Screening and identification of BSA bound ligands from Puerariae lobata flower by BSA functionalized Fe₃O₄ magnetic nanoparticles coupled with HPLC-MS/MS.

Puerariae lobata flower (Willdenow) has a long history used to treat alcoholic intoxication in China, which contains a series of isoflavones as its chief pharmacologically active constituents. In this study, bovine serum albumin (BSA) functionalized iron oxide magnetic nanoparticles (Fe₃O₄ MNPs) coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were used to screen and identify active compounds from ethanolic extract of P. lobata flower. Thirteen active isoflavones were screened and identified as glycitin (1), tectoridin (2), daidzin (3), 3'-methoxy daidzin (4), ononin (5), 3'-hydroxyl daidzein (6), tectorigenin (7), biochanin A (8), prunetin (9), genistein (10), 3'-methoxy daidzein (11), irisolidone (12) and 5,7-dihydroxy-3',4'-methylenedioxyisoflavone (13), while compounds 4, 6, 9, 11 and 13 were identified from this plant for the first time. Furthermore, the activity of each bound ligand was evaluated on-line. The results indicated that the binding affinities of compounds with BSA were highly dependent on chemical structures and the methoxylation and hydroxylation on B-ring could improve the activity. The effective method could be widely applied for rapid screening and identification of active compounds from complex mixtures.