Screening and isolation of cyclooxygenase‐2 inhibitors from Trifolium pratense L. via ultrafiltration, enzyme‐immobilized magnetic beads, semi‐preparative high‐performance liquid chromatography and high‐speed counter‐current chromatography

Abstract

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.

Total Triterpenoid Extraction from Inonotus Obliquus Using Ionic Liquids and Separation of Potential Lactate Dehydrogenase Inhibitors via Ultrafiltration High-Speed Countercurrent Chromatography

“…The mixture was then centrifuged at 10,000× g and 25 °C for 10 min with an ultrafiltration membrane (molecular weight cut-off, 100 kDa) to remove any unbound compounds. Thereafter, 100 μL MeOH: water mixture (50:50, v / v , pH 3.3) was added to the UF membrane and centrifuged at 10,000× g for 10 min (×3) to release the bound ligand [ 29 ]. The LDH inhibition rate was calculated using the equation (Aa−Ab)/Aa × 100%, where Aa and Ab are the absorbances of the blank and the experimental group, respectively [ 30 ].…”

Insights into the molecular mechanisms of Huangqi decoction on liver fibrosis via computational systems pharmacology approaches

“…MOL2 alleviated carbon tetrachloride-induced liver injury partly due to downregulate the expression of pro-inflammatory mediators, including PTGS2 [86]. MOL10 was identified as a PTGS2 inhibitor by using ultrafiltration, enzyme-immobilized magnetic beads, high-performance liquid chromatography, and electrospray-ionization mass spectrometry [87]. In addition, it was reported that MOL10 treatment significantly reduced the overexpression of PTGS2 mRNA [88].…”

Screening and isolation of potential lipoxidase and superoxide dismutase inhibitors from Scutellaria baicalensis Georgi using high‐speed countercurrent chromatography target‐guided by ultrafiltration‐liquid chromatography‐mass spectrometry

“…HSCCC also has high recovery, high efficiency, and easy scale‐up potential. It is an excellent separation technique that has been widely used for the isolation and purification of active compounds from natural products [21].…”