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.
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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 |
|(−)-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 H2O2; (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.
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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.
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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.
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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 |
|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).