Multi-parametric cellular imaging coupled with multi-component quantitative profiling for screening of hepatotoxic equivalent markers from Psoraleae Fructus

•A strategy was proposed to discover hepatotoxic equivalent markers from Psoraleae Fructus.•Multi-parametric cytotoxic imaging and multi-component quantitative profiling of Psoraleae Fructus were conducted.•Hepatotoxic equivalent markers were screened out and further verified in vitro and in vivo.•The potential hepatotoxic mechanisms of Psoraleae Fructus were related to oxidative stress, mitochondrial dysfunction and cellular apoptosis. The hepatotoxicity of Chinese herbal medicine (CHM) is an important reason for its restrictive application. Psoraleae Fructus (PF), a commonly used CHM for treatment of osteoporosis and vitiligo etc., has caused serious concern due to the frequent occurrence of liver injury incidents. To date, its hepatotoxic equivalent markers (HEMs) and potential mechanisms are still unclear. To discover and validate the HEMs of PF and further explore the potential mechanisms of hepatotoxicity. Multi-parametric cellular imaging was performed by high content screening, and multi-component quantitative profiling was conducted by ultra-high performance liquid chromatography coupled with triple-quadrupole mass spectrometry. The correlations between hepatotoxic features and component contents were modeled by chemometrics including partial least square regression, back propagation-artificial neural network, and hierarchical cluster analysis. Then the candidate HEMs of PF were screened out and subjected to hepatotoxic equivalence assessment in primary hepatocytes, zebrafish, and mice, and the hepatotoxic mechanisms of PF were investigated. The chemical combination of psoralen and isopsoralen was discovered as the HEMs of PF through pre-screening and verifying process. PF was demonstrated to induce oxidative stress, mitochondrial dysfunction and cellular apoptosis. This study not only provides a rational strategy for screening HEMs from CHMs like PF, but also contributes to understanding the underlying mechanisms of PF hepatotoxicity. [Display omitted]