ARTICLE
Auteur(s) : Ines
Dammak1, Fatma Ben Abdallah1, Sonia
Boudaya2, Laila Keskes1, Souhail
Besbes3, Amel El Gaied1, Hamadi
Attia3, Hamida Turki2, Basma
Hentati1
1Unité de recherche, Pathologies Humaines et stress
oxydatif, Institut Supérieur de Biotechnologie de Sfax, Route sokra
km 4,5, BP 261; 3038 Sfax, TunisiaFax: (+ 216) 74 67 43 64
2Service de Dermatologie, Centre Hospitalo-universitaire
Hedi-Chaker, Sfax, Tunisia
3Unité d’Analyses Alimentaires, Département de biologie,
École Nationale d’Ingénieurs de Sfax, Tunisia
accepté le 4 Juillet 2007
The date (Phoenix dactylifera L), has played an important role in
the economy and social life of the people of arid and semi-arid
regions of the world. Date seeds may have extractable high
value-added components. It has been revealed that DSO presents a
high antioxidant capacity due to its richness in polyphenols and
tocopherol compounds [1-4]. Besbes et al. reported that the
oxidative stability of DSO was better than that of most vegetable
oils [2]. Data about DSO composition and physico-chemical
properties have encouraged us to test it. In fact, the skin is
constantly exposed to pro-oxidant environmental stresses. Among a
great variety of reactive oxygen species (ROS),
H2O2 plays a pivotal role because it is
generated from nearly all sources of oxygen radicals. Recent
studies have shown that H2O2 can induce both
necrosis and apoptosis. H2O2 is capable of
oxidizing lipids, proteins, or DNA, leading to the formation of
oxidized products which have been implicated in the onset of skin
damage [5]. Endogenous enzymatic and non-enzymatic antioxidants
protect the skin from H2O2-induced oxidative
damage. However, if the antioxidant defense component of the skin
is overwhelmed by the presence of ROS, it can lead to oxidative
damage of cellular constituents [5].The aim of this study was to
investigate to what extent DSO provides protection against
H2O2-induced oxidative stress in terms of
lipid peroxidation and depletion of endogenous antioxidant defense
enzymes.
Material and methods
Plant material: Date seed oil extraction and preservation
The seeds of dates, “namely Deglet Nour” (National Institute of
Arid Zone, Degach, Tunisia) were collected at the “Tamr stage”.
Lipid extraction was carried out with an SER 148 Solvent Extractor
equipped with six Soxhlet posts. Powdered date seeds were used for
oil extraction with thimbles immersed in boiling petroleum ether.
The solvents from seed oils were removed under a stream of nitrogen
and then stored in a freezer at – 20 °C until use [2].
Organ culture
All skin samples (n = 10) were obtained from healthy adults (25 to
40 years of age), undergoing abdominal plastic surgery. Immediately
after being excised, explants were cleaned in ethanol 70% for 30
seconds, and directly immersed in cold minimum Eagle’s medium (MEM)
for transport (maximum 30 minutes) to the laboratory. The
subcutaneous fat was removed and samples were cut. Skin specimens
were placed with the dermal side down in 24 well plates at the
air-liquid interface with DMEM (Dulbecco’s minimum Eagle’s medium)
complemented with 10% FCS, 200 µg/mL glutamine, and
penicillin/streptomycin (respectively 100 U/mL and 100 µg/mL)
and kept in a humidified incubator containing 5% CO2 at 37 °C
during 48 h. Medium was changed after 24 h. In order to dissolve
the different compounds of DSO in the medium, part of this oil was
dissolved in dimethyl sulfoxide (DMSO) to obtain a concentration of
100 µg/mL (stock solution).
In vitro application of date seed oil and
H2O2
We prepared four different wells (controls: non-
H2O2-exposed skin “n
H2O2 –”, non-
H2O2-exposed skin plus DSO “n
H2O2 +”, H2O2-exposed
skin “H2O2 –”,
H2O2-exposed skin plus DSO
“H2O2 +”). In our previous studies, we tested
a range of concentrations of DSO (1%, 3%, 5%, 8%, 10%, 12%, 14% and
16% of the total volume of the culture medium), in order to choose
an optimal concentration which gives the maximum antioxidant
protective effect. We found that the maximum protection
manifested at 12% of the total volume of the culture medium. DSO
12%, which was found optimal in previous studies analysing
different parameters, is also optimal for the protection of
antioxidant enzymes, thus we chose this concentration to continue
our experiments. To prevent the tissue from the oxidizing effects
of H2O2, dissolved DSO was included in the
culture medium for 48 h before the addition of the
H2O2 at 12% of the total volume of the
culture medium. After 48 h of culture, skin samples were
rinsed very well with PBS to make them DSO free. For cell death
induction, H2O2 (in PBS pH 7.2) was added to
the same specimens at (1 mM) for 2 h. This concentration was
selected in our previous studies, because it was the optimal dose
that generated clear damage without disastrous effects in the skin
structure. After incubation, skin samples were rinsed in PBS; then
fresh medium was added and skin samples were placed for 24 hours in
an incubator, followed by biochemical evaluation.
Antioxidant systems: catalase, superoxide dismutase and
glutathione peroxidase
Skin specimens were homogenized (5% W/v) in a 50 mM phosphate
buffer (pH 7.0) containing 0.1 M EDTA and centrifuged at 250 g for
10 min at 4 °C. The supernatant was used for
determination of the enzymatic activity of catalase (CAT) [6],
superoxide dismutase (SOD) [7] and glutathione peroxidase (GPx)
[8]. The data are expressed as U/mg proteins. Total protein content
was quantified as described by Bradford method [9]. The experiments
for antioxidant enzymes were repeated at least three times.
Determination of MDA
Samples prepared for GPx, CAT and SOD assay have been used for the
quantification of lipid peroxidation [10], which measures the
amount of malondialdehyde (MDA) obtained from the reaction of lipid
peroxides with thiobarbituric acid. The result was expressed as
nmol MDA/mg protein. The experiments for determination of MDA were
repeated at least three times.
Statistics
Statistical analysis was done using t-test ANOVA. The protective
effect of DSO was considered significant if p < 0.05. Results
are presented as means ± SD.
Results
Date seed oil prevents H2O2-induced
depletion of endogenous antioxidant defense enzymes
Our results demonstrated that exposure of human skin samples to
H2O2 significantly decreased GPx (25%, p <
0.01) compared to non- H2O2-exposed skin. The
treatment with DSO prevented H2O2-induced
depletion of endogenous antioxidant GPx in human skin ( (figure 1) ). Catalase is
another endogenous antioxidant enzyme involved in the catalytic
conversion of H2O2 to oxygen and water and
thus decreases the level of oxidative stress. The exposition of
human skin samples to H2O2 resulted in
reduction of catalase (20%, p < 0.01) compared to non-
H2O2-exposed human skin, whereas pretreatment
of skin samples with DSO restored the activities of catalase (
(figure 2) ).
Similar to other enzyme levels, H2O2
exposition of human skin samples depleted the level of SOD by 17%
compared to non-H2O2exposed skin, and
pretreatment of human skin samples with DSO restored the activity
of SOD enzyme ( (figure
3) ); thus indicating a significant protective effect of
DSO against H2O2-induced depletion of
antioxidant defence in an in vitro model. Moreover, treatment of
human skin samples with DSO alone did not significantly affect the
original levels of antioxidant enzymes.
Effect of date seed oil on H2O2-induced
MDA formation
( Figure 4 )
shows that H2O2 induced the formation of MDA
in human skin. The levels of MDA were increased four-fold in the
skin exposed to H2O2 compared to
non-H2O2-exposed skin. Pre-treatment of skin
with DSO significantly inhibited H2O2-indued
MDA formation. Moreover, treatment of skin samples with DSO alone
did not significantly affect the original levels of MDA.
Discussion
When oxidative stress overwhelms the skin antioxidant capacity, the
subsequent modification of cellular redox apparatus leads to an
alteration of cell homeostasis and a generation of degenerative
processes. On the other hand, antioxidants prevent tissue damage
and stimulate wound healing. In fact, topical application of
antioxidants has been recently suggested as a preventive therapy
for skin damage; it protects skin against oxidative injury [11]. In
this, we further attempted to define the protective effect, as well
as the antioxidant properties of DSO, using human skin organ
culture as an in vitro model.
Exposure of human skin to H2O2 induced an
increase in MDA levels and a decrease in GPx, SOD and CAT
activities. These findings contribute to increase cellular
destruction; a consequence of their consumption during oxidative
damage, by favouring free radical attack enhanced after
H2O2 exposition. Our results confirmed the
data of the literature relating to the harmful effect of
H2O2 on human skin [12-14]. Thus, typical
changes in active physiological cell death, leading to apoptosis,
are well known. Among these changes, mitochondrial transmembrane
potential collapse, caspase activation and internucleosomal DNA
fragmentation have been described.
H2O2-activated molecules oxidize cellular
components. They particularly induce a chain reaction of lipid
peroxidation in membranes having a high polyunsaturated fatty acid
levels [12]. H2O2 can diffuse and cross
biological membranes because of its small size, solubility and lack
of charges. It can be reduced to HO which is a highly reactive and
largely indiscriminate oxidant. O2– can collaborate with
H2O2 in the production of HO. The reduction
of H2O2 can occur in the presence of reduced
metal cations such as Fe (II) or Cu (I). If these metal cations are
bound to DNA or to cell membranes, then the HO will be generated
adjacent to, and will react preferentially with, these critical
targets [15].
In vitro treatment of human skin with DSO resulted in the
prevention of H2O2-induced depletion of
antioxidant defense enzymes like GPx, CAT and SOD, thus providing a
possible mechanism for the protection of DSO by the reduction of
free radical generation. ROS have both intrinsic and extrinsic
origins, and cells are protected by multiple levels of antioxidant
defences [16]. Under natural conditions, internal antioxidant
enzymes such as GPx, SOD and CAT, eliminated ROS; thereby affording
some protection for the skin. Deregulating just one of these
enzymes could significantly affect the defensive mechanisms against
ROS attack. If ROS remain without being scavenged in the biological
system they may induce biochemical alterations such as
inflammation, lipid and protein oxidation [17].
H2O2-induced MDA was used as marker of
oxidative damage in our system, and it was significantly inhibited
by the treatment with DSO. Lipid peroxidation in biological
membranes is a free radical-mediated event and is regulated by the
availability of substrates in the form of pro-oxidants which
promote peroxidation and antioxidant defences [18]. Elevated levels
of MDA have been linked to injurious effects such as inactivation
of membrane enzymes and eventually disruption of cell membrane.
After oxidative stress aggression, lipid peroxides might be
involved in intracellular pathways which activate antioxidant
mechanisms [19, 16]. Thus, inhibition of
H2O2-induced MDA by DSO, regarded as a
ROS-scavenging, should reduce the risk factors associated with the
H2O2-induced oxidative damage.
The antioxidant potential of DSO has been the subject of our
considerable interest, both because of its richness in polyphenols,
tocopherols and because it has high oxidative stability, better
than that of most vegetable oils. Our findings confirm here the
results previously published concerning the richness of this oil in
antioxidant molecules [1]. In fact, oils extracted from the seeds
of the date palm have been analysed in terms of phenolic and
tocopherol profiles [1, 4]. DSO could be considered as a rich
source of natural phenolic compounds in the Mediterranean diet. In
DSO, total phenols ranged from 520.81 mg/kg. Seven phenolic
compounds were identified: hydroxytyrosol, with the highest
relative content acid (10.21%), followed by protocatechuic acid,
tyrosol, gallic acid, caffeic acid and, to a lesser extent,
p-coumaric acid and oleuropein. This was one of the main reasons
for the better oxidative stability of this oil. For the DSO, three
peaks corresponding to α-, γ- and δ-tocopherols were identified,
and these accounted for 29.95% of the total tocopherols.
α-tocopherol was the main component and constituted about 24.97% of
the total tocopherols for DSO. Meanwhile, γ-tocopherol accounted
for 3.76% whereas the content of δ-tocopherol ranged from 1.22%
[2]. So, the synergistic interaction between these natural
antioxidants present in DSO protects against
H2O2-induced oxidative damages.
The skin protection given by DSO and its capacity to repair skin
after aggressions are properties which lead us to say that this oil
can be included in “Dermatocosmetology”. The consolidation of
cosmetic dermatology is evident in the increasing number and
quality of published clinical studies and basic research concerning
dermatocosmetic issues. Today’s symbiosis of medical dermatology
and cosmetic aspects has not only contributed to a better
understanding of the pathogenesis of cosmetic skin disorders such
as premature skin ageing, but also introduced medical procedures
for cosmetic indications and the development of new active
ingredients for cosmetic formulations. Recently, a new stage was
reached by evaluating molecular modifications in the skin in
response to the challenges of everyday life [20]. The discovery of
the efficacy of certain extracts in preventing cutaneous diseases
caused and/or aggravated by environmental factors has also
stimulated research on the exact properties of the stratum corneum
and its role in maintaining skin homeostasis. However, research
into the biological effects of dermatocosmetics not only aims at
preventing changes within the skin caused by environmental stimuli,
but increasingly also tries to enable the skin to restore its
homeostasis after disturbances as quickly as possible. The exact
role and potential of dermatocosmetics in repair processes at the
cellular and molecular level still needs to be evaluated in further
detail [20].
It seems that the protective effects of DSO are mediated, at
least, through a protection of the endogenous antioxidant defence
system, preventing damage of macromolecules such as lipids. The use
of DSO as a dietary supplement may have beneficial effects in
protecting against skin disorders in humans. Further in vivo
mechanism-based studies are needed to examine whether DSO can be
used as a safe pharmacologic agent in skin care products, such as
moisturizing creams, skin care lotions and sunscreens for the
prevention of various human skin diseases.
Acknowledgements
Financial support none. Conflict of interest : none.
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