Study On The Inhibitory Mechanism Of Resorcinol Flavonoid Derivatives On Tyrosinase

Tyrosinase is the key enzyme in the synthesis of melanin,which is widely distributed in human beings,animals,plants,and microorganisms.Flavonoids are abundant in fruits,vegetables,spices,tea,soy based food stuff,and traditional herbal medicine.It has been reported that flavonoids are tyrosinase inhibitors,but their inhibitory activities and mechanisms against tyrosinase are very different because of different secondary structures,types,numbers and locations of substituents.In this study,the inhibitory activities and mechanism differences between resorcinol flavonoid derivatives with the 2',4'-dihydroxy substituted unit and their isomers(3',4'-dihydroxy substituted flavonoids)for tyrosinase were investigated by a combination of kinetic studies,fluorescence spectroscopy,circular dichroism,computational simulations and chromatography.The enzyme kinetics studied the effects of six flavonoids on the activity of diphenolase of tyrosinase.Experimental results showed that norartocarpetin reversibly inhibited tyrosinase in a competitive manner,whereas luteolin caused reversible noncompetitive inhibition.Steppogenin and 2,4,2',4'-tetrahydroxychalcone behaved as a mixed-type inhibitory mechanism,in which the competitive inhibition played the dominant roles.The activity of tyrosinase could be activated by eriodictyol and butein.Resorcinol flavonoid derivatives with 2',4'-dihydroxy substituted units showed strong inhibitory effects on tyrosinase.The interaction of six flavonoids against tyrosinase was investigated by fluorescence spectroscopy.The results demonstrated that compounds norartocarpetin,luteolin,steppogenin,eriodictyol,2,4,2',4'-tetrahydroxychalcone and butein reduced the fluorescence intensity of tyrosinase by a static quenching mechanism.The binding reaction belongs to spontaneous molecular action process,and the number of binding site in the interaction was closed to 1.The hydrogen bonds and Van der Waals forces dominated the binding process between norartocarpetin and 2,4,2',4'-tetrahydroxychalcone with tyrosinase.The hydrogen bonds and hydrophobic interaction forces played important roles in the binding process of luteolin and tyrosinase.The enzyme could also influence the fluorescence emission spectra of steppogenin,and hydrophobic forces could promote their formation of the complex.When eriodictyol and butein interacted with tyrosinase,the thermodynamic functions ?H > 0,?S > 0 indicated that the dominant sorts of binding forces between tyrosinase and compounds were mainly hydrophobic forces and electrostatic forces.The binding contants of resorcinol flavonoid derivatives with 2',4'-dihydroxy substituted units and tyrosinase were large,indicating a strong affinity between them.The analysis of circular dichroism indicated that six flavonoids induced changes of tyrosinase secondary structure.Molecular docking techniques were used to observe the binding forms and binding sites of compounds and tyrosinase.The results showed that the hydroxyl groups of the B ring of norartocarpetin interacted with tyrosinase residues Asn81 and His85 in the active pocket,while the hydroxyl groups of the B ring of luteolin bond with residues Asn81 and Cys83.Steppogenin entered the tyrosinase hydrophobic active center,and formed hydrogen bonds with amino acid residues,including Asn81,Cys83,His85,Gly245,Met319 and Arg321.However,its isomeric eriodictyol was not bound to the active pocket of tyrosinase.2,4,2',4'-tetrahydroxychalcone could form seven hydrogen bonds with tyrosinase,meanwhile interacting with amino acid residues His85,His259 and His263,which were connected with copper ions.However,only three hydrogen bonds were formed between butein with residues Gly245 and Ala323.At the same time,it was found that the binding energies of resorcinol flavonoid derivatives with 2',4'-dihydroxy substituted units and tyrosinase were smaller and the number of hydrogen bonds were more,indicating that the complexes formed with the enzyme were more stable.The structure changes of compounds were investigated by HPLC and UPLC-MS technology.The experimental results indicated that norartocarpetin,steppogenin,2,4,2',4'-tetrahydroxychalcone and luteolin could inhibit the activity of tyrosinase to reduce the contents of L-DOPA,which was catalyzed by the enzyme,but eriodictyol and butein exhibited the properties of the activators.In addition,luteolin,steppogenin,eriodictyol and butein could be catalytically oxidized by tyrosinase to produce quinone compounds,while no new product could be found after compounds norartocarpetin and 2,4,2',4'-tetrahydroxychalcone interacting with tyrosinase.