Characterization of the mean degree of polymerization of proanthocyanidins in red wines using liquid chromatography-mass spectrometry (LC-MS).

An HPLC-MS method for the characterization of proanthocyanidins (PA) has been refined. Further application to red wines provided interesting conclusions about the composition of the flavanol fraction and PA extractability during winemaking. The yield in PA extraction increases with the length of the postfermentative maceration (PFM), as well as the mean degree of polymerization (mDP) of wine flavanols. In early winemaking events mostly monomers to trimers are extracted from grape solids, whereas PFM is required for the significant extraction of higher oligomers. Nevertheless, at the end of a regular process of elaboration the mDP is not very high and does not usually exceed a value of 2.3, dimers and trimers being the predominant flavanols in red wines. With regard to groups of compounds, gallocatechins and prodelphinidins (located in the skins) are extracted rapidly in the first stages of the winemaking. On the contrary, long postfermentative macerations are required for the extraction of galloyled derivatives from the seeds. PA extractability is also dependent on the grape variety used for winemaking. Thus, wines made with Graciano grapes were found to require a longer process of PFM than those made from Tempranillo grapes to obtain similar yields in the extraction of flavanols.

Differential modulation of the genotoxicity of food carcinogens by naturally occurring monomeric and dimeric polyphenolics.

Naturally occurring dimeric polyphenolics and their gallate esters were isolated from grape seeds, almond fruits, and apple skin, and their ability to modulate the mutagenicity of food carcinogens was studied in the Ames test, and compared to that of the monomeric green tea flavonols, (+)-catechin and (-)-epicatechin. Neither the monomeric nor the dimeric polyphenols and their galloylated derivatives influenced the mutagenic activity elicited by the indirectly acting food carcinogens benzo[a]pyrene and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), in the presence of a hepatic activation system derived from Aroclor 1254-treated rats; the only exception was the B7 dimer, which, at concentrations above 1 microM, suppressed the mutagenicity of IQ. None of the polyphenolics modulated the mutagenic activity elicited by the directly acting carcinogen N'-methyl-N'-nitro-nitrosoguanidine (MNNG). In contrast, all the dimeric polyphenols and the galloylated metabolites, at concentrations over 1 microM, potentiated the mutagenic activity induced by the indirectly acting carcinogen N-nitrosopyrrolidine, in the presence of an activation system derived from isoniazid-treated rats. In conclusion, dimeric polyphenols and galloylated derivatives of plant origin are unlikely to influence the initiation stage of the carcinogenicity of chemicals through mechanisms that involve inhibition of their cytochrome P450-mediated bioactivation or scavenging of the reactive, genotoxic intermediates.

Antioxidant properties of catechins and proanthocyanidins: effect of polymerisation, galloylation and glycosylation.

A range of catechins and oligomeric procyanidins was purified by high performance liquid chromatography (HPLC) from grape seed, apple skin, lentil and almond flesh. Catechins, galloylated epicatechin, glycosylated catechin, procyanidin dimers, galloylated dimers, trimer, and tetramer species were all identified, purified and quantified by HPLC, LC-MS and NMR. The antioxidant properties of these compounds were assessed using two methods: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; (b) scavenging of the radical cation of 2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). Antioxidant activity in the lipid phase decreased with polymerisation in contrast with antioxidant action in the aqueous phase which increased from monomer to trimer and then decreased from trimer to tetramer. Galloylation of catechin and dimeric procyanidins decreased lipid phase and increased aqueous phase antioxidant activity. Glycosylation of catechin demonstrated decreased activity in both phases.

Developmental changes in the phenol concentrations of 'golden delicious' apple fruits and leaves.

The phenolic content of apple fruit skin and leaves was determined at the developmental stage of each organ. Phenolic levels decreased on a dry weight basis during the seasonal development of fruits and leaves with respect to their ontogenesis but the single compounds did not behave uniformly. A shift in flavanol pools from monomeric to oligomeric structures during fruit growth indicated the biosynthetic tendency towards the formation of procyanidins at the end of the growing period. Among the procyanidins identified epicatechin-(4 beta,6)-epicatechin-(4 beta,6)-epicatechin has not been reported previously from apple.

Antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel.

Gallocatechins and a range of prodelphinidins were purified by high performance liquid chromatography from pomegranate peel. Gallocatechin, gallocatechin-(4-8)-catechin, gallocatechin-(4-8)-gallocatechin and catechin-(4-8)-gallocatechin were all identified, purified and quantified by LC-DAD-MS and MS-MS. The antioxidant properties of these compounds were assessed using two methods: (i) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes; and (ii) scavenging of the radical cation of 2,2-azinobis (3-ethyl-benzothiazoline-6-sulphonate, ABTS) relative to the water-soluble vitamin E analogue Trolox C (expressed as Trolox C equivalent antioxidant capacity, TEAC). The prodelphinidin dimers were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer. However, in the lipid phase, only one of the dimers (gallocatechin-(4-8)-catechin) was significantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. This study represents the first report on the antioxidant properties of prodelphinidins.

Quantitative analysis of flavan-3-ols in Spanish foodstuffs and beverages.

An HPLC method, using detection after postcolumn derivatization with p-dimethylaminocynnamaldehyde (DMACA), was developed for the quantitative analysis of individual flavanols in food. This method was applied to flavanol determination in 56 different kinds of Spanish food products, including fruit, vegetables, legumes, beverages (cider, coffee, beer, tea, and wine), and chocolate. The determined compounds corresponded to the catechins and proanthocyanidin dimers and trimers usually present in food and, therefore, they were representative of the flavanols of low degree of polymerization consumed with the diet. The data generated could be used for calculation of the dietary intake of either individual or total flavanols, which would allow the further establishment of epidemiological correlations with the incidence of chronic diseases. Similar flavanol profiles were found in the different samples of a similar type of product, even though important variations could exist in the concentrations of total and individual flavanols among them. This was attributed to factors such as sample origin, stage of ripeness, post-harvesting conservation, and processing. Total flavanol contents varied from nondetectable in most of the vegetables to 184 mg/100 g found in a sample of broad bean. Substantial amounts were also found in some fruits, such as plum and apple, as well as in tea and red wine. Epicatechin was the most abundant flavanol, followed by catechin and procyanidin B2. In general, catechins were found in all the flavanol-containing products, but the presence of gallocatechins was only relevant in pomegranate, broad bean, lentil, grape, wine, beer, and tea, and most of the berries. Galloyled flavanols were only detected in strawberry, medlar, grape, and tea.

Color and stability of pigments derived from the acetaldehyde-mediated condensation between malvidin 3-O-glucoside and (+)-catechin.

A pigment derived from the acetaldehyde-mediated condensation between (+)-catechin and malvidin 3-O-glucoside has been prepared and isolated by semipreparative HPLC, and its characteristics of color and stability have been studied and compared with that of malvidin glucoside in aqueous solutions. When the pH was increased from 2.2 to 5.5, the solution of the pigment became progressively more violet (lambda(max) = 560 nm at pH 5.5), whereas similar solutions of the anthocyanin were almost colorless at pH 4.0. This behavior indicated that the anthocyanin moiety of the pigment was more protected against water attack, and thus the formation of its quinonoidal forms was favored. The color of the pigment also showed more stability with regard to bleaching by SO(2) than that of malvidin glucoside. Nevertheless, the pigment was more sensitive to degradation in aqueous solution than the anthocyanin. The cleavage of the ethyl bridge that links the anthocyanin and the catechin constituted the first step in its degradation, as demonstrated by the formation of malvidin glucoside as a major product.

Different cardiovascular protective effects of quercetin administered orally or intraperitoneally in spontaneously hypertensive rats.

We tested whether the administration procedure of quercetin affects its metabolite profile and antihypertensive activity. Spontaneously hypertensive rats (SHR) were randomly assigned to four experimental treatments: (1) 1 mL of 1% methylcellulose by oral gavage and 2% DMSO i.p. (control group); (2) 10 mg kg⁻¹ quercetin by oral gavage once daily and 2% DMSO i.p.; (3) 10 mg kg⁻¹ quercetin by oral gavage divided in two daily doses (5 + 5 at 12 h intervals) and 2% DMSO i.p.; (4) 1 mL of 1% methylcellulose by oral gavage and 10 mg kg⁻¹ quercetin i.p. injection. Rats were treated daily for 5 weeks. Single dose and two daily doses, in a long-term oral treatment were equally efficient, both restoring the impaired aortic endothelium-dependent vasodilatation and reducing mesenteric contractile response to phenylephrine, systolic blood pressure, heart rate, and heart and kidney hypertrophy. Attenuation of vascular NADPH oxidase-driven O2⁻ production was also found in orally treated rats. Intraperitoneal administration reduced, to lesser extent than oral administration, the increased systolic blood pressure, being without effect to the endothelial dysfunction and vascular oxidative stress. In contrast, greater levels of metabolites were quantified following intraperitoneal compared to oral administration at any time point, except for higher plasma methylated quercetin aglycone in oral as compared to intraperitoneal administration at 2 but not at 8 h. In conclusion, oral quercetin was superior to intraperitoneal administration for the protection from cardiovascular complications in SHR. No differences were found between the oral administration as a single daily dose or divided into two daily doses.

Analysis of flavonoids in foods and biological samples.

Flavonoids are a major class of plant phenolics that are widely distributed in the human diet and have been related to health promotion. They may occur in their natural sources in free forms (aglycones), as glycosylated or acylated derivatives, or as oligomeric and polymerized structures. This structural diversity affects their physicochemical behaviour and complicates their analysis. Thus, there is not a single standardized procedure that can be recommended for all flavonoid groups and/or type of samples, and the procedures have to be optimized depending on the nature of the sample and the target analytes. Furthermore, when dealing with the analysis of flavonoids biological samples (i.e., human and animal fluids and tissues) some differential aspects have to be taken into account; the nature of the compounds that can be found in those samples may differ from that present in plants and food, and flavonoids and metabolites occur in much lower concentrations, which make their analysis still more challenging. In this review the main techniques for extraction and analysis of flavonoids in foodstuffs and biological fluids are revised, as well as their occurrence in foods and beverages and available databases.

Urinary excretion kinetics of p-nitrophenol following oral administration of parathion in the rabbit.

The urinary excretion kinetics of p-nitrophenol were studied in rabbits following oral administration of parathion at a dose of 3 mg/kg. Elimination of p-nitrophenol began rapidly, and of the total amount excreted during the study period, 46% was excreted in the first 3 h; 85% was excreted at 6 h after administration of the pesticide. The mean maximum excretion rate of p-nitrophenol was 111.15 +/- 61.02 micrograms/h reached in a time of 0.77 +/- 0.26 h. The formation and disappearance rate constants of the metabolite were 2.85 +/- 2.80 h-1 and 0.80 +/- 0.28 h-1, respectively. A linear relationship was observed between the plasma concentrations of parathion and the urinary excretion rate of p-nitrophenol.