Untargeted metabolomics and antioxidant responses reveal tissue- and species-specific metabolic profiles in three Epimedium species

Objectives This study systematically analyzed the antioxidant responses, untargeted metabolomic profiles, and secondary metabolic pathways of three representative Epimedium species. Methods Meteorological data (atmospheric pressure, temperature, relative humidity, and precipitation) were recorded from July 2022 to July 2025 to provide environmental background context. Antioxidant indexes including H 2 O 2 and O 2 - levels, as well as CAT, POD and SOD activities in different plant tissues were determined. Untargeted metabolomics was performed to explore metabolic differences. PCA, differential metabolite analysis, KEGG and MSEA enrichment were conducted to screen key metabolic pathways, and correlation analysis was employed to explore exploratory associations between antioxidant enzyme activities and secondary metabolite abundances. Results The levels of H 2 O 2 and O 2 - were generally higher in roots than in stems and leaves. CAT activity showed no significant differences among tissues or species, whereas POD and SOD activities presented distinct tissue-specific and species-specific patterns. A total of 2730 MS features were detected. PCA and differential metabolite analysis revealed remarkable metabolic separation and diverse metabolite changes among the three Epimedium species. KEGG and MSEA enrichment indicated that differential metabolites were mainly involved in phenylpropanoid biosynthesis, flavonoid biosynthesis, galactose metabolism and so on. 46 MS features were putatively annotated at the compound level (MSI Level 2 or 3) via MS/MS fragment interpretation and literature evidence. Correlation analysis was performed to explore the associations between antioxidant enzymes and key secondary metabolites. Conclusions The three Epimedium species exhibit distinct antioxidant characteristics and metabolic profiles under the local meteorological environment. Tissue-specific and species-specific differences are mainly concentrated in phenylpropanoid and flavonoid metabolism, with potential associations observed between these metabolites and the antioxidant system. These results provide descriptive insights into the physiological characteristics and quality formation of Epimedium species.