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

ABSTRACT 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 IC 50 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.