Modulation of cholesterol metabolism by the green tea polyphenol (-)-epigallocatechin gallate in cultured human liver (HepG2) cells

Epidemiological and animal studies have found that green tea is associated with lower plasma cholesterol. This study aimed to further elucidate how green tea modulates cholesterol metabolism. When HepG2 cells were incubated with the main green tea constituents, the catechins, epigallocatechin gallate (EGCG) was the only catechin to increase LDL receptor binding activity (3-fold) and protein (2.5-fold) above controls. EGCG increased the conversion of sterol regulatory element binding protein-1 (SREBP-1) to its active form (+56%) and lowered the cellular cholesterol concentration (-28%). At 50 microM, EGCG significantly lowered cellular cholesterol synthesis, explaining the reduction in cellular cholesterol. At 200 microM EGCG, cholesterol synthesis was significantly increased even though cellular cholesterol was lower, but there was a significant increase seen in medium cholesterol. This indicates that, at 200 microM, EGCG increases cellular cholesterol efflux. This study provides mechanisms by which green tea modulates cholesterol metabolism and indicates that EGCG might be its active constituent.     

Cellular model for induction of drip loss in meat

Drip loss from porcine muscle (M. longissimus dorsi) contained high concentrations of K(+) ( approximately 135 mM) and organic osmolytes, for example, taurine ( approximately 15 mM), as well as significant amounts of protein ( approximately 125 mg.mL(-1)). Thus, the drip reflects release of intramuscular components. To simulate events taking place at the time of slaughter and leading to release of osmolytes and subsequent formation of drip loss, C2C12 myotubes were exposed to anoxia and reduction in pH (from 7.4 to 6.0). Anoxia and acidification increased the cellular Ca(2+) concentration ([Ca(2+)](i)) at a rate of 22-32 nM.min(-)(1). The anoxia-induced increase in [Ca(2+)](i) was mainly due to influx via sarcolemmal Na(+) channels. As mammalian cells swell and release lysophospholipids during anoxia, C2C12 cells and primary porcine muscle cells were exposed to either hypotonic shock or lysophosphatidylcholine (LPC) and the release of taurine was followed. The swelling-induced taurine efflux was blocked in the presence of the anion channel blocker (DIDS), the 5-lipooxygenase inhibitors (ETH 615-139 and NDGA) but unaffected by the presence of vitamin E. In contrast, the LPC-induced taurine release was unaffected by DIDS but abolished by antioxidants (butylated hydroxytoluene and vitamin E). Thus, stress-induced taurine release from muscles may precede by two different mechanisms, one being 5-lipooxygenase dependent and the other involving generation of reactive oxygen species. A model for the cellular events, preceding formation of drip in meat, is presented.     

New phenolic components and chromatographic profiles of green and fermented teas

A standardized profiling method based on liquid chromatography with diode array and electrospray ionization mass spectrometric detection (LC-DAD-ESI/MS) was applied to establish the phenolic profiles of 41 green teas and 25 fermented teas. More than 96 phenolic compounds were identified that allowed the teas to be organized into five groups. Epigallocatechin gallate (EGCG) was the major phenolic component of green tea made from mature leaves (group 2), while green tea made from the younger buds and leaves (group 1) contained lower flavonoid concentrations. Partially fermented teas (group 3) contained one-half the EGCG content of the green tea. Fully fermented black teas (group 4) had a trace of EGCG, but contained theaflavins. Highly overfermented black tea (group 5) contained only trace amounts of flavonol glycosides and theaflavins. Over 30 phenolics are new for tea, and this is the first phenolic profile to simultaneously detect C- and O-glycosylated flavonoids, catechins, proanthocyanidins, phenolic acid derivatives, and purine alkaloids.     

Epicatechin-3-O-(3″-O-methyl)-gallate content in various tea cultivars (Camellia sinensis L.) and its in vitro inhibitory effect on histamine release

It has been reported that epigallocatechin-3-O-(3″-O-methyl)-gallate (EGCG3″Me) and the EGCG3″Me-rich green tea ( Camellia sinensis L.) cultivar 'Benifuuki' exhibit antiallergic effects. The objective of this study was to investigate the effect of various tea leaf catechins on histamine release from murine bone marrow mast cells (BMMC). At a dose of 50 μg/mL, the rank order of histamine release inhibition was observed to be epicatechin-3-O-(3″-O-methyl)-gallate (ECG3″Me) > gallocatechin-3-O-(3″-O-methyl)-gallate (GCG3″Me) > EGCG3″Me > gallocatechin-gallate (GCG) > catechin-gallate (CG) > EGCG > epicatechin-gallate (ECG) > epigallocatechin (EGC) > gallocatechin (GC). Of the various tea cultivars analyzed by HPLC, the greatest content of ECG3″Me was found in the third crop of 'Benifuuki' (1.05% dry weight). Moreover, ECG3″Me content was positively correlated with EGCG3″Me content in 'Benifuuki' tea leaves. In an assay of mixtures of ECG3″Me and EGCG3″Me, inhibitory activity (50 μg/mL in total) was increased as the content of ECG3″Me increased. This suggests that ECG3″Me might link to the antiallergic effect of 'Benifuuki' tea, as has been reported for EGCG3″Me.     

Lipophilized epigallocatechin gallate (EGCG) derivatives as novel antioxidants

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea and known to render many health benefits associated with tea consumption. EGCG was modified structurally to improve its lipophilicity, expand its application in lipophilic media, and enhance its cellular absorption in vivo. Esterification of the water-soluble EGCG with selected long-chain saturated and unsaturated fatty acids was carried out, followed by a purification process. Ester derivatives of EGCG with stearic acid (SA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were prepared, and their enhanced lipophilicity was confirmed by octanol-water partition coefficient. The chemical structures of the EGCG derivatives, determined by HPLC-MS and 1H and 13C NMR, were EGCG-3',5',3',5'-O-tetraesters of SA, EPA, and DHA. The lipophilized EGCG derivatives exhibited greater antioxidant activity in scavenging the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical than EGCG itself. The results suggest that EGCG derivatives may be used as potential lipophilic antioxidants in the food, cosmetic, and medicinal industries.     

Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate

The inhibitory effects of five tea polyphenols, namely theaflavin (TF1), theaflavin-3-gallate (TF2), theaflavin-3,3'-digallate (TF3), (-)-epigallocatechin-3-gallate (EGCG), and gallic acid, and propyl gallate (PG) on xanthine oxidase (XO) were investigated. These six antioxidant compounds reduce oxidative stress. Theaflavins and EGCG inhibit XO to produce uric acid and also act as scanvengers of superoxide. TF3 acts as a competitive inhibitor and is the most potent inhibitor of XO among these compounds. Tea polyphenols and PG all have potent inhibitory effects (>50%) on PMA-stimulated superoxide production at 20 approximately 50 microM in HL-60 cells. Gallic acid (GA) showed no inhibition under the same conditions. At 10 microM, only EGCG, TF3, and PG showed significant inhibition with potency of PG > EGCG > TF3. The superoxide scavenging abilities of these six compunds are as follows: EGCG > TF2 > TF1 > GA > TF3 > PG. PG was the most potent inhibitor of PMA-stimulated H(2)O(2) production in HL-60 cells. The order of H(2)O(2) scavenging ability was TF2 > TF3 > TF1 > EGCG > PG > GA. Therefore, the antioxidative activity of tea polyphenols and PG is due not only to their ability to scavenge superoxides but also to their ability to block XO and related oxidative signal transducers.     

Correlation analyses of phytochemical composition, chemical, and cellular measures of antioxidant activity of broccoli (Brassica oleracea L. Var. italica)

Chemical measures of antioxidant activity within the plant, such as the oxygen radical absorbance capacity (ORAC) assay, have been reported for many plant-based foods. However, the extent to which chemical measures relate to cellular measures of oxidative stress is unclear. The natural variation in the phytochemical content of 22 broccoli genotypes was used to determine correlations among chemical composition (carotenoids, tocopherols and polyphenolics), chemical antioxidant activity (ORAC), and measures of cellular antioxidation [prevention of DNA oxidative damage and of oxidation of the biomarker dichlorofluorescein (DCFH) in HepG2 cells] using hydrophilic and lipophilic extracts of broccoli. For lipophilic extracts, ORAC (ORAC-L) correlated with inhibition of cellular oxidation of DCFH (DCFH-L, r = 0.596, p = 0.006). Also, DNA damage in the presence of the lipophilic extract was negatively correlated with both chemical and cellular measures of antioxidant activity as measured by ORAC-L (r = -0.705, p = 0.015) and DCFH-L (r = -0.671, p = 0.048), respectively. However, no correlations were observed for hydrophilic (-H) extracts, except between polyphenol content and ORAC (ORAC-H; r = 0.778, p < 0.001). Inhibition of cellular oxidation by hydrophilic extracts (DCFH-H) and ORAC-H were approximately 8- and 4-fold greater than DCFH-L and ORAC-L, respectively. Whether ORAC-H has more biological relevance than ORAC-L because of its magnitude or whether ORAC-L bears more biological relevance because it relates to cellular estimates of antioxidant activity remains to be determined. Chemical estimates of antioxidant capacity within the plant may not accurately reflect the complex nature of the full antioxidant activity of broccoli extracts within cells.     

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.     

Sensitive determination of (-)-epigallocatechin gallate in tea infusion using a novel ionic liquid carbon paste electrode

This paper investigates the electrocatalytic oxidation of (-)-epigallocatechin gallate (EGCG), the main monomer flavanol found in green tea, with a novel ionic liquid, n-octylpyridinium hexafluorophosphate (OPFP) carbon paste electrode (CPE). Due to the natural viscosity and high conductivity of OPFP, this novel OPFP-CPE exhibited very attractive properties, such as high stability and electrochemical reactivity, low background current, and wide electrochemical window. Therefore, this electrode is a very good alternative to traditional chemically modified electrodes because the electrocatalytic effect can achieved without any further electrode modification. Comparative experiments were carried out using CPE and a glassy carbon electrode (GCE). With OPFP-CPE, highly reproducible and well-defined cyclic voltammograms were obtained for EGCG. Under optimal experimental conditions, the peak current of differential pulse voltammetry (DPV) response increased linearly with EGCG concentration over the range of 5.0 × 10(-7)-1.25 × 10(-5) M. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.32 × 10(-7) and 4.35 × 10(-7) M, respectively. The method was applied to the determination of EGCG in green tea infusion samples, and the recovery of the spiked EGCG to the diluted (10-fold) tea extract was from 87.62 to 99.51%.     

Stability of tea polyphenol (-)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active compound in tea, has been extensively studied for its activities related to disease prevention in animal models and in vitro. However, its stability under different experimental conditions has not been well-characterized. In the present study, the stability of EGCG in animal drinking fluid and under cell culture conditions and the factors that affect its stability under these conditions were investigated. Our results demonstrated that auto-oxidation and epimerization are the two major reactions causing the instability of EGCG. The structures of the major oxidation products, EGCG dimers, were identified. The rates of these reactions were affected by the temperature, pH, the partial pressure of oxygen, the level of antioxidants, the concentration of EGCG, and other components of tea. In future studies with EGCG, its stability should be considered in order to avoid possible artifacts.     

Determination of peroxyl radical scavenging activity of flavonoids and plant extracts using an automatic potentiometric titrator

A novel potentiometric method for evaluation of peroxyl radical scavenging activity of flavonoids and plant extracts was developed. The oxidation of potassium iodide (KI) was performed in acetonitrilephosphate buffer (1:1) containing antioxidant using 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator. The amount of iodine released from KI during a 20-min free radical oxidation was determined quantitatively using an automatic potentiometric titrator with sodium thiosulfate. The radical scavenging activity of the sample was expressed as the inhibition ratio for iodine release of the control group mediated by the radical. The results obtained from some authentic polyphenols correlated well with those of previous reports. This is a simple, time-saving method requiring less than 30 min and is useful in assessing the radical scavenging activity of antioxidants in plant extracts. We describe the radical scavenging activities of various flavonoids including 21 kinds of tea catechins and vegetable extracts by this method.     

Effect of antioxidants on oxidation during the production of whey fat concentrate

Whey fat has a relatively high level of unsaturated fatty acids, and as such, whey products with a high fat content are vulnerable to oxidation. The purposes of the present study were to assess the oxidative development in whey fat concentrate (WFC) during production and investigate the effect of the addition of antioxidants. Green tea extract (GTE) or a mixture of ascorbyl palmitate and tocopherol (AP/TOC) were used, each in two concentrations. Samples were taken before and after pasteurization of WFC and after drying. The level of volatile oxidation products decreased during processing, while dityrosine concentrations increased during drying. GTE reduced oxidation in both unpasteurized and pasteurized WFC, while the effect of AP/TOC was nonsignificant. In the WFC powder, there was no significant effect of the antioxidants. In conclusion, results indicated that GTE was able to inhibit oxidation in WFC during production and that AP/TOC addition had no effect.     

Epigallocatechin-3 gallate inhibits invasion, epithelial-mesenchymal transition, and tumor growth in oral cancer cells

Epithelial to mesenchymal transition (EMT) is critical for the progression, invasion, and metastasis of epithelial tumorgenesis. Here, we provided molecular evidence associated with the antimetastatic effect of green tea polyphenol epigallocatechin-3 gallate (EGCG) in an oral squamous cell culture system by showing a nearly complete inhibition on the invasion (P < 0.001) of squamous cell carcinoma-9 (SCC-9) cells via a reduced expression of matrix metalloproteinase-2 (P < 0.001) and urokinasetype plasminogen activator (P < 0.001). EGCG exerted an inhibitory effect on cell migration (P < 0.001), motility (P < 0.001), spread, and adhesion (P < 0.001). We performed Western blot to find that EGCG inhibited p-focal adhesion kinase (p-FAK), p-Src, snail-1, and vimentin, indicating the anti-EMT effect of EGCG in oral squamous cell carcinoma. EGCG was also sufficient to inhibit phorbol-12-myristate-13-acetate-induced cell invasion and matrix metalloproteinase-9 expression, as evidenced by its inhibition on the tumor growth of SCC-9 cells in vivo via cancer cell xenografted nude mice mode. These results suggested that EGCG could reduce the invasion and cell growth of tumor cells, and such a characteristic may be of great value in developing a potential cancer therapy.     

Polyphenol content of plasma and litter after the oral administration of green tea and tea polyphenols in chickens

Metabolic profiles of broiler chickens were examined after the ingestion of green tea, tea polyphenols, and (-)-epigallocatechin-3-gallate (EGCG). Solid-phase extraction of serum and litters yielded free catechins and their metabolites, which were then identified and quantified by liquid chromatography-tandem mass spectrometry. In plasma samples, (-)-gallocatechin, (+)-catechin, and EGCG were detected in the green tea group; pyrogallol acid, (epi)catechin-O-sulfate, 4'-O-methyl-(epi)gallocatechin-O-glucuronide, and (epi)catechin-3'-O-glucuronide were detected in the tea polyphenols group; and EGCG, (-)-gallocatechin gallate (GCG), and 4'-O-methyl-(epi)gallocatechin-O-glucuronides were detected in the EGCG group. In litters, gallic acid, EGCG, GCG, and ECG were detected in the green tea and tea polyphenols groups; EGCG and ECG were detected in the EGCG group. The conjugated metabolites, 4'-O-methyl-(epi)gallocatechin-O-glucuronide, (epi)catechin-3'-glucuronide, and 4'-O-methyl-(epi)catechin-O-sulfate, were identified in the green tea group; 4'-O-methyl-(epi)catechin-O-sulfate and 4'-O-methyl-(epi)gallocatechin-O-sulfate were identified in the tea polyphenols group; only 4'-O-methyl-(epi)gallocatechin-O-sulfate was detected in the EGCG group. The excretion of tea catechins was 95.8, 87.7, and 97.7% for the green tea, tea polyphenols, and EGCG groups, respectively.     

Interaction of tea polyphenols and food constituents with model gut epithelia: the protective role of the mucus gel layer

The luminal surface of the gastrointestinal tract is covered by a mucus gel layer that acts to protect gut epithelial cells from the harsh luminal environment. This study investigated the use of two human colonic adenocarcinoma cell lines, HT29-MTX-E12 and HT29, as a model to mimic gut epithelium with and without a mucus gel layer. The effect of adding the tea polyphenols epigallocatechin gallate (EGCG) and epicatechin (EC) to the cells with subsequent examination of cell morphology and viability was assessed. EGCG, at the concentrations tested, was very toxic to the HT29 cells, but less toxic to the HT29-MTX-E12 cells, suggesting that the mucus gel layer on the HT29-MTX-E12 cells can protect the cells against EGCG toxicity. In contrast, EC had no effect on the viability of either the HT29 or HT29-MTX-E12 cells, suggesting that proteins within the mucus gel layer on the apical surface of gut epithelial cells may bind to the galloyl ring of EGCG. The effect of adding food-related ingredients with the ability to complex with EGCG, β-casein and maltodextrin, on cell viability was also examined. The presence of β-casein was very effective in protecting the cells against the toxicity effect of EGCG, but maltodextrin, at the concentration tested, was less effective in protecting against this toxicity. In conclusion, the results demonstrate that the mucus gel layer on HT29 human colonic adenocarcinoma cells may protect these cells against EGCG toxicity. In addition, the data showing reduced toxicity of EC compared to that of EGCG suggest that the cytotoxic effects of high polyphenol levels may be associated with the ability of polyphenols to interact with cellular proteins and mucins.     

Factors influencing the antioxidant and pro-oxidant activity of polyphenols in oil-in-water emulsions

The nonenzymatic oxidation of polyphenols bearing di- and trihydroxyphenol groups results in the generation of hydrogen peroxide (H?O?), a reactive oxygen species that can potentially compromise the oxidative stability of foods and beverages. An investigation of the factors that promote the oxidation of a model polyphenol, (-)-epigallocatechin-3-gallate (EGCG), was undertaken in a model lipid-based food system. Factors affecting oxidative stability, such as exogenous iron chelators (ethylenediaminetetraacetic acid; EDTA and 2,2-bipyridine; BPY) and pH (3 and 7) were evaluated in hexadecane and flaxseed oil-in-water (o/w) emulsions. At neutral pH, H?O? levels were observed to rise rapidly in hexadecane emulsions except for EDTA-containing treatments. However, EDTA-containing samples showed the highest rate of EGCG oxidation, suggesting that H?O? was rapidly reduced to hydroxyl radicals (HO?). Conversely, at pH 3, H?O? concentrations were lower across all treatments. EDTA conferred the highest degree of EGCG stability, with no loss of the catechin over the course of the study. In order to assess whether or not the H?O? production seen in oxidatively stable hexadecane emulsions translated to pro-oxidant activity in an oxidatively labile food lipid system, the effect of EGCG on the stability of flaxseed o/w emulsions was studied. EGCG displayed antioxidant activity at pH 7 throughout the study; however at pH 3, pro-oxidant activity was seen in EGCG-containing emulsions, with and without BPY. This study attempts to provide a mechanistic understanding of the conditions wherein polyphenols simultaneously exert pro-oxidant and antioxidant behavior in lipid dispersions.     

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.     

基于高光谱成像的茶叶中EGCG分布可视化

针对目前关于表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)在茶叶中的分布缺乏可视化表达问题,该文采用高光谱成像技术以实现EGCG在茶叶中的分布可视化。通过高光谱成像仪采集茶叶的光谱信息,按照标准方法 HPLC(high performance liquid chromatography)法测量茶叶的EGCG浓度。运用化学计量学方法建立光谱与EGCG浓度之间的回归模型。为寻求相对较优的模型效果,对光谱进行不同的预处理,以确定最优的预处理方法;采用4种建模方法建立回归模型,以确定最优的建模方法;对光谱进行特征波段选择,以降低数据冗余提高模型的稳定性和运算速度。最后,将高光谱图像中像素点对应的光谱变量导入模型,从而生成EGCG浓度分布图。结果表明:可见-近红外光谱与EGCG浓度之间具有很强的相关性,其回归模型的决定系数达到0.905,利用高光谱成像技术对茶叶中EGCG分布进行可视化可行。通过对不同品种、叶位的茶叶中EGCG浓度分布进行可视化,能够为高EGCG浓度茶树品种的培育、EGCG代谢规律的分析以及茶树采摘部位的识别提供有效手段。     

Inhibitory effects of (-)-epigallocatechin gallate on the mutation,DNA strand cleavage,and DNA adduct formation by heterocyclic amines

Green tea is known to be a potential chemopreventive agent against cancer. In this study, we investigated the inhibitory activities of tea extracts, and in particular the polyphenolic component (-)-epigallocatechin gallate (EGCG), against heterocyclic amine-induced genotoxicity. The tea extracts displayed inhibition of 2-hydroxyamino-6-methyldipyrido[1,2-a,3',2'-d]imidazole (Glu-P-1(NHOH))-induced mutagenicity. This inhibition can be accounted for by the presence of EGCG in the extracts. The mutagenic effect of Glu-P-1(NHOH), which induces single-strand cleavage in supercoiled circular DNA under neutral conditions, was inhibited by EGCG. Using the Drosophila repair test, a test for gross DNA damage, and DNA adduct detection by (32)P-postlabeling, we showed that EGCG prevented 2-amino-3,8-dimethylimidazo[4,5-f]quinoline-induced DNA damage and adduct formation in insect DNA. EGCG was found to accelerate the degradation of Glu-P-1(NHOH) in vitro. This observation suggested that the inhibition by EGCG is associated with an accelerated degradation of metabolically activated heterocyclic amines.