| Xanthine Oxidase(XO) is an important enzyme in the metabolism of nucleic acid: catalyzing hypoxanthine to xanthine and then to uric acid. An elevated level of blood uric acid (hyperuricemia) is the underlying cause of gout, and the excessive deposition of uric acid in joints can induce painful inflammation. At the same time, the concomitant subversive free radicals are considered to be related with the inflammation of the catalytic pathological process. Thus, in order to inhibit the activity of XO, decreasethe production of uric acid to prevent gout and reduce the tissue damage caused by free radicals, searching for natural biological XO inhibitors with safe and high potential has been attracting the attention of scholars in the world for many years. Flavonoids are widely exist in herbal medicine, they not only have a variety of biological activities, such as antioxidant, anti-inflammatory, antiviral, anticancer, and other pharmacological effects, but also have good ability of scavenging free radicals and enzyme inhibition which are widely used in medical field. In recent years, the topics of finding and screening effective xanthine oxidase inhibitors from the plant flavonoids for gout treatment have stirred widespread attention in researchers.In this article, the inhibition kinetics and inhibitory mechanism of the common flavonoid chrysin and genistein on XO were researched by a variety of modern analytical techniques such as inhibitory kinetics, fluorescence spectroscopy, FT-IR, circular dichroism, etc., combining with molecular simulation method, respectively; The chrysin-copper complexes were synthesised and their ability of inhibiting XO were also analysised; Finally, according the different structure of flavonoids, their XO inhibitory activity and structure-activity relationship were studied.The main contents and conclusions in the thesis are summarized as follows:1. The results showed that:For the XO in this study, the optimal pH range is 7-8.5, optimum temperature range is 30?55?, frozen processing is the most suitable storage condition; When the substrate isxanthine, the production is uric acid whose characteristic UV-vis absorbance was at 290 nm which, and the reaction time was 300 s; The organic solvent of ethanol and DMSO had a certain influence on the structure and activity of XO, it was supporsed to control its volume fraction within 2% in the following reaction system. In the range of low concentrations, Mn2+, Ag+, Hg2+, Cu2+ showed activation towards XO; Ca2+ had no obvious effect on the XO activity; In a certain concentration range, Mg2+, Co2+, Zn2+, Cu2+ showed XO inhibitory.2. The inhibitory mechanism of chrysin (flavone) on XO in the system of Tris-HCl buffer (pH 7.4) has been researched by inhibitory kinetics, fluorescence spectroscopy and circular dichroism. The results of inhibitory kinetics indicated that chrysin competed with the catalytic substrate of xanthine to bind to the active center of the enzyme in a reversible and competitive manner, the half inhibitory concentration IC50 and inhibition constant Ki were (1.26 ± 0.04) ×10-6 mol/L?(5.70 ± 0.14) ×10-7 mol/L,respectively. Chrysin showed strong XO inhibitory activity. The results of fluorescence spectroscopy showed that chrysin quenched the intrinsic fluorescence of the enzyme statically, its binding site n wer near 1. Thermodynamic parameters of ?G (-31.64 kJ/mol), AS (-330.06 J/(mol-K)) and ?H (-130.07 kJ/mol) under 298 K indicated that the interaction between chrysin and XO was mainly driven by hydrogen bonding and van der Waals force, with binding constant (Ka) of 7.98 ×105 L/mol. And it probably occurred radiation energy transfer between chrysin and XO, the binding distance was 4.16 nm.The analysis of circular dichroism revealed that chrysin induced the content of ?-helix and ?-sheet increased, while the contents of P-ture and random coil reduced. Molecular simulation further revealed the binding domain and binding mode of chrysin and XO, visually. It was observed that chrysin inserted into the same active region of XO as xanthine(substrate), its ring B interacted with some amino acid residues (Leu648 Phe649 Glu802, Leu873 and Phe1013) in hydrophobic regions within the XO cavity., Its C5-OH and C7-OH and the ether group formed hydrogen bonds with Thr1010, Glu879 and Ser876. Thus, the XO inhibition activity of chrysin is likely to be attributed as:chrysin inserted into the active cavity of XO, occuping the channel of substrate xanthine leading into the catalytic center area (Mo), preventig the entry of the substrate, and chrysin induced conformation change of XO which also affected the catalytic activity of enzymes.Moreover, it was found that chrysin and apigenin have a slight synergistic inhibition effect on xanthine oxidase at the concentration of chrysin 5.0 x 10-7 moI/L, apigenin 1.0 x 10-6 mol/L, while for most of the experimental groups chrysin had no significant influence on the XO inhibitory of apigenin. This might be due to that apigenin was also a kind of competitive XO inhibitor, but they were in a different position in the active cavity of XO, thus they could independently eahibit their XO inhibitory.3. The result of enzyme kinetics experiments showed that genistein (isflavone) was a reversible competitive XO inhibitors with IC50 of (1.73 ± 0.04)× 10-6 mol/L and K1 of (1.39 ± 0.11) ×10-6 mol/L? The resultsof fluorescence spectrum experiment showed that genistein bound to XO mainly by hydrophobic force and hydrogen bond withbinding constant Ka of 5.24 × 104 L/mol (298 K). By analysising the data of synchronization, three dimensional fluorescence experiment and FT-IR and CD spectrum, the results indicated that genistein combined to XO, inducing the secondary structure changes in XO, increasing cotent of a-helix??-sheet, decresing of Loop(?-turnand random coil). All these conformational changes might make the secondary structure of XO more compact which was not conducive to the formation of the active center, on the other hand, the tertiary structures of XO such as the microenvironment around the aromatic amino acids and disulfide bond were affected too which might also influence the enzyme activity. Finally, the simulation showed that genistein bound into the XO hydrophobic cavity, its B ring inserted between Phe1009 and Phe1013 which might cause ?-? effect, and it formed hydrogen bond with Ser876 and Asn768 residues with length of 2.169 A and 2.188 A respectively.4. The chrysin-Cu complexes were synthesized and characterized by using the method of Uv-vis spectra, FT-IR spectra, nuclear magnetic resonance (NMR) and mass spectrometry analysis. Then their inhibition towards XO was measured, such as the inhibition rate, inhibition type, the binding characteristic of complex and XO and the influence of complex on XO conformation. The experimental results showed that the chrysin-Cu complexes exhibited lower ICso ((0.82±0.034) × 10-6 mol/L) than Cu2+ and chrysin, respectively, which indicated the stronger effective inhibition of complex towards XO. The complexes of chrysin-Cu exhibted a kind of reversible competitive inhibiton which was different from chrysin, indicating that they could combine with the free XO and the inhibitor-enzyme complexes with Ki value of (3.14 ± 0.26) × 10-7 mol/L and a value of 5.25. Like chrysin, chrysin-Cu complexes bound to XO mainly by hydrogen bond and Van der Waals force (?G<0, ?H=-34.71 kJ/mol??S=-20.30 J/(mol·K)), while the energy was different fromthe system of chrysin-XO, this might be due to the interaction between Cu2+ of the complex and the S/N group in the active region. Addtionally, the binding of chrysin-Cu complexes influenced the microenvironment of tryptophan and tyrosine residues in XO by enhancing its polarity obviously. The binding of Chrysin-Cu complexes also induced the content of secondary structure in XO by incresing the level of a-helix (36.8% to 56.2%) obviously, and decreasing flexible random turn curve (25.1% to 6.3%) content significantly. The increasing level of a-helix might induce the structure of XO becoming more compact which was not conducive to form the active center of the enzyme, thus, reduced the contact between XO and the substrate; in addition, the change of the flexibility of active cavity in XO was also a reason of decreasing enzyme activity. Above all, the Cu2+ in complexes bridged chrysin molecule and XO, it made inhibitor insert into the active cavity easier, occupying the leading channel of substrate into the active Mo-pt, inducing the conformational change of active region, finally, exhibited higer XO inhibition ability than the single ligand (Cu2+ and chrysin).5. By referring to previous documents and analyzing the experimental results, some conclusion about structure-activity relationship were concluded:The inhibitory activities increased generally with the increasing affinities within the class, especially for flavones and flavonols, which might because the higher affinity increases the chance to enter the catalytic gorge, but the inhibition finally depends on the direct interaction between the flavonoid and the active site; The C2= C3 was both important for their affinity and XO inhibition activity; The hydrogenation of the C2=C3 double bond on flavonoids, the hydroxylation of C3 (ring C) and the glycosylation of 3C-OH, the hydroxylation and methoxylation on ring B and the hydroxylation of C6 (ring A) and glycosylation of 7C-OH were all unfavorable for the XO inhibition; The C2=C3 and 4-oxo group maintain a planar structure of flavonoids which was essential for potent inhibitory activity on XO; The hydroxyl moiety at C7 and C5 contributed favorable hydrogen bonds and interactions between inhibitors and the active site, which was favorable for XO inhibition. |
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