酸枣叶缩合单宁的分离鉴定及其对黑色素生成的抑制机制

目的:研究酸枣叶缩合单宁的结构特征及其对酪氨酸酶的效应和黑色素生成的抑制机制。方法:利用Sephadex LH-20凝胶柱色谱技术分离制备酸枣叶缩合单宁纯化物，采用硫醇降解结合高效液相色谱-电喷雾电离质谱（Thiolysis-HPLC-ESI-MS）解析酸枣叶缩合单宁的结构单元组成，运用酶动力学、紫外光谱、荧光猝灭等方法系统研究酸枣叶缩合单宁对酪氨酸酶的抑制作用机理，应用CCK-8法、多巴氧化法、NaOH裂解法和实时荧光定量PCR检测酸枣叶缩合单宁对小鼠黑色素瘤细胞（B16F10）的增殖、酪氨酸酶活性、黑色素合成及其相关基因表达水平的影响。结果:构成酸枣叶缩合单宁的主要结构单元是（表）儿茶素和（表）棓儿茶素；酸枣叶缩合单宁具有较强的酪氨酸酶单酚酶和二酚酶抑制活性，是二酚酶可逆的混合型抑制剂；酸枣叶缩合单宁可有效抑制B16F10细胞的增殖、酪氨酸酶活性、黑色素生成量以及酪氨酸酶（TYR）、酪氨酸酶相关蛋白-1（TRP-1）、小眼畸形相关转录因子（MITF）等基因的表达。结论:通过在酶水平和细胞水平的综合分析，阐明酸枣叶缩合单宁对B16F10细胞黑色素合成的分子抑制机制，为后期酸枣叶缩合单宁开发为新的美白化妆品添加剂和果蔬保鲜剂提供理论依据。

Isolation and Identification of Condensed Tannins from Ziziphus jujuba Leaves and Their Inhibition Mechanism for Melanogenesis

Objective: To explore the structural characteristics of condensed tannins from Ziziphus jujuba leaves and their effect on tyrosinase and the inhibition mechanism for melanogenesis. Methods: The condensed tannins from Z. jujuba leaves were isolated and purified on the basis of Sephadex LH-20 gel column chromatography, and their structural features were identified by thiolysis coupled with high-performance liquid chromatography-electrospray ionization mass spectrometry (Thiolysis-HPLC-ESI-MS). Besides, the inhibitory mechanism of Z. jujuba leaves condensed tannins on tyrosinase was also systematically investigated by enzyme kinetics, UV-vis absorption and fluorescence spectroscopy. Finally, CCK-8 assay, L-DOPA oxidation assay, NaOH cleavage assay and real-time fluorescence quantitative PCR were employed to probe into the effects of Z. jujuba leaves condensed tannins on the proliferation, tyrosinase activity, melanogenesis and the level of melanogenesis-related gene expression of mouse melanoma cells (B16F10). Results: The condensed tannins from Z. jujuba leaves were mainly composed of (epi)catechin and (epi)gallocatechin. Z. jujuba leaves condensed tannins were found to strongly inhibit on both monophenolase and diphenolase of tyrosinase, and the inhibition against diphenolase was proved to be reversible and mixed-type. It was also observed that the condensed tannins from Z. jujuba leaves could effectively inhibit the cell viability, tyrosinase activity, melanin content, as well as the mRNA expression of TYR, TRP-1 and MITF genes in B16F10 cells. Conclusion: Based on the comprehensive analysis of the biological effects of condensed tannins from Z. jujuba leaves at enzyme level and cell level, we could clarify the molecular inhibition mechanism of Z. jujuba leaves condensed tannins on melanogenesis in B16F10 cells. This study would provide a theoretical basis for the later development of Z. jujuba leaves condensed tannins as new whitening cosmetic additives and preservatives for fruits and vegetables.