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Model of inhibition of Thermus aquaticus polymerase and Moloney murine leukemia virus reverse transcriptase by tea polyphenols (+)-catechin and (-)-epigallocatechin-3-gallate.

Non-nutritional polyphenolic compounds such as (+)-catechin and (-)-epigallocatechin-3-gallate (EGCG) are known as anticancer chemopreventive agents and have been utilised for medical purposes in form of tea drinking. Documented anticancer properties of these compounds result from their antioxidant effects. However, also direct alteration of an enzyme performance has been reported and deserves more attention. In this paper, a direct effect of catechin and EGCG on the performance of reverse transcription (RT) and/or polymerase chain reaction (PCR) was studied. Both tea polyphenolic compounds were added into real-time RT-PCR reactions and the fluorescence data obtained were fitted with a mathematical model. Several parameters of PCR performance were compared, obtained from the mathematical model for reactions with and without addition of (+)-catechin and EGCG. Addition of EGCG to enzyme reaction seems to inhibit the RT reaction (p < 0.05) and to slow down the DNA polymerase reaction (p < 0.001). Similarly, (+)-catechin inhibited the DNA amplification (p < 0.01) but had no effect on the RT reaction. The effects could be observed in physiological flavanol concentrations ranging from 10(-5) to 10(-8)M.

Animals↗

Potential polyphenol markers of phase change in apple (Malus domestica).

In order to identify potential biochemical markers that can be used as indicators for phase change, the dynamics of polyphenolic compounds across apple seedlings (Malus domestica, Jonathan x Golden Delicious) were analyzed in this study by high performance liquid chromatography. Precocious flowering was induced by foliar sprays of plant growth regulators. Qualitative changes in the concentration of polyphenols were observed at node nos. 50, 80 and 120. Spontaneous and induced flowering was found at node nos. 122 and 77. It was reasonable to conclude that node no. 77 represented the point of transition between the juvenile phase and the adult vegetative phase, which was marked by the presence of phloridzin in the buds. The disappearance of myricitrin in the bark and the absence of caffeic acid in the aboveground tissues were qualitative markers of the reproductive phase, which was reached at node no. 122.

Biomarkers↗

In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L.

Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods--DPPH assay, beta-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2-scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2-. The O2- scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection.

Animals↗

Cecal parameters of rats fed diets containing grapefruit polyphenols and inulin as single supplements or in a combination.

OBJECTIVE: We compared the effects of grapefruit flavonoids and inulin, as single dietary components or in a combination, on cecal fermentation in rats adapted to a semipurified diet. METHODS: The experimental diets contained 0.3% flavonoid extract and 5% or 10% inulin and a combination of both supplements. The large bowel metabolism assessment was based on cecal parameters: bulk effect, pH, microbial enzymes activity, and short-chain fatty acid production. RESULTS: Both supplements induced significant enlargement of the cecal digesta weight. Acidification of cecal digesta was more pronounced, with a higher inulin addition to the diet. Cecal pH was the highest with the flavonoid-rich diets and lowest in the case of a simultaneous addition of flavonoids and a high content of inulin. The flavonoid extract applied as a single dietary supplement was observed to decrease the activity of bacterial beta-glucosidase and beta- and alpha-galactosidases in the cecal digesta. In contrast, addition of the grapefruit extract to inulin-containing diets increased the activity of alpha-glucosidase, alpha-galactosidase, and beta-galactosidase. Great accumulation of cecal digesta in rats consuming the flavonoid-diet caused a considerable increase in the short-chain fatty acid pool, mainly acetic acid. Inulin added to the diet decreased the excessive enlargement of digesta caused by dietary flavonoids. Dietary addition of inulin to the flavonoid-diet also normalized hydration of cecal digesta and significantly decreased the pH of digesta. The presence of polyphenols in the inulin-containing diets did not change total short-chain fatty acid production in the cecum of rats. CONCLUSION: Our results suggested that simultaneous intake of inulin and polyphenols can decrease the detrimental effects of the latter on cecal fermentation.

Adaptation, Physiological↗

Effect of silymarin and its polyphenolic fraction on cholesterol absorption in rats.

This study evaluated the influence of silymarin (SM) and polyphenolic fraction (PF) of silymarin on cholesterol absorption in rats fed on high cholesterol diet (HCD). HCD induced a remarkable increase in hepatic, plasma, VLDL and LDL cholesterol, a decrease in HDL cholesterol and an elevation in triacylglycerol (TAG) levels in plasma, VLDL and in the liver. SM and PF were administered as dietary supplements (1.0%) in HCD for 18 days. Intestinal cholesterol absorption was measured by dual-isotope plasma ratio method, which calculates percent of cholesterol absorption from the ratio of two labelled cholesterol doses, one given intragastrically (14C) and one intravenously (3H). Silymarin and PF significantly reduced cholesterol absorption in rats fed on HCD and caused significant decreases in plasma and VLDL cholesterol and content of cholesterol and TAG in the liver. The level of HDL cholesterol was significantly increased after silymarin, but not after administration of PF. The levels of TAG in plasma and VLDL were not affected by either silymarin or PF. These results suggest that the inhibition of cholesterol absorption caused by silymarin and its polyphenolic fraction could be a mechanism contributing to the positive changes in plasma cholesterol lipoprotein profile and in lipid content in liver.

Animals↗

Effects of tannins and related polyphenols on methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) often acquires multi-drug resistance and is involved in many cases of disease in hospitals. We investigated natural substances directly effective against MRSA or that influence antibiotic resistance. Aloe-emodin, an anthraquinone, and several licorice flavonoids showed potent antibacterial effects against MRSA. Like some hydrolysable tannins (corilagin and tellimagrandin I) and a tea polyphenol [(-)-epicatechin gallate], the licorice flavonoid licoricidin also restored the effects of oxacillin, a beta-lactam antibiotic against MRSA. Further study revealed that theasinensin A, a polyphenol formed from (-)-epigallocatechin gallate, proanthocyanidins obtained from fruits of Zizyphus jujuba var. inermis, and polymeric proanthocyanidins from fruit peels of Zanthoxylum piperitum also suppressed the antibiotic resistance of MRSA.

Flavonoids↗

MALDI-TOF mass spectrometry of oligomeric food polyphenols.

The structural heterogeneity of polyphenols from cranberries, grape seed extracts, sorghum and pomegranate was characterized by MALDI-TOF MS. Polyphenolics were isolated by liquid chromatography and subjected to MALDI-TOF MS using trans-3-indoleacrylic acid as the matrix. Spectrometric analysis gave information on degree of polymerization, monomeric substitution, and the nature of intermolecular bonds. Cranberry polyflavan-3-ols had variation in interflavan bonds (A- and B-types) and contained polyflavan-3-ols linked to anthocyanins through a CH3-CH bridge. Polygalloyl-polyflavan-3-ols in grape seed extract had large variation in the degree of galloyl substitution. Sorghum polyflavans had structural heterogeneity in glycosylation and hydroxylation. Pomegranate hydrolyzable tannins that correspond to previously described structures were detected, such as punicalagin, but others that correspond to oligomeric ellgitannins in which two to five core glucose units are cross-linked by dehydrodigalloyl and or valoneoyl units were also observed. Results demonstrate that large heterogeneity occurs in degree of polymerization, intermolecular bonds, pattern of hydroxylation, and substitution with monosaccharides and gallic acid.

Flavonoids↗

Anticlastogenic effects of black tea polyphenols theaflavins and thearubigins in human lymphocytes in vitro.

Black tea accounts for nearly 80% of total World tea production. It contains dimeric flavanols and polymeric polyphenols known as theaflavins (TF) and thearubigins (TR). TR is exclusively present in black tea. On the basis of our previous potent antimutagenic and anticlastogenic effects of TF and TR in vitro in bacterial system and in vivo in mouse bone marrow cells, we have decided to extend our study in human cells in vitro. This study investigated the anticlastogenic effects of black tea polyphenols TF and TR as measured by chromosomal aberrations (CA) and micronuclei formation (MN) against two known mutagens/carcinogens i.e. benzo[a]pyerne (B[a]P) and aflatoxin B1(AFB1) with S9 activation. A significant decrease in both CA and MN were observed in the human lymphocyte cultures treated with either TF or TR pretreated with either B[a]P or AFB1 (250, 500, 1000 microg/ml) when compared with B[a]P or AFB1 treated cultures alone. TF shows more protective effects than TR in this in vitro system. These results indicate that both TF and TR have significant anticlastogenic effects in vitro in human lymphocytes.

Animals↗

Suppression of lipopolysaccharide-induced nuclear factor-kappaB activity by theaflavin-3,3'-digallate from black tea and other polyphenols through down-regulation of IkappaB kinase activity in macrophages.

We investigated the inhibition of IkappaB kinase (IKK) activity in lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 cell line) by various polyphenols including (-)-epigallocatechin-3-gallate, theaflavin, a mixture of theaflavin-3 gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate (TF-3), pyrocyanidin B-3, casuarinin, geraniin, and penta-O-galloyl-beta-D-glucose (5GG). TF-3 inhibited IKK activity in activated macrophages more strongly than did the other polyphenols. TF-3 strongly inhibited both IKK1 and IKK2 activity and prevented the degradation of IkappaBalpha and IkappaBbeta in activated macrophage cells. The results suggested that the inhibition of IKK activity by TF-3 could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. Furthermore, geraniin, 5GG, and TF-3 all blocked phosphorylation of IKB from the cytosolic fraction, inhibited nuclear factor-kappaB (NFkappaB) activity, and inhibited increases in inducible nitric oxide synthase levels in activated macrophages. These results suggest that TF-3 may exert its anti-inflammatory and cancer chemopreventive actions by suppressing the activation of NFkappaB through inhibition of IKK activity.

Animals↗

Differential effects of natural polyphenols on neuronal survival in primary cultured central neurons against glutamate- and glucose deprivation-induced neuronal death.

Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.

Animals↗

Automated sample preparation by pressurized liquid extraction-solid-phase extraction for the liquid chromatographic-mass spectrometric investigation of polyphenols in the brewing process.

The analysis of polyphenols from solid plant or food samples usually requires laborious sample preparation. The liquid extraction of these compounds from the sample is compromised by apolar matrix interferences, an excess of which has to be eliminated prior to subsequent purification and separation. Applying pressurized liquid extraction to the extraction of polyphenols from hops, the use of different solvents sequentially can partly overcome these problems. Initial extraction with pentane eliminates hydrophobic compounds like hop resins and oils and enables the straightforward automated on-line solid-phase extraction as part of an optimized LC-MS analysis.

Automation↗

Study of the polyphenol content of red and white grape varieties by liquid chromatography-mass spectrometry and its relationship to antioxidant power.

There is considerable interest in the analysis of phenolic compounds due to their involvement in the organoleptic characteristics of foods and drinks, and their contribution to protection against cardiovascular diseases and cancers through their antioxidant activity. This article describes studies of the polyphenol content, both total (by means of the Folin-Ciocalteu method) and individual (by means of HPLC, LC-MS and LC-MS-MS) and the antioxidant power, of six varieties of grape over the course of their maturation. A good correlation is found to exist between the total polyphenol content of the samples and their antioxidant power, and both seem diminish along the maturation.

Antioxidants↗

Calculation of the molar absorptivity of polyphenols by using liquid chromatography with diode array detection: the case of carnosic acid.

Antioxidant activity of vegetable extracts is related to the nature and the amount of active components, mainly polyphenols; therefore, a correct quantification of these molecules should be required to define their concentration in such kind of vegetable extracts. A fast and accurate method to calculate molar absorption coefficients (epsilon), by using HPLC, has been tested on standard polyphenols and caffeine, and should be widely adapted for standardless quantitative analysis. Molar absorptivity (epsilon) of carnosic acid (CA) was determined from 200 to 300 nm, by the proposed method and those values were compared to tert-butyl-hydroxytoluene (BHT) ones for further comparative quantification.

Abietanes↗

Rapid, direct determination of polyphenols in tea by reversed-phase column liquid chromatography.

Column liquid chromatography on a C18-bonded silica column with water-methanol-acetic acid as eluent was used to determine polyphenols and caffeine in tea. Without any pretreatment, catechin, epicatechin gallate, epigallocatechin gallate, epigallocatechin, epicatechin and caffeine were separated successfully within 15 min. The detection limits (S/N = 3) of polyphenols studied were 1.8-24 mg/l at a detection wavelength 270 nm. The linear range of the peak area calibration curves for the analytes were over two orders of magnitude with a correlation coefficient of 0.996-0.999. Using this method, some Chinese tea samples were analyzed with a good reproducibility (RSD are below 5%).

Caffeine↗

Determination of polyphenols by high-performance liquid chromatography with inhibited chemiluminescence detection.

A chemiluminescence reaction detector was developed for the detection of polyphenols separated by HPLC based on the inhibition of chemiluminescence from the luminol-potassium hexacyanoferrate(III) reaction by polyphenols. The separation was carried out on a RP-C18 column at 37 degrees C by using stepwise gradient elutions. The detection limits are in the range of 6.8 x 10(-7)-2.0 x 10(-9) g/ml for catechol, protocatechuic acid, chlorogenic acid, rutin, resorcinol, hydroquinone and p-tert.butylpyrocatechol. The method is sensitive, selective, fast and simple. It has been successfully applied to the determination of chlorogenic acid and rutin in real tobacco samples.

Chromatography, High Pressure Liquid↗

Study of polyphenols in grape berries by reversed-phase high-performance liquid chromatography.

Several polyphenols have been tested in grape berries from Spain. The flavonoid content is important because of the pharmacological properties of these compounds, whereas resveratrol has been proved to be an antifungal, antiinflammatory and an anticarcinogenic compound. A reversed-phase HPLC method has been developed and applied to determine resveratrol, quercetine, quercitrine and rutine content in several grape berries samples in a single analysis. Covering the grapes with a preservative paper yields a healthier product, but one which has a lower polyphenol content than unprotected grapes.

Chromatography, High Pressure Liquid↗

Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin, the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages.

epsilon-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 microM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of epsilon-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 microM vs. 10 to 100 microM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids.

Alcoholic Beverages↗

Dietary supplementation of grape polyphenols and chronic ethanol administration on LDL oxidation and platelet function in rats.

Polyphenolic compounds have been implicated as the active ingredients for the cardiac protective effect in red wine. We tested the effects of dietary supplementation of polyphenols from grape (GP) and chronic ethanol administration on low-density-lipoprotein (LDL) oxidation and platelet function in rats. Four groups of young male Sprague-Dawley rats were fed the following diets for 2 months: (I) a high fat Lieber-DeCarli liquid diet with an isocaloric amount of maltose, (II) with 5% ethanol (w/v), (III) with 5 mg/dL of GP, and (IV) ethanol plus GP. Platelet aggregation was induced by thrombin and phorbol myristate acetate (PMA) and LDL oxidation was induced by Cu2+. Chronic ethanol administration resulted in a significant increase in LDL oxidation and this effect was partially protected by supplementation with GP. Although platelet number was not affected by either ethanol or GP administration, platelet aggregation induced by thrombin was reduced in ethanol, GP and ethanol plus GP groups as compared to controls. On the other hand, platelet aggregation induced by PMA was not altered in any groups, suggesting that protein kinase C was not a causal factor for the reduction of aggregatory response induced by thrombin. These results show similar effects of ethanol and GP on platelet aggregation but different effects on LDL oxidation. It can be concluded that dietary supplementation with GP may exert partial protection on oxidative insults such as those elicited by chronic ethanol ingestion.

Animals↗