Food Chemistry 141 (2013) 788–794

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Rapid high-throughput assay to assess scavenging capacity index using

DPPH
Fatima Abderrahim, Silvia M. Arribas, M. Carmen Gonzalez, Luis Condezo-Hoyos ⇑
Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Fisiología, Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: A new microplate-adapted DPPH rapid assay was developed to assess the antioxidant capacity of pure
Received 30 July 2012 compounds and foods. The assay was carried out in buffered medium (methanol: 10 mmol/l Tris buffer
Received in revised form 12 April 2013 pH 7.5, 1:1 v/v) and reaction was completed at 10 min. The scavenging capacity index (SCI), a theoretical
Accepted 20 April 2013
antioxidant parameter directly related to the antioxidant capacity of samples, was calculated. SCI for pure
Available online 30 April 2013
compounds: gallic acid (6.76 ± 0.08), quercetin (7.89 ± 0.24), catechin (6.05 ± 0.23), trolox (2.32 ± 0.03),
ascorbic acid (2.52 ± 0.15) and gluthatione (1.08 ± 0.08) and foods (lmol DPPH scavenged/100 ml): trop-
Keywords:
ical juice (655.62 ± 12.18), mediterraneo juice (702.87 ± 11.13), apple juice (212.52 ± 17.22), pomegran-
Antioxidant capacity
DPPH
ate juice (319.83 ± 9.45), red grape nectar (1093.05 ± 18.69), Don Simon orange juice (632.94 ± 17.22)
Rapid assay and date syrup (15992.34 ± 250.7) were comparable to those in previous reports using the classic DPPH
High-throughput assay assay. The relative standard deviation (RSD) for the SCI on the same and different days was less than
Scavenging capacity index 8.12% in all cases.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction seconds when semi-aqueous medium is used, at a physiological

DPPH assay is commonly used to assess antioxidant capacity of
foods and plants, providing reliable results under different DPPH
concentrations, organic solvents, antioxidant(s)/free radical vol-
ume ratios and reaction times (Luo, Wang, Wang, Ma, & Li, 2012;
Müller, Fröhlich, & Böhm, 2012; Ordoudi et al., 2012; Wootton-
Beard, Moran, & Ryan, 2012). Antioxidant capacity by DPPH assay
is usually expressed as: EC50, antiradical power or stoichoimetric
value (Brand-Williams, Cuvelier, & Berset, 1995); free radical inhi-
bition or remaining antiradical activity (Baydar, Özkan, & Yasar,
2007); or ascorbic acid equivalent antioxidant capacity (Lim, Lim,
& Tee, 2007). Recently, antioxidant activity index (AAI) and antiox-
idant activity unit (AAU) have both been proposed as parameters
to standardize antioxidant capacity of pure compounds and ex-
tracts (Deng, Cheng, & Yang, 2011; Scherer & Godoy, 2009). How-
ever, AAI does not reflect the stoichiometry of the reaction and
lacks theoretical meaning and, while AAU does reflect a stoichoi-
metric relationship, is not intuitive and is restricted to pure
compounds.
DPPH assay has been carried out using many different organic
solvents (Brand-Williams et al., 1995; Cheng, Moore, & Yu, 2006;
Mrazek, Watla-iad, Deachathai, & Suteerapataranon, 2012; Scherer
& Godoy, 2009). Curiously, the reaction can be completed in
pH, and the in vivo predictability of the antioxidant properties of
the analysed pure compound samples is improved (Bartasiute,
Westerink, Verpoorte, & Niederländer, 2007). Nevertheless, the
DPPH assay using semi-aqueous medium has not been completely
developed and applied to foods. The scavenging capacity index
(SCI) proposed in this paper was obtained by theoretical deduction.
Data were acquired using a microplate reader in a semi-aqueous
medium that reduced the analysis time to 10 min. These character-
istics resulted in a high-throughput assay that can ascertain SCI
and allow speedy comparison of the antioxidant strength of pure
compounds and foods.
2. Materials and methods
2.1. Reagents
1,1-Diphenyl-2-picrylhydrazyl (DPPH), (±)-catechin hydrate,
gallic acid, reduced glutathione, quercetin dihydrate and 6-hydro-
xy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) and tri-
zmaÒ base were purchased from Sigma–Aldrich (Madrid, Spain).
Ascorbic acid, hydrochloride acid and methanol were acquired
from Merck (Barcelona, Spain). Grade milliQ water (<18.2 mO)
was used to prepare solutions.

⇑ Corresponding author. Address: Universidad Autónoma de Madrid, Facultad de 2.2. Pure compounds and foods
Medicina, Departamento de Fisiología, Arzobispo Morcillo 2, Madrid 28029, Spain.
Tel.: +34 91 497 5417; fax: +34 91 497 5478. Stock solution (10 mmol/l) of gallic acid, quercetin, catechin and
E-mail address: luisalberto.condezo@uam.es (L. Condezo-Hoyos). trolox were prepared daily in methanol, and ascorbic acid and

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.04.055
 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794
Fig. 1. DPPH absorption spectrum of: () blank (e) 60 lmol/l DPPH in MT buffer
and after reaction with excess antioxidants (h) gallic acid/DPPH molar ratio = 6.3
(s) trolox/DPPH molar ratio = 3.2. Inset shows DPPH absorption spectrum in MT
buffer at different concentrations. Data represent the mean of four replicates
measurement.
glutathione in 10 mmol/l Tris buffer (pH 7.5). Working solutions at
the required concentration were prepared by serial dilution in
Fig. 2. Reaction kinetic of pure compound against 60 lmol/l DPPH in MT buffer (free radical/antioxidant ratio volume = 10). Experimental data was fitted to the two-phase
decay model by nonlinear regression. A is the absorbance at different time and Ao is the initial absorbance. Data represent the mean of four replicates measurement.
789
methanol or 10 mmol/l Tris buffer pH 7.5 and were used to assess
SCI. Tropical, mediterraneo and apple juice (Hacendado, Valencia,
Spain), pomegranate juice (Thai Agri Food Company limited, Thai-
land) and Don Simon orange juice (J. Garcia Carrion S.A, Murcia,
Spain), red grape nectar (Marin Montejano S.A., Murcia, Spain)
and date syrup (BAK Kardsler GmbH, Mannheim, Germany) were
also evaluated. Serial dilutions of the foods were prepared in
10 mmol/l Tris buffer pH 7.5 to assess SCI.
2.3. Experimental conditions
2.3.1. Buffered medium
The effect of adding Tris buffer to the reaction medium on
absorption of both DPPH and its reduced form (DPPH-H) was eval-
uated. DPPH was dissolved in methanol: 10 mmol/l Tris buffer pH
7.5, 1:1 v/v (MT buffer). Spectra of both DPPH (0, 30, 60, 90 and
120 lmol/l) and DPPH-H, occurring after the reaction between
the free radical and excess antioxidants was completed, were as-
sessed every 10 nm over the 400–700 nm range in a multi-mode
microplate reader (Synergy™ HT, Biotek Instruments, Potton, UK).
2.3.2. Reaction kinetics
10 ll of pure antioxidant compound, at five concentrations,
were put in each 96-well plate and then 200 ll of DPPH in MT
buffer was added using a repetitive pipette. The final methanol/
buffer ratio was adjusted to 1:1 v/v taking into account whether
the antioxidant was dissolved in methanol or in 10 mmol/l Tris
790 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794

buffer pH 7.5. The absorbance at 520 nm was monitored for 10 min [DPPH] are the concentration of the pure compound and DPPH in
every 1 min in a multi-mode microplate reader at 25 °C. The rela- mmol/l, respectively.
tive absorbance, calculated as At=t/At=0, versus the reaction time was
fitted to the exponential decay biphasic model by non-linear
2.5. SCI of pure compounds and foods
regression analysis.

Five working solutions of pure compounds or diluted foods

2.4. SCI calculation
(10 ll) were put in each 96-well plate in quadruplicate and then
200 ll of DPPH (60 lmol/l in MT buffer) was added with a repeti-
SCI was defined as the number of DPPH mol that could be scav-
tive pipette. The absorbance at 520 nm after 10 min of incubation
enged by 1 mol of pure compound or DPPH lmol scavenged by
at 25 °C was used to calculate SCI, which was not influenced by
100 ml of food (Eq. (1)).
the incubation temperature or the initial DPPH concentration (data
SCI ¼ N ¼ B ð1Þ not shown).

where B is the slope obtained by linear (for samples with fast and
medium kinetic behaviour) or polynomial (for samples with slow
reaction kinetics) regression from At/Ai versus [pure compound]/
[DPPH] or ml food/[DPPH]. At and Ai are the absorbance at 520 nm
at 10 or 0 min of reaction and, respectively. [Pure compound] and
2.6. Statistical analysis
Linear and nonlinear regression analysis was done with Graph-
Pad Prism 5.0 statistical software (GraphPad, Inc., California, USA).

Fig. 3. Scavenging capacity index theoretical deduction (A) experimental variable and (B) DPPH standard curve.
 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794 791

Table 1
Scavenging capacity index of pure compounds and foods.

Day 1 Day 2 Day 3 Day 4

2 2 2
SCI R SCI R SCI R SCI R2 SCI RSD (%
Pure compounds
Gallic acid 6.79 ± 0.07 0.9965 6.76 ± 0.08 0.9931 6.88 ± 0.10 0.9931 6.68 ± 0.09 0.9941 6.76 ± 0.08 1.23
Quercetin 7.77 ± 0.21 0.9954 8.08 ± 0.19 0.9903 8.08 ± 0.25 0.9945 7.59 ± 0.22 0.9929 7.89 ± 0.24 3.08
Catechin 5.77 ± 0.23 0.9933 6.08 ± 0.24 0.9997 6.00 ± 0.21 0.9897 6.33 ± 0.26 0.9983 6.05 ± 0.23 3.85
Trolox 2.28 ± 0.04 0.9977 2.32 ± 0.05 0.9914 2.34 ± 0.03 0.9944 2.35 ± 0.03 0.992 2.32 ± 0.03 1.41
Ascorbic acid 2.35 ± 0.10 0.9944 2.46 ± 0.12 0.9936 2.68 ± 0.12 0.9903 2.60 ± 0.14 0.9969 2.52 ± 0.15 5.81
Glutathione 0.99 ± 0.06 0.9956 1.18 ± 0.05 0.9984 1.10 ± 0.06 0.998 1.06 ± 0.07 0.9998 1.08 ± 0.08 7.25
Foods
Tropical juice 667.80 ± 16.8 0.998 655.2 ± 19.32 0.9952 639.03 ± 15.54 0.9952 660.45 ± 15.75 0.9965 655.62 ± 12.18 1.86
Mediterraneo 714.42 ± 25.2 0.9976 702.24 ± 18.72 0.9933 688.17 ± 16.38 0.9891 706.65 ± 17.64 0.9942 702.87 ± 11.13 1.57
juice
Apple juice 194.04 ± 4.83 0.9976 234.15 ± 9.03 0.9997 204.96 ± 11.34 0.9996 217.14 ± 9.03 0.9956 212.52 ± 17.22 8.12
Pomegranate 332.22 ± 5.46 0.9988 313.95 ± 7.56 0.997 321.72 ± 6.72 0.9986 311.01 ± 5.88 0.9994 319.83 ± 9.45 2.97
juice
Red grape 1113.00 ± 10.71 0.9964 1101.24 ± 20.79 0.9928 1088.43 ± 23.52 0.9939 1069.32 ± 40.32 0.9969 1093.05 ± 18.69 1.71
nectar
Don Simon 627.06 ± 11.76 0.9969 649.95 ± 18.9 0.9909 643.23 ± 10.92 0.9953 611.31 ± 33.6 0.9975 632.94 ± 17.22 2.73
juice
Date syrub 16226.28 ± 151.2 0.9957 15809.43 ± 479.22 0.991 16189.53 ± 450.66 0.9932 15744.33 ± 742.98 0.996 15992.34 ± 250.7 4.94

Experimental data for glutathione were fitted to a second order polynomial model.
SCI of the foods were expressed as lmol DPPH scavenged/100 ml sample.
RSD is relative standard deviation.

3. Results [DPPH]scavenged can be relate to the absorbance decrease At

(At = Ai  Af) from DPPH standard curve A = K  [DPPH] (Fig. 3B),
3.1. DPPH rapid assay i.e.:
At
3.1.1. Effect of MT buffer on the DPPH spectrum ½DPPHscavenged ¼ ð5Þ
K
Tris buffer did not affect the DPPH absorption spectrum. In fact,
the free radical showed maximum absorbance at 520 nm, even dis- Substituting Eq. (5) into Eq. (4) generates:
playing a plateau at 520–530 nm (Fig. 1, inset). The conversion of At ¼ N  K  ½samplei ð6Þ
DPPH to DPPH-H by a reaction with excess antioxidant (trolox
and gallic acid) only showed low absorption, i.e. DPPH-H did not K can be expressed as:
affect the absorbance of DPPH at 520 nm (Fig. 1). Therefore, DPPH Ai
reduction by antioxidants in MT buffer can be monitored at K¼ ð7Þ
½DPPHi
520 nm with a broad absorbance window.
Substituting Eq. (8) in Eq. (7) generates:
3.1.2. Reaction kinetics in MT buffer
At ½samplei
Pure antioxidant compounds can be grouped into those show- ¼N ð8Þ
Ai ½DPPHi
ing a rapid, medium or slow kinetic behaviour against DPPH when
MT buffer was used as reaction medium. Ascorbic acid showed a The slope (B) of linear region, obtained by regression of At/Ai
rapid kinetic, completing the reaction within 120 s (Fig. 2). Querce- versus [sample]i/[DPPH]i, is equal to N and represents SCI. [Sample]i
tin and gallic acid showed intermediate kinetics and the reaction and [DPPH]i are the initial concentration of the sample and DPPH in
was completed at 600 s (Fig. 2). Glutathione showed slow kinetic mmol/l, respectively.
behaviour (Fig. 2).
3.2.2. Foods
3.2. SCI theoretical deduction Establishing that N is lmoles of DPPH can be scavenged by
100 ml of sample, from Eq. (8), we propose:
The experimental variables used in the deduction of SCI for pure
compounds and foods are shown in Fig. 3A. At N ml samplei
¼  ð9Þ
Ai V t ½DPPHi
3.2.1. Pure compounds The slope (B) of linear region, obtained by regression of At/Ai
Establishing that N is moles of DPPH that can be scavenged by versus ml samplei/[DPPH]i, is equal to N/Vt (Vt is the volume of reac-
1 mol of sample, we propose: tion in litre) and N represents SCI. [DPPH]i is the initial concentra-
tion of DPPH in mmol/l.
nscavenged DPPH ¼ N  nsample ð2Þ
Dividing Eq. (2) by total volume of reaction Vt (Vt = Vsample + - 3.3. SCI for pure compounds and foods
VDPPH) (Fig. 3A)
In descending order the SCI values were: quercetin > gallic
nscavenged DPPH nsample
¼N ð3Þ acid > ascorbic acid > trolox > glutathione (Table 1). Regarding the
Vt Vt
food sample, SCI values followed the descending order: date syr-
Then: up > red grape nectar > mediterraneo juice > tropical juice > Don
Simon orange juice > pomegranate juice > apple juice (Table 1).
½DPPHscavenged ¼ N  ½samplei ð4Þ
The relative standard deviation (RSD) was below 6.6% for the pure
792 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794

antioxidant and food samples on the same day, and between days
the RSD was less than 8.1% for the rapid DPPH high-throughput as-
say (Table 1). All the evaluated samples showed a linear relation-
ship (R2 > 0.99) between At/Ai and molar ratio ([antioxidant]/
[DPPH]) or ml of sample/[DPPH] (Fig. 4).
4. Discussion
4.1. Effect of MT buffer on the DPPH spectrum
There was no significant effect of DPPH-H on the DPPH spec-
trum after a complete reaction between the free radical and the
excess gallic acid and trolox in semi-aqueous MT buffer. In fact,
the window for absorbance with the Tris buffer was comparable
to window obtained with ammonium citrate buffer, which has
been described as the best buffer for DPPH assay in a semi-aqueous
medium (Bartasiute et al., 2007), being the most frequently used in
biological experiments due to its high water solubility, buffering
capacity and lack of interaction with other components. Likewise,
as already noted by other authors (Friaa & Brault, 2006), the MT
buffer produces an acid–base equilibrium (DPPH-H M DPPH + -
H+), which is reflected by the presence of a band between 400
and 440 nm.

Fig. 4. Relationship between DPPH scavenging ratio (At/Ai) and antioxidant molar ratio for pure antioxidants or ml sample/mmol/l DPPH for foods. The slope was obtained by
linear and non-linear regression analysis. Data represent the mean of four replicates measurement.
 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794
4.2. Reaction kinetics in MT buffer
We observed a similar very fast kinetic behaviour in MT buf-
fer with trolox and with ascorbic acid (completed in few seconds
<50 s). A very fast sequential electron proton transfer (SEPT) be-
tween ArOH and DPPH has been described in semi-aqueous
medium, which occurs firstly through antioxidant deprotonation
previous to the electron transfer process (Galano, 2011; Lit-
winienko & Ingold, 2003, 2004). Trolox phenol group deprotona-
tion (pKa = 11.9–11.7) (Friaa & Brault, 2006) and ascorbate
monoanion formation (pKa = 4.2) (May, 1999) in semi-aqueous
medium could favour a SEPT reaction. In fact, SEPT is strongly
dependent on the ionisation constant of the antioxidant (Lit-
winienko & Ingold, 2004). On the basis of B3LYP/6-311++G⁄⁄,
the acidity value of quercetin (316.5 kcal/mol) is higher than gal-
lic acid (317.9 kcal/mol) (Leopoldini, Russo, & Toscano, 2011),
and it can be expected that quercetin will show a slightly higher
rate constant than gallic acid (data not shown). The reaction was
completed in 10 min in MT buffer, which is less in comparison
to the time needed to complete the reaction in methanol about
55 min (Deng et al., 2011). The reaction kinetic for glutathione in
a semi-aqueous medium (ethanol: saline solution 0.9% w/v; 1:1
v/v) has been defined as slow, since the time required to reach
a steady state was TEC50 = 40 min (Ehrlich et al., 2007). It is a
key aspect in the calculation of SCI (estimated from a non-linear
regression by applying a polynomial equation) since the reaction
is not completed in these media (Mishra, Ojha, & Chaudhury,
2012). When linearity is not achieved, antioxidant consumption
can be increased by extending the reaction time. However, this
could also increase background noise in spectrophotometric
methods (Barton, 2010).
4.3. SCI of pure compounds
Antioxidant: DPPH stoichoimetric ratios (SR) have been re-
ported for several pure compounds in a semi-aqueous medium
ethanol/water 1:1 v/v (Stasko, Brezová, Biskupic, & Misík,
2007). For instance, trolox – an antioxidant used as standard
in several assays – has an SR of 0.47 ± 0.04 in a semi-aqueous
medium compared with the SCI (2.32 ± 0.03) found in our study,
since SR = 1/SCI. Ascorbic acid has an SR value of 0.50 ± 0.02 in
semi-aqueous medium (Stasko et al., 2007), which is similar to
our results where SCI = 2.52 ± 0.15. However, SCI for ascorbic
acid was no comparable to the antioxidant activity unit (AAU,
AAU = 5.38), defined as mol of antioxidant that react with
1 mol DPPH (Deng et al., 2011); i.e. a parameter similar to SR
(Stasko et al., 2007) and to stoichoimetric value (Brand-Williams
et al., 1995). It is well-established that 1 mol trolox and ascorbic
acid are able to scavenge 2 mol DPPH each (Brand-Williams
et al., 1995; Friaa & Brault, 2006). The SCI of gallic acid
(6.78 ± 0.08) in a semi-aqueous medium showed a slight differ-
ence with respect to that calculate from Stasko et al. (2007),
which was 5.26. Paradoxically, the AAU for gallic acid (3.03)
was different from the SR (Stasko et al., 2007) and SCI. When
AAU for ascorbic acid and gallic acid were divided by 10, it
was comparable to the SCI and SR values. This factor allows to
correct the differences between units of DPPH (g/ml) and antiox-
idant (mg/ml) concentrations and to convert the DPPH scaveng-
ing from percentage to decimal fraction. With respect to
glutathione, the EC50 value corresponding to the SCI value
(1.08 ± 0.08) was found to be similar to the previously reported
23.6 lmol/l (Ehrlich et al., 2007). SCI value for quercetin
(7.88 ± 0.24) was nearly 1.2 times higher than for gallic acid in
agreement with that estimated from data reported by Scherer
and Godoy (2009).
793
4.4. SCI of foods
The SCI values for foods obtained using the rapid DPPH assay
were comparable to those found using DPPH classic assay when
SCI for Trolox was taking account. Thus, antioxidant capacity of
117–301 lmol trolox/100 ml for orange juice (Fiore et al., 2005),
107.3 lmol ascorbic acid/100 g for apple juice (Floegel, Kim,
Chung, Koo, & Chun, 2011) and 586.6 lmol ascorbic acid/100 g
for grape drink (Floegel et al., 2011) has been reported.
5. Conclusions
DPPH rapid assay was shown to be appropriate for the compar-
ison of the antioxidant strength of pure compounds and foods by
means of an SCI theoretical parameter. The use of a semi-aqueous
MT buffer medium made it possible to reduce reaction time to
10 min or less, which, in addition to its adaptation to a microplate
reader, makes this assay attractive from a cost-affectivity basis for
routine measurements.
References
Bartasiute, A., Westerink, B. H. C., Verpoorte, E., & Niederländer, H. A. G. (2007).
Improving the in vivo predictability of an on-line HPLC stable free radical
decoloration assay for antioxidant activity in methanol buffer medium. Free
Radical Biology and Medicine, 42(3), 413–423.
Barton, H. J. (2010). A ‘‘Zero Sample Concentration Approach’’: Standardization of
methods for the estimation of total antioxidant activity by the use of
extrapolation to zero sample concentration. A novel standard. 1. ABTS cation
radical scavenging. Journal of Agriculture and Food Chemistry, 58(16),
8918–8926.
Baydar, N. G., Özkan, G., & Yasar, S. (2007). Evaluation of the antiradical and
antioxidant potential of grape extracts. Food Control, 18(9), 1131–1136.
Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free-radical method
to evaluate antioxidant activity. LWT–Food Science and Technology, 28(1), 25–30.
Cheng, Z. H., Moore, J., & Yu, L. L. (2006). High-throughput relative DPPH radical
scavenging capacity assay. Journal of Agricultural and Food Chemistry, 54(20),
7429–7436.
Deng, J., Cheng, W. Y., & Yang, G. Z. (2011). A novel antioxidant activity index (AAU)
for natural products using the DPPH assay. Food Chemistry, 125(4), 1430–1435.
Ehrlich, K., Viirlaid, S. D., Mahlapuu, R., Saar, K. L., Kullisaar, T., Zilmer, M., et al.
(2007). Design, synthesis and properties of novel powerful antioxidants,
glutathione analogues. Free Radical Research, 41(7), 779–787.
Fiore, A., La Fauci, L., Cervellati, R., Guerra, M. C., Speroni, E., Costa, S., et al. (2005).
Antioxidant activity of pasteurized and sterilized commercial red orange juices.
Molecular Nutrition and Food Research, 49(12), 1129–1135.
Floegel, A., Kim, D.-O., Chung, S.-J., Koo, S. I., & Chun, O. K. (2011). Comparison of
ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich
US foods. Journal of Food Composition and Analysis, 24(7), 1043–1048.
Friaa, O., & Brault, D. (2006). Kinetics of the reaction between the antioxidant Trolox
and the free radical DPPH in semi-aqueous solution. Organic and Biomolecular
Chemistry, 4(12).
Galano, A. (2011). On the direct scavenging activity of melatonin towards hydroxyl
and a series of peroxyl radicals. Physical Chemistry Chemical Physics, 13(15),
7178–7188.
Leopoldini, M., Russo, N., & Toscano, M. (2011). The molecular basis of working
mechanism of natural polyphenolic antioxidants. Food Chemistry, 125(2),
288–306.
Lim, Y. Y., Lim, T. T., & Tee, J. J. (2007). Antioxidant properties of several tropical
fruits: A comparative study. Food Chemistry, 103(3), 1003–1008.
Litwinienko, G., & Ingold, K. U. (2003). Abnormal solvent effects on hydrogen atom
abstractions. 1. The reactions of phenols with 2,2-diphenyl-1-picrylhydrazyl
(dpph) in alcohols. The Journal of Organic Chemistry, 68(9), 3433–3438.
Litwinienko, G., & Ingold, K. U. (2004). Abnormal solvent effects on hydrogen atom
abstraction. 2. Resolution of the curcumin antioxidant controversy. The role of
sequential proton loss electron transfer. The Journal of Organic Chemistry, 69(18),
5888–5896.
Luo, L., Wang, R., Wang, X., Ma, Z., & Li, N. (2012). Compounds from Angelica keiskei
with NQO1 induction, DPPH scavenging and a-glucosidase inhibitory activities.
Food Chemistry, 131(3), 992–998.
May, J. M. (1999). Is ascorbic acid an antioxidant for the plasma membrane? The
FASEB Journal, 13(9), 995–1006.
Mishra, K., Ojha, H., & Chaudhury, N. K. (2012). Estimation of antiradical properties
of antioxidants using DPPH (center dot) assay: A critical review and results.
Food Chemistry, 130(4), 1036–1043.
Mrazek, N., Watla-iad, K., Deachathai, S., & Suteerapataranon, S. (2012). Rapid
antioxidant capacity screening in herbal extracts using a simple flow injection-
spectrophotometric system. Food Chemistry, 132(1), 544–548.
794 F. Abderrahim et al. / Food Chemistry 141 (2013) 788–794
Müller, L., Fröhlich, K., & Böhm, V. (2012). Comparative antioxidant activities of
carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS
bleaching assay (TEAC), DPPH assay and peroxyl radical scavenging assay. Food
Chemistry, 129(1), 139–148.
Ordoudi, S. A., Tsermentseli, S. K., Nenadis, N., Assimopoulou, A. N., Tsimidou, M. Z.,
& Papageorgiou, V. P. (2012). Structure–radical scavenging activity relationship
of alkannin/shikonin derivatives. Food Chemistry, 124(1), 171–176.
Scherer, R., & Godoy, H. T. (2009). Antioxidant activity index (AAI) by the 2,2-
diphenyl-1-picrylhydrazyl method. Food Chemistry, 112(3), 654–658.
Stasko, A., Brezová, V., Biskupic, S., & Misík, V. (2007). The potential pitfalls of using
1,1-diphenyl-2-picrylhydrazyl to characterize antioxidants in mixed water
solvents. Free Radical Research, 41(4), 379–390.
Wootton-Beard, P. C., Moran, A., & Ryan, L. (2012). Stability of the total antioxi-
dant capacity and total polyphenol content of 23 commercially available
vegetable juices before and after in vitro digestion measured by FRAP, DPPH,
ABTS and Folin–Ciocalteu methods. Food Research International, 44(1), 217–
224.