酪氨酸酶是黑色素生成的主限速酶,抑制酪氨酸酶活力是减少黑色素生成,从而达到美白作用的有效途径之一。该研究以蘑菇酪氨酸酶为靶点,采用酶抑制动力学分析、紫外光谱分析、红外光谱分析、荧光光谱分析、圆二色光谱分析、结合分子对接模拟分析,全面研究了矢车菊素-3-O-葡萄糖苷(cyanidin-3-O-glucoside,C3G)和矢车菊素(cyanidin,Cy)对酪氨酸酶的抑制作用及机理。结果表明,C3G(IC50 =124.8 μmol/L)和Cy(IC50 =278.1 μmol/L)对酪氨酸酶的抑制活性强于阳性对照曲酸(IC50 =536.4 μmol/L)。C3G和Cy对酪氨酸酶的抑制作用都是可逆的,C3G对酪氨酸酶的抑制作用为竞争性抑制,Cy对酪氨酸酶的抑制作用为混合性抑制。紫外、红外和圆二色光谱结果均显示,C3G和Cy与酪氨酸酶结合导致酪氨酸酶的构象发生改变,进而抑制酶的活力。荧光猝灭结果显示,C3G和Cy与酪氨酸酶结合改变Tyr和Trp残基的微环境,从而产生静态猝灭。同步荧光结果显示,C3G和Cy与酪氨酸酶结合使Tyr和Trp残基的微环境极性增强,疏水能力降低。三维荧光光谱结果显示,C3G和Cy都可以使得酪氨酸酶的光谱特征峰强度减弱。多光谱分析结果表明,C3G对酪氨酸酶的抑制作用强于Cy。分子对接模拟结果显示,C3G与酪氨酸酶的氨基酸残基HIS178、ASN174、GLN41、GLN44之间形成6个氢键,与氨基酸残基GLU173、LYS180产生π-π相互作用,Cy与酪氨酸酶结合时与氨基酸残基ASN174、GLU173、GLN41、GLN44之间形成了4个氢键,与氨基酸残基GLU173之间产生π-π相互作用。分子对接结果进一步表明,与Cy相比,C3G与酪氨酸酶活力腔中的残基紧密结合,对酪氨酸酶具有更高的亲和力。进一步补充了酶促动力学和光谱学实验的结论。该研究为C3G和Cy这类多酚类化合物在酪氨酸酶抑制剂领域的进一步研发提供了参考,为开发天然高效酪氨酸抑制剂用于化妆品美白添加剂提供了科学支持。
Tyrosinase is the main rate-limiting enzyme of melanogenesis, and inhibition of tyrosinase activity is one of the effective ways to reduce melanogenesis and thus achieve a whitening effect.In this experiment, the inhibitory effects and mechanisms of cyanidin-3-O-glucoside (C3G) and cyanidin (Cy) on tyrosinase were comprehensively investigated using enzyme inhibition kinetic analysis, ultraviolet spectroscopy, infrared spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking simulation analysis while using mushroom tyrosinase as the target.Results showed that the inhibitory activity of C3G (IC50 =124.8 μmol/L) and Cy (IC50 =278.1 μmol/L) on tyrosinase were better than that of the positive control kojic acid (IC50 =536.4 μmol/L).The inhibitory effect of C3G and Cy on tyrosinase was reversible, and the inhibitory effect of C3G on tyrosinase was competitive inhibition, while the inhibitory effect of Cy on tyrosinase was mixed inhibition.Results of ultraviolet, Fourier transform infrared spectroscopy, and circular dichroism spectra showed that the binding of C3G and Cy to tyrosinase resulted in the conformation change of tyrosinase, which led to the inhibition of the enzyme activity.Fluorescence quenching results showed that C3G and Cy combined with tyrosinase changed the microenvironment of Tyr and Trp residues, resulting in static quenching.Synchronous fluorescence results showed that the combination of C3G and Cy with tyrosinase enhanced the microenvironmental polarity of Tyr and Trp residues and reduced the hydrophobic ability.Three-dimensional fluorescence spectra showed that both C3G and Cy could reduce the intensity of spectral characteristic peaks of tyrosinase.Results of the multispectral analysis showed that the inhibitory effect of C3G on tyrosinase was stronger than that of Cy.Molecular docking simulation results showed that C3G formed six hydrogen bonds with amino acid residues HIS178, ASN174, GLN41, and GLN44 of tyrosinase, and π-π interaction was generated with amino acid residues GLU173 and LYS180.Cy bound to tyrosinase and formed four hydrogen bonds with amino acid residues ASN174, GLU173, GLN41, and GLN44, resulting in π-π interaction with amino acid residues GLU173.Molecular docking results further demonstrated that C3G bound tightly to residues in the active cavity of tyrosinase and had a higher affinity for tyrosinase compared to Cy.The conclusions of the inhibitory enzyme activity experiment and multispectral experiment were further supplemented.This study provides a reference for further research and development of polyphenols such as C3G and Cy in the field of tyrosinase inhibitors and acts as scientific support for the development of natural high-efficiency tyrosine inhibitors for cosmetic whitening additives.
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