Study On The Inhibition Mechanism Of Luteolin On Xanthine Oxidase And α-glucosidase

In recent years, the incidence of gout and diabetes are rising trend year byyear,and xanthine oxidase (XO) and α-glucosidase as the main target of high uric acidhematic disease and hyperglycemia, to explore the low toxicity of XO andα-glucosidase inhibitor and its inhibition mechanism has gradually become the focusof research. This paper used fluorescence, Ultraviolet visible (UV–vis) absorption,circular dichroism (CD) and Fourier transform infrared spectroscopy (FT IR)combined with the molecular simulation software to discuss the inhibition effect ofluteolin on XO and α-glucosidase.The inactivation and interaction mechanism weremeasured, and for a detailed study of the influence of enzyme conformation, finallyusing molecular docking technology clarify the interaction luteolin with XO andα-glucosidase.The main contents and conclusions in the thesis are summarized as follows:1. The structure, physiological function, and biological characteristics of XO andα-glucosidase were briefly stated at first in this chapter. And then, the research statusand methods of inhibition mechanism of luteolin on XO and α-glucosidase weresummarized, and expounded the application of molecular simulation technique in theenzyme inhibition.2. With XO and α-glucosidase as the model target, the inhibition effect ofluteolin on XO and α-glucosidase in PBS bu er (37℃) were investigated byUV-2450Ultraviolet spectrophotometer dynamics software/time, and the halfinhibitory concentration (IC50), inhibition type, inhibition constant, and inactivationrate constant were obtained. Results showed that luteolin reversibly inhibited XO in acompetitive manner with Kivalue of (2.38±0.05)×106mol L1(n=3), IC50value of4.79±0.02×106mol L1(n=3). analysis of Inactivation kinetics indicated that luteolincould inhibit the activity of XO quickly. Luteolin reversibly inhibited α-glucosidasein a noncompetitive manner with IC50and Kivalue of (1.72±0.05)×104mol L1and(1.40±0.02)×104mol L1, and the inhibition followed a multi-phase kinetic processwith a first-order reaction. 3. The binding mechanism of luteolin with XO and α-glucosidase undersimulated physiological conditions was investigated by fluorescence and molecularsimulation technique. Results showed that luteolin can quench the fluorescence of XOthrough a static quenching procedure with a single binding site in XO for luteolin.The calculated values of enthalpy change (31.48±0.05kJ mol1) and entropy change(178.42±0.16J mol1K1) suggested that the binding of luteolin to XO was drivenmainly by hydrophobic interactions; The molecular docking results revealed luteolinbound to the molybdenum (Mo) atomic domain of XO, and the bicyclicbenzopyranone ring of luteolin was sandwiched between Phe914and Phe1009andmade aromatic interactions (π π effects) with the two phenylalanines which might beimportant for ligand recognition by XO. Also, the ether group on C ring and onehydroxyl group on A ring could form hydrogen bonds with active-site residuesThr1010and Arg880, and the3′,4′-dihydroxyphenyl moiety at B ring of luteolin wasinserted into the hydrophobic region to interact with residues Glu802, Leu873,Val1011, Leu1014, and Pro1076. The docking simulation provided supportive datafor luteolin-induced inhibition by allowing us to predict the binding site in the activesite pocket, this may be the principal mechanism of inhibition. Luteolin had a strongability to quench the intrinsic fluorescence of α-glucosidase through a staticquenching procedure. The positive values of enthalpy (17.58±0.1kJ mol1) andentropy (39.23±0.04J mol1K1) change elucidated that the binding of luteolin toα-glucosidase was driven mainly by hydrophobic interactions, and the bindingdistance was estimated to be4.56nm. The molecular docking results showed that thebinding site for luteolin was close to the active site pocket, and luteolin wassurrounded by the residues, namely, Phe303, Ser304, His305, Val308, Gly309,Thr310, Ser311and Pro312. Moreover, one hydroxyl group on A ring could formhydrogen bonds with residue Thr310. In addition, the3′,4′-dihydroxyphenyl moiety atB ring of luteolin was inserted into the hydrophobic region to interact with residuesPhe303, Ser311, Pro312and His351which were closed to the pocket of active site,and might contribute to inducing the cleft closure to avoid entrance of the substrate.4. Conformational changes of XO and α-glucosidase by luteolin wereinvestigated by synchronous fluorescence, circular dichroism, and Fourier transform infrared spectra. Analysis of synchronous fluorescence and CD spectra demonstratedthat the microenvironment and secondary structure of XO were altered uponinteraction with luteolin, and the polarity around the tyrosine residues decreased andthe hydrophobicity increased, but the microenvironment around the tryptophanresidues has no discernable change during the binding process. With increasing themolar ratio of luteolin to XO, the contents of α-helix increased, which implied thatthe active site of XO was not easy formed and the chance for xanthine binding to theactive site of XO was decreased eventually leading to the reduction of catalytic action.And the binding of luteolin to α-glucosidase induced a partial unfolding ofα-glucosidase, and the polarity around the tyrosine and tryptophan residues increasedand the hydrophobicity decreased during the binding process. a partial unfolding ofα-glucosidase might be part of the mechanism of the enzymatic inhibition required toprevent the hydration of the substrate binding site and induce the cleft closure toavoid entrance of the substrate.