Screening and Validation of In Vivo Active Metabolites of Erianin Based on Functional Metabolomics

This study aims to systematically screen the in vivo‑formed bioactive metabolites of erianin and validate their anti‑breast‑carcinoma activities, so as to furnish experimental evidence for elucidating the material basis responsible for the in vivo pharmacological efficacy of erianin. Mice were intravenously injected with erianin (4 mg/kg), and serum samples were collected 1 h post-administration. Non-targeted metabolomics analysis was performed using UPLC-Q-TOF-MS (ultra-performance liquid chromatography coupled with quadrupole time-offlight mass spectrometry). PCA (principal component analysis) and OPLS-DA (orthogonal partial least squares discriminant analysis) were employed to screen differential metabolites, and KEGG pathway enrichment analysis was conducted to identify perturbed biological processes. Targets of differential metabolites were predicted using TCMSP, ChEMBL and STITCH databases, and breast cancer-related targets were retrieved from GeneCards, OMIM and TTD databases. Intersection analysis was performed to obtain common targets, followed by PPI (proteinprotein interaction) network construction and core target identification. Candidate metabolites were selected basedon “metabolite-target-pathway” network analysis, and their effects on proliferation, migration and EMT (epithelial mesenchymal transition) of triple-negative breast cancer MDA-MB-468 and MDA-MB-231 cells were validated using CCK-8 assay, Transwell assay, wound healing assay and Western blot. Metabolomics analysis identified 226 differential metabolites, which were significantly enriched in amino acid metabolism, ABC transporters and protein digestion and absorption pathways. Network pharmacology analysis yielded 209 common targets of metabolites and breast cancer, with core targets including p53, EGFR and STAT3, et al. Seven candidate metabolites were selected through “metabolite-target-pathway” network analysis. In vitro functional validation demonstrated that lusianthridin exerted dose-dependent inhibitory effects on proliferation of both breast cancer cell lines, significantly suppressed cell migration, down-regulated Fibronectin and Snail expression, and up-regulated E-cadherin levels. The remaining six candidate metabolites showed no significant activity or required excessively high effective concentrations. This study successfully employed a functional metabolomics strategy, identified lusianthridin as an in vivo metabolite of erianin from serum samples after in vivo administration, and further validated that lusianthridin exerts potent anti-breast cancer effects. Although the in vitro activity of lusianthridin was lower than that of the parent compound erianin, it retained a consistent anti-tumor functional profile, suggesting that the core pharmacophore of erianin is preserved during metabolism. This study provides clues for structure-activity relationship research of erianin and suggests the feasibility of functional metabolomics in discovering bioactive metabolites of natural products.

CSTR: 32200.14.cjcb.2026.09.0003