Peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) potently suppresses dextran sulfate sodium-induced colitis and colon tumorigenesis in mice

Previous studies reported that peracetylated (-)-epigallocatechin-3-gallate (AcEGCG) has antiproliferative and anti-inflammatory activities. Here, we evaluated the chemopreventive effects and underlying molecular mechanisms of dietary administration of AcEGCG and EGCG in dextran sulfate sodium (DSS)-induced colitis in mice. The mice were fed a diet supplemented with either AcEGCG or EGCG prior to DSS induction. Our results indicated that AcEGCG administration was more effective than EGCG in preventing the shortening of colon length and the formation of aberrant crypt foci (ACF) and lymphoid nodules (LN) in mouse colon stimulated by DSS. Our study observes that AcEGCG treatment inhibited histone 3 lysine 9 (H3K9) acetylation but did not affect histone acetyltransferase (HAT) activity and acetyl- CREB-binding protein (CBP)/p300 levels. In addition, pretreatment with AcEGCG decreased the proinflammatory mediator levels by down-regulating of PI3K/Akt/NFκB phosphorylation and p65 acetylation. We also found that treatment with AcEGCG increased heme oxygenase-1(HO-1) expression via activation of extracellular signal-regulated protein kinase (ERK)1/2 signaling and acetylation of NF-E2-related factor 2 (Nrf2), thereby abating DSS-induced colitis. Moreover, dietary feeding with AcEGCG markedly reduced colitis-driven colon cancer in mice. Taken together, these results demonstrated for the first time the in vivo chemopreventive efficacy and molecular mechanisms of dietary AcEGCG against inflammatory bowel disease (IBD) and potentially colon cancer associated with colitis. These findings provide insight into the biological actions of AcEGCG and might establish a molecular basis for the development of new cancer chemopreventive agents.     

Pharmacokinetics of (-)-epigallocatechin-3-gallate in conscious and freely moving rats and its brain regional distribution

A liquid chromatography technique coupled with tandem mass spectrometry (LC-MS/MS) electrospray ionization was used to measure (-)-epigallocatechin-3-gallate (EGCG) in rat plasma. This method was applied to investigate the pharmacokinetics of EGCG in a conscious and freely moving rat by an automated blood sampling device. Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z of 457 [M - H]- to the product ion 169 for EGCG and the m/z of 187 to 164 for the internal standard. The limit of quantification (LOQ) of EGCG in rat plasma was determined to be 5 ng/mL, and the linear range was 5-5000 ng/mL. The protein binding of EGCG in rat plasma was 92.4 +/- 2.5%. The brain distribution result indicated that EGCG may potentially penetrate through the blood-brain barrier at a lower rate. The disposition of EGCG in the rat blood was fitted well by the two-compartmental model after intravenous administration (10 mg/kg, iv). The elimination half-life of EGCG was 62 +/- 11 and 48 +/- 13 min for intravenous (10 mg/kg) and oral (100 mg/kg) administration, respectively. The pharmacokinetic data indicate that the oral bioavailability of EGCG in a conscious and freely moving rat was about 4.95%.     

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.     

Design, semisynthesis, and evaluation of O-acyl derivatives of (-)-epigallocatechin-3-gallate as antitumor agents

The partially purified catechin fraction isolated from green tea extract was treated with a variety of acylating agents (acyl anhydrides/chloride) to obtain (-)-epigallocatechin-3-gallate (EGCG) O-acyl derivatives in 20-25.4% yields. The (-)-EGCG O-acyl derivatives were characterized by physical data and spectral studies. These compounds were evaluated for their antitumor activity by use of a two-stage carcinogenesis model in 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA)--induced cancer in Swiss albino mice. The study showed that there was a significant decrease in the antitumor activity with the increase in size and branching of the chain length of acyl groups. The results indicated that these O-acyl derivatives of (-)-EGCG have the potential to be developed as cancer chemopreventive agents. Keywords: Green tea; catechins; (-)-EGCG O-acyl derivatives; antitumor activity.     

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.     

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.     

Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate,suppresses FcepsilonRI expression in human basophilic KU812 cells

We previously found that the O-methylated derivative of (-)-epigallocatechin-3-O-gallate (EGCg), (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG' '3Me), has potent antiallergic activity. The high-affinity IgE receptor, FcepsilonRI, is found at high levels on basophils and mast cells and plays a key role in a series of acute and chronic human allergic reactions. To understand the mechanism of action for the antiallergic EGCG' '3Me, the effect of EGCG' '3Me on the cell surface expression of FcepsilonRI in human basophilic KU812 cells was examined. Flow cytometric analysis showed that EGCG' '3Me was able to decrease the cell surface expression of FcepsilonRI. Moreover, immunoblot analysis revealed that total cellular expression of the FcepsilonRI alpha chain decreased upon treatment with EGCG' '3Me. FcepsilonRI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains. The level of mRNA production of each subunit in KU812 cells was investigated. EGCG' '3Me reduced FcepsilonRI alpha and gamma mRNA levels. The cross-linkage of FcepsilonRI causes the activation of basophils, which leads to the secretion of inflammatory mediators including histamine. EGCG' '3Me treatment inhibited the FcepsilonRI cross-linking-induced histamine release. These results suggested that EGCG' '3Me can negatively regulate basophil activation through the suppression of FcepsilonRI expression.     

Characterization, antimicrobial activity, and mechanism of a high-performance (-)-epigallocatechin-3-gallate (EGCG)-CuII/polyvinyl alcohol (PVA) nanofibrous membrane

(-)-Epigallocatechin-3-gallate (EGCG) exhibited strong antimicrobial activity. However, the easy oxidation of EGCG has limited its application. To increase the antimicrobial activity and stability of EGCG, the EGCG-Cu(II) complex was formed by chelating copper ions and then electronspun into polyvinyl alcohol (PVA) nanofibers. Electronspun nanofibrous membranes were investigated by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM), which showed that the average fiber diameter was 210 nm. Minimal inhibitory concentrations (MICs) of EGCG-Cu(II)/PVA membranes were tested against the tested strains. The bactericidal activity of EGCG-Cu(II) was suppressed by ethylenediaminetetraacetic acid (EDTA). Cell killing was accompanied by the leakage of intracellular proteins, indicating that the cytoplasmic membrane was badly damaged after exposure to the EGCG-Cu(II)/PVA membrane. We observed the process of cell damage by SEM. On the basis of experimental evidence and theoretical analyses, the mechanism proposed that copper ions played a cooperative role in the bactericidal process of EGCG. To evaluate the antimicrobial activity of the EGCG-Cu(II)/PVA membrane, we developed a rapid detection method by labeling cells with water-soluble CdTe quantum dots.     

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.     

Inhibitory effects of tea catechins and O-methylated derivatives of (-)-epigallocatechin-3-O-gallate on mouse type IV allergy

The inhibitory effects of tea catechins, the O-methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG), and the polyphenol extracts from tea leaves (Camellia sinensis L.) on oxazolone-induced type IV allergy in male ICR mice were investigated. Four major tea catechins and two O-methylated derivatives, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG3' 'Me) and (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG4' 'Me), showed significant inhibitory effects on mouse type IV allergy after a percutaneous administration at a dose of 0.13 mg/ear. Among tea catechins, the compounds including galloyl moieties, such as EGCG and (-)-epicatechin-3-O-gallate (ECG), showed the strongest inhibitory activities on mouse type IV allergy. The inhibitory activities of EGCG3' 'Me and EGCG4' 'Me were higher than that of EGCG at a dose of 0.05 mg/ear. Polyphenol extract from tea leaves of Benihomare cultivar, which includes EGCG3' 'Me, strongly inhibited mouse type IV allergy after percutaneous administration in comparison with that from Yabukita cultivar, which does not include EGCG3' 'Me, at doses of 0.05 and 0.13 mg/ear. EGCG3' 'Me is thought to contribute, at least in part, to the inhibitory ability of Benihomare tea leaves on mouse type IV allergy. EGCG and the polyphenol extracts from Benihomare and Yabukita tea leaves also inhibited mouse type IV allergy by oral administration at 1 h before the sensitization and at 1 h before the challenge with oxazolone. Therefore, daily intake of tea drinks could have potential to prevent type IV allergy.     

Inhibitory effect of (-)-epigallocatechin 3-gallate, a polyphenol of green tea, on neutrophil chemotaxis in vitro and in vivo

The effect of (-)-epigallocatechin 3-gallate (EGCG), a major polyphenol of green tea, on neutrophil migration has been studied using multiwell-type Boyden chambers in vitro and a fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA)-induced rat allergic inflammation model in vivo. EGCG inhibited rat neutrophil chemotaxis toward cytokine-induced neutrophil chemoattractant-1 (CINC-1) in a concentration-dependent manner. In addition, CINC-1-induced neutrophil chemotaxis was suppressed by the pretreatment of rat neutrophils with EGCG at the concentration over 15 microg/mL. EGCG caused concentration-dependent suppression of the transient increase in CINC-1-induced intracellular free calcium level in both rat neutrophils and rat CXC chemokine receptor 2 (CXCR2)-transfected HEK 293 cells. EGCG inhibited CINC-1 production by IL-1beta-stimulated rat fibroblasts (NRK-49F cells) and lipopolysaccharide-stimulated rat macrophages at the concentration over 50 microg/mL, a comparatively high concentration. Oral administration of EGCG (1.0 mg or 1.5 mg/rat) at 1 h before the challenge with FITC-OVA suppressed neutrophil infiltration into the air pouch (inflammatory site) in the air-pouch type FITC-OVA-induced allergic inflammation in rats. Chemokine levels in the pouch fluids, however, were not influenced by EGCG administration. The results suggest that EGCG suppressed neutrophil infiltration by a direct action on neutrophils, but not by indirect actions, including the suppression of chemokine production at the inflammatory site.     

Epimerization of tea catechins and O-methylated derivatives of (-)-epigallocatechin-3-O-gallate: relationship between epimerization and chemical structure

Epimerization at C-2 of O-methylated catechin derivatives and four major tea catechins were investigated. The epimeric isomers of (-)-epicatechin (I), (-)-epicatechin-3-O-gallate (II), (-)-epigallocatechin (III), (-)-epigallocatechin-3-O-gallate (IV), and (-)-epigallocatechin-3-O-(3-O-methyl)gallate (V) in green tea extracts increased time-dependently at 90 degrees C. The epimerization rates of authentic tea catechins in distilled water are much lower than those in tea infusion or in pH 6.0 buffer solution. The addition of tea infusion to the authentic catechin solution accelerated the epimerization, and the addition of ethylenediaminetetraacetic acid, disodium salt (Na(2)EDTA) decreased the epimerization in the pH 6.0 buffer solution. Therefore, the metal ions in tea infusion may affect the rate of epimerization. The proportions of the epimers to authentic tea catechins [III, IV, V, and (-)-epigallocatechin-3-O-(4-O-methyl)gallate (VI)] in pH 6.0 buffer solution after heating at 90 degrees C for 30 min were 42.4%, 37.0%, 41.7%, and 30.4%, respectively. These values were higher than those of I and II (23.5% and 23.6%, respectively). The O-methylated derivatives at the 4'-position on the B ring of IV and VI were hardly epimerized. These results suggest that the hydroxyl moiety on the B ring of catechins plays an important role in the epimerization in the order 3',4',5'-triol type > 3',4'-diol type > 3',5'-diol type.     

Dietary combination of conjugated linoleic acid (CLA) and pine nut oil prevents CLA-induced fatty liver in mice

Conjugated linoleic acid (CLA) strongly prevents fat accumulation in adipose tissue of mice, even if hepatic fat deposition and insulin resistance are concomitantly observed. This study investigated the possibility of maintaining the antiadiposity properties of CLA while preventing adverse effects such as liver steatosis and hyperinsulinemia. To this end, mice were divided into three groups and fed a standard diet (control) or a diet supplemented with 1% CLA (CLA) or a mixture of 1% CLA plus 7.5% pine nut oil (CLA + P). The combination of CLA + P preserved the CLA-mediated antiadiposity properties (70% fat reduction), preventing hepatic steatosis and a sharp increase in plasmatic insulin starting from the eighth week of CLA treatment. The assay of both fatty acid synthesis and oxidation in the CLA + P mice revealed a time-dependent biphasic behavior of the corresponding enzymatic activities. A sudden change in these metabolic events was indeed found at the eighth week. A strong correlation between the changes in key enzymes of lipid metabolism and in insulin levels apparently exists in CLA-fed mice. Furthermore, lower levels of lipids, in comparison to values found in CLA-fed mice, were observed in the liver and plasma of CLA + P-fed animals.     

Inhibition of estrogen-mediated mammary tumorigenesis by blueberry and black raspberry

We previously demonstrated the protective effects of blueberry (BB) and black raspberry (BRB) supplemented at 2.5% dose in an ACI rat mammary tumor model. Here, we assessed a dose-related alteration in tumor indices with diet supplemented with 5% BB or BRB powder. The diet was well tolerated. Tumor palpation from 12 weeks revealed first tumor appearance by 84 days in the control group, that was delayed by 24 and 39 days with the BB and BRB diets, respectively (p = 0.04). Ellagic acid detected in the plasma of rats fed the BRB diet was in the range of 96.6-294.2 ng/mL. While the BB diet showed better efficacy in reducing mammary tissue proliferation and tumor burden, tumor latency was delayed efficiently by BRB. Furthermore, BB was effective in downregulating CYP1A1 expression, while BRB downregulated ERα expression effectively. Distinct anticarcinogenic effects of the two berries correspond to their distinct phytochemical signatures.     

Wheat bran oil and its fractions inhibit human colon cancer cell growth and intestinal tumorigenesis in Apc(min/+) mice

This study was designed to investigate the cancer preventive activities of wheat bran (WB) oil. We studied the colon cancer preventive effects of WB oil and its subfractions in the Apc(min/+) mouse model, a recognized mouse model for human colorectal cancer, and used human colon cancer cell lines (HCT-116 and HT-29) to identify possible active fractions in WB oil. Our results showed that the oil fraction of WB was more active than the water fraction against the growth of human colon cancer cell lines and that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7% (p < 0.0001) in the Apc(min/+) mouse model. The WB oil was further fractioned into nonpolar lipids and phytochemicals and the phytochemical fraction was fractionated into phytosterols and phytosterol ferulates, 5-alk(en)ylresorcinols, and unidentified constituents by normal phase silica gel column chromatography. Results on cell culture showed that the phytochemical fraction had a higher inhibitory effect on HCT-116 human colon cancer cells than that of WB oil, whereas the nonpolar lipid fraction had less growth inhibitory effectiveness. However, neither fractions showed a stronger inhibition than WB oil in the Apc(min/+) mouse model. The current results demonstrate, for the first time, the intestinal cancer preventive activity of WB oil. The active ingredients, however, remain to be identified.     

Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabetic KK-Ay mice

Fucoxanthin is a marine carotenoid found in edible brown seaweeds. We previously reported that dietary fucoxanthin attenuates the weight gain of white adipose tissue (WAT) of diabetic/obese KK- A(y) mice. In this study, to evaluate the antiobesity and antidiabetic effects of fucoxanthin and fish oil, we investigated the effect on the WAT weight, blood glucose, and insulin levels of KK- A(y) mice. Furthermore, the expression level of uncoupling protein 1 (UCP1) and adipokine mRNA in WAT were measured. After 4 weeks of feeding, 0.2% fucoxanthin in the diet markedly attenuated the gain of WAT weight in KK- A(y) mice with increasing UCP1 expression compared with the control mice. The WAT weight of the mice fed 0.1% fucoxanthin and 6.9% fish oil was also significantly lower than that of the mice fed fucoxanthin alone. In addition, 0.2% fucoxanthin markedly decreased the blood glucose and plasma insulin concentrations in KK- A(y) mice. The mice fed with the combination diet of 0.1% fucoxanthin and fish oil also showed improvements similar to that of 0.2% fucoxanthin. Leptin and tumor necrosis factor (TNFalpha) mRNA expression in WAT were significantly down-regulated by 0.2% fucoxanthin. These results suggest that dietary fucoxanthin decreases the blood glucose and plasma insulin concentration of KK- A(y) along with down-regulating TNFalpha mRNA. In addition, the combination of fucoxanthin and fish oil is more effective for attenuating the weight gain of WAT than feeding with fucoxanthin alone.     

Regiospecific identification of 2-(12-ricinoleoylricinoleoyl)-1,3-diricinoleoyl-sn-glycerol in castor (Ricinus communis L.) oil by ESI-MS(4)

(12-Ricinoleoylricinoleoyl)diricinoleoylglycerol (RRRR), a tetraacylglycerol, was identified earlier in castor oil. Using ESI-MS (4), 95% of the 12-ricinoleoylricinoleoyl chain was identified at the sn-2 position of the glycerol backbone of RRRR. Regiospecific location of the 12-ricinoleoylricinoleoyl chain of RRRR on the glycerol backbone was identified and quantified by the ions from the losses of the acyl chains at the sn-2 position as alpha,beta-unsaturated fatty acids from the lithium adduct of RRRR. The regiospecific location was confirmed by hydrolysis of RRRR using sn-1,3 specific lipase. By comparison to the mass spectrum of 1- O-palmityl-2,3-palmitoyl- rac-glycerol containing one ether bond, the 12-ricinoleoylricinoleoyl chain of RRRR is indeed the ester bond between the two ricinoleoyl chains, not the ether bond formed from the two hydroxyl groups of the two ricinoleoyl chains. The structure of RRRR is 2-(12-ricinoleoylricinoleoyl)-1,3-diricinoleoyl- sn-glycerol.