Effects of Catechins and Their Related Compounds on Cellular Accumulation and Efflux Transport of Mitoxantrone in Caco‐2 Cell Monolayers

The ability of catechins and their related compounds to inhibit breast cancer resistance protein (BCRP) function in Caco‐2 cell monolayers was investigated with mitoxantrone as a BCRP substrate. The gallate or pyrogallol moiety on the catechin structure seemed to promote increased cellular accumulation and inhibit efflux transport of mitoxantrone. The ability of gallate catechins such as (?)‐epigallocatechin gallate (EGCG) and (?)‐epicatechin gallate (ECG) to increase cellular accumulation and inhibit efflux transport of mitoxantrone was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased intracellular mitoxantrone accumulation. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a long carbon chain for BCRP inhibition. Cellular accumulation and reduced efflux transport of mitoxantrone were greater with epigallocatechin 3‐(3″‐O‐butyl) gallate than with EGCG. EGCG inhibition of BCRP seemed to be restricted by hydrophobicity. The co‐administration of catechins, particularly EGCG and related compounds, with greater hydrophobicity may increase the therapeutic activities of BCRP substrates such as mitoxantrone.     

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.     

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).     

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.     

Polyphenolic composition and in vitro antioxidant activities of native‐ and tannase‐treated green tea extracts

The antioxidant activities of native‐ and tannase‐treated green tea extracts along with their major polyphenol components were investigated. The polyphenolic content and composition of the tea before and after tannase treatment were determined by liquid chromatography coupled with mass spectrometry (LC‐MS). Approximately 99% of the (?)‐epigallocatechin gallate (EGCG) and (?)‐epicatechin gallate (ECG) in green tea extract were converted by tannase to (?)‐epigallocatechin (EGC) and (?)‐epicatechin (EC), respectively, after 30 min. Biotransformed green tea exhibited a significantly higher DPPH˙ radical scavenging activities than native green tea (EC₅₀ value of 0.024 ± 0.001 and 0.044 ± 0.001 mg mL^?1, respectively). Kinetic parameters such as scavenging rate and stoichiometry were calculated. The rate of DPPH˙ radical scavenging activities for tannase‐treated green tea extract was shown to be higher than native green tea extract.     

Influence of Gallate and Pyrogallol Moieties on the Intestinal Absorption of (−)‐Epicatechin and (−)‐Epicatechin Gallate

Abstract: The cellular accumulation of individual catechins was measured as an index of intestinal absorption to clarify the interactions among catechins. The cellular accumulation of (?)‐epicatechin (EC) increased in the presence of other catechins. The ability of gallate catechin such as (?)‐epigallocatechin gallate (EGCG) and (?)‐epicatechin gallate (ECG) to increase the cellular accumulation of EC was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased the cellular accumulation of EC by 426% as compared with that in untreated cells. Conversely, the cellular accumulation of ECG was not influenced by other catechins, but it increased by 54% in the presence of GAO. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a catechol group and a neighboring carbonyl group, whereas the pyrogallol moiety, without a neighboring carbonyl group, required 3 hydroxyl groups to increase the cellular accumulation of EC. Furthermore, gallate esters required long carbon chains to increase the same. The experiment using EGCG, GAO, or their derivatives indicated that the ability of gallate or pyrogallol moiety to increase the cellular accumulation of EC was restricted by their hydrophobicity. These results suggest that the co‐administration of foods containing functional materials such as gallate or pyrogallol moieties, increases the intestinal absorption of catechin. Practical Application: The cellular accumulation of (?)‐epicatechin increased by the gallate or pyrogallol moiety in catechin structure. The interaction among catechins appeared to affect intestinal absorption of catechin. The bioavailability of catechin may be improved by co‐administration of functional foods.     

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     

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.     

Epimerisation of tea polyphenols in tea drinks

The present study was carried out to quantify green tea epicatechin (GTE) derivatives and to investigate the origin of epicatechin epimers present in 18 selected canned or bottled tea drinks. The major GTEs present in tea are (?)‐epigallocatechin gallate (EGCG), (?)‐epigallocatechin (EGC), (?)‐epicatechin gallate (ECG) and (?)‐epicatechin (EC). HPLC analysis showed that the content of total GTEs was lower (16.4–268.3 mg l^?1) in the canned and bottled tea drinks than in tea traditionally prepared as a beverage in a cup or teapot (3–5 g l^?1). The major finding was that they contained higher levels of epicatechin epimers, namely (?)‐gallocatechin gallate (GCG), (?)‐gallocatechin (GC), catechin gallate (CG) and (?)‐catechin (C), than of GTEs, ranging from 7.6 to 331.8 mg l^?1. To investigate the origin of these epimers, GTEs were extracted from longjing green tea and autoclaved at various temperatures for 10–60 min. It was found that at least 50% of GTEs were epimerised to their corresponding epimers when autoclaved at 120 °C for 20 min. It is concluded that epicatechin epimers in tea drinks are not originally present in green tea leaf but are instead derived from thermal conversion of GTEs. Copyright © 2003 Society of Chemical Industry     

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.     

EGCG通过激活AMPK、抑制PPARγ调控3T3-L1脂肪细胞中脂滴蓄积

研究表没食子儿茶素没食子酸酯（epigallocatechin gallate,EGCG）对3T3-L1成熟脂肪细胞脂滴蓄积的影响。通过体外诱导分化培养3T3-L1前脂肪细胞,经EGCG处理后,脂肪细胞脂滴大小减小,并表现为时间和浓度依赖性;不会引起成熟脂肪细胞的凋亡。与对照组相比,EGCG处理组显著降低与脂代谢、转运和吸收相关基因的mRNA的表达水平;EGCG处理可以显著降低过氧化物酶体增殖物激活受体（peroxisome proliferators-activated receptor-γ,PPARγ）的表达并激活腺苷一磷酸（adenosine monophosphate,AMP)活化蛋白激酶（AMP-activated protein kinase,AMPK）。使用AMPK抑制剂Dorsomorphin处理成熟脂肪细胞,发现与单独抑制剂组相比,EGCG能提高磷酸化-AMPK的表达水平,并抑制PPARγ的表达。结论：EGCG可能通过激活AMPK从而抑制PPARγ的表达并显著降低与脂代谢、转运和吸收相关基因的mRNA的表达水平,从而减少3T3-L1成熟脂肪细胞的脂滴蓄积。     

Soil moisture stress‐induced alterations in bioconstituents determining tea quality

The impact of water stress on the biochemical constituents that determine black tea quality was investigated. Phenylalanine ammonia lyase (PAL) activity was highest in the drought tolerant ‘Assam’ cultivar UPASI‐2, followed by UPASI‐8 and UPASI‐9, under non‐stress conditions. Under soil moisture stress a reduction in PAL activity was found in all three clones investigated. A strong positive correlation was observed between an increase in soil moisture deficit and a decrease in PAL activity. Lower PAL activity correlated well with lower synthesis of flavanols such as epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), which are important precursors of theaflavin‐3,3′‐digallate that determines final tea quality. Altered synthesis of EGCG and ECG could be due to their molecular rearrangement at elevated leaf temperature during drought. Synthesis of quality constituents such as gallic acid and caffeine declined significantly owing to both drought and waterlogging stress. The reduction in gallic acid due to water stress could lead to lower synthesis of theaflavin fractions such as epitheaflavic acid, epitheaflavic acid‐3′‐gallate and theaflavic acid and, thereby, quality deterioration. Similarly to drought, flooding stress was also found to alter the biochemical constituents necessary for tea quality. Copyright © 2003 Society of Chemical Industry     

“紫娟”茶中的EGCG3"Me成分研究

"紫娟"是云南大叶群体种中的一种稀有茶树品种,因富含花青素成分其新鲜嫩梢的芽、叶、茎均呈现紫色,从而具有很高的科研价值和经济开发潜力,当今其功能化学成分的研究已成为了茶学研究热点之一。本文采用HPLC以及LC-MS技术首次报道分析了茶树特异品种"紫娟"中的表没食子儿茶素-3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)成分及高EGCG3"Me含量"紫娟"茶的加工工艺。结果表明,茶树特异品种"紫娟"鲜叶中含有EGCG3"Me成分(含量为1.05%);在一芽五叶新梢不同嫩度的鲜叶中,第三叶的EGCG3"Me含量最高(含量为1.24%);采用蒸青绿茶或者晒青绿茶的加工工艺可以使成茶有效保留鲜叶中的EGCG3"Me成分。此外,还分析比较了普通炒青绿茶与"紫娟"绿茶中主要儿茶素成分的含量差异,发现较高的ECG和EC含量而较低的EGCG含量是茶树特异品种"紫娟"绿茶中儿茶素组成的主要特点。     

Characterisation of phenolic acids,flavonoids, proanthocyanidins and antioxidant activity of water extracts from seed coats of marama bean [Tylosema esculentum] – an underutilised food legume

Freeze‐dried aqueous phenolic extracts with possible application as natural antioxidant functional food ingredients were prepared from marama bean seed coats by extracting with water. Phenolic acids, flavonoids and proanthocyanidins in the extracts were characterised by HPLC/MS. The major flavonoids were the flavanols methyl (epi)afzelechin‐3‐O‐gallate (40%) and methyl (epi)catechin‐3‐O‐gallate (28%), and the major phenolic acid was gallic acid (10%). Proanthocyanidins in the extracts were predominantly prodelphinidins composed of epicatechin‐3‐O‐gallate and epigallocatechin present as major terminal and extension units and epigallocatechin‐3‐O‐gallate and epicatechin present as minor extension unit constituents. The polymer structure was found to be unique compared with other legumes because of the high percentage of galloylated units. Extracts showed a high DPPH free radical scavenging activity (707 μmol TE g^?1), protective effect against AAPH‐induced human red blood cell haemolysis and copper‐catalysed human LDL oxidation suggesting that the extracts may have potential health benefits.     

EGCG‐rich green tea extract stimulates sRAGE secretion to inhibit S100A12‐RAGE axis through ADAM10‐mediated ectodomain shedding of extracellular RAGE in type 2 diabetes

The receptor for advanced glycation of end products (RAGE) plays a critical role in the progression of type 2 diabetes (T2D). Soluble RAGE (sRAGE) is one of the RAGE variants, which acts as a decoy domain receptor and competes with RAGE, thus contributing to prevention of T2D. In this study, we conducted clinical trials of (–)‐epigallocatechin‐3‐gallate (EGCG) rich green tea extract (300–900 mg/day) to investigate the effect of EGCG on relationship between S100A12 RAGE ligand and diverse sRAGE in T2D. Moreover, mechanism of sRAGE production also confirmed in vitro. Our data indicated that EGCG could stimulate sRAGE circulation but inhibited RAGE ligand in T2D, and ADAM10‐mediated ectodomain shedding of extracellular RAGE was mainly involved in EGCG‐stimulated sRAGE circulation. The present evidence indicates that EGCG has a potential to block S100A12‐RAGE axis by stimulating sRAGE production through ADAM10‐mediated ectodomain shedding of extracellular RAGE. Therefore, EGCG contributes to nutritional strategies for diabetes, not only because of its efficient antioxidant activity to scavenge free radicals, but also because of its ability stimulating sRAGE release in the circulation. Additionally, ADAM10‐induced ectodomain shedding of extracellular RAGE leading to sRAGE circulation should be a potential of passive mechanism of sRAGE production to block S100A12‐RAGE axis‐related pathogenesis of proinflammation and diabetes.     

SEPARATION OF INDIVIDUAL CATECHINS FROM GREEN TEA USING SILICA GEL COLUMN CHROMATOGRAPHY AND HPLC

Crude catechin mixtures from green tea were separated into six fractions using a silica gel column chromatography and a chloroform-methanol-water (65:35:10, v/v/v, lower phase) solvent system. Fraction I was free of catechins, fraction II contained epicatechin (EC), fraction III had epicatechin and epigallocatechin (EGC), fraction IV possessed EGC, fraction V contained EGC, epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), and fraction VI had EGCG. EC and EGC were separated from fractions II, III and IV using HPLC with a RP-18 semipreparative column and a water-dimethylformamide-methanol-acetic acid (157:40:2:1, v/v/v/v) solvent system. For isolation of EGC, ECG and EGCG from fractions V and VI a water-acetonitrile-methanol-acetic acid (159:36:4:1, v/v/v/v) solvent system was employed. Chemical structures of purified catechins were further confirmed by ESI-MS.     

ANTIOXIDANT PROPERTIES OF POLYPHENOLS EXTRACTED FROM GREEN AND BLACK TEAS

The catechins, including epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG), and the theaflavins, including theaflavin (TF), theaflavin monogallate (TF-1), and theaflavin digallate (TF-2), were extracted from green tea and black tea, respectively. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability, superoxide-scavenging efficiency, and lipid oxidation-inhibition ability of the pure compounds listed above as well as epicatechin (EC), carnosol, carnosic acid, and butylated hydroxytoluene (BHT) were investigated.
The DPPH radical-scavenging ability of the catechins was EGCG > ECG > EGC > EC and of the theaflavins was TF-2 > TF-1 > TF. EGCG, ECG, EGC, TF-2, TF-1, and TF showed higher DPPH radical- and superoxide-scavenging abilities than carnosol, carnosic acid, and BHT. EGCG, ECG, EGC, carnosol, and carnosic acid showed higher lipid oxidation-inhibition activity, as measured by the Rancimat method, than BHT and theaflavins.     

4 种天然抗氧化剂对纯油和人造奶油氧化稳定性的影响

对脂溶性天然抗氧化剂迷迭香、VE、抗坏血酸棕榈酸酯和水溶性天然抗氧化剂表没食子儿茶素没食子酸酯,在以棕榈油为主体的纯油产品和油包水型人造奶油产品中的抗氧化效果进行研究。结果表明：亲水亲油性不同的抗氧化剂在两种体系中的抗氧化性有明显差异。在人造奶油体系中,0.02%水溶性表没食子儿茶素没食子酸酯的抗氧化性最好。在纯油体系中,0.07%脂溶性迷迭香的抗氧化性最好。人造奶油体系的氧化稳定性明显低于纯油体系。     

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.     

Potent antioxidative activity of non‐polyphenolic fraction of green tea (Camellia sinensis) – association with pheophytins a and b

The antioxidative activity of green tea‐derived polyphenols known as catechins has been extensively studied. However, the antioxidative activity of the non‐polyphenolic fraction of green tea has been poorly elucidated and is the subject of the present study. The non‐polyphenolic fraction of green tea showed a significant dose‐dependent suppressive effect against the autooxidation of linoleic acid. The chlorophyll‐related compounds pheophytins a and b showed similar antioxidative activities in the same assay, much higher than those of α‐tocopherol and the green tea catechin (−)‐epigallocatechin‐3‐gallate (EGCG). The non‐polyphenolic fraction of green tea and pheophytins a and b exhibited suppressive activities against superoxide anion (${\bf O}_{2}ˆ{‐}$ ) generation in mouse macrophages induced by 12‐O‐tetradecanoylphorbol‐13‐acetate, showing higher activities than that of EGCG. These results suggest that the non‐polyphenolic fraction of green tea leaves has potent antioxidative activity and that this activity is associated with pheophytins a and b. © 2000 Society of Chemical Industry