Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

Interaction of β‐casein with (−)‐epigallocatechin‐3‐gallate assayed by fluorescence quenching: effect of thermal processing temperature

The interactions between the flavan‐3‐ol (?)‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐casein in phosphate‐buffered saline (PBS) of pH 6.5 subjected to thermal processing at various temperatures (25–100 °C) were investigated using fluorescence quenching. The results indicated that different temperatures had different effects on the structural changes and EGCG‐binding ability of β‐casein. At temperatures below 60 °C, the β‐casein–EGCG interaction changed little (P > 0.05) with increasing temperature. At temperatures above 80 °C, native assemblies of β‐casein in solution dissociated into individual β‐casein molecules and unfolded, as demonstrated by a red shift of the maximum fluorescence emission wavelength (λ_max) of up to 8.8 nm. The highest quenching constant (K_q) and the number of binding sites (n) were 0.92 (±0.01) × 10¹³ m ^?1 s^?1 and 0.73 (±0.02) (100 °C), respectively. These results provide insight into the potential of interactions between β‐casein–EGCG that may modulate bioactivity or bioavailability to be altered during thermal process.     

Studies on the interaction of ‐epigallocatechin‐3‐gallate from green tea with bovine β‐lactoglobulin by spectroscopic methods and docking

The binding interaction between‐epigallocatechin‐3‐gallate (EGCG) and bovine β‐lactoglobulin (βLG) was thoroughly studied by fluorescence, circular dichroism (CD) and protein–ligand docking. Fluorescence data revealed that the fluorescence quenching of βLG by EGCG was the result of the formation of a complex of βLG–EGCG. The binding constants and thermodynamic parameters at two different temperatures and the binding force were determined. The binding interaction between EGCG and βLG was mainly hydrophobic and the complex was stabilised by hydrogen bonding. The results suggested that βLG in complex with EGCG changes its native conformation. Furthermore, preheat treatment (90 °C, 120 °C) and emulsifier (sucrose fatty acid ester) all boosted the binding constants (K_a) and the binding site values (n) of the βLG‐EGCG complex. This study provided important insight into the mechanism of binding interactions of green tea flavonoids with milk protein.     

Albumin stabilizes (–)‐epigallocatechin gallate in human serum: Binding capacity and antioxidant property

(–)‐Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although it can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in human serum and that human serum albumin (HSA) stabilizes EGCg under aerobic condition. Although EGCg is usually decomposed within 1 h in aqueous solution at neutral pH, EGCg in serum and phosphate buffer (pH 7.4) containing HSA was stable over 1 h, even at neutral and slightly alkaline pH. Under these conditions, EGCg binds to HSA non‐covalently. The sulfhydryl group acts as an antioxidant for EGCg oxidation. Incubation of EGCg with HSA is accompanied by the oxidation of a free sulfhydryl group in HSA. These results suggest that the antioxidant property and the binding capacity of HSA contribute to the stabilization of EGCg in human serum.     

Presence of two forms of methylated (–)‐epigallocatechin‐3‐gallate in green tea

Crude catechins extract from Chinese green tea were fractionated using Sephadex LH‐20 column chromatography. The fraction containing (–)‐epigallocatechin‐3‐gallate (EGCG) was then subjected to a semipreparative high‐performance liquid chromatography (HPLC). Using a mobile phase of water : dimethyl formamide : methanol : acetic acid (157 : 49 : 2 : 1 v/v/v/v( the mixture of two methylated catechins was separated and isolated. According to mass spectrometry (MS) and nuclear magnetic resonance (¹H‐NMR) date, these compounds were identified as (–)‐epigallocatechin‐3‐(3‐O‐methylgallate) and (–)‐epigallocatechin‐3‐(4‐O‐methylgallate).     

The development of a suitable manufacturing process for ‘Benifuuki’ green tea beverage with anti‐allergic effects

Epigallocatechin‐3‐O‐(3‐O‐methyl) gallate (EGCG3″Me) has been reported to inhibit type I allergy better than epigallocatechin gallate (EGCG), a major catechin in tea leaves (Camellia sinensis L). We examined the effects of extraction and sterilization on the catechin content and histamine release from mast cells, as a representative reaction of early phase allergy, in the manufacture of ‘Benifuuki’ green tea beverage. Among various varieties of tea, the cultivar ‘Benifuuki’ contains approximately 2% of EGCG3″Me. Ester‐type catechins and their epimers increased with the increased extraction temperature of the tea. A tea infusion, extracted at 90 °C, strongly inhibited histamine release from mast cells. Furthermore, sterilization affected the catechin content in the manufactured green tea beverage. Sterilization at high temperature promoted the isomerization of catechins and the sterilized green tea beverage had a strong inhibitory effect. When EGCG3″Me, EGCG, epicatechin‐3‐O‐gallate (ECG) and their epimers, GCG3″Me (gallocatechin‐3‐O‐(3‐O‐methyl) gallate), GCG (gallocatechin‐3‐O‐gallate) and CG (catechin‐3‐O‐gallate) were compared, the anti‐allergic effect of GCG3″Me was strongest, and the order of activity was GCG3″Me > EGCG3″Me > GCG > EGCG. We consequently suggest that it was necessary to extract components from tea at the highest temperature possible, and to pasteurize under retort conditions (118.1 °C, 20 min), to manufacture functional green tea beverage with an anti‐allergic action. Copyright © 2005 Society of Chemical Industry     

Antioxidant and Antimicrobial Activities of (‐)‐Epigallocatechin‐3‐gallate (EGCG) and its Potential to Preserve the Quality and Safety of Foods

Quality deterioration of fresh or processed foods is a major challenge for the food industry not only due to economic losses but also due to the risks associated with spoiled foods resulting, for example, from toxic compounds. On the other hand, there are increasing limitations on the application of synthetic preservatives such as antioxidants in foods because of their potential links to human health risks. With the new concept of functional ingredients and the development of the functional foods market, and the desire for a “clean” label, recent research has focused on finding safe additives with multifunctional effects to ensure food safety and quality. (‐)‐Epigallocatechin‐3‐gallate (EGCG), a biologically active compound in green tea, has received considerable attention in recent years and is considered a potential alternative to synthetic food additives. EGCG has been shown to prevent the growth of different Gram‐positive and Gram‐negative bacteria responsible for food spoilage while showing antioxidant activity in food systems. This review focuses on recent findings related to EGCG separation techniques, modification of its structure, mechanisms of antioxidant and antimicrobial activities, and applications in preserving the quality and safety of foods.     

The effects of green tea (–)‐epigallocatechin‐3‐gallate on reactive oxygen species in 3T3‐L1 preadipocytes and adipocytes depend on the glutathione and 67 kDa laminin receptor pathways

Green tea (–)‐epigallocatechin‐3‐gallate (EGCG) is known as to regulate obesity and fat cell activity. However, little information is known about the effects of EGCG on oxidative reactive oxygen species (ROS) of fat cells. Using 3T3‐L1 preadipocytes and adipocytes, we found that EGCG increased ROS production in dose‐ and time‐dependent manners. The concentration of EGCG that increased ROS levels by 180–500% was approximately 50 μM for a range of 8–16 h of treatment. In contrast, EGCG dose‐ and time‐dependently decreased the amount of intracellular glutathione (GSH) levels. EGCG was more effective than (–)‐epicatechin, (–)‐epicatechin‐3‐gallate, and (–)‐epigallocatechin in changing ROS and GSH levels. This suggests a catechin‐specific effect. To further examine the relation of GSH to ROS as altered by EGCG, we observed that exposure of preadipocytes and adipocytes to N‐acetyl‐L ‐cysteine (a GSH precursor) blocked the EGCG‐induced increases in ROS levels and decreases in GSH levels. These observations suggest a GSH‐dependent effect of EGCG on ROS production. While EGCG was demonstrated to alter levels of ROS and GSH, its signaling was altered by an EGCG receptor (the so‐called 67 kDa laminin receptor(67LR)) antiserum, but not by normal rabbit serum. These data suggest that EGCG mediates GSH and ROS levels via the 67LR pathway.     

High Concentration of Epigallocatechin‐3‐Gallate Increased the Incidences of Arrhythmia and Diastolic Dysfunction Via β2‐adrenoceptor

Epigallocatechin‐3‐gallate (EGCG) is the major and most potent representative in green tea, which has been proved to modulate myocardial contractility. Whether EGCG has some negative effects on cardiac function is not known. In the present study, we investigated the effects of EGCG at different doses on cardiac contraction and explored whether β₂‐adrenoceptor (β₂AR) was involved in EGCG‐induced cardiac effects. Isolated rat hearts were mounted on the Langendorff system and perfused with different concentrations of EGCG in low or normal calcium Krebs–Henseleit (KH) buffer. The contraction of hearts was measured. Ventricular myocytes were cultured with EGCG and isoprenaline (ISO, 10^?7 M) for 12 h. ICI118,551 (55 nM) was used to inhibit β₂AR. Cardiomyocyte shortening, viability, and responsiveness to ISO (10^?9 M) were measured. EGCG dose dependently enhanced contractility of perfused heart in low calcium KH buffer. In the normal calcium KH buffer, EGCG at low dose (20 μM) increased heart contraction, while at high dose (50 μM), it increased the incidences of arrhythmia and diastolic dysfunction. In isolated ventricular myocytes, EGCG at the concentration of 0.001 to 1.0 μΜ did not affect their contraction. However, the responsiveness to ISO and the survival of myocytes were increased by EGCG (0.01 μM). The increased responsiveness was partially abolished by ICI118,551. The data obtained in this study demonstrated that EGCG at low dose conferred cardioprotection, yet at high dose increased the incidences of arrhythmia and diastolic dysfunction. β₂AR was involved in EGCG‐induced cardiac effects.     

The (1–3, 1–4)‐β‐Glucanases in Malting Barley: Enzyme Survival and Genetic and Environmental Effects

Eighteen barley genotypes used in Brazilian malting barley breeding programs were characterized in relation to (1–3, 1–4)‐β‐glucanase activity in green and kilned malt. They were tested to determine the loss of enzyme activity during kilning in the malting process and the environmental effects on enzyme activity were measured. The genotypes analyzed showed great variation regarding the enzyme activity in both kinds of malt, in a range from 531.94 to 934.31 U/kg in green malt, and from 187.02 to 518.40 U/kg in dry malt. The mean enzyme activity loss during kilning was close to 60%, very similar to the results obtained in other studies. The loss among genotypes varied from 8.04% to 71.54%. The enzyme activity varied significantly under the different environments tested, showing existence of environmental effects on the genotypes analyzed. Embrapa 127 was the genotype that exhibited the highest enzyme activity in finished malt although it had shown a low activity in green malt, reflecting a negligible loss of activity during kilning. The data indicate promising results to malting barley breeding due to the wide variability exhibited by genotypes as to enzyme activity and levels of isoenzyme with high thermostability.     

Extraction of Epigallocatechin Gallate and Epicatechin Gallate from Tea Leaves Using β‐Cyclodextrin

Use of organic solvents to extract phenolic compounds from plants may result in environmental pollution and cause health problems in persons. Replacing organic extraction solvents by green extracting agents without affecting the extraction yield is one of the most pressing problems to be solved. The aim of this study is to evaluate the capacity of β‐cyclodextrin (β‐CD) to recover phenolic compounds from tea leaves. The extract obtained using the ethanol/water mixture presented the highest total phenolic content, followed by those obtained using β‐CD solution and water. HPLC analysis of the extracts showed that the addition of β‐CD to the extracting agent had a selective effect on the extraction of epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). The extraction yield of EGCG and ECG using 15 g/L β‐CD were higher than that obtained using water and 50% ethanol. Molecular docking results indicated that the molecules of EGCG and ECG were more inclined to interact with β‐CD than epigallocatechin, epicatechin, and gallocatechin. The impact of β‐CD concentration, temperature, and time on EGCG and ECG extraction from tea leaves was investigated and the maximum amount of EGCG (118.7 mg/g) and ECG (54.6 mg/g) were achieved when extracted with 25 g/L aqueous β‐CD solution at 60 °C for 60 min. The present study indicates that aqueous β‐CD can be used as an alternative to organic solvents to recover EGCG and ECG from tea leaves.