Efficiency and capacity of antioxidant rich foods in trapping peroxyl radicals: A full evaluation of radical scavenging activity

Food Research International 44 (2011) 269–275

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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

Efficiency and capacity of antioxidant rich foods in trapping peroxyl radicals: A full evaluation of radical scavenging activity Paola Vanzani a,b, Monica Rossetto a,b, Veronica De Marco a, Adelio Rigo a,b,⁎, Marina Scarpa c a b c

Department of Biological Chemistry, University of Padova, v. G. Colombo, 3, 35131 Padova, Italy Consortium INBB, section of Padova, v. Medaglie d'Oro, 305, 00136, Roma, Italy Department of Physics, University of Trento, v. Sommarive, 14, 38050, Povo, Trento, Italy

a r t i c l e

i n f o

Article history: Received 30 July 2010 Accepted 14 October 2010 Keywords: Beverage Fruit Vegetable Polyphenols Lipid peroxidation Peroxyl radical scavenging

a b s t r a c t For the first time a variety of foods, characterized by high antioxidant activity, have been tested for the reactivity by which the system of antioxidants present in these foods competes for peroxyl radicals with poly-unsaturated fatty acids. The oxygraphic method we have used, on the basis of a rigorous kinetic model, permits to obtain the reactivity that is the Peroxyl Radical Trapping Efficiency (PRTE), beyond the Peroxyl Radical Trapping Capacity (PRTC) and to assign to each food characteristic values of these parameters, so facilitating their inter-comparison. In the analyzed foods the PRTE/PRTC ratio spans more than one order of magnitude, so reflecting the quality of antioxidants present in foods. According to the PRTE values and on the basis of the serving size the ranking of antioxidant food efficiency in trapping peroxyl radicals was blueberryN red chicoryN coffee N pineapple≈ red wine ≥ orange≈ dark chocolate ≈ apple ≥ tea N pomegranate. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction “Healthy lifestyle” is a dictate of the incoming years and, as a consequence, the nutraceutical potentiality of food is of primary interest for consumers and care providers. For instance polyphenols present in foods may act as antioxidants in the inhibition of lipid peroxidation process, that may occur very easily under the conditions present in the gastrointestinal tract (GI) (Gorelik et al., 2005; Lapidot, Granit, & Kanner, 2005a,b; Ursini et al., 1998). In this context, the correct ranking of the antioxidant properties of food appears important. To this regards we should consider two parameters: the capacity, that is the amount of peroxyl free radicals scavenged by a given amount of food, and the efficiency, that is the reactivity of foods in trapping these radicals (Roginsky, 2003) before they damage critical biological molecules (Frankel & Meyer, 2000). The first

Abbreviations: ABIP, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride; DCA, deoxycholic acid sodium salt; DPPH, diphenylpicrylhydrazyl; GI, gastrointestinal; IC50, concentration of the antioxidant which provides 50% inhibition; IH, antioxidant; LG, rac1-lauroylglycerol; LH, linoleic acid; LO•2, peroxyl radical; LT, lag-time; PRTE, Peroxyl Radical Trapping Efficiency; ePRTE, equivalent Peroxyl Radical Trapping Efficiency; ePRTEss, equivalent Peroxyl Radical Trapping Efficiency per serving size; PRTC, Peroxyl Radical Trapping Capacity; PRTCss, Peroxyl Radical Trapping Capacity per serving size; PUFA, poly-unsaturated fatty acids; R0, rate of oxygen consummation due to azo-compound; RIN, rate of initiation of peroxidation; ss, serving size; TP, total phenol content; TPss, total phenols per serving size. ⁎ Corresponding author. Department of Biological Chemistry, University of Padova, v. G. Colombo, 3, 35131 Padova, Italy. Tel.: +39 049 8276107; fax: +39 049 8073310. E-mail address: [email protected] (A. Rigo). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.10.022

parameter depends on the stoichiometry of the trapping process while the second parameter is related to its kinetic rate constant. However, all the methods until now proposed measure only the capacity of foods (Roginsky & Lissi, 2005), since also the tests that report IC50 of foods are static methods being the electron-donating or H-donating capacity of an antioxidant (i.e. the amount of diphenylpicrylhydrazyl, DPPH, scavenged) expressed as IC50. In other words they measure in some way the stoichiometry of the inhibition process. To evaluate the capacity and efficiency of antioxidants we set up a simple oxygraphic method that, based on a rigorous treatment of the kinetic data relative to the free radical peroxidation of fatty acids, permits to calculate the Peroxyl Radical Trapping Capacity (PRTC), that is the amount of peroxyl radicals trapped by a given amount of antioxidant (usually micromoles of peroxyl radicals trapped by one gram of antioxidant), and Peroxyl Radical Trapping Efficiency (PRTE) that is the reciprocal of IC50 (Zennaro et al., 2007), where IC50 is the concentration of antioxidant which provides 50% inhibition. These two parameters can be calculated simultaneously from a single experiment. In this article we have demonstrated that the method can be applied to a variety of foods with well-recognized antioxidant properties assigning them typical values of PRTE and PRTC so facilitating the comparison of their scavenging properties. To obtain significant and reproducible measurements, which are fundamental for data comparison, the PRTE and PRTC values were measured in a simplified micellar system, which is easy to reproduce and mimics physiological conditions occurring in the upper part of small intestine during lipid assimilation (Hernell, Staggers, & Carey, 1990; Staggers, Hernell, Stafford, & Carey, 1990), where the peroxidation of poly-

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unsaturated fatty acids (PUFA) present in foods may occur with high rates. The measured PRTE and PRTC values were correlated with the polyphenol content emphasizing the deep diversities of the free radical scavenging properties of foods resulting from the different type and content of these compounds. On these bases, diverse ranking of foods was evidenced when their ability to compete for peroxyl radicals rather than their capacity is taken into account.

2. Materials and methods 2.1. Chemicals Rac1-lauroylglycerol (LG) was obtained from Sigma-Aldrich (Milano, Italy). Trolox for HPLC, linoleic acid (LH) and deoxycholic acid sodium salt monohydrate (DCA) were purchased from Fluka (Buchs, Switzerland). 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (ABIP) was a kind gift of Wako Chemicals (Germany). Folin-Ciocalteu phenol reagent was obtained from Sigma-Aldrich (Milano, Italy). Quartz distilled water was used to prepare all the aqueous solutions. Buffers were equilibrated in batch with Chelex-100 (BioRad, Richmond, CA) to minimize the concentration of heavy metal ions. Vegetables, fruits, wine, coffee, tea, and chocolate were purchased from local markets.

2.2. Sample preparation The extracts of fresh foods were prepared selecting 50 g of edible part of vegetables or fruits: only leaves in the case of chicory, pulp and skin in the case of apple, the whole fruit in the case of berries, pulp in the case of pineapple and orange, arils in the case of pomegranate. The randomly sampled raw material was homogenized in 200 mL of ethanol/water solution (85:15 v/v) containing 0.12 M HCl (Rossetto et al., 2005). The homogenates were centrifuged and the solutions were stored at −80 °C until activity measurements. Dry food extracts were prepared as follows: black coffee was prepared by an Italian Moka coffee machine (5 g of Arabic coffee powder with 25 mL of distilled water). American coffee was prepared by an automatic percolator using 40 g of coffee powder in 800 mL of water. Commercial dark chocolate (2 g), 100% cocoa blend, was dissolved in 200 mL of distilled water at 100 °C. Green and black teas were prepared by infusion of 2 g of commercial dry leaves in 200 mL of distilled water at 100 °C for 2 min (according to the manufacturer's instructions). For the various foods, on the basis of the indications of some authors (Halvorsen et al., 2006; Lee, Kim, Lee, & Lee, 2003; Waterhouse, Shirley, & Donovan, 1996), we considered the following serving size: 1 cup of coffee (5 g of powder), 1 cup of tea (2 g of powder), 1 glass of red wine (140 g), 1 cup of dark chocolate (7.3 g of cocoa), 1 bowl of red chicory (100 g), 1 cup of blueberry (100 g), 1 apple (140 g), 1 orange (140 g), 1 cup of pomegranate arils (100 g), and 1 slice of pineapple (140 g).

2.4. Measurement of the peroxyl radical scavenging activity The rate of LH peroxidation was measured from the rate of O2 disappearance by a Metrohm 663 VA stand equipped with a Yellow Spring Oxygen electrode, inserted into a thermostated oxygraphic cell. The current was recorded by a personal computer equipped with a data acquisition board (DAQ PCI-6221, M series, National Instruments, Austin, TX). The working electrode was poised at −800 mV vs Ag/AgCl. The reaction mixture, as described by Rossetto et al. (2008), was prepared by drying a solution of LG in dichloromethane and by dissolving the obtained film in 20 mM phosphate buffer containing 5 mM DCA, pH 7.4. Linoleic acid (2 mM final concentration) was then added and the solution vigorously vortexed. The micelle containing solution was equilibrated with atmospheric oxygen by continuous stirring in the oxygraphic cell, thermostated at 37 ± 0.1 °C. After thermal equilibrium, ABIP, used as a constant source of peroxyl radicals, was added (4 mM final concentration) and the rate of oxygen consumption due to the uninhibited peroxidation of linoleic acid was recorded for some minutes. At the end of this period the food extract to be examined was injected into the test solution and the rate of the oxygen consumption was recorded until the oxygen disappearance, see Fig. 1. Each food extract was analyzed at four different concentrations. 2.4.1. Computational procedure to calculate PRTC and PRTE values The experimental oxygraphic traces were automatically processed by means of a computational procedure to obtain oxygen consumption rates and PRTC and PRTE values on the basis of a rigorous treatment of the kinetic data, see Zennaro et al. (2007) and Rossetto et al. (2008). 2.4.2. A simplified procedure to calculate PRTC and PRTE values Alternatively to the computational procedure the following procedure can be followed to obtain the PRTC and PRTE values from an oxygraphic plot like that reported in Fig. 1: i) Measure the slopes of the plot at the times t0, tj, t1, t2, t3,…ti… that is α0, αj, α1, α2, α3,…αi where α0, αj, are the slopes before and just

2.3. Total phenol content The total phenol content (TP) was measured according to the Folin-Ciocalteu method after incubation of the reaction mixture for 30 min at room temperature (Singleton, Orthofer, & LamuelaRaventós, 1999). The absorbance measurements were carried out by a Varian Cary 50 spectrophotometer at 770 nm. The total phenol content of extracts was expressed in milligrams of gallic acid equivalent per gram of food. All measurements were performed in triplicate.

Fig. 1. Representative oxygraphic records of linoleic acid peroxidation in a micelle system in the presence and in the absence of an antioxidant. The peroxidation of 2 mM LH was carried out at 37 °C by decomposition of 4 mM ABIP in a micelle system containing 5 mM DCA and 0.5 mM LG, in the absence of antioxidant (trace a) and after injection (arrow) of a green tea extract (0.58 mL/L, see MM) (solid trace b). The slopes αi at the times ti were measured. Inset: plot of F(xi) versus (ti − tj). The trace c) represents the oxygraphic trace obtained during the decomposition of 4 mM ABIP in the micelle system 5 mM DCA and 0.5 mM LG, in the absence of LH.

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after the injection of the extract at the time tj, respectively, see Fig. 1. The measure of these slopes can be done graphically. ii) Calculate the function:       −1 −1 + ln 1−xj = 1 + xj F ðxi Þ = xj −xi + lnð1 + xi Þ = ð1 − xi Þ where xj = (αj − R0) / (α0 − R0 ), and xi = (αi − R0 / (α0 − R0 ), R0 being the rate of O2 consumption due to the formation of the peroxyl radicals of ABIP, see Fig. 1 (trace c). iii) Plot the values of F(xi) vs (ti − tj). iii) Calculate the slope C of this plot. Usually a linear plot characterized by R2 values N0.99, is obtained. iv) The PRTC value is calculated from the slope C and the xj value according to the following equation   −1 PRTC = xj −xj × RIN = ðzj d CÞ where zj is the concentration of the food (g L−1) in the oxygraphic cell just after the injection, and RIN is the rate of initiation of peroxidation. RIN was calculated according to equation RIN = 2½α−Tocopherol = LT where LT is the lag-time, that is the period of time when LH peroxidation is fully inhibited by addition to the peroxidation system of α-tocopherol at concentrations in the micromolar range. v) The PRTE values are calculated according to the following equation −1

PRTE = PRTC⋅C = ð1:5⋅RIN Þ = IC50

The PRTE value, which according to the kinetic equations corresponds to IC−1 50 , is directly proportional to the kinetic rate constant of the reaction between peroxyl radicals and the system of antioxidants, and is calculated as liters of testing system/gram of food. This procedure may be adopted when the perturbation of the inhibited oxygraphic trace, following the injection of the food extract lasts less than 1 min. This perturbation may be due to the different oxygen concentration between the injected solution and the reaction system and to the kinetic of distribution of the antioxidants present in the extract between the aqueous and micelle phases. As a consequence of this simplified procedure there is a slight increase of the standard deviations of PRTC and PRTE values. 3. Results and discussion Foods, which have been a matter of debate in the last years for their interesting antioxidant properties, have been tested according to the oxygraphic method mentioned in the previous sections. By this method it was possible to assign typical PRTC and PRTE values to each food we have tested like those assigned to antioxidant compounds (Zennaro et al., 2007), so facilitating food inter-comparison. The calculated PRTC and ePRTE values have been reported in Table 1. Fig. 2 shows the oxygraphic plots obtained in the cases of pineapple and dark chocolate, which are characterized by high efficiency but low capacity and vice versa, respectively. The value of R2 higher than 0.99 calculated from the fit of oxygraphic data, see insets of Fig. 2, indicates a very good agreement between the experimental data and the theory, Zennaro et al. (2007). The good matching between the experimental data and the kinetic model developed for a single antioxidant appears a distinguishing behaviour of foods we have tested, despite that these foods contain several antioxidants. This behaviour may trivially be due to the

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overwhelming activity of one antioxidant with respect to the others or to the close values of IC50 of the antioxidants present in food. However the main mechanism leading to the observed behaviour should be the cross-reactions among the radical species generated by the reaction between the highly reactive peroxyl radicals and the antioxidants present in foods (Rossetto et al., 2002). This is in accord to literature data, which demonstrate that, during lipid peroxidation, one-electron transfer reactions involving couple of antioxidants occur very easily according to their equilibrium constants (as uric acid, ascorbate, Trolox, methoxy- and dimethoxy-phenol) (Jovanovic & Simic, 1989; Simic & Jovanovic, 1989). The kinetic behaviour of the system of antioxidants present in foods as a single antioxidant is quite general since it was observed for most of the foods independently of the system where we have carried out the lipid peroxidation process (for example independently of the micelle nature and composition). The total phenol content (TP) of various foods, measured according to the Folin-Ciocalteu method, is also reported in Table 1. The TP values range from about 1 mg of TP/g of fresh weight food in the case of fruits and vegetables to about tens of milligrams of TP/g in the case of dry foods such as tea and coffee. Also in the case of ePRTE and PRTC particularly high values were obtained for dry foods and, as a consequence, the ratios between the maximum and the minimum value of the efficiency and of the capacity, that is (ePRTE)max/(ePRTE)min and (PRTC)max/(PRTC)min, are very high, that is 240 and 600, respectively. These large fluctuations of the reactivity and capacity, due in part to the water content of foods, decrease if we consider the ratios ePRTE/TP and PRTC/TP, which measure the efficiency and the capacity of foods, respectively, normalised to the polyphenol content, calculated according to the Folin-Ciocalteu method. In this case the variations of the ratios ePRTE/TP (about 9 times) and of PRTC/TP (about 14 times) are much smaller, as shown in Table 2, but enough high to highlight the different quality of the polyphenols present in the foods we have examined. In fact, while the ratios PRTC/TP are particularly high in the case of red wine, coffee, red chicory and apple (about 30–45), we must observe that the polyphenols present in red chicory and blueberry are characterized by higher efficiencies (ratios ePRTE/TP about 5–6 mg Trolox/mg of TP) than those present in blackberries, in some red wines and apple cultivars (ratios ePRTE/TP about 1–2). In the case of red wine, chocolate and blackberry the low efficiency with respect to the polyphenol content may be due to the presence in these foods of polymerized polyphenols. In fact in these foods most of the polyphenols are present as condensation polymers, where the polyphenol reactive structures are still present (Afaq, Saleem, Krueger, Reed, & Mukhtar, 2005; Gonzalez-Manzano, Santos-Buelga, Prez-Alonso, Rivas-Gonzalo, & Escribano-Bailn, 2006; Hager, Howard, Rohana, Lay, & Prior, 2008; Natsume et al., 2000; Vrhovsek, Rigo, Tonon, & Mattivi, 2004). As expected the presence of polymerized polyphenols does not modify essentially the capacity of these foods in trapping reactive radicals, since the experimental PRTC values result close to those calculated on the basis of the molarity of the monomer units present in the polymers. On the contrary we found that the experimental ePRTE values of the polymer are lower than those expected on the monomer molarity basis. In fact the kinetic rate constants of polymerized polyphenols towards peroxyl radical should drop due to stereo hindrance factors and to the lower diffusion coefficients of these polymers. This finding is in accord with the literature data (Arteel & Sies, 1999) from which it appears that some oligomers are less effective in protecting against oxidation reactions than monomers, when the sum of these repeating units in oligomers is considered. No correlation appears to exist between the ePRTE and PRTC values since the ratio ePRTE/PRTC ranges from a minimum value of about 0.02 in the case of dark chocolate to values higher than 0.69 in the case of pineapple, see last column of Table 2, where the reactivity of various foods is compared on the basis of equal trapping capacity.

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Table 1 Total phenol content, ePRTE and PRTC of some antioxidant rich foods. TP (mg/g)a

Food

Coffee

Tea Red Wine

Chocolate Chicory

Berry

Apple

Citrus

Pomegranate Pineapple

d

Coffee (Columbia) Coffee (Kenya)d Coffee (Brazil)d American coffee Black tea Green tea Pinot noir Cabernet sauvignon Merlot Dark chocolate (brand A)e Dark chocolate (brand B)e Verona red chicory Treviso red chicory Wild chicory Blueberryf (Vaccinium myrtillus) Blueberry f (Vaccinium corymbosum) Blackberry Golden delicious Morgenduft Pink lady Navel orange Tarocco orange Clementine Pomegranatef (Israel) Pomegranatef (Greece) Pineapple (Costa Rica, brand A) Pineapple (Costa Rica, brand B) Pineapple (Brazil)

ePRTE (mgTrolox/g)b

Value

Average value

44.8 45.0 49.2 48.0 39.4 42.0 1.27 1.84 1.27 36.5 45.2 2.51 1.40 2.30 3.29 3.62 3.00 0.85 1.23 0.78 0.84 1.54 1.04 1.77 1.43 0.64 0.93 0.72

PRTC (μmolLO2•/g)c

Rank

Value

Average value

46.8

1

177.8

1

40.7

3

145 191 189 186 70.3 69.4 1.38 3.88 2.03 30.8 37.1 15.9 9.7 9.48 17.5 17.2 3.23 1.12 1.85 0.80 1.57 2.11 1.21 1.29 1.19 3.66 2.16 2.11

69.9

2

1.46

40.9

7

2

2.07

5

3.30

4

0.95

9

1.14

8

1.60

6

0.76

10

2.43

Rank

7

34.0

3

11.7

5

12.6

4

1.26

9

1.63

8

1.24

10

2.64

6

Value

Average value

1721 1704 2003 1592 726 724 39.4 82.3 55.2 1050 1506 110 50.0 87.3 48.5 70.2 65.6 33.7 46.0 21.6 3.78 6.26 3.31 22.6 12.9 5.33 5.62 5.22

1755

1

725

3

59.0

1278

Rank

6

2

82.4

4

61.4

5

33.8

7

4.45

17.8 5.39

10

8 9

a Total phenols from the Folin-Ciocalteu method expressed as milligrams of gallic acid per gram of food. The values reported are the average of 3 measurements (standard deviation b 5%). b ePRTE is value of equivalent Peroxyl Radical Trapping Efficiency of each food in terms of milligrams of Trolox in 1 g of food. The values reported are the average of 4 measurements at different concentrations (standard deviation b7%). c PRTC is Peroxyl Radical Trapping Capacity expressed as micromoles of peroxyl radicals trapped by 1 g of food. The values reported are the average of 4 measurements at different concentrations (standard deviation b7%). d Coffee prepared using an Italian Moka coffee machine. e Chocolate 100% cocoa blend of different commercial brands. f Foods which fitting is characterized by a value of R2 ≈ 0.98.

In the case of the ePRTE and PRTC values calculated on the basis of serving size, that is ePRTESS and PRTCSS, the differences between the maximum and minimum values are lower than those reported in Table 1. These differences are still large (see Fig. 3), in particular if we consider that we are comparing foods with high antioxidant characteristics. In fact from Fig. 3 it appears the high values of PRTESS of red chicory and blueberry (about 1300–1700 mg Trolox/serving size) and the low values of this parameter in the case of apple, tea and pomegranate (about 200 mg Trolox/serving size). As regards the PRTCSS, the highest values were measured in the case of dark chocolate, coffee, red wine, and red chicory (8–10 mmol/serving size), while the lowest values were obtained in the case of pineapple, orange, and tea (≈ 1 mmol/serving size). In other words the ratio between the highest and lowest values of the PRTC and of the ePRTE is about one order of magnitude. However this ratio decreases by a factor of three if polyphenol content is considered. This is due to the difference in quality of polyphenols present in the foods we have tested. We have considered also the influence of the extraction process on the PRTE and PRTC. In particular a set of experiments was carried out on some foods, which are representative of the various classes we have examined, to simulate in a simple way the processes occurring during the gastrointestinal digestion. In particular in Table 3 we have compared the results obtained using as extraction medium i) water; ii) aqueous 0.12 M HCl; iii) aqueous ethanol (15% water) containing 0.12 M HCl and iv) aqueous 0.12 M HCl followed by neutralization of the homogenate and treatment with an aqueous solution containing 50 g/L of sodium cholate, to aid the extraction of more hydrophobic

molecules and to simulate the processes occurring in the GI tract. From the results reported in Table 3 it appears the importance of acidic conditions similar to those occurring in our stomach (0.12 M HCl in the gastric juice after stimulation), to achieve a high extraction yield of polyphenols. This is in accord with the results we reported in a previous article on red chicories (Rossetto et al., 2005), demonstrating the importance of low pH values to achieve a high extraction yield of phenolics with antioxidant properties. Furthermore the extraction experiments carried out by aqueous acidic ethanol that we have used in this work or by acidic water followed by treatment with sodium cholate, so reproducing the condition occurring in the GI tract, produced similar results. Because of the high relevance of phenolics in small intestine (Kerem, Chetrit, Shoseyov, & Regev-Shoshani, 2006) the knowledge of PRTC and PRTE values of foods under the conditions present in this body compartment permits to evaluate the protection afforded by the antioxidants before lipids are adsorbed. According to the data reported in Fig. 3 the rank of foods changes significantly if we consider the ePRTESS or PRTCSS values. As regards the capacity to trap peroxyl radicals, the ranking is dark chocolate ≥ coffee≥ red wine≥ red chicory N blueberry ≥ apple N pomegranate ≈ tea N pineapple ≈ orange. This order agrees broadly with those reported by other authors (Halvorsen et al., 2002; Lee et al., 2003; Pellegrini et al., 2003), who measured the antioxidant capacity of foods with various types of assays such as FRAP, TEAC, and DPPH. In general, with reference to the serving size, the PRTC values we have obtained are two–three up to ten times higher, as in the case of apples, with respect to the capacity values reported by some authors. This difference could be explained by the different reactivities

P. Vanzani et al. / Food Research International 44 (2011) 269–275

273

Table 2 ePRTE/TP, PRTC/TP and ePRTE/PRTC ratios of some foodstuffs.

Fig. 2. Inhibition of LH peroxidation by pineapple and dark chocolate. Representative oxygraphic records of linoleic acid peroxidation in a micelle system in the presence of food extracts. Panel A: pineapple extract was added (arrow) in the oxygraphic cell at final concentration of 0.25 g fresh fruit/L. Panel B: dark chocolate extract was added (arrow) in the oxygraphic cell at final concentration of 0.012 chocolate g/L. Insets: plot of F(xi) versus time according to the described procedure. The peroxidation of LH was carried out in a solution containing 5 mM DCA, 0.5 mM LG, 2 mM LH and 4 mM ABIP at 37 °C, pH 7.4.

of the titrating molecule (peroxyl radical in our case) that are used to carry out the various assays, and/or by the acidic conditions of extraction procedure and/or by the correct calculation procedure we have followed (Zennaro et al., 2007). As regards the efficiency of foods that is the reactivity in trapping reactive radicals, which has never been measured before, the ranking of foods is blueberry N red chicory N coffee N pineapple ≈ red wine ≥ orange ≈ dark chocolate ≈ apple ≥ tea N pomegranate. The efficiency appears as an important property, for some aspects more informative than the amount of radicals trapped, since it indicates the possibility of foods, or better of the system of antioxidants present in foods, to compete with other molecules such as PUFA, proteins etc. for peroxyl radicals and therefore to preserve these molecules from oxidative damages. An example of the relative importance of PRTE and of PRTC could be the protection afforded by the foods of Table 2 in the GI tract, where peroxyl radicals are generated by the reaction between PUFA and iron containing compounds (Lapidot et al., 2005a,b; Vulcain, Goupy, Caris-Veyrat, & Dangles, 2005). To verify the possibility that lipid peroxidation occurs also in the small intestine under the conditions occurring in this body compartment, PRTC–PRTE experiments, were carried out saturating the LH micelle system with N2–O2 atmosphere, characterized by O2 partial pressures in the range 10– 50 Torr. The addition of 0.1–1 μM bovine Hb, incubated for 1 hour at 37 °C in 50 mM HCl to simulate the passage through the stomach, leads to a very fast peroxidation process characterized by a constant

Food

ePRTE/TP PRTC/TP ePRTE/PRTC (mg Trolox/mg) (μmol LO2•/mg) (mg Trolox/μmol

Coffee (Columbia) Coffee (Kenya) Coffee (Brazil) American coffee Black tea Green tea Pinot noir Cabernet sauvignon Merlot Dark chocolate (brand A) Dark chocolate (brand B) Verona red chicory Treviso red chicory Wild chicory Blueberry (Vaccinium myrtillus) Blueberry (Vaccinium corymbosum) Blackberry Golden delicious Morgenduft Pink lady Navel orange Tarocco orange Clementine Pomegranate (Israel) Pomegranate (Greece) Pineapple (Costa Rica, brand A) Pineapple (Costa Rica, brand B) Pineapple (Brazil)

3.23 4.24 3.84 3.88 1.78 1.65 1.09 2.11 1.60 0.84 0.82 6.33 6.93 4.12 5.32

38.4 37.9 40.7 33.2 18.4 17.2 31.0 44.7 43.5 28.8 33.3 43.8 35.7 38.0 14.7

0.08 0.11 0.09 0.12 0.10 0.10 0.04 0.05 0.04 0.03 0.02 0.14 0.19 0.11 0.36

4.75

19.4

0.25

1.08 1.32 1.50 1.03 1.87 1.37 1.16 0.73 0.83 5.72

21.9 39.7 37.4 27.7 4.50 4.06 3.18 12.8 9.02 8.33

0.05 0.03 0.04 0.04 0.42 0.34 0.37 0.06 0.09 0.69

2.32

6.04

0.38

2.93

7.25

0.40

LO2•)

TP: total phenols from the Folin-Ciocalteu method expressed as milligrams of gallic acid per gram of food. ePRTE: equivalent Peroxyl Radical Trapping Efficiency of each food in terms of milligrams of reference compound (Trolox) in 1 g of food. PRTC: Peroxyl Radical Trapping Capacity expressed as micromoles of peroxyl radicals trapped by 1 g of food.

rate of oxygen consumption of the order of 2–3 μmol O2 s−1, which brought in a few seconds the disappearance of the oxygen. This demonstrates that under the conditions present in small intestine (low O2 partial pressure and pH values around 7), lipid peroxidation occurs and is a very fast process. According to the data of Fig. 3, the amount of peroxyl radicals which potentially may be trapped after ingestion of a serving size of these foods is of the order of a millimole or higher. This amount is by far higher than the amount of peroxyl radicals that can be generated on the basis of the limited amount of oxygen present in the initial part of GI tract, that is the stomach and upper part of the small intestine where the absorption of PUFA occurs (Gorelik, Ligumsky, Kohen, & Kanner, 2008). As a consequence the foods we have considered appear equivalent in the protection of the GI tract on a capacity basis that is on the basis of the PRTC values. However, if we consider the reactivity, there are large differences in the fraction of peroxyl radicals escaping the food antioxidants, and consequently the possibility of radical damages decreases increasing the PRTESS, and therefore also this parameter should be considered to evaluate the protection afforded by foods. 4. Conclusions With the aid of a simple kinetic model, which is rigorously followed, foods particularly rich of antioxidants have been classified on the basis of their ability to trap peroxyl radicals according to

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Fig. 3. Efficiency, capacity and total phenol content of antioxidant rich foodstuffs on the basis of typical serving sizes. The average values and intervals of ePRTEss as milligrams of Trolox/serving size (A), PRTCss as mmol of peroxyl radical/serving size (B), and TPss as g gallic acid/serving size of foodstuffs calculated according to the data of Table 1.

stoichiometric (PRTC) or to kinetic parameters (PRTE). This permits a full and more accurate estimate of their activity as antioxidant in protecting the GI tract. In fact important differences in the ranking of the antioxidant activity of foods exist if the capacity (PRTC) or the efficiency (PRTE) is considered. The efficiency appears particularly

important since only this parameter gives information on the competition for peroxyl radicals between the antioxidants present in food and biomolecules, preserving them from oxidative damages.

References Table 3 Relative PRTE and PRTC values of some foods obtained using various extracting solutions. Food Pink lady

Dark chocolate

Treviso red chicory

a

Reference values.

Extracting solutions Ethanol/Water 85:15 (v/v), 0.12 M HCl 0.12 M HCl; sodium cholate 50 g/L 0.12 M HCl Water Ethanol/Water 85:15 (v/v), 0.12 M HCl 0.12 M HCl; sodium cholate 50 g/L 0.12 M HCl Water Ethanol/Water 85:15 (v/v), 0.12 M HCl 0.12 M HCl; sodium cholate 50 g/L 0.12 M HCl Water

PRTE

PRTC

a

1.00a 1.00 0.85 0.47 1.00a 1.03 0.87 0.85 1.00a 1.03 0.44 0.09

1.00 1.13 1.02 0.69 1.00a 1.01 0.90 0.96 1.00a 0.94 0.69 0.04

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