茶黄素制备与纯化条件的初步研究
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摘要
本文研究一条以成本较低且多酚含量较高的绿茶粉为原料,利用生物酶法制备TFs并采用大孔吸附脂树脂法纯化TFs的工艺。工艺流程主要分为多酚氧化酶(PPO)酶源物质的筛选：提取梨、苹果等七种果蔬中PPO并测定其活性,选择活性高的果蔬作为酶源物质；不同金属离子对PPO活性的影响：测定不同浓度的金属离子对PPO活性的影响,选择PPO的激活剂；制备TFs的反应条件的优化选择：对TFs的合成条件做单因素试验和正交试验,选择最佳条件；纯化TFs的条件的优化选择。
(1)选择梨、苹果、马铃薯、茶鲜叶、茄子、甘薯和藕等七种不同的果蔬为酶源物质,测定其PPO活性。通过对其活性的比较,筛选活性最高者作为制备茶黄素的最佳酶源。各种酶源物质活性依次为：茄子中PPO的酶活为174U；马铃薯中PPO的酶活为101.43U；甘薯中PPO的酶活为58U；梨中PPO的酶活为37U；藕中PPO的酶活为32U；苹果中PPO的酶活为24.67U；茶鲜叶中PPO的酶活为10U。选择茄子中PPO作为最佳酶源。
(2)选择铜离子、锰离子、镁离子、钙离子、钠离子、铝离子和铁离子七种离子作为研究对象,通过在粗酶液中加入不同浓度的七种金属离子,研究七种金属离子对茄子PPO活性的影响。研究表明,浓度为3mmol/L的铜离子对PPO有显著激活作用,而钙离子、钠离子、铝离子和铁离子对PPO则有抑制作用。其中,浓度为5mmol/L的铁离子是PPO的最佳抑制剂。在此条件下PPO的活性可被抑制96.6%。
(3)通过对合成TFs的不同条件(pH值、温度、底物浓度、酶添加量和反应时间)的研究,在单因素试验和正交试验的基础上,确定合成TFs的最佳条件。结果表明,反应体系的最佳条件为：pH值为4.5,温度为25℃,底物浓度为2.5g/L,酶添加量为35m1,反应时间为40分钟。其中,pH值和底物浓度是反应体系中重要的两个因素。
(4)采用大孔吸附树脂法对在最佳条件下制备到得的TFs进行纯化,通过对不同吸附条件(树脂型号、pH值、上样速度、除杂用洗脱液浓度、洗脱液体积和洗脱液浓度)的选择,确定纯化TFs的最佳工艺条件。结果表明,纯化的最佳条件为：pH值为3,上样速度为2BV/h,除杂用洗脱液浓度为15%洗脱液体积为3BV,洗脱液浓度为90%。pH值和上样速度是影响纯化效果的重要因素。
This article reported a new process applied bio-enzyme method that green tea p owder with low cost and high polyphenol as raw material to synthesize theaflavins, then macroporous adsorptive resin was done to purify theaflavins in earlier studies. The technological process was divided into polyphenol oxidase(PPO) selection, extra cted PPO from seven plants such as pear、apple and so on and measured their acti vity, choose the material with highest activity as the best enzyme source; the effects of different metal ions on PPO, different metal ion with different concentration wer e measured to choose activator of PPO; the selection of reaction conditions of synth esizing theaflavins, optimum synthesis condition were determined on the basis of sin gle factor and orthogonal experiments; the selection of optimum condition to purify theaflavins.
(1) Pear, apple, potatoes, tea leaves, eggplant, sweet potato and lotus root seve n plants were chosen as enzyme source materials, the buffer solution method were used to extract and measure the activity of PPO. By comparing the activity of diffe rent enzyme source materials, the material with the highest activity was chosen as t he best enzyme source material for theaflavin synthesis. The PPO activity in differe nt source material was:174U in eggplant,101.43U in potato,58U in sweet potatoes, 37U in pear,32U in lotus roots,24.67U in apple,10U in tea leaves. Eggplant wa s choosen as the best enzyme source material.
(2) Copper ion, manganese ion, magnesium ion, calcium ion, sodium ion,alumi num ion and ferric ion at different concentrations were added into crude eggplant P PO enzyme.The results showed that copper ion at a concentration of 3mmol/L can s ignificantly activate PPO, while calcium ion, sodium ion, aluminum ion and ferric i on inhibited the activity of PPO. Among them, ferric ion at concentration of 5mmol /L can significantly inhibit the activity of PPO, which can be inhibited 96.6% on th is condition.
(3) Different synthesis conditions (pH value, temperature, concentration of subs trate, enzyme concentration and reaction time) were determined on the basis of singl e factor and orthogonal experiments. The results showed that the best conditions of reaction System were a:pH value of 4.5, temperature of 25℃, substrate concentrati on of 2.5g/L, enzyme additive amount of 35ml and a reaction time of 40min. Am ong these conditions, pH value and concentration of substrate were two the most i mportant factors in this reaction system.
(4) Macroporous adsorptive resin was selected to putify theaflavins that synthesi zed on the best conditions included the type of resin, pH value, concentration of sa mple, the flow rate, the effluent volume and the concentration of effluent.The results showed that the best condition to purify the theaflavins were:pH valve of 3, samp le filling rate of 2BV/h,15% effluent of eliminating impurity volume, effluent volu me of 3 BV and an effluent concentration of 90%. pH value and sample filling rat e were two important factors in the purification system.
(1)选择梨、苹果、马铃薯、茶鲜叶、茄子、甘薯和藕等七种不同的果蔬为酶源物质,测定其PPO活性。通过对其活性的比较,筛选活性最高者作为制备茶黄素的最佳酶源。各种酶源物质活性依次为：茄子中PPO的酶活为174U；马铃薯中PPO的酶活为101.43U；甘薯中PPO的酶活为58U；梨中PPO的酶活为37U；藕中PPO的酶活为32U；苹果中PPO的酶活为24.67U；茶鲜叶中PPO的酶活为10U。选择茄子中PPO作为最佳酶源。
(2)选择铜离子、锰离子、镁离子、钙离子、钠离子、铝离子和铁离子七种离子作为研究对象,通过在粗酶液中加入不同浓度的七种金属离子,研究七种金属离子对茄子PPO活性的影响。研究表明,浓度为3mmol/L的铜离子对PPO有显著激活作用,而钙离子、钠离子、铝离子和铁离子对PPO则有抑制作用。其中,浓度为5mmol/L的铁离子是PPO的最佳抑制剂。在此条件下PPO的活性可被抑制96.6%。
(3)通过对合成TFs的不同条件(pH值、温度、底物浓度、酶添加量和反应时间)的研究,在单因素试验和正交试验的基础上,确定合成TFs的最佳条件。结果表明,反应体系的最佳条件为：pH值为4.5,温度为25℃,底物浓度为2.5g/L,酶添加量为35m1,反应时间为40分钟。其中,pH值和底物浓度是反应体系中重要的两个因素。
(4)采用大孔吸附树脂法对在最佳条件下制备到得的TFs进行纯化,通过对不同吸附条件(树脂型号、pH值、上样速度、除杂用洗脱液浓度、洗脱液体积和洗脱液浓度)的选择,确定纯化TFs的最佳工艺条件。结果表明,纯化的最佳条件为：pH值为3,上样速度为2BV/h,除杂用洗脱液浓度为15%洗脱液体积为3BV,洗脱液浓度为90%。pH值和上样速度是影响纯化效果的重要因素。
This article reported a new process applied bio-enzyme method that green tea p owder with low cost and high polyphenol as raw material to synthesize theaflavins, then macroporous adsorptive resin was done to purify theaflavins in earlier studies. The technological process was divided into polyphenol oxidase(PPO) selection, extra cted PPO from seven plants such as pear、apple and so on and measured their acti vity, choose the material with highest activity as the best enzyme source; the effects of different metal ions on PPO, different metal ion with different concentration wer e measured to choose activator of PPO; the selection of reaction conditions of synth esizing theaflavins, optimum synthesis condition were determined on the basis of sin gle factor and orthogonal experiments; the selection of optimum condition to purify theaflavins.
(1) Pear, apple, potatoes, tea leaves, eggplant, sweet potato and lotus root seve n plants were chosen as enzyme source materials, the buffer solution method were used to extract and measure the activity of PPO. By comparing the activity of diffe rent enzyme source materials, the material with the highest activity was chosen as t he best enzyme source material for theaflavin synthesis. The PPO activity in differe nt source material was:174U in eggplant,101.43U in potato,58U in sweet potatoes, 37U in pear,32U in lotus roots,24.67U in apple,10U in tea leaves. Eggplant wa s choosen as the best enzyme source material.
(2) Copper ion, manganese ion, magnesium ion, calcium ion, sodium ion,alumi num ion and ferric ion at different concentrations were added into crude eggplant P PO enzyme.The results showed that copper ion at a concentration of 3mmol/L can s ignificantly activate PPO, while calcium ion, sodium ion, aluminum ion and ferric i on inhibited the activity of PPO. Among them, ferric ion at concentration of 5mmol /L can significantly inhibit the activity of PPO, which can be inhibited 96.6% on th is condition.
(3) Different synthesis conditions (pH value, temperature, concentration of subs trate, enzyme concentration and reaction time) were determined on the basis of singl e factor and orthogonal experiments. The results showed that the best conditions of reaction System were a:pH value of 4.5, temperature of 25℃, substrate concentrati on of 2.5g/L, enzyme additive amount of 35ml and a reaction time of 40min. Am ong these conditions, pH value and concentration of substrate were two the most i mportant factors in this reaction system.
(4) Macroporous adsorptive resin was selected to putify theaflavins that synthesi zed on the best conditions included the type of resin, pH value, concentration of sa mple, the flow rate, the effluent volume and the concentration of effluent.The results showed that the best condition to purify the theaflavins were:pH valve of 3, samp le filling rate of 2BV/h,15% effluent of eliminating impurity volume, effluent volu me of 3 BV and an effluent concentration of 90%. pH value and sample filling rat e were two important factors in the purification system.
引文
[1]宛晓春主编.茶叶生物化学[M].北京：中国农业出版社,2003,6-7.
[2]Roberts E A H. The phenolic substance off manufactured tea (Ⅰ) [J]. Sci Food Agric,1957,8: 72-80.
[3]Roberts E A H.The phenolic substance off manufactured tea (Ⅱ) [J]. Sci Food Agric,1958,9: 212-216.
[4]Roberts E A H. The phenolic substance off manufactured tea (Ⅲ) [J]. Sci Food Agric,1958,9: 217-223.
[5]Roberts E A H.The phenolic substance off manufactured tea (IV) [J]. Sci Food Agric,1959,10: 176-179.
[6]熊昌云,彭远菊.红茶色素与红茶品质关系及其生物学活性研究进展[J].福建茶叶,2006(3)：10-11.
[7]Http://food.icxo.com/htmlnews/2004/07/29/278482.htm.
[8]Gremwrod C. Campbell D. Tea and health:A research programe tea & Coffee Trade.1996,168:32.
[9]张建勇,江和源,江用文.茶黄素的酸性氧化形成研究[J].食品科学,2008,29(1)：50-54.
[10]李适,刘仲华,黄建安,等.毛栓菌多酚氧化酶酶学性质及茶黄素的酶促合成研究[J].茶叶科学,2008,28(5)：326-330.
[11]Kruger J E. Changes in the polyphnol oxidase of wheat during kernelgrowth and maturation[J]. Cereal Chem,1976, (53):201-213.
[12]Ya-Lun Su, Jin-Ze, Chi Ho Ng, et.al. Antioxidant Activity of Tea Theaflavins and Methylated Catechins in Canola oil.2004,81(3):269-274.
[13]Http://www.xoe.cn.
[14]Chen YC, Liang YC, et al. Inhibition of TPA-induced protein kinase C and transcription activator protein-1 binding activities by theaflavin-3,3'-digallate from black tea in NIH3T3 cells.journal of Agricultural and Food Chemistry.1999,47(4):1416-1421.
[15]P W Thygesen, I B Dry, S P Robinson. Polyphenol oxidase in potato[J]. Plant Physiol,1995, (109): 525-531.
[16]Arghya Adhikary, Suchismita Mohanty, Lakshmishri Lahiry, Dewan Md. Sakib Hossain, Samik Chakraborty, Tanya Das.Theaflavins retard human breast cancer cell migration by inhibiting NF-кB via p53-ROS cross-talk. FEBS Letters,2010,584(1):7-14.
[17]Neetu Kalra, Kavita Seth, Sahdeo Prasad, Madhulika Singh, Aditya B.Pant, Yogeshwer Shukla.Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways. Life Sciences, 2007,80(23):2137-2146.
[18]李保聚.李凤云.黄瓜不同抗性品种感染黑星病菌后过氧化物酶和多酚氧化酶的变化[J].中国农业科学,1998,31(1)：86-88.
[19]傅冬和,毛清黎,郑海涛,等.茶色素药理作用研究进展[J].茶叶通讯,2003(2)：11-15.
[20]Dry IB, Robinson SallyM-Newman S P, Eannetta N T, et al. Molecular cloning andcharacterization of grape berry polyphenoloxidase gene[J]. PlantMol. Biol,1994,26:495-502.
[21]张建军,李祥,候明生.小麦品种对梭条斑花叶病毒病抗病特性的研究Ⅱ抗病性与植株内多酚氧化酶活性、过氧化物酶活性及其同工酶酶谱的关系[J].华中农业大学学报,996,15(5)：420-425.
[22]Nakayama M, Suzuki K, Toda M. Inhibition of the ineffective of influenza virus by tea polyphenols [J]. Antiviral Research,1993,21(4):289.
[23]Masao H, Tomoco K 1, Tsuneo N, et, al. Effect of tea polyphenols on glucan synthesis byglucosyltransferase from streptococcus mutans[J]. Chem, Pharm, Bull.1990,38:717-720.
[24]王清,王蒂.温度、pH对马铃薯多酚氧化酶活性的影响[J].中国马铃薯,2003,17(3)：157-161.
[25]梁燕.微生物和水果源的多酚氧化酶固定化合成茶黄素研究[D].杭州：浙江大学,2008.
[26]Clark KJ, Grant PG, Sarr AB, et al.An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronaviras infections. Veterinary Microbiology.1998, 63(2-4):147-157.
[27]黄健,唐学玺,短德麟,等.不同光照条件下海带体内各种化合物的含量及光合作用和呼吸作用的变化[J].海洋科学,2002,26(4)：55258.
[28]于新,黄小丹,冯彤,等.草菇多酚氧化酶及过氧化物酶特性的研究[J].仲恺农业技术学院学报,1998,11(3)：27-33.
[29]杨博,韩振海,张永,等.不同光照强度对玫瑰组织培养中初代培养物褐化的影响[J].园艺园林科学,2003,19(6)：194-196.
[30]孟雅,李刚,崔焱,等.马铃薯多酚氧化酶的提取纯化条件对其活性影响的研究[J].化学与生物工程,2006,23(10)：47-49.
[31]李立祥,萧伟祥.茶色素及茶黄素药理作用研究进展[J].福建茶叶,2002(4)：35-37.
[32]Lin JK, Ho CT, Lin-Shiau SY,et al.Inhibition of xanthine oxidase and suppression ofintracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechirr-3-gallate, and propyl gallate Journal of Agricultural and Food Chemistry.2000,48(7):2736-2743.
[33]Vasyl M.Sava, Swen-Ming Yang, Meng-Yen Hong, et al. Esolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry.2001,73(2):177-184.
[34]冀德富,郭东艳,崔春利,等.纯化叶下珠总多酚的大孔树脂筛选研究[J].山西中医学院学报,2009,10(25)：1-2.
[35]李立祥,萧伟祥.茶黄素制取与分离研究研究进展[J].中国茶叶加工,2000(4)：29-33.
[36]王坤波,刘仲华,黄建安.茶黄素的提取分离与纯化研究[J].湖南农业大学学报,2002,28(4)：355-358.
[37]刘静,李湘利,冯磊,等.鲜切马铃薯多酚氧化酶酶学特性及褐变抑制剂的研究[J].安徽农业科学,2009,37(34)：17067-17069.
[38]Robertson Alastair. Effects of physical and chemical conditions on the in vitro oxidation of tea leaf catechins [J]. Phytochemistry,1983,22(4):889-896.
[39]李胜华,伍贤进,牛友芽,等.大孔树脂纯化多穗柯总黄酮的工艺研究[J].江苏农业科学,2009(5)：266-268.
[40]朱显峰,丁涛.常见金属离子对漆酶酶活的影响[J].化学研究,2003,14(3)：50-51.
[41]王曼玲,胡中立,周明全,等.植物多酚氧化酶的研究进展[J].植物学通报,2005,22(2)：215-222.
[42]代丽,宫长荣,史林,等.植物多酚氧化酶研究综述[J].中国农学通报,2007,23(6)：312-315
[43]薛超彬,王勤,贺量,等.金属离子对菜青虫酚氧化酶活力的影响[J].厦门大学学报,2005(44)：120-122.
[44]马晓春,韩宏岩,许维岸.家蚕表皮多酚氧化酶的分离纯化及酶学性质研究[J].安徽农业科学,2009,37(17)：7870-7871.
[45]段玉权,董维,张明晶,等.中华寿桃多酚氧化酶的特性研究[J].中国农业科学,2008,41(3)：795-799.
[46]Jimenz M, Garcia-Carmona F.Measurement of latent polyphenol oxidase activity in the presence of the divalent cations Ca2+, Mg2+and Mn+. PhytochemicalAnalysis,1993,4: 149-151.
[47]杨晓萍,郭大勇,袁芳亭.茶色素的研究现状与应用[J].茶叶机械杂志,2002(4)：1-3.
[48]Dix M.A., et al. Fermeatatio of tea in Aqueous influence of Tea Peroxidase [J]. J.Sci. Food Agric, 1981,32; 923-932.
[49]Shaun C Opie, Michael N Clifford and Alastair Robertson. The role of (-)-epicatechin and polyphenol oxidative breakdown of theaflavins [J]. J.Sci. Food Agric,1993,64:435-438.
[50]Finger A. In-vitro studies on the effect of polyphenol oxidase and peroxidase on theformation of polyphenolic black tea constituents [J]. J Sci Food Agric,1994,66:293-305.
[51]Koastsn Y. Suematsu S, Hisanobu Y.et al. Effects of pH and temperature on reaction kinetics of catechin in green infusion. Biosci.Biotech Bioche [J].1993,57(6):907-910.
[52]Lin JK, Ho CT, Lin-Shiau SY, et al. Inhibition of xanthine oxidase and suppression ofintracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate,(-)-epigallocatechirr-3-gallate, and propyl gallate, Journal of Agricultural and Food Chemistry.2000,48(7):2736-2743.
[53]Vasyl M.Sava, Swen-Ming Yang, Meng-Yen Hong, et al. Esolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry.2001,73(2):177-184.
[54]夏涛.红茶色素形成机理的研究[J].茶叶科学,1999,19(2)：139-144.
[55]Clark KJ, Grant PG, Sarr AB, et al. An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections. Veterinary Microbiology.1998, 63(2-4):147-157.
[56]龚新明,崔颜红,关军锋,等.皇冠果皮多酚氧化酶的酶学性质[J].安徽农业科学,2008,36(31)：13541-13543.
[57]李适,黄建安,刘仲华.茶黄素体外氧化制备方法的影响因子研究进展[J].茶叶通讯,2005,32(6)：36-39.
[58]丁晓芳.茶黄素组分分离以及与制茶技术品质的关系[J].茶叶通报,1991(2)：8-11.
[59]Cloughley J B. The effect of fermentation temperature on the quality preparation and price evaluation of central African black teas. J.S ci. Food Agric.1980(31):911-919.
[60]Our P O, Orchard J, McDowell I. Changes in the quality parameters of clonal black tea due to fermentation time[J]. J Sci Food Agric,1994,64:319-324.
[61]李大祥,宛晓春,萧伟祥.儿茶素化学氧化条件的研究简报[J].茶叶通报.2000,22(2)：17-18.
[62]Collier P D, Mallows R, Korver O, er al. The theaflavins of black tea [J]. Tetrahedon.1973,29: 125-142.
[63]Crispin DJ. The separation of theaflavins on sephadex LH-20[J]. Chromatogr,1971,54:133-135.
[64]Op ieClifford, Roberton.The role of (2)2EC andployphenol oxidase in the coup led oxidative breakdown oftheaflavins [J]. J Sci Food A gric,1993,63:4352438.
[65]Lea A G H. The separation of theaflavins onsephadex LH220 [J]. J Chromatogr,1968,37:118.
[66]Qizhen Du, Heyuan jiang, Yoich irolto.Separation of theaflavins of black tea:High speed countercurrentchromatographyVS.sephadex LH-20 gelco lumnchromatography[J]. Journal of Liquid Chromatographyand Related Technologles,2001,24(15):2363-2369.
[67]WANG Kun-bo, LIU zhong-hua, HUANG jian-an.Research progress on Extraction, separation and purification of theaflavins [J]. Journal of hunan Agricultural University (Natural science),2002, 28(4):355-358.
[68]江和源,程启坤,杜琪珍.高速逆流色谱分离纯化茶黄素[J].天然产物研究与开发,2000,12(4)： 30-35.
[69]戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001,29(5)：586-591.
[70]Changjun Yang, Daxiang Li, Xiaochun Wan.Combination of HSCCC and Sephadex LH-20 methods: An approach to isolation and purification of the main individual theaflavins from black tea. Journal of Chromatography B,2008,861(1):140-144.
[71]Kunbo Wang, Zhonghua Liu, Jian-an Huang, Xinrong Dong, Lubing Song, Yu Pan, Fang liu. Preparative isolation and purification of theaflavins and catechins by high-speedcountercurrent chromatography. Journal of Chromatography B,2008,867(2):282-286.
[72]芦金清,刘尚桃,刘再刚,等.吸附树脂法提取绞股蓝皂苷[J].1992,14(4)：2-3.
[73]何琦,及乔元,丁立生,等.D140大孔吸附树脂银杏黄酮提取纯化性能研究[J].天然产物研究与开发,2001,13(1)：56.
[74]戴文娟,郭璇华.大孔树脂吸附纯化火龙果茎黄酮类化合物的研究[J].食品研究与开发2009,30(11)：65-68.
[75]Andrew P.Neilson, Rodney J. Green, Karl V. Wood, Mario G. Ferruzzi. High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection. Journal of Chromatography A,2006,1132(1-2):132-140.
[76]李大祥,宛晓春,刘莉.茶色素中茶黄素类的HPLC定量[J].茶叶科学,2004,24(2)：124-128.
[77]王坤波,刘仲华,赵淑娟,等.梨多酚氧化酶同工酶组成及其对茶黄素合成的影响[J].湖南农业大学学报,2007,33(4)：459-462.
[78]谷计平,刘仲华,黄建安,等.酸性氧化合成茶黄素条件优化的研究[J].茶叶科学,2006,26(4)：285-290.
[79]王坤波,刘仲华,赵淑娟,等.儿茶素组成和理化条件对茶黄素酶催化合成的影响[J].茶叶科学,2007,27(3)：192-200.
[80]张建勇,江和源,崔宏春,等.茶黄素的提取制备技术[J].中国茶叶,2009(6)：10-11.
[81]萧伟祥,宛晓春,胡耀武,等.茶儿茶素体外氧化产物分析[J].茶叶科学,1999,19(2)：145-149.
[82]魏好程,汤凤霞,沈塞群.茄子多酚氧化酶特性及控制研究[J].食品研究与开发,2009,20(6)：48-51.
[83]刘云,刘良赕,朱欣婷.D140型大孔树脂纯化荷叶总黄酮的研究[J].安徽农业科学,2009,37(32)：16163-16164,16184.
[84]葛淑兰,陈龙华,李中文.大孔吸附树脂及其在黄酮类成分分离纯化中的应用进展[J].中国药师,2008,11(6)：702-705.
[85]唐巧玉,周毅峰,任甜甜,等.大孔树脂对水芹总黄酮的分离纯化研究[J].时珍国医国药,2009,20(10)：2480-2482
[86]丁阳平,刘仲华,黄建安.聚酰胺分离纯化茶黄素类物质研究[J].食品科学,2007,28(12)：55-57.
[87]黄月纯,黄樱华,刘翠玲,等.S-8型大孔吸附树脂纯化泽兰总黄酮(酚酸)的工艺研究[J].中国现代应用药学,2010,27(8)：694-698.
[88]吕远平,姚开,何强,等.树脂法纯化茶多酚的研究[J].中国油脂,2003,28(10)：64-66.
[89]甘永祥,陈文,张华,等.D-301大孔树脂富集刺山柑总酚酸工艺研究[J].石河子大学学报,2009,27(5)：630-632.
[90]江和源.程启坤,杜琪珍.调节pH值对HSCCC分离茶黄素的影响[J].茶叶科学,2003(增),23：88-91.
[91]方达任,张志.正交试验法优选大孔树脂对黄芪总皂苷动态吸附工艺的研究[J].中国药师,2009,12(12)：1747-1748.
[92]李坤平,潘天玲,贲永光,等.均匀设计法优选大孔树脂纯化七叶莲总皂苷工艺研究[J].林产化学与工业,2009,29(2)：110-114.
[93]唐杰,王秀峰,李娟,等.茶黄素提纯的研究现状[J].微量元素与健康研究,2007,24(4)：58-60.
[94]徐懿,王奕,屠幼英,等.茶黄素复合物中单体对照品的制备研究[J].中草药,2009,40(12)：1919-1921.
[2]Roberts E A H. The phenolic substance off manufactured tea (Ⅰ) [J]. Sci Food Agric,1957,8: 72-80.
[3]Roberts E A H.The phenolic substance off manufactured tea (Ⅱ) [J]. Sci Food Agric,1958,9: 212-216.
[4]Roberts E A H. The phenolic substance off manufactured tea (Ⅲ) [J]. Sci Food Agric,1958,9: 217-223.
[5]Roberts E A H.The phenolic substance off manufactured tea (IV) [J]. Sci Food Agric,1959,10: 176-179.
[6]熊昌云,彭远菊.红茶色素与红茶品质关系及其生物学活性研究进展[J].福建茶叶,2006(3)：10-11.
[7]Http://food.icxo.com/htmlnews/2004/07/29/278482.htm.
[8]Gremwrod C. Campbell D. Tea and health:A research programe tea & Coffee Trade.1996,168:32.
[9]张建勇,江和源,江用文.茶黄素的酸性氧化形成研究[J].食品科学,2008,29(1)：50-54.
[10]李适,刘仲华,黄建安,等.毛栓菌多酚氧化酶酶学性质及茶黄素的酶促合成研究[J].茶叶科学,2008,28(5)：326-330.
[11]Kruger J E. Changes in the polyphnol oxidase of wheat during kernelgrowth and maturation[J]. Cereal Chem,1976, (53):201-213.
[12]Ya-Lun Su, Jin-Ze, Chi Ho Ng, et.al. Antioxidant Activity of Tea Theaflavins and Methylated Catechins in Canola oil.2004,81(3):269-274.
[13]Http://www.xoe.cn.
[14]Chen YC, Liang YC, et al. Inhibition of TPA-induced protein kinase C and transcription activator protein-1 binding activities by theaflavin-3,3'-digallate from black tea in NIH3T3 cells.journal of Agricultural and Food Chemistry.1999,47(4):1416-1421.
[15]P W Thygesen, I B Dry, S P Robinson. Polyphenol oxidase in potato[J]. Plant Physiol,1995, (109): 525-531.
[16]Arghya Adhikary, Suchismita Mohanty, Lakshmishri Lahiry, Dewan Md. Sakib Hossain, Samik Chakraborty, Tanya Das.Theaflavins retard human breast cancer cell migration by inhibiting NF-кB via p53-ROS cross-talk. FEBS Letters,2010,584(1):7-14.
[17]Neetu Kalra, Kavita Seth, Sahdeo Prasad, Madhulika Singh, Aditya B.Pant, Yogeshwer Shukla.Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways. Life Sciences, 2007,80(23):2137-2146.
[18]李保聚.李凤云.黄瓜不同抗性品种感染黑星病菌后过氧化物酶和多酚氧化酶的变化[J].中国农业科学,1998,31(1)：86-88.
[19]傅冬和,毛清黎,郑海涛,等.茶色素药理作用研究进展[J].茶叶通讯,2003(2)：11-15.
[20]Dry IB, Robinson SallyM-Newman S P, Eannetta N T, et al. Molecular cloning andcharacterization of grape berry polyphenoloxidase gene[J]. PlantMol. Biol,1994,26:495-502.
[21]张建军,李祥,候明生.小麦品种对梭条斑花叶病毒病抗病特性的研究Ⅱ抗病性与植株内多酚氧化酶活性、过氧化物酶活性及其同工酶酶谱的关系[J].华中农业大学学报,996,15(5)：420-425.
[22]Nakayama M, Suzuki K, Toda M. Inhibition of the ineffective of influenza virus by tea polyphenols [J]. Antiviral Research,1993,21(4):289.
[23]Masao H, Tomoco K 1, Tsuneo N, et, al. Effect of tea polyphenols on glucan synthesis byglucosyltransferase from streptococcus mutans[J]. Chem, Pharm, Bull.1990,38:717-720.
[24]王清,王蒂.温度、pH对马铃薯多酚氧化酶活性的影响[J].中国马铃薯,2003,17(3)：157-161.
[25]梁燕.微生物和水果源的多酚氧化酶固定化合成茶黄素研究[D].杭州：浙江大学,2008.
[26]Clark KJ, Grant PG, Sarr AB, et al.An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronaviras infections. Veterinary Microbiology.1998, 63(2-4):147-157.
[27]黄健,唐学玺,短德麟,等.不同光照条件下海带体内各种化合物的含量及光合作用和呼吸作用的变化[J].海洋科学,2002,26(4)：55258.
[28]于新,黄小丹,冯彤,等.草菇多酚氧化酶及过氧化物酶特性的研究[J].仲恺农业技术学院学报,1998,11(3)：27-33.
[29]杨博,韩振海,张永,等.不同光照强度对玫瑰组织培养中初代培养物褐化的影响[J].园艺园林科学,2003,19(6)：194-196.
[30]孟雅,李刚,崔焱,等.马铃薯多酚氧化酶的提取纯化条件对其活性影响的研究[J].化学与生物工程,2006,23(10)：47-49.
[31]李立祥,萧伟祥.茶色素及茶黄素药理作用研究进展[J].福建茶叶,2002(4)：35-37.
[32]Lin JK, Ho CT, Lin-Shiau SY,et al.Inhibition of xanthine oxidase and suppression ofintracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechirr-3-gallate, and propyl gallate Journal of Agricultural and Food Chemistry.2000,48(7):2736-2743.
[33]Vasyl M.Sava, Swen-Ming Yang, Meng-Yen Hong, et al. Esolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry.2001,73(2):177-184.
[34]冀德富,郭东艳,崔春利,等.纯化叶下珠总多酚的大孔树脂筛选研究[J].山西中医学院学报,2009,10(25)：1-2.
[35]李立祥,萧伟祥.茶黄素制取与分离研究研究进展[J].中国茶叶加工,2000(4)：29-33.
[36]王坤波,刘仲华,黄建安.茶黄素的提取分离与纯化研究[J].湖南农业大学学报,2002,28(4)：355-358.
[37]刘静,李湘利,冯磊,等.鲜切马铃薯多酚氧化酶酶学特性及褐变抑制剂的研究[J].安徽农业科学,2009,37(34)：17067-17069.
[38]Robertson Alastair. Effects of physical and chemical conditions on the in vitro oxidation of tea leaf catechins [J]. Phytochemistry,1983,22(4):889-896.
[39]李胜华,伍贤进,牛友芽,等.大孔树脂纯化多穗柯总黄酮的工艺研究[J].江苏农业科学,2009(5)：266-268.
[40]朱显峰,丁涛.常见金属离子对漆酶酶活的影响[J].化学研究,2003,14(3)：50-51.
[41]王曼玲,胡中立,周明全,等.植物多酚氧化酶的研究进展[J].植物学通报,2005,22(2)：215-222.
[42]代丽,宫长荣,史林,等.植物多酚氧化酶研究综述[J].中国农学通报,2007,23(6)：312-315
[43]薛超彬,王勤,贺量,等.金属离子对菜青虫酚氧化酶活力的影响[J].厦门大学学报,2005(44)：120-122.
[44]马晓春,韩宏岩,许维岸.家蚕表皮多酚氧化酶的分离纯化及酶学性质研究[J].安徽农业科学,2009,37(17)：7870-7871.
[45]段玉权,董维,张明晶,等.中华寿桃多酚氧化酶的特性研究[J].中国农业科学,2008,41(3)：795-799.
[46]Jimenz M, Garcia-Carmona F.Measurement of latent polyphenol oxidase activity in the presence of the divalent cations Ca2+, Mg2+and Mn+. PhytochemicalAnalysis,1993,4: 149-151.
[47]杨晓萍,郭大勇,袁芳亭.茶色素的研究现状与应用[J].茶叶机械杂志,2002(4)：1-3.
[48]Dix M.A., et al. Fermeatatio of tea in Aqueous influence of Tea Peroxidase [J]. J.Sci. Food Agric, 1981,32; 923-932.
[49]Shaun C Opie, Michael N Clifford and Alastair Robertson. The role of (-)-epicatechin and polyphenol oxidative breakdown of theaflavins [J]. J.Sci. Food Agric,1993,64:435-438.
[50]Finger A. In-vitro studies on the effect of polyphenol oxidase and peroxidase on theformation of polyphenolic black tea constituents [J]. J Sci Food Agric,1994,66:293-305.
[51]Koastsn Y. Suematsu S, Hisanobu Y.et al. Effects of pH and temperature on reaction kinetics of catechin in green infusion. Biosci.Biotech Bioche [J].1993,57(6):907-910.
[52]Lin JK, Ho CT, Lin-Shiau SY, et al. Inhibition of xanthine oxidase and suppression ofintracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate,(-)-epigallocatechirr-3-gallate, and propyl gallate, Journal of Agricultural and Food Chemistry.2000,48(7):2736-2743.
[53]Vasyl M.Sava, Swen-Ming Yang, Meng-Yen Hong, et al. Esolation and characterization of melanic pigments derived from tea and tea polyphenols. Food Chemistry.2001,73(2):177-184.
[54]夏涛.红茶色素形成机理的研究[J].茶叶科学,1999,19(2)：139-144.
[55]Clark KJ, Grant PG, Sarr AB, et al. An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections. Veterinary Microbiology.1998, 63(2-4):147-157.
[56]龚新明,崔颜红,关军锋,等.皇冠果皮多酚氧化酶的酶学性质[J].安徽农业科学,2008,36(31)：13541-13543.
[57]李适,黄建安,刘仲华.茶黄素体外氧化制备方法的影响因子研究进展[J].茶叶通讯,2005,32(6)：36-39.
[58]丁晓芳.茶黄素组分分离以及与制茶技术品质的关系[J].茶叶通报,1991(2)：8-11.
[59]Cloughley J B. The effect of fermentation temperature on the quality preparation and price evaluation of central African black teas. J.S ci. Food Agric.1980(31):911-919.
[60]Our P O, Orchard J, McDowell I. Changes in the quality parameters of clonal black tea due to fermentation time[J]. J Sci Food Agric,1994,64:319-324.
[61]李大祥,宛晓春,萧伟祥.儿茶素化学氧化条件的研究简报[J].茶叶通报.2000,22(2)：17-18.
[62]Collier P D, Mallows R, Korver O, er al. The theaflavins of black tea [J]. Tetrahedon.1973,29: 125-142.
[63]Crispin DJ. The separation of theaflavins on sephadex LH-20[J]. Chromatogr,1971,54:133-135.
[64]Op ieClifford, Roberton.The role of (2)2EC andployphenol oxidase in the coup led oxidative breakdown oftheaflavins [J]. J Sci Food A gric,1993,63:4352438.
[65]Lea A G H. The separation of theaflavins onsephadex LH220 [J]. J Chromatogr,1968,37:118.
[66]Qizhen Du, Heyuan jiang, Yoich irolto.Separation of theaflavins of black tea:High speed countercurrentchromatographyVS.sephadex LH-20 gelco lumnchromatography[J]. Journal of Liquid Chromatographyand Related Technologles,2001,24(15):2363-2369.
[67]WANG Kun-bo, LIU zhong-hua, HUANG jian-an.Research progress on Extraction, separation and purification of theaflavins [J]. Journal of hunan Agricultural University (Natural science),2002, 28(4):355-358.
[68]江和源,程启坤,杜琪珍.高速逆流色谱分离纯化茶黄素[J].天然产物研究与开发,2000,12(4)： 30-35.
[69]戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001,29(5)：586-591.
[70]Changjun Yang, Daxiang Li, Xiaochun Wan.Combination of HSCCC and Sephadex LH-20 methods: An approach to isolation and purification of the main individual theaflavins from black tea. Journal of Chromatography B,2008,861(1):140-144.
[71]Kunbo Wang, Zhonghua Liu, Jian-an Huang, Xinrong Dong, Lubing Song, Yu Pan, Fang liu. Preparative isolation and purification of theaflavins and catechins by high-speedcountercurrent chromatography. Journal of Chromatography B,2008,867(2):282-286.
[72]芦金清,刘尚桃,刘再刚,等.吸附树脂法提取绞股蓝皂苷[J].1992,14(4)：2-3.
[73]何琦,及乔元,丁立生,等.D140大孔吸附树脂银杏黄酮提取纯化性能研究[J].天然产物研究与开发,2001,13(1)：56.
[74]戴文娟,郭璇华.大孔树脂吸附纯化火龙果茎黄酮类化合物的研究[J].食品研究与开发2009,30(11)：65-68.
[75]Andrew P.Neilson, Rodney J. Green, Karl V. Wood, Mario G. Ferruzzi. High-throughput analysis of catechins and theaflavins by high performance liquid chromatography with diode array detection. Journal of Chromatography A,2006,1132(1-2):132-140.
[76]李大祥,宛晓春,刘莉.茶色素中茶黄素类的HPLC定量[J].茶叶科学,2004,24(2)：124-128.
[77]王坤波,刘仲华,赵淑娟,等.梨多酚氧化酶同工酶组成及其对茶黄素合成的影响[J].湖南农业大学学报,2007,33(4)：459-462.
[78]谷计平,刘仲华,黄建安,等.酸性氧化合成茶黄素条件优化的研究[J].茶叶科学,2006,26(4)：285-290.
[79]王坤波,刘仲华,赵淑娟,等.儿茶素组成和理化条件对茶黄素酶催化合成的影响[J].茶叶科学,2007,27(3)：192-200.
[80]张建勇,江和源,崔宏春,等.茶黄素的提取制备技术[J].中国茶叶,2009(6)：10-11.
[81]萧伟祥,宛晓春,胡耀武,等.茶儿茶素体外氧化产物分析[J].茶叶科学,1999,19(2)：145-149.
[82]魏好程,汤凤霞,沈塞群.茄子多酚氧化酶特性及控制研究[J].食品研究与开发,2009,20(6)：48-51.
[83]刘云,刘良赕,朱欣婷.D140型大孔树脂纯化荷叶总黄酮的研究[J].安徽农业科学,2009,37(32)：16163-16164,16184.
[84]葛淑兰,陈龙华,李中文.大孔吸附树脂及其在黄酮类成分分离纯化中的应用进展[J].中国药师,2008,11(6)：702-705.
[85]唐巧玉,周毅峰,任甜甜,等.大孔树脂对水芹总黄酮的分离纯化研究[J].时珍国医国药,2009,20(10)：2480-2482
[86]丁阳平,刘仲华,黄建安.聚酰胺分离纯化茶黄素类物质研究[J].食品科学,2007,28(12)：55-57.
[87]黄月纯,黄樱华,刘翠玲,等.S-8型大孔吸附树脂纯化泽兰总黄酮(酚酸)的工艺研究[J].中国现代应用药学,2010,27(8)：694-698.
[88]吕远平,姚开,何强,等.树脂法纯化茶多酚的研究[J].中国油脂,2003,28(10)：64-66.
[89]甘永祥,陈文,张华,等.D-301大孔树脂富集刺山柑总酚酸工艺研究[J].石河子大学学报,2009,27(5)：630-632.
[90]江和源.程启坤,杜琪珍.调节pH值对HSCCC分离茶黄素的影响[J].茶叶科学,2003(增),23：88-91.
[91]方达任,张志.正交试验法优选大孔树脂对黄芪总皂苷动态吸附工艺的研究[J].中国药师,2009,12(12)：1747-1748.
[92]李坤平,潘天玲,贲永光,等.均匀设计法优选大孔树脂纯化七叶莲总皂苷工艺研究[J].林产化学与工业,2009,29(2)：110-114.
[93]唐杰,王秀峰,李娟,等.茶黄素提纯的研究现状[J].微量元素与健康研究,2007,24(4)：58-60.
[94]徐懿,王奕,屠幼英,等.茶黄素复合物中单体对照品的制备研究[J].中草药,2009,40(12)：1919-1921.