Separation And Identification Of The Tyrosinase Inhibitor From Camellia Pollen And Studies Of Inhibition Mechanism

To investigate the whitening function of camellia pollen and its action mechanism,the compounds with tyrosinase inhibitory activity had been screen out by active-guide method and separated by high-speed countercurrent chromatography?HSCCC?,then the mechanism of tyrosinase inhibition was further discussed in this study.The obtained results would provide new evidence and theoretical basis to elucidate the unique molecular mechanism of Camellia pollen.The ethanol extract of Camellia pollen was extracted by petroleum ether,ethyl acetate and n-butanol successively,the ethyl acetate extraction was then preliminary separated by macroporous adsorption resin.All the solvent extractions and eluents from resin were tested for their active ingredient content and inhibitory tyrosinase activity,the correlation between active ingredient content and activity were analyzed by Pearson's correlation test.Results showed that the tyrosinase inhibitory activity and flavonoids content of ethyl acetate extraction?inhibition rate 65.61%?were all stronger than that of other solvent extractions.After separation by HP-20macroporous resin,the activity compounds in ethyl acetate had gathered in 50%ethanol elution phase?inhibition rate 86.45%?,the tyrosinase inhibitory activity and flavonoids content of eluent fractions from resin revealed strong positive linear correlation with the correlation coefficient as0.91?R²=0.82?,which means that flavonoid are one of the main component responsible for the tyrosinase inhibitory.Fractions with stronger tyrosinase inhibitory activity?ethyl acetate extraction and 50%ethanol eluent fraction from resin?were subjected acid hydrolysis,the main active compounds in hydrolysated was then separated by HSCCC,three compounds with high purity were separated by HSCCC,identification by HPLC,LC-MS and NMR showed that these components were kaempferol,levulinic acid and 5-hydroxymethyl furaldehyde?5-HMF?,respectively.All the three fractions revealed tyrosinase inhibitory activity,with the IC₅₀ as 0.05,4.02,2.03 mg/mL,respectively,and the activity of kaempferol was far stronger than that of arbutin(IC₅₀=0.21mg/mL).The molecular mechanisms of kaempferol and 5-HMF on tyrosinase were elucidated by means of enzyme kinetics,spectroscopy techniques,and fluorescent spectroscopy.Kaempferol and 5-HMF were the competitive and non-competitive inhibitors of tyrosinase respectively.Quenching mechanism of tyrosinase by kaempferol and 5-HMF were influence the microenvironment of the fluorescent groups near the tyrosinase,instead of changing the hydrophobic environment of amino acid residues?tryptophan?with fluorescence quenching in tyrosinase.5-HMF don't coordinate to Cu²⁺,and kaempferol could chelate to with tyrosinase dinuclear copper ion,form a more stable structure than the substrate-enzyme complex configuration,and achieve the purpose of inhibiting tyrosinase catalytic activity.