TRANSPARENT TESTA10 encodes a laccase-like enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat

The Arabidopsis thaliana transparent testa10 (tt10) mutant exhibits a delay in developmentally determined browning of the seed coat, also called the testa. Seed coat browning is caused by the oxidation of flavonoids, particularly proanthocyanidins, which are polymers of flavan-3-ol subunits such as epicatechin and catechin. The tt10 mutant seeds accumulate more epicatechin monomers and more soluble proanthocyanidins than wild-type seeds. Moreover, intact testa cells of tt10 cannot trigger H2O2-independent browning in the presence of epicatechin and catechin, in contrast with wild-type cells. UV-visible light detection and mass spectrometry revealed that the major oxidation products obtained with epicatechin alone are yellow dimers called dehydrodiepicatechin A. These products differ from proanthocyanidins in the nature and position of their interflavan linkages. Flavonol composition was also affected in tt10 seeds, which exhibited a higher ratio of quercetin rhamnoside monomers versus dimers than wild-type seeds. We identified the TT10 gene by a candidate gene approach. TT10 encodes a protein with strong similarity to laccase-like polyphenol oxidases. It is expressed essentially in developing testa, where it colocalizes with the flavonoid end products proanthocyanidins and flavonols. Together, these data establish that TT10 is involved in the oxidative polymerization of flavonoids and functions as a laccase-type flavonoid oxidase.     

Rhizobia catabolize nod gene-inducing flavonoids via C-ring fission mechanisms.

Gas chromatographic and mass spectrometric analyses of derivatized culture medium extracts were used to identify the products of flavonoid metabolism by rhizobia. A number of Rhizobium species and biovars degraded their nod gene-inducing flavonoids by mechanisms which originated in a cleavage of the C-ring of the molecule and which yielded conserved A- and B-ring products among the metabolites. In contrast, Pseudomonas putida degraded quercetin via an initial fission in its A-ring, and Agrobacterium tumefaciens displayed a nonspecific mode of flavonoid degradation which yielded no conserved A- or B-ring products. When incubated with rhizobia, flavonoids with OH substitutions at the 5 and 7 positions yielded phloroglucinol as the conserved A-ring product, and those with a single OH substitution at the 7 position yielded resorcinol. A wider range of structures was found among the B-ring derivatives, including p-coumaric, p-hydroxybenzoic, protocatechuic, phenylacetic, and caffeic acids. The isoflavonoids genistein and daidzein were also degraded via C-ring fission by Rhizobium fredii and Rhizobium sp. strain NGR234, respectively. Partially characterized aromatic metabolites with potential nod gene-inducing activity were detected among the products of naringenin degradation by Rhizobium leguminosarum bv. viciae. The initial structural modification of nod gene-inducing flavonoids by rhizobia can generate chalcones, whose open C-ring system may have implications for the binding of inducers to the nodD gene product.     

Oxidative modification of quercetin by hemeproteins

The ability of a number of hemeproteins to oxidize the flavonoid quercetin has been shown. It was found that quercetin undergoes chemical modification in the presence of cytochrome c, myoglobin, and hemoglobin but not cytochrome b(5). In the range of investigated proteins the most effective oxidant appears to be cytochrome c. Chromatographic analysis of the reaction mixture revealed a number of quercetin oxidation products. The main oxidation product was purified and characterized by means of LC-MS and NMR analyses. It has a dimeric structure similar to the product of quercetin oxidation by horseradish peroxidase and is formed during radical-driven reactions. Our results indicate that a number of hemeproteins can react and modify biologically active flavonoids. However, these reactions might also lead to the generation of active species with deleterious consequences for the cellular macromolecules.     

Protection by flavonoids against the peroxynitrite-mediated oxidation of dihydrorhodamine

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by Co2/HCO3-, which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC50 of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.     

Altered regioselectivity of a poplar O-methyltransferase, POMT-7

O-Methylated flavonoids are biosynthesized by regioselective flavonoid O-methyltransferases (OMTs), which may account for the limited number of naturally occurring flavonoids in nature. It was previously shown that poplar POMT-7 regioselectively methylates the 7-hydroxyl group of flavones, whereas rice ROMT-9 regioselectively methylates the 3'-hydroxyl group of the substrate. We co-expressed both OMT genes (POMT-7 and ROMT-9) in E. coli and carried out biotransformation experiments of some flavonoids with the transformed E. coli strain. Contrast to the predicted regioselectivity of both POMT-7 and ROMT-9, unexpected methylation reaction products, i.e. 3',4'-O-methylated flavonoids, in addition to the predicted ones, were obtained with luteolin (5,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) as substrates. Reactions using the 3'-O-methyl derivative of luteolin and quercetin by POMT-7 revealed that the enzyme has altered its regioselectivity from the 7- to the 4'-hydroxyl groups. These results are discussed in terms of molecular modeling of POMT-7 in relation to its methyl donor.     

An essential difference between the flavonoids monoHER and quercetin in their interplay with the endogenous antioxidant network

Antioxidants can scavenge highly reactive radicals. As a result the antioxidants are converted into oxidation products that might cause damage to vital cellular components. To prevent this damage, the human body possesses an intricate network of antioxidants that pass over the reactivity from one antioxidant to another in a controlled way. The aim of the present study was to investigate how the semi-synthetic flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER), a potential protective agent against doxorubicin-induced cardiotoxicity, fits into this antioxidant network. This position was compared with that of the well-known flavonoid quercetin. The present study shows that the oxidation products of both monoHER and quercetin are reactive towards thiol groups of both GSH and proteins. However, in human blood plasma, oxidized quercetin easily reacts with protein thiols, whereas oxidized monoHER does not react with plasma protein thiols. Our results indicate that this can be explained by the presence of ascorbate in plasma; ascorbate is able to reduce oxidized monoHER to the parent compound monoHER before oxidized monoHER can react with thiols. This is a major difference with oxidized quercetin that preferentially reacts with thiols rather than ascorbate. The difference in selectivity between monoHER and quercetin originates from an intrinsic difference in the chemical nature of their oxidation products, which was corroborated by molecular quantum chemical calculations. These findings point towards an essential difference between structurally closely related flavonoids in their interplay with the endogenous antioxidant network. The advantage of monoHER is that it can safely channel the reactivity of radicals into the antioxidant network where the reactivity is completely neutralized.     

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.     

Protection by Flavonoids Against the Peroxynitrite-mediated Oxidation of Dihydrorhodamine

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by [Formula: See Text] which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC₅₀ of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.     

Peroxidase-catalyzed formation of quercetin quinone methide-glutathione adducts

The oxidation of quercetin by horseradish peroxidase/H(2)O(2) was studied in the absence but especially also in the presence of glutathione (GSH). HPLC analysis of the reaction products formed in the absence of GSH revealed formation of at least 20 different products, a result in line with other studies reporting the peroxidase-mediated oxidation of flavonoids. In the presence of GSH, however, these products were no longer observed and formation of two major new products was detected. (1)H NMR identified these two products as 6-glutathionylquercetin and 8-glutathionylquercetin, representing glutathione adducts originating from glutathione conjugation at the A ring instead of at the B ring of quercetin. Glutathione addition at positions 6 and 8 of the A ring can best be explained by taking into consideration a further oxidation of the quercetin semiquinone, initially formed by the HRP-mediated one-electron oxidation, to give the o-quinone, followed by the isomerization of the o-quinone to its p-quinone methide isomer. All together, the results of the present study provide evidence for a reaction chemistry of quercetin semiquinones with horseradish peroxidase/H(2)O(2) and GSH ultimately leading to adduct formation instead of to preferential GSH-mediated chemical reduction to regenerate the parent flavonoid.     

Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3'-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2 microM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3'-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide > quercetin > quercetin-3-glucuronide = quercetin-3-glucoside > catechin > quercetin-4'-glucuronide > isorhamnetin-3-glucuronide > quercetin-3'-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3'-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3'-sulfate > quercetin-7-glucuronide > quercetin-3-glucuronide > quercetin-4'-glucuronide = isorahmnetin-3-glucuronide). Kq values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3'-sulfate (approximately 4 microM)= quercetin > or = quercetin-7-glucuronide > quercetin-3-glucuronide = quercetin-3-glucoside > isorhamnetin-3-glucuronide > quercetin-4'-glucuronide (approximately 20 microM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

Metabolic conversion of dietary flavonoids alters their anti-inflammatory and antioxidant properties

The notion that dietary flavonoids exert beneficial health effects in humans is often based on in vitro studies using the glycoside or aglycone forms of these flavonoids. However, flavonoids are extensively metabolized in humans, resulting in the formation of glucuronide, methyl, and sulfate derivatives, which may have different properties than their parent compounds. The goal of this study was to investigate whether different chemical modifications of the same flavonoid molecule affect its biological and antioxidant activities. Hence, we studied the anti-inflammatory effects of several major human metabolites of quercetin and (-)-epigallocatechin-3-O-gallate (EGCG) by assessing their inhibitory effects on tumor necrosis factor α (TNFα)-induced protein expression of cellular adhesion molecules in human aortic endothelial cells (HAEC). HAEC were incubated with 1-30 μM quercetin, 3'- or 4'-O-methyl-quercetin, quercetin-3-O-glucuronide, and quercetin-3'-O-sulfate or 20-100 μM EGCG, 4'-O-methyl-EGCG, and 4',4'-di-O-methyl-EGCG, prior to coincubation with 100 U/ml of TNFα. 3'-O-Methyl-quercetin, 4'-O-methyl-quercetin, and their parent aglycone compound, quercetin, all effectively inhibited expression of intercellular adhesion molecule-1 (ICAM-1) with IC(50) values (concentration required for 50% inhibition) of 8.0, 5.0, and 4.4 μM, respectively; E-selectin expression was suppressed to a somewhat lesser but still significant degree by all three compounds, whereas vascular cell adhesion molecule-1 (VCAM-1) was not affected. In contrast, quercetin-3-O-glucuronide (20-100 μM), quercetin-3'-O-sulfate (10-30 μM), and phenolic acid metabolites of quercetin (20-100 μM) did not inhibit adhesion molecule expression. 4',4'-Di-O-methyl-EGCG selectively inhibited ICAM-1 expression with an IC(50) value of 94 μM, whereas EGCG (20-60 μM) and 4'-O-methyl-EGCG (20-100 μM) had no effect. The inhibitory effects of 3'-O-methyl-quercetin and 4',4'-di-O-methyl-EGCG on adhesion molecule expression were not related either to inhibition of NF-κB activation or to their antioxidant reducing capacity. Our data indicate that flavonoid metabolites have different biological and antioxidant properties than their parent compounds, and suggest that data from in vitro studies using nonmetabolites of flavonoids are of limited relevance in vivo.     

Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Flavonoid oxidation kinetics in aqueous and aqueous organic media in the presence of peroxidase,tyrosynase, and hemoglobin

The kinetics of oxidation reactions of flavonoids, quercetin, dihydroquercetin, and epicatechin has been studied in the presence of biocatalysts of different natures: horseradish peroxidase, mushroom tyrosinase, and hemoglobin from bull blood. Comparison of the kinetic parameters of the oxidation reaction showed that peroxidase appeared to be the most effective biocatalyst in these processes. The specificity of the enzyme for quercetin increased with increasing the polarity of the solvent in a series of ethanol–acetonitrile–dimethyl sulfoxide.     

Two-electron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16 g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: E_P/2 = 0.97 V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.     

A reevaluation of the peroxynitrite scavenging activity of some dietary phenolics

Peroxynitrite is implicated in many diseases. Hence, there is considerable interest in potential therapeutic peroxynitrite scavengers. Diet-derived phenolics have been claimed to be powerful peroxynitrite scavengers. However, the reactivity of peroxynitrite can be significantly modified by bicarbonate and this has not been considered in evaluations of the scavenging activity of phenols. Bicarbonate (25 mM) significantly decreased the ability of several phenolic compounds (caffeic acid, o- and p-coumaric acid, gallic acid, ferulic acid) but not others (catechin and epicatechin) to inhibit peroxynitrite-mediated tyrosine nitration. Bicarbonate (25 mM) also decreased the ability of catechin, epicatechin, quercetin and ferulic acid but not chlorogenic acid, gallic acid, caffeic acid and o-coumaric acid to inhibit peroxynitrite-mediated alpha(1)-antiproteinase inactivation. These results show that physiological concentrations of bicarbonate substantially modify the ability of dietary phenolics to prevent peroxynitrite-mediated reactions. When assessing compounds for peroxynitrite scavenging, experiments should be conducted in the presence of bicarbonate to avoid misleading results.     

Common gut microbial metabolites of dietary flavonoids exert potent protective activities in β-cells and skeletal muscle cells

Flavonoids are dietary compounds with potential anti-diabetes activities. Many flavonoids have poor bioavailability and thus low circulating concentrations. Unabsorbed flavonoids are metabolized by the gut microbiota to smaller metabolites, which are more bioavailable than their precursors. The activities of these metabolites may be partly responsible for associations between flavonoids and health. However, these activities remain poorly understood. We investigated bioactivities of flavonoid microbial metabolites [hippuric acid (HA), homovanillic acid (HVA), and 5-phenylvaleric acid (5PVA)] in primary skeletal muscle and β-cells compared to a native flavonoid [(−)-epicatechin, EC]. In muscle, EC was the most potent stimulator of glucose oxidation, while 5PVA and HA simulated glucose metabolism at 25 μM, and all compounds preserved mitochondrial function after insult. However, EC and the metabolites did not uncouple mitochonndrial respiration, with the exception of 5PVA at10 μM. In β-cells, all metabolites more potently enhanced glucose-stimulated insulin secretion (GSIS) compared to EC. Unlike EC, the metabolites appear to enhance GSIS without enhancing β-cell mitochondrial respiration or increasing expression of mitochondrial electron transport chain components, and with varying effects on β-cell insulin content. The present results demonstrate the activities of flavonoid microbial metabolites for preservation of β-cell function and glucose utilization. Additionally, our data suggest that metabolites and native compounds may act by distinct mechanisms, suggesting complementary and synergistic activities in vivo which warrant further investigation. This raises the intriguing prospect that bioavailability of native dietary flavonoids may not be as critical of a limiting factor to bioactivity as previously thought.     

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.     

Transgenic rice lines expressing maize C1 and R-S regulatory genes produce various flavonoids in the endosperm

Flavonoids, compounds that possess diverse health-promoting benefits, are lacking in the endosperm of rice. Therefore, to develop transgenic lines that produce flavonoids, we transformed a white rice cultivar, Oryza sativa japonica cv. Hwa-Young, with maize C1 and R-S regulatory genes. Expression of these transgenes was restricted to the endosperm using the promoter of a rice prolamin gene. The pericarp of the C1/R-S homozygous lines became dark brown in accordance with their maternal genotype, whereas the endosperm turned chalky, similar to the opaque kernel phenotype. Analysis via high-performance liquid chromatography (HPLC) revealed that numerous kinds of flavonoids were produced in these transgenic kernels. To identify individual flavonoids, the number of HPLC peaks was reduced through moderate acid hydrolysis, followed by ethyl acetate partitioning. Amongst the major flavonoids, dihydroquercetin (taxifolin), dihydroisorhamnetin (3'-O-methyl taxifolin) and 3'-O-methyl quercetin were identified through liquid chromatography/mass spectrometry/mass spectrometry and nuclear magnetic resonance analyses. Fluorescence labelling with diphenylboric acid showed that the flavonoids were highly concentrated in the cells of four to five outer endosperm layers. More importantly, a high fluorescence signal was present in the cytosol of the inner endosperm layers. However, the overall signal in the inner layers was significantly lower because starch granules and protein bodies occupied most of the cytosolic space. Our estimate of the total flavonoid content in the transgenic kernels suggests that C1/R-S rice has the potential to be developed further as a novel variety that can produce various flavonoids in its endosperm.     

Flavonoid metabolites and susceptibility of rat lipoproteins to oxidation

Flavonoids are ingested with vegetables and beverages and exert a beneficial effect on cardiovascular disease. Studies in animals in vitro and in humans ex vivo on the resistance of lipoproteins to oxidation are not consistent and the mechanisms by which flavonoids protect against atherosclerosis are a matter of debate. In the present study, we investigated the effects of administering diets containing 0.3% (wt/wt) quercetin, 0.3% (wt/wt) catechin, or 35% (vol/wt) dealcoholated red wine (DRW) for 10 days in healthy rats on markers of oxidative damage in lipoproteins and in plasma. The antioxidant levels in low-density lipoproteins (LDL) or the lag phase, oxidation rate, and maximum level of conjugated dienes during ex vivo LDL oxidation did not differ between control and treated rats. Plasma levels of alpha-tocopherol and retinol were similar in all groups. The total antioxidant status of the plasma from rats fed either quercetin or DRW diet was higher than in control rats. Only glucuronide and sulfate compounds of quercetin were detected in plasma from rats fed the quercetin-rich diet, and no flavonoids or their metabolites were detected in plasma or LDL from rats fed the catechin- or the DRW-rich diet. No significant differences in malondialdehyde or in conjugated dienes in plasma were observed. These results indicate that although metabolites from quercetin are present in plasma, they are not detected in lipoproteins and do not modify the level of other antioxidants. In conclusion, in the absence of any pathology or of oxidative stress the intake of quercetin, catechin, or DRW did not protect lipoproteins from oxidation ex vivo.