表没食子儿茶素没食子酸酯的提取分离工艺研究

采用无水乙醇对茶叶进行浸提,用乙酸乙酯和蒸馏水分别对浸提液进行萃取后浓缩包含表没食子儿茶素没食子酸酯[epigallocatechin gallate,（-）-EGCG]在内的黄酮类物质。利用聚酰胺装填的层析柱对茶叶中的（-）-EGCG进行分离,并对分离产品结果进行分析。本实验以活性炭为脱色剂,考察了活性炭的用量、脱色时间、脱色次数、脱色后溶液pH、乙酸乙酯与水的配比以及洗脱荆中乙醇浓度等条件对实验结果产生的影响。在各项条件最优的情况下,每10g绿茶可以提取出纯度大于98％的（-）-EGCG单体0．39g。     

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.     

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     

利用活性真菌转化简单儿茶素为酯型儿茶素

研究从茶树的鲜叶、茎部和茶树土壤中分离出活性真菌,通过利用天然微生物的转化能力,使简单儿茶素转化为功能性较强的复杂型酯型儿茶素。实验结果表明:筛选出了具有转化能力的活性菌种,能有效地将简单儿茶素转化为酯型儿茶素,而且其转化能力良好。并对此种活性真菌进行形态学和生理生化的初步检测,判断其属于丝状真菌藻状菌纲,毛霉目,内囊霉科,内囊霉属。     

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     

EGCG乙酰化分子修饰取代度影响因素分析

为改善表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)的脂溶性和生物利用度,在乙酸乙酯体系中化学合成乙酰化EGCG。研究酰基供体乙酸酐用量、催化剂吡啶用量、溶剂乙酸乙酯用量、反应温度、反应时间对EGCG乙酰化分子修饰取代度的影响。结果表明,以0.19 g的EGCG为原料,乙酸酐用量0.04 m L、吡啶用量0.02 m L、乙酸乙酯用量20~60 m L、反应温度20~25℃、反应时间1~3 h有利于1~3取代度乙酰化EGCG生成;乙酸酐用量0.12~0.20 m L、吡啶用量0.06~0.20 m L、乙酸乙酯用量10~20 m L、反应温度17~25℃、反应时间5~9 h有利于4~6取代度乙酰化EGCG生成;乙酸酐用量0.40~0.80 m L、吡啶用量0.10~0.20 m L、乙酸乙酯用量5~10 m L、反应温度25~40℃、反应时间5~9 h有利于7~8取代度乙酰化EGCG生成。     

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     

Novel decaffeination of green tea using a special picking method and shortening of the rolling process

We have recently succeeded in manufacturing low-caffeine tea (LCT) by employing a special picking method in the 3rd leaf period and shortening the leaf-rolling process. In the present study, the effect of this special method on the content of other physiologically active substances, such as catechins, theanine, and vitamin C, as well as the mechanism of reduction of caffeine content in the LCT were investigated using capillary electrophoresis. By comparing the various components of tea leaves at different picking periods with or without shortening of the rolling process, it was found that the delayed leaf picking period and shortening of the rolling process used in the manufacture of LCT selectively reduced the caffeine content while retaining catechins, theanine, and vitamin C at a sufficient level. Therefore, our study demonstrated that this modified method may be useful in the manufacture of decaffeinated green tea.     

儿茶素和表儿茶素异构体的高效逆流色谱分离制备

应用高效逆流色谱法(HPCCC)对茶叶中的儿茶素和表儿茶素两种同分异构体的分离制备方法进行研究。结果表明：以正己烷-乙酸乙酯-水(体积比1:20:20)为两相溶剂系统,上相为固定相,下相为流动相,可以实现儿茶素和表儿茶素的完全分离。采用138mL的分离柱、1g儿茶素粗提物,在120min内一次制备即可得到纯度在95%以上的表儿茶素约35mg,纯度在99%以上的儿茶素325mg,纯度90%～99%的儿茶素100mg。     

Comparison of extraction and isolation efficiency of catechins and caffeine from green tea leaves using different solvent systems

Catechins from green tea (Camellia sinensis L.) have received considerable attention due to their beneficial effects on human health, such as antioxidant and anticancer activities. Optimisation of extraction conditions of the catechins from green tea leaves was performed using different solvents (ethanol or distilled water), different extraction methods (ultrasound‐assisted, room temperature or reflux extractions) and various extraction times (0.5–24 h). The optimal extraction conditions were determined using 40% ethanol with ultrasound‐assisted extraction method for 2 h at 40 °C. In addition, two isolation methods for the recovery of catechins from green tea extracts were compared using different solvent combinations (chloroform/ethyl acetate versus ethyl acetate/dichloromethane). The results showed that the ethyl acetate/dichloromethane system could achieve much higher content of catechins than the other isolation approaches, indicating the method that extract catechins first with organic solvent such as ethyl acetate before removing caffeine is much effective than removing caffeine first when organic solvents are used for the recovery of catechins without caffeine from green tea extracts.     

Effect of pH on cream particle formation and solids extraction yield of black tea

Effect of pH on tea solids extraction yield was significant in increasing instant tea yield. Solids extraction yield was doubled when tea was extracted at pH 1.2 compared with that extracted in boiling distilled water with pH 6.8. H⁺ encouraged black tea cream particle formation by either releasing more solids into the infusion or stimulating polyphenols to interact with polysaccharides and nucleophilic groups on protein in tea infusions. HPLC results revealed that theaflavins and tea catechins were leading substances affecting the formation of cream particles and infusion colour. Theaflavins and some tea catechins, such as EGCG, ECG and EGC, were dissociated or degraded under alkaline conditions and thus tea cream particles tended to dissolve and tea infusion became dark in colour. Feasibility of stepwise extraction of instant teas used for iced tea and normal drinking tea, by controlling temperature and pH, is also considered in the present paper.     

Antioxidant and Cytotoxic Activities of Lipophilic and Hydrophilic Fractions of Mentha Spicata L. (Lamiaceae)

Total antioxidant activity (TAA) and cytotoxic effect of four solvent fractions of ethanol extract of Mentha spicata L. were estimated. Relative antioxidant activity (RAA) was also determined relative to quercetin and L-ascorbic acid. Polyphenolics (phenolics and flavonoids) and pigments (chlorophylls and carotenoids) were quantitated and expressed as mg/g of the ethanol fraction. The ABTS/HRP/H₂O₂ decoloration method was used to estimate the total antioxidant activity. Cytotoxic effect on human prostate cancer cell line (PC-3) was assessed by MTT assay. The TAA was highest for ethyl acetate fraction (83%) followed by aqueous (75%), chloroform (51%) and hexane (47%) fractions. RAAs of ethyl acetate and aqueous fractions were equal to quercetin and ascorbic acid but less in hexane and chloroform fractions. Phenolics and flavonoids were higher in ethyl acetate (54 and 22 mg/g), aqueous (32 and 24 mg/g) and chloroform (30 and 16 mg/g) fractions compared to the hexane (14 and 15 mg/g) fraction. Chlorophyll and carotenoids were higher in chloroform fraction (29 and 7 mg/g) than in ethyl acetate (13 and 5 mg/g), hexane (14 and 3 mg/g) and aqueous (5 and 0.9 mg/g) fractions. Cytotoxic effect against PC-3 cell line was found to be highest for the chloroform fraction and lower for the aqueous fraction. Polyphenolic content and TAAs of the ethanol fractions were positively correlated. Similarly pigment content and cytotoxic effect of PC-3 cells were positively correlated.     

Antioxidant and Xanthine Oxidase Inhibitory Activities of Persicaria hydropiper

Five different polarity fractions of methanolic extract from Persicaria hydropiper, which are consumed as vegetables, were evaluated for its total phenolic content, and antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl radical scavenging, ferric thiocyanate, and xanthine oxidase inhibition assays. Particularly, higher phenolic content was exhibited by butanol and ethyl acetate fractions with the values of 224.38 and 68.95 mg GAE/100 g dry extract, respectively. Both butanol and ethyl acetate fractions exhibited higher 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with IC₅₀ values of 28.61 and 25.55 μg/ml. Meanwhile, both fractions also were shown to inhibit xanthine oxidase activity compared to other fractions with IC₅₀ values of 28.72 and 165.25 μg/ml. As for the ferric thiocyanate method, all the fractions except hexane fraction showed similar activity against lipid peroxidation and were comparable to butylated hydroxyl toluene, with percentage of inhibition from 95 to 98%.     

Milk β-lactoglobulin complexes with tea polyphenols

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K_C–β-LG = 2.2 (±0.8) × 10³ M⁻¹, K_EC–β-LG = 3.2 (±1) × 10³ M⁻¹, K_ECG–β-LG = 1.1 (±0.6) × 10⁴ M⁻¹ and K_EGCG–β-LG = 1.3 (±0.8) × 10⁴ M⁻¹. The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.     

Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays,Especially on Different Superoxide Anion Radical Generation Systems

Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot‐water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot‐water extract was similarly demonstrated among these extracts. Varying concentrations of the hot‐water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC₅₀ 28.1 μg/mL), 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) cation radical (EC₅₀ 23.7 μg/mL), galvinoxyl radical (EC₅₀ 3.06 μg/mL), and chlorpromazine cation radical (EC₅₀ 475 μg/mL). The yacon tea‐leaf extract further demonstrated superoxide anion (O₂^?) radical scavenging effects in the phenazine methosulfate‐NADH‐nitroblue tetrazolium (EC₅₀ 64.5 μg/mL) and xanthine oxidase assay systems (EC₅₀ 20.7 μg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea‐leaf extract could suppress the cellular O₂^? radical generation (IC₅₀ 65.7 μg/mL) in a phorbol 12‐myristate 13‐acetate‐activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O₂^? radical‐mediated disorders.     

Flavanol Composition and Caffeine Content of Green Leaf as Quality Potential Indicators of Kenyan Black Teas

The flavanol composition and caffeine content of green tea leaf, black tea quality parameters of theaflavins, thearubigins, liquor brightness and total colour varied more among clones than with time of the year. In green leaf, either (-)epicatechin gallate or (-)epigallocatechin gallate was the dominant flavanol present. Regression analysis of tasters' preferences for black teas against green leaf chemical components showed positive and significant correlations for (-)epicatechin gallate (r = 0·498, P ⩽ 0·05 for taster A; r = 0·665, P ⩽ 0·01 for taster B, and r = 0·678, P ⩽ 0·01 for both tasters' overall ranking), (-)epigallocatechin gallate (r = 0·513, P ⩽ 0·05 for taster B; r = 0·532, P ⩽ 0·05 for both tasters' overall ranking and caffeine (r = 0·523, P ⩽ 0·05 for taster A; r = 0·657, P ⩽ 0·01 for taster B; and r = 0·686, P ⩽ 0·01 for both tasters' overall ranking). Similar regressions against black tea theaflavins, thearubigin content, liquor brightness and total colour were not significant. The results suggest that the green leaf chemical components, (-)epicatechin gallate, (-)epigallocatechin gallate and caffeine could be used as quality potential indicators during clonal selection and propagation. © 1997 SCI.     

辣木叶提取物的制备及抗氧化活性

采用16 种提取方法制备辣木叶提取物,考察不同提取方法对辣木叶蛋白、糖类、多酚类物质提取率和抗氧化剂溶出率的影响。采用乙酸乙酯萃取辣木叶提取物,研究水相中蛋白（水解蛋白）、糖类和乙酸乙酯相中多酚类物质相互作用对辣木叶提取物抗氧化活性的影响。结果表明：采用酶法以及酶法耦合乙醇提取法可以制备蛋白（水解蛋白）、糖类和多酚类物质含量高且抗氧化活性强的辣木叶提取物。采用Ns37071蛋白酶、胰酶时,所得辣木叶提取物蛋白、糖类、多酚类物质提取率最高,抗氧化活性最强。蛋白（水解蛋白）和糖类物质对辣木叶提取物抗氧化活性的贡献大于多酚类物质,是辣木叶提取物发挥抗氧化活性的重要物质基础。此外,辣木叶提取物中蛋白（水解蛋白）、糖类、多酚类物质存在协同增效或拮抗作用。     

巫山茶多酚提取物体外抗氧化活性研究

摘要：目的 本实验对巫山茶多酚提取物的体外抗氧化活性进行了初步评价。方法 通过使用70%乙醇提取巫山茶得到巫山茶粗提物,再依次使用不同极性溶剂萃取巫山茶粗提物得到石油醚相、乙酸乙酯相、正丁醇相和水相4个萃取相,分别测定各萃取相的总酚含量和体外抗氧化活性,选取总酚含量最高且体外抗氧化活性最强的萃取相通过HP-20大孔树脂进行富集,测定富集后的巫山茶多酚提取物总酚含量和体外抗氧化活性。结果 乙酸乙酯相的总酚含量最高,为378.1mg/g;巫山茶粗提物及其不同萃取相均具有一定的抗氧化活性,以乙酸乙酯相的体外抗氧化活性最强,DPPH?、ABTS+?、? OH的IC50值分别为0.63mg/mL、0.46mg/mL和2.35mg/mL;将乙酸乙酯相通过HP-20大孔树脂富集后得到的巫山茶多酚提取物总酚含量为637.5mg/g,总酚含量显著提高（p <0.05）,DPPH?、ABTS+?、?OH的IC50值分别为0.26mg/mL、0.16mg/mL和1.05mg/mL,体外抗氧化活性显著增强（p <0.05）。相关性分析结果显示,巫山茶粗提物及各萃取相、富集后的总酚含量与其体外抗氧化活性呈正相关。结论 巫山茶多酚提取物具有较好的体外抗氧化活性,可考虑对其进行多酚成分分析和抗氧化功能研究。     

Isolation of a hydroxytyrosol-rich extract from olive leaves (<Emphasis Type="Italic">Olea Europaea</Emphasis> L.) and evaluation of its antioxidant properties and bioactivity

In this research, a phenol extract of high hydroxytyrosol (OLPE) content was obtained from olive leaves (Olea europaea L.), and subsequently tested under different contexts. The method used to obtain the OLPE basically involved two steps: the use of strongly-acid aqueous steam, generated from 10% HCl (v/v) at 100°C, to directly hydrolyse the native complex phenols from integral olive leaves, and OLPE recovery by liquid–liquid extraction with ethyl acetate. Hydrolysis time was 1 h. Finally, the dried extract was dissolved in distilled water. The OLPE total phenols were determined by Folin–Ciocalteu’s method and by HPLC analysis. Hydroxytyrosol was about 92% of the total phenols present in OLPE, and the yield was about 0.2% on fresh leaves. OLPE showed antioxidant effects on different food lipids and did not inhibit lactic acid bacteria growth; however, it showed cytotoxicity on NIH/3T3 fibroblasts and human umbilical vein endothelial cells at concentrations higher than 0.32 mM (as hydroxytyrosol).     

基于自组装技术的EGCG多层乳液的制备及其微胶囊化

针对表没食子儿茶素没食子酸酯(EGCG,Epigallocatechin gallate)稳定性差、易氧化降解的问题,采用层层自组装技术对其进行包载,建立EGCG稳态体系的制备工艺,并对产品微观形态及离心稳定性进行考察,进而通过喷雾干燥对其微胶囊化包埋,得到了粒径均一、稳定性较好的EGCG微胶囊。结果表明:在辛烯基琥珀酸淀粉钠(SSOS,sodium starch octenyl succinate)添加量70.0 g、水相油相比8:1 (g/g),剪切速率9000 r/min,剪切时间3 min条件下制备EGCG单层乳液,其离心稳定性系数可达0.138;在阿拉伯胶浓度15%,9000 r/min剪切3 min条件下,进一步制备而得的EGCG多层乳液稳定性较高。经喷雾干燥所得微胶囊的粒径在1~10 μm,其包封率达53.5%±0.5%,呈表面有褶皱无裂痕的椭球状,具有较好热力学稳定性。本研究为提高茶多酚稳定性,拓宽茶多酚产品应用领域提供了理论参考。