Antioxidant and cardioprotective effects of Ilex brasiliensis: A comparative study with Ilex...

Food Research International xxx (2009) xxx–xxx

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Antioxidant and cardioprotective effects of Ilex brasiliensis: A comparativestudy with Ilex paraguariensis (yerba mate)

Guillermo Schinella a, Juliana C. Fantinelli b, Horacio Tournier a, José María Prieto c, Etile Spegazzini d,Silvia Debenedetti d, Susana M. Mosca b,*

a Cátedra de Farmacología Básica, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, CIC Provincia de Buenos Aires, La Plata, Buenos Aires, Argentinab Centro de Investigaciones Cardiovasculares, Universidad Nacional de La Plata-CONICET, La Plata, Buenos Aires, Argentinac Department of Pharmacognosy and Phytochemistry, School of Pharmacy University of London, UKd LABRAM, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina

a r t i c l e i n f o

Article history:Received 27 April 2009Accepted 8 July 2009Available online xxxx

Keywords:Ilex paraguariensisIlex brasiliensisIschemiaReperfusionAntioxidant capacityTBARSGSH

0963-9969/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodres.2009.07.004

* Corresponding author. Address: Centro de InveUniversidad Nacional de La Plata, 60 y 120, 1900 La P221 425 5861.

E-mail address: smosca@atlas.med.unlp.edu.ar (S.M

Please cite this article in press as: Schinella, G.,ensis (yerba mate). Food Research International (

a b s t r a c t

Our objective was to compare the antioxidant properties and cardiovascular effects in ischemia–reperfu-sion of leaves aqueous extracts of Ilex brasiliensis (B) and Ilex paraguariensis (P). In vitro systems were usedto assess the antioxidant properties of the extracts. Isolated rat hearts were treated with both extractsbefore ischemia and myocardial function was assessed. Thiobarbituric acid reactive substances (TBARS)concentration and reduced glutathione (GSH) were also measured. B extract showed higher total phenolsand ascorbic acid contents and a higher scavenging activity of peroxynitrite and ferric reducing antioxi-dant power (FRAP) than P extract. Postischemic systolic and diastolic functions were improved after Band P treatment. Both extracts decreased TBARS and preserved GSH content.

The present study demonstrates that an aqueous extract of I. brasiliensis, similar to I. paraguariensis,protects the myocardium against ischemia–reperfusion injury and attenuates oxidative damage. Theseeffects may be attributed to the potent antioxidant properties of the extract.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Reactive oxygen species (ROS) are involved in the pathogenesisof several human diseases and there is growing evidence that con-sumption of certain foods, dietary supplements and traditionalbeverages leads to a reduction in some parameters of oxidativedamage in biological systems (Aruoma, Bahorun, & Jen, 2003).

The burst of ROS is considered one of the key factors in thedevelopment of myocardial stunning and other alterations derivedfrom ischemia and reperfusion (Bolli, 1998). Myocardial stunningis an entity described by Braunwald and Kloner (1982) as the tran-sitory postischemic dysfunction of viable tissue salvaged by reper-fusion which requires hours to days before function is fullyrestored. The basic scenario requires a discrete episode of ischemia.Taking into account that antioxidant therapy decreases the postis-chemic myocardial dysfunction (Mellin et al., 2005) much atten-tion has been focused on the actions of naturally occurringantioxidants contained in plant extracts (Bixby, Spieler, Menini, &Gugliucci, 2005).

ll rights reserved.

stigaciones Cardiovasculares,lata, Argentina. Tel./fax: +54

. Mosca).

et al. Antioxidant and cardiopro2009), doi:10.1016/j.foodres.20

Mate tea o simply ‘‘mate” is a traditional beverage prepared asinfusions or decoctions of the dried and minced leaves and twigs ofthe native South America Ilex paraguariensis (St. Hill), Aquifoliaceae(yerba mate). In the last decades, the admiration for mate proper-ties and history has spread to many areas including the MiddleEast, particularly Syria, Lebanon, Israel, countries which approxi-mately import a 65% of total Argentinian yerba mate exportation.

Recently, we and others researchers had demonstrated a highantioxidant activity of aqueous extracts of I. paraguariensis (Filip,Lotito, Ferraro, & Fraga, 2000; Gugliucci, 1996; Schinella, Troiani,Dávila, de Buschiazzo, & Tournier, 2000). This extract also pro-duced a significant attenuation of myocardial stunning and lipidperoxidation in an ischemia and reperfusion model (Schinella, Fan-tinelli, & Mosca, 2005).

Other species of the genus Ilex (Choi et al., 2005) than paraguari-ensis share with it the same wild distribution area in South Amer-ica and are commonly used as adulterants or substitutes of yerbamate. Although some of these species have been researched interms of their biological activities (Filip & Ferraro, 2003), to ourknowledge, there is not yet studies about Ilex brasiliensis.

In the present paper we assessed the antioxidant activity andthe actions on systolic and diastolic alterations induced by ische-mia and reperfusion in isolated rat heart of aqueous extracts ofI. brasiliensis compared to I. paraguariensis.

tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004

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2. Materials and methods

2.1. Plant material

Aerial parts of I. brasiliensis (Sprengel) Loes and I. paraguariensisSt. Hilarie, both species of Aquifoliaceae family, were collected inCerro Azul (Misiones, Argentina) in April 2002. A voucher specimenof both samples are deposited in the herbarium of the Museo deBotánica y Farmacognosia ‘‘Carlos Spegazzini” (Universidad Nac-ional de La Plata, Argentina) under the numbers LPE 1005 and938, respectively.

2.2. Preparation of the extracts

The dried and powdered leaves of both Ilex species were ex-tracted (10% w/v) with hot water (90 �C), left standing for 20 min,filtered and lyophilised (yield: 9.0% w/w). The dry matter wasmaintained at �20 �C until it was used. Both extracts were dis-solved in distilled water immediately before performing all tests.

2.3. Determination of total phenol and ascorbic acid contents

Ascorbic acid and total phenol contents of the extracts weredetermined by colorimetric methods. The total phenol contentwas determined as caffeic acid equivalents using Folin Ciocalteureagent. Ascorbic acid content was determined after derivatizationwith 2,4-dinitrophenylhydrazine (Van Boekel & Meeuwissen,1983).

2.4. Analytical high-performance liquid chromatography (HPLC)-diodearray detector (DAD)

HPLC-DAD analysis was performed on a Merck-Hitachi systemequipped with a Pump L-6200, L-7455 Diode Array Detector andAuto Sampler L-7200, injection valve (Reodyne), loop of 100 ll,precolumn Lichrospher� C18 (4 � 4 mm, 5 lm, Merck), and col-umn Lichrospher� C18 (250 � 4 mm, 5 lm, Merck). The data werecollected and processed with the software HSM-7000 (Merck-Hitachi).

The standardization of the extracts was carried out followingthe indications of a previous paper of Giner et al. (1993). Thelyophilised aqueous extracts were reconstituted in demineralizedwater and extracted three times with ethyl acetate. The organiclayers were brought back together, dried under vacuum and madeup in methanol to a concentration of 10 mg/ml. Standards of purecaffeine (Sigma, USA), quercetin-3-O-glucoside (Extrasynthese,France), 3,5- and 4,5-di-O-caffeoylquinic acid (obtained as de-scribed by Góngora et al., 2002) were injected prior to obtain a cal-ibration curve. Each injection was made in triplicate. Data wereexpressed as percentage of dried weight of the extracts.

2.5. Reduction of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)

1.5 ml of a 20 mg/L DPPH solution in methanol was added to0.75 ml of a solution of the extracts in methanol. Absorbance at517 nm was determined after 30 min and the scavenging activitywas calculated as percentage of the radical reduction. BHT wasused as reference compound (Schinella et al., 2000).

2.6. 2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS�+)decolorization assay

ABTS�+ was prepared as described Re et al. (1999). This assaywas performed in presence of the extracts and the scavenging

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activity calculated as percentage of the radical reduction usingascorbic acid as reference compound.

2.7. FRAP assay

Reducing properties of the extracts was estimated using the‘‘ferric reducing antioxidant power” (FRAP) assay (Benzie & Strain,1996). The result was expressed as Equivalent lmol ascorbic acid/mg extract.

2.8. Superoxide radical generation

Superoxide was generated by enzymatic oxidation of hypoxan-thine with xanthine oxidase by determining the nitroblue tetrazo-lium reduction followed spectrophotometrically at 560 nm. Thisassay was performed in presence of the extracts as was previouslydescribed (Schinella et al., 2000).

2.9. Scavenging of peroxynitrite anion

Peroxynitrite was synthesized from sodium nitrite/H2O2 acidi-fied with HCl and the residual H2O2 was removed by passing thesolution through granular MnO2. The yellowish stock solutionwas stored at �80 �C and its concentration was evaluated immedi-ately before its use by measuring the absorbance at 302 nm(e = 1670 M�1 cm�1). The measurements of relative antioxidantcapacity were determined by using Pyrogallol Red as detectingmolecule (Balavoine & Geletii, 1999).

2.10. Nitration of blood serum albumin (BSA) induced by peroxynitrite

Reaction between ONOO� and BSA was carried out as describedby Jiao, Mandapati, Skipper, Tannenbaum, and Wishnok (2001).BSA (1 mg/ml in 150 mM potassium phosphate, pH 7.2) was incu-bated in presence of 2 mM ONOO� with or without 25 mM sodiumbicarbonate at room temperature. After incubation, the extent ofBSA nitration was determined by Western blot using 3-nitrotyro-sine (Bixby et al., 2005).

2.11. Hydroxyl radical generation

Hydroxyl radical was generated by incubating a reaction mix-ture containing 20 lM FeCl3, 1.4 mM H2O2, 2.8 mM deoxyribose,2 mM EDTA and 50 lM ascorbate in 1 ml 10 mM KH2PO4–KOHbuffer, pH 7.4 for 60 min at 37 �C. The inhibition of deoxyribosedegradation by both extracts (at 100 lg/ml final concentration),was estimated by using the thiobarbituric acid method. Dimethylsulfoxide (DMSO, 20 mM) was used as reference compound (Schi-nella et al., 2000).

2.12. Non-enzymatic and enzymatic lipid peroxidation

Lipid peroxidation induced by Fe2+/ascorbate (non-enzymatic)and CCl4/NADPH (enzymatic) in rat liver microsomes as previouslydescribed (Schinella et al., 2000).

2.13. LDL oxidation

LDL was oxidised using the classical copper-induced LDL auto-oxidation. Incubations were carried out at 37 �C measuring theconjugated diene formation, the TBARS production and electropho-retic mobility change of LDL during different times up to 3 h. Pro-bucol was used as a positive control as previously described(Schinella et al., 2007).

tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004

Table 1HPLC-UV standardization of the extracts.

Ilex paraguariensis (%) Ilex brasiliensis (%)

Caffeine 1.2 03,5 Di-O-caffeoylquinic acid 2.0 1.44,5 Di-O-caffeoylquinic acid 0.5 0.4Quercetin-3-O-glucoside Traces 0.4

Table 2IC50 (lg/ml) values of the aqueous extract of Ilex brasiliensis (B), Ilex paraguariensis (P)and reference drugs in radical scavenging and microsomal lipid peroxidation systems.

Radical Lipid peroxidation

ABTS�+ DPPH Fe2+/ascorbate Cl4C/NADPH

B 12.2 ± 1.0* 8.6 ± 1.8 35.9 ± 1.1* 13.5 ± 2.4P 17.2 ± 0.5 7.7 ± 1.1 31.7 ± 1.3 12.7 ± 3.0Ascorbate 1.4 ± 0.6 nd nd ndQuercetin 3-O-glucoside nd 3.1 ± 0.4 nd ndBHT nd nd 1.4 ± 0.2 1.2 ± 0.2

nd: not determined.* p < 0.05 with respect to P.

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2.14. Isolated heart preparation

Wistar rats were anesthetized with an intraperitoneal injectionof sodium pentobarbital (60 mg/kg body wt.). The heart was rap-idly excised and perfused by the non-recirculating Langendorfftechnique with Ringer’s solution (pH 7.4) and at 37 �C. The rightventricle was paced at 280 ± 10 beats/min. A latex balloon wasintroduced into the left ventricle and the opposite end of the tubewas then connected to a Statham P23XL pressure transducer. Theballoon was filled with water to provide an end-diastolic pressure(LVEDP) of 8–12 mm Hg, after which the ballon volume was keptconstant for the rest of experiment to record isovolumic pressure.Coronary flow was 11 ± 2 ml/min. Left ventricular pressure (LVP)was acquired by using an analog-to-digital converter and acquisi-tion software (Chart V4.2.3 ADInstruments).

2.15. Experimental protocols

After 10-min stabilization, the following experimental protocolswere performed:

Untreated hearts (C, n = 14): Hearts were subjected to 20 min ofnormothermic global ischemia followed by 30 min of reperfusion.Global ischemia was induced by stopping the perfusate inflow line,and the heart was placed in a saline bath held at 37 �C.

Treated hearts (B: I. brasiliensis, P: I. paraguariensis, n = 16):Hearts were treated 10 min before ischemia and the first 10 min ofreperfusion with a dose of 0.30 mg/min of P (n = 8) and B (n = 8).The final concentration of B and P in the perfusate was 30 lg/ml.

2.16. Systolic and diastolic function

Myocardial contractility was assessed by the left ventriculardeveloped pressure (LVDP) and maximal rise velocity of left ven-tricular pressure (+dP/dtmax) values. The diastolic function wasevaluated by maximal decrease velocity of left ventricular pressure(�dP/dtmax) values and LVEDP.

2.17. Assessment of lipid peroxidation

At the end of reperfusion the hearts were homogenized in phys-iological saline solution. After the samples were centrifuged, in thesupernatant the concentration of thiobarbituric reactive sub-stances (TBARS) was determined by a spectroscopic technique(Schinella et al., 2005).

2.18. Reduced glutathione (GSH)

Reduced glutathione (GSH) in cardiac tissue was determined byEllman’s method (Sedlak & Lindsay, 1968) and presented as lg/gtissue weight.

2.19. Statistical analysis

Data were expressed as means ± SD. Statistical analysis wasperformed by one-way analysis of variance (ANOVA) followed byTurkey–Kramer multiple comparisons test. Differences were con-sidered significant at p < 0.05. The inhibitory concentration of50% (IC50) was calculated from concentration/effect regression line.

3. Results

3.1. Determination of total phenol and ascorbic acid contents

The total phenol and ascorbic acid contents, expressed as mgper g of dry extract, were significantly greater in B compared with

Please cite this article in press as: Schinella, G., et al. Antioxidant and cardioproensis (yerba mate). Food Research International (2009), doi:10.1016/j.foodres.20

P (156 mg/g and 24 mg/g for B vs. 111 mg/g and 11 mg/g for P,respectively).

3.2. Analytical high-performance liquid chromatography

The extracts were analyzed by HPLC-UV for their caffeine con-tent, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acidsand quercetin-3-O-glucoside, the characteristic main phenoliccompounds in the extracts here studied. The values are reportedin Table 1.

Briefly, B is virtually free of caffeine, while in P is the main com-pound by far. The content on 3, 5-di-O-caffeoylquinic acid was 30%lower in B whilst the content in 4, 5-di-O-caffeoylquinic acid wasapproximately the same compared to P. As for quercetin-3-gluco-side, its concentration in B is twice than in P.

3.3. Antioxidant activity

The scavenger capacity of extracts was measured by bleachingthe stable radicals DPPH� and ABTS�+. There was no difference be-tween B and P for DPPH� scavenging. However, in ABTS�+ scaveng-ing B was more effective than P (Table 2).

FRAP assay measures the antioxidant effect of any substance inthe reaction medium as reducing ability. The B extract showed sig-nificantly higher FRAP antioxidant activity than P (211 vs.140 EQ lmol ascorbic acid/mg extract).

IC50 for scavenging activity of superoxide and peroxynitrite forboth Ilex species are depicted in Fig. 1. B and P showed similarsuperoxide radical scavenging activities and B showed a signifi-cantly higher scavenging activity of peroxynitrite compared with P.

The nitration of BSA with peroxynitrite was evaluated by usingwestern blotting. B and P inhibited the nitration of BSA in concen-tration-dependent manner in absence and presence of bicarbonate(Fig. 2). B showed higher inhibition capacity than P.

Both extracts (100 lg/ml, final concentration) were unable toscavenge the hydroxyl radical produced in the Fe3+-EDTA + H2O2

system in presence or absence of ascorbate. Moreover, a significantincrease in deoxyribose degradation by B in both systems was de-tected (Fig. 3).

Lipid peroxidation of rat hepatic microsomes was evaluated byusing enzymatic and non-enzymatic generation systems. B and Pinhibited lipid peroxidation in both systems. In non-enzymatic sys-tem B was significantly less active than P (Table 2).

tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004

0

30

60

90

120

150

180

210

*

ONOO_

PB

O2_ .Scavenging

activity of

IC50

(µg/

ml)

Fig. 1. IC50 values (lg/ml) for scavenging activity of superoxide ðO��2 Þ andperoxynitrite (ONOO��) by Ilex paraguariensis (P) and Ilex brasiliensis (B) extracts.Both extracts similarly scavenged O��2 , but B showed greater scavenging activity ofONOO�� than P. �p < 0.01 with respect to P.

A

B

300 100 30 10 300 100 30 10 0 µg/ml

Ilex paraguariensis Ilex brasiliensis

Fig. 2. Western blot analysis showing the inhibition of BSA nitration after differentconcentration of aqueous extracts of Ilex brasiliensis and Ilex paraguariensis (A)without bicarbonate; and (B) with bicarbonate.

0.0

0.2

0.4

0.6

0.8

1.0

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1.4

*

*

Fe3+-EDTA / H2O2/ ascorbate

DMS O C B P

0.0

0.2

0.4

0.6

0.8

1.0

Abso

rban

ce a

t532

nm

Abso

rban

ce a

t532

nm

*

*

Fe3+-EDTA / H2O

2

DMS O C B P

Fig. 3. Effects of Ilex paraguariensis (P) and Ilex brasiliensis (B) extracts ondeoxyribose degradation stimulated by Fe3+-EDTA + H2O2 in presence or in absenceof ascorbate. Dimethyl sulfoxide (DMSO) was used as reference compound. �p < 0.01with respect to C hearts.

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In copper-induced LDL auto-oxidation system both extracts in-creased the elapsed time before the onset of rapid formation ofconjugated lipid hydroperoxides (lag-phase) in a dose-dependentmanner. At 10 lg/ml, both extracts prevented the conjugated dieneformation. Also, there were not significant differences of IC50 val-ues of both extracts (approximately 3.0 lg/ml) in the TBARS con-tents in Cu2+-induced LDL oxidation.

After treatment with 10 lM CuSO4, the relative electrophoreticmobility of LDL increased compared to native LDL, and the modu-lation to electronegative LDL was observed. These modulationswere similarly inhibited by addition of both extracts in a dose-dependent manner up to 10 lg/ml (data not shown).

3.4. Myocardial systolic and diastolic function

Fig. 4 (upper panel) shows the effects of B and P on systolicfunction in our preparation of 20 min of global ischemia followedby 30 min of reperfusion. A decrease in LVDP to values of 57 ± 6%from baseline was detected at the end of the reperfusion periodin untreated hearts. The infusion of 30 lg/ml of B and P improvedpostischemic recovery, reaching LVDP values of 110 ± 3% and96 ± 8%, respectively, in contrast to 57 ± 6% in C hearts (p < 0.05against each treated group). A similar pattern was obtained when+dP/dtmax values were analyzed. Fig. 4 (lower panel) shows that

Please cite this article in press as: Schinella, G., et al. Antioxidant and cardioproensis (yerba mate). Food Research International (2009), doi:10.1016/j.foodres.20

B and P significantly improved this parameter of contractilityreaching at the end of reperfusion period higher values than thoseobserved in untreated hearts (110 ± 3% and 95 ± 10% vs. 53 ± 6%,p < 0.05). Although there was not statistical difference betweenboth treatments, the improvement of postischemic systolic func-tion after B was slightly higher than P.

When examining �dP/dtmax an improvement of relaxationvelocity was achieved by B and P treatment (118 ± 5% and90 ± 12% vs. 57 ± 8% in untreated hearts, p < 0.05) (Fig. 5, upper pa-nel). The increase of diastolic stiffness detected during reperfusionin untreated hearts was significantly attenuated by B and P treat-ment. At the end of reperfusion LVEDP reached values significantlylower than that detected in untreated hearts (10 ± 1 mm Hg for Band 20 ± 6 mm Hg for P, in contrast to 43 ± 4 mm Hg in controlhearts, Fig. 5, lower panel). The ischemic contracture observed inuntreated hearts was not modified after B administration, but itsignificantly decreased after P treatment (LVEDP = 28 ± 5 mm Hgvs. 47 ± 4 mm Hg in untreated hearts, p < 0.05).

3.5. Oxidative damage of cardiac tissue

TBARS content was significantly diminished and GSH levelswere significantly higher when hearts were treated with B and Pcompared to untreated hearts (Fig. 6).

4. Discussion

A high intake of foods and beverages rich in antioxidants likepolyphenols and ascorbic acid have been associated with a

tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004

B or PNormoperfusion Normoperfusion

*

*****

Is 20B or P

CBP

LVD

P(%

)

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120

140NormoperfusionNormoperfusion

Time (min)

Is 20B or PB or P

**

**

*

*

CBP

+dP/

dtm

ax(%

)

Fig. 4. Time course of left ventricular developed pressure (LVDP, upper panel) andmaximal rise velocity of left ventricular pressure (+dP/dtmax, lower panel) duringischemia and reperfusion from hearts treated with B and P. Note that bothparameters were significantly improved by both treatments. �p < 0.05 with respectto C hearts.

0 10 30 50

NormoperfusionB orP

NormoperfusionB orP

**

**

*Is20

CBP

-dP/

dtm

ax(%

)

20 40 60 700

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NormoperfusionIs20

Time (min)

CBP

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B orP

LVED

P (m

mH

g)

Fig. 5. Changes of maximal decrease velocity of left ventricular pressure(�dP/dtmax, upper panel) and left ventricular end-diastolic pressure (LVEDP, lowerpanel) during ischemia and reperfusion from hearts treated with B and P. Bothtreatments increased relaxation velocity and attenuated the increase of diastolicstiffness detected during reperfusion in C hearts. A decrease in ischemic contracturewas only observed after P treatment. �p < 0.05 with respect to C hearts.

0

10

20

30

40TBARS (nmol/g)GSH (µg/g)

**

**

C P B

Fig. 6. Effects of Ilex paraguariensis (P) and Ilex brasiliensis (B) extracts on tissuethiobarbituric acid reactive substances concentration (TBARS) and reduced gluta-thione content (GSH). Both extracts attenuated lipid peroxidation and preservedGSH levels compared with C hearts. �p < 0.05 with respect to C hearts.

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decreased risk of chronic degenerative diseases which have beenlinked to oxidative stress (Frei, Stocker, & Ames, 1988).

In our study the treatment of isolated hearts with an aqueousextract of I. brasiliensis attenuates myocardial stunning, and there-fore it acts as protectively by enhancing the ischemic tolerance ofcardiac tissue.

In this study, B presented higher total phenols and ascorbic acidcontent compared to P. However, the antioxidant activity was sim-ilar or different according to in vitro assays used. Thus, both ex-tracts (B and P) showed a similar attenuation of enzymatic andnon-enzymatic microsomal lipid peroxidation and LDL oxidationinduced by Cu2+ and scavenging activity of DPPH� and ABTS�+,whereas B presented a higher scavenging activity of peroxynitriteand the prooxidant effect detected in Fe3+-EDTA–H2O2 systemand FRAP than P. These last effects would be attributed to the rec-ognized efficacy of ascorbic acid to scavenge peroxynitrite (Squad-rito, Jin, & Pryor, 1995) and to redox recycling of iron (Burkitt &Gilbert, 1990).

It is known that the peroxynitrite anion can attack a wide rangeof biological molecules. Recent data (Bixby et al., 2005) demon-strate that I. paraguariensis displayed the highest inhibition of pro-tein nitration when compared with the same concentrations ofgreen tea or red wines. In our experimental conditions the inhibi-tion of BSA nitration was higher after B than after P treatment.

In the isolated rat heart model B- similar to P- attenuated themyocardial stunning, improving systolic and diastolic myocardialfunction after ischemia and reperfusion and reducing the oxidativedamage of cardiac tissue.

It is well known that brief episodes of myocardial ischemia andreperfusion are associated with the generation of reactive oxygen

Please cite this article in press as: Schinella, G., et al. Antioxidant and cardioproensis (yerba mate). Food Research International (2009), doi:10.1016/j.foodres.20

species (ROS) like superoxide radical ðO��2 Þ, H2O2 and hydroxylradical (OH), which produce structural changes and functionalalterations that contribute to reversible or irreversible cell injury

tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004

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(Bolli, 1998). The conditions present at ischemia and reperfusionalso stimulate an increase in nitric oxide (NO) radical formationwhich may prevent or promote cell death, depending on cell typeand concentration (Bolli, 2001). NO combines with O��2 . at a veryfast rate to form peroxynitrite (ONOO�) which reacts with a widevariety of molecules including deoxyribose, cellular lipids, and pro-tein sulfhydryls producing oxidative tissue damage (Kim, Ohshima,Pediaditakis, & Lemasters, 2004).

Taking into account these results, the attenuation of myocardialpostischemic dysfunction obtained after B and P treatments wouldbe attributed to the capacity of both extracts to remove O��2 ,unmasking the recognized beneficial effects of NO. A slight(approximately 15%) but not significantly higher postischemicrecovery of contractility in hearts treated with B compared withP would be correlated to the higher scavenging activity of ONOO�

of B, indicating that ONOO� would be playing an injurious role inour experimental conditions.

The interaction between NO and O��2 plays a key role in vascularpathophysiology (Kodja & Harrison, 1999). Endothelial function iscontrolled by a balance between the production of NO and ROS(Cai & Harrison, 2000; Kodja & Harrison, 1999). In presence of nor-mal NO production its bioavailability may be reduced because ofthe oxidative inactivation by an excessive production of O��2 inthe vascular wall. The high scavenging activity of O��2 of B wouldbe associated to a higher bioavailability of NO leading to a normal-ization of endothelial function and improvement of postischemicmyocardial function, as was previously suggested for P (Schinellaet al., 2005).

A diminution of ischemic contracture by P but not by B was alsoobtained. Considering that the level of contracture during ischemiahas been related to the fall in ATP stores (Steenbergen, Murphy,Watts, & London, 1990), a low contracture after P treatment wouldbe indicating a preservation of ischemic ATP levels. This possibilityis in accordance with recent data showing that chronic ingestion ofB decreases nucleotide hydrolysis in the rat blood (Gorgen et al.,2005).

ROS cause lipid peroxidation first leading to reversible damage,and eventually to necrosis and/or apoptosis. The administration ofantioxidants or free radicals scavengers is able to limit the evolu-tion of myocardial damage reducing ROS-induced lipid peroxida-tion (Tavazzi et al., 1998). B diminished peroxidation ofmembrane lipids and preserved GSH levels, demonstrating anattenuation of oxidative damage provoked by ischemia andreperfusion.

Taking into account that a misbalance between production ofROS and antioxidant defence mechanisms may lead to a dysfunc-tion of myocardial contractility, an attenuation of oxidativestress would be the mechanism of the protection conferred by B.

Our study clearly demonstrates the protective action of theinfusion of I. brasiliensis on reperfusion injury in isolated rat heart.Considering that the cardiac adverse effects of caffeine, as were re-cently demonstrated in two experimental models in humans (Rik-sen et al., 2006), the fact that I. brasiliensis infusion is virtuallydeprived of that alkaloid has a pharmacologically added value.From the point of view of food industry, these results may openthe way to label/market I. brasiliensis as a healthier source of anti-oxidants with the same cardioprotective action of mate.

Acknowledgments

This study was in part supported by Grants 11/X446 and PICT01-11987. The authors thank to José Luis Ríos, Professor of theDepartment de Farmacologia, Facultat de Farmàcia, Universitatde València, for the expert assistance and for kindly providing uswith the reactive to perform some determinations.

Please cite this article in press as: Schinella, G., et al. Antioxidant and cardioproensis (yerba mate). Food Research International (2009), doi:10.1016/j.foodres.20

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tective effects of Ilex brasiliensis: A comparative study with Ilex paraguari-09.07.004