Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 1212, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2022.123515
Keywords
Equilibrium dialysis; Bovine serum albumin; Flavonoids; Multi-components; Binding constant
Funding
- National Natural Science Foundation of China (NSFC) [21974015, 21705012]
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This study developed a method combining equilibrium dialysis with HPLC and UPLC-MS/MS to study the interactions between multi-components and protein in traditional Chinese medicine (TCM). The experiment showed that the binding affinity of flavones and flavonols with protein was stronger than dihydroflavonoids, and glycoside substitution reduced the binding affinity. Competitive interactions were found among the four flavonoids studied. The method was successfully applied to study the binding of multiple components in Radix Scutellariae (RS) extract with protein.
The therapeutic function of traditional Chinese medicine (TCM) is based on the combination effect of multiple active ingredients. However, the current pharmacological studies mainly focus on the protein binding of the single component from TCM, which is difficult to explain the overall therapeutic mechanism. Thus in this work the equilibrium dialysis method combined with high performance liquid chromatography (HPLC) and ultra -performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to study the interactions between multi-components and protein. Firstly, the binding constants of seven different structural types of flavonoids with bovine serum albumin (BSA) were determined. The results showed that the binding affinity of flavones and flavonols with BSA was stronger than that of dihydroflavonoids, and the substitution of glycosides would reduce the binding affinity with BSA. The results of competitive displacement experiment showed that there existed competitive interactions among the four flavonoids (rutin, luteolin, hesperetin and kaempferol). The binding constants of flavonoids to BSA were significantly changed under the condition of multi -components coexistence. Especially, the binding constant of hesperetin to BSA increased from 9.46 x 104 L/mol to 1.49 x 106 L/mol under the coexistence of rutin. The results of fluorescence spectroscopy showed that the reason for competitive binding was that the four flavonoids were mainly bound to the IIA region of BSA. Finally, the method was successfully applied to study the binding of multiple components in Radix Scutellariae (RS) extract with BSA. Five flavonoids in RS extract were identified by UPLC-MS/MS, they had different degrees of binding to BSA, among which oroxylin A had the strongest binding degree. In conclusion, the equilibrium dialysis was reliable and sufficiently accurate for study of the interaction between multi-components or TCM extract and protein, which can provided a theoretical basis for the scientific explanation of the overall treatment mechanism of TCM.
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