Spectroscopic Studies On The Interactions Of Insulin With Active Components In Traditional Chinese Medicine For The Treatment Of Diabetes Complications And Influence Of Glucose On The Binding

Insulin plays important roles in blood glucose, and influences lipid metabolism, protein degradation, protein synthesis, and growth, which is a polypeptide hormones secreted by pancreatic β cell. It often combined with diabetes drugs in the treatment of type Ⅱ diabetes. Active components of traditional Chinese medicine injection for the treatment of diabetes complications: TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Ferulic Acid, Hydroxysafflor yellow A. Insulin often combined with these drugs, drugs will generally bind to insulin with varying degrees, which will affect the absorbance, metabolism, efficiency, pharmacological effects of drugs and insulin. The knowledge on the interaction between drug and insulin will be of great significance on pharmacodynamics, pharmacokinetics and pharmacology. Glucose is necessary nutritions of body supplied everyday and common intravenous infusion solution. The studies about the influence of glucose on the binding have practical significance, further clinical pharmacology studies and provide theoretical basis for assessing the efficacy of insulin and glucose in blood glucose or venous infusion solution.Objective: We study the spectroscopy characteristics to speculate change of interactions of drugs with insulin and discuss the quenching mechanism, binding constants, Hill coefficient, and the binding forces. We calculate the the change of conformation, content of secondary structure, content of bioactive insulin and investigate the influence of glucose on the interactions.Methods: The interactions of active components in Chinese medicine injection for the treatment of diabetes complications(TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Ferulic Acid, Hydroxysafflor yellow A) with insulin under simulating physiological p H condition were studies by fluorescence spectra, ultraviolet spectroscopy and ATR-FTIR spectra at different temperatures(298K and 310K), respectively. Quenching mechanism, binding constants(Ka) and Hill coefficient(nH) were obtained from the calculated results. The dominant binding forces were estimated according to thermodynamic parameters. We calculate the change conformation and the content of secondary structure of insulin via synchronous fluorescence spectra, three-dimensional and ATR infrared spectrometry. The contents of bioactive insulin were measured from Insulin ELISA kit. The effect of glucose on the interactions between drugs and insulin, binding constants and Hill coefficient were obtained at the same time, and calculate the change conformation and the content of secondary structure of insulin.Results: The interactions of TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Ferulic Acid, Hydroxysafflor yellow A and insulin resulted in the quenching of the intrinsic fluorescence of insulin. The quenching constants of TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Hydroxysafflor yellow A decreased with the increasing temperature, while Ferulic Acid increased with the increasing temperature. According to thermodynamic equations, the enthalpy change(ΔH) and entropy change(ΔS) were derived to be negative values for the interactions of TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Hydroxysafflor yellow A with insulin, and the positive ΔH and positive ΔS for Ferulic Acid with insulin. Synchronous fluorescence spectra and three-dimensional fluorescence spectra indicated that the drugs binding to insulin induced some change in the intensity and position of fluorescent peak. ATR-FTIR spectra indicated the content of secondary structure of insulin changed after binding with drugs. Insulin ELISA kit indicated that the content of activity insulin decreased with the increasing concentration of drugs, the greater changes of α-helices content, the lower of bioactive insulin. The presence of glucose increased binding constants and the Hill coefficient of TanshinoneⅠ, Salvianolic acid A, and Ferulic Acid, and decreased of TanshinoneⅡA, Salvianolic acid B and Hydroxysafflor yellow. Meanwhile, glucose influenced the intensity and position of fluorescent peak of three-dimensional fluorescence spectra, and lead to the change of secondary structure since the change of ATR-FTIR spectra.Conclusion: TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Hydroxysafflor yellow A could quench the intrinsic fluorescence of insulin via static quenching, while Ferulic Acid via dynamic quenching caused by the diffusion and collision. Vander Waals force and hydrogen bond play the major role in the binding of TanshinoneⅠ, TanshinoneⅡA, Salvianolic acid A, Salvianolic acid B, Hydroxysafflor yellow A with insulin, and hydrophobic interaction was the dominant intermolecular forces in the binding of Ferulic Acid with insulin. Synchronous fluorescence spectra and three-dimensional fluorescence spectra revealed that the interactions of TanshinoneⅠ, TanshinoneⅡA, Hydroxysafflor yellow A, Ferulic Acid with insulin influenced the micro-environment of amino acid residues and induced the changes in the conformation of insulin. ATR-FTIR spectra indicated that the content of secondary structure of insulin changed by all these drugs. The contents of α-helices and β-sheets could be changed by TanshinoneⅠ, TanshinoneⅡA, Hydroxysafflor yellow A, Ferulic Acid, thus influenced the active binding sites of insulin. Insulin ELISA kit indicated that the greater change of α-helices content, the lower of bioactive insulin. The physiological function of insulin can be influenced when the structure of active parts of insulin changed. In addition, the presence of glucose could increase the binding capacity between TanshinoneⅠ, Salvianolic acid A, Ferulic Acid and insulin, and decrease the binding capacity between TanshinoneⅡA, Salvianolic acid B, Hydroxysafflor yellow A and insulin. The hydrophobicity of micro-environment of insulin tyrosine residues were lower after TanshinoneⅠ, TanshinoneⅡA, Hydroxysafflor yellow A binding with insulin in the presence of glucose, and higher after Salvianolic acid A, Salvianolic acid B, Ferulic Acid binding with insulin. Compared with absence of glucose, the α-helices content of insulin were changed, which influenced the physiological function of insulin.