Human red blood cells as a natural flavonoid reservoir

Quercetin is rapidly and avidly taken up by human red blood cells (RBC) via a passive diffusion mechanism, driven by flavonoid binding to haemoglobin and resulting in an almost quantitative accumulation of the flavonoid. Heamoglobin-free resealed ghosts accumulated quercetin exclusively in the membrane fraction. Cell-associated quercetin was biological active and could be quantitatively utilised to support the reduction of extracellular oxidants mediated by a transplasma-membrane oxido-reductase. Additional experimental evidence revealed that quercetin uptake declined in the presence of albumin and that, under these conditions, the amount of cell-associated quercetin is enhanced by increasing the RBC number. Quercetin release from flavonoid-preloaded RBC was observed only in the presence of albumin (or in human plasma) and this response was progressively inhibited upon incubation in solutions containing albumin previously exposed to increasing concentrations of quercetin and cleared of the unbound fraction of the flavonoid. Furthermore, exposure to quercetin pre-saturated albumin promoted accumulation of the flavonoid in fresh RBC and this response was a direct function of the extent of albumin saturation. These results, indicating a flow of quercetin from albumin to haemoglobin, and vice versa, are therefore consistent with the possibility that human RBC play a pivotal role in the distribution and bioavailability of circulating flavonoids.     

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.     

Flavonoid 5-glucosides from the cocoon shell of the silkworm, Bombyx mori

The flavonoid 5-glucosides, quercetin 5,4'-di-O-beta-D-glucopyranoside and quercetin 5,7,4'-tri-O-beta-D-glucopyranoside, together with the known quercetin 5-O-beta-D-glucopyranoside, were isolated from the cocoon shell of the silkworm, Bombyx mori. The structures were identified by spectroscopic analysis. These flavonoid glucosides were not present in mulberry leaves, the silkworm's only food, and they are considered to be metabolites produced by the silkworm.     

Quercetin protects mouse oocytes against chromium-induced damage in vitro and in vivo

BackgroundChromium (Cr) is a naturally-occurring element that is used in various fields of industry. Humans may be exposed to hexavalent chromium [Cr(VI)], which is one of the stable valence states of the chromium through contaminated soil, air, and water. Exposure to Cr(VI) through contaminated drinking water, soil and air causes various cancers and also fertility problems in animals and humans. Quercetin (QCT), a common flavonoid compound, has numerous biological effects as an antioxidant and free radical scavenger, but its function and mechanisms in reproductive processes in various species remain unclear. This study aims to determine the chromium effects on mice oocyte quality and the ameliorative effect of QCT in both in vitro and in vivo experimental models.MethodsFor the in vitro experiment, oocytes were collected and divided into the control, sham, QCT-treated, Cr(VI) (potassium dichromate), and treatment [Cr(VI)+QCT] groups. Collected oocytes were cultured in maturation medium with or without 10 µM quercetin and 10 µM Cr(VI) for 14 h based on the defined experimental design. For the in vivo experiment, the mice were randomly divided into the control, sham, QCT-treated, Cr(VI), and Cr(VI) + QCT groups. Control and sham mice received regular drinking water and diet. Cr(VI) group received Cr(VI) (50 ppm in drinking water) and Cr(VI) + QCT group received 50 ppm Cr(VI) with QCT (20 mg/kg body wt, through i.p) for a period of 21 days and then oocytes were collected and cultured for 14 h for in vitro maturation. For both experiments, at the end of the culture period, we examined the ameliorative effect of QCT on oocyte maturation, spindle formation, ROS production, mitochondrial function, and apoptosis.ResultsOur in vitro and in vivo results showed that Cr(VI) disrupt the oocyte maturation and spindle formation (P < 0.001). Furthermore, we found that exposure to Cr(VI) significantly increased ROS levels and decreased mitochondrial membrane potential (P < 0.001). In addition, exposure to Cr(VI) induced early apoptosis and downregulated the Bcl-2 mRNA expression and upregulated the Caspase-3 and Bax mRNAs expression (P < 0.01). Finally, quercetin significantly restored the detrimental effects of Cr(VI).ConclusionThe results indicated that quercetin protects the oocytes against Cr(VI) toxicity through the suppression of oxidative stress and apoptosis. The conclusions drawn from our study's findings suggest that quercetin might be useful agent for oocyte maturation in case of possible exposure to toxic substances such as chromium.     

Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Polyphenolics in <Emphasis Type="Italic">Rhizophora mangle</Emphasis> L. leaves and their changes during leaf development and senescence

The chemical defenses in Rhizophora mangle L. are largely carbon based. The family has long been exploited for the high proanthocyanidin (condensed tannin) content of its wood, bark and leaves. In this paper, we quantify the overall pools of plant phenolics in R. mangle leaves, identify the major constituents of these pools and document their changes during leaf maturation and senescence. Overall, polyphenolics account for approximately 23% of the total leaf dry weight. The leaves contain at least seven flavonoid glycosides, five of them based on quercetin. Additional minor constituents are myricetin and kaempferol diglucosides. The aglycone, quercetin, was found only in senescing leaves. Also during senescence, a new compound, 5,4-dimethoxy-7,3,5-trihydroxyflavone, appeared. The flavonoids were accompanied by a complex mixture of condensed tannins based mainly on (+)-catechin and (–)-epicatechin with A-type and B-type linkages; this pool is also distinguished by having previously unreported, high contributions of (+)-catechin and (–)-epicatechin glycosides. During senescence, but prior to leaf abscission, the polyphenolic pools become simplified: flavonol glycosides and low oligomeric tannins largely disappear, leaving only the largest tannin polymers. The ecological and physiological significance of these compounds as they appear in R. mangle is discussed.     

Flavonoids in flowers of 16 Kalanchoë blossfeldiana varieties

Kalancho? blossfeldiana varieties with orange, pink, red and magenta flowers were found to contain 3,5-O-beta-D-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Pink, red and magenta varieties contained relatively high amounts of quercetin based flavonols. Four distinct quercetin flavonols were identified, namely quercetin 3-O-beta-D-glucoside and three that were quercetin 3-O-alpha-L-rhamnoside based, with either glucose, xylose or arabinose attached to position 2 of the rhamnose. In addition, the presence of at least three kaempferol based diglycosides was suggested from LC-MS analyses. Orange varieties contained very low amounts of flavonol co-pigments and of delphinidin derivatives. The flower extracts of the varieties 'Diva' (magenta) and 'Molly' (red) had identical anthocyanin ratios but differed significantly in flavonol content. The magenta variety contained four times as much quercetin relative to anthocyanidin as the red variety. This difference was mainly due to a larger content of quercetin 3-O-(2'-O-beta-D-glucopyranosyl-alpha-L-rhamnopyranoside). Based on pigment and co-pigment analyses, approaches for molecular breeding towards blue flower colour are discussed.     

Quercetin significantly decreased cyclosporin oral bioavailability in pigs and rats

Cyclosporin, an immunosuppressant with a narrow therapeutic window, is a substrate for both CYP3A4 and P-glycoprotein (Pgp). Quercetin is an inhibitor of CYP3A4 and a modulator of Pgp. This study aimed to measure the effect of quercetin on the absorption and disposition of cyclosporin in pigs and rats. Cyclosporin (Sandimmune, 10 mg/kg) was orally administered with and without a concomitant dose of quercetin (50 mg/kg) to pigs and rats. Cyclosporin concentrations in blood samples were determined by a specific monoclonal fluorescence polarization immunoassay. The pharmacokinetic parameters were calculated by noncompartmental analysis using WINNONLIN. A paired Student's t-test was conducted for statistical comparison. A study using the everted intestinal sac was carried out to evaluate the effect of quercetin on the function of intestinal Pgp. The coadministration of quercetin significantly decreased cyclosporin AUC(0-3) (area under the concentration-time curve from time zero to 3 h) by 56% and AUC(0-t) (area under the concentration-time curve from time zero to the last point) by 43% in pigs and rats, respectively, indicating that the coadministration of quercetin significantly decreased cyclosporin oral bioavailability. However, the inverted sac study showed that quercetin significantly inhibited the function of intestinal Pgp. It is suggested that concurrent use of quercetin or quercetin-containing dietary supplement or herbs with cyclosporin or other medications whose absorption and metabolism are mediated by Pgp and/or CYP3A4 should require close monitoring.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.