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 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.     

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).     

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.     

Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable epidemiological evaluation of these compounds, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 24 types of fruits, 27 types of vegetables and legumes, some staple foods, and processed foods commonly consumed in The Netherlands. Most fruits, chocolate, and some legumes contained catechins. Levels varied to a large extent: from 4.5 mg/kg in kiwi fruit to 610 mg/kg in black chocolate. (+)-Catechin and (-)-epicatechin were the predominant catechins; GC, EGC, and ECg were detected in some foods, but none of the foods contained EGCg. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Catechin contents of foods commonly consumed in The Netherlands. 2. Tea, wine, fruit juices, and chocolate milk

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable an epidemiological evaluation of catechins, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 8 types of black tea, 18 types of red and white wines, apple juice, grape juice, iced tea, beer, chocolate milk, and coffee. Tea infusions contained high levels of catechins (102-418 mg of total catechins/L), and tea was the only beverage that contained GC, EGC, ECg, and EGCg in addition to (+)-catechin and (-)-epicatechin. Catechin concentrations were still substantial in red wine (27-96 mg/L), but low to negligible amounts were found in white wine, commercially available fruit juices, iced tea, and chocolate milk. Catechins were absent from beer and coffee. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Different effects of five catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Five catechins [(-)-epigallocatechins gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (+)-catechin (C)] were compared with regard to their effects on 6-hydroxydopamine (OHDA)-induced apoptosis in PC12 cells--the vitro model of Parkinson's disease. Measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 6-OHDA inhibited cell viability in a time- and concentration-dependent manner. When PC12 cells were pretreated with the five catechins for 30 min before exposure to 250 microM 6-OHDA, MTT results showed that the five catechins had different effects: EGCG and ECG had obvious concentration-dependent protective effects at 50-400 microM; EC and (+)-C had almost no effects; and EGC especially decreased cell viability. Catechins also had different effects on apoptotic morphology. Only 200-400 microM EGCG and ECG kept cells adhering well. When pretreated with other catechins at any concentration, PC12 cells became round and some of them were detached as when treated with 6-OHDA. In addition, typical apoptotic characteristics of PC12 cells were determined by fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis after the cells were treated with 250 microM 6-OHDA for 24 h or pretreated with catechins before it. Preincubation with 200-400 microM EGCG and ECG led to significant inhibitory effects against PC12 cell apoptosis, as shown by flow cytometry. The other catechins have little protective effect. Therefore, at 200-400 microM, the classified protective effects of the five catechins were in the order ECG > EGCG > EC > (+)-C > EGC. The data also indicated that EGCG and ECG might be potent neuroprotective agents for Parkinson's disease. The results of fluorescence microscopy and DNA fragment analysis supported the conclusion.     

Green tea polyphenols inhibit metalloproteinase activities in the skin,muscle, and blood of rainbow trout

We have investigated the inhibitory effects of polyphenols from natural products, such as green tea, bilberry, grape, ginkgo, and apple, on rainbow trout gelatinase activities. Gelatinases from the skin, muscle, and blood of rainbow trout contained serine proteinase, metalloproteinase, and other proteinase activities as measured by gelatin zymography. The polyphenols of green tea caused the strong inhibition of some gelatinase activities when compared with those of the other products. This inhibition was quite similar to that of metalloproteinase by ethylenediaminetetraacetic acid, suggesting that the effects of green tea polyphenols on proteinase activities are specific for metalloproteinases. The major catechins of green tea polyphenols were then separated and identified by reverse-phase chromatography to be (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin. The effects of these catechins on gelatinase activities were examined; the most potent inhibitor of metalloproteinase activities was found to be EGCG. These results have indicated that green tea polyphenols including EGCG are useful for regulating metalloproteinase activities of fish meat.     

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.     

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.     

Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin.

Oxidative modification of low-density lipoproteins (LDL) may play an important role in the development of atherosclerosis. alpha-Tocopherol functions as a major antioxidant in human LDL. The present study was to test whether green tea catechins (GTC) would protect or regenerate alpha-tocopherol in human LDL. The oxidation of LDL incubated in sodium phosphate buffer (pH 7.4, 10 mM) was initiated by addition of 1.0 mM of 2,2'-azobis(2-amidinopropane) dihydrochloride at 40 degrees C. It was found that alpha-tocopherol was completely depleted within 1 h. Under the same experimental conditions, the longjing GTC extracts demonstrated a dose-dependent protective activity to alpha-tocopherol in LDL at concentrations ranging from 2 to 20 microM. Four pure epicatechin derivatives showed varying protective activity against depletion of alpha-tocopherol in LDL with (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) being less effective than (-)-epicatechin (EC) and (-)-epicatechin gallate (ECG). The results showed that addition of longjing GTC extracts, EC, ECG, and EGCG at 5, 10, and 15 min to the incubation mixture demonstrated a gradual regeneration of alpha-tocopherol in human LDL.     

Inhibition of radical reaction of apolipoprotein B-100 and alpha-tocopherol in human plasma by green tea catechins

(-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), and Trolox inhibited the decreases of apolipoprotein B-100 (apoB) and alpha-tocopherol in a radical reaction of human plasma initiated by Cu(2+). The concentrations of EC, EGC, ECg, EGCg, and Trolox for 50% inhibition (IC50) of apoB fragmentation were 39.1, 42.2, 14.6, 21.3, and 36.2 microM, respectively. Similar IC50 values were observed for alpha-tocopherol consumption, indicating the close relationship between apoB fragmentation and alpha-tocopherol consumption. These results demonstrate that tea catechins serve as an effective antioxidant in plasma and that the gallate group has a strong antioxidative activity.     

Simultaneous determination of seven bioactive components in Oolong tea Camellia sinensis: quality control by chemical composition and HPLC fingerprints

A simple and reliable method of high-performance liquid chromatography (HPLC) was developed for the quality control of oolong tea (the dry leaves of Camellia sinensis ): the quality control included the HPLC fingerprint and the quantitative determination of seven bioactive compounds chemicals, namely, (-)-gallocatechin, (-)-epigallocatechin, (-)-epigallocatechin gallate, caffeine, (-)-epicatechin, gallocatechin gallate, and (-)-epicatechin gallate. The developed analyses of the chemicals excelled in quantifying the chemicals in oolong tea. The chemical fingerprint of oolong tea was established using the raw materials of three main production sites in China, that is, Fujian (southern and northern parts), Taiwan, and Guangdong. The fingerprints from different cultivated sources were analyzed by hierarchical cluster analysis, similarity analysis, principal component analysis (PCA), analysis of variance (ANOVA), and discriminant analysis. The results indicated that the combination of chromatographic fingerprint and quantification analysEs could be used for the quality assessment of oolong tea and its derived products.     

β-carotene radical cation addition to green tea polyphenols. Mechanism of antioxidant antagonism in peroxidizing liposomes

Green tea polyphenols, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), all showed antioxidative effect in liposomes for lipid oxidation initiated in the lipid phase (antioxidant efficiency EC > EGCG > ECG > EGC) or in the aqueous phase (EC ? EGC > EGCG > ECG) as monitored by the formation of conjugated dienes. For initiation in the lipid phase, β-carotene, itself active as an antioxidant, showed antagonism with the polyphenols (EC > ECG > EGCG > EGC). The Trolox equivalent antioxidant capacity (TEAC EGC > EGCG > ECG > EC) correlates with the lowest phenol O-H bond dissociation enthalpy (BDE) as calculated by density functional theory (DFT). Surface-enhanced Raman spectroscopy (SERS) was used to assess the reducing power of the phenolic hydroxyls in corroboration with DFT calculations. For homogeneous (1:9 v/v methanol/chloroform) solution, the β-carotene radical cation reacted readily with each of the polyphenol monoanions (but not with the neutral polyphenols) with a rate approaching the diffusion limit for EC as studied by laser flash photolysis at 25 °C monitoring the radical cation at 950 nm. The rate constant did not correlate with polyphenol HOMO/LUMO energy gap (DFT calculations), and β-carotene was not regenerated by an electron transfer reaction (monitored at 500 nm). It is suggested that the β-carotene radical cation is rather reacting with the tea polyphenols through addition, as further evidenced by steady-state absorption spectroscopy and liquid chromatography-mass spectroscopy (LC-MS), in effect preventing regeneration of β-carotene as an active lipid phase antioxidant and leading to the observed antagonism.     

Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2)

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins.     

Electrofocusing of methanolic extracts for identification of individual flavonol biomolecules in Camellia species

An effort has been made to isolate individual catechin compounds from green tea leaves in their pure form by electrophoresis. In the present study total polyphenol extraction was carried out initially and estimated through spectrophotometric and HPLC methods. Extracted polyphenol was separated on 0.7% agarose gel and visualized at 360 nm. Fragmented individual compounds were gel eluted with methanol and confirmed as (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) by HPLC. The method developed describes a suitable method for the isolation of valuable molecules in tea.     

Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate

It has been known that tea catechins, (-)-epicatechin (1), (-)-epigallocatechin (2), (-)-epicatechin gallate (3), and (-)-epigallocatechin gallate (4) are epimerized to(-)-catechin (5), (-)-gallocatechin (6), (-)-catechin gallate (7), and (-)-gallocatechin gallate (8), respectively, during retort pasteurization. We previously reported that tea catechins, mainly composed of 3 and 4, effectively inhibit cholesterol absorption in rats. In this study, the effect of heat-epimerized catechins on cholesterol absorption was compared with tea catechins. Both tea catechins and heat-epimerized catechins lowered lymphatic recovery of cholesterol in rats cannulated in the thoracic duct and epimerized catechins were more effective than tea catechins. The effect of purified catechins on micellar solubility of cholesterol was examined in an in vitro study. The addition of gallate esters of catechins reduced micellar solubility of cholesterol by precipitating cholesterol from bile salt micelles. Compounds 7 and 8 were more effective to precipitate cholesterol than 3 and 4, respectively. These observations strongly suggest that heat-epimerized catechins may be more hypocholesterolemic than tea catechins.     

The apoptotic effect of green tea (-)-epigallocatechin gallate on 3T3-L1 preadipocytes depends on the Cdk2 pathway

This study was designed to investigate the effect of green tea catechins, especially (-)-epigallocatechin gallate (EGCG), on the apoptosis of 3T3-L1 preadipocytes. Preadipocyte apoptosis as indicated by formation of DNA fragments was induced by EGCG in dose-dependent manners. While EGCG was demonstrated to decrease Cdk2 expression and activity and increase caspase-3 activity, overexpression of Cdk2 and treatment with the caspase-3 inhibitor respectively prevented preadipocytes from induction of DNA fragmentation and caspase-3 activity by doses of 100-400 muM of EGCG. This suggests the Cdk2- and caspase-3-dependent apoptotic effects of EGCG. Moreover, EGCG was more effective than EC, ECG, and EGC in changing the apoptotic signals. Results of this study may relate to the mechanism by which EGCG modulates body weight.     

Effects of tea components on the response of GABA(A) receptors expressed in Xenopus Oocytes

To study the effects of tea components on ionotropic gamma-aminobutyric acid (GABA) receptor response, ionotropic GABA receptors (GABA(A) receptors) were expressed in Xenopus oocytes by injecting cRNAs synthesized from cloned cDNAs of the alpha(1) and beta(1) subunits of the bovine receptors, and their electrical responses were measured by a voltage clamping method. Extracts of green tea, black tea, and oolong tea in an aqueous solution induced the GABA-elicited response, which showed that these teas contain GABA, whereas coffee does not. Caffeine weakly inhibited the response in a competitive manner (K(i) = 15 mM), and (+)-catechin inhibited it in a noncompetitive one (K(i) = 1.7 mM). Especially, two catechin derivatives, (-)-epicatechin gallate and (-)-epigallocatechin gallate, inhibited the response strongly. Alcohols such as leaf alcohol or linalool potentiated the response, possibly because their binding to the potentiation site enhances the GABA-binding affinity to GABA(A) receptors when they bind. Extracts of green tea made with ethyl ether, which must contain lipophilic components of green tea, inhibited the response elicited by GABA, possibly because the amounts of caffeine and catechin derivatives were much larger than fragrant alcohols in such extracts of tea.