Antioxidant Capacity of pure compounds and complex mixtures evaluated by the ORAC-pyrogallol red assay in the presence of Triton X-100 micelles

Article abstract of DOI:10.3390/molecules15096152

The protective effect of different antioxidants and complex mixtures on the consumption of pyrogallol red (PGR) induced by peroxyl radicals was studied in the absence and presence of Triton X-100 micelles. The presence of micelles decreased significantly the protection of PGR afforded by lipophilic antioxidants (β-carotene, octyl gallate), while no effect of micelles was observed for hydrophilic antioxidants such as Trolox, caffeic acid, gallic acid, and ascorbic acid. In the presence of complex mixtures a clear effect of Triton X-100 micelles was also observed in the protection afforded by wines, tea infusions, and seed extracts of Eugenia jambolana and Myrciaria cauliflora. On the other hand, no effect of micelles was observed for orange juice and pulp fruit extracts. The ORAC (Oxygen Radical Absorbance Capacity) index was evaluated in the absence (ORAC-PGR) and presence of Triton X-100 micelles (ORAC-PGR_MIC). Triton X-100 micelles affect ORAC-PGR values of antioxidants in a lipophilicity-dependent way. From the obtained results, we conclude that ORAC-PGR and ORAC-PGR_MIC assays could be considered as an alternative to estimate the antioxidant ability (ORAC-PGR) and to infer the association to Triton X-100 micelles (ORAC-PGR/ORAC-PGR_MIC) of pure antioxidants and their complex mixtures.

Full text of DOI:10.3390/molecules15096152

Molecules 2010, 15, 6152-6167; doi:10.3390/molecules15096152
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antioxidant Capacity of Pure Compounds and Complex
Mixtures Evaluated by the ORAC-Pyrogallol Red Assay in the
Presence of Triton X-100 Micelles
Max Romero 1, Benjamin Rojano 2, Jaime Mella-Raipán 1, Carlos David Pessoa-Mahana 1,
Eduardo Lissi 3 and Camilo López-Alarcón 1,*
1
Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile; C.P.
782 0436, Santiago, Chile; E-Mails: msromero@uc.cl (M.R.); predestinar@gmail.com (J.M.);
cpessoa@uc.cl (C.D.P.M.)
2
Escuela de Química, Universidad Nacional de Colombia, Sede Medellin; AA 1027 Medellin,
Colombia; E-Mail: brojano@gmail.com
3
Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile;
E-Mail: eduardo.lissi@usach.cl
* Author to whom correspondence should be addressed; E-Mail: clopezr@uc.cl
Received: 22 July 2010; in revised form: 18 August 2010 / Accepted: 27 August 2010 /
Published: 1 September 2010
Abstract: The protective effect of different antioxidants and complex mixtures on the
consumption of pyrogallol red (PGR) induced by peroxyl radicals was studied in the
absence and presence of Triton X-100 micelles. The presence of micelles decreased
significantly the protection of PGR afforded by lipophilic antioxidants (β-carotene, octyl
gallate), while no effect of micelles was observed for hydrophilic antioxidants such as
Trolox, caffeic acid, gallic acid, and ascorbic acid. In the presence of complex mixtures a
clear effect of Triton X-100 micelles was also observed in the protection afforded by
wines, tea infusions, and seed extracts of Eugenia jambolana and Myrciaria cauliflora. On
the other hand, no effect of micelles was observed for orange juice and pulp fruit extracts.
The ORAC (Oxygen Radical Absorbance Capacity) index was evaluated in the absence
(ORAC-PGR) and presence of Triton X-100 micelles (ORAC-PGRMIC). Triton X-100
micelles affect ORAC-PGR values of antioxidants in a lipophilicity-dependent way. From
the obtained results, we conclude that ORAC-PGR and ORAC-PGRMIC assays could be
considered as an alternative to estimate the antioxidant ability (ORAC-PGR) and to infer

Molecules 2010, 15
the association to Triton X-100 micelles (ORAC-PGR/ORAC-PGRMIC) of pure
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antioxidants and their complex mixtures.
Keywords: ORAC; pyrogallol red; micelles; Triton X-100; antioxidants
1. Introduction
It is recognized that reactive oxygen specie (ROS) are implicated in all stages of lipid peroxidation
(initiation, propagation, and termination) [1]. Depending of the nature of the lipid, these oxidative
reactions could modify oil composition, cell membranes, or the integrity of lipoproteins [1–3]. There is
abundant evidence that polyphenols present in foods and beverages can inhibit lipid peroxidation
processes acting as chain breaking antioxidants [1,4], being the efficiency of a particular antioxidant
mainly determined by:
- its reactivity towards ROS,
- the stoichiometry of the reaction (number of ROS molecules that an antioxidant molecule can
remove),
- its ability to reach lipidic phases, and
- its capacity to generate, after its reaction with ROS, products with low reactivity towards
lipids.
There is increasing interest concerning the measurement of the antioxidant capacity of polyphenols
such as flavonoids, and related compounds. Several methodologies have been developed to allow these
evaluations, with the ORAC (Oxygen Radical Absorbance Capacity) assay being one of the most
frequently employed. This method is based on the use of a target molecule that is exposed to a peroxyl
radical source in the absence and presence of the tested sample. Usually fluorescein (ORAC-FL), and
2,2’-azo-bis(2-amidinopropane) dihydrochloride (AAPH) are employed as probe and peroxyl radical
source, respectively [5,6]. To evaluate the antioxidant capacity of lipophilic antioxidants, the
ORAC-FL assay has been validated employing randomly methylated β-cyclodextrin (RMC) as
solubilizating agent (L-ORAC-FL) [7–9]. The consumption of fluorescein, either in ORAC-FL or
L-ORAC-FL assays, usually is easily inhibited by single antioxidants and/or complex mixtures
including very reactive compounds, generating kinetics profiles characterized by the presence of neat
induction times. This would imply that ORAC values evaluated employing fluorescein as probe would
be more related to stoichiometric factors than to the reactivity of the antioxidants. In this context, we
have previously proposed that ORAC values obtained employing pyrogallol red (PGR) as probe
(ORAC-PGR) would be more related to the reactivity of the additives toward peroxyl radicals than to
the stoichiometry of the reaction [10,11]. In fact, in contrast with fluorescein, the kinetic profiles of the
PGR consumption do not present induction times, even in the presence of very reactive polyphenols. In
particular, ascorbic acid was the only antioxidant, among the tested compounds, that generated a clear
induction time [11,12]. The ORAC-PGR method has been used, as a complementary methodology of
ORAC-FL, to estimate the antioxidant capacity of complex mixtures including beverages [13–15],
wines [10,11], fermented foods [16] fruit extracts [12,17], and human fluids [18,19].

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Taking into account the relevance of the hydrophilicity/lipophilicity balance of antioxidants in the
inhibition of lipid peroxidation processes, and the advantages of the use of PGR as probe in
ORAC-like assays, in the present work we evaluated the ORAC-PGR index of hydrophilic and
lipophilic antioxidants in Triton X-100 micelles. For this purpose, antioxidants with different degree of
hydro/lipophilicity were used. In addition, the antioxidant capacity of complex mixtures such as tea
infusions, berry extracts, wines, and orange juice was evaluated.
2. Kinetic Scheme
ORAC-PGR methodology has been used to evaluate the antioxidant capacity of antioxidants,
beverages, fruit extracts, and human fluids in aqueous homogeneus medium [10-14]. The minimal set
of reactions to consider in the ORAC-PGR assay is given in Scheme 1, where XH stands for the added
antioxidant(s).
Scheme 1:
AAPH + O2 → 2ROO• + N2
ROO• + PGR →bleaching
ROO• + XH → X • + ROOH
(1)
(2)
(3)
(4)
•
2ROO → non radicals
products
For simplicity self-reactions and cross-reactions of the radicals produced in steps (2) and (3), and
the formation of alcoxyl radicals in reaction (4), are not included. The addition of Triton X-100
micelles to a solution containing AAPH, PGR and an antioxidant (or a complex mixture) could modify
the rate of the reactions depicted in Scheme 1. Since all processes could take place in the external
phase and/or in the micellar pseudophase, the relevance of these processes is going to be determined
by the distribution of the compounds between the aqueous phase (phosphate buffer) and the less polar
micellar pseudophase. Then, in presence of Triton X-100 micelles, besides the reactions of Scheme 1
the equilibria presented in Scheme 2 should also be considered.
Scheme 2:
PGR + micelles←→PGRMIC
XH + micelles←→ XHMIC
(5)
(6)
Considering the hydrophilic character and the positive charge (in phosphate buffer) of AAPH [6], it
can be assumed that all peroxyl radicals are generated (and remain) in the phosphate buffer pseudo-
phase. Therefore, the reaction of PGR and the antioxidants with peroxyl radicals would take place in
the aqueous phase and/or the interface micelle-phosphate buffer.

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3. Results and Discussion
3.1. Consumption of PGR Induced by Peroxyl Radicals in Presence of Triton X-100 Micelles
Figure 1. Influence of Triton X-100 micelles on the UV-visible spectrum of PGR (A) and
its consumption mediated by peroxyl radicals (B). A: UV-visible spectrum of PGR (5 µM)
in phosphate buffer (75 mM, pH 7.4, solid line), and in the presence of Triton X-100
(50 mM) (dashed line); B: Time-course of the consumption of PGR induced by AAPH
derived peroxyl radicals in the absence and presence of Triton X-100 micelles. PGR
(5 µM) was incubated in phosphate buffer (75 mM, pH 7.4) at 37 °C with AAPH (10 mM)
in the absence (ꢀ), and presence of Triton X-100 (50 mM, ꢀ).
0.2
A
0.1
0.0
300
40 0
500
6 00
700
λ
/n m
1 .0
0 .5
0 .0
B
0
5 0 0
1 0 0 0
1 5 0 0
T im e /s
Figure 1A shows UV-visible spectra of PGR in the absence and presence of Triton X-100 micelles
(50 mM). As can be seen, in the absence of Triton X-100 micelles, PGR presented the typical
UV-visible spectrum (solid line in Figure 1), characterized by a band in the visible region with a
maximum at 540 nm (ε = 36,000 M-1cm-1). In the presence of Triton X-100 micelles, the spectrum of
PGR was slightly altered (dashed line in Figure 1), displacing the maximum of the visible band to
544 nm (ε = 33600 M-1cm-1). This behavior would imply that PGR was, at least partially, associated to
the micellar pseudophase. In fact, by an ultrafiltration separation process it was observed that, in our
experimental conditions (Triton X-100 50 mM, and PGR 5 µM), near 72% of PGR would be
associated to Triton X-100 micelles. Nevertheless, the association of PGR to Triton X-100 micelles did

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not modify the kinetic profile of PGR consumption induced by peroxyl radicals. As is shown in
Figure 1B, the consumption of PGR mediated by AAPH derived peroxyl radicals (evaluated by its
absorbance decay at 540 nm) was not affected by the presence of Triton X-100 micelles. This is an
unexpected result if it is considered that AAPH and its derived free radicals are mostly in the aqueous
phase, while a significant fraction of PGR is associated to the micelles. However, the PGR/peroxyl
radical process (Reaction 2, Scheme 1) is in the zero order kinetic limit [10,11] and hence its
consumption rate is independent of the PGR concentration present in the aqueous phase. In fact,
similar initial rate values (≈ 0.4 µM/min) of PGR decay were estimated in the absence and presence of
Triton X-100 micelles. Considering that peroxyl radicals are generated (APPH = 10 mM, at 37 °C) at a
rate of 0.8 µM/min [6], and that each PGR molecule reacts with two peroxyl radicals [11], the
estimated rates of PGR consumption in the absence and presence of Triton X-100 micelles supports the
proposal that PGR is in the zero order kinetic limit. Furthermore, PGR adsorbed at the micelles
interface could retain its reactivity toward the peroxyl radicals.
3.2. Effect of Single Antioxidants on the PGR Consumption in Presence of Triton X-100 Micelles
To evaluate the effect of Triton X-100 micelles on the protection of PGR afforded by pure
compounds, different antioxidants with different hydro/lipophilicity were employed. Figure 2 shows
the consumption of PGR (5 µM) induced by AAPH derived peroxyl radicals in the presence of Trolox,
and ascorbic acid with or without Triton X-100 micelles. As is shown in Figure 2A, Trolox
(50 and 100 µM) inhibited the consumption of PGR in a concentration-dependent way, showing a
similar behaviour to that observed in previous studies in aqueous media [10,11]. Interestingly, the
addition of Triton X-100 micelles did not modify the protection of PGR afforded by Trolox, a result
that would be related to the well known hydrophilic property of this antioxidant. In the same way, the
addition of Triton X-100 micelles did not alter the protective effect afforded by ascorbic acid,
characterized by a neat induction time (Figure 2B). These similarities can be attributed to the fact that,
even in presence of Triton X-100 micelles, all relevant processes take place almost exclusively in the
aqueous phase. In contrast with this, when a lipophilic antioxidant as β-carotene was added to a
solution containing Triton X-100 micelles, PGR and AAPH, no PGR protection by the antioxidant
took place (Figure 3). In fact, as it is shown in Figure 3, the absorption band corresponding to
β-carotene (at 460 nm) was not affected by peroxyl radicals during the consumption of PGR.
Nonetheless, when PGR was almost totally consumed (c.a. 37 minutes), the visible band (at 460 nm)
associated to β-carotene, decayed significantly. These results imply that β-carotene (present in the
micelles) was initially protected by PGR (present in the aqueous phase and/or in the micelle-aqueous
interface), and only after the total consumption of PGR, β-carotene was consumed by peroxyl radicals
(probably in the interface micelle-phosphate buffer). Similarly, β-carotene was efficiently protected by
Trolox and ascorbic acid (data not shown), implying that hydrophilic antioxidants react with peroxyl
radicals in the aqueous phase, minimizing its interaction with micelle associated targets (as
β-carotene). Control experiments with β-carotene micellar solutions revealed no changes in the original
absorbance intensity at 460 nm (data not shown) in the time period considered in the presence work.

Molecules 2010, 15
Figure 2. Effect of antioxidants on the consumption of PGR induced by AAPH derived
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peroxyl radicals in the absence (closed symbols) and presence of Triton X-100 micelles
(open symbols). PGR (5 µM) was incubated with AAPH (10 mM) in the absence (circles)
and presence of. A: Trolox: 50 (squares), and 100 µM (triangles); B: Ascorbic acid: 5
(squares), and 10 µM (triangles).
1.0
0.5
0.0
1.0
0.5
0.0
B
A
0
500
1000
1500
2000
0
1000
2000
Time/s
3000
Time/s
Figure 3. Effect of β-carotene on the consumption of PGR induced by AAPH derived
peroxyl radicals. PGR (5 µM) was incubated with AAPH (10 mM) and Triton X-100
(50 mM) in the absence and presence of β-carotene (5 µM). The reaction was followed by
the consumption of PGR (ꢁ, 550 nm) and β-carotene (ꢀ, 460 nm). Control experiment
without β-carotene (ꢀ).
1.0
0.5
0.0
0
900
1800
2700
3600
Time/s

Molecules 2010, 15
Figure 4. Effect of gallate derivatives (A gallic acid; B propyl gallate; C octyl gallate) on
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the consumption of PGR induced by AAPH derived peroxyl radicals in the absence and
presence of Triton X-100 micelles. PGR (5 µM) was incubated with AAPH (10 mM) and
the gallate derivatives (at 5 µM) in micellar (ꢀ) and phosphate solutions (ꢁ). Control
experiments without gallate derivatives were carried out in the presence (ꢀ) and absence
(ꢀ) of Triton X-100 micelles.
B
1.0
A
1.0
0.5
0.5
0.0
0.0
0
500
1000
1500
2000
0
500
1000
1500
2000
Time/s
Time/s
1.0
0.5
0.0
C
0
500
1000
1500
2000
Time/s
To evaluate the influence of Triton X-100 micelles on the protection of PGR afforded by others
antioxidants, compounds with a similar reactive centre, but with different degree of lipophilicity, were
used. For this purpose, gallic acid and gallate derivatives with different alkyl chain lengths, such as
methyl gallate, propyl gallate, and octyl gallate, were employed as model antioxidants. As can be seen
in Figure 4A, the consumption of PGR in the presence of gallic acid at 5 µM concentration was very

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similar in the absence and presence of Triton X-100 micelles. This result is compatible with those
obtained employing Trolox and ascorbic acid (Figure 2). However, in the case of propyl, and octyl
gallate (Figure 4B, and 4C, respectively) a clear effect of Triton X-100 micelles was observed. In the
presence of Triton X-100 micelles the protection afforded by propyl gallate was lower than that the
observed in buffer. This would imply that the incorporation of propyl gallate into the micellar
pseudophase leads to a lower availability of propyl gallate to react with peroxyl radicals. An extreme
case was observed for octyl gallate. The presence of Triton X-100 micelles significantly decreased the
observed protection of PGR afforded by this antioxidant in homogeneous medium, indicating that
octyl gallate was mostly incorporated in Triton X-100 micelles (Figure 4C). This corroborates the
results obtained with β-carotene (Figure 3) indicating that the free radical scavengers incorporated into
the micelles do not protect PGR from its consumption mediated by peroxyl radicals. Likewise, we
assessed the effect of caffeic acid and ethyl caffeate on the consumption of PGR induced by AAPH
derived peroxyl radicals. As shown in Figure 5A, the protective effect of caffeic acid on the
consumption of PGR was independent of the presence of Triton X-100 micelles. However, the
protection of PGR afforded by ethyl caffeate decreased significantly in the micellar solution
(Figure 5B). A similar behaviour was observed for quercetin (Figure 6). Quercetin, in phosphate buffer
protected efficiently PGR, while in presence of Triton X-100 micelles, its protective effect
decreased considerably.
Figure 5. Effect of caffeic acid (A) and ethyl caffeate (B) on the consumption of PGR
induced by AAPH derived peroxyl radicals in the absence and presence of Triton X-100
micelles. PGR (5 µM) was incubated with AAPH (10 mM) and the caffeic acid derivatives
(at 250 µM) in micellar (ꢀ) and phosphate solutions (ꢁ). Control experiments without
caffeic acid derivatives were carried out in the presence (ꢀ) and absence (ꢀ) of Triton X-
100 micelles.
1.0
0.5
0.0
1.0
0.5
0.0
A
B
0
500
1000
1500
2000
2500
3000
0
400
800
Time/s
1200
1600
Time/s

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Figure 6. Effect of quercetin (7 µM) on the consumption of PGR (5 µM) induced by
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AAPH derived peroxyl radicals in the absence (ꢁ) and presence of Triton X-100 micelles
(ꢁ). Control experiments without quercetin were carried out in the presence (ꢀ) and
absence (ꢀ) of Triton X-100 micelles.
1.0
0.5
0.0
0
500
1000
1500
Time/s
2000
2500
The above results imply that the protection of PGR is strongly influenced by the distribution of the
antioxidants in Triton X-100 micelles. In particular, protection by water soluble substrates is similar to
that measured in the absence of Triton X-100 micelles. On the other hand, highly hydrophobic
substrates do not protect PGR in the presence of Triton X-100 micelles. Antioxidants that partition
between both pseudophases (such as quercetin) present an intermediate behaviour. Thus, in the
presence of complex mixtures containing antioxidants, the observed effect of Triton X-100 micelles on
the kinetic profiles of PGR consumption could be directly related to the percentage of lipophilic
antioxidants included in the sample. To study the latter, we estimated the inhibition of PGR
consumption induced by AAPH derived peroxyl radicals by complex mixtures such as wines, juices,
berry extracts, and tea infusions in the absence and presence of Triton X-100 micelles. Figure 7 shows
the protection of PGR afforded by orange juice (Figure 7A) and a seed extract of Eugenia jambolana
(Figure 7B). As can be seen in this figure, orange juice protected PGR throughout a clear induction
time. This induction time, originated by the presence of ascorbic acid, was not altered by the presence
of Triton X-100 micelles. This result is in accord to those obtained regarding the antioxidant capacity
of pure ascorbic acid and implies that the protection of orange juice is almost exclusively related to this
antioxidant. Seed extracts of Eugenia jambolana inhibited efficiently the consumption of PGR induced
by peroxyl radicals (Figure 7B). However, in contrast to orange juice, the effect of the seed extract was
dependent of the presence of Triton X-100 micelles. Thus, in the presence of micelles, a lower effect
was registered, implying that lipophilic antioxidants contribute to the antioxidant capacity of
this extract.

Molecules 2010, 15
Figure 7. Effect of complex mixtures on the consumption of PGR (5 µM) induced by
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AAPH (10 mM) derived peroxyl radicals. A: orange juice (6.7 µL/mL) in the absence (ꢁ)
and presence of Triton X-100 micelles (ꢀ). B: seed extract of Eugenia jambolana (10
µL/mL) in the absence (ꢁ) and presence of Triton X-100 micelles (ꢀ). Control
experiments (without additives) were measured in the absence (ꢀ) and presence (ꢀ) of
Triton X-100 micelles.
1.0
0.5
0.0
1.0
0.5
0.0
B
A
0
1000
2000
3000
0
1000
2000
3000
Time/s
Time/s
3.3. ORAC Values of Single Antioxidants and Complex Mixtures in the Absence and Presence of Triton
X-100 Micelles
In order to quantify the effect of Triton X-100 micelles, ORAC-PGR values of pure antioxidants
and complex mixtures were estimated in the absence (ORAC-PGR) and presence of Triton X-100
micelles (ORAC-PGRMIC). Gallate derivatives showed similar ORAC-PGR values, being 11.7, and 9.8
the ORAC-PGR value of gallic acid, and octyl gallate, respectively (Table 1). The similarity of
ORAC-PGR values of gallic acid and gallate derivatives of different hydrophobicity can be related to
their common reactive centre. ORAC-PGRMIC values of the gallate derivatives are also given in
Table 1. It is observed an inverse dependence with the chain length. Thus, ORAC-PGRMIC index of the
gallate derivatives follows the order: Gallic acid > methyl gallate > propyl gallate > octyl gallate.
Table 1. ORAC-PGR and ORAC-PGRMIC values of pure antioxidants.
Compound
Gallic acid
ORAC-PGR
11.7 0.5
12.0 0.4
12.3 0.2
9.8 0.3
0.4 0.05
0.4 0.06
11.9 0.4
10.3 0.3
----
ORAC-PGRMIC
10.9 0.4
7.0 0.6
6.3 0.5
1.6 0.2
0.4 0.07
0.2 0.01
4.1 0.2
10.7 0.5
≈ 0.1
ORAC-PGR/ORAC-PGRMIC
1.1 0.1
1.7 0.2
1.9 0.2
6.1 0.9
1.0 0.3
2.0 0.4
2.9 0.2
1.0 0.08
---
Methyl gallate
Propyl gallate
Octyl gallate
Caffeic acid
Ethyl caffeate
Quercetin
Ascorbic acid
β-carotene
Trolox
1
1
1

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This result can be related to the lipophilicity of gallate derivatives and hence is related to their
distribution upon Triton X-100 micelles. Thus, ORAC-PGRMIC values are inversely related to the
distribution of the antioxidants in Triton X-100 micelles. In fact, the reported values for the partition
constant of gallate derivatives between Triton X-100 micelles and aqueous solutions range from 97 to
440 M-1 for methyl and octyl gallate, respectively [20]. A similar effect of Triton X-100 micelles was
observed for the couple caffeic acid / ethyl caffeate (Table 1). While the ORAC-PGR value of caffeic
acid was not altered by Triton X-100 micelles (0.4 in both media), ORAC-PGRMIC of ethyl caffeate
was only one half of its ORAC-PGR value (ORAC-PGRMIC = 0.2). In addition, ORAC-PGR and
ORAC-PGRMIC values of quercetin and ascorbic acid showed that while the antioxidant capacity of
quercetin decreased significantly in presence of Triton X-100 micelles, no effect of micelles was
observed for the ascorbic acid antioxidant ability. These results imply that the comparison of
ORAC-PGR and ORAC-PGRMIC values reflects the distribution of the antioxidant in Triton X-100
micelles. Thus, the ratio ORAC-PGR/ORAC-PGRMIC can be considered as an index related to the
lipophilicity of the tested antioxidant(s). As shown in Table 1, ORAC-PGR/ORAC-PGRMIC values of
gallate derivatives between 1.0 and 6.1 for gallic acid and octyl gallate, respectively, were obtained. In
addition, ORAC-PGR/ORAC-PGRMIC values higher than one were obtained for antioxidants such as
ethyl caffeate, and quercetin, compounds that can be considered to partition between the micelles and
the dispersium solvent.
The obtained results (collected in Table 1) employing single antioxidants suggest that ORAC-PGR
and ORAC-PGRMIC assays could be used to estimate the antioxidant capacity and the distribution of
antioxidants in Triton X-100 micelles of complex mixtures such as wines, tea infusion, fruit extracts,
and juices. As can be seen in Table 2, ORAC-PGR values of these mixtures ranged from 2 to 36 mM
Trolox equivalents, values that are related to the antioxidant activity of the samples. These
ORAC-PGR values were influenced in different grades by the presence of Triton X-100 micelles. In
fact, ORAC-PGRMIC values of wines, tea infusions, and seed fruit extracts, were lower than
ORAC-PGR values, implying that a fraction of the antioxidants contained in the samples were
associated to Triton X-100 micelles. A different result was obtained for orange juice and fruit pulp
extracts, where no effect of micelles was observed, indicating minimal incorporation of their
antioxidants into the micellar pseudophase. This result is compatible with a predominance of
hydrophilic antioxidants such as ascorbic acid in orange juice and fruit extracts.
Table 2. ORAC-PGR and PRAC-PGRMIC values of complex mixtures a.
Complex mixture
ORAC-PGR ORAC-PGRMIC
ORAC-PGR / ORAC-PGRMIC
36
2
24
3
1.5 0.3
Red wine
White wine
4
1
3
0.5
1
1.3 0.5
1.5 0.3
1.3 0.2
Tea extract
16
10
2
1
11
Seed extract of Eugenia
jambolana
8
0.6
Pulp extract of Eugenia
jambolana
2
0.3
1
2
0.4
2
1.0 0.3
1.3 0.3
Seed extract of
Myrciaria cauliflora
15
12

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Table 2. Cont
.
Pulp extract of
5
1
6
1
0.8 0.3
0.9 0.2
Myrciaria cauliflora
Orange juice
19
3
21
2
a
Values represent the concentration (mM) of a Trolox solution that produces the same
effect than the tested beverage.
4. Materials and Methods
4.1. Chemicals
2,2’-Azo-bis(2-amidinopropane) dihydrochloride, (AAPH), was used as peroxyl radical source.
Pyrogallol red (PGR), Trolox (6-hydroxy-2,5,8-tetramethylchroman-2-carboxylic acid), all tested
polyphenols (excluding ethyl caffeate), β-carotene, ascorbic acid, Triton X-100, and AAPH were
purchased from Sigma-Aldrich (St. Louis, MO) and employed as received (Figure 8).
Figure 8. Chemical structure of pyrogallol red (PGR), Trolox, AAPH, caffeic and gallic
acid derivatives.
COOR
SO3H
HO
OH
Compounds
R
O
O
OH
(caffeic acid derivatives)
OH
OH
Pyrogallol Red (PGR)
Caffeic acid
H
Ethyl caffeate
-CH2-CH3
CH3
H3C
H3C
NH2
H2N
HO
HO
COOR
1
N
N
2HCl
HN
NH
CH3
AAPH
OH
CH3
Compounds
R1
H
HO
(gallic acid derivatives)
Gallic acid
CH3
H3C
O
COOH
Ethyl gallate
-CH2-CH3
CH3
Propyl gallate
Octyl gallate
-(CH2)2-CH3
-(CH2)2-CH3
Trolox

Molecules 2010, 15
.2. Synthesis of Ethyl Caffeate
Caffeic acid (90 mg, 0.5 mmol) was refluxed in ethanol (80 mL) in presence of concentrated HCl
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4
(0.5 mL), during 8 hours. The reaction mixture was diluted with 500 mL of a saturated aqueous
solution of NaHCO3 and extracted with three volumes of ethyl acetate (100 mL). The organic layer
was dried with sodium sulfate and evaporated in vacuum to yield the ethyl caffeate (Figure 8) as a
1
pale brown solid (76 mg ,80% yield). IR (KBr): 3436.5, 1659.0, 1282.0 cm-1; H-NMR (400 MHz,
DMSO-d6) δ ppm: 9.53 (s, 1H), 9.15 (s, 1H), 7.46 (d, J = 15.91 Hz, 1H), 7.03 (s, 1H), 6.99 (d,
J = 8.14 Hz, 1H), 6.75 (d, J = 8.14 Hz, 1H), 6.25 (d, J = 15.91 Hz, 1H), 4.14 (q, J = 7.01Hz, 2H), 1.23
(t, J = 7.01Hz, 3H).
4.3. Sample Preparation
Polyphenols: Stock solutions of the polyphenols were prepared in ethanol immediately before
their use.
Tea infusion: Black tea bags were Chilean commercial products. Infusions were prepared by adding
150 mL of distilled water (95–100 °C) to the bags (each containing 2 g of dried tea leaves material).
The infusions were brewed for 5 min. Upon withdrawing the bags, the resulting solutions were cooled
to 20 °C and immediately used to assess their antioxidant properties.
Wines: Red wine, diluted with phosphate buffer (75 mM) at pH 7.4 (1:10), or undiluted white wine
were added directly to the working solution (final volume 3 mL).
Berry extracts: Fruits of Eugenia jambolana and Myrciaria cauliflora were harvested at the
commercial ripe stage. The samples were frozen and stored at −20 °C until their analysis. The extracts
were obtained as follows: 20 g of each sample (seed or pulp) were homogenized in a methanol/HCl 1%
(6/1, v/v) mixture. The extracts were filtered (employing Whatman N°4 filter), dried in a rotatory
evaporator at 40 °C, and dissolved in methanol. The obtained concentrations of the extracts were:
Myrciaria cauliflora: fruit pulp (38 g dried extract/L), and seed (35 g of dried extract/L). Eugenia
jambolana: fruit pulp (25 g dried extract/L), and seed (34 g dried extract/L).
4.4. Working Solutions
Phosphate buffer (75 mM, pH 7.4) with or without Triton X-100 (50 mM) was employed as
reaction media. A reaction mixture containing AAPH (10 mM), PGR (5 µM), and the tested sample,
was incubated in the reaction media at 37 °C. PGR consumption was evaluated from the progressive
absorbance decrease measured at 540 nm in the thermostatized cuvette of a Hewlett Packard 8453
(Palo Alto, CA, USA) UV-visible spectrophotometer. Control experiments were carried out employing
a PGR plus AAPH solution in the absence of the tested samples.
4.5. ORAC-PGR Determinations
The consumption of PGR, associated to its incubation in the presence of AAPH was estimated from
absorbance (A) measurements. Values of (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) reached a value of 0.2. These

Molecules 2010, 15
6165
areas were employed to obtain ORAC values, according to Equation [1], and Equation [2], for
complex mixtures and pure compounds, respectively:
(AUC − AUC0 )
(AUCTrolox − AUC0 )
ORAC =
ORAC =
f
[
Trolox
]
(AUCad − AUC0 )
(AUCTrolox − AUC0 )
[Trolox
]
]
[
additive
where:
AUC = Area under curve in the presence of the tested complex mixture.
AUCad = Area under curve in the presence of additives (pure antioxidants).
AUCº = Area under curve of control (PGR plus AAPH solution).
AUCTrolox = Area under curve in the presence of Trolox.
f = Dilution factor, equal to the ratio between the total volume of the working solution (target
molecules plus AAPH plus the sample aliquot) and the added sample volume.
[Trolox] and [Additive] = Molar concentration of Trolox and additive, respectively.
4.6. Association of PGR to Triton X-100 Micelles Assessed by Ultrafiltration
A solution (25 mL) of PGR (5 µM) in phosphate buffer (75 mM, pH 7.4) with Triton X-100
micelles (50 mM) was ultracentrifugated through a Microcom membrane (cut off = 30,000 Da). Free
PGR in the ultrafiltrate was determined by UV-visible spectroscopy. All measurements were carried
out at 20 1 °C. Control experiments carried out in the absence of Triton X-100 micelles did not show
any capture of PGR by the membrane. All experiments were carried out in triplicate.
5. Conclusions
Triton X-100 micelles affect the values of the ORAC-PGR index of antioxidants in a
lipophilicity-dependent way. In particular, ORAC-PGR values obtained in the presence of micelles
(ORAC-PGRMIC) decreased when the lipophilicity of the compounds increased. The ratio
ORAC-PGR/ORAC-PGRMIC can then be considered as a measure of the tested compound
lipophilicity. This approach is sustained by results obtained employing a series of gallate derivatives
and caffeic acid and ethyl caffeate. It is proposed that this approach, applied to complex mixtures, can
afford a semi-quantitative estimation of the average hydrophobicity of the compounds contributing to
the antioxidant activity of the tested sample.
Acknowledgments
This work was supported by FONDECYT (grants n°11060323 and 1100659).
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Sample Availability: Samples of the compounds are available from the authors.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).