【作者】 王坤波；

【导师】 刘仲华； 黄建安；

【作者基本信息】 湖南农业大学 ， 茶学， 2007， 博士

【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,¹³CNMR,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+21.359X₁-0.683X₂-1.841X₃（Y:茶黄素总量,X₁:pH,X2:温度,X₃: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,¹³NMR,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。更多还原

【Abstract】 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 H₂O₂ 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 oxidaseThe 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 theaflavinsAn 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+21.359X₁-0.683 X₂-1.841X3.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 ¹³C 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）MethodHTS 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.更多还原