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.     

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.     

Effect of tea catechins on cellular lipid peroxidation and cytotoxicity in HepG2 cells

Tea catechins inhibited TBARS accumulation in HepG2 cells, the order of effectiveness being (-)-epigallocatechin gallate (EGCG) > (-)-epigallocatechin (EGC) > or = (-)-epicatechin gallate (ECG) > (-)-epicatechin (EC). EGCG and EGC protected the depletion of alpha-tocopherol in the cells, and the glutathione content was enhanced by all four catechins. Moreover, all four catechins suppressed the formation of glutathione disulfide and the activation of glutathione peroxidase induced by tert-butylated hydroperoxide.     

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.     

Enhancement of phagocytic activity of macrophage-like cells by pyrogallol-type green tea polyphenols through caspase signaling pathways

We investigated the phagocytosis-enhancing activity of green tea polyphenols, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) catechin (+C) and strictinin, using VD3-differentiated HL60 cells. EGCG, EGC, ECG and strictinin, but not EC and +C, increased the phagocytic activity of macrophage-like cells, and a caspase inhibitor significantly inhibited phagocytic activities. These results suggest that the pyrogallol-type structure in green tea polyphenols may be important for enhancement of the phagocytic activity through caspase signaling pathways.     

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.     

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).     

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.     

Overexpression of miR-7-1 Increases Efficacy of Green Tea Polyphenols for Induction of Apoptosis in Human Malignant Neuroblastoma SH-SY5Y and SK-N-DZ Cells

Neuroblastoma is an extracranial solid tumor that usually occurs in infants and children. Malignant neuroblastomas remain mostly refractory to currently available chemotherapeutic agents. So, new therapeutic agents and their molecular mechanisms for induction of cell death must be explored for successful treatment of human malignant neuroblastomas. Two polyphenolic compounds, which are abundant in green tea, are (?)-epigallocatechin (EGC) and (?)-epigallocatechin-3-gallate (EGCG) that possess impressive anti-cancer properties. It is not known yet whether EGC and EGCG can modulate the levels of expression of specific microRNAs (miRs) for induction of apoptosis in human malignant neuroblastomas. In this investigation, we revealed that treatment with EGC or EGCG caused induction of apoptosis with significant changes in expression of specific oncogenic miRs (OGmiRs) and tumor suppressor miRs (TSmiRs) in human malignant neuroblastoma SH-SY5Y and SK-N-DZ cell lines. Treatment of both cell lines with either 50 μM EGC or 50 μM EGCG decreased expression of the OGmiRs (miR-92, miR-93, and miR-106b) and increased expression of the TSmiRs (miR-7-1, miR-34a, and miR-99a) leading to induction of extrinsic and intrinsic pathways of apoptosis. Our data also demonstrated that overexpression of miR-93 decreased efficacy while overexpression of miR-7-1 increased efficacy of the green tea polyphenols for induction of apoptosis in both cell lines. In conclusion, our current investigation clearly indicates that overexpression of miR-7-1 can highly potentiate efficacy of EGCG for induction of apoptosis in human malignant neuroblastoma cells.     

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.     

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.     

Measurement of catechin and gallic acid in tea wine with HPLC

First a kind of fermented tea wine was prepared from Dancong tea. The content of four major catechins and gallic acid (EC, EGC, EGCG and ECG) in tea wine was measured with HPLC. The results showed that the content of EC, EGC, EGCG, ECG and catechins in tea wine decreased when compared with that before fermentation. The content of EC decreased the most, reaching 26.79%, while the content of GA changed the least with a decrease of only 13.56%. Nevertheless, tea wine still contains a relatively large amount of catechins, thus proper consumption of tea wine may be salubrious.     

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.     

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.     

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.     

Metabolism of green tea catechins by the human small intestine

Numerous studies have shown that green tea polyphenols can be degraded in the colon, and there is abundant knowledge about the metabolites of these substances that appear in urine and plasma after green tea ingestion. However, there is very little information on the extent and nature of intestinal degradation of green tea catechins in humans. Therefore, the aim of this study was to examine in detail the microbial metabolism and chemical stability of these polyphenols in the small intestine using a well-established ex vivo model. For this purpose, fresh ileostomy fluids from two probands were incubated for 24 h under anaerobic conditions with (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin 3-O-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatchin 3-O-gallate (EGCG) and gallic acid (GA). After lyophilisation and extraction, metabolites were separated, identified and quantified by high performance liquid chromatography-photodiode array detection (HPLC-DAD) and HPLC-ESI-tandem mass spectrometry. Two metabolites of EC and C (3', 4', 5'-trihydroxyphenyl-γ-valerolactone and 3', 4'-dihydroxyphenyl-γ-valerolactone) were identified. In addition, 3', 4', 5'-trihydroxyphenyl-γ-valerolactone was detected as a metabolite of EGC, and (after 24-h incubation) pyrogallol as a degradation product of GA. Cleavage of the GA esters of EGCG and ECG was also observed, with variations dependent on the sources (probands) of the ileal fluids, which differed substantially microbiotically. The results provide new information about the degradation of green tea catechins in the gastrointestinal tract, notably that microbiota-dependent liberation of GA esters may occur before these compounds reach the colon.     

Enhancement of inward Ca(2+) currents in bovine chromaffin cells by green tea polyphenol extracts.

Green tea contains four major polyphenol compounds: they are (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC). Although all four polyphenol compounds are known to affect tumor suppression, little is known about whether they alter membrane properties. In this study, we examined the effects of ECG and EGCG on ionic currents and secretion. Membrane capacitance changes were used to monitor secretion in bovine chromaffin cells. ECG had the ability to reversibly enhance the inward Ca(2+) current by 21%, and inhibited the peak sodium current by 34%. EGCG had no effect on Ca(2+) current even though it differs from ECG by just a hydroxyl group. The EC(50) of ECG in enhancing Ca(2+) current was 7.6 microM. The maximum enhancement of Ca(2+) current was observed at 0 mV and the maximum current was shifted approximately 10 mV in the hyperpolarizing direction. When cells were stimulated by trains of depolarizations, the exocytosis elicited was enhanced by ECG treatment and the largest enhancement of secretion was observed in later stimulations. EGCG, although it had no significant effect on Ca(2+) current, enhanced exocytosis and slowed endocytosis. These results suggest that green tea polyphenol compounds modulate stimulus-secretion coupling in bovine chromaffin cells.     

Tea flavanols inhibit cell growth and DNA topoisomerase II activity and induce endoreduplication in cultured Chinese hamster cells

Tea polyphenols are promising chemopreventive anticancer agents, the properties of which have been studied both in vitro and in vivo, providing evidence that - within this group of compounds - the tea flavanols are able to inhibit carcinogenesis, an effect that in some cases could be correlated with increased cell apoptosis and decreased cell proliferation. Of four main tea flavanols, namely (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (+)-catechin (CA) and (-)-epicatechin (EC), it was found that EGCG was the most potent to inhibit dose dependently the topoisomerase II (TOPO II) catalytic activity isolated from hamster ovary AA8 cells. In the range of concentrations that caused TOPO II inhibition, a high level of endoreduplication, a rare phenomenon that consists in two successive rounds of DNA replication without intervening mitosis, was observed, while neither micronuclei nor DNA strand breaks (Comet assay) were detected at the same doses. We propose that the anticarcinogenic effect of tea flavanols can be partly explained by their potency and effectiveness to induce endoreduplication. Concerning such an induction, maximum effect seems to require a pyrogallol structure at the B-ring. Additional substitution with a galloylic residue at the C3 hydroxyl group leads to further augmentation of the effect. Thus, we suggest that the chemopreventive properties of tea flavanols can be at least partly due to their ability to interfere with the cell cycle and block cell proliferation at early stages of mitosis.     

Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−) epicatechin-3-gallate (ECG), (−) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 μM) caused a significant decrease in cell viability to 4.1±0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 μM) also caused a slight decrease in cell viability (70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.