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.     

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.     

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.     

Inhibition of gap junctional intercellular communication by the green tea polyphenol (-)-epigallocatechin gallate in normal rat liver epithelial cells

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.     

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.     

Identification of biliary metabolites of (-)-epigallocatechin gallate in rats

After oral administration of (-)-epigallocatechin gallate (EGCg) to rats, its biliary metabolites were examined. Although a large part of the biliary metabolites was found to exist in conjugated forms, it was difficult to separate the conjugated forms. Thus the free form of biliary metabolites was prepared by beta-glucuronidase/sulfatase treatment and was purified by HPLC. Six compounds purified were subjected to FABeta-MS and NMR analyses. The six metabolites thus obtained were shown to be EGCg, 3'-O-methyl-EGCg, 4'-O-methyl-EGCg, 3' '-O-methyl-EGCg, 4' '-O-methyl-EGCg, and 4',4' '-di-O-methyl-EGCg, respectively. The six EGCg metabolites and their conjugates excreted during a 4-h period were estimated to be roughly 0.1% and 3.3% of the administered EGCg, respectively. In addition, 4' '-O-methyl-EGCg and 4',4' '-di-O-methyl-EGCg were estimated to exist only in the sulfate form, but the other four metabolites existed in both glucuronide (and/or sulfoglucuronide) and sulfate forms.     

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%.     

Inhibitory effect of phenolic acids on the proliferation of 3T3-L1 preadipocytes in relation to their antioxidant activity

Obesity is an important topic in the world of public health and preventive medicine. Inhibition of preadipocyte proliferation plays an important role in the mechanisms of proposed antiobesity. In this in vitro study, the inhibitory effect of phenolic acids on 3T3-L1 preadipocytes was evaluated, and a relationship analysis was then conducted. The results showed that the addition of phenolic acids to the growth medium decreased the cell population growth of 3T3-L1 preadipocytes. The IC50 values of chlorogenic acid, gallic acid, o-coumaric acid and m-coumaric acid on 3T3-L1 preadipocytes were 72.3, 43.3, 48.2, and 49.2 microM, respectively. A relationship analysis indicated that there is a significant linear correlation between the influence of phenolic acids on cell population growth and their antioxidant activity (r = 0.77, p < 0.01). The cell cycle assay indicated that the treatment of 3T3-L1 preadipocytes with chlorogenic acid, o-coumaric acid, and m-coumaric acid caused cell cycle arrest in the G1 phase. Gallic acid did not affect the cell cycle profile; however, it increased the number of apoptotic cells (sub-G1 phase) in a time- and dose-dependent manner. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometric assay showed that gallic acid increased the number of early apoptotic (annexin V-FITC+/PI-) and late apoptotic cells (annexin V-FITC+/PI+) but not necrotic cells (annexin V-FITC-/PI+). The treatment of cells with gallic acid caused the loss of mitochondrial membrane potential (delta psi(m)). These results indicate that the inhibition of preadipocyte population growth by some phenolic acids might have further implication in in vivo antiobesity effects.     

Adsorption characteristics of (-)-epigallocatechin gallate and caffeine in the extract of waste tea on macroporous adsorption resins functionalized with chloromethyl, amino, and phenylamino groups

According to the Friedel-Crafts and amination reaction, a series of macroporous adsorption resins (MARs) with novel structures were synthesized and identified by the Brunauer-Emmett-Teller (BET) method and Fourier transform infrared (FTIR) spectra, and corresponding adsorption behaviors for (-)-epigallocatechin gallate (EGCG) and caffeine (CAF) extracted from waste tea were systemically investigated. Based on evaluation of adsorption kinetics, the kinetic data were well fitted by pseudo-second-order kinetics. Langmuir, Freundlich, Temkin-Pyzhev, and Dubinin-Radushkevich isotherms were selected to illustrate the adsorption process of EGCG and CAF on the MARs. Thermodynamic parameters were adopted to explain in-depth information of inherent energetic changes associated with the adsorption process. The effect of temperature on EGCG and CAF adsorption by D101-3 was further expounded. Van der Waals force, hydrogen bonding, and electrostatic interaction were the main driving forces for the adsorption of EGCG and CAF on the MARs. This study might provide a scientific reference point to aid the industrial large-scale separation and enrichment of EGCG from the extracts of waste tea using modified MARs.     

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.     

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.     

Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells

Adipocyte differentiation (adipogenesis) is a complex process including the coordinated changes in hormone sensitivity and gene expression in response to various stimuli. Natural compounds are known to be involved in the regulation of this process. Here we investigated the effects of dietary apigenin, a plant flavonoid, on adipogenesis. Apigenin suppressed adipocyte differentiation of mouse adipocytic 3T3-L1 cells and reduced the accumulation of intracellular lipids. Quantitative PCR and Western blot analyses revealed that apigenin decreased the levels of peroxisome proliferator-activated receptor γ and its target genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase. Apigenin decreased or had no effect on the expression of lipolytic genes such as adipose triglyceride lipase, hormone sensitive lipase, and monoacyl glyceride lipase, thereby reducing glycerol release from adipocytes. Noteworthily, apigenin activated 5'-adenosine monophosphate-activated protein kinase (AMPK) in an apigenin dose-dependent manner, which activation is known to suppress adipogenesis. These results provide a novel insight into the molecular mechanism involved in the action of apigenin: the apigenin-induced activation of AMPK leads to decreased expression of adipogenic and lipolytic genes, thus suppressing adipogenesis in 3T3-L1 cells. Thus, dietary apigenin may contribute to lower body-fat content and body-weight gain through the activation of AMPK.     

Metabolic fate of (-)-[4-(3)H]epigallocatechin gallate in rats after oral administration

After oral administration of [4-(3)H]EGCg to rats, the radioactivity in blood, major tissues, urine, and feces was measured over time. The radioactivity in blood and most tissues remained low for 4 h postdose, began to increase after 8 h, peaked at 24 h, and then decreased. Major urinary excretion of radioactivity occurred in the 8-24 h period, and the cumulative radioactivity excreted by 72 h was 32.1% of the dose. The radioactivity in the feces was 35.2% of the dose within 72 h postdose. In the case of rats pretreated with antibiotics (antibiotic-pretreated rats), the radioactivity levels of the blood and urine were definitely lower than those in rats not pretreated with antibiotics (normal rats). The radioactivity recovered in the antibiotic-pretreated rat urine was estimated to be only (1)/(100) of that in the normal rat urine. These results clearly demonstrated that the radioactivity detected in the blood and urine of normal rats mostly originated from degradation products of EGCg produced by intestinal bacteria. Furthermore, a main metabolite in the normal rats was purified and identified as 5-(5'-hydroxyphenyl)-gamma-valerolactone 3'-O-beta-glucuronide (M-2). In feces of the normal rats, EGC (40.8% of the fecal radioactivity) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M-1, 16.8%) were detected. These results suggested that M-1 was absorbed in the body after degradation of EGCg by intestinal bacteria, yielding M-1 with EGC as an intermediate. Furthermore, M-2 was thought to be formed from M-1 in the intestinal mucosa and/or liver, then to enter the systemic circulation, and finally to be excreted in the urine. Taking into account all of the above findings, a possible metabolic route of EGCg orally administered to rats is proposed.     

Interaction of cinnamic acid derivatives with commercial hypoglycemic drugs on 2-deoxyglucose uptake in 3T3-L1 adipocytes

Hydroxycinnamic acid derivatives are naturally occurring substances found in fruits, vegetables, and flowers and are consumed as dietary phenolic compounds. The effect of cinnamic acid, ferulic acid, p-coumaric acid, eugenol, chlorogenic acid, and caffeic acid, alone and in combination with two commercial oral hypoglycemic drugs (OHD), namely, thiazolidinedione (THZ) and metformin, on the uptake of 2-deoxyglucose (2DG) by 3T3-L1 adipocytes is studied. All of the phytochemicals other than cinnamic acid show synergistic interaction in 2DG uptake with both of the OHDs. THZ (20 μM) in combination with ferulic acid (25 μM) or p-coumaric acid (25 μM) increases 2DG uptake by 7- or 6.34-fold, respectively, with respect to control, whereas metformin (20 μM), along with ferulic acid (25 μM) or cinnamic acid (25 μM), increases 2DG uptake by 6.45- or 5.87-fold, respectively, when compared to control. Chlorogenic and cinnamic acids increased the expression of PPARγ, whereas other hydroxycinnamic acids enhanced the expression of PI3K, indicating different mechanisms of action between these compounds. These phytochemicals were able to reduce the expressions of the fatty acid synthase and HMG CoA reductase genes, indicating that they may be able to reduce the secondary complications caused by the accumulation of lipids. These studies suggest that hydroxycinnamic acid derivatives may be beneficial for the treatment of diabetes mellitus. They may act as a supplement with commercial drugs and may reduce the secondary complications caused by OHDs.     

Activity of (-)-epigallocatechin 3-O-gallate against oxidative stress in rats with adenine-induced renal failure

Methylguanidine (MG) is widely recognized as a strong uremic toxin. The hydroxyl radical (*OH) specifically plays an important role in the pathway of MG production from creatinine (Cr). In this study, we investigated whether oral administration of (-)-epigallocatechin 3-O-gallate (EGCg) suppresses MG production in rats with chronic renal failure after intraperitoneal Cr injection. MG production from Cr was significantly increased in rats with adenine-induced renal failure, which was more vulnerable to oxidative stress, compared with that in normal rats. However, oral administration of EGCg 30 min before and after Cr injection effectively inhibited MG production. Our findings suggest that EGCg, an excellent antioxidant from green tea, exerts protective activity in rats with chronic renal failure, resulting in suppression of Cr oxidation influenced by *OH.     

Identification of benzophenone C-glucosides from mango tree leaves and their inhibitory effect on triglyceride accumulation in 3T3-L1 adipocytes

A 70% ethanol-water extract from the leaves of Mangifera indica L. (Anacardiaceae) inhibited triglyceride (TG) accumulation in 3T3-L1 cells. From the active fraction, seven new benzophenone C-glycosides, foliamangiferosides A (1), A(1) (2), A(2) (3), B (4), C(1) (5), C(2) (6), and C(3) (7), together with five known compounds were isolated and the structures were elucidated on the basis of chemical and physicochemical evidence. The effects of these compounds on TG and the free fatty acid level in 3T3-L1 cells were determined, and the structure-activity relationship was discussed. On the basis of the AMPK signaling pathway, several compounds were found to increase the AMPK enzyme expression and down-regulate lipogenic enzyme gene expression such as SREBP1c, FAS, and HSL.     

Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes

Obesity has become a global epidemic in both developed and developing countries, and it is a significant risk factor for various diseases such as diabetes, cancer, heart disease, and hypertension. In the present study, the effect of naturally occurring antioxidants (flavonoids and phenolic acids) on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. The results showed that o-coumaric acid and rutin had the highest inhibition on intracellular triglyceride (61.3 and 83.0%, respectively) among 15 phenolic acids and 6 flavonoids tested. However, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 adipocytes was not influenced by those compounds. For glycerol-3-phosphate dehydrogenase (GPDH) activity, the data indicated that o-coumaric acid and rutin had the highest inhibition on GPDH activity (54.2 and 66.8%, respectively) among the compounds tested. o-Coumaric acid and rutin also inhibited the expression of PPARgamma, C/EBPalpha and leptin and then up-regulated expression of adiponectin at the protein level. Some naturally occurring antioxidants efficiently suppressed adipogenesis in 3T3-L1 adipocytes. These results suggest that o-coumaric acid and rutin targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome.