Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme

The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50% inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine tannins with an IC50 <200 microM for ACE inhibitors were identified belonging to three tannin classes: caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor. Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose were non-specific inhibitors because also reduced the activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose was also reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-O-galloyl-beta-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these tannins may explain the hypotensive effects of some traditional Chinese herbs.     

Quince (Cydonia oblonga Miller) peel polyphenols modulate LPS-induced inflammation in human THP-1-derived macrophages through NF-κB, p38MAPK and Akt inhibition

Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis and cancer. A wide range of anti-inflammatory chemicals have been used to treat such diseases while presenting high toxicity and numerous side effects. Here, we report the anti-inflammatory effect of a non-toxic, cost-effective natural agent, polyphenolic extract from the Tunisian quince Cydonia oblonga Miller. Lipopolysaccharide (LPS) treatment of human THP-1-derived macrophages induced the secretion of high levels of the pro-inflammatory cytokine TNF-α and the chemokine IL-8, which was inhibited by quince peel polyphenolic extract in a dose-dependent manner. Concomitantly, quince polyphenols enhanced the level of the anti-inflammatory cytokine IL-10 secreted by LPS-treated macrophages. We further demonstrated that the unexpected increase in IL-6 secretion that occurred when quince polyphenols were associated with LPS treatment was partially responsible for the polyphenols-mediated inhibition of TNF-α secretion. Biochemical analysis showed that quince polyphenols extract inhibited the LPS-mediated activation of three major cellular pro-inflammatory effectors, nuclear factor-kappa B (NF-κB), p38MAPK and Akt. Overall, our data indicate that quince peel polyphenolic extract induces a potent anti-inflammatory effect that may prove useful for the treatment of inflammatory diseases and that a quince-rich regimen may help to prevent and improve the treatment of such diseases.     

Wine polyphenols and ethanol do not significantly scavenge superoxide nor affect endothelial nitric oxide production

Epidemiological studies have shown that moderate intake of red wine reduces the risk of coronary heart disease. It has been proposed that the antiatherogenic effect be due to the scavenging of reactive oxygen species by polyphenols and ethanol or an effect on endothelial nitric oxide (NO) production. We have determined the reaction rates of superoxide with four different polyphenols and ethanol. The superoxide reaction rates were determined at 37 degrees C and pH 7.4 using competitive spin trapping and electron paramagnetic resonance (EPR) spectroscopy. Ethanol did not scavenge superoxide. For the polyphenols catechin, epicatechin, gallic acid, and quercetin, we find rate constants of respectively 2.3*10(4), 2.2*10(4), 2.3*10(3) and 1.9*10(4)(mole per second)(-1). Polyphenols can only exert a significant scavenging effect, if the plasma concentration reach sufficiently high levels. At concentrations found in vivo (low nanomolar range), the scavenging of superoxide by polyphenols and ethanol is negligible in comparison with endogenous protection against superoxide. Incubation of cultured endothelial cells with 5 micromol/L of catechin, epicatechin, gallic acid, quercetin, or ethanol 0.05% (v/v) did not influence the maximal production of NO by these cells as measured by fluorescent nitric oxide cheletropic traps (FNOCT). The observed antiatherogenic effects must be caused by a mechanism other than direct scavenging of superoxide or influence on maximal endothelial NO production.     

Potent inhibitory action of red wine polyphenols on human breast cancer cells

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.     

Characterization of polymerized polyphenols by size-exclusion HPLC

Various kinds of high-molecular-mass polyphenols such as condensed tannins, hydrolyzable tannins, and polymerized anthocyanins, were readily characterized by a new size-exclusion HPLC method. This rapid analytical method may also be useful for the profiling of molecular mass distribution of polyphenolic constituents in many kinds of food materials.     

Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress

Polyphenols represent a large family of plant secondary metabolites implicated in the prevention of various diseases such as cancers and cardiovascular diseases. The potato is a significant source of polyphenols in the human diet. In this study, we examined the expression of thirteen genes involved in the biosynthesis of polyphenols in potato tubers using real-time RT-PCR. A selection of five field grown native Andean cultivars, presenting contrasting polyphenol profiles, was used. Moreover, we investigated the expression of the genes after a drought exposure. We concluded that the diverse polyphenolic profiles are correlated to variations in gene expression profiles. The drought-induced variations of the gene expression was highly cultivar-specific. In the three anthocyanin-containing cultivars, gene expression was coordinated and reflected at the metabolite level supporting a hypothesis that regulation of gene expression plays an essential role in the potato polyphenol production. We proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression. This study provides information on key polyphenol biosynthetic and regulatory genes, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.     

Down-regulation of NF-κB signalling by polyphenolic compounds prevents endotoxin-induced liver injury in a rat model

Activation of NF-κB has been reported to play a key role in causing endotoxin-induced hepatic damage through enhanced production of reactive oxygen species and pro-inflammatory mediators. In this context, the potential of polyphenolic phytochemicals in preventing endotoxin-induced liver damage remains unclear. Here, we demonstrate that catechin and quercetin have the potential to down-regulate the initial signalling molecule NF-κB which may further inhibit the downstream cascade including TNF-α and NO. These results were confirmed using N-nitro-L-arginine methyl ester (L-NAME), the inhibitor of inducible nitric oxide synthase (iNOS) along with the biochemical and histological alterations occurring in the presence and absence of supplementation with both the polyphenols. However, catechin was found to be more effective than quercetin against endotoxin-induced liver injury. These findings suggest that these polyphenols may form a pharmacological basis for designing a therapeutic agent against endotoxin-mediated oxidative damage.     

High-performance liquid chromatographic separation and identification of polyphenolic compounds from the infusion of Davilla elliptica St. Hill

The isolation of polyphenolic compounds from an infusion of the Brazilian plant Davilla elliptica (Dilleniaceae), used as tea by virtue of its digestive properties, is described. An improved preparative HPLC method was used in order to isolate pure polyphenols from the complex mixture. Liquid-liquid extraction and solid-phase extraction were employed to minimise the interference of polymeric compounds and to provide an enriched fraction of the compounds of interest. The identification of the isolated compounds was performed using analytical HPLC as well as direct injection electrospray ionisation ion trap tandem mass spectrometry (ESI-IT-MS/MS). The high flavonoid content suggests that D. elliptica may be a promising source of compounds to produce natural phytomedicines.     

Ethanol and oxidative mechanisms in the brain

There is strong evidence showing that chronic and excessive ethanol consumption may enhance oxidative damage to neurons and result in cell death. Although not yet well understood, ethanol may enhance ROS production in brain through a number of pathways including increased generation of hydroxyethyl radicals, induction of CYP2E1, alteration of the cytokine signaling pathways for induction of iNOS and sPLA(2), and production of prostanoids through the PLA(2)/COX pathways. Since many neurodegenerative diseases are also associated with oxidative and inflammatory mechanisms in the brain, it would be important to find out whether chronic and excessive ethanol consumption may exacerbate the progression of these diseases. There is evidence that the polyphenolic antioxidants, especially those extracted from grape skin and seed, may protect the brain from neuronal damage due to chronic ethanol administration. Among the polyphenols from grapes, resveratrol seems to have unique antioxidant properties. The possible use of this compound as a therapeutic agent to ameliorate neurodegenerative processes should be further explored.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

Antileishmanial activity and immune modulatory effects of tannins and related compounds on Leishmania parasitised RAW 264.7 cells

The antileishmanial and immunomodulatory potencies of a total of 67 tannins and structurally related compounds were evaluated in terms of extra- and intra-cellular leishmanicidal effects and macrophage activation for release of nitric oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)-like activities. Their effects on macrophage functions were further assessed by expression analysis (iNOS, IFN-alpha, IFN-gamma, TNF-alpha, IL-1, IL-10, IL-12, IL-18). With few exceptions, e.g., caffeic acid derivatives, these polyphenols revealed little direct toxicity for extracellular promastigote Leishmania donovani or L. major strains. In contrast, many polyphenols appreciably reduced the survival of the intracellular, amastigote parasite form in vitro. Upon activation, e.g., by immune response mediators such as IFN-gamma, macrophages may transform from permissive host to leishmanicidal effector cells. Our data from functional bioassays suggested that the effects of polyphenols on intracellular Leishmania parasites were due to macrophage activation rather than direct antiparasitic activity. Gene expression analyses not only confirmed functional data, they also clearly showed differences in the response of infected macrophages when compared to that of noninfected cells. Conspicuously, infected macrophages showed augmented and prolonged activation of host defense mechanisms, indicating that parasitised macrophages were exquisitely predisposed or "primed" to react to activating molecules such as polyphenols. This promotive effect may be of special benefit, e.g., stimulation of the non-specific immune system selectively at the site of infection and when needed. Although these data provide the basis for an immunological concept of plant polyphenols for their beneficial effects in various infectious conditions, in vivo experiments are essential to prove the therapeutic benefits of polyphenolic immunomodulators.     

Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin (EGC), (−) epicatechin-3-gallate (ECG), (−) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 μM) caused a significant decrease in cell viability to 4.1±0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 μM) also caused a slight decrease in cell viability (70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.     

Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1–10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.     

High purity tocotrienols attenuate atherosclerotic lesion formation in apoE-KO mice

Previous studies have demonstrated that tocotrienol (T3) has antiatherogenic effects. However, the T3 preparations used in those studies contained considerable amounts of tocopherol (Toc), which might affect the biological activity of T3. There is little information on the effect of highly purified T3 on atherosclerosis formation. This study investigated the effect of high-purity T3 on atherosclerotic lesion formation and the underlying mechanisms. Male apolipoprotein E knockout (apoE-KO) mice were fed a cholesterol-containing diet either alone or supplemented with T3 concentrate (Toc-free T3) or with α-Toc for 12 weeks. ApoE-KO mice fed the 0.2% T3-supplemented diet showed reduced atherosclerotic lesion formation in the aortic root. The 0.2% T3 diet induced Slc27a1 and Ldlr gene expression levels in the liver, whereas the α-Toc-supplemented diet did not affect those expression levels. T3 was predominantly deposited in fat tissue in the T3 diet-fed mice, whereas α-Toc was preferentially accumulated in liver in the α-Toc diet-fed mice. Considered together, these data demonstrate that dietary T3 exerts anti-atherosclerotic effect in apoE-KO mice. The characteristic tissue distribution and biological effects of T3, that are substantially different from those of Toc, may contribute to the antiatherogenic properties of T3.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.     

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-α induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with l-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-α and DAMC resulted in increased production of NO as compared to TNF-α alone. This increased NO production was attenuated by l-Nω-nitro-l-arginine methyl ester (l-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-α treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-α mediated release of IL-6 from peripheral blood mononuclear cells.     

The hidden face of food phenolic composition

Plant polyphenols are extremely diverse, due to the occurrence of several basic structures, numerous substitutions and, for some groups, of polymers (tannins). Plant polyphenol composition depends on the plant species and organ, with some molecules specific of particular plant families while others are ubiquitous. The polyphenol content is classically assessed by global analysis methods, which lack specificity and accuracy. These methods have been replaced with high performance liquid chromatography (HPLC), that enables accurate determination of individual molecules, provided they can be unambiguously identified and calibration curves can be established. However, HPLC analysis is restricted to simple compounds and difficult to apply in the case of complex extracts. Further difficulties encountered in the case of polymers include irreversible adsorption on the stationary phases. Proanthocyanidin analysis by HPLC after acid-catalysed depolymerisation in the presence of a nucleophile permits to overcome these problems and shows that proanthocyanidins predominate in the polyphenol composition of most plants. Large varietal differences in tannin quantitative and qualitative composition were observed for all plant species studied. Moreover, analysis is usually performed after extraction, which may lead to significant underestimation of the polyphenol content, since a large proportion is not extracted by usual solvents. This may be due to covalent binding to other plant constituents and to non-covalent adsorption on plant solids. Such matrix effect also influences the taste perception of polyphenols and their fate in the digestive tract, from in-mouth interactions with salivary proteins to their metabolism by colon microflora, with potential influence on bioavailability.