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Red wine polyphenols protect n−3 more than n−6 polyunsaturated fatty acid from lipid peroxidation
Food Research International 44 (2011) 3065–3071
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Red wine polyphenols protect n−3 more than n−6 polyunsaturated fatty acid from lipid peroxidation Roberta Cazzola ⁎, Benvenuto Cestaro Department of Clinical Sciences “L. Sacco”, Faculty of Medicine and Surgery, University of Milan, Via G. B. Grassi, 74-20157 Milan, Italy
a r t i c l e
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Article history: Received 13 May 2011 Accepted 20 July 2011 Keywords: Grape polyphenols Antioxidants Polyunsaturated fatty acids n−3 fatty acids Lipid peroxidation
a b s t r a c t Moderate red wine consumption is associated with decreased risk for cardiovascular disease; however, the underlying mechanisms are not completely understood. The main aim of this study was to investigate the effects of red wine polyphenols (WP) on the oxidizability of human plasma fatty acids, in particular those most involved in the inflammatory response — archidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The oxidizability of the major fatty acids of plasma was determined by measuring their loss gaschromatographycally during peroxidation kinetics induced by 2′-azobis(2methylpropionamidine)-dihydrochloride. The capacity of WP to scavenge 1,1,diphenyl-2-picryl-hydrazyl (DPPH), superoxide anion and hydroxyl radicals, and trap total peroxyl radicals in plasma (TRAP) was also measured. WP (1.75–5 μg/mL) inhibited DPPH, superoxide anion and hydroxyl radicals and increased TRAP in a dose-dependent manner. WP (1.75 μg/mL) significantly protected all plasma PUFA from peroxidation but the protection of EPA and DHA was higher than that of AA. These results suggest that the association of WP to apolipoproteins makes EPA and DHA less accessible to hydro-soluble radicals than AA, thus providing a biochemical rationale for future ‘in vivo’ studies on the benefits to health of moderate red wine consumption. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Red wine is a significant natural source of polyphenols in Mediterranean-type diets, contributing as much as one gram of polyphenols per day from moderate wine consumption (Soleas, Diamandis, & Goldberg, 1997). Epidemiological studies suggest that consumption of grape products, especially red wine, and other foods rich in polyphenols is associated with decreased risk for cardiovascular disease, cancer and other chronic diseases (Iriti & Faoro, 2009; Leifert & Abeywardena, 2008). In particular, moderate red wine consumption (up to 300 mL wine per day) has been shown to be inversely associated with mortality due to cardiovascular diseases (Agarwal, 2002; Leifert & Abeywardena, 2008). Red wine phenols have been reported to possess a broad spectrum of pharmacological and therapeutic effects that, in most cases, are linked to their anti-oxidant properties (Brown et al., 2009). These are conducted through various mechanisms — inhibition of oxidizing enzymes, free-radical scavenger activity, transition-metalchelating action and, consequently, stabilization of lipid peroxidation (Bors, Heller, Michel, & Saran, 1990; Shan, Cai, Sun, & Corke, 2005). Lipid
Abbreviations: AA, arachidonic acid; AAPH, 2,2′-azobis(2-methylpropionamidine) dihydrochloride; DHA, docosahexaenoic acid; DPPH, 1,1,diphenyl-2-picryl-hydrazyl radical; EPA, eicosapentaenoic acid; TRAP, peroxyl radical-trapping potential of plasma; WP, red wine polyphenols. ⁎ Corresponding author. Tel.: + 39 0250319690; fax: + 39 0250319697. E-mail addresses: [email protected] (R. Cazzola), [email protected] (B. Cestaro). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.07.029
peroxidation in plasma lipoproteins may induce and propagate chronic inflammatory processes in the vasculature, a condition that plays an integral role in the pathophysiology of many chronic diseases including atherosclerotic cardiovascular disease. Among lipids, polyunsaturated fatty acids (PUFA) are easily attacked by free radicals that react with their double bonds, yielding several bioactive compounds – such as short-chain aldehydes, conjugated dienes and lipid peroxides – able to promote and exacerbate the inflammatory response and oxidative stress (Schnitzer, Pinchuk, & Lichtenberg, 2007; Spiteller, 2005). PUFA are also the precursors of several lipid mediators with pro- or antiinflammatory activity, such as eicosanoids, lipoxins, resolvins and docosatrienes (Calder, 2001). Lipid mediators derived from long-chain n−6 PUFA (e.g. arachidonic acid) have pro-inflammatory and immunoactive functions, whereas those derived from long-chain n−3 PUFA (e.g. eicosapentaenoic acid and docosahexaenoic acid) have antiinflammatory properties (Wall, Ross, Fitzgerald, & Stanton, 2010). The alteration of the ratio between n−6 and n−3 PUFA during peroxidation could amplify the inflammatory response promoted by lipid peroxidation. The susceptibility of PUFA to peroxidation depends on the kind of oxidant, the milieu in which PUFA react with oxidants (model systems, cell membranes, lipoproteins, etc.) and has been thought to be directly dependent on their degree of unsaturation. Some in vitro (Visioli, Colombo, & Galli, 1998; Yazu, Yamamoto, Ukegawa, & Niki, 1996) and in vivo (Mas et al., 2010) studies, however, suggest that individual PUFA oxidize at different rates and originate different oxidation products in such a way that the relation between the PUFA chemical structure and susceptibility to oxidation is not as simple as has been hypothesized
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from theoretical viewpoints (Visioli et al., 1998). Consequently, the ability of different antioxidants to protect various PUFA from peroxidation cannot be deduced either just by considering their chemical features. Red wine contains a very complex mixture of phenolic antioxidant compounds that includes flavonols such as myricetin, kaempferol and quercetin, the flavan-3-ol monomers catechin and epicatechin, the oligomeric and polymeric flavan-3-ols or proanthocyanidins, anthocyanins, phenolic acids (gallic, caftaric, caffeic and pcoumaric acids) and the stilbene resveratrol (Soleas et al., 1997). The action of a blend so rich in antioxidants may protect the various n−6 and n−3 PUFA from lipid peroxidation differently, thus also influencing the production of lipid mediators of inflammation by modulating the concentration of the PUFA precursors. Previous studies have investigated the effects of wine phenols (WP) on the lipid peroxidation of isolated low-density lipoprotein (LDL) by using various oxidant systems (e.g. transition metals, heme-protein, UV light exposition, etc.) and measuring one or more of the numerous compounds directly or indirectly formed during fatty acid peroxidation (e.g. conjugated dienes, 4hydroxy-nonenal, malondialdehyde, thiobarbituric acid reactive substances, etc.) (Aviram & Fuhrman, 2002; Faustino, Clark, Sobrattee, Czubryl, & Pierce, 2004; Frankel, Kanner, German, Parks, & Kinsella, 1993). However, to our knowledge, the effects of WP on the oxidizability of the major fatty acids of whole plasma have not yet been thoroughly investigated. Therefore, the main aim of this study was to investigate the effects of red wine polyphenols (WP) on the oxidizability of the major fatty acids of human plasma by gas chromatography technique measuring their loss during peroxidation kinetics. The antioxidant activity of WP was also defined by evaluating their ability to trap total peroxyl radicals in plasma (TRAP) and scavenge free radicals (DPPH, superoxide anion radicals and hydroxyl radicals). 2. Materials and methods 2.1. Materials Analytical grade chemicals, gradient grade solvents and doublydistilled water were used. Solvents were from Merck (VWR International, Milan, Italy), butylated hydroxytoluene (BHT), deoxyribose, nitrobluetetrazolium chloride, xanthine and xanthine oxidase, superoxide dismutase, quercetin, mannitol, trolox, 1,1,diphenyl-2-picryl-hydrazyl radical (DPPH), diethylene triamine pentaacetic acid (DTPA), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox ©), 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), dichlorofluorescindiacetate, polyphenol standards and fatty acid methyl esters were from Sigma (Sigma-Aldrich, Milan, Italy). Fasting venous blood samples were withdrawn in heparin from five normo-lipidemics, non-smoking, 25 to 45 years of age, male volunteers, in agreement with the ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983). Each subject signed a consent form that stated the purpose of the study and the sampling to be done. The blood was immediately centrifuged (1000 × g for 15 min at 4 °C), the plasma samples were pooled, and then aliquots were immediately frozen in liquid nitrogen and stored at −80 °C until use. 2.2. Red wine extract Red wine polyphenols were obtained from the red wine “Buttafuoco” (a blend of croatina, barbera and uva rara grape varieties, from Oltrepò Pavese, Italy). Red wine was filtered to remove sediment and concentrated under vacuum (b30 °C). The concentrated wine was passed through an absorbent polystyrene resin column Diaion HP-20 Relite (Supelco, Sigma-Aldrich, Milan, Italy), which was then washed with double-distilled water. The polyphenols were eluted with 45– 50% ethanol. The eluate was evaporated under vacuum (b30 °C) and
then the extract was solubilized in double-distilled water (10 mg/mL), divided into 1 mL aliquots and stored under nitrogen at 4 °C in the dark until use (max. 2 weeks). The concentrations of the major polyphenols of the extracts were determined by reverse-phase HPLC as described by Price et al. (Price, Breen, Valladao, & Watson, 1995). Samples were analyzed on a Perkin Elmer HPLC system using a Nova-Pak C18 column (4 μm, 4.6 × 250 mm) of Waters. The concentrations of polyphenols were calculated from the calibration curves made with standard solutions. The polyphenol composition of red wine extract was: Phenolic compound
mg/g
Anthocyanin glycosides Catechin Epicatechin Gallic acid Polymeric phenols Polymeric anthocyanidins Procyanidin dimers Quercetin Quercitin glycosides trans Resveratrol
73 7 16 0.3 198 7 63 1.1 20 2.1
2.3. Total phenols The total phenol concentration of the extract was determined by the Folin–Ciocalteu method (Singleton & Rossi, 1965), using quercetin as reference standard. 2.4. Radical scavenger activities Red wine extract was suitably diluted so as to obtain desired concentrations of total wine polyphenols (WP) in 100 μl of doublydistilled water (final concentration range: 0–15 μg/mL). Reduction of DPPH radical was performed as previously described (Cazzola, Camerotto, & Cestaro, 2011). In brief: to 3 mL of DPPH 60 μM in ethanol were added 100 μL of WP extract and absorbance was monitored at 517 nm (Abs 517 nm extract). The absorbance of a control (100 μL distilled water instead of WP) was also recorded at the same wavelength (Abs 517 nm control). Therefore, the percentage of inhibition was calculated by the formula:% inhibition = [(Abs 517 nm control − Abs 517 nm extract) / Abs 517 nm control] × 100. The scavenger activity of the extract was compared with the activity of quercetin (10 μg/mL). Superoxide was generated by oxidation of xanthine (30 mM) with xanthine oxidase (5 U) in 60 mM phosphate buffer pH 7.4 EDTA 30 mM, and was detected by nitroblue tetrazolium (3 mM) followed spectrophotometrically at 560 nm. Superoxide radical scavenger activity of WP was determined by the formula:% inhibition = [(Abs 560 nm control − Abs 560 nm extract) / Abs 560 nm control] × 100. Quercetin and superoxide dismutase were used as reference standard. Hydroxyl radical was generated by incubation for 60 min at 37 °C of a reaction mixture containing 100 μΜ Fe Cl3, 100 μΜ ascorbate, 1 mM hydrogen peroxide, 2.8 mM deoxyribose in phosphate buffer 20 mM, pH 7.4. Deoxyribose degradation by hydroxyl radical occurred in presence of WP or control (distilled water) was estimated by using the thiobarbituric acid (TBA) assay (532 nm absorbance). Therefore, the percentage of inhibition was calculated by the formula: % inhibition = [(Abs 532 nm control − Abs 532 nm extract)/ Abs 532 nm control] × 100. Quercetin and mannitol were used as reference standards. 2.5. Total peroxyl radical-trapping antioxidant potential of plasma (TRAP) Plasma diluted to 5% in phosphate-buffered saline solution pH 7.4 (PBS) was used for the determination of the effect of WP on total
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peroxyl radical-trapping potential. The total peroxyl radical-trapping potential (TRAP) was determined as described by Valkonen and Kuusi (Valkonen & Kuusi, 1997) with slight modifications. Briefly, to diluted plasma were added increasing amounts of WP (1–5 μg/mL), 10 μmol/L of the free radical probe dichlorofluorescin-diacetate (DCF-DA), 50 mmol/L of the metal chelator diethylene triamine pentaacetic acid (DTPA), and 25 mmol/L of the free radical generator 2,2′diazobis-(2-amidinopropane)-dihydrochloride (AAPH). The conversion of (DCF-DA) to the high fluorescent dichlorofluorescein (DCF) induced by the thermal decomposition of AAPH was measured at 37 °C by recording DCF fluorescence (λex 510 nm and λem 530 nm) every 15 min for 2 h. Changes in fluorescence were monitored by a Perkin-Elmer LS-50B spectrofluorometer. Trolox (5 μmol/L) was used as reference standard, each molecule of trolox is able to neutralize two molecules of peroxyl radicals. The lag-time of each DCF formation curves was calculated. The peroxyl radical-trapping potential has been quantified by using the following equation: TRAP = Tplasma = Ttrolox × plasma dilution f actor × 2 × 5 μmol = L where Tplasma is the lag time of plasma obtained in presence of increasing amounts of WP and TTrolox is the lag time of plasma with 5 μmol/L of trolox. Values are expressed as micromoles/L of peroxyl radicals trapped. The speed of DCF fluorescence development (slope) in the propagation phase of peroxidation kinetics was also calculated. 2.6. Kinetics of plasma fatty acid peroxidation The kinetics of plasma fatty acid peroxidation were measured by determining the changes of plasma fatty acid concentrations induced by AAPH 25 mM. Plasma was diluted to 10% in PBS pH 7.4 containing 50 mmol/L of the metal chelator DTPA and then divided in two aliquots: to one aliquot were added WP dissolved in 100 μL of distilled water (final concentration 1.25 μg/mL) while to the other 100 μl of distilled water, (CT). After the addition of AAPH, each sample was incubated at 37 °C for 3 h. Aliquots were taken from the reaction mixtures before the addition of AAPH and after 45, 90, 150, 180, 210 and 240 min of peroxidation and immediately transferred into cool (4 °C) chloroform: methanol 2:1 (v/v) containing 0.2% butylated hydroxytoluene to block the reaction. Plasma fatty acid composition was determined on lipid extracted according to Folch (Folch, Lees, & Sloane Stanley, 1957). The lipid extracts were evaporated under nitrogen stream and then methylated with anhydrous methanol-HCl at 90° for 2 h. Fatty acid methyl esters were analyzed using capillary gas chromatography as previously described (Cazzola, Rondanelli, Russo-Volpe, Ferrari, & Cestaro, 2004). The fatty acid methyl esters were identified according to their retention time in comparison to known standards and were quantified by using heptadecanoic acid as the internal standard. Total PUFA were calculated by summing the concentrations of linoleic acid (18:2 n−6), di-omo-γ-linolenic acid (20:3 n−6.), arachidonic acid (20:4 n−6, AA), eicosapentaenoic acid (20:5 n−3, EPA) and docosahexaenoic acid (22:6 n−3, DHA). The loss of fatty acids was estimated by the formula: ½ðF0 –Ft Þ = F0 × 100: where F0 is the concentration of the fatty acid at zero time of peroxidation and Ft is the residual concentration for a given peroxidation time. For each PUFA, the duration of lag-phase (lagtime) and the rate of fatty acid degradation in the propagation phase (slope) of the peroxidation kinetic were also calculated. 2.7. Statistical analysis Experiments were performed in triplicate and repeated three times. Data are expressed as mean of all replicates. Standard deviation
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was always less than 5% of the corresponding mean value. Comparison between data were performed by the Student's t test (two-tailed) for unpaired samples. All statistical analyses were performed by using StatistiXL software (version 1.5; StatistiXL, Bradway-Nedlands, Western Australia). 3. Results 3.1. Radical scavenger activities The scavenger action of increasing amounts (0.75–15 μg/mL) of red wine polyphenols (WP) against DPPH, hydroxyl and superoxide radicals is shown in Fig. 1. WP inhibited the formation of these free radicals in a dose-dependent manner. At each WP concentration, the inhibition of DPPH radicals was greater than that of superoxide radicals, and this was greater than that of hydroxyl radicals. WP activity was also compared with that of quercetin, the main flavonol in our diet, or other standard antioxidants, such as mannitol for hydroxyl radicals and superoxide dismutase (SOD) for superoxide radicals. In our experimental conditions, the inhibitory actions of 5 μg/mL WP against DPPH, hydroxyl and superoxide radicals were 53.3%, 8.2% and 39.6%, respectively; whereas those of 5 μg/mL of quercetin were 49.0%, 5.1% and 15.0%, respectively. Moreover, 5 μg/mL of mannitol inhibited 32.3% hydroxyl radicals and 2.5 U SOD inhibited 60.0% superoxide radical formation. 3.2. Total peroxyl radical-trapping antioxidant potential of plasma (TRAP) The effect of red wine extract on TRAP was investigated using WP concentrations ranging between 0.75 and 5 μg/mL. Fig. 2 depicts the dose dependency of the TRAP increase. The dose-dependent decrease of the rate of propagation phase (slope), measured on the same peroxidation curves used for TRAP calculation, is also shown in the same figure. The addition of 1.25 μg/mL WP to plasma increased the TRAP value by 47% and decreased the slope by 22.6%. 3.3. Kinetics of plasma fatty acid peroxidation The proportions of total saturated fatty acids, monounsaturated fatty acids and PUFA in native pooled plasma used in these experiments were 47.3%, 16.0% and 36.7%, respectively. The peroxidation of plasma, induced by peroxyl radicals derived from the thermal decomposition of AAPH, resulted in negligible losses of saturated (16:0 and 18:0) and mono-unsaturated (16:1 and 18:1) fatty acids both with and without 1.25 μg/mL WP (data not shown). The hydro-soluble peroxyl radicals derived from AAPH were able to degrade appreciable quantities of plasma PUFA. The kinetics of PUFA degradation were represented by sigmoid-shaped curves noting the three known consecutive phases — the lag, propagation, and termination. When we took into consideration the peroxidation kinetic of total PUFA we found that the addition to plasma of 1.25 μg/mL WP determined a significant increase in lag-time (from 85± 5.1 min to 118 ± 9.7 min, p b 0.01) and a significant decrease in the propagation rate (from 0.21 ± 0.02 to 0.12 ± 0.02, p b 0.01). After 150 min of peroxidation, the extent of PUFA degraded with or without 1.25 μg/mL WP were respectively 8.5% and 15.7% of their initial concentrations. The effects of WP on the lag-time and the propagation rate of the peroxidation kinetics of the most polyunsaturated PUFA of the n−6 (AA) and n−3 (EPA and DHA) series are shown in Fig. 3. In the absence of WP, EPA and DHA showed similar values of lag-time, while the lagtime of AA was significantly lower than those EPA and DHA (Fig. 3, panel A). These results indicate that in our experimental conditions the resistance of peroxidation of high polyunsaturated PUFA of plasma is influenced more by the location of double bonds on the acyl chain than from their number. Conversely, as shown in the panel B of Fig. 3, the
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100 90 80
& inhibition
70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
µg WP /ml Fig. 1. Scavenger activity of red wine polyphenols towards DPPH (–●–), superoxide anion (–▲–), and hydroxyl radicals (–■–). Experiments were performed in triplicate and repeated three times. Data are expressed as mean of all replicates. Standard deviation was always less than 5% of the corresponding mean value.
the underlying mechanisms are not completely understood. This study was primarily on the investigation of the effects of polyphenols extracted from the red wine ‘Buttafuoco’ on the peroxidation of plasma polyunsaturated fatty acid, in particular those mostly involved in the modulation of the inflammatory response. Moreover, the antioxidant capacity of the extract was also determined by assays more commonly used at this purpose — the ability to scavenge free radicals (DPPH, superoxide anion radical and hydroxyl radical). We used only one type of red wine but the antioxidant effect found in this extract can probably be generalized to other types of red wine with a similar polyphenol content. The addition of 1.75–5 μg/mL WP to human plasma significantly increased its total peroxyl radical-trapping antioxidant potential (TRAP) in a dose-dependent manner: at the lower concentration used (1.75 μg/mL), the TRAP value was yet increased by approx. 50%. This higher antioxidant activity is also likely due to the ability of WP both to interact with albumin and to synergize with low molecular-weight plasma antioxidants, such as vitamin C and E, glutathione, etc.… These concentrations of WP are of the same order of magnitude as the
speed of the peroxidation reaction in the propagation phase (slope) was strongly influenced by the number of double bonds (DHAN EPAN AA). The addition to plasma of 1.25 μg/mL WP significantly improved the lagtime and the slope of the peroxidation kinetics of all these PUFA. In particular, WP determined a significant increase in lag-time of AA, EPA and DHA of 17.3%, 45.8% and 50% and reduced the slope by 15%, 26% and 34%, respectively. Lastly, Fig. 4 shows the peroxidation-induced changes over time of the ratio between AA and EPA, the two PUFA most involved in the modulation of inflammatory response. Without WP, the ratio between AA and EPA remained almost unchanged in the first 90 min of peroxidation, and then greatly increased up to 180 min; with 1.25 μg/mL WP, this increase was delayed by approx. 60 min. 4. Discussion The antioxidant and anti-inflammatory action of red wine and/or its bioactive components has been studied extensively both ‘in vitro’ (Frankel et al., 1993; Lourenco, Gago, Barbosa, de Freitas, & Laranjinha, 2008) and ‘in vivo’ (Gresele et al., 2011; Soleas et al., 1997); however,
2500
120
100
2000
1500 60 1000
TRAP (µmol/l)
slope (F.U./min)
80
40
500 20
0
0 0
1
2
3
4
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6
µg WP/ml Fig. 2. Effects of red wine polyphenols on peroxyl radical-trapping potential (TRAP, –●–) and peroxidation rate (slope, -▲-) of human plasma. Experiments were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates.
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A 180
A 160
*
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*
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b
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a
60 40 20 0 AA
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EPA
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Slope (% FA degraded/min)
1
c b
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*
*
a
* 0.4
0.2
0 AA
EPA
DHA
Fig. 3. Lag-time (A) and slope (B) of the peroxidation kinetics of plasma fatty acids. Peroxidation was induced by AAPH 25 mM without (CT, colored bars) or with 1.25 μg/mL WP (white bars). AA: arachidonic acid; EPA: eicosapentaenoic acid; DHA docosahexaenoic acid. Measurements were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates. In each panel, values denoted by different letters are significantly different from one another (p b 0.05). * Significantly different from CT (p b 0.05).
increase in the concentration of total phenols observed in the plasma after ingestion of 100–300 mL of red wine (Duthie et al., 1998; Serafini, Maiani, & Ferro-Luzzi, 1998); however it is important to highlight that the composition of polyphenols of the extract used “in vitro” might not be the same as that found in plasma after oral intake of wine. Further, 1.75 μg/mL of WP significantly protected the plasma PUFA from AAPH-induced peroxidation. With reference to the results of the experiments on peroxidation of plasma PUFA, we consider the protective effects exerted by 1.75 μg/mL WP on the most polyunsaturated fatty acids of the n−6 and n−3 family [AA (25:4, n−6), EPA (20:5, n−3) and DHA (20:6, n−3)] of particular interest. In the absence of WP, we found that the lag-times of EPA and DHA were similar and both significantly higher than that of AA, whereas the propagation rate of peroxidation kinetic increased according to the number of double bonds of these PUFA: DHA N EPA N AA. The above results concerning the changes in the lag-time indicate that peroxyl radicals from the aqueous phase of plasma react with AA more easily and earlier than with EPA and DHA. One possible explanation is that in the phospholipid envelope of plasma lipoproteins, EPA and DHA may
form a tighter intermolecular packing than AA, as previously shown in liposomal membranes (Araseki, Yamamoto, & Miyashita, 2002). The higher reactivity of EPA and DHA in comparison with AA that we found in the propagation phase may thus be compensated by the difficulty of hydrosoluble free radicals in attacking this tighter conformation of n−3 phospholipids in the lag phase. With WP, the lag-time of all these PUFA significantly increased, but the increase in lag-time of DHA (+50%) and EPA (+46%) was higher than that of AA (+17%). WP can associate with lipoprotein surface by both ionic interactions and hydrogen bonds;and when associated in this way to the surface they probably carry out their protective action at the interface between the hydrophobic core and the hydrophilic surface of lipoproteins (Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). The association of WP with apolipoprotein may thus further limit the interaction of free radicals with phospholipid PUFA (Terao & Piskula, 1999). Further, WP were also able to decrease the propagation rate of peroxidation kinetics of DHA (− 34%) and EPA (− 26%) more intensively that that of AA (−16%). A possible explanation is that the lipidic radicals derived from these n−3 are more polar than those
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25
AA/EPA
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Time (min) Fig. 4. Changes of the ratio between arachidonic acid (AA) and eicosapentaenoic acid (EPA) during plasma peroxidation. Peroxidation was induced by AAPH 25 mM without (black circles) or with 1.25 μg/mL WP (white circles). Measurements were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates.
of n−6 (Yazu et al., 1996) and the former are likely to diffuse more rapidly from the hydrophobic core to the hydrophilic surface of the lipoproteins, thus better interacting with the WP associated with apolipoprotein. All this evidence on the protective effects of WP may thus form one of the molecular mechanisms for the explanation of the higher plasma and erythrocytes levels of EPA and DHA found in moderate wine drinkers (Di Giuseppe et al., 2009). Finally, it is important to highlight that WP were also able to retard the alteration of the ratio between AA and EPA by approximately 60 min. Since it is well known that this ratio is fundamental in regulating the production of pro-inflammatory (AA cascade) and antiinflammatory (EPA cascade) eicosanoids, this evidence may form one of the most determinant molecular mechanisms at the basis of the anti-inflammatory effects associated with moderate red wine consumption (Vazquez-Agell et al., 2007). 5. Conclusion The results of the present study indicate that red wine polyphenols protect n−3 PUFA more than n−6 PUFA of plasma. Even the study has the limitation of being conducted using only ‘in vitro’ and ‘ex vivo’ models which can deviate from the ‘in vivo’ situation because the pool of phenols that reaches the blood may, at least in part, be different from that in wine, however, these results provide new biochemical rationales for the programming of future studies to investigate the potential health benefits of moderate consumption of red wine ‘in vivox more thoroughly. Conflict of interest None. Acknowledgments This work was founded by FIRST grant provided by the Italian Ministry of University to Roberta Cazzola and Benvenuto Cestaro. References Agarwal, D. P. (2002). Cardioprotective effects of light–moderate consumption of alcohol: a review of putative mechanisms. Alcohol and Alcoholism, 37, 409–415. Araseki, M., Yamamoto, K., & Miyashita, K. (2002). Oxidative stability of polyunsaturated fatty acid in phosphatidylcholine liposomes. Bioscience, Biotechnology, and Biochemistry, 66, 2573–2577.
Aviram, M., & Fuhrman, B. (2002). Wine flavonoids protect against LDL oxidation and atherosclerosis. Annals of the New York Academy of Sciences, 957, 146–161. Bors, W., Heller, W., Michel, C., & Saran, M. (1990). Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods in Enzymology, 186, 343–355. Brown, L., Kroon, P. A., Das, D. K., Das, S., Tosaki, A., Chan, V., et al. (2009). The biological responses to resveratrol and other polyphenols from alcoholic beverages. Alcoholism, Clinical and Experimental Research, 33, 1513–1523. Calder, P. C. (2001). Polyunsaturated fatty acids, inflammation, and immunity. Lipids, 36, 1007–1024. Cazzola, R., Camerotto, C., & Cestaro, B. (2011). Anti-oxidant, anti-glycant, and inhibitory activity against α-amylase and α-glucosidase of selected spices and culinary herbs. International Journal of Food Sciences and Nutrition, 62, 175–184. Cazzola, R., Rondanelli, M., Russo-Volpe, S., Ferrari, E., & Cestaro, B. (2004). Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes. Journal of Lipid Research, 45, 1846–1851. Di Giuseppe, R., De Lorgeril, M., Salen, P., Laporte, F., Di Castelnuovo, A., Krogh, V., et al. (2009). Alcohol consumption and n−3 polyunsaturated fatty acids in healthy men and women from 3 European populations. American Journal of Clinical Nutrition, 89, 354–362. Duthie, G. G., Pedersen, M. W., Gardner, P. T., Morrice, P. C., Jenkinson, A. M., McPhail, D. B., et al. (1998). The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. European Journal of Clinical Nutrition, 52, 733–736. Faustino, R. S., Clark, T. A., Sobrattee, S., Czubryl, M. P., & Pierce, G. N. (2004). Differential antioxidant properties of red wine in water soluble and lipid soluble peroxyl radical generating systems. Molecular and Cellular Biochemistry, 263, 211–215. Folch, J., Lees, M., & Sloane Stanley, G. (1957). A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry, 226, 497–509. Frankel, E. N., Kanner, J., German, J. B., Parks, E., & Kinsella, J. E. (1993). Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet, 341, 454–457. Gresele, P., Cerletti, C., Guglielmini, G., Pignatelli, P., de Gaetano, G., & Violi, F. (2011). Effects of resveratrol and other wine polyphenols on vascular function: An update. The Journal of Nutritional Biochemistry, 22, 201–211. Iriti, M., & Faoro, F. (2009). Bioactivity of grape chemicals for human health. Natural Products Communication, 4, 611–634. Leifert, W. R., & Abeywardena, M. Y. (2008). Cardioprotective actions of grape polyphenols. Nutritional Research, 28, 729–737. Lourenco, C. F., Gago, B., Barbosa, R. M., de Freitas, V., & Laranjinha, J. (2008). LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion. Journal of Agriculture and Food Chemistry, 56, 3798–3804. Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols: Food sources and bioavailability. American Journal of Clinical Nutrition, 79, 727–747. Mas, E., Woodman, R. J., Burke, V., Puddey, I. B., Beilin, L. J., Durand, T., et al. (2010). The omega-3 fatty acids EPA and DHA decrease plasma F(2)-isoprostanes: Results from two placebo-controlled interventions. Free Radic Research, 44, 983–990. Price, S. F., Breen, P. J., Valladao, M., & Watson, B. T. (1995). Cluster sun exposure and quercetin in pinot noir grapes and wine. American Journal of Enology and Viticulture, 46, 187–194. Schnitzer, E., Pinchuk, I., & Lichtenberg, D. (2007). Peroxidation of liposomal lipids. European Biophysics Journal, 36, 499–515. Serafini, M., Maiani, G., & Ferro-Luzzi, A. (1998). Alcohol-free red wine enhances plasma antioxidant capacity in humans. Journal of Nutrition, 128, 1003–1007. Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice extracts and characterization of their phenolic constituents. Journal of Agriculture and Food Chemistry, 53, 7749–7759.
R. Cazzola, B. Cestaro / Food Research International 44 (2011) 3065–3071 Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American Journal of Enology and Viticulture, 16, 144–158. Soleas, G. J., Diamandis, E. P., & Goldberg, D. M. (1997). Wine as a biological fluid: history, production, and role in disease prevention. Journal of Clinical Laboratory Analysis, 11, 287–313. Spiteller, G. (2005). The relation of lipid peroxidation processes with atherogenesis: a new theory on atherogenesis. Molecular Nutrition & Food Research, 49, 999–1013. Terao, J., & Piskula, M. K. (1999). Flavonoids and membrane lipid peroxidation inhibition. Nutrition, 15, 790–791. Valkonen, M., & Kuusi, T. (1997). Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum. Journal of Lipid Research, 38, 823–833.
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Vazquez-Agell, M., Sacanella, E., Tobias, E., Monagas, M., Antunez, E., Zamora-Ros, R., et al. (2007). Inflammatory markers of atherosclerosis are decreased after moderate consumption of cava (sparkling wine) in men with low cardiovascular risk. Journal of Nutrition, 137, 2279–2284. Visioli, F., Colombo, C., & Galli, C. (1998). Oxidation of individual fatty acids yields different profiles of oxidation markers. Biochemical and Biophysical Research Communications, 245, 487–489. Wall, R., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2010). Fatty acids from fish: the antiinflammatory potential of long-chain omega-3 fatty acids. Nutrition Review, 68, 280–289. Yazu, K., Yamamoto, Y., Ukegawa, K., & Niki, E. (1996). Mechanism of lower oxidizability of eicosapentaenoate than linoleate in aqueous micelles. Lipids, 31(3), 337–340.
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Red wine polyphenols protect n−3 more than n−6 polyunsaturated fatty acid from lipid peroxidation Roberta Cazzola ⁎, Benvenuto Cestaro Department of Clinical Sciences “L. Sacco”, Faculty of Medicine and Surgery, University of Milan, Via G. B. Grassi, 74-20157 Milan, Italy
a r t i c l e
i n f o
Article history: Received 13 May 2011 Accepted 20 July 2011 Keywords: Grape polyphenols Antioxidants Polyunsaturated fatty acids n−3 fatty acids Lipid peroxidation
a b s t r a c t Moderate red wine consumption is associated with decreased risk for cardiovascular disease; however, the underlying mechanisms are not completely understood. The main aim of this study was to investigate the effects of red wine polyphenols (WP) on the oxidizability of human plasma fatty acids, in particular those most involved in the inflammatory response — archidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The oxidizability of the major fatty acids of plasma was determined by measuring their loss gaschromatographycally during peroxidation kinetics induced by 2′-azobis(2methylpropionamidine)-dihydrochloride. The capacity of WP to scavenge 1,1,diphenyl-2-picryl-hydrazyl (DPPH), superoxide anion and hydroxyl radicals, and trap total peroxyl radicals in plasma (TRAP) was also measured. WP (1.75–5 μg/mL) inhibited DPPH, superoxide anion and hydroxyl radicals and increased TRAP in a dose-dependent manner. WP (1.75 μg/mL) significantly protected all plasma PUFA from peroxidation but the protection of EPA and DHA was higher than that of AA. These results suggest that the association of WP to apolipoproteins makes EPA and DHA less accessible to hydro-soluble radicals than AA, thus providing a biochemical rationale for future ‘in vivo’ studies on the benefits to health of moderate red wine consumption. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Red wine is a significant natural source of polyphenols in Mediterranean-type diets, contributing as much as one gram of polyphenols per day from moderate wine consumption (Soleas, Diamandis, & Goldberg, 1997). Epidemiological studies suggest that consumption of grape products, especially red wine, and other foods rich in polyphenols is associated with decreased risk for cardiovascular disease, cancer and other chronic diseases (Iriti & Faoro, 2009; Leifert & Abeywardena, 2008). In particular, moderate red wine consumption (up to 300 mL wine per day) has been shown to be inversely associated with mortality due to cardiovascular diseases (Agarwal, 2002; Leifert & Abeywardena, 2008). Red wine phenols have been reported to possess a broad spectrum of pharmacological and therapeutic effects that, in most cases, are linked to their anti-oxidant properties (Brown et al., 2009). These are conducted through various mechanisms — inhibition of oxidizing enzymes, free-radical scavenger activity, transition-metalchelating action and, consequently, stabilization of lipid peroxidation (Bors, Heller, Michel, & Saran, 1990; Shan, Cai, Sun, & Corke, 2005). Lipid
Abbreviations: AA, arachidonic acid; AAPH, 2,2′-azobis(2-methylpropionamidine) dihydrochloride; DHA, docosahexaenoic acid; DPPH, 1,1,diphenyl-2-picryl-hydrazyl radical; EPA, eicosapentaenoic acid; TRAP, peroxyl radical-trapping potential of plasma; WP, red wine polyphenols. ⁎ Corresponding author. Tel.: + 39 0250319690; fax: + 39 0250319697. E-mail addresses: [email protected] (R. Cazzola), [email protected] (B. Cestaro). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.07.029
peroxidation in plasma lipoproteins may induce and propagate chronic inflammatory processes in the vasculature, a condition that plays an integral role in the pathophysiology of many chronic diseases including atherosclerotic cardiovascular disease. Among lipids, polyunsaturated fatty acids (PUFA) are easily attacked by free radicals that react with their double bonds, yielding several bioactive compounds – such as short-chain aldehydes, conjugated dienes and lipid peroxides – able to promote and exacerbate the inflammatory response and oxidative stress (Schnitzer, Pinchuk, & Lichtenberg, 2007; Spiteller, 2005). PUFA are also the precursors of several lipid mediators with pro- or antiinflammatory activity, such as eicosanoids, lipoxins, resolvins and docosatrienes (Calder, 2001). Lipid mediators derived from long-chain n−6 PUFA (e.g. arachidonic acid) have pro-inflammatory and immunoactive functions, whereas those derived from long-chain n−3 PUFA (e.g. eicosapentaenoic acid and docosahexaenoic acid) have antiinflammatory properties (Wall, Ross, Fitzgerald, & Stanton, 2010). The alteration of the ratio between n−6 and n−3 PUFA during peroxidation could amplify the inflammatory response promoted by lipid peroxidation. The susceptibility of PUFA to peroxidation depends on the kind of oxidant, the milieu in which PUFA react with oxidants (model systems, cell membranes, lipoproteins, etc.) and has been thought to be directly dependent on their degree of unsaturation. Some in vitro (Visioli, Colombo, & Galli, 1998; Yazu, Yamamoto, Ukegawa, & Niki, 1996) and in vivo (Mas et al., 2010) studies, however, suggest that individual PUFA oxidize at different rates and originate different oxidation products in such a way that the relation between the PUFA chemical structure and susceptibility to oxidation is not as simple as has been hypothesized
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from theoretical viewpoints (Visioli et al., 1998). Consequently, the ability of different antioxidants to protect various PUFA from peroxidation cannot be deduced either just by considering their chemical features. Red wine contains a very complex mixture of phenolic antioxidant compounds that includes flavonols such as myricetin, kaempferol and quercetin, the flavan-3-ol monomers catechin and epicatechin, the oligomeric and polymeric flavan-3-ols or proanthocyanidins, anthocyanins, phenolic acids (gallic, caftaric, caffeic and pcoumaric acids) and the stilbene resveratrol (Soleas et al., 1997). The action of a blend so rich in antioxidants may protect the various n−6 and n−3 PUFA from lipid peroxidation differently, thus also influencing the production of lipid mediators of inflammation by modulating the concentration of the PUFA precursors. Previous studies have investigated the effects of wine phenols (WP) on the lipid peroxidation of isolated low-density lipoprotein (LDL) by using various oxidant systems (e.g. transition metals, heme-protein, UV light exposition, etc.) and measuring one or more of the numerous compounds directly or indirectly formed during fatty acid peroxidation (e.g. conjugated dienes, 4hydroxy-nonenal, malondialdehyde, thiobarbituric acid reactive substances, etc.) (Aviram & Fuhrman, 2002; Faustino, Clark, Sobrattee, Czubryl, & Pierce, 2004; Frankel, Kanner, German, Parks, & Kinsella, 1993). However, to our knowledge, the effects of WP on the oxidizability of the major fatty acids of whole plasma have not yet been thoroughly investigated. Therefore, the main aim of this study was to investigate the effects of red wine polyphenols (WP) on the oxidizability of the major fatty acids of human plasma by gas chromatography technique measuring their loss during peroxidation kinetics. The antioxidant activity of WP was also defined by evaluating their ability to trap total peroxyl radicals in plasma (TRAP) and scavenge free radicals (DPPH, superoxide anion radicals and hydroxyl radicals). 2. Materials and methods 2.1. Materials Analytical grade chemicals, gradient grade solvents and doublydistilled water were used. Solvents were from Merck (VWR International, Milan, Italy), butylated hydroxytoluene (BHT), deoxyribose, nitrobluetetrazolium chloride, xanthine and xanthine oxidase, superoxide dismutase, quercetin, mannitol, trolox, 1,1,diphenyl-2-picryl-hydrazyl radical (DPPH), diethylene triamine pentaacetic acid (DTPA), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox ©), 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), dichlorofluorescindiacetate, polyphenol standards and fatty acid methyl esters were from Sigma (Sigma-Aldrich, Milan, Italy). Fasting venous blood samples were withdrawn in heparin from five normo-lipidemics, non-smoking, 25 to 45 years of age, male volunteers, in agreement with the ethical standards as formulated in the Helsinki Declaration of 1975 (revised 1983). Each subject signed a consent form that stated the purpose of the study and the sampling to be done. The blood was immediately centrifuged (1000 × g for 15 min at 4 °C), the plasma samples were pooled, and then aliquots were immediately frozen in liquid nitrogen and stored at −80 °C until use. 2.2. Red wine extract Red wine polyphenols were obtained from the red wine “Buttafuoco” (a blend of croatina, barbera and uva rara grape varieties, from Oltrepò Pavese, Italy). Red wine was filtered to remove sediment and concentrated under vacuum (b30 °C). The concentrated wine was passed through an absorbent polystyrene resin column Diaion HP-20 Relite (Supelco, Sigma-Aldrich, Milan, Italy), which was then washed with double-distilled water. The polyphenols were eluted with 45– 50% ethanol. The eluate was evaporated under vacuum (b30 °C) and
then the extract was solubilized in double-distilled water (10 mg/mL), divided into 1 mL aliquots and stored under nitrogen at 4 °C in the dark until use (max. 2 weeks). The concentrations of the major polyphenols of the extracts were determined by reverse-phase HPLC as described by Price et al. (Price, Breen, Valladao, & Watson, 1995). Samples were analyzed on a Perkin Elmer HPLC system using a Nova-Pak C18 column (4 μm, 4.6 × 250 mm) of Waters. The concentrations of polyphenols were calculated from the calibration curves made with standard solutions. The polyphenol composition of red wine extract was: Phenolic compound
mg/g
Anthocyanin glycosides Catechin Epicatechin Gallic acid Polymeric phenols Polymeric anthocyanidins Procyanidin dimers Quercetin Quercitin glycosides trans Resveratrol
73 7 16 0.3 198 7 63 1.1 20 2.1
2.3. Total phenols The total phenol concentration of the extract was determined by the Folin–Ciocalteu method (Singleton & Rossi, 1965), using quercetin as reference standard. 2.4. Radical scavenger activities Red wine extract was suitably diluted so as to obtain desired concentrations of total wine polyphenols (WP) in 100 μl of doublydistilled water (final concentration range: 0–15 μg/mL). Reduction of DPPH radical was performed as previously described (Cazzola, Camerotto, & Cestaro, 2011). In brief: to 3 mL of DPPH 60 μM in ethanol were added 100 μL of WP extract and absorbance was monitored at 517 nm (Abs 517 nm extract). The absorbance of a control (100 μL distilled water instead of WP) was also recorded at the same wavelength (Abs 517 nm control). Therefore, the percentage of inhibition was calculated by the formula:% inhibition = [(Abs 517 nm control − Abs 517 nm extract) / Abs 517 nm control] × 100. The scavenger activity of the extract was compared with the activity of quercetin (10 μg/mL). Superoxide was generated by oxidation of xanthine (30 mM) with xanthine oxidase (5 U) in 60 mM phosphate buffer pH 7.4 EDTA 30 mM, and was detected by nitroblue tetrazolium (3 mM) followed spectrophotometrically at 560 nm. Superoxide radical scavenger activity of WP was determined by the formula:% inhibition = [(Abs 560 nm control − Abs 560 nm extract) / Abs 560 nm control] × 100. Quercetin and superoxide dismutase were used as reference standard. Hydroxyl radical was generated by incubation for 60 min at 37 °C of a reaction mixture containing 100 μΜ Fe Cl3, 100 μΜ ascorbate, 1 mM hydrogen peroxide, 2.8 mM deoxyribose in phosphate buffer 20 mM, pH 7.4. Deoxyribose degradation by hydroxyl radical occurred in presence of WP or control (distilled water) was estimated by using the thiobarbituric acid (TBA) assay (532 nm absorbance). Therefore, the percentage of inhibition was calculated by the formula: % inhibition = [(Abs 532 nm control − Abs 532 nm extract)/ Abs 532 nm control] × 100. Quercetin and mannitol were used as reference standards. 2.5. Total peroxyl radical-trapping antioxidant potential of plasma (TRAP) Plasma diluted to 5% in phosphate-buffered saline solution pH 7.4 (PBS) was used for the determination of the effect of WP on total
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peroxyl radical-trapping potential. The total peroxyl radical-trapping potential (TRAP) was determined as described by Valkonen and Kuusi (Valkonen & Kuusi, 1997) with slight modifications. Briefly, to diluted plasma were added increasing amounts of WP (1–5 μg/mL), 10 μmol/L of the free radical probe dichlorofluorescin-diacetate (DCF-DA), 50 mmol/L of the metal chelator diethylene triamine pentaacetic acid (DTPA), and 25 mmol/L of the free radical generator 2,2′diazobis-(2-amidinopropane)-dihydrochloride (AAPH). The conversion of (DCF-DA) to the high fluorescent dichlorofluorescein (DCF) induced by the thermal decomposition of AAPH was measured at 37 °C by recording DCF fluorescence (λex 510 nm and λem 530 nm) every 15 min for 2 h. Changes in fluorescence were monitored by a Perkin-Elmer LS-50B spectrofluorometer. Trolox (5 μmol/L) was used as reference standard, each molecule of trolox is able to neutralize two molecules of peroxyl radicals. The lag-time of each DCF formation curves was calculated. The peroxyl radical-trapping potential has been quantified by using the following equation: TRAP = Tplasma = Ttrolox × plasma dilution f actor × 2 × 5 μmol = L where Tplasma is the lag time of plasma obtained in presence of increasing amounts of WP and TTrolox is the lag time of plasma with 5 μmol/L of trolox. Values are expressed as micromoles/L of peroxyl radicals trapped. The speed of DCF fluorescence development (slope) in the propagation phase of peroxidation kinetics was also calculated. 2.6. Kinetics of plasma fatty acid peroxidation The kinetics of plasma fatty acid peroxidation were measured by determining the changes of plasma fatty acid concentrations induced by AAPH 25 mM. Plasma was diluted to 10% in PBS pH 7.4 containing 50 mmol/L of the metal chelator DTPA and then divided in two aliquots: to one aliquot were added WP dissolved in 100 μL of distilled water (final concentration 1.25 μg/mL) while to the other 100 μl of distilled water, (CT). After the addition of AAPH, each sample was incubated at 37 °C for 3 h. Aliquots were taken from the reaction mixtures before the addition of AAPH and after 45, 90, 150, 180, 210 and 240 min of peroxidation and immediately transferred into cool (4 °C) chloroform: methanol 2:1 (v/v) containing 0.2% butylated hydroxytoluene to block the reaction. Plasma fatty acid composition was determined on lipid extracted according to Folch (Folch, Lees, & Sloane Stanley, 1957). The lipid extracts were evaporated under nitrogen stream and then methylated with anhydrous methanol-HCl at 90° for 2 h. Fatty acid methyl esters were analyzed using capillary gas chromatography as previously described (Cazzola, Rondanelli, Russo-Volpe, Ferrari, & Cestaro, 2004). The fatty acid methyl esters were identified according to their retention time in comparison to known standards and were quantified by using heptadecanoic acid as the internal standard. Total PUFA were calculated by summing the concentrations of linoleic acid (18:2 n−6), di-omo-γ-linolenic acid (20:3 n−6.), arachidonic acid (20:4 n−6, AA), eicosapentaenoic acid (20:5 n−3, EPA) and docosahexaenoic acid (22:6 n−3, DHA). The loss of fatty acids was estimated by the formula: ½ðF0 –Ft Þ = F0 × 100: where F0 is the concentration of the fatty acid at zero time of peroxidation and Ft is the residual concentration for a given peroxidation time. For each PUFA, the duration of lag-phase (lagtime) and the rate of fatty acid degradation in the propagation phase (slope) of the peroxidation kinetic were also calculated. 2.7. Statistical analysis Experiments were performed in triplicate and repeated three times. Data are expressed as mean of all replicates. Standard deviation
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was always less than 5% of the corresponding mean value. Comparison between data were performed by the Student's t test (two-tailed) for unpaired samples. All statistical analyses were performed by using StatistiXL software (version 1.5; StatistiXL, Bradway-Nedlands, Western Australia). 3. Results 3.1. Radical scavenger activities The scavenger action of increasing amounts (0.75–15 μg/mL) of red wine polyphenols (WP) against DPPH, hydroxyl and superoxide radicals is shown in Fig. 1. WP inhibited the formation of these free radicals in a dose-dependent manner. At each WP concentration, the inhibition of DPPH radicals was greater than that of superoxide radicals, and this was greater than that of hydroxyl radicals. WP activity was also compared with that of quercetin, the main flavonol in our diet, or other standard antioxidants, such as mannitol for hydroxyl radicals and superoxide dismutase (SOD) for superoxide radicals. In our experimental conditions, the inhibitory actions of 5 μg/mL WP against DPPH, hydroxyl and superoxide radicals were 53.3%, 8.2% and 39.6%, respectively; whereas those of 5 μg/mL of quercetin were 49.0%, 5.1% and 15.0%, respectively. Moreover, 5 μg/mL of mannitol inhibited 32.3% hydroxyl radicals and 2.5 U SOD inhibited 60.0% superoxide radical formation. 3.2. Total peroxyl radical-trapping antioxidant potential of plasma (TRAP) The effect of red wine extract on TRAP was investigated using WP concentrations ranging between 0.75 and 5 μg/mL. Fig. 2 depicts the dose dependency of the TRAP increase. The dose-dependent decrease of the rate of propagation phase (slope), measured on the same peroxidation curves used for TRAP calculation, is also shown in the same figure. The addition of 1.25 μg/mL WP to plasma increased the TRAP value by 47% and decreased the slope by 22.6%. 3.3. Kinetics of plasma fatty acid peroxidation The proportions of total saturated fatty acids, monounsaturated fatty acids and PUFA in native pooled plasma used in these experiments were 47.3%, 16.0% and 36.7%, respectively. The peroxidation of plasma, induced by peroxyl radicals derived from the thermal decomposition of AAPH, resulted in negligible losses of saturated (16:0 and 18:0) and mono-unsaturated (16:1 and 18:1) fatty acids both with and without 1.25 μg/mL WP (data not shown). The hydro-soluble peroxyl radicals derived from AAPH were able to degrade appreciable quantities of plasma PUFA. The kinetics of PUFA degradation were represented by sigmoid-shaped curves noting the three known consecutive phases — the lag, propagation, and termination. When we took into consideration the peroxidation kinetic of total PUFA we found that the addition to plasma of 1.25 μg/mL WP determined a significant increase in lag-time (from 85± 5.1 min to 118 ± 9.7 min, p b 0.01) and a significant decrease in the propagation rate (from 0.21 ± 0.02 to 0.12 ± 0.02, p b 0.01). After 150 min of peroxidation, the extent of PUFA degraded with or without 1.25 μg/mL WP were respectively 8.5% and 15.7% of their initial concentrations. The effects of WP on the lag-time and the propagation rate of the peroxidation kinetics of the most polyunsaturated PUFA of the n−6 (AA) and n−3 (EPA and DHA) series are shown in Fig. 3. In the absence of WP, EPA and DHA showed similar values of lag-time, while the lagtime of AA was significantly lower than those EPA and DHA (Fig. 3, panel A). These results indicate that in our experimental conditions the resistance of peroxidation of high polyunsaturated PUFA of plasma is influenced more by the location of double bonds on the acyl chain than from their number. Conversely, as shown in the panel B of Fig. 3, the
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100 90 80
& inhibition
70 60 50 40 30 20 10 0 0
2
4
6
8
10
12
14
µg WP /ml Fig. 1. Scavenger activity of red wine polyphenols towards DPPH (–●–), superoxide anion (–▲–), and hydroxyl radicals (–■–). Experiments were performed in triplicate and repeated three times. Data are expressed as mean of all replicates. Standard deviation was always less than 5% of the corresponding mean value.
the underlying mechanisms are not completely understood. This study was primarily on the investigation of the effects of polyphenols extracted from the red wine ‘Buttafuoco’ on the peroxidation of plasma polyunsaturated fatty acid, in particular those mostly involved in the modulation of the inflammatory response. Moreover, the antioxidant capacity of the extract was also determined by assays more commonly used at this purpose — the ability to scavenge free radicals (DPPH, superoxide anion radical and hydroxyl radical). We used only one type of red wine but the antioxidant effect found in this extract can probably be generalized to other types of red wine with a similar polyphenol content. The addition of 1.75–5 μg/mL WP to human plasma significantly increased its total peroxyl radical-trapping antioxidant potential (TRAP) in a dose-dependent manner: at the lower concentration used (1.75 μg/mL), the TRAP value was yet increased by approx. 50%. This higher antioxidant activity is also likely due to the ability of WP both to interact with albumin and to synergize with low molecular-weight plasma antioxidants, such as vitamin C and E, glutathione, etc.… These concentrations of WP are of the same order of magnitude as the
speed of the peroxidation reaction in the propagation phase (slope) was strongly influenced by the number of double bonds (DHAN EPAN AA). The addition to plasma of 1.25 μg/mL WP significantly improved the lagtime and the slope of the peroxidation kinetics of all these PUFA. In particular, WP determined a significant increase in lag-time of AA, EPA and DHA of 17.3%, 45.8% and 50% and reduced the slope by 15%, 26% and 34%, respectively. Lastly, Fig. 4 shows the peroxidation-induced changes over time of the ratio between AA and EPA, the two PUFA most involved in the modulation of inflammatory response. Without WP, the ratio between AA and EPA remained almost unchanged in the first 90 min of peroxidation, and then greatly increased up to 180 min; with 1.25 μg/mL WP, this increase was delayed by approx. 60 min. 4. Discussion The antioxidant and anti-inflammatory action of red wine and/or its bioactive components has been studied extensively both ‘in vitro’ (Frankel et al., 1993; Lourenco, Gago, Barbosa, de Freitas, & Laranjinha, 2008) and ‘in vivo’ (Gresele et al., 2011; Soleas et al., 1997); however,
2500
120
100
2000
1500 60 1000
TRAP (µmol/l)
slope (F.U./min)
80
40
500 20
0
0 0
1
2
3
4
5
6
µg WP/ml Fig. 2. Effects of red wine polyphenols on peroxyl radical-trapping potential (TRAP, –●–) and peroxidation rate (slope, -▲-) of human plasma. Experiments were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates.
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A 180
A 160
*
*
140
Lagtime (min)
120
*
100 80
b
b
a
60 40 20 0 AA
B
DHA
EPA
1.2
Slope (% FA degraded/min)
1
c b
0.8
0.6
*
*
a
* 0.4
0.2
0 AA
EPA
DHA
Fig. 3. Lag-time (A) and slope (B) of the peroxidation kinetics of plasma fatty acids. Peroxidation was induced by AAPH 25 mM without (CT, colored bars) or with 1.25 μg/mL WP (white bars). AA: arachidonic acid; EPA: eicosapentaenoic acid; DHA docosahexaenoic acid. Measurements were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates. In each panel, values denoted by different letters are significantly different from one another (p b 0.05). * Significantly different from CT (p b 0.05).
increase in the concentration of total phenols observed in the plasma after ingestion of 100–300 mL of red wine (Duthie et al., 1998; Serafini, Maiani, & Ferro-Luzzi, 1998); however it is important to highlight that the composition of polyphenols of the extract used “in vitro” might not be the same as that found in plasma after oral intake of wine. Further, 1.75 μg/mL of WP significantly protected the plasma PUFA from AAPH-induced peroxidation. With reference to the results of the experiments on peroxidation of plasma PUFA, we consider the protective effects exerted by 1.75 μg/mL WP on the most polyunsaturated fatty acids of the n−6 and n−3 family [AA (25:4, n−6), EPA (20:5, n−3) and DHA (20:6, n−3)] of particular interest. In the absence of WP, we found that the lag-times of EPA and DHA were similar and both significantly higher than that of AA, whereas the propagation rate of peroxidation kinetic increased according to the number of double bonds of these PUFA: DHA N EPA N AA. The above results concerning the changes in the lag-time indicate that peroxyl radicals from the aqueous phase of plasma react with AA more easily and earlier than with EPA and DHA. One possible explanation is that in the phospholipid envelope of plasma lipoproteins, EPA and DHA may
form a tighter intermolecular packing than AA, as previously shown in liposomal membranes (Araseki, Yamamoto, & Miyashita, 2002). The higher reactivity of EPA and DHA in comparison with AA that we found in the propagation phase may thus be compensated by the difficulty of hydrosoluble free radicals in attacking this tighter conformation of n−3 phospholipids in the lag phase. With WP, the lag-time of all these PUFA significantly increased, but the increase in lag-time of DHA (+50%) and EPA (+46%) was higher than that of AA (+17%). WP can associate with lipoprotein surface by both ionic interactions and hydrogen bonds;and when associated in this way to the surface they probably carry out their protective action at the interface between the hydrophobic core and the hydrophilic surface of lipoproteins (Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). The association of WP with apolipoprotein may thus further limit the interaction of free radicals with phospholipid PUFA (Terao & Piskula, 1999). Further, WP were also able to decrease the propagation rate of peroxidation kinetics of DHA (− 34%) and EPA (− 26%) more intensively that that of AA (−16%). A possible explanation is that the lipidic radicals derived from these n−3 are more polar than those
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25
AA/EPA
20
15
10
5
0 0
30
60
90
120
150
180
210
240
270
Time (min) Fig. 4. Changes of the ratio between arachidonic acid (AA) and eicosapentaenoic acid (EPA) during plasma peroxidation. Peroxidation was induced by AAPH 25 mM without (black circles) or with 1.25 μg/mL WP (white circles). Measurements were performed in triplicate and repeated three times. Data are expressed as mean ± SD of all replicates.
of n−6 (Yazu et al., 1996) and the former are likely to diffuse more rapidly from the hydrophobic core to the hydrophilic surface of the lipoproteins, thus better interacting with the WP associated with apolipoprotein. All this evidence on the protective effects of WP may thus form one of the molecular mechanisms for the explanation of the higher plasma and erythrocytes levels of EPA and DHA found in moderate wine drinkers (Di Giuseppe et al., 2009). Finally, it is important to highlight that WP were also able to retard the alteration of the ratio between AA and EPA by approximately 60 min. Since it is well known that this ratio is fundamental in regulating the production of pro-inflammatory (AA cascade) and antiinflammatory (EPA cascade) eicosanoids, this evidence may form one of the most determinant molecular mechanisms at the basis of the anti-inflammatory effects associated with moderate red wine consumption (Vazquez-Agell et al., 2007). 5. Conclusion The results of the present study indicate that red wine polyphenols protect n−3 PUFA more than n−6 PUFA of plasma. Even the study has the limitation of being conducted using only ‘in vitro’ and ‘ex vivo’ models which can deviate from the ‘in vivo’ situation because the pool of phenols that reaches the blood may, at least in part, be different from that in wine, however, these results provide new biochemical rationales for the programming of future studies to investigate the potential health benefits of moderate consumption of red wine ‘in vivox more thoroughly. Conflict of interest None. Acknowledgments This work was founded by FIRST grant provided by the Italian Ministry of University to Roberta Cazzola and Benvenuto Cestaro. References Agarwal, D. P. (2002). Cardioprotective effects of light–moderate consumption of alcohol: a review of putative mechanisms. Alcohol and Alcoholism, 37, 409–415. Araseki, M., Yamamoto, K., & Miyashita, K. (2002). Oxidative stability of polyunsaturated fatty acid in phosphatidylcholine liposomes. Bioscience, Biotechnology, and Biochemistry, 66, 2573–2577.
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