In vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract

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

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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 Laboratory of Phytochemistry and Medicinal Chemistry, Department of Pharmacy, Alfenas Federal University, Alfenas, Minas Gerais, Brazil 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 b c

a r t i c l e

i n f o

Article history: Received 18 September 2013 Received in revised form 4 December 2013 Accepted 7 January 2014 Available online 17 January 2014 Keywords: Garcinia brasiliensis Antioxidant Anti-inflammatory Cytotoxicity Photoprotection Photochemoprotection In vitro SPF

a b s t r a c t The damaging effects of sunlight to the skin has triggered studies that involve the synthesis and extraction of organic compounds from natural sources that can absorb UV radiation, and studies on polyphenolic compounds with antioxidant and anti-inflammatory properties that can be used as photochemopreventive agents for reducing skin damage. We investigated the in vitro and in vivo photoprotective/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 solution or the GbEE formulation. The in vivo photoprotective effect of the GbEE formulation was evaluated by measuring the UVB damage-induced decrease in endogenous reduced glutathione (GSH), the increase in myeloperoxidase (MPO) activity and secretion of cytokines IL-1b and TNF-a. The in vitro methodology using fibroblasts showed that the photoprotective properties of the GbEE solutions and 10% GbEE formulation were similar to the commercial sunscreen (SPF-15). In vivo results demonstrated of the GbEE formulation in decreasing UVB induced-damage such as GSH depletion, an increased in MPO activity and 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 induce adverse effects on human skin [1–3]. UVB radiation induces damages including inflammation (erythema or sunburn), pigmentation, 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 absorb 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: [email protected] (M.J.V. Fonseca). 1011-1344/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2014.01.004

Over the last decades, plants of the Garcinia species have received considerable attention because of the chemical composition of their extracts, which are rich in polyisoprenylated benzophenone derivatives as well as polyphenols, biflavonoids and xanthones [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 diseases [10]. Two natural polyisoprenylated benzophenones have been isolated from G. brasiliensis: 7-Epiclusianone and Guttiferone-A. 7-Epiclusianone, a tetraprenylated benzophenone, was isolated 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. brasiliensis epicarp extract (GbEE). To test the efficacy of GbEE we evaluated the cell viability of the L929 fibroblasts cell line after UVB exposure using a quartz plate containing the extract solution or

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the GbEE formulation on top of the cell microplate. In addition, the in vivo photoprotective effect of the GbEE formulation was evaluated by measuring the UVB damage-induced decrease in endogenous 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 cultures showed that GbEE extract and the GbEE formulation increased the viability of L929 cells exposed to UVB radiation at the same rate as was observed when a commercial sunscreen formulation 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 cytokines 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 temperature, filtered and then dried, using a rotary evaporator under reduced pressure at 45 °C to obtain the G. brasiliensis epicarp extract (GbEE).

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 concentration 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 xanthine/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% propylparaben, 0.175% methylparaben, 0.075% EDTA (ethylenediamine tetraacetic 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.

2.2. 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 reagent (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 polyphenol 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 absorbance was measured at 420 nm. The standard curve for total flavonoids 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 particle size. The mobile phases consisted of eluent A (0.5 mM/L aqueous 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 software was used for data collection [9].

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 tissue 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 humidified 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 medium was replaced with Hank’s buffer. A quartz plate of exactly the same dimensions was placed on top of the cell microplate and covered with the samples [19,20]. This assay was based on the measurement 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 unprotected 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.

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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 assess the photoprotection provided by different solutions of the extract, 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 formaldehyde 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 measure 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

20–30 g, were housed in a temperature-controlled room with access 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 = nonirradiated 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 formulations topically to the back of the animals one hour before irradiation. 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 fluorescence assay as previously described by Hissin and Hilf [24]. The dorsal skin of hairless mice (1:3, w/w dilution) was homogenized 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 supernatant was measured in a Hitachi F-4500 fluorescence spectrophotometer. 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 fluorescence 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% Hexadecyltrimethylammonium bromide (HTAB). The skins were then homogenized using a T25 digital Ultra-TurraxÒ. The homogenates were

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centrifuged at 16,100g for 2 min, and the resulting supernatant was assayed spectrophotometrically for MPO activity determination 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 determined by triplicate analysis. The antixiodant activity, cell viability percentage and in vivo tests related to the potential photoprotective were analised using GraphPad PrismÒ software. Data were statistically analyzed by Student’s t-test or one-way ANOVA followed by Tukey’s test of multiple comparisons, and the level of significance 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. livingstonei (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 polyisoprenylated benzophenones, 7-Epiclusianone and Guttiferone-A. We compared the obtained GbEE chromatographic profile with standards solutions of these compounds and with the chromatographic profiles obtained by Martins et al. [10]. The 7-Epiclusianone was the major component identified and quantified in the extract, which corresponded to 57% of the total constituents, separated 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 provided the greatest survival of cells after UVB radiation exposure (Fig. 3A). The sunscreens with SPF 15 (brands A and B) provided increases in cell viability of 17.74% and 11.64%, respectively. However, it was observed that the brand B commercial sunscreen with SPF-15 was not statistically significantly different in comparison 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 increased 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 coefficients 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 absorption 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 suggest 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 photoprotective 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 dissolved in caprylic/capric acid triglyceride (Fig. 3B). The GbEE solutions 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 calculated SPF values for GbEE solutions of 50 and 100 mg/mL were 15.9% and 18.5%, respectively (Table 1).

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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) GbEE solution (10 mg/mL) GbEE solution (50 mg/mL) GbEE solution (100 mg/mL) SPF-15 Commercial sunscreen (brand A) 2% GbEE formulation 10% GbEE formulation 20% GbEE formulation

– 5.7 28.5 57.0 – 11.4 57.0 114.0

16.6 0.0 16.3 20.1 16.0 0.0 19.1 30.8

16.2 0.0 15.9 18.5 15.8 0.0 17.8 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 spectra of the solutions of the GbEE (100 lg/mL), 7-Epiclusianone (100 lg/mL), and 3-benzophenone (20 lg/mL) dissolved in caprylic/capric acid triglyceride, as shown in Fig. 4C. Spectral anal-

ysis of the solutions showed UV absorption in the UVB (280– 320 nm) and UVA-2 (320–340 nm) range. The GbEE and 7-Epiclusianone solutions were five times more concentrated than the benzophenone-3 solution and presented absorption spectra in a single

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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 analyzing 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 absorb UVB radiation. Topical formulations supplemented with 2%, 10% and 20% of GbEE, containing 11.4, 57.0 and 114.0 mg/g of 7Epiclusianone (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 placebotreated 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 compound present in this extract. In addition, the developed method for photoprotective potential evaluation also proved to be accurate. Samples of SPF-15 commercial 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

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 photoprotective effect observed in vitro of the solutions and formulations containing GbEE. The formulation containing 10% of GbEE was selected to be used in in vivo assays because the application of this formulation did not alter the natural skin color. The photoprotective potential was evaluated in vivo using hairless mice by measuring GSH level and myeloperoxidase activity and by determining the inflammatory 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 increased myeloperoxidase activity, which were dose dependent responses 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 addition, 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, maintaining 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 untreated-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 inflammatory 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-irradiated 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 inflammatory effects induced by UVB radiation. 3.6. GbEE formulation prevents the increase of UVB-induced proinflammatory mediators TNF-a and IL-1b

Fig. 5. Percentage of cell viability of fibroblasts (L929 line) treated with formulations 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% = formulation 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.

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 interleukin-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, respectively, in the irradiated control group (IC) when compared with the non-irradiated control group (NIC).

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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 exposure 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 untreated 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 multilayered structure constituted by a highly keratinized outer epidermal layer, whilst the epidermis and dermis are constituted by primary cells such keratinocytes, melanocytes, Langerhans cell, mast cells, and infiltrating leukocytes, which participate of inflammation in physiological mechanisms [30]. Additionally, the skin is the most exposed organ to the deleterious effects of UV solar radiation, which has turned into a major environmental carcinogen. UVB radiation (290–320 nm) represents 5% of the solar radiation 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 mitochondrial 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 phospholipase 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 induces 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 multiple signaling pathways that are triggered by direct UVB action as well as by ROS generated in the damaged tissue by UVB. The activation of the mitogen activated protein kinase (MAPK) family members (ERK, JNK and p38MAPK) leads to activation of transcription factors activator protein-1 (AP-1) and nuclear factor kappa B (NFjB). It induces the up-regulation of pro-inflammatory cytokines and growth factors such as tumor necrosis factor-a (TNFa), 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, vasodilatation 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, inflammation, immunosuppression, photoaging, and carcinogenesis. Therefore, 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

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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 photoprotective 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 reflect 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-Epiclusianone, 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 products 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. Previous 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 applied 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 7Epiclusianone might make it more resistant to water. Thus, the lower skin penetration and greater water resistance may contribute 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 commercial sunscreen (SPF-15). This commercial sunscreen presents in the composition organic UVB filters such as ethylhexyl salicylate, ethylhexyl triazone, butyl methoxydibenzoylmethane and octocrylene and organic UVA/UVB filters such as BP-3, bis-ethylhexylphenol and methoxyphenyl triazene as well as inorganic filters. The in vivo photoprotective properties of GbEE were also demonstrated. Firstly, GSH depletion induced by UVB radiation was totally inhibited by GbEE formulation treatment, which suggests that GbEE might have prevented the generation of ROS and/or scavenged the generated reactive species. In addition, irradiated skin treated with GbEE formulation presented similar myeloperoxidase activity to the non-irradiated skin. This result suggests that the extract components, especially 7-Epiclusianone, were able to absorb the UVB radiation preventing the inflammatory process induced by radiation. The UVB radiation absorption by GbEE components could prevent the installation of oxidative stress and, consequently, the lipid peroxidation. In this case, the in vitro inhibition of lipid peroxidation 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 inhibition 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 antioxidant 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 extract’s efficiency to prevent an increase in MPO activity, which suggests 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. However, 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 topical formulations in addition to synthetic UV filters contributing to the reduction of synthetic filters in the formulations. It is noteworthy 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 photoprotective 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). References [1] F. Afaq, V.M. Adhami, H. Mukhtar, Photochemoprevention of ultraviolet B signaling and photocarcinogenesis, Mutation Res./Fundam. Mol. Mechanisms Mutagenesis 571 (2005) 153–173. [2] G.T. Bowden, Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling, Nat. Rev. Cancer 4 (2004) 23–35. [3] N. Khan, D.N. Syed, H.C. Pal, H. Mukhtar, F. Afaq, Pomegranate fruit extract inhibits UVB-induced inflammation and proliferation by modulating NF-jB and MAPK signaling pathways in mouse skin, Photochem. Photobiol. 88 (2012) 1126–1134. [4] M.N. Chrétien, E. Heafey, J.C. Scaiano, Reducing adverse effects from UV sunscreens by zeolite encapsulation: comparison of oxybenzone in solution and in zeolites, Photochem. Photobiol. 86 (2010) 153–161. [5] M. Hupel, N. Poupart, E. Ar Gall, Development of a new in vitro method to evaluate the photoprotective sunscreen activity of plant extracts against high UV-B radiation, Talanta 86 (2011) 362–371. [6] S. F’guyer, F. Afaq, H. Mukhtar, Photochemoprevention of skin cancer by botanical agents, Photodermatol. Photoimmunol. Photomed. 19 (2003) 56–72. [7] U.M. Acuña, K. Dastmalchi, M.J. Basile, E.J. Kennelly, Quantitative highperformance liquid chromatography photo-diode array (HPLC–PDA) analysis of benzophenones and biflavonoids in eight Garcinia species, J. Food Compos. Anal. 25 (2012) 215–220. [8] I.O. Pereira, M.J. Marques, A.L.R. Pavan, B.S. Codonho, C.L. Barbiéri, L.A. Beijo, A.C. Doriguetto, E.C. D’Martin, M.H. dos Santos, Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits, Phytomedicine 17 (2010) 339–345.

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