The document examines the scavenging capacity and synergistic effects of combinations of natural antioxidants vitamin C, gallic acid, and tannic acid on the DPPH● free radical. It finds that a binary mixture of vitamin C (5.0 μM) and tannic acid (5.0 μM) had the highest synergistic effects. Experiments measured the antioxidant activity of individual compounds and mixtures using the DPPH● assay. Results demonstrated that the antioxidant property of certain combinations was substantially superior to the sum of the individual components, indicating synergistic interactions that could enhance effectiveness in functional foods.

1. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
8
THE SCAVENGING CAPACITY AND
SYNERGISTIC EFFECT OF SOME NATURAL
ANTIOXIDANTS MIXTURES ON THE DPPH●
FREE RADICAL
Fouad A. Ahmed1*
, Mohamed M. Rashed1
, Nasser S. A. M. Khalil2
and Mahmoud A. A. M. Hashem2
1
Faculty of Agriculture, Cairo University, Giza, Egypt
2
Regional Center for Food and Feed, Agricultural Research Center, Giza, Egypt
ABSTRACT
The biological activity of the natural antioxidant vi-
tamin C can be enhanced by the presence of some other
active natural antioxidants, such as gallic and tannic acid.
Since many of these natural antioxidants are consumed
sometimes together in foods, the potency for synergistic
interactions is high in the human diet. The current study
was conducted to determine what concentrations and com-
binations of antioxidants (vitamin C, gallic acid and tannic
acid) were capable to produce synergistic antioxidant ef-
fects. Scavenging capacities of solutions of the selected
compounds alone and in different combinations were meas-
ured using the stable free radical 1,1-diphenyl-2-picrylhy-
drazyl (DPPH●
) method. Different levels of each antioxidant
were used. A comparison of the scavenging capacity
(antioxidant activity) of these combinations to antioxi-
dants arithmetic sum of the scavenging capacities of the
individual antioxidants was used to calculate the synergis-
tic effects (SEs) between the used antioxidants. The results
showed that the mixture that had the highest SEs was the
binary mixture combining vitamin C (5.0 µM) and tannic
acid (5.0 µM). The results obviously demonstrated that
the antioxidant property of this combination was substan-
tially superior to the sum of the individual antioxidant
effects, and these interactions can enhance the antioxidant
effectiveness of these natural antioxidants. The results could
guide in the formulation and development of functional
food products that have high antioxidant potency.
KEYWORDS: Antioxidant; vitamin C; DPPH
●
; scavenging capac-
ity; synergistic effect
1. INTRODUCTION
Antioxidants are considered to be important nutraceu-
ticals on account of many health benefits [1-3]. Phenolic
compounds like gallic acid, tannic acid, vitamin C, or
vitamin E exist naturally in different plants and fruits. These
compounds possess the ability to reduce oxidative damage,
that are believed to cause many diseases including cancer,
cardiovascular diseases, cataracts, atherosclerosis, diabetes,
arthritis, immune deficiency diseases and ageing [4-6]. Re-
cently, the ability of phenolic substances including fla-
vonoids and phenolic acids to act as antioxidants have been
extensively investigated [7]. Natural antioxidants exist in
nature in combination, and a combination of different anti-
oxidants might act additively and even synergistically [8].
The radical-scavenging capacity was determined by the
DPPH●
assay which is a rapid, easy and inexpensive way.
DPPH●
(1,1-diphenyl-2-picrylhydrazyl) is a stable radical
of organic nitrogen, characterized by a typical deep purple
color and a maximum absorbance in the range of 515–
520 nm [9]. Scavenging of DPPH●
radical is the basis of
the popular DPPH●
antioxidant assay [10-12]. The DPPH●
method is technically simple and needs only a UV–VIS
spectrophotometer to perform in the presence of a hydro-
gen/electron donor (free radical scavenging antioxidant).
The absorption intensity is decreased, and the radical solu-
tion is discolored according to the number of electrons
captured [13]. The DPPH●
-based method was first reported
by Blois [14] who observed the reduction of the DPPH●
radical by the thiol-containing amino acid cysteine and
other active compounds. Later, Williams et al. [15] revised
the original method and the DPPH●
radical scavenging test
became a reference point to evaluate the in vitro antioxi-
dant capacity [16]. The purpose of this thesis was to com-
pare the antioxidant activity of vitamin C, gallic acid and
tannic acid at different concentrations, alone and in com-
binations, as well as the synergistic effects of these com-
binations, using (DPPH●
) free radical scavenging capacity
assay.

2. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
9
2. MATERIALS AND METHODS
2.1. Materials
DPPH-radical and ethanol absolute extra pure were ob-
tained from Sigma–Aldrich (U.S.A). All natural antioxi-
dants (gallic acid, tannic acid and vitamin C) were used in
pure form and purchased from Sigma-Aldrich. All reagents
used in this study were of analytical grade.
2.2. Methods
a. Design of work
This study has been designed to determine what con-
centrations and combinations of antioxidants vitamin C,
gallic acid and tannic acid were capable to produce syner-
gistic antioxidant effects. Scavenging capacities of solutions
of the selected compounds alone and in different combina-
tions were measured using the stable free radical (DPPH●
)
method.
Different levels of each antioxidant were used. A com-
parison of the scavenging capacity (antioxidant activity) of
these combinations to antioxidants arithmetic sum of the
scavenging capacities of the individual antioxidants was
used to calculate the synergistic effects (SEs) between the
used antioxidants.
b. Preparation of samples
Several parameters were investigated in this thesis:
1 - Preparation of individual antioxidant solutions
2 - Preparation of binary mixture antioxidant solutions
3 - Preparation of tertiary mixture antioxidant solutions
4 - Determination of antioxidants activity against DPPH●
free-radical
5 - Calculation of synergistic effects (SEs) of antioxidant
mixtures
c. Preparation of individual antioxidant solutions
Stock solutions were prepared in ethanol and stored at
0 °C in tightly closed dark brown glass bottles. All proce-
dures and all stock solutions were stirred at room tem-
perature for 10 min before further use.
The composition and concentration of individual an-
tioxidant solutions were as follows:
a) The individual antioxidant solution concentrations
of vitamin C were 1, 2, 5, 10 and 20 µM.
b) The individual antioxidant solution concentrations
of tannic acid were 1, 2, 5, 10 and 20 µM.
c) The individual antioxidant solution concentrations
of gallic acid were 1, 2, 5, 10 and 20 µM.
d. Preparation of binary mixture antioxidant solutions
The composition and concentration of the binary an-
tioxidant mixture solutions are shown in Table 1.
TABLE 1 - Composition and concentration (µM) of binary antioxi-
dant mixture solutions.
Gallic acidTannic acidVitamin CCode
1.01.0Mix1
1.01.0Mix2
1.01.0Mix3
2.02.0Mix4
2.02.0Mix5
2.02.0Mix6
5.05.0Mix7
5.05.0Mix8
5.05.0Mix9
10.010.0Mix10
10.010.0Mix11
10.010.0Mix12
e. Preparation of tertiary mixture antioxidant solutions
Composition and concentration of the tertiary anti-
oxidant mixture solutions are shown in Table 2.
TABLE 2. Composition and concentration (µM) of tertiary antioxi-
dant mixture solutions.
Gallic acidTannic acidVitamin CCode
1.01.01.0Mix13
2.02.02.0Mix14
5.05.05.0Mix15
10.010.010.0Mix16
1.02.02.0Mix17
2.01.02.0Mix18
2.02.01.0Mix19
10.05.05.0Mix20
5.05.010.0Mix21
5.010.05.0Mix22
10.05.02.0Mix23
5.010.02.0Mix24
10.02.05.0Mix25
2.010.05.0Mix26
2.05.010.0Mix27
5.02.010.0Mix28
5.010.020.0Mix29
10.05.020.0Mix30
5.020.010.0Mix31
20.05.010.0Mix32
20.010.05.0Mix33
10.020.05.0Mix34
10.010.020.0Mix35
10.020.010.0Mix36
20.010.010.0Mix37
a) DPPH free radical-scavenging capacity assay
The radical-scavenging capacity was determined by
The DPPH●
assay to evaluate the antioxidant activity. To
measure the scavenging capacity of a single antioxidant or
a mixture of antioxidants, the required amount of antioxi-
dant was pipetted into a cuvette. These antioxidant mix-
tures were subjected to preincubation at room temperature
for 5 min in the dark. To start the reaction, 1 ml of DPPH
in ethanol (0.3 mmol/L, stored at 0 C°) was added to 2 ml
of the antioxidant mixture in a teflon-capped cuvette at
room temperature. The mixtures were shaken quickly,
placed into the cell holder and the absorbance was measured
at 540 nm with UV-VIS Double Beam Spectrophotometer
Model UVD-3500. Each cuvette was removed from the
spectrophotometer and incubated at room temperature. To
make sure that they were not exposed to light, the cuvettes

3. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
10
were covered with an opaque container. Absorbance at
540 nm was measured every 10 min over a 60-min period.
b) Calculation of synergistic effects (SEs) of antioxidant
mixtures
The experimental scavenging capacity (ESC) of anti-
oxidant mixtures was calculated using the following equa-
tion:
%ESC =100-((Abs control – Abs sample) ×100/Abs control))
where: Abs sample is the absorbance value of the sam-
ple (2.0 ml antioxidant(s) solution plus DPPH●
solution
0.3 mM, in ethanol), and at each time interval, absolute
ethanol was used instead of the sample solution as blank.
Abs control is the absorbance value of control (DPPH●
solu-
tion 0.3 mM, in ethanol). The theoretical scavenging capac-
ity (TSC) is the sum of the scavenging capacities of each
antioxidant, calculated using the individual scavenging
capacity in the following equation [8]:
%TSC = 100 - [(100 – ESC A1) × (100 – ESC A2/100)
× (100 – ESC A3/100) × (100 – ESC A4/100)]
where: ESC A1- ESC A4 represents the percentage
ESC of the individual antioxidant.
A modified version of %TSC = 100 - [(100 – ESC A1)
× (100 – ESC A2/100) × (100 – ESC A3/100)]
The synergistic effects (SEs) of a combination of an-
tioxidants were based on the ratios of the experimental
ESCs and the theoretical TSCs, and were calculated using
the following equation [8]:
SE = ESC/TSC
where: synergism was shown when SE was greater
than 1 (SE >1).
c) Statistical analysis
A randomized complete block design was used for
analysis of all data with three replications for each parame-
ter. The treatment means were compared by least signifi-
cant differences (L.S.D.) test as given by Snedecor and
Cochran [17].
3. RESULTS AND DISCUSSION
Interest in the search for new synergistic antioxidant
effects and scavenging capacities of natural antioxidants
against reactive oxygen species (ROS) production and
oxidative stress has been shown to be linked to aging-
related illnesses [18], and a large number of other illnesses.
So, the aim of this study was to determine what concentra-
tions and combinations of vitamin C, gallic acid and tan-
nic acid were capable to produce synergistic antioxidant
effects in vitro and in vivo in order to find new potential
sources of natural antioxidants.
1. In vitro antioxidant scavenging activity by DPPH
.
radical
model system
The experimental scavenging capacity (ESC) kinetic
curves of DPPH●
free radicals by single, binary and terti-
ary antioxidants at various concentrations of the 49 sam-
ples were determined spectrophotometrically. DPPH●
is a
more stable free radical. Antioxidants, on interaction with
DPPH●
, either transfer electrons or hydrogen atoms to
DPPH●
, thus neutralizing free radical character [19]. The
color of the reaction mixture changes from purple to yel-
low, and its absorbance at 517 nm decreases. The scav-
enging effect of 49 samples on DPPH●
radical changed
significantly (P < 0.05) at different concentrations and com-
binations, shown in Table 3, 4, 5a and 5b as well as graphi-
cally illustrated in Figs. (1-5).
a. Antioxidant activity of single compounds
The changes in scavenging capacity of single antioxi-
dants measured with the DPPH●
free radical is shown in
Figs. 1 a, b and c (using 1, 2, 5, 10 and 20 µM) of each
individual antioxidant.
Tannic acid was a very efficient antioxidant, showing
a large increase in scavenging capacity at all concentra-
tions, where the single tannic acid at a concentration of
20.0 µM reached maximum experimental scavenging ca-
pacity (96.98 and 97.24 %) after 30 and 60 min, respec-
tively. The experimental scavenging capacities for the con-
centrations 5.0 and 10.0 µM showed 95.99 and 96.98 %
after 30 min, respectively, for % ESC (Fig. 1c). In all
cases, the scavenging capacity had not strong increases
after the first 10 min of incubation period.
TABLE 3 - Experimental scavenging capacity percentages (% ESC) for individual antioxidant at 30 and 60 minutes.
30 min 60 minConcentration
(µM) V.C G.A T.A V.C G.A T.A
1.0 µM 63.32e
± 0.25 65.67e
± 0.44 70.52d
± 0.87 63.29d
± 0.18 66.46e
± 0.15 72.54d
± 0.61
2.0µM 68.55b
± 0.58 67.98d
± 0.28 78.60c
± 4.33 69.29a
± 0.09 68.66d
± 0.12 82.11c
± 0.31
5.0µM 64.64d
± 0.39 72.57c
± 0.72 92.38b
± 3.56 65.29c
± 0.12 73.68c
± 0.23 95.85b
± 0.26
10.0µM 69.14a
± 0.14 79.06b
± 1.41 95.99a
± 0.47 69.55a
± 0.14 80.97b
± 0.35 96.35b
± 0.16
20.0µM 65.47c
± 0.37 89.27a
± 2.7 96.98a
± 0.39 66.21b
± 0.15 91.82a
± 0.29 97.24a
± 0.05
LSD 0.27 1.61 2.92 0.27 0.19 0.53
Each value in the table was obtained by calculating the average of the three experiments ±S.D. The superscript letters indicated statistically significant
differences, with P <0.05.

4. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
11
62.00
63.00
64.00
65.00
66.00
67.00
68.00
69.00
70.00
71.00
0 10 20 30 40 50 60
Time (min.)
E
S
C
%
60.00
65.00
70.00
75.00
80.00
85.00
90.00
95.00
0 10 20 30 40 50 60
Time (min.)
E
S
C
%
68.00
72.00
76.00
80.00
84.00
88.00
92.00
96.00
100.00
0 10 20 30 40 50 60
Time (min.)
E
S
C
%
a) Vitamin C
b) Gallic acid
c) Tannic acid
FIGURE 1 - Experimental scavenging capacity (ESC) kinetic curves
of DPPH●
free radicals by single antioxidants at various concentra-
tions. Data represent means ±S.E. for n = 3. a) Vitamin C, (b) gallic
acid and (c) tannic acid by concentrations of (●, 20.0µM; ,10.0µM;
▲,5.0µM; ■,2.0µM and ♦,1.0µM).
Studying the experimental scavenging capacity of vi-
tamin C at the same concentrations (1, 2, 5, 10 and 20 µM)
revealed an independent character, since the highest vita-
min C concentration (20.0 µM) did not produce the high-
est scavenging capacity (% ESC = 65.47%; Table 3). Low
vitamin C concentration (2.0 µM) was used to keep the
scavenging capacity at reasonable levels (% ESC = 68.55
%). On the other hand, vitamin C (2.0 and 10.0 µM) had
insignificant difference, so their results were better than
with 1.0, 5.0 and 20.0 µM of vitamin C. Similar results
were reported by Liu et al. [20] where the reaction of
vitamin C with DPPH●
was completed before 1 min from
starting. At tannic acid concentrations 1 and 2 µM, experi-
mental scavenging capacity was 70.42 and 79.96 %, re-
spectively. The reaction of gallic acid with DPPH●
was
similar to that with tannic acid (reaction almost complete
after 10 min) and revealed dose dependence (Figs. 1b and c).
In both cases, the highest antioxidant level produced the
highest scavenging capacity values.
b. Antioxidant activity of binary-antioxidant mixture
Further studies were achieved to investigate the ex-
perimental scavenging capacity of gallic acid combination
with other antioxidants. Thus, among 12 possible combi-
nations containing 2 antioxidants, only one combination
(Mix. 7, Table 4), which contained vitamin C (5.0 µM)
and tannic acid (5.0 µM) had the highest significant syn-
ergistic effect SE (SE >1). Under these conditions, it is
possible to say that tannic acid is regenerating vitamin C in
Mix.7 (Fig. 2). At any case of binary mixtures containing
tannic acid at the same conditions, the experimental scav-
enging capacity was higher than the others. Tannic acid
was a very efficient antioxidant, showing a large increase in
scavenging capacity in a dose-dependent effect, explaining
the importance of tannic acid and its strong scavenging
activity against DPPH●
free radical. The synergistic effect of
tannic acid with vitamin C and gallic acid was clearly ob-
served among the binary mixtures at the same conditions
(gallic acid with vitamin C showed lower effect than tannic
acid with vitamin C in binary mixtures under the same
conditions; Table 4).
TABLE 4 - Experimental scavenging capacity (% ESC), theoretical scavenging capacity
percentages (% TSC) and synergistic effect (SE) for binary antioxidant mixtures at 30 and 60 min.
30 min. 60 min.Code
%ESC %TSC SE %ESC %TSC SE
Mix 1 73.62e
± 1.10 89.19g
± 0.39 0.82cd
± 0.01 75.40g
± 0.24 89.91h
± 0.19 0.84d
± 0.00
Mix 2 68.62g
± 0.86 75.88h
± 0.12 0.91b
± 0.01 69.34j
± 0.05 75.52i
± 0.06 0.92bc
± 0.01
Mix 3 72.14f
± 1.52 89.87f
± 0.41 0.80de
± 0.01 74.07h
± 0.28 90.79f
± 0.25 0.82de
± 0.01
Mix 4 74.91d
± 1.19 93.58d
± 0.87 0.80de
± 0.01 77.37f
± 0.45 94.51c
± 0.11 0.82de
± 0.01
Mix 5 66.95h
± 0.53 89.93f
± 0.25 0.75f
± 0.01 67.66k
± 0.13 90.38g
± 0.06 0.75f
± 0.00
Mix 6 79.65c
± 2.17 93.47d
± 0.79 0.85c
± 0.02 82.32d
± 0.37 94.39c
± 0.12 0.87cd
± 0.00
Mix 7 93.20a
± 3.04 92.78e
± 0.92 1.01a
± 0.02 95.22a
± 0.00 93.79d
± 0.13 1.02a
± 0.01
Mix 8 68.26g
± 1.58 90.30f
± 0.36 0.76ef
± 0.01 70.56i
± 0.35 90.87f
± 0.11 0.78ef
± 0.01
Mix 9 91.94ab
± 1.92 97.89c
± 1.05 0.94b
± 0.01 92.90b
± 0.00 98.91b
± 0.08 0.94b
± 0.00
Mix 10 92.47ab
± 0.05 98.62b
± 0.17 0.94b
± 0.01 92.54b
± 0.00 98.77b
± 0.05 0.94b
± 0.00
Mix 11 78.39c
± 1.22 92.77e
± 0.57 0.84cd
± 0.01 80.39e
± 0.33 93.57e
± 0.15 0.86d
± 0.00
Mix 12 91.61b
± 0.10 99.16a
± 0.16 0.92b
± 0.01 91.50c
± 0.04 99.31a
± 0.02 0.92bc
± 0.00
LSD 1.29 0.49 0.05 0.94 0.15 0.05
Each value in the table was obtained by calculating the average of the three experiments ±S.D. The superscript letters indicate statistically significant differences, with P <0.05.

5. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
12
As shown in Fig. 2, only antioxidant Mix 7 had
ESC/TSC ratios >1 showing a significant SE >1.01. When
vitamin C was omitted from Mix 7 and replaced by gallic
acid (Mix 9), the SE was decreased to 0.94. On the other
hand, the mixture of tannic acid and vitamin C (Mix 10)
which has the proper antioxidant composition, (tannic acid
10.0 µmol/L and vitamin C 10.0 µmol/L) produced SE
ratio just like that of Mix 9, i.e. 0.94.
c. Antioxidant activity of tertiary antioxidant mixture
As shown in Tables 5a and 5b, there were not any ter-
tiary antioxidant mixtures with ESC/TSC ratios >1. Mix
19 showed a significant synergistic effect and SE = 0.96.
When vitamin C was omitted from Mix 19 as in the case
of Mix 6 (Fig. 3), SE decreased to 0.87.
0
20
40
60
80
100
120
M
ix.1
M
ix.2
M
ix.3
M
ix.4
M
ix.5
M
ix.6
M
ix.7
M
ix.8
M
ix.9
M
ix.10
M
ix.11
M
ix.12
Binary antioxidant mixtures
(%)Scavengingcapacity
ESC% TSC%
FIGURE 2 - Comparison of observed experimental scavenging capacity (□: ESC) and theoretical scavenging capacity (■: TSC) of antioxi-
dant in binary-antioxidant mixtures at 60 min. Values are means ± S.E.
TABLE 5a - Experimental scavenging capacity percentages (% ESC), theoretical scavenging capacity percentages (% TSC) and synergistic
effect (SE) for tertiary antioxidant mixtures at 30 min.
30 min.Code
%ESC %TSC SE
Mix 13 73.92j
± 1.67 96.29o
± 0.18 0.77ef
± 0.02
Mix 14 79.59i
± 2.24 97.94kl
± 0.29 0.81de
± 0.02
Mix 15 88.43g
± 0.02 99.25hi
± 0.38 0.89c
± 0.01
Mix 16 90.40f
± 0.14 99.71cde
± 0.06 0.91bc
± 0.00
Mix 17 78.66i
± 2.42 97.79lm
± 0.33 0.81de
± 0.02
Mix 18 71.29k
± 1.34 97.02n
± 0.15 0.74f
± 0.01
Mix 19 93.83a
± 0.14 97.60m
± 0.31 0.96a
± 0.00
Mix 20 93.16abc
± 0.08 99.42gh
± 0.32 0.94ab
± 0.00
Mix 21 92.33b-e
± 0.28 99.27hi
± 0.37 0.93abc
± 0.00
Mix 22 92.68a-e
± 0.14 99.61d-g
± 0.06 0.93abc
± 0.00
Mix 23 92.70a-e
± 0.13 99.48fg
± 0.30 0.93abc
± 0.00
Mix 24 93.06a-d
± 0.02 99.65c-f
± 0.06 0.93abc
± 0.00
Mix 25 93.40ab
± 0.18 98.48j
± 0.30 0.95ab
± 0.01
Mix 26 93.83a
± 0.14 99.54efg
± 0.06 0.94ab
± 0.00
Mix 27 91.51ef
± 1.08 99.16i
± 0.41 0.93abc
± 0.01
Mix 28 83.02h
± 3.37 98.06k
± 0.30 0.85d
± 0.03
Mix 29 91.75def
± 0.22 99.66c-f
± 0.05 0.92abc
± 0.00
Mix 30 94.08a
± 1.45 99.50fg
± 0.28 0.95ab
± 0.01
Mix 31 91.91cde
± 0.08 99.71cde
± 0.05 0.92abc
± 0.00
Mix 32 93.16abd
± 0.08 99.92ab
± 0.05 0.93abc
± 0.00
Mix 33 92.33b-e
± 0.28 99.96a
± 0.01 0.92abc
± 0.01
Mix 34 92.68a-e
± 0.14 99.78a-d
± 0.05 0.93abc
± 0.00
Mix 35 92.70a-e
± 0.13 99.74b-e
± 0.05 0.93abc
± 0.00
Mix 36 93.06a-d
± 0.02 99.78a-d
± 0.05 0.93abc
± 0.00
Mix 37 93.40ab
± 0.18 99.85abc
± 0.06 0.93abc
± 0.00
LSD 1.41 0.19 0.05
Each value was obtained by calculating the average of the three experiments ±S.D. The superscript letters indicated statistically significant differences, with P <0.05.

6. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
13
TABLE 5b - Experimental scavenging capacity percentages (% ESC), theoretical scavenging capacity percentages (% TSC) and synergistic
effect (SE) for tertiary antioxidant mixtures at 60 min.
60 min.Code
%ESC %TSC SE
Mix 13 75.94k
± 0.34 96.62r
± 0.08 0.79fg
± 0.01
Mix 14 82.75i
± 0.55 98.28n
± 0.04 0.84de
± 0.01
Mix 15 88.57j
± 0.06 99.62ij
± 0.03 0.89bcd
± 0.00
Mix 16 90.51f
± 0.05 99.77def
± 0.01 0.91abc
± 0.00
Mix 17 81.54j
± 0.41 98.16o
± 0.05 0.83ef
± 0.00
Mix 18 73.28l
± 0.33 97.36q
± 0.08 0.75g
± 0.01
Mix 19 93.87ab
± 0.02 97.94p
± 0.04 0.96a
± 0.00
Mix 20 93.16bc
± 0.02 99.73fg
± 0.03 0.93abc
± 0.00
Mix 21 92.17e
± 0.02 99.63ij
± 0.03 0.93abc
± 0.01
Mix 22 92.62cde
± 0.00 99.67hi
± 0.01 0.93abc
± 0.00
Mix 23 92.74cde
± 0.04 99.76efg
± 0.02 0.93abc
± 0.00
Mix 24 93.07bcd
± 0.00 99.71gh
± 0.00 0.93abc
± 0.00
Mix 25 93.27bc
± 0.00 98.82l
± 0.05 0.94ab
± 0.00
Mix 26 93.87ab
± 0.02 99.60jk
± 0.02 0.94ab
± 0.00
Mix 27 92.19de
± 0.02 99.56k
± 0.03 0.93abc
± 0.00
Mix 28 86.64h
± 0.42 98.41m
± 0.05 0.88cde
± 0.00
Mix 29 91.93e
± 0.06 99.71gh
± 0.01 0.92abc
± 0.00
Mix 30 94.72a
± 0.04 99.76efg
± 0.02 0.95a
± 0.00
Mix 31 91.89e
± 0.02 99.76efg
± 0.01 0.92abc
± 0.00
Mix 32 93.16bc
± 0.02 99.96a
± 0.00 0.93abc
± 0.00
Mix 33 92.17e
± 0.02 99.97a
± 0.01 0.92abc
± 0.00
Mix 34 92.62cde
± 0.00 99.82cd
± 0.01 0.93abc
± 0.00
Mix 35 92.74cde
± 0.04 99.79cde
± 0.01 0.93abc
± 0.00
Mix 36 93.07bcd
± 0.00 99.82c
± 0.01 0.93abc
± 0.00
Mix 37 93.27bc
± 0.00 99.90b
± 0.00 0.93abc
± 0.00
LSD 0.90 0.05 0.05
Each value was obtained by calculating the average of the three experiments ±S.D. The superscript letters indicated statistically significant differ-
ences, with P <0.05.
0
20
40
60
80
100
120
M
ix
13
M
ix
14
M
ix
15
M
ix
16
M
ix
17
M
ix
18
M
ix
19
M
ix
20
M
ix
21
M
ix
22
Tertiary antioxidant mixtures
(%)Scavengingcapacity
%ESC %TSC
FIGURE 3 - Comparison of observed experimental scavenging capacity (□: ESC) and theoretical scavenging capacity (■: TSC) of antioxidant
in tertiary-antioxidant mixtures at 60 min. Values are means ± S.E.

7. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
14
75
80
85
90
95
100
105
M
ix
23
M
ix
24
M
ix
25
M
ix
26
M
ix
27
M
ix
28
M
ix
29
M
ix
30
Tertiary antioxidants mixtures
(%)Scavengingcapacity
%ESC %TSC
FIGURE 4 - Comparison of observed experimental scavenging capacity (□: ESC) and theoretical scavenging capacity (■: TSC) of antioxidant
in tertiary-antioxidant mixtures at 60 min. Values are means ± S.E.
86
88
90
92
94
96
98
100
102
M
ix
31
M
ix
32
M
ix
33
M
ix
34
M
ix
35
M
ix
36
M
ix
37
Tertiary antioxidants mixtures
(%)Scavengingcapacity
%ESC %TSC
FIGURE 5 - Comparison of observed experimental scavenging capacity (□: ESC) and theoretical scavenging capacity (■: TSC) of antioxidant
in tertiary-antioxidant mixtures at 60 min. Values are means ± S.E.
Even more, Mix 30, which has the proper antioxidant
composition, produced SE ratio <1. The lack of agreement
of such results in the present work with other studies might
be due to different factors, such as the present model sys-
tem utilized for the free radical DPPH●
that reacts with
different antioxidants at different rates [21]. Vitamin C reacts
immediately with DPPH●
, which would indicate that vitamin
C reacted predominantly with DPPH●
, and not with the
antioxidant free radicals in some cases. The reaction mix-
ture solvent in this study was ethanol. It was suggested
that water-soluble chain radicals, such as vitamin C, func-
tion as a primary defense against aqueous radicals [22].
Although Mix 37 (Fig. 5) had the highest levels of anti-
oxidant mixtures, it produced a similar value (SE=0.93) at
the same conditions as Mix 10 (SE = 0.94) which did not
contain gallic acid. Comparison of the tertiary mixtures
Mix 17, Mix 18 and Mix 20 to binary mixtures (one anti-
oxidant omitted) at the same conditions showed that the
results of these synergistic effects were almost-similar
and had no strong differences (Tables 5a and 5b).
There are many factors that might contribute to SEs
of mixed antioxidants in a biological system. The concen-
tration and combination ratio of mixed antioxidants are the
important factors. It has been shown that specific concen-
trations and antioxidants combinations in mixtures were
more effective than the corresponding single antioxidants
in the scavenging of DPPH●
. In this study, a significant
SE was produced only when tannic acid was present in a
mixture. For the mixtures of two antioxidants, tannic acid

8. © by PSP Volume 33 – No 1. 2011 Advances in Food Sciences
15
with vitamin C showed the highest SEs. For the mixtures
of three antioxidants, any of them did not show syner-
gism. The synergistic effect appeared to be based, in part,
on the reducing potential of the various antioxidants and
their ability to convert the antioxidant free radicals to their
native form. The results indicate that an optimum combina-
tion of antioxidants may play a significant role in enhance-
ment of the oxidative status of biological systems.
REFERENCES
[1] Droge, W. (2002). Free radicals in the physiological control
of cell function. Physiological Reviews, 82, 47–95.
[2] Lee, J., Koo, N. and Min, D. B. (2004). Reactive oxygen spe-
cies, aging, and antioxidative nutraceuticals. Comprehensive
Reviews in Food Science and Food Safety, 3, 21–33.
[3] Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M.
and Telser, J. (2007). Free radicals and antioxidants in normal
physiological functions and human disease. International Jour-
nal of Biochemistry and Cell Biology, 39, 44–84.
[4] Lee, K. G., Mitchell, A. E. and Shibamoto, T. (2000). Determi-
nation of antioxidant properties of aroma extracts from various
beans. Journal of Agricultural and Food Chemistry, 48, 4817–
4820.
[5] Middleton, E., Kandaswamy, C. and Theoharides, T. C. (2000).
The effects of plant flavonoids on mammalian cells: Implica-
tions for inflammation,heart disease, and cancer. Pharmacol-
ogical Reviews, 52, 673–751.
[6] Pietta, P., Simonetti, P. and Mauri, P. (1998). Antioxidant ac-
tivity of selected medicinal plants. Journal of Agricultural
and Food Chemistry,46, 4487–4490.
[7] Rice-Evans, C. A., Miller, N. J. and Paganda, G. (1996). Struc-
ture-antioxidant activity relationships of flavonoids and pheno-
lic acids. Free Radical Biology and Medicine, 20, 933–956.
[8] Fuhrman, B., Volkova, N., Rosenblat, M. and Aviram, M.
(2000). Lycopene synergistically inhibits LDL oxidation in
combination with vitamin E, glabridin, rosmarinic acid, car-
nosic acid, or garlic. Antioxidants and Redox Signaling, 2:
491–505.
[9] Eklund, P. C., Langvik, O. K., Warna, J. P., Salmi, T. O.,
Willfor, S. M. and Sjoholm, R. E. (2005). Chemical studies
on antioxidant mechanisms and free radical scavenging prop-
erties of lignans. Organic and Bimolecular Chemistry, 21,
3336–3347.
[10] Alma, M.H., Mavi, A., Yildirim, A., Digrak, M. and Hirata,
T. (2003). Screening chemical composition and in vitro anti-
oxidant and antimicrobial activities of the essential oils from
Origanum syriacum L. growing in Turkey. Biological and
Pharmaceutical Bulletin, 26, 1725–1729.
[11] Karioti, A., Hadjipavlou-Litina, D., Mensah, M. L., Fleischer,
T. C. and Skaltsa, H. (2004). Composition and antioxidant ac-
tivity of the essential oils of Xylopia aethiopica (Dun) A. Rich.
(Annonaceae) leaves, stem bark, root bark, and fresh and dried
fruits, growing in Ghana. Journal of Agricultural and Food
Chemistry, 52, 8094–8098.
[12] Kordali, S., Cakir, A., Mavi, A., Kilic, H. and Yildirim, A.
(2005). Screening of chemical composition, antifungal and
antioxidant activities of the essential oils from three Turkish
Artemisia species. Journal of Agricultural and Food Chemis-
try, 53, 1408–1416.
[13] Markowicz Bastos, D. H., Saldanha, L. A., Catharino, R. R.,
Sawaya, A. C. H. F., Cunha, I. B. S. and Carvalho, P. O.
(2007). Phenolic antioxidants identified by ESI-MS from
Yerba Maté (Ilex paraguariensis) and green tea (Camelia si-
nensis) extracts. Molecules, 12, 423–432.
[14] Blois, M.S. (1958). Antioxidant determination by the use of
stable free radical, Nature, 181, 1199–1200.
[15] Williams, B., Cuvelier, M.E. and Berset, C. (1995). Use of a
free radical method to evaluate antioxidant activity, Le-
bensm.-Wiss. Technol, 28, 25–30.
[16] Gil, M. I., Tomás-Barberán, F. A., Hess-Pierce, B., Holcroft,
D. M. and Kader, A. A. (2000). Antioxidant activity of po-
megranate juice and its relationship with phenolic composi-
tion and processing. Journal of Agricultural and Food Chem-
istry, 48, 4581–4589.
[17] Snedecor, G. A. and Cochran, W. G. (1976). Statistical Me-
thod. Iowa State Univ. Press, Ames.
[18] Finkel, T. and Holbrook, N. J. (2000). Oxidants, oxidative
stress and the biology of ageing. Nature, 408: 239.
[19] Naik, G. H., Priyadarsini, K. I., Satav, J. G., Banavalikar, M.
M., Sohoni, P. P., Biyani, M. K. and Mohan, H. (2003). Com-
parative antioxidant activity of individual herbal components
used in Ayurvedic medicine. Phytochemistry, 63: 97.
[20] Liu, D., Shi, J., Ibarra, A.C. and Kakuda, Y. S.J. (2008). The
scavenging capacity and synergistic effects of lycopene, vi-
tamin E,vitamin C, and β-carotene mixtures on the DPPH●
free radical. Food Science and Technology, 41, 1344–1349.
[21] Huang, D., Ou, B. and Prior, R. L. (2005). The chemistry be-
hind antioxidant capacity assays. Journal of Agricultural and
Food Chemistry, 53, 1841–1856.
[22] Sato, K., Niki, E. and Shimasaki, H. (1990). Free radical-
mediated chain oxidation of low-density lipoprotein and its
synergistic inhibition by vitamin E and vitamin C. Archives
of Biochemistry and Biophysics, 279, 402–405.
Received: August 11, 2010
Accepted: November 02, 2010
CORRESPONDING AUTHOR
Fouad A. Ahmed
Faculty of Agriculture
Cairo University
Giza
EGYPT
Phone: +002 0101577175
E-mail: fouad_ahmed46@yahoo.com
AFS/ Vol 33/ No 1/ 2011 – pages 8 - 15