Structure-activity relationships of tea compounds against human cancer cells

The content of the biologically active amino acid theanine in 15 commercial black, green, specialty, and herbal tea leaves was determined as the 2,4-dinitrophenyltheanine derivative (DNP-theanine) by a validated HPLC method. To define relative anticarcinogenic potencies of tea compounds and teas, nine green tea catechins, three black tea theaflavins, and theanine as well as aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). The growth of normal human lung (HEL299) cells was not inhibited. The destruction of cancer cells was also observed visually by reverse phase microscopy. Statistical analysis of the data showed that (a) the anticarcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent over the ranges from 50 to 400 microg/mL of tea compound and from 50 to 400 microg/g of tea solids; (b) the different cancer cells varied in their susceptibilities to destruction; (c) 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and (d) flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. The findings extend related observations on the anticarcinogenic potential of tea ingredients and suggest that consumers may benefit more by drinking both green and black teas.     

Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins.

It has been reported earlier that when macerated tea leaf is fermented at lower pH, the resultant black tea contains increased levels of theaflavin, an important quality marker in black tea. In an attempt to investigate the biochemistry and chemistry underlying this observation, in vitro oxidation experiments using polyphenol oxidase (PPO) from fresh tea leaves, horseradish peroxidase (POD), and tea catechins, precursors for theaflavins, were carried out. In vitro oxidation experiments using crude tea PPO resulted in higher content of theaflavins at pH 4.5 in comparison with pH 5.5, the normal pH of the macerated tea leaf. When purified PPO was used in the in vitro system, surprisingly a reversal of this trend was observed, with more theaflavins being formed at the higher pH. A combination of pure tea PPO and POD led to an observation similar to that with the crude enzyme preparation, suggesting a possible role for POD in the formation or degradation of theaflavin. POD was observed to oxidize theaflavins in the presence of H(2)O(2), leading to the formation of thearubigin, another black tea pigment. This paper demonstrates that tea PPO, while oxidizing catechins, generates H(2)O(2). The amount of H(2)O(2) produced is greater at pH 5.5, the optimum pH for PPO activity, than at pH 4.5. Hence, an observed increase of theaflavins in black teas fermented at pH 4.5 appears to be due to lower turnover of formed theaflavins into thearubigins.     

Factors affecting the levels of tea polyphenols and caffeine in tea leaves

An isocratic HPLC procedure was developed for the simultaneous determination of caffeine and six catechins in tea samples. When 31 commercial teas extracted by boiling water or 75% ethanol were analyzed by HPLC, the levels of (-)-epigallocatechin 3-gallate (EGCG), and total catechins in teas were in the order green tea (old leaves) > green tea (young leaves) and oolong tea > black tea and pu-erh tea. Tea samples extracted by 75% ethanol could yield higher levels of EGCG and total catechins. The contents of caffeine and catechins also have been measured in fresh tea leaves from the Tea Experiment Station in Wen-Shan or Taitung; the old tea leaves contain less caffeine but more EGCG and total catechins than young ones. To compare caffeine and catechins in the same tea but manufactured by different fermentation processes, the level of caffeine in different manufactured teas was in the order black tea > oolong tea > green tea > fresh tea leaf, but the levels of EGCG and total catechins were in the order green tea > oolong tea > fresh tea leaf > black tea. In addition, six commercial tea extracts were used to test the biological functions including hydroxyl radical scavenging, nitric oxide suppressing, and apoptotic effects. The pu-erh tea extracts protected the plasmid DNA from damage by the Fenton reaction as well as the control at a concentration of 100 microg/mL. The nitric oxide suppressing effect of tea extracts was in the order pu-erh tea >/= black tea > green tea > oolong tea. The induction of apoptosis by tea extract has been demonstrated by DNA fragmentation ladder and flow cytometry. It appeared that the ability of tea extracts to induce HL-60 cells apoptosis was in the order green tea > oolong > black tea > pu-erh tea. All tea extracts extracted by 75% ethanol have stronger biological functions than those extracted by boiling water.     

Potent inhibitory effects of black tea theaflavins on off-odor formation from citral

The present study was conducted to investigate the ability of black tea theaflavins to inhibit the off-odor formation from citral under acidic aqueous conditions. Acidic buffer solutions (pH 3.0) containing citral (10 mg/L) and an inhibitor (0-5 mg/L) were stored at 40 degrees C for 2 weeks. The formation of possible off-odorants p-cresol and p-methylacetophenone in the citral solutions was monitored by high-performance liquid chromatography. A mixture of the theaflavins showed inhibitory effects on the formation of both p-cresol and p-methylacetophenone with 50% inhibitory concentrations (IC(50)) of 0.18 and 0.10 mg/L, respectively. Individual theaflavins and a structurally related compound, purpurogallin, also inhibited the formation of both off-odorants, with the lowest IC(50) values for theaflavin 3,3'-digallate (0.17 and 0.06 mg/L for p-cresol and p-methylacetophenone, respectively). On the other hand, a mixture of green tea catechins and its major constituent, (-)-epigallocatechin gallate, showed relatively high IC(50) values for the formation of p-methylacetophenone (1.29 and 1.28 mg/L, respectively) and showed no inhibitory effect on the formation of p-cresol. The results of the sensory evaluation showed that the off-odor intensity of the stored citral solution was significantly decreased by the addition of the theaflavin mixture at concentrations of 0.5 mg/L and above. In addition, the calculation of the odor activity values (OAVs) for the volatile compounds detected by a gas chromatographic analysis indicated that the total OAVs of the major volatile compounds in the citral solution were significantly decreased by the addition of the theaflavins.     

Tea enhances insulin activity

The most widely known health benefits of tea relate to the polyphenols as the principal active ingredients in protection against oxidative damage and in antibacterial, antiviral, anticarcinogenic, and antimutagenic activities, but polyphenols in tea may also increase insulin activity. The objective of this study was to determine the insulin-enhancing properties of tea and its components. Tea, as normally consumed, was shown to increase insulin activity >15-fold in vitro in an epididymal fat cell assay. Black, green, and oolong teas but not herbal teas, which are not teas in the traditional sense because they do not contain leaves of Camellia senensis, were all shown to increase insulin activity. High-performance liquid chromatography fractionation of tea extracts utilizing a Waters SymmetryPrep C18 column showed that the majority of the insulin-potentiating activity for green and oolong teas was due to epigallocatechin gallate. For black tea, the activity was present in several regions of the chromatogram corresponding to, in addition to epigallocatechin gallate, tannins, theaflavins, and other undefined compounds. Several known compounds found in tea were shown to enhance insulin with the greatest activity due to epigallocatechin gallate followed by epicatechin gallate, tannins, and theaflavins. Caffeine, catechin, and epicatechin displayed insignificant insulin-enhancing activities. Addition of lemon to the tea did not affect the insulin-potentiating activity. Addition of 5 g of 2% milk per cup decreased the insulin-potentiating activity one-third, and addition of 50 g of milk per cup decreased the insulin-potentiating activity approximately 90%. Nondairy creamers and soy milk also decreased the insulin-enhancing activity. These data demonstrate that tea contains in vitro insulin-enhancing activity and the predominant active ingredient is epigallocatechin gallate.     

Molecular binding of black tea theaflavins to biological membranes: relationship to bioactivities

Molecular dynamics simulations were used to study the interactions of three theaflavin compounds with lipid bilayers. Experimental studies have linked theaflavins to beneficial health effects, some of which are related to interactions with the cell membrane. The molecular interaction of theaflavins with membranes was explored by simulating the interactions of three theaflavin molecules (theaflavin, theaflavin-3-gallate, and theaflavin-3,3'-digallate) with a mixed bilayer composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE). The simulations show that the theaflavins evaluated have an affinity for the lipid bilayer surface via hydrogen bonding. The molecular structure of theaflavins influenced their configuration when binding to the bilayer surface, as well as their ability to form hydrogen bonds with the lipid headgroups. The theaflavin-bilayer interactions studied here help to define structure-function relationships of the theaflavins and provide a better understanding of the role of theaflavins in biological processes. The significance of the results are discussed in the context of black tea composition and bioactivity.     

Black tea polyphenols, theaflavins, prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.

Tea polyphenols have been demonstrated as chemopreventive agents in a number of experimental models. However, less is known about the mechanism of chemoprevention by black tea compared with that of green tea. Some beneficial properties of theaflavins, the black tea polyphenols, were investigated in the present study. Theaflavins showed inhibitory effects on H(2)O(2)- and tert-butyl hydroperoxide (tBuOOH)-induced cytotoxicity (evaluated by tetrazolium bromide reduction), cellular oxidative stress (detected by oxidation of 2', 7'-dichlorofluorescin), and DNA damage (measured by amount of 8-OHdG and comet assay) in rat normal liver epithelium cell RL-34 cell lines. In addition, theaflavins also exhibited suppression of cytochrome P450 1A1 induced by omeprazole in the human hepatoma HepG2 cell line. Furthermore, when HepG2 cells were pretreated with omeprazole to induce CYP1A1, then exposed to benzo[a]pyrene (B[a]P), DNA damage was observed using the comet assay. However, theaflavins could inhibit this DNA damage. These results indicated that theaflavins could prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures.     

Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry

Black tea contains two major groups of pigments, theaflavins (TFs) and thearubigins (TRs). TFs contain a bis-flavan substituted 1,2-dihydroxy-3,4-benzotropolone moiety. Unlike the TFs, TRs have not yet been characterized. The chemical structure of the TRs remains a mystery. The present paper reports our effort to study the structure of TFs and TRs using delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption ionization (MALDI) technique, on line with a Linear time-of-flight (TOF) mass spectrometer. Spectra of standard TFs show not only pseudomolecular ions but also ions resulting from fragmentation. The analysis of MALDI-TOF spectra of black tea fractions shows the structure of some TRs, which are similar to those of TFs because the same loss of mass is observed.     

Microwave heating of tea residue yields polysaccharides, polyphenols, and plant biopolyester

Microwave heating was used to produce aqueous-soluble components from green, oolong, and black tea residues. Heating at 200-230 degrees C for 2 min extracted 40-50% of polysaccharides and 60-70% of the polyphenols. Solubilization of arabinose and galactose by autohydrolysis occurred with heating above 170 degrees C, whereas heating above 200 degrees C was necessary to solubilize xylose. Catechins were soluble in water by heating at low temperature (110 degrees C); however, new polyphenols having strong antioxidant activity were produced above 200 degrees C. The amount of solubilized materials and antioxidant activity increased with increased fermentation of harvested tea leaves (green tea < oolong tea < black tea). Cutin, a plant biopolyester, remained in the residue after heating as did cellulose and lignin/tannin. The predominant cutin monomer that was recovered was 9,10-epoxy-18-hydroxyoctadecanoic acid, followed by dihydroxyhexadecanoic acid and 9,10,18-trihydroxyoctadecanoic acid.     

New phenolic components and chromatographic profiles of green and fermented teas

A standardized profiling method based on liquid chromatography with diode array and electrospray ionization mass spectrometric detection (LC-DAD-ESI/MS) was applied to establish the phenolic profiles of 41 green teas and 25 fermented teas. More than 96 phenolic compounds were identified that allowed the teas to be organized into five groups. Epigallocatechin gallate (EGCG) was the major phenolic component of green tea made from mature leaves (group 2), while green tea made from the younger buds and leaves (group 1) contained lower flavonoid concentrations. Partially fermented teas (group 3) contained one-half the EGCG content of the green tea. Fully fermented black teas (group 4) had a trace of EGCG, but contained theaflavins. Highly overfermented black tea (group 5) contained only trace amounts of flavonol glycosides and theaflavins. Over 30 phenolics are new for tea, and this is the first phenolic profile to simultaneously detect C- and O-glycosylated flavonoids, catechins, proanthocyanidins, phenolic acid derivatives, and purine alkaloids.     

Breast cancer prevention by green tea catechins and black tea theaflavins in the C3(1) SV40 T,t antigen transgenic mouse model is accompanied by increased apoptosis and a decrease in oxidative DNA adducts

Tea consumption is associated with a reduced risk of mammary cancer as reflected by epidemiological studies and experiments in carcinogen-induced rodent models of mammary carcinogenesis. We tested the hypothesis that green tea catechins (GTC) or theaflavins from black tea (BTT) interfere with mammary carcinogenesis in C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma (TAg) mice and that GTC/BTT affect tumor survival or oxidation status. TAg mice received GTC/BTT (0.05%) in drinking water for their lifetime. As compared to control mice, they survived longer and had smaller tumors. On microscopic inspection, the size of the largest tumor per mouse was decreased by 40-42% (p<0.01). GTC (0.01%) and BTT (0.05%) increased levels of cleaved caspase 3 in tumor tissue by 67 and 38%, respectively (p<0.05), intimating increased apoptosis. Tumor levels of the malondialdehyde-DNA adduct M1dG in mice receiving GTC or BTT (0.05%) were reduced by 78 (p<0.001) or 63% (p<0.05), respectively, as compared to controls. The results render the exploration of the breast cancer chemopreventive properties of tea preparations in humans worthwhile.     

PROLONGED LEACHING OF ORTHIC BLACK Ah MATERIAL WITH WATER AND AQUEOUS EXTRACTS OF POPULUS TREMULOIDES AND P. BALSAMIFERA LEAVES

Cylinders filled with Orthic Black Ah material were leached over a 2-year period with distilled water or with aqueous extracts of aspen or black poplar leaves that were collected late in the growing season. Actual losses from the soil and chemical analyses of the leached soil indicated that the aqueous extract of aspen poplar leaves imposed greater changes on the chemical properties of the soil than did the extract of black poplar leaves. These changes were particularly noticeable for pH, total and exchangeable Ca, and 0.1N NaOH-extractable C. The aqueous extract of aspen poplar leaves acidified the soil more strongly than the extract of black poplar leaves, even though there was little difference between the two extracts in polyphenol and polysaccharide content and the total acidity of the black poplar extract was about twice that of the aspen poplar extract. Aqueous extract of weathered aspen poplar leaves was no more effective than distilled water.     

采用PLS-DA分析毛火方式对工夫红茶品质的影响

为探究不同毛火方式对工夫红茶品质的影响,明确新型电磁内热式滚筒-热风耦合干燥设备的毛火效果,该研究以一芽一二叶初展嫩度的"福鼎大白"品种为原料进行工夫红茶加工,设定电磁滚筒-热风耦合(Rotary pot-Hot air coupling First-Drying with electromagnetic heat,RHFD)、链板热风(Chain plate Hot air First-Drying,CHFD)、箱式热风(Box Hot air First-Drying,BHFD)、滚筒式滚炒(Rotary pot First-Drying,RFD)等4种毛火方式,比较所制茶样的茶多酚、儿茶素、茶色素、可溶性糖、咖啡碱、氨基酸等29个非挥发性指标,114个气相色谱-质谱技术(GasChromatography-Mass Spectrometry,GC-MS)检测的挥发性香气指标,10个外形和汤色色泽客观评价指标,同时进行了毛火方式的热效率、生产效率、生产成本等性能指标的分析比较,通过偏最小二乘判别统计(PartialLeastSquaresDiscriminationAnalysis,PLS-DA)分析毛火方式对优质工夫红茶品质的影响,并获得标志性差异化合物。结果表明:电磁内热式滚筒-热风耦合毛火处理下茶多酚和儿茶素总量显著最低(P<0.05),简单儿茶素含量较高,茶红素和可溶性糖含量、茶黄素综合指标TDE和茶色素综合指标10TFRB最高(P<0.05),毛火方式对茶黄素总量影响不显著(P>0.05);挥发性化合物总量以RHFD方式最高,RFD方式次之,CHFD方式最低;RHFD毛火方式芳香类、萜烯类等化合物含量最高。电磁内热式滚筒-热风耦合毛火升温快、温度分布均匀且稳定性好,热效率和生产效率高(分别为50.0%、220 kg/h),生产成本较低(仅0.32元/kg),预热时间仅14min;所制红茶在汤色透亮度、香气甜久度、滋味甜醇度等方面均得到提升,感官总分最高(P<0.05),达88.1。PLS-DA分析从挥发性和非挥发性角度均可将工夫红茶4种毛火方式显著区分,并分别获得了43种和18种差异化合物,结合差异性分析获得标志性差异化合物,2,4,6-三(1,1-二甲基乙基)-4-甲基环己-2,5-二烯-1-酮、香叶醇、3-辛酮、水杨酸甲酯、茶黄素、茶褐素、可溶性糖、表儿茶素等,可作为区分工夫红茶毛火方式,以及定向加工甜香、甜醇、高亮等优质工夫红茶的指标物质。该研究为红茶加工基础和品质提升提供技术参考和理论指导。     

Identification and comparison of phenolic compounds in the preparation of oolong tea manufactured by semifermentation and drying processes

Oolong tea manufactured via a semifermentation process possesses a taste and color somewhere between green and black teas. Alteration of constituents, particularly phenolic compounds, in the infusion of oolong tea resulting from its manufacture, was analyzed by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The identified constituents contained 2 alkaloids, 11 flavan-3-ols, 8 organic acids and esters, 11 proanthocyanidin dimers, 3 theaflavins, and 22 flavonoid glycosides, including 6 novel acylated flavonol glycosides. The tentative structures of these 6 novel compounds were depicted according to their mass fragmentation patterns in MS(n) (n = 1-4). In comparison with caffeine as an internal standard, relative contents of the constituents in the infusions of fresh tea shoot and different oolong tea preparations were examined. Approximately, 30% catechins and 20% proanthocyanidins were oxidized during the manufacture of oolong tea from fresh tea shoots, and 20% of total flavonoids were decomposed in a follow-up drying process. Gallocatechin-3-O-gallate and theaflavins putatively produced in the semifermentation process of oolong tea were not detected in fresh tea shoots, and the majority of theaflavins were presumably transformed into thearubigins after drying.     

Immunostimulating activity of a crude polysaccharide derived from green tea (Camellia sinensis) extract

Green tea extract is well-known to reduce the risk of a variety of diseases. Here, we investigated the immunostimulating activity of tea polysaccharide (TPS), one of the main components in green tea extract. The water extracts from mature or immature tea leaves were precipitated by using ethanol at room temperature. The sediment was washed with ethanol and acetone alternately and then dried. We used the phagocytic activity of macrophage-like cells as an indicator of immune function activation. Chemical components were analyzed by HPLC. The immunostimulating activity of TPS from immature leaves extract was higher than that of TPS from mature leaves, and its activities were dependent on the content of strictinin in the leaf extract. Futhermore, a mixture of catechin and TPS that removed polyphenols did not increase the immunostimulating activity. These results suggest that the catechin-polysaccharide complex is a very important molecule in the immunomodulating activity of tea extracts.     

Countercurrent supercritical fluid extraction and fractionation of alcoholic beverages

A procedure for the recovery of aromatic extracts from distilled alcoholic beverages by means of a countercurrent supercritical fluid extraction (CC-SFE) on a pilot plant scale is studied. The beverage is directly in contact with the carbon dioxide current in a packed column, and the extracts are recovered in two different fractionation cells, where the depressurization occurs. The proposed method allows the selective extraction of aromatic components of the brandy flavor, rendering a high-value concentrated extract and a colored residue without brandy aroma. The content in ethanol of the aromatic extract can be modified by tuning the extraction/fractionation conditions, rendering from 15 to 95% recovery. The effect of the main variables, including extraction pressure and quality of extracting CO(2), has been tested.     

Free radical scavenging effect of Pu-erh tea extracts and their protective effect on oxidative damage in human fibroblast cells

In the present study, we successively extracted the Pu-erh tea with acetone, water, chloroform, ethyl acetate, and n-butanol, and the extracts were then isolated by column chromatography. Our study demonstrates that the Pu-erh tea ethyl acetate extract, n-butanol extract, and their fractions had superoxide anion and hydroxyl radical scavenging activity: fractions 2 and 8 from the ethyl acetate extract and fractions 2, 4, and 5 from the n-butanol extract showed protective effects against hydrogen peroxide-induced damage in human fibroblast HPF-1 cells and increased the cells' viability under normal cell culture conditions. In addition, it is found that these fractions, except fraction 5 from the n-butanol extract, decreased the accumulation of intracellular reactive oxygen species in hydrogen peroxide-induced HPF-1 cells. Interestingly, the antioxidant effect of fraction 8 from the ethyl acetate extract on the above four systems was much stronger than that of the typical green tea catechin (-)-epigallocatechin-3-gallate, but there were almost no monomeric polyphenols, theaflavins, and gallic acid in fraction 8.     

Analysis of tea catechins in human plasma by high-performance liquid chromatography with solid-phase extraction

Accurate monitoring of tea catechins in biological samples might provide a means of better evaluation of their benefits. The aim of the present study was to develop a rapid method for extracting tea catechins from human plasma samples with a solid-phase extraction technique and to subsequently measure their concentrations using an HPLC system. A human plasma sample spiked with known concentrations of the analyte standards was passed through a Waters Oasis HLB cartridge. After repeated washing, tea catechins were eluted with 70% dimethylformamide containing 0.1% phosphoric acid, and the resulting eluate was injected into an HPLC system. Analytes were separated on a reverse-phase C18 column using an isocratic mobile phase and detected electrochemically. The coefficient of variation for inter- and intraday reproducibility was less than 5.0% and 6.4%, respectively. Linearity was established for the concentration range of 0.01-1.0 microM. The method was successfully applied to measure tea catechin concentrations in the plasma of two healthy subjects who received a single ingestion of a green tea beverage. The proposed method enables the rapid and accurate quantitation of plasma tea catechins and might prove useful for the evaluation of beneficial health effects of tea consumption.     

茶叶加工与样品制备对同位素分馏和测定的影响

为考察不同加工方式和样本制备目数对茶叶同位素分馏与测定的影响,建立茶叶中4种同位素δ¹³C、δ¹⁵N、δ²H、δ¹⁸O的元素分析-稳定同位素比率质谱(EA-IRMS)分析测试方法,研究红茶和绿茶加工过程中同位素分馏及不同目数样本制备的测试影响。结果表明,通过两点法或三点法构建的校准曲线具有很好的相关性,能够满足测试精度要求;相同原料所加工红茶和绿茶中4种同位素比率无显著差异,未发现同位素分馏效应;红茶和绿茶通过研磨后过80目、100目和120目筛与未过筛4种茶叶样品之间同位素比率无显著差异(P<0.05)。因此,相同原料的非发酵(绿茶)和发酵(红茶)加工以及不同目数样本制备对4种稳定同位素测定均无显著影响。本研究结果为茶叶稳定同位素数据库构建以及样品制备提供了方法基础。     

Simultaneous determination of neonicotinoid insecticides in agricultural samples by solid-phase extraction cleanup and liquid chromatography equipped with diode-array detection

Effective sample pretreatment procedures based on solid-phase extraction (SPE) for multiresidue determination of seven neonicotinoid insecticides in agricultural products were investigated. After extraction with acetone and concentration, the insecticides in aqueous sample extracts were transferred into organic solvent phases with a Chem Elut SPE cartridge. Finally, the eluate from the cartridge was cleaned up with a SPE cartridge packed with graphitized carbon black and aminopropyl silica gel, which showed a higher cleanup efficiency than the classical silica gel SPE cartridge. Seven insecticides were separated on a reversed-phase C18 column and a gradient system of methanol and phosphate solution based on high-performance liquid chromatography. The established multiresidue determination has been applied to several artificially spiked agricultural samples, with the result that the average recoveries were excellent, with the exception of nitenpyram. The limit of detection of the method ranged from 0.01 to 0.03 mg/kg for the insecticides.