Studies On The Interactions Of Flavonoids With Lysozyme And Influence Of B Group Vitamins On The Binding

Lysozyme (LYSO), as a small alkalescent protein in living organisms, can combine with exogenous and endogenous substances and carry many drugs. Flavonoids are natural products which exist widely in the plant kingdom and exhibit important bioactivities such as antibiosis, anti-inflammatory, antivirus, antioxidant and antitumor. Studies on the interactions of flavonoids with LYSO have significant meanings for explaining the mechanisms of drug effect at molecular level and getting the active groups in pharmacokinetics. B group vitamins are necessary nutritions of body supplied everyday. We could learn the influence of daily intake nutritions on drug metabolism in practical via investigating the effect of B group vitamins on the binding actions.Objective: To study the spectroscopy characteristics of interactions of flavonoids with LYSO, and discuss the quenching mechanism, binding constants, the numbers of binding sites, binding distance, thermodynamic parameters, the change of LYSO conformation and effect of B group vitamins on the interactions.Methods: The interactions of flavones (Luteolin, Luteoloside, Acacetin, Acacipetalin, Baicalein, Baicalin) and flavonols (Quercetin, Quercitrin, Rutin, Hyperoside) with LYSO under simulating physiological pH condition were studied by fluorescence and UV-vis spectroscopy at different temperatures (298 K, 304 K and 310 K), respectively. Quenching mechanism, binding constants (K_a), 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 LYSO with the addition of flavonoids. The effect of VB1, VB2, VB3, VB5 and VB6 on the interactions between flavonoids and LYSO were studied at 310 K, binding constants and the numbers of binding sites were obtained at the same time. Results: The complex formation between Luteolin, Luteoloside,Acacetin, Acacipetalin, Baicalein, Baicalin, Quercetin, Quercitrin, Rutin or Hyperoside and LYSO resulted in the quenching of the intrinsic fluorescence of LYSO, and the quenching constants decreased with increasing temperature. The order of magnitude of binding constants was 103~106 and the numbers of binding sites were all approximately equal to 1. The binding distance between LYSO and ten flavonoids were 4.32, 3.83, 4.14, 4.07, 4.22, 3.51, 4.56, 4.07, 4.02, 4.28 nm respectively, which were all less than 7 nm. According to thermodynamic equations, the enthalpy change (ΔH) and entropy change (ΔS) were derived to be negative values for the interactions of Luteolin, Luteoloside, Acacetin Acacipetalin, Quercetin, Quercitrin, Rutin or Hyperoside with LYSO, and the negativeΔH and positiveΔS for Baicalein or Baicalin with LYSO. Synchronous fluorescence and three-dimensional fluorescence spectra indicated that the flavonoids binding to LYSO induced some change in the intensity and position of fluorescent peak. The presence of VB1, VB2, VB3, VB5 or VB6 increased binding constants and the numbers of binding sites of flavonoids-LYSO complexes. The order of magnitude of binding constants increased to 10~5~10~8 and the numbers of binding sites maintained around 1. Meanwhile, VB1, VB2, VB3, VB5 and VB6 influenced the blue-shift or red-shift of fluorescent peak of LYSO.Conclusion: Luteolin, Luteoloside, Acacetin, Acacipetalin, Baicalein, Baicalin, Quercetin, Quercitrin, Rutin and Hyperoside could all quench the intrinsic fluorescence of LYSO via static quenching under simulating physiological pH condition, and the energy transfer from donor (LYSO) to acceptor (flavonoids) occurred with higher possibility. Hydrogen bonds and van der Waals forces played the major role in stabilizing the complexes of Luteolin-LYSO, Luteoloside-LYSO, Acacetin-LYSO, Acacipetalin-LYSO, Quercetin-LYSO, Quercitrin-LYSO, Rutin-LYSO and Hyperoside-LYSO. Electrostatic interactions were the dominant intermolecular forces in the binding of Baicalein or Baicalin with LYSO. Synchronous and three-dimensional fluorescence spectra revealed that the interactions of flavonoids and LYSO influenced the environments of tryptophan residues and induced the change of LYSO conformation. In addition, the presence of VB1, VB2, VB3, VB5 or VB6 could increase the binding constants and the numbers of binding sites between flavonoids and LYSO, which was beneficial to the process of interactions.