Dichloromethane extract is responsible for the Evodiae Fructus induced hepatotoxicity via PI3K-AKT/MAPK/p53/NF-κB...

Tetradium ruticarpum (A.Juss.) T.G. Hartley (syn. Evodia rutaecarpa (Juss.) Benth., Rutaceae), known as Wuzhuyu in Chinese and Evodiae Fructus (EF) in English, is a traditional Chinese medicine (TCM) with a history of over two thousand years. It was first documented in Shen Nong's Herbal Classic for its efficacy in dispersing cold, relieving pain, and alleviating nausea, vomiting, and diarrhea, leading to its historical use in managing gastrointestinal ailments. However, the clinical application of EF has been limited due to its associated hepatotoxicity. Existing studies have confirmed that evodiamine, rutaecarpine and other alkaloids induce hepatotoxicity via multi-pathways, but the core toxic fraction, multi-component synergistic effects and systematic toxic network of EF remain unclear.

This study aims to identify the key hepatotoxic fraction of Evodiae Fructus (EF) and elucidate its underlying mechanisms.

A high-throughput zebrafish model was used to screen EF extracts with different polar solvents. The hepatotoxicity of the target fraction was validated in mice. UPLC-Q-TOF-MS/MS, integrated network pharmacology, proteomic and transcriptomic analyses were performed in zebrafish and mice to screen toxic pathways, with western blot and qRT-PCR validation.

High-throughput zebrafish screening of five polar extracts of EF identified the dichloromethane extract (DEEF) as the core hepatotoxic fraction. Both zebrafish and mouse models consistently indicated that DEEF induced a dose-dependent elevation of serum ALT, AST, and TBA levels, disrupted the architecture of hepatic tissue, and was accompanied by marked hepatocyte apoptosis. In mice, Masson's trichrome staining further revealed abnormal collagen deposition, along with extensive infiltration of inflammatory cells. UPLC-Q-TOF-MS/MS analysis identified 73 compounds in DEEF, with 48 alkaloids (indole and quinolone types) being the dominant components, in addition to flavonoids, limonoids, and organic acids. Integrated network pharmacology, transcriptomic and proteomic analyses revealed dose-dependent global alterations in hepatic gene and protein expression profiles. Mechanistically, DEEF upregulated bile acids (BAs) synthesis enzymes (CYP7A1, CYP27A1) and downregulated efflux transporters (BSEP, MRP2). These changes were associated with activation of PI3K-AKT/MAPK/p53/NF-κB pathways and modulation of the Bax/Bcl-2/Caspase-3 apoptotic axis, as indicated by multi-omics enrichment and validated by protein and gene expression analyses.

This study is the first to confirm that DEEF represents the core toxic fraction of EF and to disclose that its hepatotoxic mechanism is driven by the crosstalk between BAs metabolism disorder and multi-signaling cascade. Our findings fill the research gap between studies on crude extract and monomers, and provide a critical foundation for targeted detoxification and safe clinical application of EF.