Investigation on the binding of （-）-epigallocatechin-3-O-gallate to bovine serum albumin by molecular spectroscopy

The binding of （-）-epigallocatechin-3-O-gallate （EGCG） to bovine serum albumin （BSA） were investigated for the first time with spectral methods, including fluorescence and absorption spectrometry under simulative physiological conditions. A strong fluorescence quenching reaction of EGCG to BSA was observed. It was proved that the fluorescence quenching of BSA by addition of EGCG was a result of the formation of EGCG-BSA complex. The binding constant K and the number of binding sites n were determined at physiological conditions and three different temperatures with fluorescence quenching method. The binding distance R and transfer efficiency E between BSA and EGCG were also obtained according to Forster theory of non-radiation energy transfer. The effects of Al^3＋, Cu^2＋, Mg^2＋ and Fe^2＋ on the binding constant between EGCG and BSA were studied at 298 K. The effect of EGCG on the conformation of BSA was also analyzed by synchronous fluorescence spectroscopy. PACS. 21. 10. Dr; 32. 50. ＋d; 32. 30. Jc; 82.80. Dx

Suppression of esophageal cancer cell growth using curcumin,(-)-epigallocatechin-3-gallate and lovastatin

AIM:To determine the effects of curcumin,(-)-epigallocatechin-3-gallate (EGCG),lovastatin,and their combinations on inhibition of esophageal cancer.METHODS:Esophageal cancer TE-8 and SKGT-4 cell lines were subjected to cell viability methyl thiazolyl tetrazolium and tumor cell invasion assays in vitro and tumor formation and growth in nude mouse xenografts with or without curcumin,EGCG and lovastatin treatment.Gene expression was detected using immunohistochemistry and Western blotting in tumor cell lines,tumor xenografts and human esophageal cancer tissues,respectively.RESULTS:These drugs individually or in combinations significantly reduced the viability and invasion capacity of esophageal cancer cells in vitro.Molecularly,these three agents reduced the expression of phosphorylated extracellular-signal-regulated kinases (Erk1/2),c-Jun and cyclooxygenase-2 (COX-2),but activated caspase 3 in esophageal cancer cells.The nude mouse xenograft assay showed that EGCG and the combinations of curcumin,EGCG and lovastatin suppressed esophageal cancer cell growth and reduced the expression of Ki67,phosphorylated Erk1/2 and COX-2.The expression of phosphorylated Erk1/2 and COX-2 in esophageal cancer tissue specimens was also analyzed using immunohistochemistry.The data demonstrated that 77 of 156 (49.4%) tumors expressed phosphorylated Erk1/2 and that 121 of 156 (77.6%) esophageal cancers expressed COX-2 protein.In particular,phosphorylated Erk1/2 was expressed in 23 of 50 (46%) cases of esophageal squamous cell carcinoma (SCC) and in 54 of 106 (50.9%) cases of adenocarcinoma,while COX-2 was expressed in 39 of 50 (78%) esophageal SCC and in 82 of 106 (77.4%) esophageal adenocarcinoma.CONCLUSION:The combinations of curcumin,EGCG and lovastatin were able to suppress esophageal cancer cell growth in vitro and in nude mouse xenografts,these drugs also inhibited phosphorylated Erk1/2,c-Jun and COX-2 expression.

Inhibition of Invasion and Up-regulation of E-cadherin Expression in Human Malignant Melanoma Cell Line A375 by(-)-Epigallocatechin-3-gallate

The inhibitory effects of （-）-epigallocatechin-3-gallate （EGCG） on the invasion of human malignant melanoma cell line A375 and the possible molecular mechanisms of this effect were investiaged. A375 cells were pretreated with 20 μg/mL EGCG for 24, 48 and 72 h respectively and the E-cadherin expression was detected by Western blot analysis. A375 cells were also pretreated with different concentrations of EGCG （1, 5, 10 and 20 μg/mL） for 72 h and the expression of E-cadherin was measured by RT-PCR. The adhesion and invasion of A375 cells were tested by cell-matrigel adhesion assay and matrigel invasion assay respectively. The results showed that EGCG could significantly up-regulate the expression of E-cadherin time-and concentration-dependently （both P〈0.05）. Statistical analysis showed that A375 cells invasion was inhibited by EGCG and correlated with the up-regulation of E-cadherin expression. It was suggested that EGCG strongly inhibited invasion of A375 cells, and the inhibition mechanism was possibly associated with the up-regulation of E-cadherin expression.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes

AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Protective effects of (-)-epigallocatechin-3-gallate on D-galactose-induced neuronal apoptosis in mice

BACKGROUND： The neuroprotective effects of （-）-epigallocatechin-3-gallate （EGCG）, the main polyphenolic constituent of green tea, have been widely reported. However, the action mechanisms, in particular in D-galactose-induced aging mice, remain poorly understood. OBJECTIVE： The present study investigated the protective effects of EGCG on D-galactose-induced hippocampus neuronal apoptosis in aging mice, as well as the relationship with expression of p751CD, JNK2, and p53 proteins. DESIGN, TIME AND SETTING： A randomized, controlled, molecular biological, animal experiment was performed at the Laboratory of Pharmacology, Pharmaceutical College of China Medical University, China, from September 2006 to July 2008. MATERIALS： D-galactose and EGCG （Sigma, USA）, as well as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling （TUNEL） In Situ Cell Apoptosis Detection Kit （Promega, USA）, were used in this study. METHODS： A total of 64 mice were equally and randomly divided into D-galactose model, low-dose EGCG, high-dose EGCG, and control groups. Mice in the D-galactose model, low-dose EGCG, and high-dose EGCG groups were subcutaneously injected with 3% D-galactose （150 mg/kg）, daily for 6 weeks, to establish a mouse model of aging. Mice in the control group were treated with saline （5 mL/kg）. At 3 weeks following injection, mice in the low-dose EGCG and high-dose EGCG groups were orally administered EGCG at a dose of 2 mg/kg and 6 mg/kg, respectively, once a day, for 4 consecutive days. Mice in the control and D-galactose model groups received distilled water （5 mL/kg）. MAIN OUTCOME MEASURES： Memory function was evaluated using a step-through passive avoidance test. Neuronal apoptosis in the mouse hippocampus was detected using TUNEL staining. Expression levels of the intracellular domain of the p75 neurotrophin receptor （p75NTR）-p751CD, JNK2, and p53 proteins in the hippocampus were determined using Western blot analysis. RESULTS： The aging mouse model was induced by subcutaneous injection of D-galactose, which resulted in obvious memory impairment, increased apoptotic index, and increased protein expression levels of p751CD, JNK2, and p53 in the hippocampus, compared with control mice （P 〈 0.01）. Oral EGCG administration （2 or 6 mg/kg） for 4 weeks significantly improved levels of memory deficit in the aging mice and reduced apoptotic indices and protein expression levels of p751CD, JNK2, and p53 in the mouse hippocampus （P 〈 0.01）. CONCLUSION： Results from this study demonstrated increased protein expression levels of p751CD, JNK2, and p53, as well as increased hippocampal neuronal apoptosis in a D-galactose-induced mouse model of aging. EGCG provided protective effects against D-galactose-induced neuronal apoptosis in the hippocampus by reducing protein expression levels of p751CD, JNK2, and p53 proteins in the hippocampus of aging mice.

Integrating network pharmacology and pharmacological evaluation for deciphering the mechanism of(-)-epigallocatechin-3-gallate alleviating ethanol-induced endothelial cells injury

Objective To investigate the potential therapeutic targets and pharmacological mechanism of(-)-epigallocatechin-3-gallate(EGCG)based on network pharmacology and experimental verification.METHODS The druggability of EGCG was measured by the traditional Chinese medicine systems pharmacology(TCMSP)server,and potential targets of EGCG were identified by Pharm Mapper and Drug Repositioning and Adverse drug Reaction via Chemical-Protein Interactome(DRAR-CPI).The potential targets were imported into GeneMANIA database to obtain the protein-protein direct interaction network,and target physical interaction,co-expression,prediction,genetic interaction,and shared protein domains.The biological process,molecular functions,cellular components and KEGG signaling pathways of potential targets were analyzed using DAVID database.For further study,ethanol was used to establish a model of endothelial injury in vitro.The cell viability was assayed by MTT method,the cellular apoptosis was stained by Annexin V/PI,and the expression levels of Bcl-2,Bax and cleved-caspase-3 were tested by Western blotting.Then,JC-1 and nuclear translocation of NF-κB experiments were used to study the mitochondrial membrane potential and nuclear translocation.RESULTS The oral availability of EGCG was 55.09%(≥30%)and drug-like index was 0.77(≥0.18),which were considered pharmacokinetically active.17 potential targetable proteins of EGCG were predicted by Pharm Mapper and DRAR-CPI.Further research showed that 68.13%displayed similar co-expression characteristics,26.11%physical interactions,and 2.74%shared the same protein domain.The depth network analysis results showed that the biofunctions of EGCG were mainly by regulating glutathione derivative biosynthetic process,glutathione metabolic process,nitrogen compound metabolic process etc..via drug binding,catalytic activity,glutathione transferase activity,anion binding etc..in sarcoplasmic reticulum,spindle pole,microtubule cytoskeleton and cytoplasm.KEGG enrichment analysis showed that Glutathione metabolism,IL^(-1)7 signaling pathway,EGFR tyrosine kinase inhibitor resistance,PI3K-Akt signaling pathway and other pathways were involves in the biofunction of EGCG.The above analyses indicated that EGCG exerts its biofunction through antioxidant and anti-inflammatory mechanisms.The experimental results showed that ethanol 20.0 mmol·L^(-1) decreased cell viability,Bcl-2 expression,and increased cell apoptosis,the intracellular ROS,as well as the expression of Bax and cleaved-caspase-3 of human endothelial cells.However,treatment of the cells with EGCG can significantly alleviate ethanol induced endothelial cells injury.Further study showed that EGCG significantly alleviates ethanol induced mitochondrial depolarization and nuclear translocation of NF-κB.CONCLUSIONS EGCG exerts pharmacological efficacies on ethanol induced endothelial cell injury through multi-target,multi-function and multi-pathway mode.Protective effect of EGCG on ethanol induced cell injury was mainly through alteration of mitochondrial function and NF-κB translocation.Therefore,EGCG have great potential in protecting against endothelial dysfunction of the persons who are chronically abuse of ethanol.This study also provides a new understanding of EGCG in clinical application on cardiovascular and cerebrovascular diseases.

Influence of (-)-epigallocatechin-3-gallate on the expression of connexin43 and gap junction intercellular communication of the bladder cancer cell lines BIU-87 in vitro

Objective： The aim of our study was to investigate the effects of （-）-epigallocatechin-3-gallate （EGCG, the major phytochemistry component in green tea） on the expression of connexin43 （Cx43） gene and detect the intercellular communication of the human bladder cancer cell lines BIU-87, and explore its possible mechanisms of prevention and cure for the bladder tumor. Methods： The methyl thiazolyl tetrazolium and Annexin-V/PI double-labeled flow cytometry methods were used to observe the growth inhibitory rate （IR） and apoptosis rate （AR） of BlU-87 cells treated by EGCG at different concentrations （0, 5, 10 and 20 mg/L）, respectively. The reverse transcription-polymerase chain reaction （RT-PCR） and Western Blotting analysis were employed to detect the relative expression levels of the Cx43 mRNA and its protein. The scrape-loading fluorescence dye transfer method was used to assess the gap junction intercellular communication （GJIC） under fluorescence microscope. Results： EGCG at concentrations （10 and 20 mg/L） both could significantly inhibit the proliferation and induce the apoptosis of BIU-87 cells. The IR and AR were （15.67 ± 1.15）%, （18.33 ± 1.53）% and （42.00 ± 4.34）%, （27.33 ± 3.21）%, respectively. And compared with the control groups of 0 mg/L and 5 mg/L （P 〈 0.05）, EGCG could significantly up-regulate the expression of Cx43 mRNA and its protein and enhance the function of BIU-87 cells. The effects had the significant correlation with the dose-dependent of EGCG. Conclusion： EGCG （10, 20 mg/L） could effectively up-regulate Cx43 expression and enhance the GJIC of BlU-87 cells. The results may indicate the effects of EGCG inducing bladder tumor cells apoptosis and inhibiting its growth which provides the experimental evidence for further demonstrating the mechanism of chemical prevention and cure for the bladder tumor by EGCG.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.

Effects of (-)-epigallocatechin-3-gallate on expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in fibroblasts irradiated with ultraviolet A

Background It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways ( ) epigallocatechin 3 gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet induced damage In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro Methods Transcription factor Jun protein levels were measured by Western blot Matrix metalloproteinase 1 (MMP 1) and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA were studied by reverse transcription polymerase chain reaction (RT PCR) analysis in conjunction with computer assisted image analysis MMP 1 and TIMP 1 proteins were quantified by enzyme linked immunosorbent assay (ELISA) Results EGCG decreased transcription activity of Jun protein after induction by UVA Both the mRNA and protein levels of MMP 1 were increased by UVA irradiation, while no significant changes were observed in TIMP 1 levels The ratio of MMP 1 to TIMP 1 showed statistically significant differences compared with the control EGCG decreased the ratio of MMP 1 to TIMP 1 by inhibiting UVA induced MMP 1 expression ( P <0 05) Conclusion EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP 1 The ratio of MMP 1 to TIMP 1, rather than the levels of MMP 1 or TIMP 1 alone, may play a significant role in human skin photodamage

Synergic Inhibition of Lung Carcinoma 95-D Cell Proliferation and Invasion by Combination with(—)-Epigallocatechin-3-Gallate and Ascorbic Acid

In this study, the anti-invasion effects of（-）-epigallocatechin-3-gallate（EGCG） mixed with ascorbic acid（Vc） on human lung carcinoma 95-D cells in vitro were examined and the synergism of the combination of EGCG and Vc was evaluated. Soft agar colony formation assay, cell migration assay, invasion assay, western blot analysis of NF-κB, in situ detection of cellular oxidative stress, and statistical analysis were assessed. The results showed that combining EGCG with Vc could inhibit clone forming rate of 95-D cell by 73.2%, reduce the migration ability of 95-D cell by 65.9%, and decrease the intracellular reactive oxygen species（ROS） level by 76.8%. The results of western blot proved that Vc enhanced the activity of EGCG in inhibiting NF-κB localization. It is speculated that the combination of EGCG and Vc can strongly suppress the proliferation and metastasis of lung carcinoma cells in a synergic manner, possibly with a mechanism associated with the scavenging of reactive oxygen species.

EGCG对糖尿病肾病模型大鼠肾小管间质性损害的治疗作用及机制

目的探讨(-)-Epigallocatechin 3-O-Gallate(EGCG)对大鼠糖尿病肾病模型小管间质性损害以及纤维化的治疗作用。方法取60只Wistar大鼠,高糖、高脂饲料喂养4周后16只作为对照组,另44只组一次性腹腔内注射STZ 30 mg/kg制作糖尿病肾病模型,将其中39只存活、成膜的大鼠随机分为模型组(n=20)和治疗组(n=19)。各组均予高蛋白饲料喂养,治疗组予EGCG 1 g/d灌胃。第12周测定各组血糖、糖化血红蛋白(HbA1c)、Cr、BUN,尿β2微球蛋白(β2-MG),ELISA法测定血清TGF-β1水平,荧光分光光度法测定血清血管收缩素转换酶(AGEs)水平;取肾组织行病理检查,免疫组化或免疫荧光法检测肾小管间质TGF-β1、晚期糖基化终产物受体(RAGE)、VEGF表达。结果治疗组上述血清学及尿液指标均明显优于模型组;肾小管间质病理损害明显轻于模型组;肾小管间质中TGF-β1、RAGE、VEGF的表达明显低于模型组;P均<0.05。结论 EGCG能减轻糖尿病肾病模型大鼠肾小管间质损害并下调肾脏纤维化相关因素的表达,其作用机制可能与拮抗氧化应激有关。

Separation of epigallocatechin-3-gallate from crude tea polyphenols by using Cellulose diacetate graft β-cyclodextrin copolymer asymmetric membrane

This study demonstrates a new Cellulose diacetate graft b-cyclodextrin(CDA-b-CD)copolymer asymmetric membrane prepared by a phase inversion technique for the separation of(–)-epigallocatechin-3-gallate(EGCG)from other polyphenols in crude tea.The graft copolymer,CDA-b-CD,was synthesized by pre-polymerization of cellulose diacetate(CDA)and 1,6-hexamethylene-diisocyanate(HDI),which was then grafted with b-cyclodextrin(b-CD).Surface and cross-section morphologies of the CDA-b-CD membranes were analyzed by using scanning electron microscopy(SEM).Fourier transform infrared spectroscopy(FT-IR)indicated that the b-CD was grafted onto the CDA by chemical bonding.The influences of the HDI/CDA mass ratio and the catalyst mass fraction on the b-CD graft yield were investigated.The optimum conditions of a HDI/CDA mass ratio of 0.35 g$g–1 and a catalyst mass fraction of 0.18 wt-%produced ab-CD graft yield of 26.51 wt-%.The effects of the b-CD graft yield and the concentration of the polymer cast solution on the separation of EGCG were also investigated.Under optimum conditions of a b-CD graft yield of 24.21 wt-%and a polymer concentration of 13 wt-%,the purity of EGCG increased from 26.51 to 86.91 wt-%.

Natural compounds as anticancer agents: Experimental evidence

Cancer prevention research has drawn much attention worldwide. It is believed that some types of cancer can be prevented by following a healthy life style. Cancer chemoprevention by either natural or synthetic agents is a promising route towards lowering cancer incidence.In recent years, the concept of cancer chemoprevention has evolved greatly. Experimental studies in anima models demonstrate that the reversal or suppression of premalignant lesions by chemopreventive agents is achievable. Natural occurring agents such as dietary phytochemicals, tea polyphenols and resveratrol show chemopreventive activity in animal models. Moreover,clinical trials for testing the safety and efficacy of a variety of natural agents in preventing or treating human malignancy have been ongoing. Here, we summarize experimental data on the chemopreventive or tumor suppressive effects of several natural compounds including curcumin,(-)-epigallocatechin-3-gallate, resveratrol, indole-3-carbinol, and vitamin D.

Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells

Tea polyphenols have been shown to have anticancer activity in many studies.In the present study,we investigated effects of theaflavin-3-3'-digallate(TF3),one of the major theaflavin monomers in black tea,in combination with ascorbic acid(AA),a reducing agent,and(-)-epigallocatechin-3-gallate(EGCG),the main polyphenol presented in green tea,in combination with AA on cellular viability and cell cycles of the human lung adenocarcinoma SPC-A-1 cells.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay showed that the 50% inhibition concentrations(IC50) of TF3,EGCG,and AA on SPC-A-1 cells were 4.78,4.90,and 30.62 μmol/L,respectively.The inhibitory rates of TF3 combined with AA(TF3+AA) and EGCG combined with AA(EGCG+AA) at a molar ratio of 1:6 on SPC-A-1 cells were 54.4% and 45.5%,respectively.Flow cytometry analysis showed that TF3+AA and EGCG+AA obviously increased the cell population in the G0/G1 phase of the SPC-A-1 cell cycle from 53.9% to 62.8% and 60.0%,respectively.TF3-treated cells exhibited 65.3% of the G0/G1 phase at the concentration of its IC50.Therefore,TF3+AA and EGCG+AA had synergistic inhibition effects on the proliferation of SPC-A-1 cells,and significantly held SPC-A-1 cells in G0/G1 phase.The results suggest that the combination of TF3 with AA or EGCG with AA enhances their anticancer activity.