Catechin degradation with concurrent formation of homo- and heterocatechin dimers during in vitro digestion

Catechins were subjected to in vitro gastric and small intestinal digestion. EGCG, EGC, and ECG were significantly degraded at all concentrations tested, with losses of 71-91, 72-100, and 60-61%, respectively. EC and C were comparatively stable, with losses of 8-11 and 7-8%, respectively. HLPC-ESI-MS/MS indicated that EGCG degradation under simulated digestion resulted in production of theasinensins (THSNs) A and D (m/z 913) and P-2 (m/z 883), its autoxidation homodimers. EGC dimerization produced the homodimers THSN C and E (m/z 609) and homodimers analogous to P-2 (m/z 579). ECG homodimers were not observed. EGCG and EGC formed heterodimers analogous to the THSNs (m/z 761) and P-2 (m/z 731). EGCG and ECG formed homodimers analogous to the THSNs (m/z 897). This study provides an expanded profile of catechin dimers of digestive origin that may potentially form following consumption of catechins. These data provide a logical basis for initial screening to detect catechin digestive products in vivo.     

Tea polyphenol (-)-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascade signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.     

Bitter gourd suppresses lipopolysaccharide-induced inflammatory responses

Bitter gourd ( Momordica charantia L.) is a popular tropical vegetable in Asian countries. Previously it was shown that bitter gourd placenta extract suppressed lipopolysaccharide (LPS)-induced TNFalpha production in RAW 264.7 macrophage-like cells. Here it is shown that the butanol-soluble fraction of bitter gourd placenta extract strongly suppresses LPS-induced TNFalpha production in RAW 264.7 cells. Gene expression analysis using a fibrous DNA microarray showed that the bitter gourd butanol fraction suppressed expression of various LPS-induced inflammatory genes, such as those for TNF, IL1alpha, IL1beta, G1p2, and Ccl5. The butanol fraction significantly suppressed NFkappaB DNA binding activity and phosphorylation of p38, JNK, and ERK MAPKs. Components in the active fraction from bitter gourd were identified as 1-alpha-linolenoyl-lysophosphatidylcholine (LPC), 2-alpha-linolenoyl-LPC, 1-lynoleoyl-LPC, and 2-linoleoyl-LPC. Purified 1-alpha-linolenoyl-LPC and 1-linoleoyl-LPC suppressed the LPS-induced TNFalpha production of RAW 264.7 cells at a concentration of 10 microg/mL.     

Catechin and caffeine content of green tea dietary supplements and correlation with antioxidant capacity

The health benefits associated with tea consumption have resulted in the wide inclusion of green tea extracts in botanical dietary supplements, which are widely consumed as adjuvants for complementary and alternative medicines. Tea contains polyphenols such as catechins or flavan-3-ols including epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate (EGCG), as well as the alkaloid, caffeine. Polyphenols are antioxidants, and EGCG, due to its high levels, is widely accepted as the major antioxidant in green tea. Therefore, commercial green tea dietary supplements (GTDS) may be chemically standardized to EGCG levels and/or biologically standardized to antioxidant capacity. However, label claims on GTDS may not correlate with actual phytochemical content or antioxidant capacity nor provide information about the presence and levels of caffeine. In the current study, 19 commonly available GTDS were evaluated for catechin and caffeine content (using high-performance liquid chromatography) and for antioxidative activity [using trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays]. Product labels varied in the information provided and were inconsistent with actual phytochemical contents. Only seven of the GTDS studied made label claims of caffeine content, 11 made claims of EGCG content, and five specified total polyphenol content. Caffeine, EGCG, and total polyphenol contents in the GTDS varied from 28 to 183, 12-143, and 14-36% tablet or capsule weight, respectively. TEAC and ORAC values for GTDS ranged from 187 to 15340 and from 166 to 13690 mumol Trolox/g for tablet or capsule, respectively. The antioxidant activities for GTDS determined by TEAC and ORAC were well-correlated with each other and with the total polyphenol content. Reliable labeling information and standardized manufacturing practices, based on both chemical standardization and biological assays, are recommended for the quality control of botanical dietary supplements.     

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.     

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.     

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

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

Effect of flavonoids on stress responses in myotube cultures

Effects of flavonoids on stress response of myotube cultures was studied by monitoring the release of [14C] taurine, leukotriene production, and 2',7'-dichlorodihydroflourescein (DCFH2) oxidation. Stress was induced by hypotonic shock, which was accompanied by cell swelling leading to increased leukotriene production and a concomitant increase in reactive oxygen species and osmolyte release. In this model system, addition of the flavonoids catechin and quercetin decreased leukotriene production, DCFH2 oxidation, and taurine efflux, indicating a reduction of cellular stress. High concentrations of epigallocatechin gallate (EGCG) and tea extract increased leukotriene production and initial DCFH2 oxidation, indicating an increased cellular stress (possibly toxicity). However, taurine efflux was reduced, and also longer exposure time as well as lower concentrations of EGCG and tea reduced DCFH2 oxidation. Trolox and alpha-tocopherol did not significantly affect taurine efflux or leukotriene production, and it was therefore concluded that suppression of these responses was not confined to redox activity in a myotube culture.     

Degradation kinetics of catechins in green tea powder: effects of temperature and relative humidity

The stability of catechins in green tea powders is important for product shelf life and delivering health benefits. Most published kinetic studies of catechin degradation have been conducted with dilute solutions and, therefore, are limited in applicability to powder systems. In this study, spray-dried green tea extract powders were stored under various relative humidity (RH) (43-97%) and temperature (25-60 °C) conditions for up to 16 weeks. High-performance liquid chromatography (HPLC) was used to determine catechin contents. Catechin degradation kinetics were affected by RH and temperature, but temperature was the dominant factor. Kinetic models as functions of RH and temperature for the individual 2,3-cis-configured catechins (EGCG, EGC, ECG, and EC) were established. The reaction rate constants of catechin degradation also followed the Williams-Landel-Ferry (WLF) relationship. This study provides a powerful prediction approach for the shelf life of green tea powder and highlights the importance of glass transition in solid state kinetics studies.     

Investigation of adsorption behavior of (-)-epigallocatechin gallate on bovine serum albumin surface using quartz crystal microbalance with dissipation monitoring

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the interactions between (-)-epigallocatechin gallate (EGCG) and bovine serum albumin (BSA) surface. The adsorbed mass, thickness, and viscoelastic properties of EGCG adlayer on BSA surface at various EGCG concentrations, temperatures, sodium chloride concentrations, and pH values have been determined by QCM-D in combination with the Voigt model. The adsorption isotherm of EGCG on BSA surfaces can be better described by the Freundlich model than the Langmuir model, indicating that EGCG adsorption on BSA surfaces is dominated by nonspecific hydrophobic interactions, as supported by stronger EGCG adsorption at higher temperature. Shifts in the Fourier transform infrared spectra of the BSA surface with and without EGCG adsorption disclose that hydrogen bonding might also be involved in EGCG adsorption on BSA surfaces. The addition of salt and change of pH can also influence the EGCG adsorption on BSA surfaces. Usually, higher EGCG adsorption leads to higher values of viscosity and shear elastic modulus of EGCG adlayer, which can be explained by the aggregation of BSA through EGCG bridges. Compared with EGCG, nongalloylated (+)-catechin shows much lower adsorption capacity on BSA surfaces, suggesting the importance of the galloyl group in polyphenol/protein interactions.     

Reaction kinetics of degradation and epimerization of epigallocatechin gallate (EGCG) in aqueous system over a wide temperature range

(-)-Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea, which has been linked with many health benefits. To ensure the conceivable health benefits from thermally processed products, a kinetic study on the stability of (-)-EGCG in aqueous system was carried out using a HPLC-UV system and Matlab programming. Simultaneous degradation and epimerization of (-)-EGCG were characterized during isothermal reactions at low temperatures (25-100 degrees C) combined with previously conducted experimental results at high temperature (100-165 degrees C); the degradation and epimerization complied with first-order reaction and their rate constants followed Arrhenius equation. Mathematical models for the stability of (-)-EGCG were established and validated by the reactions at 70 degrees C and with varied concentrations from different catechin sources. Two specific temperature points in the reaction kinetics were identified, at 44 and 98 degrees C, respectively. Below 44 degrees C, the degradation was more profound. Above 44 degrees C, the epimerization from (-)-gallocatechin gallate (GCG) to (-)-EGCG was faster than degradation. When temperature increased to 98 degrees C and above, the epimerization from (-)-GCG to (-)-EGCG became prominent. Our results also indicated that the turning point of 82 degrees C reported in the literature for the reaction kinetics of catechins would need to be re-examined.     

Evaluation of the antioxidant and pro-oxidant effects of tea catechin oxypolymers

Tea catechin oxypolymers (TCOP) were prepared by oxidizing tea catechin (TC, the content of EGCG was >85%) with H2O2. Their antioxidant and pro-oxidant effects were tested using a deoxyribose assay, a photoreduction of NBT assay, a lipoxygenase assay, a POV assay, and animal tests. The scavenging effects of TCOP to both the hydroxyl radical and superoxide radical were stronger than that of TC, and also they had no pro-oxidant effect; the rate constant for reactions of TC and TCOP for hydroxyl radical were 1.0 x 10(10) and (1.4-2.8) x 10(10) M(-1) x S(-1), respectively. TCOP can inhibit lipid peroxidation and lipoxygenase effectively, and it also can activate red cell SOD and reduce the MDA content in serum of mice very significantly. These results suggested that the antioxidant activity of TCOP was not less than or even more notable than that of TC.     

茶树特异成分对辐照诱导自由基的作用

研究了茶树体内特异成分对辐照诱导自由基的作用。结果表明，辐照茶籽内自由基的增长趋势表现为乔木型大叶品种大于灌木型中、小叶品种，１０个品种茶籽内自由基的耗照剂量效应模型为ｙ＝ａｅ（－ｂ／ｘ）。茶树体内儿茶素、茶多酚、黄酮苷、咖啡碱等特异成分与其辐射敏感性及自由基含量变化密切相关。影响辐照茶树体内自由基消长的主要因子是儿茶素，其中（－）－没食子儿茶素没食子酸酯（ＥＧＣＧ）与自由基反应有浓度效应，在低浓度条件下，具有诱发产生自由基的作用，而在高浓度条件下，则有消除自由基的作用     

Topoisomerase I and II enzyme inhibitory aqueous extract of Ardisia compressa and ardisin protect against benomyl oxidation of hepatocytes

Tea preparations of Ardisia compressa (AC) have been used in folk medicine against liver disorders. The objective of this study was to evaluate the in vitro topoisomerase I and II enzyme inhibition and the antioxidant effect of an aqueous extract from dry leaves of AC and a pure component (ardisin) purified from AC on benomyl (Be)-induced cytotoxicity in primary culture rat hepatocytes. Lipid peroxidation (malondialdehyde), antioxidant enzyme activities of glutathione reductase, glutathione peroxidase, and superoxide dismutase, and glutathione levels were studied. Topoisomerase I and II enzyme inhibition was used to guide purification of ardisin, which was purified using TLC, MPLC, and preparative and analytical HPLC methods. Benomyl increased malondialdehyde (58% change in comparison to the control) and glutathione peroxidase (10%), producing a significant consumption of endogenous antioxidant glutathione (65%, P < 0.05). A 94% hepatocyte protection was observed when cells were first exposed to ardisin (0.27 microg/mL), followed by Be (35 microg/mL). Cell protection by the tea extract of AC (AE) was greater than that by (-)-epigallocatechin 3-gallate (EGCG). Ardisin showed a clear inhibition of topoisomerases I and II catalytic activity in Saccharomyces cerevisiae mutant cells JN 394, JN394t(-)(1), and JN394t-(2)(-)(5). The potency of ardisin was superior to that of AE and EGCG as an antioxidant, protecting rat hepatocytes when exposed to Be. On the basis of the effective concentrations of equivalents to [+]catechin found in the present study, it can be estimated that, in order to gain antioxidative protection, a person would need to ingest approximately 1 L of AC tea per day, with a total content of 10.8 g of plant material.     

HPLC analysis of naturally occurring methylated catechins, 3' '- and 4' '-methyl-epigallocatechin gallate, in various fresh tea leaves and commercial teas and their potent inhibitory effects on inducible nitric oxide synthase in macrophages

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, undergoes substantial biotransformation to species that includes the methylated compounds. Recent studies have demonstrated that the methylated EGCG has many biological activities. In this study, we have investigated the composition of the three O-methylated EGCG derivatives, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (3' '-Me-EGCG), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (4' '-Me-EGCG) and (-)-4'-methyl epigallocatechin-3-O-(4-O-methyl)gallate (4',4' '-di-Me-EGCG) in tea leaves which were picked from various species and at various seasons, ages of leaves, locations, and fermentation levels. Higher levels of 3' '-Me-EGCG and 4' '-Me-EGCG were detected in Chinshin-Kanzai (a species of Camellia sinensis) cultivated in the mountain area of Sansia, Taipei County, Taiwan. Also, these O-methylated EGCG levels were found to be higher in autumn and winter than in spring and summer. The young leaves were found to be richer in the O-methylated compounds than old leaves and the amount of O-methylated EGCG was higher in unfermented longjin green tea than in semifermented oolong tea. However, the fermented black tea and puerh tea did not contain these compounds. 4',4' '-diMe-EGCG could not be detected in either fresh tea leaves or commercial tea leaves. We also found that 3' '-Me-EGCG has a higher inhibitory effect on the nitric oxide generation and inducible nitric oxide synthase (iNOS) expression as compared with EGCG, while 4' '-Me-EGCG and 4',4' '-di-Me-EGCG were less effective.     

Relationship between the biological activities of methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities

It was previously reported that (-)-epigallocatechin-3-O-gallate (EGCG) suppresses the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic cells and that this suppressive effect is associated with EGCG binding to the cell surface. This study examined the effects of five methylated derivatives of EGCG, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG 3' 'Me), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4' 'Me), (-)-4'-O-methyl-epigallocatechin-3-O-gallate (EGCG 4'Me), (-)-epigallocatechin-3-O-(3,4-O-methyl)gallate (EGCG 3' '4' 'diMe), and (-)-4'-O-methyl-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4'4' 'diMe) on FcepsilonRI expression and ERK1/2 phosphorylation, and each of their cell surface binding activities was measured. Of these five methylated derivatives, three that are methylated at the 3' '- and/or 4' '-position, EGCG 3' 'Me, EGCG 4' 'Me, and EGCG 3' '4' 'diMe, suppressed FcepsilonRI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG 4'Me and EGCG 4'4' 'diMe, both of which are methylated at the 4'-position, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG 3' 'Me, EGCG 4' 'Me, EGCG 3' '4' 'diMe, and EGCG 4'Me, which are methylated at the 3' '- and/or 4' '-positions or the 4'-position, could bind to the cell surface even though their binding activities were lower than that of EGCG. Only EGCG 4'4' 'diMe, which is methylated at both the 4'- and 4' '-positions, could not bind. These results suggest that the trihydroxyl structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4'-position in the B ring and the 4' '-position in the gallate are crucial for the cell surface binding activity of EGCG.