Pone.0032673 1.8

Glucuronidated Quercetin Lowers Blood Pressure inSpontaneously Hypertensive Rats via Deconjugation Pilar Galindo1, Isabel Rodriguez-Go´mez2, Susana Gonza´lez-Manzano3, Montserrat Duen˜as3, Rosario Jime´nez1, Carmen Mene´ndez4,5, Fe´lix Vargas2, Juan Tamargo4, Celestino Santos-Buelga3, Francisco Pe´rez-Vizcaı´no4,5, Juan Duarte1* 1 Department of Pharmacology, School of Pharmacy, University of Granada, Granada, Spain, 2 Department of Physiology, School of Medicine, University of Granada, Granada, Spain, 3 Grupo de Investigacio´n en Polifenoles, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain, 4 Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos, Madrid, Spain, 5 Ciber Enfermedades Respiratorias, Background: Chronic oral quercetin reduces blood pressure and restores endothelial dysfunction in hypertensive animals.
However, quercetin (aglycone) is usually not present in plasma, because it is rapidly metabolized into conjugated, mostlyinactive, metabolites. The aim of the study is to analyze whether deconjugation of these metabolites is involved in theblood pressure lowering effect of quercetin.
Methodology/Principal Findings: We have analyzed the effects on blood pressure and vascular function in vitro of theconjugated metabolites of quercetin (quercetin-3-glucuronide, Q3GA; isorhamnetin-3-glucuronide, I3GA; and quercetin-39-sulfate, Q3'S) in spontaneously hypertensive rats (SHR). Q3GA and I3GA (1 mg/kg i.v.), but not Q3'S, progressively reducedmean blood pressure (MBP), measured in conscious SHR. The hypotensive effect of Q3GA was abolished in SHR treated withthe specific inhibitor of b-glucuronidase, saccharic acid 1,4-lactone (SAL, 10 mg/ml). In mesenteric arteries, unlike quercetin,Q3GA had no inhibitory effect in the contractile response to phenylephrine after 30 min of incubation. However, after1 hour of incubation Q3GA strongly reduced this contractile response and this effect was prevented by SAL. Oraladministration of quercetin (10 mg/Kg) induced a progressive decrease in MBP, which was also suppressed by SAL.
Conclusions: Conjugated metabolites are involved in the in vivo antihypertensive effect of quercetin, acting as moleculesfor the plasmatic transport of quercetin to the target tissues. Quercetin released from its glucuronidated metabolites couldbe responsible for its vasorelaxant and hypotensive effect.
Citation: Galindo P, Rodriguez-Go´mez I, Gonza´lez-Manzano S, Duen˜as M, Jime´nez R, et al. (2012) Glucuronidated Quercetin Lowers Blood Pressure inSpontaneously Hypertensive Rats via Deconjugation. PLoS ONE 7(3): e32673. doi:10.1371/journal.pone.0032673 Editor: Joao B. Calixto, Universidad Federal de Santa Catarina, Brazil Received November 28, 2011; Accepted February 2, 2012; Published March 12, 2012 Copyright: ß 2012 Galindo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants from the Spanish Ministerio de Ciencia e Innovacio´n (AGL2007-66108, SAF2008-03948, AGL2009-12001 andSAF2010-22066) and Consolider-Ingenio 2010 Programme (CSD2007-00063), Junta de Andalucia (Proyecto de Excelencia P06-CTS-01555), and Ministerio deSanidad y Consumo, Instituto de Salud Carlos III (Red HERACLES RD06/0009 and Red de Investigacio´n Renal, REDinREN RD06/0016/0017). The funders had no rolein study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] exerts systemic and coronary vasodilatation and antiaggreganteffects in vitro [6–8] and reduces blood pressure, oxidative status Flavonoids are polyphenolic compounds that occur ubiquitously and end-organ damage in animal models of hypertension [9,10], in plants and are consumed in the form of fruits, vegetables, nuts including spontaneously hypertensive rats (SHR) [9–12]. Chronic and derived products such as wine and chocolate. The average quercetin also reduces blood pressure in stage 1 hypertensive daily intake in the western diet of flavonols plus flavones (two main subjects [13]. However, there are not studies analyzing the acute classes of flavonoids) is estimated to be <23 mg, with quercetin effects on blood pressure of oral quercetin.
(3,39,49,5,7-pentahydroxyflavone) contributing 60–75% of the Many previous in vitro studies have exposed tissues or cultured total [1,2]. Quercetin is a prime example of such a flavonoid cells to commercially available aglycones or the glycosylated group and it is found in foods bound to sugars, mainly as b- compounds which are present at extremely low concentrations in glycosides. Quercetin glycosides occur in broccoli, apples, and plasma [14]. Upon ingestion with the diet, quercetin glycosides are especially in onions, with an abundance as high as 0.25–0.5 g/kg rapidly hydrolyzed during their passage across the small intestine [3]. Prospective studies have shown an inverse correlation between or by bacterial activity in the colon to generate quercetin aglycone.
dietary flavonoid intake and mortality from coronary heart disease Absorbed quercetin is rapidly conjugated with glucuronic acid [1,4]. Several studies using various animal models provide support and/or sulfate during first-pass metabolism (intestine-liver) and a for the observed protective effects of dietary flavonoids with portion of the metabolites are also methylated and, therefore, the respect to cardiovascular diseases [5]. For example, quercetin major metabolites of quercetin in rat and human plasma are PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR quercetin-3-glucuronide (Q3GA), quercetin-39-sulfate (Q3'S) and Blood pressure measurement isorhamnetin-3-glucuronide (I3GA) (Figure 1) while the aglycone Direct blood pressure was measured in conscious SHR. For this is usually undetectable [15–18]. The biological activity of purpose, the rats were anaesthetised with 2.5 mL/kg i.p.
quercetin is generally attenuated after its conversion into the equitensin (500 mL contain 43% w/v chloral hydrate in 81 mL metabolites. However, antioxidant activity for various quercetin ethanol; 4.86 mg pentobarbitone; 198 mL propylene glycol; metabolites has been reported [19–21]. This may lead in vascular 10.63 g MgSO4; distilled water) and the carotid artery was beds to an improvement of endothelial function, while the cannulated to obtain direct measurements of arterial blood conjugated metabolites have no direct vasorelaxant effect in rat pressure. The catheter was exteriorised through the skin on the aorta [21]. Moreover, injured/inflamed arteries, as occur in dorsal side of the neck and protected with a silver spring. A hypertension and atherosclerosis, with activated macrophages are cannula was also introduced into the left jugular vein for the potential targets of the metabolites of dietary quercetin [22]. Some administration of quercetin metabolites and blood sampling. Upon previous studies have shown that quercetin glucuronides can be completion of the surgical procedure, rats were fasted and allowed deconjugated in vitro in cultured macrophages [22] and in to recover for 6 h and, after connecting the catheter to a homogenates from human liver and small intestine [23]. Q3GA transducer and a two-channel recorder (TRA-021 and Letigraph can be also slowly deconjugated within the vascular wall [24].
2000, respectively; Letica SA, Barcelona, Spain), blood pressure We hypothesized that the antihypertensive effects of quercetin and heart rate (HR) were continuously recorded. Animals received could be mediated by the conjugated derivatives that are present either Q3GA (0.2, 0.02 or 1 mg/kg), Q3'S or I3GA (1 mg/kg), or in the circulating blood. These metabolites would reduce vascular drug vehicle (100 mL of phosphate buffered saline). The acute tone after deconjugation in the vascular tissue. Therefore, the aim effect of an oral dose of quercetin (10 mg/kg) administered by of the present study was to analyse the long term in vitro effects of gavage on blood pressure and heart rate were also analysed.
the main plasma quercetin conjugates in resistance mesenteric In another set of experiments, SHR rats were daily given i.p. for arteries, their in vivo effects given intravenously on blood pressure 3 days either isotonic solution (1 mL) or D-saccharic acid 1.4- in SHR and the role of deconjugation via glucuronidase.
lactone (SAL), a specific inhibitor of beta-glucuronidase, (10 mg/ Moreover, we tested whether deconjugation is required for the mL in 1 mL) [25] before the administration of the flavonoids.
antihypertensive effects of oral quercetin aglycone.
Analysis of quercetin metabolites in rat plasma Materials and Methods Blood was collected into heparinized tubes and centrifuged. The plasma samples (300 mL) were extracted with 300 mL of methanol/0.5 M acetic acid (80:20, v/v) for 30 min at 25uC in All the experiments were performed in accordance with an ultrasonic bath, and then centrifuged for 3 min at 3500 g. The Institutional Guidelines for the ethical care of animals, and ethic supernatant was collected and the pellet was submitted to the same committee of the University of Granada approved this study (ref.
process two further times assisted by sonication (1 min) using a 2066/10). Twenty four-week old, male spontaneously hypertensive MicrosonTM ultrasonic cell disruptor (New York, USA). The rats (SHR) were obtained from Harlan Laboratories (Barcelona, methanolic extracts were combined and dried in a centrifugal Spain). All rats were maintained five per cage at a constant concentrator micVac (GeneVac, Ipswich, United Kingdom). The temperature (2461uC), with a 12-hour dark/light cycle and on residue was dissolved in 120 mL acetonitrile/water (30:70 v/v) and standard rat chow.
centrifuged (5 min, 3500 g) previous to its injection (100 mL) in theHPLC-DAD-ESI/MS system.
Analyses were carried out in a Hewlett-Packard 1100 chromatograph (Agilent Technologies, Waldbronn, Germany)with a quaternary pump and a DAD coupled to an HP ChemStation (rev. A.05.04) data-processing station. An AscentisTM RP-Amide 3 mm (2.16150 mm) column at 30uC was used. Thesolvents used were: (A) 0.1% formic acid, and (B) acetonitrile. Anelution gradient was established from 15 to 50% B over 15 min,isocratic 50% B for 10 min, from 50 to 75% B over 3 min,isocratic 75% B for 10 min, and re-equilibration of the column, ata flow rate of 0.2 mL/min. Double online detection was carriedout in the DAD using 370 nm as a preferred wavelength and in amass spectrometer connected to HPLC system via the DAD celloutlet. MS detection was performed in an API 3200 Qtrap(Applied Biosystems, Darmstadt, Germany) equipped with an ESIsource and a triple quadrupole-ion trap mass analyzer that wascontrolled by the Analyst 5.1 software. Zero grade air served as thenebulizer gas (30 psi) and turbo gas for solvent drying (400uC,40 psi). Nitrogen served as the curtain (20 psi) and collision gas(medium). The quadrupoles were set at unit resolution. The ionspray voltage was set at 24500 V in the negative mode. Precursorion analysis was employed to detect all the precursor ions thatfragment to a common product ion (i.e., m/z 301 correspondingto quercetin). Settings used were: declustering potential (DP) Figure 1. Structure of quercetin and its metabolites isorham- 240 V, entrance potential (EP) 210 V, collision energy (CE) netin, quercetin 3-glucuronide (Q3GA), isorhamnetin 3-glucu-ronide (I3GA) and quercetin 39-sulfate (Q3'S).
250 V, and cell exit potential 23 V. Enhanced product ion mode was further performed in order to obtain the fragmentation PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR pattern of the parent ion(s) of the studied transition in the previous (25 mM, pH 5.5 and pH 7.2) or ultra-pure water, containing in experiment using the following parameters: DP 250 V, EP 26 V, either case 10 mM MgCl2, UDP-glucuronic acid (8 mM) and CE 225 V, and collision energy spread 0 V. Quantitative analysis UDP-glucosamine (4 mM). I3G was isolated by semipreparative of the assayed flavonols and conjugated metabolites was performed HPLC. All other drugs were from Sigma (Tres Cantos, Madrid, from their chromatographic peaks recorded at 370 nm by comparison with calibration curves obtained by injection ofincreasing concentrations of quercetin, I3GA, and Q3GA.
Statistical analysis Results are expressed as the mean 6 SEM and n describes the Vascular reactivity in vitro number of measurements made (i.e., from different animals).
SHR were stunned and killed by cervical dislocation. The Differences between experimental groups were treated using mesentery was removed and placed in cold Krebs solution unpaired Student's t-test or, for multiple comparisons, using (composition in mmol/L: NaCl 118, KCl 4.75, NaHCO3 25, one-way analysis of variance followed by a Dunnett's post hoc test.
MgSO4 1.2, CaCl2 2, KH2PO4 1.2, and glucose 11). Third-order P values,0.05 were considered statistically significant.
arteries were cleaned of surrounding fat and mounted in anautomated tension myograph (Danish Myotechnology, Denmark) containing Krebs solution maintained at 37uC and gassed with 5%CO2 in O2. After an equilibration period of 45 min, vessels were Effects of plasma metabolites on blood pressure normalized according to published protocols and vessel diameter SHR showed a basal MBP of 18165 mm Hg and HR of determined [26]. Following normalization, relaxation of phenyl- 424614 bpm. Q3GA and I3GA (1 mg/kg i.v.) progressively ephrine (3 mM)-precontracted vessels to acetylcholine (Ach, 1 mM) reduced mean blood pressure (MBP) in SHR, while Q3'S was was used to determine endothelial integrity (vessels that relaxed by without effect. This hypotensive effect induced by both metabolites at least 50% were considered endothelium-intact).
was statistically significant after 1 and 2 h, respectively, of the In order to analyze the effects on vascular function, in metabolite injection. The maximum effects observed at 3 h were endothelium-intact rings a concentration–response curve was 14.961.8% and 11.461.8%, respectively (Figure 2A). No constructed by cumulative addition of phenylephrine (1027– significant changes in heart rate (HR) were observed with any 1024 M). Then vessels incubated in the absence or presence of metabolite (Figure 2B). Q3GA also decreases MBP at low quercetin, isorhamnetin, Q3'S, Q3GA or I3GA (10 or 25 mM) for concentrations (0.02 and 0.2 mg/kg) (Figure 2C), being also 30–120 min and a second concentration–response curve was without effects on HR (Figure 1D).
performed. In some arteries SAL (1 mM) was added 1 hour beforeand during the incubation period with Q3GA.
Time-course of the Q3GA concentrations in plasma When SHR were treated with Q3GA, 1 mg/kg i.v., there was b-glucuronidase activity an increase in the plasma concentration of this metabolite b-glucuronidase activity was measured by a colorimetric reaching 23.261.8 mM at 1 min and decreased rapidly (,1 mM analysis using phenolphthalein mono-b-glucuronide as the sub- at 30 min) (Figure 3A, 3B). Moreover, free quercetin aglycone and strate [22]. Briefly, 30 mg of protein of vascular mesenteric bed I3GA was detected in plasma after Q3GA injection.
homogenates from SHR were mixed with 0.6 mM phenolphtha-lein mono-b-glucuronide in 100 mL of 0.1 mM sodium phosphate Effects of metabolites in the reactivity of mesenteric buffer at pH 5. After incubation at 37uC for 30 min followed by adding 200 mL of 0.1 M sodium phosphate buffer pH 11, theabsorbance at 540 nm indicating the formation of phenolphtha- Phenylephrine induced a maximal contractile effect in mesen- lein aglycone was measured. In some experiments, SAL (1 mM) teric vessels from SHR of 19.460.9 mN (n = 20). When was added 1 hour before phenolphthalein mono-b-glucuronide mesenteric arteries from SHR were incubated with the aglycones quercetin or isorhamnetin for 30 min a significant concentration-dependent decrease in the vasoconstrictor response to phenyleph- rine was observed (Figure 4) while Q3GA at this time had no effect(Figure 5A). However, when the incubation of 25 mM Q3GA was Q3GA was isolated from green bean pods and stored as prolonged to 1 and 2 hours a significant reduction in the described [27]. Briefly, defated pods were homogenized in 70% MeOH, the concentrated extract was fractionated on a polyamide (Figure 5B and 5C).
column and washed firstly with phosphate buffer, then withmethanol and finally with methanol/ammonia (99.5:0.5 v/v) toelute the acidic flavonols (e.g. glucuronides). The glucuronide was Role of glucuronidase activity in the hypotensive and purified by semipreparative-HPLC. Q3'S was synthesized by an vascular effects induced by Q3GA adaptation of the method described by Jones et al. [28]. Briefly, To explore the possible role of deconjugation of Q3GA on the dehydrated quercetin was dissolved in dioxane and allowed to observed effects a specific inhibitor of beta-glucuronidase (SAL) react at 40uC for 90 min with a 10-fold molar excess of sulfur was used, which was administered i.p. during the 3 days before the trioxide-N-triethylamine complex under a nitrogen atmosphere.
blood pressure recordings. Interestingly, the hypotensive effect of Precipitated products of sulfation were redissolved in 10% Q3GA was abolished in SHR treated with SAL (Figure 6A). We methanol in water and the mixtures of quercetin sulfates were confirmed the inhibitory glucuronidase activity of SAL (1 mM) in fractioned on a Sephadex LH-20 column and Q3'S further homogenates from the mesenteric bed in in vitro conditions, by purified by semipreparative HPLC. I3GA was produced enzy- incubating during 1 h and measuring glucuronidase activity matically using pig liver microsomal enzymes with a modification (Figure 6C). We also found that the inhibitory effects of Q3GA of the methodology described by Plumb et al. [29]. Briefly, a post- in the contractile response induced by phenylephrine were lysosomal fraction was obtained from a pig liver extract, incubated suppressed when mesenteric arteries were incubated with SAL with isorhamnetin at 37uC for 240 min, in a Hepes buffer (Figure 6D), but not those of quercetin (Figure 6E).
PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR Figure 2. Effects of intravenous Q3GA, Q3'S, and I3GA (1 mg/kg) and Q3GA (0.02, 0.2 mg/kg) on mean arterial blood pressure (A, C)and heart rate (B, D) measured by direct carotid artery recording in a conscious rat. Results are means 6 SEM of 4–6 experiments.
* P,0.05 vs. saline.
doi:10.1371/journal.pone.0032673.g002 Role of glucuronidase activity in the hypotensive and in vitro effect of quercetin on the contractile response to vascular effects induced by quercetin phenylephrine in isolated mesenteric arteries.
The above results prompted us to analyze whether deconjuga- tion was also required for the antihypertensive effect of orally administered quercetin. Administration of quercetin (10 mg/Kg Fruit and vegetable consumption is associated with a decrease in using an intragastric gavage) induced a progressive decrease in blood pressure, which is an important cardiovascular risk factor MBP and HR during 6 hours of register. These reductions were [30]. Quercetin, the most important dietary flavonol, present in significant, as compared to vehicle, after 2.5 h of administration, multiple fruits and vegetables, reduces blood pressure in and reached a maximum of 2864% and 1862%, respectively at hypertensive animals and human after chronic consumption 6 h (Figure 7A, 7B). Importantly, when SHR were treated with [9,13,31–37]. Herein, we show for the first time that the SAL, oral quercetin was unable to induce changes in both MBP conjugated derivatives Q3GA and I3GA can exert antihyperten- and HR (Figure 7C, 7D). However, SAL was unable to modify the sive effects when administered intravenously. As previously Figure 3. Concentrations of quercetin, I3GA, and Q3GA measured in plasma from SHR treated with 1 mg/kg Q3GA. (A) HPLCchromatograms recorded at 370 nm of plasma samples taken at 1 min. (B) Time-concentration relationship. Results are means 6 SEM of 4experiments.
doi:10.1371/journal.pone.0032673.g003 PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR Figure 4. Effects of (A) quercetin and (B) isorhamnetin (10 or 25 mM, incubated for 30 min) on the contractile responses tophenylephrine in mesenteric resistance arteries. Control is treated with vehicle (DMSO). Results are means 6 SEM of 4–8 experiments.
* P,0.05 and **P,0.01 vs. control.
doi:10.1371/journal.pone.0032673.g004 reported Q3GA had no acute effect in vitro (at 30 min), however it published papers from other groups [15–18], the concentrations of developed with more prolonged incubations. Both the in vitro and methylated forms of quercetin are in the same range or higher the in vivo effects were prevented by the b-glucuronidase inhibitor than non methylated ones in rats and humans supplemented with SAL. Taken together these data strongly suggest that deconjuga- quercetin, suggesting that both forms may contribute to the tion is required for the effect of quercetin metabolites. Moreover, oral quercetin reduced blood pressure by almost 30% in SHR, When we analyzed the time course of the antihypertensive effect being this effect persistent at least during 6 hours and, importantly, and compared it to the plasma concentrations of Q3GA we found this effect was also prevented by SAL, indicating that the sequence a clear dissociation (Fig. 2A vs Fig. 3B). In our experimental of liver-intestine conjugation and local (vascular) deconjugation conditions, the dose of 1 mg/kg of Q3GA intravenously induced a processes is required for the antihypertensive effect of quercetin.
peak plasma concentration of ,25 mM which is higher than that Both human and rat tissues, except for the cells lining the previously reported by da Silva et al. [39] of 9.6 mM 6 h after intestine tract, are exposed to quercetin via the blood. However, 10 mg/kg quercetin delivered via oral gavage. However, Q3GA the free forms of quercetin and its methylated metabolite rapidly disappeared from the plasma, indicating that the two isorhamnetin are barely detected in plasma, which raises the modes of administration result in a completely different pharma- question of which is/are the compound(s) responsible for the cokinetic profile. The fast decay of Q3GA concentration in plasma antihypertensive activity. Because glucuronidated and sulfated is not compatible with renal excretion, suggesting that Q3GA is compounds are the only detectable metabolites, it is suggested that metabolized, accumulated in tissues or both. In a recent in vitro conjugated metabolites must play a decisive role in the possible study [24], the perfusion of Q3GA through the rat mesenteric beneficial effects [38]. Our results support this hypothesis, because vascular bed resulted in a partial accumulation of Q3GA in the we showed that Q3GA and I3GA, the main plasma metabolites of tissue and a progressive process of deconjugation. The resulting quercetin exerted an antihypertensive effect. Doses of Q3GA as aglycone was partly found in the extracellular buffer and mostly low as 0.02 mg/kg also significantly reduced blood pressure. In retained intracellularly. The beta-glucuronidase inhibitor SAL contrast, Q3'S was without effect. Herein, we show that both increased the tissue Q3GA and reduced the aglycone. Deconjuga- Q3GA and I3GA metabolites show a similar effect. Previous tion by beta-glucuronidase is expected to occur intracellularly Figure 5. Effects of Q3GA (10 or 25 mM) on the contractile responses to phenylephrine in mesenteric resistance arteries, after 30 (A),60 (B) or 120 (C) min of incubation. Results are means 6 SEM of 4–8 experiments. * P,0.05 and **P,0.01 vs. control.
doi:10.1371/journal.pone.0032673.g005 PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR Figure 6. Effects of Q3GA in arterial blood pressure (A) and heart rate (B) in SHR treated with SAL (10 mg/rat/day for 3 days) orvehicle (means ± SEM of 4 experiments). Panel (C) shows the bands of b-glucuronidase expression by Western blot and the b-glucuronidaseactivity and its inhibition by SAL (1 mM) in vascular bed homogenates (means 6 SEM of 8 experiments). (D) Effects of Q3GA (25 mM) on thecontractile responses to phenylephrine in mesenteric arteries after 120 min in the presence of SAL (1 mM) (means 6 SEM of 5 experiments). (E)Effects of quercetin (25 mM) on the contractile responses to phenylephrine in mesenteric arteries after 30 min in the presence of SAL (1 mM) (means6 SEM of 5 experiments). * P,0.05 and **P,0.01 vs. control.
doi:10.1371/journal.pone.0032673.g006 because this enzyme is located in the lysosomes and the endothelial cells and interact with the subintimal cells, such as the microsomal fraction. Therefore, the aglycone is formed within macrophages and smooth muscle cells [22,47]. Deconjugation of the vessel and probably in the cytosol of smooth muscle cells where the glucuronide metabolites of the flavonoids by increased b- it is expected to interact with its targets to exert vascular smooth glucuronidase activity at the site of inflammation has been muscle relaxation. The most plausible targets for this effect include suggested as a plausible mechanism for the protective effects of the protein kinases involved in the regulation of myosin-actin flavonoids in vivo [23,48]. Accordingly, the release of b-glucuron- interactions including protein kinase C, myosin light chain kinase idase is considered an index of lysosomal membrane integrity [49].
or Rho kinase and possibly potassium channels [8,9,40,41].
In fact, mesenteric bed from SHR expresses b-glucuronidase and Quercetin aglycone released to the plasma is likely to be rapidly its activity was significantly inhibited by SAL, a specific inhibitor.
re-conjugated in the liver explaining its low levels.
Vascular tissues from SHR showed increased expression of The vasorelaxant effects of quercetin and related metabolites proinflammatory markers, altered endothelial function, and have been widely assessed in vitro in aorta and perfused mesenteric increased macrophage infiltration than normotensive animals bed [7,8,42]. Increased alpha-adrenergic response in small [50,51], which could facilitate metabolite accumulation and mesenteric arteries has been involved in increased blood pressure deconjugation in this inflamed tissue. In our experiments, the in SHR [43,44]. As expected, both quercetin and isorhamnetin antihypertensive effect of Q3GA was abolished by b-glucuronidase incubated during 30 min, inhibited the contractile response inhibition, which suggests that this effect requires b-glucuronidase- induced by the alpha-adrenergic receptor agonist phenylephrine.
mediated deconjugation. Moreover, the inhibitory effect in the In the same experimental conditions, Q3GA did not modify this contractile response to phenylephrine in mesenteric arteries response. These results are consistent with previous data showing induced by Q3GA was also suppressed by SAL, showing that that conjugation of flavonoids results in a decreased biological Q3GA requires deconjugation to exert this inhibitory effect.
activity [45,46] and that conjugated metabolites have no direct Given the role of b-glucuronidase in the effects of Q3GA we vasorelaxant effect in isolated rat aorta at physiological concen- aimed to analyze whether it was also relevant for the antihyper- trations [21]. However, when small mesenteric arteries were tensive effect of quercetin. Surprisingly, despite several chronic incubated for 1 or 2 h with Q3GA, at 25 mM, the vasoconstriction studies, to our knowledge the effects of acute oral quercetin induced by phenylephrine was significantly reduced, suggesting administration on blood pressure in hypertensive animals had not that quercetin accumulates in this vascular bed and it is been studied. We found a slow developing but long lasting responsible of the reduced vascular tone. A similar scenario has antihypertensive effect. Remarkably, the effects of oral quercetin been described previously in which quercetin metabolites in were also abolished by b-glucuronidase inhibition with SAL.
circulating blood can permeate through the injured/activated However, as expected, the in vitro effects of quercetin were PLoS ONE www.plosone.org March 2012 Volume 7 Issue 3 e32673 Glucuronidated Quercetin in SHR Figure 7. Effects of oral quercetin (10 mg/kg) on arterial blood pressure (A, C) and heart rate (B, D) in SHR treated with SAL (10 mg/rat/day for 3 days) or vehicle (1 ml isotonic solution). Results are means 6 SEM of 4 experiments. *P,0.05 and **P,0.01 vs. quercetin vehicle(1 ml of 1% methylcellulose).
doi:10.1371/journal.pone.0032673.g007 unaffected by SAL. Thus, our data suggest that the biological hypercontractile response in resistance arteries. Quercetin could activity of quercetin is dependent on the conjugation-deconjuga- be initially inactivated by a conjugation metabolism during tion processes. Although decreased glucuronidation often results in absorption and then safely be delivered to inflamed arterial wall, increased activity and/or toxicity of drugs, paradoxically, and the recruited metabolites are incorporated and converted to glucuronidation seems to be required for the activity of quercetin.
the aglycone in vascular smooth muscle cells and exert the Therefore, glucuronidation may protect quercetin from its inhibitory activity on vascular tone. These results are in agreement metabolism via other pathways and help to carry the flavonoid with the hypothesis that flavonoid glucuronides appear to serve as to the tissues where the free aglycone is released [52]. Our data plasma transport metabolites to target cells rather than solely as also suggest that polymorphisms of UDP-glucuronosyltransferases (encoded by the UGT1 and UGT2 loci), which are common inhumans [53] and changes in the b-glucuronidase activity, might Author Contributions result in a variable response to quercetin.
In conclusion, we show that glucuronides of quercetin and its Conceived and designed the experiments: FP-V JD. Performed the methylated metabolite isorhamnetin are involved in the antihy- experiments: PG IR-G SG-M MD RJ CM. Analyzed the data: RJ FV JTCS-B JD. Wrote the paper: FP-V JD.
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In Pharmacy, IMS MAT Jan 2011 To Fever and Pain INCLUDES DR. KEELY S TIPS Effective relief you can trust A Parent's Guide to Fever and Pain The content of this guide has been drafted in conjunction with Dr. JimKeely, who has spent 6 years working in Paediatrics at three of the mainteaching hospitals in Ireland. Dr Keely entered general practice in 1994and currently works as a GP at the Seabury Medical Centre in Malahidewith a special interest in Paediatrics. He is also the father of five childrenand gives us his personal top tips on how to deal with pain and fever.

Microsoft word - laboratory rotations researchtopics msc iandi - 100827

Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics IMMUNOLOGY – LABROTATIONS & RESEARCH TOPICS Researchmaster Infection & Immuntiy Laboratory rotations & Reaearch topics Title: (Immuno)pathogenesis of chronic lymphocytic leukemia Workgroupleader: dr. A.W. Langerak T: 010-704 4089 E: [email protected] W: http://www.erasmusmc.nl/immunologie/onderzoek/moleculaireimmunologie/mid/?lang=en Background Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemic proliferation in the Western world. CLL is found in adults and typically associated with age. The majority of CLL cases is of B-cell type, while a minority derives from T lymphocytes (also called T-cell large granular lymphocyte leukemia, T-LGL). Over the last years it has become increasingly clear that CLL is a heterogeneous disease, with a variable clinical course and differences in survival. CLL is an example of a multi-factorial disease, in which both genetic and micro-environmental factors contribute to leukemogenesis. Although in recent years many studies have focused on prognostic markers, there is still no complete picture of the factors that are involved in the (immuno)pathogenesis and that are determining for the prognosis.