Single‐step screening and isolation of potential lipoxidase inhibitors from Trifolium repens by stepwise flow rate high‐speed countercurrent chromatography and semipreparative high‐performance liquid chromatography target‐guided by ultrafiltration‐LC‐MS

An efficient method based on ultrafiltration high-performance liquid chromatography coupled with photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and identified of the ligands for activated from the extract of Trifolium repens L. Five major compounds, namely ononin, daidzein, genistein, formononetin, and biochanin A, were identified as potentially effective inhibitors. Subsequently, the specific binding ligands were separated by stepwise flow rate high-speed countercurrent chromatography and semipreparative high-performance liquid chromatography. This is the first report that T. repens extracts contain potent lipoxidase inhibitors. In summary, we systematically studied the active components in T. repens, evaluated their activity, separated and purified them, and identified their structure. This method is simple, fast, and efficient. It is suitable for the separation and purification of active compounds in T. repens, and provides a theoretical basis and technical platform for the development of natural medicines.