Natural flavonoid in green tea

(-)-Epigallocatechin (EGC) is a strong in vitro inhibitor of the development of amyloid cystatin I66Q amyloid fibrils. According to computational study, (-)-epigallocatechin stabilizes the molecule in its original state instead of causing aggregation to reroute into disordered, amorphous aggregates, which prevents the production of amyloid cysteine protease fibrils [1]. The population of cancer stem cell-like cluster of differentiation 44 (CD44)-positive cells was decreased by combination therapy with curcumin and EGCG. Curcumin and (-)-epigallocatechin (EGC) significantly suppressed STAT3 phosphorylation and retained the STAT3-NFkB connection, as demonstrated by Western blot and immunoprecipitation study [2]. (-)-Epigallocatechin (EGC) effectively removes Enterococcus faecalis biofilms, with MIC and MBC values of 5 μg/mL and 20 μg/mL, respectively. Enterococcus faecalis produces hydroxyl radicals when exposed to (-)-epigallocatechin. the DIP-protected E addition. faecalis through the antibacterial actions of EGCG. Enterococcus faecalis virulence genes are significantly downregulated at sub-MIC levels by (-)-epigallocatechin [3].

ThT Fluorescence Assay[1]
To determine the formation of amyloid fibrils, phosphate buffer (50 mM Na2HPO4, 50 mM NaH2PO4, pH 7.0) was used to prepare a ThT stock solution of 1 mM. Aliquots of cC I66Q samples taken at different times were diluted with phosphate buffer, followed by the addition of 30 μL of ThT stock solution. ThT fluorescence measurement was conducted by exciting samples at 440 nm and recording the emission signals at 485 nm over 120 s using a Cary Eclipse fluorescence spectrophotometer.

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ANS Binding Assay[1]
A 0.4 mM stock solution of ANS was prepared by dissolving ANS in PBS (pH 7.0). The ANS stock solution was stored at 4 °C. Aliquots of cC I66Q solution with or without EGCG taken at different times were mixed with an aliquot of ANS solution, followed by the addition of PBS (pH 7.0) to a final volume of 3 mL. After incubation at room temperature in the dark for 30 min, the samples were subjected to fluorescence assay using an excitation wavelength of 380 nm and an emission wavelength between 400 and 600 nm. Both the ANS fluorescence intensity and the average emission wavelength were recorded to account for the changes in intensity and spectrum.

PC12 cells were purchased from American Type Culture Collection. PC12 cells were maintained in DMEM with 10% horse serum, 5% fetal bovine serum, and 1% penicillin/streptomycin antibiotics. Cells were cultured in a 5% CO2 atmosphere at 37 °C and then harvested and plated in 96-well at a density of 104 cells/well. The plates were incubated at 37 °C for 24 h. Subsequently, cC I66Q with and without EGCG was incubated at 65 °C for 35 days, and aliquots of the samples were collected after centrifugation at 6000g for 1 h. The concentrated cC I66Q samples were dissolved in PBS buffer (pH 7.0, 50 mM), and the protein concentration in each sample was determined by BCA assay. The cC 166Q samples, with and without ECGC, were separately added to the cells to give a final concentration of cC 166Q in the cells ranging from 0 to 500 ng/mL. The final concentrations of cC I66Q samples (both with and without EGCG) in each well were 0, 1, 5, 50, and 500 ng/mL. The plates were then incubated with the protein samples for 48 h at 37 °C, and cell viability was determined using MTT toxicity assay by adding 10 μL of 5 mg/mL MTT (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) reagent to each well, followed by further incubation for 3 h. After that, the medium was removed and replaced with 100 μL of DMSO. After 10 min of shaking at room temperature, the absorbance of the plate was measured at 490 nm using an iMarkMicroplate Reader.[1]
To suppress the CSC phenotype, two breast cancer cell lines (MDA-MB-231 cells and MCF7 cells transfected with HER2) were treated with curcumin (10 μM) with or without EGCG (10 μM) for 48 h. We used tumor-sphere formation and wound-healing assays to determine CSC phenotype. To quantify CSC populations, Fluorescence-activated cell sorting profiling was monitored. STAT3 phosphorylation and interaction with Nuclear Factor-kB (NFkB) were analyzed by performing western blot and immunoprecipitation assays. [2]
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG on E. faecalis were determined. The efficacy of EGCG on E. faecalis biofilms was tested by exposing 7-day old E. faecalis biofilm to EGCG. Flow cytometry analysis of hydroxyphenyl fluorescein (HPF) labelled E. faecalis was used to determine if EGCG induced intracellular hydroxyl radical formation. Co-treatment of EGCG with the iron chelator 2,2-dipyridyl (DIP) was carried out to determine if hydroxyl radical generated through Fenton reaction played a role in EGCG-mediated killing of E. faecalis. Furthermore, the effects of EGCG on the expression of virulence genes in E. faecalis were evaluated by quantitative polymerase chain reaction.
Results: EGCG exhibited a MIC and MBC of 5 μg/mL and 20 μg/mL respectively and effectively eradicated E. faecalis biofilms. EGCG induced the formation of hydroxyl radicals in E. faecalis. The addition of DIP protected E. faecalis against EGCG-mediated antibacterial effects. At sub-MIC, EGCG induced significant down-regulation of E. faecalis virulence genes.
Conclusions: EGCG is an effective antimicrobial agent against both the planktonic and biofilm forms of E. faecalis, inhibiting bacterial growth and suppressing the expression of specific genes related to virulence and biofilm formation. The antimicrobial action of EGCG on E. faecalis occurred through the generation of hydroxyl radical[3].

Metabolism / Metabolites
(-)-Epigallocatechin has known human metabolites that include (-)-Epigallocatechin, 3p-hydroxy-glucuronide.

[1]. (-)-Epigallocatechin-3-gallate Inhibits Fibrillogenesis of Chicken Cystatin. J Agric Food Chem. 2015 Jan 26.

[2]. Curcumin and Epigallocatechin Gallate Inhibit the Cancer Stem Cell Phenotype via Down-regulation of STAT3-NFκB Signaling. Anticancer Res. 2015 Jan;35(1):39-46.

[3]. Effects of Epigallocatechin gallate against Enterococcus faecalis biofilm and virulence. Arch Oral Biol. 2015 Mar;60(3):393-9.

(-)-epigallocatechin is a flavan-3,3',4',5,5',7-hexol having (2R,3R)-configuration. It has a role as an antioxidant, a plant metabolite and a food component. It is a flavan-3,3',4',5,5',7-hexol and a catechin. It is an enantiomer of a (+)-epigallocatechin.
Epigallocatechin has been reported in Camellia sinensis, Eschweilera coriacea, and other organisms with data available.
Epigallocatechin is a metabolite found in or produced by Saccharomyces cerevisiae.
See also: Crofelemer (monomer of).

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Previous studies have reported that (-)-epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, can bind to unfolded native polypeptides and prevent conversion to amyloid fibrils. To elucidate whether this antifibril activity is specific to disease-related target proteins or is more generic, we investigated the ability of EGCG to inhibit amyloid fibril formation of amyloidogenic mutant chicken cystatin I66Q, a generic amyloid-forming model protein that undergoes fibril formation through a domain swapping mechanism. We demonstrated that EGCG was a potent inhibitor of amyloidogenic cystatin I66Q amyloid fibril formation in vitro. Computational analysis suggested that EGCG prevented amyloidogenic cystatin fibril formation by stabilizing the molecule in its native-like state as opposed to redirecting aggregation toward disordered and amorphous aggregates. Therefore, although EGCG appears to be a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves such inhibition may be specific to the target fibril-forming polypeptide.[1]

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The cancer stem cell (CSC) model postulates the existence of a small proportion of cancer cells capable of sustaining tumor formation, self-renewal and differentiation. Signal Transducer and Activator of Transcription 3 (STAT3) signaling is known to be selectively activated in breast CSC populations. However, it is yet to be determined which molecular mechanisms regulate STAT3 signaling in CSCs and what chemopreventive agents are effective for suppressing CSC growth. The aim of this study was to examine the potential efficacy of curcumin and epigallocatechin gallate (EGCG) against CSC and to uncover the molecular mechanisms of their anticancer effects. Results: Combined curcumin and EGCG treatment reduced the cancer stem-like Cluster of differentiation 44 (CD44)-positive cell population. Western blot and immunoprecipitation analyses revealed that curcumin and EGCG specifically inhibited STAT3 phosphorylation and STAT3-NFkB interaction was retained. Conclusion: This study suggests that curcumin and EGCG function as antitumor agents for suppressing breast CSCs. STAT3 and NFκB signaling pathways could serve as targets for reducing CSCs leading to novel targeted-therapy for treating breast cancer.[2]

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Epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in green tea (Camellia sinesis) has been shown to exert antimicrobial effects on numerous bacterial pathogens. However its efficacy against Enterococcus faecalis biofilm, which is associated with persistent root canal infection is unknown. The aims of this study were to investigate the effects of EGCG against E. faecalis biofilm and virulence.[3]

C15H14O7

306.27

306.073

C, 58.82; H, 4.61; O, 36.57

970-74-1

(-)-Epigallocatechin Gallate;989-51-5;(-)-Gallocatechin;3371-27-5;(+)-Gallocatechin;970-73-0

72277

White to off-white solid powder

1.7±0.1 g/cm3

685.6±55.0 °C at 760 mmHg

208-210°C

368.5±31.5 °C

0.0±2.2 mmHg at 25°C

1.776

-0.1

6

7

1

22

380

2

C1[C@H]([C@H](OC2=CC(=CC(=C21)O)O)C3=CC(=C(C(=C3)O)O)O)O

WMBWREPUVVBILR-WIYYLYMNSA-N

InChI=1S/C22H18O11/c23-10-5-12(24)11-7-18(33-22(31)9-3-15(27)20(30)16(28)4-9)21(32-17(11)6-10)8-1-13(25)19(29)14(26)2-8/h1-6,18,21,23-30H,7H2/t18-,21-/m1/s1

[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate

EGC; NSC 674039; (-)-Epigallocatechin; Epigallocatechin; 970-74-1; Epigallocatechol; L-Epigallocatechin; epi-Gallocatechin; Antiscurvy factor C2; (-)-Epigallocatechol; epiGallocatechin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~66.67 mg/mL (~217.68 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)