Browning Reaction between Phospholipids and Acetaldehyde

Tea catechins have strong bitterness and influence the taste of tea. Among the 25 human bitter-taste receptors (TAS2Rs), we found that hTAS2R14 responded to catechins, in addition to hTAS2R39, a known catechin receptor. Although hTAS2R14 responded to (?)-epicatechin gallate and (?)-epigallocatechin gallate, it did not respond to (?)-epicatechin and (?)-epigallocatechin.     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Activation of human bitter taste receptors by polymethoxylated flavonoids

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.     

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.     

Bitter peptides activate hTAS2Rs, the human bitter receptors

Fermented food contains numerous peptides derived from material proteins. Bitter peptides formed during the fermentation process are responsible for the bitter taste of fermented food. We investigated whether human bitter receptors (hTAS2Rs) recognize bitterness of peptides with a heterologous expression system. HEK293 cells expressing hTAS2R1, hTAS2R4, hTAS2R14, and hTAS2R16 responded to bitter casein digests. Among those cells, the hTAS2R1-expressing cell was most strongly activated by the synthesized bitter peptides Gly-Phe and Gly-Leu, and none of the cells was activated by the non-bitter dipeptide Gly-Gly. The results showed that these bitter peptides, as well as many other bitter compounds, activate hTAS2Rs, suggesting that humans utilize these hTAS2Rs to recognize and perceive the structure and bitterness of peptides.     

Human Bitter Taste Receptors hTAS2R8 and hTAS2R39 with Differential Functions to Recognize Bitter Peptides

The strong bitter peptide, Phe-Phe-Pro-Arg, activated cultured cells expressing either of the known human bitter taste receptors, hTAS2R8 and hTAS2R39. The partial structure of Pro-Arg activated hTAS2R39, but did not activate hTAS2R8. These receptors may not indiscriminately recognize bitter peptides, but have a differential function in their recognition.     

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H₂O₂-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3′-O-gallate (TF3′G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3′G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H₂O₂-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3′G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p?<?0.05), and improved to approximately 100%. Only TF3′G did not significantly increase cell viability. It was suggested that the inhibitory effect on H₂O₂-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.     

The human taste receptor hTAS2R14 responds to a variety of different bitter compounds

The recent advances in the functional expression of TAS2Rs in heterologous systems resulted in the identification of bitter tastants that specifically activate receptors of this family. All bitter taste receptors reported to date exhibit a pronounced selectivity for single substances or structurally related bitter compounds. In the present study we demonstrate the expression of the hTAS2R14 gene by RT-PCR analyses and in situ hybridisation in human circumvallate papillae. By functional expression in HEK-293T cells we show that hTAS2R14 displays a, so far, unique broad tuning towards a variety of structurally diverse bitter compounds, including the potent neurotoxins, (-)-alpha-thujone, the pharmacologically active component of absinthe, and picrotoxinin, a poisonous substance of fishberries. The observed activation of heterologously expressed hTAS2R14 by low concentrations of (-)-alpha-thujone and picrotoxinin suggests that the receptor is sufficiently sensitive to caution us against the ingestion of toxic amounts of these substances.     

Combination of HSCCC and Sephadex LH-20 methods An approach to isolation and purification of the main individual theaflavins from black tea

In order to separate the main individual theaflavin monomers from black tea, high-speed countercurrent chromatography (HSCCC) and Sephadex LH-20 column chromatography were applied. The results showed that theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B) and theaflavin-3,3'-digallate (TF3) can be obtained by HSCCC using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:1:6, v/v/v/v), but the TF1 was containing epicatechin-3-gallate (ECG). Similarly, Sephadex LH-20 can also effectively separate TF2A(B) and TF3, but epigallocatechin-3-gallate (EGCG) contaminated TF1, too. Combination of HSCCC and Sephadex LH-20, the preferably purified TF1, TF2A(B) and TF3 were obtained than single separation technique. In addition, ECG and EGCG were also suggested to be able to be comprehensively separated by combination of the two techniques.     

Theaflavins inhibit the ATP synthase and the respiratory chain without increasing superoxide production

Four dietary polyphenols, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3,3'-digallate (TF3), have been isolated from black tea, and their effects on oxidative phosphorylation and superoxide production in a model system (Escherichia coli) have been examined. The esterified theaflavins were all potent inhibitors of the membrane-bound adenosine triphosphate (ATP) synthase, inhibiting at least 90% of the activity, with IC(50) values in the range of 10-20 μM. ATP-driven proton translocation was inhibited in a similar fashion, as was the purified F(1)-ATPase, indicating that the primary site of inhibition was in the F(1) sector. Computer modeling studies supported this interpretation. All four theaflavins were also inhibitory towards the electron transport chain, whether through complex I (NDH-1) or the alternative NADH dehydrogenase (NDH-2). Inhibition of NDH-1 by TF3 appeared to be competitive with respect to NADH, and this was supported by computer modeling studies. Rates of superoxide production during NADH oxidation by each dehydrogenase were measured. Superoxide production was completely eliminated in the presence of about 15 μM TF3, suggesting that inhibition of the respiratory chain by theaflavins does not contribute to superoxide production.     

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.     

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.     

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

Background

Theaflavins 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 Findings

In 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.

Conclusions

The 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.     

Functions of human bitter taste receptors depend on N-glycosylation

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs. Non-glycosylated receptors exhibit substantially lower cell surface localization and reduced association with the cellular chaperone calnexin. As the auxiliary factors receptor transporting proteins 3 and 4 are able to restore the function of non-glycosylated hTAS2R16 partially, we conclude that glycosylation is important for receptor maturation but not for its function per se .     

Genetic signature of differential sensitivity to stevioside in the Italian population

The demand for diet products is continuously increasing, together with that for natural food ingredients. Stevioside and other steviol glycosides extracted from the leaves of the plant Stevia rebaudiana Bertoni are the first natural high-potency sweeteners to be approved for consumption in the United States and the European Union. However, the sweetness of these compounds is generally accompanied by aversive sensations, such as bitter and off-tastes, which may constitute a limit to their consumption. Moreover, consumers’ differences in sensitivity to high-potency sweeteners are well known, as well as difficulties in characterizing their aftertaste. Recently, TAS2R4 and TAS2R14 have been identified as the receptors that mediate the bitter off-taste of steviol glycosides in vitro. In the present study, we demonstrate that TAS2R4 gene polymorphism rs2234001 and TAS2R14 gene polymorphism rs3741843 are functional for stevioside bitterness perception.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0401-y) contains supplementary material, which is available to authorized users.     

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.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

Structure–function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Syntheses of procyanidin B2 and B3 gallate derivatives using equimolar condensation mediated by Yb(OTf)3 and their antitumor activities

Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 3″-O-gallate, and 3,3″-di-O-gallate, were synthesized using equimolar condensation mediated by Yb(OTf)₃. Synthesized compounds showed significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than well-known EGCG and prodelphinidin B3.