Effect of solvent and certain food constituents on different antioxidant capacity assays
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), quantification of products formed during the lipid peroxidation (TBARS, LDLs oxidation), etc. (Aruoma, 2003, Frankel and Meyer, 2000, Sánchez-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 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 different 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 and Ong, 1999). Therefore, the method was modified (Re et al., 1999), so that the radical is formed by chemical reaction with potassium persulphate prior to addition of the sample.
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 modified at our laboratory to measure kinetic parameters (Sánchez-Moreno, Larrauri, & Saura-Calixto, 1998): the absorbance was measured until the reaction reached the plateau, instead of taking a fixed point. A calibration curve at that wavelength was made to calculate the remaining DDPH. The parameter EC50, which reflects 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 efficiency (AE = 1/EC50tEC50) were also calculated. This method has been recently modified 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′-azobi(2-amidinopropane) dihydrochloride) and attacks a fluorescent molecule, generating a decrease in the emission of fluorescence, 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 fluorescent molecule was β-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-specifically with some polyphenols, causing overestimation of antioxidant capacity. It was therefore substituted with fluorescein (3,6′-dihydroxy-spiro [isobenzofuran-1 [3H], 9′[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 antioxidants, causing an increase in absorbance at 595 nm. The absorbance reached at a fixed 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 different proportions (Gray et al., 2002, Yu et al., 2002), acetone/water in different proportions (Ou et al., 2001, Yilmaz and Toledo, 2006), methanol/water in different proportions (Yilmaz & Toledo, 2006) or acidic methanol/water followed by acetone/water (Saura-Calixto & Goñi, 2006). During the last few years, some researchers have noted the influence of the extraction solvent in the ORAC (Fernández-Pachón et al., 2004, Villaño et al., 2005, Zhou and Yu, 2004), DPPH (Barclay et al., 1999, Pinelo et al., 2004, Valgimigli et al., 1999, Zhou and 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 effect of the solvent on the antioxidant capacity of a polyphenol solution tested by these four assays.
Type of solvent and polarity may affect 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 affect the results. This work addresses both factors.
The first 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 flavonoid, 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).
Another aspect considered was the possibility that certain common food constituents interfere in antioxidant capacity assays. Antioxidant capacity is usually measured in aqueous–organic 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 different 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 effect of the polyphenols and the possible effects of the food constituents were cumulative, or whether new interactions appear when mixing them.
Section snippets
Chemicals
Trolox (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analogue of vitamin E, 2,2-dyphenyl-1-picryhydrazil (DPPH), potassium persulfate, fluorescein (3,6′-dihydroxy-spiro [isobenzofuran-1 [3H], 9′[9H]-xanthen]-3-one), AAPH (2,2′-azobi(2-amidinopropane) dihydrocloride) and 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) (Sigma–Aldrich Química, S. A., Madrid, Spain).
2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) was from Fluka Chemicals, Madrid, Spain.
Iron
Solvent effect
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 influenced these four antioxidant capacity assays, but not all in the same way.
ORAC is the assay in which the solvent influence 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 different to the ones obtained in the other
Conclusions
The ranking of the interference effect 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 different antioxidant capacity value to that produced by the polyphenols
Acknowledgements
The present research was performed under the financial support of the Spanish Ministry of Education and Science (project AGL 2004-07579-C04-01/ALI). J. Pérez-Jiménez thanks the Consejo Superior de Investigaciones Científicas for granting her an I3P scholarship, financed by the European Social Fund.
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2022, Food ChemistryCitation Excerpt :However, after a slight increase in the recovery, there is no significant difference in the results between 150 W and 200 W (P > 0.05). Reportedly, different solvents have different impacts on the ingredients of extracts, which may also lead to this difference (Pérez-Jiménez & Saura-Calixto, 2006; Pulido et al., 2000). Finally, the peak value of recovery and ln K has appeared at the extraction time of 40 min (Fig. 1F).