Effects of external factors on the interaction of tea catechins with lipid bilayers

Green tea contains a high concentration of such catechins as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.     

Determination of green tea catechins in human plasma using liquid chromatography-electrospray ionization mass spectrometry

A method for the sensitive and specific determination of eight green tea catechins, consisting of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin-3-gallate (CG), epicatechin-3-gallate (ECG), gallocatechin-3-gallate (GCG) and epigallocatechin-3-gallate (EGCG), in human plasma was established. For optimization of conditions for LC-ESIMS, the separation of the eight catechins was achieved chromatographically using Inertsil ODS-2 column combined with a gradient elution system of 0.1M aqueous acetic acid and 0.1M acetic acid in acetonitrile. Detection using a mass spectrometer was performed with selected ion monitoring at m/z=289 for E and EC, 305 for GC and EGC, 441 for CG and ECG, and 457 for GCG and EGCG under negative ESI. A preparative procedure, consisting of the addition of perchloric acid and acetonitrile to the plasma for deproteinizing and the subsequent addition of potassium carbonate solution to remove excess acid, was developed. In six different plasma with the eight catechins spiked at two different concentrations, the average recoveries were in the range between 72.7 and 84.1%, which resulted from the matrix effect and preparative loss, with coefficients of variance being 8.2-19.8% among individuals. The levels of the catechins in prepared plasma solutions that were kept at 5 degrees C within 24h were stable, which allows us to simply analyze many prepared plasma solutions using an autosampler overnight. When using this method to analyze the eight catechins in human plasma after oral ingestion of a commercial green tea beverage, we detected all the catechins absorbed into human blood for the first time. This also suggested that extremely small amounts of the eight catechins orally ingested may be absorbed based on each absorptive property for the catechins. The method should enable pharmacokinetic studies of green tea catechins in humans.     

Effect of green tea powder (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L. cv. Benifuuki) particle size on <Emphasis Type="Italic">O</Emphasis>-methylated EGCG absorption in rats; The Kakegawa Study

Tea polyphenols, e.g., (-)-epigallocatechin-3-O-(3-O-methyl gallate (EGCG3”Me), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate (ECG), and (-)-epicatechin (EC), are believed to be responsible for the beneficial effects of tea. ‘Benifuuki’, a tea (Camellia sinensis L.) cultivar grown in Japan, is rich in the anti-allergic molecule epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me). Pulverized Benifuuki green tea powder (BGP) is more widely distributed than leaf tea in Japan. Japanese people mix their pulverized tea with water directly, whereas it is common to drink leaf tea after extraction. However, few studies of the effects of BGP particle size on polyphenol bioavailability have been performed. This study was conducted to investigate the absorption of catechins in rats after the intragastric administration of Benifuuki green tea. Therefore, we assessed the plasma concentrations of catechins following the ingestion of BGP with different mean particle sizes (2.86, 18.6, and 76.1 μm) or Benifuuki green tea infusion (BGI) as a control in rats. The bioavailabilities of EGCG3”Me, EGCG, ECG, EGC, and EC were analyzed after the oral administration of a single dose of Benifuuki green tea (125 mg/rat) to rats. The plasma concentrations of tea catechins were determined by HPLC analysis combined with of electrochemical detection (ECD) using a coulometric array. The AUC (area under the drug concentration versus time curve; min μg/mL) of ester-type catechins (EGCG3”Me, EGCG, and ECG) for the BGP 2.86 μm were significantly higher than those in the infusion and 18.6 and 76.1 μm BGP groups, but the AUC of free-type catechins (EGC and EC) showed no differences between these groups. Regarding the peak plasma level of EGCG3”Me adjusted for intake, BGP 2.86 μm and BGI showed higher values than the BGP 18.6 and 76.1 μm groups, and the peak plasma levels of the other catechins displayed the same tendency. The present study demonstrates that the bioavailability of ester-type catechins (EGCG and ECG) can be improved by reducing the particle size of green tea, but the plasma level of EGCG3”Me in the BGI group was similar to that in the BGP 2.86 μm group. This result suggests that drinking Benifuuki green tea with a particle size of around 2 μm would deliver the anti-allergic EGCG3”Me and the anti-oxidant EGCG efficiently.     

The impact of the 67kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins

Here, we investigated the structure-activity relationship of major green tea catechins and their corresponding epimers on cell-surface binding and inhibitory effect on histamine release. Galloylated catechins; (−)-epigallocatechin-3-O-gallate (EGCG), (−)-gallocatechin-3-O-gallate (GCG), (−)-epicatechin-3-O-gallate (ECG), and (−)-catechin-3-O-gallate (CG) showed the cell-surface binding to the human basophilic KU812 cells by surface plasmon resonance analysis, but their non-galloylated forms did not. Binding activities of pyrogallol-type catechins (EGCG and GCG) were higher than those of catechol-type catechins (ECG and CG). These patterns were also observed in their inhibitory effects on histamine release. Previously, we have reported that biological activities of EGCG are mediated through the binding to the cell-surface 67 kDa laminin receptor (67LR). Downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. These results suggest that both the galloyl moiety and the B-ring hydroxylation pattern contribute to the exertion of biological activities of tea catechins and their 67LR-dependencies.     

Measurement of catechin and gallic acid in tea wine with HPLC

First a kind of fermented tea wine was prepared from Dancong tea. The content of four major catechins and gallic acid (EC, EGC, EGCG and ECG) in tea wine was measured with HPLC. The results showed that the content of EC, EGC, EGCG, ECG and catechins in tea wine decreased when compared with that before fermentation. The content of EC decreased the most, reaching 26.79%, while the content of GA changed the least with a decrease of only 13.56%. Nevertheless, tea wine still contains a relatively large amount of catechins, thus proper consumption of tea wine may be salubrious.     

Inhibition of tyrosinase by green tea components

The pigment melanin in human skin is a major defense mechanism against ultraviolet light of the sun, but darkened skin color, which is the result of increased and redistributed epidermal melanin, could be a serious aesthetic problem. Epidemiologically, it is well known that the consumption of green tea may help prevent cancers in humans and also reduce several free radicals including peroxynitrite. In the present study, to assess the efficacy of the inhibition of mushroom tyrosinase (monophenol monooxygenase EC 1.14.18.1), ten kinds of Korean traditional teas were screened for their tyrosinase inhibitory activity. Green tea was the strongest inhibitor, and the major active constituents in the tea are (-)-epicatechin 3-O-gallate (ECG), (-)-gallocatechin 3-O-gallate (GCG), and (-)-epigallocatechin 3-O-gallate (EGCG). All are catechins with gallic acid group as an active site. The kinetic analysis for inhibition of tyrosinase revealed a competitive nature of GCG with this enzyme for the L-tyrosine binding at the active site of tyrosinase.     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

Effects of black tea consumption on plasma catechins and markers of oxidative stress and inflammation in patients with coronary artery disease

We previously demonstrated that black tea consumption reverses endothelial dysfunction in patients with coronary artery disease. To investigate potential mechanisms of this effect, we examined plasma catechins and systemic markers of oxidation, inflammation, and antioxidant protection from 66 subjects enrolled in that study. We collected samples at baseline, 2 h after 450 ml of black tea (acute), after 4 weeks of 900 ml of black tea per day (chronic), and after acute and chronic consumption of water. Total catechins increased 33% after acute tea (P < 0.05) and 29% after chronic tea (P < 0.05). Of individual catechins, plasma epicatechin gallate (ECG) concentration significantly increased with acute tea consumption, and plasma epicatechin (EC) increased with chronic tea consumption. Tea consumption did not improve plasma antioxidant capacity and did not reduce urinary 8-hydroxy-2'-deoxyguanosine, or urinary 8-isoprostane levels. Changes in catechin levels did not correlate with changes in endothelial function, plasma markers of oxidative stress, or C-reactive protein. In contrast, endothelial function at baseline correlated with dietary flavonoid intake (beta = 0.32, P = 0.02) and with baseline plasma EC concentration after adjusting for confounding variables (beta = 0.39, P = 0.03). These findings suggest that the benefits of black tea consumption on endothelial function may not be attributable to tea catechins or a systemic antioxidant or anti-inflammatory effect. Chronic dietary flavonoid status appears to relate to endothelial function, possibly suggesting that other flavonoids or polyphenolic components of tea favorably influence vascular health and risk for cardiovascular disease.     

Green tea catechins can bind and modify ERp57/PDIA3 activity

Background

Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model.

Methods

The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated.

Results

A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity.

Conclusions

Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected.

General significance

This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.     

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols

Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 microg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CH(R)C5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CH(R)C5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.     

Antimicrobial Activities of Tocklai Vegetative Tea Clones

Thirty-one Tocklai vegetative (TV) tea clones contained caffeine and total catechin 44.39 and 227.55 mg/g dry weight of leaves, respectively. The (−)-epigallocatechin gallate (EGCG) was the most abundant (109.60 mg/g) followed by -(−)-epigallocatechin (EGC, 44.54 mg/g), (−)-epicatechin gallate (ECG, 41.74 mg/g), (−)-epicatechin (EC, 27.42 mg/g) and +catechin (4.25 mg/g). Total catechins were highest in TV 20 (509.7 mg/g) and lowest in TV 6 (71.7 mg/g). The tea clones that contain high level of total catechin exhibited the strongest antimicrobial activity. Among caffeine and flavanol compounds, theaflavins (TF) present in black tea possess a similar antimicrobial potency as EC present in fresh leaves, and that the conversion of catechins to TF during fermentation in making black tea tends to alter their antimicrobial activities. The bioactive molecules other than catechins present in tea leaves may also contribute towards antimicrobial activity.     

Evidence for alpha-tocopherol regeneration reaction of green tea polyphenols in SDS micelles

The synergistic antioxidant mechanism of alpha-tocopherol (vitamin E) with green tea polyphenols, i.e., (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA), was studied by assaying the kinetics of the reaction of alpha-tocopheroxyl radical with green tea polyphenols by stopped-flow electron paramagnetic resonance, the inhibition of linoleic acid peroxidation by these antioxidants, and the decay of alpha-tocopherol during the peroxidation. It was found that the green tea polyphenols could reduce alpha-tocopheroxyl radical to regenerate alpha-tocopherol with rate constants of 0.45, 1.11, 1.31, 1.91, and 0.43 x 10(2) M(-1) s(-1) for EC, EGC, ECG, EGCG, and GA, respectively, in sodium dodecyl sulfate micelles. In addition, these second-order rate constants exhibited a good linear correlation with their oxidation potentials, suggesting that electron transfer might play a role in the reaction.     

Green tea catechins: a fresh flavor to anticancer therapy

Green tea catechins have been extensively studied for their cancer preventive effects. Accumulating evidence has shown that green tea catechins, like (?)-epigallocatechin-3-gallate, have strong anti-oxidant activity and affect several signal transduction pathways relevant to cancer development. Here, we review the biological properties of green tea catechins and the molecular mechanisms of their anticancer effects, including the suppression of cancer cell proliferation, induction of apoptosis, and inhibition of tumor metastasis and angiogenesis. We summarize the efficacy of a single catechin and the synergetic effects of multiple catechins. We also discuss the enhanced anticancer effects of green tea catechins when they are combined with anticancer drugs. The information present in this review might promote the development of strategy for the co-administration of green tea catechins with other anticancer drugs to increase the potency of currently available anticancer medicine. This new strategy should in turn lower the cytotoxicity and cost of anticancer treatment.     

Combined effect of epigallocatechin gallate and triclosan on enoyl-ACP reductase of Mycobacterium tuberculosis

Among the various inhibitors known for enoyl-acyl carrier protein (ACP) reductases, triclosan and green tea catechins are two promising candidates. In the present study, we show, for the first time that epigallocatechin gallate (EGCG), a major component of green tea catechins, inhibits InhA, the enoyl-ACP reductase of Mycobacterium tuberculosis with an IC50 of 17.4 μM. EGCG interferes with the binding of NADH to InhA. We also demonstrate that EGCG increased the inhibitory activity of triclosan towards InhA and vice versa. Direct binding assay using [³H]EGCG and fluorescence titration assay support the spectrophotometric/kinetic inhibition data. The biochemical data has been explained by docking simulation studies.     

茶多酚及其儿茶素单体对过氧化氢诱导的线粒体通透性改变孔道开放的影响

线粒体是细胞内重要的细胞器,是生成ATP的主要场所.线粒体通透性改变孔道（PT孔道）的开放会引起线粒体许多功能的紊乱而导致细胞死亡.对茶多酚及其单体儿茶素对过氧化氢诱导的线粒体膨胀及膜电势变化过程中PT孔开放的影响进行了研究.实验结果表明茶多酚及其儿茶素单体对PT孔开放的影响显著不同：茶多酚及其主要成分EGCG和ECG能够有效地抑制PT孔道的开放;而ECG,（+）-C和EGC却加速PT孔道的开放过程.从总体效果来看,茶多酚及其单体EGCG和ECG对线粒体的保护作用占主导地位.     

Green tea as inhibitor of the intestinal absorption of lipids: potential mechanism for its lipid-lowering effect

Animal and epidemiological studies suggest that green tea catechins may reduce the risk of cardiovascular diseases [e.g., coronary heart disease (CHD)]. The health benefit of green tea has been attributed to its antioxidant and anti-inflammatory properties; however, considerable evidence suggests that green tea and its catechins may reduce the risk of CHD by lowering the plasma levels of cholesterol and triglyceride. Although the mechanism underlying such effect of green tea is yet to be determined, it is evident from in vitro and in vivo studies that green tea or catechins inhibit the intestinal absorption of dietary lipids. Studies in vitro indicate that green tea catechins, particularly (-)-epigallocatechin gallate, interfere with the emulsification, digestion, and micellar solubilization of lipids, critical steps involved in the intestinal absorption of dietary fat, cholesterol, and other lipids. Based on the observations, it is likely that green tea or its catechins lower the absorption and tissue accumulation of other lipophilic organic compounds. The available information strongly suggests that green tea or its catechins may be used as safe and effective lipid-lowering therapeutic agents.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

Metabolism of green tea catechins by the human small intestine

Numerous studies have shown that green tea polyphenols can be degraded in the colon, and there is abundant knowledge about the metabolites of these substances that appear in urine and plasma after green tea ingestion. However, there is very little information on the extent and nature of intestinal degradation of green tea catechins in humans. Therefore, the aim of this study was to examine in detail the microbial metabolism and chemical stability of these polyphenols in the small intestine using a well-established ex vivo model. For this purpose, fresh ileostomy fluids from two probands were incubated for 24 h under anaerobic conditions with (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin 3-O-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatchin 3-O-gallate (EGCG) and gallic acid (GA). After lyophilisation and extraction, metabolites were separated, identified and quantified by high performance liquid chromatography-photodiode array detection (HPLC-DAD) and HPLC-ESI-tandem mass spectrometry. Two metabolites of EC and C (3', 4', 5'-trihydroxyphenyl-γ-valerolactone and 3', 4'-dihydroxyphenyl-γ-valerolactone) were identified. In addition, 3', 4', 5'-trihydroxyphenyl-γ-valerolactone was detected as a metabolite of EGC, and (after 24-h incubation) pyrogallol as a degradation product of GA. Cleavage of the GA esters of EGCG and ECG was also observed, with variations dependent on the sources (probands) of the ileal fluids, which differed substantially microbiotically. The results provide new information about the degradation of green tea catechins in the gastrointestinal tract, notably that microbiota-dependent liberation of GA esters may occur before these compounds reach the colon.     

The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention

Green tea (Camellia sinensis) is rich in catechins, of which (−)-epigallocatechin-3-gallate (EGCG) is the most abundant. Studies in animal models of carcinogenesis have shown that green tea and EGCG can inhibit tumorigenesis during the initiation, promotion and progression stages. Many potential mechanisms have been proposed including both antioxidant and pro-oxidant effects, but questions remain regarding the relevance of these mechanisms to cancer prevention. In the present review, we will discuss the redox chemistry of the tea catechins and the current literature on the antioxidant and pro-oxidative effects of the green tea polyphenols as they relate to cancer prevention. We report that although the catechins are chemical antioxidants which can quench free radical species and chelate transition metals, there is evidence that some of the effects of these compounds may be related to induction of oxidative stress. Such pro-oxidant effects appear to be responsible for the induction of apoptosis in tumor cells. These pro-oxidant effects may also induce endogenous antioxidant systems in normal tissues that offer protection against carcinogenic insult. This review is meant point out understudied areas and stimulate research on the topic with the hope that insights into the mechanisms of cancer preventive activity of tea polyphenols will result.