Study on the inhibition to tyrosinase in Xinjiang plants

酪氨酸酶广泛存在于微生物，动植物及人体中，是一种多亚基的含铜的氧化还原酶。酪氨酸酶是合成黑色素的重要酶类，通过改变酪氨酸酶的活性可以控制黑色素生成量，从而对生物体内黑色素水平及相关代谢途径产生影响。 本文以新疆植物为研究对象，从新疆植物中提取了一批对蘑菇酪氨酸酶具有抑制作用的天然活性物质。多种植物都是首次研究，还没有文献报道。在理论上从酶学角度系统阐述了天然活性物质与蘑菇酪氨酸酶的作用机理及其生物活性。 本文经过粗提的植物，再经石油醚，乙酸乙酯，氯仿，正丁醇萃取，得到不同提取层的物质，冻干之后样品进行研究，根据各物质在不同溶剂中的溶解度不同，从而得到有效分离。再对不同有机层的物质进行，进一...Tyrosinase is an important enzyme containing oxido-reductase, existing widely in microorganisms, plants and animals and the human body. Tyrosinase is the key enzyme in organisms about melanin synthesis, controlling melanin content by regulating its activity,thereby affecting the melanin levels in organisms and related metabolic pathway. This paper takes Xinjiang plant as the research object, and...学位：理学硕士院系专业：生命科学学院_生物化学与分子生物学学号：2162012115233

Study of kinetics with respect to action of flavonoid from mulberry leaves on inhibition of activity of tyrosinase

采用超声波辅助法,以体积分数为60%的乙醇水溶液从新疆桑叶粉末中提取桑叶黄酮。以l-多巴为底物,采用酶动力学方法研究该提取物对酪氨酸酶的抑制作用。实验结果表明,桑叶黄酮提取物对酪氨酸酶有较强的抑制作用,其IC50为0.2 g·l-1。其对酪氨酸酶的抑制作用表现为可逆效应,抑制类型为混合型抑制,对游离酶的抑制常数(kI)和对酶-底物络合物的抑制常数(kIS)分别为366和873 Mg·l-1。dPPH实验显示新疆桑叶黄酮提取物具有一定的抗氧化作用,对dPPH自由基氧化作用相对活性的IC50为25 Mg·l-1,与VC效果相当。Flavonoid was extracted from powder of mulberry leaves planted in Xinjiang with 60%( vol) ethanol aqueous solution under assistance of ultrasonic wave.Adopting enzymatic kinetic method,inhibitory action of the extract on tyrosinase activity was studied using L- dopa as the substrate.Results showed that the flavonoid extract displays rather strong inhibitory action on activity of tyrosinase.Its IC50 was estimated as 0.2 g·L- 1.The inhibition mechanism was studied and it was found that the reaction is reversible and type of the inhibition belongs to the mixed type,with the inhibition equilibrium constants for inhibitor binding with free enzyme KI and with enzyme- substrate complex KISwere determined as 366 and 873 mg·L- 1respectively.The DPPH( 1,1-diphenyl-2-picrylhydrazyl) radical test results showed that the flavonoid extract has the antioxidant activity to a certain extent.The IC50 is 25 mg·L- 1that is similar to vitamin C.新疆维吾尔自治区高校科研计划科学研究重点资助项目(XJEDU2012I30); 新疆师范大学校级重点学科招标课题资助项目(XJMB-1

Inhibition of mushroom tyrosinase by flavonoid from Sorbus tianschanica Ruper in Xinjiang

以新疆天山花楸的茎为材料,采用超声波辅助法提取得到天山花楸黄酮类物质。以l-多巴为底物,运用酶动力学方法研究黄酮类物质对酪氨酸酶的抑制作用。结果表明:天山花楸黄酮类物质对酪氨酸酶有较强的抑制作用,导致酪氨酸酶二酚酶活力下降50%所需的提取物浓度(IC50)值为0.27 Mg/M l。对酪氨酸酶的抑制显示可逆的效应,抑制类型为混合型,对游离酶的抑制常数(kI)和酶-底物配位化合物的抑制常数(kIS)分别为0.218和0.313Mg/M l。1,1-二苯基-2-三硝基苯肼自由基(dPPH·)实验显示天山花楸黄酮类物质具有一定的抗氧化作用,引起dPPH·活性下降50%的黄酮类物质浓度(IC50)为0.18 Mg/M l。本研究为进一步开发利用天山花楸中的果蔬保鲜及美白成分提供了理论依据。Flavonoid was extracted from powder of Sorbus tianschanica Ruper stems under assistance of ultrasonic wave.Adopting enzymatic kinetic method,inhibitory action of flavonoid extract on the tyrosinase activity was studied using L-DOPA as substrate.Results showed that flavonoid extract from Sorbus tianschanica Ruper inhibited tyrosinase.Its IC50 was estimated as 0.27 mg / m L.The inhibition mechanism was studied and it was found that the reaction was reversible and type of the inhibition belonged to the mixed-type.The inhibition equilibrium constants for inhibitor binding with free enzyme KI and with enzyme-substrate complex KISwere determined as 0.218 and 0.313 mg / m L,respectively.The DPPH( 1,1-diphenyl-2-picrylhydrazyl) radical test results showed that flavonoid extract from Sorbus tianschanica Ruper had the antioxidant activity to a certain extent and the inhibitor concentration leading to 50% DPPH· free radical activity loss( IC50) was determined to be 0.18 mg /m L.The above study provides a basis for further development of the fruits and vegetables preservatives and whitening ingredient in the Sorbus tianschanica Ruper.国家自然科学基金(31260213); 新疆维吾尔自治区高校科研计划科学研究重点项目(XJEDU2012I30); 新疆维吾尔自治区教育厅重点实验室开放基金项目(XJSD2013-06

Experimental Study of the Abalone Shell on Cataract induced by Oxidative Stress

目的研究石决明提取物对体外培养的晶状体氧化应激性白内障形成的作用及机制。方法离体培养小鼠晶状体,应用不同浓度的石决明提取物预孵育晶状体24h后,加入1mm过氧化氢,继续培养3小时后恢复正常培养,72小时后观察小鼠晶状体混浊程度,测定晶状体组织培养液中的乳酸脱氢酶(LDH)含量,晶状体组织中还原型谷胱甘肽(GSH)含量及超氧化物歧化酶(SOD)活力。结果石决明提取物在1~2 mg/ml浓度范围内减轻氧化应激造成的晶状体混浊,减少晶状体LDH的释放,提高组织内GSH含量和SOD活力。结论石决明提取物可减轻氧化应激白内障的形成,其作用主要与石决明提取物提高内源性抗氧化系统有关。Objective To study the effect of the abalone shell extract on oxidative stress induced cataract formation and its mechanism in cultured mouse lens in vitro. Methods The cultured mouse lens were pretreated with the abalone shell extract in different concentrations for24 hours,and then 1mm hydrogen peroxide was added and continued incubating for 3 hours,and they were changed to normal culture media.After 72 hours,the opacity of each lens was observed under an anatomical microscope,the content of lactate dehydrogenase( LDH)leakages,the content of the reduced glutathione( GSH) and activity of superoxide dismutase( SOD) in lens tissue were assayed. Results Abalone shell extract in the concentration range of 1 ~ 2 mg / ml reduced the lens opacity caused by oxidative stress,alleviated the release of LDH,and increased GSH content and SOD activity in cultured lens. Conclusion Abalone shell extract can alleviate the oxidative stress induced cataract formation,and this effect is mainly related to its improvement of the endogenous antioxidant system in lens.2012年福建省卫生厅中医药科研专项课题(No.WST201210);; 2013年福建省卫生厅中医药科研专项课题(No.wzhw201302);; 2014年厦门市科技局科技惠民项目课题(No.3502Z20144030

罗非鱼hepcidin 1-5成熟肽在毕赤酵母中的表达和活性研究

为获得重组酵母分泌表达的莫桑比克罗非鱼hepcidin并进行其功能研究,将罗非鱼hepcidin 1-5（TH1-5）成熟肽cDNA插入表达载体pPIC9K,获得重组分泌表达质粒pPIC9K-TH1-5,电转化毕赤酵母GS115,阳性克隆经甲醇诱导获得表达上清；表达上清冻干品经Dot-ELISA检测,并分析了表达产物对指示菌和鸡外周血淋巴细胞的影响。结果表明,TH1-5成熟肽可通过表达质粒pPIC9K在毕赤酵母中实现分泌型表达,Dot-ELISA检测表达上清中含有TH1-5。重组表达的TH1-5对大肠埃希菌（CMCC44102）、金黄色葡萄球菌（ATCC25923）、铜绿假单胞菌（ATCC27853）、粪肠球菌（ATCC29212）具有明显的抑菌活性；含重组表达TH1-5的上清冻干品（6.25mg/mL）可协同ConA（0.125&mu;g/mL）或LPS（0.125&mu;g/mL）促进鸡外周血淋巴细胞的增殖。</p

罗非鱼hepcidin 1-5成熟肽在毕赤酵母中的表达和活性研究

为获得重组酵母分泌表达的莫桑比克罗非鱼hepcidin并进行其功能研究,将罗非鱼hepcidin 1-5（TH1-5）成熟肽cDNA插入表达载体pPIC9K,获得重组分泌表达质粒pPIC9K-TH1-5,电转化毕赤酵母GS115,阳性克隆经甲醇诱导获得表达上清；表达上清冻干品经Dot-ELISA检测,并分析了表达产物对指示菌和鸡外周血淋巴细胞的影响。结果表明,TH1-5成熟肽可通过表达质粒pPIC9K在毕赤酵母中实现分泌型表达,Dot-ELISA检测表达上清中含有TH1-5。重组表达的TH1-5对大肠埃希菌（CMCC44102）、金黄色葡萄球菌（ATCC25923）、铜绿假单胞菌（ATCC27853）、粪肠球菌（ATCC29212）具有明显的抑菌活性；含重组表达TH1-5的上清冻干品（6.25mg/mL）可协同ConA（0.125μg/mL）或LPS（0.125μg/mL）促进鸡外周血淋巴细胞的增殖

液-液-液三相萃取苯氧甲基青霉素的研究

液-液-液三相萃取是针对复杂体系分离纯化的一种新技术．本文对比考察了传统两相体系与聚乙二醇-硫酸铵-乙酸丁酯构筑的三相体系对苯氧甲基青霉素滤液萃取行为及效果．重点考察了体系pH对苯氧甲基青霉素在三相萃取体系相间的分配规律。结果表明采用三相体系萃取苯氧甲基青霉素发酵滤液不需加入破乳剂．下相中乙酸丁酯的含量明显降低．而且色素在中间相富集，可以达到纯化有机相，简化工艺流程的效果。三相体系的pH对苯氧甲基青霉素在相间的分配有显著的影响．随着体系pH的降低．上相与下相分配系数Dt/b、上相与中相分配系数Dt/m显著增加．而中相与下相分配系数Dm/b呈缓慢下降趋势；当pH降低到3．0以下时．苯氧甲基青霉素在上相的质量分数达到90％以上．萃取率的变化比较平缓

液-液-液三相萃取苯氧甲基青霉素的研究

液-液-液三相萃取是针对复杂体系分离纯化的一种新技术．本文对比考察了传统两相体系与聚乙二醇-硫酸铵-乙酸丁酯构筑的三相体系对苯氧甲基青霉素滤液萃取行为及效果．重点考察了体系pH对苯氧甲基青霉素在三相萃取体系相间的分配规律。结果表明采用三相体系萃取苯氧甲基青霉素发酵滤液不需加入破乳剂．下相中乙酸丁酯的含量明显降低．而且色素在中间相富集，可以达到纯化有机相，简化工艺流程的效果。三相体系的pH对苯氧甲基青霉素在相间的分配有显著的影响．随着体系pH的降低．上相与下相分配系数Dt/b、上相与中相分配系数Dt/m显著增加．而中相与下相分配系数Dm/b呈缓慢下降趋势；当pH降低到3．0以下时．苯氧甲基青霉素在上相的质量分数达到90％以上．萃取率的变化比较平缓

液-液-液三相萃取苯氧甲基青霉素的研究

液-液-液三相萃取是针对复杂体系分离纯化的一种新技术,本文对比考察了传统两相体系与聚乙二醇-硫酸铵-乙酸丁酯构筑的三相体系对苯氧甲基青霉素滤液萃取行为及效果,重点考察了体系pH对苯氧甲基青霉素在三相萃取体系相间的分配规律。结果表明采用三相体系萃取苯氧甲基青霉素发酵滤液不需加入破乳剂,下相中乙酸丁酯的含量明显降低,而且色素在中间相富集,可以达到纯化有机相,简化工艺流程的效果。三相体系的pH对苯氧甲基青霉素在相间的分配有显著的影响,随着体系pH的降低,上相与下相分配系数Dt/b、上相与中相分配系数Dt/m显著增加,而中相与下相分配系数Dm/b呈缓慢下降趋势;当pH降低到3.0以下时,苯氧甲基青霉素在上相的质量分数达到90%以上,萃取率的变化比较平缓