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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Flavor characteristics of lapsang souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking process

The major volatile constituents of lapsang souchong, smoked lapsang souchong, and smoked souchong, a group of special black teas in China, were analyzed with GC and GC-MS analyses. Forty-nine constituents were identified. Longifolene and alpha-terpineol were the most abundant compounds in the aroma. Due to its special production process, the compounds longifolene, longicyclene, guaiacol, 4-methylguaiacol, 4-ethylguaiacol, etc., were identified only in this kind of black tea. The aroma constituents of tea origin decreased during the smoking process, whereas the pine terpenoids and the thermal pyrolysis products of the pine wood increased markedly. The pine material for smoking was also analyzed for the volatile constituents showing apparent selectivity of the tea leaves to absorb. There were apparent differences among the tea samples in the contents of aroma constituents. The characteristics of the aroma of the teas are discussed with their processes and the features of sensory qualities.     

微波、超声波对茶叶主要化学成分浸提效果的研究

浸提工艺是影响茶饮料品质的关键环节之一。如何在低温短时浸提条件下保持茶叶良好的品质和较高的内含成分浸出率，是茶饮料生产工艺急待解决的难题。尝试将微波、超声波应用于茶饮料浸提过程中，研究了两种浸提方法对茶叶主要化学成分浸出的影响，结果显示微波、超声波对茶多酚、氨基酸、咖啡碱的浸出率与常规浸提法相当,对蛋白质、果胶等大分子物质的浸出有抑制作用。分析了不同浸提方式茶汤中主要儿茶素的含量，结果表明超声波浸提茶汤中主要儿茶素的含量高于其它浸提方式。微波、超声波处理适合于茶饮料的浸提。     

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.