ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers.
The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)) and their corresponding epimers ((-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O-.2) generated in the irradiated riboflavin system, singlet oxygen(1O2) generated in the photoradiation-hemoporphyrin system, the free radicals generated from 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5' position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O-.2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed (aN=15.6 G and aHbeta=21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O-.2 and 1O2, especially in the case with EGCG and GCG with more bulky steric hindrance. (+info)
Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).
1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed. (+info)
Inhibition of benzo[a]pyrene-induced mutagenesis by (-)-epigallocatechin gallate in the lung of rpsL transgenic mice.
Epigallocatechin gallate (EGCG) is a major water-soluble component of green tea. The antimutagenic activity of EGCG against benzo[a]pyrene (B[a]P)-induced mutations was assessed by using transgenic mice carrying the rpsL gene as a monitor of mutations. Seven-week-old male mice were given drinking water containing EGCG for 3 weeks. On day 7, mice were treated with a single i.p. injection of B[a]P (500 mg/kg body wt). Two weeks after the injection, the mutations in the rpsL gene were analyzed. B[a]P treatment resulted in an approximately 4-fold increase of mutation frequency at the rpsL gene in the lung. An approximately 60% reduction in the B[a]P-induced mutations in the lung was observed when mice were given EGCG at concentrations >0.005%. B[a]P-induced mutations mainly occurred at G:C basepairs in the several specific nucleotide sequences of the rpsL gene. These were AGG, CGG, CGT, TGG, TGC and GGT: all of them contained a guanine residue. Mutations seen similarly in the human Ki-ras codon 12 or p53 codons 157, 248, and 273 of lung tumor were also found in the rpsL gene, and the mutations were suppressed by the EGCG treatment. In conclusion, the antimutagenic effects of EGCG for B[a]P-induced mutagenesis in vivo suggest that drinking green tea may reduce the tumor-initiating potency of B[a]P in the lung. (+info)
Methylation of tea catechins by rat liver homogenates.
Methylation of (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) was carried out with a rat liver homogenate and S-adenosyl-L-methionine. A structural analysis of the reaction products by MS and NMR showed that 4'-O-methyl EGC, 4"-O-methyl ECg, and 4"-O-methyl EGCg had been formed from EGC, ECg, and EGCg, respectively. These results suggest that methylation may be one of the metabolic pathways to the catechins. (+info)
Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).
Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea. (+info)
Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea.
Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-gamma, and tumor necrosis factor alpha in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-gamma and tumor necrosis factor alpha-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis. (+info)
Cancer chemopreventive mechanisms of tea against heterocyclic amine mutagens from cooked meat.
Cooking meat and fish under normal conditions produces heterocyclic amine mutagens, several of which have been shown to induce colon tumors in experimental animals. In our search for natural dietary components that might protect against these mutagens, it was found that green tea and black tea inhibit the formation of heterocyclic amine-induced colonic aberrant crypt foci (ACF) in the rat. Since ACF are considered to be putative preneoplastic lesions, we examined the inhibitory mechanisms of tea against the heterocyclic amines. In the initial studies using the Salmonella mutagenicity assay, green tea and black tea inhibited according to the concentration of tea leaves during brewing and the time of brewing; a 2-3-min brew of 5% green tea (w/v) was sufficient for >90% antimutagenic activity. N-hydroxylated heterocyclic amines, which are direct-acting mutagens in Salmonella, were inhibited by complete tea beverage and by individual components of tea, such as epigallocatechin-3-gallate (EGCG). Inhibition did not involve enhanced mutagen degradation, and EGCG and other catechins complexed only weakly with the mutagens, suggesting electrophile scavenging as an alternative mechanism. Enzymes that contribute to the metabolic activation of heterocyclic amines, namely microsomal NADPH-cytochrome P450 reductase and N, O-acetyltransferase, were inhibited by tea in vitro. Studies in vivo established that tea also induces cytochromes P450 and Phase II enzymes in a manner consistent with the rapid metabolism and excretion of heterocyclic amines. Collectively, the results indicate that tea possesses anticarcinogenic activity in the colon, and this most likely involves multiple inhibitory mechanisms. (+info)
Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (-)epicatechin.
The present study investigated the involvement of endothelial nitric oxide in relaxation induced by purified green tea (-)epicatechin in rat isolated mesenteric arteries. (-)Epicatechin caused both endothelium-dependent and -independent relaxation. NG-Nitro-L-arginine methyl ester (L-NAME, 100 microM) and methylene blue (10 microM) significantly attenuated (-)epicatechin-induced relaxation in endothelium-intact tissues. L-Arginine (1 mM) partially antagonized the effect of L-NAME. (-)Epicatechin-induced relaxation was inhibited by Rp-guanosine 3',5'-cyclic monophosphothioate triethylamine. In contrast, indomethacin and glibenclamide had no effect. (-)Epicatechin (100 microM) significantly increased the tissue content of cyclic GMP and NG-nitro-L-arginine (100 microM) or removal of the endothelium abolished this increase. (-)Epicatechin (100 microM) induced an increase in intracellular Ca2+ levels in cultured human umbilical vein endothelial cells. Iberiotoxin at 100 nM attenuated (-)epicatechin-induced relaxation in endothelium-intact arteries and this effect was absent in the presence of 100 microM L-NAME. In summary, (-)epicatechin-induced endothelium-dependent relaxation is primarily mediated by nitric oxide and partially through nitric oxide-dependent activation of iberiotoxin-sensitive K+ channels. In addition, there may be a causal link between increased Ca2+ levels and nitric oxide release in response to (-)epicatechin. (+info)