Influence of the Target Molecule

Chapter 24

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Influence of the Target Molecule on the ORAC Index Camilo López-Alarcón,*,1 Alexis Aspée,2 and Eduardo Lissi2 1Departamento

de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile. Av. Vicuña Mackenna 4860, Santiago, Chile 2Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile *E-mail: [email protected]

The ORAC (Oxygen Radical Absorbance Capacity) assay has been widely employed to evaluate the antioxidant ability of pure compounds, foods and beverages. The method usually employs fluorescein and AAPH (2,2’-Azo-bis(2-amidinopropane) dihydrochloride) as target molecule (TM) and peroxyl radical source, respectively. According to a simple competition between TM and additives (antioxidants) for peroxyl radicals, the ORAC values (relative to a reference antioxidant), should be independent on the TM employed. In the present work we present ORAC values obtained employing different TM, such as fluorescein, alizarin red, pyrogallol red and pyranine. The results clearly show that the ORAC index of pure compounds and complex samples strongly depends on the TM used. Contradictory results on the antioxidant capacity of herbal and tea infusions can be obtained if fluorescein and pyrogallol red ORAC assays are compared. The dependence of the ORAC index on the TM emphasize the role of secondary radical reactions on the ORAC derived indexes.

1. Introduction It has been demonstrated that the consumption of rich polyphenolic diets leads to a decreasing risk of cardiovascular diseases, hypertension, certain forms of cancer, type II diabetes, and other degenerative or age-related diseases © 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


(1–6). These associations have been partially explained in terms of the ability of polyphenols to act as antioxidants through their reaction with Reactive Oxygen or Nitrogen Species (ROS, or RNS, respectively) (1–6). Due to the complexity that supposes in vivo or ex-vivo experiments, different methodologies have been developed to estimate the in vitro antioxidant capacity of complex mixtures such as foods, beverages and human fluids (7–12). The methods, that involve different experimental conditions, evaluate: i) the consumption of stable free radicals induced by antioxidants (13, 14); ii) the capacity of antioxidants to reduce cupric or ferric ions (15, 16); iii) the ability of antioxidants to protect a target molecule exposed to a free radical source (17, 18); iv) the capacity of antioxidants to inhibit lipoperoxidation processes (19), v) the antioxidant capacity of polyphenols in cell cultures (20), and the effect of antioxidants on the free radical steady state concentration (21).

2. ORAC (Oxygen Radical Absorbance Capacity) Assay Among the above mentioned in vitro methodologies, ORAC is one of the most employed. This method estimates the ability of a particular sample to inhibit the bleaching of a target molecule (TM) induced by peroxyl radicals. Usually, Trolox or gallic acid are employed as reference antioxidants and 2,2’-Azo-bis(2amidinopropane) dihydrochloride (AAPH) is employed as peroxyl radical source (22). To quantify the protection given by an additive it is estimated the area under the curve (AUC) of the kinetic profiles associated to the bleaching of the TM in the absence and presence of the tested sample (Figure 1). These data are compared to that of a reference antioxidant and the ORAC values estimated according to Equation 1 [eq 1], and Equation 2 [eq 2], for pure compounds and complex mixtures respectively:

where: AUCad = Area under curve in the presence of additives (pure antioxidants). AUC = Area under curve in the presence of the tested complex mixture. AUCº = Area under curve of control (TM plus AAPH solution). AUCRa = Area under curve in the presence of a reference antioxidant (usually Trolox or gallic acid). f = Dilution factor, equal to the ratio between the total volume of the working solution (TM plus AAPH plus the sample aliquot) and the added sample volume. [Ra] and [Additive] = Molar concentration of the reference antioxidant and additive, respectively. 418 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Kinetic profile of the TM consumption mediated by peroxyl radicals in the abscence (●) and presence of an antioxidant (○).

The higher AUC of the time-integrated bleaching of TM obtained in the presence of antioxidants (XH) can be explained by the minimal set of reactions presented in Scheme 1. This scheme represents a simple competition between TM and XH by peroxyl radicals. 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. According to this over simplified scheme, the AUC associated with reactions [1] to [4] will only depend on the difference of rate constant of process [2] and [3] and stoichiometric factors, following the reactions of TM and XH.

Scheme 1

419 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The ORAC method was firstly reported by Cao and co-workers employing R-phycoerythryn (PE) as TM (ORAC-PE) (17). ORAC-PE was used to estimate the antioxidant capacity of vitamin C, uric acid and human serum (17, 23–26). However, it has been demonstrated that PE interacts with polyphenols and is a photounstable protein (27). Considering the latter, Ou and co-workers (27), reported an improved ORAC assay employing fluorescein (FL) as TM (ORAC-FL). FL does not interact with polyphenols, is photostable and it is considerable less expensive than PE. FL has been widely employed being, at present, the TM of choice. In addition to the use of PE and FL, we have explore the use of other TMs, such as pyrogallol red (PGR) (28, 29), pyranine (PY) (30, 31), alizarin red (AR) (32), and c-phycocyanin (33, 34) in ORAC-like methodologies. In this chapter it is discussed how the selection of the TM could affect the ORAC index. Results regarding the ORAC index of complex mixtures, employing PGR and FL as TM, are also presented.

3. Influence of the Target Molecule (TM) on the ORAC Index of Pure Compounds Figure 2 shows the Trolox inhibition on FL consumption-induced by peroxyl radicals. The presence of Trolox clearly inhibes the bleaching of FL. This protective effect is characterized by kinetic profiles with the presence of clear lag times. This behavior has been commonly obtained for different antioxidants (even for antioxidants of low reactivity) and would imply that FL is easily protected by additives almost irrespectively of their capacity to remove peroxyl radicals (29, 35). Similar results have been obtained when pyranine (PY) is used as TM (ORACPY). In Figure 3 it is shown the decay of the fluorescence of PY mediated by AAPH derived peroxyl radicals in the absence and presence of Trolox or gallic acid. As in Figure 2, the protection of PY is characterized by the presence of neat lag times in the kinetic profiles. Alizarin red (AR) can also be used as TM (ORAC-AR) and its consumption can be easily followed by UV-visible spectroscopy. As in the case of FL and PY, the addition of antioxidants to a solution containing AR plus AAPH usually leads to kinetic profiles with lag times (Figure 4) (32). Among the possible aspects that should be considered to explain lag times as those shown in Figures 2-4, are; i) a lower reactivity of the TM toward peroxyl radicals than the antioxidants, and ii) the presence of repair mechanisms (30, 35). Nonetheless, independently of the origin of the lag time, its presence in the kinetic profiles implies that the antioxidant(s) is (are) totally consumed before the TM consumption. Thus, the lag times would be directly related to the stoichiometry of the reaction between antioxidant(s) and peroxyl radicals. Thereby, the AUC of kinetic profiles with lag times, and then, the related ORAC values, would be mainly associated to the stoichiometry of the antioxidant-peroxyl radicals reaction. Therefore, in many cases ORAC-FL, ORAC-PY and ORAC-AR values would be more influenced by the stoichiometry of the reaction (defined as the number 420 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


of radicals that each additive molecule can remove) than by the reactivity of the additives toward peroxyl radicals.

Figure 2. Time-course of the FL (70 nM) consumption induced by AAPH derived peroxyl radicals in the absence and presence of Trolox. Trolox concentrations: 1 µM (□); 5 µM (○); 7.5 µM (4); 10 µM (5). Control experiment (●). [AAPH] = 10 mM. Data taken from ref. (29).

Figure 3. Time course of PY (5 µM) consumption induced by AAPH derived peroxyl radicals in the absence (●) and presence of Trolox (○) or gallic acid (◊). [Antioxidants] = 5 µM, [AAPH] = 10 mM. Phosphate buffer 10 mM, pH 7, 37ºC. 421 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 4. Consumption of AR (30 µM) elicited by AAPH (30 mM) derived peroxyl radicals in the presence of caffeic (4 µM, 5) and sinapic acid (12 µM, ◊). Control experiment (in the absence of cinnamic acids, ○). The reaction was followed by visible spectroscopy at 520 nm. Data taken from ref. (32).

We have proposed that pyrogallol red (PGR) can be considered as TM in an ORAC-like methodology (ORAC-PGR) more sensitive to reactivity and less influenced by stoichiometric factors than FL or PY based methods (28, 29). The time profiles associated to PGR consumption present clear differences with those depicted in Figures 2-4. PGR quickly reacts with AAPH-derived peroxyl radicals, and the reaction can easily be followed by the decrease of the absorbance of the sample measured at 540 nm. As it is shown in Figure 5, in parallel with the decrease at 540 nm (and at 280 nm), a new band at 380 nm is formed, a process that is characterized by the presence of isosbestic points at 325 and 435 nm. In contrast to FL, PY and AR results, the protection of PGR afforded by Trolox is characterized by the absence of lag times (Figure 6). This behavior has been observed for different antioxidants, ascorbic acid being the only exception since its addition produces lag times in the kinetic profiles of the PGR consumption (36, 37). The extent of the lag time can be considered as a measure of ascorbic acid concentration in tested complex samples. The absence of lag times in the kinetic profiles of the consumption of PGR in the presence of antioxidants would imply that the AUC are mostly affected by the reactivity of the antioxidants toward peroxyl radicals. In spite of ORAC-PGR index would be more related to antioxidant reactivity than stoichiometric factors it can not be completely discarded the influence of secondary reactions. In fact, we have reported that the protection of PGR afforded by cinnamic acid derivatives is considerably smaller than those predicted from the reactivity of these antioxidants toward peroxyl radicals (29, 38). This lack of protection could be interpreted in 422 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


terms of secondary reactions, in which cinnamic acid derived radicals react with PGR (39).

Figure 5. Bleaching of PGR (30 µM) induced by AAPH-derived peroxyl radicals. Curves represent the time course of the reaction between 0 and 50 minutes (28).

Figure 6. Time-course of the PGR consumption mediated by peroxyl radicals in the prescence of different Trolox concentrations. PGR (5 µM) was incubated with AAPH (10 mM) in the presence of Trolox at: 10 µM (4); 30 µM (○); 50 µM (□); 75 µM (◊); 100 µM (5). Control experiment (in the abscence of Trolox, ●). Data taken from ref. (29). 423 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Table I shows ORAC values of pure compounds obtained employing FL, PY, PGR and AR as TM. As it is shown in this Table, the ORAC values are very dependent on the TM employed. For example, if the case of gallic acid is analyzed, ORAC-FL and ORAC-PY values of 1.2 and 1.4 were obtained, respectively. These values imply that the antioxidant capacity of gallic acid, estimated employing FL and PY as TM is close to that of Trolox. However, in contrast to ORAC-FL and ORAC-PY, the ORAC-PGR value of gallic acid is near to 11, implying that its antioxidant is much more efficient than Trolox.

Table I. ORAC values of pure compounds estimated using FL, PY, PGR and AR as TM. Data taken from ref. a (29), b (27), c (40), d (31), e (32) ORAC-PYd

ORAC-PGRa

ORAC-ARe

Compound

ORAC-FL

Quercetin

10.7 ± 0.4a 7.28 ± 0.22b

6.4 ± 0.5

11.5 ± 0.4

---

Kaempferol

10.2 ± 0.3a

---

8.8 ± 0.7

---

Gallic acid

1.2 ± 0.03a

1.4 ± 0.1

11.1 ± 0.7

---

Caffeic acid

4.37 ± 0.24a 6.63 ± 0.24c

3.2 ± 0.3

≈ 0.2

2.1 ± 0.1

Ferulic acid

3.5 ± 0.1a 4.47± 0.1c

1.0 ± 0.1

≈ 0.1

0.2 ± 0.03

Sinapic acid

3.0 ± 0.1a

2.7 ± 0.3

≈ 0.4

1.9 ± 0.1

1

1

1

1

Trolox

The data depicted in Table I, clearly show that different TMs give different ORAC values, even for pure compounds. Furthermore, these results appear relevant if it is consider that all ORAC values are relative to the same reference antioxidant (Trolox). The latter can not be interpreted in terms of the simplified reactions depicted in Scheme 1 and emphasize the role of secondary reactions of TM or phenolic (additives) derived radicals (35, 39). The bleaching of a particular target molecule induced by peroxyl radicals involves, as a first step, an electron or hydrogen transfer in which a TM derived (TM•) radical is produced. If this radical is able to react with an antioxidant (XH) a repair mechanism would operative, according to:



Thus, as is shown in reaction [5], the reduction of TM• (mediated by XH) reduces the rate of TM bleaching. If this process is efficient, it could explain the presence of lag times in the kinetic profiles when FL and PY are used as target molecule (30, 35). On the other hand, if XH reacts with peroxyl radicals, a XH secondary free radical (X•) is formed (reaction [3]). If X• reacts with TM, a secondary damage of the target molecule would take place, according to:

This reaction would imply bleaching of TM induced by X• , as has been postulated in the protection of PGR elicited by cinnamic acids (39).

4. ORAC Values of Complex Mixtures Employing FL or PGR as Target Molecules (TM) The ORAC-FL and ORAC-PGR methodologies have been applied to estimate the antioxidant capacity of complex mixtures such as herbal and tea infusions, and fruit berry extracts.

ORAC-FL and ORAC-PGR of Herbal and Tea Infusions Herbal and tea infusions inhibited the consumption of FL through kinetic profiles characterized by the presence of lag times. However, the protection of PGR afforded by the same samples leads to changes in PGR initial consumption rate (without lag times). Figure 7 shows the dependence of ORAC values (ORACFL in plot A, and ORAC-PGR in plot B) with the total phenol content, evaluated by Folin assay, of herbal and tea infusions. As it is shown in Figure 7, according to ORAC-FL method (Figure 7A), some herbal infusions presented higher antioxidant capacity than that of tea infusions with similar Folin values. However, all tea infusions show higher ORAC-PGR values than those of all herbal infusions (Figure 7B). Therefore, as in the case of pure compounds, ORAC values for different infusions depend upon the employed methodology. Thus, depending on the chosen TM opposite conclusions could be deduced. Considering the characteristic of ORAC-FL and ORAC-PGR assays, we have proposed that the ratio between both indexes would indicate the quality of the antioxidants present in a particular sample. In Table II are presented the values of the ORAC-PGR/ORAC-FL ratio obtained for herbal and tea infusions.



Figure 7. ORAC-index versus total phenolic content plots. Correlation between ORAC-FL (A) and ORAC-PGR (B) with total phenolic content of herbal (○) and tea (□) infusions. Data taken from ref. (41).

Table II. ORAC-FL/ORAC-PGR ratios of herbal and tea infusions. Data taken from refs. (41, 42) Infusion

ORAC-PGR / ORAC-FL

Infusion

ORAC-PGR / ORAC-FL

Chenopodium ambrosioides

0.008a

Mentha piperita

0.025

Buddleia globosa

0.011a

Plantago major

0.045

Erythroxylum coca

0.012

Tilia spp

0.055

Black tea (1)

0.10a

Aloysia citriodora

0.021

(0.011a)

Matricaria chamomilla

0.021a

Black tea (2)

0.14a

Peumus boldus

0.019a

Green tea

0.15a

Rosa moschata

0.10

White tea

0.17a

Haplopappus baylahuen

0.020a

As can be seen in Table II, the ORAC-PGR/ORAC-FL ratio depends on the tested sample. For example, the ratio of Peumus boldus is close to 8 times smaller than that of green tea. This would indicate that the antioxidants present in green tea are more efficient that the antioxidants present in the Peumus boldus infusion. In this context, a similar approach, employing PY and PGR as TM, has been proposed by Niki and co-workers to estimate the antioxidant capacity of beverages and foods (43–45). 426 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


ORAC-FL and ORAC-PGR of Berry Extracts Blue and black berry extracts protect PGR from its consumption mediated by AAPH derived peroxyl radicals. This protection is characterized by the absence of lag times. On the other hand, kinetic profiles related to the protection of PGR afforded by raspberry extract (Figure 8) are characterized by the presence of clear lag times. Considering previous studies that indicated that ascorbic acid generates lag times in the protection of PGR (28), experiments in the presence of ascorbate oxidase were carried out. The preincubation of raspberry extract with this enzyme completely eliminated the lag time, without modifying the slope observed after the lag time (36). The raspberry extract protected FL throughout kinetic profiles with neat lag time, but the addition of ascorbate oxidase did not modify these kinetic profiles. The latter implies a minimal contribution of ascorbic acid on the AUC when FL is employed as TM (36).

Figure 8. Effect of ascorbate oxidase on the kinetic profiles of the PGR consumption in the presence of raspberry extract. Previously to PGR (5 µM) and AAPH (10 mM) addition, the raspberry solution (33 µL of extract / mL in phosphate buffer, 75mM, pH 7.4) was incubated with ascorbate oxidase (0.09 U/mL) during 40 minutes. Solution of PGR in the presence of both, raspberry extract (33 µL / mL) and AAPH (10 mM) = (4). Pre-incubated solution with ascorbate oxidase (□). Control experiment (○). Data taken from ref. (36).

These results show that in rich ascorbic acid mixtures, the use of PGR as TM allows an estimation, throughout a simple methodology, of the ascorbic acid concentration (from the lag time) and the ORAC-PGR value of the sample. This allows to estimate the contribution of ascorbic acid to the antioxidant capacity of a particular complex mixture. In raspberry extracts an ORAC-PGR value of 1453 427 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

micromolar Trolox equivalents was estimated. However, without the contribution of ascorbic acid, an ORAC-PGR value of 489 was determined. The presence of ascorbate oxidase enzyme did not modify the AUC of the kinetic profiles of FL consumption. This imply that the ORAC-FL value of raspberry extract does not reflect the antioxidant capacity given by ascorbic acid. In fact, the ORAC-FL value of raspberry extract was 2870 micromolar Trolox equivalents in the absence and presence of ascorbate oxidase (36).


5. Conclusions ORAC values of pure compounds and complex samples strongly depend on the target molecule. This dependence emphasize the role of secondary radical reactions on the ORAC derived indexes. These reactions could partially explain contradictory ORAC values obtained for berry fruits and herbal and tea infusions employing fluorescein or pyrogallol red as target molecules..

Acknowledgments This work was supported by FONDECYT (no. 1100659).

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Influence of the Target Molecule

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