Mechanistic insights into Licorice-Evodiae Fructus interaction: UPLC-MS/MS-based hepatotoxic metabolite detection and CYP3A4 regulation study

Abstract

Evodiae Fructus (EF) is a hepatotoxic herbal medicine whose toxicity is linked to CYP3A4-mediated metabolic activation of evodiamine (EVD), as identified in our previous study using high-resolution MS platform. In clinic, EF is often combined with Licorice to enhance efficacy and reduce toxicity, but the detoxification mechanism is unknown. In this study, we developed an integrated analytical strategy combining multi-mass spectrometry techniques and biological experiments to investigate the detoxification mechanism of Licorice-EF combination. The changes in EVD serum contents, biochemical indexes and pathological sections in rats were monitored after Licorice administration, and correlation analysis was performed to clarify the detoxification effect of Licorice. In addition, the effect of the major components in Licorice on P450 enzymes and the electrophilic metabolites derived from EVD were investigated by combined application of UPLC-QQQ-MS/MS and UPLC-Q/TOF-MS/MS. Our study showed that the combined use of Licorice and EF in the ratio of 1:1 could significantly ameliorate EF-induced liver injury. The Licorice extract mainly inhibited the activities of CYP3A4, with isoliquiritigenin (ILG) exhibiting the most inhibitory potency with enzyme kinetics parameters k_inact and K_I at 0.034 min⁻¹ and 0.63 μM, respectively. Furthermore, ILG not only significantly alleviated EVD-induced liver injury, but also obviously reduced the production of EVD-derived reactive metabolites. These results suggested that Licorice alleviated EF-induced liver injury by inhibition of metabolic activation of EF mediated by cytochrome P450s, providing stringent and scientific evidence for such combination from perspectives of metabolism-based interaction.