Pharmacodynamic Material Basis of the Components of Four Epimedium Species with Activities Against Hepatocellular Carcinoma Based on Biological Target Networks and Multi-Omics Analysis

Aim: Epimedium plants are used in traditional Chinese medicine due to their medicinal properties and can be clinically used for the treatment of liver cancer.Using network pharmacology and HPLC, we identified a key anti-HCC complex, CMPLX (containing icariin and kaempferol), from four Epimedium species. In vitro and in vivo studies demonstrated that CMPLX suppresses HCC proliferation by downregulating p-Akt, p-PI3K, and Bcl-2 expression. Untargeted metabolomics and gut microbiome analysis revealed significant negative correlations between serum levels of lignin/kaempferol derivatives and Escherichia coli abundance. These findings highlight CMPLX as a promising candidate for HCC drug development. Purpose: Investigate the pharmacodynamic material basis of the components of four Epimedium species with activities against hepatocellular carcinoma based on biological target networks and multi-omics analysis. Materials and Methods: We first screened four Epimedium extracts for anti-HCC activity using HepG2 cells. Shared bioactive compounds were identified through network pharmacology and HPLC, defining core target AKT1 and key complex CMPLX (icariin and kaempferol). Molecular docking/dynamics simulations confirmed CMPLX-AKT1 binding. In vitro assays (CCK-8, wound healing, colony formation, Annexin V/PI, Western blot) demonstrated CMPLX inhibits proliferation, migration, and induces apoptosis via PI3K/AKT/Bcl-2 pathway. In vivo validation in H22 tumor-bearing mice showed tumor suppression, corroborated by histology, serum metabolomics and gut microbiota analysis. Results: CMPLX suppressed hepatocellular carcinoma proliferation in vitro and in vivo. Mechanistically, it downregulated p-Akt, p-PI3K, and Bcl-2 expression, inhibiting growth and promoting apoptosis in HepG2 cells. Integrated multi-omics revealed CMPLX treatment elevated flavonoid/kaempferol derivatives while reducing Enterobacteriaceae_A/Escherichia abundance, with Marinifilaceae dominating the gut microbiota. Crucially, lignan/kaempferol derivatives showed significant negative correlation with Escherichia levels. Conclusion: CMPLX demonstrated synergistic anti-HCC efficacy in vitro and in vivo. Multi-omics analysis revealed its modulation of tumor-related pathways and gut microbiota composition, collectively contributing to tumor suppression. Keywords: Epimedium , hepatocellular carcinoma, metabolomics, gut microbiota