Discovery of Pancreatic Lipase Inhibitory and Antimicrobial Constituents in Crataegi Folium by High-Performance Thin-Layer Chromatography-Bioautography-Mass Spectrometry.

Crataegi Folium (CF), a traditional Chinese medicine widely used for promoting blood circulation and regulating lipid metabolism, contains diverse bioactive constituents with potential health-promoting effects. However, the rapid localization and identification of its functional compounds remain challenging due to the complexity of its chemical constituents. This study aimed to establish an integrated approach using high-performance thin-layer chromatography (HPTLC) coupled with bioactivity assay and mass spectrometry (MS) for the rapid screening and identification of pancreatic lipase inhibitory and antibacterial constituents in CF. Two HPTLC developing systems with different polarity ranges were optimized to comprehensively separate the chemical constituents, enabling effect-directed analysis. The results revealed that the pancreatic lipase inhibitory activity originated primarily from low-polarity triterpenoids. One active zone, consisting of co-migrating isomers, was successfully separated by in-situ iodine derivatization and characterized as maslinic acid and corosolic acid through comparison with reference standards. Another active zone was identified as arjunolic acid by using the exclusion method. Based on the Vibrio fischeri bioassay, six observed V. fischeri bioluminescence-inhibitory zones were mainly associated with high-polarity compounds, among which five were identified as isoquercitrin, hyperoside, chlorogenic acid, vitexin-2''-O-rhamnoside, and vitexin-4''-O-glucoside. Our findings provide new insights into bioactive constituents of CF, and demonstrate that HPTLC-bioautography-MS is an efficient and integrated platform for the rapid discovery and identification of markers for quality assessment of bioactive compounds in complex botanical matrices.