Research ArticleReceived: 17 August 2013 Revised: 8 February 2014 Accepted article published: 14 February 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6620

Onion extract (Allium cepa L.), quercetinand catechin up-regulate paraoxonase 1activity with concomitant protection againstlow-density lipoprotein oxidation inmaleWistar rats subjected to oxidative stressNidhi Jaiswala and Syed Ibrahim Rizvib*

Abstract

BACKGROUND: Paraoxonase 1 (PON1) protects the oxidative modification of low-density lipoprotein (LDL) and is a majoranti-atherosclerotic protein component of high-density lipoprotein. We explored the effect of onion extract and flavonoids(quercetin and catechin) in the regulation of PON1 expression and correlating with oxidised LDL levels in male Wistar ratssubjected to mercuric chloride (HgCl2) induced oxidative insult. Rats were divided into eight groups: Control, Experimental(HgCl2), Experimental+onion/catechin/quercetin, Positive control (Normal+onion/catechin/quercetin). Treatment continuedfor 4 weeks.

RESULTS: PON1 activity and radical scavenging activity decreased in the Experimental group (P < 0.001) with increasedsusceptibility of LDL for oxidation and plasma malondialdehyde levels (P < 0.001). Onion extract significantly attenuated theadverse effects of HgCl2 by up-regulating PON1 activity (P< 0.05), radical scavenging activity (P< 0.01), and protected againstLDL oxidation (P < 0.001) and lipid peroxidation (P < 0.01). Similar effects were observed with quercetin and to a lesser extentwith catechin.

CONCLUSIONS: The findings may explain the anti-atherosclerotic effect of onion and also foods containing quercetin andcatechins.© 2014 Society of Chemical Industry

Keywords: onion; quercetin; catechin; oxidative stress; paraoxonase 1; low-density lipoproteins

INTRODUCTIONFlavonoids are a diverse group of low molecular weight naturallyoccurring phenolic constituents, ubiquitously present in plants,which have been reported to exert health beneficial effects indegenerative processes associated with oxidative stress.1,2 Theyhave been reported to scavenge reactive oxygen species, chelatemetal ions, act as chain-breaking antioxidants by scavenginglipid peroxyl radicals, or integrate into the lipid bilayer to pre-vent lipid damage.3–5 Quercetin (abundant in onion, apple, broc-coli and berries) and catechin (abundant in tea and fruit) areamong the most widely distributed flavonoids. These compoundshave been linked to a variety of pharmacological effects, such ascardiovascular protection, anti-cancer activity, anti-ulcer effects,anti-allergic activity, cataract prevention, antiviral activity andanti-inflammatory effects.6,7 Onion (Alliumcepa L.) is a good sourceof activated phytomolecules such as phenolic acids, flavonoidsespecially quercetin, cepaenes, thiosulfinate, organosulfur com-pounds and anthocyanin,8 with proven antioxidant properties andprotective effects against different degenerative pathologies suchas cardiovascular and neurological diseases, cancer and other dys-functions based on oxidative stress.9

Oxidative imbalance in the body has been implicated in theaetiology of several degenerative diseases including cancers, car-diovascular diseases, renal diseases, neurological disorders such asAlzheimer or Parkinson disease, and diabetes.10,11 Atherosclerosisis initiated by oxidative modification of low-density lipopro-tein (LDL) in the arterial wall cells including macrophages.High-density lipoprotein (HDL) can remove excess cholesterolfrom arteries and its serum levels are inversely related to the riskof developing atherosclerosis.12 Serum paraoxonase1 (PON1), acalcium-dependent esterase synthesised in the liver and closelyassociated to HDL, containing both apolipoprotein A1 andapolipoprotein J (apoA1 and apoJ), is known to be related to theanti-atherogenic properties of HDL.13

∗ Correspondence to: Syed IbrahimRizvi, Department of Biochemistry, Universityof Allahabad, Allahabad 211002, India. E-mail: sirizvi@gmail.com

a Centre of Food Technology, University of Allahabad, Allahabad 211002, India

b Department of Biochemistry, University of Allahabad, Allahabad 211002,India

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www.soci.org N Jaiswal, S I Rizvi

Experimental evidence has shown that PON1 functions as anantioxidative enzyme that inhibits the oxidation of LDL, thuslowering the risk of developing atherosclerosis.14 It has beenshown that PON1 expression and activity can be modulated bydietary polyphenols.15,16 The present study has been undertakento explore the relationship between PON1 activity and the suscep-tibility of LDL oxidation in a rat model of experimental oxidativestress and the effect of supplementation with onion extract andflavonoids (quercetin and catechin).

EXPERIMENTALChemicalsQuercetin, (+)-catechin, and 2,2-diphenyl-1-picrylhydrazyl freeradical (DPPH) were obtained from Sigma–Aldrich (St Louis, MO,USA). All other chemicals were of analytical grade procured fromMerck (Mumbai, India) or Himedia (Mumbai, India).

Preparation of extractFresh onion bulbs were purchased from a local market in Alla-habad, India. Among the various local varieties of onion in India,the red onion (Pusa cultivar) was selected because of its reportedhigh antioxidant potential.17 The plant was collected, its botanicalidentification and authentication was confirmed, the herbariumsheets were sent to the herbarium at the Department of Botany,University of Allahabad and a voucher specimen number (refer-ence number 25840) was obtained. The procedure for the prepa-ration of extract was based on a reported methodology.18 In brief,the bulbs were carefully dressed and chilled (4 ∘C). About 100mLof chilled, distilled water per 100 g of onion were added andcrushed in a grinding machine. The resultant slurry was squeezedand filtered through a fine cloth and the filtrate was quickly chilled(4 ∘C) until given to rats through gavage.

Animal model and study protocolThe experiment was carried out with 48 male Wistar rats(5± 0.5months and body weight 150± 20 g). They were housedin a temperature controlled room (25± 5 ∘C) with 12 h light–darkcycles for at least 1 week. After the stabilisation period of 1week, the rats were randomly divided into eight groups, con-taining six animals each. Onion extracts and flavonoids (catechinand quercetin) were administered separately by gavage for 30days. Flavonoids under certain reaction conditions, can displaypro-oxidant activity,19 thus, we selected optimal dosages for theflavonoid treatments as reported in previous studies.5

• Group I: Control, receiving no treatment/supplementation• Group II: Onion extract treated group (1mL 100 g−1 bodyweight

per day) for 30 days• Group III: Quercetin-only group; quercetinwas dissolved in 0.5%

DMSO and rats were treated per day via gavage (20mg kg−1

body weight) for 30 days• Group IV : Catechin-only group; catechin was dissolved in 0.5%

DMSO and rats were treated per day via gavage (20mg kg−1

body weight) for 30 days• Group V : (negative control): Rats were injected intraperitoneally

with mercuric chloride (HgCl2) 5mg kg−1 body weight in 0.9%NaCl (Augusti et al.20)

• Group VI: Onion extract plus HgCl2-treated group; onion extract(1mL 100 g−1 body weight per day) was given via gavage 10days before HgCl2 injection and onion extract continued fornext 30 days

• Group VII: Quercetin plus HgCl2-treated group, quercetin dis-solved in 0.5%DMSO (20mg kg−1 bodyweight day−1)was givenvia gavage 10 days before HgCl2 injection and quercetin contin-ued for up to 30 days

• Group VIII: Catechin plus HgCl2-treated group; catechin dis-solved in 0.5%DMSO (20mg kg−1 bodyweight day−1)was givenvia gavage 10days beforeHgCl2 injection and catechinwas con-tinued for up to 30 days

Collection of blood, isolation of red blood cells and plasmaAfter the experimental period, rats were sacrificed under lightanesthaesia. Blood samples were collected by cardiac punctureinto anticoagulant syringes rinsed with 10 U mL−1 heparin, andthen red blood cells were pelleted by centrifugation at 800× g for10min at 4 ∘C. The plasma was immediately frozen at −80 ∘C forbiochemical assays. The protocol of the studywas approved by theAnimal Care and Ethics Committee of the University of Allahabad.

Paraoxonase 1 activityThe PON1 assay was performed by the method developed byAyub et al.21 Enzyme activity towards phenyl acetate (arylesteraseactivity) was determined by measuring the initial rate of substratehydrolysis in the assay mixture (3mL) containing 2mmol L−1 sub-strate (phenyl acetate), 2mmol L−1 CaCl2 and 10 μL of plasma in100mmol L−1 Tris-HCl (pH 8.0). The absorbance wasmonitored for3min at 270 nm and the activity was calculated from E270 = 1310per mol cm−1. The results are expressed in U mL−1, 1 U ofarylesterase hydrolyses 1 μmol phenyl acetate min−1.

Low-density lipoprotein oxidationThis assay was performed according to the method developedby Schnitzer et al.22 The rate of LDL oxidation was measured inthe assay mixture (2mL) containing 0.72mmol L−1 sodium citrate,90 μmol L−1 copper chloride and 40 μL of plasma in 10mmol L−1

phosphate buffer (pH 7.4). Absorbance was monitored at 245 nmfor 3000 s and a graphwas plotted for absorbance versus time. LDLoxidation was obtained by measuring oxidation at 3000 s.

Radical scavenging activity of plasmaThis assay was performed according to the method proposedby Szabo et al.23 One hundred microlitres of plasma was addedto 10mmol L−1 phosphate buffer (1.9mL), 0.1mmol L−1 DPPH inmethanol (2.0mL) with a control having 2mL of 10mmol L−1

phosphate buffer with same amount of DPPH solution. It was keptfor incubation for 30min at 21 ∘C and centrifuged for 5min at1000× g. Absorbance was measured at 517 nm with methanol asa blank. Values were compared for control (A0) and plasma (A) and% radical scavenging activity was calculated as [(A0 −A)/A0]× 100.

Plasma lipid peroxidationPlasma lipid peroxidation was measured according to the methodof Esterbauer and Cheeseman,24 with slight modification. Plasma(0.2mL) was added to 1mL of 10% trichloroacetic acid and2mL of 0.67% thiobarbituric acid boiled for 20min at 90–100 ∘C,cooled, the mixture was centrifuged at 1000× g for 5min and theabsorbance of supernatant was read at 532 nm. The concentra-tion of malondialdehyde (MDA) in plasma was calculated usingextinction coefficient (� = 31 500) and is expressed as nmoL mL−1

of plasma.

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Figure 1. Effect of onion extract, quercetin and catechin on HgCl2 induced oxidative stress on paraoxonase 1 (PON 1) arylesterase activity in vivo in Wistarrats. (A) *P< 0.05 and **P< 0.001 as compared to control. (B) #P < 0.0001 as compared to the control, *P < 0.05 and **P< 0.001 as compared to the HgCl2group.

Figure 2. Effect of onion extract, quercetin and catechin on HgCl2-induced oxidative stress on induced LDL oxidation measured as a function of increasein absorbance at 245 nm for 3000 s.

Statistical analysisAll data are presented as means± SEM and statistical analyseswere conducted using the software PRISM version 5.01. To assessrelationships between parameters and differences among treat-ments were determined using a t-test.

RESULTSTreatment with onion extract caused an increase (7.7%) in PON1activity (P< 0.05), treatment with quercetin resulted in signif-icantly higher PON1 activity (20.5%) as compared to control(P< 0.001) (Fig. 1A). We observe a significant decrease (28.3%) inPON1 activity (P< 0.0001) in HgCl2-induced oxidatively stressedrats (Fig. 1B). However, supplementation of onion extract (P< 0.05)and quercetin (P< 0.001) to oxidatively stressed rats significantlyimproved the PON 1 levels (18.06% and 31.5%) as compared to theHgCl2 group (Fig. 1B). Catechin showed no significant increase inPON 1 activity when given alone and also upon supplementationto oxidatively stressed rats.Supplementation of quercetin and catechin alone, significantly

(P< 0.05) decreased the plasma ox-LDL levels as compared to con-trol, onion extract showed little effect on plasma ox-LDL levels(Fig. 2A). An increase in susceptibility of LDLwas observed inHgCl2

treated oxidatively stressed rats (Pearson’s r= 0.9970, P< 0.001);however, supplementation of onion extract, quercetin and cate-chin significantly (P< 0.05) decreased the plasma ox-LDL levels inoxidatively stressed rats as compared to the HgCl2 group (Fig. 2B).Significantly elevated levels of radical scavenging activity of

plasma (measured by using the DPPH radical) (P< 0.05 andP< 0.01) (Fig. 3A), with no statistically significant change in MDAlevels in plasma were observed (Fig. 4A), when treated alone withonion, quercetin and catechin groups. However, HgCl2 treatmentsignificantly depleted the radical scavenging activity (Fig. 3B) andincreased theplasmaMDA levels (P< 0.001) (Fig. 4B). Co-treatmentof onion extract, quercetin and catechin with HgCl2 significantly(P< 0.01) improved the radical scavenging activity as comparedto HgCl2 group (Fig. 3B). The plasma MDA level was significantlydecreased at the end of the fourth week on supplementation ofonion extract, quercetin and catechin in oxidatively stressed ratscompared with the HgCl2 group (P< 0.05, P< 0.001 and P< 0.01)(Fig. 4B).

DISCUSSIONPrevious studies have shown that HgCl2 treatment of rats resultsin the production of reactive oxygen species which in turn

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Figure 3. Effect of onion extract, quercetin and catechin on HgCl2-induced oxidative stress on DPPH radical scavenging activity in vivo in Wistar rats. (A)**P < 0.05 and *P < 0.01 as compared to the control. (B) #P < 0.001 as compared to control, *P < 0.01 as compared to the HgCl2 group.

Figure 4. Effect of onion extract, quercetin and catechin on HgCl2-induced oxidative stress on plasma malondialdehyde (MDA) content as a free radicalmediated oxidative stress marker in vivo in Wistar rats. (A) No significant difference as compared to control. (B) #P < 0.001 as compared to control, *P <

0.05, **P < 0.001 and †P < 0.01 as compared to the HgCl2 group.

generates oxidative stress.20 Increased oxidative stress can leadto lipid, protein and DNA oxidation and alter the redox statusof the cell. Oxidative modification of LDL takes place in thesub-endothelial space of the arterial wall and a certain amountof ox-LDL is released into the circulation with increased intra-cellular formation of reactive oxygen species resulting intoox-LDL-induced formation of atherosclerotic plaques.25,26

In the present study, PON1 activity was significantly reduced inthe HgCl2-treated group when compared with the control group.However, an increased ox-LDL was observed in rats treated withHgCl2. The decreased serum PON1 activity could be related to theoxidative stress generated by HgCl2. It is well known that PON1loses its activity in an oxidative environment.27 Therefore, any fac-tor that affects the status of oxidative stress will also affect PON1activity.28 Reduced PON1 activity is observed in several chronicdiseases, including type 1 and type 2 diabetes, hypercholestero-laemia and during human ageing.29,30 The age-dependent effectof the PON1 genotype has been investigated recently, showingthe association of human longevity with PON1 status and itsrole in delaying the onset of the major age-related diseases,including cardiovascular diseases.31 Seres and colleagues32 inves-tigated the factors influencing PON1 activity and as a function ofage. They reported a significant decrease in PON1 activity withage without a change in its serum concentration. It was alsoobserved that HDL from elderly subjects was more susceptible to

oxidation than HDL from young subjects measured in termsof lipid peroxidation rate.32 Reduced serum PON1 activity andincreased oxidative stress was also shown in E0 mice and in dys-lipidaemic obese mice.33 Thus, PON1 activity has been suggestedto play an antioxidant defence role.34 Our study suggests thatthe increase in the susceptibility of LDL for oxidation is due tothe decrease in serum PON1 arylesterase activity subsequent tooxidative stress generated by HgCl2.Under oxidative stress, lipid peroxidation affects not only

lipoproteins but also cellular lipids.35 Oxidative stress-inducedperoxidation of membrane lipids can be very damaging becauseit leads to alterations in the biological properties of the mem-brane, such as the degree of fluidity, and can lead to inactivation ofmembrane-bound receptors or enzymes,which in turnmay impairnormal cellular function and increase tissue permeability.36,37 Lipidperoxidation generates a variety of relatively stable decomposi-tion end products, such as MDA which can then be measured inplasma as an indirect index of oxidative stress. In this study, HgCl2treatment increased lipid peroxidation by generating free radicalsresulting in significantly elevated levels of MDA in plasma.The present study demonstrates for the first time that the oxida-

tively stressed rats supplemented with onion extracts showedincreased PON1 activity, though not to the levels of the con-trol group. These results demonstrate that the constituents ofonion extracts (mainly quercetin) may be able to modulate the

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expression level of PON1 and enhance its activity, which is inacti-vatedbyoxidative stress.Ourpresent study alsodemonstrates thatquercetin significantly up-regulates PON1 activity to the greaterlevel in oxidatively stressed rats. Our findings are supported bythe report showing that dietary supplementation with quercetinup-regulates PON1 expression in the liver of laboratory mice.38

In vivo studies have revealed an increase of PON1 serum activityfollowing treatment with flavonoids.15,39,40 Unlike quercetin, cat-echin showed little effect on PON1 activity, despite the fact thatcatechins are known to be relatively potent free radical scavengersin vitro,4 indicating that the free radical scavenging properties offlavonoids in vitro do not seem to be positively associated withtheir PON1-inducing activity. Among various dietary polyphenoliccompounds, catechin was a poor inducer of PON1 mRNA andPON1 transactivation as indicated in studies.15,41

The protection of lipoproteins (LDL and HDL) from oxidativemodification is probably the result of the ability of paraoxonaseto hydrolyze specific oxidised lipids in oxidised lipoproteins andhuman atherosclerotic lesions.42 It was shown previously thatcovalent linkage of lipid peroxidation products to the LDL protein,as well as the accumulation of lipid peroxides in LDL, is diminishedin thepresenceof PON1.43 Dietary supplementationwithpolyphe-nolic antioxidants inhibits LDL oxidation and macrophage foamcell formation and attenuates development of atherosclerosis inanimals.27 The inhibition of LDL oxidation by polyphenols couldbe related, at least in part, to a direct effect of the polyphenolson the LDL, since both quercetin and catechin were found to bindto the LDL particle via the formation of an ether bond. We thusconclude that dietary consumption of onion or its polyphenolicflavonoids quercetin and, to a lesser extent, catechin is associatedwith reduced susceptibility of LDL to oxidation and aggregation.We have shown in a recent study that supplementation of

onion as well as flavonoids improves antioxidant potential inHgCl2-treated rats.44 Oral administration of flavonoids (quercetin)enhanced the antioxidative ability of rat plasma, indicating thatconjugated metabolites participate in the antioxidant defence.45

Thus, the combination of potent antioxidants (as indicated byincreased radical scavenging activity of plasma and inhibition oflipid peroxidation with reduced plasma MDA levels) and activePON1 could possibly play a major role in reducing ox-LDL. Previ-ous studies have shown that licorice-derived glabridin can pre-serve paraoxonase activity during lipoprotein oxidation,27 andpomegranate juice consumption also increases PON1 arylesteraseactivity, in associationwith a reduction in LDL susceptibility to cop-per ion-induced oxidation.39 These results provide evidence of thepositive effect of onion extract on PON1 activity and preventionof LDL oxidation during periods of oxidative insult, with improve-ment in the antioxidant capability of rats, suggesting its value asa dietary antioxidant food.44,46 Moreover, quercetin has been pre-viously shown to exert protective effects on ageing of specifictissues in mouse and other cellular models.47 Quercetin and itsderivative, namely quercetin caprylate as a proteasome activatorwith antioxidant properties has been reported to influence cellu-lar lifespan, survival and viability of Human foetal lung fibroblast,cell lines derived from a 16–18week old human foetus (HFL-1) pri-mary human fibroblasts. Moreover, when these compounds weresupplemented to senescent fibroblasts, a rejuvenating effect wasobserved.48 Additionally, quercetin has been shown to increaseoxidative stress resistance and longevity in Saccharomyces cere-visiae49 and to extend lifespan in Caenorhabditis elegans.50 Giventhe antioxidant properties of onion and quercetin and the link

between ageing and oxidative stress,51 the quercetin-mediatedprotection of PON1 levels may also be an anti-ageing strategy.

CONCLUSIONSThe present study highlights the radical-scavenging capacity ofonion. We show that onion extract can provide protection againstHgCl2-induced oxidative damage by up-regulating or preservingPON1 activity and also can protect LDL oxidation and lipid peroxi-dation. Dietary flavonoids show similar effects either by enhancingPON1 activities (in the case of quercetin) and antioxidant defencesor reducing susceptibility to LDLoxidation (in the caseof catechin),when given to oxidatively stressed rats. We provide a mechanismwhich may account for the observed protective effects of thesecompounds with respect to atherosclerosis.

ACKNOWLEDGEMENTSNidhi Jaiswal acknowledges the Senior Research Fellowship (SRF)from the University Grants Commission (UGC), New Delhi, India.

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