Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells

The protective effects of three tea extracts (green tea, GTE; oolong tea, OTE; and black tea, BTE) and five tea polyphenols (epicatechin, EC; epicatechin gallate, ECG; epigallocatechin, EGC; epigallocatechin gallate, EGCG; and theaflavins, THFs) on benzo[a]pyrene (B[a]P)-induced DNA damage in Chang liver cells were evaluated using the comet assay. B[a]P-induced DNA damage in Chang liver cells was significantly (p < 0.05) inhibited by GTE and OTE at a concentration of 10 microg/ml and by BTE at 25 microg/ml. At a concentration of 100 microg/ml, the % tail DNA was reduced from 33% (B[a]P treated only) to 10, 9, 13%, by GTE, OTE and BTE, respectively. EC and ECG did not cause DNA damage in cells according to the results of the comet assay; however, EGC, EGCG and theaflavins caused DNA damage in cells at a concentration of 100 microM. The results indicated that EC and ECG had protective effects against B[a]P-induced DNA damage in cells at a concentration of 10-100 microM. Although EGC, EGCG and the theaflavins caused DNA damage at a high concentration, but they had protective effects against B[a]P-induced DNA damage in cells at a low concentration of 10-50 microM. The results also showed that the DNA damage in cells induced by EGC, EGCG, and the theaflavins was due to the generation of superoxide during incubation with cells at a higher concentration. Therefore, tea catechins and THFs play an important role in enabling tea extracts to inhibit DNA damage in Chang liver cells.     

Epigallocatechin gallate-induced apoptosis does not affect adipocyte conversion of preadipocytes

In the present study, we examined the effect of epigallocatechin gallate (EGCG) on the growth and differentiation of human preadipocyte cells, AML-I. EGCG exhibited cytotoxic activity on AML-I cells, accompanied by the appearance of characteristics of apoptosis by Annexin V-FITC staining method. Among apoptosis-related proteins examined, loss of NF-kappaB and p-Akt, and accumulation of Bad were displayed in EGCG-treated cells by Western blot analysis. Among 6 structure-related catechins including catechin (C), epicatechin (EC), catechin gallate (CG), epigallocatechin (EGC), epicatechin gallate (ECG) and EGCG, the catechins containing galloyl moiety exhibited apoptotic capacity. Interestingly, exposure of AML-I to EGCG increased the amounts of cytoplasmic lipid droplets as well as the expression of fatty acid synthase and peroxisome proliferator activated receptor-gamma proteins. Our results suggest that EGCG induces growth arrest and apoptosis, but does not affect adipocyte conversion of preadipocytes.     

Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450

Catechins, major polyphenol constituents of green tea, are potent chemopreventive agents against cancers caused by chemical carcinogens in rodents. The effects of four epicatechin derivatives, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC), on the metabolic activation of benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) and aflatoxin B(1) (AFB(1)) by human cytochrome P450 (CYP) were examined. B[a]P, PhIP and AFB(1) were activated by respective human CYP1A1, CYP1A2 and CYP3A4 expressed in the membrane fraction of genetically engineered Salmonella typhimurium (S. typhimurium) TA1538 cells harboring the human CYP and human NADPH-CYP reductase (OR), when the membrane fraction was added to S. typhimurium TA98. Galloylated catechins, ECG and EGCG inhibited the mutagenic activation potently, while EGC and EC showed relatively weak inhibitory effects. Catechins also inhibited the oxidations of typical substrates catalyzed by human CYPs, namely ethoxycoumarin O-deethylation by CYP1A1, ethoxyresorufin O-deethylation by CYP1A2 and midazolam 1'-hydroxylation by CYP3A4. The IC(50) values of catechins for the inhibition of human CYP were roughly the same as those seen in the mutagenic activation. EGCG inhibited other forms of human CYP such as CYP2A6, CYP2C19 and CYP2E1, indicating the non-specific inhibitory effects of EGCG toward human CYPs. Furthermore, EGCG inhibited human NADPH-cytochrome CYP reductase (OR) with a K(i) value of 2.5 microM. These results suggest that the inhibition of the enzyme activity of CYP is accounted for partially by the inhibition of OR.     

Structure-activity relationship of the tocopherol-regeneration reaction by catechins

The reaction rates (k(r)) of 5,7-diisopropyl-tocopheroxyl radical (Toc) with catechins (epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG)) and related compounds (methyl gallate (MG), 4-methylcatechol (MC), and 5-methoxyresorcinol (MR)) have been measured by stopped-flow spectrophotometer. The k(r) values increased in the order of MR < < MG < EC < MC approximately ECG < EGC < EGCG in ethanol and 2-propanol/H(2)O (5/1, v/v) solutions, indicating that the reactivity of the OH groups in catechins increased in the order of resorcinol A-ring < < gallate G-ring < catechol B-ring < pyrogallol B-ring. The catechins which have lower oxidation potentials show higher reactivities. The rate constants for catechins in micellar solution showed notable pH dependence with one or two peaks around pH 9-11, because of the dissociation of various phenolic hydroxyl protons in catechins. The structure-activity relationship in the free-radical-scavenging reaction by catechins has been clarified by the detailed analyses of the pH dependence of k(r) values. The reaction rates increased remarkably with increasing the anionic character of catechins, that is, the electron-donating capacity of catechins. The mono anion form at catechol B-and resorcinol A-rings and dianion form at pyrogallol B-and gallate G-rings show the highest activity for free-radical-scavenging. It was found that catechins (EC, ECG, EGC, and EGCG) have activity similar to or higher than that of vitamin C in vitamin E regeneration at pH 7-12 in micellar solution.     

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

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

Adsorption affinity of tea catechins onto polymeric resins: Interpretation from molecular orbital theory

Adsorption of certain tea catechins such as (+) catechin (C), (−) epicatechin (EC), (+) gallocatechin (GC), (−) epigallocatechin (EGC), (+) catechin gallate (CG), (−) epicatechin gallate (ECG), (+) gallocatechin gallate (GCG), and (−) epigallocatechin gallate (EGCG) have been studied using three types of polymeric resins as adsorbents. Adsorption affinity expressed as the slope of the linear region of the isotherm for a solute is found to be different for different adsorbents, and this difference can be interpreted from the chemical nature of the sorbents. Molecular interactions on polymeric resins have been studied based on molecular orbital theory. Electronic states of adsorbent and adsorbate were calculated using the semiempirical molecular orbital (MO) method from which energy of adsorption in aqueous solution was estimated. The adsorptive interaction on the polymeric resins computed on the basis of frontier orbital theory seems to correlate well with the experimentally measured adsorption affinity and enthalpy.     

Catechins delay lipid oxidation and alpha-tocopherol and beta-carotene depletion following ascorbate depletion in human plasma

Blood plasma was incubated with 50 mM AAPH [2, 2'-azobis-(2-amidinopropane) hydrochloride] in the absence or presence of catechins (5-100 microM). Lipid oxidation was evaluated by measuring the formation of 2-thiobarbituric acid reactive substances (TBARS). The concentration of alpha-tocopherol (AT), beta-carotene (BC), ascorbic acid (AA), and catechins was determined by reverse phase high performance liquid chromatography (HPLC) with electrochemical detection. All the assayed catechins inhibited plasma TBARS formation. Based on the calculated IC50, the order of effectiveness was: epicatechin gallate (ECG) > epigallocatechin gallate (EGCG) > epigallocatechin (EGC) > epicatechin (EC) > catechin (C). Catechins protected plasma AT and BC from AAPH-mediated oxidation. The order of effectiveness for AT protection was ECG > EGCG > EC = C > EGC; and for BC protection, the order was EGCG > ECG > EGC > > EC > C. The addition of catechins modified the kinetics of TBARS formation and AT depletion, but the rate of AA depletion was not affected. Catechin oxidation did not start until the complete depletion of AA, and it preceded AT depletion. These results indicate that catechins are effective antioxidants in human blood plasma, delaying the lipid oxidation and depletion of endogenous lipid-soluble antioxidants (AT and BC).     

人工接种冠突散囊菌对白茶主要呈味物质的影响

本文为了排除其他微生物的干扰,首次以人工接种的方式研究了冠突散囊菌Eurotium cristatum对白茶主要呈味物质的作用。采用高效液相色谱、分光光度计等方法,分析表明冠突散囊菌能够显著降低白茶中呈苦涩味的表没食子儿茶素没食子酸酯（EGCG）和表儿茶素没食子酸酯（ECG）,并提高表没食子儿茶素（EGC）、Asp、His、咖啡碱和山奈酚的含量;灭菌压制的过程中EGCG可能异构化成为更稳定的没食子儿茶素没食子酸酯（GCG）,且二者以相近的含量共存。冠突散囊菌可以降低人工发花白茶饼中呈苦涩味的化合物含量,从而达到减少白茶饼苦涩味的效果;灭菌压制过程也能够降低白茶饼的苦涩味物质的含量。“发花”处理为白茶带来了新的风味,可以丰富白茶产品种类,同时为促进粗老原料白茶的综合利用提供新思路。     

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells

Summary The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B₄ (LTB₄), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (−)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (−)-epicatechin (EC) had no effect, (−)-epigallocatechin (EGC) and (−)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB₄ release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG>ECG>EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.     

Inhibitory Effects of Persimmon (Diospyros kaki) Extract and Related Polyphenol Compounds on Growth of Human Lymphoid Leukemia Cells

We have investigated the effects of persimmon (Diospyros kaki) extract (PS) and related polyphenol compounds such as catechin (C), epicatechin (EC), epicatechingallate (ECG), epigallocatechin (EGC), and epigallocatechingallate (EGCG) on the growth of human lymphoid leukemia Molt 4B cells. We found that PS, ECG, EGC, and EGCG strongly inhibited the growth of the cells in a dose-dependent manner, while C and EC inhibited the growth of the cells only moderately. Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, was inhibited by 10–20% by these polyphenol compounds. The morphology of the Molt 4B cells indicated severe damage 3 days after treatment with PS, ECG, EGC, and EGCG. Irregular shape of the cells and DNA fragmentation were observed in PS, ECG, EGC, or EGCG-treated cells. These results suggest that PS, ECG, EGC, and EGCG induce apoptosis (programmed cell death) of Molt 4B cells.     

Determination of catechins in human urine subsequent to tea ingestion by high-performance liquid chromatography with electrochemical detection

The title determination was conducted by HPLC with electrochemical detection using an ODS column and a mobile phase of acetonitrile: 0.1 M phosphate buffer (pH 2.5) (15:85, v/v). The eight catechins, gallocatechin (GC), epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin gallate (EGCg), gallocatechin gallate (GCg), epicatechin gallate (ECg), and catechin gallate (Cg), were detected at 0.6 V vs Ag/AgCl. Good linear relationships between current and amount were noted for 0.5-250 pmol of each catechin, with a correlation coefficient of 0.999 in each case. The detection limit for any one was 0.5 pmol (signal to noise ratio, S/N = 3). After the ingestion of 340 ml canned green tea, GC, EGC, C, and EC, mostly in conjugated form, were determined in urine samples. Conjugated catechins were hydrolyzed by enzymes using sulfatase and beta-glucuronidase. The time courses of the above four catechins showed a maxima at 1-3 h after tea ingestion. (+), (-)-EC and (+), (-)-C were present in canned tea.     

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

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

Effect on the epigallocatechin gallate/epigallocatechin ratio in a green tea (Camellia sinensis L.) extract of different extraction temperatures and its effect on IgA production in mice

We found that the epigallocatechin gallate (EGCG)/epigallocatechin (EGC) ratio in a green tea (Camellia sinensis L.) extract was affected by the extraction temperature. The EGCG/EGC ratio in the 4 °C extract was around 1:3-4, whereas in the 100 °C extract, it was around 1:0.7. Oral administration of the mixture with a high EGC ratio (1:2-3 = EGCG/EGC) resulted in greater IgA production by murine Peyer's patch cells.     

Synthesis and biological activity of the tea catechin metabolites, M4 and M6 and their methoxy-derivatives

Syntheses are reported for metabolites M4 (1) and M6 (2) of the green tea polyphenols epicatechin (EC) and epigallocatechin (EGC) and their gallate derivatives. Several methoxy-derivatives of 1 and 2 were also prepared. Compounds 1 and 2 were evaluated for growth inhibitory activity against a panel of immortalized and malignant human cell lines with 1 being the more active compound. The possible antiinflammatory activity of 1 and its trimethoxy derivative was also evaluated. Neither compound inhibited the release of arachidonic acid, although 1 inhibited NO production by 50% at 20 microM.     

N-Acylation of N-Desulfated Heparin

We found that the epigallocatechin gallate (EGCG)/epigallocatechin (EGC) ratio in a green tea (Camellia sinensis L.) extract was affected by the extraction temperature. The EGCG/EGC ratio in the 4 °C extract was around 1:3-4, whereas in the 100 °C extract, it was around 1:0.7. Oral administration of the mixture with a high EGC ratio (1:2-3 = EGCG/EGC) resulted in greater IgA production by murine Peyer’s patch cells.     

Antioxidant synergism of green tea polyphenols with alpha-tocopherol and L-ascorbic acid in SDS micelles

The synergistic antioxidant effect of polyphenols extracted from green tea, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), with alpha-tocopherol (vitamin E) and l-ascorbic acid (vitamin C) against the peroxidation of linoleic acid has been studied in sodium dodecyl sulfate (SDS) micelles. The peroxidation was initiated thermally by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), and the reaction kinetics were studied by monitoring the formation of linoleic acid hydroperoxides and consumption of the antioxidants. It was found that the mixture of the green tea polyphenol, vitamin E and vitamin C could act synergistically to protect lipid peroxidation. Kinetic and mechanistic studies on the antioxidation process revealed that this antioxidant synergism was due to the regeneration of vitamin E by the green tea polyphenol and the regeneration of the latter by vitamin C.     

Effects of green tea catechins on gramicidin channel function and inferred changes in bilayer properties

Green tea's health benefits have been attributed to its major polyphenols, the catechins: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), and epicatechin (EC). Catechins (especially EGCG) modulate a wide range of biologically important molecules, including many membrane proteins. Yet, little is known about their mechanism(s) of action. We tested the catechins' bilayer-modifying potency using gramicidin A (gA) channels as molecular force probes. All the catechins alter gA channel function and modify bilayer properties, with a 500-fold range in potency (EGCG>ECG?EGC>EC). Additionally, the gallate group causes current block, as evident by brief downward current transitions (flickers).     

Antioxidant effects of green tea polyphenols on free radical initiated peroxidation of rat liver microsomes

Antioxidative effects of the principal polyphenolic components extracted from green tea leaves, i.e. (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), and gallic acid (GA), against free radical initiated peroxidation of rat liver microsomes were studied. The peroxidation was initiated by a water-soluble azo compound 2,2′-azobis(2-amidinopropane hydrochloride (AAPH). The reaction kinetics was monitored by oxygen uptake and formation of malondialdehyde (MDA). Kinetic analysis of the antioxidation process demonstrates that these green tea polyphenols (GOHs), especially EC and ECG which bear ortho-dihydroxyl functionality, are good antioxidants for microsomal peroxidation. The antioxidant synergism of these GOHs with the endogenous -tocopherol (TOH) (vitamin E) is also discussed.     

Antioxidative effects of green tea polyphenols on free radical initiated and photosensitized peroxidation of human low density lipoprotein

Antioxidative effects of the main polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either thermally by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or photochemically by a triplet sensitizer benzophenone (BP). The reaction kinetics was monitored by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL. Kinetic analysis of the antioxidation process demonstrates that these green tea polyphenols are effective antioxidants against both AAPH-initiated and BP-photosensitized LDL peroxidation. The antioxidative action of the green tea polyphenols includes trapping the initiating and/or propagating peroxyl radicals with the activity sequence EC>EGCG>ECG>EGC>GA for the AAPH initiated peroxidation, and reducing the alpha-tocopheroxyl radical to regenerate alpha-tocopherol with the activity sequence of ECG>EC>EGCG>EGC>GA and ECG>EGCG>GA>EC>EGC for the AAPH-initiated and BP-photosensitized peroxidations respectively.