catalytic site. Molecular docking suggests that OEL-F polyphenols may stabilize HIF-1α through PHD2 inhibition, proposing a novel natural product-driven strategy for safer hypoxia intervention.