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.     

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。     

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.     

Solid-state NMR analysis of the orientation and dynamics of epigallocatechin gallate, a green tea polyphenol, incorporated into lipid bilayers

Catechins are the principle polyphenolic compounds in green tea; the four major compounds identified are epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg). Tea catechins tend to attach externally to their targets, such as viral envelopes, cell membranes, or the surface of low-density lipoproteins. In order to further our understanding of the molecular mobility of these compounds in cells, we examined the interaction of tea catechins with lipid membranes using solid-state NMR techniques. Our previous work indicated that the EGCg molecule is incorporated into lipid bilayers in a unique orientation. However, the detailed configuration, orientation, and dynamics of EGCg in lipid bilayers have not been well-characterized. Here, we investigated the orientation and dynamics of EGCg incorporated into multi-lamellar vesicles (MLVs) and bicelles using solid-state NMR spectroscopy.     

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

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On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

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.     

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.     

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.     

Determination of green-tea catechins in cosmetic formulations and in in-vitro skin extracts by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry

Summary A simple, rapid and reproducible method is presented for the analysis of green-tea extracts in different cosmetic formulations and in in-vitro skin extracts. Cosmetically active principal components were used for determination of complex assembled green-tea extracts. The catechins selected were catechin, epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate, because of their efficacy and their concentrations in green-tea extracts. The determination was performed by high-performance liquid chromatography (HPLC), on a reversed-phase (RP) column, coupled to a single-quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the negative selected-ion-monitoring (SIM) mode. A detection limit between 5 and 15 ng g⁻¹ was achieved in methanol-water-ascorbic acid extracts from different emulsions. A routine analytical procedure could be established with good quantitative reliability. During validation, the repeatabilities (relative standard deviation) for catechin standard solutions were found to be 1.1–2.7% (within one day) and 2.2–4.3% (day-to-day). Recoveries from spiked placebos were 98–105%. The method was successfully used to determine the storage stability of green-tea extract in cosmetic formulations and the in-vitro penetration of green-tea extract into the skin.     

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.     

Hybrid E‐Tongue for the Evaluation of Sweetness and Bitterness of Soft Drinks Fortified with Epigallocatechin Gallate

A home‐made hybrid electronic tongue was set up, validated and applied to discriminate soft drinks fortified with plant extracts of green tea. The e‐tongue consists of a flow injection system equipped with two electrochemical and one optical sensors. Different formulations of soft drinks composed of glucose and epigallocatechin gallate were then discriminated by principal component analysis. Furthermore, two partial least squares regression models were developed to estimate the “sweetness” (r² of 0.992) and “bitterness” (r² of 0.993) of the model solutions and commercial soft drinks, before and after their fortification with epigallocatechin gallate.     

Magnetic molecularly imprinted polymers based on silica modified by deep eutectic solvents for the rapid simultaneous magnetic‐based solid‐phase extraction of Salvia miltiorrhiza bunge,Glycine max (Linn.) Merr and green tea

Novel magnetic molecularly imprinted polymers (MMIPs) with multiple‐template based on silica were modified by four types of deep eutectic solvents (DESs) for the rapid simultaneous magnetic solid‐phase extraction (MSPE) of tanshinone Ⅰ, tanshinone ⅡA, and cryptotanshinone from Salvia miltiorrhiza bunge; glycitein, genistein, and daidzein from Glycine max (Linn.) Merr; and epicatechin, epigallocatechin gallate, and epicatechin gallate from green tea, respectively. The synthesized materials were characterized by Fourier transform infrared spectroscopy and field emission scanning electron microscopy. Single factor experiments were to explore the relationship between the extraction efficiency and four factors (the sample solution pH, amount of DESs for modification, amount of adsorbent, and extraction time). It was showed that the DES4‐MMIPs have better extraction ability than the MMIPs without DESs and the other three DESs‐modified MMIPs. The best extraction recoveries with DES4‐MMIP were tanshinone Ⅰ (85.57%), tanshinone ⅡA (80.58%), cryptotanshinone (92.12%), glycitein (81.65%), genistein (87.72%), daidzein (92.24%), epicatechin (86.43%), epigallocatechin gallate (80.92%), and epicatechin gallate (93.64%), respectively. The novel multiple‐template MMIPs materials modified by DES for the rapid simultaneous MSPE of active compounds were proved to reduce the experimental steps than single‐template technique, and increase the extraction efficiency.     

Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

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.     

Identification of ligands from natural products as inhibitors of glutathione S‐transferases using enzyme immobilized mesoporous magnetic beads with high‐performance liquid chromatography plus quadrupole time‐of‐flight mass spectrometry and molecular docking

Multidrug resistance is recognized as one of the main reasons leading to the failure of chemotherapy. Studies have shown that glutathione S‐transferase inhibitors could be regarded as multidrug resistance reversal agents. Herein, a method of applying enzyme immobilization, molecular docking, and high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was employed to screen glutathione S‐transferase inhibitors from natural products. Magnetic mesoporous silica microspheres were synthesized and modified with a poly(dopamine) layer, which has a large quantity of amino, enabling further non‐covalent binding with glutathione S‐transferase. Moreover, the immobilization conditions, namely, potential of hydrogen, catalase concentration, reaction temperature and reaction time, were optimized. In total, six potential compounds were isolated and identified from Perilla frutescens (L.) Britt leaves and green tea and molecular docking was applied to identify the binding site. Rosmarinic acid, (?)‐epigallocatechin‐3‐O‐gallate and (?)‐epicatechin‐3‐gallate showed higher binding affinity than the compounds, and their half maximal inhibitory concentration values were further determined. The results suggested that this proposed method was effective and convenient for identifying glutathione S‐transferase inhibitors from natural products.     

Application of amino‐based chitosan cyclodextrin derivatives for the extraction of catechins in green tea with high‐performance liquid chromatography

Chitosan derivatives have been studied widely, but poor solubility in water restricts their applications. In this study, four types of amine‐based chitosan derivatives were prepared and modified further with beta‐cyclodextrin. The sequential microextraction of catechins ((+)‐catechin and (?)‐epigallocatechin gallate) from green tea powder by an optimized solid‐phase extraction method using these four derivatives was investigated. The optimal conditions for the extraction of catechins were 60°C for a 40 min extraction period. The purity and amount of each catechin were determined by high‐performance liquid chromatography. The different amines strengthened the extraction capacity of chitosan. Among the four types of amines, ethylene diamine grafted chitosan beta‐cyclodextrin had the highest extraction capacity to catechins. Therefore, this material was used in the extraction assay, and the standard curves of (+)‐catechin and (?)‐epigallocatechin gallate were linear over the concentration range, 0.25–500 µg/mL, after assaying five data points in duplicate. Solid‐phase extraction with the amino‐based chitosan beta‐cyclodextrin system is a new application of chitosan, which has potential applications in the extraction of bioactive compounds from plant materials or the removal of different impurities from specific extracts.     

Cyclodextrin encapsulation of the functional group diminishes an antioxidant's free radical scavenging rates

Scavenging rates of cyclodextrin‐solubilized lipophilic antioxidants, namely catechin, epicatechin, epigallocatechin gallate, and resveratrol, against alkoxyl (RO^?) radical were measured with the use of electron paramagnetic resonance (EPR) spin‐trapping method. Results indicated that the scavenging rates of catechin and resveratrol were notably dependent on the solubilizer used, i.e., native β‐cyclodextrin (β‐CD) or heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DM‐β‐CD). But, epicatechin and epigallocatechin gallate showed almost no dependence on the cyclodextrin used. Catechin's scavenging rate in β‐CD was 66% lower than in DM‐β‐CD; in contrast, resveratrol in β‐CD showed 45% higher rate than in DM‐β‐CD. Based on the reported solution‐NMR structure of the inclusion complex of these antioxidants, it was concluded that the scavenging rate is decreased when the cyclodextrin cavity preferentially encapsulates the antioxidant‐function bearing group, i.e., O‐ and p‐quinolinol group in catechin and resveratrol, respectively. The depth of inclusion of the functional group determines the extent of the scavenging rate difference, suggesting that the cavity wall of the cyclodextrin acts like a barrier that hinders the approach of attacking free radicals. © 2012 Wiley Periodicals, Inc. Int J Chem Kinet 44: 598–603, 2012