Inhibition Effect And Mechanism Of Four Flavones On Xanthine Oxidase

Xanthine oxidase is mainly distributed in liver and kidney of human body,which is responsible for regulating purine catabolic metabolism,promoting the conversion of hypoxanthine and xanthine to uric acid,and accompanied by the generation of superoxide anion.When the XO activity in the body is abnormally increased,not only the metabolic disorder leads to excessive uric acid secretion,but also the body lacks uric acid oxidase,uric acid can't be further decomposed into soluble allantoin,leading to hyperuricemia eventually.Excessive urate crystals are deposited in the tissues,causing damage and developing gout.In addition,excess O2-free radicals can lead to a variety of diseases,including cell damage,stroke,diabetes,atherosclerosis and liver disorders.Nowadays,gout has become the second major metabolic disease,seriously endangering the health of Chinese people.In view of the key role of XO in the output of uric acid,the inhibition of XO activity has become the main treatment method for hyperuricemia and gout.Studies have shown that currently commonly used drugs in clinical practice,such as febuxostat and allopurinol,can cause certain toxic and side effects on the human body.Therefore,it is of great practical significance to find and screen new XO inhibitors with low toxicity and high efficiency.Flavones are a kind of secondary metabolites of polyphenols,widely distributed in many edible plants.They have anti-inflammatory,heart protection,anticancer,anti-diabetes and other biological activities,and can effectively inhibit the activity of XO,which has attracted great attention.In this paper,baicalein,baicalin,fisetin and hesperetin were selected as the research objects.The in vitro inhibitory effect of the above four flavonoids on XO,inhibition kinetics,binding properties and their effects on the structure of enzymes were studied.The inhibition effect of baicalein(baicalin,fisetin or hesperidin)in combination with allopurinol on XO activity was studied.The effects of several soluble sugars(fructose,sucrose,or stachyose)on fisetin inhibiting enzyme activity and mechanism analysis were explored.In addition,using hesperetin as a raw material,hesperetin copper complex was successfully synthesized and compared with the ability and mechanism of hesperetin to suppress enzyme activity.The main outcomes are as follows:1?The inhibitory effects of baicalein,baicalin,fisetin and hesperitin on XO were studied by enzymatic kinetics,which simulated normal human physiological acidity conditions(pH 7.4,37?).The results indicated that four flavones(baicalein,baicalin,fisetin and hesperetin)equipped strong XO activity inhibitory ability,and the IC50 values of their half inhibitory concentrations that inhibited uric acid production were(7.54±0.06)×10-6?(1.23±0.03)×10-4?(3.93±0.12)×10-4?(1.17 ±0.12)×10-5 and(2.53±0.04)×10-6 mol L-1,respectively.The inhibitory ability was from strong to weak:fisetin>baicalein>hesperitin>baicalin.Baicalein,baicalin and fisetin showed a mixed type of XO inhibition,while hesperetin is a competitive XO inhibitor.In addition,the isobolographic analysis method was used to find that baicalein,fisetin and hesperetin combined with allopurinol showed a certain synergistic effect when XO activity was inhibited.Among them,hesperitin and allopurinol had the strongest synergy effect.When baicalin and allopurinol were used together,except for the synergistic effect when they inhibited the activity of 50%XO,they all showed antagonistic effect.2?All of selected four flavones could significantly inhibit the formation of O2-free radicals in the XO reaction system,their IC50 values are 6.97×10-6?10.21×10-6?2.96×104 and 2.66×10-6 mol L-1,respectively,and the inhibitory ability was from strong to weak:hesperitin>fisetin>baicalein>baicalin.By comparing and analyzing the ability of inhibitors to scavenge O2-free radicals of non-enzymatic systems and DPPH free radicals,indicating that the promotion from the reduced form of XO molecules and the inhibition of the uric acid formation led in the inhibition of baicalein or fisetin on O2-.By reducing the enzyme molecules,baicalin and hesperitin enabled the FADH·/FADH2 electron pair to equip a higher reduction potential,thus promoting the regeneration of the reduced iron center and the intramolecular electron of Mo center.The other electron was provided to O2-free radical to form H2O2,which ultimately led to the reduction of O2-production.3?Fluorescence titration experiments combined with molecular docking technology were applied to further discuss the binding effect of flavones and XO.Baicalein,fisetin and hesperitin could statically quench the endogenous fluorescence of XO,while static and dynamic quenching were both existed in the interaction between baicalin and XO.Under the combined action of hydrophobic force and hydrogen bonds,all of four flavones could bind to XO spontaneously,and only had a binding site on XO.At 298 K,the binding constants Ka of the four flavones to form a complex with XO were from large to small:baicalein[(9.10±0.03)×104 L mol-1]>baicalin[(8.93±0.02)×104L mol-1]>hesperitin[(8.27± 0.03)×104L mol-1]>fisetin[(5.97±0.02)×104 L mol-1].Synchronous fluorescence experiment showed that baicalein or fisetn has little effect on the polarity and hydrophobicity around tyrosine and tryptophan residues,while the combination of baicalin or hesperitin increased the polarity around tryptophan residues and decreased the hydrophobicity.Meanwhile,the tryptophan residues were closer to the binding sites of baicalein(baicalin,fisetin or hesperitin)and XO,contributing more to the endogenous fluorescence quenching of XO.Molecular simulation showed that baicalein,baicalin or fisetin could successfully enter the hydrophobic cavity of XO,while hesperitin was directly inserted into the active center of catalytic substrates transformation.The combination of four flavones and XO induced a conformational change.Baicalein,baicalin or hesperitin could increase the ?-helix and random coil contents in XO,decrease the?-fold and ?-turn contents,while fisetin increased thea-helix content and ?-fold contents,decreased the contents of ?-turn content and random coil.4?According to the above experimental results,it was speculated that the mechanism of the four flavones selected to inhibit XO activity:baicalein,baicalin or fisetin entered the FAD active center of XO and binded to the amino acid residues around the isosine ring,inhibiting the release of O2-free radicals from FAD sites,leading to the transfer of another electron from FADH2 to O2-free radical to form H2O2,interrupting the enzyme catalyzed reaction so as to achieve the purpose of inhibiting the catalytic activity of XO.Whereas,hesperitin entered the Mo-pt active center of XO in a competitive manner,which blocked the substrate from entering the active center,affecting the normal operation of electron transfer chain,reducing the rate of XO catalyzing xanthine.At the same time,the combination of flavones and XO induced the conformational change of XO,which made its secondary structure tend to be tight,which was not conducive to exposing active cavity,reducing the binding probability of enzyme and xanthine,thus weakening the catalytic function of enzyme.5?Fructose,sucrose or stachyose neither interacted with XO nor had the ability to inhibit the activity of XO,but both could reduce the inhibitory effect of fisetin on XO.The ability to affect the IC50 value of fisetin was ranked as fructose>sucrose>stachyose.Meanwhile,soluble sugars did not change the type of inhibition and quenching of fisetin on XO,but significantly reduced the binding constant and quenching constant of fisetin-XO complex,and the decrease trend was positively related to the ability of lutein to inhibit enzyme activity.In addition,fructose/sucrose/stachyose-fisetin could spontaneously combine with XO under hydrophobic forces and hydrogen bonds.It was speculated that soluble sugars could bind fisetin to weake its inhibitory ability on XO activity.With the increase of monosaccharide structure in individual sugar molecules,the reduction of binding effect resulted in the decreased activity of soluble carbohydrate protective enzymes.6?The inhibitory effect of the hesperetin copper complex on XO activity was synthesized and determined.The results displayed that the ability of complex to inhibit XO was significantly enhanced,and its IC50 value[(1.07±0.08)×10-6 mol L-1]was smaller than the monomer hesperetin[(1.17±0.12)×10-5 mol L-1]and copper ion[(4.93±0.21)×10-6mol L-1].Hesperetin-Cu complex was a reversible mixed XO inhibitor,with the inhibitory constant Ki of 1.18×10-6mol L-1 and apparent coefficient a of 3.63.Compared with hesperidin that only complexed with XO,the complex could not only compete for the binding site with xanthine,but also binded with xanthine-XO complex,indicating that chelating copper ions made hesperidin-Cu complex obtained more methods to inhibit the activity of XO.Although the antioxidant capacity of the complex was lower than that of the hesperetin,it could still inhibit the formation of O2-radicals in the enzymatic reaction by inhibiting the formation of uric acid.The hesperidin-Cu complex could significantly statically quench the endogenous fluorescence of XO,and its Ksv and Ka value were higher than that of hesperetin at the same temperature,indicating that there was a good and stable affinity between the complex and XO,which was mainly driven by hydrophobic forces and hydrogen bonding.The same concentration of copper ligand reduced much more fluorescence intensity of tryptophan residues,indicating that they were closer to the binding site of the complex with XO,but had little effect on the polarity and hydrophobicity of tyrosine and tryptophan in the XO molecule.In addition,the complex induced an increase in the ?-sheet and ?-turn contents in XO,while the?-helix and random coil contents were decrease,indicating that the structure of XO became loose,which was not conducive for the substrate xanthine to enter the reactive center,thereby XO lost its catalytic activity.