A new potent natural antioxidant mixture provides global

protection against oxidative skin cell damage

A. T. S. Jorge, K. F. Arroteia, J. C. Lago, V. M. de Sa-Rocha, J. Gesztesi and P. L. Moreira
Natura Innovation and Product Technology Ltda, Cajamar, Sao Paulo 07750-000, Brazil
Received 23 November 2009, Accepted 23 February 2010
Keywords: ageing, antioxidant, natural extracts, photoprotection, skin
Synopsis
Oxidative stress occurs when there is an over production of free
radicals and cells are not able to neutralize them by their own
antioxidant mechanisms. These excess of free radicals will attack
cellular macromolecules leading to cell damage, function impair-
ment or death. Because of that, antioxidant substances have been
largely used in products to offer complementary protection. In this
study a new mixture of three known antioxidants (cocoa, green
tea and alpha-tocopherol) was evaluated and its antioxidant pro-
tection was assessed focusing on its capacity to protect main cell
macromolecules. Results have shown that it has a high antioxi-
dant capacity by protecting lipids, DNA and proteins against oxi-
dative damage. The antioxidant effect of the mixture on cells was
also investigated and it was able to reduce oxidative stress gener-
ated by lipopolisacharide in human broblasts. Finally, as the
mixture has proved to be highly antioxidant, its effect on cell
senescence was evaluated, and it was demonstrated that bro-
blasts in culture had delayed senescence when treated with these
actives on a mixture. All results together provide important data
about a new antioxidant mixture that uses a small amount of
actives and is able to protect cell against oxidative damages in a
global way.
Re sume
Le stress oxidatif a lieu quand il y a une surproduction de radicaux
libres et que les cellules ne sont pas capables de les neutralizer, par
leur propres mecanismes anti-oxidant. Ces exce`s de radicaux libres
vont aller attaquer les macromolecules cellulaires, conduisant a` des
dommages cellulaires, a` des defauts de fonctionnement, voir, a` la
mort cellulaire. De ce fait, les substances antioxydantes sont large-
ment utilisees dans les produits car elles offrent une protection
complementaire. Dans cette etude, une nouvelle mixture composee
de trois antioxidants connus (cacao, cafe vert et alpha-tocopherol)
est evaluee, et sa protection antioxidante a ete testee en fonction de
sa capacite a` proteger les principales macromolecules. Les resultats
ont montre que la grande capacite antioxidante de ce melange pro-
vient de sa capacite a` proteger les lipides, lADN et les proteines des
dommages oxidatifs. Leffet antioxidant de la mixture a aussi ete
teste sur les cellules et il a ete montre que le melange etait capable
de reduire le stress oxidatif genere par le LPS sur des broblastes
humains. Finalement, comme la mixture a prouve etre un puissant
antioxidant, son effet sur la senescence a ete evalue et il a ete
montre que des broblastes en culture ont un vieillissement retarde
quand ils sont traites avec la mixture. Tous ces resultats fournis-
sent dimportantes donnees sur un nouveau melange antioxidant
qui utilize une petite quantite dactifs et qui est capable de proteger
les cellules contre les dommages oxidatifs de facon general.
Introduction
Cells produce energy by reducing molecular oxygen to water. Dur-
ing this process, intermediate reactive oxygen species (ROS) are
formed, being the main ones: hydroxyl radicals (OH

), superoxide
anion (O
2
)
) and hydrogen peroxide (H
2
O
2
) [1]. There are numer-
ous cellular mechanisms that act to avoid an uncontrolled increase
in ROS amount. These mechanisms are classied as enzymatic
superoxide dismutase and catalase, which remove superoxide anion
and hydrogen peroxide from the cytoplasm, gluthatione peroxidase
and gluthatione, which reduces hydrogen peroxide to water and
non-enzymatic that comprises vitamin E and C and act impairing
lipid peroxidation chain reaction [2]. Oxidative stress occurs when
these defense mechanisms are not able to scavenge ROS amount
that is uncontrolled generated in situations as UV exposure, for
example [3]. This stress status is responsible for many diseases and
implicates directly on ageing progress [4].
Reactive oxygen species substances that are highly oxidative
because they have an impaired electron and they take it from other
molecules to stabilize themselves. In a cell system, ROS can damage
lipids, nucleic acids (DNA) and proteins [2]. Lipids are the main cell
membrane constituents and have an important structural function.
Lipid oxidative attack is called lipid peroxidation and initiates with
hydrogen abstraction from a methylene group (-CH
2
-) leaving one
impaired electron on the carbon (-

CH-) and starting a chain reac-

tion that damage cell membrane by the production of chemical
intermediates such as malondialdehyde and hydroxynonenal [5].
Furthermore, there are at least 100 different DNA oxidative dam-
ages described [6]. They are classied as base or deoxyribose
lesions, strand breaks or cross-links and are considered the most
serious ROS-induced cellular modications because of their capacity
to lead to mutation. In addition to DNA and lipids damage, accu-
mulation of oxidized proteins is being recognized as one of the
markers of oxidative stress [7]. It has been shown that protein oxi-
dation occurs mainly as a result of the action of lipid peroxidation
products such as peroxides and aldehydes [7].
Correspondence: Adriano Tadeu Siqueira Jorge, Rodovia Anhanguera,
S/N, Km 30,5, Cajamar, SP 07750-000, Brazil. Tel.: (55-11) 4446-
2669, fax: (55-11) 4446-2138; e-mail: adrianojorge@natura.net
International Journal of Cosmetic Science, 2011, 33, 113119 doi: 10.1111/j.1468-2494.2010.00595.x
2010 Natura Innovation and Product Technology. Journal compilation
2010 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie 113
Oxidative stress has been shown to accelerate cellular senes-
cence. Fibroblasts cultured from accelerated senescence-prone
SAMP11 mice produce more ROS then cells from accelerated senes-
cence resistant SAMR1 mice [8]. Besides that, some oxidative con-
ditions, as UV and peroxide hydrogen exposure accelerate
senescence in human cultured broblasts [9, 10]. All these ndings
together are important to show that oxidative stress status contrib-
utes to accelerate ageing. In fact, some parameters as lipid and pro-
tein oxidation are used as biomarkers to determine the oxidation
and so the ageing status of a person [11].
Many chemical substances have special interest in biology
because of their capacity to neutralize ROS and thus protect cell
structures from their attack. For this study, three of them have
been selected [by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and lipid
peroxidation in vitro assay data not shown] because of their
higher antioxidant capacity. Cacao (Theobroma cacao) has high
amounts of xantines and polyphenols and is described as having
antioxidant properties, protecting against lipid peroxidation, keep-
ing cardiovascular health and preventing photodamage, including
wrinkle formation and histological alterations (dermal connective
tissue and collagen accumulation) [12, 13]. Green Tea (Camellia
sinensis) is rich in polyphenols and inhibits lipid peroxidation in
mouse skin [14]. Following oral administration, it was also able to
inhibit protein oxidation in mouse skin (it was also observed in cul-
tured human broblasts) and it has inhibited UVB induced MMPs
expression, which is related to ROS increase [15]. In other experi-
ment, green tea protective effect against H
2
O
2
DNA oxidation was
evaluated, but, no positive result was shown [16]. Tocopherol is a
lipophilic molecule that is well known as having antioxidant prop-
erties. Its molecular organization is supposed to be the main
responsible for its important activity against lipid peroxidation [17].
It acts impairing lipid peroxidation chain reaction and damage
propagation [18]. Because of its property, tocopherol is highly pro-
tective against membrane oxidative attack.
Usually, studies are published to show substances or molecules
antioxidant activity and they select a few in vitro or in vivo methods
to prove it. When considering that ROS increase leads to a multi
oxidative damage in cells, it is very important to consider a global
evaluation on which different oxidation parameters are assessed.
The aim of this study was to evaluate the synergic action of these
three components mixed together on protecting cells against oxida-
tive damage. For this purpose, protection against oxidation of main
cell macromolecules (DNA, proteins and lipids) was assessed in vitro
by specic methodologies. To conrm the antioxidant activity of
the mixture, a cell system was used and the oxidative stress status
was assessed. Finally, to link antioxidant capacity and macromole-
cules protection to global cellular protection, a senescence study
was conducted in broblasts.
Materials and methods
Materials
Cocoa dry extract was produced and obtained at Natura Cosmetico
Brazil through an ethanolic extraction. The extract (pH 34) has
4.5% theobromine, 1.3% caffeine, 5.0% catechin, 12,5% epicatechin
(active compounds), besides 30% total glycides, 20% total proteins,
10% water, 6% ethereal extract and 2% mineral material. Green Tea
dry extract was obtained from Cognis. The extract contains 95%
polyphenols with catechins representing 50% of total mass (40.92%
caffeine, 24.95% epigallocatechin gallate, 12.61% epicatechin gal-
late, 6.0% gallocatechin gallate, 4.49% theobromine, 3.61% epicate-
chin, 1.59% catechin gallate, 1.5% catechin, 1.27% gallocatechin,
0.41% ellagic acid and 0.43% epigallocatechin). Alpha-tocopherol
(96% purity), DPPH, ()-6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid (trolox), 2,2-azobys(2-methylpropionamide)dihy-
drochloride (AAPH), l-a-phosphatidylcholine from egg yolk type
XVI-E (99%), xylenol orange, riboavin and 5-bromo-4-chloro-3-
indolyl-d-d-galactopyranoside (X-gal) were purchased from Sigma
Aldrich (St Louis, MA, U.S.A.). The human dermal broblasts were
obtained from skin biopsies. Human dermal broblasts were
obtained from skin biopsies by Excellion Laboratories in Rio de
Janeiro, Brazil, under approbation of a Brazilian Medical Ethics
Research Committee and in conformity with the principles of Decla-
ration of Helsinki.
Lipid peroxidation
A phosphatidylcholine solution was prepared in a 100 mM metha-
nol solution and used in control and blank assays. The tested solu-
tions were also prepared in methanol and mixed with
phosphatidylcholine solution to generate liposomes with actives.
Solutions were transferred to a tube and completely nitrogen dried.
Enough 50 mM Tris buffer solution was added to this mixture to
have 10 mM phosphatidylcholine liposomes on the nal solution.
A Kit (liposofast basic) consisting on two syringes and a 100 nm
membrane was used to produce the liposomal phosphatidylcholine
structure according to the procedure described on its manual. A
volume of 250 lL of these liposomes (phosphatidylcholine only or
phosphatidylcholine + actives) were mixed with 10 lL 10 mM
AAPH and 240 lL distilled water and heated up to 37C, being
constantly mixed in a thermomixer. After 3 h of reaction, an
aliquot (20 lL) was taken and added to 50 lL of 5 mM ferrous
sulfate, 50 lL of 2 mM xylenol orange and 880 lL of distilled
water. Solutions were incubated during 30 min protected from
light. This nal product reaction was measured in a spectropho-
tometer at 560 nm. Oxidized liposomes produce hydroperoxides
which oxidize Fe
2+
to Fe
3+
and, the last one reacts with xylenol
forming a brown-orange compound. Percentage of protection is
related to decrease in colour (oxidation avoidance) compared with
control without actives.
DPPH
Sample solution (50 lL, prepared at different concentrations in eth-
anol), trolox (positive control, prepared at 0.005% in ethanol) and
solvent (negative control) were mixed with 450 lL of DPPH (pre-
pared at 0.004% in ethanol). Preparations were then incubated
during 30 min protected from light and after this time 150 lL of
each test solution was transferred to a 96-well plate and read at
517 nm. DPPH is a free radical that produces a purple colour solu-
tion in ethanol. Antioxidants are supposed to decrease the colour
intensity by donating a hydrogen atom to this free radical and
their potency is measured by their capacity on decreasing solution
absorbance at this wavelength.
Protein oxidation
Bovine serum albumin (BSA) at 10 mg mL
)1
was incubated with
10 mM hydrogen peroxide (H
2
O
2
) plus 3 mM ferrous sulfate and
actives during 2 h and then washed with 10% triuoroacetic acid
followed by centrifugation. After repeated washings, the protein
was solubilized in water and 20 lL was added to a mixture
(980 lL) containing ferrous sulfate, xylenol orange and water. This
2010 Natura Innovation and Product Technology. Journal compilation
2010 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 113119 114
Global protection against oxidative skin cell damage A. T. S. Jorge et al.
nal mixture was kept protected from light during 30 min and the
xylenolFe
3+
complex formation was assessed by reading it at
560 nm in a spectrophotometer. The rst step reaction is carried
out to generate hydroxyl radicals (OH

) that lead to protein hydro-

peroxides formation (BSAOOH). At the second step, BSAOOH
formed is able to oxidize Fe
2+
to Fe
3+
that reacts with xylenol form-
ing a colour complex. The protection against protein oxidation is
given by actives capacity to avoid protein hydroperoxides formation
and it is observed by absorbance decreasing.
Plasmid DNA oxidation
For this assay, 6 lL of Pblue script plasmid 0.6 lg mL
)1
, extracted
from transformed Escherichia coli HB 101, was added to 50 lL of
actives at tested concentration, 10 lL of riboavin and 34 lL of
PBS. This sample was kept under UVA irradiation during 20 min
(1.83 10
)3
W, 7 cm, Bio-Sun ref 313/365, Vilber Loumart).
Samples not irradiated were kept as test control. An electrophoresis
in agarose gel was performed to evaluate DNA breakage. Riboavin
is a photo sensitizer that generates ROS in presence of UVA and
these ROS are responsible for breaking plasmid DNA by oxidation.
The result is the comparison between irradiated and non-irradiated
samples (qualitative analysis). If there is protection against DNA
oxidative breakage, electrophoresis prole between irradiated and
non-irradiated samples will be identical. If there is DNA breakage,
fragments will be noted on irradiated sample.
Cell senescence assay
Dermal human broblasts were isolated from a face skin fragment
coming from a female, 51-year-old donor. The procedure was per-
formed according to a standard two-step dissociation protocol by
which the skin layers are dissociated with dispase and the collagen-
ases are used to release broblasts from dermis. After the isolation,
broblasts were cultured in Dulbeccos modied Eagles medium
(DMEM) containing 10% of fetal bovine serum (FBS) at 37C in 5%
CO
2
humidied atmosphere. Medium was changed every 24 h and
cells were sub-cultured every 48 h. After six passages, cells were
plated in 24 wells plates with DMEM with 2% FBS. Four plates
were prepared for each assay, being two of them used for senes-
cence induction via UVB exposition (test groups) and the other two
mirror plates were used as irradiation negative control (control
group, unexposed to UVB). In both groups (test and control), the
rst plate was treated with the actives mixture diluted on the cul-
ture medium (treated cells), and the second one was treated with
medium alone (untreated cells). To induce senescence, cells in the
test group were exposed daily to UVB irradiation (75 mJ day
)1
,
Bio-Sun ref 313/365, Vilber Loumart) during 4 days (one exposi-
tion per day). Just before each irradiation, cells in the four plates
were washed twice and incubated with PBS. The UVB irradiation
was carried out to the test groups (treated and untreated with the
mixture) at room temperature, whereas the plates of control group
were also kept at the same temperature levels but totally protected
from light incidence. After each exposition session, the PBS of all
plates was removed and replaced again with medium alone or
medium containing the mixture. After the fourth irradiation, cells
were left to rest 72 h and then exposed to a X-gal solution during
12 h. X-gal is the substrate of the b-galactosidase enzyme, which is
converted to a blue compound inside of the cytoplasm of senescent
cells. As a standard result, the cultured broblasts not exposed to
UVB nor to any treatment are supposed to present on average 10%
b-galactosidase-positive cells, whereas cells exposed to UVB are sup-
posed to present 4565% b-galactosidase-positive cells. This event
is directly linked to ageing and the present protocol allows us to
evaluate whether an active is able to decrease b-galactosidase over
expression on human broblasts.
Antioxidant activity assay using cells
Dermal human broblasts were grown in DMEM containing 10%
of FBS at 37C in 5% CO
2
humidied atmosphere. Cells were trea-
ted with different actives concentrations during 4 h. After this per-
iod, ROS inductors 100 ng mL
)1
lipopolisacharide (LPS) and
25 lM of the uorescent compound 2,7-dichlorouorescein diace-
tate (DCFH-DA) were added to cells and incubated during 25 min.
Cells were then washed with Hanks buffered salt solution and cen-
trifuged during 8 min. Samples were analysed by ow cytometry.
This assay evaluates ROS increase by assessing uorescence inten-
sity. Antioxidant actives are supposed to decrease this intensity
when compared with untreated control.
Statistical analysis
All results are expressed as the mean SE. Statistical analysis was
performed for each test and are indicated on each gure legend.
Values were determined to be signicant when P < 0.05.
Results
Antioxidant potential of the mixture (cell free assays)
Three actives have been selected for this study: a green tea extract
rich in polyphenols, a cocoa extract rich in catechins and a-tocoph-
erol. The main objective was to evaluate if a mixture composed of
equal quantities of each substance would have a higher antioxi-
dant capacity. The rst step was to test the three substances alone
vs. the mixture composed of same total actives concentration. For
this purpose, we have selected lipid peroxidation assay because this
methodology gives consistent results about antioxidant capacity
and when compared with other in vitro antioxidant assays it is
closer to the biological interaction that occurs between ROS and
cell macromolecules. Results show that when we mix 10 lg mL
)1
cocoa extract, 10 lg mL
)1
green tea extract and 10 lg mL
)1
a-tocopherol (total active concentration 30 lg mL
)1
) we have
signicant less hydroperoxides formed than when we test each
substance individually at 30 lg mL
)1
(Fig. 1).
As the results from lipid peroxidation assay had shown that
there was a signicant advantage on using the mixture instead of
using the isolated actives, this methodology was also used to
assess in vitro mixture capacity to protect lipophilic membranes
against oxidative damage. Phosphatidylcholine liposomes were pre-
pared and exposed to an oxidative compound (AAPH) with or
without the tested mixture. The percentage of protection of the
mixture was obtained in relation to a negative control and data
were collected after 1, 2 and 3 h reaction. After 1 h being exposed
to the oxidative chemical, the mixture is signicantly able to pro-
tect against lipid peroxidation, because the protection was
73.98 0.86% compared with control without actives. This pro-
tection is kept high and constant up to 2 h reaction
(71.4 1.27%, no statistical difference compared to 1 h protec-
tion) and presents a little decrease after 3 h reaction
(68.15 0.94%, signicantly different compared with 1 h protec-
tion). All data were signicantly different from the control without
actives (P < 0.05; n = 3; t-test).
2010 Natura Innovation and Product Technology. Journal compilation
2010 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 113119 115
Global protection against oxidative skin cell damage A. T. S. Jorge et al.
One of the most classical assays used to evaluate antioxidants is
DPPH because it is fast and gives consistent results. As described
on Materials and methods (pg. 3), it evaluates sample capacity to
donate an electron to DPP

radical compared with a negative con-

trol without actives. Results were obtained for the mixture on its
original concentration (10 lg mL
)1
cocoa extract; 10 lg mL
)1
green tea extract and 10 lg mL
)1
a-tocopherol), a mixture that
was 10 times concentrated (10 ), and other mixtures that were
10 and 100 times diluted (1/10 and 1/100, respectively). Results
show that antioxidant protection of the mixture is dose-dependant
until its original concentration, reaching a constant protection
from this point to higher concentrations (Fig. 2).
Proteins are also target for free radicals attack and thus, they
are macromolecules that must be protected. To evaluate this pro-
tection, a method on which BSA is the protein source was used. As
described previously on Materials and methods (pg. 3), BSA is
exposed to hydroxyl radicals (OH

) generated by Fenton reaction

and the protein hydroperoxides concentration is measured (Fig. 3).
The mixture was tested on its original concentration, 10 times con-
centrated and 10 times diluted. Results show that on all concentra-
tions tested, the mixture reduces signicantly the amount of
hydroperoxides formed in comparison with control without actives.
This protection is not dose-dependant, because there is no statisti-
cal difference between the three samples tested.
Besides protein and lipids, DNA is one of the most important
macromolecules in cells. It is also target for oxidative damage. Once
affected, if the cell is not able to repair its damage, it will progress
leading to tumorigenesis in worst cases. In this experiment, Pblue
script plasmid (bacteria DNA) extracted from transformed E. coli HB
101 was exposed to ROS generated from Riboavin (photo sensi-
tizer) irradiated with UVA. Results are qualitative (Fig. 4) and repre-
sent the comparison between electrophoresis prole on irradiated
(UV+) and non-irradiated samples (UV)). Control samples irradiated
or not irradiated did not show any breakage, what was expected,
because there is no photo sensitizer at these samples and therefore
no ROS source. Riboavin alone has no effect in the DNA integrity
(data not shown). Mixtures at its original and 10 concentration
were able to protect plasmid oxidative breakage, and it is noted no
differences between irradiated and no irradiated samples. The 10
times diluted mixture was not able to protect plasmid breakage
because it was possible to observe a clear visual difference on elec-
trophoresis prole between irradiated and non-irradiated samples.
Antioxidant protection (cell assays)
It was also important to conrm these results checking if this mix-
ture could decrease oxidative stress in a cell culture. For that,
human broblasts were treated with LPS that is responsible for
inducing oxidative stress in cells by free radicals over generation. A
substance that is uorescent when oxidized (DCFH-DA) was added
and nal uorescence is proportional to the amount of free radicals
generated. Figure 5 shows that LPS increases uorescence on both
conditions (control and mixture treatments). On LPS-treated (LPS+)
groups, mixture signicantly reduces the uorescence compared to
control what means that it neutralizes part of free radicals gener-
ated. If there are less free radicals available, cells are more pro-
tected against oxidative damage.
Figure 1 Lipid peroxidation assay. Hydroperoxides concentration generated
in phosphatidylcholine liposomes after 3 h reaction with AAPH. Comparison
among control without actives, three actives at 30 lg mL
)1
and a mixture
composed of equal quantities )10 lg mL
)1
- of each active tested. One aster-
isk (*) indicates that all values are signicantly different from control and
two asterisks (**) indicate that mixture is signicantly different from control
and also from actives. Mean +/) SD, P < 0.05; n = 3; T-test.
Figure 2 DPPH assay. Antioxidant protection (%) conferred by the antioxi-
dant mixture tested on DPPH assay. Mixture was tested at its original con-
centration (10 lg mL
)1
green tea extract + 10 lg mL
)1
cocoa extract +
10 lg mL
)1
alpha-tocopherol), 10 times concentrated, 10 and 100 times
diluted. The antioxidant protection of the mixture is dose-dependant until its
original concentration, reaching a constant protection from this point to
higher concentrations. Protection reached by 10 (*) and 100 (**) times
diluted mixture was signicantly different among them and compared with
10 times concentrated mixture and original concentration. Mean +/) SD,
P < 0.05; n = 2; T-test.
Figure 3 Protein oxidation assay. Mixture protection against protein oxida-
tion. Fenton reaction was used to generate hydroxyl radicals that attack and
oxidize proteins leading to hydroperoxides formation. Mixture was tested at
its original concentration (10 lg mL
)1
green tea extract + 10 lg mL
)1
cocoa extract + 10 lg mL
)1
alpha-tocopherol), 10 times concentrated and
10 times diluted. Asterisk (*) indicates that protein hydroperoxides concen-
tration is signicantly different from control at all tested concentrations. No
difference was shown between different concentrations of the mixture. Mean
+/) SD; P < 0.05; n = 3; T-test.
2010 Natura Innovation and Product Technology. Journal compilation
2010 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 113119 116
Global protection against oxidative skin cell damage A. T. S. Jorge et al.
After conrming that the mixture had the capacity to decrease
oxidative stress in cells and also to protect cell main macromole-
cules, the impact of these ndings on cell senescence was investi-
gated. Here, human broblasts were exposed to UVB radiation to
induce their senescence. b-Galactosidase (b-Gal) enzyme was used
as a senescence marker and it was assessed in cells that were trea-
ted or untreated with the mixture. Both treated and untreated cells
that were unexposed to UVB had an expected basal percentage of
b-Gal-positive cells (respectively 11.67 0.6% and 12.2 0.5%)
showing that the mixture per se does not have any b-Gal inhibitory
effect alone. The results showed that the basal percentage of b-Gal
expression was signicantly increased when the untreated cells
were exposed to UVB (51.67 1.36%), However, when the UVB-
exposed cells were treated with the mixture, the percentage of
b-Gal-positive cells reached 38.81 0.91% instead of 51.67%
detected on untreated cells. The statistical analysis indicates that
the mixture reduces signicantly the percentage of b-Gal-positive
cells on UVB-exposed group compared with cells unprotected with
the mixture (P < 0.01, n = 4, t-test).
Discussion
There are a number of factors responsible for ageing. Despite the
biological programmed ageing, different external aggressors, includ-
ing skin exposition to solar radiation, can accelerate this phenome-
non [19]. One of the major consequences of the exposition to
external aggressors is the increase in deleterious oxidation process,
so-called oxidative stress. Oxidative stress has been shown to be
one of the mechanisms that accelerate cellular senescence and gen-
eral ageing. Being aware of the biological complexity involved in
oxidative stress, the aim of this study was to investigate a possible
global protection provided by a mixture of different natural an-
tioxidants. For this proposal, we designed an experimental protocol
including a pool of in vitro strategies able to demonstrate the global
antioxidant efciency of our selected plant extracts.
The three substances that had been selected for this study (cocoa
extract, green tea extract and alpha-tocopherol) are widely
described for their antioxidant capacity [1218, 20, 21] but there
is no description on their antioxidant behaviour when mixed all
together. Because of that, the rst strategy was to assess if there
was any advantage on mixing them. This issue was rstly assessed
by measuring the amount of hydroperoxides formed on a lipid per-
oxidation assay, once that lipid peroxidation is one of the conse-
quences of UV irradiation and also one of the causes of direct
damage on cell membranes. The obtained results allowed us to
conclude that those mixed substances are more effective than each
one alone, once it is possible to reach a higher antioxidant protec-
tion using the mixture compared with each substance individually
in the same concentration. Therefore, we were able to show a posi-
tive synergy effect when mixing them. Moreover, its high protec-
tion capacity (almost 70%) is kept during a 3 h period, which
strongly means that it is able to effectively protect cell membranes
for a longer time.
It has been widely described that oxidative stress is responsible
for damaging cell structures by lipid, proteins and DNA oxidation
[27]. At this study, several experiments were carried out to asses
the protection potential of the mixture against oxidation of these
macromolecules. As described in the results section, the performed
in vitro assays had shown that this proposed mixture is able to pro-
tect bacteria DNA against oxidative attack and consequent break-
age. In the same way, the results of protein oxidation assay
showed a signicant decrease in hydroperoxides formation, mean-
ing that the proposed mixture also protects proteins against oxida-
tion. Taken together, the data clearly show that this new mixture
has a high global antioxidant activity and protect main cell macro-
molecules: lipids, proteins and DNA.
Once knowing that the mixture had a high antioxidant capacity,
it was important to evaluate how it could behave in a cell system.
For that, human broblasts were exposed to an oxidative environ-
ment (with LPS) and free radicals formation was assessed. The
results showed that the mixture decreased the amount of generated
Figure 4 Protection against DNA oxidative breakage. Pblue script plasmid (bacteria DNA) was exposed to reactive oxygen species and mixture protection was
assessed comparing UV exposed sample with UV non-exposed sample. Results are qualitative in duplicate and show that mixture at its original concentration
and 10 times concentrated are able to protect DNA (same electrophoresis prole on UV exposed and non exposed samples). Mixture diluted 10 times is not able
to protect DNA as demonstrated by different prole at UV+ sample.
Figure 5 Antioxidant protection assessed in human broblasts. Cells were
treated with DCFH-DA, a substance that oxidizes in presence of free radicals.
Once oxidized, this substance becomes uorescent. White and gray bars indi-
cate LPS non-treated or LPS treated cultures, respectively. Non-treated LPS
cells show constant basal uorescence intensity. Cells treated with LPS plus
the mixture signicantly decrease (**) uorescence intensity compared to
control without actives (*). Mean +/) SD P < 0.01; n = 3; ANOVA followed
by Tukey-Kramer test.
2010 Natura Innovation and Product Technology. Journal compilation
2010 Society of Cosmetic Scientists and the Societe Francaise de Cosmetologie
International Journal of Cosmetic Science, 33, 113119 117
Global protection against oxidative skin cell damage A. T. S. Jorge et al.
free radicals. As the receptor to LPS is a Toll-like one with very
high specicity [25], it is reasonable to assume that the mixture is
not able to inhibit the LPS binding to its receptor. The observed
antioxidant effect would occur as a result of the previously demon-
strated ability of the mixture of scavenging the free radicals.
Cell senescence is a multi-factor biological process that leads cel-
lular growth arrest and stop of cellular division, directing affecting
the cellular renovation ability [22, 23]. Related to human skin, cel-
lular senescence directly decreases skin rmness and healthy
appearance. Oxidative stress caused mainly by UV exposition is also
described as being partly responsible for accelerate the cell senes-
cence process [8, 9, 11, 24]. Because of that, the mixture capacity
to delay the in vitro broblast senescence process was evaluated. It
has been shown that human broblasts exposed to a long-term
subcytotoxic doses of UVB irradiation display some biomarkers of
replicative senescence, like the b-galactosidase. This phenomenon is
called stress-induced pre-mature senescence [9, 10]. Our results
showed that the proposed mixture could decrease the number of
pre-mature-induced-senescent cells formed after the UVB exposi-
tion. This fact demonstrates that the mixture is able to avoid or to
neutralize the free radicals generated by stress conditions, blocking
an important pathway that leads to cellular senescence and skin
ageing.
We described here a new potent antioxidant alternative concern-
ing a mixture of cocoa extract, green tea extract and alpha-tocoph-
erol. UV exposure, pollution and other external and internal
conditions increase free radicals production and consequently are
responsible for cell damage, which are cumulative and lead to age-
ing. That is why it is very important to offer extra protection
against oxidative stress by using antioxidant substances. The pro-
posed mixture tested at this study was proven to have a high anti-
oxidant potential able to prevent cellular damage and protect main
cell macromolecules. Furthermore, its effect on delaying cell senes-
cence shows that it is able to neutralize free radicals attack and
decrease the cell ageing process. The advantage of using these
plant extracts in a specic mixture, was deeply corroborated by this
study and allow us to present it as a highly antioxidant approach
with real global protective effect and anti- ageing activity.
Acknowledgments
We are grateful to Paolo di Mascio who has supported Natura as a
consultant for in vitro antioxidant assays development and imple-
mentation. The authors own institution, Natura, provided funding
support and resources to carry out this study.
Conict of interest
The authors state no conict of interest.
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