Online HPLC-DPPH-Guided Isolation and Free Radical-Scavenging Evaluation of C-4″-Epimeric Quercetin Diglycosides From Saxifraga tangutica.

Activity-guided separation of medicinal plants requires linking activity signals and chromatographic resolution in complex matrices. In this study, based on a previously established HPLC-DPPH platform, medium-pressure prefractionation and preparative chromatographic purification were applied for the targeted isolation of free radical scavengers from Saxifraga tangutica (S. tangutica). Medium-pressure prefractionation reduced sample complexity and enriched active fractions, while Click XION chromatography concentrated the target fraction. Subsequent purification on the ReproSil-Pur C18 AQ column resolved this fraction into two baseline-separated active peaks, affording compounds with purities greater than 95%. The isolated compounds were identified as quercetin-3-O-β-D-glucoside-7-O-β-D-glucoside and quercetin-3-O-β-D-galactoside-7-O-β-D-glucoside, a pair of C-4″-epimeric quercetin 3,7-di-O-glycosides. Both compounds exhibited DPPH radical-scavenging activity, with IC50 values of 68.94 ± 2.77 and 75.59 ± 2.14 μM, respectively. DFT calculations, molecular docking, and ADMET prediction further supported the comparative interpretation of their electronic features, antioxidant-related interaction potential, and predicted pharmacokinetic profiles. Overall, this study achieved the activity guided isolation and activity evaluation of a pair of high-purity C-4″-epimeric flavonol diglycosides from S. tangutica, providing a practical chromatographic strategy for the targeted characterization of structurally similar bioactive constituents from medicinal plants.