Accumulation of orally administered quercetin in brain tissue and its antioxidative effects in rats

Quercetin is widely distributed in vegetables and herbs and has been suggested to act as a neuroprotective agent. Here, we demonstrate that quercetin can accumulate enough to exert biological activity in rat brain tissues. Homogenates of perfused rat brain without detectable hemoglobin contaminants were treated with β-glucuronidase/sulfatase and the released quercetin and its methylated form were analyzed using high-performance liquid chromatography (HPLC) with three different detection methods. Both quercetin and the methylated form were detected in the brain of quercetin-administered rats using HPLC-UV and HPLC with electrochemical detection and were further identified using HPLC-tandem mass spectrometry. Oral administration of quercetin (50 mg/kg body wt) attenuated the increased oxidative stress in the hippocampus and striatum of rats exposed to chronic forced swimming. The possible transport of quercetin derivatives into the brain tissue was reproduced in vitro by using a rat brain capillary endothelial cell line, a model of the blood-brain barrier. These results show that quercetin could be a potent nutrient that can access the brain and protect it from disorders associated with oxidative stress.     

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.     

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

Modification of quercetin with l-cysteine by horseradish peroxidase

Horseradish peroxidase is a well-known member of the peroxidase family that catalyzes oxidation of flavonoids and phenolic substrates to free phenoxyl or semiquinone radicals. Aim of this study was to investigate in vitro oxidation of quercetin by horseradish peroxidase in the presence of l-cysteine as nucleophilic agent, and its influence on previously formed semiquinone- and quinone-type metabolites. The obtained results showed that in the reaction without l-cysteine several products were present, such as quercetin quinone methide, phloroglucinol carboxylic acid, protocatechuic acid, as well as quercetin heterodimer and derivates of quercetin heterodimer. On the other hand, in the presence of l-cysteine only three products were obtained, quercetin quinone methide and two new isomeric mono-cysteine derivatives of quercetin with mass exp. m/z 420.04?±?0.1 [quercetin?+?cysteine–H]^– (theor. m/z 420.0389 [quercetin?+?cysteine–H]^–).     

Isolation and measurement of quercetin glucosides in flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.)

Three quercetin glucosides were isolated from flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.) together with caffeic acid as the ingredients that had DPPH radical scavenging activity, using the DPPH-HPLC method for measuring the radical scavenging activity. These quercetin glucosides were identified as quercetin 3-O-beta-D-glucoside, quercetin 3-O-beta-D-6'-O-acetylglucoside, and rutin, and the amounts of the glucosides in flower buds were also examined by HPLC. The flower buds were harvested from four different sites, the total amount of quercetin glucosides in each site was 100-170 mg/100 g fr. wt., and there were no great differences of the amounts between growing fields.     

Solid-phase extraction and gas chromatography–mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography–mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was ±9.40% for kaempferol and ±7.34% for quercetin (n=6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with β-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Bioavailability and metabolism of the flavonol quercetin in the pig

During the last years, much data pointing to putative health-promoting effects of dietary plant-derived flavonoids (stemming mainly from epidemiological and in vitro studies) have been published. Our knowledge, however, concerning the systemic availability of these substances after ingestion with food is only sketchy. In the present study, we have investigated the bioavailability of the flavonol quercetin after intravenous and oral application in pigs equipped with a permanent jugular catheter. Each animal received a single intravenous dose of quercetin (0.4 mg/kg body weight) and one week later an oral dose of 50 mg/kg. A single animal additionally received an oral dose of 500 mg/kg one week after the lower oral dose. Blood samples were drawn at defined intervals over a total period of three days following the application of quercetin. Analysis of quercetin and some of its metabolites (isorhamnetin, tamarixetin, kaempferol) in plasma samples were performed by HPLC. The calculated apparent bioavailability of free, unchanged quercetin after intake of 50 mg quercetin/kg body weight was 0.54+/-0.19%. Bioavailability was, however, considerably increased to 8.6+/-3.8% after additionally taking into account conjugated quercetin and further increased to 17.0+/-7.1% by including quercetin's metabolites. Our results further indicate, that the conjugation of orally administered quercetin with glucuronic and sulfuric acid appears to preferentially occur in the intestinal wall.     

Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin

In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon 1 h incubation with 100 microM quercetin or 2 h incubation with 25 microM quercetin, whereas 1 and 10 microM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LC-MS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LC-MS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47.     

Metabolic activation of quercetin mutagenicity

The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test. The mutagenicity of quercetin was enhanced by the cytosolic fraction of liver extract (S100), or by ascorbate, and even more by the complete liver supernatant (S9) in the presence of cofactors (NADP and glucose-6-phosphate). The formation of metabolites by the S9 enzymes was demonstrated by reverse-phase HPLC.     

Caffeine,quercetin and alizarin stimulate the exhalation of metabolic products of [14C]-N-nitrosodiethylamine in mice

Naturally occurring plant products belonging to different chemical classes namely alizarin, an anthraquinone, caffeine, a methylxanthine derivative and quercetin, a flavonol were studied for their effect on elimination of metabolites of [14C]-N-nitrosodiethylamine (14C-NDEA) through respiration in mice. Treatment with caffeine, quercetin and alizarin at doses of 200, 9 and 9 microg/ml respectively, in drinking water enhanced the exhalation of 14CO2, one of the major end products of NDEA metabolism. Radioactive CO2 exhaled in 60 min increased by 2, 1.61 and 1.4-folds in animals treated with caffeine, quercetin and alizarin for 8 weeks respectively. This increase in exhalation in caffeine-treated animals was achieved even in 2 weeks. These compounds had no adverse effects on the absorption of radioactive NDEA from the gut of the animals as shape and time of 14CO2 peak was similar in i.p. and orally administered [14C-NDEA]. Increased detoxification/elimination of the carcinogen could be one of the mechanisms for the anticarcinogenic properties of these phytochemicals in lung tumorigenesis induced by orally administered NDEA.     

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Human metabolism of dietary flavonoids: Identification of plasma metabolites of quercetin

The position of conjugation of the flavonoid quercetin dramatically affects biological activity in vitro, therefore it is important to determine the exact nature of the plasma metabolites. In the present study, we have used various methods (HPLC with diode array detection, LCMS, chemical and enzymic synthesis of authentic conjugates and specific enzymic hydrolysis) to show that quercetin glucosides are not present in plasma of human subjects 1.5 h after consumption of onions (a rich source of flavonoid glucosides). All four individuals had similar qualitative profiles of metabolites. The major circulating compounds in the plasma after 1.5 h are identified as quercetin-3-glucuronide, 3′-methyl-quercetin-3-glucuronide and quercetin-3′-sulfate. The existence of substitutions in the B and/or C ring of plasma quercetin metabolites suggests that these conjugates will each have very different biological activities.     

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.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

槲皮素与DNA的相互作用

有机小分子与DNA相互作用机理研究已经成为药物作用机理研究与新药筛选的重要手段之一。槲皮素 (Quercetin) 是一种多羟基黄酮类化合物,具有抗癌、抗炎、抗菌、抗病毒、降糖降压、免疫调节及保护心血管的作用。实验研究的目的是发现与确认槲皮素与DNA之间是否具有相互作用,以及确定其相互作用的类型。根据荧光光谱法和共振散射荧光光谱法的分析结果,发现槲皮素与鲱鱼精DNA之间存在相互作用;使用紫外-可见分光光度法和荧光偏振分析,发现槲皮素与鲱鱼精DNA之间的相互作用模式不属于嵌插作用,而是沟槽嵌合或者静电相互作用;最后通过分子对接实验,成功佐证槲皮素与鲱鱼精DNA之间的相互作用模式是沟槽结合。该工作有利于理解槲皮素与DNA之间的体外作用方式,助力于相应疾病的药物开发。     

Degradation of dibenzothiophene by Brevibacterium sp.DO

Dibenzothiophene, a polycyclic aromatic sulfur heterocycle, represents as a model compound the organic sulfur integrated in the macromolecular coal matrix. A pure culture of a Brevibacterium species was isolated, which is able to use dibenzothiophene as sole source of carbon, sulfur and energy for growth. During dibenzothiophene utilization sulfite was released in a stoichiometrical amount and was further oxidized to sulfate. Three metabolites of dibenzothiophene degradation were isolated and identified as dibenzothiophene-5-oxide, dibenzothiophene-5-dioxide and benzoate by cochromatography, UV spectroscopy and gas chromatographymass spectrometry analyses. Based on the identified metabolites a pathway for the degradation of dibenzothiophene by Brevibacterium sp. DO is proposed.Non-standard abbreviations DBT dibenzothiophene - PASH polycyclic aromatic sulfur heterocycle - PAH polycyclic aromatic hydrocarbons - GC-MS gas chromatography-mass spectrometry - HPLC high pressure liquid chromatography - IC ion chromatography