Achievable Central Nervous System Concentrations of the Green Tea Catechin EGCG Induce Stress in Glioblastoma Cells in Vitro

The polyphenolic compounds present in green tea are preventative against cancer in several animal tumor models. However, direct cytotoxic effects on cancer cells have also been reported. In order to determine whether drinking of green tea has chemopreventive or cytotoxic effects on brain cancer cells, we investigated the effect of the major green tea polyphenol EGCG as a pure substance and as tea extract dietary supplement on primary human glioblastoma cell cultures at the CNS-achievable concentration of 100?nM reported in the literature. We compared this with the effect of the cytotoxic concentration of 500?μM determined to be specific for the investigated primary glioblastoma cultures. After treatment with 500?µM EGCG, strong induction of autophagy and apoptosis was observed. Under treatment with 100?nM EGCG, glioblastoma cells proliferated over the entire observation period of 6 days without any detectable signs of cell death. Only within the first 12?h of treatment was increased accumulation of autophagic vacuoles and increased reactive oxygen species production as a stress response demonstrated. Mild forms of stress, such as treatment with 100?nM EGCG, activate different endogenous repair mechanisms to protect cells. Our data imply that drinking of green tea may have chemopreventive effects, but no direct cytotoxic properties.     

绿茶及其有效成分对肾性高血压大鼠左室肥厚抑制作用的研究

目的研究绿茶及其提取物对肾性高血压大鼠左室肥厚的抑制作用机制。方法雄性Wistar大鼠160只,体重180~220g,随机分为5组,建立二肾一夹法大鼠肾性高血压左室肥厚模型:假手术组(SHAM)和手术组(2K1C)正常饮水;手术+绿茶组(2K1C+GT)饮用2%绿茶水;手术+茶多酚组(2K1C+TP)饮用0.1%茶多酚水;手术+EGCG组(2K1C+EGCG)饮用0.05%EGCG水,术后即给予不同浓度的绿茶及其提取物,持续8周直至实验结束。结果与假手术组比较,手术组和各饮茶组血压、左心室与体重比值(LVW/BW)和左心室壁厚度(LVWT)均显著升高(P<0.01)。而与手术组相比,不同浓度的绿茶及其有效成分(2%绿茶、0.1%茶多酚、0.05%EGCG)左心室重/体重(LVW/BW)、左心室壁厚度显著降低,抗氧化酶(SOD和GSH-Px)的活性升高,活性氧自由基(ROS)以及Ras和ERK1/2蛋白表达降低,但对大鼠收缩压无显著性影响。结论绿茶及其提取物对肾性高血压大鼠左室肥厚有抑制作用,其机制可能是由于茶及其有效成分具有抗氧化活性,能够清除ROS,以及对Ras-MAPK信号传导通路的调控有关。     

Reduction of Hexavalent Chromium by Green Tea Polyphenols and Green Tea Nano Zero-Valent Iron (GT-nZVI)

This study reports on the direct reduction of hexavalent chromium [Cr(VI)] by green tea polyphenols, including a green tea solution and pure epigallocatechin gallate (EGCG) solution. A linear trend was observed between the amount of reduced Cr(VI) and the amount of added polyphenols. The green tea solution showed a continued decrease in the observed stoichiometry with increasing pH, from a maximum of 1.4 mol per gallic acid equivalent (GAE) of green tea at pH 2.5, to 0.2 mol/GAE at pH 8.8. The EGCG solution exhibited different behavior, with a maximum stoichiometry of 2 at pH 7 and minimum of 1.6 at pH 4.4 and 8.9. When green tea was used to first react with Fe³⁺ and form GT-nZVI, the amount of Cr(VI) reduced by a certain volume of GT-nZVI was double compared to green tea, and 6 times as high considering that GT-nZVI only contains 33 % green tea.     

Piperine enhances the bioavailability of the tea polyphenol (-)-epigallocatechin-3-gallate in mice

(-)-Epigallocatechin-3-gallate (EGCG), from green tea (Camellia sinensis), has demonstrated chemopreventive activity in animal models of carcinogenesis. Previously, we reported the bioavailability of EGCG in rats (1.6%) and mice (26.5%). Here, we report that cotreatment with a second dietary component, piperine (from black pepper), enhanced the bioavailability of EGCG in mice. Intragastric coadministration of 163.8 micromol/kg EGCG and 70.2 micromol/kg piperine to male CF-1 mice increased the plasma C(max) and area under the curve (AUC) by 1.3-fold compared to mice treated with EGCG only. Piperine appeared to increase EGCG bioavailability by inhibiting glucuronidation and gastrointestinal transit. Piperine (100 micromol/L) inhibited EGCG glucuronidation in mouse small intestine (by 40%) but not in hepatic microsomes. Piperine (20 micromol/L) also inhibited production of EGCG-3"-glucuronide in human HT-29 colon adenocarcinoma cells. Small intestinal EGCG levels in CF-1 mice following treatment with EGCG alone had a C(max) = 37.50 +/- 22.50 nmol/g at 60 min that then decreased to 5.14 +/- 1.65 nmol/g at 90 min; however, cotreatment with piperine resulted in a C(max) = 31.60 +/- 15.08 nmol/g at 90 min, and levels were maintained above 20 nmol/g until 180 min. This resulted in a significant increase in the small intestine EGCG AUC (4621.80 +/- 1958.72 vs. 1686.50 +/- 757.07 (nmol/g.min)). EGCG appearance in the colon and the feces of piperine-cotreated mice was slower than in mice treated with EGCG alone. The present study demonstrates the modulation of the EGCG bioavailablity by a second dietary component and illustrates a mechanism for interactions between dietary chemicals.     

Green tea catechins suppress xanthine oxidase activity in dairy products: An improved HPLC analysis

A new rapid isocratic HPLC method was developed to quantify xanthine oxidase activity in several dairy products by determining both substrate (xanthine) and product (urate) concentrations at short retention times (6.76 min and 4.92 min, respectively) with high values for linearity and reliability. The impact of added green tea catechins (1000 ppm) on xanthine oxidase activity was also examined in dairy products containing fat globules using the developed HPLC method. The addition of (+)-catechin, (−)-epigallocatechin gallate (EGCG), and green tea extract significantly (p ≤ 0.05) decreased the xanthine oxidase activity of these products. The effect of EGCG on decreasing xanthine oxidase activity of milk fat globules was stronger than for (+)-catechin, and can be attributed to different structures of green tea polyphenols, such as different numbers of OH groups in catechins. The results of this study suggest possible associations between milk fat globule surfaces and green tea catechins.     

Absorption and pharmacokinetics of green tea catechins in beagles

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12.35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 0-2, 2-6, 6-8 and 8-24 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for ( - )-epigallocatechin-3-gallate (EGCG), ( - )-epicatechin-3-gallate (ECG), ( - )-epigallocatechin glucuronide (EGC-glucuronide), ( - )-epicatechin glucuronide (EC-glucuronide), ( - )-epicatechin sulphate (EC-sulphate) were 0.3 (range 0.1-1.9), 0.1 (range 0-0.4), 0.8 (range 0.2-3.9), 0.2 (range 0.1-1.7) and 1 (range 0.3-3.4) micromol/l, respectively. The areas under the plasma concentration v. time curves (AUC0 --> 24) were 427 (range 102-1185) micromol/l x min for EGC-glucuronide, 112 (range 53-919) micromol/l x min for EC-sulphate, 71 (range 26-306) micromol/l x min for EGCG, 40 (range 12-258) micromol/l x min for EC-glucuronide and 14 (range 0.1-124) micromol/l x min for ECG. The values of mean residence time (MRT0 --> 24) were 5 (range 2-16), 2 (range 1-11), 10 (range 2-13), 3 (range 2-16) and 2.4 (range 1-18) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC-sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.     

Genotoxic effects of green tea extract on human laryngeal carcinoma cells in vitro

Green tea (Camellia sinensis) contains several bioactive compounds which protect the cell and prevent tumour development. Phytochemicals in green tea extract (mostly flavonoids) scavenge free radicals, but also induce pro-oxidative reactions in the cell. In this study, we evaluated the potential cytotoxic and prooxidative effects of green tea extract and its two main flavonoid constituents epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) on human laryngeal carcinoma cell line (HEp2) and its cross-resistant cell line CK2. The aim was to see if the extract and its two flavonoids could increase the sensitivity of the cisplatin-resistant cell line CK2 in comparison to the parental cell line. The results show that EGCG and green tea extract increased the DNA damage in the CK2 cell line during short exposure. The cytotoxicity of EGCG and ECG increased with the time of incubation. Green tea extract induced lipid peroxidation in the CK2 cell line. The pro-oxidant effect of green tea was determined at concentrations higher than those found in traditionally prepared green tea infusions.     

The influence of domestic culinary processes on the Trolox Equivalent Antioxidant Capacity of green tea infusions

This paper presents an investigation into the influence of several culinary factors, such as water temperature, infusion time, stirring and dosage form, on polyphenol content and antioxidant capacity (TEAC values) during the domestic preparation of green tea. The results obtained show that water temperature and infusion time strongly influence total polyphenol levels and the antioxidant capacity of green tea. Temperatures of 70-80 °C together with infusion times of 3-5 min produced greatest effect, in this respect. At 90 °C, extraction was faster and more effective. However, prolonged infusion at this temperature may cause a loss of polyphenol compounds and, consequently, of antioxidant capacity. Factors such as agitation and dosage form do not seem to have much influence. Furthermore, it was found that pure green tea infusions have higher antioxidant properties than do blends of green tea with aromatic herbs and fruits.     

The impact of the catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract

The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The GG [corrected] group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.     

Green tea polyphenol (–)-epigallocatechin-3-gallate enhances the inhibitory effect of huperzine A on acetylcholinesterase by increasing the affinity with serum albumin

Abstract

The green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) was investigated for its enhancement effect of huperzine A on inhibiting acetylcholinesterase (AChE). The inhibitory effect of huperzine A on acetylcholinesterase is quite weak in the whole phase. EGCG hardly inhibits the AChE activity within the range 10–300 mg/kg. However, upon addition of EGCG to the huperzine A groups, a remarkably enhanced inhibitory effect was observed. The EGCG also can largely prolong the inhibitory time. These results indicate that addition of EGCG to huperzine A can reduce the dose of huperzine A required compared with huperzine A alone. The enhancement and complementary effect of EGCG on huperzine A activity may partly be due to the antioxidant property of EGCG. One of the beneficial effects of green tea is to induce a feeling of relief. It is conceivable that this function may be regulated by EGCG in the central nervous system since EGCG is distributed in the brain after oral administration. EGCG can be used as an enhanced supplement for huperzine A to treat Alzheimer's disease.     

Comparison of antioxidant activity and bioavailability of tea epicatechins with their epimers

Canned and bottled tea drinks contain not only green tea epicatechins (GTE), namely (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC), but also four GTE epimers, namely (-)-gallocatechin gallate (GCG), (-)-catechin gallate (CG), (-)-gallocatechin (GC) and (-)-catechin (C). In the present study we examined the antioxidant activity and bioavailability of these epimers compared with their corresponding precursors. The epimerisation reaction was induced by autoclaving GTE extract derived from longjing green tea at 120 degrees C for 20 min. Isolation and purification of each GTE and epimer were accomplished by various column chromatographic and semi-preparative HPLC techniques. The antioxidant activity of each epimer with its corresponding GTE precursor was conducted in the three in vitro systems, namely human LDL oxidation, ferric reducing-antioxidant power (FRAP), and anti-2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assays. The results of all three assays demonstrated that CG had similar antioxidant activity with its precursor ECG, while GC was less potent as an antioxidant than its precursor EGC. Regarding EGCG and GCG, the antioxidant potency was similar for both LDL oxidation and DPPH free radical assays, but GCG was statistically less effective than EGCG in the FRAP assay. For EC and C, the latter had less anti-free radical activity in the DPPH assay, but in LDL oxidation and FRAP assays the antioxidant activity was similar. Oral and intravenous dosing of GTE-epimer mixture led to increase in total plasma antioxidant capacity in rats. In general, both epicatechins and epimers had low bioavailability (0.08-0.31) and most of the observed differences between epicatechins and their corresponding epimers were small, even if they were statistically significant in some cases. It was concluded that the epimerisation reaction occurring in manufacturing canned and bottled tea drinks would not significantly affect antioxidant activity and bioavailability of total tea polyphenols.     

Green tea and its catechins inhibit breast cancer xenografts

Investigators have shown that green tea may decrease the risk of cancer. It is widely accepted that the main active component of green tea is epigallocatechin-3-gallate (EGCG). In this study, we examined the effect of green tea on breast cancer growth and endothelial cells in in vitro assays and in animal models. Furthermore, we compared the potency of the different catechin components of green tea extract (GTE), including EGCG. Our data showed that mixed GTE and its individual catechin components were effective in inhibiting breast cancer and endothelial cell proliferation. In mouse experiments, GTE suppressed xenograft size and decreased the tumor vessel density. Our results demonstrated the value of all catechins and argued for the use of a mixed GTE as a botanical dietary supplement, rather than purified EGCG, in future clinical trials.     

Green tea polyphenols attenuate glial swelling and mitochondrial dysfunction following oxygen-glucose deprivation in cultures

Abstract

Astrocyte swelling is a major component of cytotoxic brain edema in ischemia. Oxidative stress and mitochondrial dysfunction have been hypothesized to contribute to such swelling in cultures. We investigated the protective effects of polyphenol-rich green tea extract (GTE) on key features of ischemic injury namely cell swelling, nitric oxide (NO) production, and depolarization of the inner mitochondrial membrane potential (Δψ_m). C6 glial cultures were subjected to 5-h oxygen-glucose deprivation (OGD) and cell volume was determined using the 3-O-methyl-glucose method. At 90 min after the end of OGD, cell volume increased by > 33% and this increase was attenuated by GTE but not by the individual polyphenol components including catechin, epicatechin, or epigallocatechin gallate (EGCG). However, a combination of catechin, epicatechin and EGCG prevented swelling. OGD-induced increase in NO was further increased by GTE. OGD-induced decline in Δψ_m was also attenuated by green tea extract, EGCG and a combination of catechin, epicatechin and EGCG but not by catechin or epicatechin alone. Our findings indicate a protective effect of GTE in cell swelling in ischemic injury and such protective effects may be mediated by its effect on the mitochondria. It appears that effects on cell swelling are mediated by the concerted action of more than one of the individual components of GTE.     

In vivo antioxidant effect of green tea

OBJECTIVE: The object of this study was to investigate the in vivo antioxidant effect of green tea and dosage effect of green tea on antioxidant effect. DESIGN: We tested 10 healthy subjects (aged 23-25 y, five women and five men) with overnight fasting. The total antioxidant capacity of plasma was measured at baseline and 60 min and 120 min after ingestion of 150 ml green tea. Green tea was prepared by infusing 2.5 g of dried green tea leaves for 2 min at 80 degrees C in 150 ml of water. In the second week, they took 300 ml of tea (5.0 g of green tea leaves) and, in the third week, 450 ml of tea (7.5 g of green tea leaves). The total antioxidant capacities of plasma were determined with a Total Antioxidant Kit (Randox Laboratories Ltd, UK) using a Cobas Mira analyser (Roche Diagnostic Systems Inc., Switzerland). The mean intra-assay coefficient of variation was 1.2%. RESULTS: The total antioxidant capacity of plasma increased by 1.1% at 60 min and 2.1% at 120 min over baseline value in subjects consuming 150 ml of green tea, which was statistically not significant. However, total antioxidant capacity of plasma after consuming 300 ml of green tea showed a significant increase of 7.0% after 60 min and 6.2% after 120 min (P<0.0001), and after consuming 450 ml 12.0% after 60 min and 12.7% after 120 min over baseline value (P<0.0001). CONCLUSIONS: Total antioxidant capacity of plasma was significantly increased after taking green tea in amounts of 300 and 450 ml. A positive increment according to green tea dosage was also observed. SPONSORSHIP: This work was funded by the Pacific Corporation (Korea).     

Influence of steeping conditions (time,temperature, and particle size) on antioxidant properties and sensory attributes of some white and green teas

Abstract

The influence of commonly used steeping times and temperatures, as well as leaf size on the antioxidant activity and sensory attributes of tea were studied. Five unblended white and green tea samples from China and Malawi, infused in hot (70?°C and 90?°C; 7?min) or cold water (room temperature: 15, 30, 60, or 120?min) either as whole leaves or as milled, were analyzed. Total phenolic and flavonoid contents as well as antioxidant power (ABTS assay) were measured. The results show that the maximum extraction efficiency occurs with cold water for 120?min and with hot water at 90?°C and that only in the case of teas from whole, large leaves, the extraction was greater in cold than in hot infusions. Moreover, tea infusions prepared from milled leaves have the greatest antioxidant activity. In the sensory evaluation of some of the tea infusions, white teas were perceived more fragrant than green ones and were judged as the most favorite by the majority of the judges, especially for the brew prepared in cold water from whole leaves; all infusions obtained from the milled leaves in fact have a more bitter and astringent taste.     

Nongallated compared with gallated flavan-3-ols in green and black tea are more bioavailable

Green tea and black tea (BT) contain gallated [(-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate] and nongallated [(-)-epicatechin, (-)-epigallocatechin (EGC)] tea polyphenols (PP). During BT production, PP undergo oxidation and form larger polymers such as theaflavins (THE) and thearubigins, which contribute to the health benefit of BT. This article gives an overview of the role of chemical characteristics and endogenous metabolism of tea PP and their bioavailability in humans and describes attempts to increase their bioavailability. At pH close to neutral, EGCG and EGC form homo- and heterodimers generating hydrogen peroxide. To confirm the pH instability of EGCG, EGC, and THE in cell culture medium, their antiproliferative activity was determined in the presence and absence of catalase. The antiproliferative activity in LNCaP prostate cancer cells was decreased when incubated with catalase prior to EGCG, EGC, and THE treatment. In addition, new findings demonstrated that the formation of methyl-EGC increased the stability at neutral pH compared with EGC. Approaches to increase the bioavailability of flavan-3-ols are reviewed, which include the administration of tea in combination with fruit juices, coadministration with piperine, and peracetylation of EGCG. Future intervention studies will need to focus on the bioactivity not only of green tea and BT PP but also of their metabolites and biotransformation products.     

(-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin.

Recently, we and others showed that the components of green tea may be useful cancer chemopreventive agents. It has been suggested that (-)-epigallocatechin-3-gallate (EGCG), the major constituent in green tea, may possess antitumor-promoting and/or anticarcinogenic effects in rodent tumor bioassay systems. During the chemical analysis of various green tea products, we found a traditionally preserved preparation of green tea used by tribes in the Himalayan region of Sikkim, India that was rich in EGCG. EGCG was isolated from this tea product, and its inhibitory effects were evaluated against the binding of topically applied 3H-labeled polycyclic aromatic hydrocarbons (PAHs) to epidermal DNA and 12-O-tetradecanoylphorbol-13-acetate (TPA) caused induction of epidermal ornithine decarboxylase (ODC) activity in Sencar mice, the short-term markers of tumor initiation and tumor promotion, respectively. Preapplication of EGCG resulted in significant inhibition (p less than 0.05) in the binding of [3H]PAH to epidermal DNA. Similarly, the topical application of EGCG resulted in significant inhibition (p less than 0.005) in TPA-caused induction of epidermal ODC activity. In further studies, we assessed the anti-skin tumor-initiating effect of EGCG in Sencar mice in an initiation-promotion protocol. The application of EGCG before challenge with 7,12-dimethylbenz[a]anthracene as tumor initiator resulted in significant reduction both in percentage of mice with tumors and number of tumors per mouse compared with a non-EGCG-pretreated group of animals. The results of the present study suggest that the green tea preparation from Sikkim may be a good source for the isolation of EGCG and that this compound may have significant potential as a cancer chemopreventive agent.