Food Chemistry 129 (2011) 139148

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Food Chemistry
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Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (aTEAC), DPPH assay and peroxyl radical scavenging assay
Lars Mller, Kati Frhlich, Volker Bhm
Institute of Nutrition, Friedrich Schiller University Jena, Dornburger Strae 25-29, 07743 Jena, Germany

a r t i c l e

i n f o

a b s t r a c t
The purpose of this study was to assess the antioxidant activity of carotenes and xanthophylls measured by various methods, compared to a-tocopherol, BHA and BHT. Four assays were selected to achieve a wide range of technical principles. Besides aTEAC, which uses ABTS+ radical cation, ferric reducing activity (measured by using FRAP assay), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay were used. In addition, a luminol-chemiluminescence based peroxyl radical scavenging capacity (LPSC) assay, was used. Most of the compounds showed signicant differences in their activity of scavenging radicals depending on the assay used. Of the 22 compounds tested, only a few such as lutein, zeaxanthin and capsanthin gave comparable results in the various assays. Surprisingly, in contrast to a-tocopherol, BHA and BHT, carotenoids did not show any DPPH scavenging activity. To standardise the relative contribution of the assays used, weighted means of the values obtained in aTEAC, FRAP, DPPH and LPSC assay were calculated. This strategy was used to assess the antioxidant capacity of several juices and oil samples. The highest lipophilic antioxidant capacity in all assays was observed for sea buckthorn berry juice, followed by tomato juice, carrot juice and orange juice. Within the oil samples, the order of antioxidant capacity depended on the assay used. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 1 September 2010 Received in revised form 17 February 2011 Accepted 18 April 2011 Available online 24 April 2011 Keywords: Lipophilic antioxidant activity Lycopene Carotenes Xanthophylls

1. Introduction Carotenoids, a class of natural fat-soluble compounds mainly de novo synthesised by plants (Rodriguez-Amaya, Kimura, Godoy, & Amaya-Farfan, 2008), are one class of major food micronutrients in human diet (Maiani et al., 2009). In plants, they have potential antioxidant properties due to their chemical structure (Stahl & Sies, 2003). In the human organism, carotenoids are part of the antioxidant defense system, too. The quantitative most important carotenoids in human diet are b-carotene, lycopene, lutein, b-cryptoxanthin, zeaxanthin, and astaxanthin (Riccioni, 2009). Numerous observational studies have supported the hypothesis that antioxidants like carotenoids and vitamin E or metabolites of these nutrients are associated with cardiovascular diseases (CVD) (Lichtenstein, 2009). Carotenoids could be used as an inexpensive mean of prevention, and as a possibly treatment, even though human intervention trials showed controversial results, with some positive ndings, many null ndings, and some suggestion of harm in certain high-risk populations (Riccioni, 2009). Recent smaller interventional studies with carefully chosen populations, such as
Corresponding author. Tel.: +49 (0) 3641 949633; fax: +49 (0) 3641 949702.
E-mail address: volker.boehm@uni-jena.de (V. Bhm). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.04.045

those under high levels of oxidative stress, have yielded largely positive results (Lichtenstein, 2009). Carotenoids play a role in protecting plants against photooxidative processes. They are efcient antioxidants, e.g. in scavenging singlet molecular oxygen (Di Mascio, Kaiser, & Sies, 1989) and peroxyl radicals (Stahl & Sies, 2003). Certain convenient methods were developed for a quick, simple and reliable quantication of the antioxidant capacity. In general, the methods to determine the total antioxidant capacity were divided into two major groups: assays based on the single electron transfer (SET) reaction, displayed through a change in colour as the oxidant is reduced, and assays based on a hydrogen atom transfer (HAT) (Huang, Ou, & Prior, 2005), which measure the activity of the antioxidant to scavenge peroxyl radicals, such as the total radical trapping antioxidant parameter (TRAP) assay, the oxygen radical absorbance capacity (ORAC) assay and the luminol-chemiluminescence based peroxyl radical scavenging capacity (LPSC) assay (Alho & Leinonen, 1999; Huang et al., 2005; Ou, Hampsch-Woodill, & Prior, 2001). The ferric reducing antioxidant power (FRAP), the a-tocopherol/ Trolox equivalent antioxidant capacity (aTEAC/TEAC) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays include electron transfer reaction (Benzie & Strain, 1996; Brand-Williams, Cuvelier, & Berset, 1995; Huang et al., 2005; Re et al., 1999).

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L. Mller et al. / Food Chemistry 129 (2011) 139148 Table 1 Absorptivity values at specic wavelength maxima and used solvents, and solvents used for stock solutions of analysed carotenoids and a-tocopherol (Franke, Murphy, Lacey, & Custer, 2007; Rodriguez-Amaya, 2001). Carotenoid Antheraxanthin Astaxanthin a-Carotene b-Carotene Canthaxanthin Capsanthin b-Cryptoxanthin Echinenone Lutein Lycopene Neoxanthin Neurosporene Phytoene Phytouene Rubixanthin Violaxanthin Zeaxanthin Bixin Crocetine
DL-

Solvent Ethanol n-Hexane n-Hexane n-Hexane Petroleum Benzene Petroleum Petroleum Ethanol Petroleum Ethanol n-Hexane Petroleum n-Hexane Petroleum Ethanol Ethanol Petroleum Petroleum Ethanol

Wavelength (nm) 444 478 445 450 466 487 449 458 445 470 442 440 286 347 460 440 450 456 422 292

Absorptivity value (E1%,1 cm) 2350 2100 2710 2590 2200 2070 2400 2160 2550 3450 2380 2920 915 1580 2750 2550 2480 4200 4320 75.8

Solvent used for stock solution Methanol Acetone T + CH (1 + 4, T + CH (1 + 4, T + CH (1 + 4, Methanol T + CH (1 + 4, Ethanol Ethanol T + CH (1 + 4, Methanol Methanol T + CH (1 + 4, T + CH (1 + 4, T + CH (1 + 4, Methanol Ethanol Ethanol Ethanol Ethanol

ether ether ether ether

v/v) v/v) v/v) v/v)

v/v)

ether ether

v/v) v/v) v/v)

ether ether

a-Tocopherol

T + CH: toluene + cyclohexane.

The purpose of this study was to compare the antioxidant activity values obtained by different methods (FRAP, aTEAC, DPPH, LPSC) of a variety of carotenes and xanthophylls, known as antioxidants, and found in human diet. The second aim was to standardise the reporting on antioxidant activity for each single compound by calculating a meanful standardised value based on the results observed in all the antioxidant capacity assays used. In addition, the proposed approach was tested on several juices (tomato, carrot, sea buckthorn berry, and orange) and oil samples (sunower, olive, walnut, and sh). 2. Material and methods 2.1. Materials 2.1.1. Chemicals 2,20 -Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), K2S2O8, 2,4,6-tripyridyltriazine (TPTZ), and 2,6-di-tert-butyl4-hydroxytoluene (BHT) were obtained from SigmaAldrich (Taufkirchen, Germany). 2,20 -Azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from Acros Organics (Schwerte, Germany). Manganese dioxide was obtained from Merck KGaA (Darmstadt, Germany). Luminol and 2-tert-butyl-4-hydroxyanisol (BHA) were purchased from Fluka (Buchs, Switzerland). DL-atocopherol (a-T), b-tocopherol (b-T), c-tocopherol (c-T) and d-tocopherol (d-T) were purchased from Calbiochem (Darmstadt, Germany) with purities of 97100% shown by GC. a-Tocotrienol (a-T3), b-tocotrienol (b-T3), c-tocotrienol (c-T3) and d-tocotrienol (d-T3) were obtained from Davos Life Sciences (Singapore). Rubixanthin was purchased from Apin Chemicals Ltd. (Abingdon, UK). Bixin and crocetine were obtained from Extrasynthse (Genay, France). All other carotenoids used were obtained from CaroteNature (Lupsingen, Switzerland) with a purity of 9498% by HPLC. All solvents used were of HPLC grade. HPLC grade water (18 MX) was prepared using a Millipore Milli-Q purication system (Millipore GmbH, Schwalbach, Germany). Buffer salts and all other chemicals were of analytical grade. 2.1.2. Food samples Tomato and carrot juice, as well as sunower, olive, and walnut oil were bought in a local supermarket. Sea buckthorn berry juice

was obtained in a local health food store. The sh oil was from Croda (Nettetal, Germany).

2.2. Sample preparation Stock solutions of each carotenoid (ca. 100 lg/ml) and a-T (ca. 1 mg/ml) were prepared in the specic solvent of each compound (see Table 1) and stored at 30 2 C until analysis. Exact concentrations of the stock solutions were determined spectrophotometrically using the absorptivity at the specic wavelength of each compound (see Table 1), except of the two synthetic antioxidants BHA and BHT, in which initial weight was used. Lipophilic extracts of the various juices were prepared using nhexane (Rsch, Bergmann, Knorr, & Kroh, 2003). To assess the antioxidant capacity of the oils used, 1 g of the samples was diluted with n-hexane to a nal volume of 25 ml and centrifuged (5 min, 16,900g).

2.3. Evaluation of the antioxidant capacity All experiments were done under subdued light. Before analysis, a dened volume of each carotenoid stock solution was evaporated to dry under a stream of nitrogen at 30 1 C and the residue was resolved in n-hexane (for FRAP and aTEAC), ethanol/n-hexane 1 + 1 (v/v, for DPPH assay), or MTBE/DMSO 1 + 19 (v/v, for LPSC assay), respectively. All carotenoid samples were analysed in triplicate at four different concentrations (1, 5, 10, 20 lM). The extract solutions of juices and oil samples were analysed in triplicate on different days, too. One assay used to assess the antioxidant capacity was the ferric reducing antioxidant power (FRAP) assay, in order to determine the ferric reducing activity of carotenoids and food samples. The procedure was based on the work recently published by our research group (Mller, Theile, & Bhm, 2010). In a reaction tube, 100 ll of carotenoid or extract solution, standard (ca. 4.5 114 lmol a-T/l), or blank (n-hexane) and 600 ll of FRAP reagent, consisting of ferric chloride and TPTZ in acetate buffer (pH 3.6), were shaken on a thermoshaker (25 1 C, 1400 rpm). After 6 min of shaking, the mixtures were transferred into half microcuvettes (1.5 ml, PS), and centrifuged for 30 s at 1000g to separate the layers. Finally, the absorbances of the lower layer of samples,

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antioxidant activity [mol -TE/mol]

standards and blank at 595 nm were measured exactly 8 min after starting shaking. In addition, an a-tocopherol equivalent antioxidant capacity (aTEAC) assay, on the basis of scavenging the synthetic radical ABTS+ (Bhm, Puspitasari-Nienaber, Ferruzzi, & Schwartz, 2002), was used. The procedures for measuring aTEAC values were carried out in accordance to Mller et al. (2010). An ABTS+ stock solution was prepared by passing a ABTS solution through a lter paper coated with manganese dioxide, followed by membrane ltration (0.2 lm). Before analysis, this stock solution was diluted with phosphate buffered saline (PBS) to obtain an absorbance of 0.70 0.05 at 734 nm. In the assay, 100 ll carotenoid solution or sample extract, standard (ca. 4.5114 lmol a-T/L), or blank (nhexane) and 600 ll ABTS+ working solution were vortexed for 30 s in a reaction tube. Thereafter, the mixture was transferred completely into half micro-cuvettes, centrifuged 30 s at 1000g to separate the layers. The absorbance of the lower layer was determined at 734 nm exactly 2 min after starting shaking. The antioxidant capacity was also determined by scavenging the DPPH radical as described by Liu, Shi, Colina Ibarra, Kakuda, and Xue (2008). The procedures followed the description of Mller et al. (2010). In the assay, 1.0 ml of carotenoid or sample solution, standard (ca. 4.5114 lmol a-T/L), or blank (ethanol/n-hexane 1 + 1, v/v) and 0.5 ml of 0.3 mM ethanolic DPPH solution were shaken in a thermoshaker (25 1 C, 1000 rpm). The absorbance of samples, standards, and blanks at 540 nm was determined after 15 min. The luminol-chemiluminescence based peroxyl radical scavenging assay (LPSC) was carried out on a BMG Labtech FluoStar Optima (Offenburg, Germany) plate reader in white 96-well micro-plates. The temperature of the incubator was set at 37.0 0.2 C. The procedure was based on the CL method of Mller et al. (2010). Briey, AAPH was used as a peroxyl radical generator, a-T as the standard, and luminol as the chemiluminescent dye. Fifty micro-litres of carotenoid or extract solution, a-T calibration solution (12240 lmol a-T/l) or blank (MTBE/DMSO 1 + 19, v/v) were mixed with 50 ll of luminol (10 mM) and 100ll DMSO/borax buffer (1+9,v/v) and incubated for 10 min at 37.0 0.2 C prior to addition of 150 ll AAPH solution (60 mM). The chemiluminescence was measured every 2 min for 1.5 h. The nal LPSC values were calculated using the area under the curves technique. For these four methods, a-tocopherol (a-T) was used as a standard and the antioxidant capacity was calculated as mol a-T equivalents (a-TE) per mol carotenoid, and in lmol a-TE/100 g for the juices and oil samples, respectively. 2.4. Quantication of carotenoids and vitamin E in juices and oils For vitamin E quantication using the HPLC procedure, the oils were dissolved in a mobile phase (n-hexane/MTBE 98 + 2, v/m) to initial weights of approx. 5 mg sh oil per ml and 20 mg per ml (for sunower, olive, walnut), respectively, with a-tocopherol acetate as internal standard. After a centrifugation (5 min, 3000g), the HPLC procedures using normal phase Eurospher DIOL-column (Knauer, Berlin, Germany) followed the descriptions of Franke, Frhlich, Werner, Bhm, and Schne (2010). For the juice samples a combined extraction with ethanol, MTBE and petroleum ether was used prior to chromatographic separation (Seybold, Frhlich, Bitsch, Otto, & Bhm, 2004). To determine the carotenoid contents, 500 mg of the oil samples were dissolved in MTBE to a nal volume of one millilitre, with b-apo-80 -carotenal as internal standard. After centrifugation (5 min, 3000g), the solutions were analysed using a RP-HPLCDAD method with C30 column (Trentec, Rutesheim, Germany) and a gradient of methanol and MTBE for elution as described by Franke et al. (2010). The carotenoids of the juices were extracted

using ultra-turrax (typ T-25, IKA, Staufen, Germany) method with methanol/THF 1 + 1 (v/v, +0.1% BHT) as extractant (Seybold et al., 2004). Due to the high content of xanthophyll esters the extracts of sea buckthorn berry juice and orange juice were saponied for 30 min with KOH (100 g/l in methanol) at room temperature using echinenone as internal standard. All the extract solutions were evaporated to dryness under a stream of nitrogen at 30 1 C. The resulting residues were redissolved in methanol/THF (1 + 1, v/v, +0.1% BHT) and centrifuged (5 min, 16,900g) prior to HPLC analysis as described above (Seybold et al., 2004).

antioxidant activity [mol -TE/mol]

1
A A A B C D D D B B B E E B D F G B H I J A

antioxidant activity [mol -TE/mol]

1
A B B B C B B C C C D D E C F G H I I A B D

35 30 25 20 15 10 5 0
A B B B C D D D D D E D E E G F D E H D I I

Fig. 1. Antioxidant activities (mol a-TE/mol) of standard compounds (10 lM) determined by using aTEAC, FRAP and LPSC assay. Different subscript letters indicate a signicant difference, determined by ANOVA (p < 0.05) with post hoc S NK. ACA, a-carotene; ANX, antheraxanthin; AT, a-tocopherol; ASX, astaxanthin; BCA, b-carotene; BCX, b-cryptoxanthin; BHA, 2-tert-butyl-4-hydroxyanisol; BHT, 2,6-di-tert-butyl-4-hydroxytoluene; BIX, bixin; CAN, canthaxanthin; CAP, capsanthin; CRC, crocetine; ECH, echinenone; LUT, lutein; LYC, lycopene; NEX, neoxanthin; NSP, neurosporene; PHT, phytoene; PHF, phytouene; RUX, rubixanthin; VIX, violaxanthin; ZEA, zeaxanthin.

AT PHT PHF NSP LYC BCA ACA BCX LUT ZEA ECH CAN ASX CAP RUX VIX NEX ANX BIX CRC BHT BHA

AT PHT PHF NSP LYC BCA ACA BCX LUT ZEA ECH CAN ASX CAP RUX NEX VIX ANX BIX CRC BHT BHA

AT PHT PHF NSP LYC BCA ACA BCX LUT ZEA ECH CAN ASX CAP RUX NEX VIX ANX BIX CRC BHT BHA

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2.5. Statistics Each sample analysis was performed in triplicate. All results presented are means (SD) of at least three independent experiments. Statistical analysis (ANOVA with a statistical signicance level set at p < 0.05 with post-hoc StudentNewmanKeuls (SNK) procedure) was carried out with SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Correlations between the contents of vitamin E and the carotenoids and antioxidant capacities were made using the Pearson procedure (p < 0.01). 3. Results 3.1. Antioxidant activity of standard compounds The antioxidant activities of carotenes, xanthophylls, BHA and BHT were evaluated using FRAP, aTEAC, DPPH and LPSC assay. The results varied according to the assay used (Fig. 1). 3.1.1. FRAP assay Ten of the compounds (ve carotenes, three xanthophylls, BHA, BHT) reduced the ferric di-TPTZ complex used in the FRAP assay less than a-tocopherol (Fig. 1). Even the ve carotenes as well as BHT did not show any noteworthy ferric reducing activity. Of the remaining compounds, rubixanthin showed signicantly higher antioxidant activity (3.3 mol a-TE/mol) than all other tested compounds. Most of the xanthophylls as well as lycopene and the apocarotenoids bixin and crocetine displayed FRAP values similar to a-tocopherol or up to maximum twice higher. 3.1.2. aTEAC assay Using the aTEAC assay, phytoene, phytouene and neoxanthin showed the same activity as a-tocopherol, whereas all other tested carotenes and most of the xanthophylls showed higher activities (Fig. 1). Within the carotene group, lycopene presented the highest activity (four times higher than that of a-tocopherol). No

difference was observed between a- and b-carotene. b-Cryptoxanthin and rubixanthin showed the highest ABTS.+ bleaching activity amongst the oxygenated carotenoids. Within the apocarotenoids, the aTEAC value of crocetine differed signicantly from a-tocopherol (2.4 mol a-TE/mol), whereas bixin as well as the tested synthetic antioxidant BHA showed rather similar antioxidant activity, or at least one-third (BHT) of the antioxidant activity of a-tocopherol. 3.1.3. DPPH assay Only the synthetic antioxidants BHA (0.3 mol a-TE/mol) and BHT (0.1 mol a-TE/mol) as well as the reference substance a-tocopherol scavenged the DPPH radical (Table 2). Surprisingly, neither the carotenes nor the xanthophylls showed any DPPH scavenging activity. Additionally, the apocarotenoids bixin and crocetin did not react in this assay. We observed that the absorbance values of the reaction mixtures, which consisted of DPPH and carotenoid dissolved in ethanol/n-hexane (1 + 1, v/v), increased with increasing the concentration of the carotenoid compound. After subtracting the self-absorbance of the carotenoid at the specic concentration used in the assay at 540 nm, from the absorbance of the mixture of DPPH and carotenoid, the resulting absorbance value was in compliance with the absorbance of the DPPH blank value. This observation indicates that there was no reaction between the DPPH radical and the carotenoid within the 15 min of shaking. Prolongation of the reaction time up to 60 min and changing the solvents to THF/PBS(1+1,v/v) or methanol did not lead to a decrease in the DPPH absorbance caused by the presence of carotenoids. 3.1.4. Luminol-chemiluminescence peroxyl radical scavenging capacity (LPSC) assay For the carotenoids and the fat-soluble synthetic antioxidants, the antioxidant activities measured by the LPSC assay (Fig. 1) were considerably higher compared to a-tocopherol (1319 times for the carotenes, 926 times for the xanthophylls, 2029 times for

Table 2 Antioxidant activities standardised with respect to a-tocopherol measurement [mol a-TE/mol]. Compound Carotenes ACA BCA LYC NSP PHF PHT ANX ASX BCX CAN CAP ECH LUT NEX RUX VIX ZEA AT BHA BHT BIX CRC FRAP 0.0 0.0 2.1 0.0 0.0 0.1 1.8 0.5 2.1 0.3 2.1 0.3 2.1 1.3 3.3 1.5 2.0 1.0 0.2 0.0 1.7 0.9 1.1

aTEAC
3.3 3.1 3.9 2.1 1.0 1.0 2.0 0.8 3.2 0.8 2.0 2.2 2.0 1.1 3.3 1.6 1.9 1.0 0.9 0.3 1.3 2.4 1.9

DPPH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.0 0.3 0.1 0 0 0.1

LPSC 19.0 19.1 13.3 0.0 0.1 0.1 24.7 26.3 19.3 21.0 25.3 26.3 19.6 20.6 8.9 22.5 20.3 1.0 5.6 5.5 29.1 19.5 15.8

Weighted average incl. DPPH 0.7 0.7 1.2 0.3 0.1 0.2 1.1 0.6 1.2 0.5 1.2 0.8 1.1 0.8 1.4 0.9 1.1 4.3 1.4 0.5 1.0 0.9

Weighted average excl. DPPH 1.4 1.4 1.9 0.7 0.3 0.3 1.5 0.9 1.8 0.7 1.5 1.3 1.4 1.0 1.8 1.2 1.4 0.5 0.4 0.2 1.3 1.3

Xanthophylls

Others

Average

The right-hand columns show the weighted averages obtained by the following calculation: (1) dividing the antioxidant activity of each compound, as determined using the specied method, by the average activity determined for the whole set of compounds in the same method (last row), (2) summing the four results (FRAP, aTEAC, DPPH, and LPSC) of this calculation, and (3) dividing the sum by four. Abbreviations: see legend of Fig. 1.

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143

A
antioxidant capacity [mol -TE/100 g]

75 50 25 0
DPPH FRAP TEAC LPSC

carrot juice sea buckthorn berry juice orange juice

antioxidant capacity [mol -TE/100 g]

800 600 400 200

tomato juice

1050 1000 400 300 200 100 0

DPPH FRAP TEAC LPSC

sunflower oil olive oil walnut oil fish oil

Fig. 2. Evaluation of the antioxidant capacities of various juices (A) and oil samples (B).

18 16 14 12 10 8 6 4 2 0
1 tomato juice

l z b-cr b-c a-c l

sea 2 3 4 carrot buckthorn orange juice berry juice juice

Fig. 3. Contents of carotenoids (lmol/100 g) of various juices.

the apocarotenoids, 56 times for BHA and BHT). The only exceptions were phytoene, phytouene and neurosporene, which did not show any antioxidant activity using this method. The antioxidant activities of most of the tested compounds ranged between 20 and 25 mol a-TE/mol, far higher than most of the aTEAC and FRAP values. It is noteworthy that lycopene (a carotene) and rubixanthin (a xanthophyll), behaving as strongest antioxidants in the aTEAC and FRAP assay, showed the lowest antioxidant activity of all tested carotenoid compounds in the LPSC assay, expect phytoene, phytouene, and neurosporene. 3.1.5. Antioxidant capacity and contents of vitamin E and carotenoids of various food samples Juices of tomatoes, carrots, sea buckthorn berries, and oranges, as well as oil samples of sunower, olive, walnut and sh were tested for their lipophilic antioxidant capacities by the four different methods. Results for each sample varied extensively (Fig. 2). The results detected for the different samples depended on the assay used, but in general the antioxidant capacities of the analysed juices were lower than those of the oil samples. Only in the LPSC assay the juices presented much higher values than the oils. However, we observed that olive oil was the only sample which did not follow these tendencies. In the group of oil samples, the FRAP assay obtained the lowest antioxidant capacity values (e.g. sh oil: 6.5 lmol a-TE/100 g),

whereas the DPPH assay was the most active assay with values up to 1027 lmol a-TE/100 g (sh oil). In contrast, the DPPH assay showed the lowest activity in the analysis of the extracts of the four juice samples (e.g. 1.3 lmol a-TE/100 g in orange juice). The most potent assay in the analysis of the juice extracts was the peroxyl radical scavenging based LPSC assay, which showed a-TE values up to 738 lmol a-TE/100 g (in sea buckthorn berry juice). The aTEAC assay and the FRAP assay generally yielded quite similar results in each of the juice samples. In contrast, the results of the aTEAC assay of the oil samples were 210 times higher than in the FRAP assay. Within the group of juice extracts, tomato and carrot juice displayed similar results in all four assays; in each assay the lipophilic extract of orange juice was the less active one and the lipophilic extract of sea buckthorn berry juice was always the most active juice extract, 20 times more active than orange juice. As expected, no carotenoids were detected in sunower, walnut and sh oil. Only in olive oil small amounts of lutein (0.83 0.03 lmol/100 g) and zeaxanthin (0.076 0.002 lmol/ 100 g) were found. In contrast, the juices contained high amounts of carotenoids. The highest content was quantied in sea buckthorn berry juice, with 15.8 0.8 lmol total carotenoids/100 g, at which lutein, zeaxanthin, b-carotene and lycopene dominated with contributions of 33%, 27%, 23% and 13%, respectively (Fig. 3). The vegetable juices, tomato and carrot juice, contained similar total amounts of carotenoids (11.2 0.1 lmol/100 g and 10.1 0.2 lmol/100 g, respectively); however, with large differences in the qualitative composition. As expected, lycopene was predominant in the tomato juice, with a contribution of more than 98%, while b-carotene, and a-carotene were the major carotenoids in carrot juice, with contributions of 70% and 29%, respectively. By far, the lowest carotenoid content was observed in the orange juice (0.1 lmol/100 g) containing similar low amounts of lutein, zeaxanthin, and b-cryptoxanthin. Summarised, the order of increasing carotenoid content in the juices was: orange ( carrot 6 tomato < sea buckthorn berry. As known from the literature, natural oils in general, and sh oil in particular, contain high amounts of vitamin E. In our studies, the measured vitamin E content ranged from 66 lmol/100 g in olive oil up to 491 lmol/100 g in sh oil (Fig. 4.B). Summarising, the order

carotenoid [mol/100 g]

vitamin E [mol/100 g]

A 18
16 4 3 2 1 0
tomato carrot sea buck- orange juice juice thorn berry juice juice

vitamin E [mol/100 g]

-T -T -T3 -T3 -T3

B 490
200 150 100 50 0
sunflower olive oil oil walnut oil fish oil

-T d T -T b T -T a T -T g T

Fig. 4. Contents of vitamin E (lmol/100 g) of various juices (A) and oil samples (B).

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of increasing amount of vitamin E in the oil samples was: olive < walnut < sunower ( sh. The contribution of the different vitamin E compounds differed largely between the oil samples. In the sunower oil as well as in the olive oil sample a-T was the dominant vitamer, whereas c-T was predominant in walnut oil and sh oil with a contribution of 6070%. The a-T content was low in both samples. Additionally, signicant amounts of d-T were only detected in the sh oil. As expected, the amounts of vitamin E compounds were much lower in the juice samples compared to the oil samples. The concentrations ranged from 1.3 lmol/100 g in orange juice up to 17.1 lmol/100 g in sea buckthorn berry juice. Besides its high carotenoid content (Fig. 3), sea buckthorn berry juice also presented the highest vitamin E content. The composition of vitamin E compounds was quite similar for the juice samples. In all juices, a-T was predominant, followed by small amounts of b-T3 (Fig. 4.A). In contrast to the tendencies observed for the carotenoid content described above, the total vitamin E content of the orange juice was similar to the amounts founds in the vegetables juices. The following order of the vitamin E content within the juice samples was obtained: orange < carrot < tomato < sea buckthorn berry.

as a-tocopherol. For each method, differences between the carotenoids can be explained largely by the specic structure of each compound. 4.1.1. FRAP assay In contrast to the results of Pulido, Bravo, and Saura-Calixto (2000) using only b-carotene and zeaxanthin, most of the carotenoids analysed in our studies showed ferric reducing activity; some of them even on a higher level than a-tocopherol. In the group of carotenes, only lycopene was an effective ferric reducing compound. The ferric reducing activity is mainly inuenced by the size of the conjugated double bond (CDB) system. The colourless acyclic intermediate carotenoids of the plant biosynthesis (phytoene, phytouene, neurosporene) did not show any signicant activity to reduce ferric ions due to their low number of CDB (3, 5, and 9, respectively). However in lycopene, an acyclic carotenoid with 11 CDB, the orbital overlap in the chromophor is great enough to form a stable carotenoid radical (Mortensen & Skibsted, 1997) and consequently to display FRAP activity. A comparison to the cyclic structures of b-carotene (11 CDB) and a-carotene (10 CDB) demonstrate the strong inuence of the b-ionone ring system. Despite the similar number of CDB, the b-ionone ring element on both sides of the molecule hinder these carotenoids to react with the ferric di-TPTZ complex. The steric hindrance between the methyl substituent at C5 and the hydrogen atom at C8 of the chain in b-carotene hinder the molecule to achieve coplanarity (Jimenez-Escrig, Jimenez-Jimenez, Sanchez-Moreno, & Saura-Calixto, 2000; Miller, Sampson, Candeias, Bramley, & Rice-Evans, 1996), so that in consequence, the orbital overlap is reduced and the contribution of the ring double bonds to the conjugated systems tends to zero (Mortensen & Skibsted, 1997). This steric hindrance in cyclic carotenes is passed over by the substitution of the hydroxyl functions in the 3 (30 )-position at the ring system, forming b-cryptoxanthin, lutein and zeaxanthin, respectively. A similar result was found for the epoxidised hydroxyl carotenoids (antheraxanthin, neoxanthin, violaxanthin), whereas the antioxidant capacity decreased with increasing number of epoxidations of ring double bonds in the order (number of epoxidised elements in brackets): lutein (0) = zeaxanthin (0) > antheraxanthin (1) > violaxanthin (2) > neoxanthin (2). The hydroxyl function in the vicinity to the CDB system is indispensible for the FRAP activity of the carotenoids containing two b-ionone rings. This strong inuence of the combination of CDB and hydroxyl functions was postulated decades ago for phenolic compounds like vitamin E, phenolic acids and avonoids (Burton & Ingold, 1981; Mller et al., 2010; Rice-Evans & Miller, 1996). The insertion of carbonyl groups at the 4 (40 )-position in the b-ionone ring led to a strong suppressive effect on the ferric reducing activity response, but in comparison to the cyclic carotenes, a slight increase was observed. The electron-withdrawing characters of the carbonyl oxygen affects the decrease of the FRAP value of these ketocarotenoids (echinenone, canthaxanthin and astaxanthin) down to 0.2 mol a-TE/mol. Rubixanthin combines the structural elements of the highly FRAP active carotenoids lycopene and b-cryptoxanthin. The combinations of an acyclic chain and a hydroxylated b-ionone ring led to the signicantly highest FRAP value (3.3 mol a-TE/mol) amongst all analysed compounds. As mentioned by many authors (Rodriguez-Amaya et al., 2008; Schiedt & Liaaen-Jensen, 1995) carotenoids are sensitive to various conditions. Isomerisation of (all-E)-carotenoids, the usual conguration in nature, to (Z)-isomers is promoted by heat treatment, exposure to light and contact with acids, and results in the loss of provitamin A activity and the alteration of bioavailability and metabolism (Rodriguez-Amaya et al., 2008). The highly unsaturated carotenoid molecule is also susceptible to oxidation. Hence,

4. Discussion 4.1. Antioxidant activity of the standard compounds with the different methods To date, only a very small number of publications using assays under varying conditions from author to author are available concerning the assessment of the antioxidant activity of carotenoids, which makes it difcult to compare the results. Some of the carotenoids analysed in the present study were measured for the rst time in an antioxidant activity assay. FRAP and aTEAC are commonly used to assess antioxidant activity in vitro. Both, aTEAC and FRAP assay based on SET reactions, in which the redox potential of the compounds analysed is important. The determined aTEAC values and the redox potentials of the main carotenoids, published by El-Agamey and McGarvey (2008), correlated well (R = 0.780). An increase of the redox potential of the carotenoid led to a decrease of the aTEAC values. In contrast, only a slight correlation between FRAP values and redox potentials was observed (R = 0.491), due to the very low activity of b-ionone ring containing carotenoids and ketocarotenoids. As mentioned above, both assays are based on the same mechanism. Additionally, the redox potential of Fe(II)/(III) (0.70 V) is comparable to that of the redox couple ABTS/ABTS+ (0.68 V). Thus, compounds should react similar in aTEAC and FRAP assay, which should lead to a good correlation between their results, as mentioned by Prior, Wu, and Schaich (2005) related to phenolic antioxidants. However, the reaction conditions between these assays differ, especially the pH-value (FRAP: 3.6; aTEAC: 7.4) and steric claims of the oxidising molecules and ferric di-TPTZ and ABTS. Consequently, in the case of carotenoids we calculated only a slight correlation between FRAP and aTEAC results (R = 0.444). Only a low correlation was observed between either aTEAC and the FRAP data and the LPSC data (R = 0.191, and R = 0.333 respectively), because the results in the LPSC assay are reecting more than just radical scavenging. The peroxyl radicals were generated from AAPH by thermal degradation throughout the whole test. The LPSC assay is the only method, except of the ORAC assay often used for the determination of the antioxidant capacity of hydrophilic compounds and food extracts, combining both the inhibition time and the degree of inhibition. With this test, all the carotenoid compounds (except of phytoene, phytouene, neurosporene) tested displayed an antioxidant activity more than eight times as high

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the antioxidant activity of the compounds could also be inuenced. In general, the FRAP assay is performed in acetic buffer (pH 3.6) to maintain iron solubility (Prior et al., 2005). For this reason, HPLC analysis of a lutein and a lycopene solution treated with acetic acid buffer was performed to detect possible oxidation and isomerisation. The HPLC-DAD conditions used followed the description of Frhlich, Kaufmann, Bitsch, and Bhm (2006). No signicant change in the composition of the lutein solution (10 lM in n-hexane) was observed after 8 min of shaking with acetic buffer (pH 3.6) compared to the untreated solution. Before and after the treatment, the lutein solution contained 9597% of the (all-E)-isomer. 35% of the solution was determined as lutein-(Z)-isomers, and oxidation products, respectively. A similar result was observed for the lycopene analysis, but the contributions of (Z)-isomers and oxidation products were higher compared to lutein, due to the higher sensitivity of this carotene against oxidising conditions. Two-third of the lycopene solution were identied as the (all-E)isomer. About 30% of the lycopene solution consisted of mono(Z)-isomers, with (5Z), (13Z) and (9Z) lycopene as the major ones. Oxidation products were identied on a level of approximately 5%, before, as well as after the acetic treatment. In conclusion, the acetic conditions used in the FRAP assay did not lead to isomerisation or oxidative degradation during the assay. Finally, our in vitro results support the in vivo observation of Matos et al. (2006), that lycopene prevents oxidation induced by ferric ions. They could show that lycopene injected in rats (10 mg/kg/day, 5 days) protect from ferric iron-induced oxidative damage in the prostate tissue and lipoperoxidation (LPO) (Matos et al., 2006). 4.1.2. aTEAC assay The six C40 carotenes studied showed different ability to scavenge ABTS+, with lycopene being the most efcient carotenoid. The activity of the carotenes increased with increasing the number of conjugated double bonds (CDB) in the order (number of CDB in brackets): phytoene (3) = phytouene (5) < neurosporene (9) < acarotene (10) = b-carotene (11) < lycopene (11). A comparison of their structures shows that both acyclic lycopene (aTEAC: 3.9 mol a-TE/mol) and b-carotene (3.1 mol a-TE/mol) have 11 CDB, but the latter is bicyclic. The inuence of the steric hindrance between the methyl group at the b-ionone ring and the chain hydrogen led to a low contribution of the ring double bonds to the conjugated systems, as described above. The ability of carotenes to scavenge ABTS+ increases with extension of the chromophore and a maximum overlap of the carboncarbon double bond molecular orbitals, as mentioned by Miller et al. (1996). The presence of functional groups on the terminal rings as displayed by the xanthophylls modulates the ABTS+ reducing activity. The incorporation of carbonyl functions in the ring has a profound suppressive effect on the aTEAC value. The carbonyl oxygen atom presented an electron-withdrawing effect and reduced therefore the unpaired electron density in the conjugated systems, which led to a higher energy of the intermediate and the transition states for the reactions with free radicals (Mortensen et al., 1997). Echinenone has less than two-third of the antioxidant activity of b-carotene despite a longer chromophore of 12 CDB. The extension of the conjugated system to 13 CDB by insertion of a further keto function, as in canthaxanthin, and further hydroxylation, as in astaxanthin, reduced the aTEAC value to 0.8 mol a-TE/mol. The decrease of reactivity was explained by Mortensen et al. (1997) as follows. If the free radical adds to the conjugated system close to the cyclohexenone ring, the carbonyl group will not be part of the oddelectron-conjugated system, and the intermediate will be more stable. In contrast, the insertion of a single hydroxyl group at C3 of the b-ionone ring of b-carotene, forming b-cryptoxanthin, did not led

to a signicant change in the ability to scavenge ABTS+. However, the presence of hydroxyl groups in each ring as in zeaxanthin signicantly decreased the aTEAC value. A similar modication at the structure of a-carotene forming lutein had a similar effect. The xanthophyll capsanthin, a 3,30 -dihydroxy-b,j-carotene6-one, combines the structural elements of one hydroxylated b-ionone ring and a carbonyl function together with a hydroxylcyclopentyl element. This may have led to the reduced antioxidant function, which is comparable to that of echinenone. For rubixanthin, a b,w-carotene-3-ol, an ABTS+ reducing activity comparable to the related carotenoids lycopene (w,w-carotene) and b-cryptoxanthin (b,b-carotene-3-ol) was observed. The insertion of a single epoxy group at the place of the double bond in the hydroxylated b-ionone ring of zeaxanthin, forming antheraxanthin, did not greatly modulate the ability to reduce ABTS+ radical cations. However, the presence of one epoxy group in each ring (forming violaxanthin) decreased the activity by about 10%, due to the reduction of the CDB system. The degradation of one epoxy group to a hydroxyl function, forming neoxanthin, led to a further decrease down to the activity of a-T. The ABTS+ reducing activity of apo-carotenoids increased with the dimension of the conjugated double bond system, whereas the number of carbonyl function in these molecules led to a decrease of antioxidant activity as described above. Consequently, the order of increasing activity was the following (in brackets: number of CDB + number of carbonyl groups): bixin (7 + 2) < crocetin (10 + 2). In the reaction with free radicals derived from chloroform and phenol (Mortensen et al., 1997) and with ABTS+ (Miller et al., 1996), the same order of reactivity was observed as observed in our studies: carotenes > hydroxycarotenoids > ketocarotenoids. Summarising, the order of increasing reactivity of the analysed compounds against ABTS+ follows the decrease of the ionisation potentials for these carotenoids in non-polar solvents (e.g. benzene). Soffers et al. (1999) found a good correlation (r = 0.914) between the EHOMO (energy of the highest occupied molecular orbital) and the TEAC value of carotenoids measured by Miller et al. (1996). The aTEAC values measured in the present work support the hypothesis that the ABTS+ bleaching activity mainly depends on the ionisation potential of the carotenoid. Excellent correlations were calculated between the aTEAC value and the EHOMO published by Soffers et al. (1999) and Galano (2007) (r = 0.929 and r = 0.923 respectively). 4.1.3. DPPH scavenging assay In contrast to the achievements of other research groups (Jimenez-Escrig et al., 2000) using the DPPH scavenging assay, we could not detect any antioxidant activity of carotenoids. Only a-tocopherol, BHA and BHT showed reactions. Mendez-Robles et al. (2006) detected lower DPPH scavenging activities for two apocarotenoids from b-carotene (ditaxin, heteranthin) compared to b-carotene and lycopene. The reason for the measurable, but low DPPH scavenging activities could be seen in the concentrations used, which ranged in high non-physiological doses up to 1500 lM in methanol. Liu et al. (2008) determined the activity of a-T and some carotenoids in this assay too, using the same reaction conditions as we did (reaction time, solvents, concentrations, used wavelength). A concentration-dependent increase of DPPH scavenging activity was detected for a-T, and the reaction between the antioxidant and the DPPH was nished after 10 min incubation. In contrast, b-carotene and lycopene did not react in such a dose-dependent manner. For lycopene, an inverse relationship between concentration (515 lM) and scavenging activity was observed, and the measured DPPH values were on a very low level compared to the reaction of a-T (Liu et al., 2008). In our studies, when a carotenoid solution was mixed with DPPH solution, a dark purple brown

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colour was produced (as rst described by Liu et al., 2008) which interfered with the absorbance readings at 515 nm, which is the wavelength at the absorbance maximum of DPPH. To reduce this interference between the absorbance of DPPH and carotenoids, the wavelength was changed to 540 nm, as mentioned by Liu et al. (2008), where DPPH still has signicant absorbance. However, some of the carotenoids (e.g. lycopene, astaxanthin) still absorb at this wavelength. As described above, after subtraction of the self-absorbance (at 540 nm) of the specic carotenoid used from the absorbance of the mixtures of DPPH and carotenoid, the resulting absorbance value was equal to the DPPH blank value. This observation led to the conclusion that carotenoids are not able to scavenge the DPPH. 4.1.4. LPSC assay Conventional chain-breaking antioxidants, such as tocopherols trap peroxyl (ROO) radicals by donating a hydrogen atom. However, carotenoids seem to exert an antioxidant activity by a mechanism in which the chain-propagating peroxyl radical is trapped by the addition to the conjugated polyene system of the carotenoid rather than the mechanism of hydrogen-donation (Krinsky & Johnson, 2005). The radical scavenging activity of carotenoids is based on the resulting carbon-centred radical, which is resonance-stabilized because of the delocalization of the unpaired electron in the conjugated polyene system, leading to chain termination of LPO (Terao, 1989). In the present studies, using a luminol based chemiluminescence assay to determine the peroxyl radical scavenging capacity (LPSC), the amounts of ROO radicals (generated by AAPH) consumed by a-tocopherol and carotenoids were similar to the published data by Tsuchiya, Scita, Freisleben, Kagan, and Packer (1992). They achieved ROO radical scavenging values of approximately 2732 mol ROO per mol carotenoid for the most important carotenoids (2 mol ROO/mol a-T, respectively), even though using 2,20 -azobis(2,4-dimethylvaleronitrile) (AMVN) for radical generation. The present results support their hypothesis that carotenoids containing keto groups at the 4 (40 )-position in the b-ionone ring system (echinenone, canthaxanthine, astaxanthin) can serve as more effective antioxidants than b-carotene in the ROO radicaldependent assays. The substitution of a hydrogen atom by a carbonyl function at the 4 (40 )-position, but not by the corresponding substitute of a hydroxyl group at the 3 (30 )-position, increased the efciency of the ROO radical-trapping ability of carotenoids containing the b-ionone ring system in our studies, as a result of an expansion of the polyene system. Echinenone (b,b-carotene4-one) and astaxanthin (3,30 -dihydroxy-b,b-carotene-4,40 -dione) presented an approximately 25% higher ROO scavenging activity than b-carotene. In contrast, the hydroxylation in 3 (30 )-position of carotenoids (as in b-cryptoxanthin, lutein, and zeaxanthin) did not lead to a signicant change in ROO radical trapping value compared to b-carotene due to the unchanged size of the polyene system. This absent inuence of the hydroxylation was observed by Naguib (2000) in past studies using BODIPY uorescence and AMVN as radical generator, too. He found a similar order of peroxyl radical scavenging activity: ketocarotenoids > hydroxycarotenoids % b-ionone carotenes > lycopene. Bixin, an acyclic diapocarotenoid (C25) found in Bixa orellana L., was the best antioxidant in this assay; twice as active as lycopene. The combination of a large hydrocarbon polyene system including electron-withdrawing terminal functions (COOH and COCH3), which led to negative mesomeric effects, reduce the electron density in the conjugated chain and raise the reactivity to add peroxyl radicals. Crocetin, a diapocarotenoid (C20) presented a 25% higher LSPC value than lycopene despite a polyene system of only 9 CDB compared to lycopene with 11 CDB, due to the conjugated carboxylic functions on both sides of the molecule, which increase the activity to form carotenoidradical adducts.

4.2. A standardised antioxidant activity: weighted average based on four methods As recommended by certain research groups (Frankel & Meyer, 2000; Hengst, Werner, Mller, Frhlich, & Bhm, 2009; Huang et al., 2005), the use of more than one assay to determine the antioxidant potential of food extracts or single compounds is necessary, since different methods can yield widely diverging results. Various methods, based on different mechanisms, should be used. In the present study, we used assays based on the main reaction mechanisms of antioxidants: FRAP (activity to reduce metal ions), aTEAC (activity to reduce synthetic radical cations), DPPH (activity to scavenge free radicals) and LPSC (activity to scavenge peroxyl radicals). Of course, a ranking of all compounds within each assay is possible (Kranl et al., 2005). However, for summarising the results of one compound within all assays used a standardisation is recommended. Tabart, Kevers, Pincemail, Defraigne, and Dommes (2009) proposed to use a weighted average of the results obtained in all assays used based on the reference compound. However, a simple mathematical mean is not adequate, because one of the four methods (LPSC) gave much higher values due to the poor potential of a-tocopherol in this assay. This would give this assay undue preponderance in the mean. On the other hand, carotenoids did not show any activity to scavenge DPPH radicals. This would lead to an underestimation of the antioxidant potential of carotenoids when calculating a simple mean. Therefore, we calculated a compensing antioxidant activity as a weighted mean including the results achieved by the FRAP, aTEAC, DPPH and LPSC assays. As the antioxidants analysed in this paper are the major carotenoids found in human diet, we calculated a weighted average for each compound where the weighting factor was achieved by dividing the antioxidant activity of a compound determined using a given method by the average activity determined for the whole set (all) of compounds in the same method. Then, summing the four activity results (FRAP, aTEAC, DPPH, and LPSC) and nally dividing the sum by four followed. The following equation describes the calculation of the weighted average for lycopene (LYC):

Weighted av erage LYC

FRAP LYC FRAPall x

TEAC LYC aaTEAC DPPHLYC LPSC LYC x xDPPH xLPSC

all all

all

Results of these calculations are shown in Table 2 (last two columns). As the carotenoids did not show any potential to scavenge DPPH radical, weighted averages, including the results obtained in the DPPH assay and without these results, were performed. On this basis of excluding the DPPH results, the xanthophylls b-cryptoxanthin and rubixanthin, as well as lycopene appeared as the best antioxidants, with a weighted average of 1.81.9. Some other carotenoids also showed a high antioxidant activity, when calculated in this way. However, when calculating the weighted average by including the results achieved in the DPPH assay, a-tocopherol showed the highest antioxidant potential (4.3), followed by BHA (1.4), and rubixanthin (1.4) due to the absence of DPPH scavenging activity in carotenoids. 4.3. Antioxidant capacity of juices and oil samples As mentioned above, the assessment of the antioxidant capacity of food samples requires the use of various methods (Frankel & Meyer, 2000; Tabart et al., 2009). In the present study, we have applied the FRAP, aTEAC, DPPH and LPSC assay to several juices and oil samples. The carotenoid rich juices (tomato, carrot, and sea buckthorn berry) gave high LPSC values, whereas the vitamin E rich oils (sunower, olive, walnut and especially sh oil) caused much higher DPPH values than the juices. We observed that the lipophilic antioxidant capacity of the four analysed juices was

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mainly inuenced by the content of vitamin E and carotenoids. Quite good correlations were evaluated between the sum of vitamin E and carotenoids, and the results determined in the DPPH, aTEAC and FRAP assay (R = 0.999, R = 0.981, and R = 0.942). Only the peroxyl radical scavenging capacity, measured by the LPSC assay, showed a lower correlation (R = 0.862) between the content of these antioxidants and the antioxidant capacity detected, due to the lower LPSC activity of lycopene (see Fig. 1), the main carotenoid found in tomatoes and one of the main carotenoids found in sea buckthorn berries (Fig. 3). Of all juices tested, sea buckthorn berry juice showed the highest antioxidant capacity regardless of the method used. The high contents of carotenoids and vitamin E detected in our analysis and published in the literature (Andersson, Olsson, Johansson, & Rumpunen, 2009; Beveridge, Li, Oomah, & Smith, 1999; Andersson, Rumpunen, Johansson, & Olsson, 2008) can explain this high antioxidant capacity of the sea buckthorn berry juice sample, as already mentioned by Krinsky and Johnson, (2005). Of all oil samples tested, sh oil showed some inconsistent results. On one hand, sh oil presented the highest DPPH value adapted to the high vitamin E content, which is often added to protect the x3-fatty acids against LPO. On the other hand, this maritime oil displayed the lowest ferric reducing capacity in the FRAP assay. Fish oil is rich in polyunsaturated fatty acids (PUFA) especially EPA (C20:5) and DHA (C22:6) (Kouba & Mourot, 2011), which are sensitive to LPO, possibly initiated by transition metals ions such as Fe3+ and Cu2+. The addition of the FRAP reagent, containing ferric chloride, to the oil could have induced the initiation of LPO chain reactions of these PUFAs, whereas vitamin E acted as protector. The protection of PUFAs against LPO by vitamin E might be a competitive reaction to the direct interactions between vitamin E and the FRAP reagent. In contrast, such a competitive reaction was not estimated in the reactions between FRAP reagent and the plant oil samples (sunower, olive and walnut) due to the absence of x3-fatty acids in these oils. The DPPH values of the oil samples correlated well with their vitamin E contents (R = 0.999). However, aTEAC, FRAP, and LPSC values did not show any (good) correlation to this main antioxidant of oil samples (R = 0.589, 0.232, and 0.032, respectively) caused by the high antioxidant capacity of olive oil despite its low vitamin E content. When excluding the olive oil sample from the calculation of correlations between the vitamin E content and antioxidant capacity, both parameters correlated well with each other (R = 0.9710.999). The small amounts of lutein in the olive oil, a carotenoid known as a good antioxidant in the literature (Krinsky et al., 2005; Miller et al., 1996) and shown also above, could have caused this effect. It would be interesting to know the type of further compounds responsible for the high FRAP, aTEAC, and LPSC values of olive oil. Possibly, apolar polyphenols could cause the high ABTS+ bleaching and ferric reducing capacity of this Mediterranean oil. Jemai, Bouaziz, and Sayadi (2009) observed a quite good correlation between the content of hydroxytyrosol, tyrosol esters and o-diphenol and the DPPH and TEAC values, respectively. These major phenolic compounds might be the cause for the enhanced antioxidant capacity of olive oil. As discussed above the use of several methods to assess the antioxidant capacity of food matrices is advised. On the one hand, the LPSC assay gave high results for the juices, determined as rich in carotenoids and low in vitamin E (tomato, carrot, sea buckthorn berry). On the other hand, the assays based on the redox mechanisms (aTEAC, FRAP) as well as the DPPH scavenging assay showed 10 times lower results. A similar relationship was observed in the group of the oil samples, in which the DPPH assay obtained high values. By calculating the weighted averages of the results obtained in the four assays, it was possible to rank the antioxidant capacity of the food samples as follows: orange juice (0.1 lmol

a-TE/100 g) < walnut oil (0.7 lmol a-TE/100 g) = sunower oil (0.7 lmol a-TE/100 g) = tomato juice (0.7 lmol a-TE/100 g) < carrot juice (0.8 lmol a-TE/100 g) < sea buckthorn berry juice (1.2 lmol a-TE/100 g) < olive oil (1.8 lmol a-TE/100 g) < sh oil (2.1 lmol a-TE/100 g).
From this study, it is clear that neither one of the antioxidant capacity assays nor the calculation of the weighted averages will truly reect the total antioxidant capacity/activity of the food samples or the carotenoids themselves. These components, which act as antioxidants have also other more specic functions. However, using different kinds of in vitro assays to assess the antioxidant activity of single compounds could give an idea of the possible reactions of carotenoids in vivo and in food samples. The results from the FRAP assay for example could give an idea of the possible ferric reducing activity of dietary carotenoids against oxidants within the gastro-intestinal tract (GIT). Additionally, the activity of carotenoids to reduce Fe3+ to Fe2+ could also play a role in mammalian tissues to inhibit LPO initiated by transition metal ions such as Fe3+ and Cu2+, which consequently indicates a protective role in vivo. The strong activity of carotenoids in scavenging peroxyl radicals, in our in vitro studies generated by thermal degradation of the diazo compound AAPH, indicates a possible inhibition ability of carotenoids on the propagation of LPO chain reactions, which are known to occur in food samples as well as in human tissues (Negre-Salvayre et al., 2010). Finally, it has to be kept in mind that given the complex composition of food, separating each antioxidant compound and studying it individually is inefcient, without understanding the possible synergistic or antagonistic interactions amongst the antioxidant compounds within a food matrix. For instance, lycopene and b-carotene, known as major antioxidants in tomato and carrot, show strong antioxidant activity but may also act as prooxidants (Young & Lowe, 2001). More studies analysing the possible interactions between different antioxidants found in food and biological tissues have to be performed. 5. Conclusions To assess the antioxidant activities of single compounds or the antioxidant capacity of food extracts, a variety of methods based on different mechanistic principles must be used in parallel, because different methods often give different results. We showed in the present study widely ranging results. Lycopene as well as hydroxy carotenoids were the most effective carotenoids in reducing ferric ions (FRAP assay) due to steric hindrance and the low chemical reactivity of cyclic carotenes and their carbonyl substituted derivatives. The observations in the aTEAC assay demonstrated that the quantitative important carotenes found in the human diet (lycopene, a- and b-carotene) were more efcient quenchers of ABTS+ than most of the xanthophylls (with the exception of rubixanthin) and that this was maybe inuenced by the increasing polarities or electron-withdrawing effects of the functional groups in the terminal rings. In contrast, the keto carotenoids demonstrated the highest activity in scavenging peroxyl radicals due to their large conjugated double bond systems. Additionally, none of the analysed carotenoids showed any activity to scavenge DPPH. A weighted average, on the basis of the results obtained in the different assays, was calculated to summarise the potential of the analysed carotenoids in reducing metal ions (FRAP) or synthetic radical dyes (aTEAC), in scavenging free radicals (DPPH) and peroxyl radicals (LPSC) into a comprehensive value. Still, big differences amongst the carotenoid compounds were observed. When excluding the results achieved in the DPPH assay, most of them showed a greater antioxidant activity than a-tocopherol, BHA and BHT, due to the high peroxyl scavenging activity of

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