Proteomics analysis of rat brain protein modulations by grape seed extract

Dietary supplements such as grape seed extract (GSE) enriched in proanthocyanidins (PA) (oligomeric polyphenols) have been suggested to have multiple health benefits, due to antioxidant and other beneficial activities of the PA. However, a systematic analysis of the molecular basis of these benefits has not been demonstrated. Because the brain is vulnerable to age-related oxidative damage and other insults including inflammation, it was hypothesized that rats ingesting GSE would experience changes in expression or modifications of specific brain proteins that might protect against pathologic events. Normal adult female rats were fed diets supplemented with 5% GSE for 6 weeks. Proteomics analysis (2D electrophoresis and mass spectrometry) of brain homogenates from these animals identified 13 proteins that were altered in amount and/or charge. Because many of these changes were quantitatively in the opposite direction from previous findings for the same proteins in either Alzheimer disease or mouse models of neurodegeneration, the data suggest that these identified proteins may mediate the neuroprotective actions of GSE. This is the first identification and quantitation of specific proteins in mammalian tissues modulated by a dietary supplement, as well as the first to demonstrate links of such proteins with any disease.     

90-day oral toxicity study of a grape seed extract (IH636) in rats

To assess the safety of grape seed extract with less than 5.5% catechin monomers (IH636), 4 groups of male and female Sprague-Dawley rats were provided grape seed extract in the diet at levels of 0 (control), 0.5, 1.0, or 2.0% for a period of 90 days. All animals survived the duration of the study, and no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings were observed. A significant increase in food consumption was observed in male and female rats provided the grape seed extract diets compared to that of the control rats, especially in male rats consuming 2.0% grape seed extract. This effect was not accompanied by increases in body weight gains. Grape seed extract appeared to increase the insoluble fraction of the diet. Male rats in the high-dose group exhibited decreased serum iron levels and decreased serum iron/total iron binding capacity ratio compared to those of the controls, although all values were within historical ranges for Sprague-Dawley rats. In conclusion, administration of the grape seed extract IH636 to male and female Sprague-Dawley rats in the feed at levels of 0.5, 1.0, or 2.0% for 90 days did not induce any significant toxicological effects.     

Antioxidant activity of a proanthocyanidin-rich extract from grape seed in whey protein isolate stabilized algae oil-in-water emulsions

Algae oil-in-water emulsions stabilized with 0.2% whey protein isolate (WPI) at pH 3.0 and 7.0 were chosen to evaluate antioxidant activity of a proanthocyanidin-rich extract from grape seed. In this emulsion system, (+)-catechin and ascorbic acid (620 microM) were found to be prooxidative at pH 3.0 and ineffective at pH 7.0. Grape seed extract was not able to effectively inhibit both lipid hydroperoxides and propanal formation when added to the emulsion at 124 microM. However, increasing the concentration of the grape seed extract to 620 microM resulted in inhibition of both lipid hydroperoxide and propanal formation at pH 3.0 and 7.0. None of the antioxidants tested had any effect on the physical stability of the WPI-stabilized emulsion. The superior antioxidant activity of the grape seed extract is likely due to the presence of oligomeric procyanidins which are better antioxidants compared to their monomeric counterparts.     

Antigenotoxic effect of grape seed procyanidin extract in Fao cells submitted to oxidative stress

The protective effects of grape seed procyanidin extract on the repair of H(2)O(2)-induced DNA lesions were tested using Fao cells. Cells were exposed to 600 microM H(2)O(2) for 3 or 21 h. A procyanidin extract from grape seed (PE) was incubated or preincubated (1 h) during the exposure to H(2)O(2). The ability of procyanidins to protect against the genotoxicity of H(2)O(2) was compared with those of the monomeric flavanols (+)-catechin and (-)-epicatechin and the flavonol quercetin. After treatment, DNA damage was monitored using alkaline single-cell gel electrophoresis (the comet assay) (Aherne, S. A.; O'Brien, N. M. Nutr. Cancer 1999, 34, 160-166). At the end of the experiment, PE significantly decreased the damage caused by H(2)O(2). The results also showed that quercetin was the most effective of the flavonoids tested, which is consistent with its powerful antioxidant character. The results indicate that procyanidins are more effective than the corresponding individual monomers, catechin and epicatechin, at preventing DNA lesions in hepatocytes and that this protection is higher after preincubation than after co-incubation.     

Endogenous formation of N'-nitrosonornicotine in F344 rats in the presence of some antioxidants and grape seed extract

N'-Nitrosonornicotine (NNN) is one of the most abundant strong carcinogens in unburned tobacco and cigarette smoke and is classified by the International Agency for Research on Cancer as carcinogenic to humans. Human exposure to NNN mainly occurs upon use of tobacco products. It is also possible that additional amounts of NNN are formed endogenously. The goal of this study was to evaluate the inhibitory effect of some antioxidants, including ascorbic acid and grape seed extract (GSE), on endogenous NNN formation in rats treated with nornicotine and sodium nitrite by gavage twice daily for 3 days. The study included four groups of rats: (1) negative control group A, to which no chemical was administered; (2) negative control group B, treated with nornicotine alone (2.5 micromol per gavage); (3) positive control group, to which both nornicotine (2.5 micromol per gavage) and sodium nitrite (7.5 micromol per gavage) were administered; and (4) rats treated with nornicotine (2.5 micromol per gavage), inhibitor (7.5 or 37.5 micromol per gavage), and sodium nitrite (7.5 micromol per gavage). The mean (+/-SD) total amount of NNN in the 3-day urine of rats treated with both nornicotine and sodium nitrite was 4.78 +/- 2.88 nmol. The order of inhibition of endogenous NNN formation in rats at the molar ratio [nitrite]:[inhibitor] 1:5 was as follows: ascorbic acid (91%) > dihydroxyfumaric acid (86%) approximately catechin (85%) > resveratrol (no inhibition). Treatment of rats with grape seed extract did not produce statistically significant inhibition of endogenous nornicotine nitrosation. This is the first study that demonstrates endogenous NNN formation in rats treated with nornicotine and sodium nitrite and effective inhibition of this process by ascorbic acid, dixydroxyfumaric acid, and catechin.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of polygalloyl polyflavan-3-ols in grape seed extract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to characterize the polygalloyl polyflavan-3-ols (PGPF) in grape seed extracts. Masses corresponding to a series of PGPF units inclusive of nonamers were observed in the positive-ion reflectron mode. Masses of PGPF inclusive of undecamers were observed in the positive-ion linear mode, providing the first known evidence of PGPF of this size. Soluble PGPF of grape seed extracts were precipitated by complexation with Yb(3+). The PGPF were then recovered by dissolving the precipitate in water and removing the Yb(3+) by a weakly acidic cation-exchange resin (Amberlite IRP-64). Comparisons of HPLC chromatograms of the crude grape seed extract prior to precipitation with Yb(3+) and after recovery of the PGPF indicated that 96% of the phenolic compounds were precipitated and 99% of the precipitated PGPF were recovered by cation-exchange resin. These results indicate that MALDI-TOF MS is able to determine the mass distribution of complex mixtures of oligomeric PGPF and that precipitation of PGPF by Yb(3+) is useful for isolation and quantification.     

葡萄种籽中原花色素提取方法的研究

以赤霞珠种籽为试材,研究了不同溶剂、不同浓度、不同时间、不同次数、不同温度对原花色素的提取效率影响.结果表明,丙酮/水(7/3,V/V)为最佳提取液,25℃下,提取6h,重复提取2次,原花色素产率最佳.     

Metabolism of grape seed polyphenol in the rat

The metabolism of grape seed polyphenol (GSP) has been investigated in rats by high-performance liquid chromatography analysis of the serum and urinary concentrations of the GSP metabolites (+)-catechin (CT), (-)-epicatechin (EC), 3'-O-methyl-(+)-catechin, and 3'-O-methyl-(-)-epicatechin. The serum concentration of these four metabolites reached a maximum 3 h after the oral administration of GSP. The urinary excretion of these GSP metabolites accounted for 0.254% (w/w) of the administered dose of GSP (1.0 g/kg), and the majority of these metabolites were excreted within 25 h of oral administration. The serum concentration and urinary excretion of these metabolites were also compared after the oral administration of different GSP monomers (gallic acid, CT, and EC), normal GSP, and the high molecular weight components of GSP (GSPH). No metabolites were detected in the serum of rats given GSPH. The urinary percentage excretion of the GSP metabolites derived from the respective monomers (CT or EC) did not vary with the administration of different substances (CT or EC, GSP, or GSPH). Taken together, these results suggest that only the monomers of GSP are absorbed and metabolized.     

Rapid fractionation of grape seed proanthocyanidins.

A rapid method that permits separation of grape seed proanthocyanidins according to their polymerization degrees has been developed. This method was based on liquid/liquid extraction and relative solubility of these compounds in different solvents (water, ethyle acetate, methanol, and chloroform). The different fractions obtained were then analyzed by various HPLC techniques (normal phase, reversed phase after thiolysis, and gel permeation) to determine their mean degree of polymerization (DP) and molecular weight profiles. Results show that this method can be used on a preparative scale to separate these polymers according to their sizes.     

Pre- and post-mortem use of grape seed extract in dark poultry meat to inhibit development of thiobarbituric acid reactive substances

Diets containing grape seed extract (GSE)-control, GSE [low GSE, low GSE + methionine, high GSE, and high GSE + methionine], or alpha-tocopherol-were fed to broiler chicks to estimate the antioxidative activity of GSE in processed meat. GSE was detrimental to the growth of chicks, and methionine did not reverse the detrimental effect. GSE with 85.4 g of gallic acid equiv/100 g (GAE 85.4) was added to ground dark turkey meat to obtain treatments with no GSE, 1.0% GSE, and 2.0% GSE and then processed as unsalted or salted and unheated or heated. Processed treatments were analyzed for thiobarbituric acid reactive substances (TBARS) and percent expressible moisture (%EM). GSE at 1.0 and 2.0% decreased TBARS values nearly 10-fold as compared to the control. GSE (1.0%) had a %EM value significantly greater than that of the control. GAE 85.4 decreased TBARS values more than GAE 88.9.     

Kinetics and thermodynamics of the thermal inactivation of polyphenol oxidase in an aqueous extract from Agaricus bisporus

The kinetics and thermodynamics of the thermal inactivation of polyphenol oxidase (PPO) in an aqueous extract from mushroom Agaricus bisporus (J.E. Lange) Imbach was studied, using pyrocatechol as a substrate. Optimal conditions for enzymatic studies were determined to be pH 7.0 and 35-40 °C. The kinetics of PPO-catalyzed oxidation of pyrocatechol followed the Haldane model with an optimum substrate concentration of 20 mM. Thermal inactivation of PPO was examined in more detail between 50 and 73 °C and in relation to exposure time. Obtained monophasic kinetics were adequately described by a first-order model, with significant inactivation occurring with increasing temperature (less than 10% preserved activity after 6 min at 65 °C). Arrhenius plot determination and calculated thermodynamic parameters suggest that the PPO in aqueous extract from Agaricus bisporus mushroom is a structurally robust yet temperature-sensitive biocatalyst whose inactivation process is mainly entropy-driven.     

Interactions of grape seed tannins with salivary proteins

To evaluate the amount and type of condensed tannins binding salivary proteins, which are supposed to be involved in astringent sensation, model systems allowing further analyses of proteins and condensed tannins were developed. The precipitates formed after addition of grape seed tannins to salivary proteins indicate that a binding interaction occurs. Dissociation of insoluble complexes was achieved by sodium dodecyl sulfate treatment. Thiolysis reaction allowed the quantification and characterization of proanthocyanidins on both the resulting pellet and the supernatant. Binding proteins were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The higher polymerized tannins predominantly precipitated together with the salivary proteins. The condensed tannins remaining in solution were low molecular weight polymers.     

Immunomodulatory and antitumor activities of grape seed proanthocyanidins

Proanthocyanidins are naturally occurring compounds that are widely available in many kinds of plants; particularly, the grape seeds are a rich source of proanthocyanidins. Grape seed proanthocyanidins (GSPs) have been demonstrated to possess a wide range of health beneficial properties. This study was carried out to elucidate the molecular mechanisms involved in the antitumor therapeutic and immunomodulating effects of GSPs through in vivo and in vitro models. The results showed that GSPs could significantly inhibit the growth of Sarcoma 180 tumor cells in vivo and remarkably increase thymus and spleen weight of Sarcoma 180-bearing mice and upgrade the secretion level of tumor necrosis factor-α (TNF-α) in serum. Moreover, GSPs could stimulate lymphocyte transformation, enhance lysosomal enzyme activity and phagocytic capability of peritoneal macrophages, and remarkably promote the production of TNF-α. These results suggested that GSPs could improve functional activation of the immune system, and the antitumor effects of GSPs were achieved by immunostimulating properties.     

Contamination of grape seed oil with mineral oil paraffins

The contamination of 11 commercial grape seed oils with paraffins of mineral oil origin was analyzed by online-coupled HPLC-HPLC-GC-FID and ranged from 43 to 247 mg kg(-1). The analysis of the marc and seeds indicated that the contamination is primarily from the peels. Since superficial extraction of the seeds with hexane removed most of the mineral paraffins, the contamination of the seeds is largely on the surface, perhaps transferred from the peels during storage of the marc. Mechanical purification of the seeds combined with washing with hexane reduced the contamination of the oil by a factor of about 10. The refining process removed 30% of the mineral paraffins, primarily the more volatile components. Oil obtained from the seeds of fresh grapes, including grapes not having undergone any phytochemical treatment, contained clearly less mineral paraffins (up to 14 mg kg(-1)), and the peels were less contaminated, suggesting an environmental background contamination. To this an additional contamination might be added by a treatment of the grapes used for wine making.     

Ethanol and polyphenolic free wine matrix stimulate the differentiation of human intestinal Caco-2 cells. Influence of their association with a procyanidin-rich grape seed extract

The effect of daily contact with ethanol on Caco-2 cell differentiation was investigated. Pure ethanol (1%) and a polyphenolic free wine matrix (polyphenol-free wine containing 1% ethanol) associated or not with a procyanidin-rich grape seed extract (GSE) were added to Caco-2 cells from confluency for 2 h a day after successive incubation in salivary, gastric, and pancreatic media. Treatment with 1% ethanol did not appear to be cytotoxic to cells, but it also stimulated Caco-2 cell differentiation, particularly in the first days following confluency, and this effect was more marked when associated with polyphenolic free wine matrix constituents. This activation resulted in an increase in microvillar density, organization, and elongation (+70%) and was associated with strong stimulation of sucrase-isomaltase (+780%) and a concomitant regular increase in cell protein content (+50-88%). While the presence of GSE in alcoholic solutions did not modify the morphological pattern observed in cells subjected to ethanol and polyphenolic free wine matrix alone, it had a clear reducing effect on their microvillus elongation (-30%). However, these stimulating effects of ethanol on morphological differentiation were attenuated from day 10 postconfluency, which could suggest cell cytoprotection against ethanol. These are the first results in support of the notion that moderate concentration of ethanol may stimulate the differentiation of Caco-2 cells, particularly when integrated with a polyphenolic free wine matrix.     

Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.     

Comparison of proanthocyanidins in commercial antioxidants: grape seed and pine bark extracts

The major constituents in grape seed and pine bark extracts are proanthocyanidins. To evaluate material available to consumers, select lots were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), gel permeation chromatography (GPC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Atmospheric pressure chemical ionization (APCI) LC/MS was used to identify monomers, dimers, and trimers present. GC/MS analyses led to the identification of ethyl esters of hexadecanoic acid, linoleic acid, and oleic acid, as well as smaller phenolic and terpene components. The GPC molecular weight (MW) distribution indicated components ranging from approximately 162 to approximately 5500 MW (pine bark less than 1180 MW and grape seed approximately 1180 to approximately 5000 MW). MALDI-TOF MS analyses showed that pine bark did not contain oligomers with odd numbers of gallate units and grape seed contained oligomers with both odd and even numbers of gallate. Reflectron MALDI-TOF MS identified oligomers up to a pentamer and heptamer, and linear MALDI-TOF MS showed a mass range nearly double that of reflectron analyses.     

微波辅助提取葡萄皮中花色苷的动力学和热力学特性

为拓展微波辅助提取技术在食品工业领域的应用,研究了微波辅助技术对提取葡萄皮中花色苷的作用,并对其动力学和热力学进行了评价。结果表明：柠檬酸浓度、液料比、提取时间和微波功率可以显著影响总花色苷提取率。采用高浓度柠檬酸水溶液（1.00?mol/L）提取的总花色苷含量（172.99?mg/100?g）是低浓度柠檬酸水溶液（0.02?mol/L）提取的总花色苷含量（43.48?mg/100?g）的4倍。此外,在实验选取的动力学模型中,指数衰减模型可以更好的拟合提取动力学数据（R2＞0.9875）。微波辅助提取葡萄皮中花色苷的有效扩散系数（2.12～4.87×10-11?m2/s）随着微波功率的增加而增大。微波辅助提取过程的热力学参数表明微波提取方式有利于传质过程。所得研究结果将为微波辅助提取技术的工业化应用提供理论依据。     

葡萄籽提取物对无淀粉火腿肠保质期的影响

为了探讨葡萄籽提取物对无淀粉火腿肠保质期的影响,将葡萄籽提取物配成质量分数为0.05%、0.10%、0.15%和0.20%的溶液,添加于火腿肠中,于0～4℃贮藏,测定细菌总数、pH值等的变化。结果表明：在贮藏过程中,添加葡萄籽提取物火腿肠的感官品质明显好于对照样;处理样的细菌总数、pH值、挥发性盐基氮（TVB-N值）、硫代巴比妥酸（TBA值）和黄色值（b*）明显低于对照组,亮度（L*）和红色值（a*）明显高于对照组;葡萄籽提取物的浓度越高,对细菌总数、pH值、L*的影响越明显,但对TVB-N值、TBA值、a*和b*的影响没有明显规律;从生产成本和使用效果综合考虑,建议生产中使用质量分数为0.20%的葡萄籽提取物。