Thearubigins/Polymeric black tea polyphenols (PBPs) do not prevent benzo[a]pyrene (B[a]P) induced lung tumors in A/J mice.

OBJECTIVES: Previously we have demonstrated the chemopreventive effect of Thearubigins/Polymeric Black-tea Polyphenols (PBPs) upon pre-treatment to a combination of carcinogens, that is, Benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which are present in Tobacco smoke (TS). However, the chemopreventive effect in response to B[a]P as a single carcinogen remains unexplored. B[a]P is a universal carcinogen and an important constituent of particulate matter 2.5 (PM2.5) found in the environment and in TS. METHODS: We investigated the pre-treatment of Thearubigins/PBPs as a chemopreventive agent at three doses (1.5, 5, 10%) against B[a]P-induced lung carcinogenesis at early & late time points. We also investigated the effect of PBPs at early time points to understand molecular changes by employing western blotting in xenobiotic metabolism pathways, DNA damage, inflammation, apoptosis, and proliferation as they are modulated in response to carcinogens. We used 6-8 weeks male A/J mice for tumorigenicty and western blotting to probe the molecular biomarkers. RESULTS: We report no decrease in tumor incidence and multiplicity upon pre and concurrent treatment of Thearubigins/PBPs. Further, we also report no changes in molecular markers at early time points, in agreement with former observations. CONCLUSION: Our results suggest that chemopreventive agents need to be tested with different combinations of carcinogens and regimens to fully understand the complex interplay between carcinogenesis and the efficacy of chemopreventive agents. Studies like these will provide meaningful data before initiating large-scale clinical trials.

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.

Metagenomics analysis of the polymeric and monomeric phenolic dynamic changes related to the indigenous bacteria of black tea spontaneous fermentation.

Spontaneous fermentation during black tea production involves several reactions, including the oxidation of phenolic compounds. This process has usually been studied without considering the potential involvement of indigenous tea microorganisms. This work utilised a metagenomic technique targeting bacterial 16S rRNA genes and evaluated the profile of phenolic compounds generated during the production of black tea. The resulting data were used to develop correlational and predictive functional analyses related to bacterial dynamics and the syntheses of various phenolic compounds. In particular, the genera Methylobacterium and Devosia were correlated with gallic acid and quercetin. Concurrently, the genera Sphingomonas, Chryseobacterium and Aureimonas were correlated with kaempferol, theaflavins, thearubigins and theabrownins. These results, supported by predicted functional analysis based on 16S rRNA genes associated with phenolic compounds, indicated that yfiH (polyphenol oxidase) and katG (catalase-peroxidase) are likely the dominant genes of the bacterial community involved in the black tea production process. This research suggests that bacteria could potentially contribute to the production process of black tea.

Gut Microbiota as a Novel Tool to Dissect the Complex Structures of Black Tea Polymers.

Thearubigins, polymers of tea catechins, account for more than 20% of the black tea polyphenols and have been reported to be the active components in black tea. However, the chemical structures and underlying mechanisms regarding how the thearubigins, being poorly bioavailable, generate in vivo health benefits are still largely unknown. Using germ-free and specific pathogen-free husbandry conditions combined with LC/MS-based nontargeted and targeted metabolomic analyses, we investigated the role of intestinal bacteria in thearubigin metabolism. Theaflavins and theasinensins were identified as the major microbial metabolites of thearubigins, suggesting that these molecules are the building units for the complex thearubigins. To further confirm this, thearubigin depolymerization was done using menthofuran in an acidic condition. Menthofuran-conjugated theaflavins, theasinensins, and catechins as well as their free forms were detected as the major degradation products of thearubigins. This indicated that theaflavins and theasinensins could be further polymerized through B-type proanthocyanidin linkages. Furthermore, four microbial degradation products were able to be detected in urine samples, suggesting that they can be absorbed into the circulatory system. Using the combination of microbial degradation, metabolomics, and chemical degradation, our results demonstrate that thearubigins are the complex polymers of theaflavins, theasinensins, and catechins and can be metabolized by gut microbiota to their corresponding bioactive and bioavailable smaller molecular metabolites.

Macro-composition quantification combined with metabolomics analysis uncovered key dynamic chemical changes of aging white tea.

It is a common belief in China that aging could improve the quality of white tea. However, the stored-induced compositional changes remain elusive. In this study, ten subsets of white tea samples, which had been stored for 1-, 2-, 3-, 4-, 5-, 6-, 7-, 10-, 11- and 13- years, were selected. Macro-compositions were quantified firstly. As the results showed, it was interesting to find total flavonoids, thearubigins (TRs), and theabrownines (TBs) increasing, accompanied with a gradual decrease of total polyphenols, which suggest a conversion of phenolic component in the aging process. Then, nontargeted metabolomics was further conducted on selected subsets of samples, including 1-, 7- and 13- years stored to profile their conversion. As a result, most different metabolites were related to flavonol glycosides and flavone glycosides, suggesting dynamic phenolic component changes were vital in aging. The partial least-squares-discriminant analysis (PLS-DA) also identified them as markers in distinguishing.

Tea chemistry - What do and what don't we know? - A micro review.

Progress in analytical tools have led to a deeper insight into the chemical constitution and reaction pathways during the tea manufacture. However, the challenges have also changed as "new" teas are traded internationally which makes the authentication much more complicated. This micro-review demonstrates that despite all the achievements in the field of validated methods, authenticity, non-targeted methods we still have some gaps. New reactions products have been detected and those might be useful for authenticity purposes. As regards definitions of certain types of tea it makes sense to combine compositional data generated by validated targeted methods with non-targeted work to get a clearer view. Some more work seems to be necessary to get e.g. a deeper insight in the fate of proanthocyanidins during different types of processing and to develop a concept to quantify the thearubigins. There was progress in our knowledge of the thearubigin fraction in the last decade, however, there are still concepts to develop.

Corrigendum: Effect of Black Tea Extract and Thearubigins on Osteoporosis in Rats and Osteoclast Formation in vitro.

[This corrects the article DOI: 10.3389/fphys.2018.01225.].

Effect of Black Tea Extract and Thearubigins on Osteoporosis in Rats and Osteoclast Formation in vitro.

Background: Osteoporosis is a major health problem that is closely related to substantial morbidity, mortality and decline in life quality for the aging population. Although previous studies and epidemiological evidence have demonstrated an association between black tea consumption and the prevention of bone loss, the underlying mechanism remains unclear. So, the effect of black tea extract (BTE) and thearubigins (TRs) on osteoporosis in rats and osteoclast formation in vitro were investigated. Methods:In vivo, ovariectomized (OVX) rats were used to establish osteoporosis models. To validate the model and study the effects of BTE and TRs on osteoporosis, the female Wistar rats were divided into a sham-operated group and five OVX groups including model, Xian-Ling-Gu-Bao (XLGB) (as a positive control), BTE, TRs low-dose, and TRs high-dose group. The rats in the four treatment groups were given the corresponding test sample for 12 weeks. Then, the body weight, femur indices, and serum biomarkers were examined and analyzed. In vitro, RAW264.7 murine macrophages were used as model of osteoclast formation. The effects of BTE and TRs on osteoclasts formation and the specific genes and protein levels of osteoclasts were determined. Results: Although there was no significant effect on the OVX-induced body weight gain by BTE or TRs, the levels of maximum bending force, cortical bone thickness and biomarker of bone resorption (acid phosphatase) can be significantly ameliorated by BTE or TRs in OVX rats. Furthermore, both of BTE and TRs can inhibit the osteoclastogenesis and diminish the expression levels of the related genes and proteins.

A new method to prepare and redefine black tea thearubigins.

Thearubigins (TRs) are the major components of black tea, which are formed during the fermentation reactions. Although anti-inflammatory and anti-cancer activities of TRs have been reported, the prepared TRs according to the literature methods still contain many floating peaks. It is puzzling whether the observed activities are from TRs or these floating peaks. Thus, it is urgent to develop a method to prepare pure TRs and redefine them. In the present study, we developed a new method, the combination of caffeine precipitation and Sephadex LH-20 column chromatography, to prepare pure TRs. The floating peaks on the hump of the crude TRs were removed, and pure TRs were prepared. The chemical profile of the floating peaks was established using LC/MS, and the major compounds in this fraction were identified as apigenin glycosides, quercetin glycosides, kaempferol glycosides, theaflavins, theasinensin, and galloylglucoses based on the analysis of their tandem mass spectra and in comparison with literature data. This study will pave the way to further study the chemistry and biological activities of TRs and the health effects of black tea consumption.

Exploring the potential of black tea based flavonoids against hyperlipidemia related disorders.

BACKGROUND: In recent decade, Hyperlipidemia related disorders like obesity, hypercholesterolemia and diabetes are considered as the leading killers for mankind. Fundamental nexus between nutrition and health diverting the consumers focus towards plant based natural products as a remedy against various metabolic syndrome. Considering this, present study was conducted to explicate the role of black tea polyphenols such as Theaflavins and thearubigins therapeutic potential to tackle targeted maladies especially oxidative stress related disorders like hypercholesterolemia and diabetes. METHODS: The mandate of current investigation was to explore the hypoglycemic and hypocholestrolemic perspective of isolated theaflavin and thearubigins through a model feeding trial. For the purpose, theaflavin & thearubigins were isolated from black tea through solvent partition method and utilize to form three types of nutraceutical drinks (theaflavin, thearubigins & theaflavin + thearubigins based) alongside control to be further utilized in bioefficacy trial. In bioefficacy trial, three types of independent studies were design on the bases of diet by involving 20 male wistar rats in each study (5 for each group). In study I, normal diet was administrated while, in study II & III high cholesterol and high sucrose diet was given, respectively along with prepared nutraceutical drinks to synchronize their therapeutic effect for a period of 56 days. At the termination of trial, Feed & drink intakes, body weight, total cholesterol, LDL, HDL, triglycerides, glucose and insulin levels were measured. RESULTS: The results indicated reduction in cholesterol, LDL and triglycerides levels of experimental rats in all studies with significant increase in HDL. In this context, theaflavin based drink imparted maximum reduction in cholesterol (3.75, 11.03 & 10.39%), LDL (3.84, 14.25& 10.84%) & triglycerides (2.99, 8.54 & 6.65%) in respective studies compared to thearubigins and theaflavin + thearubigins based drinks. However, theaflavin+ thearubigins based drink caused highest glucose decline and maximum insulin increase in all studies as compared to other nutraceutical drinks. The reported value for the insulin increase were 13.02 ± 1.02 & 14.55 ± 1.13, 10.09 ± 0.15 & 11.59 ± 0.86 for Hyperglycemic and Hypocholestrolemic rats respectively compared to control (7.84 ± 0.45 & 9.10 ± 0.41) for study I and II. CONCLUSIONS: In the nutshell, theaflavin and thearubigins based dietary interventions are helpful to alleviate the hypercholestrolemia and hyperglycemia and should be promoted as parallel therapy to combat these disorders.

Quality, antioxidant activity and composition of Indian black teas.

A comparative study was undertaken to determine the difference in quality, antioxidant activity, and mineral content of northern and southern Indian tea varieties. The results showed that the antioxidant activity, theaflavin and thearubigin content, color particle size and total soluble solids influence the tea leaf and tea dust. Assam leaf showed higher values for all the physico-chemical properties followed by Darjeeling leaf. In addition, the mineral composition of the tea infusion was also influenced. The preparation method, including the amounts of tea and water used, infusion time was shown to be a major determinant of the component concentrations of tea beverages as consumed. An illustration of the variation introduced by these varieties of tea is provided by comparing solids, caffeine, minerals and antioxidant activity of black teas available locally.

Spatial variability of theaflavins and thearubigins fractions and their impact on black tea quality.

The spatial distribution of theaflavin and thearubigin fractions and their impact on black tea quality were investigated using multivariate and geostatistics techniques. Black tea samples were collected from tea gardens of six geographical regions of Assam and West Bengal, India. Total theaflavin (TF) and its four fractions of upper Assam, south bank and North Bank teas were higher than the other regions. Simple theaflavin showed highest significant correlation with tasters' quality. Low molecular weight thearubigins of south bank and North Bank were significantly higher than other regions. Total thearubigin (TR) and its fractions revealed significant positive correlation with tasters' organoleptic valuations. Tea tasters' parameters were significantly and positively correlated with each other. The semivariogram for quality parameters were best represented by gaussian models. The nugget/sill ratio indicated a strong/moderate spatial dependence of the studied parameters. Spatial variation of tea quality parameters may be used for quality assessment in the tea growing areas of India.

Assessing biochemical changes during standardization of fermentation time and temperature for manufacturing quality black tea.

In black tea manufacturing, one of the most important steps is fermentation which influences the quality of tea. The macerated tea leaves were fermented at various temperatures (20, 25, 30, 35 °C) for different duration. Changes in polyphenoloxidase and peroxidase activities, depletion patterns of individual catechins, differences in individual theaflavin levels and formation of thearubigins were measured in leaves during fermentation. Higher stability of polyphenoloxidase and peroxidase enzymes was observed at lower temperature (20 °C), and increase in temperature, led to enzyme instability. The rate of degradation of all the catechins was found to be fastest at 35 °C and slowest at 20 °C. The formation and depletion of individual theaflavins varied with temperature and fermentation duration. The time required for the formation of maximum total theaflavins (TF) was highest at lower temperature and this time duration also varied for different theaflavins formation. Maximum amount of thearubigins (TR) content and liquor colour development was observed at 35 °C, and decrease in temperature reduced thearubigins accumulation. However, maximum brightness as well as TF/TR ratio was obtained at 20 °C, which suggests that fermentation at lower temperature is suitable for manufacturing quality black tea.

Model system-based mechanistic studies of black tea thearubigin formation.

Thearubigins are the most abundant pigments found in black tea, comprising polyphenolic oxidation products, whose composition and chemical nature have remained unresolved until recently. In the course of studying the mechanism of thearubigin formation from green tea flavan-3-ols, a model system, based on electrochemical oxidation of one of the main tea flavan-3-ol substrates, epigallocatechin gallate (EGCG), was employed. Reaction intermediates and products were subsequently analysed using mass spectrometry techniques, allowing for the identification of key intermediates and products. The results provided, for the first time, spectroscopic evidence for the structures of primary oxidation products, and led to the conclusion that oxidation is mainly taking place on the B-ring and the galloyl group, where the oxidized components undergo oxidative coupling for the formation of theaflavins, theasinensins and polyhydroxylated flavan-3-ols, all precursors for thearubigin formation. Furthermore, density functional theory (DFT) calculations were carried out to support key findings.

Investigation of isomeric flavanol structures in black tea thearubigins using ultraperformance liquid chromatography coupled to hybrid quadrupole/ion mobility/time of flight mass spectrometry.

Ultra performance liquid chromatography (UPLC) when coupled to ion mobility (IMS)/orthogonal acceleration time of flight mass spectrometry is a suitable technique for analyzing complex mixtures such as the black tea thearubigins. With the aid of this advanced instrumental analysis, we were able to separate and identify different isomeric components in the complex mixture which could previously not be differentiated by a conventional high performance liquid chromatography/tandem mass spectrometry. In this study, the difference between isomeric structures theasinensins, proanthocyanidins B-type and rutin (quercetin-3O-rutinoside) were studied, and these are present abundantly in many botanical sources. The differentiation between these structures was accomplished according to their acquired mobility drift times differing from the traditional investigations in mass spectrometry, where calculation of theoretical collisional cross sections allowed assignment of the individual isomeric structures. The present work demonstrates UPLC-IMS-MS as an efficient technology for isolating and separating isobaric and isomeric structures existing in complex mixtures discriminating between them according to their characteristic fragment ions and mobility drift times. Therefore, a rational assignment of isomeric structures in many phenolic secondary metabolites based on the ion mobility data might be useful in mass spectrometry-based structure analysis in the future.

Identification of novel homologous series of polyhydroxylated theasinensins and theanaphthoquinones in the SII fraction of black tea thearubigins using ESI/HPLC tandem mass spectrometry.

Thearubigins are the most abundant phenolic pigments found in black tea, produced by enzymatic oxidation of green tea flavan-3-ols in tea fermentation of until recently unknown composition. In this study electrospray ionization tandem LC-MS(n) experiments have been applied for the characterization of crude thearubigins isolated from black tea not exceeding 1000 Da. The aim of this study is to confirm the oxidative cascade hypothesis of tea fermentation. The data revealed the presence of two novel classes of compounds in thearubigin fractions. The first class of compounds revealed the presence of polyhydroxylated dimers of the theanaphthaquinone and theasinensin C structures, which were consistent with the polyhydroxylation hypothesis previously formulated. Furthermore, new classes of peroxo-/epoxy- compounds in the series of theasinensin A were identified, thus indicating the presence of H2O2 and its important contribution as a nucleophile in the tea fermentation process.

Black tea extract and its thearubigins relieve the sildenafil-induced delayed gut motility in mice: a possible role of nitric oxide.

In this study we hypothesize that a standardized black tea aqueous extract (BTE) and thearubigins, its main polyphenolic pigments, will improve sildenafil-induced delay in gastric emptying (GE) and small intestinal transit (SIT) in mice. Twenty groups of mice (n = 8) were given a phenol red meal, and three sets of experiments were performed. In the first and second sets, effects of different concentrations of BTE, thearubigins (TRs), and sildenafil (SLD), alone and in combinations, on GE and SIT were measured. In the third set, influence of nω -Nitro-l-arginine methyl ester hydrochloride (l-NAME) pretreatment on effects of these treatments was tested. Black tea extract (3% and 4.5%) and thearubigins (50 and 60 mg/kg) dose-dependently increased GE and SIT, whereas BTE 6% and thearubigins 70 mg/kg did not affect them. Sildenafil dose-dependently reduced both GE and SIT. Combination of metoclopramide, BTE 4.5%, thearubigins 60, or l-NAME with sildenafil (5 mg/kg) reversed its motility-delaying effects. Pretreatment with l-NAME followed by BTE 4.5%, thearubigins 60, BTE 4.5% + sildenafil 5, or thearubigins 60 + sildenafil 5 only partially affected the accelerating effects of BTE 4.5% and thearubigins 60. In conclusion, a standardized BTE and its thearubigins improve the sildenafil-induced delayed gut motility in mice. This improvement was partially blocked by l-NAME suggesting a possible role of nitric oxide. Thus, BTE 4.5% or TRs 60 mg/kg solution could be considered a reliever therapy for the sildenafil-induced dyspepsia.

Comparative assessment of tea quality by various analytical and sensory methods with emphasis on tea polyphenols.

Attempts were made to evolve an efficient technique for quality assessment of tea (Camellia sinensis) using a tyrosinase based biosensor to detect polyphenols (PP). Tyrosinase catalyzes the polymerization of PP to form theaflavins (Tf) and thearubigins (Tr) contributing to the colour and astringency of tea, which determine tea quality. Variation in biosensor response of tea infusions gave an indication of differential amount of Tf and Tr. A comparative study of quality attributes of 8 varieties of commercially available brands of tea (A-H) was done using biosensor and results were compared with conventional techniques such as spectrophotometry, high performance liquid chromatography (HPLC), Commission Internationale de I'Eclairage (CIE) system and sensory evaluation. Considerable correlation was observed among the biosensor, sensory and spectrophotometric evaluation of tea samples. Sample A showed high Tf content and also showed a relative high biosensor response whereas sample G showed relatively poor response. Application of biosensors would serve as a basis for the evaluation of market value of tea in the near future.