Journal of Food Composition and Analysis 24 (2011) 10431048

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original article

Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular

antioxidant-rich US foods
Anna Floegel a,1, Dae-Ok Kim b, Sang-Jin Chung c, Sung I. Koo a, Ock K. Chun a,*
a
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, United States
b
Department of Food Science and Technology and Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea
c
Department of Foods and Nutrition, Kookmin University, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: To evaluate the comparability of the two most common radical scavenging assays using 2,20 -azino-bis-3-
Received 29 October 2010 ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the 50
Received in revised form 7 January 2011 most popular antioxidant-rich fruits, vegetables and beverages in the US diet were identied and
Accepted 14 January 2011
analyzed for their antioxidant capacities, total phenolics and avonoids content. SpearmansRho
Available online 5 March 2011
correlation coefcients were calculated in order to characterize the relationship between antioxidant
capacities, total phenolics and avonoids content. Antioxidant capacity showed a strong positive
Keywords:
relationship comparing both assays (r = 0.949, p < 0.001). Antioxidant capacity detected by ABTS assay
ABTS
DPPH
was stronger positively associated with the oxygen radical absorbance capacity (ORAC) from USDA
Total antioxidant capacity database (for ABTS: r = 0.593, p < 0.001; for DPPH: r = 0.539, p < 0.001, respectively), phenolics (for
Foods ABTS: r = 0.946, p < 0.001; for DPPH: r = 0.897, p < 0.001, respectively) and avonoids content (for
Polyphenols ABTS: r = 0.718, p < 0.001; for DPPH: r = 0.708, p < 0.001, respectively). Antioxidant capacity detected
Flavonoids by ABTS assay was signicantly higher for fruits, vegetables and beverages compared to that by DPPH
Fruits assay. The high-pigmented and hydrophilic antioxidants were better reected by ABTS assay than DPPH
Vegetables assay. These data suggest that ABTS assay may be more useful than DPPH assay for detecting antioxidant
Beverages
capacity in a variety of foods.
Food analysis
2011 Elsevier Inc. All rights reserved.
Food composition

1. Introduction scavenge free radicals. This concept provides a broader picture of

Different assays have been introduced to measure antioxidant
capacity of foods and biological samples. The concept of
antioxidant capacity rst originated from chemistry and was later
adapted to biology, medicine, epidemiology and nutrition (Cao and
Prior, 1998; Floegel et al., 2010; Pellegrini et al., 2003). It describes
the ability of redox molecules in foods and biological systems to
Presented in part at the 2010 Experimental Biology Conference in Anaheim, CA,
April 2010.
Abbreviations: ABTS, 2,20 -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; DPPH,
1,1-diphenyl-2-picrylhydrazyl; ORAC, oxygen radical absorbance capacity; FRAP,
ferric reducing ability of plasma; TEAC, Trolox equivalent antioxidant capacity;
VCEAC, vitamin C equivalent antioxidant capacity; NHANES, National Health and
Nutrition Examination Survey; VCE, vitamin C equivalent; GAE, gallic acid
equivalent; CE, catechin equivalent; CV, coefcient of variation.
* Corresponding author at: Department of Nutritional Sciences, University of
Connecticut, 3624 Horsebarn Road Extension Unit 4017, Storrs, CT 06269-4017,
United States. Tel.: +1 860 486 6275; fax: +1 860 486 3674.
E-mail addresses: anna.oegel@dife.de (A. Floegel), ock.chun@uconn.edu
(O.K. Chun).
1
Present address: Department of Epidemiology, German Institute of Human
Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany.
the antioxidants present in a biological sample as it considers the
additive and synergistic effects of all antioxidants rather than the
effect of single compounds, and may, therefore, be useful to study
the potential health benets of antioxidants on oxidative stress-
mediated diseases (Brighenti et al., 2005; Puchau et al., 2009).
In recent years, a wide range of spectrophotometric assays has
been adopted to measure antioxidant capacity of foods, the most
popular being 2,20 -azino-bis-3-ethylbenzthiazoline-6-sulphonic
acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay,
among others such as oxygen radical absorbance capacity (ORAC)
and ferric reducing ability of plasma (FRAP) assay (Brand-Williams
et al., 1995; Kim et al., 2002; Ou et al., 2002; Re et al., 1999;
Thaipong et al., 2006; van den Berg et al., 1999; van den Berg et al.,
2001). Most of the assays employ the same principle: a synthetic
colored radical or redox-active compound is generated; and the
ability of a biological sample to scavenge the radical or to reduce
the redox-active compound is monitored by spectrophotometer,
applying an appropriate standard to quantify antioxidant capacity,
e.g. as Trolox equivalent antioxidant capacity (TEAC) or vitamin C
equivalent antioxidant capacity (VCEAC). Furthermore, there are
two types of assays. One approach is based on an electron transfer

0889-1575/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2011.01.008
1044 A. Floegel et al. / Journal of Food Composition and Analysis 24 (2011) 10431048
and involves reduction of a colored oxidant, e.g. in ABTS, DPPH and
FRAP assay. The other approach involves a hydrogen atom transfer,
like ORAC assay, in which antioxidants and substrate compete for
thermally generated peroxyl radicals (Dudonne et al., 2009;
Rodriguez-Amaya, 2010). In specication, the ABTS assay is based
on the generation of a blue/green ABTS+ that can be reduced by
antioxidants; whereas the DPPH assay is based on the reduction
of the purple DPPH to 1,1-diphenyl-2-picryl hydrazine. Both assays
are convenient in their application and thus most popular;
nevertheless they are limited as they use non physiological radicals.
In contrast, the ORAC assay detects chemical change in a uorescent
molecule caused by a free radical attack. It is based on peroxyl
radicals that reect physiological relevant perturbations. The FRAP
assay is different from the others as there are no free radicals
involved but the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+)
is monitored. When applied to food analysis, the antioxidant
capacity measurements may be different depending on the assay
used. Many studies for the analysis of assay comparability
previously focused on only few, selected or exotic foods (Gorinstein
et al., 2010; Kim et al., 2003; Pellegrini et al., 2003; Samaniego
Sanchez et al., 2007). Food analyses that based their sample selection
on population intake data are currently rare. Thus, information on
the comparability of different antioxidant capacity assays is not
readily available for a variety of foods commonly consumed in the
United States (US). In this population not only fruits and vegetables,
but also beverages contribute strongly to antioxidant intake.
Thus, the purpose of this study was to evaluate the compara-
bility of antioxidant capacity measurements obtained by ABTS and
DPPH assay, when applied to a large number of food samples
containing a variety of fruits, vegetables and beverages that are
major contributors to antioxidant intake in the US diet.
2. Materials and methods
2.1. Reagents and standards
ABTS, (+)-catechin, DPPH, FolinCiocalteus phenol reagent, and
gallic acid were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Ascorbic acid was obtained from Fisher Scientic (Fair Lawn,
NJ, USA). 2,2-Azobis(2-amidinopropane) dihydrochloride was
purchased from Wako Chemicals Inc. (Richmond, VA, USA). All
chemicals used were of analytical grade. Standard solutions were
prepared with distilled deionized water.
2.2. Selection of food samples
To cover a variety of antioxidant-rich foods that are widely
consumed in the US, the top 100 foods in terms of (1) antioxidant
content and (2) amount of consumption were identied. Therefore,
the National Health and Nutrition Examination Survey (NHANES)
19992000 and 20012002 datasets (NCHS, 2002, 2004) were
linked to the USDA avonoid, isoavone and proanthocyanidin
databases and the USDA database for standard references
(AgricResServ, 2004, 2005, 2007a, 2008). A total of 8809
individuals aged >19 years who had completed a 24-h dietary
recall (24-h DR) and provided information on antioxidant
supplement use were included in this preliminary study (Chun
et al., 2007). Antioxidant content of each food was multiplied by
the average amount of consumption of this food in the US
population and food items were ranked according to their
contribution to antioxidant intake in this population.
From the top 100 foods, the 50 fruits, vegetables and beverages
that contribute most to antioxidant intake in the US were
identied. For fruits and vegetables, raw foods were included
and prepared foods were excluded, such as chili con carne with
beans. Four exceptions of processed liquid foods were included:
tomato catsup; canned pinto beans; canned red kidney beans;
and canned cranberries, because they were convenient to analyze.
White potato chips and white potato home fries were previously
excluded. Instead, their shared ingredient, raw potato, was
included on the list. Tea was split into three items on the nal
list: black tea, caffeinated, green tea, caffeinated and green tea,
decaffeinated. Any beverages that were enriched with vitamin C
were excluded, except for grape juice because it was the only kind
available in the supermarket. For all juices, pure juice or 100%
from concentrate and no pulp was selected for analysis.
2.3. Sample collection and preparation
All foods and beverages were purchased fresh at local super-
markets or liquor stores in Tolland and Manseld, CT, and were
stored in the fridge until extraction up to three days. For each food
item, a diverse sample was obtained, including three different
brands per food which was possible for 60% of the sample. For 40%
of the sample only two or one brands were available at the
supermarket. At least three pieces of each brand were collected. If
available, one organic brand was included for analysis. Apples, for
example, 2 kg of each brand Gala Washington, Empire NY and
McIntosh were purchased. Similar for beverages, 3 brands and at
least 1 L per brand for each drink were included into the analysis.
Orange juice, for example, 2 L of each Floridas Natural, Tropicana
and Simply Orange, were collected. Fruits and vegetables were
washed with tap water, dried and chopped. Different brands of one
item were mixed, weighed and extracted. For the hot beverages,
2 g tea or coffee were brewed with 150 mL boiling tap water for
2 min. Different brands were mixed and analyzed after cooling to
room temperature. Cold beverages were mixed, weighed and
analyzed immediately. Drink from powder was prepared according
to instructions on the package and analyzed immediately.
2.4. Sample extraction
Samples were extracted with methanol (Kim et al., 2003).
Briey, chopped fresh samples (50 g) were mixed with 100 mL
absolute methanol and blended for 2 min under nitrogen gas using
the Waring Laboratory Blender LB10 (Waring Laboratory, Torring-
ton, CT, USA). The mixture was then ltered through Whatman #2
lter paper (Whatman International Limited, Maidstone, UK) with
a chilled Buchner funnel and rinsed with additional methanol. The
rst extract obtained was stored in a 20 8C freezer. The solid lter
cake was resolved in 100 mL of 80% (v/v) methanol, blended for
2 min and ltered again (second extract). Both extracts were
combined and the solvent evaporated using a rotary evaporator
under reduced pressure at 45 8C. The extract of about 40 mL was
dissolved with additional 50 mL absolute methanol and made up
to a total volume of 100 mL with distilled deionized water. For each
food item, extraction was done in triplicate. Extracts were stored at
20 8C until analysis up to three days.
2.5. Measurement of antioxidant capacity expressed as vitamin C
equivalents
Antioxidant capacity analysis was performed immediately for
beverages or within one to three days for food extracts. For both
ABTS and DPPH assays, samples were diluted appropriately with
50% (v/v) methanol. Ascorbic acid was used as an antioxidant
standard.
The ABTS assay was based on the method of van den Berg et al.
(1999) slightly modied by Kim et al. (2003). Briey, 2.5 mM of
ABTS was mixed with 1 mM of 2,2-azobis(2-amidinopropane)
dihydrochloride in 10 mM phosphate buffered saline (PBS) solution,
pH 7.4. The mixture was heated in a water bath at 68 8C for 40 min.
 A. Floegel et al. / Journal of Food Composition and Analysis 24 (2011) 10431048
The blue-green ABTS+ solution was cooled to room temperature,
ltered through nylon syringe lters, and diluted with fresh PBS
buffer until absorbance of 0.650  0.020 at 734 nm. Then, 20 mL of
vitamin C standard or sample were mixed with 980 mL of ABTS+
solution and incubated for 10 min in 37 8C water bath. The decrease of
absorbance was monitored at 734 nm after 10 min. A control consisted
either of 20 mL of acidied distilled deionized water in 980 mL of radical
solution for vitamin C standard or 50% (v/v) methanol in 980 mL of
radical solution for samples. The coefcient of variation (CV) for ABTS
assay was found to be 4.72% within day (n = 10).
The DPPH assay was performed according to the method
developed by Brand-Williams et al. (1995) slightly modied by
Kim et al. (2002). A solution of 1 mM DPPH in 80% (v/v) methanol
was stirred for 40 min. Absorbance of the solution was adjusted to
0.650  0.020 at 517 nm using fresh 80% (v/v) methanol. Then, 50 mL
of standard or sample were mixed with 2.95 mL of DPPH solution and
incubated for 30 min in the dark covered with aluminum foil.
Decrease of absorbance was monitored at 517 nm at 30 min. A control
consisted either of 50 mL of acidied distilled deionized water in
2.95 mL of DPPH solution for vitamin C standard or 50 mL of 50% (v/v)
methanol in 2.95 mL of DPPH solution for samples.
Standard curves for both assays were obtained by measuring
the ABTS+ and DPPH scavenging activities of 10, 30, 60 and 100 mg
vitamin C/L. The ABTS+ and the DPPH scavenging activities of food
extracts were expressed on the fresh weight basis as mg vitamin C
equivalent (VCE)/100 g. For beverages, antioxidant capacity was
expressed per 100 g based on the fresh weight of each beverage.
Each sample was measured in triplicate. Mean and standard
deviation (n = 3) were calculated.
2.6. Total phenolics and total avonoids content
Total phenolics content of foods extracts and beverages was
measured by FolinCiocalteus phenol reagent (Kim et al., 2003;
Singleton and Rossi, 1965). First, 200 mL of appropriately diluted
sample or gallic acid standard were added to 2.6 mL of distilled
deionized water. Then, 200 mL of FolinCiocalteus phenol reagent
was added at time zero and mixed. After 6 min, 2 ml of 7% (w/v)
Na2CO3 solution was added and mixed. After incubation for 90 min
at room temperature, absorbance was measured at 750 nm versus a
prepared blank. The blank consisted of 200 mL 50% (v/v) methanol
instead of sample. Gallic acid in 50% (v/v) methanol solution in
concentrations of 10, 30, 60 and 100 mg/L was used as a standard
and a calibration curve was drawn for each day of analysis. The
content of total phenolics was expressed as mg gallic acid equivalent
(GAE)/100 g of fresh sample. All samples were analyzed in triplicate.
Total avonoids content of food extracts and beverages was
determined using a spectrophotometric method (Floegel et al.,
2010). Briey, 500 mL of sample or standard (catechin) were mixed
with 3.2 mL of distilled deionized water. At time zero, 150 mL of 5%
(w/v) sodium nitrite (NaNO2) solution were added and mixed.
After 5 min, 150 mL of 10% (w/v) aluminum chloride (AlCl3) was
added and mixed. After 6 min, 1 mL of 1 M sodium hydroxide
(NaOH) was added and mixed. Absorbance of the colored
avonoidaluminum complex was measured immediately at
510 nm versus a blank. The blank consisted of 500 mL of 50% (v/
v) methanol instead of sample. For standard catechin was
dissolved in 50% (v/v) methanol to concentrations of 10, 30, 60
and 100 mg/L. A calibration curve was drawn. Total avonoid
content of the foods was expressed as mg catechin equivalent (CE)/
100 g fresh sample. All samples were analyzed in triplicate.
2.7. Statistical analysis
Data from the NHANES 19992000 and 20012002 and USDA
databases were analyzed using Statistical Analysis System (SAS)
1045
software, release 8.1, 2000 (SAS Institute Inc., Cary, NC) and the
Survey Data Analysis (SUDAAN) for multi-stage sample designs
professional software package, release 8.0.2, 2003 (Research
Triangle Institute, Research Triangle Park, NC). Statistical Package
for the Social Sciences (SPSS) 17.0 software, release 2008 (SPSS Inc.,
Headquarters, Chicago, IL) was used for statistical comparison of
the two assays. Data was reported as mean, standard deviation and
coefcient of variation (CV). Food items were ranked according to
their antioxidant capacities, total phenolics and total avonoids
concentrations. As the data was not normally distributed, but
right-skewed, SpearmansRho correlation coefcients were calcu-
lated in order to characterize the relationship between antioxidant
capacities detected by different assays, phenolics and avonoids
content. Additionally, ORAC from USDA database (AgricResServ,
2007b) could be obtained and matched to 44 foods detected by
ABTS and DPPH assay in our analysis. Mean antioxidant capacities
were log-transformed and students paired t-test was used to
compare antioxidant capacities determined by ABTS and DPPH
assays. The level of statistical signicance was set at p < 0.05, for
two-sided testing.
3. Results
Among the 50 most popular foods in the US diet with high
antioxidant capacity were 18 fruits, 13 vegetables and 19 beverages.
The antioxidant capacities of foods are summarized in Table 1. The
highest antioxidant capacities were detected for strawberry by DPPH
assay (520.7  39.3 mg VCE/100 g) and for blueberry by ABTS assay
(476.6  28.9 mg VCE/100 g). Although their ranking differed slightly,
both assays identied the top ten foods according to their antioxidant
capacities as blueberry, black plum, strawberry, red cabbage, red wine,
red grape, sweet cherry, green tea, broccoli and apple. Lowest
antioxidant capacity was shown for fruit drink from powder (for ABTS
assay: 4.5  0.5 mg VCE/100 g and for DPPH assay: 6.8  0.9 mg VCE/
100 g, respectively). The ORAC database reported highest antioxidant
capacity for cranberry, red beans, kidney beans, black plums, blueberry,
red wine, strawberry, sweet cherry, apple and pear. Lowest antioxidant
capacity was found for lemon by ORAC database.
Table 2 shows that antioxidant capacity by ABTS assay was
strongly positively correlated to that by DPPH assay (r = 0.949,
p < 0.001). Compared with antioxidant capacity by DPPH assay,
antioxidant capacity measured by ABTS assay showed a stronger
correlation with ORAC from the USDA database (for ABTS: r = 0.593
and for DPPH: r = 0.539, p < 0.001, respectively). Table 2 sum-
marizes the SpearmansRho correlation coefcients. Furthermore,
antioxidant capacity was strongly positively correlated with total
phenolics content (for ABTS: r = 0.946 and for DPPH: r = 0.897,
p < 0.001, respectively) and moderately positively correlated with
total avonoids content of 50 foods (for ABTS: r = 0.718 and for
DPPH: r = 0.708, p < 0.001, respectively). Antioxidant capacity by
ABTS assay was signicantly and consistently higher than antioxi-
dant capacity by DPPH assay in the total sample (n = 50, p < 0.001),
as compared by students paired t-test.
Among the three food groups, fruits showed the highest
antioxidant capacity, followed by vegetables and beverages
(Fig. 1). ABTS assay showed higher antioxidant capacity than
DPPH assay in the 18 fruits (p < 0.05), 13 vegetables (p < 0.01) and
19 beverages (p < 0.05), as compared by students paired t-test.
The correlation between antioxidant capacities detected by ABTS
and DPPH assays was highest in beverages and fruits, and lower in
vegetables (Table 3).
4. Discussion
The present study showed that relative to DPPH assay, the ABTS
assay was more strongly correlated with ORAC from USDA
1046 A. Floegel et al. / Journal of Food Composition and Analysis 24 (2011) 10431048

Table 1
Antioxidant capacities of the 50 most popular antioxidant-rich US foods as determined by three different methods.

ABTSa DPPHa ORACe

mg VCE/100 g CV Rank mg VCE/100 g CV Rank mmol TE/100 g Rank

Fruits
Apple 158.7  35.6 22.5% 10 143.7  47.3 32.9% 9 3082 9
Avocado 70.0  10.1 14.4% 28 41.2  6.4 15.5% 39 1933 13
Banana 122.7  43.4 35.3% 17 135.6  42.0 31.0% 11 879 31
Blueberry 476.6  28.9 6.1% 1 383.5  16.0 4.2% 2 6552 5
Cantaloupe 49.3  6.1 12.4% 37 46.1  5.1 11.1% 35 315 42
Cherry, sweet 194.2  22.0 11.3% 7 165.1  12.8 7.7% 8 3365 8
Cranberry, can. 119.6  7.5 6.3% 19 86.8  7.0 8.1% 17 9584 1
Grape, red 214.4  48.5 22.6% 6 182.9  36.8 20.1% 6 1260 20
Grapefruit 97.9  1.7 1.7% 21 65.2  2.9 4.4% 24 1548 16
Lemon 145.8  13.7 9.4% 12 101.2  2.0 2.0% 16 82 44
Mango 63.9  3.4 5.3% 35 72.4  6.4 8.9% 20 1002 29
Nectarine 35.7  3.0 8.4% 43 38.9  5.7 14.6% 41 750 33
Orange 141.1  20.4 14.4% 14 81.4  13.2 16.2% 19 1819 14
Peach 70.0  8.5 12.1% 29 64.6  9.2 14.2% 25 1814 15
Pear 94.3  8.6 9.1% 23 68.3  4.7 6.8% 22 2941 10
Plum, black 372.0  25.8 6.9% 2 287.6  14.6 5.1% 3 7581 4
Strawberry 273.5  21.8 8.0% 3 520.7  39.3 7.6% 1 3577 7
Watermelon 14.7  1.3 8.8% 48 6.8  0.5 7.9% 49 142 43

Vegetables
Bean, pinto, can. 73.5  4.1 5.6% 26 50.7  3.7 7.3% 31 7779 3
Bean, kidney, can. 68.6  4.5 6.6% 31 50.0  2.0 4.1% 32 8459 2
Broccoli 160.4  20.6 12.8% 9 139.8  7.4 5.3% 10 1362 19
Cabbage, red 268.5  13.6 5.0% 4 187.4  8.4 4.5% 5 2252 12
Cabbage, green 64.5  2.9 4.5% 34 54.9  1.6 3.0% 29 508 37
Lettuce 69.7  22.6 32.5% 30 43.4  15.0 34.5% 37 1447 18
Onion 29.6  4.0 13.4% 45 12.5  1.8 14.6% 47 1034 28
Pepper chili 136.9  21.8 15.9% 15 126.4  20.9 16.5% 13 n.a.
Pepper sweet 97.5  8.4 8.6% 22 86.3  5.9 6.9% 18 923 30
Potato 41.4  1.4 3.4% 41 39.0  1.0 2.6% 40 1058 27
Spinach 147.7  6.8 4.6% 11 71.6  3.8 5.3% 21 1515 17
Tomato 39.1  3.1 7.9% 42 44.2  1.1 2.4% 36 367 41
Tomato catsup 48.1  1.2 2.6% 39 35.2  0.8 2.4% 42 578 36

Beverages
Apple juice 22.8  2.2 9.8% 47 18.9  1.1 5.8% 45 408 39
Beer 46.1  4.8 10.4% 40 30.2  1.4 4.7% 44 n.a.
Beer, lite 29.3  0.3 1.0% 46 12.2  0.5 4.1% 48 n.a.
Cranberry juice 99.7  3.1 3.1% 20 67.2  6.2 9.3% 23 865 32
Cranapple juice 81.2  0.5 0.6% 25 57.9  1.7 3.0% 28 637 35
Coffee, regularb 59.6  2.9 4.8% 36 61.7  1.2 1.9% 27 n.a.
Fruit punch 48.2  1.8 3.8% 38 48.0  1.0 2.1% 33 n.a.
Fruit drink powderc 4.5  0.5 11.2% 50 6.8  0.9 12.9% 50 n.a.
Grape juiced 120.7  7.0 5.8% 18 102.6  1.5 1.5% 15 2377 11
Grapefruit juice 65.1  4.7 7.2% 33 50.8  2.0 4.0% 30 1238 23
Lemonade 8.3  0.1 1.3% 49 16.7  0.1 0.6% 46 1225 24
Lemon Juice 70.3  2.6 3.6% 27 41.8  0.1 0.2% 38 1225 24
Orange juice 65.2  1.4 2.2% 32 47.4  0.1 0.2% 34 726 34
Tea, blackb 126.4  8.9 7.1% 16 106.3  9.5 8.9% 14 1128 26
Tea, green, decafb 145.2  8.2 5.7% 13 133.5  5.6 4.2% 12 1253 21
Tea, greenb 184.7  6.6 3.6% 8 165.8  5.8 3.5% 7 1253 21
Tomato juice 81.4  7.0 8.7% 24 62.6  0.1 0.2% 26 486 38
Wine, table, red 223.0  0.2 0.1% 5 196.4  0.1 0.1% 4 3873 6
Wine, table, white 32.7  2.0 6.0% 44 30.6  1.9 6.0% 43 392 40

Abbreviations: can.: canned; CV: coefcient of variation; VCE: vitamin C equivalent; TE: Trolox equivalent; n.a.: not available.
a
Each value is presented as mean  SD (n = 3), based on fresh weight.
b
Brewed according to instructions (2 g in 150 mL of boiling tap water for 2 min).
c
Prepared with cold tap water according to instructions on the package.
d
With added vitamin C.
e
Values were obtained from USDA ORAC database (AgricResServ, 2007b) that provided data for 44 of the 50 food items. No data was available for chili pepper, beer, lite
beer, regular coffee, fruit punch, and fruit drink from powder.

database, phenolics and avonoids content of the 50 most popular
antioxidant-rich foods in the US diet. The results suggest that ABTS
assay better reects the antioxidant contents in a variety of foods
than DPPH assay.
It has been previously reported that antioxidant capacity
determined by in vitro assays differs (Ou et al., 2002; Wootton-
Bearda et al., 2010; Wu et al., 2004). Ou et al. (2002) conducted a
large scale vegetable analysis using two different in vitro assays,
FRAP and ORAC, and obtained very different antioxidant capacities
from these methods. In their study antioxidant capacities as
determined by FRAP and ORAC assays were only weakly correlated.
Pellegrini et al. (2003) reported that rankings of several fruits,
vegetables and beverages differed based on antioxidant capacity
measured by FRAP and ABTS assays suggesting that caution should
be exercised when interpreting antioxidant capacities from
different assays. Gorinstein et al. (2010) recently reported a high
 A. Floegel et al. / Journal of Food Composition and Analysis 24 (2011) 10431048 1047

Table 2 Table 3
SpearmansRho coefcients for the correlation between antioxidant capacities SpearmansRho coefcients for the correlation between antioxidant capacities
measured by ABTS, DPPH and ORAC assays, total phenolics and avonoids content measured by ABTS and DPPH assays, stratied by food group.
of 50 US foods**.
N r p
ABTS DPPH ORACa Phenolics Flavonoids
Fruits 18 0.953 <0.001
ABTS 1 0.949 0.593 0.946 0.718 Vegetables 13 0.896 <0.001
DPPH 0.949 1 0.539 0.897 0.708 Beverages 19 0.956 <0.001
ORACa 0.593 0.539 1 0.547 0.653 Total samples 50 0.949 <0.001
Phenolics 0.946 0.897 0.547 1 0.665
Flavonoids 0.718 0.708 0.653 0.665 1
a Among the food groups, the correlation between antioxidant
Values were obtained from USDA ORAC database (AgricResServ, 2007b) that
provided data for 44 of the 50 food items. No data was available for chili pepper, capacities detected by ABTS and DPPH assays was strong in fruits
beer, lite beer, regular coffee, fruit punch, and fruit drink from powder. and beverages, but lower in vegetables (Table 3). Most vegetables
**
Correlation is signicant at p < 0.001. analyzed showed much lower antioxidant capacities as measured
by DPPH assay relative to ABTS assay. The ABTS assay is based on
the generation of a blue/green ABTS+, which is applicable to both
correlation between polyphenols content in three exotic fruits and hydrophilic and lipophilic antioxidant systems; whereas DPPH
antioxidant capacities measured by ABTS, DPPH and FRAP assays. assay uses a radical dissolved in organic media and is, therefore,
Dudonne et al. (2009) reported a strong positive correlation between applicable to hydrophobic systems (Kim et al., 2002). The
ABTS and DPPH assays with a Pearson correlation coefcient of difference between antioxidant capacities determined by the
r = 0.906 when used for 30 aqueous plant extracts. The ndings from two assays was greater in highly pigmented foods such as cherry,
the aforementioned studies are in this respect in agreement with the spinach, plums and red cabbage. Strawberries, in contrast, showed
results of the present study which found a SpearmansRho a much higher antioxidant capacity detected by DPPH assay. It has
correlation coefcient of r = 0.949 for the relationship between been previously reported that strawberries are among the foods
ABTS and DPPH assays. In addition, the present study found a with the highest antioxidant potential (Battino and Mezzetti,
stronger correlation between total phenolics content and ABTS assay 2006). It has to be pointed out that although there was an overall
than DPPH assay in agreement with previous studies (Dudonne et al., moderate association between antioxidant capacity measured by
2009; Samaniego Sanchez et al., 2007). ABTS/DPPH assays and retrieved from ORAC database, antioxidant
Antioxidant capacities by both assays were more strongly capacity for individual foods diverged strongly for few foods, e.g.
correlated with total phenolics than with total avonoids content cranberries and beans were ranked very high in the ORAC database
as reported previously (Chun et al., 2003; Kim et al., 2003). Kim but much lower in ABTS and DPPH assay. This may be explained by
et al. (2003) found that antioxidant capacity measured by ABTS the fact, that the ORAC database was based on a different food
assay was highly correlated with total phenolics content in plums, sample than the ABTS and DPPH analysis. In the case of cranberries
with a weaker correlation between antioxidant capacity and total and beans, antioxidant capacity measured by ABTS and DPPH assay
avonoids content. The correlation coefcients they reported were were based on canned foods, whereas the ORAC values were based
of similar magnitude as found in this study (ABTS and total on fresh foods, because no other information was available. This
phenolics: r2 = 0.977 vs. r = 0.946 and ABTS and total avonoids fact may explain the diverse ranking. However, for many foods, e.g.
r2 = 0.762 vs. r = 0.718, respectively). These ndings taken plum, strawberry, blueberry, cherry, apple, orange, watermelon,
together suggest that phenolic compounds are a major contributor tomato, cabbage, orange juice, lemon juice, grape juice and wine
to antioxidant capacity in these foods; however, this is presumably among others, the antioxidant ranking according to the three
due to not only avonoids but also non-avonoid phenolics. methods was in good agreement.

300
p<0.05

250

200
Antioxidant capacity

p<0.01 p<0.05
mg VCE/100 g

150 ABTS
DPPH

100

151
138
50 96
72 80
66

0
Fruits Vegetables Beverages
Fig. 1. Comparison of antioxidant capacities measured by ABTS and DPPH assays, stratied by food group (Results are based on students paired t-test with log transformed
data and presented as mean  SD (fruits n = 18, vegetables n = 13, and beverages n = 19). Abbreviations: VCE, vitamin C equivalent.
1048 A. Floegel et al. / Journal of Food Composition and Analysis 24 (2011) 10431048
The unique strength of the present study was that a large
variety of fresh foods were included into the analysis. For each food
item, a diverse sample was obtained made up of different cultivars
and brands to achieve most representative data. The foods
included were, furthermore, the major contributors of antioxidant
capacity in the US diet.
The present study, however, comprised several limitations.
First, it has to be taken into account that ABTS and DPPH assay are
in vitro models and do not assess all of the antioxidant activities in
foods. Second, for the extraction of the antioxidants, methanol was
used. Especially the fat soluble antioxidants might not have been
extracted to the full extent; although, the majority of the foods
analyzed in the present study contained low amounts of fat. A
previous study reported that antioxidant capacity depends on the
solvent used (Perez-Jimenez and Saura-Calixto, 2006). Third, the
comparison of antioxidant capacities measured by ABTS and DPPH
assay to ORAC from the USDA database was limited to only 44
foods, which were not necessarily of the same cultivar but only the
best match. Last, antioxidant capacity of foods may differ
depending on the growing season, geographical origin, and
agricultural practices (Chun et al., 2005). It was not possible to
consider these variables when selecting food samples.
5. Conclusions
The ndings of the present study based on the analysis of a large
number of food samples rich in antioxidants strongly suggest that
compared to DPPH assay the ABTS assay better estimates the
antioxidant capacity of foods, particularly fruits, vegetables and
beverages. The data show that the antioxidant capacities measured
by ABTS assay are strongly correlated with the ORAC from USDA
database, phenolics and avonoids content in the 50 most popular
antioxidant-rich foods of the US diet. These ndings suggest that
ABTS assay is superior to DPPH assay when applied to a variety of
plant foods containing hydrophilic, lipophilic, and high-pigmented
antioxidant compounds.
Acknowledgments
This study was supported by the University of Connecticut
Hatch Project No. CONS00846 from the United States Department
of Agriculture.
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