Precise recognition and isolation of pancreatic lipase inhibitors from Rubus irritans fruits

Abstract

Functional food research is hindered by the complex natural food matrices, which demand accurate, rapid methods for identifying bioactive compounds. Here, we developed an activity-guided online HPLC-pancreatic lipase recognition system to simultaneously recognize and monitor active chromatographic peaks in Rubus irritans fruit extracts. Four compounds, namely cinnamic acid (1), 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (2), 1-hydroxy-2,3,5-trimethoxyxanthone (3), and salvigenin (4), were first isolated from these fruits via medium- and high-pressure liquid chromatography. In vitro assays showed that all inhibited pancreatic lipase, with IC₅₀ values of 444.90 ± 0.19, 142.64 ± 0.93, 168.70 ± 0.64, and 118.84 ± 0.89 μM, respectively. Salvigenin (4) acted via reversible competitive inhibition. Molecular docking revealed the binding modes of the four compounds in the pancreatic lipase active site, with salvigenin exhibiting the lowest binding energy (-7.3 kcal/mol), followed by compounds 1–3 (-5.4, −6.8, and −6.9 kcal/mol). DFT calculations indicated that salvigenin had a smaller HOMO-LUMO gap and a higher dipole moment, suggesting favorable reactivity. Overall, our study's primary contribution is the establishment of an efficient activity-guided online recognition system for precisely recognizing and isolating natural pancreatic lipase inhibitors from edible fruits, with R. irritans fruits and their derived salvigenin identified as promising functional food ingredients.