Two types of oxidative dimerization of the black tea polyphenol theaflavin.

Theaflavin and its galloyl esters are polyphenolic pigments of black tea. In the course of studies on the oxidation mechanism of tea polyphenols, two theaflavin oxidation products named bistheaflavins A and B were isolated, and their structures were elucidated on the basis of MS and NMR spectroscopic analyses. Treatment of a mixture of (-)-epicatechin and (-)-epigallocatechin with banana fruit homogenate yielded bistheaflavin A together with theaflavin and theanaphthoquinone. The symmetrical structure of bistheaflavin A suggested that this compound was formed by oxidative C [bond] C coupling of two theaflavin molecules. In contrast, theaflavin in phosphate buffer (pH 7.3) was gradually oxidized to give bistheaflavin B and theanaphthoquinone. Bistheaflavin B possesses a bicyclooctane skeleton probably formed by intermolecular cyclization between dehydrotheaflavin and dihydrotheanaphthoquinone.     

A novel convenient process to obtain a raw decaffeinated tea polyphenol fraction using a lignocellulose column

Lignocellulose prepared from sawdust was investigated for its potential application in obtaining a raw decaffeinated tea polyphenol fraction from tea extract. Tea polyphenols having gallate residues, namely, (-)epigallocatechin gallate (EGCg) and (-)epicatechin gallate (ECg), were adsorbed on the lignocellulose column, while caffeine was passed through it. Adsorbed polyphenols were eluted with 60% ethanol, and the elute was found to consist mainly of EGCg and ECg. The caffeine/EGCg ratio was 0.696 before lignocellulose column treatment, but it became 0.004 after the column treatment. These results suggest that the lignocellulose column provides a useful and convenient process of purification of tea polyphenol fraction accompanied by decaffeination.     

Characterization of tea cultivated at four different altitudes using 1H NMR analysis coupled with multivariate statistics

The taste of black tea differs according to the different areas in which the tea is grown, even for the same species of tea. A combination of (1)H NMR spectroscopy and partial least-squares discriminate analysis (PLS-DA) was used to assess the quality differences of tea leaves from four cultivation areas with different elevations, RAN > 1800 m, UDA = 1200 m, MEDA = 600 m, and YATA < 300 m, in Sri Lanka. As a result of a statistical analysis, PLS-DA showed a separation between high- and low-quality black teas derived from the four different tea cultivation areas. RAN from the highest elevation showed characteristic trends in the levels of theaflavin and theaflavin 3,3'-digallate that were found only in RAN, and the levels of theanine and caffeine were higher, and the levels of thearubigins, especially thearubigin 3,3'-digallate, were lower in RAN than in UDA, MEDA, and YATA. The structures of these components were determined by 1D and 2D NMR analyses. These results demonstrate that this method can be used to evaluate black tea quality according to the chemical composition or metabolites, which are characteristic of the tea leaves cultivated in four regions with different elevations in Sri Lanka.     

茶树施用控释氮肥的产量和品质效应

采用盆栽和大田试验,研究了控释氮肥对茶树生长发育、茶叶产量和品质的影响。结果表明,控释氮肥能明显促进茶树的生长发育,茶树新梢数、百芽重、成熟叶片数量及叶绿素含量、树高和主杆直径等均有明显提高,从而使盆栽茶树根、茎和叶各部位的生物产量比施普通尿素分别提高了58.3%、36.9%和17.2%。10个大田试验的茶叶产量增加了4.2%￣24.1%,平均9.6%。茶叶品质成分氨基酸、咖啡碱和水浸出物含量有不同程度提高,其中氨基酸增幅明显,但对茶多酚含量的影响不大,茶叶酚氨比明显降低。可见,控释氮肥具有明显的增产提质效果。     

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.     

HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea

Tea is a complex mixture containing a range of compounds from simple phenolics to complex thearubigins, many of which have well-recognized antioxidant properties. This paper describes the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS(n)) methods for the rapid and routine analysis of more than 30 phenolics in tea. Green and black tea infusions were injected directly onto a reversed phase HPLC column, and the phenolics eluted using two different mobile phase gradients, one optimized to resolve catechin derivatives and the other, flavonols and theaflavins. Compounds, identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern, included (+)-catechin, (-)-epicatechin, theaflavin and their various gallate derivatives, quercetin and kaempferol mono-, di-, and triglycosides, quinic acid esters of gallic acid and hydroxycinnamates, and the purine alkaloids, caffeine and theobromine.     

Tea polyphenols inhibit the transport of dietary phenolic acids mediated by the monocarboxylic acid transporter (MCT) in intestinal Caco-2 cell monolayers

It was previously reported that a fluorescent marker dye, fluorescein, is transported via the monocarboxylic acid transporter (MCT). Fluorescein transport was competitively inhibited by MCT substrates such as ferulic and salicylic acids. Tea polyphenols, in particular, epigallocatechin gallate (EGCg) and epicatechin gallate (ECg), inhibited the transport of fluorescein. Tea polyphenols also inhibited the transport of salicylic and ferulic acids, suggesting tea polyphenols might be substrates of MCT. However, the transepithelial flux of tea polyphenols was much lower than that of the MCT substrates and was inversely correlated with the paracellular permeability of Caco-2 cell monolayers. These findings suggest that tea polyphenols are not substrates but inhibitors of MCT. Furthermore, the transepithelial transport of these polyphenols is mainly via paracellular diffusion. However, directional transport of ECg and EGCg from the basolateral to the apical side was observed, indicating that the behavior of tea polyphenols in the intestinal epithelium is complex.     

Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells

The effects of tea polyphenols on the invasion of highly metastatic human fibrosarcoma HT1080 cells through a monolayer of human umbilical vein endothelial cells (HUVECs) and the accompanying basal membrane were investigated. Among the tea polyphenols tested, epicatechin gallate (ECg), epigallocatechin gallate (EGCg), and theaflavin strongly suppressed the invasion of HT1080 cells into the monolayer of HUVECs/gelatin membrane, whereas epicatechin, epigallocatechin, tea flavonols, tea flavones, and gallate derivatives had no effect. Both theaflavin-digallate and theasinensin D showed a weak invasion inhibitory effect. ECg significantly inhibited the invasion without cytotoxicity against cancer cells and HUVECs. Ester-type catechins (ECg and EGCg) and theaflavin strongly suppressed the gelatin degradation mediated by matrix metalloproteinase (MMP) 2 and MMP-9, which were secreted into the conditioned medium of HT1080 cells. In conclusion, among the tea polyphenols tested, ECg was considered to be the agent with the most potential antimetastasis activity because it inhibited invasion in the absence of cytotoxicity.     

机械超微粉碎与不同粒度常规粉碎对红茶理化特性的影响

为探究机械超微粉碎对茶叶物性及功能成分浸出特性的影响,该文通过超微粉碎和常规粉碎2种方法制备超微红茶粉和4种粒度的粗粉红茶粉,研究了5种茶粉的粒度分布、微观形貌、比表面积、结晶度、表面氧碳元素比、中红外图谱和功能成分的浸出特性。结果表明:常规粉碎不同粒度茶粉间差异较小,而超微粉碎较大程度地改变了茶叶的物料性质。超微粉碎后,茶粉粒度减小至微米级,比表面积增大,茶粉的结晶度降低,表面暴露的纤维素和半纤维素相对含量增多,而中红外谱图显示超微粉碎茶粉的官能团结构并没有改变。沸水浸提过程中茶多酚、咖啡因、可溶性糖和水浸出物的溶出结果表明:5种茶粉功能成分的浸出量都随时间的增加而增加,经过一段时间后达到平衡。在达到浸出平衡时,不同粒度茶粉的茶多酚和咖啡因含量差异不显著,超微茶粉的可溶性糖含量为4种粗粉的2倍左右,水浸出物比粗粉茶粉提高10%以上。上述研究结果表明:超微粉碎能够有效减小茶粉的粒度,破坏植物的细胞壁结构,使内部成分暴露于表面,此外,组织结构和细胞壁的破坏使不溶的纤维成分转化成可溶性成分,可溶性糖和水浸出物显著增多,超微粉碎能够在一定程度上提高茶粉的有效利用率。     

Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate

The inhibitory effects of five tea polyphenols, namely theaflavin (TF1), theaflavin-3-gallate (TF2), theaflavin-3,3'-digallate (TF3), (-)-epigallocatechin-3-gallate (EGCG), and gallic acid, and propyl gallate (PG) on xanthine oxidase (XO) were investigated. These six antioxidant compounds reduce oxidative stress. Theaflavins and EGCG inhibit XO to produce uric acid and also act as scanvengers of superoxide. TF3 acts as a competitive inhibitor and is the most potent inhibitor of XO among these compounds. Tea polyphenols and PG all have potent inhibitory effects (>50%) on PMA-stimulated superoxide production at 20 approximately 50 microM in HL-60 cells. Gallic acid (GA) showed no inhibition under the same conditions. At 10 microM, only EGCG, TF3, and PG showed significant inhibition with potency of PG > EGCG > TF3. The superoxide scavenging abilities of these six compunds are as follows: EGCG > TF2 > TF1 > GA > TF3 > PG. PG was the most potent inhibitor of PMA-stimulated H(2)O(2) production in HL-60 cells. The order of H(2)O(2) scavenging ability was TF2 > TF3 > TF1 > EGCG > PG > GA. Therefore, the antioxidative activity of tea polyphenols and PG is due not only to their ability to scavenge superoxides but also to their ability to block XO and related oxidative signal transducers.     

Catechin and caffeine content of green tea dietary supplements and correlation with antioxidant capacity

The health benefits associated with tea consumption have resulted in the wide inclusion of green tea extracts in botanical dietary supplements, which are widely consumed as adjuvants for complementary and alternative medicines. Tea contains polyphenols such as catechins or flavan-3-ols including epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCG), as well as the alkaloid, caffeine. Polyphenols are antioxidants, and EGCG, due to its high levels, is widely accepted as the major antioxidant in green tea. Therefore, commercial green tea dietary supplements (GTDS) may be chemically standardized to EGCG levels and/or biologically standardized to antioxidant capacity. However, label claims on GTDS may not correlate with actual phytochemical content or antioxidant capacity nor provide information about the presence and levels of caffeine. In the current study, 19 commonly available GTDS were evaluated for catechin and caffeine content (using high-performance liquid chromatography) and for antioxidative activity [using trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays]. Product labels varied in the information provided and were inconsistent with actual phytochemical contents. Only seven of the GTDS studied made label claims of caffeine content, 11 made claims of EGCG content, and five specified total polyphenol content. Caffeine, EGCG, and total polyphenol contents in the GTDS varied from 28 to 183, 12-143, and 14-36% tablet or capsule weight, respectively. TEAC and ORAC values for GTDS ranged from 187 to 15340 and from 166 to 13690 mumol Trolox/g for tablet or capsule, respectively. The antioxidant activities for GTDS determined by TEAC and ORAC were well-correlated with each other and with the total polyphenol content. Reliable labeling information and standardized manufacturing practices, based on both chemical standardization and biological assays, are recommended for the quality control of botanical dietary supplements.     

Determination of tea polyphenols and caffeine in tea flowers (Camellia sinensis) and their hydroxyl radical scavenging and nitric oxide suppressing effects

The native occurrence of tea polyphenols, namely, (-)-epicatechin, (+)-catechin, (-)-epigallocatechin 3-gallate, (-)-epicatechin, and (-)-epicatechin 3-gallate, and caffeine in tea flowers was assessed by an isocratic HPLC procedure. The levels of total catechins and caffeine were determined in tea flowers collected from 10 different species of Camellia sinensis. The results showed the levels of total catechin ranged from 10 to 38 mg/g, whereas the level of caffeine ranged from 3 to 8 mg/g. Levels of catechins and caffeine in tea leaves and various teas were also determined and ranged from 2 to 126 mg/g and from 23 to 49 mg/g, respectively. Both tea flower and tea leaf extracts exert their strong hydroxyl radical scavenging effects in the Fenton reaction system and nitric oxide suppressing effects in LPS-induced RAW 264.7 cells. Most tea flowers contain less caffeine, but comparable amounts of total catechins, compared to tea leaves and teas. The present study demonstrates that both tea flowers and tea leaves contain appreciable amounts of catechins and caffeine. It is likely that tea flowers might be useful for making alternative tea beverages.     

Effects of tea polyphenols on emulsification of olive oil in a small intestine model system

Tea catechins have been shown to reduce plasma cholesterol and suppress hypertriacylglycerolemia by reducing triglyceride absorption. However, the mechanism is not yet clear. One of the possible mechanisms is that tea polyphenols may modify dietary fat emulsification in the gastrointestinal tract. The digestive enzyme (lipase) acts on specific emulsion interface properties (droplet size and surface area). Therefore, changes in these properties may modify emulsification and lead to changes in dietary fat digestion and absorption. In this study, the effect of both green and black tea on the changes of emulsification was examined by measuring the droplet size and the surface area. A model emulsion system containing olive oil, phosphatidylcholine (PC), and bile salt was developed to simulate small intestinal conditions. Initial changes in droplet size (from 1.4 to 52.8 microm and from 1.4 to 25.9 microm) of the emulsion were observed in the presence of 1.04 mg/mL and 0.10 mg/mL of total catechins prepared from green and black tea, respectively. Both teas caused similar changes on the emulsion properties; however, black tea was more effective than green tea. The underlying mechanisms of actions of tea polyphenols are discussed.     

Comparison of the chemical constituents of aged pu-erh tea, ripened pu-erh tea, and other teas using HPLC-DAD-ESI-MSn

Pu-erh tea is a popular beverage in southwestern China and South Asian countries. To explain the differences of aged pu-erh tea and ripened pu-erh tea, the chemical constituents of these teas were identified by HPLC-DAD-ESI-MS(n). In addition, HPLC was used to determine the contents of the major polyphenols, gallic acid, caffeine, and theobromine, in various types of teas. These results showed that the majority of chemical constituents in ripened pu-erh tea and aged pu-erh tea were similar, but the contents of catechins and gallic acid presented significant differences between these two teas. After fermentation by microorganism, the levels of catechins in ripened pu-erh tea were decreased, but the contents of gallic acid and caffeine were conversely elevated compared with aged pu-erh tea.     

Enhancement in growth and quality parameters of tea [Camellia sinensis (L.) O. Kuntze] through inoculation with arbuscular mycorrhizal fungi in an acid soil

The present study was undertaken to determine the effect of inoculation with arbuscular mycorrhizal fungi (AMF) from natural and cultivated tea rhizospheres (NTR and CTR, respectively) on growth as well as on quality parameters of tea [Camellia sinensis (L.) O. Kuntze]. Seeds (after germination) and cuttings were inoculated with AMF consortia from NTR and CTR in non-sterilized acid soil (pH 5.0). Seedlings and cuttings were grown under net-house conditions and the data on growth and quality parameters were recorded up to 2 years. Tea plants were found to be highly colonized by AMF (74–83%) after 1 year. The significant increase in most of the growth parameters (root/shoot length, dry weight; r/s ratio) was recorded in AMF-treated plants, which also showed better tea quality parameters than noninoculated plants. Maximum significant increases up to 31% and 100%, over control, were recorded in amino acids and total protein content, respectively. Similarly, maximum increase in total polyphenols (15%) and caffeine content (34%) over control was also significant. Enhanced sugar content (maximum 16% in total soluble sugars, maximum 45% in reducing sugars, and maximum 69% in non-reducing sugars) was recorded in AMF-inoculated plants. In most cases, better response was observed in tea plants inoculated with AMF consortia from NTR. These results indicate that growth and quality of tea can be improved by AMF inoculations in acid soils.     

茶园施肥模式对茶叶黄酮类及糖苷类代谢物含量的影响

【目的】研究施肥模式对茶叶生化成分含量和黄酮类及糖苷类代谢物累积的影响,为实现茶叶优质高产提供科学依据。【方法】田间试验设在四川茶树种植典型区域—雅安,试验连续进行了3年,设置不施肥(CK)、常量化肥(N 585 kg/hm2,TF)、有机肥替代25%化肥氮(OF)、减施25%氮肥(SF) 4个处理。分析了不同处理茶叶主要品质成分含量,并基于非靶向代谢组学检测结果分析了黄酮类及糖苷类代谢物累积差异。【结果】与C K处理相比,T F、 O F、 S F处理茶氨酸、游离氨基酸含量分别提高了7.2 2%～1 3.4 0%、23.15%～25.50%,可溶性糖和茶多酚含量分别降低了8.17%～13.86%、6.08%～11.49%,表儿茶素、表没食子儿茶素、芸香苷等10种代谢物累积水平显著下降。与TF处理相比,SF处理茶氨酸、咖啡碱和水浸出物含量分别降低了5.45%、5.97%、8.91%,茶多酚、可溶性糖、游离氨基酸含量和酚氨比无显著差异;芍药素-3-O-葡萄糖苷、肉桂萜醇C1-19-葡萄糖苷、查耳酮樱花素等7种代谢物累积水平显著降低,川陈皮素、牡荆素-4’-O-α-L-吡喃鼠李糖苷、(S)...     

Synthesis of theaflavin from epicatechin and epigallocatechin by plant homogenates and role of epicatechin quinone in the synthesis and degradation of theaflavin

Oxidation products of (-)-epicatechin and (-)-epigallocatechin by treatment with homogenates of 62 plants belonging to 49 families were compared. Forty-six plants were capable of synthesizing theaflavin, a black tea pigment, regardless of whether they contained catechins. Loquat, Japanese pear, and blueberry had activities higher than that of fresh tea leaves after 5 h of treatment; furthermore, these plants oxidized theaflavin to theanaphthoquinone. An additional new metabolite, dehydrotheasinensin, was generated on treatment with fresh tea leaves, eggplant, and unripened Japanese orange. Evidence for the oxidation of epigallocatechin and theaflavin by electron transfer to epicatechin quinone was demonstrated in a time course study using bananas and trapping the quinone intermediates as glutathione conjugates.     

Preparative separation of polyphenols from tea by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) was applied to the separation of polyphenols from tea leaves (Camellia sinensis L.). The capability of HSCCC to isolate pure tea polyphenols from complex mixtures on a preparative scale was demonstrated for catechins, flavonol glycosides, proanthocyanidins, and strictinin from green and black tea. The purity and identity of isolated compounds was confirmed by (1)H NMR and HPLC-ESI-MS/MS. Gram quantities of polyphenols from tea can be isolated with the procedure described.     

便携式茶鲜叶品质光谱检测装置研制

品质监测对茶鲜叶适时采摘和茶叶加工品控具有重要意义。该研究基于可见/近红外光谱技术,研发了便携式茶鲜叶品质无损检测装置。该装置分为主机和手柄2部分,主机大小约240 mm×250 mm×240 mm,包括光谱仪、光源、可充电锂电池、稳压板和散热风扇;手柄大小约130 mm×100 mm×30 mm,包括光纤探头、金属灯杯、白参考板和外触发按钮。基于该设备,采集了茶鲜叶500～900 nm范围内可见/近红外漫反射光谱,对比了归一化（Normalize,NOR）、一阶导数（First Derivative,FD）、标准正态变量变换（Standard Normal Variable Transformation,SNV）和概率商归一化（Probabilistic Quotient Normalization,PQNOR）等不同光谱预处理方法对茶叶光谱的处理结果,建立了茶鲜叶干物质含量、水浸出物含量、茶多酚含量的偏最小二乘定量预测模型。结果表明,PQNOR预处理后建立的偏最小二乘预测模型精度最好,干物质、水浸出物和茶多酚含量预测模型在验证集的相关系数分别为0.905、0.896和0.747,均方根误差分别为0.856%、0.559%和0.549%。在茶园对装置的精度进行了现场测试,单片茶鲜叶检测时间约为1 s,干物质、水浸出物和茶多酚含量预测值与测量值的均方根误差分别为0.903%、0.634%和0.551%。该装置可以实现茶鲜叶光谱原位采集和干物质含量、水浸出物、茶多酚的定量分析,对及时掌握茶树生长情况、辅助决策采茶方案和保障茶叶品质具有重要作用。