Theaflavin-3-gallate Research Articles

Active compounds and derivatives of camellia sinensis responding to erosive attacks on dentin

This research explored the potential of Camellia sinensis-derived teas and active compounds to be used as treatments to prevent dentin wear. Human root dentin slabs were randomly assigned to 5 groups (n = 10) as follows: distilled water (DW, control), epigallocatechin-3-gallate (EGCG), theaflavin gallate derivatives (TF), commercial green tea (GT), and commercial black tea (BT). The samples were submitted to a pellicle formation and an erosive cycling model (5x/day, demineralization using 0.01 M hydrochloric acid/60 s) followed by remineralization (human stimulated saliva/60 min) for three days. The samples were treated for 5 min using the test group solutions between the erosive cycles. Dentin changes were assessed with profilometry analysis and FT-Raman spectroscopy. The data regarding wear were analyzed by ANOVA followed by Tukey's test (p < 0.05). EGCG, TF derivatives, and both regular teas significantly suppressed erosive dentin loss (38-47%, p < 0.05). No obvious changes in the Raman spectra were detected in the specimens; however, the DW group had a minor relationship of 2880/2940 cm-1. The phenolic contents in both green and black tea and the important catechins appear to have protective effects on dentin loss.

The role of gut microbiota on the metabolism of black tea theaflavins

The main bioactive polyphenols in black tea are theaflavins, including theaflavin (TF), theaflavin‐3‐gallate (TF3G), theaflavin‐3¢‐gallate (TF3¢G), and theaflavin‐3,3¢‐digallate (TFDG). Theaflavins have been reported as anti‐inflammatory and cancer preventive agents in different in vitro and in vivo models. Because of the poor systemic bioavailability of theaflavins, it is still unclear how these compounds can exert their biological functions. Studies have shown that higher molecular weight polyphenols are metabolized by the microbiota and their metabolites may play an important role in chronic disease prevention. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans. Purified TF and TFDG were oral gavaged to specific pathogen free (SPF) mice, germ free (GF) mice, and incubated with human fecal slurries in vitro. Microbial bioconversion was monitored using liquid chromatography/mass spectrometry (LC/MS) by analyzing the MSn (n = 1–3) spectra. Our results indicated that TFDG can be metabolized by gut microbiota to generate gallic acid, pyrogallol, TF, TF3G, and TF3'G, and TF can be further broke down to small molecular metabolites by gut microbiota. Furthermore, we used selective culturing techniques to probe which bacterial groups are responsible for theaflavins metabolism in vitro. Fecal bacteria from SPF‐born 129SvEv mice were anaerobically cultured on blood agar (non‐selective agar), MacConkey agar (selective for gram‐negatives; G−) and Columbia colistin and nalidixic acid agar (gram‐positives; G+). The cultures were then anaerobically incubated in fermentation media containing theaflavins, and the resulting metabolites were analyzed by LC/MS. Lactobacillus plantarum and Escherichia coli NC101 were similarly assessed for theaflavins fermentation abilities. Our results revealed that the G+ bacteria (including L. plantarum on its own) instead of G− bacteria (including E. coli NC101 on its own) were able to produce these metabolites.Support or Funding InformationThis work was supported by NIH R01 grant AT008623.

The Microbiota Is Essential for the Generation of Black Tea Theaflavins-Derived Metabolites

BackgroundTheaflavins including theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3′-gallate (TF3′G), and theaflavin-3,3′-digallate (TFDG), are the most important bioactive polyphenols in black tea. Because of their poor systemic bioavailability, it is still unclear how these compounds can exert their biological functions. The objective of this study is to identify the microbial metabolites of theaflavins in mice and in humans.Methods and FindingsIn the present study, we gavaged specific pathogen free (SPF) mice and germ free (GF) mice with 200 mg/kg TFDG and identified TF, TF3G, TF3′G, and gallic acid as the major fecal metabolites of TFDG in SPF mice. These metabolites were absent in TFDG- gavaged GF mice. The microbial bioconversion of TFDG, TF3G, and TF3′G was also investigated in vitro using fecal slurries collected from three healthy human subjects. Our results indicate that TFDG is metabolized to TF, TF3G, TF3′G, gallic acid, and pyrogallol by human microbiota. Moreover, both TF3G and TF3′G are metabolized to TF, gallic acid, and pyrogallol by human microbiota. Importantly, we observed interindividual differences on the metabolism rate of gallic acid to pyrogallol among the three human subjects. In addition, we demonstrated that Lactobacillus plantarum 299v and Bacillus subtilis have the capacity to metabolize TFDG.ConclusionsThe microbiota is important for the metabolism of theaflavins in both mice and humans. The in vivo functional impact of microbiota-generated theaflavins-derived metabolites is worthwhile of further study.

Metabolism of black tea theaflavins by gut microbiota

As a popular beverage, tea (Camellia sinensis, Theaceae) has been associated with many beneficial health effects, including the prevention of cancer and heart disease, a phenomenon mostly attributed to the presence of polyphenolic compounds. The main bioactive polyphenols in black tea are theaflavins, which are formed by the oxidation and polymerization of catechins in green tea leaves during fermentation. Theaflavins including theaflavin (TF), theaflavin‐3‐gallate (TF3G), theaflavin‐3′‐gallate (TF3′G), and theaflavin‐3,3′‐digallate (TFDG). We and others have found that theaflavins have poor systematic bioavailability. Therefore, it is still unclear how these compounds could exert their biological functions. It has been reported that higher molecular weight polyphenols are metabolized by the microbiota and their metabolites may play an important role in chronic disease prevention. In the present study, we investigated the microbial bioconversion of theaflavins in specific pathogen free (SPF) mice, germ free (GF) mice, and in human fecal microbiota in vitro. Microbial bioconversion was monitored using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MSn (n = 1–3) spectra. Our results indicated that TFDG, TF3G and TF3′G can be metabolized by gut microbiota to generate gallic acid, pyrogallol, and TF. Glucuronidated and sulfated, instead of methylated metabolites of TF3G, TF3′G and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. To our knowledge, this is the first report on the microbial metabolism of theaflavins.

Structural identification of mouse fecal metabolites of theaflavin 3,3′-digallate using liquid chromatography tandem mass spectrometry

Black tea consumption has been associated with many health benefits including the prevention of cancer and heart disease. Theaflavins are the major bioactive polyphenols present in black tea. Unfortunately, limited information is available on their biotransformation. In the present study, we investigated the metabolic fate of theaflavin 3,3′-digallate (TFDG), one of the most abundant and bioactive theaflavins, in mouse fecal samples using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MS n ( n = 1–3) spectra. Four metabolites theaflavin, theaflavin 3-gallate, theaflavin 3′-gallate, and gallic acid were identified as the major mouse fecal metabolites of TFDG. Glucuronidated and sulfated, instead of methylated metabolites of theaflavin 3-gallate, theaflavin 3′-gallate, and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. Our results indicate that TFDG can be degraded in mice. Further studies on the formation of those metabolites in TFDG-treated mice in germ-free conditions are warranted. To our knowledge, this is the first report on the biotransformation of TFDG in mice.

Black-Tea Polyphenols Decrease Micellar Solubility of Cholesterol in Vitro and Intestinal Absorption of Cholesterol in Rats

Administration of black-tea polyphenols (BTP) simultaneously reduced lymphatic recovery of both (3)H-cholesterol and (14)C-trioleoylglycerol in rats that were cannulated in the thoracic duct. BTP decreased the in vitro micellar solubility of cholesterol in a dose-dependent manner. When purified theaflavins, which are components of BTP, were used, theaflavin-monogallates (TFMGs), theaflavin-3-gallate (TF3G), and theaflavin-3'-gallate (TF3'G) were effective in eliminating cholesterol from bile salt micelles in vitro. Theaflavin (TF) and theaflavin-3,3'-digallate (TFDG) had no effect on the micellar solubility of cholesterol. The concentration of bile acid in the micelles was not influenced by the addition of any BTPs or theaflavins. These results suggest that the reduction of micellar cholesterol by BTP could be important to reducing cholesterol absorption.

Increase of Theaflavin Gallates and Thearubigins by Acceleration of Catechin Oxidation in a New Fermented Tea Product Obtained by the Tea-Rolling Processing of Loquat (Eriobotrya japonica) and Green Tea Leaves

In a project to produce a new fermented tea product from non-used tea leaves harvested in the summer, we found that kneading tea leaves ( Camellia sinensis ) with fresh loquat leaves ( Eriobotrya japonica ) accelerated the enzymatic oxidation of tea catechins. The fermented tea obtained by tea-rolling processing of tea and loquat leaves had a strong, distinctive flavor and a plain aftertaste, which differed from usual black, green, and oolong teas. The phenolic constituents were similar to those of black tea. However, the concentrations of theaflavin 3-O-gallate, theaflavin 3,3'-di-O-gallate, and thearubigins were higher in the tea leaves kneaded with loquat leaves than in tea leaves kneaded without loquat leaves. The results from in vitro experiments suggested that acceleration of catechin oxidation was caused by the strong oxidation activity of loquat leaf enzymes and a coupled oxidation mechanism with caffeoyl quinic acids, which are the major phenolic constituents of loquat leaves.

TLC separation of catechins and theaflavins on polyamide plates

Summary The potential of polyamide as adsorbent for separation of nine compounds – (±)-catechin (DL-C), (–)-epicatechin (EC), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECg), (–)-epigallocatechin gallate (EGCg), theaflavin (TF1), theaflavin 3-gallate (TF2A), theaflavin 3’-gallate (TF2B), and theaflavin 3,3’-digallate (TF3) – was studied. Polyamide TLC plates dried with a hair dryer, application as bands, and development in a horizontal chamber gave the best results. Detection was performed using iron(III) chloride– ethanol reagent. Twofold development with chloroform–methanol 2:3 (v/v) as mobile phase resulted in the separation of TF1, TF2A, TF2B, and TF3 from all the other compounds. EGCg, ECg, and EGC were separated but EC and DL-C were not separated from each other. The best mobile phase for each of the compounds is reported. The best separation of the five main catechins (EC, DL-C, EGC, ECg, and EGCg) was achieved by use of n-butanol–acetone–acetic acid 5:5:3 (v/v). In addition, separation of the flavonols myricetin, quercetin, kaempferol, and rutin and the phenolic acids gallic acid, chlorogenic acid, and caffeic acid was achieved by twofold development with chloroform–methanol 2:3 (v/v). The applicability of the method was checked by screening of extracts of green, black, oolong, and pu-erh tea.

Polymer-Like Polyphenols of Black Tea and Their Lipase and Amylase Inhibitory Activities

Lipase and amylase inhibitory activities of black tea were examined. After solvent partitioning of a black tea extract with the ethyl acetate and n-butanol, the two soluble fractions showed comparable inhibitory activities. Activity in the ethyl acetate fraction was mainly attributable to polyphenols with low-molecular weights, such as theaflavin gallates. On the other hand, the active substance in the n-butanol layer was ascertained to be a polymer-like substance. 1H- and 13C-NMR spectra showed signals arising from the flavan A-ring and galloyl groups, although signals due to flavan B-rings were not detected, suggesting that the polymer-like substances were generated by oxidative condensation of flavan B-rings, a result which was previously deduced from our results of in vitro catechin oxidation experiments. Enzymatic oxidation of epicatechin 3-O-gallate produced a similar polymer-like substance and suggested that condensation between a B-ring and galloyl groups was involved in the polymerization reaction.

Theadibenzotropolone A, a New Type Pigment from Enzymatic Oxidation of (‐)‐Epicatechin and (‐)‐Epigallocatechin Gallate and Characterized from Black Tea Using LC/MS/MS.

Abstract Theaflavins and thearubigins are major pigments of black tea. In the course of studies on the oxidation mechanism of tea polyphenols, a new type of tea pigment, theadibenzotropolone A, together with theaflavin 3-gallate were formed by the reaction of (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) with horseradish peroxidase in the presence of H 2 O 2 . The structure of theadibenzotropolone A was elucidated on the basis of MS and 2D NMR spectroscopic analyses. The observation that galloyl ester groups of theaflavins can be oxidized to form dibenzotropolone skeletons strongly implied that this type of oxidation as an important pathway to extend the molecular size of thearubigins. The existence of this compound in black tea was characterized by LC/ESI-MS/MS. Theadibenzotropolone A is the first theaflavin type trimer of catechins in black tea.

Theadibenzotropolone A, a new type pigment from enzymatic oxidation of (−)-epicatechin and (−)-epigallocatechin gallate and characterized from black tea using LC/MS/MS

Theaflavins and thearubigins are major pigments of black tea. In the course of studies on the oxidation mechanism of tea polyphenols, a new type of tea pigment, theadibenzotropolone A, together with theaflavin 3-gallate were formed by the reaction of (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) with horseradish peroxidase in the presence of H 2O 2. The structure of theadibenzotropolone A was elucidated on the basis of MS and 2D NMR spectroscopic analyses. The observation that galloyl ester groups of theaflavins can be oxidized to form dibenzotropolone skeletons strongly implied that this type of oxidation as an important pathway to extend the molecular size of thearubigins. The existence of this compound in black tea was characterized by LC/ESI-MS/MS. Theadibenzotropolone A is the first theaflavin type trimer of catechins in black tea.

Effect of black and green tea polyphenols on c-jun phosphorylation and H(2)O(2) production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction.

The biological activities of theaflavin (TF), theaflavin gallate (TFG) and theaflavin digallate (TFdiG) from black tea and (-)-epigallocatechin 3-gallate (EGCG) and (-)-epigallocatechin (EGC) from green tea were investigated using SV40-immortalized (33BES) and Ha-ras gene transformed (21BES) human bronchial epithelial cell lines. Growth inhibition and cell viability were measured by trypan blue dye exclusion assay following 24 h treatment with the tea polyphenols. TFdiG, EGC and EGCG displayed comparable inhibitory effects on the growth of 21BES cells, with estimated IC(50) values of 22-24 microM. TFG exhibited a lower inhibitory activity (IC(50) 37 microM) and TF was even less effective (IC(50) 47 microM) in this cell line. A similar effect was also observed in 33BES cells. These results suggest that the gallate structure of theaflavins is important for growth inhibition. Exposure of 21BES cells to 25 microM TFdiG, EGC and EGCG for 24 h led to induction of cell apoptosis/death as determined by the Annexin V apoptosis assay. With TFdiG treatment cell death occurred early, and quickly peaked at 8-12 h. Morphological observations showed that TFdiG-treated cells appeared irregular in shape, with cytoplasmic granules, suggesting a cytotoxic effect. On the other hand, EGC and EGCG showed a lag phase before a rapid increase in apoptosis between 16 and 24 h, without any marked morphological changes, which was similar to that induced by H(2)O(2). TFdiG, EGC and EGCG induced similar amounts of H(2)O(2) formation in 21BES cells. Exogenously added catalase significantly prevented EGC- and EGCG-induced cell apoptosis, but did not prevent TFdiG-induced cell death, suggesting that H(2)O(2) is involved in the apoptosis induced by EGCG and EGC, but not in TFdiG-induced cell death. EGCG and TFdiG were shown to decrease c-jun protein phosphorylation in 21BES cells. Such inhibition is expected to result in lowered AP-1 activity, which may contribute to the growth inhibitory activity of tea polyphenols.

Suppression of lipopolysaccharide-induced nuclear factor-κB activity by theaflavin-3,3′-digallate from black tea and other polyphenols through down-regulation of IκB kinase activity in macrophages

We investigated the inhibition of IκB kinase (IKK) activity in lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 cell line) by various polyphenols including (−)-epigallocatechin-3-gallate, theaflavin, a mixture of theaflavin-3 gallate and theaflavin-3′-gallate, theaflavin-3,3′-digallate (TF-3), pyrocyanidin B-3, casuarinin, geraniin, and penta- O-galloyl-β- d-glucose (5GG). TF-3 inhibited IKK activity in activated macrophages more strongly than did the other polyphenols. TF-3 strongly inhibited both IKK1 and IKK2 activity and prevented the degradation of IκBα and IκBβ in activated macrophage cells. The results suggested that the inhibition of IKK activity by TF-3 could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. Furthermore, geraniin, 5GG, and TF-3 all blocked phosphorylation of IκB from the cytosolic fraction, inhibited nuclear factor-κB (NFκB) activity, and inhibited increases in inducible nitric oxide synthase levels in activated macrophages. These results suggest that TF-3 may exert its anti-inflammatory and cancer chemopreventive actions by suppressing the activation of NFκB through inhibition of IKK activity.

Influence of polyphenol oxidase activity and polyphenol content of tea shoot on quality of black tea

AbstractVariation in polyphenot oxidase activity and levels of total polyphenols and catechins with respect to different clones and shoot components, and its effect on quality of black tea (Camellia sinensis (L) 0 Kuntze, were studied. There was a wide variation in polyphenol oxidase activity of the different clones tested. The optimum fermentation time and polyphenol oxidase activity of different clones exhibited a hyperbolic relationship, viz y = 2.36 + 1129/x, where y = optimum fermentation time in minutes and x = polyphenol oxidase activity in μM catechol oxidised g−1 acetone powder min−1, with an r value of −0.98, which is signi Jicant at P ≤ 0.001. A good non‐linear relationship was found between polyphenol oxidase activity of ji‐esh tea shoots of different clones and the theajavins content of corresponding black teas. Among different shoot components, bud and first leaf had higher levels of polyphenols and catechins than internodes. However, the polyphenol oxidase activity showed a reverse trend: the internodes exhibited a higher enzyme activity compared with other components. Formation of theajavins during fermentation of different shoot components was in good agreement with polyphenol oxidase activity. High performance liquid chromatographic analysis of the theaflavins fraction in tea brew of black teas made from different components of tea shoot showed that buds resulted in black tea with the highest amount of theaflavin gallates, whereas teas produced from internodes had the lowest amount of theaflavin gallates. A new factor, viz theaflavin digallate equivalent, was developed, and the significance of this factor for chemical evaluation of black tea quality is discussed in this paper.

In vitro oxidation of flavanols from tea leaf

The six major flavanols present in fresh shoots of tea ( Camellia sinensis L.) were extracted, purified, and oxidized in vitro with a soluble and partially purified polyphenol oxidase preparation, also extracted from fresh tea shoots. Besides different forms of theaflavin gallate, two species of theaflavin were observed. When the six flavanols were oxidized together, epigallocatechin limited total theaflavin production.

The phenolic substances of manufactured tea. V.—Hydrolysis of gallic acid esters by Aspergillus Niger

AbstractWhen tea extracts are used as culture media for Aspergillus niger, theaflavin gallate is converted into theaflavin, substances A and B into substance C, and one of the components of Q into substance R. Flavanol gallates are also converted into the corresponding flavanols. These changes arc probably all due to the action of an esterase developed by the mould, and indicate that theaflavin gallate and substances A, B and Q are gallic acid esters, as previously deduced.

The phenolic substances of manufactured tea. VI.–The preparation of theaflavin and of theaflavin gallate

AbstractImproved methods are described for the preparation of theaflavin gallate and of theaflavin from black tea extracts Both substances were obtained chromatographically pure and the theaflavin was crystallized.

The chemistry of tea manufacture

AbstractMany of the substances occurring in freshly plucked tea shoots are listed and the chemical changes taking place during withering and fermentation are discussed. In fermentation (—)‐epigallocatechin and its gallate are the only polyphenols to undergo appreciable chemical change. A scheme for the oxidation of these substances is developed which suggests that substances A, B and C are bisflavanols, and that theaflavin and theaflavin gallate are derivatives of purpurogallin. Thearubigins are probably formed as a result of coupled oxidation of theaflavins. Structures are also suggested for the minor components P, Q, R and Z. It is probable that theaflavins and thearubigins account for the colour and strength of tea liquors, and that theaflavins also contribute towards quality and briskness.

The phenolic substances of manufactured tea. III.—ultra‐violet and visible absorption spectra

AbstractThe absorption spectra of thearubigin fractions are all very similar and differ appreciably from those of the theaflavins. Theaflavin and theaflavin gallate have similar absorption spectra, which also resemble that of purpurogallin. Substances A, B and C have absorption bands at 275 mμ. The spectrum of Q differs from that of purpurogallincarboxylic acid. The identification of P as a 5:7:3′:4′:5′‐pentahydroxyflavylium salt is suggested. Substance 1C absorbs similarly to gallic acid, and the presumed p‐coumarylquinic acids to p‐coumaric acid.