Rosmarinic acid, the key dietary antioxidant in Rosmarinus officinalis L. identified by integrated screening, targets PPARγ to activate Nrf2/NQO1 axis and mitigate oxidative stress

Although Rosmarinus officinalis L. (rosemary) is widely consumed as a prominent source of natural antioxidants, its specific bioactive constituents and precise molecular targets against oxidative stress remain elusive. This study aimed to identify the core functional ingredients of rosemary and elucidate their underlying protective mechanisms. Through an integrated screening strategy combining untargeted metabolomics, spectrum-effect analysis, and a component knock-out method, rosmarinic acid (RA) was identified as the pivotal dietary antioxidant. The efficacy of RA was validated in hydrogen peroxide (H₂O₂)-challenged HepG2 cells and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-challenged zebrafish models, where RA effectively mitigated oxidative damage by suppressing reactive oxygen species (ROS) accumulation and apoptosis. Mechanistically, RA upregulated peroxisome proliferator-activated receptor gamma (PPARγ) expression and activated downstream nuclear factor erythroid 2-related factor 2 (Nrf2)/NAD(P)H quinone dehydrogenase 1 (NQO1) pathway. Concurrently, RA attenuated mitogen-activated protein kinase 1 (MAPK1) and signal transducer and activator of transcription 1 (STAT1) phosphorylation and inhibited cleaved-Caspase-3 activation. Crucially, molecular docking (binding affinity: −9.038 kcal/mol) and cellular thermal shift assay (CETSA) confirmed the direct physical binding of RA to PPARγ. In conclusion, RA is the core bioactive compound in rosemary that exerts potent cytoprotective effects by directly targeting PPARγ to activate the antioxidant Nrf2/NQO1 axis. These findings provide a robust scientific basis for utilizing rosemary and RA as functional food ingredients to combat oxidative stress-related pathologies.