Studies On The Interactions Of Flavonoids With Trypsin And Influence Of Glucose And Linoleic Acid On The Binding

Trypsin is an important proteolytic enzyme in human and animal small intestinal, it is excreted by the pancreas and takes part in digestive deconstruction of food proteins and other biological processes. Flavonoids are a broad class of compounds in the nature which exist widely in the medicinal plants, vegetables and fruits and exhibit various pharmacological and physiological activities such as anti-microbial, anti-inflammatory, anticancer, antivirus, antioxidant and protect the cardiovascular system. The application of flavonoids is widely concerned because of its wide range of biological activities. If flavonoids enter into gastrointestinal via oral, it will must be get in touch with Trypsin in intestinal.Therefore, studies on the interactions between flavonoids and Trypsin at molecular level have an important meaning for exploring the influence which Trypsin impact of small s of flavonoids as well Trypsin molecule conformation functional variations. Mealwhile, the researches have important guiding significance for the clinical use of drugs, new drug development and design. Glucose and Linoleic acid are necessary nutritions of body supplied everyday. The studies that influence of Glucose and Linoleic acid on the binding have practical significance for assessing the affection which pharmacokinetics of the drug by food nutrients and further clinical pharmacology studies.Objective:To study the spectroscopy characteristics of interactions of flavonoids with Trypsin, and discuss the quenching mechanism, binding constants, the numbers of binding sites, binding distance, thermodynamic parameters, and the conformation change of Trypsin and effect of Glucose and Linoleic acid on the interactions.Methods:The interactions of flavones (Luteolin, Luteoloside, Acacetin, Acacipetalin, Baicalein, Baicalin) and flavonols (Quercetin, Quercitrin, Hyperoside) with Trypsin under simulating physiological pH condition were studied by fluorescence and UV-vis spectroscopy at different temperatures298K(Indoor temperature) and310K(The body’s normal physiological temperature), respectively. Quenching mechanism, binding constants (Ka), the numbers of binding sites (n) and binding distance (r) were obtained from the calculated results. The dominant binding forces were estimated according to thermodynamic parameters. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the conformation change of Trypsin with the addition of flavonoids. The effect of Glucose and Linoleic acid on the interactions between flavonoids and Trypsin were studied at310K, binding constants and the numbers of binding sites were obtained at the same time.Results:The interactions of Luteolin, Luteoloside, Acacetin, Acacipetalin, Baicalein, Baicalin, Quercetin, Quercitrin, and Hyperoside with Trypsin resulted in the quenching of the intrinsic fluorescence of Trypsin.The order of range of binding constants was5.01×103～2.10×106and the numbers of binding sites were all approximately equal to1. The binding distance between Trypsin and nine flavonoids were all less than7nm. According to thermodynamic equations, the enthalpy change (△H) and entropy change (△S) were derived to be negative values for the interactions of Luteolin, Luteoloside, Quercetin, Quercitrin or Hyperoside with Trypsin, and the negative△H and positive AS for Baicalein, Baicalin, Acacetin or Acacipetalin with Trypsin. Synchronous fluorescence and three-dimensional fluorescence spectra indicated that the flavonoids binding to Trypsin induced some change in the intensity and position of fluorescent peak. The presence of Glucose and Linoleic acid increased binding constants and the numbers of binding sites of flavonoids-Trypsin complexes.Conclusion:Luteolin, Luteoloside, Baicalein, Baicalin, Acacetin, Acacipetalin, Quercetin, Quercitrin and Hyperoside could all quench the intrinsic fluorescence of Trypsin via static quenching under simulating physiological pH condition, and the energy transfer from donor (Trypsin) to acceptor (flavonoids) occurred with higher possibility. Hydrogen bonds and van der Waals forces played the major role in stabilizing the complexes of Luteolin-rypsin, Luteoloside-Trypsin, Quercetin-Trypsin, Quercitrin-Trypsin and Hyperoside-Trypsin. Electrostatic interactions were the dominant intermolecular forces in the binding of Baicalein, Baicalin, Acacetin or Acacipetalin with Trypsin. Synchronous and three-dimensional fluorescence spectra revealed that the interactions of flavonoids and Trypsin influenced the environments of tryptophan residues and induced the change of Trypsin conformation. In addition, the presence of Glucose and Linoleic acid could increase the binding constants and the numbers of binding sites between flavonoids and Trypsin, which was beneficial to the process of interactions.