A Principle-Guided Multistep Workflow for Screening Potential Quality Marker Candidates of Sanqi Shangyao Tablet Based on UHPLC-Q-Orbitrap MS, In Silico Screening, and In Vitro Validation.

Abstract

Quality marker (Q-marker) screening is crucial for the quality control of traditional Chinese medicine (TCM) formulas but remains challenging. In this study, a principle-guided multistep strategy was established for the rational identification of potential Q-marker candidates in Sanqi Shangyao tablet (SST). Ultrahigh-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry was employed for comprehensive chemical profiling, followed by traceability assessment and relative specificity estimation. A total of 124 compounds were characterized, among which 66 were confirmed using authentic standards. After successive filtering based on traceability, relative specificity, and drug-like properties, 25 candidate compounds were retained and associated with 163 osteoarthritis-related targets. Enrichment analysis identified the TNF signaling pathway as a key regulatory axis, and network topology analysis prioritized prunetin for further investigation. In IL-1β-stimulated chondrocytes, prunetin showed no apparent cytotoxicity at concentrations up to 50 μM, significantly reduced nitric oxide production, suppressed pro-inflammatory mediators, and attenuated extracellular matrix degradation. Molecular dynamics simulations provided supportive structural evidence for favorable interactions between prunetin and representative targets, including MAPK14, PTGS2, and AKT1. Based on integrated chemical, biological, and TCM theory considerations, six compounds were proposed as potential Q-marker candidates. This study provides a practical workflow for preliminary Q-marker candidate prioritization in complex herbal formulations.