| Walnut is rich in polyphenols and has better anti-ultraviolet, antioxidant, scavenging free radicals and other function. Apart of polyphenols monomers and extracts can change melanin content by affecting the tyrosinase activity. Melanogenesis has importantly related to chloasma and leukoderma. It is useful to study the effect and mechanism of walnut polyphenols on tyrosinase activity and melanogenesis in order to seek for tyrosinase inhibitors to develop freckle cosmetic materials and drug of anti-leukoderma.The main conclusions of this study were as follows:1. The polyphenol content of extracts from walnut dreg was 9.09%. The IC50 value of extracts from walnut dreg on tyrosinase activity with was 16.2 mg/mL. The extracts from walnut dreg behaved as a non-competitive inhibitor of tyrosinase, and equilibrium constant was 3.97 mg/mL. In addition, the extracts from walnut dreg could reduce ferric ions well and had stronger antioxidant capacity of scavenging DPPH radical, hydroxyl radical, superoxide radical, and ABTS+ radical with ICso values of 64.43 (μg/mL,104.31μg/mL,7.12μ.g/mL,149.45μg/mL, respectively.2. The polyphenol content of extracts from walnut testa was 42.3%, and the extracts were served as a mixed type inhibitor of tyrosinases, and values of IC50, Kis and Ki were 3.74mg/mL,4.67mg/mL and1.76 mg/mL, respectively. The extracts from walnut testa could generate fluorescence quenching of tyrosinase and changed the hydrophobic environment of amino acid residues within tyrosinase by means of fluorescence quenching, which was consistent with the result of ANS-tyrosinase fluorescence. In addition, the extracts from walnut testa exercised a little influence on the structural and conformational changes of tyrosinase with method of circular dichroism spectroscopy.3. The inhibition of salidroside on tyrosinase by enzyme kinetics was 33.04%, and salidroside was also a competitive inhibitor of tyrosinase which was served as a substrate analogue to combine with enzyme to produce quinones with stronger light absorption. Furthermore, salidroside couldn’t react with copper ions located in the active center of tyrosinase. The docking results implied that inhibitory mechanisms might be attributed to the weak interactions between salidroside and tyrosinase such as hydrogen bonds and vander Waals. In conclusion, it could be conjectured that the inhibition ability to inhibitors of tyrosinase was not only related to the similarity of substrates, but also associated with the extent of chelation between inhibitor and copper ions located in the active center of tyrosinase.4. The walnut polyphenol monomers of ferulic acid, caffeic acid and vanillic acid had activation on proliferation in mouse B16 cells. Ferulic acid and caffeic acid mainly increased melanin content through activating tyrosinase activity, while the activation of vanillic acid on the activity of tyrosinase was weak. Morin and p-hydroxybenzoic acid could inhibit the proliferation in mouse B16 cells. Effect of morin on tyrosinase activity in cells was very significant. When the concentration of morin got to 400uM/L, the inhibition rate of tyrosinase activity and melanin content in cells both reached 45.87% and 33.47%, respectively. However, the inhibition of p-hydroxybenzoic acid on tyrosinase activity in mouse B16 cells had no dependent relationship with concentrations, but melanin content presented positive correlation with concentrations of p-hydroxybenzoic acid. The polyphenol content of extracts from walnut leaves, shell, and green husk was 10.26%,13.92%,9.93%, respectively. Walnut extracts all had certain inhibition on melanin content. The extracts from walnut leaves exerted the greatest influence on cell proliferation. The extracts from walnut shell showed lower inhibition melanin content in mouse B16 cells, but had the weakest effect on cell proliferation. The extracts from walnut testa presented the most obvious inhibition on melanin synthesis, and mainly reduced melanin content through inhibiting tyrosinase activity 5. The macula-removing emulsion with walnut polyphenols as the main effective components had been prepared, and meanwhile the experiment of evaluation effectiveness had also been done. The results showed that tyrosinase inhibition rate from macula-removing diluent with walnut polyphenols reached 92.86%, and the DPPH radical scavenging rate also got to 88.46%, which indicated that macula-removing emulsion with walnut polyphenols had better inhibition of melanogenesis and cavenging effect on free radicals. In a word, the macula-removing emulsion with walnut polyphenols was a safe and effective freckle cosmetic. |
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