Extraction And Isolation 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 tyrosinase inhibitors were extracted from camellia pollen and 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.Based on the results of single factor experiment,the extraction conditions were optimized by the response surface methodology?RSM?with the index of anti-tyrosinase rate.The best extraction condition was 52oC,ratio of material to liquid 1:10?m/v?,with the concentration of ethanol 79%.Under the above condition,the inhibition rate of tyrosinase by the crude extract of camellia pollen was?51.54±1.34?%?n=3?.41.09%of anti-tyrosinase rate was increased after optimized.Macroporous adsorptive resins and High-speed counter-current chromatography?HSCCC?were used to separate tyrosinase inhibitors of ethyl acetate extracts from camellia pollen with bioassay-guided method.Through the comparison of two methods,a more efficient and faster method which was HSCCC solvent gradient method was filtered to separate camellia pollen.Component E-1 which has simpler composition and with higher anti-tyrosinase activity was isolation by HSCCC.The IC₅₀0 values for E-1 on the inhibition of monophenolase activity was41.57±2.45?g/mL,which was lower than V_C and arbutin.A main compound whose molecular weight was 462.3573 was found by UPLC-TOF-MS technique,and the formula of this compound was C₂₁H₁₈O₁₂.Compared with the known compound isolated from pollen before,the compound with the same molecular weight represented scutellarin.The molecular mechanisms of component E-1 from camellia pollen on tyrosinase were elucidated by means of enzyme kinetics,spectroscopy techniques,and fluorescent spectroscopy were used for interaction model establishment between inhibitors and tyrosianse.The inhibition of E-1 on the monophenolase of tyrosinase was reversible and belonged to competitive,but it facilitated catalytic action on diphenolase of tyrosinase.Quenching mechanism of tyrosinase by E-1 was influence the microenvironment of the fluorescent groups near the tyrosinase,instead of changing the hydrophobic environment of amino acid residues?tryptophan,et al?with fluorescence quenching in tyrosinase.E-1 could form complexes with tyrosinase by forming hydrogen bonds with residues near the catalytic core domain and hydrophobic associations with residues in the hydrophobic pocket of tyrosinase.And these interactions extended the lag time of tyrosinase for oxidating L-tyrosine and decreased the initial reaction velocity of monophenolase of tyrosinase.