International Journal of Cosmetic Science, 2010, 32, 7380

doi: 10.1111/j.1468-2494.2008.00483.x

Antioxidant kinetics of plant-derived substances and

extracts
A. R. Silva, P. F. C. Menezes, T. Martinello, G. F. L. Novakovich, C. E. O. Praes and I. H. S. Feferman
O Boticario, Av. Rui Barbosa n 3450, 83055-900 Sao Jose dos Pinhais, Brazil

Received 5 June 2008, Accepted 14 September 2008

Keywords: anti-ageing, antioxidant, DPPH, free radical, kinetics, plant extracts

Synopsis
The antioxidant activity (AA) of substances present in several plant species has been widely studied which reflects their fundamental role in the
protection of skin tissue against the harmful action
of reactive oxygen species. Given the importance
of effective and long-lasting protection against
ultraviolet radiation, we studied the AA of several
plant derivatives and extracts over time. Several
chemical in vitro methods may be used to evaluate
antioxidant capability, among which the 1,1diphenyl-2-picrylhydrazyl (DPPH) method stands
out, despite its unspecificity, as the most cited and
described method in the literature. In this work
the AA was evaluated by measuring their capacity
to reduce DPPH in 30 min, which is suggested in
the literature, and additionally at different times
up to 8 h from the baseline reading. The methodology used to evaluate the AA over time was validated. It is important to emphasize that this study
proposes to modify the conventional DPPH
method, although considered to be non-specific, to
be used to test new antioxidant agents. This represents a considerable advantage because some substances show no significant activity during the
first 30 min of reaction. Among other plant products, we tested a proantocyanidin-rich grapeseed
extract, a hesperidin derivative, a rutin-containing
ginkgo extract, a polyphenol-containing yerba
mate extract and tocopheryl acetate, all of which
were properly standardized. As they have different
Correspondence: Carlos Eduardo de Oliveira Praes, O Boticario, Av. Rui Barbosa n 3450, 83055-900 Sao Jose
dos Pinhais, Brazil. Tel.: 55 41 3381 7506; fax: 55 41
3381 7987; e-mail: carlosep@boticario.com.br

antioxidant profiles, each ingredient showed a specific behaviour over time, which may promote the
selection of anti-radical compounds capable of
offering protection against external agents. Combining extracts and plant derivatives that present
fast, medium and slow antioxidant kinetic it is possible to create complexes capable of offering an
effective protection from the moment of application
up to several hours later. It is a perfectly feasible
method, and such combinations prove to be more
effective and have more durable effect.
Resume
Lactivite antioxydante de substances presentes
dans differentes espe`ces de plantes a ete largement
etudiee. Elle refle`te leur role fondamental dans la
protection des tissus cutane contre laction alterante des espe`ces reactives de loxyge`ne (ROS). Etant
donne limportance dune protection efficace et
durable contre les effets des radiations UV, nous
avons mene une etude portant sur lactivite antioxydante de plusieurs derives de plantes et dextraits en fonction du temps. Plusieurs methodes
chimiques in vitro ont ete utilisees pour evaluer la
capacite antioxydante, parmi lesquelles la methode
bien connue au 1,1-diphenyl-2-picrylhydrazyl
(DPPH) qui est malgre sa non specificite, la plus
citee et la plus decrite dans la litterature. Dans ce
travail, lactivite antioxydante a ete evaluee en
mesurant la capacite a` reduire le DPPH en
30 min, ce qui est evoque dans la litterature, et de
facon complementaire, en la mesurant, a` differents
temps allant jusqua` 8 heures apre`s la premie`re
lecture. La methodologie utilisee pour evaluer lactivite antioxydante en fonction du temps a ete

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Antioxidant kinetics of plant-derived substances and extracts

validee. Il est important de signaler que cette etude

propose de modifier la methode conventionnelle
DPPH qui, bien que consideree comme non specifique, peut etre utilisee pour tester de nouveaux
agents antioxydants. Ceci represente un avantage
considerable car certaines substances ne montrent
aucune activite specifique pendant les 30 premie`res minutes de reaction. Parmi les differents
derives de plantes, nous avons teste un Extrait de
Pepins de Raisins riches en Proantocyanidine, un
Derive dHesperidine, un Extrait de Ginkgo contenant de la rutine, un Extrait de Mate Yerba contenant un Polyphenol et lAcetate de Tocopheryle,
tous etant correctement standardises. Puisque tous
ces produits posse`dent un profil antioxydant different, chacun dentre eux presente un comportement specifique en fonction du temps qui peut
promouvoir la selection de melanges danti-radicalaires capables doffrir une protection contre les
agents exterieurs. La combinaison dextraits et de
derives de plantes qui presentent des cinetiques
antioxydantes rapide, moyenne et lente rend possible la creation de complexes capables doffrir une
protection efficace depuis le moment de lapplication jusqua` plusieurs heures apre`s. Cest une
methode parfaitement realisable et de telles combinaisons save`rent plus efficaces et ont des effets
plus durables.
Introduction
Intrinsic or chronological, ageing is natural and
occurs with time. External agents are capable of
accelerating this process, particularly ultraviolet
(UV) radiation, which is responsible for the type of
ageing known as photoageing. A reduction in the
endogenous antioxidant defences and an increase
in the amounts of reactive oxygen species (ROS)
have also been reported to occur after excessive
exposure to sunlight, and may contribute to
destructuring of proteins and lipids in cells and
tissues [1, 2].
The free radicals are reactive chemical species
that contain one or more unpaired electrons. The
body normally maintains a state of equilibrium
between production and decay ROS. An unbalance
occurring between the reactive species produced
by the body and its endogenous antioxidant capacity generates a condition known as oxidative stress
[3].
Oxidative stress involves the formation of reactive species of oxygen, such as the superoxide

A. R. Silva et al.

(O2) and hydroxyl (OH) radicals, in addition to

hydrogen peroxide (H2O2) and singlet oxygen
(1O2), which are not completely neutralized, and
may react with substances, such as lipids, proteins,
carbohydrates, RNA, DNA, etc. and modify their
structure and functions. The presence of transition
metals catalyses the formation of these free radicals [4, 5].
As previously mentioned, the body and especially the skin, are routinely exposed to stressful
environmental factors such as pollutants and UV
radiation, which produce a large number of
aggressive oxidants that damage all the biological
skin cell membranes [6]. Skin is a tissue that consists of several cell layers containing proteins, lipids and DNA, which makes it one of the primary
targets for assault by ROS [7].
The body presents an antioxidant defence system that includes both enzymatic and non-enzymatic components. This system is essential for the
protection of the body against ROS attacks generated by the environment, because it enables the
antioxidants to effectively remove free radicals
from the body [8].
Therefore, it is important to combine several
antioxidant substances with different, but complementary, characteristics for a broader protection of
the skin (topical application) and the body (oral
application). There are three groups of antioxidants that may constitute the anti-radical protection system: the essential nutrients (vitamins),
enzymes and phytochemical substances. The latter
group includes a large number of plants that may
show an important antioxidant activity (AA)
because of their high content of active compounds
[810].
Plants produce a great variety of organic compounds and can be classified into three major
groups: terpenoids, alkaloids and phenolic compounds [11]. Flavonoids and phenolics also have
anti-tumour, antiviral and antibacterial activities,
and anti-radical and antioxidative activities [11].
The flavonoid rutin and other phenolic compounds
(hesperidin derivative) present in plant species are
widely described as natural antioxidants that are
capable of helping to neutralize ROS and acts in
the lipid peroxidation assay [12]. Tannins like proanthocyanidin found in the grapeseed extract, on
the other hand, display a capability to protect erythrocytes against oxidative stress [13]. Studies
have confirmed the activities of polyphenols
obtained from numerous plants such as green tea

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A. R. Silva et al.

Antioxidant kinetics of plant-derived substances and extracts

and Yerba Mate. Among the characteristics of

these polyphenols, the following deserve special
mention: chemopreventive and therapeutic activities in cancer treatment [14]; lipoperoxidation prevention in mammals [15]; prevention of adverse
effects caused by UV radiation, with a reduction of
oxidative damage and in metalloproteinase production [1618].
The phenolic compounds are characterized by
presenting in its chemical structure an aromatic
ring linked to a hydroxyl group, which has a great
ability to donate electron and hydrogen. This
explains their exceptional AA [19]. Vitamin E or
tocopheryl acetate acts against the lipoperoxidation removing free radicals. This vitamin is widely
used because of anti-tumoral, anti-inflammatory
and photoprotection properties [20].
In this work, we studied ginkgo, yerba mate
and grapeseed extracts as well as hesperidin derivatives, which are phenolic compounds, and tocopheryl acetate (vitamin).
In view of the beneficial properties attributed to
plant extracts and derivatives, companies in the
pharmaceutical (oral medicines), cosmetic (antiageing and sunscreen products) and food (cereal
bar, beverages) sectors have been increasingly
interested in using such ingredients in their products. Therefore, it is useful to consider the possibility of associating different substances with different
antioxidant profiles, which would provide a complementary of effects and lead to the cosmetics formulated for high effectiveness and long-lasting
action.
Several in vitro methods based on chemical reactions with 1,1-diphenyl-2-picrylhydrazyl (DPPH),
2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid)
and 2-thiobarbituric acid, among others, are being
used to evaluate antioxidant activity of raw materials and even cosmetic products.
Among the above methods, the DPPH radical
scavenging activity is the most used method as
it shows faster response and is less expensive
than the others, thus being useful as a screening
technique [12, 2130]. Antioxidants agents are
able to protect against free radical injuries and,
consequently, reduce the ageing process maintaining the skin homeostasis [2130].
In this study, the AA of extracts and plantderived substances were investigated by measuring
their capacity to reduce DPPH, a free radical stable
at room temperature, which produces a purple
solution in ethanol when reduced in the presence

of an antioxidant (hydrogen donor) molecule. In

the DPPH method (DPPH radical scavenging activity), the AA is determined by measuring the absorbance of the solution at a specific time and
wavelength [30].
The antioxidant kinetics of plant-derived substances and extracts were evaluated by the DPPH
method during 480 min (8 h) to select sufficient
kinetic parameters needed to determine their antioxidant efficiency. This represents a considerable
advantage because some substances do not show
significant activity during the first 30 min of
reaction.
The purpose of this work is to discuss the modification of the original DPPH method [21, 22, 30]
in relation to the time for analysis (30 min or a
kinetic study) as well as to simplify the methodology. It is already known that many antioxidant
actives do not demonstrate significant activity in
(after/within) 30 min (traditional DPPH protocol).
The new method is innovative because of its capability in identifying antioxidant substances in a
long-term test. This methodology is reliable as it
produces precise and reproducible results, and
when combined with other analytical methods
could be useful for the correct AA measurement.
Materials and methods
Reagents
The DDPH used in these experiments was
acquired from Sigma-Aldrich (Sao Paulo, Brazil).
The rutin-containing ginkgo extract and the proanthocyanidin-rich grapeseed extract were supplied by Croda (Sao Paulo, Brazil); hesperidin
derivative was obtained from Coletica (Sao Paulo,
Brazil); polyphenol-containing yerba mate wxtract
was from Sanrisil (Sao Paulo, Brazil) and tocopheryl acetate from BASF (Sao Paulo, Brazil).
The samples were prepared with ethanol (analytical grade minimum 960 GL of high purity) supplied by Usina Acucareira Ester S.A. (Sao Paulo,
Brazil). It is important to point out that the plant
extracts and derivatives used in this study had at
least one chemical marker that was standardized
and measured for each batch.
Antioxidant activity determination (EC50)
Each substance was analysed according to the
steps described below. A 0.3 mM DPPH ethanol

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75

A. R. Silva et al.

Antioxidant kinetics of plant-derived substances and extracts

solution prepared at the time of use and as

described in the literature [30] was used for the
preparation of the sample and control tubes. In
order to determine the AA of the analytes based
on their reaction with DPPH in ethanol solution,
the samples were first diluted in ethanol. Solutions
prepared with different concentrations of each
plant extract and derivative were used to determine the concentration needed to reduce the
amount of free radicals present in the medium by
50% in 30 min (EC50). After the ingredients of
interest were dissolved for analysis, 2.5 mL of each
solution was made to react with 1 mL of the
DPPH ethanol solution. Thirty minutes after the
beginning of the reaction, the absorbance of each
solution was read on a UV-Vis spectrophotometer
(Varian Cary 50 Conc: Varian, Palo Alto, CA,
USA), using a 1-cm optical path quartz cell and a
wavelength (k) of 518 nm.
For the EC50 determination, solutions were prepared for each extract in different concentrations.
Next, the AA percentage of each concentration
was determined using Equation 1:

and averaged results were used to calculate the

percentage of AA for each different time point in
the analysis (30 to 480 min), determining the
kinetic profile of each extract. All analyses were
carried out in triplicate.
Methodology validation
The DDPH method used in this paper to evaluate
the long-term antioxidant capability was validated
through the analysis of specificity, linearity, precision and accuracy parameters described in the
United States Pharmacopeia (USP) [31].
Statistical analysis
The data are presented as mean values SD. Differences between the EC50 of plant-derived substances and extracts were determined using
ANOVA combined with Students t-test (Student
NewmanKeuls test). For all analyses, P < 0.05
values were considered statistically significant.
Results and discussion

AA%
100sample absorbance - blank absorbance100
control absorbance
1
Using the AA% as a function of different concentration, it is possible to obtain the EC50 using a
linear fit (AA% vs. extracts concentration). All
analyses were performed in triplicate.

The EC50 values resulting from three trials performed during different days on each test ingredient are shown in Table I below. The EC50 values
of plant-derived substances and extracts demonstrated in Table I were considered statistically significant (P < 0.05). As observed in Table I, the
Table I EC50 values for the investigated plant-derived
substances and extracts

Determination of antioxidant activity over time

Raw materials tested

In order to select the appropriate concentration for

the study of time-dependent AA, the EC50 (the
required concentration to reduce free radicals by
50% within 30 min) was first established for each
plant extract and derivative, as described in the
previous section. The concentrations were then
adjusted to handle the same range of AA, which
made it possible to compare the data measured.
In contrast to the traditional DPPH method,
absorbance readings at the 518 nm wavelength
were taken every 5 min, during 8 h. As a result,
this investigation revealed the kinetics, and subsequently, the antioxidant profile of the plant
extracts/derivatives under analysis. All samples
were evaluated as described above (Equation 1)

Proantocianidin-rich
grapeseed extract (1)
Hesperidin derivative (2)

Rutin-containing ginkgo
extract (3)
Yerba mate extract (4)

Tocopheryl acetate (5)

EC50
(lg mL)1)

2185
1707
2032
42 332
50 057
45 717
3249
3050
2983
656
748
704
60 068
59 841
42 192

Average EC50
(lg mL)1) SD

1975 17

46 035 12

3094 5

703 9

54 034 19

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Antioxidant kinetics of plant-derived substances and extracts

extract (4) presented a lower EC50 than the other

extracts and derived substances being an indicative of high AA. The extracts (1) and (3) in comparison with substance (2) also show a higher
antioxidant potential being able to neutralize 50%
of the free radicals present in the reaction medium
in 30 min with approximately 18-fold less in concentrated solutions. The tocopheryl acetate, a wellknown antioxidant, showed a lower EC50 value
than the other extracts.
Although the structures of the active markers in
the test samples are mostly polyphenolic, the
remainder of the phytochemical composition contains important structural differences, which provided a positive contribution to the antioxidant
efficacy evaluated in this study with the DPPH
method.
Table II presents the AA differences (%)
observed after 0.5, 1.0, 2.0, 4.0 and 8.0 h from
the beginning of reaction measured using the initial value obtained by each plant derivatives and
extracts. The AA% and increases in percentage of
AA [D (%) AA] values of plant-derived substances
and extracts demonstrated in Table II were considered statistically significant (P < 0.05).
Tocopheryl acetate is an ingredient commonly
applied in cosmetic products and it is also frequently used as a standard in various antioxidant
determination methods. In Table II, it can be
observed that it demonstrated low values with
regard to increased percentages of AA, giving it a
fast and stable profile over time.
The ginkgo extract, the grapeseed extract and
the yerba mate extract demonstrated a satisfactory
and rising AA during 8 h, but they did not reach
higher values than the hesperidin derivative,
thereby giving them the characteristics of intermediate profile behaviour.

The hesperidin derivative presented higher values (%AA) than the other extracts at all analysis
times, indicating a slow and long-term profile. It is
important to emphasize that the hesperidin derivative results obtained after 4 h are higher than the
results obtained after 8 h for the other extracts,
thus justifying its effective antioxidant action.
Figure 1 illustrates the AA profiles of a proanthocyanidin-rich grapeseed extract, a hesperidin
derivative, a rutin-containing ginkgo extract, a
polyphenol-containing yerba mate extract and tocopheryl acetate. It is important to say that Fig. 1
was constructed based on the data obtained in
Table II. In order to facilitate the comprehension
regarding error bar values in the Fig. 1, we present them in Table III. The average results and
their SD values demonstrated in Table III were
considered statistically significant (P < 0.05).
The antioxidant kinetics represented in the Fig. 1
shows that a significant difference exists between
the profile of the hesperidin derivative and the profiles of the other two ingredients. The hesperidin
derivative shows high and increasing values even
after 8 h, which indicates a continuous and significant action during this entire period. This behaviour is defined as slow antioxidant kinetics.
On the other hand, the proanthocyanidin-rich
grapeseed extract, the rutin-containing ginkgo
extract and the polyphenol-containing yerba mate
extract show a less effective protection profile than
the hesperidin derivative. Nonetheless, this may be
considered satisfactory, as their AA values reached
a peak 6 h after the beginning of the reaction.
This behaviour suggests that these ingredients may
provide an important contribution in achieving
long-lasting protection, which is consistent with
an intermediate type of antioxidant kinetics. Considering the high AA of yerba mate extract in the

Table II Percentage of antioxidant activity (AA%) and increase of this activity over time [D (%) AA]
Rutin-containing
ginkgo extract

Hesperidin
derivative

Proantocianidin-rich
grapeseed extract

Tocopheryl
acetate

Polyphenol-containing
yerba nate extract

Time
(h)

% AA

D (%)
AA

% AA

D (%)
AA

% AA

D (%)
AA

% AA

D (%)
AA

% AA

D (%)
AA

0.5
1
2
4
8

48.74
53.69
60.13
66.59
71.20

0
10.16
23.38
36.63
46.10

52.69
64.95
75.34
82.96
87.89

0
23.28
43.01
57.46
66.82

49.60
54.90
60.34
65.67
70.60

0
10.67
21.65
32.39
42.32

58.24
59.33
60.74
62.47
64.80

0
1.88
4.30
7.27
11.26

56.91
60.63
65.24
70.94
77.87

0
6.55
14.64
24.66
36.83

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A. R. Silva et al.

Antioxidant kinetics of plant-derived substances and extracts

100
90
80
70

%AA

60
50
40

Hesperidin derivative
Rutin-containing ginkgo extract

30

Proanthocyanidin-rich grapeseed extract

Polyphenol-containing yerba mat extract

20

Tocopheryl acetate

10
0
0

0.5

Time (h)

Figure 1 Plant-derived ingredient

antioxidant kinetics obtained from
calculated EC50 values and presented in Table I. AA, antioxidant
activity.

Table III Standart deviation of antioxidant activity (AA) for three independent experiments over time
Antioxidant activity % (Mean SD)

Time
(h)

Rutin-containing
ginkgo extract

Hesperidin
derivative

Proantocianidin-rich
grapeseed extract

Tocopheryl
acetate

Polyphenol-containing
yerba mate extract

0.5
1
2
3
4
5
6
7
8

48.74
53.68
60.13
64.04
66.59
68.33
69.52
70.47
71.20

52.68
64.95
75.34
80.15
82.96
84.79
86.12
87.11
87.89

49.60
54.90
60.34
63.49
65.67
67.32
68.62
69.68
70.60

58.24
59.33
60.74
61.72
62.47
63.13
63.74
64.29
64.79

56.91
60.63
65.24
68.38
70.94
73.05
74.85
76.47
77.87

2.81
3.41
5.85
7.62
8.67
9.37
9.78
10.05
10.17

0.80
1.60
1.78
1.58
1.38
1.22
1.09
0.99
0.94

beginning of the reaction (To) compared with the

ginkgo and grapeseed extracts, it is possible to
observe that there is an immediate activity such as
showed by tocopheryl acetate; however, its kinetic
profile could be classified as intermediate.
Tocopheryl acetate which showed a profile consistent with fast antioxidant kinetics as it resulted
in somewhat lower antioxidant values and did not
demonstrate significant growth rates during the
test. It should be noted that although quite different from the other profiles described above, this
behaviour does not characterize a low antioxidant
potential rather it indicates that this ingredient
produces an immediate AA (much sooner than
30 min) that remains constant over time.
It should be pointed out that the antiradical and
chelating properties of flavonoids, phenolic acids
and tannins result from their polyphenolic struc-

1.33
1.63
2.13
2.46
2.64
2.79
2.93
3.00
3.07

2.73
2.71
2.83
2.96
3.05
3.14
3.18
3.22
3.29

1.07
0.96
0.89
0.86
0.93
0.97
1.07
1.17
1.26

ture, which is known for its great ability to donate

an electron and hydrogen, and thus stabilize free
radicals. However, as mentioned before, the chemical composition of plant-derived substances and
extracts shows differences that may completely
change the profile of both their immediate and longterm antioxidant responses. This specificity contributes to the occurrence of different types of antioxidant kinetics and generates a sort of fingerprint for
each evaluated plant extract and derivative.
The kinetic profile can or cannot be associated
to chemical profile as each extract presents
several photochemical compounds in different
concentrations in its composition, characterizing
a specific AA as well as an individual kinetic
profile. The statement mentioned above can be
proved because the four extracts studied (ginkgo,
grapeseed and yerba mate extracts and hesperidin

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Antioxidant kinetics of plant-derived substances and extracts

derivative), containing phenolic compounds in

their composition, showed different kinetic
profiles.

testing, cell culture trials and in vivo (human) efficacy evaluations.

Acknowledgements

Methodology validation
The analytical method used to evaluate the AA
over time is according to the parameters required
by United States Pharmacopeia (USP) [31]. The
specificity analysis revealed that there is no interference of the extract components in the wavelength used. The method was demonstrated to be
linear, with a correlation coefficient of 0.9992.
The recovery results in the accuracy test varied
between 97.7% and 100.4% and the RSD was
under 2.86% [31].
Conclusion
The DPPH method was found to be adequate for a
preliminary study of antioxidant substances. This
study demonstrates that substances screened by
the traditional methodology, which only involves
a reading after 30 min, may be considered as
potential antioxidants when evaluated by the modified DPPH method proposed here (antioxidant
kinetics).
In addition, this study identified antioxidant profiles that may be complementary, given the possibility of combining plant extracts and derivatives
with different kinetics to build complexes capable
of providing effective and long-lasting protection.
However, any combination of substances with fast,
intermediate and slow kinetics should be carefully
investigated to ensure that it will deliver the
desired potential activity.
Although plant extracts and derivatives contain
phenolic phytochemical classes in their composition, such ingredients may display very different
behaviours regarding the immediate and long-term
protection they offer. The diversity of phytochemical species and their concentration are known to
promote antioxidant behaviour, which is consistent with the results of this study. The use of hesperidin derivatives, proanthocyanidin-rich extracts
and rutin-containing extracts alone or in association with other cosmetic substances is promising
for protective or therapeutic skin care when
applied in emulsions, gels, lotions, etc. Based on
the results of this study, additional specific tests
will be conducted in the future to determine longterm AA by means of further in vitro chemical

The authors would like to thank Professor Claudio

H. Sibata from Department of Radiation Oncology,
Brody School of Medicine, East Carolina University,
Greenville (NC), U.S.A. for important comments
and suggestions and O Boticario for the financial
support provided for this research.
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