【作者】 官兴丽；

【导师】 曾亮；

【作者基本信息】 西南大学 ， 茶学， 2011， 硕士

【摘要】 儿茶素是2-苯基苯并吡喃的衍生物,属于黄烷醇类化合物。它大量存在于茶树新稍中,占茶叶干重的12%-24%,是多酚类的主要成分,约为总量的70%-80%。根据结构不同,儿茶素分为表儿茶素(EC)、表没食子儿茶素(EGC)、表儿茶素没食子酸酯(ECG)、表没食子儿茶素没食子酸酯(EGCG)。由于结构中具有连或邻苯酚基,儿茶素具有良好的抗氧化能力,其相同物质的量浓度的儿茶素抗氧化能力依次为L-EGCG>L-EGC>L-ECG>L-EC。黑色素的过速增长或分布不均会造成皮肤局部过黑及色素沉着,从而产生雀斑、老年斑、黑斑病等病症,影响美观。酪氨酸酶(EC,1.14.18.1)又称多酚氧化酶,是一种铜蛋白,其活性中心的双核铜离子在酶催化中起重要作用,是黑色素生成的关键酶。随着化妆品及药品行业的发展,天然产物化妆品及药品顺应了回归大自然的发展潮流和人们的消费需求,将成为化妆品及药品开发中的主题。本文初步探讨儿茶素对黑色素形成的影响,包括儿茶素对马铃薯酪氨酸酶活性的抑制作用；儿茶素对蘑菇酪氨酸酶活性的抑制作用；EGCG对B16小鼠黑素瘤细胞的形态、细胞增殖率、酪氨酸酶活性及黑色素生成量的影响。取得结果如下：一、采用分光光度计法测定酶液加入量、底物浓度、温度、pH值对酶活力的影响,确定最适反应条件。在30℃、pH 6.8的Na2HPO4-NaH2PO4缓冲体系中,采用酶动力学方法研究了儿茶素对马铃薯酪氨酸酶的抑制效应。结果表明：反应最适条件为酶液加入量0.5 mL、底物浓度2 mmol/L、温度30℃、pH 6.8。儿茶素对马铃薯酪氨酸酶有抑制作用,抑制率达到50%的儿茶素质量浓度(IC50)约为0.27 mg/mL; Lineweaver-Burk图显示儿茶素对马铃薯酪氨酸酶的抑制作用表现为竞争型抑制,抑制常数(K1)为0.16 mg/mL。二、在30℃,pH 6.8的Na2HPO4-NaH2PO4缓冲体系中,采用酶动力学方法研究了儿茶素对蘑菇酪氨酸酶单酚酶和二酚酶的抑制效应。结果表明,儿茶素对酪氨酸酶单酚酶和二酚酶均有抑制作用,其半数抑制浓度(IC50)约分别为0.89 mg/mL和1.13 mg/mL; Lineweaver-Burk图显示儿茶素对二酚酶的抑制作用表现为竞争型抑制,抑制常数(K1)为0.53 mg/mL。三、采用倒置显微镜观察EGCG对B16细胞形态的影响,亚甲基蓝法测定EGCG对B16细胞增殖率的影响,L-多巴氧化法测定EGCG对B16细胞酪氨酸酶活力的影响,氢氧化钠裂解法测定EGCG对B16细胞黑色素生成量的影响。结果表明：EGCG作用24 h,高浓度(>60μM)组细胞胞膜融合现象明显；作用72 h,高浓度组大部分细胞死亡。EGCG对B16细胞增殖率、酪氨酸酶活性、黑色素生成量均有显著抑制,呈明显的剂量效应关系；且EGCG抑制B16细胞增殖,IC50为33.99μM。以上研究表明,儿茶素具有抑制体外酪氨酸酶的作用,表现为竞争型抑制；EGCG能够显著抑制B16细胞形态、细胞增殖率、酪氨酸酶活性及黑色素生成量。儿茶素对抑制黑色素形成有一定作用,有望开发成为化妆品及药品。更多还原

【Abstract】 Catechins are 2-phenyl benzopyran derivatives, the compound of flavanol. The large numbers of catechins are in the new shoot of tea tree, which content is 12% to 24% of the dry weight, about 70%-80% of the total tea polyphenols. According to different structures, catechin include epicatechin (EC), epigallocatechin(EGC), epicatechin gallate (ECG), Epigallocatechin gallate(EGCG). As the structure has phenolic base, catechins have excellent antioxidant activity, the same amount of substance concentration catechins of antioxidant activity were L-EGCG> L-EGC> L-ECG> L-EC. Melanin is too fast growth or uneven distributions that lead to black skin and pigmentation, resulting in freckles, age spots, melasmaand other diseases. Tyrosinase (EC,1.14.18.1) is polyphenol oxidase and a copper protein, which the binuclear copper ions play an important role in enzyme catalysis. It is a key enzyme in melanin synthesis. With the development of cosmetics and medicine industry, natural products as cosmetics and medicine conform to the development trend of returning to nature and people’s consumption demand, which will become the subject of cosmetic and medicine development.This article preliminarily study effect of catechins on the formation of melanin, including inhibitory effect of catechins on potato tyrosinase; inhibitory effect of catechins on mushroom tyrosinase; effect of EGCG on the B16 melanoma cell morphology, proliferation rate, tyrosinase activity and melanogenesis. We get the following results:一、The effect of the quantity of enzyme solution, substrate concentation, temperature and pH on the activity of potato tyrosinase was determined by spectrophotometer method. The inhibitory kinetics of catechins on the activity of potato tyrosinase was studied by enzymological kinetic method in a Na2HPO4-NaH2PO4 buffer solution (pH 6.8) and at 30℃. The results showed that the optimal reaction conditions is quantity of enzyme solution 0.5 mL, substrate concentation 2 mmol/L, temperature 30℃, pH 6.8. Catechins can inhibit the activity of potato tyrosinase, and catechins concentration to 50% inhibitory rate (IC50) is 0.27 mg/mL. It was found that the inhibitory effect of catechins on potato tyrosinase is competitive inhibition mode and inhibition constant (KI) of catechins on potato tyrosinase is 0.16 mg/mL by Lineweaver-Burk plots.二、The inhibitory kinetics of catechins on the activity of mushroom tyrosinase monophenolase and diphenolase was studied by enzymological kinetic method in a Na2HPO4-NaH2PO4 buffer solution (pH 6.8) and at 30℃. The results showed that catechins can inhibit both the activity of monophenolase and diphenolase, and catechins concentration to 50% inhibitory rate (IC50) are 0.89 mg/mL and 1.13 mg/mL. It was found that the inhibitory effect of the catechins on diphenolase is in a competitive inhibition mode, and inhibition constant (KI) is 0.53 mg/mL by Lineweaver-Burk plots.三、Inverted microscope was used to observe B16 cell morphology; methylene bluemethod, L-DOPA oxidation method, NaOH decomposition method respectively was used to study the effect of EGCG on B16 cell proliferation rate, tyrosinase activity and melanogenesis. The results showed that EGCG act on the B16 at 24 h, the high concentration (>60μM) appeared the phenomenon of cell membrane fusion; after 72 h, most of cell were death at this group. EGCG have significantly inhibited B16 on cell proliferation rate, tyrosinase activity, melanogenesis, which is obvious dose-response relationship. And EGCG have inhibited B16 on cell proliferation, catechins concentration to 50% inhibitory rate (IC50) is 33.99μM.These studies have shown that catechins can inhibit tyrosinase in vitro, showed competitive inhibition; EGCG could significantly inhibit the B16 murine melanoma cell morphology, cell proliferation rate, tyrosinase activity and melanogenesis. Catechins have a certain role on the inhibition of melanin synthesis, which is expected to develop as cosmetics and medicines.更多还原