Antioxidant capacity of phenolic compounds in acidic medium: A pyrogallol red-based ORAC (oxygen radical absorbance capacity) assay

Journal of Food Composition and Analysis 32 (2013) 116–125

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Original Research Article

Antioxidant capacity of phenolic compounds in acidic medium: A pyrogallol red-based ORAC (oxygen radical absorbance capacity) assay E. Atala a, A. Aspe´e b, H. Speisky c, E. Lissi b, C. Lo´pez-Alarco´n a,* a b c

Departamento de Farmacia, Facultad de Quı´mica, Pontificia Universidad Cato´lica de Chile, C.P. 782 0436, Santiago, Chile Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Santiago, Chile Instituto de Nutricio´n y Tecnologı´a de los Alimentos, Universidad de Chile, Santiago, Chile

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 April 2013 Received in revised form 23 September 2013 Accepted 24 September 2013

A novel ORAC (oxygen radical absorbance capacity) assay to assess antioxidant capacity of phenolic compounds in near-gastric conditions (pH 2.0) is presented. AAPH (2,20 -azo-bis(2-amidinopropane)dihydrochloride) was used as peroxyl radicals source, and fluorescein, pyranine and pyrogallol red were employed as target molecules. Only pyrogallol red (PGR) showed a behavior compatible with an ORAC assay under acidic conditions (ORAC-PGRa). Excepting Trolox and ascorbic acid, phenolic compounds protected PGR, giving kinetic profiles without the presence of an induction time. ORAC-PGRa values, which reflect the reactivity of the antioxidants toward peroxyl radicals, ranged from 0.2 (caffeic acid) to 29.1 (myricetin) gallic acid equivalents. The ORAC-PGRa method showed analytical parameters in agreement with other ORAC-like assays and was applied to wines, teas, commercial juices and herb infusions, peach juice being the sample with the highest ORAC-PGRa value (7.1 mM gallic acid equivalents). In addition, ascorbic acid concentration in complex mixtures can be determined from kinetic profiles. ß 2013 Elsevier Inc. All rights reserved.

Keywords: Pyrogallol red Peroxyl radicals Antioxidant capacity Acid medium ORAC Phenolic compounds Beverages Food analysis Food composition

1. Introduction The human gastrointestinal tract is commonly exposed to substances capable of inducing oxidative stress, such as foods (mainly meats) that contain large amounts of lipids, hydroperoxides, free metals and myoglobin (Kanner, 1994). In the stomach cavity, free metals or myoglobin are known to catalyze hydroperoxides decomposition generating reactive species that are able to induce lipid peroxidation (Lapidot et al., 2005a,b). These reactions are favored by the low pH of the stomach fluid that promotes, among others, the generation of peroxyl radicals and carbonyl compounds (Lapidot et al., 2005b). These processes have been associated with vitamin oxidation and postprandial modification of low density lipoproteins and with their consequent deleterious effects on human health (Gorelik et al., 2005; Kanner et al., 2012). On the other hand, human diets often comprise rich-phenolic foods and beverages such as fruits, vegetables, wines and teas, whose consumption is expected to lead to high concentration of

* Corresponding author. Tel.: +56 223544838. E-mail address: [email protected] (C. Lo´pez-Alarco´n). 0889-1575/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jfca.2013.09.007

phenolic compounds in the stomach fluid (Halliwell et al., 2000). For example, after the intake of 80 mL of red wine, quercetin concentration in the stomach can reach values close to 3 mM (a value estimated considering a quercetin concentration in red wine close to 30 mM and a ten time dilution factor in the stomach cavity) (Neveu et al., 2010; Burton et al., 2005). Under gastric conditions, phenolic compounds have shown to inhibit lipid peroxidation (Lapidot et al., 2005b), an effect associated with their reaction with peroxyl radicals. This phenolic activity is related to their capacity to donate a hydrogen atom or a single electron to a damaging free radical. Nonetheless, the low stomach pH could influence the ability of phenolic compounds to neutralize peroxyl radicals via hydrogen donation. In spite of the relevance of these processes, it has been few attempts to evaluate the antioxidant capacity of phenolic compounds in acid medium. In this context, Di Majo et al. (2011) compared throughout competitive reactions, the antioxidant activity of phenolic compounds (flavonoids and phenolic acids) toward peroxyl radicals at pH 3.5 and 7.4. The capacity of phenolic compounds to inhibit the peroxyl radical-induced consumption of a probe like crocin was strongly influenced by media pH. In fact, increasing the pH of the media implied an increase in antioxidant activity (Di Majo et al., 2011).

E. Atala et al. / Journal of Food Composition and Analysis 32 (2013) 116–125

The present work was designed to develop an ORAC (oxygen radical absorbance capacity) assay capable of evaluating the antioxidant capacity of single phenolic compounds and their complex mixtures (wines, fruit juices and teas) under stomach-like acidic conditions. To such purpose, the peroxyl radicals-induced oxidative consumption of fluorescein, pyranine and pyrogallol red, three probes widely used in ORAC assays run at physiological neutral pH was evaluated under acidic conditions using a simulated gastric fluid as medium. 2. Materials and methods 2.1. Chemicals 2,20 -Azo-bis(2-amidinopropane) dihydrochloride (AAPH) was used as peroxyl radical source. Pyrogallol red (PGR) fluorescein (Fl), pyranine (Py), Trolox (6-hydroxy-2,5,8-tetramethylchroman-2carboxylic acid), ascorbic acid, AAPH, and all phenolic compounds employed were purchased from Sigma–Aldrich (St. Louis, MO, USA). All compounds were analytical grade and employed as received. 2.2. Solutions Unless otherwise indicated, experiments were carried out in simulated gastric fluid without pepsin (SGF), comprising a solution of sodium chloride (17 mM) adjusted to pH 2.0 with concentrated hydrochloric acid (The United States Pharmacopoeia). Stock solutions of PGR, Fl or Py (300 mM) were prepared daily in phosphate buffer 75 mM, pH 7.4. Stock solutions of phenolic compounds (1 mM) were prepared daily in ethanol. AAPH stock solutions (0.6 M) were prepared daily in SGF. 2.3. Samples Herb and tea bags (green and black tea, Rosa moschata, Mentha piperita, and Peumus boldus) were Chilean commercial products. Infusions were prepared by adding 150 mL of distilled water (95– 100 8C) to one bag (containing 2 g of dry material) and were brewed for 5 min. After withdrawing the bags, the resulting solution was cooled to 20 8C and immediately used to assess its total phenolic content and antioxidant properties (see below). Wines (red and white) and commercial juices were centrifuged at 10,000  g during 2 min (20 8C) and aliquots (10–400 mL) were taken and directly added to the working solutions (3 mL, final volume). 2.4. Oxygen consumption Solutions of AAPH (10 mM) in SGF or phosphate buffer (75 mM, pH 7.4) were incubated in a thermostatized cell at 37 8C. Oxygen consumption was assessed employing an ISO-OXY-2 electrode (WPI Inc., Sarasota, FL, USA) and registered in a TBR4100, Free Radical Analyzer instrument (WPI Inc., Sarasota, FL, USA). 2.5. Working solutions 2.5.1. Reaction of probes with AAPH-derived peroxyl radicals Reaction mixtures containing AAPH (10 mM), PGR, Fl, or Py were incubated at 37 8C in SGF in the thermostatized cuvette of an Agilent 8453 (Palo Alto, CA, USA) UV-visible spectrophotometer. Probe consumption was followed at 465, 437, and 403 nm, for PGR, Fl, and Py, respectively. In some experiments (i.e. for Fl or Py at concentrations lower than 5 mM) probe consumption was followed by fluorescence spectroscopy. For Fl, 437 and 515 and for Py, 403 nm and 504 nm were employed as excitation and

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emission wavelengths, respectively. Fluorescence measurements were carried out using a Perkin Elmer LS-55 spectrofluorimeter (Beaconsfield, U.K). 2.5.2. Effects of antioxidants on the PGR consumption induced by peroxyl radicals Solutions containing AAPH (10 mM), PGR (5 mM) and the tested samples were incubated in SGF at 37 8C. Addition of the samples (single antioxidants or beverages) did not modify the pH of solutions (2.0). PGR consumption was evaluated from the progressive absorbance decrease measured at 465 nm in the thermostatized cuvette of either, an Agilent 8453 (Palo Alto, CA, USA) or a Unicam Helios-g (Cambridge, U.K) UV-visible spectrophotometer. In some cases, the addition of beverages resulted in a small contribution at 465 nm. This absorbance was subtracted from the absorbance intensity of PGR at such wavelength. 2.6. ORAC determinations Values of the relative absorbance (A/A0) were plotted as a function of time. Integration of the area under the curve (AUC) was performed up to a time such that (A/A0) equal to 0.2. These areas were employed to obtain ORAC values, according to Eqs. (1) and (2) for single antioxidants and beverages, respectively: ORAC ¼

ORAC ¼

ðAUCXH  AUC0 Þ ½GA ðAUCGA  AUC0 Þ XH ðAUC  AUC0 Þ ðAUCGA  AUC0 Þ

f ½GA

(1)

(2)

where AUC(XH) is the area under curve in the presence of the tested sample, integrated between time zero and that corresponding to 80% of the probe consumption; AUC0 is the area under curve for control experiment (in the absence of antioxidants); AUCGA is the area under curve obtain in the presence of gallic acid; f is the dilution factor, equal to the ratio between the total volume of the AAPH-PGR solution and the added beverage volume and [GA] is the gallic acid molar concentration. 2.7. High performance liquid chromatography (HPLC) experiments 2.7.1. Reaction of PGR with AAPH-derived peroxyl radicals PGR solutions (10–30 mM) were incubated at 37 8C in the presence of AAPH 10 mM in SGF, under aerobic conditions. At different times, aliquots were extracted and immediately injected into the HPLC system. No concentration changes were observed in control experiments carried out in the absence of AAPH. All experiments were carried out in duplicate or triplicate. Method A: Chromatograms were obtained using an Agilent 1100 Series HPLC (Palo Alto, CA, USA), equipped with a Phorospher STAR RP18e (5 mm) 4.6 mm  250 mm HPLC column (Merck), and a diode array detector (DAD G1315A). Method B: Aliquots of the PGR-AAPH solution were analyzed by HPLCDAD-MS/MS employing a Shimadzu HPLC system (Tokyo, Japan) equipped with a quaternary LC-10ADVP pump with a FCV-10ALVP elution unit, a DGU-14A degasser unit, a CTO-10AVP oven, and an UV-vis diode array detector (model SPD-M10AVP) coupled in tandem with a QTrap LC/MS/MS 3200 Applied Biosystems MDS Sciex (Foster City, CA, USA). Instrument control and data collection system were carried out using a CLASS-VP DAD Shimadzu Chromatography Data System and Analyst software (version

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1.5.2) for MS/MS analysis. The analyses were carried out employing a C18 column (Kromasil 250 mm  4.6 mm, 5 mm) with a C18 precolumn (Nova-Pak Waters, 22 mm  3.9 mm, 4 mm) (Milford, MA, USA) at 30 8C. Chromatographic separation was achieved with an isocratic flow of a mobile phase constituted by 75% of formic acid 0.1% in water and 25% acetonitrile, at a flow rate of 0.4 mL min1. The sample injection volume was 20 mL and the column oven temperature was set at 30 8C. Compounds were identity under the following optimized ESI-MS/MS conditions: negative ionization mode, 12 V of collision energy, 4500 V of ionization voltage, capillary temperature at 300 8C and N2 as nebulization and drying gas at 30 and 25 psi respectively.

2.7.2. Phenolic compounds consumption induced by peroxyl radicals All phenolic compounds (2–900 mM) were incubated at 37 8C in the presence of AAPH 10 mM in SGF, under aerobic conditions. At different times, aliquots were withdrawn and immediately injected into the HPLC system. No concentration changes were observed in control experiments carried out in the absence of AAPH. Phenolic compound consumption was evaluated by HPLC-DAD. Chromatograms were obtained using method A with phosphate (KH2PO4, 10 mM adjusted to pH 2.6 with HCl)/acetonitrile; (70/30, V/V) for cinnamic acids, (65/35) for flavonols and luteolin, (90/10) for gallic acid, (60/40) for Trolox and (80/20) for PGR, as mobile phases (isocratic elution). Depending on the tested phenolic compound, chromatograms were obtained at detector wavelengths ranging from 265 to 376 nm. The flow rate was 0.8 mL min1, and all experiments were carried out in duplicate or triplicate. Results were analyzed with the HPLC-computercoupled ChemStation LC 3D program. Initial consumption rates were obtained from the slope, at t = 0, of the best fitting curve of concentration versus time plots.

Fig. 1. Dependence of the initial consumption rate of Trolox with its initial concentration in phosphate buffer at pH 7.4 (*) and SGF (^). [AAPH] = 10 mM, 37 8C.

Analytical parameters of the assay were obtained employing a microplate reader. Briefly, the consumption of PGR was evaluated at 465 nm in the thermostatized wells of a Multi-Mode microplate reader (SynergyTM HT, Biotek instruments, Winooski, VT, USA). The reaction mixture (final volume 250 mL in SGF) containing PGR (30 mL, 5 mM final concentration), gallic acid (40–100 mL of stock solutions) or red wine (20–100 mL) was placed in the well of the microplate. Solutions were preincubated for 30 min at 37 8C. AAPH solution (30 mL, 10 mM final concentration), previously incubated at 37 8C, was added using a multichannel pipette. The microplate was immediately placed in the reader, automatically shaken and absorption units (A) registered every 60 s for 240 min. Red wine samples showed a small absorption intensity at 465 nm which was subtracted from the absorbance intensity of PGR at such wavelength.

a clear decrease in the concentration of oxygen was registered during the incubation of AAPH (10 mM) at 37 8C (Fig. 1S, Supplementary data). From the oxygen consumption data, a rate of peroxyl radicals formation of 0.8 mM/min was estimated to have taken place in both, SGF and phosphate buffer media; such value is in agreement with that previously reported by Niki, using AAPH at the same concentration (Niki, 1990). To gain more insights regarding the rate of peroxyl radicals formation from AAPH in SGF, we evaluated the consumption of Trolox, an antioxidant able to trap two peroxyl radicals per molecule at pH 7.4 (n = 2) (Halliwell and Gutteridge, 2007). As Fig. 1 shows, at pH 7.4, Trolox was oxidized by peroxyl radicals in a concentration-independent way. In fact, an initial consumption rate close to 0.45 mM/min was determined between 3 and 150 mM Trolox concentrations. On the other hand, in SGF, for Trolox concentrations below than 15 mM, the initial consumption rate was found to increase when the concentration increases. Beyond 15 mM, the rate of Trolox consumption reached a plateau at 0.48 mM/min (Fig. 1). Considering the above-mentioned stoichiometric factor of Trolox (n, defined as the number of peroxyl radicals trapped per each Trolox molecule), in the zero-order kinetic limit in Trolox, the initial consumption rate implies a peroxyl radical formation close to 0.9 mM/min. This estimated value is similar to that obtained from oxygen consumption measurements, and shows therefore, that AAPH generates peroxyl radicals at nearly the same rate at both pH values (7.4 and 2.0). This statement is in line with data reported by Hanlon and Seybert (1997) who found similar rates of AAPH decomposition at pH 5.5 and 7.5.

2.9. Data expression and analysis

3.2. The choice of target

Data presented in Tables and Figures represent the mean values of at least three independent experiments, each conducted in triplicate.

Taking into account the use of Fl, Py, and PGR as target molecules in ORAC-like methodologies at pH 7.4 (Lo´pez-Alarco´n and Lissi, 2006; Omata et al., 2008; Ou et al., 2001), we studied the possible use of these probes in SGF. As presented in Fig. 2A, all probes showed visible spectroscopic bands compatible with their use in ORAC assays in SGF. Fl, Py, and PGR showed visible bands with maxima at 437, 403, and 465 nm, respectively (Fig. 2A). When these target molecules were incubated in SGF with AAPH (10 mM), different consumption rates were observed (Fig. 2B). The absorption visible intensity of Fl was unaltered by the presence of AAPH, implying that Fl did not react with peroxyl radicals in SGF. In

2.8. Analytical parameters of the ORAC-PGRa index

3. Results and discussion 3.1. Generation of AAPH-derived peroxyl radicals in SGF By monitoring oxygen and Trolox consumption, we studied the generation of peroxyl radicals by AAPH in both, SGF and a phosphate buffer (at pH 7.4). Under both experimental conditions,

E. Atala et al. / Journal of Food Composition and Analysis 32 (2013) 116–125

AAPH+O2

ROO• +PGR

2ROO•

119

2ROO• +N2

bleaching

non radicals products Scheme 1.

Fig. 2. (Graphic A) UV–visible spectra of Py (dot line), Fl (solid line) and PGR (dash line) at 5 mM concentration in SGF. (Graphic B) Consumption of Fl (&); Py (*) and PGR (4) mediated by peroxyl radicals derived from AAPH (10 mM) in SGF at 37 8C. [probe] = 5 mM. Consumption of Py, Fl and PGR was followed at 403, 437, and 465 nm, respectively.

contrast, a clear decrease in the visible bands of Py and PGR was observed in the presence of AAPH-derived peroxyl radicals. However, the initial consumption rate of Py was considerably lower than that of PGR. In fact, the initial consumption rate of PGR (0.33 mM/min) was three times higher than that of Py. PGR was almost totally consumed after 25 min incubation while, at the same incubation time, only a 40% of the added Py was consumed (Fig. 2B). In agreement with the latter, the zero order kinetic limit in Py was reached at 60 mM (data not shown) while, for PGR it was reached at 10 mM (vide infra), indicating a lower reactivity of Py than PGR toward AAPH-derived peroxyl radicals in SGF. This result contrasts with that observed at pH 7.4, where Py shows a higher reactivity toward peroxyl radicals than PGR (Lo´pez-Alarco´n and Lissi, 2005; Pino et al., 2003). The differences observed of the reactivity of probes between pH 2.0 and pH 7.4 could be explained by acid–base equilibria of each target molecule. At pH 7.4 all probes are partially deprotonated, and therefore, their reactivity at this pH could be explained by the reactivity of both, neutral and ionic species. At pH 2.0, hydroxyl groups are protonated, and in consequence, obtained results in SGF should reflect differences in the ability of the targets to donate a hydrogen atom to peroxyl radicals. The higher reactivity showed by PGR at pH 2.0 is probably associated with its capacity to form a quinone derivative as product of the reaction (vide infra). Considering the latter (higher

reactivity of PGR than Py in SGF), and that PGR showed a visible band at higher wavelengths than that shown by Py, we selected PGR as the more suitable probe to be used thereafter in an ORAClike assay in SGF (ORAC-PGRa). Taking into account a simple kinetic scheme of reactions, the following set of reactions should be consider: From these reactions, at low PGR concentrations, reaction (3) should compete with PGR bleaching (reaction (2)). However, at high PGR concentrations, all peroxyl radicals react with this target, disregarding the occurrence of reaction (3). As Fig. 3 shows, the initial consumption rate of PGR depended on its initial concentrations up to 10 mM. From these data, Log–Log dependence was plotted leading a value of 0.75 for the PGR kinetic order; probably implying a more complex mechanism than that of the expected for a first-kinetic order. Beyond 10 mM, initial PGR consumption rates reached a maximum value of 0.5 mM/min showing a zero order kinetic limit at higher PGR concentrations. In comparison with the consumption of PGR at pH 7.4, in SGF the zero order kinetic limit was reached at higher PGR concentrations. In fact, in the SGF media, the initial consumption rate of PGR at 3 mM concentration was 2.8 times lower than that seen at pH 7.4. On the other hand, in SGF, the maximum consumption rate (zero kinetic order limit) involves a n value of 1.7; i.e. 1.7 peroxyl radicals are trapped per each PGR molecule. This value is similar to the stoichiometry earlier reported by us at pH 7.4 (n = 1.8) (Lo´pez-Alarco´n et al., 2007b). To establish if PGR consumption in SGF follows the same mechanism of reaction than that seen at pH 7.4, we studied the oxidation of PGR induced by peroxyl radicals in SGF employing an HPLC separation technique coupled to a diode array and mass detection system. As Fig. 4 shows (chromatogram a), in SGF, in

Fig. 3. Dependence of the initial consumption rate of PGR with its initial concentration in SGF. [AAPH] = 10 mM, 37 8C.

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Fig. 4. Reaction of PGR with AAPH-derived peroxyl radical followed by HPLC-DAD. PGR solutions (15 mM) were incubated at 37 8C in the presence of AAPH (10 mM) in SGF (chromatogram a) and phosphate buffer at pH 7.4 (chromatogram b). Chromatograms were obtained (l = 380 nm) after c.a. half PGR consumption.

addition to the chromatographic peaks of AAPH and PGR (at 2.9 and 11.4 min, respectively), two products, at 5.3 and 5.5 min, were registered. These products were the same that those generated at pH 7.4, as is presented in the chromatogram b of Fig. 4. These peaks showed the same UV-visible spectrum, as well as the same mass pattern (m/z = 433, 389 and 361), and have been recently identified as a quinone derivative of PGR (Atala et al., 2013). These results strongly support that PGR is oxidized at pH 2.0 by the same mechanism of reaction than that taking place at pH 7.4, in agreement with Scheme 2. As mentioned above, the observed differences in the SGF and pH 7.4 regarding the rate of PGR oxidation would be mainly related to differences in the oxidation rate due to acid–base equilibria of hydroxyl groups of the PGR chemical structure (Ivanov and Mamedov, 2006).

Fig. 5. (Graphic A) Consumption profile of PGR in the absence (4) and presence of gallic acid at: 10 (&); 25 (*), 50 (5); 100 (^); and 150 (v) mM. (Graphic B) Dependence of AUC with [XH]. Myricetin (4); sinapic acid (5); ascorbic acid (&); kaempferol (^); and gallic acid (*).

3.3. Protection of PGR by single antioxidants Solutions containing PGR (5 mM), AAPH (10 mM) and single antioxidants in SGF were incubated at 37 8C, and the reaction was followed at 465 nm. Fig. 5A shows typical results obtained in the presence of gallic acid. In the presence of this antioxidant, a lower PGR consumption rate was evidenced in a gallic acid concentration-dependent way. In the presence of a single antioxidant, reaction (4) should be considered together with Scheme 1. ROO þ XH ! X þ ROOH

(4)

In addition, it should be taken into account all self-reactions and cross-reactions of the radicals produced in reactions (3) and (4).

As Fig. 5A presents, this protection takes place without an induction time in the kinetic profiles. A similar behavior was observed for all the (eleven) antioxidants studied excepting ascorbic acid and Trolox which protected PGR through clear induction times (vide infra). The presence of such induction times could be related to a high efficient of these antioxidants to protect PGR or to the presence of repair mechanisms. From plots as those presented in Fig. 5A, the area under the curve of the kinetic profiles was obtained; a parameter that linearly increased with the antioxidant concentration as depicted in Fig. 5B. From these data, employing Eq. (1), ORAC-PGRa (suffix a indicates acid medium) values for single antioxidants were calculated. Since induction

PGR oxidation mediated by peroxyl radicals Scheme 2.

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Table 1 ORAC-pyrogallol red (ORAC-PGR) values and kXH/kGA ratio (corrected by n) of single antioxidants in simulated gastric fluid (SGF). Compound

ORAC-PGRaa

nb

(kXH/kGA)(nGA/nXH)c

Caffeic acid Coumaric acid Ferulic acid Sinapic acid Quercetin Apigenin Luteolin Myricetin Kaempferol Gallic acid Trolox1 Ascorbic acid PGR

0.2  0.1 0.6  0.1 2.9  0.4 22.1  2.2 2.6  0.5 1.6  0.2 2.0  0.1 29.1  3.8 1.7  0.4 1 22.8  3.2 7.6  0.8 –

4.1 5.3 2.3 1.0 – – – 0.9 6.2 3.7 1.7 2.3 1.7

0.59 0.26 0.81 11.47 – – – 22.94 0.93 1 77.70 – 44.77

a

ORAC-PGRa values expressed as gallic acid equivalents. n = stoichiometric factor, number of peroxyl radical molecules trapped by each antioxidant molecule. n values were determined from maxima consumptions rates of antioxidants assessed by HPLC. c (kXH/kGA)(nGA/nXH) ratio. kXH/kGA was assessed from initial consumption rate versus phenol (XH) concentration plots (assessed by HPLC technique). kGA and nGA represent the kinetic rate constant, and the stoichiometry of the reaction between gallic acid and peroxyl radicals, respectively. b

times were registered in the kinetic profiles associated with the PGR protection given by Trolox (Fig. 2S, Supplementary data), gallic acid was selected as reference antioxidant. ORAC-PGRa values are presented in Table 1. Among all antioxidants studied, the highest ORAC-PGRa value was found for myricetin (29.1 gallic acid equivalents), and the lowest one for caffeic acid (0.2 gallic acid equivalents). These values imply that the ORAC-PGRa value of myricetin was 145 times higher than that of caffeic acid, showing a high discrimination of the ORAC-PGRa assay. The order of the ORAC-PGRa values presented in Table 1 is:

Myricetin > Trolox > sinapic acid > ascorbic acid > ferulic acid  quercetin > kaempferol  apigenin > gallic acid > coumaric acid > caffeic acid From this ORAC-PGRa order, and considering the absence of induction times in the kinetic profiles (expecting for ascorbic acid and Trolox), a relationship between the reactivity of the compounds toward peroxyl radicals and the ORAC-PGRa index is expected. To support this contention we carried out kinetic experiments aimed to evaluate, throughout HPLC technique, the initial consumption rate of antioxidants induced by AAPH-derived peroxyl radicals in SGF. Fig. 6A shows the kinetic profiles of ferulic acid consumption. From the kinetic data initial consumption rates (mM/min) of ferulic acid were estimated. Fig. 6B shows the dependence of these initial consumption rates with ferulic acid concentration. As can be seen in this figure, between 2 and 300 mM ferulic acid concentration, the initial consumption rate increased in a concentration-dependent manner. Beyond 300 mM the initial consumption rate reached a plateau at 0.35 mM/min. This implies a first order kinetics at low ferulic acid concentrations (lower than 300 mM) and a zero order kinetics at concentrations higher than 300 mM. Two parameters can be estimated from this kind of plots; the relative kinetic rate constant (estimated from the slope of the fitted curved without antioxidant and normalized by gallic acid, kXH/kGA) and the stoichiometry of the reaction (n, evaluated from rate values in the zero order kinetic limit and the rate of peroxyl radicals production from AAPH). This analysis consider a reaction kinetic model as ri = kXH [XH][ROO]ss and assume that the steady state concentration of peroxyl radicals ([ROO]ss) is similar in the presence of XH or gallic acid (GA). Table 1 shows n and kXH/kGA

Fig. 6. (Graphic A) Consumption profile of ferulic acid induced by peroxyl radicals in SGF. [Ferulic acid] = 100 (&); 300 (4); 500 (5); and 800 (*) mM. Reactions were carried out in SGF at 37 8C, and followed by HPLC (see experimental section). Area = area under the chromatographic peak of ferulic acid. [AAPH] = 10 mM. (Graphic B) Dependence of the initial consumption rate of ferulic acid with its initial concentration in SGF. [AAPH] = 10 mM, 37 8C.

(corrected by n, (kXH/kGA)(nGA/nXH)) values of most of the antioxidants studied (quercetin, apigenin and luteolin showed low solubility at high concentrations precluding to reach the zero order kinetic limit). As depicted in the table, n values varied from 0.9 to 6.2, and (kXH/kGA)(nGA/nXH) values from 0.26 to 77.7. High n values obtained for antioxidants with low reactivity, as coumaric acid, toward peroxyl radicals could be associated with complex mechanisms involving self-reactions of peroxyl radicals. In particular, the presence of alcoxyl radicals generated from reaction (5) cannot been disregarding. 2ROO ! 2RO þ O2

(5)

Highly reactive compounds (as Trolox) at high concentrations, readily reacts with peroxyl (ROO) and/or alcoxyl (RO) radicals, leading to n = 2.0. On the other hand, low reactive phenolic compounds such as coumaric acid, are unable to trap peroxyl radicals, and even in the plateau only reacts with alcoxyl radicals. Occurrence of reactions (3) and (5) in this conditions leads to very high ‘‘apparent’’ n values. Excluding ascorbic acid and Trolox, which protected PGR with clear induction times, the ORAC-PGRa of all other antioxidants showed a linear regression dependence with (kXH/kGA)(nGA/nXH) in agreement with: y = 0.035 + 0.702x, r = 0.9736 (Fig. 7). In contrast, the ORAC-PGRa values showed an inverse correlation

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1.0

A/A0

A

0.5

0.0 0

5000

10000

Time / s Fig. 7. Dependence of ORAC-PGRa values with (kXH/kGA)(nGA/nXH). y = 0.035 + 0.702x, correlation coefficient = 0.9736.

3.4. Protection of PGR by complex mixtures The ORAC-PGRa assay was applied to commonly consumed beverages. Thus, aliquots of black and green tea, red and white wines, herbal infusions and commercial juices were added to solutions containing PGR (5 mM) and AAPH (10 mM) in SGF. Figs. 9 and 10 present kinetic profiles of PGR consumption in the presence of the tested beverages. As Fig. 9A shows, green tea efficiently inhibited the consumption of PGR mediated by peroxyl radicals, without generating clear induction times in the kinetic profiles. A similar behavior was observed for black tea, white wine, P. boldus and M. piperita infusions (data not shown). Red wine protected PGR throughout kinetic profiles with the presence of short induction times (Fig. 9B), associated with the presence of low concentrations of highly reactive phenolic compounds (as evidenced from experiments carried out in the presence of ascorbate oxidase) (Lo´pez-Alarco´n and Lissi, 2005). In contrast, R. moschata infusion and commercial juices (Fig. 10) protected PGR giving kinetic profiles with induction times pointing to the presence of ascorbic acid in the latter foodstuff. In fact, when these samples were preincubated with ascorbate oxidase, the induction times were practically abolished. In agreement with the reported presence of

B

10000

Induction time / s

with n, y = 4.5  0.119x; r = 0.7997. These results would support the conclusion that ORAC-PGRa index is more associated with the rate (reactivity) than the stoichiometry of the antioxidant-peroxyl radical reaction. Caffeic acid, whose secondary reactions are different to those of other cinnamic acid derivatives, present an ORAC-PGRa index lower than that the expected from its reactivity toward free radicals. This emphasizes the contribution of the secondary reactions to ORAC-like values (Lo´pez-Alarco´n et al., 2007a,b, 2009; Bisby et al., 2008; Pino et al., 2003). Ascorbic acid was the only natural antioxidant that protected PGR by kinetic profiles characterized by the presence of clear induction times (Fig. 8A). This behavior has been also evidenced at pH 7.4 and has been proposed as the basis to assess ascorbic acid concentrations in fruits and human fluids by the ORAC-PGR assay (Atala et al., 2009; Torres et al., 2008). Interestingly, in contrast with the results obtained at pH 7.4, where a down curvature was observed, at pH 2.0 (SGF medium), the induction times showed a linear dependence with ascorbic acid concentration between 6 and 50 mM, with a n = 2.3, implying that no ascorbic acid chain decomposition is taking under these experimental conditions (Fig. 8B).

15000

5000

0 0

20

40

60

[ASC] / µM Fig. 8. (Graphic A) Consumption profile of PGR in the absence (&) and presence of ascorbic acid at: 6 (*); 15 (4); 20 (5); 30 (^); and 40 (v) mM. SGF at 37 8C, [AAPH] = 10 mM. (Graphic B) Dependence of induction time with ascorbic acid concentration. Induction times were estimated as the time at which intercept the straight lines drawn to data corresponding to the slow and fastest consumption rates. This criterion is somehow arbitrary (Atala et al., 2009) but it was chosen for its simplicity.

ascorbic acid in both types of beverages, these results gave ascorbic acid concentrations of 7, 226 and 184 mg/L for R. moschata infusion, peach and pineapple juices, respectively. The presence of small amounts of ascorbic acid in R. moschata infusions also agrees with previously reported data (Gomez et al., 1993; Mannino and Cosio, 1997). The area under the curve of the kinetic profiles of the PGR protection given by the complex samples showed a linear dependence with the initial sample concentration (Fig. 11). From these data ORAC-PGRa values were estimated (Table 2). The highest ORAC-PGRa value was 7.1 mM of gallic acid equivalents (commercial peach juice), while the lowest one was 0.18 mM (P. boldus infusion) of gallic acid equivalents. The order of the ORACPGR values presented in Table 2 was:

Commercial peach juice > commercial pineapple juice > red wine > green tea > black tea > Rosa moschata infusion > white wine > Mentha pi perita > Peumus boldus Interestingly, the Folin index of most samples was higher than ORAC-PGRa values implying that the tested beverage samples contain antioxidants with low reactivity toward peroxyl radicals

E. Atala et al. / Journal of Food Composition and Analysis 32 (2013) 116–125

123

A/A0

1.0

A

0.5

0.0 0

4000

8000

Time / s

B

A/A0

1.0

0.5

0.0 0

2000

4000

6000

Time / s

Fig. 9. (Graphic A) Effect of green tea on PGR consumption induced by AAPH in SGF. PGR (5 mM) was incubated in the presence of AAPH (10 mM) and green tea infusion. Control experiment (&); [green tea]: 8 (*); 17 (4); 25 (5); 33 (^); and 42 (v) mL/ mL. (Graphic B) Effect of red wine on the PGR consumption induced by AAPH in SGF. PGR (5 mM) was incubated in the presence of AAPH (10 mM) and red wine. Control experiment (&); [red wine]: 5 (4); 8 (^); and 12 (") mL/mL. The reaction was followed by the decrease in absorbance intensity at 465 nm in SGF at 37 8C.

Fig. 10. (Graphic A) Effect of commercial peach juice on the PGR consumption induced by AAPH in SGF. PGR (5 mM) was incubated in the presence of AAPH (10 mM) and commercial peach juice at 3 (*); 7 (4); 10 (5); 13 (^); 17 (v); and 20 (") mL/mL. Control experiment (&). (Graphic B) Effect of Rosa moschata infusion on the PGR consumption induced by AAPH in SGF. PGR (5 mM) was incubated in the presence of AAPH (10 mM) and Rosa moschata at; 67 (4); 133 (^); and 167 (*) mL/ mL. Control experiment: (&). The reaction was followed by the decrease in the absorbance intensity at 465 nm in SGF at 37 8C.

(in SGF) and then, with a low ability to protect PGR. In addition, a poor correlation was observed between ORAC-PGRa and Folin index (y = 2.38 + 0.066x; r = 0.045, Fig. 3S, Supplementary data). This emphasizes the relevance of kinetic factors on the ORAC-PGRa indexes. 3.5. Analytical parameters of the ORAC-PGRa index In addition to the above presented and discussed results, we performed experiments aimed to obtain analytical parameters regarding the ORAC-PGRa assay. As we obtained similar ORACPGRa values employing UV-visible spectrophotometers and a microplate reader, we decided to study the analytical parameters of the assay employing the latter instrument. For this purpose, ORAC-PGRa assay was applied to gallic acid and red wine in a microplate reader to obtain linearity, precision and accuracy of the method. Linearity: AUC, obtained from kinetic profiles in the presence of ten gallic acid concentrations (between 0 and 90 mM), were employed to build calibration curves. Mean obtained data from eighteen calibration curves gave the following linear regression:

Fig. 11. Dependence of AUC with beverage addition. Commercial peach juice (*); red wine (v); green tea (^); black tea (5); and Rosa moschata infusion (4).

E. Atala et al. / Journal of Food Composition and Analysis 32 (2013) 116–125

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Table 2 ORAC-pyrogallol red under acidic conditions (ORAC-PGRa) values obtained in SGF and Folin index of complex mixtures (beverages). Complex sample

ORAC PGRa

Folinb

Green tea Black tea Red wine White wine Rosa moschata Peumus boldus Mentha piperita Commercial pineapple juice Commercial peach juice

2.7  0.5 0.9  0.1 5.5  0.6 0.32  0.04 0.42  0.05 0.18  0.02 0.26  0.03 5.9  0.06 7.1  0.6

3.22  0.35 5.06  0.66 7.16  0.06 1.67  0.27 2.34  0.13 3.34  0.11 1.94  0.04 1.83  0.11 1.03  0.11

a ORAC-PGRa values (n = 3) are expressed as mM gallic acid equivalents. They represent the concentration (mM) of a gallic acid solution that produces the same effect that the tested sample. b Total phenolic content is expressed as mM gallic acid equivalents.

AUC ¼ ð2269  158Þ þ ð23  1Þ ½gallic acid; mM with a regression coefficient of 0.9856  0.0056, n = 18. These data imply RSD values of 6.9 and 4.3% for the intercept and slope, respectively. These results are in agreement with the analytical parameters recently reported for the ORAC-PGR index in phosphate buffer at pH = 7.4 (Ortiz et al., 2011, 2012). Precision and accuracy: quality control (QC) experiments employing gallic acid at 20, 40 and 60 mM were developed. Obtained results are presented in Table 3. As is depicted in such table, polled run data show RSD values of 10.5, 7.0 and 3.6 for 20, 40 and 60 mM gallic acid concentrations, respectively. These values show, that in agreement with the analytical parameters of the ORAC-PGR assay at pH 7.4, lower RSD values at 40 and 60 mM were estimated than 20 mM gallic acid concentrations. The latter would imply a better precision at the highest gallic acid concentration employed. On the other hand, unlike Ortiz et al. (2011, 2012), similar accuracy was determined for all the gallic acid concentrations employed.

AUC ¼ ð1955  94Þ þ ð154  12Þ ½red wine; mL=mL with a regression coefficient of 0.9995  0.0025, n = 15. From these data, RSD values of 4.8 and 7.8% for the intercept and slope, respectively, were estimated. In addition, ruggedness experiments carried out during four days, with four different red wine solutions, gave RSD a value of 7.7%. 4. Conclusions In contrast with fluorescein and pyranine, pyrogallol red retains its potential as a peroxyl radical-target molecule suitable to be used in an ORAC-like assay under acid conditions. The ORAC-PGRa method could be employed to assess an antioxidant capacity index related to the reactivity of phenolic compounds and their complex samples toward peroxyl radicals in simulated gastric conditions. The assay showed analytical parameters in agreement with other ORAC-like methods, and also could be used to estimate the protection afforded by ascorbic acid under acidic conditions. Acknowledgments This work was supported by FONDECYT (grant 1100659). Elias Atala gratefully acknowledges CONICYT fellowship (Chilean Commission of Scientific Research and Technology, 24121097). Authors thank Dr. Patricio Cumsille for his assistance in statistical analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfca.2013.09.007. References

Table 3 Precision and accuracy of quality control (QC).

Nominal gallic acid (mM)

To gain more insights about the analytical parameters of the ORAC-PGRa index, we determined linearity and ruggedness employing red wine as complex sample. Fifteen calibration curves gave a mean linear regression of:

QC1

QC2

QC3

20

40

60

Run (1) Intra-mean (mM) SD RSD (%) REC (%) n=4

19.4 2.2 11.3 97.0

43.3 3.4 7.9 108.3

64.8 4.2 6.5 108.0

Run (2) Intra-mean (mM) SD RSD (%) REC (%) n=4

24.4 1.3 5.3 122.0

43.5 3.8 8.7 108.8

66.2 1.1 1.7 110.3

Run (3) Intra-mean (mM) SD RSD (%) REC (%) n=4

20.3 3.0 14.8 101.5

41.6 1.8 4.3 104.0

60.9 1.6 2.6 101.5

Pooled runs Inter-mean (mM) SD RSD (%) REC (%) n = 12

21.4 2.2 10.5 106.8

42.8 3.0 7.0 107.0

64.0 2.3 3.6 106.6

Atala, E., Vasquez, L., Speisky, H., Lissi, E., Lopez-Alarcon, C., 2009. Ascorbic acid contribution to ORAC values in berry extracts: an evaluation by the ORACpyrogallol red methodology. Food Chemistry 113, 331–335. Atala, E., Vela´zquez, G., Vergara, C., Mardones, C., Reyes, J., Tapia, R., Quina, F., ˜ artu, M., Aspee, A., Lopez-Alarcon, C., Mendes, M., Speisky, H., Lissi, E., Ureta-Zan 2013. Mechanism of pyrogallol red oxidation induced by free radicals and reactive oxidant species. A kinetic and spectroelectrochemistry study. Journal of Physical Chemistry B 117, 4870–4879. Bisby, R.H., Brooke, R., Navaratnam, S., 2008. Effect of antioxidant oxidation potential in the oxygen radical absorption capacity (ORAC) assay. Food Chemistry 108, 1002–1007. Burton, D.D., Kim, H.J., Camilleri, M., Stephens, D.A., Mullan, B.P., O’Connor, M.K., et al., 2005. Relationship of gastric emptying and volume changes after a solid meal in humans. American Journal of Physiology – Gastrointestinal and Liver Physiology 289, G261–G266. Di Majo, D., La Neve, L., La Guardia, M., Casuccio, A., Giammanco, M., 2011. The influence of two different pH levels on the antioxidant properties of flavonols, flavan-3-ols, phenolic acids and aldehyde compounds analysed in synthetic wine and in a phosphate buffer. Journal of Food Composition and Analysis 24, 265–269. Gomez, R.G., Malec, L.S., Vigo, M.S., 1993. Chemical composition of ripe fruits of Rosa rubiginosa L. Anales de la Asociacion Quimica Argentina 81, 9–14. Gorelik, S., Lapidot, T., Shaham, I., Granit, R., Ligumsky, M., Kohen, R., et al., 2005. Lipid peroxidation and coupled vitamin oxidation in simulated and human gastric fluid inhibited by dietary polyphenols: health implications. Journal of Agricultural and Food Chemistry 53, 3397–3402. Halliwell, B., Zhao, K.C., Whiteman, M., 2000. The gastrointestinal tract: a major site of antioxidant action? Free Radical Research 33, 819–830. Halliwell, B., Gutteridge, J.M.C., 2007. Free Radicals in Biology and Medicine. Oxford University Press, New York, USA. Hanlon, M.C., Seybert, D.W., 1997. The pH dependence of lipid peroxidation using water-soluble azo initiators. Free Radical Biology and Medicine 23, 712–719.

E. Atala et al. / Journal of Food Composition and Analysis 32 (2013) 116–125 Ivanov, V.M., Mamedov, A.M., 2006. 3,4,5-Trihydroxyfluorones as analytical reagents. Journal of Analytical Chemistry 61, 1040–1062. Kanner, J., 1994. Oxidative processes in meat and meat-products – quality implications. Meat Science 36, 169–189. Kanner, J., Gorelik, S., Roman, S., Kohen, R., 2012. Protection by polyphenols of postprandial human plasma and low-density lipoprotein modification: the stomach as a bioreactor. Journal of Agricultural and Food Chemistry 60, 8790–8796. Lapidot, T., Granit, R., Kanner, J., 2005a. Lipid hydroperoxidase activity of myoglobin and phenolic antioxidants in simulated gastric fluid. Journal of Agricultural and Food Chemistry 53, 3391–3396. Lapidot, T., Granit, R., Kanner, J., 2005b. Lipid peroxidation by free iron ions and myoglobin as affected by dietary antioxidants in simulated gastric fluids. Journal of Agricultural and Food Chemistry 53, 3383–3390. Lo´pez-Alarco´n, C., Lissi, E., 2005. Interaction of pyrogallol red with peroxyl radicals. A basis for a simple methodology for the evaluation of antioxidant capabilities. Free Radical Research 39, 729–736. Lo´pez-Alarco´n, C., Lissi, E., 2006. A novel and simple ORAC methodology based on the interaction of pyrogallol red with peroxyl radicals. Free Radical Research 40, 979–985. Lo´pez-Alarco´n, C., Aspe´e, A., Lissi, E., 2007a. Competitive kinetics in free radical reactions of cinnamic acid derivatives. Influence of phenoxyl radicals reactions. Free Radical Research 41, 1189–1194. Lo´pez-Alarco´n, C., Aspe´e, A., Lissi, E., 2007b. Antioxidant reactivity evaluated by competitive kinetics: influence of the target molecule concentration. Food Chemistry 104, 1430–1435. Lo´pez-Alarco´n, C., Aspe´e, A., Lissi, E., 2009. Relevance of secondary processes in ORAC values obtained employing pyrogallol red as target molecule. Journal of the Chilean Chemical Society 53, 1718–1719.

125

Mannino, S., Cosio, M.S., 1997. Determination of ascorbic acid in foodstuffs by microdialysis sampling and liquid chromatography with electrochemical detection. Analyst 122, 1153–1154. Neveu, V., Perez-Jimenez, J., Vos, F., Crespy, V., Du, C.L., Mennen, L., et al., 2010. Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database (Oxford) 2010, bap024. Niki, E., 1990. Free-radical initiators as source of water-soluble or lipid-soluble peroxyl radicals. Methods in Enzymology 186, 100–108. Omata, Y., Saito, Y., Yoshida, Y., Niki, E., 2008. Simple assessment of radical scavenging capacity of beverages. Journal of Agricultural and Food Chemistry 56, 3386–3390. Ortiz, R., Antile´n, M., Speisky, H., Aliaga, M.E., Lo´pez-Alarco´n, C., 2011. Analytical parameters of the microplate-based ORAC-pyrogallol red assay. Journal of AOAC International 94, 1562–1566. Ortiz, R., Antile´n, M., Speisky, H., Aliaga, M.E., Lo´pez-Alarco´n, C., Baugh, S., 2012. Application of a microplate-based ORAC-pyrogallol red assay for the estimation of antioxidant capacity: first action. Journal of AOAC International 95, 1558– 1561. Ou, B.X., Hampsch-Woodill, M., Prior, R.L., 2001. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry 49, 4619–4626. Pino, E., Campos, A.M., Lissi, E., 2003. 8-Hydroxy-1,3,6-pyrene trisulfonic acid (pyranine) bleaching by 2,20 -azobis(2-amidinopropane) derived peroxyl radicals. International Journal of Chemical Kinetics 35, 525–531. The United States Pharmacopoeia, Inc., Rockville, MD, 2000. Torres, P., Galleguillos, P., Lissi, E., Lopez-Alarcon, C., 2008. Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe. Bioorganic & Medicinal Chemistry 16, 9171–9175.