Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins,three purine alkaloids,and gallic acid in tea by high-performance liquid chromatography with diode array detection

Monomers of (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) and (−)-3-O-methyl epicatechin gallate (ECG3′Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (−)-catechin (C), (−)-gallocatechin (GC), (−)-gallocatechin gallate (GCG), and (−)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C₁₈ reversed-phase column, fourteen compounds were rapidly separated within 15 min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5–7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40–105 min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1–1.0 ng for most components at the applied wavelength of 280 nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92–106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.     

Epicatechin-copper 9II) complexes: Damage of small intestinal epithelium

Four epicatechins [(−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCg)] and their corresponding copper complexes were compared with regard to their effect on the viability of Caco-2 colon cancer cells in vitro, measured by 3-(4,5-dimethylthyazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. The viability of Caco-2 cells exposed to EC (1 mM), ECg (1 mM) or EGC (1mM) respectively, for 30 min, was comparable to that of the saline control group, while EGCg (1 mM) apparently enhanced cellular activity. in contrast, the cells treated with epicatechin-copper complexes were killed. Bivalent copper 91 mM), in similar conditions, did not affect the cells. No cell leakage or other histological differences were observed, implying a rapid cell death. The suggested mechanism of killing is by OH radical attack, produced in the presence of epicatechin-copper complexes, but not in the presence of either of the epicatechins or copper alone. The reaction sites are discussed.     

Determination of catechins and catechin gallates in biological fluids by HPLC with coulometric array detection and solid phase extraction

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β-d-glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10-2000 and 10-5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.     

Characteristics of the OH radical scavenging activity of tea catechins

Reaction rate constants of (−)-epigallocatechin gallate (EGCG) and (+)-catechin with the hydroxyl radical (·OH) were measured using the rapid flow ESR method. The rate constant of EGCG was larger twice than that of the pyrogallol or gallic acid, they are the model compounds of the B ring of EGCG. It was explained by the quantum-chemical calculation of the bond dissociation energy (BDE) of the phenolic hydroxyl group (ϕ-OH) and the spin densities of EGCG radical. The energy of the EGCG radical was lowered by the hydrogen bonding between the radical part on the B ring and the hydroxyl group on the gallate group, leading to the lowering of BDE. Linear relationship between the relative activation energy and BDE of all the polyphenols measured was observed (Evans-Polanyi equation), showing that the reaction with ·OH occurs in the same manner.     

Inhibition of lipid peroxidation induced by tritiated water due to tea catechins

Inhibiting effect of tea catechins on the lipid peroxidation induced by tritiated water was studied. In our previous reports, it was found that the inhibiting effects of (−)-epigallocatechin gallate (EGCg) was decreased in the concentration range of above 5.0·10⁻⁵ M. In this study, the reason of decrease was revealed using fluorescence analysis and electron spin resonance, and its inhibiting mechanism of EGCg was considered. The decrease of the inhibiting effect was mainly attributed to the interaction between lipid membranes and EGCg. EGCg induced aggregation of liposomes with interacting with membranes at its concentration range of above 5.0·10⁻⁵ M. Finally, EGCg broke the membrane at its concentration range of above 1.0·10⁻³M. Furthermore, autoxidation of EGCg did not influence on the inhibiting effects of the lipid peroxidation under these conditions. Thus, inhibiting effect of EGCg was attributed to the amount of EGCg adsorbed on the membranes, but membrane structure was broken by adsorption of large amount of EGCg on the membranes.     

Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectrometry

(-)-Epigallocatechin gallate (EGCG) of catechins changes from non-colored at around neutral pH to yellow at higher pH region in aqueous solution. The pH-dependent oxidation of EGCG was analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). LC/MS/MS analysis of EGCG and its related compounds, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC), successfully elucidated the structure relationship of EGCG solution involving the color change reaction at different pH conditions. The oxidation species produced at alkaline pH was detected at a different retention time from EGCG in the chromatograms of the EGCG sample. The oxidation species was found to correspond to M+14 (where M is the molecular weight of EGCG), which has two hydrogen atoms removed and addition of one oxygen atom to the gallyl moiety in the B-ring of EGCG.     

高效液相色谱法分析元宝枫叶中儿茶素类物质

本文建立了元宝枫树叶中儿茶素种类及其含量的高效液相色谱(HPLC)测定方法。采用反相C18色谱柱,以甲醇/水(含0.5%乙酸)=25/75(V/V)为流动相,对没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)和没食子儿茶素没食子酸酯(GCG)进行定性、定量分析;以甲醇/水(含0.5%乙酸)=35/65(V/V)为流动相,对表儿茶素没食子酸酯(ECG)和儿茶素没食子酸酯(CG)进行定性分析,柱温均为35℃,检测波长为278 nm,流速为1.0mL/min。结果表明:元宝枫叶中有EGC、EC和GCG,其它五种则无。EGC平均含量为0.0389 mg/g,方法精密度(RSD)为0.42%(n=6);EC平均含量为0.0289 mg/g,方法RSD为1.5%(n=6);GCG平均含量为0.284 mg/g,方法RSD为0.32%(n=6)。该方法简便、准确、分离效果好,为元宝枫叶开发成茶叶、饮料以及医疗保健品提供重要依据。     

Microemulsion electrokinetic chromatography for the analysis of green tea catechins: effect of the cosurfactant on the separation selectivity

Microemulsion electrokinetic chromatography (MEEKC) was applied to the separation of six catechins and caffeine, the major constituents of the green tea. The developed methods involved the use of sodium dodecyl sulfate (SDS) as surfactant, n-heptane as organic solvent and an alcohol as cosurfactant. The separations were performed under acidic conditions (pH 2.5 phosphate buffer, 50 mM) to ensure good stability of the catechins, with reversed polarity (anodic outlet). The effect of the alcohol nature on the MEEKC selectivity was evaluated; nine alcohols were used as cosurfactant: 1-butanol, tert-butanol, 1-pentanol, 2-pentanol, 3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, and cyclohexanol. The migration order of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-gallocatechin (GC), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), caffeine and theophylline was significantly affected by the alcohol used as cosurfactant. Using nine microemulsions, four different selectivities were achieved: A (cyclohexanol); B (2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol); C (1-butanol, 1-pentanol, cyclopentanol); D (tert-butanol). MEEKC methods, based on 2-hexanol and cyclohexanol as cosurfactant were validated and successfully applied to the analysis of catechins and caffeine in commercial green tea products.     

Full Validation of a Simple Method for Determination of Catechins and Caffeine in Brazilian Green Tea (Camellia sinensis var. assamica) Using HPLC

This paper describes the validation of an HPLC method for the assay of a green tea brew. The method employs a RP-18 column with water:methanol:ethyl acetate elution and UV detection at 280 nm. Specificity was evaluated using a photodiode array detector. The validation data showed that the assay is specific, accurate, precise, and reproducible for determination of six catechins and caffeine simultaneously. The response was linear over a range of 37–185 μg mL^?1 for caffeine, 99–500 μg mL^?1 for (?)-epigallocatechin (EGC), 20–100 μg mL^?1 for (+)-catechin (C), 30–150 μg mL^?1 for (?)-epicatechin (EC), 150–800 μg mL^?1 for (?)-epigallocatechin gallate (EGCG), 20–105 μg mL^?1 for (?)-gallocatechin gallate (GCG) and 40–205 μg mL^?1 for (?)-epicatechin gallate (ECG) (r > 0.9999 for all compounds). The range of recoveries was 96.12–110.48% according to substances. The RSD values for intra- and inter-day precision studies were <2.07 and <6.65%, respectively. The composition of samples assayed suggests that the summer is the best season for extract a major content of EGCG and caffeine. This assay can be readily utilized as quality controlled method for major green tea compounds.     

绿茶多酚对自由基诱导的红细胞膜过氧化的抑制作用

采用水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在37引发入血红细胞膜的过氧化,通过测定氧气吸收及维生素E的消耗研究了过氧化过程的动力学,并对从绿茶中提取的主要多酚类化合物的抗氧化活性做了定量研究。使用的绿茶多酚有:(-)-表儿茶素(EC),(-)-表儿茶素(EGC),(-)-表儿茶素酸酯(ECG)和(-)-表儿茶素培酸酯(EGCG)。结果表明,这些绿茶多酚能够显著缩短过氧化反应的动力学链长,有效地抑制红细胞膜的过氧化。抗氧化活性顺序为:EC〉GCG〉EGCG〉EGC。     

Borate complexation-assisted field-enhanced sample injection for on-line preconcentration of cis-diol-containing compounds in capillary electrophoresis

A new on-line preconcentration technique called borate complexation-assisted field-enhanced sample injection (BCA-FESI) was proposed for preconcentrating cis-diol-containing compounds (CDCCs) in capillary electrophoresis (CE). The principle relies on amplification of the difference in the electrophoretic mobilities of CDCC in sample matrix and background electrolyte (BGE) through complexation of CDCC with borate in a sample matrix of basic pH and dissociation of the complex in a BGE of acidic pH. Meanwhile, CDCC and borate ions electro-injected into the capillary are finally in neutral state, which maintains the pre-filled low conductivity zone and thus allows for longer injection time. With catechol as a test compound, the principle and effectiveness of BCA-FESI was verified. As compared to conventional sample injection, BCA-FESI allowed for sensitivity enhancement of 1850-fold. The established method was further evaluated with three catechins, including (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), and (−)-epigallocatechin (EGC), in a standard mixture of trace content. The limit of detection (LOD) was found to be 1.4, 3.8, 17.5 nM (S/N = 3) for ECG, GCG, EGC, respectively. Finally, the BCA-FESI method was applied to a real sample of diluted tea beverage, in which the three catechins were detected.     

Development of HPLC Method for Catechins and Related Compounds Determination and Standardization in Miang (Traditional Lanna Fermented Tea Leaf in Northern Thailand)

High performance liquid chromatography (HPLC) for catechins and related compounds in Miang (traditional Lanna fermented tea leaf) was developed to overcome the matrices during the fermentation process. We investigated a variety of columns and elution conditions to determine seven catechins, namely (+)-catechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, (−)-epicatechin gallate, as well as gallic acid and caffeine, resulting in the development of reproducible systems for analyses that overcome sample matrices. Among the three reversed-phase columns, column C (deactivated, with extra dense bonding, double endcapped monomeric C18, high-purity silica at 3.0 mm × 250 mm and a 5 µm particle size) significantly improved the separation between Miang catechins in the presence of acid in the mobile phase within a shorter analysis time. The validation method showed effective linearity, precision, accuracy, and limits of detection and quantitation. The validated system was adequate for the qualitative and quantitative measurement of seven active catechins, including gallic acid and caffeine in Miang, during the fermentation process and standardization of Miang extracts. The latter contain catechins and related compounds that are further developed into natural active pharmaceutical ingredients (natural APIs) for cosmeceutical and nutraceutical products.     

Simultaneous determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC with diode array detection

A simple high-performance liquid chromatography method using a diode array detector (DAD) is developed for the simultaneous analysis of five major catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GCT), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and the phenolic plant metabolites gallic acid (GA) and rutin (RT) in lyophilized extracts of Cistus species. The optimal analytical conditions are investigated to obtain the best resolution and the highest UV sensitivity for the quantitative detection of catechins. The optimized conditions (acetonitrile-phosphate buffer 50mM, pH 2.5, gradient elution system on a C(18) reversed-phase column with a flow rate of 1 mL/min and UV absorbance at 210 nm) allowed a specific and repeatable separation of the studied analytes to be achieved. All compounds are successfully separated within 32 min. Calibration curves are linear in the 2-50 microg/mL range for GCT, C, and EGCG and in the 5-50 microg/mL range for GA, EGC, EC, and RT. The limit of detection values ranged from 0.24 to 0.74 microg/mL. The limit of quantitation limit values ranged from 0.77 to 1.94 microg/mL. The validated method is applied to the determination of the specific phytochemical markers GA, GCT, C, and RT in Cistus incanus and Cistus monspeliensis lyophilised extracts. The recovery values ranged between 78.7% and 98.2%. The described HPLC method appears suitable for the differentiation and determination of the most common catechins together with the glycoside rutin and the phenolic compound gallic acid and can be considered an effective and alternative procedure for the analyses of this important class of natural compounds.     

Sample stacking for the analysis of catechins by microemulsion EKC

In this study, an on-line concentration method, ASEI (anion-selective exhaustive injection)-sweeping technology which was coupled with microemulsion EKC (MEEKC), was used to analyze and detect six catechins ((-)-epicatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-gallocatechin). In addition to the effects of the buffer pH and electrolyte concentration on stacking, the compositions of microemulsion (types of oil phase, and types and levels of cosurfactant) also dominated the stacking effect of catechins. In MEEKC, the effect of the type of oil in microemulsion on separation mechanism is often unclear. This study had demonstrated that the oil type in microemulsion indeed altered the affinity of oil droplets with analytes. Finally, this proposed ASEI-sweeping MEEKC method was able to detect trace level of catechins in food products that was not previously possible by a normal MEEKC method.     

Reactions of tea polyphenol four constituents with hydroxyl radical: A pulse radiolytic study

Ｆｒｅｅｒａｄｉｃａｌｂｉｏｌｏｇｙａｎｄｍｅｄｉｃｉｎｅｈａｖｅｄｅｍｏｎｓｔｒａｔｅｄｔｈａｔｆｒｅｅｒａｄｉｃａｌｄｉｒｅｃｔｌｙｒｅｌａｔｅｓｔｏｔｈｅｐａｔｈｏｇｅｎｅｓｉｓｏｆｖａｒｉｏｕｓｂｉｏｌｏｇｉｃａｌｄｉｓｏｒｄｅｒ,ｅ.ｇ.ｃａｒｃｉｎｏｇｅｎｅｓｉｓ,ｃａｒｄｉｏｖａｓｃｕｌａｒｄｉｓｅａｓｅｓ,ｒａｄｉａｔｉｏｎｉｎｄｕｃｅｄｂｉｏｌｏｇｉｃａｌｄａｍａｇｅｓａｎｄａｇｉｎｇ[1],ｔｈｅｓｅｒａｄｉｃａｌｓ,ｉｎｃｌｕｄｉｎｇＯＨ·,Ｏ·-2,ｅｔｃ.,ｃａｎｂｅｐｒｏｄｕｃｅｄｂ…     

Health Benefits and Chemical Composition of Matcha Green Tea: A Review

Japanese matcha is a type of powdered green tea, grown in a traditional way. Shading of the plants during the growth period enhances the processes of synthesis and accumulation of biologically active compounds, including theanine, caffeine, chlorophyll and various types of catechins. Green tea contains four main catechins, i.e., (−)-epicatechin (EC), (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin-3-gallate (EGCG), of which the latter is the most active and abundant and matcha is their best condensed source. Due to its unique chemical composition and prized flavour, which sets it apart from other tea beverages, it is considered the highest quality tea. Its health-promoting properties are attributed to the high content of antioxidant and anti-inflammatory substances. Studies confirming the high antioxidant potential of tea beverages claim that it originates from the considerable content of catechins, a type of phenolic compound with beneficial effects on human health. Due to its potential for preventing many diseases and supporting cognitive function, regular consumption of matcha may have a positive effect on both physical and mental health. The aim of this review was to compile the health benefits of matcha tea. It is the first such review to be undertaken, and presents its main bioactive compounds in a systematic manner.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.     

Inhibition of gut bacterial β-glucuronidase by chemical components from black tea: Inhibition interactions and molecular mechanism

Gut bacterial β-glucuronidase is regarded as an important molecular target for several therapeutic applications. Inhibitors of β-glucuronidase can effectively alleviate the drug-induced gastrointestinal tract toxicity. In this study, the ethanol extracts of black tea was found to display significant inhibitory activities against Escherichia coli β-glucuronidase (EcGUS), and seven polyphenols including catechins and theaflavins were identified as the key components responsible for the strong inhibitory potency of black tea towards EcGUS. Among these seven identified naturally occurring inhibitors, (−)-catechin gallate (CG), theaflavin-3-monogallate (TF-3-G), theaflavin-3′-monogallate (TF-3′-G) and theaflavin-3,3′-digallate (TFDG) were more potent inhibitors of EcGUS compared with (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG), and (−)-epigallocatechin gallate (EGCG). Furthermore, molecular docking and molecular dynamics simulation results further indicated that TFDG could bind in the cavity of EcGUS and interacted with key residues Ser360, Glu413 and Ile560 of EcGUS through hydrogen bonds. Taken together, these data offer important information for efficient development of black tea and its catechins and theaflavins constituents for treating drug-induced enteropathy.     

Proanthocyanidins ofZiziphus jujuba

The catechin and proanthocyanidin compositions of the leaves and bark ofZiziphus jujuba have been studied over the vegetation periods. This has led to the isolation of 16 compounds, including 8 monomeric catechins — (–)-epiafzelechin, (–)-epicatechin, (–)-epigallocatechin, (–)-epicatechin gallate, (–)-epigal-locatechin gallate, (+)-catechin, (+)-catechin gallate, and (+)-gallocatechin; 4 dimeric proanthocyanidins — (–)-epiafzelechin-(4-8)-(–)-epicatechin, proanthocyanidin B-2, (–)-epicatechin-(4-8)-(–)-epigallocatechin, and (–)-epiafzelechin-(4-8)-(–)-epigallocatechin; and 4 oligomeric proanthocyanidins consisting of epiafzelechin, epigallocatechin, catechin, and epicatechin with different degrees of polymerization. Their structures have been established by a study of PMR and¹³C NMR spectra and the products of chemical transformation.The materials of this paper were presented at the Second International Symposium on the Chemistry of Natural Compounds (SCNC, Eskiehir, Turkey, October 22–24, 1996).Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 221–231, March–April, 1997.