Liu_2026_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1284_125322

BACKGROUND: Translating biological recognition into chromatographic information that can continuously guide targeted isolation remains challenging in natural products. METHODS: HPLC coupled with fluorescence detection (HPLC-FLD) was combined with multimodal preparative chromatography to recognize, track, and isolate candidate pancreatic lipase (PL)-affinity ligands from Saxifraga tangutica. Their effects on PL were evaluated by activity and kinetic assays, with DFT calculations and molecular docking as complementary analyses. RESULTS: Three flavonoid glycosides with chromatographic purities above 95% were isolated: Q3XG (160.8 mg), Q3AG (167.9 mg), and Q3GG (623.1 mg). Q3XG activated PL with an EC50 of 4.78 +/- 0.95 microg/mL, whereas Q3AG and Q3GG inhibited PL with IC50 values of 38.33 +/- 1.97 and 43.86 +/- 2.87 microg/mL, respectively. Kinetic analysis showed that Q3XG decreased the apparent Km and slightly increased the apparent Vmax, whereas Q3AG and Q3GG showed the opposite changes, consistent with mixed-type inhibition. DFT and docking analyses indicated differences in electronic properties and predicted PL-ligand interaction patterns. CONCLUSION: Integration of HPLC-FLD with multimodal chromatography preserved the link between affinity signals and target peaks throughout chromatographic tracking, selectivity optimization, and preparative isolation, providing a practical route to functionally validated ligands from complex natural products.