An Integrated Workflow for Activity-Associated Constituent Prioritization and Candidate Quality Marker Evaluation in Complex Herbal Medicines

Herbal medicines (HMs) are chemically complex multi-component systems, yet analytical strategies that systematically connect chemical composition, biological activity, target prioritization, and quantitative analysis remain limited. Here, we established an integrated analytical workflow combining ultra-high-performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS), bioactivity-guided fractionation, network pharmacology, molecular docking, surface plasmon resonance (SPR), and quantitative 1H NMR (qHNMR) to investigate Fu’an Granules. UPLC-QTOF-MS/MS provided a broad constituent-annotation profile of the formulation. Bioactivity-guided fractionation using an LPS-stimulated RAW 264.7 macrophage model showed that fractions FA3 and FA4 exhibited relatively stronger inhibition of nitric oxide (NO) production at the tested screening concentration. Corilagin, rutin, and isochlorogenic acid A were subsequently selected as representative activity-associated constituents according to their distribution in the chromatographic fractions and showed reduced NO production without detectable cytotoxicity under the tested conditions. Network pharmacology, protein–protein interaction analysis, pathway enrichment, and molecular docking prioritized EGFR and PTGS2 as candidate binding targets. SPR further demonstrated concentration-dependent interactions between the three representative constituents and both proteins, providing experimental support for ligand-target binding. An exploratory internal-standard-based qHNMR analysis estimated corilagin content at 5.7% (w/w) of the analyzed sugar-depleted extract. Collectively, this study presents an integrated workflow linking chemical profiling, activity-guided constituent prioritization, computational target analysis, biophysical binding assessment, and complementary quantitative analysis. The results support corilagin as a representative activity-associated quantitative constituent for Fu’an Granules while providing a general analytical framework for activity-oriented quality marker evaluation in complex herbal medicines.