Tracing novel hemostatic compounds from heating products of total flavonoids in Flos Sophorae by spectrum-effect relationships and column chromatography

FlosSophorae and its processed product have been clinically used to treat hemorrhage. In this study, the total ion chromatographic fingerprints of the heating products of total flavonoids in FlosSophorae were established by high-performance liquid chromatography with tandem mass spectrometry and the hemostatic activities were studied by hemostatic screening tests in vivo. The spectrum-effect relationships between fingerprints and hemostatic activities were investigated using canonical correlation analysis to trace the peaks responsible for the hemostatic effects. The predicted active peaks in fingerprints were isolated by column chromatography and their structures were identified by NMR spectroscopy and mass spectrometry. The hemostatic activities of them were verified by platelet aggregation and procoagulation assays in vitro. Canonical correlation analysis results showed that peak 8 and peak 11 were correlated most closely, thus probably being the main hemostatic compounds. Through column chromatography separation, peak 8 (compound I) and peak 11 (compound II) were obtained with purities of 95.61 and 93.38%, respectively, and were discovered new hemostatic compounds named as huaicarbon A (I) and huaicarbon B (II), respectively. This study provides a universal model to trace the active compounds of other herbs which have bioactivity enhancement after processing by spectrum-effect relationships and column chromatography.