摘要
茶黄素是指红茶中溶于乙酸乙酯呈橙黄色的物质,由茶多酚(主要是儿茶素类及其衍生物)在多酚氧化酶存在下氧化缩合而来。红茶中茶黄素的含量约为0.5%～3%,但茶黄素不仅是红茶的重要品质成分,而且具有多种药理功能与保健功效,如降血脂、抗氧化、抗衰老、抗突变、抗癌防癌等,在某些功能方面甚至优于儿茶素。红茶茶黄素类物质的研究与开发是继茶多酚、儿茶素之后近年来兴起的又一热点,不仅具有重要的理论价值,而且在天然药物、保健品、化妆品和日化用品等领域具有广阔的产业化前景。但是,茶黄素的化学研究不能满足医药领域研究的要求,如茶黄素的提纯难度较大、性质欠稳定等限制了药用研究的深入。所以,到目前为止,茶黄素提制的产业化工艺技术尚不成熟,表现为产品纯度低、成本高、产能低,严重阻碍了以茶黄素为原料的天然药物及功能性食品的研究开发。同时,由于结构的相似性,茶黄素类物质单体的分离非常困难,直接从红茶中分离茶黄素单体更加困难。由于红茶中茶黄素的含量低,依靠红茶为原料来提制茶黄素成本过高,必须研究如何采用儿茶素为原料,筛选特异多酚氧化酶酶源,利用酶促氧化合成茶黄素,研究茶黄素合成机理,具有重要的理论与实际意义。因此,本研究设计了一种利用植物或微生物多酚氧化酶氧化儿茶素混合物体外合成茶黄素复合物的方法。并通过调控底物儿茶素的组成比例来调控氧化产物中茶黄素各组分的组成比例。采用聚酰胺柱色谱结合制备液相色谱、高速逆流色谱,对茶黄素复合物进行分离,拟得到4种茶黄素单体,其化学结构由~1HNMR,~(13)CNMR,UV,IR和MS表征。并采用高通量筛选模型对茶黄素单体的降血脂、抗炎和抗肿瘤活性方面进行了系统深入的研究。
1多酚氧化酶的优选及合成茶黄素的研究
采用聚丙烯酰胺凝胶垂直平板电泳技术,研究了不同来源(茶叶、苹果、梨、蘑菇和漆酶)的多酚氧化酶同工酶的酶带特征。结果表明,不同来源多酚氧化酶同工酶具有相似的酶带,但对酶带数目、Rf值及分子量均存在种间差异。梨有11条同工酶带,苹果4条,茶叶有5条,蘑菇5条,而漆酶只有1条。PPO同工酶带Rf值集中于0.40～0.70的区间内。茶叶多酚氧化酶同工酶的相对分子量大于94 000,而梨、苹果、蘑菇和漆酶多酚氧化酶同工酶的相对分子量介于45000～94 000。同时研究了多酚氧化酶活性和同工酶组成对茶黄素合成的影响。合成茶黄素能力大小顺序为:梨PPO,茶叶PPO,微生物PPO,苹果PPO,漆酶PPO,蘑菇PPO。其中,在所有梨品种中,丰水梨果实多酚氧化酶同工酶谱带最多,酶活性最大,合成茶黄素的能力最强。
2茶黄素的酶催化合成
利用丰水梨果实多酚氧化酶,在单因素试验基础上,设计正交试验,进行酶性合成茶黄素,研究儿茶素组成、浓度和理化条件对茶黄素合成的影响,以确定最佳合成条件。结果表明,可以通过氧化不同儿茶素组成比例的底物来调控氧化产物中茶黄素各组分的组成比例。以儿茶素混合物C(EGC>200 mg/g,EGCG>200mg/g,儿茶素总量>500mg/g)为材料,茶黄素酶促合成的最佳条件为:反应体系的最佳pH值为5.5,温度为30℃,底物浓度为5mg/ml,酶添加量为49162.50U,最佳反应时间40min。pH值和儿茶素浓度是反应体系中两个重要的影响因子(P<0.05)。应用多元线性回归模型,研究了儿茶素混合物C酶性氧化参数对制备茶黄素复合物的综合效应,以茶黄素为目标,建立了儿茶素混合物C酶促氧化制备茶黄素复合物的参数模型,即Y=-23.758+21.359X_1-0.683X_2-1.841X_3(Y:茶黄素总量,X_1:pH,X2:温度,X_3:儿茶素底物浓度)。根据回归模型,并结合各单因素试验,综合优化方案与上述正交实验结果相吻合。
3茶黄素单体的分离及结构鉴定
用高速逆流色谱分离纯化茶黄素,以正己烷.乙酸乙酯.甲醇.水.冰醋酸(1:5:1:5:0.25,v/v/v/v/v)为溶剂系统,上相为固定相,下相为流动相,流速为2mL/min,仪器转速700r/min,进样量30mg。尤其使得两种茶黄素同分异构体(茶黄素-3-没食子酸酯、茶黄素-3'-没食子酸酯)达到较好的分离。从茶黄素粗提物中分离纯化得到茶黄素、茶黄素-3-没食子酸酯、茶黄素-3'-没食子酸酯和茶黄素-3,3'-双没食子酸酯。经高效液相色谱分析,纯度分别为94.2%,95.8%,93.7%和96.4%。其化学结构由~1HNMR,~(13)NMR,UV,IR和MS鉴定。
用制备型反相高效液相色谱结合聚酰胺柱色谱对茶黄素提取物进行了制备分离。色谱柱为PRC-ODS C18(20.0mm×250mm i.d.,10um),流动相为ACN.EtOAc-2%ttAc(21:3:76,v/v),检测波长280nm,流速15mL/min,进样量400μl(30mg/ml)。在此条件下分离得到4个单体化合物,经理化反应和光谱分析(HPLC、HPTLC、Uv、IR、MS、NMR)分别鉴定为茶黄素、茶黄素-3-没食子酸酯、茶黄素-3'-没食子酸酯和茶黄素-3,3'-双没食子酸酯。经高效液相色谱分析,纯度分别为96.9%,98.8%,95.9%和98.9%。
采用聚酰胺为分离材料,对茶黄素及红茶酚性色素进行高效薄层色谱分离。结果表明,展开剂为甲醇.三氯甲烷(1:1,v/v),二次展开,四种主要茶黄素类物质达到基线分离,可进行茶黄素的定性定量分析。本实验采用二次展开分离红茶乙酸乙脂层酚性色素,第一次在甲醇.醋酸(1:1,v/v)体系中展开,然后在甲醇-丙酮-甲酸(2:1:1,v/v)体系中二次展开,茶黄素类物质和儿茶素类物质可以达到分离。根据对照品的光谱特性和Rf值,分别鉴定为茶黄素-3,3'-双没食子酸酯、茶黄素-3'-没食子酸酯,茶黄素-3-没食子酸酯和茶黄素、EGCG、ECG和EC。
4茶黄素降血脂、抗炎、抗肿瘤活性的高通量筛选
首次应用PPARs、FXR、LXR、TNFα、IL-1、SGC-7901、A549、K562、HepG2为靶点的高通量筛选模型,研究茶黄素的降血脂、抗炎、抗肿瘤活性。结果表明,TFs在LXR受体上呈现微弱的活性,激活倍数为1.55,其次是TFDG,其激活顺序为:TFs>TFDG>TF3G≈TF>TF3'G。TF、TF3G、TF3'G和TFDG及TFs都呈现出微弱的激活FXR受体的活性,其激活顺序为:TFDG>TF3'G≈TF3G>TF>TFs;TFDG的激活效果最佳,激活倍数达2.35,其次是TF3'G,激活倍数达2.22,激活作用明显。TFDG和TFs样品有增加FXR受体敏感性的作用。茶黄素单体(TF、TF3G、TF3'G和TFDG)及茶黄素混合物(TFs)在TNFα和IL-1分泌抑制模型上抑制活性不显著。TF、TF3G、TF3'G和TFDG及TFs在高浓度下(100μg/ml)均对SGC-7901及A549肿瘤细胞株生长有重度抑制作用,其中TF3G对四种肿瘤细胞(SGC-7901、A549、K562和HepG2)均有抑制作用(50μg/ml浓度以上),TF在高浓度下(100μg/ml)对HepG2有微弱的抑制作用。
Theaflavins are orange or orange-red in color and possess a benzotropolone skeleton that is formed from cooxidation of selected pairs of catechins,one with a victrihydroxyphenyl moiety,and the other with an orthodihydroxyphenyl structure.It is known that theaflavins,which account for 0.5-3%of the dry weight of solids in black tea,contribute to astringency and briskness of black tea.theaflavin 3,3'-digallate has the most astringency taste(6.4 times higher than theaflavin).Thus, theaflavin composition has been regarded as an important factor that can affect color and briskness of black tea.Recently,theaflavins have attracted considerable interest because of their potential benefits for human health,including antimutagenicity, suppression of cytochrome P450 1A1 in cell culture,anticlastogenic effects in bone marrow cells of mice,suppression of extracellular signals and cell proliferation,and anti-inflammatory and cancer chemopreventive action.Theaflavins also scavenge H_2O_2 and have been shown to inhibit lipid oxidation,LDL oxidation,DNA oxidative damage,and xanthine oxidase activity.Researching and developing theaflavins has important theoretical value and wonderful future of industrial production,especially in natural medicine,health care products,makeup and so on.However,the separation of theaflavins from black tea is both times consuming and tedious.In the face of the limited information available on the biological activities of individual theaflavins,we have devised a simple method for synthesizing theaflavins from tea catechins mixtures using enzymatic oxidation methods.The resulting extracts were subjected to to column chromatography(polyamide),preparative HPLC and HSCCC.After successive chromatography,it was expected to recover theaflavins monomers.We then examined the theaflavins so created for anti-hyperlipemia,anti-inflammatory and anti-cancer activities.
1 Selection of polyphenol oxidase
The isoenzymes of polyphenol oxidase of different source(pear,apple,tea, mushroom and fungal laccase)were studied by means of the polyacrylamide gel vertical slab electrophoresis.Similar bands were observed in different source,but the number and width of the bands & Rf value and molecular weight were different.11 bands of PPO activity were visible in pear extract.4 bands of PPO activity were visible in apple extract.5,5 and 1 bands were present in tea,mushroom extract and laccase,respectively.Rf value of PPO isoenzymes was between 0.40 and 0.70. Molecular weight of tea PPO isoenzymes was more than 94 000,while that of pear,mushroom,apple and laccase PPO isoenzymes ranged from 45 000 to 94 000.An in vitro model fermentation system,containing tea catechins and crude pear polyphenol oxidase from different source has been used to determine the effect of PPO activity and isoenzyme forms on the formation of theaflavins.The ability to synthesis of theaflavins was in the order:pear PPO,tea PPO,microorganism PPO, apple PPO,laccase and mushroom PPO.The bands of fengshui pear were more than that of other cultivars and its PPO activity is the highest among pear cultivars.So its ability to synthesis of theaflavins was the highest among pear cultivars.
2 Enzymatic synthesis of theaflavins
An in vitro model fermentation system,containing tea catechins and crude pear polyphenol oxidase from pear fruits has been used to determine the effect of tea catechin mixtures of different proportions & physico-chemical conditions on the formation of theaflavin under single factor and orthogonal experimental design.We could synthesize theafalvins complex of different proportions by oxidizing tea catechins mixture of different proportions.The optimum temperature for theaflavins formation is 30℃and the pH optima for theaflavin formation were 5.5.The concentration of tea catechins mixture(EGC>200mg/g,EGCG>200 mg/g,total catechins>500 mg/g)for theaflavins formation is 5 mg/ml and the concentration of enzyme for theaflavins formation is 49162.50U.pH and temperature in the model fermentation system are the most important factors(P<0.05).By applying the stepwise linear regression,the synthetical effects of preparing parameters of theaflavins complex from tea catechins mixture using enzymatic oxidation methods were investigated.The parameter model were set up,Y=-23.758+21.359X_1-0.683 X_2-1.841X3.The optimum of preparing parameter of theaflavins complex was as same as above optimum parameter.
3 Separation,purification and structural identification of theaflavins
A preparative high-speed counter-current chromatography(HSCCC)method for the isolation and purification of theaflavins from the crude extract was successfully established by using hexane-ethyl acetate-methanol-water-acetic acid as the two-phase solvent system at a flow rate of 2ml/min and a revolution speed of 700rpm.The upper phase of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25,v/v/v/v/v)was used as the stationary phase of HSCCC.The mobile phase of HSCCC was the lower phase of hexane-ethyl acetate-methanol-water -acetic acid(1:5:1:5:0.25,v/v/v/v/v).Four kinds of theaflavins including theaflavin(TF),theaflavin 3-gallate(TF3G),theaflavin 3'-gallate(TF3'G)and theaflavin 3,3'-digallate(TFDG)were obtained from crude extract,with the purity of 94.2%,95.8%,93.7%and 96.4%,respectively,as determined by high performance liquid chromatography(HPLC).The structures of the isolated compounds were positively confirmed by ~1H NMR and ~(13)C NMR,UV,IR,and MS analysis.
A preparative reversed phase liquid chromatography(PRPLC)combined with polyamide column chromatography was used to isolate the chemical constituents from the theaflavins extract.The separation condition was optimized as follows.the column was PRC-ODSC18(20.0mm×250mm i.d.,10um).The mobile phase for the HPLC consisted of acetonitrile,ethylacetate,and 2%acetic acid at the ratio:21:3:76.The flow rate was set at 15ml/min.The detection wavelength and sample size were set at 280nm and 400μl(30mg/ml).Under the optimized conditions,four compounds (theaflavin,theaflavin 3-gallate,theaflavin 3-gallate and theaflavin 3,3'-digallate) were isolated with the purity of 96.9%,98.8%,95.9%and 98.9%,respectively,as determined by high performance liquid chromatography.Their chemical structures were identified as theaflavin,theaflavin 3-gallate,theaflavin 3-gallate and theaflavin 3,3'-digallate by spectroscopic methods including ultraviolet,infrared,electrospay mass spectrometry and nuclear magnetic resonance(NMR),as well as by comparison with published spectral data.
High-performance Thin layer chromatography of theaflavins was performed on polyamide with chloroform-methanol 1:1(two fold development).TLC of EtOAc phase was performed on polyamide with acetic acid-methanol 1:1 and methanol-acetone-formic acid 2:1:1(two fold development).The following constituents were identified on the basis of their Rf value and absorbance spectrum,included epigallocatechin gallate,epicatechin gallate,epicatechin,theaflavins and their various gallate derivatives.
4 studying the anti-hyperlipemia,anti-inflammation and anti-cancer of theaflavins by High Throughput Screening(HTS)Method
HTS models of PPARs,FXR,LXR,TNFa and IL-1 were used to evaluate the pharmacological activity of theaflavins.TFs exhibited the highest inhibitory activity on LXR.The active times was 1.55.The second active one was TFDG.The results were in the following order:TFs>TFDG>TF3G≈TF>TF3'G TFs and TF,TF3G, TF3'G and TFDG had inhibition on FXR,in which TFDG exhibited the highest inhibitory activity.The active value is 2.35.The second active one was TF3'G which active value was 2.22.The results were in the following order:TFDG>TF3'G≈TF3G>TF>TFs.The results showed that the effects of theaflavins(TFs,TF,TF3G, TF3'G and TFDG)on TNFa and IL-1 were insignificant.
The inhibition effects of theaflavins complex(TFs),theaflavin, theaflavin-3-gallate,theaflavin-3'-gallate,and theaflavin-3-3l-digallate on the growth of human liver cancer HepG2 cells,gastric cancer SGC-7901 cells,lung cancer A549 cells and acute promyelocytic leukemia K562 cells were investigated.The effects of theaflavins(TFs,TF,TF3G,TF3'G and TFDG)on SGC-7901 and A549 were significant at the concentration of 100μg/ml.TF3G exhibited inhibitory effect against SGC-7901,K562,A549 and HepG2 at the concentration of 50μg/ml and TF exhibited little inhibitory effect against HepG2 at the concentration of 100μg/ml.It implied that TF3G had more multifaceted bioactivity functions whereas TFs,TF, TF3'G and TFDG had less.
引文
[1]杨贤强,王岳飞,陈留记等著.茶多酚化学[M].上海科学技术出版社,2003,上海
[2]陈宗道,周才琼,童华荣.茶叶化学工程学[M].西南师范大学出版社,1999,重庆
[3]宛晓春主编.茶叶生物化学[M].中国农业出版社,2003
[4]Roberts E A H.The phenolic substances off manufactured tea(I)[J].J Sci Food Agric,1957,8:72-80,1958,9:212-216,1958,9:217-223,1959,10:176-179
[5]Takino,Y.,Imagawa H.,Horikawa,et al.studies on the machanism of catecins by tea oxidase.Formation of a crystalline reddish orange pigment of benzotropolone nature[J].J.Agric.Biol.Chem.,1963,27,319-321
[6]Takino,Y.,Imagawa H.,Horikawa,et al.studies on the machanism of tea catecins:Parts Ⅲ,Formation of a reddish orange pigment and its special relationship to some benzotropolone derivatives[J].J.Agric.Biol.Chem.,1964,28,64-71
[7]Takino,Y.,lmagawa H.,Horikawa,et al.studies on the machanism of tea catecins:Parts IV,Formation of a categallin and pyrogallin,new compounds of benzotropolone nature derived from epigallocatechin[J].J.Agric.Biol.Chem.,1964,28,125-130
[8]Coxon D.T.,Holms A,Ollis W D.,et al.The constitution and configuration of the theaflavin pigments of black tea[J].Tetrahederon Lett.,1970,60:5237-5240
[9]Coxon D.T.,Holms A.Ollis W D.,et al.Theaflavic and epitheaflavic acid[J].Tetrahedron Lett.,1970,60:5247-5250
[10]滝野庆则,力泽哲夫,真田宏夫,等.Studies on the mechanism ofthe oxidation oftea catechins:part V[J].农芸化学会志(日本),1971,45:176-183
[11]Berkowitz J.E.,et al Formation of epitheaflavic acid and its transformation to thearubigins during tea fermentation[J].Phytockemistry,1971,10:2271-2278
[12]Sanderson,G W.the chemistry of tea and tea manufacturing[J].Recent advances in phytochemistry,1972,5:247-316
[13]Coggon P.,Fairley C J.,Millin D.J.,et al.The biochemistry of tea fermentation:oxidative degallation and epimerization of the tea flavanol gallates[J].J.Agric.Food chem.1973,21:727-733
[14]Collier P.D.,et al.The theaflavins of black tea[J].Tetrahedron,.1973,29:125-142
[15]Dix M.A.,et al.Fermeatatio of tea in Aqueous lnfluence of Tea Peroxidase[J].J.Sci.Food Agric,1981,32:923-932
[16]Takeo T.Photochemistry[J],1981,20(9):2145-2147
[17]Nonaks R.G.,et al.The chemical oxidation of catechins and other phenolics:a study of the formation of black tea pigments[J].J.Sci.Food Agric.1993,63:455-464
[18]Davis A P,Y cai,A L Davis,et al.Ployphenol Dimmers of.Black tea[J].1997,中国国际茶技术与茶文化交流会:8-10
[19]Lewis J R,Davis A L,Ya Cai,et al.Theaflavate B,isotheaflavin-3'-O-gallate and Neoheaflavin-3'-O-gallate:three polyphenolic pigments from black tea[J].phytochemistry,1998,49(8):2511-2519
[20]Xiaochun Wan,Harry E Nursten,Ya Cai,et al.A new type of tea pigment -from the chemical oxidation of epicatechin gallate and isolated from tea[J].J Sci Food Agric[J].,1997,74:401-108.
[21]Bajaj K L,Anan T,Tsushida T,et al.The effect of(-)-EC or the oxidation of TF by PPO from tea leaves[J].Agric Biol Chem,1987,51:1767-1772
[22]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
[23]P.J.Hilton.In vitro oxidation of flavanols from tea leaf[J].Phytochemistry.1972,Vol.11:1243-1248
[24]Robertson Alastair.Effects of catechin concentration on the formation of black tea polyphenols during in vitro oxidation[J].Phytochemistry,1983,22(4):897-903
[25]Robertson Alastair.Effects of physical and chemical conditions on the in vitro oxidation of tea leaf catechins[J].Phytochemistry,1983,22(4):889-896
[26]Millin D,J.Swaine D.Fermentation of tea in aqueous suspension[J].J Sci Food Agric,1981,32:905-919
[27]Finger A.In-vitro studies on the effect of ployphenol oxidase and peroxidase on the formation of ployphenolic black tea constituents.J Sci Food Agric[J],1994,66:293-305
[28]N.Subramanion,Purna Venkatesh,Shovan Ganguli et al.Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins[J].J Sci Food Chem,1999,47,2571-2578
[29]Shengmin Sang,Joshua D.Lambert,Shiying Tian et al.Enzymatic synthesis of tea theaflavin derivatives and theiranti-inflammatory and cytotoxic activities[J].Bioorganic & Medicinal Chemistry,2004,12:459-467
[30]谷纪平.茶黄素酶促氧化制备技术的研究[D].湖南农业大学2004硕士论文,湖南长沙,2004,06
[31]吴红梅.多酚氧化酶酶源筛选及酶法制取茶色素研究[D].安徽农业大学2004届硕士论文,安徽合肥,2004,06
[32]萧伟祥,钟瑾,胡耀武.双液相系统酶化学技术制取茶色素[J].天然产物研究与开发,2001,13(5):49-52
[33]Komatsn Y.Suematsu S,Hisanobu Y.et al.Effects of PH and temperature on reaction kinetics of catechin in green infusion[J].Biosci.Biotech.Biochem,1993,57(6):907-910
[34]夏涛.红茶色素形成机理的研究[J].茶叶科学,1999,19(2):139-144
[35]Owour P O.Orchard J,McDowell I.Changes in the quality parameters of clonal black tea due to fermentation time[J].J Sci Food Agric[J],1994,64:319-326
[36]萧伟祥,钟瑾,萧慧,等.茶红色素形成机理与制取[J].茶叶科学,1997,17(1):1-8
[37]夏涛,童启庆,萧伟祥.悬浮发酵红茶与传统红茶品质比较研究[J].茶叶科学,2000,20(2):105-109
[38]魏锋,马双成,金红军等.天然产物化学研究现状[J].国外医药:植物药分册,1999,14(3):93-101
[39]程启坤.红茶中的茶黄素[J].国外农学—茶叶,1983(1):1-14
[40]Crispin D J.The separation of theaflavins on sephadex LH-20[J].J Chromatogr.,1971,54:133-135
[41]竹尾忠一.茶黄素的分离与定量[J].茶叶技术研究,1973(45):46
[42]Bailey R G,Nursten H E,et al.Use of an HPLC photodiode-array detector in a study of the nature of a black tea liquor[J].J Sci food Agric[J],1990,52:509-525
[43]Bailey R.G.,et al Journal of Chromatography[J],1991,542:115-128
[44]Bee-Lan Lee,Choon-Nam Ong.Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillary electrophoresis[J].Journal of Chromatography A,881(2000):439-447
[45]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
[46]Qizhen Du,Heyuan Jiang,Yoichiro Ito.Separation of theaflavins of black tea:High-speed countercurrent chromatography vs sephadex LH-20 gel column chromatography[J].Journal of Liquid Chromatography & Related Technologies,2001,24(15):2363-2369
[47]Xueli Cao,John R.Lewis,and Yoichiro Ito.Application of high-Speed countercurrent Chromatography to the separation of black tea Theaflavins[J].Journal Of Liquid Chromatography & Related Technologies,2004,Vol.27,No.12,pp.1893-1902
[48]中国专利,CN1345899A;中国专利,CN1074459A;中国专利,CN1285152A;中国专利,CN1403580A;中国专利,CN1328092
[49]王坤波.茶黄素的生化调控和分离纯化技术研究[D].湖南农业大学硕士论文,2002,06
[50]丁阳平.茶黄素类单体分离及对唾液淀粉酶、脂肪酶和环氧和酶的影响研究[D].湖南农业大学硕士论文,2004,06
[51]原征彦等.農芸化學會志,1987,61:803-808
[52]Zhen Yong-su,Chen Zongmao,Cheng Shu-jun et al.Tea bioactivity and therapeutic potential[M].Taylor & Francis.CRC press Inc.,2002
[53]David J.Maron,Guo Ping Lu,Nai Sheng Cai,Zong Gui Wu,et al.Cholesterol-Lowering Effect of a Theaflavin-Enriched Green Tea Extract[J].Archive Internanl Medicine,2003,163:1448-1453
[54]谢笔钧,等.绿茶、乌龙茶、红茶中的主要组分和酚类化合物抑制氧合酶活性和抗油脂自动氧化特性的研究[J].天然产物研究与开发,1994,16(4):19-26
[55]Yoshino K,et al.Antioxidative effects of black tea theaflavins and thearubigin on lipid of rat liver homogenates induces by tert-butyl hydroperoxide[J].Biol pharm Bull,1994,17(1):146-149
[56]Yoshida H.et al.Inhibitory effect of tea theaflavins on the ability of cells to oxidize low density lipoprotein[J].Biochemical Pharmacology 1999,58(11):1695-1703
[57]Miller,N.J.,Castelluccio,C,Tijburg,L,Rice-Evans,C.The antioxidant properties of theaflavins and their gallate esters radical scavengers or metal chelators[J].FEBS letters,1996,392(l):40-44
[58]南条文雄.红茶的机能性[J].New Food Industry 2000,42(5):49-55
[59]胡香芳等.茶多酚抗氧化的构效关系[M].由基生命科学进展(第八集),北京:原子能出版社,200l:60-69
[60]仲川清隆,等.食品机能研究法[M].东京,光琳书院,2000,241-245
[61]Masao H,Tomoco K I,Tsuneo N,et al,Effect of tea polyphenols on glucan synthesis by glucosyltransferase from streptococcus mutans[J].Chem.Pharm.Bull..1990,38:717-720
[62]Yoshino K.,Nakamura,Y.,Ikeya,H.,Sei.T.,M.,Tomita.Antimicrobical activity of tea extracts on cariogenic bacterium(Streptococcus mutans)[J].Journal-food hygienic society of Japan,1996,37(2):104-108
[63]Tanaka N.et al.on the factors affecting variation of a-amylase activity in saliva of normal subjects:Ⅱ effects of tannin(polyphenol)in a black tea solution on a-amylase activity in saliva[J].Nippon Kasei Gakkaishi,1989,40(7):587-594
[64]李立祥,萧伟祥.茶色素及茶黄素理作用研究进展[J].福建茶叶,2002(4):35-37
[65]Toda M.et al.antibacterial and bactericidal activities of tea extracts and catechins against methicillin-resistant staphylococcusaureus[J].Nippon saikingaku zasshi-Japanese journal of bacteriology,September 1991,46:839-845
[66]杜琪珍,江和源.茶色素的药理及功效.中国茶叶[J],1997,19(5):42-43
[67]安藤寿.含有茶黄素的酶氧化绿茶提取物及儿茶素氧化生成物抑制淀粉酶的作用[J].国外医学:中医中药分册,2004(6):372
[68]Clark,K.J.,Grant,P.G,Sarr,A.B.,Belakere,J.R.,Swaggerty,C.L.,Phillips,T.D.,Woode,G N.An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections[J].Veterinary microbiology,1998.63(2-4):147-157
[69]Nakayama M.,Toda M.,Okubo S.,Shimamura,T.inhibition of influenza virus infection by tea[J].Letters in Appl microbio,1990(11):38-40
[70]Mikio Nakayama,Kenji Suzuki,Masako Toda,Sachie Okuboc,Yukihiko Hara and Tadakatsu Shimamurac.Inhibition of the infectivity of influenza virus by tea polyphenols[J].Antiviral Research,1993,Vol 21(4):289-299
[71]Nakano H.et al.Proceeding of international symposium on tea sci[J].Shizuoka Japan/The orgariging committee of ISTS,1992:282
[72]Shuwen Liu,Hong Lu,Qian Zhao,Yuxian He,et ah Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41[J].Biochimica et Biophysica Acta 1723(2005)270-281
[73]Chia-Nan Chen,Coney P.C.Lin1,Kuo-Kuei Huangl,Wei-Cheng Chenl et al.lnhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate[J].eCAM 2005;2(2)209-215 doi:10.1093/ecam/neh081
[74]Guang-Yu Yang,Jie Liao et al.Effect of black and green tea polyphenols on c-jun phosphorylation and H_2O_2 production in transformed and non-transformed human bronchial cell)lines:possible mechanisms of cell growth inhibition and apoptosis induction[J].Carcubigenesis,2002,No 11,2035-2039
[75]Jie BoLu,Chi-Tang Ho,Geetha Ghai and Kuang Yu Chen et al.Differential effects of theaflavin monogallates on cell growth,apoptosis,and Cox-2 gene expression in cancerous versus normal cell[J].Cancer Research,2000,60(22):6455-6471
[76]Hibasamj H Komiya T,et al.Black tea theaflavins induce programmed cell death in cultured human stomach cancer cells[J].International Journal of Molecular Medicine 1998,1(4);725-727
[77]More M.A.,et al.effects of theaflavins on N-nitrosomethylbengy-lamine induced esophageal tumorigenesis[J].Nutrition and cancer,1997,29(1):7-12
[78]Yang-Guang Yus et al.black tea constituents,theaflavins,inhibit 4-(methyl nitrosamino)-1-(3-pyridy)-l-butanone(NNK)-induced lung tumorigenesis in A/J mice[J].Carcinogenesis,1997,18(12):2361
[79]You-Ying TU,An-Bin TANG,and Naoharu Watanabe.The Theaflavin Monomers Inhibit the Cancer Cells Growth in Vitro[J].Acta Biochimica et Biophysica Sinica 2004,36(7):508-512
[80]Saeki K.,et al.Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells[J].Biosci.Biotechnol.Biocheml,1999,63(3):585-587
[81]大泽俊彦.应用植物成分进行化学防癌[J].日本医学介绍,2004,Vol.25(1)26-27
[82]Y C Liang,S Y Lin.Shiau,C T Ho.Inhibition of TPA-induced Protein kinase C and transcription activator Protein-1 binding activities by theaflavin-3-3'-digal late from black tea in NIH3T3 cells[J].J.Agric Food Chem.1999 47(4):1416-1421
[83]Chung J.K.Huang C.S.Inhibition of activator protein 1 activity ob cell growth by purified green tea and black tea polyphebols in H-rat-transformed cells structure-activity relationship and mechanisms involved[J].Cancer Research 1999,59(18):4610-4617
[84]Yu-Li Lin,Shu-Huei Tsai,Shoei-Yn Lin-Shiau,Chi-Tang Ho,et al.Theaflavin-3,3X-digallate from black tea blocks the nitric oxide synthase by down-regulating the activation of NF-kB in macrophages[J].European Journal of Pharmacology,1999(367):379-388
[85]Liang Yuchih et al.Suppression of extracellar signals and cell proliferation by the black tea polyphenols,theaflavin-3,3'-digallate[J]Carcinogenesis 1999,20(4):733-736
[86]Chen Yenchou,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[J].J Agric food chem.,1999,47(4):1416-1421
[87]Maeda-Yamamoto M.et al.effects of thearubigin and genistein on human prostate tumor cell(PC-3)growth via cell cycle arrest.Cancer letters,2000,15(1):103-109
[88]Apostolides Z.V.Balentine D.A.lnhibition of 2-amino-l-methyl-6-phenylimidazo[4,5,6]pyridine(PHIP)mutagenicity by black and green tea extracts and polyphenols[J].Mutaion Research,1996,359:159-163
[89]Sazuka M,Imazawa H.,Shoji Y,Mita T.,Hara Y.,Isemura M..Inhibition of collagenases from mouse lung carcinoma cells by green tea catechins and black tea theaflavinsfJ].Biosci.biotechnol.biochem.1997,vol.61:1504-1506
[90]Man MY,Hiroharu K,Nobufumi T,Kenkou T,Yukihiko H,Mamoru I(1999).Effects of tea polyphenols on the invasion and matrix metalloproteinase activities of human fibrosarcoma HT1080 cells[J].J.Agric.Food Chem.47:2350-2354
[91]中华药学会重点推广工程《茶色素推广中心》编著.北京,中国茶色素临床科研论文专辑,1998
[92]http://food.icxo.com/htmlnews/2004/07/29/278482.htm
[93]http://www.xoe.cn/
[94]http://www.nashai.com/index.php
[95]Zhen Yong-su,Chen Zongmao,Cheng Shu-jun et al.Tea bioactivity and therapeutic potential[M].Taylor & Francis,CRC press Inc.,2002.
[96]山根横夫著.生化反应工程[M](周斌编译).西北大学出版社,1992年
[97]巫光密,詹福建,黄卓烈等.马占相思树两家系过氧化物酶、多酚氧化酶活性及同工酶比较研究[J].亚热带植物科学,2002,31(2):15
[98]辛广,侯冬岩,张兰杰等.南果梨果皮、果肉、果心多酚氧化酶同工酶的研究[J].鞍山师范学院学报,2004,6(6):50-52
[99]袁艺.侧耳(平菇)的酯酶、多酚氧化酶及过氧化酶同工酶的电泳分析[J].安徽农业太学学报,1997,24(1):88-92
[100]萧伟祥.茶的多酚氧化酶和过氧化物酶的研究进展[J].茶业通报,1983,(6):3-7
[101]刘仲华,施兆鹏.红茶制造中多酚氧化酶同工酶谱与活性的变化[J].茶叶科学,1989,9(2):141-150
[102]Takashi Tanaka,chie mine,kyoko inoue,miyuki matsuda,and isao kouno.Synthesis of Theaflavin from Epicatechin and Epigallocatechin by Plant Homogenates and Role of Epicatechin Quinone in the Synthesis and Degradation of Theaflavin[J].J.Agric.Food Chem.2002,50,2142-2148
[103]Jhoo Jin-woo.Chemistry of black tea(camellia sinensis)polyphenols and blue cohosh(caulophyllum thalictroides)saponins(D).Rutgers,the state university of new Jersy,2003,October.
[104]李适.微生物多酚氧化酶酶源筛选及其在茶黄素合成中的应用[D].湖南农业大学2006届硕士论文,湖南长沙,2006,06
[105]商业部茶叶畜产局,商业部杭州茶叶加工研究所编著.茶叶品质理化分析[M],1989,471-47
[106]林向东,张琪,李冀新等.无核白葡萄多酚氧化酶特性研究[J].食品科学,2000,21(12):43-45
[107]王坤范,陈秀芳。桃果实多酚氧化酶性质的研究[J].北京农业太学学报,1995,21(4):370-376
[108]李立祥.茶色素形成机理及制取新工艺研究[D].安徽农业大学博士论文,安徽合肥,2002,10
[109]CattellDJ,NurstenHE.Separation of thearubigins on sephadex LH-20[J].Phytochenistry,1977,16:1269-12723
[110]OzawaT.Separation of the Components in Black Tea Infusion by Chromatography on Toyopearl[J].AgricBiol.Chem,1982;46(4):1079-1814
[111]Daniele DEl Rio;Amanda J.Stewart;William Mullen;Jennifer Burns;Michael E.J.Lean;Furiio Brighenti;Alan Crozier.HPLC-MSn Analysis of Phenolic Compounds and Purine Alkaloids in Green and Black Tea[J].Journal of Agricultural and Food Chemistry,2004,Vol.52,2807-2815
[112]Roberts EAH,CartwrightRA,01dschoolM.The Phenolic Substances of Manufactured Tea.J Sci Food Agric,1957;2:72-80
[113]Bailey RG,Nursten HE,Mc Dowelll.Isolation and Analysis of a Polymeric Thearubigin Fraction form Tea[J].J Sci Food Agric,1992;59:365-375
[114]Bailey RG,Nursten HE,McDowell I.Use of an HPLC Photodiode-array Detector in a Study of the Nature of Black Tea Liquor[J].J Sci Food Agric,1990,52:509-525
[115]Opie S.C,RobertsonA,Clifford M N.Black Tea Thearubigins-Their Separation and Preparation during in vitro Oxidation[J].J Sci Food Agric,1990;50:547-5617
[116]Bailey R G Nursten H E and Ian McDowell.Comparative study of the reverse-phase high-performance liquid chromatography of black tea liquors with special reference to the thearubigins[J].Journal of Chromatography,1991,542:115-128.
[117]Degenhardt A,Engelhardt U.H,Wendt A.S,Winterhalter P.Isolation of black tea pigments using high-speed countercurrent chromatography and studies on properties of black tea polymers[J].Journal of Agricultural and Food Chemistry,2000,V.48,no.11:5200-5205
[118]徐向群,Clifford M.N.红茶酚性色素的分离[M].茶叶科学,1998,18(1):155-156
[119]邵宛芳,M.N Clifford,C.Powell.红茶及普洱茶主要成分差异的初步研究[J].云南农业大学学报,1995,Vol.10 No.4:285-291
[120]K.霍斯泰特曼,A.马斯顿,M.霍斯泰特曼编著.制备色谱技术在天然产物分离中的应用[M].科学出版社,北京,2000
[121]袁黎明编著.制备色谱技术及应用[M].北京,化学工业出版社,2004
[122]魏莉,张煜,方岩雄,赵肃清.制备型高效液相色谱技术的研究进展[J].广东化工,2005(11):5-7
[123]曹学丽编著.高速逆流色谱分离技术及应用[M].北京,化学工业出版社,2005.02
[124]张天佑。逆流色谱技术[M].北京科技出版社,1991.03
[125]匡海学,董小萍,石任兵编著.中药化学[M].北京,中国中医药科技出版社,2003.01
[126]Rober M.Silverstein,Francis X..Webster,David J.Kiemle著,药明康德新药研究开发优秀公司分析部译.有机化合物的波谱解析[M].上海,华东理工大学出版社,2007.01
[127]Broach J R,Throner J.High through screening for drug discovery[J].Nature,1996,384:14
[128]刘文尧,栾新慧,赵毅民.高通量筛选的检测原理及应用[J].解放军药学学报,2003,19(1):63-66
[129]J Issemann I,Green S.Activation of the steroid hormone receptor srperfamily by peroxisome pmlifemtom[J].Nature,1990,347(6294):645-650
[130]Okuno A,Tamemoto H,Tobe K,et al.Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats[J].Clin Invest,1998,101(6):1354-1361
[131]Auboeuf,Rietmset J,Fajas L,et al.Tissue distribution and quantification the expression of mRNAs of peroxisome proliferators-activated receptors and liver X receptor-alpha in humans:no alteration in adipose tissue of obese and NIDDM patients[J].Diabetes,1997,46:1319-1327
[132]Di P N,Michalik L,Tan N S,et al.The anti-apoptotic role of PPAR β contributes to efficient skin wound healing[J].J Steroid Biochem Mol Biol,2003,85(225):257-65
[133]Guan Y,Breyer M D.Proliferators-activated receptors(PPARs):novel therapeutic targets in renal disease[J].Kidney Int,2001,60:14-30
[134]Wang Y X,Lee C H,Tiep S,et al.Peroxisome proliferators-activated receptor 5 activates fatmetabolism to prevent obesity[J].Cell,2003,l 13(2):159-170
[135]Hihi A K,Michalikl,Wahlt W.PPARs:transcriptional effectors of fatty acids and their derivatives[J].Cell Mol Life Sci,2002,59:790-798
[136]Dressel U,Allen T L,Pippal J B,et al.The peroxisome-prolif erator-activated receptor β/δ Agonist,GW501516,regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells[J].Mol Endocrinol,2003,17(12):2477-2493
[137]谢谆怡,彭家和,江渝.PPAR γ与代谢性疾病[J].生命的化学,2004,24(2):152-154
[138]Makishima M.Okamoto A,Repa J,et al.ldentification of a nuclear receptor for bile acid[J].Science,1999,284:1362-1365
[139]Forman B M,Goode E,Chen J,et al.ldentification of a nuclear receptor that is activated by farnesol metabolites[J].Cell,1995,81:687-693
[140]Zhang Y Q,Kast-Woelbem H R,Edwards P A.Natural strucrural variants of the nuclear receptor FXR afect transcriptional activation[J].Biol Chem,2002,278:104-110
[141]杨光锐,吴静,陈丽红.代谢性核受体及其与代谢综合征的关系[J].基础医学与临床,2006,26(1):35-41
[142]黄路平.生物活性成分的高通量筛选(第一版)[M].第二军医大学出版社,2002
[143]Coggon Philip,Moss Gerald A,Sanderson Gary W.Tea catechol oxidase:isolation,purification and kinetic characterization[J].Phytochemstry,1973,12:1947-1955
[144]Trina Kundu,Subhabrata Dey,Madhumita Roy,M.Siddiqi,R.K.Bhattacharya.Induction of apoptosis in human leukemia cells by black tea and its polyphenol theaflavin[J].Cancer Letters,2005(230):111-121
[145]李伟,屠幼英.茶叶提取物对抗肺癌细胞(A549)体外试验研究[J].茶叶,2006,32(1):28-30
[146]周艳,顾星星,胡亚娥等.三氧化二砷对肝癌细胞株SMMC-7721、HepG2抑制作用的研究[J].南通医学院学报,2005,25(1):9