Green Tea Polyphenol Extract Research Articles

One-step purification of epigallocatechin gallate from crude green tea extracts by mixed-mode adsorption chromatography on highly cross-linked agarose media

(−)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.2 mg/ml Superose). An EGCG purity of 99% with recoveries in the range 60–65% was achieved in one step directly from the crude GTP extract. Full column regeneration was obtained using solvents in the following order: 0.5 M NaOH, distilled water and 30% acetic acid.

Green Tea Polyphenol Extract Regulates the Expression of Genes Involved in Glucose Uptake and Insulin Signaling in Rats Fed a High Fructose Diet

Green tea has antidiabetic, antiobesity, and anti-inflammatory activities in animal models, but the molecular mechanisms of these effects have not been fully understood. Quantitative real-time polymerase chain reaction (PCR) was used to investigate the relative expression levels and the effects of green tea (1 and 2 g solid extract/kg diet) on the expression of glucose transporter family genes (Glut1/Slc2a1, Glut2/Slc2a2, Glut3/Slc2a3, and Glut4/Slc2a4) and insulin signaling pathway genes (Ins1, Ins2, Insr, Irs1, Irs2, Akt1, Grb2, Igf1, Igf2, Igf1r, Igf2r, Gsk3b, Gys1, Pik3cb, Pik3r1, Shc1, and Sos1) in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance and oxidative stress. Glut2 and Glut4 were the major Glut mRNAs in rat liver and muscle, respectively. Green tea extract (1 g) increased Glut1, Glut4, Gsk3b, and Irs2 mRNA levels by 110, 160, 30, and 60% in the liver, respectively, and increased Irs1 by 80% in the muscle. Green tea extract (2 g) increased Glut4, Gsk3b, and Pik3cb mRNA levels by 90, 30, and 30% but decreased Shc1 by 60% in the liver and increased Glut2, Glut4, Shc1, and Sos1 by 80, 40, 60, and 50% in the muscle. This study shows that green tea extract at 1 or 2 g/kg diet regulates gene expression in the glucose uptake and insulin signaling pathway in rats fed a fructose-rich diet.

Glutathione as a Mediator of the In Vitro Cytotoxicity of a Green Tea Polyphenol Extract

ABSTRACTThe 24-hr cytotoxicities of a green tea polyphenol (GTP) extract to cell lines derived from the human oral cavity were assessed using the neutral red (NR) assay. The sequence of sensitivity was carcinoma HSC-2 cells > immortalized gingival GT1 fibroblasts > normal gingival HGF-2 fibroblasts. The GTP extract generated hydrogen peroxide (H2O2) in cell culture medium and in phosphate buffer, albeit to a lesser extent. A 3-hr exposure to the GTP extract lowered the intracellular glutathione (GSH) content of the HSC-2 cells, but stimulated that of the GT1 and HGF-2 fibroblasts. The cytotoxicity of a 4-hr exposure of the GTP extract to the HSC-2 and GT1, but not to the HGF-2, cells was lessened in the presence of 2.5 mM GSH. Conversely, a 0.5 hr preexposure to the glutathione depleter, 1-chloro-2-dinotrobenzene (CDNB) at 25 μM, potentiated the 24-hr cytotoxicity of the GTP extract to the HSC-2 and GT1, but not to the HGF-2, cells. Using a cell-free system, it was shown that the GTP extract quickly depleted GSH, with depletion greatly enhanced at an alkaline pH, thus, correlating with the enhanced generation of H2O2 by the GTP extract observed at alkaline pH. Apparently, a mode of cytotoxicity of the GTP extract, in particular to the carcinoma HSC-2 cells, was to induce oxidative stress, as noted by the generation of H2O2, the depletion of intracellular GSH, the protection afforded by extracellular GSH, and cell hypersensitivity after pretreatment with CDNB.

Green tea polyphenol extract attenuates colon injury induced by experimental colitis

Inflammatory bowel disease (IBD) is characterised by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of green tea extract in rats subjected to experimental colitis induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). At 4 days after DNBS administration the rats were sacrificed. Treatment with green tea extract significantly attenuated diarrhoea and loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture, significant reduction of colonic myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) production. Green tea extract also reduced the appearance of nitrotyrosine immunoreactivity in the colon and reduced the up-regulation of ICAM-1.

Protective effects of green tea polyphenol extracts against ethanol-induced gastric mucosal damages in rats: Stress-responsive transcription factors and MAP kinases as potential targets

There are multiple lines of compelling evidence from epidemiologic and laboratory studies supporting that frequent consumption of green tea is inversely associated with the risk of chronic human diseases including cancer. The chemopreventive and chemoprotective effects of green tea have been largely attributed to antioxidative and anti-inflammatory activities of its polyphenolic constituents, such as epigallocatechin gallate. The present study was designed to evaluate the efficacy of green tea polyphenols in protecting against alcohol-induced gastric damage and to elucidate the underlying mechanisms. Intragastric administration of ethanol to male Sprague–Dawley rats caused significant gastric mucosal damage, which was accompanied by elevated expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as transient activation of redox-sensitive transcription factors, such as NF-κB and AP-1, and mitogen-activated protein kinases (MAPKs). Oral administration of the green tea polyphenolic extract (GTE) significantly ameliorated mucosal damages induced by ethanol and also attenuated the ethanol-induced expression of COX-2 and iNOS. Inactivation of MAPKs, especially p38 and ERKl/2, by GTE might be responsible for inhibition of ethanol-induced expression of COX-2 and iNOS.

Green tea polyphenol extract attenuates lung injury in experimental model of carrageenan-induced pleurisy in mice

Here we investigate the effects of the green tea extract in an animal model of acute inflammation, carrageenan-induced pleurisy. We report here that green tea extract (given at 25 mg/kg i.p. bolus 1 h prior to carrageenan), exerts potent anti-inflammatory effects in an animal model of acute inflammation in vivo.Injection of carrageenan (2%) into the pleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many neutrophils (PMNs), an infiltration of PMNs in lung tissues and increased production of nitrite/nitrate, tumour necrosis factor alpha. All parameters of inflammation were attenuated by green tea extract treatment. Furthermore, carrageenan induced an up-regulation of the adhesion molecule ICAM-1, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) formation, as determined by immunohistochemical analysis of lung tissues. Staining for the ICAM-1, nitrotyrosine, and PARS was reduced by green tea extract.Our results clearly demonstrate that treatment with green tea extract exerts a protective effect and offers a novel therapeutic approach for the management of lung injury.

Peripheral Nerve Allografts Stored in Green Tea Polyphenol Solution

We previously demonstrated the successful 1-month storage of peripheral nerve segments in a green tea polyphenol extract. We investigated whether this method could reduce the donor-host immune reaction associated with peripheral nerve allotransplantation. Sciatic nerve segments (20 mm long) were harvested from Dark Agouti (DA) rats, stored in polyphenol solution (1 mg/mL) for 1 month, and transplanted into recipient major histocompatibility complex-mismatched Lewis rats to bridge 15-mm-long sciatic nerve gaps (polyphenol-treated allograft group). The controls were an isograft group (nerve segments harvested from Lewis rats were immediately transplanted into Lewis rats), a polyphenol-treated isograft group (nerve segments harvested from Lewis rats were treated by polyphenol in the same method and transplanted into Lewis rats), and a fresh allograft group (nerve segments harvested from DA rats were transplanted into Lewis rats without storage). To investigate the origins of the cells in the transplanted nerves, sciatic nerve segments harvested from the male DA rat donors were transplanted into female Lewis rat recipients; genomic DNA was extracted from each nerve segment and amplified by polymerase chain reaction using primers specific for the rat sex-determining region of the Y-chromosome (Sry). Nerve regeneration in the polyphenol-treated allograft group was similar to that in the isografted group. Sry-specific bands were detected in all samples in the sex-mismatched polyphenol-treated allograft specimens despite their major histocompatibility complex incompatibility. Storage in green tea polyphenol solution can reduce both ischemic damage to nerve tissue and donor-host immune reactions after allotransplantation.

Mediation of the in vitro cytotoxicity of green and black tea polyphenols by cobalt chloride

The effects of Co 2+ (as CoCl 2) on the cytotoxicity of green tea polyphenol (GTP) and black tea polyphenol (BTP) extracts towards proliferation of immortalized human gingival epithelial-like S-G cells were studied. The 24 h potencies of GTP and BTP extracts, as determined with the neutral red (NR) cell viability assay, were greatly reduced in the presence of 250, but not of 50, μM Co 2+ . The cytotoxicities of the GTP and BTP extracts were due, in part, to their generation of hydrogen peroxide (H 2O 2) in the cell culture medium (DMEM). Progressively increasing the concentration of Co 2+ in the tea polyphenol-amended cell culture medium resulted in a lowering of the level of H 2O 2. The cytotoxicity of freshly added H 2O 2 to S-G cells was abolished in the presence of 250 μM Co 2+ and the level of freshly added H 2O 2 to cell culture medium was progressively lowered as the concentration of Co 2+ was increased. Apparently, under the conditions of these studies, the decreases in the cytotoxicity of GTP and BTP extracts in the presence of CoCl 2 were due to the rapid catalytic decomposition by Co 2+ of the H 2O 2 generated in the tea polyphenol-amended cell culture medium.

Kinetics of Reduction of Ferrylmyoglobin by (−)-Epigallocatechin Gallate and Green Tea Extract

The hypervalent heme pigment ferrylmyoglobin, a potential prooxidant in muscle tissue and meat, is efficiently reduced by epigallocatechin gallate (EGCG) from green tea and by green tea polyphenol extract (GTP) in neutral or moderately acidic aqueous solution (0.16 M NaCl) to yield metmyoglobin in two parallel processes. The second-order rate constant for direct reduction at pH 7.4 and 25 degrees C was found to have the value 1170 +/- 83 M(-1).s(-1) and activation parameters DeltaH(#) = 70.6 +/- 7.2 kJ.mol(-1) and DeltaS(#) = 50.7 +/- 24.1 J.mol(-1).K(-1) for EGCG and the value 2300 +/- 77 M(-1).s(-1) and parameters DeltaH(#) = 60.6 +/- 2.6 kJ.mol(-1) and DeltaS(#) = 23 +/- 9 J.mol(-1).K(-1) for GTP (based on EGCG concentration). For decreasing pH, the rate increased moderately due to a parallel reduction of protonated ferrylmyoglobin. At physiological pH, EGCG is more efficient in deactivating ferrylmyoglobin than other plant phenols investigated, and the relatively high enthalpy and positive entropy of activation suggest an outer-sphere electron transfer mechanism. The interaction between EGCG and other tea catechins in GTP could be responsible for the even stronger ability for GTP to deactivate ferrylmyoglobin.

Enhancement of inward Ca 2+ currents in bovine chromaffin cells by green tea polyphenol extracts

Green tea contains four major polyphenol compounds: they are (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epicatechin (EC). Although all four polyphenol compounds are known to affect tumor suppression, little is known about whether they alter membrane properties. In this study, we examined the effects of ECG and EGCG on ionic currents and secretion. Membrane capacitance changes were used to monitor secretion in bovine chromaffin cells. ECG had the ability to reversibly enhance the inward Ca 2+ current by 21%, and inhibited the peak sodium current by 34%. EGCG had no effect on Ca 2+ current even though it differs from ECG by just a hydroxyl group. The EC 50 of ECG in enhancing Ca 2+ current was 7.6 μM. The maximum enhancement of Ca 2+ current was observed at 0 mV and the maximum current was shifted ∼10 mV in the hyperpolarizing direction. When cells were stimulated by trains of depolarizations, the exocytosis elicited was enhanced by ECG treatment and the largest enhancement of secretion was observed in later stimulations. EGCG, although it had no significant effect on Ca 2+ current, enhanced exocytosis and slowed endocytosis. These results suggest that green tea polyphenol compounds modulate stimulus-secretion coupling in bovine chromaffin cells.

Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans

Oxidation of low density lipoprotein (LDL) plays crucial roles in atherogenesis. We previously reported that green tea polyphenols (flavan 3-ols), especially epigallocatechingallate (EGCg) and epicatechingallate, exerted potent inhibitory effects on LDL oxidation in vitro. To examine whether intake of green tea polyphenols renders LDL resistant to ex vivo oxidation in humans, 22 male volunteers aged between 22 and 32 years were recruited and assigned the same dietary regimen for 2 weeks. After a 1-week baseline period, they were equally divided into two groups: control and tea. The tea group ingested 300 mg of green tea polyphenol extract twice daily for 1 week. Plasma EGCg concentration at the end of the experiment was 56 nmol/L on average (56% in free form) in the tea group; no EGCg was detected before the experiment. Plasma concentrations of lipids, ascorbate, α-tocopherol, and lipid peroxides did not change before and after the experiment in either group, but β-carotene was higher in the tea group ( P< 0.01 by paired Student’s t-test). LDL (0.1 mg/mL) was incubated with 5 μM Cu 2+ and the oxidation was measured by absorbance at 234 nm. The lag time was significantly prolonged by 13.7 min in the tea group ( P < 0.05 by paired Student’s t-test, before versus after), whereas such a change was not observed in the control group. These results suggest that daily consumption of seven to eight cups (approximately 100 mL each cup) of green tea may increase resistance of LDL to in vivo oxidation, leading to reduction in the risk of cardiovascular diseases.

Inhibitory effect of jasmine green tea epicatechin isomers on LDL-oxidation

Oxidative modification of low-density lipoproteins (LDL) is thought to play an important role in the development of atherosclerosis. The present study examined inhibitory effect of jasmine green tea polyphenol (GTP) extracts and its individual epicatechin isomers on Cu +2-mediated oxidation of human LDL. The jasmine tea GTP extracts consisted mainly of four epicatechin isomers with (−) epigallocatechin gallate (EGCG) being most abundant (51.2%) followed by (−) epigallocatechin (EGC, 18.7%), (−) epicatechin (EC, 12.3%), and (−) epicatechin gallate (ECG, 11.8%). Jasmine tea GTP as a mixture or its four epicatechin isomers demonstrated strong antioxidant activity in Cu +2-mediated oxidation of human LDL. The inhibitory effect of these epicatechin isomers on LDL oxidation was dose-dependent at the concentrations ranging from 5 to 40 μM. EC and EGC were less protective against LDL oxidation than their gallate derivatives, ECG and EGCG, respectively. ECG and EGCG seemed to have a similar antioxidant activity. In contrast, EGC was less effective than EC as an antioxidant against LDL oxidation. The inhibitory effect of jasmine green tea epicatechin isomers on Cu +2-mediated oxidation was also characterized by protecting docosahexaenoic, arachidonic, α-linolenic, and linoleic acid in LDL against oxidative degradation. We conclude that, in addition to their hypocholesterolemic effect, jasmine tea epicatechin isomers may serve as a source of natural antioxidative agents in human diet.

Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression.

Green tea polyphenols, major constituents of green tea, are potent chemopreventive agents in a number of experimental models of cancer in animals. The mechanisms of cancer protection by these agents are not clear, but may involve modulation of the enzyme systems responsible for the detoxification of chemical carcinogens. The present studies show that a green tea polyphenol extract (GTP) induces chloramphenicol acetyltransferase (CAT) activity in human heptoma HepG2 cells transfected with a plasmid construct which contains an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene. This indicates that GTP stimulates the transcription of Phase II detoxifying enzymes through the ARE. To explore the upstream signaling pathways leading to gene expression, we studied the involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1). Potent activation of ERK2 was seen following treatment of HepG2 cells with different concentrations of GTP. Similar to ERK2, JNK1 was also strongly activated by treatment with GTP, although to a lesser extent and in a different dose-dependent fashion. Kinetic studies revealed that GTP activation of JNK1 was delayed and sustained, whereas ERK2 activation was rapid and transient. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos, as determined by reverse transcriptase-coupled polymerase chain reaction. Taken together, these studies provide insights into the action of GTP and suggest that the stimulation MAPKs may be the potential signaling pathways utilized by GTP to activate ARE-dependent genes.

Black tea, green tea, and tea polyphenols. Effects on trace element status in weanling rats.

Previous studies have shown that tea consumption can impair trace element metabolism, particularly iron status, and increase the risk of anemia in humans and animals. More recently, however, evidence has been accumulating to show that, in animals, consumption of green tea or its polyphenols is associated with a reduction of the incidence and severity of a variety of experimentally induced cancers. In this study we have monitored the growth, trace element status, including hematological parameters of weanling rats given either (1) water, (2) 1% black tea, (3) 1% green tea, or (4) 0.2% crude green tea extract as their sole drinking fluid while consuming diets containing either adequate or low amounts of iron. With the exception of manganese, none of the trace elements studied (iron, copper, zinc, and manganese) or the hematological indices measured were affected by the type of beverage supplied, even though the polyphenol extract was shown to chelate metals in vitro and all the animals fed the low iron diet were shown to be anemic. There appeared to be an effect of black and green teas on manganese balance in both the first and last weeks of the study. A lower level of brain manganese was associated with green tea consumption, and a higher level of this element in the kidneys of animals fed black tea. The results demonstrate that both black and green teas and a green tea polyphenol extract do not represent a risk to animals consuming the beverages as their sole fluid intake with respect to iron availability, although the interactions with manganese deserve further study.