Theaflavin-3,3'-digallate Research Articles

Inhibition of digestive enzymes by tea polyphenols: enzymological and in silico studies (1045.34)

Tea (Camellia sinensis) has shown anti‐obesity effects in animals. Tea polyphenols can inhibit digestive enzymes such as pancreatic lipase (PL), phospholipase A2 (PLA2), and trypsin in vitro. Our objective was to explore the mechanisms of inhibition by tea polyphenols by enzymology and in silico modeling. Theaflavin‐3,3’‐digallate (TFdiG), theaflavin‐3’‐gallate, theaflavin‐3‐gallate, and theaflavin inhibit PL with IC50 = 1.9, 4.2, 3.0, and 32.9 µM, respectively, indicating that the location of the galloyl ester is essential for inhibitory potency. TFdiG exhibits a mixed mode of inhibition. Theaflavins were found to bind to a pocket adjacent to the active site in PL; galloyl‐containing theaflavins are able to form a hydrogen bond with His264, which forms part of the catalytic triad. Similar interactions are predicted with green tea catechins. Catechins also inhibit PLA2. Modeling studies show that these compounds occupy a tunnel that leads to the active site and are stabilized by van der Waals bonds. This interaction is expected to result in noncompetitive inhibition. We found that (‐)‐epigallocatechin‐3‐gallate (EGCG) inhibited trypsin (IC50 = 193 µM) in a non‐competitive manner. Modeling studies show that EGCG interacts with amino acids around the active site and hinders substrate binding. These findings provide insight into the mechanisms by which tea polyphenols can inhibit key digestive enzymes.Grant Funding Source: NIH AT004678

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

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

Germplasm appraisal of western Himalayan tea: a breeding strategy for yield and quality improvement

Western Himalayan tea represents the status of Geographical Indication in the form of “Kangra Tea” which is unique to the tea grown world over due to its delicate flavor and high quality. The tea germplasm resources of western Himalayan region include selections from the commercial tea estates, abandoned tea gardens of Kangra valley and elite tea clones of CSIR-IHBT, Palampur. Data were recorded for different morphological traits contributing to yield and the biochemical parameters were evaluated using standard HPLC procedures. Multivariate clustering of the data differentiated the germplasm resources into distinct groups based on comparisons among the clusters for leaf size and biochemical parameters. Leaf size differentiated the tea accessions into six phenotypic groups, while shoot density which is an important yield parameter was observed to be independent of leaf size. On the basis of total catechin and caffeine contents, tea accessions were differentiated into nine groups of which Group I in comparison with other groups, recorded the highest total catechin content, moderate to high caffeine level and high astringency factor (AF). Epicatechin gallate (ECG) had a significantly high correlation with AF, implying that high levels of ECG are critical along with corresponding levels of Epigallocatechin gallate (EGCG) for the production of Theaflavin 3,3′digallate (TFDG) which is an important quality constituent providing astringency and briskness to black tea liquor. Sustainability of tea breeding programme can be achieved through organization of germplasm resources and by utilizing elite tea genotypes in breeding programme.

Metabolism of black tea theaflavins by gut microbiota

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

Tea Polyphenols Inhibit Rat Osteoclast Formation and Differentiation

Matrix metalloproteinases (MMPs) play an important role in degeneration of the matrix associated with bone and cartilage. Regulation of osteoclast activity is essential in the treatment of bone disease, including osteoporosis and rheumatoid arthritis. Polyphenols in green tea, particularly epigallocatechin-3-gallate (EGCG), inhibit MMPs expression and activity. However, the effects of the black tea polyphenol, theaflavin-3,3′-digallate (TFDG), on osteoclast and MMP activity are unknown. Therefore, we examined whether TFDG and EGCG affect MMP activity and osteoclast formation and differentiation in vitro. TFDG or EGCG (10 and 100 μM) was added to cultures of rat osteoclast precursors cells and mature osteoclasts. Numbers of multinucleated osteoclasts and actin rings decreased in polyphenol-treated cultures relative to control cultures. MMP-2 and MMP-9 activities were lower in TFDG- and EGCG-treated rat osteoclast precursor cells than in control cultures. MMP-9 mRNA levels declined significantly in TFDG-treated osteoclasts in comparison to control osteoclasts. TFDG and EGCG inhibited the formation and differentiation of osteoclasts via inhibition of MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Thus, TFDG and EGCG may be suitable agents or lead compounds for the treatment of bone resorption diseases.

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

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

Black tea polyphenol inhibits CXCL10 production in oncostatin M-stimulated human gingival fibroblasts

CXC chemokine ligand 10 (CXCL10) plays an important role in the infiltration of Th1 cells and thus in the exacerbation of periodontal disease. Theaflavin-3,3′-digallate (TFDG), polyphenol in black tea, has some beneficial effects but the effect of TFDG on CXCL10 production from human gingival fibroblasts (HGFs) is uncertain. In this study, we investigated the mechanisms by which TFDG may inhibit oncostatin M (OSM)-induced CXCL10 production in human gingival fibroblasts. TFDG prevented OSM-mediated CXCL10 production by HGFs in a dose dependent manner. TFDG significantly inhibited OSM-induced phosphorylation of c-Jun N terminal kinase (JNK), protein kinase B (Akt) (Ser473) that are related to CXCL10 production from OSM-stimulated HGFs. In addition, TFDG suppressed OSM receptor (OSMR) β expression on HGFs. These data provide a novel mechanism where the black tea flavonoid, theaflavin, could provide direct benefits in periodontal disease.

Green Tea Catechin, Epigallocatechin Gallate, Suppresses Signaling by the dsRNA Innate Immune Receptor RIG-I

BackgroundThe Innate immune system constitutes the first line of defense against pathogen infections. The Retinoic acid-inducible gene I (RIG-I) receptor recognizes triphosphorylated ssRNAs and dsRNA to initiate downstream signaling of interferon response. However, unregulated activity of these receptors could lead to autoimmune diseases. We seek to identify small molecules that can specifically regulate RIG-I signaling.Methodology/Principal FindingsEpigallocatechin gallate (EGCG), a polyphenolic catechin present in green tea, was identified in a small molecule screen. It was found to bind RIG-I and inhibits its signaling at low micromolar concentrations in HEK293T cells. Furthermore, EGCG dose-dependently inhibited the ATPase activity of recombinant RIG-I but did not compete with RIG-I interaction with RNA or with ATP. EGCG did not inhibit signaling by Toll-like receptors 3, 4, 9 or constitutive signaling by the adapter protein IPS-1. Structure activity relationship analysis showed that EGCG, its epimer GCG and a digallate-containing compound, theaflavin 3,3′ digallate (TFDG) were potent RIG-I inhibitors. EGCG also inhibited IL6 secretion and IFN- β mRNA synthesis in BEAS-2B cells, which harbors intact endogenous RIG-I signaling pathway.Conclusions/SignificanceEGCG and its derivatives could have potential therapeutic use as a modulator of RIG-I mediated immune responses.

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

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

Preventive effects of black tea theaflavins against mouse type IV allergy

Tea (Camellia sinensis L.), one of the most popular beverages, contains various beneficial constituents. We investigated the preventive effects of black tea theaflavins, theaflavin-3-gallate (3-TF) and theaflavin-3,3'-digallate (TFDG), on oxazolone-induced type IV allergy in male ICR mice. Percutaneous administration of both 3-TF and TFDG at 0.2 mg ear(-1) showed significant preventive effects against mouse type IV allergy. Oral administration of these agents at 50 mg kg(-1) body weight also showed significant preventive effects against mouse type IV allergy. Oral administration of 3-TF and TFDG at a dose of 50 mg kg(-1) body weight prevented the increases in levels of some proinflammatory cytokines, interleukin-12 (IL-12), interferon-gamma (IFN-gamma), and tumour necrosis factor-alpha (TNF-alpha), in the sera and/or ears of mice with type IV allergy. Lowering of serum antioxidant activity in mice with allergic symptoms was also prevented by oral administration of these theaflavins at a dose of 50 mg kg(-1) body weight. The anti-allergic mechanisms of action of theaflavins involve inhibition of the fluctuations of cytokines and maintenance of antioxidant status in allergic mice. These results suggest that the theaflavins as well as catechins contribute to the anti-allergic effects of black tea.

Tea polyphenols inhibit IL‐6 production in tumor necrosis factor superfamily 14‐stimulated human gingival fibroblasts

IL-6 is well recognized to be a potent bone resorptive agent and thus in the development of periodontal disease. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), the major catechins in green tea, and theaflavin-3,3'-digallate (TFDG), polyphenol in black tea, have multiple beneficial effects, but the effects of catechins and theaflavins on IL-6 production in human gingival fibroblasts (HGFs) are not known. In this study, we investigated the mechanisms by which EGCG, ECG, and TFDG inhibit tumor necrosis factor superfamily 14 (TNFSF14)-induced IL-6 production in HGFs. We detected TNFSF14 mRNA expression in human diseased periodontal tissues. TNFSF14 increased IL-6 production in HGFs in a concentration-dependent manner. EGCG, ECG, and TFDG prevented TNFSF14-mediated IL-6 production in HGFs. EGCG, ECG, and TFDG prevented TNFSF14-induced extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappaB activation in HGFs. Inhibitors of ERK, JNK, and nuclear factor-kappaB decreased TNFSF14-induced IL-6 production. In addition, EGCG, ECG, and TFDG attenuated TNFSF14 receptor expression on HGFs. These data provide a novel mechanism through which the green tea and black tea polyphenols could be used to provide direct benefits in periodontal disease.

Black-Tea Polyphenols Suppress Postprandial Hypertriacylglycerolemia by Suppressing Lymphatic Transport of Dietary Fat in Rats

Administration of black-tea polyphenols (BTP) at 100 and 200 mg/kg of body weight in rats suppressed postprandial hypertriacylglycerolemia in a dose-dependent manner. Administration of BTP also suppressed lymphatic recovery of (14)C-trioleoylglycerol in rats that were cannulated in the thoracic duct. BTP dose-dependently inhibited the activity of pancreatic lipase in vitro with an IC50 of 0.254 mg/mL. When purified theaflavins, which are components of BTP, were used, theaflavins with galloyl moieties, but not those without galloyl moiety, inhibited the activity of pancreatic lipase. Theaflavin-3,3'-digallate (TFDG) was more effective in inhibiting the activity of pancreatic lipase than epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and a mixture of EGCG and ECG. BTP and TFDG had a similar effect in inhibiting the activity of pancreatic lipase when the total polyphenol amount was adjusted to the same. BTP had no effect on micellar solubility of hydrolysis products of triacylglycerol. These results suggest that BTP suppressed postprandial hypertriacylglycerolemia by reducing triacylglycerol absorption via the inhibition of pancreatic lipase activity.

Green and black tea are equally potent stimuli of NO production and vasodilation: new insights into tea ingredients involved

Epidemiological studies suggest that consumption of tea is associated with beneficial cardiovascular effects. Since different types of tea are consumed throughout the world, a question of much interest is whether green tea is superior to black tea in terms of cardiovascular protection. We therefore compared the effects of green and black tea on nitric oxide (NO) production and vasodilation and elucidated the tea compounds involved. We chose a highly fermented black tea and determined concentrations of individual tea compounds in both green and black tea of the same type (Assam). The fermented black tea was almost devoid of catechins. However, both teas stimulated eNOS activity and phosphorylation in bovine aortic endothelial cells (BAEC) as well as vasorelaxation in rat aortic rings to a similar extent. In green tea, only epigallocatechin-3-gallate (EGCG) resulted in pronounced NO production and NO-dependent vasorelaxation in aortic rings. During tea processing to produce black tea, the catechins are converted to theaflavins and thearubigins. Individual black tea theaflavins showed a higher potency than EGCG in NO production and vasorelaxation. The thearubigins in black tea are highly efficient stimulators of vasodilation and NO production. Green and black tea compounds induced comparable phosphorylation of eNOS and upstream signalling kinases. Whereas stimulation of eNOS activity by EGCG was only slightly affected by pretreatment of cells with various ROS scavengers, TF3(theaflavin-3',3-digallate)-induced eNOS activity was partially inhibited by PEG-catalase. These results implicate that highly fermented black tea is equally potent as green tea in promoting beneficial endothelial effects. Theaflavins and thearubigins predominantly counterbalance the lack of catechins in black tea. The findings may underline the contribution of black tea consumption in prevention of cardiovascular diseases.

Protection from experimental colitis by theaflavin‐3,3′‐digallate correlates with inhibition of IKK and NF‐κB activation

Inflammatory bowel disease (IBD) is associated with activation of nuclear factor kappa B (NF-kappaB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin-3,3'-digallate (TFDG), the most potent anti-oxidant polyphenol of black tea, down-regulates NF-kappaB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines. The in vivo efficacy of TFDG was assessed in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Both mRNA and protein levels of proinflammatory cytokines and iNOS were analyzed in colon tissue treated with or without TFDG. NF-kappaB activation was determined by electrophoretic mobility shift assay and levels of NF-kappaB inhibitory protein (IkappaBalpha) were analyzed by Western blotting. Oral administration of TFDG (5 mg kg(-1) daily i.g.) significantly improved TNBS-induced colitis associated with decreased mRNA and protein levels of TNF-alpha, IL-12, IFN-gamma and iNOS in colonic mucosa. DNA binding and Western blotting revealed increase in NF-kappaB activation and IkappaBalpha depletion in TNBS-treated mice from Day 2 through Day 8 with a maximum at Day 4, which resulted from increased phosphorylation of IkappaBalpha and higher activity of IkappaB kinase (IKK). Pretreatment with TFDG markedly inhibited TNBS-induced increases in nuclear localization of NF-kappaB, cytosolic IKK activity and preserved IkappaBalpha in colon tissue. TFDG exerts protective effects in experimental colitis and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.

The theaflavin monomers inhibit the cancer cells growth in vitro.

The inhibition effects of tea theaflavins complex (TFs), theaflavin-3-3'-digallate (TFDG), theaflavin-3'-gallate (TF2B), and an unidentified compound (UC) on the growth of human liver cancer BEL-7402 cells, gastric cancer MKN-28 cells and acute promyelocytic leukemia LH-60 cells were investigated. TFs was obtained through the catalysis of catechins with immobilized polyphenols oxidase. TFDG, TF2B and UC were isolated from TFs with high speed countercurrent chromatography (HSCCC). The results showed that TF2B significantly inhibited the growth of all three kinds of cancer cells, TFs, TFDG and UC had some effect on BEL-7402 and MKN-28, but little activity on LH-60. The inhibition effects of TF2B, TFDG, and UC on BEL-7402 and MKN-28 were stronger than TFs. The relationship coefficients between monomer concentration and its inhibition rate against MKN-28 and BEL-7402 were 0.87 and 0.98 for TF2B, 0.96 and 0.98 for UC, respectively. The IC50 values of TFs, TF2B, and TFDG were 0.18, 0.11, and 0.16 mM on BEL-7402 cells, and 1.11, 0.22, and 0.25 mM on MKN-28 cells respectively.

Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols (-)-epigallocatechin-3-gallate and theaflavin-3,3'-digallate.

Our previous study showed that tea polyphenols inhibited MAP kinase and AP-1 activities in mouse epidermal JB6 cells and the corresponding H-ras-transformed cell line 30.7b Ras 12. The present study investigated the mechanisms of this inhibition. The cells were incubated with (-)-epigallocatechin-3-gallate (EGCG) or theaflavin-3,3'-digallate (TFdiG) (20 mM) for different times, and the cell lysate was analyzed by immunoblotting. EGCG treatment decreased the levels of phospho-Erk1/2 and -MEK1/2 time-dependently (by 60% at 60 min). TFdiG lowered their levels by 38%-50% at 15 min. TFdiG effectively decreased total Raf-1 protein levels, most likely through lysosomal degradation. EGCG did not affect protein levels or the activity of Raf-1 significantly but decreased its association with MEK1 as determined by co-immunoprecipitation. In addition, EGCG and TFdiG (10 mM) inhibited the phosphorylation of Elk-1 by isolated phospho-Erk1/2 in vitro. This inhibition of Erk1/2 activity is Elk-1 concentration-dependent and ATP concentration-independent, which suggests that EGCG and TFdiG interfere with the binding of the protein substrate to the kinase. The presently demonstrated specific mechanisms of inhibition of MAP kinases by EGCG and TFdiG may help us to understand the effects of tea consumption on cancer, inflammatory diseases, and cardiovascular diseases.