Synthesis,Stability, and Antidiabetic Activity Evaluation of (−)-Epigallocatechin Gallate (EGCG) Palmitate Derived from Natural Tea Polyphenols

This work describes a novel approach for the synthesis of (−)-epigallocatechin gallate (EGCG) palmitate by a chemical-synthesis method, where the elevated stability of the EGCG derivative is achieved. Various parameters affecting the acylation process, such as the base, solvent, as well as the molar ratio of palmitoyl chloride, have been studied to optimize the acylation procedure. The optimized reaction condition was set as follows: EGCG/palmitoyl chloride/sodium acetate was under a molar ratio of 1:2:2, with acetone as the solvent, and the reaction temperature was 40 °C. Under the optimized condition, the yield reached 90.6%. The EGCG palmitate (PEGCG) was isolated and identified as 4′-O-palmitoyl EGCG. Moreover, the stability of PEGCG under different conditions was proved significantly superior to EGCG. Finally, PEGCG showed better inhibition towards α-amylase and α-glucosidase, which was 4.5 and 52 times of EGCG, respectively. Molecular docking simulations confirmed the in vitro assay results. This study set a novel and practical synthetic approach for the derivatization of EGCG, and suggest that PEGCG may act as an antidiabetic agent.     

Colloidal complexes from associated water soluble cellulose derivative (methylcellulose) and green tea polyphenol (Epigallocatechin gallate)

The present work deals with the preparation and characterisation of colloidal complexes from association of water soluble cellulose derivative (methylcellulose) and green tea polyphenol-EGCG (Epigallocatechin gallate). Colloidal complexes with well defined size range of 95-300 nm (polydispersity index<0.15) and a negative surface potential (-25 to -45 mV) were obtained by mixing solutions of methylcellulose and EGCG under vigorous stirring. The binding stoichiometry of 21 molecules of EGCG per one molecule of polymer was obtained from isothermal titration calorimetry. The free energy of binding (-31 kJ mol(-1)) is dominated by the binding enthalpy suggesting that the non-covalent complex is preferentially formed due to the hydrogen bonding. Transmission electron microscopy revealed almost spherical particle morphology of the formed colloidal complexes. Further, sustained release of EGCG from the complex in simulated in vitro media was observed which resulted in protecting the antioxidant property of EGCG in alkaline pH.     

Biological Effects of EGCG@MOF Zn(BTC)4 System Improves Wound Healing in Diabetes

Tea contains high levels of the compound epigallocatechin gallate (EGCG). It is considered an important functional component in tea and has anti-cancer, antioxidant, and anti-inflammatory effects. The eight phenolic hydroxyl groups in EGCG’s chemical structure are the basis for EGCG’s multiple biological effects. At the same time, it also leads to poor chemical stability, rendering EGCG prone to oxidation and isomerization reactions that change its original structure and biological activity. Learning how to maintain the activity of EGCG has become an important goal in understanding the biological activity of EGCG and the research and development of tea-related products. Metal–organic frameworks (MOFs) are porous materials with a three-dimensional network structure that are composed of inorganic metals or metal clusters together with organic complexes. MOFs exploit the porous nature of the material itself. When a drug is an appropriate size, it can be wrapped into the pores by physical or chemical methods; this allows the drug to be released slowly, and MOFs can also reduce drug toxicity. In this study, we used MOF Zn(BTC)₄ materials to load EGCG and investigated the sustained release effect of EGCG@MOF Zn(BTC)₄ and the biological effects on wound healing in a diabetic mouse model.     

(−)-Epigallocatechin gallate/gelatin layer-by-layer assembled films and microcapsules

A new type of protein/polyphenol microcapsules on the basis of naturally occurring polyphenol (−)-epigallocatechin gallate (EGCG) and gelatin, type A, was obtained using the layer-by-layer (LbL) assembly method. The microcapsules show a more pronounced dependence of permeability on molecular weight of permeating substances than commonly used polyallylamine/polystyrene sulfonate capsules. The regularities of EGCG adsorption in alternation with type A and B gelatins have been investigated using quartz crystal microbalance and electrophoretic mobility measurements on microparticles and found to be dependent on gelatin properties. EGCG in the LbL assemblies retains its antioxidant activity. The kinetics of the reaction of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cation-radicals with films consisting of 1–10 gelatin/EGCG bilayers is affected by film structure. The EGCG content in the protein/polyphenol film material is as high as 30% w/w. Encapsulation of EGCG via its alternated adsorption with gelatins can be a perspective way to new formulations containing the polyphenol for drug delivery applications.     

Efficient Separation and Purification of Epigallocatechin Gallate (EGCG) Based on EGCG-Imprinted Polymer Prepared with Chitosan as Matrix

A molecularly imprinted polymer (MIP), which was suitable for recognizing epigallocatechin gallate (EGCG), was prepared by using EGCG as template molecule and biocompatible chitosan as a functional matrix in aqueous medium. Molecular recognition ability of the EGCG-imprinted polymer (EIP) was evaluated by high performance liquid chromatography (HPLC). The results show that the EIP has a high imprinting factor (1.32) for EGCG and was used to purify EGCG from crude tea polyphenol efficiently. The percentage of EGCG can be improved from 78.6% in crude tea polyphenol (TP) to 90.1% in product and the adsorption quantity per unit can reach 4.02 mg · g^?1. EIP shows potential excellent prospect in the application of separating and purifying EGCG from TP.     

Determination of (-)-epigallocatechin gallate in rat blood by microdialysis coupled with liquid chromatography

A liquid chromatographic method coupled with microdialysis was used to determine the protein-unbound (-)-epigallocatechin-3-gallate (EGCG) in rat blood. EGCG and dialysates were separated using a Merck RP-18e column maintained at ambient temperature, and a mobile phase comprised of acetonitrile-10 mM monopotassium phosphate (pH 3.82) (20:80, v/v) with a flow rate of 1.0 ml/min. The UV detector wavelength was set at 206 nm. The detection limit for EGCG was 10 ng/ml. The concentration-response relationship was linear (r2 > 0.995) over a concentration range of 0.05-10 microg/ml; intra- and inter-assay precision and accuracy of EGCG fell within predefined limits. Pharmacokinetic parameters of EGCG were assessed using compartmental models. The disposition of EGCG in the rat blood suggests that EGCG was fitted by two-compartmental model. The distribution and elimination half-lives were 6 and 72 min respectively, after the dosage of 30 mg/kg.     

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin

Identification of natural products capable of affording protection against UVB radiation-induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure-induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB-induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB-induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun-protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB-induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB-induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB-exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB-exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB-induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti-inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB-induced ROS-associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.     

Plasma-Induced Oxidation Products of (–)-Epigallocatechin Gallate with Digestive Enzymes Inhibitory Effects

(−)-Epigallocatechin gallate (EGCG), the chief dietary constituent in green tea (Camellia sinensis), is relatively unstable under oxidative conditions. This study evaluated the use of non-thermal dielectric barrier discharge (DBD) plasma to improve the anti-digestive enzyme capacities of EGCG oxidation products. Pure EGCG was dissolved in an aqueous solution and irradiated with DBD plasma for 20, 40, and 60 min. The reactant, irradiated for 60 min, exhibited improved inhibitory properties against α-glucosidase and α-amylase compared with the parent EGCG. The chemical structures of these oxidation products 1–3 from the EGCG, irradiated with the plasma for 60 min, were characterized using spectroscopic methods. Among the oxidation products, EGCG quinone dimer A (1) showed the most potent inhibitory effects toward α-glucosidase and α-amylase with IC₅₀ values of 15.9 ± 0.3 and 18.7 ± 0.3 μM, respectively. These values were significantly higher than that of the positive control, acarbose. Compound 1, which was the most active, was the most abundant in the plasma-irradiated reactant for 60 min according to quantitative high-performance liquid chromatography analysis. These results suggest that the increased biological capacity of EGCG can be attributed to the structural changes to EGCG in H₂O, induced by cold plasma irradiation.     

Structural identification of novel glucoside and glucuronide metabolites of (-)-epigallocatechin-3-gallate in mouse urine using liquid chromatography/electrospray ionization tandem mass spectrometry

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and most biologically active polyphenolic compound in tea, has been proposed to have many health beneficial effects. The metabolic fate of EGCG, however, is not well understood. In the present study, we identified a novel EGCG metabolite, 7-O-beta-D-glucopyranosyl-EGCG-4'-O-beta-D-glucupyranoside, in a mouse urine sample using liquid chromatography/electrospray ionization tandem mass spectrometry. The structure of this metabolite was confirmed by analyzing the MSn (n = 1-4) spectra as well as comparing the MS/MS spectra of its product ions with those from EGCG and EGCG-4'-O-beta-D-glucupyranoside standards. To our knowledge, this is the first report of the identification of a glucoside metabolite of EGCG in mammals. Our results indicate that glucosidation represents a novel pathway in the metabolism of EGCG in mice.     

Evaluation of renal protective effects of the green-tea (EGCG) and red grape resveratrol: role of oxidative stress and inflammatory cytokines

Epigallocatechin-gallate (EGCG) and resveratrol (RSVL) are two of the most promising natural medicines. We verified their capacity to ameliorate cisplatin (CP)-induced disruption of renal glomerular filtration rate (GFR) in rats, and sought the mediatory involvement of lipid peroxidation (malondialdehyde [MDA]-level) and inflammatory cytokine (TNF-α) therein. CP (10 mg kg?1), a single i.p. dose, disrupted GFR (11-fold-rise in proteinuria, 2-5-fold rise in serum creatinine/urea levels) after 7 days, and killed all animals after 10 days. Kidney-homogenates from CP-treated rats displayed higher MDA and TNF-α, but lower reduced-glutathione (GSH) levels. Rats treated with EGCG (50 mg kg?1, but not 25 mg kg?1) had no fatalities and showed significantly-recovered GFR; while their kidney-homogenates had markedly reduced MDA, TNF-α and enhanced GSH levels at 7 days. Conversely, RSVL or quercetin (25, 50 mg kg?1) neither improved GFR nor reduced (MDA)/TNF-α levels after 7 days. Resuming treatment with 50 mg kg?1 for 10 days rescued only 25% of animals (p > 0.05). Correlation studies showed a significant association between creatinine level, and each of MDA (r = 0.91), GSH (r = -0.87), and TNF-α (0.91). The study showed for the first time that EGCG, unlike RSVL, can protect against CP-induced nephrotoxicity. At the molecular level, CP triggers a high level of oxidative stress and systemic inflammation, events that were all abrogated with EGCG; better than RSVL or quercetin.     

Systematic characterization of the covalent interactions between (−)‐epigallocatechin gallate and peptides under physiological conditions by mass spectrometry

(?)‐Epigallocatechin gallate (EGCG) is a major bioactive component in leaves of green tea, and has been widely investigated for its anti‐tumor activity. The interaction between EGCG and the key peptides or proteins (e.g. glutathione, enzymes) in vivo is thought to be involved in the toxicity and anti‐cancer mechanism of EGCG. However, the true anti‐tumor mechanism of EGCG is not clear, and few studies have focused on the reactivity of EGCG toward peptides or proteins under physiological conditions (pH 7.4, 37°C). In this work, the covalent interactions between EGCG and model peptides containing one or more nucleophilic residues (i.e. Arg, Cys, Met, and α‐NH₂ of the N‐terminus of peptides) under physiological condition were fully characterized using mass spectrometry. It was found that EGCG can react with the thiol groups of peptides to form adducts under physiological conditions (pH 7.4, 37°C), even in the absence of the peroxidase/hydrogen peroxide system. Besides the thiol groups of peptides, it is firstly reported that EGCG also reacts with α‐NH₂ of the N‐terminus or arginine residues of model peptides to form Schiff base adducts, and the methionine residues of model peptides can be easily oxidized by hydrogen peroxide (H₂O₂) generated during the process of EGCG auto‐oxidation to form methionine sulfoxide products. The preference for the reaction of nucleophlic residues of peptides with EGCG was determined to have the following order: Cys > α‐NH₂ of the N‐terminus > Arg. The neutral loss ions of [M+H–170]⁺ and [M+H‐138]⁺ were detected in all tandem mass spectra of the EGCG adducts of peptides, which indicates that these two neutral loss ions can be considered as the characteristic neutral loss ions of peptides modified by EGCG. Results of the present research provide insights into the toxicology and anti‐tumor mechanism of EGCG in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Cancer preventive mechanisms of the green tea polyphenol (-)-epigallocatechin-3-gallate

Accumulating evidence indicates that consumption of tea, especially green tea, is good for preventing cancer. To elucidate the cancer preventive mechanisms of green tea, much effort has been devoted to investigating the anticancer effects of (-)-epigallocatechin-3-gallate (EGCG), the major component of green tea. It has been revealed that EGCG restrained carcinogenesis in a variety of tissues through inhibition of mitogen-activated protein kinases (MAPK), growth factor-related cell signaling, activation of activator protein 1 (AP-1) and nuclear factor-B (NF-kappaB), topoisomerase I, matrix metalloproteinases and other potential targets. Therefore, EGCG is a multipotent anticancer agent, which not only provides solid evidence to support the anticancer potential of green tea, but also offers new clues for discovering multiple-targeted anticancer drugs.     

Synthesis of a 3,4,5-trimethoxybenzoyl ester analogue of epigallocatechin-3-gallate (EGCG): a potential route to the natural product green tea catechin, EGCG

The synthesis of a trimethoxybenzoyl ester (D-ring) analogue of epigallocatechin-3-gallate (EGCG) is described. The versatile synthesis route can be used to synthesize A, B, and D ring analogues of EGCG and involves a key cyclization of the chalcone to the 3-flavene. This synthesis provides a possible route to the polyphenolic green tea natural product EGCG.     

Nanochemoprevention by encapsulation of (-)-epigallocatechin-3-gallate with bioactive peptides/chitosan nanoparticles for enhancement of its bioavailability

Nanochemoprevention by oral consumption was developed by the encapsulation of (-)-epigallocatechin-3-gallate (EGCG) with nanoparticles that were electrostatically assembled from bioactive caseinophosphopeptides and chitosan, which was highly biocompatible and able to enhance the bioavailability of EGCG.     

Mixed-mode retention mechanism for (-)-epigallocatechin gallate on a 12% cross-linked agarose gel media

The adsorption behaviour of (-)-epigallocatechin gallate (EGCG), the major polyphenolic substance in green tea extracts, on the cross-linked agarose gel Superose 12 HR 10/30, has been studied using a variety of solvent systems and shown to be based on a mixture of hydrogen bonding and hydrophobic interaction. The hydrogen bonding was studied in acetonitrile in the presence of different co-solvents possessing varying hydrogen bond donor (HBD) and/or hydrogen bond acceptor (HBA) characteristics. The HBA-value of the co-solvent had the highest effect whereas the HBD-value played a subordinate role. Retention due to hydrophobic interaction could be demonstrated when mobile phases containing high water content were applied. The retention of EGCG, and its analogues (-)-epigallocatechin (EGC) and (-)-catechin (C) were thus shown to be dependent on the polarity of the organic modifiers added. However, the elution order of EGC and C, was inversed to that observed in reversed phase chromatography, indicating that some hydrogen bonding was still in effect. The retardation of EGCG in the presence of a wide concentration range of acetonitrile in water confirmed the interpretation that the retention mechanism is of mixed-mode character based on both hydrogen bonding and hydrophobic interaction.     

Solid‐Phase Synthesis of a Combinatorial Methylated (±)‐Epigallocatechin Gallate Library and the Growth‐Inhibitory Effects of these Compounds on Melanoma B16 Cells

We report on the solid‐phase synthesis of a combinatorial methylated (±)‐epigallocatechin gallate (EGCG) library and its biological evaluation. Epigallocatechin gallate (EGCG) and its methylated derivatives, which are members of the catechin family, exhibit various anti‐cancer effects. The solid‐phase synthesis of methylated EGCG involves the preparation of the α‐acyloxyketone by the coupling of a solid‐supported aldehyde with a ketone and an acid. The subsequent release and reductive etherification reaction of the solid‐supported α‐acyloxyketone provide the protected EGCG in good total yields. Sixty‐four methylated EGCGs were successfully prepared. The growth‐inhibitory effects of the methylated EGCG library were also examined. Although methylation of EGCG generally causes reduced growth inhibition, the growth‐inhibitory effect of 7‐OMe EGCGs was comparable to that of EGCG. The 7‐OMe EGCGs are attractive drug candidates because of their enhanced bioavailability.     

Selective inhibitory effect of epigallocatechin-3-gallate on migration of vascular smooth muscle cells

In order to prevent restenosis after angioplasty or stenting, one of the most popular targets is suppression of the abnormal growth and excess migration of vascular smooth muscle cells (VSMCs) with drugs. However, the drugs also adversely affect vascular endothelial cells (VECs), leading to the induction of late thrombosis. We have investigated the effect of epigallocatechin-3-gallate (EGCG) on the proliferation and migration of VECs and VSMCs. Both cells showed dose-dependent decrease of viability in response to EGCG while they have different IC(50) values of EGCG (VECs, 150 mM and VSMCs, 1050 mM). Incubating both cells with EGCG resulted in significant reduction in cell proliferation irrespective of cell type. The proliferation of VECs were greater affected than that of VSMCs at the same concentrations of EGCG. EGCG exerted differential migration-inhibitory activity in VECs vs. VSMCs. The migration of VECs was not attenuated by 200 mM EGCG, but that of VSMCs was significantly inhibited at the same concentration of EGCG. It is suggested that that EGCG can be effectively used as an efficient drug for vascular diseases or stents due to its selective activity, completely suppressing the proliferation and migration of VSMCs, but not adversely affecting VECs migration in blood vessels.     

Electroanalytical Sensing of Green Tea Anticarcinogenic Catechin Compounds: Epigallocatechin Gallate and Epigallocatechin

A novel electroanalytical methodology for the sensing of anticarcinogenic catechin compounds epigallocatechin gallate (EGCG) and epigallocatechin (EGC) is presented. The protocol is based on the electrochemical oxidation of aminophenol in aqueous solutions, where the quinoneimine oxidation product chemically reacts with the EGCG or EGC compounds resulting in consumption of quinoneimine and consequently results in a reduction in the magnitude of the quinoneimine reduction wave, which is found to provide an analytical signal from which to indirectly detect EGCG and/or EGC.     

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50=15.8 microgram/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.     

Novel Perbutyrylated Glucose Derivatives of (–)-Epigallocatechin-3-Gallate Inhibit Cancer Cells Proliferation by Decreasing Phosphorylation of the EGFR: Synthesis,Cytotoxicity, and Molecular Docking

Lung cancer is one of the most commonly occurring cancer mortality worldwide. The epidermal growth factor receptor (EGFR) plays an important role in cellular functions and has become the new promising target. Natural products and their derivatives with various structures, unique biological activities, and specific selectivity have served as lead compounds for EGFR. D-glucose and EGCG were used as starting materials. A series of glucoside derivatives of EGCG (7–12) were synthesized and evaluated for their in vitro anticancer activity against five human cancer cell lines, including HL-60, SMMC-7721, A-549, MCF-7, and SW480. In addition, we investigated the structure-activity relationship and physicochemical property–activity relationship of EGCG derivatives. Compounds 11 and 12 showed better growth inhibition than others in four cancer cell lines (HL-60, SMMC-7721, A-549, and MCF), with IC₅₀ values in the range of 22.90–37.87 μM. Compounds 11 and 12 decreased phosphorylation of EGFR and downstream signaling protein, which also have more hydrophobic interactions than EGCG by docking study. The most active compounds 11 and 12, both having perbutyrylated glucose residue, we found that perbutyrylation of the glucose residue leads to increased cytotoxic activity and suggested that their potential as anticancer agents for further development.