Attomole catechins determination by capillary liquid chromatography with electrochemical detection.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

茶叶及茶多酚中儿茶素的高效液相色谱分析方法研究

 筛选出HypersilBDSC18和ZorbaxSBC18两种适合同时分离茶叶和茶多酚中 7种儿茶素和咖啡因的反相柱。采用甲醇 水 醋酸 (或三氟醋酸 )作流动相 ,分别以等强度洗脱和梯度洗脱 (均在 30min内 )分离测定了我国 6种不同产地茶叶样品和 3种茶多酚样品中 7种儿茶素的含量。考察了 7种儿茶素和咖啡因的保留值与流动相组成及柱温的关系 ,优化了色谱条件及样品前处理方法。用电喷雾电离质谱 (ESI MS)定性确认没食子儿茶素没食子酸酯(GCG)和儿茶素没食子酸酯 (CG)两组分 ,并用高效液相色谱制备两对照品用于定量分析。     

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.     

Camellia sinensis: Insights on its molecular mechanisms of action towards nutraceutical,anticancer potential and other therapeutic applications

The Camellia sinensis plant provides a wide diversity of black, green, oolong, yellow, brick dark, and white tea. Tea is one of the majorly used beverages across the globe, succeeds only in the water for fitness and pleasure. Generally, green tea has been preferred more as compared to other teas due to its main constituent e.g. polyphenols which contribute to various health benefits. The aim of this updated and comprehensive review is to bring together the latest data on the phytochemistry and pharmacological properties of Camellia sinensis and to highlight the therapeutic prospects of the bioactive compounds in this plant so that the full medicinal potential of Camellia sinensis can be realised. A review of published studies on this topic was performed by searching PubMed/MedLine, Scopus, Google scholar, and Web of Science databases from 1999 to 2022. The results of the analysed studies showed that the main polyphenols of tea are the four prime flavonoids catechins: epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) along with the beneficial biological properties of tea for a broad heterogeneity of disorders, including anticancer, neuroprotective, antibacterial, antiviral, antifungal, antiobesity, antidiabetes and antiglaucoma activities. Poor absorption and low bioavailability of bioactive compounds from Camellia sinensis are limiting aspects of their therapeutic use. More human clinical studies and approaching the latest nanoformulation techniques in nanoparticles to transport the target phytochemical compounds to increase therapeutic efficacy are needed in the future.     

Development of rapid and simultaneous quantitative method for green tea catechins on the bioanalytical study using UPLC/ESI‐MS

A rapid and quantitative analytical method for the simultaneous determination of green tea catechins using ultra‐performance liquid chromatography/electrospray ionization–mass spectrometry was developed. Total analytical run time was 3.5 min for the detection of (?)‐epicatechin (EC), (?)‐epicatechin‐3‐O‐gallate (ECG), (?)‐epigallocatechin (EGC), (?)‐epigallocatechin‐3‐O‐gallate (EGCG) and myricetin as the internal standard (IS) in rat plasma. The calibration curves were linear over the range of 10–5000 ng/mL for all the catechins. The inter‐ and intra‐day precision (relative standard deviation) and accuracy (percentage deviation) of the method were both lower than 10%. The average extraction recoveries in plasma ranged from 68.5 to 86.5%, and the lower limits of quantification of EC, EGC, ECG and EGCG were 10 ng/mL with a signal‐to‐noise ratio of >10. The assay developed was successfully applied to a pharmacokinetic study of catechins following intravenous and intragastric administrations of green tea extract in rats. Plasma concentrations of four catechins were detected up to 5–24 h after administration, and the pharmacokinetic parameters of catechins were in agreement with previous studies. From these findings, taken together with the high productivity and precision, the developed method could be a reliable and reproducible tool for the evaluation of pharmacokinetic properties of catechins. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of tea samples with a multisensor system and capillary electrophoresis

Potential of an “electronic tongue” multisensor system in identification of various tea samples and in quantitative analysis of separate tea components was studied. As a reference method for quantitative analysis of antioxidants of the polyphenolic type served micellar electrokinetic chromatography with a UV detector. The contents of epicatechin gallate, epigallocatechin gallate, epicatechin, epigallocatechin, gallic acid, gallocatechin gallate, and caffeine alkaloid in various tea samples were quantitatively estimated.     

Analysis of natural flavonoids by microchip-micellar electrokinetic chromatography with pulsed amperometric detection

Catechins (catechin and other derivatives) are naturally occurring flavonoids present in a number of plants and foods. They are also part of numerous nutraceutical formulations because they are believed to have antioxidant, cancer chemo-preventative, anti-inflammatory and antimicrobial properties. The determination of catechins has traditionally been performed by HPLC. However, this methodology is both time and sample intensive and generates large amounts of organic solvent waste. In the current report, an application of MEKC using a PDMS microchip is presented for the analysis of catechins. The system uses pulsed amperometric detection for direct analysis of important naturally occurring catechins. The effect of pH, surfactant concentration, detection potential and signal stability were analyzed. Linear relationships were found between the concentration and peak current, with good stability and limits of detection of 8 [micro sign]M for catechin, epigallocatechin gallate and epicatechin, and 14 [micro sign]M for epicatechin gallate. Optimum conditions were applied to the detection of selected catechins in a commercially available green tea extract nutraceutical and the results were compared to HPLC analysis. The analysis using microchip micellar electrokinetic chromatography and pulsed amperometric detection was completed in 4.5 min, 10 times faster than the HPLC analysis.     

Preparation,evaluation and characterization of quercetin-molecularly imprinted polymer for preconcentration and clean-up of catechins

Molecularly imprinted polymer (MIP) for solid extraction and preconcentration of catechins have been successfully prepared by a thermal polymerization method using quercetin as template, 4-vinylpyridine as functional monomer and ethylene glycol dimethacrylate as crosslinker. A solution mixture of acetone and acetonitrile was used as porogen. Systematic investigations of the influence of monomer, cross-linker, porogen, as well as polymerization conditions on the properties of the MIPs were carried out. The quercetin MIPs were evaluated according to their selective recognition properties for quercetin, structurally related compounds (catechin, epigallocatechin gallate and epicatechin) and a unrelated compound of similar molecular size (α-tocopherol). Good binding was observed for quercetin, catechin and epigallocatechin gallate with an optimized MIP in a solid phase extraction system. Adsorption and kinetic characteristics were evaluated for catechins which indicated that the synthesized polymer had high adsorption capacity and contained homogeneous binding sites. Chemical and morphological characterization of the MIP was investigated by FTIR, SEM and BET, which confirmed a high degree of polymerization. Finally, the MIP was successfully applied to the clean-up and preconcentration of catechins from several natural samples.     

茶中茶多酚的高效液相色谱法分离分析

用改进的Ａｇａｒｗａｌ方法萃取不同种类茶叶和茶饮料中的茶多酚,建立了用高效液相色谱（ＨＰＬＣ）法对茶多酚进行分离分析方法。ＨＰＬＣ可有效分离ＧＴＰｓ主要组成成分ＥＣ、ＥＧＣ、ＥＣＧ和ＥＧＣＧ并精确定量,相对标准偏差小于５％。茶叶加工过程对ＧＴＰｓ含量有很大影响,绿茶总ＧＴＰｓ含量在６￣１５ｇ／１００ｇ干茶叶、乌龙茶总ＧＴＰｓ含量在５￣７ｇ／１００ｇ干茶叶,红茶总ＧＴＰｓ含量低于２ｇ／１００ｇ干茶叶     

Rapid, direct determination of polyphenols in tea by reversed-phase column liquid chromatography.

Column liquid chromatography on a C18-bonded silica column with water-methanol-acetic acid as eluent was used to determine polyphenols and caffeine in tea. Without any pretreatment, catechin, epicatechin gallate, epigallocatechin gallate, epigallocatechin, epicatechin and caffeine were separated successfully within 15 min. The detection limits (S/N = 3) of polyphenols studied were 1.8-24 mg/l at a detection wavelength 270 nm. The linear range of the peak area calibration curves for the analytes were over two orders of magnitude with a correlation coefficient of 0.996-0.999. Using this method, some Chinese tea samples were analyzed with a good reproducibility (RSD are below 5%).     

Isomeric differentiation of green tea catechins using gas-phase hydrogen/deuterium exchange reactions

Hydrogen/deuterium exchange reactions in a quadrupole ion trap mass spectrometer are used to differentiate galloylated catechin stereoisomers (catechin gallate and epicatechin gallate; gallocatechin gallate and epigallocatechin gallate) and the nongalloylated analogs (catechin and epicatechin, gallocatechin and epigallocatechin). Significant differences in the hydrogen/deuterium exchange behavior of the four pairs of deprotonated catechin stereoisomers are observed upon reaction with D(2)O. Interestingly, the nongalloylated catechins undergo H/D exchange to a much greater extent than the galloylated species, incorporating deuterium at both aromatic/allylic and active phenolic sites. Nongalloylated catechin isomers are virtually indistinguishable by their H/D exchange kinetics over a wide range of reaction times (0.05 to 10 s). Our experimental results are explained using high-level ab initio calculations to elucidate the subtle structural variations in the catechin stereoisomers that lead to their differing H/D exchange kinetics.     

Development and validation of a high‐throughput assay for the quantification of multiple green tea‐derived catechins in human plasma

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed‐phase liquid chromatography–tandem mass spectrometry (LC–MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra‐batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high‐throughput sample analysis studies.     

高效液相色谱-质谱法及模式识别法用于鉴别普洱生茶与熟茶

采用高效液相色谱-质谱联用技术及高效液相色谱法对生熟普洱茶中的主要成分进行定性和定量分析。鉴定出普洱茶水溶液中8种主要成分,分别为没食子酸(GA)、没食子酸儿茶素(GC)、表没食子酸儿茶素(EGC)、儿茶素(C)、咖啡因(CAF)、表儿茶素(EC)、表没食子酸儿茶素没食子酸酯(EGCG)和表儿茶素没食子酸酯(ECG)。以这8种成分的含量为指标,对普洱生茶和熟茶各20批进行主成分分析、聚类分析和判别分析,能准确地区分普洱生茶与熟茶。     

半制备型液相色谱法分离纯化茶叶鲜叶中7种儿茶素类化合物

建立了从茶叶鲜叶中分离纯化7种儿茶素类化合物(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)、表没食子儿茶素3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)和表儿茶素没食子酸酯(ECG))的半制备色谱法。铁观音鲜叶经甲醇超声浸提、浓缩、氯仿萃取后,向水相中加入碱式醋酸铅沉淀,得到茶多酚粗品。分别以甲醇-水、乙腈-水作为流动相,采用半制备色谱法纯化7种儿茶素类化合物,纯度均达到90%。此外,利用同样的方法分离纯化另外两种茶叶鲜叶中的7种儿茶素类化合物,得到相似的结果。该方法以溶剂提取、离子沉淀结合半制备色谱,适于简单、高效地同时分离制备多种儿茶素类化合物。     

Analysis of catechins and condensed tannins by thermally assisted hydrolysis/methylation-GC/MS and by a novel two step methylation

The objective of this study is to observe high molecular weight markers of catechins and condensed tannins by thermally assisted hydrolysis/methylation (THM)-GC/MS. Techniques for formation of intact methylated flavanols of catechins using THM in the presence of trimethylsulfonium hydroxide (TMSH), and, of a dimer marker of condensed tannins using a novel two step methylation technique, are presented. The two step methylation procedure involves pre-methylation of the sample with trimethylsilyl diazomethane (TMS-diazomethane) followed by THM. The dimer marker, a methylated product containing the C–C linkage between adjacent flavanol units, has a molecular weight of 540. Intact methylated flavanols of catechins were also successfully observed as high molecular weight compounds including partially methylated catechin and epicatechin (3-flavanol, 3′,4′,5,7-tetramethoxy, cis/trans; m/z = 346), epigallocatechin and gallocatechin (3-flavanol, 3′,4′,5,5′,7-pentamethoxy, cis/trans; m/z = 376). These techniques were successfully applied to the analysis of series of condensed tannins isolated from plants, and catechins and other phenolics present in (hot water) extracts of tea leaves. In green tea the major catechins were identified as epicatechin and epigallocatechin along with flavonols and tannin dimers.     

Extraction of Catechin Compounds from Green Tea with a New Green Solvent

An emerging green solvent called a deep eutectic solvent（DES） was applied to the extraction and determination of catechin（C）,（＋）epicatechin gailate（ECG） and （-）epigallocatechin gallate（EGCG） from Chinese green tea.After evaluating different combinations of them by extraction methods and DESs,a DES-based extraction method was established and optimized by a systematic investigation of the influencing factors.As a result,a total of 3.629,35.25 or 114.2 mg/g catechin,（＋）epicatechin gallate or （-）epigaliocatechin gallate were extracted respectively under optimal conditions with extraction efficiencies of 82.7％,92.3％ and 97.0％,respectively.By comparing with other common used solvents for extracting catechin compounds,DESs were proved to be potential extraction solvents for bioactive ingredients.