Analysis of interaction property of bioactive components in Danggui Buxue Decoction with protein by microdialysis coupled with HPLC–DAD–MS

Interaction of a commonly used combined prescription of Danggui Buxue Decoction (CPDBD) with protein was studied by microdialysis coupled with HPLC–DAD–MS. Nine compounds in CPDBD were unequivocally identified by comparing with their tR, MS data and UV spectra with those of reference compounds, and simultaneously quantified. Microdialysis recoveries and binding degrees of 20 compounds in CPDBD with bovine serum albumin (BSA) were determined. Recoveries of microdialysis sampling ranged from 66.9 to 91.5% with RSD below 3.0%, and the binding degrees of those to BSA ranged from 6.3 to 59.8% (0.3 mM BSA) and from 6.9 to 86.6% (0.6 mM BSA). The results were determined at pH 7.4 and the influence of different pH value was investigated. Furthermore, the binding degrees of eight reference compounds were determined separately under the same conditions, indicating a significant effect of the interaction of compounds with each other on their binding degrees to BSA. By comparing their binding degrees with BSA with those of proven active compounds in CPDBD, i.e. chlorogenic acid (3), ferulic acid (6), ononin (12) and calycosin (16), other five compounds were found to possess potential activities, which were tentatively identified as calycosin-7-O-β-d-glucoside-6-O-malonate (9), senkyunolide I or H (10), formononetin-7-O-β-d-glucoside-6-O-malonate (17), and two unknown compounds.