Investigating the Material Basis and Mechanisms of Toxicity Reduction in Processing and Compatibility of Euodiae Fructus Based on UPLC‐MS/MS Quantitative Analysis and UHPLC‐Q‐TOF‐MS Metabolomics

ABSTRACT Introduction Determining the hepatotoxic potential of Euodiae Fructus (EF) and exploring the methods and mechanisms of detoxification after processing and compatibility are critical for its rational use. The changes in components and endogenous metabolites after administration might provide a pathway to resolve the above issues. Objective This study aims to investigate whether boiling water washing (BWW), drying after BWW, or compatibility in Wuzhuyu decoction (WZYD) can mitigate the hepatotoxicity of EF, and to explore the underlying mechanisms through chemical composition and metabolomics analysis. Methods The hepatotoxicity of EF, processed EF, and WZYD were evaluated in normal mice, then the hepatotoxicity of WZYD was evaluated in migraine model mice. General physical signs (e.g., weight loss, reduced activity, and dull fur), biochemical markers (e.g., ALT, AST, TBIL, and ALP levels), and histopathological examination were observed. Ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) was used to quantify the change of 14 specific ingredients in EF after processing and compatibility, including alkaloids (e.g., evodiamine, rutaecarpine), phenolic acids (e.g., chlorogenic acid), and flavonoids. Metabolomics based on ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UHPLC‐Q‐TOF‐MS) was employed to identify key pathways, and protein expression in these pathways was assessed to confirm detoxification mechanisms. Results The decocted extract of raw EF (SEF) induced liver injury in normal mice at a human equivalent dose, while the decocted extracts of boiling water‐washed EF (TEF) and dried boiling water‐washed EF (GEF) reduced this injury. WZYD did not induce liver injury in normal and migraine model mice, further supporting its safety profile. Significant differences in chemical composition were observed among SEF, TEF, GEF, and WZYD, including a reduction in phenolic acids (e.g., chlorogenic acid and caffeic acid) and an increase in alkaloids (e.g., evodiamine and rutaecarpine). Metabolomics analysis revealed that both boiling water washing (BWW) processing and WZYD compatibility influenced linoleic acid metabolism, oxidative stress, and inflammation. Specifically, CYP2E1 expression was significantly reduced in the TEF and WZYD groups, accompanied by decreased oxidative markers (MDA) and inflammatory cytokines (TNF‐α and IL‐6). Conclusion In summary, BWW processing and WZYD compatibility reduce EF‐induced hepatotoxicity by modulating linoleic acid metabolism, CYP2E1 activity, oxidative stress, and inflammation. These processes are interconnected and play a central role in the detoxification of EF. By altering the chemical composition of EF, these processes reduce the levels of potential hepatotoxic components such as phenolic acids, while increasing the concentration of hepatoprotective alkaloids like evodiamine and rutaecarpine. These changes, combined with the modulation of key metabolic pathways, provide a scientific basis for the safer clinical use of EF.