Food Research International 39 (2006) 791800 www.elsevier.

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Eect of solvent and certain food constituents on dierent antioxidant capacity assays
Jara Perez-Jimenez *, Fulgencio Saura-Calixto
Department of Metabolism and Nutrition, Instituto del Fro, CSIC, c/Jose Antonio Novais, 10, CP 28040 Madrid, Spain Received 28 November 2005; accepted 26 February 2006

Abstract The eect of the sample solvent in the antioxidant capacity of foods measured by the four most widely used procedures (ABTS, FRAP, DPPH and ORAC) has not been systematically studied. This was the aim of this work. The antioxidant capacity of catechin:gallic acid solutions in dierent solvents (water, methanol/water, methanol, acetone/water) were determined. Signicant dierences were found between the values obtained by the same method in dierent solvents, as well as between the ones obtained by the dierent method in the same solvent (ranging from 10,000 to 30,000 lmol Trolox/g dw). In addition, certain non-antioxidant food constituents, such as amino acids and uronic acids, also showed an interfering eect on these assays. ORAC was the assay in which all these factors had a greatest interfering eect. Antioxidant capacity values should only be compared when the measurements have been made by the same method in the same solvent. 2006 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant capacity; Solvent eect; ABTS; FRAP; DPPH; ORAC

1. Introduction Growing epidemiological evidence of the role of food antioxidants in the prevention of certain diseases (Stanner, Hughes, & Buttriss, 2004) has led to the development of a wide number of assays to determine antioxidant capacity. These methods can be based on peroxyl radical scavenging (ORAC, TRAP), metal reducing power (FRAP, CUPRAC), hydroxyl radical scavenging (deoxyrribose assay), organic radical scavenging (ABTS, DPPH), quantication of products formed during the lipid peroxidation (TBARS, LDLs oxidation), etc. (Aruoma, 2003; Frankel & Meyer, 2000; Sanchez-Moreno, 2002). Of all these methods, ABTS, FRAP, DPPH and ORAC are some of the most widely used. The ABTS or TEAC (Trolox equivalent antioxidant capacity) assay is based on the ability of the antioxidants
*

Corresponding author. Tel.: +34 915492300; fax: +34 915493627. E-mail address: jara@if.csic.es (J. Perez-Jimenez).

to scavenge the long-life radical cation ABTS+. This scavenging produces a decrease in the absorbance at 658 nm. The absorbance readings of the mixture of the radical and the antioxidant at dierent times are represented graphically alongside those of a blank. Then, the area under the curve generated by this inhibition of the absorbance is calculated. The results are interpolated in a Trolox calibration curve and expressed as Trolox equivalents. In the original method (Miller et al., 1993), the radical was formed through the reaction between ferrylmyoglobin and ABTS, and the sample was added to the medium before generation of the radical. However, the faster reacting antioxidants could also contribute to the reduction of the ferrylmyoglobin radical, causing an overestimation of the antioxidant capacity of some compounds (Re et al., 1999; Yu & Ong, 1999). Therefore, the method was modied (Re et al., 1999), so that the radical is formed by chemical reaction with potassium persulphate prior to addition of the sample.

0963-9969/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2006.02.003

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List of abbreviations DPPH 2,2-diphenyl-1-picrylhydrazyl ABTS 2,2 0 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) FRAP ferric reducing/antioxidant power ORAC oxygen radical absorbance capacity CUPRAC cupric reducing/antioxidant capacity TBARS thiobarbituric acid reactive substances LDLs low density lipoproteins TEAC trolox equivalents antioxidant capacity TPTZ 2,4,6-tri(2-pyridyl)-s-triazine EC50 concentration of antioxidant needed to reduce the original amount of radical by 50% tEC50 time needed for the EC50 to reach 50% of the original amount of radical scavenged AE antiradical eciency (AE = 1/(EC50tEC50)) SET single electron transfer HAT hydrogen atom transfer

The DPPH method (Brand-Williams, Cuvelier, & Berset, 1995) is based on scavenging of the radical DPPH from the antioxidants, which produces a decrease in absorbance at 515 nm. This method was modied at our labora tory to measure kinetic parameters (Sanchez-Moreno, Larrauri, & Saura-Calixto, 1998): the absorbance was measured until the reaction reached the plateau, instead of taking a xed point. A calibration curve at that wavelength was made to calculate the remaining DDPH. The parameter EC50, which reects depletion of the free radical to 50%, was expressed in terms of g dry weight/g DDPH. The time taken to reach the steady state at EC50 (tEC50) and the antiradical eciency (AE = 1/EC50tEC50) were also calculated. This method has been recently modied to be measured by HPLC instead of spectrophotometrically (Chandrasekar, Madhusudhana, Ramakrishna, & Diwan, 2006). The ORAC assay is another method based on the scavenging of free radicals, but in this case the peroxyl radical, which is generated by oxidative processes in the human body. In the assay, the peroxyl radical is generated from the organic molecule AAPH (2,2 0 -azobi(2-amidinopropane) dihydrochloride) and attacks a uorescent molecule, generating a decrease in the emission of uorescence, which is monitored. The area under the curve is measured and is interpolated in a Trolox curve, and the results are expressed as Trolox equivalents. In the original method, the uorescent molecule was b-phycoerithrin (Cao, Alessio, & Culter, 1993), a protein isolated from Porphyridium cruentum. However, the results were not reproducible enough, the compound was degraded after a certain amount of exposure to light and it could react non-specically with some polyphenols, causing overestimation of antioxidant capacity. It was therefore substituted with uorescein (3,6 0 -dihydroxy-spiro [isobenzofuran-1 [3H], 9 0 [9H]-xanthen]-3-one) (Ou, Hampsch-Woodill, & Prior, 2001), a molecule that does not present these problems. Finally, the FRAP assay (Benzie & Strain, 1996) is the only one of these methods that is based not on free radical scavenging capacity but on reducing ability. In an acidic medium, the ferric-tripyridyltriazine complex is reduced to its ferrous, coloured form in the presence of antioxi-

dants, causing an increase in absorbance at 595 nm. The absorbance reached at a xed end-point is interpolated in a Trolox calibration curve, and the results are expressed as Trolox equivalents. In the original method the absorbance was monitored up to a time of 4 min, but this time the reaction was not completed, so it was suggested that monitoring be prolonged up to 30 min (Pulido, Bravo, & Saura-Calixto, 2000). In the literature, these methods are used to determine the antioxidant capacity of food extracts obtained with different extraction solvents, such as ethanol (Yu, Perret, Davy, Wilson, & Melby, 2002), ethanol/water in dierent proportions (Gray, Clarke, Baux, Bunting, & Salter, 2002; Yu, Haley, Perret, & Harris, 2002), acetone/water in dierent proportions (Ou et al., 2001; Yilmaz & Toledo, 2006), methanol/water in dierent proportions (Yilmaz & Toledo, 2006) or acidic methanol/water followed by acetone/water (Saura-Calixto & Goni, 2006). During the last few years, some researchers have noted the inuence of the extraction solvent in the ORAC (Fernandez-Pachon, Villan o, Garca-Parrilla, & Troncoso, 2004; Villano, Fernandez-Pachon, Troncoso, & Garca-Parrilla, 2005; Zhou & Yu, 2004), DPPH (Barclay, Edwards, & Vinqvist, ez, & Nicoli, 2004; Valgimi1999; Pinelo, Manzocco, Nun gli, Inold, Lusztyk, & Banks, 1999; Zhou & Yu, 2004), FRAP (Pulido et al., 2000) and ABTS (Zhou & Yu, 2004). However, to the authors knowledge, there has been no simultaneous study of the eect of the solvent on the antioxidant capacity of a polyphenol solution tested by these four assays. Type of solvent and polarity may aect the single electron transfer (SET) and the hydrogen atom transfer (HAT), which are key aspects in the measurements of antioxidant capacity. The presence of non-antioxidant compounds in the tested solutions, also could aect the results. This work addresses both factors. The rst aim of this work was to determine the antioxidant capacity of a mixture of gallic acid and catechin, as examples of a phenolic acid and a avonoid, respectively, in water, methanol/water (30:30 v/v), methanol/water (50:50 v/v), acidic methanol (50:50 v/v, pH 2), methanol and acetone/water (50:50 v/v).

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Another aspect considered was the possibility that certain common food constituents interfere in antioxidant capacity assays. Antioxidant capacity is usually measured in aqueousorganic extracts that may contain, not only the antioxidants, but also other non-antioxidant food constituents that may interfere in antioxidant capacity assays. To address this aspect, a number of solutions were analysed by FRAP, ABTS, DPPH and ORAC. These were: glucose, pectin and galacturonic acid as examples of dierent glucids; tyrosine and trytophan as aromatic amino acids; arginine as a nitrogenated amino acid, cysteine as a sulphurated one; and albumin as a common protein. Finally, mixed solutions of gallic acid and catechin containing each of these food constituents were tested to determine whether the eect of the polyphenols and the possible eects of the food constituents were cumulative, or whether new interactions appear when mixing them. 2. Materials and methods 2.1. Chemicals Trolox (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analogue of vitamin E, 2,2dyphenyl-1-picryhydrazil (DPPH), potassium persulfate, uorescein (3,6 0 -dihydroxy-spiro [isobenzofuran-1 [3H], 9 0 [9H]-xanthen]-3-one), AAPH (2,2 0 -azobi(2-amidinopropane) dihydrocloride) and 2,2 0 -azino-bis(3-ethylbenz-thia zoline-6-sulfonic acid) (ABTS) (SigmaAldrich Qumica, S. A., Madrid, Spain). 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was from Fluka Chemicals, Madrid, Spain. Iron III-clorure-6-hydrate was from Panreac (Castellar del Valles, Barcelona, Spain). All reagents used were of analytical grade. 2.2. Standards and food constituents A solution 1 M:1 M of gallic acid (Merck) and catechin (SigmaAldrich Qumica, S.A.) was prepared in the solvents tested. Solutions of glucose (Merck), pectin (Fluka Chemi cals), galacturonic acid (SigmaAldrich Qumica, S.A.), L-tyrosine (Merck, Darmstadt, Germany), L-trytophan (Fisher Scientic Company, USA), L-cystein (Merck), L-arginine (Merck) and albumin (SigmaAldrich Qumica, S.A.) were prepared in water and in acetone/water (50:50 v/v). Mixed solutions of gallic acid:catechin 1 M:1 M and each one of the dierent food constituents in dierent concentrations were also prepared in water and in acetone/ water (50:50 v/v). 2.3. Methods All determinations were performed in triplicate.

2.3.1. FRAP assay 900 lL of the FRAP reagent, containing TPTZ, FeCl3 and acetate buer, were mixed with 90 lL of distilled water and 30 lL of the test sample or the blank (solvent tested). Maximum absorbance values at 595 nm were taken every 15 s and at 37 C, using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA) equipped with a thermostatic auto-cell-holder (Benzie & Strain, 1996; Pulido et al., 2000). Solutions of known Trolox concentrations in the dierent solvents tested were used for calibration. 2.3.2. DPPH assay After adjusting the blank with methanol, 3.9 mL of the sample was mixed with 0.1 mL of a DDPH methanolic solution (60 lM). The absorbance at 515 nm was measured until the reaction reached the plateau. A calibration curve at that wavelength was made to calculate the remaining DDPH. The parameter EC50, which reects the depletion of free radical to 50%, was expressed in terms of g dry weight/g DDPH. The time taken to reach the steady state at EC50 (tEC50) and the antiradical eciency (AE = 1/EC50tEC50) were also calculated (Brand-Williams et al., 1995; Sanchez-Moreno et al., 1998). 2.3.3. ABTS assay After addition of 100 lL of sample or Trolox standard to 3.9 mL of diluted ABTS+ solution (10 mL of a solution of potassium persulfate containing 66 mg in 100 mL of distilled water mixed with 38.4 mg of ABTS and stirred overnight, after which the solution is diluted with methanol up to a nal absorbance of 0.7 0.02), absorbance readings were taken every 20 s using a Beckman DU-460 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA). The reaction was monitored during 6 min. The percentage inhibition of absorbance vs. time was plotted and the area below the curve (06 min) was calculated. Solutions of known Trolox concentrations in the dierent solvents tested were used for calibration. 2.3.4. ORAC assay It was followed the procedure described by Ou et al. (2001) slightly modied: 175 lL of the sample/blank were mixed with 120 lL of PBS, pH 7.4, 75 mM, 205 lL of an AAPH solution 53 mM and 3 mL of a uorescein solution 48 nM. Fluorescence was recorded until it reached zero (excitation wavelength 493 nm, emission wavelength 515 nm) in a uorescence spectrophotometer PerkinElmer LS 55, equipped with an automatic thermostatic autocell-holder at 37 C. Results are calculated using the dierences of areas under the uorescein decay curve between the blank and the sample and are expressed as Trolox equivalents. 2.4. Statistical analysis Results are expressed as mean values standard deviation. Means of three measurements were compared, using a

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signicance level of P < 0.05, by one-way analysis of variance (ANOVA). The computer system used was Statgraphic Computer System, version 5.1. 3. Results and discussion 3.1. Solvent eect Table 1 shows the results of ORAC, ABTS, FRAP and DPPH for a solution of gallic acid:catechin 1 M:1 M in different solvents. The solvent clearly inuenced these four antioxidant capacity assays, but not all in the same way. ORAC is the assay in which the solvent inuence was highest; the value of the mixture gallic acid:catechin in water is 48% lower than the value of the same sample in acetone/water (50:50 v/v), and these two values are significantly dierent to the ones obtained in the other solvents tested. There is a tendency, for the ORAC value to be higher the more apolar the solvent is (in this case, the less water there is in the mixture methanol/water). These results agree with previous works (Fernandez-Pachon et al., 2004; Villano et al., 2005), that reported that there was a relation ship between the ethanol content of the sample and the ORAC-PE value. As the same eect was observed in this work using uorescein as uorescent agent, the inuence of the solvent would seem to be intrinsic to the ORAC method regardless of the uorescent reagent used. All these run counter to the idea that the antioxidant capacity assays based on hydrogen atom transfer reactions are solventindependent, as stated by Prior, Wu, & Schaich (2005). Moreover, various authors have shown that, in polar solvents, hydrogen bonding may induce dramatic changes in the H-atom donor activities of phenolic antioxidants, reducing the antioxidant capacity of phenolics (Pedrielli, Pedulci, & Skibsted, 2001; Pinelo et al., 2004). It was also found that the presence of acid in the solvent did not seem to have any inuence on the ORAC assay, since there was no signicant dierence between the ORAC values of methanol/water (50:50 v/v) and acidic methanol/ water (50:50 v/v, pH 2). This is consistent with earlier reports (Davalos, Gomez-Cordoves, & Bartolome, 2004; Ou et al., 2001).

In the case of ABTS, the solvent also played an important role; the value of the mixture of phenolic compounds was 40% lower in methanol/water (50:50 v/v) than in water. In this case, the more polar the solvent is, the greater is the ABTS value. It is also evident a clear eect of pH, with a signicantly dierent value 61% lower in acidic methanol/water (50:50 v/v) than in methanol/water (50:50 v/v), which agrees with previous ndings (Labrinea & Georgiou, 2004). The FRAP assay also demonstrated the inuence of the solvent, as previously reported (Pulido et al., 2000). In general, the values were more similar than in ORAC or ABTS, excepting the value of methanol/water (30:30 v/v), which was much higher than the others. Since the FRAP assay is already carried out at an acidic pH (3.6), there was not a signicant dierence between the value of methanol/ water (50:50 v/v) and the same solvent acidied. DPPH was the assay in which the solvent inuence was weakest; the greatest signicant dierence was between water, with an EC50 of 0.067, and acetone/water (50:50 v/v) or methanol, with values of 0.083. The acidity of the medium did not seem to aect the DPPH values, but it did aect the kinetic; acidic methanol/water (50:50 v/v, pH 2) presented a tEC50 highest than any of the other solvents tested. All the dierences observed between the dierent solvents would be greater if the sample analysed were a food a complex matrix in which the dierent compounds may establish dierence interactions between them and with the solvent instead of a mixture of standards, as in this paper. Therefore, when doing comparisons of antioxidant capacities, these have to have been measured in extracts or solutions obtained with the same solvents, otherwise, the results might vary a lot and a direct comparison would be meaningless. To express the results of ORAC, ABTS and FRAP as Trolox equivalents, Trolox calibration curves had rst to be plotted for each of the solvents tested. The slopes, intercepts and R2 of these calibration curves are shown in Table 2. In all the methods, R2 values are high for all the solvents tested, showing a good doseresponse curve. However, there are signicant dierences in the slopes and intercepts

Table 1 Results of ORAC, ABTS, FRAP and DPPH assays of a mixture of catechin:gallic acid 1 M:1 M in dierent solvents Solvent ORAC (lmol Trolox/g) 20,836.6 2079.6a 18,284.6 1661.5a 19,818 750.4a 19,410.8 2068.4a 13,543.6 298.4b 30,217.5 2100.1c ABTS (lmol Trolox/g) 11,291.4 837a 11,148 730.3a 6763.9 65.6b 26,494.5 2011.7c 28,104 275.8c 13,894.8 240.2a FRAP (lmol Trolox/g) 9642.4 977.1a 11,149.7 565.3b 10,934 135.1b 14,622.7 1095.9c 9559.2 110.7a 10,100.1 489.7a,b DPPH EC50 (g/g DPPH) Methanol Methanol/water (50:50 v/v) Acidic methanol/water (50:50 v/v, pH 2) Methanol/water (30:70 v/v) Water Acetone/water (50:50 v/v) 0.083 0.004c 0.080 0.002b,c 0.081 0.002b,c 0.076 0.001b 0.067 0.001a 0.083 0.003c tEC50 (min) 19.72 0.73b 16.70 0.65a 28.02 2.35c 19.57 0.73b 19.73 1.45b 19.44 0.80a,b AE 0.61a 0.75b 0.44c 0.67a,b 0.75b 0.62a

Dierent letters in the same column imply signicant dierences.

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Table 2 Slope, intercept and R2 for the Trolox calibration curves (y = ax + b) in FRAP, ABTS and ORAC in dierent solvents, being x Trolox lM and y absorbance for FRAP and net area under the curve for ABTS and ORAC Solvent FRAP Slope Water Methanol/water (30:30 v/v) Methanol/water (50:50 v/v) Acidic methanol/water (50:50 v/v, pH 2) Methanol Acetone/water (50:50 v/v) 0.015 0.0014 0.0013 0.0013 0.0013 0.0016 Intercept 0.0117 0.0176 0.0383 0.0878 0.0124 0.0398 R2 0.9994 0.9998 0.9976 0.9966 0.9987 0.9895 ORAC Slope 7683.8 5364.1 7156.4 8064.6 6878.4 5141.8 Intercept 671.85 12,248 10,930 44,887 887.84 10,759 R2 0.9896 0.9925 0.987 0.9976 0.9941 0.9938 ABTS Slope 14.087 16.907 34.228 32.151 28.389 35.73 Intercept 365.67 3.4 435.04 540.18 1938.8 300.75 R2 0.9835 0.9989 0.9965 0.9992 0.9891 0.9958

of the three methods, especially in the case of ABTS and ORAC, which suggest that Trolox, like catechin and gallic acid, does not behave in the same way in all the solvents. Moreover, there is no sign of some tendencies that were observed in the mixture of catechin and gallic acid, such as higher ORAC values the more apolar the solvent is. In fact water, the most polar solvent, has one of the steepest slopes, which suggests that the solvent aects dierent compounds in dierent ways. It can be concluded that, in any antioxidant capacity assay, the results must be related to a calibration curve in the same solvent that is used for the extraction, given that the standard may behave dierently depending on the solvent. 3.2. Food constituents eect Two of the solvents previously tested, water and acetone/water (50:50 v/v), were chosen to study the eect that certain common non-antioxidant food constituents may have in the antioxidant capacity assays studied. The idea was to see whether they could generate false positives, and hence, overestimation of the antioxidant capacity of the samples. The results using water as solvent are shown in Table 3. First of all, there is a clear dierence between ORAC and ABTS on the one hand and FRAP and DPPH on the other hand. In the latter two, only cysteine gave a positive result in the FRAP assay and arginine and cysteine in the DPPH. On the contrary, in ORAC and ABTS all the amino acids and the protein gave positive values, statisti-

cally signicant in some cases. The glucids chosen presented no signicant value in any of the methods. Tyrosine and tryptophan in particular, presented high ORAC and ABTS values, even at very low concentrations. This agrees with a previous report (Yilmaz & Toledo, 2005), in which the amino acid hystidine by itself presented a high ORAC value. This seems to indicate that proteins and amino acids, which are present in high concentrations in certain plant foods such as legumes, could produce considerable interference in these antioxidant capacity assays, producing overestimated results. Cysteine presented a very high ABTS value, but a negative ORAC value. Therefore, before ABTS and ORAC are applied to a protein-rich sample, the concentration of certain amino acids should be considered. The results for the same food constituents using acetone/water (50:50 v/v) as solvent are shown in Table 4. The tendencies are the same as for water, but in this case the ORAC and ABTS values for tyrosine and tryptophan are even higher. In the case of ORAC assay, cysteine and tyrosine yields a value signicantly lower than the one of triptophane, whilst in ABTS assay the highest value is obtained with tyrosine. 3.3. Combined eect of solvent and food constituents Finally, we determined the ORAC value of a mixture of catechin and gallic acid (5 lM:5 lM for the ORAC assay and 125 lM:125 lM for the other assays, which are much less sensitive), in water and in acetone/water (50:50 v/v), when each one of the food constituents previously tested

Table 3 ORAC, ABTS, FRAP and DPPH values for aqueous solutions of dierent food constituents Sample Glucose Pectin Galacturonic acid Tyrosine Tryptophane Arginine Cystein Albumin ORAC (lm Trolox/g) 0a 18.94 0.69a 0a 4529.04 256.17b 7127.03 285.83c 0a 0a 119.711 12.4842a ABTS (lm Trolox/g) 0a 0a 0a 5360.23 270.47b 1020.7 163.87c 185.26 9.18a 22,822.1 968.37d 38.32 1.64a FRAP (lm Trolox/g) 0a 0a 0a 0a 0a 0a 396.44 90.92b 0a DPPH % inhibition* 0a 0a 0a 0a 0a 7.93 1.88b 19.2 1.86c 0a

Dierent letters in the same column imply signicant dierences. * With catechin:gallic acid 5 lM:5 lM and an initial DPPH concentration of 63 lM.

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Table 4 ORAC, ABTS, FRAP and DPPH values for acetone/water (50:50 v/v) solutions of dierent food constituents Sample Glucose Galacturonic acid Tyrosine Tryptophane Arginine Cystein ORAC (lm Trolox/g) 0a 0a 5226.73 486.67b 12,154.52 766.74c 0a 1564.68 192.66b ABTS (lm Trolox/g) 0a 0a 1727.89 27.92b 862.93 49.42c 405.02 43.18d 946.74 111.76c FRAP (lm Trolox/g) 0a 0a 0a 0a 0a 0a DPPH % inhibition* 0a 0a 0a 0a 0a 0a

Dierent letters in the same column imply signicant dierences. * With catechin:gallic acid 5 lM:5 lM and an initial DPPH concentration of 63 lM.

is added to it. The object of this experiment was to determine, in the case of ORAC and ABTS, whether the resulting value was the sum of the values of the polyphenols and the food constituents by themselves, or it changed due to new interactions between the antioxidants and the food constituents. In the case of FRAP and DPPH, the object was to see whether, although most of them produced no eect when tested alone, these food constituents when mixed with polyphenols produced new interactions that altered the original value of the polyphenols solution. It must be pointed out the fact that when arginine was mixed with the gallic acid:catechin aqueous solution, which is colourless, this turned rst green and then yellow, due to interactions between these compounds. This is an example of the kind of complex interactions that take place within the food matrix. The results of the ORAC assay for the mixture of polyphenols and food constituents in aqueous solutions can be seen in Fig. 1, where the expected value column shows the sum of the ORAC value of the polyphenol solutions plus the ORAC value of the food constituent solutions, and the real value column shows the actual results in each of these solutions. In the case of galacturonic acid,

tyrosine and tryptophan, the experimental values are not statistically dierent to the expected ones, nor to the value of the original polyphenols solution. However, in the case of glucose, arginine and cysteine, which produced no ORAC value on their own, and albumin, which produced only a low value, there was a considerable increase in the eect of the ORAC value, producing a clear overestimation of the antioxidant capacity of the polyphenol solution. This indicates the existence of polyphenol/food constituent interactions that generate new interferences in the ORAC assay. The results of the ORAC assay for the mixture of polyphenols and food constituents when using acetone/water (50:50 v/v) as solvent are shown in Fig. 2. The results for galacturonic acid and tyrosine were lower than expected; the addition of glucose and arginine produced a slightly higher ORAC value, and the addition of cysteine clearly tended to raise the ORAC value. Again, this evidences that the same compounds interact dierently when the solvent is changed. It must pointed out that only galacturonic acid and cysteine showed a signicant dierence with their expected values and with the original value of the polyphenols solution.

8000 7000 6000 5000 4000 3000 2000 1000 0

expected ORAC value-sum of the values of the polyphenols solution alone and the food constituent alone experimental ORAC value

trolox micromolar

Fig. 1. Expected and experimental ORAC values of an aqueous solution of catechin:gallic acid 5 lM:5 lM (pps sol) mixed with dierent food constituents.

pp ss ol

ga 0. la 03 ct % ur on ic ac pp id ss 0. ol 01 + % ty ro pp sin ss e0 ol .0 + 00 try 5% pt op ha ne pp 0. ss 00 ol 05 + % ar gi ni ne pp 0. 00 ss 05 ol % + cy ste in pp 0. ss 00 ol 05 + % al bu m in e0 .0 03 %

pp ss ol

lu co se +g pp ss ol

5% +

pp ss ol

pe ct in e

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10000 9000 8000

797

trolox micromolar

7000 6000 5000 4000 3000 2000 1000 0

0. 00 0 uc o 0. 00 0 pp 0. 0 00 05 % 01 % 5% % 5% so l se 0. 00 5 s 5%

expected ORAC value-sum of the values of the polyphenols solution alone and the food constituent alone
experimental ORAC value

ac id

ro si

ur on i

ar gi

so l

cy st

pp s

ac t

so l+

l+

ga l

so

so l

so l+

pp s

pp s

Fig. 2. Expected and experimental ORAC value of an acetone/water, 50:50, v/v solution of catechin:gallic acid 5 lM:5 lM (pps sol) mixed with dierent food constituents.

pp s

In general, the ORAC values obtained in acetone/water (50:50 v/v) were higher than those obtained in water, which agrees with the conclusions drawn from Table 1. It must be remarked that although only a few of these mixtures of polyphenols and food constituents produced signicantly dierent results from the of the polyphenols solution alone, some of them tyrosine, tryptophan and albumine were tested at very low concentrations, so that much greater interference might be expected at the concentrations to be found in aqueousorganic extracts. For example, the concentrations of tyrosine and tryptophan tested were 0.0005%, whereas wheat our contains 0.54%
5000 4500 4000

t r o l o x mi c r om o l a r

pp s

of tyrosine (Abdel-Aal & Hull, 2002) and rice 0.21% of tryptophan (Zhai, 2001). The ABTS values for the polyphenols and food constituents in aqueous solutions are shown in Fig. 3. All the food constituents tested, except cysteine, reduced the ABTS value, especially galacturonic acid, followed by pectin and tyrosine, which would tend to produce underestimation of the antioxidant capacity of a sample. Even food constituents that produced no eect by themselves aected the values when they were mixed with the polyphenols, so here again there are new interactions aecting the antioxidant capacity values. On the other hand, only the solution

so l+

pp s

try

pt o

ty

ph an e

ne

ni n

ei n

gl

0.

3500 3000 2500 2000 1500 1000 500 0

pp ss ol 5% in e0 .0 05 % tin e0 .0 3% bu m in e0 .0 3% 0. 00 5% uc os e ac id 0. 00 5% 0. 00 5% 1%

expected ABTS value-sum of the values of the polyphenols solution alone and the food constituent alone experimental ABTS value

ph an e

ar gi ni ne

tu ro ni c

gl

pp ss ol

ty r

try pt o

cy ste i + pp ss ol

pe c

os

n +

pp ss ol

ac

pp ss ol

Fig. 3. Expected and experimental ABTS values of an aqueous solution of a catechin:gallic acid 125 lM:125 lM (pps sol) mixed with dierent food constituents.

pp ss ol

pp ss ol

pp ss ol

pp ss ol

ga l

al

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with arginine gave a value statistically non-dierent to the one of the original polyphenols solution. The results for the same analysis using acetone/water (50:50 v/v) as solvent are shown in Fig. 4. In this case, all the experimental values were quite similar to the expected values, excepting that of the mixtures of polyphenols and galacturonic acid and cysteine. Whereas galacturonic acid reduced the ABTS value in the aqueous solution, it increased the ABTS values in the acetone/water (50:50 v/v) solution. The opposite happened in the case of cysteine, showing once again that the food constituents behave quite dierently depending on the solvent. As regards to the original polyphenols solution, only the addition of tyrosine and trytophane to it gave values nonstatistically dierent. In general, the ABTS values obtained in water were higher than the values obtained ones in acetone/water (50:50 v/v) as was the case in Table 1. At all events, these dierences between experimental and expected values were produced with a polyphenol concentration 25 times the concentration used for the ORAC assay and with 10 times the amino acid concentration, and even under these conditions the dierences were not so great as they were in the ORAC assay. Therefore, it
2500

can be said that the interference that some food constituents cause in antioxidant capacity assays, by interacting with the solvent and with the polyphenols present in the sample is less in ABTS than in ORAC. Table 5 shows the results of FRAP and DPPH for an aqueous solution of catechin and gallic acid and each of the food constituents. In the DPPH assay, there were clear interactions between the polyphenols and the food constituents, since most of the latter produced no eect by themselves but now altered the original polyphenol EC50 value. All the glucidic compounds (glucose, galacturonic acid and pectine) reduced the EC50, indicating a greater antioxidant capacity. In the case of antioxidant capacity, on the contrary, some reduced the EC50, while others increased it. Cysteine, which produced a considerable inhibitory eect by itself, was the amino acid that most reduced the EC50. There were also important changes in the tEC50 values; all the food constituents reduced the original value of 19.73 in the polyphenols solution except for galacturonic acid, which increased it to 26.62 min. These changes in the EC50 and tEC50 values produced considerable changes in the AE, for example, from an original value of 0.74 in the polyphenols solution to 1.74 when cysteine was added.

trolox micromolar

2000 1500

1000 500 0
pps sol pps sol + glucose 5% pps sol + galacturonic acid 1% pps sol + tyrosine 0.005% pps sol + tryptophane 0.005% pps sol + pps sol + cystein arginine 0.005% 0.005%

expected ABTS value-sum of the polyphenols solution and the food constituents alone experimental ABTS value

Fig. 4. Expected and experimental ABTS values of an acetone/water (50:50 v/v) solution of catechin:gallic acid 125 lM:125 lM (pps sol) mixed with dierent food constituents.

Table 5 FRAP and DPPH values of an aqueous solution of catechin:gallic acid 125 lM:125 lM (pps sol) mixed with dierent food constituents Sample FRAP (lmol Trolox/g pps) 9559.2 110.7a 9465.5 797.8a 9281.7 223.8a 10,049 412.6a 9304.1 341.1a 9298 332.3a 11,891.36 452.67a,b 30,049.76 3019.52c 9573 134.3a DPPH EC50 (g pps/g DPPH) pps pps pps pps pps pps pps pps pps sol sol + glucose 5% sol + galacturonic acid 1% sol + pectin 0.03% sol + albumine 0.03% sol + tyrosine 0.005% sol + arginine 0.005% sol + cystein 0.005% sol + tryptophane 0.005% 0.067 0.001a 0.055 0.010a 0.060 0.002a 0.062 0.010a 0.077 0.005a 0.091 0.007b 0.068 0.0034a 0.064 0.0052a 0.082 0.005a tEC50 (min) 19.73 1.45d 11.84 1.82a 26.62 1.58d 18.31 0.77b,c 14.08 3.02b 8.8 0.25a 15.79 2.99 ab,c 14.24 4.77b 15.6 1.02b,c AE 0.75a,b 1.54c 0.62b 0.88b 0.92b 0.6a 0.93b 1.74 c 0.79b

Dierent letters in the same column imply signicant dierences.

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799

Table 6 FRAP and DPPH values of an acetone/water (50:50 v/v) solution of catechin:gallic acid 125 lM:125 lM (pps sol) mixed with dierent food constituent Sample FRAP (lmol Trolox/g pps) 10,100.1 489.7a 11,513.3 694.3a 11,726.1 443a 11,302.9 429.8a 32,123.5 2119b 94,046.47 7688.8c 11,302.9 429.8a DPPH EC50 (g pps/g DPPH) pps pps pps pps pps pps pps sol sol + glucose 5% sol + galacturonic acid 1% sol + tryptophane 0.005% sol + arginine 0.005% sol + cystein 0.005% sol + tyrosine 0.005% 0.083 0.003a 0.053 0.0005b 0.071 0.002c 0.066 0.001c 0.068 0.0034c 0.051 0.0036b 0.066 0.004c tEC50 19.44 0.80a 13.68 0.75a 29.46 0.67d 16.19 0.79b 15.79 2.99c 34.48 3.25d 17.8 0.54a,b AE 0.62a 1.37b 0.48a 0.93c 0.93c 0.57a 0.85c

Dierent letters in the same column imply signicant dierences.

On the contrary, of the four assays tested, FRAP was the one in which the combined eect of polyphenols and food constituents in an aqueous solution was smallest, although there were signicant dierences between the original value of the polyphenol solution and the ones obtained when arginine and cysteine were added. This could be related to the fact that FRAP is the only one of the assays studied that is based not on radical scavenging ability, but on metal ferric-reducing ability. Anyway, when cysteine was added to the polyphenol solution, it increased the original value three-fold. The presence of cysteine should therefore be considered when conducting the FRAP assay. Finally, the ABTS and DPPH values for the acetone/ water (50:50 v/v) solution of polyphenols and the dierent food constituents are shown in Table 6. In the DPPH assay, all the food constituents tested reduced signicantly the EC50 value, increasing the antioxidant capacity, showing the dierent behaviour of the same compound in dierent media. As regards kinetics, the compounds showed the same tendencies as in water; glucose, tyrosine and trytophan reduced the tEC50, and galacturonic acid exerted a clear slowing eect, producing a higher tEC50. Again, these changes generated signicant changes in antioxidant eciency, a parameter that expresses a balance between the antioxidant capacity and the time that the sample needs to act. As in the case of the aqueous solution, in the acetone/ water (50:50 v/v) medium FRAP was the assay in which there were least dierence between the polyphenol solution and the mixture of polyphenols and most of the food constituents tested. Then again, the dierences in the FRAP assay were greater in this solvent than in water; in acetone/water (50:50 v/v) all the food constituents had the eect of increasing the FRAP value, whereas in water some of them increased the value and others reduced it. And arginine and most of all cysteine clearly tended to cause overestimation of the FRAP value, which in the case of cysteine, meant that when this amino acid was added to the polyphenol solution, it increased its original value nine-fold. 4. Conclusions The ranking of the interference eect of solvent and food constituents in the four antioxidant capacity assays

tested was: ORAC > ABTS > DPPH > FRAP. Certain non-antioxidant food constituents, especially amino acids and uronic acids, may produce a positive result in antioxidant capacity assays and, even if they do not produce a result by themselves, they may interfere with the polyphenols present in the food matrix, producing a dierent antioxidant capacity value to that produced by the polyphenols alone. There is a wealth of data in the literature on antioxidant capacity of foods and food constituents. Our ndings suggest that antioxidant capacity values should only be compared when the measurements have been made by the same method in the same solvent. In addition, the presence of certain interfering amino acids and uronic acids in the tested solution should be checked. Acknowledgements The present research was performed under the nancial support of the Spanish Ministry of Education and Science (project AGL 2004-07579-C04-01/ALI). J. Perez-Jimenez thanks the Consejo Superior de Investigaciones Cientcas for granting her an I3P scholarship, nanced by the European Social Fund. References
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