【作者】 李银花；

【导师】 刘仲华； 黄建安；

【作者基本信息】 湖南农业大学 ， 药用植物资源工程， 2013， 博士

【摘要】 EGCG是茶树中具有重要生理功能的次级代谢产物。(-)-表没食子儿茶素-3-0(3-O-甲基)没食子酸酯(EGCG3"Me)和(-)-表没食子儿茶素-3-O(4-O-甲基)没食子酸酯(EGCG4"Me)作为EGCG甲基化衍生物,研究表明其具有高效的抗过敏功能,尤其是抗花粉过敏表现出的优势引起了日本产业界的高度重视,也掀起了研究甲基化EGCG的热潮。花粉症在一些国家发病率较高,日本发病率超过1/3。目前治疗过敏性疾病的药物多为化学合成药,尚未发现有比茶叶中的EGCG3"Me和EGCG4"Me对过敏,尤其是花粉症过敏更显著功效的纯植物药品,国际医药与保健品行业均认为甲基化EGCG非常有可能发展为一个独立茶树品种,成为新的儿茶素经济增长点而为社会带来巨额财富。本论文立足于上述研究背景,以富含甲基化EGCG的茶树资源为材料,研究了茶树中甲基化EGCG的分析检测方法,分离纯化技术及其化学合成方法,全长克隆和分析了甲基化EGCG生物合成相关基因,并采用原核表达和体外酶试验对候选基因进行了功能验证,为茶树甲基化EGCG生物合成的调控及甲基化EGCG高效利用提供了有价值的理论依据与技术基础。主要研究结果如下：建立了同时测定茶叶中8种儿茶素(EGC、DL-C、EC、EGCG、 GCG、EGCG3"Me、 EGCG4"Ne、ECG)、3种生物碱(茶碱、可可碱、咖啡碱)和没食子酸的高效液相色谱方法。色谱条件如下：采用C18色谱柱；流动相为磷酸缓冲液-乙腈溶液,梯度洗脱；波长278nm；流速1.0mL/min;柱温30℃。该方法除了可以测定两种甲基化EGCG外,还能检测茶叶中其他成分,对茶树资源的筛选、茶叶甲基化EGCG的分离纯化等提供了强有力的分析工具。采用柱色谱和反相高效制备液相色谱相结合的方法从茶叶中分离制备两种甲基化EGCG (EGCG4"Me和EGCG3"Me)。用含水的乙酸乙酯为溶剂提取茶叶,粗提液浓缩后经HP-20柱色谱分离,以不同浓度的酒精为洗脱剂,收集80%酒精洗脱组分再经制备液相色谱分离,以水和乙腈为流动相进行梯度洗脱分离两种甲基化EGCG单体。经紫外光谱、质谱、核磁共振分析以及与文献比对,确定两种甲基化EGCG分别为EGCG4"Me和EGCG3"Me,纯度在98%以上。以硫酸二甲酯为甲基化试剂、EGCG为底物、丙酮为溶剂,采用化学方法合成甲基化EGCG。利用制备型高效液相色谱和超临界流体色谱对合成的粗产物进行分离,得到3种甲基化EGCG单体。经紫外光谱、核磁共振、质谱分析以及与文献比对,确定3种甲基化EGCG分别为EGCG4"Me、3’,4"-di-O-methyl-EGCG、4’,4"-di-O-methyl-EGCG,且EGCG4"Me为主要产物。所制备的3种甲基化EGCG的纯度在98%以上。利用SMART RACE技术成功克隆了甲基化EGCG合成酶(CsCOMT)的全长cDNA序列。CsCOMT基因的cDNA全长1040bp,ORF为738bp编码245个氨基酸。经过相关的生物信息学分析,CsCOMT氨基酸残基数为245,理论等电点PI5.39,分子量27553.6(Da),属于亲水性蛋白,CsCOMT蛋白氨基酸序列中可能不存在卷曲螺旋结构,存在跨膜结构域,属于跨膜蛋白,其磷酸化位点有5个,非均匀的分布在整个多肽链中。克隆甲基化EGCG合成酶基因的全长cDNA,对于改良茶树品种资源具有良好的现实意义。采用原核表达和体外酶试验法,对甲基化EGCG合成酶基因功能进行了初步验证。采用中间质粒pMD18-CsCOMT,成功构建了茶树CsCOMT蛋白的重组质粒pET-28a-CsC0MT,并转入表达载体BL21,经IPTG诱导实现了重组蛋白的表达,表达蛋白的分子量大约为28kD,其分子量与预测的一致。以EGCG为底物,利用纯化的重组蛋白进行体外酶促试验,经HPLC分析检测,成功诱导出甲基化EGCG。采用相对定量的分析方法,分析了同一茶树品种—芽二叶及第三、四叶、不同品种同一时期、同一品种不同采摘时期中的CsCOMT基因表达量。更多还原

【Abstract】 EGCG is a kind of secondary metabolite which has important physiological function in tea.(-)-epigallocatechin-3-O(3-O-methyl) gallate(EGCG3"Me) and (-)-epigallocatechin-3-O(4-O-methyl) gal late (EGCG4"Me) are O-methylated derivatives of EGCG. The experimental results show that they have potent anti-allergic effect, especially anti-pollen hypersensitivity, Japanese industrial circles have pay more attention to their advantages. Allergy has been defined as a disease of excessive immune activity, the morbidity of allergy is very high in some countries and increases with the development of civilization, for example in Japan, the morbidity of allergy is estimated to be more than30%. Now people often use chemosynthetic medicine to heal allergic disease, they have misgivings about the use of anti-allergic medicine because of mounting medical expenses and side effects, but researchers have not found other pure plant drugs whose function of anti-allergy, especially anti-pollen allergy are more than EGCG3"Me and EGCG4"Me in tea leaves. Health product and international medical industries think it is highly possible for methylated EGCG to be developed into a single variety resources and consequently become to be a new economic growth point for catechins which will bring about great wealth to the society. In this study, HPLC method was established for determination of methylated EGCG from tea leaves, methylated EGCG was isolated and purified, genes which related to the property of methylated EGCG were cloned and their functions were analyzed preliminarily. The results may show theoretical and technical support for regulating biosynthesis of metylated EGCG. The main results were as follows:A analytic method was developed to determine eight kinds of catechins, three kinds of purine alkaloids and gallic acid in tea by HPLC-PDA. The optimum condition was as follows:Welchrom Cis column; mobile phase:phosphate buffer-acetonitrile; flow rate:1.0mL/min; wavelength:278nm; column temperature:30℃. This method showed good linearity among injection and peak area. The HPLC method I have developed offers a kind of modern analytical method for screening tea germplasm resource and monitoring the course of separation and purification for methylated EGCG in tea.A separation method was established to separate and purify EGCG4"Me and EGCG3"Me in tea leaves by using column chromatography and preparative high performance liquid chromatography. Crude extracts were obtained from tea leaves by using water and ethyl acetate as extraction solvent. Then the concentracted crude extract was separated on a HP-20column by using different concentration of ethyl alcohol as eluant. The Fr-1fraction was obtained from the eluate of80%ethyl ethanol, then concentrated and subjected to a preparative high performance liquid chromatography for isolation of target components by using water-acetonitrile as mobile phase. Two monomeric compounds were acquired and identified. They were EGCG4"Me and EGCG3"Me.The purity of them was more than98%.A chemical method was established to synthesize methylated EGCG by using dimethyl sulfate as methylated reagent. Methylated EGCG product was separated by using preparative high performance liquid chromatography and supercritical fluid chromatography.Three kinds of monomeric compounds were acquired and identified. They were EGCG4"Me,3’,4"-di-O-methyl-EGCG and4’,4"-di-O-methyl-EGCG, the main product is EGCG4"Me. The purity of them was more than98%.Methylated EGCG synthase gene from tea was cloned. The full-length cDNA of methylated EGCG synthase gene was1040bp and open reading frame was738bp. By bioinformatics analysis, the amino acid residues base of methylated EGCG synthase gene was245, theoretic isoelectric point was5.39, molecular weight was27553.6Da. It is a kind of hydrophilic protein and transmenbrane protein. It has realistic meaning for improving tea cultivars resources by cloning the full-length cDN A of methylated EGCG synthase gene.The function of methylated EGCG synthase gene was confirmed by enzymatic test in vitro and Tua Prokaryotic Expression. Methylated EGCG synthase protein recombinant plasmid was constructed from pET-28-CsCOMT in tea plant. A recombinant protein in the expressive vector BL21was induced by IPTG From recombinant protein enzymatic test in vitro, the result shows methylated EGCG was induced successfully.A relative quantification’s analytic technique was used to analyze CsCOMT gene relative expression level.更多还原