Integrated Pharmacology, Correlation Analysis, and Network Pharmacology: A Combined Strategy for Discovering and Validating Antidepressant Q‐Markers of Yueju Wan

Yueju Wan (YJW), a classical Traditional Chinese medicine (TCM) formula documented since the Yuan Dynasty, is renowned for its ability to relieve all types of stagnation and exhibits antidepressant effects. However, the key active components closely associated with its antidepressant efficacy, as well as the pharmacologically validated quality markers (Q-markers) for efficacy-oriented quality control, remain to be systematically identified and validated. This study aimed to identify and validate the antidepressant Q-markers of YJW through an integrated strategy combining chemical profiling, spectrum-effect correlation, network pharmacology, molecular docking, and in vivo pharmacological validation. The 95% ethanol extract demonstrated the strongest antidepressant effect in the reserpine-induced zebrafish depression model, significantly improving zebrafish locomotion, restoring neurotransmitter 5-HT and DA levels, and suppressing inflammatory cytokines IL-6 and TNF-α (p < 0.01). A total of 42 common compounds were identified from different extracts of YJW by UPLC-Q-TOF-MS/MS, of which 14 were prioritized through Pearson bivariate correlation and Gray relational analysis (GRA). Subsequent mechanistic analyses, including pathway enrichment and molecular docking, indicated that these prioritized components were mainly involved in inflammation-related pathways. In vivo validation showed that these 4 compounds alleviated depression-like behaviors, modulated neurotransmitters and cytokines, and downregulated tnfa, il6, and rela expression. HPLC quantification confirmed their presence in YJW, with the validated method demonstrating feasibility. Collectively, these findings establish 4 pharmacologically meaningful Q-markers for YJW, provide a scientific basis for its quality standardization, and contribute to advancing the modernization of TCM-based antidepressant therapy.