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.     

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。     

Investigation of the radioprotective properties of some tea polyphenols

The influence of tea and coffee flavonoids (−)-epicatechin (EC) and (+)-catechin (C) on the effect of γ-irradiation of DNA molecules in solutions was investigated. The concentrations of these and other components in different samples of black and green tea were analyzed by the capillary electrophoresis. It was determined from the experimental data obtained by different methods (circular dichroism and UV spectra, viscosimetry and optical investigations) that C and EC do not form complexes with DNA in solutions. It was shown that C and EC have radioprotective properties, being scavengers of the water radiolysis products.     

Separation of radioactive metabolites in cultured tea cells fed with [14C]phenylalanine using high-speed counter-current chromatography

Separation of radioactive metabolites in cultured tea cells fed with [14C]phenylalanine was conducted using high-speed counter-current chromatography. Among seven components obtained our studies focused two metabolites, i.e. (-)-epicatechin and D,L-catechin. The specific radioactivity of (-)-epicatechin was 212.01 KBq/mg, amounting to 8.5% of the total radioactivity of ethyl acetate extract while that of D,L-catechin was 1.0006 MBq/mg or 5.4% of the total.     

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.     

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.     

Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition

Tea is rich in polyphenols and has a variety of biological activities. In order to better understand the biological effects of tea constituents on human health, markers for their exposure and their metabolic fates are needed. Previously, we have characterized several catechin metabolites in the blood and urine, but more information on the metabolite profile of tea polyphenols is needed. In the present study, the human urinary metabolite profile of tea polyphenols was investigated using liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. With data-dependent MS/MS analysis by collecting the MS2 and MS3 spectra of the most intense ions in the sample, we identified more than twenty metabolites of tea polyphenols from human urine samples. (-)-Epigallocatechin (EGC) glucuronide, methylated EGC glucuronide, methylated EGC sulfate, (-)-epicatechin (EC) glucruronide, EC sulfate, methylated EC sulfate, as well as the glucuronide and sulfate metabolites of the ring-fission metabolites of tea catechins, 5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4), 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M6'), were the major human urinary metabolites of tea polyphenols. To our knowledge, this is the first report of the direct simultaneous analysis of the human urinary metabolite profile of tea polyphenols using single sample analysis. This method can also be used for thorough investigations of the metabolite profiles of many other dietary constituents.     

Versatile synthesis of epicatechin series procyanidin oligomers, and their antioxidant and DNA polymerase inhibitory activity

Proanthocyanidins, known as condensed tannins or oligomeric flavonoids, exist in many edible plants and show various interesting biological activities. We have developed a simple and versatile method of synthesizing procyanidin oligomers consisting of (−)-epicatechin and (+)-catechin. This method is applicable to the synthesis of various 3-O-substituted oligomers. We report here the stereoselective and length controlled synthesis of [4-8]-condensed (−)-epicatechin series procyanidin oligomers. We described the details of the synthesis of an two tetramers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(−)-epicatechin and (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(+)-catechin (arecatannin A1), (−)-epicatechin pentamer and two 3,3″,3?-tri-O-galloyl trimers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-3,3″,3?-tri-O-gallate and (−)-epicatechin-(−)-epicatechin-(+)-catechin-3,3″,3?-tri-O-gallate with the condensation method using TMSOTf as a catalyst. The ability of DPPH radical scavenging activity and DNA polymerase inhibitory activity of these oligomeric compounds were investigated.     

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.     

Indonesian medicinal plants. XXV. Cancer cell invasion inhibitory effects of chemical constituents in the parasitic plant Scurrula atropurpurea (Loranthaceae)

Six fatty acids (1-6), two xanthines (7, 8), two flavonol glycosides (9, 10), one monoterpene glucoside (11), one lignan glycoside (12), and four flavanes (13-16) were clarified by a bioassay-guided separation as chemical constituents of Scurrula atropurpurea (Loranthaceae), a parasitic plant of the tea plant Thea sinensis (Theaceae). Among these constituents, it was found that the alkynic fatty acid octadeca-8,10,12-triynoic acid (6) exhibits a more potent inhibitory effect on cancer cell invasion in vitro than flavanes [(+)-catechin (13), (-)-epicatechin (14), (-)-epicatechin-3-O-gallate (15) and (-)-epigallocatechin-3-O-gallate (16)].     

Inhibition Effects of Dilute-Acid Prehydrolysate of Corn Stover on Enzymatic Hydrolysis of Solka Floc

Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.     

Biooxidation of (+)-catechin and (-)-epicatechin into 3,4-dihydroxyflavan derivatives by the endophytic fungus Diaporthe sp. isolated from a tea plant

The microbial transformation of (+)-catechin (1) and (-)-epicatechin (2) by endophytic fungi isolated from a tea plant was investigated. It was found that the endophytic filamentous fungus Diaporthe sp. transformed them (1, 2) into the 3,4-cis-dihydroxyflavan derivatives, (+)-(2R,3S,4S)-3,4,5,7,3',4'-hexahydroxyflavan (3) and (-)-(2R,3R,4R)-3,4,5,7,3',4'-hexahydroxyflavan (7), respectively, whereas (-)-catechin (ent-1) and (+)-epicatechin (ent-2) with a 2S-phenyl group resisted the biooxidation.     

Epicatechin B-ring conjugates: first enantioselective synthesis and evidence for their occurrence in human biological fluids

Herein, the first enantioselective total synthesis of a number of biologically relevant (-)-epicatechin conjugates is described. The success of this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate for the first time the pattern of (-)-epicatechin conjugates present in four different human biological fluids following (-)-epicatechin absorption.     

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (-)-epicatechin and (-)-epigallocatechin gallate solutions, were linear over the range of 0.1-50 microM. The detection limits (signal-to-noise ratio=3) were 0.8 pmol for (-)-epigallocatechin gallate and 1.2 pmol for (-)-epicatechin. The high-performance liquid chromatography-chemiluminescence (HPLC-CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.     

Uniformly-sized, molecularly imprinted polymers for (-)-epigallocatechin gallate, -epicatechin gallate and -gallocatechin gallate by multi-step swelling and polymerization method

Uniformly-sized, molecularly imprinted polymers (MIPs) for (-)-epigallocatechin gallate (EGCg), -epicatechin gallate (ECg) and -gallocatechin gallate (GCg) were prepared by a multi-step swelling and polymerization method using 2-vinylpyridine as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and cyclohexanol as a porogen. Molecular recognition abilities of the obtained MIPs were evaluated in liquid chromatography using a mixture of ethanol and water, or ethanol as the eluent. Each MIP gave the highest molecular recognition ability for the respective template molecule. In addition, (-)-EGCg and -ECg had the same configuration (2R,3R) at positions 2 and 3, and therefore resulting in high cross reactivity each other. However, (-)-GCg, which has different configuration at position 2 with (-)-EGCg and -ECg, showed low cross reactivity with them. On the other hand, those MIPs showed no molecular recognition against (-)-epigallocatechin and -epicatechin, which have no gallate group at position 3. These results indicate that the MIPs prepared can recognize configuration at position 2 and a gallate group at position 3. Furthermore, the MIP for (-)-GCg could be successfully used for isolating (-)-EGCg and -ECg from green tea extract.     

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.     

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.     

Simultaneous determination of catechins, caffeine and gallic acids in green, Oolong, black and pu-erh teas using HPLC with a photodiode array detector

A simple and fast HPLC method using a photodiode array detector was developed for simultaneous determination of four major catechins, gallic acid and caffeine. After multiple extractions with aqueous methanol and acidic methanol solutions, tea extract was separated within 20 min using a methanol-acetate-water buffer gradient elution system on a C(18) column. The sample extraction data demonstrated that the single extraction used in the previous studies with aqueous acetonitrile or methanol is not sufficient; the multiple extraction procedure is essential for the quantitative analysis of catechins, phenolic acids and caffeine in teas. Several green, Oolong, black and pu-erh teas were successfully analyzed by this method. The analytical results obtained indicated that green teas contain higher content of catechins [(-)-epigallocatechin gallate, (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin] than both Oolong, pu-erh and black teas because fermentation process during the tea manufacturing reduced the levels of catechins significantly. The fermentation process also remarkably elevated the levels of gallic acid in full-fermented pu-erh and black teas. Another interesting finding is the low level of caffeine in Oolong teas, especially in Fujian Oolong tea.     

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.     

Anticomplement and antimicrobial activities of flavonoids from Entada phaseoloides

Seventeen flavonoids isolated from the extracts of the stem of Entada phaseoloides were investigated for their anticomplement (both classic and alternative pathways) and antimicrobial activities against Gram-positive bacteria (MRSA, MSSA, Standard Enterococcus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeuroginosa) and the yeast-like pathogenic fungus Candida albicans. The anticomplement studies revealed a dose-dependent activity among isolated quercetin, luteolin, apigenin, galangin, 5,2',5'-trihydroxy-3,7,4'-trimethoxyflavone-2'-O-beta-D-glucoside (+)-3,3',5',5,7-pentahydroflavanone, (+)-dihydrokaempferol, (-)-epicatechin, (+)-catechin, naringenin, and 5,7,3',5'-tetrahydroxyflavanone, and the antimicrobial results indicated that quercetin, 5,7,4'-trihydroxy-3'-methoxyflavonol and galangin produced the inhibitory activities against MRSA, MSSA, and Standard Enterococcus, while luteolin and rhamnocitrin displayed inhibition against only MRSA and MSSA. To the best of our knowledge, this is the first report that describes the anticomplement and antimicrobial activities of the stem of E. phaseoloides.