【作者】 商飞；

【导师】 焉翠蔚；

【作者基本信息】 中国海洋大学 ， 生物工程， 2011， 硕士

【摘要】 酪氨酸酶（EC.1.14.18.1）是一种广泛分布于微生物、动植物以及人体当中的与黑色素形成相关的酶。酪氨酸酶在生物体中具有单酚酶活性和二酚酶活性,单酚酶活性能将单酚加氧形成二酚,多酚酶活性能将二酚等多元酚氧化形成醌,这些醌类物质多聚化,再与细胞内蛋白相互作用最后形成黑色素。酪氨酸酶对于生物体黑色素的形成以及外来物入侵时获得性免疫反应的产生有至关重要的作用。从天然海藻中筛选具有高效抑制活性的酪氨酸酶抑制剂可以应用于色素型皮肤病的治疗,研发美白化妆品、生物农药和食品添加剂等。本实验对11种海洋藻类进行了酪氨酸酶抑制活性的筛选,研究了羽藻和缘管浒苔提取物对酪氨酸酶的抑制作用。通过正交试验确定了这两种海藻提取物的最佳提取条件,采用有机溶剂萃取分离出两种海藻提取物的不同组分,通过动力学学方法分析对酪氨酸酶的抑制作用类型和机理,相关研究内容及结果如下:1对11种海洋藻类进行了酪氨酸酶抑制活性的筛选。筛选体系总体积为5mL,底物多巴含量为1.0mmol/L,磷酸缓冲液pH值为6.8,酪氨酸酶液的终浓度为1.28ug/mL,抑制物终浓度为4mg/mL,反应温度为37℃,测试反应时间为1min。结果表明,在4mg/mL的提取物浓度条件下,红藻门的叉枝藻、真江篱、蜈蚣藻、珊瑚藻、鸭毛藻和扇形拟伊藻对酪氨酸酶有不同程度的抑制作用,抑制活性均高于20%。其中叉枝藻和真江篱对酪氨酸酶的抑制率达40%以上,绿藻门的羽藻、孔石莼、缘管浒苔对酪氨酸酶表现出不同程度的抑制作用,其中羽藻的抑制率最高,达78%,其次是缘管浒苔40.24%,红藻门的细枝软骨藻和褐藻门的角叉菜对酪氨酸酶则表现为反向激活作用,激活率分别为18.18%和20.45%。2采用恒温水浴浸提法对羽藻和缘管浒苔的化学成分进行了提取,浸提溶剂为乙醇溶液、浸提料液比为1:12,浸提温度为40℃。首先通过单因素实验确定单个浸提条件的最佳值,然后利用正交实验确定出最佳的浸提条件组合。羽藻的最佳浸提条件组合为:60℃恒温水浴、60%的乙醇浓度、5h的浸提时间和1:14的料液比;缘管浒苔的最佳浸提条件组合为:60℃恒温水浴、60%的乙醇浓度、5h的浸提时间和1:10的料液比。3将两种海藻的乙醇浸提物经有机溶剂萃取依次得到乙酸乙酯萃取物、石油醚萃取物和水溶性组分,分别测定三种组分的抑制活性、抑制类型和多酚含量。结果表明:羽藻石油醚组分对酪氨酸酶的半抑制浓度IC₅₀为2.1mg/mL,属于非竞争性可逆抑制,抑制常数为K_I=0.33;羽藻乙酸乙酯组分对酪氨酸酶的半抑制浓度IC₅₀为,0.27mg/mL,属于非竞争性可逆抑制,抑制常数为K_I=0.13;羽藻水溶性组分对酪氨酸酶的半抑制浓度IC₅₀为1.0mg/mL,属于竞争性可逆抑制,抑制常数为:K_I=0.91。缘管浒苔石油醚萃取物对酪氨酸酶的半抑制浓度IC₅₀为0.29mg/mL,属于竞争性可逆抑制,抑制常数为:K_I=0.28;缘管浒苔乙酸乙酯组分对酪氨酸酶的半抑制浓度IC₅₀为0.19mg/mL,属于非竞争性可逆抑制,抑制常数为K_I=0.08;缘管浒苔水溶性组分对酪氨酸酶的半抑制浓度IC₅₀为7.02mg/mL,属于竞争性可逆抑制,抑制常数为K_I=2.95。羽藻乙酸乙酯组分对酪氨酸酶的抑制作用最强,缘管浒苔乙酸乙酯组分对酪氨酸酶抑制活性最高。更多还原

【Abstract】 Tyrosinase（EC.1.14.18.1） is widely distributed in microorganisms, plants and animals,involving in pigment biosynthesis. Tyrosinase in organisms have single phenolic enzyme activity and poly phenols enzyme activity. Single phenolic enzyme activity can make a single phenol become dipheno; polyphenols enzyme activity can make the polyphenols become quinones. After multimerizing, these quinones finally formed melanin with interacting with intracellular protein. Tyrosinase played an important role in biological melanin formation and make the body acquired immune ability when foreign substance invaded. Efficient tyrosinase inhibitor is acquired by screening natural seaweeds. The tyrosinase inhibitor can apply to developping skin care products, cosmetics, biological pesticides and food additives.In this research 11 species of marine algae were screened .The extracts of Bryopsis plumose and Enteromorpha linza have significant inhibition on tyrosinase. According to orthogonal tests, the optimum extraction conditions were determined; Using organic solvent extracting method , different components of two algaes are obtained. With kinetic analysis method ,the inhibiton mechanism and inhibition type are determined. Results are as follows:1. The screening system is 5mL volume, using 1.0mmol/L DOPA as substrate, the pH of the buffer being 6.8, the enzyme concentration 1.28ug/mL, the inhibitor final concentration 4mg/mL, and the reaction temperature and time being 37℃and 1min. The inhibition results: with the concentration of the extracts being 4mg/mL, Gymnogongrus flabelliformis, Gracilaria asiatica, Grateloupia sp., Cracilaria textorii, Symphyocladia latiuscula, and Ahnfeltiopsis flabelliformis have strong inhibition effects on tyrosinase, the inhibition ratio reaching 20%, the inhibition ratio of Gymnogongrus flabelliformis and Gracilaria asiatica reaching 40%. Bryopsis plumose , Enteromorpha prolifra and Ulva pertusa all have different inhibition effects on tyrosinase, the inhibition ratio of Bryopsis plumose reaching 78%, the inhibition ratio of Enteromorpha prolifra reaching 40.24%, Chondria tenuissima and Gymnogongrus flabelliformis show opposite effects on tyrosinase,—activating the enzyme, the activation ratio benging 18.18% and 20.45%.2. Using constant temperature water extraction method to extract the chemical composition of Bryopsis plumose and Enteromorpha. The extraction solvent is enthanol. The ratio of solid to liquid is 1:12. The extracting temperature is 40℃.First using single factor experiment to confirm the optimum extraction condition of single factor. And then through the orthogonal experiment the best extraction conditions are determined. Bryopsis plumose optimum extraction cinditions are 60℃constant temperature water extraction, 60% concentration of enthanol, 5h exracting time and 1:14 ratio of solid to liquid. Enteromorpha optimum extraction cinditions are 60℃constant temperature water extraction, 60% concentration of enthanol, 5h exracting time and 1:10 ratio of solid to liquid.3. After solvent extracting, the ethanol extract of the seaweeds were separated into three components: Ethyl acetate extract, Petroleum ether extract and water soluble components, and then separately testing the inhibition activity, inhibition type and polyphenol content. The result are as follows: The concentration corresponding to 50% inhibitory rate of Bryopsis plumose Petroleum ether extract was 2.1mg/mL, being noncompetitive reversible inhibitor and K_I being 0.33. Ethyl acetate extract was noncompetitive reversible inhibitor too, and its IC₅₀ was 0.27mg/mL, K_I being 0.13. Water soluble components was competitive reversible inhibitor, and its IC₅₀ and K_I were 1.0mg/mL, 0.91. Enteromorpha Petroleum ether extract was competitive reversible inhibitor, and its IC₅₀ and K_I were 0.29mg/mL and 0.28; Ethyl acetate extract was noncompetitive reversible inhibitor , its IC₅₀ was 0.19mg/mL and K_I was 0.08; Water soluble components was competitive reversible inhibitor, and its IC₅₀ and K_I were 7.02mg/mL, 2.95. Both the Ethyl acetate components of the seaweeds have significant inhibition activity.更多还原