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.     

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.     

Relationship between the methylation status of dietary flavonoids and their growth-inhibitory and apoptosis-inducing activities in human cancer cells

Flavonoids are polyphenolic compounds widely distributed in the plant kingdom. Compelling research indicates that flavonoids have important roles in cancer chemoprevention and chemotherapy possibly due to biological activities that include action through anti-inflammation, free radical scavenging, modulation of survival/proliferation pathways, and inhibition of the ubiquitin-proteasome pathway. Plant polyphenols including the green tea polyphenol (-)-epigallocatechin gallate or (-)-EGCG, and the flavonoids apigenin, luteolin, quercetin, and chrysin have been shown to inhibit proteasome activity and induce apoptosis in human leukemia cells. However, biotransformation reactions to the reactive hydroxyl groups on polyphenols could reduce their biological activities. Although methylated polyphenols have been suggested to be metabolically more stable than unmethylated polyphenols, the practical use of methylated polyphenols as cancer preventative agents warrants further investigation. In the current study, methylated and unmethylated flavonoids were studied for their proteasome-inhibitory and apoptosis-inducing abilities in human leukemia HL60 cells. Methylated flavonoids displayed sustained bioavailability and inhibited cellular proliferation by arresting cells in the G(1) phase. However, they did not act as proteasome inhibitors in either an in vitro system or an in silico model and only weakly induced apoptosis. In contrast, unmethylated flavonoids exhibited inhibition of the proteasomal activity in intact HL60 cells, accumulating proteasome target proteins and inducing caspase activation and poly(ADP-ribose) polymerase cleavage. We conclude that methylated flavonoids lack potent cytotoxicity against human leukemia cells and most likely have limited ability as chemopreventive agents.     

Polyphenols as potential indicators for drought tolerance in tea (Camellia sinensis L.)

Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a 'rain-out' shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r2=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars.     

Antioxidant,Anti‐inflammatory and Cytotoxic Activities of Polyphenols Extracted from Chroogomphus rutilus

Chroogomphus rutilus is a rare fungal species that grows under pine trees and is now widely used as a functional food and pharmaceutical product. However, the chemical constituents and biological activities of Chroogomphus rutilus have been relatively limited. The present study aimed at determining the total polyphenols and flavonoids contents, biological activities and main phenolic compounds of Chroogomphus rutilus from different geographical origins at the stipe and pileus. The results suggested that Chroogomphus rutilus polyphenol extracts revealed a higher antioxidant, anti‐inflammatory, and cytotoxic activities, and there were significant differences between samples from different locations and regions. Correlation analysis showed that the contents of total polyphenols and flavonoids were significantly correlated with antioxidant and anti‐inflammatory activities. However, only the content of total flavonoids was significantly correlated with cytotoxicity, which means that the cytotoxicity of Chroogomphus rutilus polyphenol extracts may be regulated by flavonoids or other compounds. HPLC‐DAD analysis revealed that the main phenolic compound was protocatechuic acid, followed by baicalin, p‐hydroxyphenylacetic acid and p‐hydroxybenzoic acid, but comparing with the pileus extracts, the stipe extracts can be considered as a higher concentration of phenolic compounds. Therefore, antioxidant, anti‐inflammatory and cytotoxic activities of Chroogomphus rutilus polyphenol extracts could be due to the identified compounds. This study investigated a deep knowledge about the constituents and activities of Chroogomphus rutilus and provided the reference for its application in food and pharmaceutical.     

Polyphenols as Potential Indicators for Drought Tolerance in Tea (Camellia sinensis L.)

Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a ‘rain-out’ shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r²=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars.     

植物多酚的微生物合成

植物多酚属于苯丙烷衍生物,包括酚酸类、茋类、姜黄素类和黄酮类等。它们具有抗氧化、扩血管、抗血凝、抗炎、抗肿瘤、抗病毒等生理药理活性,在医药、食品、化妆品、化工等领域具有巨大的应用市场。微生物具有生长快、培养简单、可工业化等优点,成为异源合成天然产物的重要宿主。近年来,合成生物学的发展促进了植物天然产物的微生物合成,并取得了实质性进展。文中综述了植物多酚代谢途径在工程大肠杆菌、酿酒酵母等微生物中的构建、表达和产物合成现状,讨论了提高产量的前体工程、动态调控、共培养等优化策略,并就未来的多酚途径工程提出展望。     

Cancer chemoprevention by tea polyphenols

Tea is one of the most widely consumed beverages, second only to water. Many experimental researches in laboratory animals demonstrated that tea components had an inhibitory effect on carcinogenesis at a number of organ sites. The inhibitory effects of tea against carcinogenesis have been attributed to the biologic activities of the polyphenol fraction in tea. This review summarizes experimental data on chemopreventive effects of tea polyphenols in various tumor bioassay systems. Many laboratory studies have demonstrated the inhibitory effects of green tea polyphenols, especially (-)-epigallocatechin-3-gallate (EGCG), on carcinogenesis in animals models. The majority of these studies have been conducted in mouse skin tumor models, where tea polyphenols were used either as oral feeding in drinking water or in direct local application. Most studies used 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV) radiation as the tumor promoter and found anticarcinogenic effects caused by green tea polyphenols. Black tea was also found to be effective, although the activity was weaker than that of green tea in some experiments. Other studies showed that black tea polyphenols-theaflavins exhibited stronger anticarcinogenic activity than did EGCG. Caffeine in tea was also important for tea to prevent tumorigenesis. The molecular mechanisms of the cancer chemopreventive effects of tea polyphenols are not completely understood. They are most likely related to the mechanisms of biochemical actions of tea polyphenols, which include antioxidative activities, modulation of xenobiotic metabolite enzymes and inhibition of tumor promotion. In addition, we have also proposed that tea polyphenols function as cancer chemopreventive agents through modulation of mitotic signal transduction. However, the molecular mechanisms involved in this modulation need further investigation.     

Production and HPLC analysis of black tea theaflavins and thearubigins during in vitro oxidation

A model fermentation system has been designed which utilizes pure catechins and partially purified polyphenol oxidase (EC 1.14.18.1 from green tea shoots. HPLC analysis of the products formed during in vitro oxidation has demonstrated a close similarity between this system and in vivo oxidation occurring during factory fermentation. Furthermore, changes in theaflavin and thearubigin levels, revealed by time courses of fermentation, show in vitro and in vivo systems to be qualitatively similar, although the former system produces considerably higher levels of both components. The model fermentation system, therefore, appears to be a suitable experimental system for studying the formation of theaflavin and thearubigin pigments under strictly controlled conditions. In preliminary experiments the theaflavins have been identified on HPLC profiles by enzymic oxidation of the relevant catechin pairs. Similarly, major coloured components other than theaflavins, which are considered to be thearubigins, have been shown to be formed by the oxidation and reaction of two gallocatechins (epigallocatechin and epigallocatechin gallate). The model fermentation system, in conjunction with HPLC as described in this paper, provides a means whereby precise data on theaflavin and thearubigin formation can be obtained and, in the case of the thearubigins, one which could yield additional structural information.     

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.     

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。     

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.     

Immunochemical detection of food-derived polyphenols in the aorta: macrophages as a major target underlying the anti-atherosclerotic activity of polyphenols

It has been suggested that polyphenol-rich diets decrease the risk of cardiovascular diseases. Although studies of the bioavailability of polyphenols, particularly their absorption and metabolism, have been reported recently, the tissue and cellular distributions underlying their biological mechanisms remain unknown. It is difficult to evaluate the specific localization of tissue and/or cellular polyphenols, because the method is limited to chromatography. To overcome these difficulties, we have developed anti-polyphenol antibodies to characterize immunohistochemically the localization of polyphenols and their metabolites in vivo. Two novel monoclonal antibodies were raised against quercetin and tea catechins, which represent flavonoid-type polyphenols distributed in foods and beverages, and are expected to exhibit anti-oxidative and anti-inflammatory activities in vivo. Using these antibodies, we identified activated macrophages as a specific target of these flavonoids during the development of atherosclerotic lesions. This review describes recent findings on the molecular actions of flavonoids that underly their anti-atherosclerotic activity in vivo.     

High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine

Polyphenols in biological fluids are generally estimated by HPLC with UV, electrochemical or fluorimetric detection. We describe herein a method specially developed to estimate biomarkers of polyphenol consumption in human urine. We simultaneously quantified 15 polyphenols and their related compounds in human urine using an HPLC coupled with electrospray ionization mass-mass (HPLC-ESI-MS-MS) method with an analytical run time of 6 min. The method has been validated with respect to linearity, precision, and accuracy in intra- and inter-day assays. It has been applied to human urine samples collected from volunteers after consumption of different polyphenol-rich beverages in controlled conditions.     

GC-MS methods for metabolic profiling of microbial fermentation products of dietary polyphenols in human and in vitro intervention studies

Flavonoids, a subclass of polyphenols, are major constituents of many plant-based foods and beverages, including tea, wine and chocolate. Epidemiological studies have shown that a flavonoid-rich diet is associated with reduced risk of cardiovascular diseases. The majority of the flavonoids survive intact until they reach the colon where they are then extensively metabolized into smaller fragments. Here, we describe the development of GC-MS-based methods for the profiling of phenolic microbial fermentation products in urine, plasma, and fecal water. Furthermore, the methods are applicable for profiling products obtained from in vitro batch culture fermentation models. The methods incorporate enzymatic deconjugation, liquid-liquid extraction, derivatization, and subsequent analysis by GC-MS. At the level of individual compounds, the methods gave recoveries better than 80% with inter-day precision being better than 20%, depending on the matrix. Limits of detection were below 0.1mug/ml for most phenolic acids. The newly developed methods were successfully applied to samples from human and in-vitro intervention trials, studying the metabolic impact of flavonoid intake. In conclusion, the methods presented are robust and generally applicable to diverse biological fluids. Its profiling character is useful to investigate on a large scale the gut microbiome-mediated bioavailability of flavonoids.     

Role of plant polyphenols in genomic stability

Polyphenols are a large and diverse class of compounds, many of which occur naturally in a range of food plants. The flavonoids are the largest and best-studied group of these. A range of plant polyphenols are either being actively developed or currently sold as dietary supplements and/or herbal remedies. Although, these compounds play no known role in nutrition (non-nutrients), many of them have properties including antioxidant, anti-mutagenic, anti-oestrogenic, anti-carcinogenic and anti-inflammatory effects that might potentially be beneficial in preventing disease and protecting the stability of the genome. However not all polyphenols and not all actions of individual polyphenols are necessarily beneficial. Some have mutagenic and/or pro-oxidant effects, as well as interfering with essential biochemical pathways including topoisomerase enzyme activities, prostanoid biosynthesis and signal transduction. There is a very large amount of in vitro data available, but far fewer animal studies, and these are not necessarily predictive of human effects because of differences in bacterial and hepatic metabolism of polyphenols between species. Epidemiological studies suggest that high green tea consumption in the Japanese population and moderate red wine consumption in the French population may be beneficial for heart disease and cancer, and these effects may relate to specific polyphenols. A small number of adequately controlled human intervention studies suggest that some, but not all polyphenol extracts or high polyphenol diets may lead to transitory changes in the antioxidative capacity of plasma in humans. However, none of these studies have adequately considered long-term effects on DNA or the chromosome and unequivocally associated these with polyphenol uptake. Furthermore, clinical trials have required intravenously administered polyphenols at concentrations around 1400mg/m(2) before effects are seen. These plasma concentrations are unlikely to be achieved using the dietary supplements currently available. More focused human studies are necessary before recommending specific polyphenolic supplements at specific doses in the human population.     

茶多酚的抗氧化和抑菌活性及其增效剂

论述了茶多酚的抗氧化和抑菌活性机理,其中起重要作用的是茶多酚分子内的多个酚羟基。在抗氧化过程中,其酚羟基具有供氢体的活性,阻断自由基的链式反应。在抑菌过程中,其酚羟基可与蛋白质分子中的氨基或羧基结合,其疏水性的苯环结构也可与蛋白质发生疏水结合,茶多酚与蛋白质之间的这种多点结合作用阻止了细菌的侵染,使其具有抑菌性。作为增效剂,有机酸、β-胡萝卜紊等能够增强茶多酚的抗氧化性能;壳聚糖、食盐等能够增强茶多酚的抑菌活性．维生素类既能够增强茶多酚的抗氧化性能又能增强其抑菌作用。     

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.     

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.