Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

In vitro and in vivo photoprotective/photochemopreventive potential

of Garcinia brasiliensis epicarp extract
Sônia Aparecida Figueiredo a, Fernanda Maria Pinto Vilela a, Claudinei Alves da Silva b,
Thiago Mattar Cunha c, Marcelo Henrique dos Santos d, Maria José Vieira Fonseca a,⇑
a
Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
b
Laboratory of Phytochemistry and Medicinal Chemistry, Department of Pharmacy, Alfenas Federal University, Alfenas, Minas Gerais, Brazil
c
Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
d
Laboratory of Organic Chemistry, Department of Chemistry, Viçosa Federal University, Viçosa, Minas Gerais, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The damaging effects of sunlight to the skin has triggered studies that involve the synthesis and extrac-
Received 18 September 2013 tion of organic compounds from natural sources that can absorb UV radiation, and studies on polyphe-
Received in revised form 4 December 2013 nolic compounds with antioxidant and anti-inflammatory properties that can be used as
Accepted 7 January 2014
photochemopreventive agents for reducing skin damage. We investigated the in vitro and in vivo photo-
Available online 17 January 2014
protective/photochemopreventive potential of Garcinia brasiliensis epicarp extract (GbEE). We evaluated
the cell viability of L929 fibroblasts after UVB exposure using a quartz plate containing the extract solu-
Keywords:
tion or the GbEE formulation. The in vivo photoprotective effect of the GbEE formulation was evaluated by
Garcinia brasiliensis
Antioxidant
measuring the UVB damage-induced decrease in endogenous reduced glutathione (GSH), the increase in
Anti-inflammatory myeloperoxidase (MPO) activity and secretion of cytokines IL-1b and TNF-a. The in vitro methodology
Cytotoxicity using fibroblasts showed that the photoprotective properties of the GbEE solutions and 10% GbEE formu-
Photoprotection lation were similar to the commercial sunscreen (SPF-15). In vivo results demonstrated of the GbEE for-
Photochemoprotection mulation in decreasing UVB induced-damage such as GSH depletion, an increased in MPO activity and
In vitro SPF secretion of cytokines IL-1b and TNF-a. The results showed that the extract has great potential for use
as a sunscreen in topical formulations in addition to UV filters.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction
Skin exposure to solar ultraviolet (UV) radiation, particularly its
UVB component (280–320 nm) due to its energetic properties is
thought to be the most harmful portion of UV radiation and to in-
duce adverse effects on human skin [1–3]. UVB radiation induces
damages including inflammation (erythema or sunburn), pigmen-
tation, hyperplasia, immunosuppression, cutaneous photoaging
and cancer [4,5].
Review of the complexity of the damaging effects of sunlight on
the skin has triggered studies which involve the synthesis and
extraction of organic compounds from natural sources that can ab-
sorb UV radiation, and the extraction of polyphenolic compounds
with antioxidant and anti-inflammatory properties that can be
used as photochemopreventive agents in topical products for
reducing UV-induced skin damage [5,6].
⇑ Corresponding author. Address: Quality Control and Photochemoprevention
Laboratory, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São
Paulo, Ribeirão Preto 14040-903, Brazil. Tel.: +55 1636024433.
E-mail address: magika@fcfrp.usp.br (M.J.V. Fonseca).
Over the last decades, plants of the Garcinia species have re-
ceived considerable attention because of the chemical composition
of their extracts, which are rich in polyisoprenylated benzophe-
none derivatives as well as polyphenols, biflavonoids and xanthon-
es [7]. Extracts of the pericarp, epicarp and seeds of Garcinia fruit
have demonstrated antioxidant, anti-inflammatory, leishmanicidal
and antiprotozoal activities [8,9] and in traditional medicine the
Garcinia fruit has been used to treat wounds, ulcers and dysentery
[7].
Garcinia brasiliensis is a native plant from the Amazon forest and
is found throughout Brazil. The fruit of this species has been used
as a folk medicine for treating peptic ulcer, urinary and tumor dis-
eases [10]. Two natural polyisoprenylated benzophenones have
been isolated from G. brasiliensis: 7-Epiclusianone and Guttifer-
one-A. 7-Epiclusianone, a tetraprenylated benzophenone, was iso-
lated as a main constituent of the hexane extract from the fruit
pericarp of G. brasiliensis [11,12].
In the present study we investigated the in vitro and in vivo
photoprotective and photochemopreventive potential of G. brasili-
ensis epicarp extract (GbEE). To test the efficacy of GbEE we evalu-
ated the cell viability of the L929 fibroblasts cell line after UVB
exposure using a quartz plate containing the extract solution or

1011-1344/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2014.01.004
66 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73
the GbEE formulation on top of the cell microplate. In addition, the
in vivo photoprotective effect of the GbEE formulation was evalu-
ated by measuring the UVB damage-induced decrease in endoge-
nous reduced glutathione (GSH), the increase in myeloperoxidase
(MPO) activity and secretion of the pro-inflammatory cytokines
IL-1b and TNF-a.
In vitro photoprotection studies employing fibroblast cell cul-
tures showed that GbEE extract and the GbEE formulation in-
creased the viability of L929 cells exposed to UVB radiation at
the same rate as was observed when a commercial sunscreen for-
mulation with an SPF of 15 was used. In vivo results demonstrated
the photoprotective effect of the GbEE formulation, which was able
to decrease the UVB induced-damage, such as depletion of GSH,
activation of in MPO, and secretion of the pro-inflammatory cyto-
kines IL-1b and TNF-a.
2. Materials and methods
2.1. Plant materials
The fruits of G. brasiliensis were collected on the campus of the
Federal University of Viçosa (UFV), Viçosa-MG, Brazil. Botanical
identification was performed in the Botanical Garden of the UFV,
and a voucher specimen was deposited under registry code
VIC2604.
Dried and powdered epicarp of the G. brasiliensis fruits (1000 g)
was extracted by maceration with 3.0 L of ethanol at room temper-
ature, filtered and then dried, using a rotary evaporator under re-
duced pressure at 45 °C to obtain the G. brasiliensis epicarp
extract (GbEE).
2.2. Total polyphenol and flavonoid contents
The total polyphenol content in GbEE was determined using the
Folin–Ciocalteau colorimetric technique [13]. Briefly, the GbEE
solution (0.5 mL) was added to 0.5 mL of the Folin–Ciocalteau re-
agent (IMBRALAB – Química e Farmacêutica, Ribeirão Preto, SP,
Brazil) and 0.5 mL of 10% Na2CO3. After incubation for 1 h at room
temperature, the absorbance was measured at 760 nm. Total poly-
phenol contents were expressed as gallic acid equivalents per gram
of dry extract (GAE/g dry extract).
The total flavonoid content of the GbEE was determined using
the method employed by Woisky and Salatino [14]. The extract
solution (0.5 mL) was added to 2% AlCl3 ethanol solution
(0.5 mL), and after 1 h at room temperature incubation, the absor-
bance was measured at 420 nm. The standard curve for total flavo-
noids was prepared using a quercetin standard solution, and the
total flavonoid content was calculated as quercetin equivalent
per gram of dry extract (QE/g dry extract) from a calibration curve.
Both the polyphenol and flavonoid contents are presented as the
means of triplicate analyses.
2.3. HPLC analysis of GbEE
High performance liquid chromatography (HPLC) analysis of
GbEE was performed on Shimadzu LC-100 equipment using a
C18 column, Shimadzu CLC-ODS (250–4.6 mm), with a 5 lm parti-
cle size. The mobile phases consisted of eluent A (0.5 mM/L aque-
ous acetic acid) and eluent B (methanol/acetic acid 0.1%). The
gradient (A:B) utilized was the following: 0 min (50:50), 10 min
(0:100) and 25 min (0:100), with a solvent flow rate of 1.2 mL/
min and observation at 254 nm. Additionally, an injection volume
of 20 lL at a concentration of 1 mg/mL was used. LC solution soft-
ware was used for data collection [9].
2.4. Antioxidant activity
The antioxidant activity of the GbEE was evaluated by studying
the H-donor activity using the DPPH radical as described by Blois
[15], by the inhibiting lipid peroxidation as described by Rodrigues
et al. [16] and by scavenging superoxide radicals produced in the
chemiluminescence assay using the xanthine/luminol/XOD system
[17].
The extract was first solubilized with ethyl alcohol and diluted
using the medium of each reaction to the following final concen-
tration ranges: 10–80 lg/mL for H-donor activity using the DPPH
radical assay, 2.5–20 lg/mL for the lipid peroxidation assay, and
1.87–15 lg/mL for the chemiluminescence assay using the xan-
thine/luminol/XOD system.
For all three different methodologies employed, the percentage
inhibition was plotted against the different concentrations of GbEE,
and the concentration that caused 50% inhibition of the system
was reported as the IC50 value.
2.5. Preparation of GbEE formulations
The present study was performed using a cream gel formulation
prepared with 1.50% commercially available self-emulsifying wax
(PolawaxÒ – cetostearyl alcohol and polyoxyethylene derived from
fatty acid ester sorbitan 2OE), 0.50% anionic hydrophilic colloid
(carboxypolymethylene – CarbopolÒ 940), 1.75% cetyl alcohol,
2.00% stearic acid, 2.75% gliceryl monostearate, 0.025% propylpar-
aben, 0.175% methylparaben, 0.075% EDTA (ethylenediamine tetra-
acetic acid), 10.00% propylene glycol, and 81.23% deionized water
[18]. GbEE was solubilized in capric/caprylic triglyceride and then
added to the formulations at room temperature at 2.0, 10.0 and
20.0% (w/w) concentrations.
2.6. In vitro photoprotective potential
2.6.1. Cell culture
The L929 fibroblast cell line was purchased from the Cell Bank
of Rio de Janeiro (BCRJ). They were routinely grown in 150 cm2 tis-
sue culture flasks in DMEM (supplemented with 1% (v/v) of an
antibiotic solution containing 10,000 I.U./mL of penicillin,
10,000 lg/mL of streptomycin and 25 lg/mL of amphotericin B),
and 10.0% (v/v) of FBS. The cells were grown at 37 °C in a humidi-
fied incubator with 5% CO2.
2.6.2. Irradiation
The UV irradiation source was a Philips TL/12 RS 40 W lamp
(Medical-Holland). This source emits in the range of 270–400 nm
with an output peak at 313 nm, resulting in an irradiation of
0.27 mW/cm2 at a distance of 20 cm as measured by an IL 1700
radiometer (Newburyport, MA, USA) equipped with UVB detector.
For the irradiation experiments, cells were seeded into six-well
microplates at an initial density of 8  105 cells/well and grown for
12 h to 80% confluency. During the irradiation procedure, the med-
ium was replaced with Hank’s buffer. A quartz plate of exactly the
same dimensions was placed on top of the cell microplate and cov-
ered with the samples [19,20]. This assay was based on the mea-
surement of cell viability after being protected against UV
exposure by photoprotective substances or not being protected
(Fig. 1).
Initially, to get a reduction of approximately 50% of the unpro-
tected L929 fibroblast cell viability, the culture cells were submitted
to different UVB radiation doses, between 0.060 and 0.500 J/cm2,
and incubated for 24 and 48 h, after irradiation. The decrease in cell
viability of 50% was achieved when the UVB dose was 0.09 J/cm2
and incubation for 48 h.
 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73
Fig. 1. Experimental design used in the photoprotection study. The L929 fibroblast cell line was seeded into six-well plates at an initial density of 8  105 cells/well. A quartz
plate with exactly the same dimensions was placed on top of the cell microplate and covered with the samples. Both plates were exposed UVB radiation.
Two different brands of commercial sunscreens (A and B) with
three different sun protection factors (15, 30 and 60) were used as
standards in the photoprotective potential assay, and the amount
of the formulations applied on top of the quartz plate was 2 mg/
cm2. The percentage of photoprotection was calculated using the
following equation:
Photoprotection ð%Þ ¼ ð% of cell viability of treated cells
 % of cell viability of irradiated controlÞ
After proving the method ability to discriminate among the
samples of sunscreen formulations with different SPF, the previous
procedure was used to evaluate the photoprotective effect of the
GbEE extract. GbEE extract solutions in the concentrations of 10,
50 and 100 mg/mL were prepared in capric/caprylic triglyceride
solvent.
Finally, once the effectiveness of the method was proven to as-
sess the photoprotection provided by different solutions of the ex-
tract, the study was continued using formulations incorporating
GbEE at concentrations of 2, 10 and 20% (w/w). The amounts of
the GbEE solutions and the GbEE formulations applied onto the
quartz plate were also 2 mg/cm2.
After the irradiation procedure (0.09 J/cm2), the quartz plate was
removed, the Hank’s buffer in the cell was exchanged for culture
medium and the cells were incubated in a CO2 incubator for 48 h.
2.6.3. Cell viability
After the irradiation and incubation of the fibroblasts for 48 h,
the medium was removed and the cells were washed with saline
solution. Cell viability was determined by neutral red assay [21].
The medium (3 mL) containing neutral red at concentration of
50 lg/mL was added to each well, and the plate was returned to
the incubator for 3 h. Thereafter, the medium was removed, the
cells were washed rapidly with an aqueous solution of 1% of form-
aldehyde and 1% of CaCl2, and then 2.0 mL of a solution of 1% acetic
acid and 50% ethanol was added to each well to extract the dye.
After agitation, the plate was transferred to a microplate reader
(iMark™, BIO-RAD, Japan) equipped with a 540 nm filter to mea-
sure absorbance.
2.6.4. Assessment of the in vivo photoprotective effect of GbEE
formulation
2.6.4.1. Irradiation of animals. In vivo experiments were performed
on 3-month-old, sex-matched hairless mice. The animals, weighing
67
20–30 g, were housed in a temperature-controlled room with ac-
cess to water and food ad libitum until use. They were housed in
cages with a 12 h light and 12 h dark cycle. All experiments were
conducted in accordance with the National Institutes of Health
guidelines for the welfare of experimental animals and with the
approval of the Ethics Committee of the Faculty of Pharmaceutical
Science of Ribeirão Preto (University of São Paulo, Ribeirão Preto,
SP, Brazil – Process n. 12.1.1367.53.0).
The animals were divided into four groups: Group NIC = non-
irradiated control, Group IC = irradiated control, Group NIF =
treated with the GbEE formulation and non-irradiated, and Group
IF = treated with the GbEE formulation and irradiated. The
treatment protocol consisted of applying 30 mg of the formula-
tions topically to the back of the animals one hour before irradia-
tion. The groups exposed to UVR were placed inside a wooden
enclosure containing the lamp and were irradiated for 2 h, which
corresponds to a total dose of 2.87 J/cm2. The mice were sacrificed
by inhalation of carbon dioxide 6 h following UVR exposure, and
full dorsal skins were removed and stored at 80 °C until analysis
[22,23].
2.6.4.2. GSH assay. GSH skin levels were determined using a fluo-
rescence assay as previously described by Hissin and Hilf [24].
The dorsal skin of hairless mice (1:3, w/w dilution) was homoge-
nized in 100 mM NaH2PO4 (pH 8.0) containing 5 mM EGTA using
a T25 digital Ultra-TurraxÒ (IKAÒ, Germany). Whole homogenates
were treated with 30% trichloroacetic acid and, then centrifuged
at 1900g for 6 min and the fluorescence of the resulting superna-
tant was measured in a Hitachi F-4500 fluorescence spectropho-
tometer. Briefly, 100 lL of the sample supernatant was mixed
with 1 mL of 100 mM NaH2PO4 (pH 8.0) containing 5 mM EGTA
and 100 lL of o-ftalaldeide (OPT) (1 mg/mL in methanol). The fluo-
rescence was determined after 15 min (k exc = 350 nm; k
em = 420 nm). The values were compared to a curve prepared with
standard GSH, and the results are presented as lmol of GSH per mg
of skin.
2.6.4.3. MPO activity. The UVB-induced leukocyte migration into
the skin was evaluated using the MPO kinetic–colorimetric assay
[25,22].
The dorsal skin (1:20 dilution) was collected and placed in
50 mM K2HPO4 buffer (pH 6.0) containing 0.5% Hexadecyltrime-
thylammonium bromide (HTAB). The skins were then homoge-
nized using a T25 digital Ultra-TurraxÒ. The homogenates were
68 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73
centrifuged at 16,100g for 2 min, and the resulting supernatant
was assayed spectrophotometrically for MPO activity determina-
tion at 450 nm (lQuantTM; BioTek Instruments Inc., Winooski,
Vermont, USA) after 10 min. Briefly, 10 lL of sample were mixed
with 200 lL of 50 mM phosphate buffer (pH 6.0) containing
0.167 mg/mL o-dianisidine dihydrochloride and 0.015% hydrogen
peroxide. The MPO activity of the samples was compared with a
standard enzyme curve, and the results are presented as units of
MPO per mg of skin.
2.6.4.4. TNF-a and IL-1b measurements. The skin samples (100 mg)
were homogenized in 500 lL of the appropriate buffer containing
protease inhibitors, and TNF-a and IL-1b levels were determined
by ELISA as described previously by Cunha et al. [26]. The results
were expressed as picograms (pg) of each cytokine per g skin
tissue.
2.7. Statistical analysis
Data were expressed as the mean ± standard deviation as deter-
mined by triplicate analysis. The antixiodant activity, cell viability
percentage and in vivo tests related to the potential photoprotec-
tive were analised using GraphPad PrismÒ software. Data were sta-
tistically analyzed by Student’s t-test or one-way ANOVA followed
by Tukey’s test of multiple comparisons, and the level of signifi-
cance was set to p < 0.05.
3. Results
The obtained GbEE was chemically characterized by quantifying
the contents of polyphenols and flavonols, as well as functionally
by measuring of antioxidant activities using different methods.
The total polyphenolic content expressed as gallic acid equivalent
per gram of dry extract (GAE/g), and the total flavonoid content
calculated as a quercetin equivalent per gram of dry extract (QE/
g), were 69.8 mg GAE/g and 3.4 mg QE/g, respectively. The results
showed that the polyphenol amount of the G. brasiliensis extract
was lower than other Garcinia fruit extracts such as G. intermedia
(476.9 ± 40.8 mg GAE/g dry fruit), G. hombroniana (326.9 ± 8.1 mg
GAE/g dry fruit), G. xanthochymus (283.6 ± 65.3 mg GAE/g dry
fruit), G. kola (248.3 ± 48.7 mg GAE/g dry fruit), G. mangostana
(263.3 ± 6.4 mg GAE/g dry fruit), G. spicata (237.6 ± 15.6 mg GAE/
g dry fruit), G. aristata (237.1 ± 6.3 mg GAE/g dry fruit) and G. liv-
ingstonei (115.5 ± 34.1 mg GAE/g dry fruit) [7].
The results of the GbEE chromatography analysis employing
HPLC-photo-diode array showed the major presence of the polyi-
soprenylated benzophenones, 7-Epiclusianone and Guttiferone-A.
We compared the obtained GbEE chromatographic profile with
standards solutions of these compounds and with the chromato-
graphic profiles obtained by Martins et al. [10]. The 7-Epiclusia-
none was the major component identified and quantified in the
extract, which corresponded to 57% of the total constituents, sepa-
rated and detected by chromatographic analysis (Fig. 2).
The antioxidant activities of GbEE obtained using different
methods were expressed as IC50 value. The IC50 values found were
47.46, 4.22 and 4.49 lg of extract per mL of reaction medium in the
reduction of DPPH, in the inhibition of lipid peroxidation and in
the inhibition of chemiluminescence generated by the xanthine/
luminol/XOD system, respectively.
3.1. In vitro photoprotective potential of GbEE
It was observed that the commercial sunscreens protected the
cells against viability reduction induced by UVB radiation that
was observed in the irradiated cells without sunscreens (irradiated
Fig. 2. GbEE HPLC chromatogram profile. The chromatogram profile showed the
major presence of the polyisoprenylated benzophenones, 7-Epiclusianone and
Guttiferone-A, in 17.5 and 19.5 min, respectively.
control). The efficiency of sunscreens in absorption, reflection and
scatter of the UVB radiation was dependent on the sun protection
factor (SPF). The commercial sunscreen with the highest SPF pro-
vided the greatest survival of cells after UVB radiation exposure
(Fig. 3A). The sunscreens with SPF 15 (brands A and B) provided in-
creases in cell viability of 17.74% and 11.64%, respectively. How-
ever, it was observed that the brand B commercial sunscreen
with SPF-15 was not statistically significantly different in compar-
ison to the irradiated control which shows that the employed
method is suitable for discriminating the photoprotective efficacy
of different formulations. The increases in the cell viability when
sunscreens with SPF-30 (brands A and B) were used 43.22% and
38.91%, respectively, whereas the sunscreens with SPF 60 in-
creased cell viability to 52.58% and 48.26% for brands A and B,
respectively.
When we plotted the percentage of protection conferred by
brand A and B formulations with different SPF (Table 1) versus
the log of SPF value, we observed a linear relationship with coeffi-
cients of determination (r) of 0.9661 and 0.9623, respectively
(Fig. 4A and B).
This result shows an advantage of the presently employed
method when compared with the in vivo method used by Nash
et al. [27] which showed that the relationship between the absorp-
tion of erythemogenic energy versus SPF is nonlinear. Sunscreens
with SPF values of 10, 15 and 30 reduced erythema formation by
approximately 88%, 92% and 94%, respectively. Therefore, we sug-
gest that the in vitro method used in the present study might be
an alternative to in vivo methods to screen different formulations
supplemented with UV filters or plant extracts with photoprotec-
tive effects during the developmental and production phases of
these products.
The standardized photoprotective potential assay was used to
evaluate the UVB radiation absorption capacity by GbEE solutions
in the concentrations of 10 mg/mL, 50 mg/mL and 100 mg/mL dis-
solved in caprylic/capric acid triglyceride (Fig. 3B). The GbEE solu-
tions of 50 mg/mL and 100 mg/mL, containing 28.5 mg/mL and
57.0 mg/mL of 7-Epiclusianone, respectively, absorbed the UVB
radiation and protected the viability of fibroblast cell by 16.3%
and 20.1%, respectively, in relation to the irradiated control (IC)
(Table 1). The caprylic/capric acid triglyceride (solvent control)
and 10 mg/mL GbEE solution in caprylic/capric acid triglyceride,
containing 5.7 mg/mL 7-Epiclusianone did not protect the cells
against UVB radiation. The viability of the cells treated with this
solution was similar to the irradiated control (Fig. 3B). The calcu-
lated SPF values for GbEE solutions of 50 and 100 mg/mL were
15.9% and 18.5%, respectively (Table 1).
 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73 69

Fig. 3. Percentage of cell viability of fibroblasts (L929 line) treated with commercial sunscreens (A) and GbEE solutions (B) exposed to UVB radiation. Where, Panel A: Brand A
and Brand B refer commercial sunscreens, respectively, with SPF-15, SPF-30 and SPF-60; NIC = non-irradiated control, IC = irradiated control. Panel B: GbEE solutions in
caprylic/capric acid triglyceride solvent in the concentrations of 10 mg/mL (10); 50 mg/mL (50) and 100 mg/mL (100); S = commercial sunscreen SPF-15 used as standard;
SC = control solvent; NIC = non-irradiated control, IC = irradiated control. The results represent the average of five independent determinations, with 3 wells per group.
*
p < 0.05 statistically significant difference compared with non-irradiated control (NIC) group. **p < 0.05 significant difference compared to the irradiated control (IC) group.

Table 1
Calculated SPF values for solutions and formulations supplemented with GbEE.

Samples Concentration of 7-Epiclusianone (mg/mL) Photoprotection (%) Calculated SPFa

SPF-15 Commercial sunscreen (brand A) – 16.6 16.2
GbEE solution (10 mg/mL) 5.7 0.0 0.0
GbEE solution (50 mg/mL) 28.5 16.3 15.9
GbEE solution (100 mg/mL) 57.0 20.1 18.5
SPF-15 Commercial sunscreen (brand A) – 16.0 15.8
2% GbEE formulation 11.4 0.0 0.0
10% GbEE formulation 57.0 19.1 17.8
20% GbEE formulation 114.0 30.8 27.7
a
Calculated SPF based on equation obtained in vitro photoprotection assays (Fig. 4A). The results represent the average of five independent determinations, with 3 wells per
group.

Fig. 4. Mathematical relationship between the in vitro photoprotective percentages versus log of SPF commercial sunscreens expressed mathematically by a linear regression
line obtained by method of least squares fit (Panels A and B). Espectrophotometric analysis of GbEE photoprotective potential (Panel C). (A) Brand A commercial sunscreen
with SPF-15, SPF-30 and SPF-60; (B) Brand B commercial sunscreen with SPF-15, SPF-30 and SPF-60. The percentage of photoprotection was calculated using the following
equation: Photoprotection (%) = % of cell viability of treated cells  % of cell viability of irradiated control (Materials and Methods item 2.6.2). (C) UV absorption spectrum of
100 lg/mL 7-Epiclusianone (7-Epi), Garcinia brasiliensis epicarp extract (GbEE) solutions, and 20 lg/mL benzophenone-3 (BP-3) solution in caprylic/capric acid triglyceride.

Based on these results, we determined the UV absorbance spec- ysis of the solutions showed UV absorption in the UVB (280–
tra of the solutions of the GbEE (100 lg/mL), 7-Epiclusianone 320 nm) and UVA-2 (320–340 nm) range. The GbEE and 7-Epiclu-
(100 lg/mL), and 3-benzophenone (20 lg/mL) dissolved in sianone solutions were five times more concentrated than the ben-
caprylic/capric acid triglyceride, as shown in Fig. 4C. Spectral anal- zophenone-3 solution and presented absorption spectra in a single
70 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73
mode with a maximum absorption plate in the range of 285–
320 nm, while benzophenone-3 presented a bimodal absorption
spectrum with one peak at 290 nm and other at 325 nm. By analyz-
ing the results in Fig. 4C, it is possible observe that the GbEE could
provide protection in the UVB range of the solar spectrum while
benzophenone-3 protects against solar radiation in the UVB and
UVA-2 ranges, as mentioned by Sambandan and Ratner [28]. The
UV-absorption spectrum of 7-Epiclusianone was similar to the
GbEE, which suggests that GbEE UV absorption capacity might be
due to the presence of this compound in the extract.
3.2. In vitro photoprotective potential of formulations added with
GbEE
The major component present in GbEE is the polyisoprenylated
benzophenone 7-Epiclusianone that corresponds to 57% of extract.
This component might be responsible for the extract’s ability to ab-
sorb UVB radiation. Topical formulations supplemented with 2%,
10% and 20% of GbEE, containing 11.4, 57.0 and 114.0 mg/g of 7-
Epiclusianone (Table 1), respectively, were prepared and evaluated
in vitro to evaluate their photoprotection effectiveness using the
method optimized and standardized.
As shown in Fig. 5, the formulation containing 2% of GbEE and
the placebo formulation (without extract) did not protect the cells
against UVB radiation since the cell viabilities were 42.0% and
42.2%, respectively, which were similar to the irradiated control
(49.0%). The similarities among the cell viabilities of the placebo-
treated cells and the irradiated control show that the formulation
components are unable to absorb the UVB radiation.
On the other hand, the formulations containing 10% and 20% of
GbEE increased cell viability by 19.1% and 30.8% compared to the
irradiated control (Fig. 5 and Table 1). The SPF values calculated
for formulations supplemented with 10% and 20% of GbEE were
17.8 and 27.7 (Table 1), respectively, using the in vitro standardized
photoprotective potential assay. The GbEE solution (100 mg/mL)
and the formulation supplemented with 10% of GbEE that contains
the same amount of 7-Epiclusianone (57.0 mg/mL) showed very
similar values for the calculated SPF of 18.5 and 17.8, respectively.
These results reinforce the observation that the UVB radiation
absorption capacity of GbEE is due to the 7-Epiclusianone com-
pound present in this extract.
In addition, the developed method for photoprotective potential
evaluation also proved to be accurate. Samples of SPF-15 commer-
cial sunscreen (brand A) analyzed on different days presented the
calculated SPF values of 16.6 and 16.0 (Table 1), with variation
coefficient of 2.6%. Moreover, different samples of GbEE solution
(100 mg/mL) and the formulation with 10% GbEE containing the
Fig. 5. Percentage of cell viability of fibroblasts (L929 line) treated with formula-
tions supplemented with different concentrations of GbEE exposed to UVB
radiation. Where, NIC = non-irradiated control, S = commercial sunscreen SPF-15
used as standard, IC = irradiated control, PLAC = placebo formulation, 2% = formu-
lation supplemented with 2% of extract, 10% = formulation supplemented with 10%
of extract and formulation supplemented with 20% of extract. The results represent
the average of five independent determinations, with 3 wells per group. *p < 0.05
significant difference compared with non-irradiated control (NIC) group. **p < 0.05
significant difference compared to the irradiated control (IC) group.
same amount of 7-Epiclusianone and analyzed in different days
have also showed approximate calculated SPF values, 18.5 and
17.5, respectively (Table 1).
3.3. In vivo photoprotective potential of topical formulation containing
10% of GbEE against UVB induced damages
The in vivo assays were performed to confirm the photoprotec-
tive effect observed in vitro of the solutions and formulations con-
taining GbEE. The formulation containing 10% of GbEE was selected
to be used in in vivo assays because the application of this formu-
lation did not alter the natural skin color. The photoprotective po-
tential was evaluated in vivo using hairless mice by measuring GSH
level and myeloperoxidase activity and by determining the inflam-
matory cytokines IL-1b and TNF-a.
Studies performed by Casagrande et al. [22] and Vicentini et al.
[23] showed that UVB radiation decreased GSH levels and in-
creased myeloperoxidase activity, which were dose dependent re-
sponses in the 0.96–3.69 J/cm2 range. The dose of 2.87 J/cm2 used
in the present study decreased GSH level by 28.3% and increased
myeloperoxidase activity by 175.0% in the irradiated animals (IC)
when compared to the non-irradiated control group (NIC). In addi-
tion, this dose of radiation (2.87 J/cm2) increased the IL-1b and
TNF-a level by approximately 463.0% and 234.0%, respectively.
Based on these results the dose of 2.87 J/cm2 was chosen in the
present study.
3.4. GbEE formulation prevents UVB-induced skin GSH depletion
Results obtained in the present study corroborate with previous
studies in the literature that describes the GSH depletion induced
by UVB radiation exposure. A depletion of 28.3% in GSH level in
the skin exposed to UVB radiation (2.87 J/cm2) was observed.
Topical treatment of mice skin with the 10% GbEE formulation
before UVB exposure resulted in inhibition of GSH depletion, main-
taining a similar level to untreated-non-irradiated control group
(NIC) (Fig. 6). In addition, UVB unexposed skin treated with 10%
GbEE formulation presented glutathione levels similar to the un-
treated-unexposed controls (NIC). This result suggests that neither
the formulation components nor the GbEE interfered with the
measurement of GSH in the skin.
3.5. GbEE formulation prevents MPO activity increase induced by UVB
Myeloperoxidase activity can be used as a marker of inflamma-
tory process. In the present study, six hours after UVB radiation
(2.87 J/cm2) an increase of 175.0% in MPO activity in the irradiated
control group (IC) was detected in comparison to the non-irradi-
ated and untreated group (NIC). Interestingly, MPO activities were
similar in the irradiated groups treated with 10% GbEE formulation
(IF), non-irradiated group treated with 10% GbEE formulation, and
non-irradiated control group (NIC) (Fig. 6). These results suggest
that the 10% GbEE formulation was very effective against inflam-
matory effects induced by UVB radiation.
3.6. GbEE formulation prevents the increase of UVB-induced pro-
inflammatory mediators TNF-a and IL-1b
One of the earliest events after UV skin exposure is the direct or
indirect activation receptors of cell surface growth factors and
cytokine receptors such as tumor necrosis factor (TNF-a) and inter-
leukin-1 (IL-1b) [29]. In this study, the results showed an increase
of 463.0% and 234.0% in the amounts of IL-1b and TNF-a, respec-
tively, in the irradiated control group (IC) when compared with
the non-irradiated control group (NIC).
 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73 71

Fig. 6. In vivo photoprotective potential of topical formulation containing 10% of GbEE against UVB induced damages. Panel A: in vivo evaluation of GSH skin levels. Panel B:
in vivo evaluation of MPO activity. Panel C and D: in vivo evaluation of inflammatory cytokines TNF-a and IL-1b, respectively. Where, group NIC = non-irradiated control,
group IC = irradiated control, group IF = treated with 10% GbEE formulation and irradiated and NIF = treated with 10% GbEE formulation and non-irradiated. Bars represent
the average of three independent determinations, with five animals per group. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test of multiple
comparisons. *p < 0.05 significant difference compared to the non-irradiated control (NIC) group. **p < 0.05 significant difference compared to the irradiated control (IC) group.

Topical treatment with 10% GbEE formulation before UVB expo-
sure induced a decreased of 64.0% and 63.0% in the levels of TNF-a
and IL-1b, respectively, when compared to the irradiated and un-
treated animals (IC) (Fig. 6). In addition, the non-irradiated group
treated with 10% GbEE formulation (NIF) presented TNF-a and
IL-1b levels similar to the basal levels present in the untreated
and non-irradiated group (NIC).
4. Discussion
Skin is considered the largest organ of the body and constitutes
a physical barrier against injuries, infections, water and electrolyte
loss; it is also an important part of the immune system. It is a mul-
tilayered structure constituted by a highly keratinized outer epi-
dermal layer, whilst the epidermis and dermis are constituted by
primary cells such keratinocytes, melanocytes, Langerhans cell,
mast cells, and infiltrating leukocytes, which participate of inflam-
mation in physiological mechanisms [30]. Additionally, the skin is
the most exposed organ to the deleterious effects of UV solar radi-
ation, which has turned into a major environmental carcinogen.
UVB radiation (290–320 nm) represents 5% of the solar radia-
tion that reaches the Earth surface. However, the reduction of the
ozone layer has been increasing the amount of UV radiation that
reaches the surface, and it is estimated that for every 1% decrease
in ozone layer there is an increase of 1–2% in the UVB levels that
reach the Earth’s surface [31].
UVB affects mainly epidermal cells, and it is more genotoxic and
approximately 1000 times more capable of causing sunburn than
UVA. Its adverse biological effects are complex because UVB acts
by direct and indirect mechanisms. The direct effects induced by
UVB include damage to DNA, protein and stratum corneum lipids,
while it is also capable of generating reactive oxygen species (ROS)
and reactive nitrogen species (RNS) by indirect mechanisms that
induce imbalance in the oxidative status in skin [32].
Oxidative stress starts a cascade of events that includes the
alteration of the protein glycosylation and nuclear and mitochon-
drial DNA modifications as well as lipid peroxidation induction
that when associated with the direct damages to lipids change
the integrity and functionality of the cellular membranes [33,34].
Therefore, the skin functions after UVB exposure are markedly
modified leading to the development of inflammatory processes
and immune suppression. The inflammatory process includes a
cascade of events. The first phase of the inflammatory process is
the vasodilatation event. In this phase, the direct effects of UVB
radiation associated with the ROS generation induced by radiation
stimulate the activity and expression of the cytosolic phospholi-
pase A2 (cPLA2) and the up-regulation of the cyclooxygenase-2
(COX-2) expression resulting in increased PGE2 production [30].
This increase in PGE2 in association with the NO generation in-
duces arteriolar vasodilatation providing blood flow increase and
leukocytes migration from the circulation to the damaged tissue
[35].
The second phase of the inflammatory process involves multi-
ple signaling pathways that are triggered by direct UVB action as
well as by ROS generated in the damaged tissue by UVB. The acti-
vation of the mitogen activated protein kinase (MAPK) family
members (ERK, JNK and p38MAPK) leads to activation of transcrip-
tion factors activator protein-1 (AP-1) and nuclear factor kappa B
(NFjB). It induces the up-regulation of pro-inflammatory cyto-
kines and growth factors such as tumor necrosis factor-a (TNF-
a), interleukin-1b (IL-1b), transforming growth factor b (TGF-b)
and interferon-c [36].
The last phase consists of the resolution of inflammation, which
is an active process that involves biochemical mediators such as
the pro-resolution mediators and signaling pathways controlling.
The resolution of inflammation is essential to maintain tissue
health after stimuli cause tissue damage, and this phase involves
key events including granulocyte recruitment reduction, vasodila-
tation and vascular permeability reversion as well as phagocytosis
of dying cells [37].
The biochemical, molecular and histological changes induced by
acute skin UVB exposure can lead to oxidative stress, inflamma-
tion, immunosuppression, photoaging, and carcinogenesis. There-
fore, to protect the skin against sunlight damages the use of
appropriate clothing and sunglasses in conjunction of the daily
use of sunscreens composed of a mixture of organic and inorganic
UV filters are recommended.
However, research about the efficacy of UV filters has shown a
decrease of their UV-protective capacity that can be attributed to
72 S.A. Figueiredo et al. / Journal of Photochemistry and Photobiology B: Biology 131 (2014) 65–73
their photo decomposition, the generation of reactive species and
their transdermal absorption. Thus, applied amounts of UV filters
on the skin would not be fully available to perform their photopro-
tective effects [38,18]. It is important to consider that UV filters
should remain on the skin’s surface and not be easily washed off
during use, and they should not be released into the aquatic
environment.
Although sunscreen products have been used for 75 years, they
are health care products that should be constantly innovating
through research of new compounds with abilities to absorb or re-
flect UV radiation at different wavelengths (UVA-I, UVA-II and
UVB) and should also be photostable, unabsorbed and not toxic.
The GbEE exhibited high amounts of the compound 7-Epiclusia-
none, a polyisoprenylated benzophenone, corresponding to 57% of
the total extract. This result sparked interest in assessing whether
the extract could present photoprotective potential. In this study,
the GbEE showed a capacity to absorb UVB radiation five times
smaller than benzophenone-3 (BP-3).
BP-3, an organic UVA and UVB filter, is one of the most common
sunscreen ingredients present in nearly 60% of sunscreens prod-
ucts marketed in world. It is characterized by a relatively low
molecular weight (228.25), is water soluble at 25 °C of 68.56 mg/
L and has octanol–water partition coefficient (Log P) of 3.600. Pre-
vious studies showed that due to these physical properties, BP-3
were able to penetrate, permeate the skin and reach the systemic
circulation after topical application [39,40]. The transdermal
absorption of this UV filter in human skin may reach 2% of the ap-
plied amount [41]. BP-3 has shown to be slightly irritating and a
high incidence of photodermatitis has been observed among BP-3
users [42].
Although 7-Epiclusianone has shown lower ability in the
absorption of UVB when compared with BP-3, this compound has
approximately two times higher values of octanol–water partition
coefficient (log P 7.147) and molecular weight than BP-3, which
may contribute to less penetration of 7-Epiclusianone into the skin
in comparison to BP-3. Additionally, the higher lipophilicity of 7-
Epiclusianone might make it more resistant to water. Thus, the
lower skin penetration and greater water resistance may contrib-
ute to a better retention on the skin, thereby compensating the
lower UV absorption efficiency of 7-Epiclusianone in relation to
BP-3.
In vitro methodology using fibroblasts cell culture showed that
the photoprotective properties of the GbEE solutions and 10% and
20% GbEE formulations were similar to the properties of the com-
mercial sunscreen (SPF-15). This commercial sunscreen presents in
the composition organic UVB filters such as ethylhexyl salicylate,
ethylhexyl triazone, butyl methoxydibenzoylmethane and octocr-
ylene and organic UVA/UVB filters such as BP-3, bis-ethylhexyl-
phenol and methoxyphenyl triazene as well as inorganic filters.
The in vivo photoprotective properties of GbEE were also dem-
onstrated. Firstly, GSH depletion induced by UVB radiation was to-
tally inhibited by GbEE formulation treatment, which suggests that
GbEE might have prevented the generation of ROS and/or scav-
enged the generated reactive species.
In addition, irradiated skin treated with GbEE formulation pre-
sented similar myeloperoxidase activity to the non-irradiated skin.
This result suggests that the extract components, especially 7-Epi-
clusianone, were able to absorb the UVB radiation preventing the
inflammatory process induced by radiation.
The UVB radiation absorption by GbEE components could pre-
vent the installation of oxidative stress and, consequently, the lipid
peroxidation. In this case, the in vitro inhibition of lipid peroxida-
tion and antioxidant properties of GbEE might also contribute to
this photoprotective effect.
Based on the evaluation of the results we suggest that GbEE
may have action against the installation of the first phase of the
inflammatory process. This suggestion is based on the protection
against oxidative stress in the skin observed in vivo by the inhibi-
tion of GSH depletion induced by UVB radiation. This protection
provided by the formulation supplemented with the extract may
be attributed to its ability to inhibit lipid peroxidation and its anti-
oxidant and free radical-scavenging capacity and mainly by
absorption of UVB radiation.
Consequently, the extract also avoided triggering the second
phase of the inflammatory process, which can be proven by the ex-
tract’s efficiency to prevent an increase in MPO activity, which sug-
gests the inhibition of neutrophil migration toward the exposed
area, and also to inhibit the synthesis of the pro-inflammatory
cytokines such as TNF-a and IL-1b induced by UVB. In addition,
in the in vivo photoprotection assay, topical formulation with
10% GbEE extract, applied on non-irradiated skin, did not cause
any alterations in the inflammatory parameters, such as MPO
activity, TNF-a and IL-1b levels. These results suggest the absence
of toxic effects of the formulation and extract on normal skin. How-
ever, additional studies about security and penetration/retention
on viable epidermis should be performed.
5. Conclusion
The results showed that the extract has a great potential to be
used as a sunscreen, suggesting that it can be incorporated in top-
ical formulations in addition to synthetic UV filters contributing to
the reduction of synthetic filters in the formulations. It is notewor-
thy that the major component of the extract, 7-Epiclusianone,
which responsible for photoprotection activity of the extract, is
present in high quantities in the peel of the fruit that is a byproduct
of G. brasiliensis, which represents a sustainable extraction. Further
studies are necessary to clarify the precise nature of this photopro-
tective effect.
Acknowledgments
The authors gratefully acknowledge the financial support of the
‘‘Conselho Nacional de Desenvolvimeto Científico e Tecnológico’’
(CNPq, Brazil), ‘‘Fundação de Amparo à Pesquisa do Estado de São
Paulo’’ (FAPESP, Brazil) and ‘‘Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior’’ (CAPES, Brazil) for financial support
and a research fellowship. Sônia Aparecida Figueiredo was the
recipiente of a CNPq fellowship (Processo # 132143/2011-9).
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