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.     

Simultaneous determination of polyphenols and major purine alkaloids in Greek Sideritis species, herbal extracts, green tea, black tea, and coffee by high-performance liquid chromatography-diode array detection

Herein, a high-performance liquid chromatography-diode array detection method has been developed for the simultaneous determination of 15 phenolic antioxidants: flavan-3-ols, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin, a phenolic acid (gallic acid), a hydroxycinnamic acid (chlorogenic acid), flavones (apigenin), flavonols (kaempferol, quercetin, and myricetin), and purine alkaloids (caffeine theophylline, theobromine) in different herb extracts, tea, and coffee varieties. The developed method was validated and successfully applied in order to determine the polyphenolic content to estimate the antioxidant activity of the Sideritis species commonly known as Greek mountain tea. To the best of our knowledge, this is the first report on the quantitative determination of catechins and other polyphenols in Greek mountain tea. Acidic hydrolysis was necessary for the simultaneous determination of the aglycones of the target analytes. According to our results, chlorogenic acid, myricetin, apigenin, catechin, and epicatechin gallate are found in the Sideritis species.     

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.     

Densitometric determination of (+)-catechin and (−)-epicatechin by 4-dimethylaminocinnamaldehyde reagent

We report the optimization of a sensitive, selective and robust derivatization method using 4-dimethylaminocinnamaldehyde (DMACA) for densitometric determination of (+)-catechin and (−)-epicatechin. The separation of these compounds was achieved by thin-layer chromatography (TLC) on cellulose plates developed with water. With DMACA in HCl, both compounds gave blue bands, while under the same conditions, vanillin produced a fast fading red coloration of bands. Quantitation at 655 nm showed that for both compounds the calibration curve was linear from 2 to 12 ng and polynomial from 2 to 30 ng, and the repeatability of chromatography of 20 ng was 3.5% (RSD, n = 6). The visible limit of detection of both standards was 1 ng, but the densitometric limit of detection was lower (0.2 ng). The optimized DMACA reagent is superior to the more frequently used vanillin reagent and is applicable also for determination of mixtures containing other catechins ((−)-catechin, (−)-epicatechin gallate, (−)-epigallocatechin gallate, procyanidin A2, procyanidin B1 and procyanidin B2).     

A new norisoprenoid and other compounds from Fuzhuan brick tea

Fuzhuan brick tea, a kind of dark tea consumed mainly in the border regions of Southwestern and Northwestern China since the 1860s, is produced from the leaves of Camellia sinensis var. sinensis by microbial fermentation. From this special fermented tea, a new norisoprenoid, 3R,9R-oxido-5-megastigmene, was isolated, together with α-linolenic acid, strictin, isovitexin, astragalin, (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (+)-gallocatechin, (-)-epigallocatechin, (-)-epigallocatechin gallate and gallic acid. The structures of the compounds were identified by spectroscopic means. The new compound didn't show any inhibition activity against the tested enteric pathogenic microorganisms at a concentration of 800 μg/mL by the hole plate diffusion method.     

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.     

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.     

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.     

Study of the catechins and proanthocyanidins ofQuercus robur

More than 20 compounds have been isolated from the bark ofQuercus robur. Monomers: (–)-epicatechin, (–)-epicatechin gallate, (+)-catechin, (+)-catechin gallate, (+)-gallocatechin, (–)-epigallocatechin, and (–)-epigallocatechin gallate; dimeric proanthocyanidins: (+)-catechin-(4-8)-(+)-catechin, 3-O-galloyl-(+)-catechin-(4-8)-3-O-galloyl-(+)-catechin, 3-O-galloyl-(+)-gallocatechin-(4-8)-(+)-gallocatechin, (–)-epicatechin-(4-8)-3-O-galloyl-(–)-epigallocatechin gallate, 3-O-galloyl-(–)-epicatechin-(4-8)-3-O-galloyl-(–)-epigallocatechin, 3-O-galloyl-(–)-epigallocatechin-(4-8)-(+)-catechin; and oligomeric proanthocyanidins: D14-D19.Materials presented at the IInd International Symposium on the Chemistry of Natural Compounds (SCNC, Eskiehir, Turkey, October, 22–24, 1996).Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 819–833, November–December, 1997.     

Direct enantioseparation of catechin and epicatechin in tea drinks by 6-O-alpha-D-glucosyl-beta-cyclodextrin-modified micellar electrokinetic chromatography

Cyclodextrin-modified micellar electrokinetic chromatography was applied to the enantioseparation of catechin and epicatechin using 6-O-alpha-D-glucosyl-beta-cyclodextrin together with sodium dodecyl sulfate and borate-phosphate buffer. Factors affecting chiral resolution and migration time of catechin and epicatechin were studied. The optimum running conditions were found to be 200 mM borate-20 mM phosphate buffer (pH 6.4) containing 25 mM 6-O-alpha-D-glucosyl-beta-cyclodextrin and 240 mM sodium dodecyl sulfate with an effective voltage of +25 kV at 20 degrees C using direct detection at 210 nm. Under these conditions, the resolution (Rs) of racemic catechin and epicatechin were 4.15 and 1.92, respectively. With this system, catechin and epicatechin enantiomers along with other four catechins ((-)-catechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate) and caffeine in tea samples were analyzed successfully. The difference of migration time between catechin and epicatechin is discussed.     

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.     

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.     

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。     

Separation of proanthocyanidins isolated from tea leaves using high-speed counter-current chromatography

The proanthocyanidin extract from tea (Camellia sinensis) leaves was purified for the further study of the biological role of proanthocyanidins in blister blight leaf disease of tea, which is caused by the fungus Exobasidium vexans. An aqueous acetone extract of proanthocyanidins prepared from healthy tea leaves was partially purified using Sephadex LH-20 chromatography. The crude proanthocyanidin extract obtained was fractionated with high-speed counter-current chromatography (HSCCC) using the solvent system n-hexane–EtOAc–MeOH–water (1:5:1:5). The purity of the each isolated fraction after a single HSCCC run was evaluated by high-performance liquid chromatography (HPLC). Seven fractions of high purity were isolated. The identity of the compound present in each fraction isolated was established using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. Five proanthocyanidins and two flavanol digallates, (−)-epigallocatechin digallate (EGCDG) and (−)-epicatechin digallate (ECDG) were isolated. Comparison of spectral data of the proanthocyanidins isolated with those previously reported indicated that all five were known B-type proanthocyanidins with 2,3-cis stereochemistry in both the upper (u-unit) and the terminal (t-unit) units, and 4R configuration of the C-ring in the u-unit. The proanthocyanidins were established to be dimers composed of (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epiafzelechin gallate (EAG) units with the following structures: EGCG-(4β → 6)-EGCG, ECG-(4β → 6)-EGCG, EGCG-(4β → 6)-ECG, EAG-(4β → 6)-EGCG, ECG-(4β → 6)-ECG by analysis of spectral data. Therefore HSCCC offers a powerful method for the separation of a group of closely related naturally occurring compounds.     

Catechins and proanthocyanidins ofAlhagi sparsifolia. I

Ten individual compounds have been isolated from the epigeal part ofAlhagi sparsifolia Shap. in various stages of vegetative growth. Their structures have been established by a study of PMR spectra, physicochemical properties, and the products of chemical transformations: (–)-epicatechin, (–)-epigallocatechin, (–)-epigallocatechin gallate, (+)-catechin, (+)-gallocatechin, proanthocyanidin B-2, (–)-epigallocatechin-(4–8)-(–)-epicatechin, epigallocatechin gallate-(4–8)-(–)-epicatechin, proanthocyanidin B-1, and (–)-epicatechin-(4–8)-gallocatechin.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. 232–237, March–April, 1997.     

Comparison of microemulsion electrokinetic chromatography and micellar electrokinetic chromatography methods for the analysis of phenolic compounds

In this study, microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) were compared for their abilities to separate and detect thirteen phenolic compounds (syringic acid, p-coumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), and two other ingredients (caffeine and theophylline) in teas and grapes. Separation of phenolic compounds was improved by changing the SDS concentration for MEEKC, but the SDS concentration rarely affected the resolution for MEKC. Organic modifier (acetonitrile or methanol) was found to markedly influence the resolution and selectivity for both MEEKC and MEKC systems. In addition, a higher voltage and a higher column temperature improved the separation efficiency without any noticeable reduction in resolution for MEEKC whereas they caused a poor resolution for the MEKC system. Although separations with baseline resolution were achieved by the optimized MEEKC and MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC.     

Catechin gallates ofRheum maximoviczii A. Los

Summary The roots ofRheum maximoviczii A. Los. contain two catechin gallates: (–)-epicatechin gallate, previously isolated from the leaves of green tea and (±)-catechin gallate found in this plant for the first time. The amount of catechin gallates in the roots is lower in the autumn than in the spring.Khimiya Prirodnykh Soedinenii, Vol. 2, No. 1, pp. 26–29, 1966     

Analyses of phenolic compounds by microemulsion electrokinetic chromatography

In this study, a microemulsion electrokinetic chromatography (MEEKC) method was developed to analyze and detect 13 phenolic compounds (syringic acid, p-cumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), which are present in many plant-derived foods. The effects of cosurfactant, organic modifier, and oil were examined in order to optimize the separation of these phenolic compounds. The amounts of cosurfactant (cyclohexanol) and organic modifier (acetonitrile) were determined as the major influence on the separation selectivity, while the type of oil partially affected the separation resolution of the phenolic compounds. A highly efficient MEEKC separation method was achieved within 14 min by using a microemulsion solution of pH 2.0 containing 2.89% w/v SDS, 1.36% w/v heptane, 7.66% w/v cyclohexanol, and 2% w/v ACN. Furthermore, the present work could demonstrate that the nature of the oil phase has a significant influence on the separation selectivity of phenolic 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.     

Stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. isolated from a tea plant

The microbial transformation of five flavans (1-5) by endophytic fungi isolated from the tea plant Camellia sinensis was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. oxidized stereoselectively at C-4 position of (+)-catechin (1) and (-)-epicatechin (2) to give the correspondent 3,4-cis-dihydroxyflavan derivatives (6, 10), respectively. (-)-Epicatechin 3-O-gallate (3) and (-)-epigallocatechin 3-O-gallate (4) were also oxidized by the fungus into 3,4-dihydroxyflavan derivatives (10, 12) via (-)-epicatechin (2) and (-)-epigallocatechin (11), respectively. Meanwhile, (-)-gallocatechin 3-O-gallate (5), (-)-catechin (ent-1) and (+)-epicatechin (ent-2), which possess a 2S-phenyl substitution, resisted the biotransformation.