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.     

Determination and comparison of γ-aminobutyric acid (GABA) content in pu-erh and other types of Chinese tea

Two previous studies have reported that pu-erh tea contains a high level of γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system and has several physiological functions. However, two other researchers have demonstrated that the GABA content of several pu-erh teas was low. Due to the high value and health benefits of GABA, analysis of mass-produced pu-erh tea is necessary to determine whether it is actually enriched with GABA. A high-performance liquid chromatography (HPLC) method was developed for the determination of GABA in tea, the results of which were verified by amino acid analysis using an Amino Acid Analyzer (AAA). A total of 114 samples of various types of Chinese tea, including 62 pu-erh teas, 13 green teas, 8 oolong teas, 8 black teas, 3 white teas, 4 GABA teas, and 16 process samples from two industrial fermentations of pu-erh tea (including the raw material and the first to seventh turnings), were analyzed using HPLC. Statistical analysis demonstrated that the GABA content in pu-erh tea was significantly lower than that in other types of tea (p < 0.05) and that the GABA content decreased during industrial fermentation of pu-erh tea (p < 0.05). This mass analysis and comparison suggested GABA was not a major bioactive constituent and resolved the disagreement GABA content in pu-erh tea. In addition, the GABA content in white tea was found to be significantly higher than that in the other types of tea (p < 0.05), leading to the possibility of producing GABA-enriched white tea.     

Comparative studies on the hypolipidemic and growth suppressive effects of oolong, black, pu-erh, and green tea leaves in rats

The four major commercial teas, oolong, black, pu-erh, and green teas, have been manufactured in southeast Asia. In this study, we evaluated the growth suppressive and hypolipidemic effect of these four different tea leaves by oral feeding to male Sprague-Dawley rats for 30 weeks. The results showed that the suppression of body weights of tea leaves-fed groups were in the order: oolong tea > pu-erh tea > black tea > green tea. Pu-erh tea and oolong tea could lower the levels of triglyceride more significantly than that of green tea and black tea, but pu-erh tea and green tea were more efficient than oolong tea and black tea in lowering the level of total cholesterol. In lipoprotein, 4% pu-erh tea could increase the level of HDL-C and decrease the level of LDL-C, but other teas simply decrease the levels of both. The activity of antioxidant enzyme SOD is increased in all tea-fed groups as compared to the basal diet-fed group. Finally, relative weight ratios of liver to epididylmal adipose tissue were lower in feeding oolong tea and pu-erh tea groups. On the basis of these findings, it seemed that the fully fermented pu-erh and black tea leaves and partially fermented oolong tea leaves were more effective on their growth suppressive and hypolipidemic effects as compared to the nonfermented green tea leaves.     

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.     

Effect of microbial fermentation on content of statin, GABA, and polyphenols in Pu-Erh tea

Besides cancer prevention, the hypolipidemic effects of tea have been well studied in animals and humans. Recently, statin has been identified in Pu-erh tea extract. Clinical trials have confirmed that statin decreases the incidence of major coronary and cerebrovascular events and this may be due to its hypolipidemic and antiinflammatory effects. Since a good Pu-erh tea needs longer storage (10 years or more) of fermentation to enhance the flavor and fragrance, we screened microorganisms from two Pu-erh teas, 20 and 25 years old. Species of fungi and bacteria strains that contributed to a good taste of Pu-erh tea were isolated. The effect of fermentation was investigated by inoculating fresh tea leaves with individual strains of isolated microorganisms. Results showed that statin, total polyphenol content, and the scavenging activities of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals increased during fermentation. Tea leaves inoculated with Streptomyces bacillaris strain R9 had the highest polyphenol content (3.3 mg/100 g) and scavenging ability to DPPH radicals (92%). Streptomyces cinereus strain Y11 was equally good for polyphenol content but yielded the highest amount of statin (1012 ng/g) after 42 days of fermentation. Interestingly, the statin content of fresh tea leaves fermented with strain R9 or Y11 after 180 days was much higher (4- and 8-fold, respectively) than that of the 25-year-old Pu-erh tea (513 ng/g) as measured by the HPLC method. Similarly, these two strains also increased the content of gamma-aminobutyric acid (GABA) 5.7- and 4.7-fold in tea fermented for 180 days as compared with the fresh leaves (1270 microg/g) and that were higher than that of the Pu-erh tea (4900 microg/g). Taken together, the present results indicate that tea short-term fermented with S. bacillaris or S. cinereus enhances the color and content of statin, GABA, and polyphenols.     

高氨基酸和高茶氨酸茶树资源筛选

为筛选高氨基酸和高茶氨酸茶树资源及探究茶树中茶氨酸与氨基酸含量之间的关系,本研究利用茚三酮显色法结合酶标仪检测技术测定218份茶树资源春季一芽二叶蒸青样的氨基酸含量,利用高效液相色谱法测定了其茶氨酸含量。结果表明,218份资源的氨基酸含量介于1.50%~8.98%之间,其中高氨基酸茶树资源(氨基酸含量≥5%)55份;茶氨酸含量介于0%~4.03%之间,其中高茶氨酸茶树资源(茶氨酸含量≥3%)13份,不含茶氨酸的茶树资源4份;茶氨酸与氨基酸含量的比值介于0~0.78之间,且集中分布于0.2~0.5之间,二者的相关性系数达0.752,呈极显著正相关。本研究筛选获得了一批氨基酸特异的茶树资源,为高氨基酸和高茶氨酸茶树新品种选育及其形成的分子机理研究奠定了基础。     

Total phenol, catechin, and caffeine contents of teas commonly consumed in the United kingdom.

Levels of total phenol, catechins, and caffeine in teas commonly consumed in the United Kingdom have been determined using reversed phase high-performance liquid chromatography. Tea bags or tea leaves were purchased from local supermarkets and extracted in boiling water for 5 min. The resulting data showed considerable variability in both total phenols [80.5-134.9 mg/g of dry matter (DM) in black teas and 87-106.2 mg/g of DM in green teas] and catechins (5.6-47.5, 51.5-84.3, and 8.5-13.9 mg/g of DM in black, green, and fruit teas, respectively); this was most probably a result of differing agronomic conditions, leaf age, and storage during and after transport, as well as the degree of fermentation. Caffeine contents of black teas (22-28 mg/g of DM) were significantly higher than in less fermented green teas (11-20 mg/g of DM). The relative concentration of the five major tea catechins ranked EGCG > ECG > EC > EGC > C. The estimated U.K. dietary intakes of total tea catechins, calculated on the basis of an average tea consumption of three cups of tea (200 mL cup, 1% tea leaves w/v), were 61.5, 92.7, and 405.5 mg/day from fruit teas, black teas, and green teas, respectively. The coefficients of variation were 19.4, 88.6, and 17.3%, respectively, indicating the wide variation in these intakes. The calculated caffeine intake ranged between 92 and 146 mg/day. In addition, many individuals will consume much larger quantities of tea, of various strengths (as determined by the brewing conditions employed). This broad spread of U.K. daily intakes further emphasizes the need for additional research to relate intake and effect in various population groups.     

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.     

Identification and quantification of free radical scavengers in Pu-erh tea by HPLC-DAD-MS coupled online with 2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) diammonium salt assay

Pu-erh tea, a well-known traditional beverage in China, has attracted more attention because of its beneficial health effects and special flavor and taste. Generally, it is believed that Pu-erh tea with a longer preservation period has better quality and taste. Antioxidant activity is one of the major beneficial activities of tea. In this study, a HPLC-DAD-MS coupled with 2,2'-azinobis (3-ethylbenzthiazolinesulfonic acid) diammonium salt (ABTS) assay was employed for identification and quantification of free radical scavengers in different samples of Pu-erh tea. Among 12 main peaks detected in Pu-erh raw tea, 11 compounds were identified as gallic acid, (-)-gallocatechin, (-)-epigallocatechin, (+)-catechin, caffeine, (-)-epicatechin, (-)-epigallocatechingallate, rutin, (-)-epicatechingallate, quercetin-3-glucoside, and kaempferol-3-glucoside by comparison of their UV and MS data with standard compounds or literature data, respectively. The contents of 12 investigated compounds were also determined or estimated using caffeine, (-)-epicatechin, or rutin as standard. ABTS assay showed that 10 out of 12 compounds were free radical scavengers. Their total amount was used as the marker for evaluation of free radical scavenging activities of different Pu-erh teas, which indicated that the activity of different Pu-erh teas varied; Pu-erh raw tea was stronger than the ripe one, and the activity decreased with the increase of preservation period.     

Capillary electrophoretic determination of theanine, caffeine, and catechins in fresh tea leaves and oolong tea and their effects on rat neurosphere adhesion and migration

Theanine, caffeine, and catechins in fresh tea leaves and oolong tea were determined by using capillary electrophoresis (CE). CE separated these tea polyphenols from three other tea ingredients, namely, caffeine, theophylline, and theanine, within 8 min. The young leaves (apical bud and the two youngest leaves) were found to be richer in caffeine, (-)-epigallocatechin gallate (EGCg), and (-)-epicatechin gallate (ECg) than old leaves (from 5th to 7th leaves). On the other hand, the old leaves (from 8th to 10th leaves) contained higher levels of theanine, (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). Results from a comparison of fresh young tea and oolong tea compositions indicated oolong tea contained more theanine and catechins than fresh young tea. Furthermore, it was found that the levels of theanine, EGC, and EGCg in young leaves rose markedly with the withering process. Caffeine did not markedly change. However, fully or partially fermented teas (oolong tea or pauchong tea) have a common initial step in the withering process. Fresh tea leaves or oolong tea extract (0.1%, w/v) markedly inhibited neurosphere adhesion, cell migration, and neurite outgrowth in rat neurospheres. Theanine (348 micrograms/mL) and caffeine at high concentration (50 micrograms/mL) did not inhibit neurosphere adhesion or migration activities, but EGCg at 20 micrograms/mL effectively inhibited neurosphere adhesion for 24 h. These results indicated that EGCg might affect neural stem cell survival or differentiation.     

Metabolite profiling using (1)H NMR spectroscopy for quality assessment of green tea, Camellia sinensis (L.)

A set of 191 green teas from different countries was collected and analyzed by (1)H NMR. It was proposed to establish if the teas could be discriminated according to the country of origin or with respect to quality. Both principal component analysis (PCA) and cluster analysis were applied to the data. Some separation of Chinese and non-Chinese teas was observed. The present results did not allow allocation of samples to individual countries, but cluster analysis suggested that it might be possible with an augmented sample set. The PCA did show a separation between the Longjing type (highest quality Chinese tea) and most other Chinese teas and indicated some metabolites that could be responsible for the difference. Longjing teas showed higher levels of theanine, gallic acid, caffeine, epigallocatechin gallate, and epicatechin gallate and lower levels of epigallocatechin when compared with other teas. These compounds have been mentioned previously in connection with quality, but it was also shown that higher levels of theogallin (5-galloyl quinic acid), theobromine, 2-O-(beta-l-arabinopyranosyl)-myo-inositol and some minor sugar-containing compounds were found in Longjing teas while higher levels of fatty acids and sucrose were found in the other teas. These new markers could prove to be useful for the authentication of bulk tea.     

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.     

不同防冻措施茶园茶汤滋味差异的电子舌检测

为探讨轻度“倒春寒”对不同防护措施下3种茶园蒸青茶样的茶汤滋味影响,遂选品种和管理措施一致的安装防霜扇的茶园、松树林围绕的茶园以及未设防护措施的普通茶园,用电子舌综合评价3种茶园茶汤滋味、高效液相色谱检测茶汤游离氨基酸组分及含量。结果表明：基于电子舌检测数据的主成分分析、软独立建模分析和聚类分析结果揭示了普通茶园的茶汤滋味与防霜扇茶园和林间茶园的差异显著（P<0.05）;从9种茶样中皆检测出18种氨基酸,游离氨基酸质量分数介于19.691～30.947 mg/g 之间,茶氨酸含量占游离氨基酸含量的55.1%～70.5%;随着3种茶园气温的降低,游离氨基酸含量和茶氨酸含量呈升高趋势,3种鲜味氨基酸（天冬氨酸、谷氨酸、茶氨酸）、5种甜味氨基酸（脯氨酸、丙氨酸、甘氨酸、苏氨酸、丝氨酸）和5种苦味氨基酸（异亮氨酸、亮氨酸、苯丙氨酸、酪氨酸、缬氨酸）共占游离氨基酸含量在防霜扇茶园和林间茶园中分别出现升高、降低和降低趋势,而在普通茶园中变化与之不同。分析认为：轻度“倒春寒”可导致无防护措施的普通茶园茶叶滋味劣变,林间茶园能够减轻冻害,防霜扇作为防护手段不仅可以减免冻害,还可避免茶叶滋味品质下降。该研究结果为后续深入研究不同防护措施茶园茶叶品质提供参考。     

Phenolic acids in berries, fruits, and beverages

The contents of soluble and total phenolic acids were analyzed in samples of 29 berries and berry products, 24 fruits and fruit peels, and 12 beverages. Variation of phenolic acids in berries was also studied. Soluble phenolic acids were extracted with methanolic acetic acid, and a tentative quantification was performed by high-performance liquid chromatography (HPLC). The total phenolic acid content was determined by HPLC after alkaline and acid hydrolyses. The content of total phenolic acids as aglycones in the above samples varied from 0 (pear cider) to 103 mg/100 g fresh weight (rowanberry). Besides rowanberry, the best phenolic acid sources among berries were chokeberry (96 mg/100 g), blueberry (85 mg/100 g), sweet rowanberry (75 mg/100 g), and saskatoon berry (59 mg/100 g). Among fruits, the highest contents (28 mg/100 g) were determined in dark plum, cherry, and one apple variety (Valkea Kuulas). Coffee (97 mg/100 g) as well as green and black teas (30-36 mg/100 g) were the best sources among beverages. Caffeic acid dominated in all of these samples except in tea brews. Variation in the phenolic acid contents of the berries was either small or moderate.     

Metabolic dependence of green tea on plucking positions revisited: a metabolomic study

The dependence of global green tea metabolome on plucking positions was investigated through (1)H nuclear magnetic resonance (NMR) analysis coupled with multivariate statistical data set. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), were employed for a finding metabolic discrimination among fresh green tea leaves plucked at different positions from young to old leaves. In addition to clear metabolic discrimination among green tea leaves, elevations in theanine, caffeine, and gallic acid levels but reductions in catechins, such as epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), glucose, and sucrose levels were observed, as the green tea plant grows up. On the other hand, the younger the green tea leaf is, the more theanine, caffeine, and gallic acid but the lesser catechins accumlated in the green tea leaf, revealing a reverse assocation between theanine and catechins levels due to incorporaton of theanine into catechins with growing up green tea plant. Moreover, as compared to the tea leaf, the observation of marked high levels of theanine and low levels of catechins in green tea stems exhibited a distinct tea plant metabolism between the tea leaf and the stem. This metabolomic approach highlights taking insight to global metabolic dependence of green tea leaf on plucking position, thereby providing distinct information on green tea production with specific tea quality.     

Metabolism of theanine, gamma-glutamylethylamide, in rats

The metabolism of theanine, one of the major amino acid components in tea (Camellia sinensis), was studied in rats. High-performance liquid chromatography (HPLC) with fluorometric detection was used to evaluate the nature of theanine's metabolites in plasma, urine, and tissues. In the urine samples collected after administration of 100, 200, and 400 mg each of theanine, intact theanine, L-glutamic acid, and ethylamine, these compounds were detected in a dose-dependent manner. When 200 mg of theanine was orally administered to rats, the plasma concentrations of theanine and ethylamine reached their highest levels about 0.5 and 2 h after administration, respectively. It seems most likely that the enzymatic hydrolysis of theanine to glutamic acid and ethylamine was accomplished in the kidney. These results indicate that orally administered theanine is absorbed through the intestinal tract and hydrolyzed to glutamic acid and ethylamine in the rat kidney.     

红茶与绿茶感官品质与其化学组分的相关性

对茶叶感官审评分属性与其化学组分的相关性进行探讨,该文以不同等级、产地红茶和绿茶为代表,利用传统感官审评方法鉴定茶叶品质（外形、香气、汤色、滋味和叶底）,并对各品质指标进行相关性分析,筛选出对茶叶品质影响最重要的品质指标,即滋味。在利用尺度评价法对茶叶滋味品质的10个分属性（浓度、厚度、甘度、鲜度、醇度、涩度、嫩度、陈味、纯正度、火工度）进行更为细致的评定和对茶叶中主要呈味物质—茶多酚、咖啡碱、茶氨酸进行定量分析的基础上,对不同等级、不同产地茶叶的滋味分属性进行差异分析,并对化学组分与滋味分属性进行相关性分析,结果表明,各种茶样之间的产地和等级间差异主要是鲜度和涩度,而与之相关的组分主要是茶氨酸和咖啡碱,茶多酚的影响还有待进一步考察。该研究实现了茶叶感官品质的定量分析,提高了感官审评结果的客观性和确定性,为茶叶原产地保护和分等分级提供核心技术和标准支撑。     

Effect of alternative anions (Cl– vs. SO$ _4^{2-} $) on concentrations of free amino acids in young tea plants

The quality of green tea is highly dependent on the concentration of free amino acids, whose profile is dominated by the unique amino acid theanine (N⁵‐ethyl‐glutamine). A high quality is associated with a high amino acid–to–catechin ratio, but previous results indicated that excessive chloride (Cl^–) supply is detrimental for amino acid accumulation. Several experiments were conducted to investigate the effect of chloride on growth and concentrations of free amino acids in young tea plants. Soil‐grown tea plants supplied with different levels of potassium (K) as K₂SO₄ or KCl exhibited increased concentrations of free amino acid in young shoots only when supplied with K₂SO₄, and the negative effect of KCl supply was mainly due to a reduced concentration of theanine. Concentrations of other nutrients in plant tissues were not influenced. The uptake of Cl^– and its interaction with nitrogen (N) uptake were further investigated in a second experiment, in which soil‐cultivated tea plants were supplied with varying amounts of Cl^–. Chloride application reduced yield of young shoots, and severity of leaf damage was related to the concentration of Cl^– in leaves. Nitrogen uptake was reduced by Cl^– addition. To verify whether the decrease of free amino acids was simply a result of inhibited NO assimilation, a third experiment was conducted, in which tea plants were NH ‐fed in the absence or presence (equivalent to the NH concentration) of Cl^–. Again, concentrations of theanine and total free amino acids in young shoots were reduced by Cl^– supply, but changes of the free–amino acid pool did not contribute to the maintenance of charge balance. However, concentration of theanine in roots, where it is synthesized, was not influenced by Cl^–. Total N concentrations of roots and mature leaves, uptake rate of NH , and activity of glutamine synthetase in fibrous roots and young leaves were all unaffected by Cl^– as well. It is suggested that translocation of theanine from root to shoot and its catabolism in young shoots might be influenced by Cl^–.