【作者】 孙晓梅；

【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,Tyrosinase)是一种结构复杂的多亚基含铜氧化还原酶,广泛存在于微生物、动植物及人体中,是生物体内合成黑色素的关键酶,具有独特的双重催化功能。多年来,对酪氨酸酶抑制剂的研究一直备受国内外学者的关注,因此,筛选酪氨酸酶的天然抑制剂可以为其在医疗美容、食品工业、农业抗虫等多个领域提供理论依据。我国海域辽阔,海洋生物资源丰富,海藻养殖技术居于世界领先水平。真江蓠和孔石莼也是黄海、渤海海域常见的大型经济海藻,其丰富的营养成分和活性物质也被逐渐应用于医疗保健、饲料养殖、生态修复等多个方面。本文深入研究了真江蓠和孔石莼乙醇提取物对酪氨酸酶的抑制作用,通过超声波提取,单因素分析和正交试验设计相结合的方法确定了这两种海藻提取物的最佳提取条件;酶动力学方法系统研究了两种海藻提取物对酪氨酸酶的抑制作用机理和抑制作用类型。相关研究内容及结果如下:1超声波提取真江蓠中酪氨酸酶的抑制活性物质,通过单因素试验与正交试验相结合的方法优化提取条件。最佳提取条件为:超声时间20min,超声功率100w,料液比1g:16mL,乙醇浓度70%。采用有机溶剂萃取真江蓠乙醇提取物,分别得到乙酸乙酯组分、石油醚组分和水溶性组分,酶活性抑制实验发现:真江蓠这三种组分对酪氨酸酶均具有不同程度的抑制作用,半数抑制浓度IC50分别为0.6mg/mL、22.32 mg/mL和7.85 mg/mL,其中,乙酸乙酯组分抑制效果显著。Lineweaver-Burk图显示:真江蓠乙酸乙酯组分、石油醚组分对酪氨酸酶具有非竞争性抑制作用,抑制常数(KI=KIS)分别为0.86 mg/mL和1.96 mg/mL;水溶性组分对酪氨酸酶具有竞争性抑制作用,抑制常数KI为0.81mg/mL。2超声波提取孔石莼中酪氨酸酶抑制活性物质的最佳提取条件为:超声时间8min,超声功率100w,料液比1g:20mL,乙醇浓度70%。有机溶剂萃取实验发现,孔石莼石油醚组分对酪氨酸酶的抑制作用不明显,乙酸乙酯组分和水溶性组分对酪氨酸酶具有一定的抑制作用,IC50分别为1.75mg/mL和11.53mg/mL,乙酸乙酯组分抑制效果显著。动力学分析表明:孔石莼乙酸乙酯组分对酪氨酸酶表现为非竞争性抑制,抑制常数KI=KIS为1.92mg/mL;水溶性组分对酪氨酸酶表现为竞争性抑制,抑制常数KI为1.11mg/mL。3综合两种海藻对酪氨酸酶的抑制作用研究,真江蓠和孔石莼提取物中均含有多种对酪氨酸酶具有抑制作用的活性成分,且抑制作用显著成分都集中在乙酸乙酯萃取物组分,其活性成分可能是同类物质。更多还原

【Abstract】 Tyrosinase(EC 1.14.18.1)is a copper-constaining redoxidase enzyme which has complex constructure, widely distributed in microorganisms, animals, plants and human beings. It is a key enzyme involved in the melanin biosynthesis, which has a unique double catalytic functions. Over these years, the study on tyrosinase inhibitors has been a major concern of domestic and international academics. So, the study of screening new naturally tyrosinase inhibitors would provide a theoretical basement for the medical cosmetology, food industry and agriculture resist insects fields. There are vast sea areas as well as rich marine resources in china, and the seaweed growing techniques are in the world leading level. Gracilaria asiatica and Ulva pertusa kjellm are large economic algae resources rich in the huanghai sea and the bohai sea areas. Their rich nutrition ingredients and active substances had applied to medical care, food culture and ecological restored aspects.This article made a deep research on the tyrosinase inhibition of the ethanol extracts that extracting from Gracilaria asiatica and Ulva pertusa kjellm. The optimum ultrasonic extracting conditions of the substances from the seaweeds were confirmed by single factor and orthogonal experiment; the inhibitory mechanism and types of the extracts on tyrosinase were studied using enzyme kinetic method. The contents and results were as follows.1 The antienzyme components were extracted from Gracilaria asiatica with the ultrasonic technology, using single factor and orthogonal experiment method to optimize the extracting conditions. The optimum extracting conditions were ultrasonic time 20min, ultrasonic power 100w, the liquid-solid ratio 16mL:1g, ethanol concentration 70%. The Gracilaria asiatica ethanol extracts was leached with organic solvents to obtain the ethyl acetate fraction, the petroleum ether fraction and the substance in water. The results showed that all of the three components had different inhibition on the activity of the tyrosinase. The concentration of Gracilaria asiatica extracts corresponding to 50% inhibition (IC50) was 0.6mg/mL, 22.32 mg/mL and 7.85 mg/mL, respectively, the ethyl acetate fraction showed a prominent inhibition. The inhibition kinetics analysis based on the Lineweaver-Burk plots showed that the inhibition of the Gracilaria asiatica ethyl acetate fraction and the petroleum ether fraction belongs to noncompetitive inhibition, the KI(KIS) was 0.86 mg/mL and 1.96 mg/mL, respectively; the substance in water belongs to competitive inhibition, the KI was 0.81mg/mL.2 The optimum extracting conditions with ultrasonic technology to extract the antienzyme components from Ulva pertusa kjellm were ultrasonic time 8min, ultrasonic power 100w, the liquid-solid ratio 20mL:1g, the ethanol concentration 70%. The organic solvents leached experiments showed that the petroleum ether fraction from Ulva pertusa kjellm had no inhibition on the activity of tyrosinase, the ethyl acetate fraction and the substance in water can inhibit the activity of tyrosinase. The concentration of extracts corresponding to 50% inhibition (IC50) was 1.75mg/mL and 11.53mg/mL, respectively, the ethyl acetate fraction showed a prominent inhibition. The Lineweaver-Burk plots showed that the ethyl acetate fraction belongs to noncompetitive inhibition, the KI(KIS) was 1.92mg/mL; the substance in water belongs to competitive inhibition, the KI was 1.11mg/mL.3 Synthesizing the tyrosinase inhibition of the two algaes, the extracts from Gracilaria asiatica and Ulva pertusa kjellm both have different antienzyme active components, and the distinct antienzyme components are both on the ethyl acetate fraction, its active ingredients may be the similar substance.更多还原