Black Tea Pigment Research Articles

Oligomerization Mechanisms of Tea Catechins Involved in the Production of Black Tea Thearubigins.

Thearubigins (TRs) are chemically ill-defined black tea pigments composed of numerous catechin oxidation products. TRs contain oligomeric components; however, the oligomerization mechanisms are poorly understood. The comparison of the 13C nuclear magnetic resonance (NMR) spectra of TRs with different molecular sizes suggested the participation of A-ring methine carbons in the oligomerization. Crushing fresh tea leaves with phloroglucinol, a mimic of the catechin A-rings, yielded the phloroglucinol adducts of the B-ring quinones of pyrogallol-type catechins and dehydrotheasinensins, indicating that intermolecular oxidative couplings between pyrogallol-type B-rings and A-rings are involved in the oligomerization. This is supported by the comparison of the 13C NMR spectra of the oligomers generated from the dehydrotheasinensins and epicatechin. Furthermore, the presence of the quinones or related structures in the catechin oligomers is shown by condensation with 1,2-phenylenediamine. The pyrogallol-type catechins account for approximately 70% of tea catechins; therefore, the B-A ring couplings of the pyrogallol-type catechins are important in the catechin oligomerization involved in TR production.

New degradation mechanism of black tea pigment theaflavin involving condensation with epigallocatechin-3-O-gallate

Theanaphthoquinone (TNQ) is the initial and main oxidation product of theaflavin, a representative black tea pigment. Nevertheless, TNQ is virtually undetected in the high-performance liquid chromatography analysis of black tea leaves using photodiode array detection. To elucidate the degradation mechanism of theaflavin in the black tea production process, this study investigated the reaction of TNQ with epigallocatechin-3-O-gallate (EGCg), which is the most abundant polyphenol in tea leaves. In citrate–phosphate buffer solution at pH 6 and room temperature, TNQ reacted nonenzymatically with EGCg to afford three products, whose structures were determined on the basis of spectroscopic data. The results indicated that the double bond of the ortho-naphthoquinone moiety in TNQ reacted with the autoxidation product of EGCg. This study demonstrates novel reactions occurring in the process of theaflavin degradation, which might be involved in the formation of thearubigins, the major black tea pigments composing oligomeric catechin oxidation products.

How black tea pigment theaflavin dyes chicken eggs: Binding affinity study of theaflavin with ovalbumin

Theaflavin (TF), which is the key pigment in black tea, is a health-promoting food component with beneficial effects on humans. However, the interactions by which these effects are transferred and exerted into protein-rich foods are unclear. Here, egg ovalbumin (OVA) was selected as a representative dietary protein to ascertain their binding mechanism. Steady-state, time-resolved fluorescence and isothermal titration calorimetric results showed that TF can interact well with OVA with an affinity magnitude of 104. The noncovalent binding was mainly driven by hydrophobic interaction and hydrogen bonds. Structural analysis displayed that the TF binding pocket significantly overlapped with one of the surrounding specific IgE-binding epitopes, thereby causing a subtle structural adjustment on the secondary conformation of OVA. The biological complexation model that was delineated here will help understand how black tea dyes egg white in tea egg products and for the development of protein-rich carriers in functional foods.

Abstract B67: Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells

Abstract Ovarian cancer ranks fifth in cancer deaths among women in the United States, accounting for approximately 5% of all cancer deaths. Acquired resistance to conventional platinum-based chemotherapy drives the development of more selective anticancer drugs. Theaflavin-3-gallate (TF2a) and theaflavin-3’-gallate (TF2b) are the major bioactive components in black tea. In this study, we evaluated TF2a and TF2b as potential anticancer agents using human ovarian cancer cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37˚C in a humidified incubator with 5% CO2. The [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry, and Western blot analysis were used to confirm that TF2a and TF2b exhibited a potent growth-inhibitory effect against cisplatin-resistant ovarian cancer A2780/CP70 cells. Treatment with TF2a and TF2b (10-40 μM) significantly decreased A2780/CP70 cell viability (p< 0.05). Treatment with TF2a and TF2b (5, 10 and 20 μM) increased the percentage of apoptotic cells in a concentration-dependent manner. Western blot analysis was used to evaluate the expression of proteins related to apoptosis in A2780/CP70 cells treated with designated concentrations (0, 5, 10, and 20 μM) of TF2a and TF2b for 24 h. The protein levels of procaspase-3/7 for TF2a-treated cells and procaspase 3 for TF2b-treated cells decreased significantly in A2780/CP70 cells (p< 0.05). Treatment with TF2a or TF2b significantly increased the protein levels of cleaved caspase-3/7, full-length PARP, and cleaved PARP in A2780/CP70 cells. Our study demonstrated that TF2a and TF2b are potential agents for treating platinum-resistant ovarian cancer by inducing apoptosis. Note: This abstract was not presented at the conference. Citation Format: Yi Charlie Chen. Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B67.

Synthesis of Theaflavins and Their Functions.

Numerous epidemiological and interventional clinical studies have consistently reported that black tea is good for human health. The polyphenolic compound, theaflavin, and its galloyl esters (theaflavins) are the primary red pigments in black tea that possess several health benefits, including fat-reducing and glucose-lowering capabilities and lifestyle-related disease prevention related to anti-obesity, anticancer, anti-atherosclerotic, anti-inflammatory, antiviral, antibacterial, anti-osteoporotic, and anti-dental caries properties. These compounds are produced by key enzymes, such as polyphenol oxidase and peroxidase, from parent green tea catechins present in fresh green tea leaves during the production of black tea leaves or the fermentation of green tea. However, theaflavins are only present in low concentrations in black tea; thus, their extraction from black tea leaves at sufficient levels for use in medical studies has been difficult. To circumvent this issue, different procedures for the synthesis of theaflavins using chemical oxidizing reagents or enzymes have been studied; however, low yields have limited their utility. Recently, however, several biosynthetic methods have been developed for the mass production of theaflavins. Using these methods, the physiological functions of theaflavins in lifestyle-related diseases in mice and humans have also been studied. In this review, we present the synthesis of theaflavins and their health benefits.

Inhibitory effect of black tea pigments, theaflavin‑3/3'-gallate against cisplatin-resistant ovarian cancer cells by inducing apoptosis and G1 cell cycle arrest.

Adverse side effects and acquired resistance to conventional chemotherapy based on platinum drive the exploration of other selective anticancer drugs. Theaflavin-3-gallate (TF2a) and theaflavin-3′-gallate (TF2b), theaflavin monomers in black tea, exhibited a potent growth inhibitory effect on cisplatin-resistant ovarian cancer A2780/CP70 cells and were less cytotoxic to normal ovarian IOSE-364 cell line. Flow cytometry analysis and western blotting indicated that TF2a and TF2b induced apoptosis and G1 cell cycle arrest in ovarian cancer A2780/CP70 cells. Hoechst 33342 staining was used to confirm the apoptotic effect. Downregulation of CDK2 and CDK4 for TF2a and CDK2 and cyclin E1 for TF2b led to the accumulation of cells in G1 phase. TF2a and TF2b induced apoptosis and G1 through p53-dependent pathways. TF2a and TF2b induced DNA damage through ATM/Chk/p53 pathway. TF2a and TF2b also induced inhibition of A2780/CP70 cells through Akt and MAPK pathways. The results of this study implied that TF2a and TF2b might help prevent and treat platinum-resistant ovarian cancer.

Nonenzymatic Biomimetic Synthesis of Black Tea Pigment Theaflavins

Theaflavins are reddish-orange black tea pigments with a benzotropolone chromophore, and their various biological activities have been reported. Theaflavins are produced by oxidative coupling between catechol-type and pyrogallol-type catechins via bicyclo[3.2.1]octane-type intermediates. In this study, a new method for nonenzymatic biomimetic synthesis of theaflavins was developed using the DPPH radical as an oxidizing agent.

Study of the Stereochemistry and Oxidation Mechanism of Plant Polyphenols, Assisted by Computational Chemistry

In recent years, plant polyphenols have attracted great attention due to their wide range of biological activities. Certain kinds of polyphenols have complex structures; therefore, it is difficult to elucidate their total structure, including stereochemistry. In this study, we reinvestigated the stereostructures of two major C-glycosidic ellagitannins contained in Quercus plants, vescalagin and castalagin, and revised their stereostructures based on theoretical calculations of spectroscopic data. We also determined the structures of quercusnins A and B, isolated from the sapwood of Quercus crispula, based on theoretical calculations of NMR data. The oxidation mechanism of polyphenols has not been entirely elucidated. Therefore, we have also studied the oxidation mechanism of tea catechins during black tea production. Our investigation of the oxidation mechanism of black tea pigment theaflavins revealed that the difference in the position of the galloyl ester affords different oxidation products of theaflavins. In addition, oxidation products of pyrogallol-type catechins could be classified into three types-dehydrotheasinensins, theacitrins, and proepitheaflagallins; their detailed production and degradation mechanisms were also examined.

Oxidation mechanism of black tea pigment theaflavin by peroxidase

A large number of black tea polyphenols remain uncharacterized because of the complexity of catechin oxidation reactions that occur during tea fermentation. In the course of our studies on black tea polyphenols, we examined the enzymatic degradation of theaflavins, which are black tea pigments having a benzotropolone chromophore. Oxidation of theaflavin with peroxidase afforded a new product named theacoumarin A together with known pigment theanaphthoquinone. The structure of the new compound was determined by spectroscopic examination and a production mechanism via theanaphthoquinone is proposed.

Production and degradation mechanism of theacitrin C, a black tea pigment derived from epigallocatechin-3- O-gallate via a bicyclo[3.2.1]octane-type intermediate

Black tea is rich in polyphenols and has been shown to have various health benefits; however, its components have not yet been clarified in detail. Enzymatic oxidation of epigallocatechin-3- O-gallate, the most abundant polyphenol in tea, is thought to contribute significantly to the production of black tea polyphenols. We identified theacitrin C, an unstable black tea pigment, as an enzymatic oxidation product of epigallocatechin-3- O-gallate. Degradation of theacitrin C afforded theacitrinin A and 2,3,5,7-tetrahydroxychroman-3- O-gallate. Furthermore, theacitrinin B, which was isolated from black tea, is deduced to be a degradation product of theacitrin A, the desgalloyl analogue of theacitrin C. The structures of theacitrinins A and B were elucidated based on spectroscopic data. This is the first time that a degradation product of theacitrin has been isolated from black tea. We also examined the influence of esterification of the epigallocatechin C-3 hydroxyl group on the decomposition of bicyclo[3.2.1]octane-type intermediates.

Reaction of the Black Tea Pigment Theaflavin during Enzymatic Oxidation of Tea Catechins

Degradation of the black tea pigment theaflavin was examined in detail. Enzymatic oxidation of a mixture of epigallocatechin and epicatechin initially produced theaflavin, while prolonged reaction decreased the product. Addition of ethanol to the reaction mixture at the point when theaflavin began to decrease afforded four new products, together with theanaphthoquinone, a known oxidation product of theaflavin. The structures of the new products were determined by spectroscopic methods. One of the products was an ethanol adduct of a theanaphthoquinone precursor, and this reacted with theaflavin to give two further products. A product generated by coupling of theaflavin with epicatechin quinone was also obtained. The structures of the products indicate that oxidation and coupling with quinones are key reactions in the degradation of theaflavins. The degradation of theaflavin probably contributes to production of thearubigins.

Production mechanism of proepitheaflagallin, a precursor of benzotropolone-type black tea pigment, derived from epigallocatechin via a bicyclo[3.2.1]octane-type intermediate

An unstable intermediate proepitheaflagallin B ( 2), a precursor of proepitheaflagallin ( 3), was isolated as an enzymatic oxidation product of (−)-epigallocatechin ( 1), and the structure of 2 was determined based on spectroscopic data. The structure and its decomposition revealed that the detailed production mechanism of proepitheaflagallin ( 3) via a bicyclo[3.2.1]octane-type intermediate was related to that of major black tea pigments, theaflavins.

Suppression of free-radicals and protection against H 2O 2-induced oxidative damage in HPF-1 cell by oxidized phenolic compounds present in black tea

Whereas catechins are the most abundant polyphenols in green tea, the typical pigments in black tea are theaflavins (TF), thearubigins (TR) and theabrownins (TB), which are derived from the oxidation of catechins and their gallates during the fermentation stage of black tea processing. In this study, oxidized phenolic compounds (TF, TR and TB) present in black tea were obtained from a model oxidation system, using immobile enzyme. The order of OH − and 2,2-diphenyl-1-picrylhydrazyl-scavenging ability of these oxidation products was TF > TB > TR. These oxidized phenolic compounds showed protection against H 2O 2-induced damage in HPF-1 cells and suppressed the accumulation of intracellular reactive oxygen species in H 2O 2-induced damage of HPF-1 cells. Interestingly, TB, as a further oxidative product from TF or TR, showed potent activity, followed TF in the above four systems.

A new mechanism for oxidation of epigallocatechin and production of benzotropolone pigments

Enzymatic oxidation of (−)-epigallocatechin gave two new quinone dimers, dehydrotheasinensin C and proepitheaflagallin. Dehydrotheasinensin C has a hydrated cyclohexenetrione structure and its oxidation–reduction dismutation reaction yielded black tea polyphenols, theasinensins C and E, and desgalloyl oolongtheanin. The structure of proepitheaflagallin was determined based on spectroscopic data of its quinoxaline derivatives prepared by condensation with o-phenylenediamine. Proepitheaflagallin was decomposed on heating to give epitheaflagallin and hydroxytheaflavin. The former is a known black tea pigment and the latter is a new pigment with 1′,2′,3′-trihydroxy-3,4-benzotropolone moiety. The results revealed a new mechanism for the production of these pigments from epigallocatechin.

A Novel Black Tea Pigment and Two New Oxidation Products of Epigallocatechin-3-O-gallate

During tea fermentation, oxidation-reduction dismutation of a number of quinone metabolites of tea catechins yields numerous minor products, which make it difficult to separate and purify black tea polyphenols. In this study, epigallocatechin-3-O-gallate was enzymatically oxidized and then the unstable quinone metabolites in the oxidation mixture were hydrogenated with 2-mercaptoethanol to reduce production of inseparable minor dismutation products. As a result, three new oxidation products including a new black tea pigment were isolated, and their structures were determined based on chemical and spectroscopic data. Dehydrotheasinensin AQ is a new reddish-orange pigment with a 1,2-diketone structure, and its presence in commercial black tea was confirmed. In addition, a new quinone dimer with a complex caged structure and a trimer of epigallocatechin-3-O-gallate were also isolated and their production mechanisms are proposed. The presence of this trimer suggested participation of galloyl quinones in production of minor polyphenols in black tea.

Isolation and analyses of polymeric polyphenol fractions from black tea

Polymeric polyphenols are the most abundant pigments in black tea. Very limited information is available on the isolation and chemical characterization of these compounds. The present paper reports the comparative yields, physicochemical properties and preliminary biological activities of five polymeric black tea polyphenol (PBP) fractions isolated by liquid–liquid and Soxhlet-based solid (tea powder)–liquid extraction methods. The two methods have also been comparatively evaluated as regards cost, time, labour and practicality of scale-up. Overall, results show that PBP fractions, isolated by both the procedures, were free of other known biologically active monomeric and oligomeric components, and have similar physicochemical properties and biological effect(s). Solid–liquid extraction results in a relatively greater yield of PBP-1 to -3, significant reduction in cost, time and labour and also makes scale-up relatively easier.

The Isolation of a Bicyclo[3.2.1] Intermediate During Formation of Benzo­tropolones, a Common Nucleus Found in Black Tea Pigments: Theaflavins

The coupling of 5-methylpyrogallol (6) with 4-methyl-o-quinone (7) in an aprotic solvent (CH2Cl2) efficiently afforded a key intermediate 8 having a bicyclo[3.2.1] ring whose structure had been proposed for fifty years. Subsequent ring-opening of 8 by the addition of H2O, air oxidation and decarboxylation produced the benzotropolone 10 in good yield.

New dibenzotropolone derivatives characterized from black tea using LC/MS/MS

Theaflavins and thearubigins are major pigments in black tea, and it is generally accepted that they are produced by oxidation of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the oxidation mechanism of tea polyphenols, especially the formation of thearubigins, a method combining the enzymatic synthesis and LC/ESI-MS/MS analysis was developed to search for new higher molecular weight polymers from black tea. Three new dibenzotropolones, theadibenzotropolone A, B, and C, together with one new tribenzotropolone, theatribenzotropolone A, were formed by the reaction of theaflavins and tea catechins with horseradish peroxidase in the presence of H 2O 2. The structures of these new benzotropolone derivatives were elucidated on the basis of MS and 2D NMR spectroscopic analyses. The existence of these compounds in black tea was characterized by LC/ESI-MS/MS. Theadibenzotropolone A and B were the first benzotropolone-type trimers of catechins found in the black tea extract. The observation that galloyl ester groups of theaflavins can be oxidized to form di- or tri-benzotropolone skeletons strongly implied that this type of oxidation is an important pathway to extend the molecular size of thearubigins.

Peroxidase-mediated oxidation of catechins

Theaflavins and thearubigins are major pigments in black tea, and they are produced by oxidation and polymerization of flavan-3-ols (catechins) during tea fermentation. In the course of studies on the mechanism of black tea polyphenol formation, we have systematically studied peroxidase-mediated oxidation of tea catechins. Individual catechins, appropriate pairs of catechins, and the combination of selected catechins and theaflavins have been used as substrates of peroxidase. Different dimers, trimers and a tetramer are formed. This work has led to the characterization of some of the new trimers in black tea by LC/ESI-MS/MS. The proposed mechanisms for the peroxidase-mediated reaction are discussed.

Structures of Two New Oxidation Products of Green Tea Polyphenols Generated by Model Tea Fermentation.

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