Fast Determination of Protein Binding Constants and Sites to Active Flavonoids and Phenolic Acids by Affinity Capillary Electrophoresis and Fluorescence Spectroscopy

The interaction of small molecule drugs with human serum albumin (HSA) after entering the human blood circulation system profoundly affects their distribution and absorption in the body, and further influences the activity. Thus, the in-depth investigation of drug interactions with HSA is of great significance for the optimization of drug candidates, the research and development of new drugs, and the risk assessment and control of drug combinations. Phenolic acids and flavonoids are valuable in antioxidant, antitumor, immunomodulatory studies, and many other fields, which is of great value in clinical medication. Here, the binding behaviors of 12 phenolic acids/flavonoids to HSA under physiological conditions were simultaneously investigated by affinity capillary electrophoresis (ACE) method and fluorescence spectroscopy for the first time. Results showed the binding behaviors of 12 substances were related directly to their structures. Even a little change, such as the amount of phenolic hydroxyl groups and an extra C=C bond, the binding would significantly change. Among them, rosmarinic acid (K_a = 3.880 × 10⁵ M⁻¹, n = 1.074, Sudlow site I) showed the best binding ability. Caffeic acid trended to bind with Sudlow site II, which meant it could cooperate with the other active phenolic acids/flavonoids (like dihydromyricetin and rosmarinic acid) in clinical medication to increase the curative effect. Fluorescence method verified the accuracy of the results determined by ACE. However, it showed some limitations such as fluorescence interference, indicating it was not suitable for some determinations. These provide a new reference for the screening of potential inhibitors for anticancer and antidiabetic agents.