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Journal of Cosmetic Dermatology,13, 151--157
Protective effect of red orange extract supplementation against
UV-induced skin damages: photoaging and solar lentigines
Carmelo Puglia, Pharm. D,
1
Alessia Offerta, Pharm. D,
1
Antonella Saija, Pharm. D,
2
Domenico
Trombetta, Pharm. D,
2
& Cardile Venera, Pharm. D
3
1
Department of Drug Sciences, University of Catania, Catania, Italy
2
Dipartimento Farmaco-Biologico, University of Messina, Messina, Italy
3
Department of Bio-medical Sciences, University of Catania, Catania, Italy
Summary Background Exposure of the skin to solar ultraviolet (UV) radiations causes important
oxidative damages that result in clinical and hystopathological changes, contributing
to premature skin aging. Hyperpigmented lesions, also known as age spots, are one
of the most visible alterations in skin photoaging. Skin is naturally equipped with
antioxidant systems against UV-induced ROS generation; however, these antioxidant
defenses are not completely efficient during exposure to sunlight. Oral antioxidants
are able to counteract the harmful effects of UV radiation and to strengthen the
physiological skin antioxidant defenses.
Aims The present study was performed to evaluate the in vivo skin photo-protecting
and anti-aging effects of a red orange (Citrus sinensis varieties Moro, Tarocco and
Sanguinello) extract supplementation. Previous studies showed that red orange
extracts possess strong in vitro free radical scavenging/antioxidant activity and photo-
protective effects on human skin.
Materials/Methods The photo-protective effects of red orange extract intake against
UV-induced skin erythema and melanin production in solar lentigo was evaluated on
healthy volunteers by an objective instrumental method (reflectance spectro-
photometry).
Results Data obtained from in vivo studies showed that supplementation of red orange
extract (100 mg/daily) for 15 days brought a significant reduction in the UV-induced
skin erythema degree. Moreover, skin age spots pigmentation (melanin content)
decreased from 27% to 7% when subjects were exposed to solar lamp during red
orange extract supplementation.
Conclusions Red orange extract intake can strengthen physiological antioxidant skin
defenses, protecting skin from the damaging processes involved in photo-aging and
leading to an improvement in skin appearance and pigmentation.
Keywords: skin, red orange, oral supplementation, anti-aging, solar lentigo
Introduction
A localized hyperpigmented lesion, also known as age
spot, solar, or senile lentigo, is one of the most visible
alterations in photoaged skin, especially in Asian and
Caucasian populations.
1
In fact, these benign-pigmented
Correspondence: Carmelo Puglia, Department of Drug Sciences, University
of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. E-mail: capu-
glia@unict.it
Accepted for publication November 17, 2013
©2014 Wiley Periodicals, Inc. 151
Journal Innovention
lesions are not only considered unattractive on visible
body areas (face, dorsum of the hand, upper back, and
extensor forearm), but they are also a clinical marker
of the degree of skin photodamage.
2,3
The molecular
mechanisms responsible for photoaged hyperpigmenta-
tion are not completely known, but they do depend on
the cumulative dose of sun exposure as well as on the
amount of protection provided by its pigmentation.
4
Skin pigmentation is a defensive response to the
production of reaction oxygen species (ROS) induced
by UV radiation. Skin is naturally equipped with anti-
oxidant systems against ROS generation; however,
these antioxidant defenses are not completely efficient
during exposure to sunlight, and this limitation
becomes gradually more pronounced during aging.
3,5
Topical application of sunscreens, which frequently
contain UV filters, has been considered the first line of
defense in protecting skin from UV-induced damage.
Chemical sunscreens are able to absorb UV radiations,
thereby protecting the skin during sun exposure. How-
ever, it has been recently recognized that their efficacy
in practice is conditioned by several limiting aspects,
such as inadequacy of application to the skin or
removal by cutaneous perspiring.
6,7
Moreover, there
are many concerns about the safety of UV filters due to
the risk of harmful effects caused by penetration
through the skin, the systemic absorption, and the
UV-mediated photodegradation.
7
To overcome these sunscreen limitations, the use of
nutricosmetics in skin photo-protection, also known as
“beauty pills”, “beauty from within” and even “oral
cosmetics”, has gained considerable attention in recent
years.
8
Among several ingredients proposed for nutri-
cosmetics, antioxidants represent the most interesting
active compounds in photo-protection due to their abil-
ity to fight-free radicals generated by solar radiation.
8,9
The present study was performed to evaluate the in
vivo skin photo-protecting and anti-aging effect
obtained from red orange extract intake (Citrus sinen-
sis varieties Moro, Tarocco and Sanguinello). Previous
studies showed that red orange extracts possess strong
in vitro free radical scavenging/antioxidant activity and
photo-protective effects on human skin.
10–12
Taken
together, these reports strongly suggested that the skin
protection by red orange extract is due to a block of
cellular oxidative stress-related events and the inhibi-
tion of UVB-induced responses associated to inflamma-
tory processes such as ICAM-1 (intercellular adhesion
molecule-1), MCP-1 (monocytes chemoattractant pro-
tein-1), and IL-8 (interleukin-8) cellular release.
10,12
Moreover, the beneficial effects of blood orange extract
intake on antioxidant bioavailability and on different
markers related to oxidative stress were already evalu-
ated in healthy volunteers, such as sportsmen and
smokers.
13–15
In these studies, the activity of red orange fruit has
mainly been attributed to the high levels of anthocya-
nins, (not present in blonde oranges), together with
other antioxidants such as flavones, hydroxycinnamic
acids, and ascorbic acid.
10–15
At the actual status of
researches, the photoprotective properties of red orange
extract cannot be attributed to a component in partic-
ular, but to the phytocomplex in its whole. The biologi-
cal properties of phytocomplexes, which are constituted
by several and different compounds, have been due not
to one or few of its active principles, rather it is deter-
mined by a combined effects of all the components.
10
The aim of this study was to investigate the in vivo
protective effect of red orange intake in skin photo-
damage and photo-aging by evaluation of UV-induced
skin erythema and hyperpigmentation in sunlamp
exposure test models.
Materials and methods
Test material
The powder extract of red orange fruit (Red Orange
Complex
â
) used in this study was supplied by Bionap
srl (Catania, Italy). It is a standardized solid extract
obtained from three pigmented orange varieties (Moro,
Tarocco and Sanguinello) containing the following
active substances: anthocyanins (cyanidin-3-O-gluco-
side) 2.8–3.2% w/w, hydroxycinnamic acids (caffeic,
cumaric, ferulic, sinapic acid) 1.8–2.2% w/w, flavone
glycosides (narirutin, hesperidin) 8.5–9.5% w/w, and
ascorbic acid 5.5–6.5% w/w. Capsules filled with red
orange extract were prepared for the in vivo studies with
a daily dosage of 100 mg of extract for each subject.
Subjects
In vivo experiments were performed on healthy volun-
teers recruited after medical screening including the
completion of a health questionnaire followed by physi-
cal examination of the application sites. Subjects exhibit-
ing such features as abnormal sensitivity to sunlight,
obesity, active smoking, occupational exposure to toxic
agents, the use of antioxidant dietary supplementation,
and vegetarian dietary habits were excluded from the
study. After they were fully informed of the nature of the
study, substances and procedures involved, they gave
their written consent. No subjects were taking medica-
tions (including vitamin and antioxidant supplements)
152 ©2014 Wiley Periodicals, Inc.
Red orange extract supplementation vs. photoaging .C Puglia et al.
and were required not to modify their dietary habits dur-
ing the course of the study. Moreover, to minimize any
environmental influence on daily doses of UV radiation
exposure, studies were started in November, and each
subject was advised to avoid exposure to the sun and
use of sunscreen on the treated skin.
In vivo evaluation of UV-induced skin erythema
The in vivo evaluation of skin erythema induced by UV
irradiation was performed on twenty Caucasian subjects
of skin types II and III (aged 26–47 years). For each
subject, two sites on the ventral surface of each forearm
were defined using a circular template (1 cm
2
) and
demarcated with permanent ink. Afterward, in each test
site, skin erythema was induced by UV-B irradiation
using an ultraviolet lamp, model UVM-57 (UVP, San
Gabriel, CA, USA) which emitted in the range 290–
320 nm with an output peak at 302 nm. The flux rate
measured at the skin surface was 0.80 mW/cm
2
and
the irradiation dose corresponded to double of the mini-
mal erythema dose (MED) of each subject. The ery-
thema induced by lamp exposure was monitored on the
skin sites at different time points for 48 h (2, 8, 12, 25,
35, and 48 h) using a reflectance visible spectropho-
tomer X-Rite model 968 (X Rite Inc. Grandville, MI,
USA), having 0°illumination and 45°viewing angle.
16
Reflectance spectra were obtained over the wavelength
range 400–700 nm using illuminant C and 2°standard
observer. After a rest period of 3 weeks, the same sub-
jects were treated with 100 mg/die of red orange
extract for a period of 15 days. At the end of red orange
extract supplementation, skin sites on the forearms of
subjects were exposed to UV irradiation, and the
induced skin erythema was monitored by reflectance
spectrophotometry for 48 h, as previously reported.
From the spectral data, skin erythema index (E.I.)
values were calculated by subtracting the logarithm of
inverse reflectance (log 1/R) values at 510 and
610 nm (mainly due to melanin absorption) from the
sum of log 1/R values at 540, 560, and 580 nm,
which represent the wavelengths of hemoglobin
absorption peaks (Eqn. 1).
16
E:I:¼100 log 1
R560 þ1;5 log 1
R540 þlog 1
R580
2 log 1
R510 þlog 1
R610
ð1Þ
Afterward, variation of the erythema index (Δ.E.I.)
for each skin sites was calculated by subtracting E.I.
baseline values from the E.I. values obtained at
different time points during the monitoring period of
the study. For each site, plotting Δ.E.I. vs. time, the
area under the curve was computed using the trapezoi-
dal rule to obtain AUC (area under curve) dimension-
less index values directly related to the degree of skin
erythema. All the regions were measured in triplicate.
In vivo evaluation of skin appearance homogeneity
during sunlamp exposure
The in vivo study was carried out for a period of
5 weeks on 25 volunteers of skin types II and IV, aged
45–70 years (mean of 56 years), recruited after der-
matological screening and all having at least five solar
lentigo (spots) on the dorsa of each hand. For each
subject, three solar spots (mean size 8–10 mm) were
selected, and one spotless area was demarcated with
permanent ink using a circular template (1 cm
2
) and
considered as a control on each hand. Experimental
design of the study was reported in Table 1. In details,
in the 1st week of the study, solar spots (A) and spot-
less sites (B) located on one hand for each subjects
were exposed to tanning treatment by a lamp simulat-
ing sunlight (Helios Italquartz srl, Milan, Italia), which
emitted the range of 300–400 nm (6.5 mW/cm
2
), for
2–8 min depending on the minimal erythema dose
(MED). To avoid induced and interfering skin erythema
events, skin exposure to the lamp was not conducted
on the third and sixth day of the week. The induced
pigmentation on spots (A) and spotless sites (B) of the
hand was monitored at the end of the 1st week and
the 2nd week, when skin reached highest pigmenta-
tion, as observed in previous research.
16
After a rest
period of 1 week (3rd week), spots (C) and spotless
sites (D) located on the other hand of the same subjects
Table 1 Experimental design of the in vivo evaluation of skin tan-
ning induced by sunlight lamp exposure of solar spots (A and C)
and spotless skin sites (B and D) located on the hands (dorsa) for
each subjects
No supplementation
1st week Tanning treatment by sunlight lamp exposure of spots
(A) and spotless sites (B) located on the same hand
and evaluation of the induced pigmentation
2nd week Evaluation of skin melanin development of spots (A)
and spotless sites (B)
3rd week Rest period
Oral supplementation with red orange extract (100 mg/die)
4th week Tanning treatment by sunlight lamp exposure of spots
(C) and spotless sites (D) located on the other hand
and evaluation of the induced pigmentation
5th week Evaluation of skin melanin development of spots (C)
and spotless sites (D)
©2014 Wiley Periodicals, Inc. 153
Red orange extract supplementation vs. photoaging .C Puglia et al.
were exposed to sunlight lamp treatment on the 4th
week of the experimental study, and the induced pig-
mentation of skin sites was monitored at the end of the
4th and the 5th week, as previously reported, during
red orange extract supplementation (100 mg/die, for
15 days). From the reflectance spectral data, the mela-
nin index (M.I.) of spots (A,C) and spotless sites (B, D)
was obtained using the following equation (Eqn. 2):
16
M:I:¼log 1
R650
log 1
R700
þ0:015 ð2Þ
where the log of inverse reflectance values (log 1/R) is
the apparent absorbance at a specific wavelength (650
and 700 nm), and 0.015 is an adjusted instrumental
factor. This index is calculated as the slope of the
apparent absorbance levels from 650 to 700 nm and
was used to measure both melanin and melanogenic
dose–response. Thereafter, the mean Δ.M.I. values (var-
iation of melanin index vs. baseline) obtained from the
solar spots were compared to Δ.M.I. values of spotless
skin sites contained on the same hand (A vs. B and C
vs. D), and the homogeneity of skin pigmentation was
expressed as tanning variation percentage (TV%),
using the following equation (Eqn. 3):
TV(%) ¼D:M:I:SD:M:I:C
D:M:I:C
100 ð3Þ
where Δ.M.I.
S
is the variation in melanin index
induced by sunlamp exposure for the solar spots (A,C),
and Δ.M.I.
c
is the variation in melanin index of spotless
sites (B,D). All the regions were measured in triplicate.
Statistical analysis
All data obtained were submitted to a statistical
analysis. All statistical comparisons in instrumental
assessment were evaluated using repeat-measure
analysis of variance (ANOVA) followed by the Bonferron-
i–Dunn post hoc pair-wise comparison procedure. A P
value of <0.05 was considered significant.
Results
The in vivo evaluation of skin erythema induced by UV
irradiation showed that supplementation of red orange
extract (100 mg/die) for 15 days brought about a sig-
nificant reduction in the skin erythema degree in sub-
jects. The trends in mean E.I. variation (Δ.E.I.) vs. time
(48 h) before and after supplementation for subjects
are reported in Fig. 1. Results showed a mean reduc-
tion of approximately 40% of the UV-induced skin ery-
thema after oral supplementation, as evidenced by
AUC values reported in Fig. 2. From the results of the
in vivo evaluation of tanning skin homogeneity, it was
observed that a very low increase in melanin content
in spot (A, C) and spotless sites (B, D) was obtained at
the end of the sunlight lamp exposure (data not
shown). As expected, skin pigmentation occurred the
week following sunlight lamp exposure, and a signifi-
cant increase in melanin content was observed in the
skin.
16
At this time point, Δ.M.I value obtained from
the solar spots A and C was calculated and compared
to the values obtained from the spotless site B and D,
respectively, for each subject. From the data obtained
(Fig. 3), it was observed that Δ.M.I. values of solar
spots A were significantly higher (P<0.05) than
Δ.M.I values of spotless sites B, whereas the increase in
melanin content of solar spots C was not significantly
higher than spotless skin sites D for the same subjects
(P<0.05). In fact, it was observed that the mean
value of TV% decreased from 27% to 7% when the
same subjects were exposed to sunlight lamp treatment
during red orange extract supplementation.
Figure 1 Trend of UV-induced skin erythema index (Δ.E.I.) vs. time (48 h) from subjects before and after red orange extract supplemen-
tation.
154 ©2014 Wiley Periodicals, Inc.
Red orange extract supplementation vs. photoaging .C Puglia et al.
Discussion
In recent years, skin care science has emphasized the
importance of food phytochemical supplements in com-
bating skin aging and disorders. The well-known rela-
tionship between nutrition and human health,
including skin condition, has recently led to the mod-
ern concept of “skin care from within”.
8
In particular,
skin protection against damage induced by sun expo-
sure has gained a great deal of interest.
1
In fact, the
use of nutricosmetics or “beauty pills” in skin photo-
protection seems to be particularly appealing as their
efficacy is not dependent on the adequacy of topical
application.
9
Many studies have already shown that
some health nutrients, such as vitamin A, E and C,
and herbal extracts, protect skin because of their anti-
oxidant activities.
8,17,18
Oral antioxidants are able to
counteract the damage processes induced by UV radia-
tion and to strengthen the physiological skin antioxi-
dant defenses.
9
In this study, the in vivo protective effects of red
orange intake against UV-induced skin damage and
skin photo-aging were evaluated. Red oranges (Moro,
Tarocco and Saguinello varieties) are characterized by
high content of antioxidant natural compounds such
as flavonoids (anthocyanin, flavones, hydroxycinnamic
acids) and ascorbic acid.
19
Red orange intake has been
shown to have several biological applications, and the
antioxidant effect is already well known in litera-
ture.
10–15
Previous studies showed that red orange extract pos-
sesses strong in vitro free radical scavenging/antioxi-
dant activity and photo-protective effect against UVB-
induced skin responses.
10–12
Cimino et al.
10
strongly
suggested that the protection by red orange extract is
exerted through an antioxidant mechanism, either by
direct shielding of UV radiation or by improving the
antioxidant cellular network. Researchers indicated
that it is potentially able to efficiently counteract UVB-
induced response, by blocking cellular oxidative stress-
related events, in cultured human keratinocytes. In
particular, events related to inflammation and apopto-
sis, such as NF-kB and AP-1 translocation and procas-
pase-3 cleavage, are significantly affected by red
orange extract pretreatment.
10
Moreover, Cardile
et al.
12
have proven that red orange extract markedly
decreased expression of membrane molecules (ICAM-1)
and the release of inflammatory soluble factors (MCP-1
and IL-8) induced by pro-inflammatory mediators in
normal human keratinocytes cell line (NCTC 2544).
12
Finally, in vivo bioavailability of the antioxidant effect
of red orange extract supplementation was already
evaluated.
13–15
Dietary supplementation with red
orange extract was able to counteract human oxidative
stress status by increasing serum thiol groups in
healthy volunteers who smoke and in sportsmen.
13–15
In this study, it was observed that supplementation
with red orange extract containing antioxidant com-
pounds of these fruits is able to protect skin against
sun exposure damage. The in vivo test models have
proven that red orange extract can inhibit skin ery-
thema processes induced by UV radiation. Moreover,
red orange intake can counteract skin hyperpigmenta-
tion of solar lentigo and improve the appearance of
skin and the homogeneity of skin tanning through the
inhibition of melanin overproduction caused by
repeated UV exposure. To this purpose, the increase of
melanin content in solar lentigenes (sun spots) has
been monitored by objective instrumental method
(reflectance spectrophotometry) in healthy volunteers
before and after oral supplementation, while undergoing
sunlamp exposure. Results showed that UV exposure
0
200
400
600
800
1.000
1.200
1.400
1.600
1.800
before aŌer
AUC values
*
Figure 2 Mean area under curve values (AUC DS) of UV-
induced skin erythema from subjects before and after red orange
extract supplementation; *P<0.05 (significantly different).
0
10
20
30
40
50
60
no supplementaƟon supplementaƟon
M.I.
Spotless sites Solar spots
*
Figure 3 Mean values of Δ.M.I. (variation of the melanin index
vs. baseline) of solar spots and spotless skin sites from one hand
treated before (A and B) and from the other hand of the same
subjects during red orange extract supplementation (C and D),
*P<0.05 significant different vs. spotless sites.
©2014 Wiley Periodicals, Inc. 155
Red orange extract supplementation vs. photoaging .C Puglia et al.
can increase pigmentation of spot sites, but a signifi-
cant variation of melanin content between spot and
spotless skin sites, used as control, was observed before
supplementation with red orange extract. As conse-
quence, higher skin color homogeneity was obtained
by oral supplementation during sunlamp exposure.
The in vivo effects of the extract intake observed in
these studies may be correlated to the antioxidant
actions of active compounds contained in orange varie-
ties and their ability to counteract the sensibility of
hyperpigmented spots to UV exposure.
It is already well known that when UV radiation pene-
trates into the human skin, several defensive molecular
events occur that result in clinical and
hystopathological changes (erythema, edema, hyperpig-
mentation, and rough texture) contributing to prema-
ture skin aging.
1,17
Even if molecular mechanisms are
still not completely understood, UV-induced oxidative
stress and ROS generation within the skin seems to play
a major role in these cutaneous changes. In particular,
ROS species can act on several skin processes such as
UV-induced melanogenesis and inflammation.
5
ROS can
directly stimulate melanocytes to produce excess mela-
nin through different molecular pathways.
4
The
increase in NO production and DAG (diacylglycerol)
intracellular content seems to be mainly correlated to
direct melanogenic effects of UV on melanocytes. More-
over, among ROS, NO radical species derived from adja-
cent keratinocytes can also induce melanogenesis by
increasing the amount of the melanogenic factors tyrosi-
nase and tyrosinase-related protein 1.
5
Oxidative stress
and ROS production in the skin seem to be strictly corre-
lated to UV-induced inflammatory processes too.
4,5
It
was observed that ROS can stimulate transcription
nuclear factor such as NF-jB, leading to an increase in
pro-inflammatory cytokines release. In turn, pro-inflam-
matory cytokines can stimulate the epidermal keratino-
cytes and dermal fibroblasts, upregulate
metalloproteases levels, and degrade dermal collagen
and elastic fibers.
9,17
Therefore, skin inflammation
induced by acute or long-term exposure to sunlight
(sunburn) can strongly contribute to the acceleration of
skin-aging processes. Furthermore, earlier studies have
shown that a correlation between skin inflammatory
processes and melanogenesis could also occur, and low-
level chronic inflammation can stimulate the develop-
ment of hyperpigmented solar letigenes in the skin.
1
On the basis of these assumptions, it is possible to
hypothesize that red orange extract intake can
strengthen physiological antioxidant skin defenses,
protecting skin from the damaging processes involved
in photo-aging and leading to an improvement in skin
appearance and pigmentation. In conclusion, the natu-
ral blend of red orange active compounds (varieties
Moro, Tarocco, and Sanguinello) can be considered as
a good candidate as a nutracosmetic ingredient in skin
photo-protection and skin care.
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