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Research Report

Quercitrin, a glycoside form of quercetin, prevents lipidperoxidation in vitro

Caroline Wagnera, Roselei Fachinettoa, Cristiane Lenz Dalla Cortea,Verônica Bidinotto Britoa, Diego Severoa, Gilvan de Oliveira Costa Diasb, Ademir F. Morelb,Cristina W. Nogueiraa, João B.T. Rochaa,⁎aCentro de Ciências Naturais e Exatas, Departamento de Química, Programa de Pós-Graduação Bioquímica Toxicológica,Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, BrazilbCentro de Ciências Naturais e Exatas, Departamento de Química, Universidade Federal de Santa Maria, RS, Brazil

A R T I C L E I N F O

⁎ Corresponding author. Fax: +21 55 220 8978E-mail address: jbtrocha@yahoo.com.br (J

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.05.084

A B S T R A C T

Article history:Accepted 26 May 2006Available online 7 July 2006

Reactive oxygen species have been demonstrated to be associated with a variety of diseasesincluding neurodegenerative disorders. Flavonoid compounds have been investigated fortheir protective action against oxidativemechanisms in different in vivo and in vitromodels,which seems to be linked to their antioxidant properties. In the present study, we examinetheprotectivemechanismof quercitrin, a glycoside formof quercetin, against theproductionof TBARS induced by different agents. TBARS productionwas stimulated by the incubation ofrat brain homogenate with Fe2+, Fe2+ plus EDTA, quinolinic acid (QA), sodium nitroprusside(SNP) and potassium ferricyanide ([Fe(CN)6]3−). Quercitrin was able to prevent the formationof TBARS induced by pro-oxidant agents tested; however, it was more effective againstpotassium ferricyanide ([Fe(CN)6]3−, IC50 = 2.5), than quinolinic acid (QA, IC50 = 6 μg/ml) andsodium nitroprusside (SNP, IC50 = 5.88 μg/ml) than Fe2+ (Fe2+, IC50 = 14.81 μg/ml), Fe2+ plusEDTA (Fe2+ plus EDTA, IC50 = 48.15 μg/ml). The effect of quercitrin on the Fenton reactionwasalso investigated (deoxyribose degradation). Quercitrin caused a significant decrease indeoxyribose degradation that was not dependent on the concentration. Taken together, thedata presented here indicate that quercitrin exhibits a scavenger and antioxidant role, andthese effects probably are mediated via different mechanisms, which may involve thenegative modulation of the Fenton reaction and NMDA receptor.

© 2006 Elsevier B.V. All rights reserved.

Keywords:QuercitrinOxidative stressQuinolinic acidTBARSDeoxyribose degradationFlavonoidsSodium nitroprussideFenton reaction

1. Introduction

Cell metabolism continuously produces reactive oxygenspecies (ROS) as by products of respiration and othermetabolicactivities (Azbill et al., 1997; Halliwell, 1994; Park et al., 2004).These reactive species can normally be handled by non-enzymatic and enzymatic antioxidant defenses (Rodriguez-Martinez et al., 2000; Santamaría et al., 2003). However, the

..B.T. Rocha).

er B.V. All rights reserved

imbalance between the antioxidant system and the overproduction of ROS has been associated with a variety ofhuman diseases such as atherosclerosis, cancer and neurode-generative diseases (Alexi et al., 2000; Ames et al., 1993;Johnson, 2004; Lohr, 1991; Halliwell, 1994; Witztum, 1994).

Of particular importance for the design of new therapeuticapproaches, natural and synthetic antioxidant compoundscan afford protection in a variety of in vitro and in vivomodels

.

Fig. 1 – Quercitrin structure.

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of toxicity (Bastianetto and Quirion, 2002; Bellé et al., 2004;Bixby et al., 2005; Burger et al., 2004, 2005; Cabrera et al., 2000;Cooke et al., 2005; Dominguez et al., 2005; Ghisleine et al. 2003;Gugliucci and Stahl, 1995; Gupta et al., 2003; Moridani et al.,2003; Nogueira et al., 2004; Pérez-Severiano et al., 2004; Steffenet al., 2005; Williams et al., 2004; Youdim et al., 2004).Therefore, the consumption of foods rich in natural antiox-idants is thought to be of preventive value to delay thedevelopment or to impede the manifestation of neurodegen-erative diseases in humans and animalmodels (Aruoma et al.,2003; Naidu et al., 2003).

Flavonoids are components of the human diet and arewidely found in vegetables (Hollman and Katan, 1997, 1999)and beverages (Filip et al., 2001; Gugliucci and Stahl, 1995).Moreover, it was recently demonstrated that the plasmaantioxidant status was significantly higher in animals treatedwith quercetin, suggesting that quercetin metabolites canretain some antioxidant activity when the o-catechol groupdoes not undergo conjugation reactions. In the same study, theauthors showed that plasmaquercetinmetabolites compete invivowithothermolecules for peroxynitrite (Justino et al., 2004).

Therefore, the study of the potential antioxidant activity offlavonoids has attracted the attention of researchers whointend to identify whether these compounds could beeffective antioxidant agents. Although literature data indicatethat different types of flavonoids are potent antioxidants, theexact mechanism(s) which underlie their protective effects isstill not completely understood. There are several points ofevidence in the literature indicating that polyphenols arereducing agents and free radical scavengers and they canparticipate in the regeneration of other antioxidants, such asvitamin E (Jovanovic et al., 1998; Rice-Evans et al., 1996).

Quercetin is the most abundant bioflavonoid found invegetable and fruits, and this compound is mainly present inthe glycoside form, for example, as quercitrin. Studies havedemonstrated that the absorption of quercetin glycosidescontained in onions was higher (52%) than that of quercetinaglycones (24%) (Hollmanet al., 1995). In linewith this, the 3-O-β-glucoside of quercetin is better absorbed than quercetin(Morand et al., 2000). Perhaps, because the glycoside formgivesphysical and chemical properties which are different fromthose of the aglycone forms. The sugar portion bound to theaglycone portion increases the solubility in polar solvents andconsequently improves absorption, through the utilization ofglucose transporters that are present in the intestinal mucosa(Gee et al., 1998). However, the majority of the studies havebeen carried out with the aglycone form and little is knownabout the biological properties of glycoside forms, due to thelack of commercial standards (Scalbert et al., 2002) (Fig. 1).

Quinolinic acid (QA), an endogenous tryptophan metabo-lite formed in the kynurenine pathway, is a potent neurotoxinand a selective NMDA subtype of the glutamate receptoragonist (Scalbert, 1993). In vitro studies have demonstratedenhanced cytosolic calcium concentrations, ATP exhaustion,GABA depletion and superoxide radical formation (Foster etal., 1983), oxidative stress and lipid peroxidation induced byQA (Bellé et al., 2004; Puntel et al., 2005; Ríos and Santamaría,1991; Vega-Naredo et al., 2005). Behavioral alterations oxida-tive stress and lipid peroxidation are important featuresobserved in vivo as a result of QA-induced neurotoxicity

(Perez-de la Cruz et al., 2005; Rossato et al., 2002; Susel et al.,1989; Stýpek et al., 1997).

Sodium nitroprusside (SNP) has been suggested to causecytotoxicity via the release of cyanide and/or nitric oxide(Bates et al., 1991; Chen et al., 1991; Dawson et al., 1991;Rauhala et al., 1998). There are several studies concerning therole of NO in the pathophysiology of strokes, traumas,seizures and Alzheimer's, and Parkinson's diseases (Bolanosand Almeida, 1999; Castill et al., 2000; Prast and Philippou,2001; Weisinger, 2001). It is known that light exposurepromotes the release of NO from SNP through a photode-gradation process (Arnold et al., 1984; Singh et al., 1995), anddata from the literature have demonstrated that after therelease of NO, SNP or [NO–Fe–(CN)5]2− is converted to ironcontaining [(CN)5–Fe]3− and [(CN)4–Fe]2− species (Loiacono andBeart, 1992). After the release of NO, the iron moiety mayreact with SNP, which could lead to the formation of highlyreactive oxygen species, such as hydroxyl radicals via theFenton reaction (Graf et al., 1984).

The aims of this study were to investigate the antioxidantaction of quercitrin, the glycoside form of quercetin, in ratbrain lipid peroxidation induced by different agents. Inaddition, we investigated whether the antioxidant mecha-nism of quercitrin involves the Fenton reaction.

2. Results

2.1. Fe2+ and Fe2+/EDTA × quercitrin

Statistical analyses revealed that Fe2+ caused a significantstimulation of brain TBARS formation (P < 0.01), whereasquercitrin caused a reduction in the TBARS productioninduced by Fe2+. Fe2+ plus EDTA caused a similar increase inTBARS production to that caused by Fe2+ alone, and quercitrinwas also able to reduce the stimulatory effects of Fe2+.Furthermore, quercitrin caused a significant reduction inTBARS production under basal conditions (Fig. 2).

2.2. Quinolinic acid × quercitrin

QAcauseda significant increase inbrainTBARSproduction, andthe effect of QA was abolished by quercitrin (P < 0.001) (Fig. 3).

Fig. 3 – Effect of different concentrations of quercitrin onbasal (control), QA (2 mM)-induced TBARS production inbrain homogenates. The homogenate was incubated for 1 hwith quinolinic acid in the presence or absence of quercitrin.Data show mean ± SEM. Values average from 3 to 4independent experiments performed in duplicate. *, **,***Represent differences in relation to induced by QA and *,***, @compared to basal (0 μg/ml).

Fig. 2 – Effect of different concentrations of quercitrin onbasal (control), Fe2+ (100 μM) or Fe2+/EDTA (100 μM)-inducedTBARS production in brain homogenates. The homogenatewas incubated for 1 h with Fe2+ or Fe2+/EDTA in the presenceor absence of quercitrin. Data show means ± SEM valuesaverage from 3 to 4 independent experiments performed induplicate. @Represent differences in relation to induced byFe2+, **differences in relation to that induced by Fe2+/EDTAand *compared to basal.

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2.3. Sodium nitroprusside × quecitrin

SNP caused a significant increase in brain TBARS production,and theeffect ofSNPwasabolishedbyquercitrin (P<0.05) (Fig. 4).

2.4. Ferricyanide, fresh and photodegraded sodiumnitroprusside × quercitrin

The pro-oxidant effect of SNP can be a result of the release ofNO, cyanide or both In order to clarify this aspect, we tested thepro-oxidant effect of photodecomposed SNP and [Fe(CN)6]3− andcompared them to that of freshly prepared SNP. The resultspresented in Figs. 5a, b and c indicate that fresh SNP was themost potent inducer of TBARS formation, followed by photo-decomposed SNP. [Fe(CN)6]3− had a modest but statisticallysignificant pro-oxidative effect. It is known that NO can reactwith the ironmoiety of the SNP,which can lead to the formationof highly reactive oxygen species, such as hydroxyl radicals viathe Fenton reaction. (Arnold et al., 1984; Graf et al., 1984;Loiacono and Beart, 1992; Singh et al., 1995). In our experiment,we used potassium ferricyanide to compare with photode-gradednitroprusside. [Fe(CN)6]3−producedan increase inTBARSlevels of around 33% in relation to the control, and quercitrinprotected against this effect in a concentration-dependentmanner. Quercitrin reduced TBARS levels induced by freshSNP, photodegraded SNP and [Fe (CN)6]3− with F(3,27) = 42,98;F(3,27) = 12,97; F(3,27) = 4,94, respectively, and P < 0.01.

2.5. Deoxyribose degradation × quercitrin

Deoxyribose degradation was stimulated by H2O2 stimulated2.5 times and Fe2+ plus H2O2 stimulated 3.5 times respectively.

Quercitrinwas able to reduce deoxyribose degradation by H2O2

(22%) and by Fe2+ plus H2O2 (33%) (Fig. 6).

3. Discussion

The results of the present study show that quercitrinprevented the lipid peroxidation in brain homogenatesinduced by different agents.

Taken together, these results can indicate that theantioxidant properties of quercitrin, a glycoside form ofquercetin, depend on the agent used to induce lipid peroxida-tion. When SNP and QA were used to induce oxidative stress,lower quercitrin concentrations (10 μg/ml) were required toreduce TBARS to basal levels, as compared with concentra-tions required in the presence of free iron or Fe2+/EDTA (50–100 μg/ml). We suppose that, in the first case, quercitrinexhibits protection via its scavenger properties acting as oneelectron donor (Bors et al., 2001; Bors and Michel, 1999;Jovanovic et al., 1998; Rice-Evans et al., 1996). In the presenceof free iron, the chelating properties of quercitrin, which inflavonoids depend on the cathecol ring (Rice-Evans et al.,1996). In line with these, the antioxidant activities offlavonoids are believed to be associated with their chemicalstructure and the two hydroxyl groups in the catechol B-ring.These groups can act by allowing donation of hydrogenstabilizing radical species. (Bors and Michel, 1999; Bors et al.,2001; Rice-Evans et al., 1996). Moreover, other structuralfeatures important to their antioxidant properties includethe presence of 2,3 unsaturation in conjugation with a 4-oxo-function in the C-ring and the presence of functional groupscapable of binding transition metal ions, such as iron andcooper (Rice-Evans et al., 1996).

Fig. 5 – Effect of different concentrations of quercitrin on (A)Fresh SNP (5 μM), (B) photodecomposed SNP (5 μM) or (C) [Fe(CN)6]3− (5 μM)-induced TBARS production in brainhomogenates. The homogenate was incubated for 1 h withdifferent indutors in the presence or absence of quercitrin.Data show mean ± SEM. Values average from 3 to 4independent experiments performed in duplicate. **,***Represent differences in relation to induced by SNP and *,**, ***compared to basal (0 μg/ml).

Fig. 4 – Effect of different concentrations of quercitrin onbasal (control) or SNP (5 μM)-induced TBARS production inbrain homogenates. The homogenate was incubated for 1 hwith SNP in the presence or absence of quercitrin. Data showmean ± SEM. Values average from 3 to 4 independentexperiments performed in duplicate. *, **, ***Representdifferences in relation to induced by SNP and *, ***,@compared to basal (0 μg/ml).

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The pro-oxidant effects of QA are attributed to sustainedstimulation of NMDA receptors, which induces free radicalproduction and posterior lipid peroxidation (Pérez-Severianoet al., 2004; Rice-Evans and Burdon, 1993; Santamaría et al.,2003). In addition, data in the literature show that flavonoidscan act as neuroprotector agents in vivo models, and thisaction can be related to NMDA antagonistic effect (Naidu et al.,2003; Xu et al., 2005).

However, literature data support that not all the neurotoxicactions produced by QA can be explained by its properties asan NMDA receptor agonist, since at least a fraction of itstoxicity is related with its pro-oxidant nature, which seems tobe independent of NMDA receptors (Behan et al., 1999;Santamaria et al., 2001). In fact, under in vitro conditions QAis able to form complexes with iron (II) (Goda et al., 1996),which in turn may stimulate the Fenton reaction (Iwahashi etal., 1999), suggesting that QA-induced lipid peroxidation isdependent on iron (Stipek et al., 1997).

Indeed, the results depicted in Fig. 5 may reinforce the ideathat sodium nitroprusside cause cytotoxicity via either releaseof cyanide and/ornitric oxide (Bates et al., 1991;Chenet al., 1991;Dawson et al., 1991; Rauhala et al., 1998), because lipidperoxidation was more pronounced after exposure of brainhomogenates to the fresh SNP than to ferrous or lightdecompose SNP. Data of literature demonstrated that after therelease of NO, sodium nitroprusside or [NO–Fe–(CN)5]2− isconverted to iron containing [(CN)5–Fe]3− and [(CN)4–Fe]2−

species (Loiacono and Beart, 1992) and the iron moiety mayreactwith SNPwhich could leads to formationof highly reactiveoxygen species, such as hydroxyl radicals via the Fentonreaction (Graf et al., 1984). In this vein, we demonstrated thatthe antioxidant and protective action of quercitrin involves themodulationofFentonreaction.Theglycosides formofquercetinreduces significantly the deoxyribose degradation.

Fig. 6 – Effect of different concentrations of quercitrin onbasal, H2O2 (1 mM) or Fe2+/H2O2-induced deoxyribosedegradation. The deoxyribose was incubated for 20 min withH2O2 or Fe2+/H2O2 in the presence or absence of quercitrin.Data shows mean ± SEM. Values average from 3 to 4independent experiments performed in duplicate.*Represents differences in relation to induced by Fe2+ plusH2O2.

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The glycosides form of quercetin was able to prevent lipidperoxidation in the presence of different inducers. Theseresults are interesting, because flavonoids are widely found inhuman foods (Hollman and Katan, 1997, 1999), and predom-inantly as beverages (Filip et al., 2001; Gugliucci and Stahl,1995). We realize that in vivo quercitrin can be a moreimportant antioxidant and neuroprotective agent than quer-cetin because its high bioavailability in at the digestive track.However, there were a few in vivo studies with this glycoside(Comalada et al., 2005; Camuesco et al., 2004).

In conclusion, the results of the present investigationsupport a scavenger and antioxidant role of quercitrin andthis action probably involves the participation of the Fentonreaction and NMDA receptors. However, the antioxidantactivity of these compounds varied considerably dependingon the agent used to induce TBARS of particular clinical andnutritional importance, quercitrin was more effective againstquinolinic acid and SNP, induced lipoperoxidation, whichmayindicate that this compound is more promising againstpathologies associated with over activation of NMDA and noformation, such as ischemia. In line with this, literature datasupport a protective dose of flavonoids against brain ischemiain rodents (Lois et al., 2004). Additional studies are necessaryfor investigate themechanism responsible for protective effectobserved in lipid peroxidation induced by QA, and moreover,the effects of quercitrin in vivo models of oxidative stress.

4. Experimental procedures

4.1. Chemicals

Tris–HCl, QA, thiobarbituric acid and malonaldehyde bis-(dimethyl acetal) (MDA) were obtained from Sigma (St.Louis, MO, USA). Sodium nitroprusside was obtained from

Merck (Darmstadt, Germany). Ferrous sulphate, ethylene-diamintetracetic (EDTA), hydrogen peroxide, chloridric acidand acetic acid were obtained from Merck (Rio de Janeiro,RJ, Brazil).

4.2. Quercitrin

Quercitrin (Fig. 1) was isolated from Solidago microglossa D.C.and tested at concentrations of 0–100 μg/ml. The purity of theisolated compound was 99.3%.

4.3. Animals

Male Wistar rats (±3 months old), weighing between 270and 320 g, from our own breeding colony (Animal House-holding, UFSM, Brazil) were kept in cages with free access tofoods and water in a room with controlled temperature(22 ± 3 °C) and in 12-h light/dark cycle with lights on at 7:00am. The animals were maintained and used in accordanceto the guidelines of the Committee on Care and Use ofExperimental Animal Resources, School of veterinary med-icine and Animal Science of the University of Sao Paulo,Brazil.

4.4. Tissue preparation

Rats were decapitated under mild ether anesthesia, and thecerebral (whole brain) tissue was rapidly dissected, placed onice and weighed. Tissues were immediately homogenized incold 10 mM Tris–HCl, pH 7.5 (1/10, w/v). The homogenatewas centrifuged for 10 min at 4000 × g to yield a pellet thatwas discarded and a low-speed supernatant (S1).

4.5. TBARS production

Just after the end of centrifugation, an aliquot of 200 μl or of S1was incubated for 1 h at 37 °C and then used for lipidperoxidation determination with pro-oxidants agents inpresence or absence of flavonoid quercitrin. TBARS productionwas determined as described by Ohkawa et al. (1979) andRossato et al. (2002).

4.6. Deoxyribose degradation

Deoxyribose degradation was determined by Halliwell et al.(1987). Deoxyribose is degraded by hydroxyl radicals with therelease of thiobarbituric acid (TBA) reactive material. Deoxy-ribose (3 mM) was incubated at 37 °C for 30 min with 50 mMpotassium phosphate(pH 7.5) plus ferrous sulphate (0.1 mM)and/or H2O2 (1 mM) to induce deoxyribose degradation, andquercitrin at a concentration of 4–20 μg/ml. After incubation,0.4 ml of TBA 0.8% and 0.8 ml of TCA 2.8% were added, andthe tubes were heated for 20 min at 100 °C and spectropho-tometric measured at 532 nm.

4.7. Statistical analysis

Data were analyzed statistically by one-way ANOVA, fol-lowed by Duncan's multiple range tests when appropriate.The results were considered statistically significant forP < 0.05.

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Acknowledgments

The financial support by CAPES, CNPq, FAPERGS is gratefullyacknowledged.

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