Food Chemistry 128 (2011) 40–48

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Food Chemistry

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Neuroprotective properties of Spanish red wine and its isolated polyphenolson astrocytes

Sara Martín, Elena González-Burgos, M. Emilia Carretero, M. Pilar Gómez-Serranillos ⇑Department of Pharmacology, Faculty of Pharmacy, Universidad Complutense de Madrid, Madrid, Spain

a r t i c l e i n f o

Article history:Received 14 December 2010Received in revised form 11 February 2011Accepted 18 February 2011Available online 23 February 2011

Keywords:NeuroprotectionU373 MGRed winePolyphenolsOxidative stressAntioxidant

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.02.074

⇑ Corresponding author. Tel.: +34 913941767; fax:E-mail address: pserra@farm.ucm.es (M.P. Gómez-

a b s t r a c t

The neuroprotective effect of Merlot red wine and its isolated polyphenols was evaluated in an oxidativestress model induced by Fenton reaction and hydrogen peroxide in the human astrocytoma U373 MG cellline. Compared with cells treated only with oxidative stress inductors, the pre-incubation with Merlot redwine for 24 h caused a significant increase in cell viability for all concentrations assayed. The most abun-dant polyphenols found in Merlot red wine were the flavonoids catechin (37.8 mg/l), epicatechin(52.3 mg/l), quercetin (5.89 mg/l) and procyanidins (15.2 mg/l), the hydroxybenzoic acid gallic acid(16.7 mg/l), and the phenolic alcohol tyrosol (31.4 mg/l). The potential protective role of these polyphe-nols when isolated was then assessed in treated Fenton reaction U373 MG cells. Polyphenols decreasedreactive oxygen species generation and increased the activity and the protein expression of the antioxi-dant enzymes catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase. Of thepolyphenols, quercetin and procyanidins showed the highest neuroprotective effect.

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1. Introduction increased the activity of the antioxidant enzymes (Mainzen Prince,

Merlot is one of the most common grape varieties used to makered wine. The name of Merlot derives from the Old French word‘‘merlot’’, a diminutive of merle, which means ‘‘blackbird’’, in refer-ence to the dark-blue colour of the grapes (Robinson, 2006). Epide-miological studies have demonstrated that a moderateconsumption of red wine is correlated with a reduction in the inci-dence of those neurodegenerative diseases associated to oxidativestress such as Alzheimer’s disease, Parkinson’s disease and amyotro-phic lateral sclerosis (Ramassamy, 2006; Rodrigo, Miranda, & Verg-ara, 2011; Sun, Wang, Simonyi, & Sun, 2008). Red wine is one ofthe major sources of polyphenols, containing concentrations of theorder of 12.6–22.4 mM (Miller & Rice-Evans, 1995). Since polyphe-nols have been shown to be strong antioxidants, there has been anincreasing interest in the potential protective role of these com-pounds against oxidative stress (Urquiaga & Leighton, 2000). Oxida-tive stress occurs as consequence of an imbalance between reactiveoxygen species (ROS) production and the inability of the antioxidantbiological system to detoxify free radicals or repair the oxidative in-jury (Halliwell, 2006). Polyphenols act as ROS scavengers, avoidingthe direct damage to lipids, proteins and DNA caused by an excessivefree radical production (Urquiaga & Leighton, 2000). Interestingly,quercetin exerted a protective effect on neuronal cells against oxida-tive stress induced by hydrogen peroxide (Heo & Lee, 2004). Gallicacid decreased products of lipid peroxidation in the brain, and also

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+34 913942276.Serranillos).

Kumar, & Selvakumari, 2010). Besides the antioxidant activity, poly-phenols have other beneficial biological effects including anti-can-cer, anti-inflammatory and cardioprotective (Pignatelli et al., 2006).

Among the different classes of cells in the nervous system, it isworth highlighting the astrocytes, since they perform a variety ofcrucial roles, including homeostasis maintenance, regulation ofneuronal signalling and providing protection to neurons againstoxidative damage induced by compounds like nitric oxide, dopa-mine, H2O2 or 6-hydroxydopamine (Montgomery, 1994; Shihet al., 2003). Glutathione (GSH) is one potential defence againstthese free radical inductors. Under oxidative stress conditions,GSH synthesis and release is increased in astrocytes. Consequently,neurons which need GSH or GSH precursors such as N-acetylcyste-ine to synthesis its own glutathione are potentially protected fromoxidative injury (Shih et al., 2003).

The aim of the present study is to evaluate the neuroprotectiveactivity of a monovarietal Spanish red wine (Merlot) and its majorpolyphenols (quercetin, catechin, epicatechin, tyrosol, gallic acidand procyanidins) on the human astrocytoma U373 MG cell lineagainst the oxidative stress induced by hydrogen peroxide andFenton reaction (FeSO4 + H2O2).

2. Materials and methods

2.1. Chemicals

Dulbecco’s modified Eagle’s medium (DMEM), foetal bovineserum (FBS), trypsin–EDTA, penicillin and streptomycin were

S. Martín et al. / Food Chemistry 128 (2011) 40–48 41

obtained from Gibco (Grand Island, NY). Polyvinylidene difluoride(PVDF) membranes were purchased from Bio-Rad (Hercules, CA).ECL-Advance detection kit was provided by Amersham PharmaciaBiotech Inc. (Piscataway, NJ). Sigma–Aldrich (St. Louis, MO) pro-vided us with all other chemicals, such as hydrogen peroxide,3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT), oxi-dised glutathione (GSSG), reduced glutathione (GSH), nicotinamideadenine dinucleotide phosphate-oxidase (NADPH), diethylenetria-mine pentaacidic acid (DTPA), ethylenediamine tetraacetate(EDTA), dimethylsulphoxide (DMSO), O-phthaldialdehyde (OPT),n-ethylmaleimide (NEM).

2.2. Wine samples and polyphenols

Merlot red wine was made from Vitis vinifera cv. Merlot grapes,which were cultivated in the experimental vineyard station of IMI-DRA, ‘‘El Encin’’, Alcala de Henares (Madrid, Spain). Merlot redwine was dealcoholised by distillation at reduced pressure (60–80 mBar) and low temperature (25–30 �C) as previously reported(Ortega et al., 2008).

Polyphenols (quercetin, catechin, epicatechin, tyrosol, gallicacid and procyanidins) were purchased from Extrasynthèse (Lyon,France) (Fig.1).

OHO

OH

OH

OH

R

HO

OH

O

OH

R3

R4

R2

R1

HO

Procyanidins

O

OH

OH

HO

OH

OH

Catechin

O

OOH

HO

OH

OH

OH

Quercetin

Fig. 1. Structures of the major poly

2.3. Analysis of individual phenolic compounds by HPLC–DAD andHPLC–MS (ESI)

The analysis of individual phenolic compounds found in Merlotwine was carried out by HPLC–DAD and HPLC–MS (ESI) as previ-ously reported (Gómez-Serranillos et al., 2009).

In brief, for the detection of non-anthocyanin polyphenoliccompounds, dealcoholised wine samples (50 mL) were extractedthree times with diethyl ether and ethyl acetate. Organic fractionswere collected and evaporated to dryness under conditions of re-duced pressure and 30 �C. The residue was dissolved in metha-nol:water (1:1), filtered through a cellulose acetate filter(0.45 lm) and analysed by high-performance liquid chromatogra-phy (HPLC) with photodiode array 2001 (Waters, Milford, MA) andmass spectrometric (MS) detection. Compounds were separated onNova-Pak C18 column (300 � 3.9 mm, 4 lm) with a mobile phasethat consisted of A: water/acetic acid (98:2 V/V) and B: water/ace-tonitrile/acetic acid (78:20:2 V/V/V). The gradient elution was 0–55 min, 100–20% A; 55–70 min, 20–10% A; 70–80 min, 10–5% A;80–100 min, 100% B. Compounds were detected from 210 to400 nm.

For the detection of anthocyanin, wine samples were filteredthrough a PVDF membrane (45 lm) and a 100 ll aliquot wasinjected onto a Nova-Pak C18 column (300 � 3.9 mm, 4 lm).

OH

HO

Tyrosol

OH

OH

OH

O

OH

Gallic acid

O

OH

OH

HO

OH

OH

Epicatechin

phenols found in Merlot wine.

42 S. Martín et al. / Food Chemistry 128 (2011) 40–48

Anthocyanins were detected at 530 nm, using malvidin 3-gluco-side as standard.

2.4. Cell culture and treatment

The human astrocytoma U373 MG cell line was obtained fromECACC (European Collection of Animal Cell Cultures, Salisbury,Wiltshire). U373 MG cells were cultured in DMEM supplementedwith 10% FBS, penicillin (10 U/mL) and streptomycin (100 U/mL)at 37 �C in a humidified 5% CO2 atmosphere.

Cells were treated with different concentrations of Merlot Encinred wine extracts (6.8, 10.2 and 13.6 mL/L) and with polyphenols(0.5, 0.25, 0.1, 0.05 and 0.025 mM) for 24 h. Then, U373 MG cellswere incubated with 1 mM H2O2 and 0.5 mM FeSO4 + 1 mM H2O2

(Fenton reaction) for 30 min at 37 �C to induce oxidative stress.

2.5. Assessment of cell viability (MTT assay)

MTT reduction assay, a method used to measure the redoxactivity of living cells, was performed as Mossman described in1983 with slight modifications (Mosmann, 1983). Cells wereseeded in 96-well plates at a density of 50,000 cells/well and trea-ted with Merlot wine and polyphenols as explained above. Then,100 ll MTT (2 mg/mL PBS) were added in each well and cells wereincubated for 1 h at 37 �C. The dark blue formazan crystals formedwere dissolved with DMSO. The absorbance was measured at awavelength of 550 nm using a microplate reader (Digiscan 340,Asys Hitech GmbH, Eugendorf, Austria). Results were expressedas percentage of cell viability (%), assuming control cells as 100%.

2.6. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging activity

The scavenging activity of wine polyphenols was evaluatedusing DPPH method (Blois, 1958; Molyneux, 2004). In brief,100 ll of different concentrations of the polyphenols (range from0.05 to 0.0125 mg/mL) and 100 ll of 1 mM DPPH in methanol weremixed and incubated for 20 min in the dark. Vitamin E was used asan antioxidant reference standard. The absorbance of samples wasmeasured at 517 nm in a microplate reader. Radical-scavengingactivity was expressed as IC50, defined as the concentration ofthe sample required for 50% scavenging of the DPPH free radical.

2.7. Measurement of intracellular reactive oxygen species formation

Intracellular reactive oxygen species (ROS) generation was eval-uated using the fluorescent probe 2,7-dichlorofluorescein diacetate(DCFH–DA), according to the method described by LeBel, Ischiro-poulos, and Bondy (1992). Cells were incubated with 0.01 MDCFH–DA diluted in PBS–glucose for 30 min at 37 �C. Then, cellswere washed twice with PBS–glucose and treated with differentconcentrations of Merlot wine samples (6.8, 10.2 and 13.6 mL/L)and polyphenols (0.25 and 0.1 mM) in combination with 1 mMH2O2 and 0.5 mM FeSO4 + 1 mM H2O2 (Fenton reaction). The fluo-rescence intensity was measured using a microplate fluorescencereader (FLx800, Bio-Tek Instruments, Winooski, VT) at an excita-tion wavelength of 485 nm and an emission wavelength of 530 nm.

2.8. Protein concentration determination

The protein concentration was determined using the bicinchon-inic acid method (Smith et al., 1985). Bovine serum albumin wasused as protein standard.

2.9. Enzyme activity assays

2.9.1. Catalase (CAT) activityThe measure of catalase activity was performed by the method

described by Aebi (1984). In brief, total extracts samples wereadded to 50 mM of phosphate buffer (pH 7.4) containing 15 mMof H2O2 in a total volume of 700 ll. The absorbance was deter-mined using a spectrophotometer (UVIKON 930, Kontron Instru-ments, Bletchley, UK) at a wavelength of 240 nm for 1 min.

2.9.2. Superoxide dismutase (SOD) activitySOD activity was measured following the method described by

Marklund and Marklund (1974). The reaction mixture containedTris–DTPA (pH 8.2), 0.15 mM pyrogallol and 10 mM HCl. The inhi-bition was measured using a spectrophotometer (UVIKON 930,Kontron Instruments) at a wavelength of 420 nm for 1 min at25 �C.

2.9.3. Glutathione peroxidase (GPx) activityGPx activity was determined using the Paglia and Valentine

method (1967). Extracts samples were incubated with 50 mMphosphate buffer (pH 7.4), 4 mM sodium azide, 1 mM EDTA,4 mM GSH, 27 U of glutathione reductase and 0.2 mM NADPH for4 min. Then, cumene was added to initiate the reaction. The GPxactivity was measured using a spectrophotometer (UVIKON 930,Kontron Instruments) at a wavelength of 340 nm for 3 min at 25 �C.

2.9.4. Glutathione reductase (GR)GR activity was determined following the method described by

Barja de Quiroga, Perez-Campo, and Lopez Torres (1990). The reac-tion mixture contained 50 mM phosphate buffer, 6.3 mM EDTA(pH 7.4), 80 mM GSSG and 6 mM NADPH. The GR activity was mea-sured using a spectrophotometer (UVIKON 930, Kontron Instru-ments) at 340 nm for 4 min with a delay time of 60 s at 25 �C.

2.10. Western blot analysis

The detection of antioxidant proteins expression (catalase,superoxide dismutase, glutathione peroxidase and glutathionereductase) by western blot has been described previously (Stark,Brown, & Kerr, 1981). After treatment, cells (200,0000 cells/dish)were collected and washed with PBS. The harvested cells were cen-trifuged at 640g for 5 min and lysed on ice for 30 min in lysis buffer(25 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 20 ll/mL leupeptine, 10 ll/mL pepstatin, 35 ll/mL PMSF). Then, extractswere centrifuged at 1600g at 4 �C for 10 min. Proteins were sepa-rated on a 10% SDS–polyacrylamide gel and transferred to polyvi-nylidene difluoride (PVDF) membranes (Bio-Rad). Themembranes were blocked for 90 min at room temperature inblocking buffer containing 10% skimmed milk in PBS. Followingthat, membranes were incubated with primary antibodies: anti-SOD (1:1000), anti-CAT (1:1000), anti-GPx (1:2000), and anti-GR(1:2000) for 1 h at room temperature. After 30 min washing withPBS–Tween (PBS and 0.1% Tween 20), membranes were incubatedwith horseradish peroxidase-conjugated anti-rabbit or anti-mouseIgG for 2 h at room temperature. Membranes were washed againwith PBS–Tween for 30 min, and transferred proteins were incu-bated with ECL–Advance detection kit (Amersham Pharmacia Bio-tech Inc.) for 1 min. Densitometric analysis of the bands was doneusing an image analyser (Syngene MultiGenius bioimaging system,Syngene, Cambridge, UK) with GenSnap and GenTools programs.

2.11. Oxidised and reduced glutathione

In order to assess redox status in cells, oxidised (GSSG) andreduced glutathione (GSH) levels were quantified using a method

Table 1Composition of Merlot red wine (mg/L). Values are expressed asmean ± SD of two independent determinations.

Compounds (mg/L) Mean ± SD

Gallic acid 16.7 ± 1.03Syringic acid 2.18 ± 0.19Cafftaric acid 22.5 ± 0.31p-Cumaric glucoside 1.23 ± 0.02cis Cutaric acid 0.60 ± 0.07trans Cutaric acid 4.20 ± 0.04(+)-Catechin 37.8 ± 3.51(�)-Epicatechin 52.3 ± 2.82Gallocatechin 1.48 ± 0.03Protocatechin acid 2.07 ± 0.02Procyanidin dimer (B1) 1.66 ± 0.05Procyanidin dimer (B2) 3.97 ± 0.06Procyanidin dimer (B5) 0.73 ± 0.03Procyanidin dimer I 1.09 ± 0.03Procyanidin dimer II 7.36 ± 0.06Procyanidin dimer III 0.39 ± 0.02

S. Martín et al. / Food Chemistry 128 (2011) 40–48 43

described by Hissin and Hilf (1976). Cells were lysed in phosphatebuffer (0.1 mM) EDTA (0.005 M) (pH 8) on ice by sonication andcentrifuged at 1600g at 4 �C. Proteins were precipitated with 1%HClO4 for 5 min on ice and centrifuged at 12000g for 10 min at4 �C.

For GSH measurement, samples were incubated for 15 min inthe dark at room temperature with 0.1 M sodium phosphatebuffer (pH 8) and O-phthaldehyde (1 mg/mL methanol). ForGSSG measurement, samples were incubated for 15 min in thedark at room temperature with N-ethylmaleimide (NEM), 0.1 NNaOH (pH 12) and O-phthaldehyde (1 mg/mL methanol). Fluo-rescent intensity was measured using a fluorometer (FLx800,Bio-Tek Instruments) at an excitation wavelength of528 ± 20 nm and an emission wavelength of 485 ± 20 nm. GSHand GSSH contents in cells were expressed in ng GSSG or GSH/mg protein. Results were expressed as Redox Index:(RI = GSSG/GSH + GSSG).

Quercetin 3-O-glucoside 2.56 ± 0.09Quercetin 3-O-rutoside 14.94 ± 0.09Quercetin 3-O-galactoside 19.69 ± 0.05Quercetin 5.89 ± 0.15Myricetin 14.36 ± 0.05trans-Resveratrol 0.84 ± 0.07Methyl gallate 1.96 ± 0.09Tyrosol 31.36 ± 0.73

2.12. Statistical analysis

Data were analysed by one-way ANOVA followed by Tukey’stest using Statgraphics Plus Version 5.1 (Statpoint Technologies,Inc., Warrenton, VA). Data of p 6 0.05 were considered statisticallysignificant.

3. Results and discussion

Previous studies have demonstrated that red wine and certainof its individual polyphenols exhibited antioxidant effects on cellsof the nervous system (Heo & Lee, 2004; Urquiaga & Leighton,2000). However, few studies have been focused on the effect ofred wine and their polyphenols on astrocytes, the major glial cellsin the central nervous system, undergoing oxidative stress condi-tions. To the better understanding of the action mechanisms ofMerlot red wine and its major polyphenols on astrocytes in an oxi-dative stress model (the effect on cell viability, ROS production,antioxidant enzymes’ activities and protein expression, and scav-enging activities were evaluated).

3.1. Merlot Encin red wine composition

The analysis of polyphenols in Merlot red wine, by high-perfor-mance liquid chromatography (HPLC) and mass spectrometricdetection (Table 1), revealed the presence of high concentrationsof catechin and epicatechin, together with quercetin. Accordingto the structural classification, these three compounds are flavo-noids. The oxidation state of the central pyran ring makes thesecompounds different, so that catechin and epicatechin are flava-nols, whereas quercetin belongs to the flavonols group. Besidesthese polyphenols, gallic acid (a hydroxybenzoic acid), tyrosol (aphenolic alcohol) and procyanidins (a polymer form of flavonols)were also abundant in the Merlot wine sample.

High amounts of flavonoids in red wine have been reported forother wines, such as Cabernet Sauvignon (Fang, Jing-Ming, Qiu-Hong, & Wei-Dong, 2007), Garnacha and Tempranillo (Gómez-Serranillos et al., 2009). Apart from red wine, other beverages(tea and juices), fruits, vegetables and some herbs are rich sourcesof these different polyphenol types. Apples, broccoli, onions andberries are quercetin-rich foods; tyrosol is the major polyphenolin olives and olive oil; procyanidins are found at significant levelsin apples, dark chocolate, berries, nuts and pomegranates and themain polyphenol in green tea and black tea is catechin (Espositoet al., 2002; Vauzour, Corona, & Spencer, 2010; Williamson &Manach, 2005).

3.2. Merlot Encin red wine protected U373 MG cells against H2O2,FeSO4 + H2O2-induced oxidative stress

The present study examines the possible protective effect of dif-ferent concentrations of Merlot wine samples (6.8, 10.2 and13.6 mL/L) on U373 MG cells treated with hydrogen peroxideand Fenton’s reagent (FeSO4 + H2O2) as inductors of oxidativestress by MTT assay (Fig.2). These concentrations of Merlot winehave been used in previous studies (Gómez-Serranillos et al.,2009) and none of them affected cell survival compared with con-trol cells.

When cells were treated with 1 mM H2O2 and Fenton’s reagent(0.5 mM FeSO4 + 1 mM H2O2) for 30 min, a decrease in cell viabilityof 30% and 44.4%, respectively, was observed as compared withcontrol cells. However, the pre-treatment of U373 MG cells for24 h with Merlot wine samples (6.8, 10.2 and 13.6 mL/L) increasedsignificantly cell viability at all concentrations assayed. Merlotwine sample at a concentration of 13.6 mL/L showed the highestprotection against the toxicity induced by both hydrogen peroxideand Fenton reaction. This protection was 26.6% compared withhydrogen peroxide and 31.8% compared with Fenton reaction.

In addition, the effect of Merlot wine samples (6.8, 10.2 and13.6 mL/L) on ROS production was evaluated using dichlorofluo-rescein method (Fig.3). Results showed that both hydrogen perox-ide and Fenton reaction yield an increased production of ROS inU373 MG. A significant reduction in ROS production was observedwhen cells were treated with Merlot wine samples at all concen-trations tested. Merlot red wine at 13.6 mL/L exhibited the highesteffect against the Fenton reaction as a free radicals inductor. Theseresults suggest that Merlot wine protects U373 MG against the tox-icity induced by hydrogen peroxide and Fenton reaction.

3.3. Scavenging activity of polyphenols

The antioxidant capacity of the major polyphenols found inMerlot red wine (quercetin, catechin, epicatechin, tyrosol, gallicacid and procyanidins) was quantified using the stable radicalDPPH. In order to compare the scavenging activity of each com-pound, the value of IC50 was calculated. The lower the IC50 value

A

Control 6.8 ml/l 10.2 ml/l 13.6 ml/l Triton

Cel

l sur

viva

l ( %

)

0

20

40

60

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1mM H2O2

Merlot + 1mM H2O2

*

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Control Fenton 6.8 ml/l 10.2 ml/l 13.6 ml/l Triton 0

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80

100

120

Cel

l sur

viva

l ( %

)

Merlot + Fenton (0.5mM FeSO4+ 1mM H2O2)

*

***

Fig. 2. Protective effect of Merlot red wine on U373 MG cells. Cells were treatedwith different concentrations of Merlot Encin (6.8, 10.2 and 13.6 mL/L) for 24 hfollowed by 30 min of (A) hydrogen peroxide and (B) Fenton reaction (0.5 mMFeSO4 + 1 mM H2O2). Cell viability was measured by MTT assay. Data are expressedas percentage of cell survival (mean ± SD) relative to control cells. Results arerepresentative of three independent experiments. ⁄p < 0.05 versus hydrogen perox-ide and Fenton reaction.

Time (min)

0 20 40 60 80 100 120

RO

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)

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1 mM H2O2

6.8 ml/l + 1 mM H2O2

10.2 ml/l + 1 mM H2O2

13.6 ml/l + 1 mM H2O2

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1800Fenton (0.5 mM FeSO4 + 1 mM H2O2)

6.8 ml/l + Fenton10.2 ml/l + Fenton13.6 ml/l + Fenton

* * * * * * *** *

* ** *

* **

** **

A

B

Fig. 3. Effect of Merlot red wine on ROS production induced by hydrogen peroxideand Fenton reaction in U373 MG cells. Cells were incubated with differentconcentrations of Merlot Encin (6.8, 10.2 and 13.6 mL/L) and (A) hydrogen peroxideor (B) Fenton’s reagent (0.5 mM FeSO4 + 1 mM H2O2). The intracellular ROSproduction was measured for 2 h using dichlorofluorescein assay. Data areexpressed as percentage of ROS production (mean ± SD) relative to control cells.Results are representative of three independent experiments. ⁄p < 0.05 versushydrogen peroxide and Fenton reaction.

Table 2DPPH radical-scavenging activities of the major poly-phenols found in Merlot wine. Results are expressed asIC50 (lg/mL) (mean ± SD) of three independentexperiments.

Polyphenols IC50 (lg/mL)

Quercetin 27 ± 0.00*

Catechin 39 ± 0.01Epicatechin 29 ± 0.00Tyrosol 86 ± 0.02Gallic acid 2 ± 0.00*

Procyanidins 34 ± 0.01Vitamin E 57 ± 0.02

* p < 0.05 versus Vitamin E.

44 S. Martín et al. / Food Chemistry 128 (2011) 40–48

the higher the radical-scavenging activity. As shown in Table 2, gal-lic acid (IC50 2 lg/mL) exhibited the most powerful radical-scav-enging activity, followed by quercetin IC50 (27 lg/mL) andepicatechin (IC50 29 lg/mL). Except for tyrosol, all polyphenols as-sayed had higher antioxidant effect than the reference compoundvitamin E.

The free radical-scavenging properties of polyphenols are due tothe hydrogen-donating capacity of their phenolic groups (Bors,Heller, Michel, & Saran, 1990). The fact that tyrosol possessed thelowest antiradical action could be explained based on its structure.Tyrosol is the only polyphenol evaluated that has one hydroxylgroup bonded to an aromatic ring, while the rest of the polyphe-nols have at least two hydroxyl groups. The greater number of hy-droxyl groups, the more effective is the antioxidant activity (Salahet al., 1995). The strong antioxidant action of catechin, epicatechin,quercetin, procyanidins and gallic acid is attributed to their cate-chol-type structures, which act as a reducing agent (Sun et al.,

2009). Quercetin has the same number of hydroxyl groups as cat-echin and epicatechin. However, the presence of the double bondand the 4-oxo group in its structure confers a higher antiradical

A Control1 mM H2O2

0.25 mM Polyphenols + 1 mM H2O2

0.1 mM Polyphenols + 1 mM H2O2

S. Martín et al. / Food Chemistry 128 (2011) 40–48 45

capacity (Salah et al., 1995). The difference in antioxidant potencybetween epicatechin and catechin may be due to the first havingmore significant charge delocalisation in its structure than the sec-ond (Saint-Cricq de Gaulejac, Provost, & Vivas, 1999).

Gallic acid Quercetin Catechin Epicatechin Tyrosol Procyanidins

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0.25 mM Polyphenols + Fenton0.1 mM Polyphenols + Fenton0.05 mM Polyphenols + Fenton0.025 mM Polyphenols + FentonTriton

***

*

* * * * * *

******

Fig. 4. Protective effect of polyphenols (quercetin, catechin, epicatechin, tyrosol,gallic acid and procynidins) on U373 MG cells. Cells were treated with differentconcentrations of polyphenols (0.25, 0.1, 0.05 and 0.025 mM) for 24 h followed by30 min of (A) hydrogen peroxide and (B) Fenton reaction (0.5 mM FeSO4 + 1 mMH2O2). Cell viability was measured by MTT assay. Data are expressed as percentageof cell survival (mean ± SD) relative to control cells. Results are representative ofthree independent experiments. ⁄p < 0.05 versus hydrogen peroxide and Fentonreaction.

3.4. Polyphenols protected U373 MG cells against H2O2, FeSO4 + H2O2-induced oxidative stress

The major polyphenols (quercetin, catechin, epicatechin, tyro-sol, gallic acid and procyanidins) present in Merlot wine sampleswere investigated to evaluate their possible protective effect onthe human astrocytoma U373 MG cell line under oxidative stressconditions using MTT assay (Fig.4). For this purpose, U373 MGcells were pre-treated with different polyphenols concentrations(0.25, 0.1, 0.05 and 0.025 mM) for 24 h, following an applicationof 1 mM H2O2 and 0.5 mM FeSO4 + 1 mM H2O2 (Fenton reaction)for 30 min. A loss of cell viability of 30% and 40.9% compared tocontrol was observed when cells were treated for 30 min with1 mM H2O2 and 0.5 mM FeSO4 + 1 mM H2O2 respectively. How-ever, a 24 h pre-incubation with polyphenols at 0.25 and0.1 mM concentrations restored cell viability, found to be signif-icant for quercetin (0.25 and 0.1 mM versus hydrogen peroxideand Fenton reaction), catechin (0.1 mM versus hydrogen peroxideand Fenton reaction), epicatechin (0.25 mM versus Fenton reac-tion and 0.1 mM versus hydrogen peroxide and Fenton reaction),tyrosol (0.25 and 0.1 mM versus Fenton reaction), gallic acid(0.1 mM versus Fenton reaction) and procyanidins (0.25 and0.1 mM versus hydrogen peroxide and Fenton reaction). Theother two concentrations tested (0.25 and 0.1 mM) did not in-duce any significant effect on U373 MG cells under oxidativestress conditions.

As the most effective protection was shown for all polyphenolsat the concentrations of 0.25 and 0.1 mM and against the Fentonreaction (0.5 mM FeSO4 + 1 mM H2O2), these experimental condi-tions have been chosen for the following experiments.

Further, we investigated the time-course effect of polyphenolson ROS production induced by Fenton reaction in U373 MG cells(Fig.5). Intracellular ROS levels were determined by dichlorofluo-rescein method. The exposition of U373 MG cells to exogenousFenton reaction resulted in a significant increase in ROS produc-tion. ROS generation was attenuated when cells were treatedwith the combination Fenton reaction and polyphenols at con-centrations of 0.25 and 0.1 mM. All polyphenols reduced signifi-cantly the percentage of ROS formation, catechin andprocyanidins being the most effective polyphenols (Fig. 5). Thisreduction in ROS generation may be due to in part to free radi-cal-scavenging activity shown by these polyphenols in DPPHassay.

According to other previous cell culture experimental modelsand in vivo studies, these findings support the fact that polyphenolsmay be used as protective agents against oxidative stress status,through their ability to scavenge reactive oxygen species. Applepeel polyphenols, such as quercetin, epicatechin and rutin havebeen shown to protect the human colonic carcinoma Caco-2 cellsagainst the oxidative damage induced by indometacin. This actionis related to its quenching superoxide anion, hydroxyl radical andperoxyl generation (Carrasco-Pozo, Gotteland, & Speisky, 2010).The gallic acid polyphenol, isolated from fruits of Pistacia lentiscus,was efficient in reducing hydrogen peroxide-induced lipid peroxi-dation and ROS production in the human erythromyeloblastoidleukaemia K562 cell line (Abdelwahed et al., 2007). Also, greentea polyphenols recovered the morphological changes and theintracellular structural damage produced in primarily culturedrat calvarial osteoblasts under hydrogen peroxide and xanthineoxidase-induced oxidative stress (Park et al., 2003).

3.5. Effect of polyphenols on antioxidants enzymes activity andexpression

The antioxidant enzymes (CAT, SOD, GPx and GR) activitieswere measured by spectrophotometric assays (Table 3) and theantioxidant enzymes’ protein expression was measured by wes-tern blot (Fig. 6). Decreases in antioxidant enzymes’ activity andprotein expression were observed in those cells treated with Fen-ton’s reagent for 30 min compared to control cells. CAT activityand protein expression were increased significantly by the pre-treatment with 0.25 mM quercetin and 0.25 mM procyanidins for24 h, compared to cells treated only with Fenton’s reagent for30 min. A significant increase in SOD activity and protein

Time (min)0 20 40 60 80 100 120

RO

S pr

oduc

tion

( % )

200

400

600

800

1000

Fenton (0.5 mM FeSO4 + 1 mM H2O2)0.25 mM Quercetin + Fenton0.1 mM Quercetin + Fenton0.25 mM Catechin + Fenton0.1 mM Catechin + Fenton0.25 mM Epicatechin + Fenton0.1 mM Epicatechin + Fenton0.25 mM Tyrosol + Fenton0.1 mM Tyrosol + Fenton0.25 mM Gallic acid + Fenton0.1 mM Gallic acid + Fenton0.25 mM Procyanidins + Fenton0.1 mM Procyanidins + Fenton

* * * * * * *

**

****

*****

*****

****

**** *

*****

Fig. 5. Effect of polyphenols (quercetin, catechin, epicatechin, tyrosol, gallic acidand procynidins) on ROS production induced by Fenton reaction in U373 MG cells.Cells were incubated with polyphenols (0.25 and 0.1 mM) and with Fenton’sreagent (0.5 mM FeSO4 + 1 mM H2O2). The intracellular ROS production wasmeasured for 2 h using dichlorofluorescein assay. Data are expressed as percentageof ROS production (mean ± SD) relative to control cells. Results are representative ofthree independent experiments. ⁄p < 0.05 versus Fenton reaction.

46 S. Martín et al. / Food Chemistry 128 (2011) 40–48

expression were observed in cells treated with 0.25 and 0.1 mM ofquercetin and procyanidins. GPx activity and expression were sig-nificant increased by 0.25 mM quercetin, 0.25 and 0.1 mM procy-anidins and 0.25 mM gallic acid, whereas GR activity andexpression were significant improved by 0.25 mM and 0.1 mMquercetin, 0.25 mM catechin and 0.25 mM and 0.1 mM procyani-dins (Table 3 and Fig. 6). Results revealed that quercetin and procy-anidins at 0.25 mM improved the activity and the proteinexpression of all antioxidant enzymes.

Table 3Effect of polyphenols on antioxidant enzymes activities (CAT, catalase; SOD, superoxide disoxidised glutathione levels (GSH and GSSG, respectively) in U373 MG cells. Cells were trereaction (0.5 mM FeSO4 + 1 mM H2O2) for 30 min. Data are expressed as mean ± SD. ResGSSG + GSH.

CAT activity (IU/minmg protein)

SOD activity (IU/mgprotein)

GR actimin/mg

Control 0.22 ± 0.01 20.53 ± 1.40 11.7 ± 2Fenton (0.5 mM

FeSO4 + 1 mM H2O2)0.19 ± 0.01 18.18 ± 0.60 10.6 ± 2

0.25 mMQuercetin + Fenton

0.22 ± 0.02* 22.27 ± 0.90 * 13.6 ± 1

0.1 mMQuercetin + Fenton

0.17 ± 0.01 23.45 ± 2.00 * 14.2 ± 2

0.25 mMCatechin + Fenton

0.18 ± 0.02 25.85 ± 1.50 * 14.5 ± 0

0.1 mM Catechin + Fenton 0.18 ± 0.03 18.55 ± 0.80 17.2 ± 00.25 mM

Epicatechin + Fenton0.19 ± 0.03 24.28 ± 3.30 * 15.7 ± 0

0.1 mMEpicatechin + Fenton

0.19 ± 0.03 18.94 ± 0.90 14.1 ± 0

0.25 mM Tyrosol + Fenton 0.22 ± 0.02 * 19.44 ± 2.20 16.5 ± 10.1 mM Tyrosol + Fenton 0.16 ± 0.02 25.02 ± 7.00 * 16.6 ± 00.25 mM Gallic

acid + Fenton0.20 ± 0.05 26.24 ± 5,00 * 17.0 ± 1

0.1 mM Gallicacid + Fenton

0.18 ± 0.01 18.11 ± 1.70 17.6 ± 0

0.25 mMProcyanidins + Fenton

0.22 ± 0.03 * 22.11 ± 1.90 * 17.3 ± 2

0.1 mMProcyanidins + Fenton

0.23 ± 0.03 * 23.90 ± 0.60 * 14.1 ± 0

* p < 0.05 versus Fenton reaction.

Previous in vitro and in vivo studies have described the enhanc-ing antioxidant enzyme activity and protein expression by poly-phenols in different oxidative stress models. Quercetin increasedGSH, SOD, CAT, GPx, and GST levels in the liver of carbon-tetrachlo-ride treated rats (Amália, Possa, Augusto, & Francisca, 2007) andcatechin increased SOD and GPX-Se activity in cultured rat brainastrocytes, by activating genes that codify for antioxidant enzymesinvolved in protection against oxidative stress (Chan et al., 2002).

3.6. Effect of polyphenols on oxidised and reduced glutathione

The effect of polyphenols on GSH and GSSG levels in a model ofoxidative stress induced by Fenton’s reagent in U373 MG cells isshown in Table 3. Results are expressed as ratio index (GSSG/GSH+GSSG). After a 30 min treatment, Fenton reaction led to an in-crease in redox index compared to control cells. When U373 MGcells were pre-treated with polyphenols (0.1 and 0.25 mM) for24 h, prior to Fenton reaction (30 min), the ratio index was de-creased, compared to cells treated only with Fenton’s reagent. Thisdecrease was significant for quercetin and catechin at 0.25 mM.

These observations are consistent with findings reported by As-sunção et al. (2007), who showed that hippocampus glutathionelevels were increased and exerted a protective effect in those ratsingesting red wine, compared to control cells. The effect of redwine polyphenols on increasing GSH levels may be explained bytheir ability to modulate the activity of those enzymes involvedin glutathione cycle, such as GR or GPx.

4. Conclusions

In the present study, we have demonstrated that Merlot redwine may exert a beneficial effect in the prevention or therapy ofneurodegenerative diseases associated with oxidative stress, suchas Alzheimers disease or Parkinsons disease. Results showed thatMerlot red wine protects the human astrocytoma U373 MG cellline against the oxidative damage induced by Fenton reaction

mutase; GPX, glutathione peroxidase and GR, glutathione reductase) and reduced andated with polyphenols (0.25 and 0.1 mM) for 24 h followed by exposure to a Fentonults are representative of three independent experiments. RI (Redox Index): GSSG/

vity (nmol NADPH/protein)

Total GPx activity (nmol NADPH/min/mg protein)

RI = (GSSG/GSH) + GSSG

.4 25.12 ± 3.20 0.37 ± 0.02

.0 23.69 ± 0.00 0.42 ± 0.01

.6 * 27.65 ± 0.80 * 0.36 ± 0.04 *

.6 * 26.60 ± 5.30 * 0.40 ± 0.01

.6 * 20.46 ± 4.50 0.32 ± 0.08 *

.5 * 34.67 ± 5.10 * 0.41 ± 0.05

.7 * 23.57 ± 4.30 0.39 ± 0.02

.6 * 22.02 ± 4.70 0.40 ± 0.01

.4 * 24.20 ± 2.60 0.36 ± 0.09

.5* 29.03 ± 3.40 * 0.40 ± 0.01

.1* 32.21 ± 4.50 * 0.41 ± 0.03

.5 * 26.85 ± 3.40 * 0.39 ± 0.03

.8 * 29.00 ± 4.40 * 0.41 ± 0.01

.7 * 26.41 ± 5.10 * 0.41 ± 0.02

º Q C E T GA PC

Con

trol

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Q C E T GA PC

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Quercetin Catechin Epicatechin Tyrosol Gallic acid Procyanidins

CA

T e

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n (

% )

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40

60

80

100

120

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180

ControlFenton (0.5 mM FeSO4 + 1 mM H2O2)

0.25 mM Polyphenols + Fenton0.1 mM Polyphenols + Fenton

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CATβ- actin

Q C E T GA PC

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Quercetin Catechin Epicatechin Tyrosol Gallic acid Procyanidins

SO

D e

xpre

ssio

n (

% )

0

50

100

150

200

250

ControlFenton (0.5 mM FeSO4 + 1 mM H2O2)

0.25 mM Polyphenols + Fenton0.1 mM Polyphenols + Fenton

*

*

*

**

Quercetin Catechin Epicatechin Tyrosol Gallic acid Procyanidins

GP

x ex

pre

ssio

n (

% )

0

20

40

60

80

100

120

140

160

ControlFenton (0.5 mM FeSO4 + 1 mM H2O2)

0.25 mM Polyphenols + Fenton0.1 mM Polyphenols + Fenton

***

* **

Quercetin Catechin Epicatechin Tyrosol Gallic acid Procyanidins

GR

exp

ress

ion

( %

)

0

50

100

150

200

250

ControlFenton (0.5 mM FeSO4 + 1 mM H2O2)

0.25 mM Polyphenols + Fenton0.1 mM Polyphenols + Fenton

*

*

*

*

*

*

Fig. 6. Effect of polyphenols on protein expression of antioxidant enzymes (CAT, catalase; SOD, superoxide dismutase; GPX, glutathione peroxidase and GR, glutathionereductase) in U373 MG cells. Cells were treated with polyphenols (0.25 and 0.1 mM) for 24 h followed by exposure to a Fenton reaction (0.5 mM FeSO4 + 1 mM H2O2) for30 min. Data are expressed as mean ± SD. Results are representative of three independent experiments. ⁄p < 0.05 versus Fenton reaction.

S. Martín et al. / Food Chemistry 128 (2011) 40–48 47

(hydrogen peroxide and iron) and hydrogen peroxide. The highestprotective effect against oxidative stress inductors was observedfor 13.6 mL/L of Merlot red wine against the Fenton reaction. Thiseffect has become evident in an increase in cell survival, comparedto cells treated only with Fenton reagent or hydrogen peroxide andin a decrease in ROS production.

We found that the major polyphenols (quercetin, catechin, epi-catechin, tyrosol, gallic acid and procyanidins) found in Merlot redwine may be in part responsible for the protective effect observedin wine samples. These polyphenols attenuate ROS generation in-duced by Fenton reaction in U373 MG by their ability to scavengefree radicals. This study also revealed a high correlation between

48 S. Martín et al. / Food Chemistry 128 (2011) 40–48

polyphenols content and antioxidant activity. In addition to thescavenging activities of polyphenols, these compounds may exerta positive effect by increasing the activity and protein expressionof the antioxidant enzymes CAT, SOD, GR and GPx.

Taken together the results of this study, Merlot red wine sampleand its polyphenols (quercetin and procyanidins) showed the high-est neuroprotective effect in the human astrocytoma U373 MG cellline. These results support the beneficial effects of red wine for itsantioxidant properties, mainly attributed to the polyphenols pres-ent in high amounts, and provide hope for the treatment and pre-vention of oxidative stress-related neurodegenerative diseases. Theneuroprotective activity of the red wine polyphenols studied maybe extensible to those polyphenols in fruits and vegetables.

Acknowledgement

This work was supported by Comunidad Autónoma de la Rioja(FPI fellowship).

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