Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells

Black tea is one of the world's most popular beverages, and its health-promoting effects have been intensively investigated. The antiobesity and hypolipidemic effects of black tea have attracted increasing interest, but the mechanisms underlying these phenomena remain unclear. In the present study, the black tea major component theaflavins were assessed for their hepatic lipid-lowering potential when administered in fatty acid overload conditions both in cell culture and in an animal experimental model. We found that theaflavins significantly reduced lipid accumulation, suppressed fatty acid synthesis, and stimulated fatty acid oxidation. Furthermore, theaflavins also inhibited acetyl-coenzyme A carboxylase activities by stimulating AMP-activated protein kinase (AMPK) through the LKB1 and reactive oxygen species pathways. These observations support the idea that AMPK is a critical component of decreased hepatic lipid accumulation by theaflavin treatments. Our results show that theaflavins are bioavailable both in vitro and in vivo and may be active in the prevention of fatty liver and obesity.     

Black tea theaflavins suppress dioxin-induced transformation of the aryl hydrocarbon receptor

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.     

Modulation of tea and tea polyphenols on benzo(a)pyrene-induced DNA damage in Chang liver cells

The protective effects of three tea extracts (green tea, GTE; oolong tea, OTE; and black tea, BTE) and five tea polyphenols (epicatechin, EC; epicatechin gallate, ECG; epigallocatechin, EGC; epigallocatechin gallate, EGCG; and theaflavins, THFs) on benzo[a]pyrene (B[a]P)-induced DNA damage in Chang liver cells were evaluated using the comet assay. B[a]P-induced DNA damage in Chang liver cells was significantly (p < 0.05) inhibited by GTE and OTE at a concentration of 10 microg/ml and by BTE at 25 microg/ml. At a concentration of 100 microg/ml, the % tail DNA was reduced from 33% (B[a]P treated only) to 10, 9, 13%, by GTE, OTE and BTE, respectively. EC and ECG did not cause DNA damage in cells according to the results of the comet assay; however, EGC, EGCG and theaflavins caused DNA damage in cells at a concentration of 100 microM. The results indicated that EC and ECG had protective effects against B[a]P-induced DNA damage in cells at a concentration of 10-100 microM. Although EGC, EGCG and the theaflavins caused DNA damage at a high concentration, but they had protective effects against B[a]P-induced DNA damage in cells at a low concentration of 10-50 microM. The results also showed that the DNA damage in cells induced by EGC, EGCG, and the theaflavins was due to the generation of superoxide during incubation with cells at a higher concentration. Therefore, tea catechins and THFs play an important role in enabling tea extracts to inhibit DNA damage in Chang liver cells.     

Black tea represents a major source of dietary phenolics among regular tea drinkers

The phenolic composition and antioxidant activities [TEAC, ORAC, FRAP] of consumer brews (1 tea bag in 230 ml for 1 min) of seven different brands of black tea from the British market were investigated. The main phenolic compounds identified were epigallocatechin gallate, four theaflavins, as well as epicatechin gallate, theogallin (tentative assignment), quercetin-3-rutinoside and 4-caffeoyl quinic acid. Thearubigins represented an estimated 75-82% of the total phenolics. Further, polyphenol fractions were in decreasing order theaflavins, flavan-3-ols, flavonols, gallic acids and hydroxycinnamates. On average, a cup of a consumer brew of black tea is providing polyphenols at the level of 262 mg GAE/serving, of which 65 mg were assigned to individual polyphenols. The antioxidant activity of black tea preparations is higher than that of most reported dietary agents on a daily basis. Correlations were observed between the antioxidant activities and the sum of all quantified polyphenols by HPLC analysis as well as with the total phenolics. Treatment of the black tea brew with simulated gastric juice resulted in a significant increase of the identified theaflavins implying a partial cleavage of thearubigins in the environment of the gastric lumen. Therefore, black tea can be considered to be a rich source of polyphenols and/or antioxidants.     

Black Tea Represents a Major Source of Dietary Phenolics Among Regular Tea Drinkers

The phenolic composition and antioxidant activities [TEAC, ORAC, FRAP] of consumer brews (1 tea bag in 230 ml for 1 min) of seven different brands of black tea from the British market were investigated. The main phenolic compounds identified were epigallocatechin gallate, four theaflavins, as well as epicatechin gallate, theogallin (tentative assignment), quercetin-3-rutinoside and 4-caffeoyl quinic acid. Thearubigins represented an estimated 75-82% of the total phenolics. Further, polyphenol fractions were in decreasing order theaflavins, flavan-3-ols, flavonols, gallic acids and hydroxycinnamates. On average, a cup of a consumer brew of black tea is providing polyphenols at the level of 262 mg GAE/serving, of which 65 mg were assigned to individual polyphenols. The antioxidant activity of black tea preparations is higher than that of most reported dietary agents on a daily basis. Correlations were observed between the antioxidant activities and the sum of all quantified polyphenols by HPLC analysis as well as with the total phenolics. Treatment of the black tea brew with simulated gastric juice resulted in a significant increase of the identified theaflavins implying a partial cleavage of thearubigins in the environment of the gastric lumen. Therefore, black tea can be considered to be a rich source of polyphenols and/or antioxidants.     

Evaluation of the antigenotoxic potential of monomeric and dimeric flavanols, and black tea polyphenols against heterocyclic amine-induced DNA damage in human lymphocytes using the Comet assay

The polyphenolic dimers, epicatechin-4beta-8-catechin (B1), epicatechin-4beta-8-epicatechin (B2), catechin-4beta-8-catechin (B3), catechin-4beta-8-epicatechin (B4), and the gallate ester epicatechin-4beta-8-epicatechin gallate (B'2G) were isolated from grape seeds, and theaflavins and theafulvins from black tea brews. The ability of these naturally-occurring polyphenols to afford protection against the genotoxicity of the heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was compared with that of the monomeric tea flavanols, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG). Genotoxic activity was evaluated in human peripheral lymphocytes using the Comet assay. At the concentration range of 1-100 microM, neither the monomeric nor the dimeric flavanols prevented the lymphocyte DNA damage induced by Trp-P-2. In contrast, both of the black tea polyphenols, theafulvins and theaflavins, at a dose range of 0.1-0.5 mg/ml, prevented, in a concentration-dependent manner, the DNA damage elicited by Trp-P-2. Finally, neither the monomeric and dimeric polyphenols (100 microM) nor the theafulvins and theaflavins (0.5mg/ml) caused any DNA damage in the human lymphocytes. These studies illustrate that black tea theafulvins and theaflavins, if absorbed intact, may contribute to the anticarcinogenic potential associated with black tea intake.     

Preparative isolation and purification of theaflavins and catechins by high-speed countercurrent chromatography

High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins. The HSCCC run was carried out with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that pure theaflavin, theaflavins-3-gallate, theaflavins-3'-gallate and theaflavin-3,3'-digallate could be obtained from crude theaflavins sample and black tea. The structures of the isolated compounds were positively confirmed by (1)H NMR and (13)C NMR, MS analysis, HPLC data and TLC data. Meanwhile, catechins including epigallocatechin gallate, gallocatechin gallate, epicatechin gallate and epigallocatechin were isolated from the aqueous extract of green tea by using the same solvent system. This study developed a modified method combined with enrichment theaflavins method by using HSCCC for separation of four individual theaflavins, especially for better separation of theaflavins monogallates.     

Analysis of tea polyphenols

Tea is the most highly consumed beverage in the world, other than water. However, unlike water, tea contains substantial amounts of polyphenols that have unique biological activities and may be responsible for many of the health benefits of tea. As a result, it is essential to be able to measure the various tea-associated polyphenols. Total polyphenol content is currently measured by using methodology based on reducing activity. Several HPLC systems with detectors that, collectively, have wide ranges in sensitivity have been developed for analysis of individual flavonoids in tea and biological samples, and for theaflavins in tea. Catechins also have been measured in plasma by solid phase extraction, addition of a chromophore, and colorimetric quantification. Except for theaflavins in tea, routine and robust methods for the measurement of polyphenol condensation products (dimers and thearubigens) in tea and biological samples have not been developed. Although in vitro and animal studies suggest substantial metabolism of flavonoids in the gastrointestinal tract, only a single HPLC procedure has been assembled for monitoring the metabolic products of quercetin in urine of human subjects.     

Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.     

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(2)O(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.     

Analysis of theaflavins in biological fluids using liquid chromatography–electrospray mass spectrometry

A HPLC–MS procedure for the sensitive and specific analysis of the black tea flavonoid theaflavin in human plasma and urine was developed. Levels were measured after enzymatic deconjugation, extraction into ethyl acetate, and separation by HPLC, using tandem mass spectrometry as a detecting system. Two healthy volunteers consumed 700 mg theaflavins, equivalent to about 30 cups of black tea. The maximum concentration detected in blood plasma was 1.0 μg l⁻¹ in a sample collected after 2 h. The concentration in urine also peaked after 2 h at 4.2 μg l⁻¹. Hence, only minute amounts of theaflavins can be detected in plasma and urine samples of healthy volunteers after ingestion.     

Anxiolytic effects of theaflavins via dopaminergic activation in the frontal cortex

Epidemiological investigations have reported that the habit of drinking tea reduces the risk of developing a mental disorder, including anxiety disorder and depression. Theaflavins, black tea polyphenols, show antibacterial and anti-oxidative effects, but their effects on brain function, especially mental condition, have not been elucidated. The present study demonstrated that theaflavins increased dopamine (DA) turnover in the frontal cortex and showed an anxiolytic effect in mice. Theaflavin consumption increased the time spent by mice in the open arms of an elevated plus maze test. Theaflavin administration increased the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and the ratios of DOPAC/DA and (DOPAC+homovanillic acids)/DA indicating DA turnover, in the frontal cortex. These results suggest that the consumption of theaflavins induced anxiolytic effects via activation of the dopaminergic system in the frontal cortex, which support the findings of previous epidemiological studies. Theaflavins in black tea may be helpful to reduce anxiety in daily life. (150/150 words).     

茶色素及其药理学功能

茶色素是茶叶多酚类及其衍生物的混合物,主要成分为茶黄素类、茶红纱类。茶色素含有大量的活性酚羟基等活性基因、具有极强的清除自由基和抗氧化作用。近代药理学研究表明,花色素具有防癌抗癌、防紫外线照射、抗氧化、抗动脉粥样硬化、抗龋护齿等多种药理功能。花色素产品的开可广泛用于医药、食品工业。     

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.     

Antiviral activity of green tea and black tea polyphenols in prophylaxis and treatment of COVID-19: A review

BackgroundThe rapid spread of novel coronavirus called SARS-CoV-2 or nCoV has caused countries all over the world to impose lockdowns and undertake stringent preventive measures. This new positive-sense single-stranded RNA strain of coronavirus spreads through droplets of saliva and nasal discharge.PurposeUS FDA has authorized the emergency use of Remdesivir looking at the increasing number of cases of COVID-19, however there is still no drug approved to treat COVID-19. An alternative way of treatment could be the use of naturally derived molecules with known antiviral properties.MethodWe reviewed the antiviral activities of two polyphenols derived from tea, epigallocatechin-3-gallate (EGCG) from green tea and theaflavins from black tea. Both green tea and black tea polyphenols have been reported to exhibit antiviral activities against various viruses, especially positive-sense single-stranded RNA viruses.ResultsRecent studies have revealed the possible binding sites present on SARS-CoV-2 and studied their interactions with tea polyphenols. EGCG and theaflavins, especially theaflavin-3,3′-digallate (TF3) have shown a significant interaction with the receptors under consideration in this review. Some docking studies further emphasize on the activity of these polyphenols against COVID-19.ConclusionThis review summarizes the available reports and evidences which support the use of tea polyphenols as potential candidates in prophylaxis and treatment of COVID-19.     

Identification of a PKCepsilon-dependent regulation of myocardial contraction by epicatechin-3-gallate

In this study, the effects of tea catechins and tea theaflavins on myocardial contraction were examined in isolated rat hearts using a Langendorff-perfusion system. We found that both tea catechins and theaflavins had positive inotropic effects on the myocardium. Of the tested chemicals, epicatechin-3-gallate (ECG) and theaflavin-3,3'-digallate (TF(4)) appear to be the most effective tea catechin and theaflavin, respectively. Further studies of ECG-induced positive inotropy revealed the following insights. First, unlike digitalis drugs, ECG had no effect on intracellular Ca(2+) level in cultured adult cardiac myocytes. Second, it activated PKCepsilon, but not PKCalpha, in the isolated hearts as well as in cultured cells. Neither a phospholipase C (PLC) inhibitor (U73122) nor the antioxidant N-acetyl cysteine (NAC) affected the ECG-induced activation of PKCepsilon. Third, inhibition of PKCepsilon by either chelerythrine chloride (CHE) or PKCepsilon translocation inhibitor peptide (TIP) caused a partial reduction of ECG-induced increases in myocardial contraction. Moreover, NAC was also effective in reducing the effects of ECG on myocardial contraction. Finally, pretreatment of the heart with both CHE and NAC completely abolished ECG-induced inotropic effects on the heart. Together, these findings indicate that ECG can regulate myocardial contractility via a novel PKCepsilon-dependent signaling pathway.     

Keemun black tea extract contains potent fatty acid synthase inhibitors and reduces food intake and body weight of rats via oral administration

The inhibitory effects of a black tea extract on fatty acid synthase were measured through inhibition kinetics. The Keemun black tea extract showed more potent inhibitory activity on fatty acid synthase than green tea extract. Additionally, the inhibitory ability of the black tea extract depended on the extracting solvent and the conditions used. Only 10-23% of the inhibitory activity from the black tea was extracted by the general method of boiling with water. The results suggested that the main fatty acid synthase inhibitors in black tea might be theaflavins. Which were more potent than epigallocatechin gallate or C75. The reaction site on the fatty acid synthase and the inhibition kinetics for the extract were different from those of epigallocatechin gallate or C75. In addition, Keemun black tea extract significantly reduced food intake, body weight and blood triglyceride of diet-induced obesity SD rats via oral administration.     

Black Tea Extract and Its Theaflavin Derivatives Inhibit the Growth of Periodontopathogens and Modulate Interleukin-8 and β-Defensin Secretion in Oral Epithelial Cells

Over the years, several studies have brought evidence suggesting that tea polyphenols, mostly from green tea, may have oral health benefits. Since few data are available concerning the beneficial properties of black tea and its theaflavin derivatives against periodontal disease, the objective of this study was to investigate their antibacterial activity as well as their ability to modulate interleukin-8 and human β-defensin (hBD) secretion in oral epithelial cells. Among the periodontopathogenic bacteria tested, Porphyromonas gingivalis was found to be highly susceptible to the black tea extract and theaflavins. Moreover, our data indicated that the black tea extract, theaflavin and theaflavin-3,3’-digallate can potentiate the antibacterial effect of metronidazole and tetracycline against P. gingivalis. Using lipopolysaccharide-stimulated oral epithelial cells, the black tea extract (100 μg/ml), as well as theaflavin and theaflavin-3,3’-digallate (50 μg/ml) reduced interleukin-8 (IL-8) secretion by 85%, 79%, and 86%, respectively, thus suggesting an anti-inflammatory property. The ability of the black tea extract and its theaflavin derivatives to induce the secretion of the antimicrobial peptides hBD-1, hBD-2 and hBD-4 by oral epithelial cells was then evaluated. Our results showed that the black tea extract as well as theaflavin-3,3’-digallate were able to increase the secretion of the three hBDs. In conclusion, the ability of a black tea extract and theaflavins to exert antibacterial activity against major periodontopathogens, to attenuate the secretion of IL-8, and to induce hBD secretion in oral epithelial cells suggest that these components may have a beneficial effect against periodontal disease.     

Identification of novel QTL for black tea quality traits and drought tolerance in tea plants (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Tea (Camellia sinensis) contains polyphenols and caffeine which have been found to be of popular interest in tea quality. Tea production relies on well-distributed rainfall which influence tea quality. Phenotypic data for two segregating tea populations TRFK St 504 and TRFK St 524 were collected and used to identify the quantitative trait loci (QTL) influencing tea biochemical and drought stress traits based on a consensus genetic map constructed using the DArTseq platform. The populations comprised 261 F₁ clonal progeny. The map consisted of 15 linkage groups which corresponds to chromosome haploid number of tea plant (2n?=?2×?=?30) and spanned 1260.1 cM with a mean interval of 1.1 cM between markers. A total of 16 phenotypic traits were assessed in the two populations. Both interval and multiple QTL mapping revealed a total of 47 putative QTL in the 15 LGs associated with tea quality and percent relative water content at a significant genome-wide threshold of 5%. In total, six caffeine QTL, 25 catechins QTL, three theaflavins QTL, nine QTL for tea taster score, and three QTL for percent relative water contents were detected. Out of these 47 QTL, 19 QTL were identified for ten traits in three main regions on LG01, LG02, LG04, LG12, LG13, and LG14. The QTL associated with caffeine, individual catechins, and theaflavins were clustered mostly in LG02 and LG04 but in different regions on the map. The explained variance by each QTL in the population ranged from 5.5 to 56.6%, with an average of 9.9%. Identification of QTL that are tightly linked to markers associated with black tea quality coupled with UPLC assay may greatly accelerate development of novel tea cultivars owing to its amenability at seedling stage. In addition, validated molecular markers will contribute greatly to adoption of marker-assisted selection (MAS) for drought tolerance and tea quality improvement.     

An improved method for the determination of green and black tea polyphenols in biomatrices by high-performance liquid chromatography with coulometric array detection

Tea polyphenols are strong antioxidants and are believed to have beneficial health effects. However, the blood and tissue levels of these compounds are not well characterized because of a lack of suitable analytical methods for the biological resolution of these compounds. Previously, we developed methods for the analysis of three green tea catechins. Now we report an improved method for the measurement of the levels of the different catechins and theaflavins in biological fluids and tissues. The method includes digestion of the plasma, urine, or tissue samples with beta-d-glucuronidase and sulfatase, followed by extraction with ethyl acetate and subsequent separation by reversed-phase high-performance liquid chromatography (HPLC). The polyphenols are identified on the basis of their retention times, spectral analysis, and electrochemical behavior across an array of electrodes. In a single HPLC run, it is possible to determine the major catechins and theaflavins as well as some of the catechin metabolites. The detection limits for catechins and theaflavins are from 5 to 10 ng/ml of saliva, plasma, or urine.