茶树中甲基化EGCG的分离纯化与相关基因的克隆及其表达研究
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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。
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.
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。
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.
引文
[1]宛晓春主编.茶叶生物化学[M].中国农业出版社.北京,2003,07
[2]Kakegawa H, Matsumoto H, Endo K, el al. Inhibitory effect of tannins on hyaluronidase activation and on the degradation from rat mesenterymast cells[J]. Chemical & Pharmaceutical Bulletin.1985,33(11):5079-5082
[3]Ohmori Y, Ito M, Kishi M, et al. Antiallergic constituents from oolong tea stem [J]. Biological & Pharmaceutical Bulletin.1995,18(5):683-686
[4]Wagner H. Search for new plant constituents with potential antiphlogistic and antiallergic activity[J]. Planta Medical.1989,55(3):235-241
[5]Matsuo N, Yamada K, Shoji K, et al. Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells:the structure-inhibitory activity relationship[J]. Allergy.1997,52(1):58-64
[6]Shiozaki T, Sugiyama K, Nakazato K, et al. Effect of tea extracts,catechin and caffeine against type-I allergic reaction[J]. Journal of the Pharmaceutical Society of Japan. 1997,117(7):448-454.
[7]Khan SG, Katiyar SK, Agarwal R, et al. Enhancement of antioxidant and phase Ⅱ enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice:Possible role in cancer chemoprevention[J]. Cancer Research.1992,52:4050-4052
[8]Serafini M, Ghiselli A, Ferro Luzzi A. In vivo antioxidant effect of green and black tea in man[J]. European Journal of Clinical Nutrition.1996,50(1):28-32
[9]Sano M, Takahashi Y, Yoshino K, et al. Effect of tea(Camellia sinensis L.) on lipid peroxidation in rat liver and kidney:a comparison of green tea and black tea feeding[J]. Biological & Pharmaceutical Bulletin.1995,18(7):1006-1008
[10]Yoshino K, Tomita I, Sano M, et al. Effects of long-term dietary supplement of tea polyphenols on lipid peroxide levels in rats[J]. Age.1994,17(3):79-85
[11]Ho CT, Chen Q, Shi H, et al. Antioxidative effect of polyphenol extract prepared from various Chinese teas [J]. Preventive Medicine.1992,21(4):520-525
[12]Yoshino K, Hara Y, Sano M, et al. Antioxidative effects of black tea theaflavins and thearubigin on lipid peroxidation of rat liver homogenates induced by tertbutyl hydroperoxide [J]. Biological & Pharmaceutical Bulletin.1994,17(1):146-149
[13]Katiyar SK, Agarwal R, Mukhtar H. Inhibition of spontaneous and photo-enhanced lipid peroxidation in mouse epidermal microsomes by epicatechin derivatives from green tea[J]. Cancer Letters.1994,79(1):61-66
[14]Gow-Chin Yen, Hui-Yin Chen. Antioxidant Activity of Various Tea Extracts in Relation to Their Antimutagenicity[J]. Journal of Agricultural and Food Chemistry.1995, 43(1):27-32
[15]Wang ZY, Cheng SJ, Zhou ZC, et al. Antimutagenic activity of green tea polyphenols[J]. Mutation Research.1989,223(3):273-285
[16]Jain AK, Shimoi K, Nakamura Y, et al. Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and intragastric tract of rats[J]. Mutation Research.1989,210(1):1-8
[17]Wang ZY, Huang MT, Ferraro T, et al. Inhibitory effect of green tea in the drinking water on tumorigenesisby ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in skin of SKH-1 mice[J]. Cancer Research.1992,52(5):1162-1170
[18]Shiraki M, Hara Y, Osawa T, et al. Antioxidative and antimutagenic effects of theaflavins from black tea[J]. Mutation Research.1994,323(1-2):29-34.
[19]Yamane T, Hagiwara N, Tateishi M, et al. Inhibition of azoxymethane-induced colon carcinogenesisin rat by green tea polyphenol fraction[J]. Japanese Journal of Cancer Research.1991,82(12):1336-1339
[20]秦杰,郑祥毅,王云彬,等.没食子儿茶素没食子酸酯对人膀胱癌T24细胞凋亡的影响[J].杭州师范学院学报(医学版).2008,28(1)：5-8
[21]Nihal M, Ahmad N, Mukhtar H, et al. Antiproliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate on human melanoma:possible implications for the chemoprevention of melanoma[J]. International Journal of Cancer.2005,114(4):513-521
[22]Naasani I, Oh-Hashi F, Oh-Hara T, et al. Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo[J]. Cancer Research.2003,63(4):824-830
[23]Yang F, Oz HS, Barve S, et al. The green tea polyphenol (-)-epigallocatechin-3-gallate blocks nuclear factor-kappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6 [J]. Molecular Pharmacology.2001,60(3):528-533
[24]Bhimani RS, Troll W, Grunberger D, et al. Inhibition of oxidative stress in Hela cells by chemoproventive agents[J]. Cancer Research.1993,53(19):4528-4533
[25]Miura S, Watanabe J, Tomita T, et al. The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low-density lipoprotein[J]. Biological & Pharmaceutical Bulletin.1994,17(12):1567-1572
[26]Sakanaka S, Juneia LR, Taniquchi M. Antimicrobial effects of green tea polyphenols on thermophilic spore-forming bacteria [J]. Journal of bioscience and bioengineering.2000,90(1):81-85
[27]华德兴,许晓玲,彭青,等.表儿茶素没食子酸酯与四环素协同抗金黄色葡萄球菌作用及机制[J].中国新药与临床杂志.2009,28(12)：935-939
[28]Yokozawa T, Oura H, Sakanaka S, et al. Depressor effect of tannin in green tea on rats with rental hypertension[J]. Bioscience, Biotechnology and Biochemistry.1994, 58:855-858
[29]Murase T, Nagasawa A, Suzuki J, et al. Beneficial effects of tea catechins on diet-induced obesity:stimulation of lipid catabolism in the liver[J]. International Journal of Obesity.2002,26(11):1459-1464
[30]Matsumoto N, Okushio K, Hara Y. Effect of black tea polyphenols on plasma lipids in cholesterol-fed rats [J]. Journal of nutritional Science and Vitaminology (Toyko).1998, 44(2):337-342
[31]Yang TTC, Koo MWL. Hypocholesterolemic effects of Chinese tea[J]. Pharmacological Research.1997,35(6):505-512
[32]肖潇,杨占秋,石丽桥,等.表没食子儿茶索没食子酸酯抗流感病毒作用的研究[J].中国中药杂志.2008,33(22)：2678-2682
[33]Sano M, Suzuki M, Miyase T, et al. Novel anti-allergic catechin derivatives isolated from oolong tea[J]. Journal of Agricultural and Food Chemistry.1999,47(5):1906-1910
[34]Maeda-Yamamoto M, Sano M, Maisuda N, et al. The Change of Epigallocatechin-3-O-(3-O-methyl) gallate Content in Tea of Different Varieties, Tea Seasons of Crop and Processing Method[J]. Journal of the Japanese Society for Food Science and Technology.2001,48(1):64-68
[35]吕海鹏,谭俊峰,林智.茶树种质资源EGCG3"Me含量及其变化规律研究[J].茶叶科学.2006,26(4)：310-314
[36]罗正飞,龚正礼,汪毅,等.茶叶中甲基化EGCG的研究[J].西南大学学报. 2008,30(3)：56-59
[37]唐娜,刘仲华,李银花.湖南地区高含量甲基化EGCG茶树资源的调查[J].农产品加工·创新版.2010,209(5)：36-38
[38]Sano M, TabataM, Suzuki M, et al. Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection[J]. Analyst.2001,126(6):816-820
[39]Maeda-Yamamoto M, Nagai H, Asai K, et al. Changes in Epigallocatechin-3-O-(3-O-methyl) Gallate and Strictinin Contents of Tea (Camellia sinensis L.) Cultivar 'Benifuki' in Various Degrees of Maturity and Leaf Order[J]. Food Science and Technology Research.2004,10(2):186-190
[40]Bing Hu, Lin Wang, Bei Zhou, et al. Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection[J]. Journal of chromatography A.2009,1216(15):3223-3231
[41]孙业良,吕海鹏,林智,等.茶叶中EGCG3"Me的分析方法研究[J].茶叶科学.2009,29(5)：379-384
[42]Amarowicz R, Maryniak A, Shahid F. TLC Separation of Methylated (-)-Epigallocatechin-3-Gallate[J]. Czech Journal of Food Sciences.2005,23(1):36-39
[43]雷宇佳,高明哲,王莉,等.制备型高效液相色谱法分离纯化川西獐芽菜提取物中的龙胆苦苷[J].色谱.2010,28(9)：902-904
[44]刘霞,唐骁爽,汪永强,等.制备型高效液相色谱法制备和厚朴酚与厚朴酚[J].中成药.2010,32(11)：2006-2008
[45]王传金,魏运洋,吕晶.制备型高效液相色谱法分离冬凌草中冬凌草甲素[J].精细化工,2007,24(7)：662-666
[46]田娜,刘仲华,黄建安,等.高效制备液相法从荷叶中分离制备黄酮类化合物[J].色谱.2007,25(1)：88-92
[47]Jankowski V, Meyer AA, Schlattman P, et al. Increased uridine adenosine tetraphosphate concentrations in plasma of juvenile hypertensives [J]. Arteriosclerosis.Thrombosis and Vascular Biology.2007,27(8):1776-1781
[48]Hagelin G, Oulie I, Raknes A, et al. Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex-a family of cyclic peptide antibiotics from Bacillus subtilis[J]. Journal of Chromatography B.2004, 811(2):243-245
[49]Sano M, Suzuki M, Miyase T, et al. Novel anti-allergic catechin derivatives isolated from oolong tea[J]. Journal of Agricultural and Food Chemistry.1999, 47(5):1906-1910
[50]周蓓,王琳,李伟,等.茶叶中甲基化儿茶素的分离、纯化和高效液相色谱法分析[J].分析化学.2008,36(4)：494-498
[51]龚正礼,罗正飞,杨坚,等.从茶叶中制备甲基化儿茶素的工艺方法：中国,101177422A[P].2008-05-14
[52]罗正飞,金孝芳,龚正礼,等.高速逆流色谱分离纯化EGCG3"Me的研究[J].分析测试学报.2011,30(7)：813-816
[53]Yanase E, Matsumoto E, Shinoda Y, et al. Synthesis of Methyl Derivatives of Epigallocatechin Gallate (EGCg) and Their Stabilities[J]. ITE Letters on Batteries, New Technologies & Medicine (with News).2005,6(1):34-37
[54]Meng XF, Sang SM, Zhu N, et al. Identification and Characterization of Methylated and Ring-Fission Metabolites of Tea Catechins Formed in Humans, Mice, and Rats[J]. Chemical Research in Toxicology.2002,15(8):1042-1050
[55]吕海鹏,孙业良,林智,等.表没食子儿茶素没食子酸酯的甲基化分子修饰[J].食品科学.2010,31(15)：139-142
[56]Utenova B T, Malterud K E, Rise F. Antioxidant activity of O-protected derivatives of (-)-epigallocatechin-3-gallate:inhibition of soybean and rabbit 15-lipoxygenases[J]. Archive for Organic Chemistry.2007,9:6-16
[57]伍妍俊,汪小钢,宛晓春.甲基化EGCG的合成及其在人工模拟胃肠液中的稳定性[J].安徽农业大学学报.2010,37(4)：688-691
[58]Aihara Y, Yoshida A, Furuta T, et al. Regioselective synthesis of methylated epigallocatechin gallate via nitro benzenesulfonyl (Ns) protecting group[J]. Bioorganic and Medicinal Chemistry Letters.2009,19(15):4171-4174
[59]Sheng BiaoWan, Q Ping Dou, Tak Hang Chan. Regiospecific and enantioselective synthesis of methylated metabolites of tea catechins[J]. Tetrahedron.2006, 62(25):5897-5904
[60]田铃,嵇保中,刘曙雯,等.甲基转移酶的功能与分类[J].生命的化学.2007, 27(5)：425-427
[61]Lambert JD, Sang S, Yang CS. Biotransformation of Green Tea Polyphenols and the Biological Activities of Those Metabolites[J]. Molecular Pharmaceutics.2007, 4(6):819-825
[62]Axelrod J. Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines[J]. Pharmacological Reviews.1966,18(1):95-113
[63]Axelrod J, Tomchick R. Enzymatic O-methylation of epinephrine and other catechols[J]. Journal of Biological Chemistry.1958,233(3):702-705
[64]Guldberg HC, Marsden CA. Catechol-O-Methyl Transferase:Pharmacological Aspects and Physiological Role[J]. Pharmacological Reviews.1975,27(2):135-206
[65]Zhu BT, Conney AH. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis[J]. Cancer Research.1998,58(11):2269-2277
[66]赵晓苗,谢梅青.儿茶酚-O-甲基转移酶基因多态性与妇科肿瘤发生风险的研究进展[J].现代妇产科进展.2005,14(5)：403-405
[67]Lakhani N J, Sarkar M A, Venitz J, et al.2-Methoxyestradiol, a promising anticancer agent[J]. Phamacotherapy.2003,23(3):165-172
[68]Zhu BT, Wang P, Nagai M, et al. Inhibition of human catechol-O-methyltransferase (COMT)-mediated O-methylation of catechol estrogens by major polyphenolic components present in coffee[J]. Journal of Steroid Biochemistry and Molecular Biology. 2009,113(1/2):65-74
[69]Salminen M, Lundstrom K, Tilgmann C, et al. Molecular cloning and characterization of rat liver catechol-O-methyltransferase [J]. Gene.1990,93(2):241-247
[70]Bertocci B, Miggiano V, Da Prada M, et al. Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form[J]. Proceedings of the National Academy Sciences of the United States of America.1991,88(4):1416-1420
[71]Lundstrom K, Salminen M, Jalanko A, et al. Cloning and characterization of human placental catechol-O-methyltransferase cDNA[J]. DNA and cell biology.1991, 10(3):181-189
[72]闰小华,邓小敏,吴怀安.高效液相色谱法测定人体红细胞中儿茶酚氧位甲基转移酶的活性[J].色谱.2001,19(3)：230-232
[73]林翠鸿,王长连,黄品芳,等.高效液相色谱-电化学法测定人红细胞儿茶酚氧 位甲基转移酶活性[J].中国医院药学杂志.2008,28(13)：1082-1085
[74]Nissinen E, Mannisto P. Determination of catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection[J]. Analytical Biochemistry.1984,137(1):69-73
[75]Schultz E, Nissnen E, Kaakkola S. Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection[J]. Biomedical Chromatography.1989,3(2):64-67
[76]Assicot M, Bohuon C. A simple and rapid fluorimetric determination of catechol-O-methyl transferase (COMT) activity[J]. Life Sciences.1969,8(2):93-100
[77]Smit NPM, Pavel S, Kammeyer A, et al. Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection[J]. Analytical Biochemistry.1990, 190(2):286-291
[78]Zurcher G, Da Prada M. Rapid and sensitive single-step radiochemical assay for catechol-O-methyltransferase[J]. Journal of Neurochemistry.1982,38(1):191-195
[79]Lautala P, Ulmanen I, Taskinen J. Radiochemical high-performance liquid chtomatographic assay for the determination of catechol O-methyltransferase activity towards various substrates[J]. Journal of Chromatography B.1999,736(1-2):143-151
[80]Bugos RC,Chiang VL,Campbell WH.cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen[J].Plant Molecular Biology.1991,17(6):1203-1215
[81]山本万里,切田雅信,佐见学.编码甲基化儿茶素生物合成酶的基因：中国.101061222A[P].2007-10-24
[82]Kirita M,Honma D, Tanaka Y, et al.Cloning of a novel O-methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity[J]. Journal of Agricultural and Food Chemistry.2010,58(12):7196-7201
[83]费冬梅,林智,吕海鹏,等.EGCG-O-甲基转移酶(EOMT)在重组大肠杆菌中的表达条件研究.茶叶科学.2011,31(4)：333-340
[84]吕海鹏,费冬梅,张悦,等.EGCG-O-甲基转移酶(EOMT)催化EGCG形成的EGCG 甲基化衍生物分析[J].茶叶科学.2012,32(2)：100-106
[85]Okushio K,Suzuki M,Matsumoto N,Nanjo F,HaraY.Methylation of tea catechins by rat liver homogenates[J]. Bioscience,biotechnology,and biochemistry.1999, 63(2):430-432
[86]Lu H,Meng XF,Yang CS.Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate[J]. Drug Metabolism and Disposition.2003,31(5):572-579
[87]Zhu BT, Patel UK,Cai MX, et al. O-Methylation of tea polyphenols catalyzed by human placental cytosolic catechol-O-methyltransferase[J]. Drug metabolism and disposition:the biological fate of chemicals.2000,28(9):1024-1030
[88]Kida K, Suzuki M, Matsumoto N, et al. Identification of biliary metabolites of (-)-epigallocatechin gallate in rats[J]. Jouranl of Agricultural and Food Chemistry.2000, 48(9):4151-4155
[89]Maeda-Yamamoto M, Ema K, Monobe M, et al. The efficacy of early treatment of seasonal allergic rhinitis with Benifuuki green tea containing O-methylated catechin before pollen exposure:an open randomized[J]. Allergology International.2009, 58(3):437-444
[90]Maeda-Yamamoto M, Kawamoto K, Matsuda N, et al. Anti-allergic catechins of tea(Camellia Sienesis)[J]. Animal Cell Technology:Basic & Applied Aspects.2002, 12:415-419
[91]Suzuki M, Yoshino K, Maeda-Yamamoto M, et al. Inhibitory effects of tea catechins and O-Methylated derivatives of (-)-epigallocatechin-3-O-gallate on mouse type IV allergy[J]. Journal of Agricultural and Food Chemistry.2000,48(11):5649-5653
[92]Maeda-Yamamoto M, Inagaki N, Kitaura J, et al. O-Methylated Catechins from Tea Leaves Inhibit Multiple Protein Kinases in Mast Cells[J]. Journal of Immunology. 2004,1 (72):4486-4492
[93]Tachibaba H, Sunada Y, Miyase T, et al. Identification of a methylated tea catechin as inhibitor of degranulation in human basophilic KU812 cells[J]. Biosciences, Biotechnology and Biochemistry.2000,64(2):452-454
[94]Fujimura Y, Tachibana H, Maeda-Yamamoto M, et al. Antiallergic Tea Catechin,(-)-Epigallocatechin-3-O-(3-O-methyl)-gallate,Suppresses FcεRI Expression in Human Basophilic KU812 Cells[J]. Journal of Agricultural and Food Chemistry.2002, 50(20):5729-5734
[95]Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, et al. Alterations of Na+ Homeostasis in hepatocyte reoxygenation injury [J]. Biochimica et Biophysica Acta. 2000,1500(3):297-305
[96]Kim JS, Southard JH. Membrane stabilizing effects of calcium and taxol during the cold storage of isolated rat hepatocytes [J]. Transplantation.1999,68(7):938-943
[97]Yreugdenhil PK, Rankin MA, Southard JH. Cold storage sensitizes hepatocytes to oxidative stress injury[J].Transplation International:official journal of the European Society for Organ Transplantation.1997,10(15):379-385
[98]Siraki AG, Pourahmad J, Chan TS, et al. Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes [J]. Free Radical Biology & Medicine.2002,32(1):2-10
[99]Kagaya N, Hara Y, Saijo R, et al. Novel Function of rare catechin, epigallocatechin-3-(3"-O-methyl) gallate, against cold injury in primary rat hepatocytes[J]. Journal of Bioscience and Bioengineering.2003,96(6):559-653
[100]Ya-Lun Su, Jin-Ze Xu, Chi Ho, et al. Antioxidant activity of Tea Theflavins and Methylated catechins in Canole oil[J]. Journal of the American Oil Chemists'Society. 2004,81(3):269-273
[101]Kawase M, Rong Wang, Shiomi T, et al. Antioxidative activity of (-)-epigallocatechin-3-(3"-O-methyl) gallate isolated from fresh tea leaf and preliminary results on its biological activity [J]. Bioscience, Biotechnology, and Biochemistry.2000, 64(10):2218-2220
[102]Landis-Piwowar KR, Wan SB, Wieqand RA, el al. Methylation suppresses the proteasome-inhibitory function of green tea polyphenols [J]. Journal of Cellular Physiology.2007,213(1):252-260
[103]Moncada S, Palmer RM, Higgs EA. Nitric oxide:Physiology,pathology and pharmacology [J]. Pharmacol ReV.1991,43(2):109-142
[104]Saleh D, Ernst P, Lim S, et al. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase:effect of inhaled glucocorticoid[J]. FASEB Journal:official Publication of the Federation of American Societies for Experimental Biology.1998,12(11):929-937
[105]Taylor-Robinson AW, Liew FY, Severn A, et al. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells[J]. European Journal of Immunology.1994,24(4):980-984
[106]Chiu FL, Lin JK. HPLC Analysis of Naturally Occurring Methylated Catechins,3-and 4-Methyl-epigallocatechin Gallate, in Various Fresh Tea Leaves and Commercial Teas and Their Potent Inhibitory Effects on Inducible Nitric Oxide Synthase in Macrophages[J]. Journal of Agricultural and Food Chemistry.2005,53 (18):7035-7042
[107]Inagaki H, Sugitani M,Setoguchi Y, et al. Effective suppression of adipose tissue weigh gain in high-fat diet-fed mice by'Benifuuki'green tea consumption[J] Journal of the Japanese Society for Food Science and Technology,2009.56(7):403-411
[108]Oritani Y,Matsui Y,Kurita I, et al. Mechanism of anti-obese effects'Benifuuki'of green tea[J].Journal of the Japanese Society for Food Science and Technology.2009, 56(7):412-418
[109]Kurita I, Maeda-Yamamoto M, Tachibana H, et al. Antihypertensive effect of Benifuuki tea containing O-methylated EGCG[J]. Journal of agricultural and food chemistry.2010,58(3):1903-1908
[110]Maeda-Yamamoto M, Nagai H, Suzuki Y, et al. Manufacturing Method for Low Caffeine 'Benifuuki','Banihomare'Green Tea remaining the Contents of Anti-Allergic Compounds[J] Journal of the Japanese Society for Food Science and Technology.2004, 51(8):435-439
[111]Ikeda I, Kobayashi M, Hamada T, et al. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechingallate[J]. Journal of Agricultural and Food Chemistry.2003,51(25):7303-7307
[112]于世林主编.高效液相色谱方法及应用[M].化学工业出版社.北京,2000,01
[113]陈鸳谊,李行诺,张翠萍,等.高效制备液相色谱在天然产物分离中的应用[J].药学进展.2010,34(8)：337-343
[114]张锐,刘建群,刘丽艳,等.制备高效液相色谱从竹根中制备苜蓿素的研究[J].中草药.2010,41(12)：1985-1987
[115]高明哲,袁晓艳,肖红斌.制备型高效液相色谱法从积雪草提取物中分离纯化积雪草甙和羟基积雪草甙对照品[J].色谱.2008,26(3)：362-365
[116]袁黎明编著.制备色谱技术及应用[M].化学工业出版社.北京,2004
[117]金灯萍,彭国平,陆晓峰.填充柱超临界流体色谱在中药中的分析与制备应用[J].中医药学刊.2006,24(3)：541-542
[118]郭亚东.超临界流体色谱在药物分析中的应用[J].天然产物研究与开发.2001,13(3)：61-64
[119]庞斐,吕惠生,刘岩,等.超临界流体色谱纯化青蒿素的研究[J].高效化学工程学报.2010,24(4)：569-573
[120]申京宇,尹花仙,沈在漠.超临界流体萃取和高效液相色谱同时测定鸡肉中4种氟喹诺酮类药物残留[J].食品科技.2007,10：210-212
[121]戴涛,黄珺珺,季红,等.超临界流体色谱拆分齐留通对映体[J].华西药学杂志.2012,27(1)：67-69
[122]刘志敏,赵锁奇,王仁安,等.黄酮醇异构体的超临界流体色谱法分离[J].色谱.1997,15(4)：288-291
[123]武洁,冯芳.超临界流体色谱法分离手性药物[J].药学进展.2004,28(7)：300-304
[124]任其龙,苏宝根,黄梅,等.超临界流体色谱法分离手性化合物的进展[J].分析化学评述与进展.2000,28(6)：772-776
[125]徐明仙,高伟亮,金丽霞,等.拟除虫菊酯类农药在超临界流体色谱中的对映体分离[J].农药.2011,50(7)：492-497
[126]王海燕,刘焕云,韩帅.茶多酚在冷却牛肉保鲜中的应用研究[J].河北科技大学学报.2011,32(2)：197-200
[127]谭明杰,张春香.冷冻干燥法茶多酚微胶囊的制备及其在烟用复合咀棒中的应用研究[J].食品工业.2011,8,57-59
[128]王文渊,唐守勇,黄光文,等.茶多酚的超滤分离及其在化妆品中的应用[J].日用化学工业.2011,41(2)：109-112
[129]杨贤强,汪叔雄.天然抗氧化剂茶多酚在油脂和食品保鲜中的应用[J].浙江农业大学学报.1995,21(5)：513-518
[130]山本万李,切田雅信,佐见学.编码甲基化儿茶素生物合成酶的基因：中国,101061222A[P].2007-10-24
[131]李娟,刘硕谦,刘仲华,黄建安*.一种改良的茶树高质量RNA快速提取方法.福建茶叶.2007,4：34-35
[2]Kakegawa H, Matsumoto H, Endo K, el al. Inhibitory effect of tannins on hyaluronidase activation and on the degradation from rat mesenterymast cells[J]. Chemical & Pharmaceutical Bulletin.1985,33(11):5079-5082
[3]Ohmori Y, Ito M, Kishi M, et al. Antiallergic constituents from oolong tea stem [J]. Biological & Pharmaceutical Bulletin.1995,18(5):683-686
[4]Wagner H. Search for new plant constituents with potential antiphlogistic and antiallergic activity[J]. Planta Medical.1989,55(3):235-241
[5]Matsuo N, Yamada K, Shoji K, et al. Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells:the structure-inhibitory activity relationship[J]. Allergy.1997,52(1):58-64
[6]Shiozaki T, Sugiyama K, Nakazato K, et al. Effect of tea extracts,catechin and caffeine against type-I allergic reaction[J]. Journal of the Pharmaceutical Society of Japan. 1997,117(7):448-454.
[7]Khan SG, Katiyar SK, Agarwal R, et al. Enhancement of antioxidant and phase Ⅱ enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice:Possible role in cancer chemoprevention[J]. Cancer Research.1992,52:4050-4052
[8]Serafini M, Ghiselli A, Ferro Luzzi A. In vivo antioxidant effect of green and black tea in man[J]. European Journal of Clinical Nutrition.1996,50(1):28-32
[9]Sano M, Takahashi Y, Yoshino K, et al. Effect of tea(Camellia sinensis L.) on lipid peroxidation in rat liver and kidney:a comparison of green tea and black tea feeding[J]. Biological & Pharmaceutical Bulletin.1995,18(7):1006-1008
[10]Yoshino K, Tomita I, Sano M, et al. Effects of long-term dietary supplement of tea polyphenols on lipid peroxide levels in rats[J]. Age.1994,17(3):79-85
[11]Ho CT, Chen Q, Shi H, et al. Antioxidative effect of polyphenol extract prepared from various Chinese teas [J]. Preventive Medicine.1992,21(4):520-525
[12]Yoshino K, Hara Y, Sano M, et al. Antioxidative effects of black tea theaflavins and thearubigin on lipid peroxidation of rat liver homogenates induced by tertbutyl hydroperoxide [J]. Biological & Pharmaceutical Bulletin.1994,17(1):146-149
[13]Katiyar SK, Agarwal R, Mukhtar H. Inhibition of spontaneous and photo-enhanced lipid peroxidation in mouse epidermal microsomes by epicatechin derivatives from green tea[J]. Cancer Letters.1994,79(1):61-66
[14]Gow-Chin Yen, Hui-Yin Chen. Antioxidant Activity of Various Tea Extracts in Relation to Their Antimutagenicity[J]. Journal of Agricultural and Food Chemistry.1995, 43(1):27-32
[15]Wang ZY, Cheng SJ, Zhou ZC, et al. Antimutagenic activity of green tea polyphenols[J]. Mutation Research.1989,223(3):273-285
[16]Jain AK, Shimoi K, Nakamura Y, et al. Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and intragastric tract of rats[J]. Mutation Research.1989,210(1):1-8
[17]Wang ZY, Huang MT, Ferraro T, et al. Inhibitory effect of green tea in the drinking water on tumorigenesisby ultraviolet light and 12-O-tetradecanoylphorbol-13-acetate in skin of SKH-1 mice[J]. Cancer Research.1992,52(5):1162-1170
[18]Shiraki M, Hara Y, Osawa T, et al. Antioxidative and antimutagenic effects of theaflavins from black tea[J]. Mutation Research.1994,323(1-2):29-34.
[19]Yamane T, Hagiwara N, Tateishi M, et al. Inhibition of azoxymethane-induced colon carcinogenesisin rat by green tea polyphenol fraction[J]. Japanese Journal of Cancer Research.1991,82(12):1336-1339
[20]秦杰,郑祥毅,王云彬,等.没食子儿茶素没食子酸酯对人膀胱癌T24细胞凋亡的影响[J].杭州师范学院学报(医学版).2008,28(1)：5-8
[21]Nihal M, Ahmad N, Mukhtar H, et al. Antiproliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate on human melanoma:possible implications for the chemoprevention of melanoma[J]. International Journal of Cancer.2005,114(4):513-521
[22]Naasani I, Oh-Hashi F, Oh-Hara T, et al. Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo[J]. Cancer Research.2003,63(4):824-830
[23]Yang F, Oz HS, Barve S, et al. The green tea polyphenol (-)-epigallocatechin-3-gallate blocks nuclear factor-kappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6 [J]. Molecular Pharmacology.2001,60(3):528-533
[24]Bhimani RS, Troll W, Grunberger D, et al. Inhibition of oxidative stress in Hela cells by chemoproventive agents[J]. Cancer Research.1993,53(19):4528-4533
[25]Miura S, Watanabe J, Tomita T, et al. The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu2+ mediated oxidative modification of low-density lipoprotein[J]. Biological & Pharmaceutical Bulletin.1994,17(12):1567-1572
[26]Sakanaka S, Juneia LR, Taniquchi M. Antimicrobial effects of green tea polyphenols on thermophilic spore-forming bacteria [J]. Journal of bioscience and bioengineering.2000,90(1):81-85
[27]华德兴,许晓玲,彭青,等.表儿茶素没食子酸酯与四环素协同抗金黄色葡萄球菌作用及机制[J].中国新药与临床杂志.2009,28(12)：935-939
[28]Yokozawa T, Oura H, Sakanaka S, et al. Depressor effect of tannin in green tea on rats with rental hypertension[J]. Bioscience, Biotechnology and Biochemistry.1994, 58:855-858
[29]Murase T, Nagasawa A, Suzuki J, et al. Beneficial effects of tea catechins on diet-induced obesity:stimulation of lipid catabolism in the liver[J]. International Journal of Obesity.2002,26(11):1459-1464
[30]Matsumoto N, Okushio K, Hara Y. Effect of black tea polyphenols on plasma lipids in cholesterol-fed rats [J]. Journal of nutritional Science and Vitaminology (Toyko).1998, 44(2):337-342
[31]Yang TTC, Koo MWL. Hypocholesterolemic effects of Chinese tea[J]. Pharmacological Research.1997,35(6):505-512
[32]肖潇,杨占秋,石丽桥,等.表没食子儿茶索没食子酸酯抗流感病毒作用的研究[J].中国中药杂志.2008,33(22)：2678-2682
[33]Sano M, Suzuki M, Miyase T, et al. Novel anti-allergic catechin derivatives isolated from oolong tea[J]. Journal of Agricultural and Food Chemistry.1999,47(5):1906-1910
[34]Maeda-Yamamoto M, Sano M, Maisuda N, et al. The Change of Epigallocatechin-3-O-(3-O-methyl) gallate Content in Tea of Different Varieties, Tea Seasons of Crop and Processing Method[J]. Journal of the Japanese Society for Food Science and Technology.2001,48(1):64-68
[35]吕海鹏,谭俊峰,林智.茶树种质资源EGCG3"Me含量及其变化规律研究[J].茶叶科学.2006,26(4)：310-314
[36]罗正飞,龚正礼,汪毅,等.茶叶中甲基化EGCG的研究[J].西南大学学报. 2008,30(3)：56-59
[37]唐娜,刘仲华,李银花.湖南地区高含量甲基化EGCG茶树资源的调查[J].农产品加工·创新版.2010,209(5)：36-38
[38]Sano M, TabataM, Suzuki M, et al. Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection[J]. Analyst.2001,126(6):816-820
[39]Maeda-Yamamoto M, Nagai H, Asai K, et al. Changes in Epigallocatechin-3-O-(3-O-methyl) Gallate and Strictinin Contents of Tea (Camellia sinensis L.) Cultivar 'Benifuki' in Various Degrees of Maturity and Leaf Order[J]. Food Science and Technology Research.2004,10(2):186-190
[40]Bing Hu, Lin Wang, Bei Zhou, et al. Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection[J]. Journal of chromatography A.2009,1216(15):3223-3231
[41]孙业良,吕海鹏,林智,等.茶叶中EGCG3"Me的分析方法研究[J].茶叶科学.2009,29(5)：379-384
[42]Amarowicz R, Maryniak A, Shahid F. TLC Separation of Methylated (-)-Epigallocatechin-3-Gallate[J]. Czech Journal of Food Sciences.2005,23(1):36-39
[43]雷宇佳,高明哲,王莉,等.制备型高效液相色谱法分离纯化川西獐芽菜提取物中的龙胆苦苷[J].色谱.2010,28(9)：902-904
[44]刘霞,唐骁爽,汪永强,等.制备型高效液相色谱法制备和厚朴酚与厚朴酚[J].中成药.2010,32(11)：2006-2008
[45]王传金,魏运洋,吕晶.制备型高效液相色谱法分离冬凌草中冬凌草甲素[J].精细化工,2007,24(7)：662-666
[46]田娜,刘仲华,黄建安,等.高效制备液相法从荷叶中分离制备黄酮类化合物[J].色谱.2007,25(1)：88-92
[47]Jankowski V, Meyer AA, Schlattman P, et al. Increased uridine adenosine tetraphosphate concentrations in plasma of juvenile hypertensives [J]. Arteriosclerosis.Thrombosis and Vascular Biology.2007,27(8):1776-1781
[48]Hagelin G, Oulie I, Raknes A, et al. Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex-a family of cyclic peptide antibiotics from Bacillus subtilis[J]. Journal of Chromatography B.2004, 811(2):243-245
[49]Sano M, Suzuki M, Miyase T, et al. Novel anti-allergic catechin derivatives isolated from oolong tea[J]. Journal of Agricultural and Food Chemistry.1999, 47(5):1906-1910
[50]周蓓,王琳,李伟,等.茶叶中甲基化儿茶素的分离、纯化和高效液相色谱法分析[J].分析化学.2008,36(4)：494-498
[51]龚正礼,罗正飞,杨坚,等.从茶叶中制备甲基化儿茶素的工艺方法：中国,101177422A[P].2008-05-14
[52]罗正飞,金孝芳,龚正礼,等.高速逆流色谱分离纯化EGCG3"Me的研究[J].分析测试学报.2011,30(7)：813-816
[53]Yanase E, Matsumoto E, Shinoda Y, et al. Synthesis of Methyl Derivatives of Epigallocatechin Gallate (EGCg) and Their Stabilities[J]. ITE Letters on Batteries, New Technologies & Medicine (with News).2005,6(1):34-37
[54]Meng XF, Sang SM, Zhu N, et al. Identification and Characterization of Methylated and Ring-Fission Metabolites of Tea Catechins Formed in Humans, Mice, and Rats[J]. Chemical Research in Toxicology.2002,15(8):1042-1050
[55]吕海鹏,孙业良,林智,等.表没食子儿茶素没食子酸酯的甲基化分子修饰[J].食品科学.2010,31(15)：139-142
[56]Utenova B T, Malterud K E, Rise F. Antioxidant activity of O-protected derivatives of (-)-epigallocatechin-3-gallate:inhibition of soybean and rabbit 15-lipoxygenases[J]. Archive for Organic Chemistry.2007,9:6-16
[57]伍妍俊,汪小钢,宛晓春.甲基化EGCG的合成及其在人工模拟胃肠液中的稳定性[J].安徽农业大学学报.2010,37(4)：688-691
[58]Aihara Y, Yoshida A, Furuta T, et al. Regioselective synthesis of methylated epigallocatechin gallate via nitro benzenesulfonyl (Ns) protecting group[J]. Bioorganic and Medicinal Chemistry Letters.2009,19(15):4171-4174
[59]Sheng BiaoWan, Q Ping Dou, Tak Hang Chan. Regiospecific and enantioselective synthesis of methylated metabolites of tea catechins[J]. Tetrahedron.2006, 62(25):5897-5904
[60]田铃,嵇保中,刘曙雯,等.甲基转移酶的功能与分类[J].生命的化学.2007, 27(5)：425-427
[61]Lambert JD, Sang S, Yang CS. Biotransformation of Green Tea Polyphenols and the Biological Activities of Those Metabolites[J]. Molecular Pharmaceutics.2007, 4(6):819-825
[62]Axelrod J. Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines[J]. Pharmacological Reviews.1966,18(1):95-113
[63]Axelrod J, Tomchick R. Enzymatic O-methylation of epinephrine and other catechols[J]. Journal of Biological Chemistry.1958,233(3):702-705
[64]Guldberg HC, Marsden CA. Catechol-O-Methyl Transferase:Pharmacological Aspects and Physiological Role[J]. Pharmacological Reviews.1975,27(2):135-206
[65]Zhu BT, Conney AH. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis[J]. Cancer Research.1998,58(11):2269-2277
[66]赵晓苗,谢梅青.儿茶酚-O-甲基转移酶基因多态性与妇科肿瘤发生风险的研究进展[J].现代妇产科进展.2005,14(5)：403-405
[67]Lakhani N J, Sarkar M A, Venitz J, et al.2-Methoxyestradiol, a promising anticancer agent[J]. Phamacotherapy.2003,23(3):165-172
[68]Zhu BT, Wang P, Nagai M, et al. Inhibition of human catechol-O-methyltransferase (COMT)-mediated O-methylation of catechol estrogens by major polyphenolic components present in coffee[J]. Journal of Steroid Biochemistry and Molecular Biology. 2009,113(1/2):65-74
[69]Salminen M, Lundstrom K, Tilgmann C, et al. Molecular cloning and characterization of rat liver catechol-O-methyltransferase [J]. Gene.1990,93(2):241-247
[70]Bertocci B, Miggiano V, Da Prada M, et al. Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form[J]. Proceedings of the National Academy Sciences of the United States of America.1991,88(4):1416-1420
[71]Lundstrom K, Salminen M, Jalanko A, et al. Cloning and characterization of human placental catechol-O-methyltransferase cDNA[J]. DNA and cell biology.1991, 10(3):181-189
[72]闰小华,邓小敏,吴怀安.高效液相色谱法测定人体红细胞中儿茶酚氧位甲基转移酶的活性[J].色谱.2001,19(3)：230-232
[73]林翠鸿,王长连,黄品芳,等.高效液相色谱-电化学法测定人红细胞儿茶酚氧 位甲基转移酶活性[J].中国医院药学杂志.2008,28(13)：1082-1085
[74]Nissinen E, Mannisto P. Determination of catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection[J]. Analytical Biochemistry.1984,137(1):69-73
[75]Schultz E, Nissnen E, Kaakkola S. Determination of catechol-O-methyltransferase activity in erythrocytes by high performance liquid chromatography with electrochemical detection[J]. Biomedical Chromatography.1989,3(2):64-67
[76]Assicot M, Bohuon C. A simple and rapid fluorimetric determination of catechol-O-methyl transferase (COMT) activity[J]. Life Sciences.1969,8(2):93-100
[77]Smit NPM, Pavel S, Kammeyer A, et al. Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection[J]. Analytical Biochemistry.1990, 190(2):286-291
[78]Zurcher G, Da Prada M. Rapid and sensitive single-step radiochemical assay for catechol-O-methyltransferase[J]. Journal of Neurochemistry.1982,38(1):191-195
[79]Lautala P, Ulmanen I, Taskinen J. Radiochemical high-performance liquid chtomatographic assay for the determination of catechol O-methyltransferase activity towards various substrates[J]. Journal of Chromatography B.1999,736(1-2):143-151
[80]Bugos RC,Chiang VL,Campbell WH.cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen[J].Plant Molecular Biology.1991,17(6):1203-1215
[81]山本万里,切田雅信,佐见学.编码甲基化儿茶素生物合成酶的基因：中国.101061222A[P].2007-10-24
[82]Kirita M,Honma D, Tanaka Y, et al.Cloning of a novel O-methyltransferase from Camellia sinensis and synthesis of O-methylated EGCG and evaluation of their bioactivity[J]. Journal of Agricultural and Food Chemistry.2010,58(12):7196-7201
[83]费冬梅,林智,吕海鹏,等.EGCG-O-甲基转移酶(EOMT)在重组大肠杆菌中的表达条件研究.茶叶科学.2011,31(4)：333-340
[84]吕海鹏,费冬梅,张悦,等.EGCG-O-甲基转移酶(EOMT)催化EGCG形成的EGCG 甲基化衍生物分析[J].茶叶科学.2012,32(2)：100-106
[85]Okushio K,Suzuki M,Matsumoto N,Nanjo F,HaraY.Methylation of tea catechins by rat liver homogenates[J]. Bioscience,biotechnology,and biochemistry.1999, 63(2):430-432
[86]Lu H,Meng XF,Yang CS.Enzymology of methylation of tea catechins and inhibition of catechol-O-methyltransferase by (-)-epigallocatechin gallate[J]. Drug Metabolism and Disposition.2003,31(5):572-579
[87]Zhu BT, Patel UK,Cai MX, et al. O-Methylation of tea polyphenols catalyzed by human placental cytosolic catechol-O-methyltransferase[J]. Drug metabolism and disposition:the biological fate of chemicals.2000,28(9):1024-1030
[88]Kida K, Suzuki M, Matsumoto N, et al. Identification of biliary metabolites of (-)-epigallocatechin gallate in rats[J]. Jouranl of Agricultural and Food Chemistry.2000, 48(9):4151-4155
[89]Maeda-Yamamoto M, Ema K, Monobe M, et al. The efficacy of early treatment of seasonal allergic rhinitis with Benifuuki green tea containing O-methylated catechin before pollen exposure:an open randomized[J]. Allergology International.2009, 58(3):437-444
[90]Maeda-Yamamoto M, Kawamoto K, Matsuda N, et al. Anti-allergic catechins of tea(Camellia Sienesis)[J]. Animal Cell Technology:Basic & Applied Aspects.2002, 12:415-419
[91]Suzuki M, Yoshino K, Maeda-Yamamoto M, et al. Inhibitory effects of tea catechins and O-Methylated derivatives of (-)-epigallocatechin-3-O-gallate on mouse type IV allergy[J]. Journal of Agricultural and Food Chemistry.2000,48(11):5649-5653
[92]Maeda-Yamamoto M, Inagaki N, Kitaura J, et al. O-Methylated Catechins from Tea Leaves Inhibit Multiple Protein Kinases in Mast Cells[J]. Journal of Immunology. 2004,1 (72):4486-4492
[93]Tachibaba H, Sunada Y, Miyase T, et al. Identification of a methylated tea catechin as inhibitor of degranulation in human basophilic KU812 cells[J]. Biosciences, Biotechnology and Biochemistry.2000,64(2):452-454
[94]Fujimura Y, Tachibana H, Maeda-Yamamoto M, et al. Antiallergic Tea Catechin,(-)-Epigallocatechin-3-O-(3-O-methyl)-gallate,Suppresses FcεRI Expression in Human Basophilic KU812 Cells[J]. Journal of Agricultural and Food Chemistry.2002, 50(20):5729-5734
[95]Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, et al. Alterations of Na+ Homeostasis in hepatocyte reoxygenation injury [J]. Biochimica et Biophysica Acta. 2000,1500(3):297-305
[96]Kim JS, Southard JH. Membrane stabilizing effects of calcium and taxol during the cold storage of isolated rat hepatocytes [J]. Transplantation.1999,68(7):938-943
[97]Yreugdenhil PK, Rankin MA, Southard JH. Cold storage sensitizes hepatocytes to oxidative stress injury[J].Transplation International:official journal of the European Society for Organ Transplantation.1997,10(15):379-385
[98]Siraki AG, Pourahmad J, Chan TS, et al. Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes [J]. Free Radical Biology & Medicine.2002,32(1):2-10
[99]Kagaya N, Hara Y, Saijo R, et al. Novel Function of rare catechin, epigallocatechin-3-(3"-O-methyl) gallate, against cold injury in primary rat hepatocytes[J]. Journal of Bioscience and Bioengineering.2003,96(6):559-653
[100]Ya-Lun Su, Jin-Ze Xu, Chi Ho, et al. Antioxidant activity of Tea Theflavins and Methylated catechins in Canole oil[J]. Journal of the American Oil Chemists'Society. 2004,81(3):269-273
[101]Kawase M, Rong Wang, Shiomi T, et al. Antioxidative activity of (-)-epigallocatechin-3-(3"-O-methyl) gallate isolated from fresh tea leaf and preliminary results on its biological activity [J]. Bioscience, Biotechnology, and Biochemistry.2000, 64(10):2218-2220
[102]Landis-Piwowar KR, Wan SB, Wieqand RA, el al. Methylation suppresses the proteasome-inhibitory function of green tea polyphenols [J]. Journal of Cellular Physiology.2007,213(1):252-260
[103]Moncada S, Palmer RM, Higgs EA. Nitric oxide:Physiology,pathology and pharmacology [J]. Pharmacol ReV.1991,43(2):109-142
[104]Saleh D, Ernst P, Lim S, et al. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase:effect of inhaled glucocorticoid[J]. FASEB Journal:official Publication of the Federation of American Societies for Experimental Biology.1998,12(11):929-937
[105]Taylor-Robinson AW, Liew FY, Severn A, et al. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells[J]. European Journal of Immunology.1994,24(4):980-984
[106]Chiu FL, Lin JK. HPLC Analysis of Naturally Occurring Methylated Catechins,3-and 4-Methyl-epigallocatechin Gallate, in Various Fresh Tea Leaves and Commercial Teas and Their Potent Inhibitory Effects on Inducible Nitric Oxide Synthase in Macrophages[J]. Journal of Agricultural and Food Chemistry.2005,53 (18):7035-7042
[107]Inagaki H, Sugitani M,Setoguchi Y, et al. Effective suppression of adipose tissue weigh gain in high-fat diet-fed mice by'Benifuuki'green tea consumption[J] Journal of the Japanese Society for Food Science and Technology,2009.56(7):403-411
[108]Oritani Y,Matsui Y,Kurita I, et al. Mechanism of anti-obese effects'Benifuuki'of green tea[J].Journal of the Japanese Society for Food Science and Technology.2009, 56(7):412-418
[109]Kurita I, Maeda-Yamamoto M, Tachibana H, et al. Antihypertensive effect of Benifuuki tea containing O-methylated EGCG[J]. Journal of agricultural and food chemistry.2010,58(3):1903-1908
[110]Maeda-Yamamoto M, Nagai H, Suzuki Y, et al. Manufacturing Method for Low Caffeine 'Benifuuki','Banihomare'Green Tea remaining the Contents of Anti-Allergic Compounds[J] Journal of the Japanese Society for Food Science and Technology.2004, 51(8):435-439
[111]Ikeda I, Kobayashi M, Hamada T, et al. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechingallate[J]. Journal of Agricultural and Food Chemistry.2003,51(25):7303-7307
[112]于世林主编.高效液相色谱方法及应用[M].化学工业出版社.北京,2000,01
[113]陈鸳谊,李行诺,张翠萍,等.高效制备液相色谱在天然产物分离中的应用[J].药学进展.2010,34(8)：337-343
[114]张锐,刘建群,刘丽艳,等.制备高效液相色谱从竹根中制备苜蓿素的研究[J].中草药.2010,41(12)：1985-1987
[115]高明哲,袁晓艳,肖红斌.制备型高效液相色谱法从积雪草提取物中分离纯化积雪草甙和羟基积雪草甙对照品[J].色谱.2008,26(3)：362-365
[116]袁黎明编著.制备色谱技术及应用[M].化学工业出版社.北京,2004
[117]金灯萍,彭国平,陆晓峰.填充柱超临界流体色谱在中药中的分析与制备应用[J].中医药学刊.2006,24(3)：541-542
[118]郭亚东.超临界流体色谱在药物分析中的应用[J].天然产物研究与开发.2001,13(3)：61-64
[119]庞斐,吕惠生,刘岩,等.超临界流体色谱纯化青蒿素的研究[J].高效化学工程学报.2010,24(4)：569-573
[120]申京宇,尹花仙,沈在漠.超临界流体萃取和高效液相色谱同时测定鸡肉中4种氟喹诺酮类药物残留[J].食品科技.2007,10：210-212
[121]戴涛,黄珺珺,季红,等.超临界流体色谱拆分齐留通对映体[J].华西药学杂志.2012,27(1)：67-69
[122]刘志敏,赵锁奇,王仁安,等.黄酮醇异构体的超临界流体色谱法分离[J].色谱.1997,15(4)：288-291
[123]武洁,冯芳.超临界流体色谱法分离手性药物[J].药学进展.2004,28(7)：300-304
[124]任其龙,苏宝根,黄梅,等.超临界流体色谱法分离手性化合物的进展[J].分析化学评述与进展.2000,28(6)：772-776
[125]徐明仙,高伟亮,金丽霞,等.拟除虫菊酯类农药在超临界流体色谱中的对映体分离[J].农药.2011,50(7)：492-497
[126]王海燕,刘焕云,韩帅.茶多酚在冷却牛肉保鲜中的应用研究[J].河北科技大学学报.2011,32(2)：197-200
[127]谭明杰,张春香.冷冻干燥法茶多酚微胶囊的制备及其在烟用复合咀棒中的应用研究[J].食品工业.2011,8,57-59
[128]王文渊,唐守勇,黄光文,等.茶多酚的超滤分离及其在化妆品中的应用[J].日用化学工业.2011,41(2)：109-112
[129]杨贤强,汪叔雄.天然抗氧化剂茶多酚在油脂和食品保鲜中的应用[J].浙江农业大学学报.1995,21(5)：513-518
[130]山本万李,切田雅信,佐见学.编码甲基化儿茶素生物合成酶的基因：中国,101061222A[P].2007-10-24
[131]李娟,刘硕谦,刘仲华,黄建安*.一种改良的茶树高质量RNA快速提取方法.福建茶叶.2007,4：34-35