Enzymatic production of epigallocatechin by using an epigallocatechin gallate hydrolase induced from Aspergillus oryzae

Catechins are a group of polyphenolic compounds that are antioxidants having beneficial biological activities. There are four main catechins in green tea, and each has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new medicine from catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, by chemical synthesis, or by biotransformation reactions with high yield and specificity. In this study epigallocatechin gallate (EGCG) can be hydrolyzed to epigallocatechin (EGC) by a hydrolase from Aspergillus oryzae after induction by addition of EGCG to the cultures. However, cultures without EGCG induction did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 70%. Thin layer chromatography and high performance liquid chromatography were applied to separate and quantify EGCG and EGC.     

Food-drug interaction of (-)-epigallocatechin-3-gallate on the pharmacokinetics of irinotecan and the metabolite SN-38

The aim of the present study was to investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on the pharmacokinetics of irinotecan (CPT-11) and its metabolite SN-38. EGCG was potentially used to modulate the ATPase activity of P-glycoprotein (P-gp). Experimental Sprague-Dawley rats were treated with EGCG (20mg/kg, i.v.) 10min before CPT-11 (10mg/kg, i.v.) administration, whereas the control group received CPT-11 (10mg/kg, i.v.) only. The biological samples were prepared by the protein precipitation and detected by HPLC-fluorescence detection which provided a good separation of CPT-11 and SN-38 within 10min. The pharmacokinetic data indicate that the area under the plasma concentration-time curves (AUC) of CPT-11 and SN-38 were increased by 57.7 and 18.3%, and AUC in bile were decreased by 15.8 and 46.8%, respectively, for the group pretreated with EGCG. The blood to bile distribution ratio (AUC(bile)/AUC(blood)) was significantly reduced after group coadministration of EGCG, it can be seen that the bile efflux transport system of CPT-11 and SN-38 may be markedly reduced by the treatment of EGCG which plays the role of P-gp inhibitor. In conclusion, EGCG was found to inhibit the transport of CPT-11 and SN-38 into the biliary elimination and their half-lives in plasma could be substantially prolonged. Based on the food-drug interaction, persons taking daily nutritional supplements should be warned of this interaction possibility.     

(-)-Epigallocatechin-3-gallate (EGCG) sensitizes melanoma cells to interferon induced growth inhibition in a mouse model of human melanoma

Melanoma incidences have increased over the last few decades and metastatic melanoma is one of the hardest malignancies to treat. Thus, novel approaches are needed for an effective management of melanoma. Interferon-α2b (IFN), an immunomodulatory cytokine commonly used in melanoma treatment, has shown marginal efficacy and often results in discontinuation of therapy due to toxicity. We earlier demonstrated that epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, caused cell cycle arrest and apoptosis of human melanoma cells via modulation in cki-cyclin-cdk machinery and Bcl-2 family proteins. This study was undertaken to determine if EGCG could enhance the anti-proliferative effects of IFN. In this study, we demonstrated that EGCG and/or IFN treatments to melanoma cells resulted in a marked i) decrease in cell proliferation and colony formation ability, and ii) induction of apoptosis. Interestingly, the combination was found to be more effective than either of the agents alone. Further, the anti-proliferative effects of EGCG and/or IFN were accompanied with an increase in FAS protein levels and a decrease in nuclear factor NF-κB/p65 in the nucleus as well as NF-κB promoter activity. EGCG and/or IFN also resulted in an increase in FAS-L mediated apoptosis. Further, EGCG and/or IFN treatments resulted in a decrease in melanoma tumor growth and protein levels of proliferation marker PCNA, in athymic nude mice implanted with melanoma tumors. The combination of the two modalities demonstrated a better response than either of them alone. Our data suggest that EGCG could impart therapeutic advantage if used in conjunction with IFN.     

Simultaneous determination of the major active components of tea polyphenols in rat plasma by a simple and specific HPLC assay

A simple and specific HPLC assay for simultaneous determination of two major active components (-) epigallocatechin-3-gallate (EGCG), and (-) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid+CH(3)CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5-300 microg/mL for EGCG and 0.1-60 microg/mL for ECG (r>0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration-time curves.     

Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia ulmodies

An open microwave-assisted extraction system was used to extract gallic acid, protocatechuic acid, chlorogenic acid and caffeic acid from Eucommia ulmodies. The effect of extraction variables, especially solvent, on the recoveries of these polyphenolic compounds was investigated using factorial design. As extracting solvent for these compounds, methanol produced a higher recovery than pure water. For straight chain alcohol solvents, the lower the carbon number, the higher the recoveries of the polyphenolic acids. The optimal ratio of methanol:water:glacial acetic acid in the solvent mixture used in microwave-assisted extraction was 2:8:0.3 (v/v) and this solvent could be directly used as the mobile phase in HPLC separation without additional intermittent treatment as reported in literature. The extraction under the condition of 50% microwave power and 30 s irradiation at a solvent:sample ratio of 10 (mL/g) was found to be the most advantageous. The repeatability test of extraction and chromatographic analysis was satisfactory for the analysis of these polyphenolic compounds.     

Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Combination of HSCCC and Sephadex LH-20 methods An approach to isolation and purification of the main individual theaflavins from black tea

In order to separate the main individual theaflavin monomers from black tea, high-speed countercurrent chromatography (HSCCC) and Sephadex LH-20 column chromatography were applied. The results showed that theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B) and theaflavin-3,3'-digallate (TF3) can be obtained by HSCCC using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:1:6, v/v/v/v), but the TF1 was containing epicatechin-3-gallate (ECG). Similarly, Sephadex LH-20 can also effectively separate TF2A(B) and TF3, but epigallocatechin-3-gallate (EGCG) contaminated TF1, too. Combination of HSCCC and Sephadex LH-20, the preferably purified TF1, TF2A(B) and TF3 were obtained than single separation technique. In addition, ECG and EGCG were also suggested to be able to be comprehensively separated by combination of the two techniques.     

Structure–activity studies of (−)-epigallocatechin gallate derivatives as HCV entry inhibitors

Preventing viral entry into cells is a recognized approach for HIV therapy and has attracted attention for use against the hepatitis C virus (HCV). Recent reports described the activity of (−)-epigallocatechin gallate (EGCG) as an inhibitor of HCV entry with modest potency. EGCG is a polyphenolic natural product with a wide range of biological activity and unfavorable pharmaceutical properties. In an attempt to identify more drug-like EGCG derivatives with improved efficacy as HCV entry inhibitors, we initiated structure–activity investigations using semi-synthetic and synthetic EGCG analogs. The data show that there are multiple regions in the EGCG structure that contribute to activity. The gallate ester portion of the molecule appears to be of particular importance as a 3,4-difluoro analog of EGCG enhanced potency. This derivative and other active compounds were shown not to be cytotoxic in Huh-7 cell culture. These data suggest that more potent, non-cytotoxic EGCG analogs can be prepared in an attempt to identify more drug-like candidates to treat HCV infection by this mechanism.     

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

Novel Epigallocatechin-3-Gallate (EGCG) Derivative as a New Therapeutic Strategy for Reducing Neuropathic Pain after Chronic Constriction Nerve Injury in Mice

Neuropathic pain is common in peripheral nerve injury and often fails to respond to ordinary medication. Here, we investigated whether the two novel epigallocatechin-3-gallate (EGCG) polyphenolic derivatives, compound 23 and 30, reduce the neuropathic pain in mice chronic constriction nerve injury (CCI). First, we performed a dose-response study to evaluate nociceptive sensation after administration of EGCG and its derivatives 23 and 30, using the Hargreaves test at 7 and 21 days after injury (dpi). We daily administered EGCG, 23 and 30 (10 to 100 mg/Kg; i.p.) during the first week post-CCI. None of the doses of compound 23 caused significant pain diminution, whereas 50mg/kg was optimal for both EGCG and 30 to delay the latency of paw withdrawal. With 50 mg/Kg, we showed that EGCC prevented the thermal hyperalgesia from 7 to 21 dpi and compound 30 from 14 to 56 dpi. To evaluate the molecular mechanisms underpinning why EGCG and compound 30 differentially prevented the thermal hyperalgesia, we studied several biochemical parameters in the dorsal horn of the spinal cord at 14 and 56 dpi. We showed that the effect observed with EGCG and compound 30 was related to the inhibition of fatty acid synthase (FASN), a known target of these polyphenolic compounds. Additionally, we observed that EGCG and compound 30 reduced the expression of CCI-mediated inflammatory proteins and the nuclear localization of nuclear factor-kappa B at 14 dpi, but not at 56 dpi. We also strongly detected a decrease of synaptic plasma membrane levels of N-methyl-D-asparte receptor 2B in CCI-mice treated with compound 30 at 56 dpi. Altogether, compound 30 reduced the chronic thermal hyperalgesia induced by CCI better than the natural compound EGCG. Thus, our findings provide a rationale for the preclinical development of compound 30 as an agent to treat neuropathic pain.     

Green Tea Catechin,Epigallocatechin Gallate,Suppresses Signaling by the dsRNA Innate Immune Receptor RIG-I

Background

The Innate immune system constitutes the first line of defense against pathogen infections. The Retinoic acid-inducible gene I (RIG-I) receptor recognizes triphosphorylated ssRNAs and dsRNA to initiate downstream signaling of interferon response. However, unregulated activity of these receptors could lead to autoimmune diseases. We seek to identify small molecules that can specifically regulate RIG-I signaling.

Methodology/Principal Findings

Epigallocatechin gallate (EGCG), a polyphenolic catechin present in green tea, was identified in a small molecule screen. It was found to bind RIG-I and inhibits its signaling at low micromolar concentrations in HEK293T cells. Furthermore, EGCG dose-dependently inhibited the ATPase activity of recombinant RIG-I but did not compete with RIG-I interaction with RNA or with ATP. EGCG did not inhibit signaling by Toll-like receptors 3, 4, 9 or constitutive signaling by the adapter protein IPS-1. Structure activity relationship analysis showed that EGCG, its epimer GCG and a digallate-containing compound, theaflavin 3,3′ digallate (TFDG) were potent RIG-I inhibitors. EGCG also inhibited IL6 secretion and IFN- β mRNA synthesis in BEAS-2B cells, which harbors intact endogenous RIG-I signaling pathway.

Conclusions/Significance

EGCG and its derivatives could have potential therapeutic use as a modulator of RIG-I mediated immune responses.     

Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC₅₀ 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC₅₀ values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.     

Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high performance liquid chromatography

A rapid isocratic method for determining the total phosphatidylcholine and disaturated phosphatidylcholine levels in lung surfactant preparations by high performance liquid chromatography (HPLC) is described. The analysis was performed on a 3.9 x 300 mm mu-Porasil column with detection by refractive index. The lipids were eluted with a solvent system of chloroform-acetonitrile-methanol-water-85% phosphoric acid 650:650:500:130:2 (v/v/v/v/v). A 4.6 x 30 mm silica guard column was used in place of an injector loop which served as a sample concentrator and purifier. Phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol, all known components of lung surfactants, were eluted from the loop column and were prevented from reaching the analytical column. Sphingomyelin and lysophosphatidylcholine elute later than the phosphatidylcholines on the analytical column. The method was developed so that phosphatidylcholines elute as a single peak regardless of the fatty acid chain length (C12-C20). When the sample was first oxidized with a potassium permanganate-potassium metaperiodate solution, and potentially interfering oxidation products were removed by extraction into a basic aqueous phase, then only the disaturated phosphatidylcholines were analyzed.     

Epigallocatechin-3-gallate protects against cisplatin-induced nephrotoxicity by inhibiting endoplasmic reticulum stress-induced apoptosis

Cisplatin (CP)-induced nephrotoxicity hampers its application in clinic. Green tea, particularly its predominant polyphenolic constituent epigallocatechin-3-gallate (EGCG), possesses anti-inflammatory, antioxidant, and anti-apoptotic properties. The present study was designed to investigate the protective effects of EGCG against CP-induced nephrotoxicity in mice. Male C57/BL6 mice in different groups received single injection of CP (20 mg/kg) and EGCG (100 mg/kg) in various sets and kidney tissues and blood were collected after killing. Then, samples were used for biochemical and immunohistochemical assay. Our results showed EGCG decreased biochemical factors and immunohistochemical damage induced by CP. Besides, expression of phosphorylated-extracellular signal-regulated kinase (p-ERK), glucose-regulated protein 78 (GRP78), caspase-12, and apoptosis of kidney were decreased by EGCG via inhibition of endoplasmic reticulum (ER) stress-induced apoptosis.     

Green tea constituent (−)-Epigallocatechin-3-gallate inhibits hep G2 cell proliferation and induces apoptosis through p53-dependent and fas-mediated pathways

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenolic compound found in green tea. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. To provide a better understanding of the preventive effect of EGCG on liver cancer, we examined EGCG for its effect on proliferation and cell cycle progression in a human liver cancer cell line, Hep G2. The results showed that EGCG inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA showed that EGCG significantly increased the expression of p53 and p21/WAF1 protein, and this contributed to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by EGCG. Taken together, our study suggests that the induction of p53 and the activity of the Fas/FasL apoptotic system play major roles in the antiproliferative activity of EGCG in Hep G2 cells.     

(-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.