In vivo sequential metabolism by UPLC/MS, network pharmacology and cell experimentation for screening Q-markers of Penthorum chinense Pursh

PCP (Penthorum chinense Pursh) is a traditional Chinese medicine of Miao nationality, and is used to prevent and treat liver diseases. Modern research shows that it has anti-hepatitis virus, anti-hepatic fibrosis and anti-oxidation effects. It has certain curative effect on cholestatic liver injury (CLI). However, the quality marker (Q-marker) of PCP in the treatment of CLI is still unclear. This study aims to explore and screen the Q-markers of PCP using a novel multi-dimensional strategy of in vivo sequential metabolism, network pharmacology and cell experimentation. Sequential metabolism in rats identified 18 prototypes and 123 metabolites, primarily flavonoids, with hydroxylation, reduction, acetylation, and glucuronidation as the main metabolic pathways. A total of 23 inherent components of PCP were identified, including 18 prototype components exposed in blood and 5 prototypes inferred from reverse tracing of their metabolites. Network pharmacology analysis revealed six potential Q-markers for the treatment of CLI: pinocembrin, apigenin, alpinetin, naringenin, pinocembrin-7-O-β-D-glucoside, and 5-methoxy pinocembroside. The core therapeutic targets were identified as TNF, AKT1, and ESR1. Molecular docking analysis demonstrated that all the six potential Q-markers exhibited binding energies below −6.5 kcal/mol with targets, suggesting their significant role in CLI treatment. Cell experiments indicated that six Q-markers had certain pharmacological activity against CLI. Furthermore, quantification of six Q-markers demonstrated that their content remained relatively stable across 12 batches of Gansu Granules. These findings provide a material basis for the quality control and development of PCP.