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Electrophoretic identification and quantitation of compounds in the polyphenolic fraction of extra-virgin olive oil.

A capillary zone electrophoresis method has been carried out to determine and quantitate some compounds of the polyphenolic fraction of virgin olive oil which have never previously been determined before using capillary electrophoresis, such as elenolic acid, ligstroside aglycon, oleuropein aglycon, and (+)-pinoresinol. The compounds were identified using standards obtained by semipreparative high-performance liquid chromatography (HPLC). A detailed method optimization was performed to separate the phenolic compounds present in olive oil using a methanol-water extract of Picual extra-virgin olive oil, and different extraction systems were compared (C18-solid phase extraction (SPE), Diol-SPE, Sax-SPE and liquid-liquid extraction). The optimized parameters were 30 mM sodium tetraborate buffer (pH 9.3) at 25 kV with 8 s hydrodynamic injection, and the quantitation was carried out by the use of two reference compounds at two different wavelengths.

Chromatography, High Pressure Liquid↗

Antimutagenicity of green tea polyphenols in the liver of transgenic medaka.

We examined the ability of a mixture of the predominant green tea polyphenolic compounds (GTP) to reduce benzo[a]pyrene (B[a]P)-induced mutations in the cII gene of the lambda transgenic medaka. Fish were treated with 50 ppb B[a]P for 24 hr, followed by exposure to 2 ppm or 10 ppm GTP for 28 days. cII mutations in livers of fish exposed to B[a]P were increased significantly, 2.6-fold above controls. In contrast, the addition of GTP significantly reduced the frequency of cII mutants by 84%, comparable to that of controls. The frequencies of mutations at G:C basepairs, mutations that are highly characteristic of B[a]P exposure, were elevated significantly in treated fish. By comparison, B[a]P-exposed fish also treated with GTP showed reductions in these mutations, demonstrating a protective effect of GTP against B[a]P-induced mutagenesis. The antioxidant mechanism of GTP possibly played an important role in the reduction of B[a]P mutagenicity. These results corroborate findings from rodent models, showing that the protective effects of green tea extend to different species, and suggesting that similar mechanisms of B[a]P mutagenesis and GTP antimutagenesis are shared among the models. These studies illustrate the utility of lambda transgenic medaka for in vivo mutation analyses and suggest that this fish may be a valuable model in chemoprevention studies.

Animals↗

Differential oxidative stress in oligodendrocytes and neurons after excitotoxic insults and protection by natural polyphenols.

Oligodendrocytes are vulnerable to overactivation of both their AMPA receptors and their high- and low-affinity kainate receptors. Depending on the intensity of the insult and the type of receptor activated, excitotoxic oligodendrocyte death mediated by these receptors has different characteristics. One important consequence at a cellular level is the ensuing oxidative stress, related to Ca2+-dependent alterations in mitochondrial functioning. We observed that oxidative stress associated with selective AMPA receptor activation is much higher than that associated with the selective activation of high- and low-affinity kainate receptors. Moreover, excitotoxic insults generate more intense oxidative stress in oligodendrocytes than in cortical neurons, though similar alterations in [Ca2+]i and mitochondrial potential were observed in both cell types. Nanomolar concentrations of mangiferin and morin, two natural polyphenols with antioxidant properties, partially protect oligodendrocytes as well as cortical neurons from mild, but not intense, insults mediated by AMPA receptors. In addition to presenting oxygen radical scavenging activity, mangiferin and morin attenuate the intracellular Ca2+ overload subsequent to the activation of AMPA receptors, a mechanism that may contribute to their protective properties. The inclusion of these antioxidant agents in therapeutic strategies for the treatment of diseases in which oligodendrocyte as well as neuron loss occurs may prove to be beneficial.

Animals↗

Green tea polyphenol stimulates cancer preventive effects of celecoxib in human lung cancer cells by upregulation of GADD153 gene.

To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (-)-epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase-2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage-inducible 153 (GADD153) gene, in human lung cancer cell line PC-9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC-9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21(WAF1) and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC-9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K-1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC-9 cells in combination with N-(4-hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC-9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG.

Anticarcinogenic Agents↗

Evaluation of alginate purification methods: effect on polyphenol, endotoxin, and protein contamination.

Alginate, a polysaccharide extracted from brown seaweed, is widely used for the microencapsulation of islets of Langerhans, allowing their transplantation without immunosuppression. This natural polymer is known to be largely contaminated. The implantation of islets encapsulated using unpurified alginate leads to the development of fibrotic cell overgrowth around the microcapsules and normalization of the blood glucose is restricted to a very short period if it is achieved at all. Several research groups have developed their own purification method and obtained relatively good results. No comparative evaluation of the efficiencies of these methods has been published. We conducted an evaluative study of five different alginate preparations: a pharmaceutical-grade alginate in its raw state, the same alginate after purification according to three different published methods, and a commercially available purified alginate. The results showed that all purification methods reduced the amounts of known contaminants, that is, polyphenols, endotoxins, and proteins, although with varying efficiencies. Increased viscosity of alginate solutions was observed after purification of the alginates. Despite a general efficiency in decreasing contamination levels, all of the purified alginates contained relatively high residual amounts of protein contaminants. Because proteins may be immunogenic, these residual proteins may have a role in persisting microcapsule immunogenicity.

Alginates↗

Protective effect of green tea polyphenol EGCG against neuronal damage and brain edema after unilateral cerebral ischemia in gerbils.

Previous studies have demonstrated that a green tea polyphenol, (-)-epigallocatechine gallate (EGCG), has a potent free radical scavenging and antioxidant effect. Glutamate leads to excitotoxicity and oxidative stress, which are important pathophysiologic responses to cerebral ischemia resulting in brain edema and neuronal damage. We investigated the effect of EGCG on excitotoxic neuronal damage in a culture system and the effect on brain edema formation and lesion after unilateral cerebral ischemia in gerbils. In vitro, excitotoxicity was induced by 24-hr incubation with N-methyl-D-aspartate (NMDA; 10 microM), AMPA (10 microM), or kainate (20 microM). EGCG (5 microM) was added to the culture media alone or with excitotoxins. We examined malondialdehyde (MDA) level and neuronal viability to evaluate the effect of EGCG. In vivo, unilateral cerebral ischemia was induced by occlusion of the right common carotid artery for 30, 60, or 90 min and followed by reperfusion of 24 hr. Brain edema, MDA, and infarction were examined to evaluate the protective effect of EGCG. EGCG (25 or 50 mg/kg, intraperitoneally) was administered twice, at 30 min before and immediately after ischemia. EGCG reduced excitotoxin-induced MDA production and neuronal damage in the culture system. In the in vivo study, treatment of gerbils with the lower EGCG dose failed to show neuroprotective effects; however, the higher EGCG dose attenuated the increase in MDA level caused by cerebral ischemia. EGCG also reduced the formation of postischemic brain edema and infarct volume. These results demonstrate EGCG may have future possibilities as a neuroprotective agent against excitotoxicity-related neurologic disorders such as brain ischemia.

Animals↗

Analytical determination of polyphenols in olive oils.

The increasing popularity of olive oil is mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of human disease. An overview of analytical methods for the measurement of polyphenols in olive oil is presented. In principle, the analytical procedure for the determination of individual phenolic compounds in virgin olive oil involves three basic steps: extraction from the oil sample, analytical separation, and quantification. A great number of procedures for the isolation of the polar phenolic fraction of virgin olive oil, utilizing two basic extraction techniques, LLE or SPE, have been included. The reviewed techniques are those based on spectrophotometric methods, as well as analytical separation (gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE)). Many reports in the literature determine the total amount of phenolic compounds in olive oils by spectrophometric analysis and characterize their phenolic patterns by capillary gas chromatography (CGC) and, mainly, by reverse phase high-performance liquid chromatography (RP-HPLC); however, CE has recently been applied to the analysis of phenolic compound of olive oil and has opened up great expectations, especially because of the higher resolution, reduced sample volume, and analysis duration. CE might represent a good compromise between analysis time and satisfactory characterization for some classes of phenolic compounds of virgin olive oils.

Antioxidants↗

Characterization of polyphenols from plant materials through their silylation and 29Si NMR spectroscopy-line assignment through 29Si, 13C spin-spin couplings.

The lines in (29)Si NMR spectra of silylated polyphenols and some other compounds are difficult to assign owing to the absence of couplings with protons outside the silyl group. The assignment can be derived through small (n)J((29)Si, (13)C) couplings (n > 1). Using a previously described method for measurements of these couplings, the assignment procedure is demonstrated here on three examples of trimethylsilylated phenols: 7-hydroxyflavone, ferulic acid, and quercetin. In some cases the procedure can be used to identify carbon atoms to which the siloxy groups are attached.

Carbon Isotopes↗

Docking studies and model development of tea polyphenol proteasome inhibitors: applications to rational drug design.

Previously, we demonstrated that natural and synthetic ester bond-containing green tea polyphenols were potent and specific non-peptide proteasome inhibitors. However, the molecular mechanism of inhibition is currently unknown. Here, we report that inhibition of the chymotrypsin activity of the 20S proteasome by (-)-epigallocatechin-3-gallate (EGCG) is time-dependent and irreversible, implicating acylation of the beta5-subunit's catalytic N-terminal threonine (Thr 1). This knowledge is used, along with in silico docking experiments, to aid in the understanding of binding and inhibition. On the basis of these docking experiments, we propose that (-)-EGCG binds the chymotrypsin site in an orientation and conformation that is suitable for a nucleophilic attack by Thr 1. Consistently, the distance from the electrophilic carbonyl carbon of (-)-EGCG to the hydroxyl group of Thr 1 was measured as 3.18 A. Furthermore, the A ring of (-)-EGCG acts as a tyrosine mimic, binding to the hydrophobic S1 pocket of the beta5-subunit. In the process, the (-)-EGCG scissile bond may become strained, which could lower the activation energy for attack by the hydroxyl group of Thr 1. This model is validated by comparison of predicted and actual activities of several EGCG analogs, either naturally occurring, previously synthesized, or rationally synthesized.

Amides↗

Polyphenols of Melaleuca quinquenervia leaves--pharmacological studies of grandinin.

Four polyphenolic acid derivatives and three ellagitannins were isolated from the leaves of Melaleuca quinquenervia (Clav.) S. T. Blake for the first time. Their structures were elucidated as gallic acid (1), ellagic acid (2), 3-O-methylellagic acid (3), 3,4,3'-tri-O-methylellagic acid (4), 2,3-O-hexahydroxydiphenoyl-(alpha/beta)-D-(4)C(1)-glucopyranose (5), castalin (6) and grandinin (7) on the basis of chemical, mass spectrometric (-ve ESI-MS) and spectroscopic (UV, (1)H-, (13)C NMR, (1)H,(1)H-, (1)H,(13)C-COSY, (1)H,(1)H-TOCSY and HMBC) analyses. Grandinin (the major compound) showed radical scavenging properties by its reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical [EC(50) = 4.3 +/- 0.3 micro g mL(-1)]. It was found to be relatively nontoxic in mice [LD(50) = 316 mg Kg(-1) b.wt.]. It exhibited a significant dose-dependent (35-70 mg Kg(-1)) hypoglycemic effect by significantly reducing blood glucose level in basal condition and after heavy glucose load in normal mice. Moreover, it reduced the elevated blood glucose level in STZ-induced diabetic mice. In addition, grandinin reduced the elevated blood urea nitrogen and serum lipid peroxides in STZ-induced diabetic mice.

Animals↗

Effect of green tea polyphenols on the genes with atherosclerotic potential.

The genomics of atherosclerosis can arise as a result of cross-talk between the genes coding for the LDL-receptor (LDL-R), LXR-alpha, PPARs (alpha, gamma), CD36 and C-myc because these genes control lipid metabolism, cytokine production and cellular activity within the arterial wall. The effect of green tea polyphenols (GTPs) upon such genomics revealed their ability to down-regulate genes coding for PPAR-gamma, CD36, LXR-alpha, C-myc coupled with up-regulation of genes coding for LDL-R and PPAR-alpha at the transcriptional level. Based upon these results, it is proposed that GTPs have the inherent capacity to inhibit the development of atherosclerotic lesions.

Arteriosclerosis↗

Immunostimulating effects of the polyphenol-rich fraction of sugar cane (Saccharum officinarum L.) extract in chickens.

The phagocytic activity of peripheral blood leukocytes (PBL) in chickens orally administered sugar cane extracts (SCE) or polyphenol-rich fraction (PRF) of SCE (500 mg/kg/day) for 3 consecutive days increased significantly, when compared with that of saline-administered control chickens. Chickens orally administered SCE or PRF (500 mg/kg/day) for 3 consecutive days showed significantly higher antibody responses against sheep red blood cells and Brucella abortus than control chickens. In addition, oral administration of SCE or PRF also resulted in a significant increase in the number of IgM- and IgG-plaque forming cell responses of PBL, intestinal leukocytes and splenocytes, when compared with those of control chickens. Furthermore, delayed type hypersensitivity responses to human gamma globulin significantly increased in chickens orally administered SCE or PRF, compared with those of control chickens when evaluated on the basis of net increased wattle thickness at 24, 48 and 72 h after challenge. These results suggest that PRF of SCE has an immunostimulating effect in chickens.

Adjuvants, Immunologic↗

Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens).

Peroxynitrite (ONOO-) has been proposed as a mediator of gut inflammation and as an inducer of cell death by apoptosis. Phytolens (PHY), a water-soluble extract of polyphenolic antioxidants from nonsoy legumes (Biotics Research Corp, patent pending), was evaluated as a cytoprotective agent in human colonic (T84) and murine macrophage (RAW 264.7) cell lines. In the antioxidant testing, PHY showed a significant free radical scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide (O2.) radicals with an IC50 of 4.44 and 5.87 microg/ml against DPPH and O2., respectively. Apoptosis (DNA fragmentation) was measured by an ELISA technique. Cells were exposed to oxidative stress by treating them with peroxynitrite (100-300 microM) for 4 h in the presence and absence of PHY. Peroxynitrite elicited a dose-dependent increase in DNA fragmentation in both cell lines compared to the control group receiving decomposed ONOO-. PHY (10, 30, or 50 microg/ml) significantly attenuated the degree of apoptosis in T84 cells induced by ONOO- (P < 0.05). PHY (10-100 microg/ml) did not directly affect T84 cell viability or induce apoptosis after 4 h or overnight exposure. RAW 264.7 cells exposed to PHY alone (>30 microg/ml) for 4 h displayed decreased cell viability (P < 0.05) and increased apoptosis (P < 0.05). Phytolens may have beneficial effects on inflammation by attenuating peroxynitrite-induced apoptosis. The sparing of epithelial cells while compromising the viability of macrophages suggests that PHY may be beneficial in autoimmune disorders.

Animals↗

Plant polyphenolic complex inhibits the reproduction of influenza and herpes simplex viruses.

A polyphenolic complex (PC), isolated from the Bulgarian medicinal plant Germanium sanguineum L., inhibited the reproduction of influenza virus types A and B in vitro and in ovo. It protected white mice in an experimental lethal influenza infection. When influenza viruses were treated with a high dose (1 mg/mL), their hemagglutination, neuraminidase and infections activities were reduced completely. It was assumed that the PC-specific inhibitory effect on influenza virus reproduction in tissue cultures (50 micrograms/mL) was mediated by partial inactivation of viral glycoproteins which prevented the initial stages of reproduction. The PC also affected the intracellular stages of viral replication. The effect was dependent on the mode of application, the viral strain, and the biological test system. The PC also inhibited the reproduction of herpes simplex virus in vitro. Infectious titers were reduced and newly synthesized virion with damaged protein envelopes were observed by electron microscopy.

Animals↗

Nutritional consequences of the reactions between proteins and oxidized polyphenolic acids.

The chemical and enzymatic browning reactions of plant polyphenols and their effects on amino acids and proteins are reviewed. A model system of casein and oxidizing caffeic acid has been studied in more detail. The effects of pH, time, caffeic acid level and the presence or not of tyrosinase on the decrease of FDNB-reactive lysine are described. The chemical loss of lysine, methionine and tryptophan and the change in the bioavailability of these amino acids to rats has been evaluated in two systems: pH 7.0 with tyrosinase and pH 10.0 without tyrosinase. At pH 10.0, reactive lysine was more reduced. At pH 7.0 plus tyrosinase methionine was more extensively oxidized to its sulphoxide. Tryptophan was not chemically reduced under either condition. At pH 10.0 there was a decrease in the protein digestibility which was responsible for a corresponding reduction in tryptophan availability and partly responsible for lower methionine availability. Metabolic transit of casein labelled with tritiated lysine treated under the same conditions indicated that the lower lysine availability in rats was due to a lower digestibility of the lysine-caffeoquinone complexes.

Amino Acids↗

Automated determination of total polyphenols in apple juice.

An automated method of total polyphenols analysis in apple juice using the Folin-Ciocalteu reagent is presented. Chlorogenic acid was selected as the standard since a similar kinetic behaviour, standard-sample, was observed. A straightforward method with good reproducibility (CV < 1%), sampling frequency (48 h-1) and correlation with the traditional batch method (r = 0.9924) is proposed.

Beverages↗

Interactive effects of polyphenols, tocopherol and ascorbic acid on the Cu2+-mediated oxidative modification of human low density lipoproteins.

BACKGROUND: Only limited knowledge is available about any interactions between phenolic compounds and other antioxidants in inhibiting LDL oxidation. Many foods and beverages contain high levels of phenolic compounds; therefore, these compounds should not be considered in isolation from each other. AIM OF THE STUDY: The aim of this study was to examine the structure-antioxidant activity relationship of quercetin, caffeic acid, epicatechin, hesperetin and phloretin as well as alpha-tocopherol and ascorbic acid through their ability to interact with copper ions. METHODS: Isolated human LDL were incubated with single antioxidants or a combination of two and the kinetics of lipid peroxidation were assessed by measurement of conjugated diene formation (lag phase) via monitoring the absorbance at 234 nm after addition of copper ions. In addition, the degree of oxidation of the LDL protein moiety was followed by tryptophan fluorescence and carbonyl content measurements. RESULTS: Alpha-tocopherol and ascorbic acid showed a lower antioxidant activity in all test systems as compared to polyphenols at equimolar concentrations. Quercetin was the most effective compound in all three systems (p < 0.001 for lag phase and carbonyl content determination). A significant (p < 0.001) prolongation of the lag phase was found when combinations of ascorbic acid/quercetin, ascorbic acid/epicatechin, epicatechin/caffeic acid, and quercetin/epicatechin were tested as compared to the sum of the individual effects. Concerning the effects on LDL protein oxidation, the results from carbonyl content and the tryptophan fluorescence measurements showed that the combination of quercetin and caffeic acid revealed the strongest inhibitory effect (p < 0.001 carbonyl content; p < or = 0.002 tryptohan fluorescence) on protein oxidation which was higher than the effect of the single compounds. CONCLUSIONS: The results of the present study indicate that a combination of different antioxidants can be superior to the action of single antioxidants in protecting LDL lipid and protein moiety against oxidation. However, the substances may act by different antioxidative mechanisms, which are not necessarily complementary.

Antioxidants↗

A strawberry fruit-specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring.

A strawberry (Fragaria x ananassa cv. Chandler) fruit cDNA (Fahyprp -cDNA) and its corresponding gene (Fahyprp) showing sequence homology to higher plant hyprp genes have been isolated. The cDNA contains an open reading frame encoding a 16 kDa protein with 156 amino acids. The peptide has an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxy-terminal region homologous to other HyPRP proteins. Northern blot and QRT-PCR analysis have shown that the strawberry transcript is specifically expressed in fruit, not being detected in other plant tissues. " In situ " hybridization and immunolocalization studies have indicated that the Fahyprp gene is strongly expressed in achene sclerenchyma cells, in the vascular and receptacle cells of immature green fruit and in the vascular cells of mature red fruits. The achenes removal from unripe green fruits induced the expression of this Fahyprp gene. This induction was reverted by treatment of deachened fruit with the auxin NAA, supporting the idea that Fahyprp gene expression is regulated by auxins. Furthermore, the HyPRP protein has been localized in parenchymatic cells of immature fruits associated to structures containing condensed tannins. The results are discussed supporting a putative role of this protein in the anchoring of polymeric polyphenols in the strawberry fruit during growth and ripening.

5' Flanking Region↗