Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.     

The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampal neurons.

Previous evidence has indicated that the neuronal toxicity of amyloid beta (betaA) protein is mediated through oxygen free radicals and can be attenuated by antioxidants and free radical scavengers. Recent studies have shown that green tea polyphenols reduced free radical-induced lipid peroxidation. The purpose of this study was to investigate whether (-)-epigallocatechin gallate (EGCG) would prevent or reduce the death of cultured hippocampal neuronal cells exposed to betaA because EGCG has a potent antioxidant property as a green tea polyphenol. Following exposure of the hippocampal neuronal cells to betaA for 48 hours, a marked hippocampal neuronal injuries and increases in malondialdehyde (MDA) level and caspase activity were observed. Co-treatment of cells with EGCG to betaA exposure elevated the cell survival and decreased the levels of MDA and caspase activity. Proapoptotic (p53 and Bax), Bcl-XL and cyclooxygenase (COX) proteins have been implicated in betaA-induced neuronal death. However, in this study the protective effects of EGCG seem to be independent of the regulation of p53, Bax, Bcl-XL and COX proteins. Taken together, the results suggest that EGCG has protective effects against betaA-induced neuronal apoptosis through scavenging reactive oxygen species, which may be beneficial for the prevention of Alzheimer's disease.     

Potentiometric and (1)H NMR studies of complexation of Al(3+) with (-)-epigallocatechin gallate, a major active constituent of green tea.

The acid dissociation of (-)-epigallocatechin gallate (abbreviated as egcg) and its complexation with Al(3+) were studied by potentiometric titrations, and were compared with those of (-)-epicatechin (ec) and (-)-epigallocatechin (egc). In Al(3+)-ec and Al(3+)-egc reaction systems, [Al(LH(-2))](+), [Al(LH(-2))(OH)](0), and [Al(LH(-2))(2)](-) are formed, as reported for Al(3+)-catechin (c). Reactions between Al(3+) and egcg at pH <4.1 yield AlLH(-2) and AlLH(-3) species. The 1H NMR studies have shown that two hydroxyl groups of the gallate (D) ring are deprotonated and coordinated to an Al(3+) ion in [Al(egcgH(-2))](+). The AlLH(-3) species of egcg is supposed to be formulated as [Al(egcgH(-3))](0) in which one hydroxyl group of the pyrogallol (B) ring and two hydroxyl groups of the D ring are deprotonated; an Al(3+) ion is coordinated to two oxygen atoms of the D ring and one oxygen atom from the B ring of the neighboring chelate molecule, resulting in the formation of a polymeric structure. In the Al(3+) complex of egcg, the gallate group forms major coordinate bonds and results in solution properties that are different from those of ec, egc and c which have no gallate group.     

DNA cleavage activities of (-)-epigallocatechin, (-)-epicatechin, (+)-catechin, and (-)-epigallocatechin gallate with various kinds of metal ions

The DNA cleavage activities of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCg) were examined with 16 different metal ions. Cu(2+) with all the catechins facilitated DNA cleavage, while Ag+ with EGC and EC showed a strong repressive effect. The other metal ions examined showed little effect.     

A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG

Green tea has been shown to have many biological effects, including effects on metabolism, angiogenesis, oxidation, and cell proliferation. Unfortunately, the most abundant green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG is very unstable in neutral or alkaline medium. This instability leads to a low bioavailability. In an attempt to enhance the stability of (-)-EGCG, we introduced peracetate protection groups on the reactive hydroxyls of (-)-EGCG (noted in text as 1). HPLC analysis shows that the protected (-)-EGCG analog is six times more stable than natural (-)-EGCG under slightly alkaline conditions. A series of bioassays show that 1 has no inhibitory activity against a purified 20S proteasome in vitro, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural (-)-EGCG, indicating an intercellular conversion. Inhibition of cellular proteasome activity by 1 is associated with induction of cell death. Therefore, our results indicate that the protected analog 1 may function as a prodrug of the green tea polyphenol proteasome inhibitor (-)-EGCG.     

Immune cell activation and subsequent epithelial dysfunction by Staphylococcus enterotoxin B is attenuated by the green tea polyphenol (-)-epigallocatechin gallate

Bacterial superantigens (SAg) are potent T cell activators and when delivered systemically elicit a self-limiting enteropathy in mice. Also, SAg-stimulated human peripheral blood mononuclear cells (PBMC) increase enteric epithelial cell monolayer permeability in vitro. Epigallocatechin gallate (EGCG), the major polyphenol component of green tea (Camilla sinesis) leaf, has been presented as an anti-inflammatory agent. We tested the hypothesis that EGCG (10-100 microM) would block PBMC activation by the SAg, Staphylococcus aureus enterotoxin B (SEB, 1 microg/ml), thus preventing disruption of the epithelial barrier. Pretreatment or co-treatment of human PBMC or murine lymphnode cells with EGCG significantly reduced SEB-induced proliferation and IL-2, IFNgamma, and TNFalpha production. ConA-induced proliferation was also inhibited by EGCG (50 microM) co-treatment. These effects of EGCG were not due to induction of immune cell apoptosis, and were independent of EGCGs anti-oxidant activity, and inhibition of NF-kappaB or AP-1 activation. Moreover, addition of exogenous IL-2 (20 ng/ml) to the cultures could not overcome the immunosuppressive effect of EGCG. Culture supernatant from PBMC stimulated in the presence of EGCG failed to increase the permeability of T84 epithelial cell monolayers: a finding consistent with the reduced IFNgamma and TNFalpha production by SAg+EGCG treated PBMC. These data promote EGCG as a suppressor of T cell activation, and given the prominent role that bacteria and T cells play in inflammatory disease we suggest that EGCG could be a useful addition to current treatments for enteric immune disorders and T cell driven immunopathologies.     

Green tea extract and its major polyphenol (-)-epigallocatechin gallate improve muscle function in a mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy is a frequent muscular disorder caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that contributes to the stabilization of muscle fiber membrane during muscle activity. Affected individuals show progressive muscle wasting that generally causes death by age 30. In this study, the dystrophic mdx^5Cv mouse model was used to investigate the effects of green tea extract, its major component (–)-epigallocatechin gallate, and pentoxifylline on dystrophic muscle quality and function. Three-week-old mdx^5Cv mice were fed for either 1 or 5 wk a control chow or a chow containing the test substances. Histological examination showed a delay in necrosis of the extensor digitorum longus muscle in treated mice. Mechanical properties of triceps suræ muscles were recorded while the mice were under deep anesthesia. Phasic and tetanic tensions of treated mice were increased, reaching values close to those of normal mice. The phasic-to-tetanic tension ratio was corrected. Finally, muscles from treated mice exhibited 30–50% more residual force in a fatigue assay. These results demonstrate that diet supplementation of dystrophic mdx^5Cv mice with green tea extract or (–)-epigallocatechin gallate protected muscle against the first massive wave of necrosis and stimulated muscle adaptation toward a stronger and more resistant phenotype. pharmacotherapy; muscular disorders; dystrophic mdx^5cv mouse; muscle mechanical properties; muscle histology     

Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate act as inhibitors of Ribonuclease A

Green tea polyphenols, which have the ability to inhibit angiogenesis, form complexes with Cu(II), a known potent stimulator of blood vessel proliferation. Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate were found to inhibit the enzymatic activity of Ribonuclease A (RNase A) as revealed by an agarose gel based assay and urea denatured gel electrophoresis. The copper complexes were found to be non-competitive inhibitors of RNase A with inhibition constants in the micromolar range. Changes in the secondary structure of the protein are found to occur due to the interaction as revealed from Fourier transform infrared and circular dichroism studies.     

Human hair growth enhancement in vitro by green tea epigallocatechin-3-gallate (EGCG)

Green tea is a popular worldwide beverage, and its potential beneficial effects such as anti-cancer and anti-oxidant properties are believed to be mediated by epigallocatechin-3-gallate (EGCG), a major constituent of polyphenols. Recently, it was reported that EGCG might be useful in the prevention or treatment of androgenetic alopecia by selectively inhibiting 5alpha-reductase activity. However, no report has been issued to date on the effect of EGCG on human hair growth. This study was undertaken to measure the effect of EGCG on hair growth in vitro and to investigate its effect on human dermal papilla cells (DPCs) in vivo and in vitro. EGCG promoted hair growth in hair follicles ex vivo culture and the proliferation of cultured DPCs. The growth stimulation of DPCs by EGCG in vitro may be mediated through the upregulations of phosphorylated Erk and Akt and by an increase in the ratio of Bcl-2/Bax ratio. Similar results were also obtained in in vivo dermal papillae of human scalps. Thus, we suggest that EGCG stimulates human hair growth through these dual proliferative and anti-apoptotic effects on DPCs.     

Antimitogenic effect of green tea (-)-epigallocatechin gallate on 3T3-L1 preadipocytes depends on the ERK and Cdk2 pathways

Green tea catechins, especially (–)-epigallocatechin gallate (EGCG), have been proposed as a chemopreventative for obesity, diabetes, cancer, and cardiovascular diseases. However, relatively little is known about the mechanism of the action of EGCG on fat cell function. This study was designed to investigate the pathways of EGCG's modulation of the mitogenesis of 3T3-L1 preadipocytes. Preadipocyte proliferation as indicated by an increased number of cells and greater incorporation of bromodeoxyuridine (BrdU) was inhibited by EGCG in dose-, time-, and growth phase-dependent manners. Also, EGCG dose and time dependently decreased levels of phospho-ERK1/2, Cdk2, and cyclin D₁ proteins, reduced Cdk2 activity, and increased levels of G₀/G₁ growth arrest, p21^waf/cip, and p27^kip1, but not p18^ink, proteins and their associations to Cdk2. However, neither MEK1, ERK1/2, p38 MAPK, phospho-p38, JNK, nor phospho-JNK was changed. Increased phospho-ERK1/2 content and Cdk2 activity, respectively, via the transfection of MEK1 and Cdk2 cDNA into preadipocytes prevented EGCG from reducing cell numbers. These data demonstrate the ERK- and Cdk2-dependent antimitogenic effects of EGCG. Moreover, EGCG was more effective than epicatechin, epicatechin gallate, and epigallocatechin in changing the mitogenic signals. The signal of EGCG in reducing growth of 3T3-L1 preadipocytes differed from that of 3T3 fibroblasts. Results of this study may relate to the mechanism by which EGCG modulates body weight. 3T3-L1 preadipocyte; mitogen-activated protein kinase; cyclin-dependent kinase     

Studies on the interaction of copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate with calf thymus DNA

The interaction of copper complexes of (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) with calf thymus DNA (ct-DNA) was investigated by UV-visible (UV-Vis), fluorescence and circular dichroism along with melting studies. It was observed that both copper complexes quench the fluorescence intensity of ethidium bromide bound ct-DNA upon binding, resulting in a ground state complex formation by a static quenching process. The binding constants evaluated from fluorescence data were supported by the UV-Vis study. The values ranged from 0.84 to 1.07 × 10⁵ M⁻¹ and 1.14 to 1.04 × 10⁵ M⁻¹ for Cu(II)-ECG and Cu(II)-EGCG, respectively for the temperature range 21-42 °C with two binding sites. Thermodynamic parameters obtained are suggestive of the involvement of different modes of interaction during binding for each complex although both were found to be intercalating in nature. Circular dichroism studies and variations in the melting temperature reveal unwinding of the ct-DNA helix with conformational changes due to binding.     

Inhibitory effect of green tea (-)-epigallocatechin gallate on resistin gene expression in 3T3-L1 adipocytes depends on the ERK pathway

Resistin (Rstn) is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. By contrast, green tea catechins, especially (-)-epigallocatechin gallate (EGCG), have been reported as body weight and diabetes chemopreventatives. Whether EGCG regulates production of Rstn is unknown. Using 3T3-L1 adipocytes, we found that EGCG at 20 and 100 microM suppressed Rstn mRNA levels by approximately 35 and 50%, respectively, after 3 h. The basal half-life of Rstn mRNA induced by actinomycin D was >12 h but shifted to 3 h in the presence of EGCG. This suggests that EGCG regulates the stability of Rstn mRNA. Treatment with cycloheximide did not prevent EGCG-suppressed Rstn mRNA levels, which suggests that the effect of EGCG does not require new protein synthesis. Intracellular Rstn protein significantly decreased in the presence of 100 microM EGCG 3 h after treatment, whereas the release of the Rstn protein did not significantly change. This suggests that EGCG may modulate the distribution of Rstn protein between the intracellular and extracellular compartments. EGCG did not affect the amounts of extracellular signal-related kinase-1/2 (ERK1/2), phospho-JNK, phospho-p38, and phospho-Akt proteins but reduced the amounts of phospho-ERK1/2 proteins. Overexpression with MEK1 blocked EGCG-inhibited Rstn mRNA expression. These data suggest that EGCG downregulates Rstn expression via a pathway that is dependent on the ERK pathway.     

Antiproliferative and apoptosis-inducing activities of alkyl gallate and gallamide derivatives related to (-)-epigallocatechin gallate

Green tea and (-)-epigallocatechin gallate (EGCG: one of the main components of green tea) are reported to have cancer-preventive activity in humans. A previous SAR study of EGCG and derivatives indicated that a galloyl group is essential for the activity. To test this hypothesis, we synthesized various alkyl gallate and gallamide derivatives and evaluated their antiproliferative effects on human leukemia HL-60 cells. Dodecyl 3,4,5-trihydroxybenzoate (6c) showed the most potent activity, being more potent than EGCG. To clarify the molecular mechanism of the antiproliferative action, we investigated the effects of 6c on various factors. Compound 6c was found to induce apoptosis mediated by endoplasmic reticulum (ER)-stress-related caspase-12. Upregulation of gadd-153, an ER-stress marker protein, was also observed. These results indicate that 6c induced apoptosis via the ER-stress-related pathway.     

Chromosome analysis of Murrah buffalo (Bubalus bubalis ) spermatozoa using zona-free hamster oocytes

An interspectific in vitro fertilization system was adopted to analyse sperm chromosomes of Murrah buffalo (Bubalus bubalis ). Superovulation was induced in mature female golden hamsters (Mesocricetus auratus ) to obtain a large number of oocytes. The zona pellucidae were digested by trypsin treatment. Zona free hamster oocytes were penetrated by buffalo spermatozoa capacitated with calcium ionophore A23187. Fertilized ova were cultured in TC 199 medium supplemented with 10% fetal calf serum (FCS). Podophyllotoxin and vinblastine were used to interrupt karyogamy and tubulin polymerization, respectively. Oocytes were fixed by modified gradual fixation air drying method. Slides were stained with 2% Giemsa for 45 min. Analyzable metaphase chromosome spreads were obtained from 22.4+/-3.8% of the penetrated oocytes. Of the 70 sexed spermatozoa, 38 were X-bearing and 32 were Y-bearing spematozoa.     

Cytogenetics of human oocytes,zygotes, and embryos after in vitro fertilization

Summary Chromosome errors, inherited or arising de novo during gametogenesis and transmitted at fertilization to the conceptus, may be a major cause of embryonic mortality. The in vitro fertilization and embryo transfer (IVF/ET) procedure provides extra material — oo-cytes, zygotes, and embryos — to investigate the contribution of chromosomal abnormality to implantation failure. This paper reviews the results of cytogenetic studies on such material. Estimates from a total of 1120 oocytes from 11 studies give an overall proportion of chromosomal abnormality of 35%. Single and multiple nullisomies and disomies are found, involving nonrandom chromosome gain or loss. Hypohaploid complements are more frequent than hyperhaploid complements. The higher rate of chromosome loss of hypohaploid karyotypes was found to be largely artifactual. The estimated overall frequency of aneuploidy is 13%. In embryos the level of chromosomal abnormality is 23%–40%. Errors of fertilization are responsible for a substantial number of triploid embryos, many of which develop into mosaics. Factors extrinsic to the conceptus, such as infertility, advanced maternal age, and ovarian hyperstimulation, may increase the level of chromosomal abnormality. More refined methods for accurately recognizing and selecting chromosomally normal embryos for transfer are needed to improve the success rate of this reproductive technology.     

Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4

A characteristic of many enteropathies is increased epithelial permeability, a potentially pathophysiological event that can be evoked by T helper (Th)-1 (i.e., IFN-gamma) and Th2 (i.e., IL-4) cytokines and bacterial infection [e.g., enteropathogenic Escherichia coli (EPEC)]. The green tea polyphenol (-)-epigallocatechin gallate (EGCG) has immunosuppressive properties, and we hypothesized that it would ameliorate the increased epithelial permeability induced by IFN-gamma, IL-4, and/or EPEC. EGCG, but not the related epigallocatechin, completely prevented the increase in epithelial (i.e., T84 cell monolayer) permeability caused by IFN-gamma exposure as gauged by transepithelial resistance and horseradish peroxidase flux; EGCG did not alleviate the barrier disruption induced by IL-4 or EPEC. IFN-gamma-treated T84 and THP-1 (monocytic cell line) cells displayed STAT1 activation (tyrosine phosphorylation on Western blot analysis, DNA binding on EMSA) and upregulation of interferon response factor-1 mRNA, a STAT1-dependent gene. All three events were inhibited by EGCG pretreatment. Aurintricarboxylic acid also blocked IFN-gamma-induced STAT1 activation, but it did not prevent the increase in epithelial permeability. Additionally, pharmacological blockade of MAPK signaling did not affect IFN-gamma-induced epithelial barrier dysfunction. Thus, as a potential adjunct anti-inflammatory agent, EGCG can block STAT1-dependent events in gut epithelia and monocytes and prevent IFN-gamma-induced increased epithelial permeability. The latter event is both a STAT1- and MAPK-independent event.     

Formation of antioxidants from (-)-epigallocatechin gallate in mild alkaline fluids, such as authentic intestinal juice and mouse plasma

The oxidative dimerization of (-)-epigallocatechin gallate (EGCG), the major catechin of tea leaves (Camellia sinensis L.), in authentic intestinal juice (pH 8.5) and mouse plasma (pH 7.8) was investigated. EGCG was unstable in the alkaline solutions over pH 7.4. The content of EGCG was decreased to 19.4% and 60.7% at 5 minutes in the intestinal juice and plasma, respectively. Three products-P-1 (theasinensin A), P-2 (a new dimerized product reported in a previous paper), and P-3 (theasinensin D, a rotational isomer of P-1)-were detected in these fluids. The sum of the molar contents of the three products formed after 5 minutes of incubation at 37 degrees C corresponded to 35.1% and 21.9% of the degraded molar content of EGCG, respectively. These dimerization products of EGCG would be formed by the dehydrogenation and decarboxylation of EGCG under oxidative conditions in alkaline solutions. The formation of P-2 was greater than that of P-1 and of P-3 at 30 minutes of incubation in the intestinal juice and mouse plasma. Fe(2+)-chelating activities of the three products were much higher than that of EGCG, and superoxide anion radical-scavenging activity of P-2 was also significantly higher than that of EGCG. The absorbance of P-2 administered to male ddY mice was studied. The content of P-2 in mouse plasma was less than that of administration of EGCG, but P-2 was absorbed quickly within 30 minutes and metabolized slowly. These dimerization products of EGCG are expected to contribute to in vivo antioxidative activities enhanced by tea drinking.     

Effects of (-)-epigallocatechin gallate on the redox reactions of human hemoglobin

The toxicity of acellular hemoglobin (Hb)-based therapeutics has been attributed in part to the uncontrolled oxidative reactions. A variety of antioxidant strategies to ameliorate potential oxidative damage in vivo have been suggested. We have examined the effects of (-)-epigallocatechin gallate (EGCG), a green tea polyphenol compound widely regarded as a chain-breaking antioxidant, on the oxidative stability of diaspirin crosslinked Hb (DBBF) and its cytotoxic ferryl intermediate. DBBF (ferrous) was rapidly oxidized to the ferric form in the presence of EGCG relative to the normal spontaneous oxidation of this Hb. The fast elimination of ferrous Hb is probably due to the ability of EGCG to produce hydrogen peroxide (H2O2) as these reactions were almost completely reversed by the addition of catalase and superoxide dismutase to the reaction medium. EGCG, however, effectively reduced ferryl back to ferric Hb in a biphasic kinetic reaction at physiological pH. At acidic pH where the autoreduction of protonated ferryl Hb is enhanced, a monophasic reduction process of the ferryl heme is achieved. A balance between pro and antioxidant properties of EGCG should be taken into account if EGCG is used in combination therapy with redox active acellular Hbs.