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.     

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.     

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.     

Determination of tea components with antioxidant activity

Levels of essential elements with antioxidant activity, as well as catechins, gallic acid, and caffeine levels, in a total of 45 samples of different teas commercialized in Spain have been evaluated. Chromium, manganese, selenium, and zinc were determined in the samples mineralized with HNO(3) and V(2)O(5), using ETAAS as the analytical technique. The reliability of the procedure was checked by analysis of a certified reference material. Large variations in the trace element composition of teas were observed. The levels ranged from 50.6 to 371.4 ng/g for Cr, from 76.1 to 987.6 microg/g for Mn, from 48.5 to 114.6 ng/g for Se, and from 56.3 to 78.6 ng/g for Zn. The four major catechins [(-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC)], gallic acid (GA), and caffeine were simultaneously determined by a simple and fast HPLC method using a photodiode array detector. In all analyzed samples, EGCG ranged from 1.4 to 103.5 mg/g, EGC from 3.9 to 45.3 mg/g, ECG from 0.2 to 45.6 mg/g, and EC ranged from 0.6 to 21.2 mg/g. These results indicated that green tea has a higher content of catechins than both oolong and fermented teas (red and black teas); the fermentation process during tea manufacturing reduces the levels of catechins significantly. Gallic acid content ranged from 0.039 to 6.7 mg/g; the fermentation process also elevated remarkably gallic acid levels in black teas (mean level of 3.9 +/- 1.5 mg/g). The amount of caffeine in the analyzed samples ranged from 7.5 to 86.6 mg/g, and the lower values were detected in green and oolong teas. This study will be useful for the appraisal of trace elements and antioxidant components in various teas, and it will also be of interest for people who like drinking this beverage.     

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.     

Effects of pu-erh tea on oxidative damage and nitric oxide scavenging

The effects of pu-erh tea, which is prepared by fermentation of tea, on oxidative damage and nitric oxide scavenging, compared with various other brands of tea were investigated. The total antioxidant activity was determined using the Trolox equivalent antioxidant capacity (TEAC) assay. The results showed that TEAC values of the 200 microg/mL water extracts of pu-erh tea (WEPT), green tea, oolong tea, and black tea were 86.3, 85.3, 87.4, and 80.3 (microg/mL), respectively, indicating that WEPT showed a significant antioxidant activity. WEPT, like green tea extract, oolong tea extract, and black tea extract, exhibited a remarkable protective effect in lipid (liposome) and nonlipid (protein and deoxyribose) model systems, implying that it is an inhibitor of lipid and nonlipid oxidative damage. It also exhibited metal-binding ability, reducing power, and scavenging effect for free radicals. Moreover, WEPT showed a decreasing effect on nitric oxide production of lipopolysaccharide-induced RAW 264.7 macrophages. In addition, the results revealed that epicatechin (EC), flavonoid, ascorbic acid, and polyphenolic compounds are present in WEPT, which may partially account for the protective effect on oxidative damage. Thus, WEPT may have potential as an antioxidant and as a nitric oxide scavenging agent.     

Development of new antioxidant active packaging films based on ethylene vinyl alcohol copolymer (EVOH) and green tea extract

Ethylene vinyl alcohol copolymer (EVOH) films containing green tea extract were successfully produced by extrusion. The films were brown and translucent, and the addition of the extract increased the water and oxygen barrier at low relative humidity but increased the water sensitivity, the glass transition temperature, and the crystallinity of the films and improved their thermal resistance. An analysis by HPLC revealed that the antioxidant components of the extract suffered partial degradation during extrusion, reducing the content of catechin gallates and increasing the concentration of free gallic acid. Exposure of the films to various food simulants showed that the liquid simulants increased their capacity to reduce DPPH(?) and ABTS(?+) radicals. The release of green tea extract components into the simulant monitored by HPLC showed that all compounds present in the green tea extract were partially released, although the extent and kinetics of release were dependent on the type of food. In aqueous food simulants, gallic acid was the main antioxidant component released with partition coefficient values ca. 200. In 95% ethanol (fatty food simulant) the K value for gallic acid decreased to 8 and there was a substantial contribution of catechins (K in the 1000 range) to a greatly increased antioxidant efficiency. Kinetically, gallic acid was released more quickly than catechins, owing to its faster diffusivity in the polymer matrix as a consequence of its smaller molecular size, although the most relevant effect is the plasticization of the matrix by alcohol, increasing the diffusion coefficient >10-fold. Therefore, the materials here developed with the combination of antioxidant substances that constitute the green tea extract could be used in the design of antioxidant active packaging for all type of foods, from aqueous to fatty products, the compounds responsible for the protection being those with the higher compatibility with the packaged product.     

基于数据融合策略的红茶发酵程度判别

发酵是红茶加工过程中关键的一道工序,对红茶的品质形成有着重要影响。该研究以大叶种英德红茶中的英红九号为研究对象,试验收集了204份不同发酵时间的红茶样品并使用便携式近红外光谱仪和工业相机获取红茶发酵中的信息,基于近红外光谱数据、图像数据和数据融合策略分别建立了红茶发酵程度判别模型。通过分析茶多酚和儿茶素类含量的变化,将红茶的发酵划分为3个阶段,即发酵不足、发酵适度和发酵过度。采用Savitzky-Golay光滑对原始光谱进行预处理,利用竞争自适应重加权采样（Competitive Adaptive Reweighted Sampling, CARS）、连续投影算法（Successive Projections Algorithm, SPA）和主成分分析（Principal Components Analysis, PCA）对近红外光谱变量进行降维处理;相应地,图像进行去阴影后提取了9个颜色特征变量,采用皮尔森（Pearson）相关分析和主成分分析进行特征变量提取。最后采用线性判别分析（Linear Discriminant Analysis, LDA）和支持向量机（Support Ve...     

Polyphenol content of plasma and litter after the oral administration of green tea and tea polyphenols in chickens

Metabolic profiles of broiler chickens were examined after the ingestion of green tea, tea polyphenols, and (-)-epigallocatechin-3-gallate (EGCG). Solid-phase extraction of serum and litters yielded free catechins and their metabolites, which were then identified and quantified by liquid chromatography-tandem mass spectrometry. In plasma samples, (-)-gallocatechin, (+)-catechin, and EGCG were detected in the green tea group; pyrogallol acid, (epi)catechin-O-sulfate, 4'-O-methyl-(epi)gallocatechin-O-glucuronide, and (epi)catechin-3'-O-glucuronide were detected in the tea polyphenols group; and EGCG, (-)-gallocatechin gallate (GCG), and 4'-O-methyl-(epi)gallocatechin-O-glucuronides were detected in the EGCG group. In litters, gallic acid, EGCG, GCG, and ECG were detected in the green tea and tea polyphenols groups; EGCG and ECG were detected in the EGCG group. The conjugated metabolites, 4'-O-methyl-(epi)gallocatechin-O-glucuronide, (epi)catechin-3'-glucuronide, and 4'-O-methyl-(epi)catechin-O-sulfate, were identified in the green tea group; 4'-O-methyl-(epi)catechin-O-sulfate and 4'-O-methyl-(epi)gallocatechin-O-sulfate were identified in the tea polyphenols group; only 4'-O-methyl-(epi)gallocatechin-O-sulfate was detected in the EGCG group. The excretion of tea catechins was 95.8, 87.7, and 97.7% for the green tea, tea polyphenols, and EGCG groups, respectively.     

Simultaneous analysis of catechins, gallic acid, strictinin, and purine alkaloids in green tea by using catechol as an internal standard

We developed a high-performance liquid chromatography-based method for simultaneous analysis of nine catechins, gallic acid, strictinin, caffeine, and theobromine in green tea by using catechol as an internal standard. Although the high cost and instability of the catechin reference standards limit the application of this method, the addition of ascorbic acid to the standard stock solution preserved the stability of the reference standards in the solution for 1 year when stored at -30 degrees C. Furthermore, we found that the slopes of the calibration curves plotted were stable for a run time of 2000 h. Our method proved to be appropriate for quantification and yielded good correlation coefficients, detection levels, repeatability, reproducibility, and recovery rates. Quantitative data revealed that the contribution of only 200 mL of brewed tea to the total dietary catechins was approximately 220-420 mg, while that of 500 mL of bottled tea was approximately 170-900 mg.     

Application of near-infrared reflectance spectroscopy to the simultaneous prediction of alkaloids and phenolic substances in green tea leaves

A near-infrared reflectance spectroscopic (NIRS) method for the prediction of polyphenol and alkaloid compounds in the leaves of green tea [Camellia sinensis (L.) O. Kuntze] was developed. Reference measurements of the individual catechins, gallic acid, caffeine, and theobromine were performed by reversed-phase HPLC. The total polyphenols were determined according to the colorimetric Folin-Ciocalteu assay. Using the partial least-squares algorithm, very good calibration statistics were obtained for the prediction of gallic acid, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, caffeine, and theobromine (R(2) > 0.85) with standard deviation/standard error of cross-validation (SD/SECV) ratio ranging from 2.00 to 6.27. Simultaneously, the dry matter content of the tea leaves can be analyzed very precisely (R(2) = 0.94; SD/SECV = 4.12). Furthermore, it is possible to discriminate tea leaves of different age by principal component analysis on the basis of the received NIR spectra. Prediction of the total polyphenol content is performed with a lower accuracy, which might be due to the lack of specificity in the colorimetric reference method. The study demonstrates that NIRS technology can be successfully applied as a rapid method not only for breeding and cultivation purposes but also to estimate the quality and taste of green tea and to control industrial processes, for example, decaffeination.     

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.     

Application of microwave energy in the manufacture of enhanced-quality green tea

Green tea manufacture was standardized with respect to the inactivation of polyphenol oxidase (PPO), rolling, and drying for quality manufacture. Inactivation of PPO by parching, steaming, microwave heating, and oven heating was monitored in tea shoots. The inactivated shoots were rolled under regimens of high and low pressures and dried by microwave heating, oven heating, or sun-drying; total phenols and catechins were estimated. Parched and sun-dried teas contained the lowest levels of total phenols and catechins, and their infusions were dull in color with a slightly burnt odor. Microwave-inactivated and-dried teas showed the highest levels of total phenols and catechins, and their infusions were bright in color and sweet in taste with a subtle pleasant odor. In steam-inactivated and oven/microwave-dried teas, total phenol and catechin contents were intermediate between parched and sun-dried teas and microwave-inactivated and microwave-dried teas, and their infusions were bright with a umami taste.     

Stability of tea catechins in the breadmaking process

A green tea extract (GTE) was incorporated into bread as a source of tea catechins. The stability of tea catechins in the breadmaking process including unfrozen and frozen dough was studied. A method was developed for the separation and quantification of tea catechins in GTE, dough, and bread samples using a RP-HPLC system. The separation system consisted of a C18 reversed-phase column, a gradient elution system of water/methanol and formic acid, and a photodiode array UV detector. Tea catechins were detected at 275 nm. GTEs at 50, 100, and 150 mg per 100 g of flour were formulated. The results obtained showed that green tea catechins were relatively stable in dough during freezing and frozen storage at -20 degrees C for up to 9 weeks. There were no further detectable losses of tea catechins in bread during a storage of 4 days at room temperature. It was also revealed that (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) were more susceptible to degradation than (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC). (-)-EGCG and (-)-ECG were normally selected as the quality indices of green tea catechins, and their retention levels in freshly baked bread were ca. 83 and 91%, respectively. One piece of bread (53 g) containing 150 mg of GTE/100 g of flour will provide 28 mg of tea catechins, which is approximately 35% of those infused from one green tea bag (2 g).     

Gallic acid metabolites are markers of black tea intake in humans

Gallic acid is one of the main phenolic components of black tea. The objective of this study was to identify urinary gallic acid metabolites with potential for use as markers of black tea intake. In an initial study, nine compounds, assessed by using gas chromatography-mass spectrometry, were found to increase in concentration in urine after 3 cups of black tea over 3 h. A subsequent study employed a controlled crossover design in which 10 subjects consumed 5 cups per day of black tea or water for 4 weeks in random order. Twenty-four hour urine samples were collected at the end of each period. Of the 9 candidate compounds identified in the initial study, only 3 were present at higher concentrations in urine of all 10 subjects during tea-drinking in comparison to water-drinking periods. These compounds were identified as 4-O-methylgallic acid, 3-O-methylgallic acid, and 3, 4-O-dimethylgallic acid, all methyl ether derivatives of gallic acid. It is suggested that these compounds have the potential to be used as markers of black tea intake.     

Optimization of fabrication parameters to produce chitosan-tripolyphosphate nanoparticles for delivery of tea catechins

This work investigated the polyanion-initiated gelation process in fabricating chitosan-tripolyphosphate (CS-TPP) nanoparticles intended to be used as carriers for delivering tea catechins. The results demonstrated that the particle size and surface charge of CS-TPP nanoparticles could be controlled by fabrication conditions. For preparation of CS-TPP nanoparticles loaded with tea catechins, the effects of modulating conditions including contact time between CS and tea catechins, CS molecular mass, CS concentration, CS-TPP mass ratio, initial pH value of CS solution, and concentration of tea catechins on encapsulation efficiency and the release profile of tea catechins in vitro were examined systematically. The study found that the encapsulation efficiency of tea catechins in CS-TPP nanoparticles ranged from 24 to 53%. In addition, FT-IR analysis showed that the covalent bonding and hydrogen bonding between tea catechins and CS occurred during the formation of CS-TPP nanoparticles loaded with tea catechins. Furthermore, studies on the release profile of tea catechins in vitro demonstrated that the controlled release of tea catechins using CS-TPP nanoparticles was achievable.     

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.     

Massive accumulation of gallic acid and unique occurrence of myricetin, quercetin, and kaempferol in preparing old oolong tea

Old oolong tea, tasting superior and empirically considered beneficial for human health, is prepared by long-term storage accompanied with periodic drying for refinement. Analyzing infusions of three old and one newly prepared oolong teas showed that significant lower (-)-epigallocatechin gallate (EGCG) but higher gallic acid contents were detected in the old teas compared to the new one. The possibility of releasing gallic acid from EGCG in old tea preparation was supported by an in vitro observation of gallic acid degraded from EGCG under heating conditions mimicking the drying process. Moreover, three minor flavonols, myricetin, quercetin, and kaempferol, that were undetectable in the new tea occurred in all of the three old teas. Converting the new oolong tea into an old one by periodic drying revealed the same characteristic observation, i.e., massive accumulation of gallic acid presumably released from EGCG and unique occurrence of flavonols putatively decomposed from flavonol glycosides.     

茶叶籽贮藏时间对毛油产率与质量的影响

为了揭示茶叶籽贮藏时间对发酵法茶叶籽毛油产率与质量的影响,每2周从贮藏的茶叶籽中取样,利用茶叶籽油发酵法生产工艺进行茶叶籽毛油生产,并对工艺中各项剩余物的含油量及毛油的重要质量指标进行了测定,结果如下。室温条件下,茶叶籽贮藏47周后,毛油产率下降了23.5%、酸值及过氧化值分别升高了44.88%及69.4%,毛油色泽基本没有变化。滤渣、发酵沉淀的质量分别升高了20.27%及23.35%;淀粉、油渣质量分别降低了6.13%及3.64%。滤渣、发酵沉淀、淀粉及油渣含油率分别升高了15.63%、22.77%、206%及12.88%。发酵沉淀质量对毛油产率的影响是正向的,淀粉、油渣及滤渣质量对毛油产率的影响是负向的;发酵沉淀及滤渣含油率对毛油产率的影响是正向的,淀粉及油渣含油量对毛油产率的影响是负向的,影响大小的排序为:油渣淀粉滤渣发酵沉淀。综合分析表明,滤渣是通过滤渣质量的增加导致毛油产率随贮藏时间下降的,其下降作用的贡献占全部下降因素的79.28%。贮藏47周后的茶叶籽仁,利用发酵法生产工艺仍然具有毛油生产价值。该研究可为茶叶籽油合理生产提供理论支撑。