Rapid screening and purification of potential inhibitors from Medicago sativa by ultrafiltration‐liquid chromatography combined with stepwise flow rate counter‐current chromatography

Abstract

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.

In‐depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.‐based receptor–ligand affinity coupled with complex chromatography

“…The principle of UF‐LC–MS screening is that ligands bound to their macromolecular receptors cannot pass through the ultrafiltered membrane 32 . However, destabilising conditions, such as the presence of an organic solvent, facilitate the release of bound ligands from the receptor, and the released ligands pass through the UF membrane 33 .…”

“…The principle of UF-LC-MS screening is that ligands bound to their macromolecular receptors cannot pass through the ultrafiltered membrane. 32 However, destabilising conditions, such as the presence of an organic solvent, facilitate the release of bound ligands from the receptor, and the released ligands pass through the UF membrane. 33 Specifically, components bound to receptors can be isolated and analysed by UF-LC-MS. On the basis of this principle, it has been widely used to screen specific active compounds from complex chemical compounds.…”

Efficient and fast screening and separation based on computer‐aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum

“…For certain mixtures with complex compositions, especially for the separation of triterpenoids, commonly used techniques include column chromatography, semi-preparative high-performance liquid chromatography (semi-preparative HPLC), and high-speed countercurrent chromatography (HSCCC), among others. 23 HSCCC and semi-preparative HPLC are widely used in the modern separation field due to their advantages of high automation and high compound purity. [24][25][26] Compared to conventional liquid-solid separation methods, HSCCC is a unique liquid-liquid partition chromatography method in which a solid support is not used.…”

Genistein—Opportunities Related to an Interesting Molecule of Natural Origin

“…Genistein is an isoflavone, isolated for the first time in 1899, from belonging to the Fabaceae family Genista tinctoria L. [ 1 ]. Since then, its presence has been confirmed in many other plants of this family, such as Medicago sativa L. [ 2 ], Lupinus albus L. [ 3 ], Apios americana Medik [ 4 ] and Pueraria montana (Lour.) Merr [ 5 ].…”