Study On The Inhibition Mechanism Of Morin On Tyrosinase And Xanthine Oxidase

For a long time, the human body skin diseases such as chloasma, age spots, pregnancy spots etc., are common diseases that troubled people, thus, the research of whitening cosmetics and foods has received the widespread attention and development. Gout, a disease caused by excessive uric acid, has been listed to be one of top 20 persistent ailment of the 21 st century by the United Nations. The incidence of gout increased year by year in our country for the improvement of living standard, speeding up of life rhythm and unreasonable of people’s diet structure, and it is now higher than the world average. It has been reported that, in human body, tyrosinase is mainly to catalyze the organism to generate melanin, and its overexpression would lead to excessive accumulation of melanin, and then cause skin disorders. Thus, it is an important mean of spot whitening to inhibit the activity of tyrosinase. Xanthine oxidase is a key enzyme in the process of producing uric acid and a important factor of generating gout in human body. Therefore, it is one of the main ways for the clinical treatment of gout that inhibit the activity of xanthine oxidase when taking it as the drug targets.In recent years, the scientific workers have made certain achievements in the synthesis, research and development of spot and anti-gout drugs. However, most of the synthetic drugs have certain side effects, which limit their application to a certain extent. Flavonoids with its wide range of biological activity by the favour of people, It has been widespread concerned by science researchers that finding and screening effective tyrosinase and xanthine oxidase inhibitors from plant flavonoids in order to provide options of new drugs for spot and gout treatment. In this article, the inhibitory kinetics and inhibitory mechanism of the common flavonoid morin on the above two kinds of enzyme were researched by a variety of modern analytical techniques such as inhibitory kinetics, fluorescence spectroscopy, circular dichroism, etc., combining with molecular simulation method, when taking tyrosinase and xanthine oxidase as the targets, respectively. Hope to provide a scientific basis for thedevelopmentandapplicationofmorinincosmeticsandanti-goutdrugs,andalsoprovidereferencemethodsandtechnicalsupportforresearchingtheinhibitingeffectofotheractivecomponentsonenzyme.themaincontentsandconclusionsinthethesisaresummarizedasfollows:1.inthischapter,thestructure,catalyticfunction,andbiologicalcharacteristicsoftyrosianseandxanthineoxidasewerebrieflystated,theresearchprogressoftyrosinaseinhibitorsandxanthineoxidaseinhibitorswereoverviewedatfirst.2.withtyrosinaseasthemodeltarget,theinhibitorymechanismofmorinontyrosinaseinthesystemofphosphatebuffer(ph6.8)undertheconditionofroomtemperaturehasbeenresearchedbyinhibitorykinetics,fluorescencespectroscopyandcirculardichroism.theresultsofinhibitorykineticsindicatedthatmorincompetedwiththecatalyticsubstrateoftyrosinaselevodopa(l-dopa)tobindtotheactivecenteroftheenzymeinareversiblemannerbymultiphasedynamicsprocess,thehalfinhibitoryconcentrationic50andinhibitionconstantkiwere(8.13±1.21)×10-5l/mol,(7.32±0.36)×10-5l/mol,respectively.morinshowedstrongtyrosinaseinhibitoryactivity.theresultsoffluorescencespectroscopyshowedthatmorinquenchedtheintrinsicfluorescenceoftheenzymestaticallysinceitboundtoaspeciallocationontyrosinaseformingaground-statecomplex,mainlythroughhydrogenbondsandvanderwaalsforces.thebindingconstantat298kwascalculatedtobe8.10×104l/mol.anditprobablyoccurredradiationenergytransferbetweenmorinandtyrosinase,thebindingdistancewas5.12 nm.theanalysisofcirculardichroismrevealedthatmorininducedthecontentofβ-sheetincreased,whilethecontentsofα-helix,β-tureandrandomcoilreduced.molecularsimulationfurtherrevealedthebindingdomainandbindingmodeofmorinandtyrosinase,visually.itwasobservedthatmorininsertedintotheactivecenteroftyrosinase,andinteractedwithsomesurroundingaminoacidresiduesashis85,his94,his244,his259,asn260,his263,phe264,met280,gly281,val283,his296,etc.,aswellasformedtwohydrogenbonds.theinhibitorymechanismofmorinontyrosinasecouldbededucedthattheinsertionofmorinintotheactivesiteoftyrosinasenotonlyoccupiedthecatalyticcenteroftyrosinasetosubstratel-dopahinderingtheentranceofthesubstrate,butalsoinducedsomeconformationalchangesresultinginsomedecreaseoftyrosinaseactivitydirectly.3.theinhibitionrateandinhibitiontypehasbeendetermined,andthebindingcharacteristicofmorinandxanthineoxidase,theinfluenceofmorinonxanthineoxidaseconformationandtheeffectsofkaempferolonmorininhibitionofxanthineoxidaseactivityhavebeenresearchedinthesystemofph7.5phosphatebuffer.theexperimentalresultsshowedthatmorinwasakindofstrong,reversiblemixed-typeinhibitor,anditsic50wascalculatedtobe1.35×10–5mol/l,inhibitionconstantskiandinhibitioncoefficientαwere1.21×10–5mol/land1.94,respectively.therewasabindingsiteinxanthineoxidaseformorin,andthebindingconstantkaat298kwascalculatedtobe3.22×104l/mol.theinteractionbetweenmorinandxanthineoxidasewasanendothermicandentropydrivenprocess,andhydrophobicforcewasthemaindrivingforce.itwasfoundfrommoleculardockingthatmorininsertedintothehydrophobiccavityofxanthineoxidase,andinteractedwithsomeprimaryaminoacidresiduesser1075,pro076,asn768,lys771,leu648,leu1014,etc.,whichlocatedaroundtheactivecenterofxanthineoxidase(moarea).theanalysisofcirculardichroismindicatedthatmorininducedsomechangesofxanthineoxidasesecondarystructure.itcouldbededucedthattheinhibitoryactivityofmorinonxanthineoxidaseontheonehandwasduetothebindingofmorintothehydrophobiccavityoftheenzymeoccupyingtheentranceofsubstrateenteringintotheactivecenter,thus,reducedthecatalyticrateofxanthineoxidasetothesubstrate.ontheotherhand,itwasduethebindingofmorininducedthestructureofxanthineoxidasemorecompactwhichisnotconducivetotheactivecenteroftheenzymeexposingtothereactionenvironment,thus,reducedthecontactbetweenxanthineoxidaseandthesubstrate.moreover,itwasfoundthatmorinandkaempferolhavegoodsynergisticinhibitioneffectonxanthineoxidaseattheconcentrationofmorin2.0 × 10–6 mol/L, kaempferol 2.0 × 10–6 or 8.0 × 10–6 mol/L, and morin 1.4 × 10–5 mol/L, kaempferol 8.0 × 10–6 mol/L. It might be that morin and kaempferol not only occupied the XO activity center, but occupied the channel for the substrate entering into, and both of them influenced the conformation of xanthine oxidase.4. The inhibitory mechanism of kaempferol on xanthine oxidase and the effects of luteolin on kaempferol inhibition of xanthine oxidase activity were investigated by multispectroscopic methods, combining with molecular simulation technique. Asa result, kaempferol was a kind of effective reversible xanthine oxidase inhibitors, and it could compete with the substrate xanthine to bind to the active site with IC50 and Ki of(2.18 ± 0.02) × 10-6 mol/L and(6.77 ± 1.02) × 10-6 mol/L. Strong static fluorescence quenching of xanthine oxidase was observed due to the formation of complex with kaempferol, and hydrophobic function was the main driving force. Moreover, kaempferol induced the polarity around the tyrosine and tryptophan residues in xanthine oxidase increased and the hydrophobicity decreased, and the contents of α-helix and random coil in xanthine oxidase reduced while the β-sheet and β-ture on the contrary. It was also observed from molecular simulation that kaempferol inserted into the hydrophobic cavity of xanthine oxidase, and interacted with some surrounding primary amino acid residues such as Glu802, Leu873, Phe914, Arg880, Phe1009, Thr1010, Val1011, Leu1014, Pro1076, etc., moreover, formed two hydrogen bonds with Asn768 and Luz1(coenzyme factor connected with Mo atoms). The inhibitory mechanism of kaempferol on xanthine oxidase was probably that kaempferol inserted into the active site of xanthine oxidase occuping the catalytic center and hindering the entrance of the substrate xanthine, and inducing some conformational changes of xanthine oxidase which lead to the decrease of the catalytic activity of xanthine oxidase. In addition, it has been found that kaempferol and luteolin have synergistic effect on inhibiting xanthine oxidase activity at the concentration of kaempferol 0.5 × 10–6 mol/L, and kaempferol 2.0 × 10–6 mol/L, luteolin 1.0 × 10–6 mol/L or 5.0 × 10–6 mol/L, and kaempferol 8.0 × 10–6 mol/L, luteolin 1.0 × 10–6 mol/L, respectively. It could be speculated that both of kaempferol and luteolin bound to the active center of xanthine oxidase that occupied the binding site of xanthine oxidase and its substrate xanthine, and it was bad for the formation of xanthine oxidase active center since both of kaempferol and luteolin induced some conformation change of xanthine oxidase.