Inhibition Of Tyrosinase Activity And Its Mechanism By Anthocyanidins From Zijuan Tea

Six anthocyanins in Zijuan tea were analyzed and determined by liquid chromatography and electrospray mass spectrometry.They were delphinidin-3-O-gala-ctoside,cyanidin-3-galactoside,delphinidin,cyanidin,pelargonidin,peonidin.Six contents of anthocyanins were 3.76,5.85,2.87,3.09,0.42 and 0.18 mg/g,respectively.And the exact molecular weights were 465.13,449.25,303.21,287.14,271.07,301.04,respectively.The in vitro inhibition experiment showed that under the conditions of p H 6.8 of phosphate buffer solution,incubation time of 45 min and reaction temperature of30℃,using levodopa as substrate,anthocyanin extract of Zijuan tea had a good inhibitory effect on tyrosinase,and the IC50 value was 1.31mg/m L.Cyanidin showed strong inhibitory effect on tyrosinase,followed by peonidin,delphinidin,delphinidin-3-O-galactoside,pelargonidin,cyanidin-3-galactoside,respectively,and the IC₅₀ values were 11.46×10^-6,30.86×10^-6,31.14×10^-6,41.94×10^-6,50.51×10^-6mol·L^-1.All the six anthocyanins showed reversible inhibition on tyrosinase.Cyanidin was a non-competitive-anti-competitive mixed inhibitor of tyrosinase,and the other five anthocyanin components were competitive-non-competitive mixed inhibitors.The experimental results showed that the six anthocyanins in Zijuan tea can effectively inhibit the activity of tyrosinase.The interaction mechanism of six anthocyanins in Zijuan tea with tyrosinase was studied by multiple spectral methods combined with molecular docking technique for the first time.The quenching effect of six anthocyanins on tyrosinase was investigated at 298 K,305 K and 312 K.The results showed that six anthocyanins have static quenching effect on tyrosinase.Gibbs free energy changeΔG is negative,indicating that six anthocyanins and tyrosinase complexes were formed spontaneously.The enthalpy changeΔH and entropy changeΔS were negative,indicating that the interaction forces between six anthocyanins and tyrosinase are mainly hydrogen bond and van der Waals force.It can be seen from the energy transfer experiment that non-radiative energy transfer is accompanied by the combination of six anthocyanins and tyrosinase.The experimental results of synchronous fluorescence,three dimensional fluorescence,UV-vis absorption spectrum and circular dichroism spectrum showed that the binding of six anthocyanins to tyrosinase changed the secondary structure of tyrosinase and the microenvironment around the chromophore,increase the polarity of the enzyme and weaken its hydrophobicity.The results of molecular docking simulation proved that the six anthocyanin components bind to the active center of tyrosinase,and the main force between six anthocyanin and tyrosinase are hydrogen bonds and van der Waals forces.