Peroxidase-mediated oxidation of catechins

Theaflavins and thearubigins are major pigments in black tea, and they are produced by oxidation and polymerization of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the mechanism of black tea polyphenol formation, we have systematically studied peroxidase-mediated oxidation of tea catechins. Individual catechins, appropriate pairs of catechins, and the combination of selected catechins and theaflavins have been used as substrates of peroxidase. Different dimers, trimers and a tetramer are formed. This work has led to the characterization of some of the new trimers in black tea by LC/ESI-MS/MS. The proposed mechanisms for the peroxidase-mediated reaction are discussed.     

Black tea is a powerful chemopreventor of reactive oxygen and nitrogen species: comparison with its individual catechin constituents and green tea

Production of black tea [BT] results in biotransformation of catechins of green tea [GT] to theaflavins and thearubigins. BT was found to be more efficient than GT and its individual catechin constituents in proportionate amounts in abrogating production of NO and O2(-) in activated murine peritoneal macrophages. In a reconstitution system of BT that is free of all catechins, stepwise addition of catechins showed that though all the constituents contributed to the overall effect of BT, theaflavin was the most powerful in abrogating NO production. RT-PCR analysis also showed theaflavin to be the most important constituent in down-regulating synthesis of iNOS. Clearly, BT containing theaflavin is an excellent chemopreventor against reactive oxygen and nitrogen species.     

Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins. The HSCCC run was carried out with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that pure theaflavin, theaflavins-3-gallate, theaflavins-3'-gallate and theaflavin-3,3'-digallate could be obtained from crude theaflavins sample and black tea. The structures of the isolated compounds were positively confirmed by (1)H NMR and (13)C NMR, MS analysis, HPLC data and TLC data. Meanwhile, catechins including epigallocatechin gallate, gallocatechin gallate, epicatechin gallate and epigallocatechin were isolated from the aqueous extract of green tea by using the same solvent system. This study developed a modified method combined with enrichment theaflavins method by using HSCCC for separation of four individual theaflavins, especially for better separation of theaflavins monogallates.     

Production and HPLC analysis of black tea theaflavins and thearubigins during in vitro oxidation

A model fermentation system has been designed which utilizes pure catechins and partially purified polyphenol oxidase (EC 1.14.18.1 from green tea shoots. HPLC analysis of the products formed during in vitro oxidation has demonstrated a close similarity between this system and in vivo oxidation occurring during factory fermentation. Furthermore, changes in theaflavin and thearubigin levels, revealed by time courses of fermentation, show in vitro and in vivo systems to be qualitatively similar, although the former system produces considerably higher levels of both components. The model fermentation system, therefore, appears to be a suitable experimental system for studying the formation of theaflavin and thearubigin pigments under strictly controlled conditions. In preliminary experiments the theaflavins have been identified on HPLC profiles by enzymic oxidation of the relevant catechin pairs. Similarly, major coloured components other than theaflavins, which are considered to be thearubigins, have been shown to be formed by the oxidation and reaction of two gallocatechins (epigallocatechin and epigallocatechin gallate). The model fermentation system, in conjunction with HPLC as described in this paper, provides a means whereby precise data on theaflavin and thearubigin formation can be obtained and, in the case of the thearubigins, one which could yield additional structural information.     

Black tea represents a major source of dietary phenolics among regular tea drinkers

The phenolic composition and antioxidant activities [TEAC, ORAC, FRAP] of consumer brews (1 tea bag in 230 ml for 1 min) of seven different brands of black tea from the British market were investigated. The main phenolic compounds identified were epigallocatechin gallate, four theaflavins, as well as epicatechin gallate, theogallin (tentative assignment), quercetin-3-rutinoside and 4-caffeoyl quinic acid. Thearubigins represented an estimated 75-82% of the total phenolics. Further, polyphenol fractions were in decreasing order theaflavins, flavan-3-ols, flavonols, gallic acids and hydroxycinnamates. On average, a cup of a consumer brew of black tea is providing polyphenols at the level of 262 mg GAE/serving, of which 65 mg were assigned to individual polyphenols. The antioxidant activity of black tea preparations is higher than that of most reported dietary agents on a daily basis. Correlations were observed between the antioxidant activities and the sum of all quantified polyphenols by HPLC analysis as well as with the total phenolics. Treatment of the black tea brew with simulated gastric juice resulted in a significant increase of the identified theaflavins implying a partial cleavage of thearubigins in the environment of the gastric lumen. Therefore, black tea can be considered to be a rich source of polyphenols and/or antioxidants.     

Black Tea Represents a Major Source of Dietary Phenolics Among Regular Tea Drinkers

The phenolic composition and antioxidant activities [TEAC, ORAC, FRAP] of consumer brews (1 tea bag in 230 ml for 1 min) of seven different brands of black tea from the British market were investigated. The main phenolic compounds identified were epigallocatechin gallate, four theaflavins, as well as epicatechin gallate, theogallin (tentative assignment), quercetin-3-rutinoside and 4-caffeoyl quinic acid. Thearubigins represented an estimated 75-82% of the total phenolics. Further, polyphenol fractions were in decreasing order theaflavins, flavan-3-ols, flavonols, gallic acids and hydroxycinnamates. On average, a cup of a consumer brew of black tea is providing polyphenols at the level of 262 mg GAE/serving, of which 65 mg were assigned to individual polyphenols. The antioxidant activity of black tea preparations is higher than that of most reported dietary agents on a daily basis. Correlations were observed between the antioxidant activities and the sum of all quantified polyphenols by HPLC analysis as well as with the total phenolics. Treatment of the black tea brew with simulated gastric juice resulted in a significant increase of the identified theaflavins implying a partial cleavage of thearubigins in the environment of the gastric lumen. Therefore, black tea can be considered to be a rich source of polyphenols and/or antioxidants.     

Improving the Polyphenol Content of Tea

Tea, prepared from the leaves of Camellia species, has one of the highest contents of flavonoids among common food and beverage products. Tea consumption has moved beyond its pleasant flavor and cultural significance since a number of health promoting properties have been ascribed to this widespread beverage (e.g., anticancer, antiobesity and hypotensive effects). The major bioactive compounds in tea are catechins (flavan-3-ols), a group of flavonoids that include, among others, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG). These compounds are also the precursors of theaflavins and thearubigins, oxidation products responsible for the taste and colour of certain tea types such as black tea. The composition of the tea leaf, and thus tea quality, is influenced by many pre-harvest factors such as the genetic make-up of the plant, region of production, horticultural and harvesting practices, and environmental conditions. Once harvested, processing, brewing, and storage conditions influence the phenolic composition and quality of tea infusions as well. In the present review we aim at outlining our current knowledge about means to increase the catechin content of teas, a cornerstone for improving the health-promoting properties of this beverage.     

茶儿茶素氧化机理

在制茶发酵过程中,儿茶素可发生苯骈环化反应而形成茶黄素,茶黄素还可转化成茶萘酚醒、脱氢茶黄素和高聚合物;同时,儿茶素可发生二聚合反应产生原花青素类以及双黄烷醇类等物质;儿茶素还可能与茶没食子素、杨梅甙、阿福豆素、维生素C、茶氨酸发生聚合反应,形成加合物。体外自由基清除实验表明,儿茶素具有优良的抗氧化特性。儿茶素在清除自由基时,自身氧化产生双黄烷醇,也可发生A环的断裂而产生羧酸类物质。     

The prevention of lung cancer induced by a tobacco-specific carcinogen in rodents by green and black Tea

A growing body of evidence from studies in laboratory animals indicates that green tea protects against cancer development at various organ sites. We have previously shown that green tea, administered as drinking water, inhibits lung tumor development in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone (NNK), a potent nicotine-derived lung carcinogen found in tobacco. The inhibitory effect of green tea has been attributed to its major polyphenolic compound, epigallocatechin gallate (EGCG), and, to a lesser extent, to caffeine. We have also demonstrated that while levels of O6-methylguanine, a critical lesion in NNK lung tumorigenesis, were not affected in lung DNA. However, the levels of 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, were significantly suppressed in mice treated with green tea or EGCG. These studies underscore the importance of the antioxidant activity of green tea and EGCG for their inhibitory activity against lung tumorigenesis. Unlike green tea, the effect of black tea on carcinogenesis has been scarcely studied, even though the worldwide production and consumption of black tea far exceeds that of green tea. The oxidation products found in black tea, thearubigins and theaflavins, also possess antioxidant activity, suggesting that black tea may also inhibit NNK-induced lung tumorigenesis. Indeed, bioassays in A/J mice have shown that black tea given as drinking water retarded the development of lung cancer caused by NNK. However, data on the relationship of black tea consumption with the lung cancer risk in humans are limited and inconclusive. There is a need for additional tumor bioassays in animal models to better examine the protective role of black tea against lung cancer. The development of adenocarcinomas and adenosquamous carcinomas in F344 rats upon chronic administration of NNK provides an important and relevant model for lung carcinogenesis in smokers. Thus far, no information was previously available regarding the effects of tea on this model. We conducted a 2-year lifetime bioassay in F344 rats to determine whether black tea and caffeine are protective against lung tumorigenesis induced by NNK. Our studies in both mice and rats have generated important new data that support green and black tea and caffeine as potential preventive agents against lung cancer, suggesting that a closer examination of the roles of tea and caffeine on lung cancer in smokers may be warranted.     

Antiviral Effect of Theaflavins on Tobacco Mosaic Virus

Theaflavins, the oxidized products formed from tea leaf catechins during black tea fermentation, showed an antiviral activity on TMV. From the survey of the interactions of theaflavins with RNA and its related substances, it was presumed that theaflavins disturbed the replication cycle of TMV through binding to TMV-RNA.     

Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells

The protective effects of three tea extracts (green tea, GTE; oolong tea, OTE; and black tea, BTE) and five tea polyphenols (epicatechin, EC; epicatechin gallate, ECG; epigallocatechin, EGC; epigallocatechin gallate, EGCG; and theaflavins, THFs) on benzo[a]pyrene (B[a]P)-induced DNA damage in Chang liver cells were evaluated using the comet assay. B[a]P-induced DNA damage in Chang liver cells was significantly (p < 0.05) inhibited by GTE and OTE at a concentration of 10 microg/ml and by BTE at 25 microg/ml. At a concentration of 100 microg/ml, the % tail DNA was reduced from 33% (B[a]P treated only) to 10, 9, 13%, by GTE, OTE and BTE, respectively. EC and ECG did not cause DNA damage in cells according to the results of the comet assay; however, EGC, EGCG and theaflavins caused DNA damage in cells at a concentration of 100 microM. The results indicated that EC and ECG had protective effects against B[a]P-induced DNA damage in cells at a concentration of 10-100 microM. Although EGC, EGCG and the theaflavins caused DNA damage at a high concentration, but they had protective effects against B[a]P-induced DNA damage in cells at a low concentration of 10-50 microM. The results also showed that the DNA damage in cells induced by EGC, EGCG, and the theaflavins was due to the generation of superoxide during incubation with cells at a higher concentration. Therefore, tea catechins and THFs play an important role in enabling tea extracts to inhibit DNA damage in Chang liver cells.     

ESI LC-MS and MS/MS characterization of antifungal cyclic lipopeptides produced by Bacillus subtilis XF-1

Bacillus subtilis XF-1 (CGMCC No. 2357), a patent strain with good effects for controlling the clubroot of crucifer and many pathogenic fungi, was predicted to produce cyclic lipopeptide (CLP) antibiotics based on its genomic analysis. In this study, the CLPs were purified and determined with the following protocol: the supernatant of XF-1 cultivating mixture was firstly precipitated, then the precipitants were extracted with methanol and further separated by Sephadex LH-20 chromatography to test its antifungal activities. Fungi-inhibiting fractions were further characterized with LC/ESI-MS and LC/ESI-MS/MS. The results show that four molecular ion peaks [M+H]? (m/z 1,464, 1,478, 1,492 and 1,506) from fungi suppression fraction were identified as fengycin A with fatty acid of C??-C??, fengycin B (C??-C??), fengycin C (C??-C??), fengycin D (C??-C??) and fengycin S (C??-C??). Fengycin C (C?? and C??), fengycin D (C??, C?? and C??) and fengycin S (C??, C?? and C??) were reported for the first time. The diversity of the fengycins that exist in this strain will help the elucidation of their biocontrol mechanisms.     

苦茶Camellia assamica var.kucha茶多酚的HPLC-DAD/MS分析

苦茶是一种特殊的茶组植物,它以1,3,7,9-四甲基尿酸为主要的嘌呤生物碱。本文采用高效液相色谱-二极管阵列检测/质谱联用(HPLC-DAD/MS)的方法对苦茶水提液中的茶多酚和嘌呤生物碱进行了定性分析,检测到3种嘌呤生物碱、7种儿茶素类化合物和2种非儿茶素类茶多酚(没食子酰奎宁酸酯和咖啡酰奎宁酸酯)。同时与传统绿茶在儿茶素类化合物的组成和含量上进行了比较,结果显示,苦茶中总儿茶素含量(13.82%)远高于传统绿茶(7.37%),但各儿茶素类化合物的相对组成比在两种茶中极其相近,均以酯型儿茶素为主。     

Characteristic fragmentation of polyunsaturated fatty acids with allylic vicinal diols in positive-ion LC/ESI-MS/MS

A characteristic fragmentation was observed for PUFAs that contain allylic vicinal diol groups (resolvin D1, D2, D4, E3, lipoxin A4, B4, and maresin 2), which were derivatized with N,N-dimethylethylenediamine (DMED), in positive-ion ESI-MS/MS. The findings indicate that when these compounds contain an allylic hydroxyl group that is located distal to the terminal DMED moiety in the case of resolvin D1, D4, and lipoxin A4, an aldehyde (-CH=O) is predominately formed, which arises from the breakdown in between vicinal diols, whereas, in the case of an allylic hydroxyl group that is located proximal to the DMED moiety, as in resolvin D2, E3, lipoxin B4, and maresin 2, an allylic carbene (-CH=CH-CH:) is formed. These specific fragmentations could be used as diagnostic ions for characterizing the above seven PUFAs. As a result, it was possible to detect resolvin D1, D2, E3, lipoxin A4, and B4 in sera (20 μl) obtained from healthy volunteers by multiple-reaction monitoring using LC/ESI-MS/MS.     

Evaluation of the antigenotoxic potential of monomeric and dimeric flavanols, and black tea polyphenols against heterocyclic amine-induced DNA damage in human lymphocytes using the Comet assay

The polyphenolic dimers, epicatechin-4beta-8-catechin (B1), epicatechin-4beta-8-epicatechin (B2), catechin-4beta-8-catechin (B3), catechin-4beta-8-epicatechin (B4), and the gallate ester epicatechin-4beta-8-epicatechin gallate (B'2G) were isolated from grape seeds, and theaflavins and theafulvins from black tea brews. The ability of these naturally-occurring polyphenols to afford protection against the genotoxicity of the heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was compared with that of the monomeric tea flavanols, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG). Genotoxic activity was evaluated in human peripheral lymphocytes using the Comet assay. At the concentration range of 1-100 microM, neither the monomeric nor the dimeric flavanols prevented the lymphocyte DNA damage induced by Trp-P-2. In contrast, both of the black tea polyphenols, theafulvins and theaflavins, at a dose range of 0.1-0.5 mg/ml, prevented, in a concentration-dependent manner, the DNA damage elicited by Trp-P-2. Finally, neither the monomeric and dimeric polyphenols (100 microM) nor the theafulvins and theaflavins (0.5mg/ml) caused any DNA damage in the human lymphocytes. These studies illustrate that black tea theafulvins and theaflavins, if absorbed intact, may contribute to the anticarcinogenic potential associated with black tea intake.     

Unraveling the structure of the black tea thearubigins

Thearubigins are the most abundant group of phenolic pigments found in black tea accounting for an estimated 60% of the solids in a typical black tea infusion. Fifty years ago the term thearubigins was first introduced and up to now the chemical nature of the thearubigins remains largely unresolved if not mysterious despite many efforts clarifying their structure. This paper summarizes some of our attempts to clarify and elucidate the chemical nature of the thearubigins, presenting for 15 commercially representative teas data obtained using combustion analysis, IR spectroscopy, NMR spectroscopy, Diffusion NMR spectroscopy, UV-vis spectroscopy, Circular Dichroism spectroscopy and atomic force microscopy, MALDI-TOF-MS and ESI-FT-ICR-MS. The thearubigin fractions from these 15 teas are remarkably similar with respect to their spectroscopic fingerprint. The data obtained are consistent with the thearubigins being structures of not more than 2000 Da with more than 5000 individual chemical entities detected that are susceptible to concentration-driven aggregation in aqueous solution, and that retain the chiral properties of the flavanols and theaflavins. By applying petrolomics-style data interpretation strategies and by developing novel data interpretation strategies a structural model for the thearubigins was developed.     

Antimicrobial Activities of Tocklai Vegetative Tea Clones

Thirty-one Tocklai vegetative (TV) tea clones contained caffeine and total catechin 44.39 and 227.55 mg/g dry weight of leaves, respectively. The (−)-epigallocatechin gallate (EGCG) was the most abundant (109.60 mg/g) followed by -(−)-epigallocatechin (EGC, 44.54 mg/g), (−)-epicatechin gallate (ECG, 41.74 mg/g), (−)-epicatechin (EC, 27.42 mg/g) and +catechin (4.25 mg/g). Total catechins were highest in TV 20 (509.7 mg/g) and lowest in TV 6 (71.7 mg/g). The tea clones that contain high level of total catechin exhibited the strongest antimicrobial activity. Among caffeine and flavanol compounds, theaflavins (TF) present in black tea possess a similar antimicrobial potency as EC present in fresh leaves, and that the conversion of catechins to TF during fermentation in making black tea tends to alter their antimicrobial activities. The bioactive molecules other than catechins present in tea leaves may also contribute towards antimicrobial activity.     

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.     

Plasma and lipoprotein levels of tea catechins following repeated tea consumption

Epidemiological studies suggest that antioxidant flavonoids in tea may reduce the risk of cardiovascular disease, possibly via protection of low-density lipoproteins (LDL) against oxidation. However, the extent of absorption of tea flavonoids and their accumulation in LDL during regular consumption of tea is not clear. Therefore we investigated plasma and lipoprotein levels of catechins during tea consumption and the impact on LDL oxidizability ex vivo. Eighteen healthy adults consumed, in an incomplete balanced cross-over design, green tea, black tea, black tea with milk or water, one cup every 2 hr (eight cups/day) for three days. Blood samples were obtained in the mornings and evenings of each day. Plasma total catechin concentration was determined in all blood samples, and the distribution of catechins among lipoproteins was determined at the end of the third day (t = 60 hr). The resistance of LDL to copper-induced oxidation ex vivo was assessed before tea consumption and at t = 60 hr. Repeated tea consumption during the day rapidly increased plasma total catechin levels whereas they declined overnight when no tea was consumed. There was a gradual increase in plasma levels in the mornings (respectively, 0.08 microM vs. 0.20 microM on first and last day of black tea consumption) and evenings (respectively, 0.29 microM vs. 0.34 microM on first and last day of black tea consumption). Green tea catechins were mainly found in the protein-rich fraction of plasma (60%) and in high-density lipoproteins (23%). Although present in LDL, the concentration of catechins in LDL was not sufficient to enhance the resistance of LDL to oxidation ex vivo. Addition of milk to black tea did not affect any of the parameters measured. In conclusion, the present study shows that catechin levels in blood rapidly increase upon repeated tea consumption. The accumulation of catechins in LDL particles is not sufficient to improve the intrinsic resistance of LDL to oxidation ex vivo.