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Introduction: Medicago sativa contains flavonoids, saponins, coumarins, sterols, monoterpenes, and organic acids, with flavonoids being the main active constituents. Flavonoids naturally contain a 2‐phenylchromone structure with antioxidant, free radical scavenging, cardiovascular, and trace estrogen‐like effects. Objective: Screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from M. sativa via ultrafiltration‐liquid chromatography‐mass spectrometry (UF‐LC–MS) combined with stepwise flow rate counter‐current chromatography (CCC). Method: Utilising the medicinal plants M. sativa as the research objects and UF‐LC–MS was used for activity screening followed by isolation and purification of the inhibitors by stepwise flow rate CCC. Finally, identification of the three active compounds was achieved by MS and nuclear magnetic resonance. Results: Three major compounds, viz. quercetin, genistein, and formononetin, were identified as potent neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors, respectively. A two‐phase solvent system of ethyl acetate/methanol/n‐butanol/water (5.0:1.5:5.0:10; v/v/v/v) was subsequently selected for separation by stepwise flow rate CCC. Conclusion: This novel approach based on UF‐LC–MS and stepwise flow rate CCC represents a powerful tool for the screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from complex matrices. Therefore, a useful platform for the large‐scale production of bioactive and nutraceutical ingredients was developed herein. An efficient method for the rapid screening and purification of potential NA, LDH, and LOX inhibitors from the M. sativa was established using UF‐LC‐MS combined with stepwise flow rate CCC.Three flavonoid compounds were successfully separated and their structures determined by MS and nuclear magnetic resonance (NMR).To the best of our knowledge, this is the first report of this technique for the screening and isolation of flavonoids from M. sativa [ABSTRACT FROM AUTHOR]

Nonsteroidal anti‐inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase‐2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase‐2 inhibitors can also act through cyclooxygenase‐independent mechanisms. In this study, using ultrafiltration, enzyme‐immobilized magnetic beads, high‐performance liquid chromatography, and electrospray‐ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase‐2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase‐2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography, and high‐speed counter‐current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors. [ABSTRACT FROM AUTHOR]