Protective Effects of Merlot Red Wine Extract and its Major Polyphenols in Pc12 Cells under Oxidative Stress Conditions

Abstract

Abstract:  The potential effect of the extracts from free-run and pressed Merlot red wine has been evaluated in PC12 cells under oxidative stress situation. Comparing both vinification process, pressed Merlot red wine extract possessed higher neuroprotective activity than the free run wine, possibly attributed to the major content in all global polyphenolic families. High performance liquid chromatography determination of individual polyphenols showed that the major compounds found in Merlot red wine extract were quercetin, catechin, epicatechin, tyrosol, gallic acid, and procyanidins. Pretreatments with these polyphenolic compounds (0.25 mM and 0.1 mM, 24 h) significantly increased cell viability of H₂O₂ and Fenton reaction treated cells. Moreover, these polyphenols attenuated ROS production and decreased the Redox Index of glutathione (RI = GSSG/GSH + GSSG) in cells treated only with Fenton reaction. Furthermore, some polyphenols induced antioxidant enzymes activity and protein expression. Quercetin was the most active. These results support the beneficial effects of red wine extracts and some of its polyphenols under oxidative stress conditions.

Practical Application:  This research provides evidences of the preventive properties of wine extracts and its major polyphenols under oxidative stress conditions.

Introduction

Wine, together with fruits, vegetables, fishes, cereals, and olive oil, is one of the essential components of the Mediterranean Diet food pyramid (Bach-Faig and others 2011). Many in vitro, in vivo and epidemiological studies have shown that a moderate wine intake possesses healthy properties for cardiovascular diseases, cancer, diabetes, and neurological disorders (Martin and others 2006). These benefits are attributed, at least, to the presence of high amounts of polyphenols, which are also responsible for color, astringency, flavor, aroma and other organoleptic wine's properties (Rastija and Medić-Šarić 2009).

The antioxidant capacity is involved in the mechanism of action of polyphenols. Polyphenols act as hydrogen-donating free radical scavengers, transferring hydrogen atoms from their phenol group to free radicals such as superoxide ion (O²⁻) and hydroxyl radical (HO ^•) (Martin and others 2006). The more number of hydroxyl groups in the structure the more powerful the antioxidant capacity (Rastija and Medić-Šarić 2009).

Increasing evidences support the negative role played by an overproduction of reactive oxygen species (ROS) in the pathophysiology of many neurodegenerative diseases including Alzheimer′ and Parkinson's diseases (Tarawneh and Galvin 2010). Postmortem brain analysis of patients who suffered from an oxidative-stress related neurodegenerative disorder have revealed lower levels of antioxidants, iron excess and an increased of free radicals (Tarawneh and Galvin 2010) in specific regions of the brain. Thus, the identification of potential compounds with antioxidant properties, particularly polyphenols, has become one of the main strategies to delay or inhibit this oxidative damage (Martin and others 2006).

The aim of the present study is evaluating the protective role of free run and pressed Merlot red wine extracts, which is one of the most popular red wine varietals, as well as its major polyphenols in neurons under oxidative stress situation. The possible protective role of this ancient beverage and the investigated dietary polyphenols could avoid the abnormal loss of neurons produced under oxidative stress conditions, becoming as potential nutritional agents with a beneficial role in those neurodegenerative diseases associated with this oxidative imbalance. For this purpose, we have employed the undifferentiated PC12 cells, and H₂O₂ and Fenton reaction as free radical generating systems.

Materials and Methods

Wine samples, polyphenols, and reagents

Merlot grapes were cultivated in the experimental vineyard station of IMIDRA, “El Encin”, situated in Alcala de Henares (Madrid, Spain) and harvested at maturity in 2004. Merlot red wine was made following the procedure of Viñedos y Bodegas de Toledo S.L. (Madrid). After fruit obtention, grapes were crushed and then, 70 mg/L of SO₂ and the yeast Saccharomyces bayanus were added. Maceration was carried out at a temperature up to 30 °C during 7 days. Two types of wine were obtained, one free run and the other one pressed. The free run wine is the juice which flows without any pressing. The pressed wine is that one released by pressing the cap (Ortega and others 2008). Finally, the wines were dealcoholized by distillation at reduced pressure (60–80 mBar) and low temperature (25–30 °C) with a rotating evaporator. Samples, concentrated to 25% of its initial volume, were kept at −20 °C until analysis.

All the polyphenols used for quantitative analysis and for the activity assays (≥ 99% high performance liquid chromatography [HPLC] purity) were from Extrasynthese (Lyon, France).

All reagents were from Sigma-Aldrich (St. Louis, Mo., U.S.A.) and cell culture products from Gibco [Grand Island, N.Y.]. Louis, Mo., U.S.A.).

Analytical methods

Analysis of global polyphenolic families in Merlot red wine By using different colorimetric methods, the content in total polyphenols (Singleton and Rossi 1965), total catechins (Swain and Hillis 1959), total proanthocyanidins (Porter and others 1986), and total anthocyanidins (Paroneto 1977) have been determined in dealcoholized samples of free run and pressed Merlot red wine.

Analysis of polyphenols in Merlot red wine extract by HPLC Samples of dealcoholized wine were extracted with diethyl ether and ethyl acetate. The organic fractions were combined and evaporated to dryness at reduced pressure and 30 °C. The residue was dissolved in MeOH/H₂O (1:1, v/v). Samples were analyzed by HPLC with photodiode array (PAD) and mass spectrometry (MS) detection following the method described in detail by Ortega and others (2008). All polyphenolic compounds presented in wine extracts have been identified by comparing retention times and spectral data, recorded with the photodiode array detector, with commercial standards. The external standard method was used for the quantitative determination.

Cell culture and treatment Rat adrenal pheochromocytoma PC12 cell line was obtained from the American Type Culture Collection (ATCC; Manassas, Va., U.S.A.). Cells were cultured in DMEM supplemented with 10% horse serum, 5% fetal bovine serum, penicillin (10 U/mL), streptomycin (100 U/mL), and 0.2 mM sodium pyruvate and maintained in a humidified atmosphere with 5% CO₂ in air at 37 °C.

Cells were incubated with dealcoholized-free run and pressed Merlot red wine residues (6.8, 10.2 and 13.6 ml/L in PBS) and with the major polyphenols (0.25, 0.1, 0.025 and 0.01 mM) for 24 h. Then, cells were exposed to oxidative stress inductors (0.1 mM H₂O₂ and 0.1 mM FeSO₄+ 0.1 mM H₂O₂) for 30 min.

Cell viability assay The effect on PC12 cell viability has been evaluated by MTT assay (Mosmann 1983). After treatments, cells were incubated with 2 mg/mL MTT for 1 h at 37 °C. The blue formazan crystals, formed in viable cells, were dissolved in DMSO. Triton X-100 was used as a positive control of cytotoxicity. Absorbance at 550 nm was measured using a microplate reader (Digiscan 340, Assys Hitech GMBH, Austria). Absorbance of control cells was considered as 100% viability.

Measurement of intracellular ROS formation This assay has been performed as previously described Lebel and others (1992). After treatments, cells were incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) for 30 min. Fluorescence intensity was measured for 2 h using a fluorometer (FLx800, Bio-Tek instruments) at 485 nm and 530 nm λ_exc and λ_em, respectively.

Preparation of total cellular extracts After treatments, cells were washed with PBS and scrapped off from the dish. Samples were centrifuged at 640 g for 5 min. Then, cells were lysed with lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 20 μL/mL leupeptine, 10 μL/mL pepstatine, 35 μL/mL PMSF) and centrifuged at 1600 g at 4°C for 10 min. The supernatant (total cellular extract) was used for western blot analysis, glutathione analysis, enzymes activity assays and protein determination.

Enzyme activity assays Catalase (CAT)—CAT activity was measured based on the method described by Abei (1984). The decrease in absorbance was determined at 240 nm for 1 min. Superoxide dismutase (SOD)—SOD activity was determined according to the method of Marklund and Marklund (1974). Absorbance of the samples was measured at 420 nm for 1 min. Glutathione peroxidise (GPx)—GPx activity was measured following the method of Paglia and Valentine (1967). The absorbance was read at 340 nm for 3 min. Glutathione reductase (GR)—GR activity was assayed spectrophotometrically by following the oxidation of NADPH to NADP⁺ for 3 min at 340 nm at 25 °C using the method of Barja de Quiroga and others (1990). All enzymatic assays were performed using total cellular extract and the spectrophotometer was UVIKON 930 (Kontron Instruments, UK).

Western blot analysis Proteins were separated on a 10% SDS-polyacrylamide gel. The membranes were blocked for 90 min at room temperature in blocking buffer containing 10% nonfat milk in PBS. Then, membranes were incubated with primary antibodies: anti-SOD (1:1000), anti-CAT (1:1000), anti-GPx (1:2000), anti-GR (1:2000) for 1 h at room temperature. After a 30-min wash with PBS-Tween (PBS and 0.1% Tween 20), membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG for 2 h at room temperature. The membranes were washed again with PBS-Tween for 30 min, and transferred proteins were incubated with ECL-Advance detection kit for 1 min. Densitometric analysis of the bands was done using an image analyzer (Syngene Multigenius) with GenSnap and GenTools programs.

Determination of intracellular oxidized and reduced glutathione Quantification of oxidized (GSSG) and reduced glutathione (GSH) has been performed following the Hissin and Hilf method (1976). The fluorescent intensity was measured using a fluorometer (FLx800, Bio-Tek instruments) at λ_exc 350 nm and at λ_em 420 nm. GSH and GSSH content in cells were calculated as ng GSSG/mg protein. The results were expressed as Redox Index (RI = GSSG/GSH + GSSG).

Statistical analysis Results were expressed as mean ± SD of at least three independent experiments. The one-way ANOVA followed by LSD's test was used to compare control and treated groups. P < 0.05 was considered statistically significant.

Results and Discussion

Effect of Merlot red wine extracts against H₂O₂, FeSO₄+ H₂O₂-induced oxidative stress

Previous to evaluate the possible protective effect of the free run and pressed Merlot red wine extracts, the effect of different concentrations (6.8, 10.2 and 13.6 ml/L) of both wines on PC12 cell viability was investigated using MTT assay. As shown in Figure 1(A) none of these concentrations altered PC12 viability, being then used for investigating their possible neuroprotective effect under oxidative stress situation. As shown in Figure 1(B), both H₂O₂ (0.1 mM) and Fenton reagent (0.1 mM FeSO₄+ 0.1 mM H₂O₂) caused a significant loss in cell viability. However, pretreatments with free run and pressed Merlot red wine extracts for 24 h, prior to both oxidative stress inductors exposition for 30 min, avoid cellular death. The protective effect was higher for pressed Merlot red wine extract than for free run Merlot red wine extract; moreover, the 3 test concentrations for pressed Merlot red wine extracts were significant.

Figure 1–. Effect of free run and pressed Merlot red wine on PC12 cell viability. (A) Cells were treated with different concentrations of 2 types of wines (6.8, 10.2. and 13.6 ml/L) for 24 h. (B) Cells were preincubated with both types of wines (6.8, 10.2, and 13.6 ml/L) for 24 h, and then exposed to H₂O₂ (0.1 mM) and Fenton reaction (0.1 mM FeSO4 + 0.1 mM H₂O₂) for 30 min. Cell viability was measured by MTT assay. Results are expressed as mean ± SD of 3 independent experiments. *P < 0.05 versus oxidative stress inductors; ^#P < 0.05 versus control.

Following, using different colorimetric methods, we analyzed the content in global polyphenolic families. As shown in Table 1A, Merlot red wine made from pressed juices contained more total phenols, catechins, proanthocyanidins and anthocyanidins than free run juices; these differences in polyphenolic content were not significant between the winemaking processes. The highest concentrations of polyphenols in red wine are found in grape skins, seeds, and stems; during the process of pressing wines, all polyphenolic compounds are extracted in high amounts (Xia and others 2010).

Table 1–.  (A) Content in global polyphenolic families of both free run and pressed Merlot red wine, determined using different colorimetric methods. (B) Polyphenolic composition of pressed Merlot red wine (mg/L) determined by HPLC.

A)
	Free run wine	Pressed wine
Total Phenols (mg gallic acid/L)	1247.2	1469.2
Total Catechins (mg catechin/L)	929.5	1976.0
Total Proanthocyanidins (mg cyanidin-3-glucoside /L)	1182.9	1724.5
Total Anthocyanidins (mg malvidin 3-glucoside /L)	133.5	370.7

B)
Compounds (mg/L)	mean ± SD
Results are expressed as mean ± SD of three independent experiments.
(−)-Epicatechin	27.81 ± 1.29
(+)-Catechin	46.97 ± 2.77
Caffeic Acid	0.29 ± 0.01
Cafftaric Acid	1.99 ± 0.03
Dihydroquercetin	2.19 ± 0.04
Dydroflavonols	2.64 ± 0.11
Ethyl gallate	1.56 ± 0.02
Gallic acid	17.01 ± 0.25
Malva	0.06 ± 0.01
Methyl gallate	0.90 ± 0.03
Myricetin	1.24 ± 0.02
p-coumaroyl hexose	0.09 ± 0.01
Procyanidins	7.78 ± 1.17
Protocatechin acid	1.66 ± 0.06
Quercetin	4.65 ± 0.23
Quercetin 3- O galactoside	0.85 ± 0.04
Quercetin 3-O glucuronid	1.84 ± 0.03
Resveratrol cis	0.20 ± 0.03
Resveratrol cis glucoside	0.17 ± 0.02
Resveratrol trans	0.38 ± 0.04
Resveratrol trans glucoside	0.13 ± 0.03
Syringic acid	1.84 ± 0.09
trans cutaric	0.77 ± 0.01
Tryptofol	1.56 ± 0.07
Tyrosol	3.46 ± 0.42
Vanillic acid	2.83 ± 0.23

Finally, analysis of individual polyphenols in pressed Merlot red wine extract was carried out by HPLC. Non-anthocyanin polyphenolic compounds such as hydroxybenzoic acids (gallic acid, vanillic acid, and syringic acid) and hydroxycinnamic acids (caffeic acid and caftaric acid), flavanols (catechin, epicatechin and procyanidins), flavonols (quercetin and myricetin) and isomers of resveratrol and its glycosides have been identified (Table 1B). Quantitatively, quercetin, catechin, epicatechin, tyrosol, gallic acid, and procyanidins were the major polyphenols found and they were chosen for further investigation assays.

Effect of polyphenols against H₂O₂, FeSO₄+ H₂O₂-induced oxidative stress

Cytotoxic effects of polyphenols (quercetin, catechin, epicatechin, tyrosol, gallic acid and procyanidins) on PC12 cells were evaluated by incubating with different concentrations (0.25, 0.1, 0.05, and 0.025 mM) for 24 h. None of these concentrations were toxic for PC12 cells (data not shown).

Following, the possible protective effect of the polyphenols on an oxidative stress model was analyzed. Exogenous application of H₂O₂ and Fenton reaction for 30 min caused a decrease in cell viability of 51.5% and 56.1%, respectively, compared to control cells (Figure 2). Different sensitivity to both oxidative stress inductors was observed for PC12 cells. The reaction kinetics of H₂O₂, which is the major ROS in living organisms, is really slow. However, its oxidative strength is markedly increased with the addition exogenous of Fe²⁺ as it produces ^•OH. Because ^•OH is an extremely reactive species, cellular structures including lipids, proteins and DNA are oxidized, triggering apoptotic and necrotic cell death. Moreover, free iron, without an added oxidant agent, produces also free radicals (Tarawneh and Galvin 2010). Thus, that free iron which has not reacted with exogenous H₂O₂ can cause additional cellular damage.

Figure 2–. Protective effect of polyphenols on an oxidative stress model in PC12 cells. Cells were treated with 0.25, 0.1, 0.05, and 0.025 mM concentrations of polyphenols for 24 h followed by 30 min of oxidative stress inductors treatments. Cell viability was measured by MTT assay. (A) PC12 cells exposed to H₂O_2; (B) PC12 cells exposed to Fenton reaction (0.1 mM FeSO₄+ 0.1 mM H₂O₂). Results are expressed as mean ± SD of 3 independent experiments. *P < 0.05 versus oxidative stress inductors; ^#p < 0.05 versus control.

Pretreatments with catechin, quercetin and procyanidins at 0.25 mM and with catechin, quercetin, and epicatechin at 0.1 mM for 24 h, prior to 0.1 mM H₂O₂ exposure, restored significantly cell survival. The other 2 concentrations tested (0.05 and 0.025 mM) did not show any significant change in cell viability compared to H₂O₂-treated cells. The most protective polyphenols were quercetin at 0.1 mM (29.2% increased cell viability compared to H₂O₂ treated cells), following by epicatechin at 0.1 mM (25.5%) and quercetin at 0.25 mM (23.3%). Interestingly, a different protection against H₂O₂-induced cellular damage has been observed depending on the polyphenol used. Cíz and others (2008) have demonstrated different specificity and efficiency of polyphenols to scavenge certain free radicals such as peroxyl radicals.

When the protective effect of polyphenols Fenton reaction-induced toxicity was evaluated, results showed that all concentrations exerted a significant increase in PC12 cell viability compared with Fenton reaction-treated cells. Quercetin was once again the most active polyphenol by increasing cell viability 51.8% (0.25 mM) and 49.9% (0.1 mM) compared to Fenton reaction treated-cells.

In agreement with previous findings (Martin and others 2006), the flavonoids showed higher antioxidant capacity than other groups of polyphenols. Comparing the chemical structure of the polyphenols, quercetin contains some important different structural features which determine its significant effect against oxidizing agents. The catechol group in ring B is implicated in the inhibition of free radical production and overspreading through its radical scavenging activity and transition-metal ions chelating ability. Moreover, the 2,3-double bond, in conjugation with the 4-oxo function, determine a higher electron-delocalization, and the presence of both 3- and 5-OH groups seem to play an essential role in free radical stabilization.

Since the antioxidant effect of polyphenols is related to both their metal-chelating properties and their scavenging activities against free radicals, many authors have attempted to establish which of these 2 activities the main action mechanism of polyphenolic compounds is. Iwahashi (2000) consider the chelating properties of polyphenols to sequester the metal ions as the main mechanism of their antioxidant properties. On the other hand, Fremont and others (1999) attributed the antioxidant activity mostly to their markedly ROS scavenging properties. Our results showed that the different polyphenols exerted much more potent protective effect against Fenton reaction than against H₂O₂. As Fenton reaction involves Fe²⁺ and H₂O₂, we suggest that both mechanisms of action of polyphenolic antioxidants are implicated and have a great importance in cellular protection. This fact could explain the additive protective polyphenol effect observed using Fenton reaction oxidative system and comparing with H₂O₂ alone.

Regarding these results and comparing both oxidative stress inductors, all polyphenols exerted the highest and significant protective effect at 0.25 mM and 0.1 mM concentrations against Fenton reaction. For these reasons, these 2 concentrations and this oxidative stress inductor were chosen for the continuous experiments.

Effect of polyphenols on ROS production induced by Fenton reaction

During 2 h, ROS generation in PC12 cells exposed to exogenous Fenton reaction was measured. At the end of the experiment, a decrease in fluorescence which reflected a decrease in ROS production was observed throughout time, suggesting that the reaction may have been fully saturated.

Exposure to Fenton reaction led to a significant increase of intracellular ROS generation. A reduction in ROS formation was observed when cells were incubated with all the test polyphenols. This reduction was significant for quercetin, epicatechin, and catechin (0.25 mM; Figure 3). One of the possible preventive mechanisms of polyphenols against oxidative stress consists of removing the ROS excess. Polyphenols act as direct free radicals scavengers, donating a hydrogen atom to the radical and being the electron stabilizers by delocalization over the aromatic structures. Previous studies claimed a link between antioxidant potency of polyphenols and the structural features (Servili and others 2009). All test polyphenols reduced Fenton reaction-induced ROS production, this being significant for procyanidins, epicatechin, and catechin at 0.25 mM. These 3 natural products, which are flavonoids, have in common the presence of a catechol group that makes them very efficient as free radicals scavengers.

Figure 3–. Effect of polyphenols on Fenton reaction induced ROS production in PC12 cells. Cells were incubated with polyphenols (0.25 and 0.1 mM) and Fenton reaction (0.1 mM FeSO₄+ 0.1 mM H₂O₂). The intracellular ROS production was measured for 2 h. Results are expressed as mean ± SD of 3 independent experiments. *P < 0.05 versus Fenton reaction.

Effect of polyphenols on antioxidants enzymes and on glutathione

In addition to the classical antioxidant mechanism of polyphenols, we observed that the neuroprotective effect of these compounds is also likely to involve its capacity for modulating the antioxidant defence system. Spectrophotometric assays and Western blot analysis revealed that Fenton reaction exposure of 30 min in PC12 cells caused a significant decrease in activity and protein expression of the antioxidant enzymes SOD and GPx compared to control cells. No significant changes were observed for CAT and GR. A significant increase in both protein expression and activity of antioxidant enzymes were detected when PC12 cells were pretreated with quercetin and gallic acid (0.25 mM) for CAT enzyme, quercetin (0.25 and 0.1 mM) for SOD enzyme, quercetin (0.25 mM), epicatechin (0.25 and 0.1 mM), and gallic acid (0.1 mM) for GPx and quercetin and catechin (0.25 mM) for GR (Figure 4 and Table 2). Quercetin was the most active compound at 0.25 mM by increasing significantly activity and protein expression of all antioxidant enzymes compared to PC12 cells Fenton-only treated.

Figure 4–. Effect of polyphenols on antioxidant enzymes protein expression in PC12 cells under oxidative stress produced via Fenton reaction. Cells were treated with polyphenols (0.25 mM and 0.1 mM) for 24 h followed by a Fenton reaction (0.1 mM FeSO₄+ 0.1 mM H₂O₂) exposure for 30 min. Protein expression was analyzed by Western Blot assays. Results are expressed as mean ± SD of 3 independent experiments. *P < 0.05 versus Fenton reaction; ^#P < 0.05 versus control. β-actine was used as a loading control. Q: quercetin, C: catechin, E: epicatechin, T: tyrosol, GA: gallic acid, and PC: procyanidins.

Table 2–.  Effect of polyphenols on antioxidant enzymes activity and on Redox Index (RI = GSSG / GSSG + GSH) in PC12 cells under oxidative stress produced via Fenton reaction. Cells were treated with polyphenols (0.25 and 0.1 mM) for 24 h followed by a Fenton reaction (0.1 mM FeSO₄+ 0.1 mM H₂O₂) exposure for 30 min.

	CAT activity (IU/min·mg protein)	SOD activity (IU/mg protein)	GR activity (nmol NADPH/min·mg protein)	Total GPx activity (nmol NADPH/min·mg protein)	RI = GSSG /GSSG + GSH
Results are expressed as mean ± SD of 3 independent experiments. *P < 0.05 versus Fenton reaction; ^#P < 0.05 versus control.
Control	7.60 ± 1.1	9.21 ± 0.6	33.82 ± 7.8	24.84 ± 4.1	0.42 ± 0.07
Fenton	7.16 ± 1.2	7.53 ± 0.5^#	31.38 ± 8.4	14.54 ± 1.7^#	0.57 ± 0.04^#
0.25 mM Quercetin + Fenton	8.21 ± 0.5*	8.74 ± 2.3*	45.63 ± 8.6*	28.28 ± 0.0*	0.48 ± 0.08*
0.1 mM Quercetin + Fenton	7.11 ± 1.4	7.85 ± 1.7*	33.10 ± 8.8	21.07 ± 1.5*	0.48 ± 0.03*
0.25 mM Catechin + Fenton	7.65 ± 1.1	7.79 ± 1.7	41.29 ± 7.1*	12.70 ± 1.0^#	0.46 ± 0.09*
0.1 mM Catechin + Fenton	6.33 ±0.5	6.03 ± 1.9^#	41.34 ± 6.3*	15.66 ± 0.4	0.46 ± 0.01*
0.25 mM Epicatechin + Fenton	7.19 ± 0.6	7.54 ± 1.3	37.15 ± 6.7	23.72 ± 5.2*	0.46 ± 0.06*
0.1 mM Epicatechin + Fenton	6.07 ± 0.2	7.76 ± 1.5	37.81 ± 5.2	25.45 ± 4.2*	0.42 ± 0.04*
0.25 mM Tyrosol + Fenton	7.69 ± 1.4	9.10 ± 3.0*	37.35 ± 6.1	20.65 ± 2.5*	0.41 ± 0.06*
0.1 mM Tyrosol + Fenton	5.68 ± 0.4^#	6.18 ± 1.0^#	33.13 ± 5.0	24.16 ± 4.8*	0.41 ± 0.08*
0.25 mM Gallic acid + Fenton	7.76 ± 0.5*	6.45 ± 1.0^#	40.44 ± 7.2*	19.12 ± 5.8*	0.44 ± 0.06*
0.1 mM Gallic acid + Fenton	5.76 ± 1.0^#	4.65 ± 0.7^#	40.40 ± 6.4*	25.97 ± 3.5*	0.43 ± 0.04*
0.25 mM Procyanidins + Fenton	7.44 ± 1.3	7.32 ± 1.8	37.66 ± 6.8	23.29 ± 4.5*	0.44 ± 0.05*
0.1 mM Procyanidins + Fenton	6.77 ± 0.6	5.27 ± 0.4^#	41.46 ± 6.1*	22.32 ± 1.4*	0.44 ± 0.08*

Previous studies have demonstrated that CAT and SOD levels were recovered when quercetin was administrated for 30 days to mice under oxidative stress conditions (Singh and others 2003). Interestingly, Echeverry and others (2010) found that quercetin, through the hydroxyl substitutions in C5 and C7 in the A-ring and in C3 in the C-ring, could exert a neuronal protection by inducing the expression of intracellular targets.

Finally, we demonstrated that these polyphenols enhanced the GSH levels. Exposure of PC12 cells to Fenton reaction (30 min) resulted in a significant increase of Redox Index (RI = GSSG/GSH+GSSG) compared to control cells. The level of GSSG was higher than the level of GSH. Changes in RI were observed with pretreatments (24 h) with all polyphenols (0.25 mM and 0.1 mM). The amounts of GSH were increased in favour to GSSG, leading to a significant decrease of RI compared to Fenton reaction cells treated (Table 2). Quercetin (0.25 mM) was once again the most active compound; however, any statistical difference has been observed between quercetin and the other polyphenols. These results support previous works that have demonstrated that those catechol-containing molecules are able to enhance GSH levels. Quercetin exerted a neuronal protection against glutamate cytotoxicity by preventing GSH oxidation (Ishiga and others 2001).

Conclusion

This study provides further evidences to support those previous findings on potential benefits of the Merlot red wine extracts and its major polyphenolic compounds (quercetin, catechin, epicatechin, tyrosol, gallic acid, and procyanidins) as protective agents of neurons under oxidative stress situation. These results showed that pressed Merlot red wine extract contained high content in all global polyphenolic families and exerted higher protective effect than free run Merlot red wine extract; these differences were not significant, suggesting that the vinification process does not influence markedly in neuroprotection. Among the individual major polyphenols found in Merlot red wine extract, quercetin was the most active, preventing PC12 cell death, ROS overproduction, changes in glutathione index and loss of antioxidant enzymes activity and protein expression.

Acknowledgment

This work was supported by Comunidad Autónoma de la Rioja (FPI fellowship). The authors thank Drs. I. Estrella and T. Hernández from CSIC, Madrid, Spain for their technical assistance with HPLC analysis.