Online HPLC-FLD recognition system drives activity-guided progressive separation to discover pancreatic lipase inhibitors from Sinacalia tangutica

Hyperlipidemia is closely linked to abnormal dietary lipid absorption, and pancreatic lipase (PL) is a key target for inhibiting intestinal lipid hydrolysis. Thus, the efficient discovery of PL inhibitors is crucial for treating the disease. However, existing recognition techniques suffer from false positives and disconnection from subsequent separation, hindering the exploration of natural product active components. This study developed an online high-performance liquid chromatography-fluorescence detection (HPLC-FLD) recognition system. It enables real-time mixing of chromatographic eluents with PL solution, forming protein-ligand complexes that yield characteristic negative peaks due to their lower fluorescence, thus enabling rapid localization of PL-binding components at corresponding retention times. Validation with orlistat as a reference standard confirmed the system's high precision, stability, and specificity. When applied to the crude extract of Sinacalia tangutica , this system recognized 15 active chromatographic peaks in a single analysis. Combined with an activity-guided progressive separation strategy, 16 compounds were obtained and exhibited PL inhibitory activity in vitro enzymatic assays. The therapeutic potential of phlorizin, one of inhibitors identified by our recognition system, was evaluated in hyperlipidemic mice. High-dose phlorizin lowered serum total cholesterol (TC) by 46.3% and triglyceride (TG) by 66.3%, reduced pancreatic PL content by 52.4%, raised fecal TG by 65.7%, alleviated hepatic steatosis and oxidative stress, with efficacy near orlistat. This integrated strategy of online recognition with activity-guided separation provides an efficient solution for the discovery of natural PL inhibitors with therapeutic potential.