茶（红茶）与茶（Camellia sinensis）花多酚类物质的分离鉴定及其抗氧化机理研究
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摘要
第1部分茶黄素单体的分离与红茶多酚衍生物质的抑菌、抗氧化活性与机制研究
多酚类物质的抗氧化作用被认为是茶叶保健抗癌最重要的作用机理。大量生物体内、体外研究和流行病学调查等都证实了绿茶及其多酚类物质—儿茶素具有良好的抗氧化功效。一直以来绿茶及其活性成分在儿茶素的研究备受关注，而红茶在世界茶消费中占80％，所以，近十年来其多酚类衍生物质—茶黄素与茶红素的生物活性也逐渐引起研究学者的重视。本研究采用体外、细胞实验评估红茶中多酚衍生物质—茶色素的抗氧化活性并利用LC-MS与NMR技术从化学结构的角度对其抗氧化机制进行讨论。
主要研究结果如下：
1．本研究首先采用固定化酶催化茶多酚氧化，所得产物为茶黄素复合物；其次利用高速逆流色谱(HSCCC)分离茶黄素单体，比较不同溶剂系统与NaHCO_3前处理对其分离效果的影响。试验结果表明：采用溶剂系统乙酸乙酯、正己烷、甲醇和水(3∶1∶1∶6，v／v)与(3∶1∶3∶4，V／V)结合分离具有较佳的分离效果；NaHCO_3前处理明显有助于茶黄素单体的分离；当流动相速度1.2ml／min、仪器转速880r／min时，能达到最好的分离效果；从250mg茶黄素复合物中分离所得57mg 97.7％茶黄素(theaflavin)、26mg 61.2％茶黄素-3'-单没食子酸酯(theaflavin-3'-gallate)、64mg 84.5％茶黄素-3，3'-双没食子酸酯(theaflavin-3，3'-gallate)与33mg of 92.3％茶黄素单食子酸酯(theaflavin-3-gallate and theaflavin-3'-gallate)。
2．采用微生物实验方法比较了茶黄素复合物和茶多酚对口腔两种主要致病菌变链菌族的S．mutans Ingbritt和S．sobrinus 6715的抑制作用及其对细菌产酸能力的影响。结果表明茶黄素具有较强的抑菌和杀菌能力，与茶多酚相比无显著性差异，且变链菌对两种多酚化合物均未产生耐药性。
体外模拟红茶中酶促氧化过程制备三类主要多酚氧化产物—茶黄素、茶红素及茶褐素，并对这三类产物的体外清除羟自由基、DPPH自由基能力，及其对HPF-1细胞氧化损伤的保护作用进行评估。结果表明茶黄素在上述体系中显示了最强的抗氧化能力，茶褐素次之，茶红素最弱，且这三类物质均可通过清除细胞内的活性氧保护HPF-1细胞。值得关注的是，对红茶品质具有消极影响的茶褐素却具有接近于茶黄素的活性，因此有必要深入研究红茶中每一种多酚氧化产物的生物活性，对正确评估其在红茶保健作用中的贡献具有重要的意义。
以EGCG为对照，对茶黄素主要单体茶黄素(TF1)、茶黄素单没食子酸酯(TF2)与茶黄素双没食子酸酯(TF3)的体外清除羟自由基、DPPH自由基能力，及其对HPF-1细胞氧化损伤的保护作用进行研究。结果表明，对羟自由基的清除能力依次为TF3＞TF2＞TF1＞EGCG；对DPPH自由基清除能力依次为TF3＞TF2＞EGCG＞TF1；茶黄素各单体对H_2O_2诱导氧化损伤的HPF-1细胞具有保护作用，且强于EGCG。除了清除细胞内的活性氧外，TF3与TF1还可能通过提高细胞抗氧化体系，保护H_2O_2诱导氧化损伤的HPF-1细胞。TF2对正常HPF-1细胞则表现出最强的增殖作用。
第2部分
茶(Camellia sinensis)花中多酚类组成分析及其抗氧化活性与机理研究
长期以来，人们只是重视茶树芽叶的生产加工，对于具有丰富资源的茶树花朵任其自生自灭，造成资源的大量浪费。已有研究表明茶花含有与茶鲜叶相似的重要生化成分如多酚、咖啡碱，多糖和蛋白质等。本研究对茶花中黄酮类等重要化学成分进行分离和鉴定，对其抗氧化活性进行评估，并利用HPLC与LC-MS技术从化学结构的角度对其抗氧化机制进行阐明。
1．采用不同的有机溶剂，分步萃取冷冻干燥茶花的70％乙醇提取物与水提物，得到氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物和萃取后剩余物。采用有机自由基二苯基苦基偕腙肼(DPPH)的分光光度法及其化学发光法，比较各提取物的抗氧化能力。结果表明茶花醇提物比水提物具有更强的清除自由基能力，且醇提物的乙酸乙酯萃取组分(EEA)在所有组分中具有最强的抗氧化活性，同时对茶花的抗氧化能力具有最大的贡献率。通过对各萃取组分的化学成分测定，表明其抗氧化活性与其多酚类物质有关。
2．通过液质联用分析从茶花乙酸乙酯层中鉴定出3类化合物：a．儿茶素类：儿茶素、表儿茶素、没食子儿茶素、表没食子儿茶素、儿茶素没食子酸酯、表儿茶素没食子酸酯、没食子儿茶素没食子酸酯、表没食子儿茶素没食子酸酯；b．儿茶素衍生物：儿茶素糖苷物与儿茶素二聚体；c．黄酮苷类：山奈酚、毛地黄黄酮与槲皮素单糖苷及双糖苷类物质。经HPLC分离与DPPH测定，确定表没食子儿茶素没食子酸酯与表儿茶素没食子酸酯为茶花中主要抗氧化活性成分，在茶花提取物的抗氧化活性中起到主要的作用。
Part 1 Autioxidant activity and mechanism of phenolic compounds present in black tea
Tea is one of the most widely consumed beverages in the world. Green tea is more popular in China, Japan, Korea and some African countries, whereas black tea is preferred in India and the Western countries. During the past decade numerous in vitro and in vivo studies have suggested the possible protective effects of green tea and catechins in cancer and cardiovascular disease development. Whereas catechins are the most abundant polyphenols in green tea, the typical pigments in black tea are theaflavins (TF), thearubigins (TR) and theabrownins (TB), which are derived from the oxidation of catechins and their gallates during fermentation stage of black tea processing. Recently, the molecular mechanism and bioactivities of polyphenols in black tea have attracted considerable interest because of its beneficial health properties.
In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The major theaflavins monomers were separated by high-speed countercurrent chromatography (HSCCC). The antioxidant activities of these products were evaluated in Fenton reaction system, 2,2-diphenyl-1- picrylhydrazyl (DPPH) system and H_2O_2-induced oxidative damage system in the HPF-1 cells. Their antioxidant mechanisms were discussed. The main results were showed as followings:
1. Separation of theaflavins monomers.
Three theaflavins monomers were separated from the oxidation product of polyphenols by immobilized polyphenol oxidase plate using HSCCC. The effects of solvent systems and NaHCO_3 on the separation were investigated. The result showed that NaHCO_3 was helpful to purify the theaflavins. Separation was performed with a two-phase solvent system composed of ethyl acetate - hexane -methanol-water (3:1: 1: 6, V/V) and (3: 1: 3: 4, V/V) by eluting the lower aqueous phase at 1.2 ml/min at a revolution speed of 880 rpm. 57 mg of 97.7% theaflavin, 26 mg of 61.2% theaflavin-3'-gallate, 64 mg of 84.5% theaflavin-3-3'-gallate and 33 mg of 92.3% theaflavin-3-gallate and theaflavin-3'-gallate complex were obtained from 250 mg of the crude TFs sample.
2. The antibacterial, and antioxidant function and mechanism of black tea pigments.
The effects of theaflavins and tea polyphenols on two major oral bacterium, namely S.mutans Ingbritt and S.sobrinus 6715 were investigated. It indicated that the two polyphenols had the potent inhibiting activity. It was no remarkable difference between them. Furthermore, the two bacterium had no medicine-resistant against the two polyphenols.
In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The order of OH·and DPPH scavenging ability of these oxidation products was TF> TB> TR. These oxidized phenolic compounds showed protection against H_2O_2-induced damage in HPF-1 cells and suppressed the accumulation of intracellular reactive oxygen species in H_2O_2-induced damage HPF-1 cells. Interestingly, TB, as a further oxidative product from TF or TR, showed the potent activity followed TF in the above four systems.
The protective effect of three major black tea polyphenols, namely theaflavin (TF1), theaflavin-3 (3')-gallate (TF2) and theaflavin-3,3'-digallate (TF3) on oxidative damage human fibroblast (HPF-1) cell were investigated by comparing with epigallocatechin gallate (EGCG). The order of OH·and DPPH scavenging ability were TF3 > TF2 > TF1 >EGCG and TF3>TF2>EGCG>TF1 respectively. TF1, TF2, and TF3 showed more effective than EGCG in protection against H_2O_2-induced damage in HPF-1 cells. Pretreated for 2h and eliminated from the cell, TF1 and TF3 still showed protection against H_2O_2-induced damage in HPF-1 cells. TF1, TF2 and TF3 suppressed the accumulation of intracellular reactive oxygen species (ROS) in H_2O_2-induced damage HPF-1 cells. Our results suggest, other than inhibition of ROS, the protective effect of TF1 and TF3 on oxidative damage HPF-1 cell may also be responsible for improvement of normal HPF-1 cell resistive ability toward radical-damage. TF2 may protect against H_2O_2-induced damage in HPF-1 cells, through enhancing HPF-1 cell proliferation.
Part 2 Analysis of chemical compositons in tea (Camellia sinensis) flowers and their antioxidant mechanism
While these properties of tea have been extensively studied, the less attention has been given to these properties of tea flower. This is due to the fact that people have been only picking the tender shoots from tea tree to make tea for a long history, but for tea flowers, the majority lets them run their courses. Compared with tea leaves, tea flowers have similar chemical compositions such as polyphenols and caffeine, polysaccharide and protein etc. The aim of the present study was to ascertain the phenolic profile of tea flower extracts, as well as to know the major active components responsible for the antioxidant activity of tea flowers. The antioxidant activity of the extracts was evaluated by DPPH assay. Phenolic compounds were separated using HPLC-DAD, and identified by means of LC-MS-ion trap.
1. Evaluation of the antioxidant compounds of extracts from tea (Camellia sinensis) flower.
The antioxidant activity of tea flower was investigated by the DPPH free radical-scavenging assay and the hydroxyl radical-induced luminol chemiluminescence assy. The tea flower was extracted with ethanol (EE) and separated by the order of chloroform, ethyl acetate (EEA), and n-butanol. The result pointed tea flower ethyl acetate extract (EEA) had the strongest antioxidant activity and the most contribution to the antioxidant activity of tea flower. The stronger scavenging abilities to free radicals were shown by EE and EEA, which might be due to the content of polyphenols contained. However, almost non free radical-scavenging activity was observed while the tea flower was extracted with water and its fractions of several organic solvents.
2. Liquid chromatographic-mass spectrometric analysis of phenolics in tea flower extract.
High performance liquid chromatography (HPLC) combined with diode array (DAD) and electrospray (ESI)-ion trap-MS detection was used to separate and identify the compounds present in EEA. Eight catechins, namely, catechin, epicatechin (EC), gallocatechin, epigallocatechin, gallocatechin gallate, epigallocatechin gallate (EGCG), catechin gallate and epicatechin gallate (ECG), catechins derivatives, namely, EC-3'-di- rhamnoside and catechins dimer, and flavonol glycosides, namely, quercetin, luteolin, kaempferide and kaempferol mono-, diglycosides (glucoside and rhamnoside) were identified. In addition, it is found that EGCG and ECG are the major active components responsible for the antioxidant activity of tea flowers by HPLC separation and DPPH assay.
多酚类物质的抗氧化作用被认为是茶叶保健抗癌最重要的作用机理。大量生物体内、体外研究和流行病学调查等都证实了绿茶及其多酚类物质—儿茶素具有良好的抗氧化功效。一直以来绿茶及其活性成分在儿茶素的研究备受关注，而红茶在世界茶消费中占80％，所以，近十年来其多酚类衍生物质—茶黄素与茶红素的生物活性也逐渐引起研究学者的重视。本研究采用体外、细胞实验评估红茶中多酚衍生物质—茶色素的抗氧化活性并利用LC-MS与NMR技术从化学结构的角度对其抗氧化机制进行讨论。
主要研究结果如下：
1．本研究首先采用固定化酶催化茶多酚氧化，所得产物为茶黄素复合物；其次利用高速逆流色谱(HSCCC)分离茶黄素单体，比较不同溶剂系统与NaHCO_3前处理对其分离效果的影响。试验结果表明：采用溶剂系统乙酸乙酯、正己烷、甲醇和水(3∶1∶1∶6，v／v)与(3∶1∶3∶4，V／V)结合分离具有较佳的分离效果；NaHCO_3前处理明显有助于茶黄素单体的分离；当流动相速度1.2ml／min、仪器转速880r／min时，能达到最好的分离效果；从250mg茶黄素复合物中分离所得57mg 97.7％茶黄素(theaflavin)、26mg 61.2％茶黄素-3'-单没食子酸酯(theaflavin-3'-gallate)、64mg 84.5％茶黄素-3，3'-双没食子酸酯(theaflavin-3，3'-gallate)与33mg of 92.3％茶黄素单食子酸酯(theaflavin-3-gallate and theaflavin-3'-gallate)。
2．采用微生物实验方法比较了茶黄素复合物和茶多酚对口腔两种主要致病菌变链菌族的S．mutans Ingbritt和S．sobrinus 6715的抑制作用及其对细菌产酸能力的影响。结果表明茶黄素具有较强的抑菌和杀菌能力，与茶多酚相比无显著性差异，且变链菌对两种多酚化合物均未产生耐药性。
体外模拟红茶中酶促氧化过程制备三类主要多酚氧化产物—茶黄素、茶红素及茶褐素，并对这三类产物的体外清除羟自由基、DPPH自由基能力，及其对HPF-1细胞氧化损伤的保护作用进行评估。结果表明茶黄素在上述体系中显示了最强的抗氧化能力，茶褐素次之，茶红素最弱，且这三类物质均可通过清除细胞内的活性氧保护HPF-1细胞。值得关注的是，对红茶品质具有消极影响的茶褐素却具有接近于茶黄素的活性，因此有必要深入研究红茶中每一种多酚氧化产物的生物活性，对正确评估其在红茶保健作用中的贡献具有重要的意义。
以EGCG为对照，对茶黄素主要单体茶黄素(TF1)、茶黄素单没食子酸酯(TF2)与茶黄素双没食子酸酯(TF3)的体外清除羟自由基、DPPH自由基能力，及其对HPF-1细胞氧化损伤的保护作用进行研究。结果表明，对羟自由基的清除能力依次为TF3＞TF2＞TF1＞EGCG；对DPPH自由基清除能力依次为TF3＞TF2＞EGCG＞TF1；茶黄素各单体对H_2O_2诱导氧化损伤的HPF-1细胞具有保护作用，且强于EGCG。除了清除细胞内的活性氧外，TF3与TF1还可能通过提高细胞抗氧化体系，保护H_2O_2诱导氧化损伤的HPF-1细胞。TF2对正常HPF-1细胞则表现出最强的增殖作用。
第2部分
茶(Camellia sinensis)花中多酚类组成分析及其抗氧化活性与机理研究
长期以来，人们只是重视茶树芽叶的生产加工，对于具有丰富资源的茶树花朵任其自生自灭，造成资源的大量浪费。已有研究表明茶花含有与茶鲜叶相似的重要生化成分如多酚、咖啡碱，多糖和蛋白质等。本研究对茶花中黄酮类等重要化学成分进行分离和鉴定，对其抗氧化活性进行评估，并利用HPLC与LC-MS技术从化学结构的角度对其抗氧化机制进行阐明。
1．采用不同的有机溶剂，分步萃取冷冻干燥茶花的70％乙醇提取物与水提物，得到氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物和萃取后剩余物。采用有机自由基二苯基苦基偕腙肼(DPPH)的分光光度法及其化学发光法，比较各提取物的抗氧化能力。结果表明茶花醇提物比水提物具有更强的清除自由基能力，且醇提物的乙酸乙酯萃取组分(EEA)在所有组分中具有最强的抗氧化活性，同时对茶花的抗氧化能力具有最大的贡献率。通过对各萃取组分的化学成分测定，表明其抗氧化活性与其多酚类物质有关。
2．通过液质联用分析从茶花乙酸乙酯层中鉴定出3类化合物：a．儿茶素类：儿茶素、表儿茶素、没食子儿茶素、表没食子儿茶素、儿茶素没食子酸酯、表儿茶素没食子酸酯、没食子儿茶素没食子酸酯、表没食子儿茶素没食子酸酯；b．儿茶素衍生物：儿茶素糖苷物与儿茶素二聚体；c．黄酮苷类：山奈酚、毛地黄黄酮与槲皮素单糖苷及双糖苷类物质。经HPLC分离与DPPH测定，确定表没食子儿茶素没食子酸酯与表儿茶素没食子酸酯为茶花中主要抗氧化活性成分，在茶花提取物的抗氧化活性中起到主要的作用。
Part 1 Autioxidant activity and mechanism of phenolic compounds present in black tea
Tea is one of the most widely consumed beverages in the world. Green tea is more popular in China, Japan, Korea and some African countries, whereas black tea is preferred in India and the Western countries. During the past decade numerous in vitro and in vivo studies have suggested the possible protective effects of green tea and catechins in cancer and cardiovascular disease development. Whereas catechins are the most abundant polyphenols in green tea, the typical pigments in black tea are theaflavins (TF), thearubigins (TR) and theabrownins (TB), which are derived from the oxidation of catechins and their gallates during fermentation stage of black tea processing. Recently, the molecular mechanism and bioactivities of polyphenols in black tea have attracted considerable interest because of its beneficial health properties.
In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The major theaflavins monomers were separated by high-speed countercurrent chromatography (HSCCC). The antioxidant activities of these products were evaluated in Fenton reaction system, 2,2-diphenyl-1- picrylhydrazyl (DPPH) system and H_2O_2-induced oxidative damage system in the HPF-1 cells. Their antioxidant mechanisms were discussed. The main results were showed as followings:
1. Separation of theaflavins monomers.
Three theaflavins monomers were separated from the oxidation product of polyphenols by immobilized polyphenol oxidase plate using HSCCC. The effects of solvent systems and NaHCO_3 on the separation were investigated. The result showed that NaHCO_3 was helpful to purify the theaflavins. Separation was performed with a two-phase solvent system composed of ethyl acetate - hexane -methanol-water (3:1: 1: 6, V/V) and (3: 1: 3: 4, V/V) by eluting the lower aqueous phase at 1.2 ml/min at a revolution speed of 880 rpm. 57 mg of 97.7% theaflavin, 26 mg of 61.2% theaflavin-3'-gallate, 64 mg of 84.5% theaflavin-3-3'-gallate and 33 mg of 92.3% theaflavin-3-gallate and theaflavin-3'-gallate complex were obtained from 250 mg of the crude TFs sample.
2. The antibacterial, and antioxidant function and mechanism of black tea pigments.
The effects of theaflavins and tea polyphenols on two major oral bacterium, namely S.mutans Ingbritt and S.sobrinus 6715 were investigated. It indicated that the two polyphenols had the potent inhibiting activity. It was no remarkable difference between them. Furthermore, the two bacterium had no medicine-resistant against the two polyphenols.
In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The order of OH·and DPPH scavenging ability of these oxidation products was TF> TB> TR. These oxidized phenolic compounds showed protection against H_2O_2-induced damage in HPF-1 cells and suppressed the accumulation of intracellular reactive oxygen species in H_2O_2-induced damage HPF-1 cells. Interestingly, TB, as a further oxidative product from TF or TR, showed the potent activity followed TF in the above four systems.
The protective effect of three major black tea polyphenols, namely theaflavin (TF1), theaflavin-3 (3')-gallate (TF2) and theaflavin-3,3'-digallate (TF3) on oxidative damage human fibroblast (HPF-1) cell were investigated by comparing with epigallocatechin gallate (EGCG). The order of OH·and DPPH scavenging ability were TF3 > TF2 > TF1 >EGCG and TF3>TF2>EGCG>TF1 respectively. TF1, TF2, and TF3 showed more effective than EGCG in protection against H_2O_2-induced damage in HPF-1 cells. Pretreated for 2h and eliminated from the cell, TF1 and TF3 still showed protection against H_2O_2-induced damage in HPF-1 cells. TF1, TF2 and TF3 suppressed the accumulation of intracellular reactive oxygen species (ROS) in H_2O_2-induced damage HPF-1 cells. Our results suggest, other than inhibition of ROS, the protective effect of TF1 and TF3 on oxidative damage HPF-1 cell may also be responsible for improvement of normal HPF-1 cell resistive ability toward radical-damage. TF2 may protect against H_2O_2-induced damage in HPF-1 cells, through enhancing HPF-1 cell proliferation.
Part 2 Analysis of chemical compositons in tea (Camellia sinensis) flowers and their antioxidant mechanism
While these properties of tea have been extensively studied, the less attention has been given to these properties of tea flower. This is due to the fact that people have been only picking the tender shoots from tea tree to make tea for a long history, but for tea flowers, the majority lets them run their courses. Compared with tea leaves, tea flowers have similar chemical compositions such as polyphenols and caffeine, polysaccharide and protein etc. The aim of the present study was to ascertain the phenolic profile of tea flower extracts, as well as to know the major active components responsible for the antioxidant activity of tea flowers. The antioxidant activity of the extracts was evaluated by DPPH assay. Phenolic compounds were separated using HPLC-DAD, and identified by means of LC-MS-ion trap.
1. Evaluation of the antioxidant compounds of extracts from tea (Camellia sinensis) flower.
The antioxidant activity of tea flower was investigated by the DPPH free radical-scavenging assay and the hydroxyl radical-induced luminol chemiluminescence assy. The tea flower was extracted with ethanol (EE) and separated by the order of chloroform, ethyl acetate (EEA), and n-butanol. The result pointed tea flower ethyl acetate extract (EEA) had the strongest antioxidant activity and the most contribution to the antioxidant activity of tea flower. The stronger scavenging abilities to free radicals were shown by EE and EEA, which might be due to the content of polyphenols contained. However, almost non free radical-scavenging activity was observed while the tea flower was extracted with water and its fractions of several organic solvents.
2. Liquid chromatographic-mass spectrometric analysis of phenolics in tea flower extract.
High performance liquid chromatography (HPLC) combined with diode array (DAD) and electrospray (ESI)-ion trap-MS detection was used to separate and identify the compounds present in EEA. Eight catechins, namely, catechin, epicatechin (EC), gallocatechin, epigallocatechin, gallocatechin gallate, epigallocatechin gallate (EGCG), catechin gallate and epicatechin gallate (ECG), catechins derivatives, namely, EC-3'-di- rhamnoside and catechins dimer, and flavonol glycosides, namely, quercetin, luteolin, kaempferide and kaempferol mono-, diglycosides (glucoside and rhamnoside) were identified. In addition, it is found that EGCG and ECG are the major active components responsible for the antioxidant activity of tea flowers by HPLC separation and DPPH assay.
引文
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