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Food and Chemical Toxicology 48 (2010) 70–75

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Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox

Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines
C.F. Zanatta a,b, M. Mitjans b, V. Urgatondo b, P.A. Rocha-Filho a, M.P. Vinardell b,*
a b

Dept. Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil Dept de Fisiologia, Facultat de Farmácia, Universitat de Barcelona, Spain

a r t i c l e

i n f o

a b s t r a c t
Considering the belief that natural lipids are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, we have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect against UVA and UVB irradiation on keratinocytes and fibroblasts. Cell treatment was divided into two steps, prior and after exposition to 30 min of UVA plus UVB radiation or to 60 min of UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing a-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the two cell lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection, especially in after sun formulations. Ó 2009 Elsevier Ltd. All rights reserved.

Article history: Received 11 May 2009 Accepted 15 September 2009

Keywords: Buriti oil Emulsion UV protection Skin irritation Cell culture

1. Introduction Exposure of human skin to UVA radiation can induce various biological responses, ranging from erythema to photoaging (Matsumura and Ananthaswamy, 2004; Wlaschek et al., 2001), because this component of the solar UV spectrum penetrates through the dermis to the subcutaneous tissue and affects both the epidermal and dermal components of skin (Tarozzi et al., 2005). At the cellular level, UVA radiation causes significant oxidative stress because it leads to the generation of reactive oxygen species (ROS), exerting a variety of harmful effects including oxidation of nucleic acids, proteins and membrane lipids (Morita and Krutmann, 2000). The UVB radiation is experimentally demonstrated to be the most effective light to induce skin cancers in animals, and can cause DNA damage, particularly cyclobutane pyridimine dimmers (CPDs) and photoproducts which induce mutations in the epidermal cells, leading to the development of cancer cells (Ichihashi et al., 2003). Fundamentally, protection against solar UV-induced oxidative damage to human skin relies on avoiding excessive sunlight expo* Corresponding author. Address: Dept de Fisiologia, Facultat de Farmácia, Av. Joan XXIII s/n, 08028 Barcelona, Spain. Tel.: +34 934024505; fax: +34 934035901. E-mail address: mpvinardellmh@ub.edu (M.P. Vinardell). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.09.017

sure and on the use of sunscreens. Topical and endogenous photoprotection by antioxidants could have the potential to complement these strategies (Chiu and Kimball, 2003; Tarozzi et al., 2005), since ROS are the main responsible for the biological effects caused by the photo-oxidative stress. The use of natural ingredients in cosmetics is steadily increasing. As a result, formulators are being offered a host of newly derived lipids, most from renewable plants sources (Fernandes and Cabral, 2007; Bogdan Allemann and Baumann, 2008). High levels of phytochemicals, especially antioxidants are commonly found in many of these plants. One of such plants is Buriti palm tree (Mauritia flexuosa), which oil is rich in carotenoids and has been frequently used in cosmetic production. The Buriti oil contains about 1706 ± 54 lg of total carotenoids/g and b-carotene is considered the major carotenoid performing 90% of total content (GarciaQuiroz et al., 2003). The oil also presents high levels of oleic acid (60.3%) and considerable amounts of a-tocopherol (643.2 mg/g) (Costa, 2007). Carotenoids are known to be powerful antioxidants and their ability to quench singlet oxygen has been studied and extensively reviewed (Chen et al., 2007; Hix et al., 2004; McNulty et al., 2008). Oxygen reactive species, such as superoxide radical, peroxide radical and hydroxyl radical, which can be generated by cytotoxic compounds and by exposure to UV radiation, are potentially damaging to cells through initiation of lipid peroxidation.

The dimethyl sulfoxide and the neutral red dye was acquired from Sigma–Aldrich (Deisenhofen. 3. Zanatta et al. 5% CO2 to the wells corresponding to post-treatment condition. Hatziantoniou et al. 10 mM HEPES buffer and 1% penicillin (10. We have established a dose of 2. assessed on monolayers cultures of human keratinocytes HaCat and 3T3 embryonic mouse fibroblast. For the emulsions preparation. formulated with Buriti oil. 1% acetic acid in distilled water was added to extract the dye. Germany). 2008) The plain Buriti oil was also tested. 2 mM l-glutamine. Differences were considered statistically significant at a p < 0.P. Buriti oil was supplied by Croda (Campinas. Germany). Brazil). To reach this irradiation. The 75 cm2 flasks and 96-well plates were obtained from TPP (Trasadingen. 5% CO2. which is known as a potent antioxidant (Bogdan Allemann and Baumann. and the D-panthenol (P) at 1% to produce the 37M. a broad range of cell and tissue culture systems has been developed for assessing the irritation and phototoxic potential of chemicals. and the ability in preventing formation of epidermal erythema during sun exposure (Stahl and Sies. Switzerland). Hix et al.5 g/l glucose) supplemented with 10% fetal bovine serum (FBS). 2. the medium was removed and Neutral Red solution (50 lg/ml) was added. the cells were washed with PBS and medium was added. the aim of this study was to investigate whether topical creams and lotions. 2000) and that carotenoids are able to protect cells against photooxidative damages (Merzlyak et al.3. Barcelona (Spain).. t stands for the time of exposure (in seconds). 713 D) at 600 rpm until they reached room temperature (25 ± °C). can exert photoprotective effect against UVA and UVB irradiation. 2004). 2007). Ceteareth-5. Materials Culture media and reagents: Dulbecco’s Modified Eagle’s Medium (DMEM). 2. for certain specific purposes. When the cells were approximately 80% confluent..VE. Cell treatment and UV irradiation Emulsions were diluted in DMEM medium supplemented with 10% FBS at a final concentration of 125 mg/ml according to previous studies of cytotoxicity (Zanatta et al. PEG-40 castor oil by Oxiteno (São Paulo. Tarozzi et al.P and the 37N. Cells were cultured in DMEM medium (4.F. To this end. D-panthenol were supplied by Sigma–Aldrich (Deisenhofen.6.VE and 37N.VE. the PBS was removed from non-treated wells and 100 ll of the emulsions with and without active ingredient (1% of a-tocopherol or panthenol) were added for 15 min at 37 °C. Belgium).000 U/ml)–streptomycin (10. Plates were then incubated at 37 °C. Materials and methods 2. the absorbance of neutral red was measured at a wavelength of 550 nm in a Bio-Rad 550 microplate reader (Riddell et al. penicillin (10. Photoprotective potential of emulsions was evaluated by one-way analysis of variance conducted by ANOVA test (Sigmastat 3. 2005). especially the beta-carotene. Cell treatment was divided into two steps. cells were washed twice with PBS and a solution containing 50% ethanol absolute. two O/W macroemulsions containing liquid crystals (29R and 51LC). legal and financial motives have banned the in vivo method for testing cosmetic ingredients and formulations. The O/W macroemulsions (51S. Several authors studied the antioxidant activity of carotenoids and vitamin E in the skin and the synergism of their association (Gaspar and Campos. / Food and Chemical Toxicology 48 (2010) 70–75 71 Consequently. commercial surfactants and vitamins. After 10 min on a microtitre-plate shaker. Brazil). 2007. 29R and 29R. Over the past few years. NRU assay Following treatment.. 51S. halfplates were seeded with HaCat and the other half with 3T3.VE. 2.P) were prepared using the emulsification by phase inversion (EPI) method (Santos et al.. When culture cells in the 96-well reached confluence. The vitamin E acetate and. one W/O/W multiple emulsion (37M) and one O/W nanoemulsion (37N).. 5% CO2 medium was removed. 2002). Human keratinocytes have become the focus of attention in skin irritation by virtue of their importance in maintaining the integrity of the stratum corneum barrier. the treated cells were washed with PBS and the culture medium of the whole plate was replaced by PBS. 2005). Irradiance was measured using a photoradiometer Delta OHM (HD2302 – Italy) to determine the time of exposure. 2006). prior and after the irradiation to determine which treatment would be more effective. after previous solubilization in DMSO (final concentration not higher than 5%). For these experiments. 51LC. 2008. there has been great interest in the antioxidant properties of carotenoids with regard to human health.VE) were obtained by the phase inversion temperature method (Tadros et al. among them human keratinocytes and fibroblasts cultures (Dijoux et al. 51LC.5 Â 104 cells/ml for fibroblasts and of 10 Â 104 cells/ml for keratinocytes and then incubated for 48 h at 37 °C.5).C.. USA). PBS was replaced by culture medium with 10% FBS from the remaining wells as described previously (Riddell et al. Cells were used at passage 3–5. After 3 h of incubation at 37 °C. and P is the light intensity. 2..000 lg/ml) mixture at 37 °C. All cell cultures proved mycoplasma-free on periodic checks using a polymerase chain reaction (PCR) assay and standard agarose gel electrophoresis... at a cell density of 8. The concentrations of Buriti oil. several in vitro test methods have been suggested as valid alternatives of the irritation tests and recently reviewed (Vinardell and Mitjans. 51LC. the medium was removed from the wells corresponding to pre-treatment condition. The capacity of absorbing the excess of energy would allow the carotenoids to absorb part of the UV radiation and also to reduce . 1986). Statistical analysis Results were expressed as a percentage of viability compared with control wells (the mean optical density of untreated cells was set to 100% viability).4. Huang et al. 2009). 2008. Stahl and Sies. 2. cells remained exposed 30 min in the case of the UVA and UVB lamps and 60 min for the UVA lamp. 51S. 2002. HEPES buffer. Removed the product. Results and discussion The photoprotective potential of the Buriti oil emulsions was based on the high levels of carotenoids present in the oil. since in vivo they are the first cells to be exposed to topical formulations. 2. calculated from the dose–response curves by linear regression analysis. Before UV exposure.05. 5% CO2. and their ability to produce a wide range of inflammatory mediators. The Steareth-2 was obtained from Beraca Sabará Ingredients (São Paulo. they were harvested with trypsin/EDTA and seeded into a 96-well plates.. 5% CO2 for 3 h to run the effects of the UV. 2008).5. 1986). After UV exposure. Ceteareth-20.VE. water and surfactants of each different systems are described in Table 1. using the following equation: E ðJ=cm2 Þ ¼ tðsÞ Â P ðW=cm2 Þ where E represents the UV energy. at a final concentration of 100 ll/ml.. is preferable to use keratinocytes. These cells were treated with 100 ll of the emulsions with and without active ingredient (1% of atocopherol or panthenol) for 15 min at 37 °C. 5% CO2.VE) and the multiple emulsions (37M and 37M.VE. Each experiment was performed at least three times using triplicates for each emulsion and condition analyzed.. Although some authors (Burlando et al.000 lg/ml) mixture and fetal bovine serum (FBS) were purchased from Bio-Whittaker (Verviers. an UV TLK 40 W lamp with UVA and UVB emission and a TL-D 15 W/10 UVA lamp (Royal Philips Eletronics – The Netherlands). 2008) argue that keratinocytes are less sensitive than fibroblasts to irritation.000 U/ml)–streptomycin (10. Each system was produced with and without an active ingredient at concentration of 1%. 2008. Considering the belief that natural lipids are safer for topical applications (Mandawgade and Patravale. 2004). The DL-a-tocopherol or vitamin E acetate (VE) at 1% was used to produce the 29R.. Sorbitan Monooleate.1. Two types of UV sources were used. Carotenoids showed a peroxide radical scavenging activity in human skin and a capacity of inhibiting lipid peroxidation (Mortensen. All emulsions were produced under constant stirring (Mechanic Mixer Fisatom Mod. diluted in DMEM 10% FBS. Products preparation Five different emulsions were evaluated. 2002). one simple O/W emulsion (51S). Culture of HaCaT and 3T3 cell line The spontaneously immortalized human keratinocyte cell line HaCaT and the mouse embryonic fibroblast cell line 3T3 were obtained from the ‘‘Banco de Células Eucariotas”. while the nanoemulsions (37N and 37N.2. Brazil) and sodium poliacrylate from Dewolf Chemical Inc (Rhode Island.5 J/cm2 to assays performed in fibroblasts and keratinocytes as reported in the literature (Offord et al. Ethical.P and 37N.

as was previously reported (Dijoux et al.VEb F51LCb F51LC. like Polysorbate 80 and Cremophor EL. in general. F51LS and F51S with and without vitamin E attributed to the high concentration of surfactants in the formulations and the higher sensitivity of fibroblasts. when applied before UV exposure in the pre-treatment condition.VE. Tatsuishi et al.VE and F37M (Table 4). all emulsions showed to be more phototoxic to the 3T3 fibroblasts.53 4.47 5.F. F51LC. but also pre. the results obtained were more significant..and post-treatment of HaCaT cells with F37N. F29. The cells that were treated after UVA irradiation exposure.30 2. although the phototoxicity test had been validated by the ECVAM (European Centre for the Validation of Alternative Methods) in fibroblasts 3T3 (Spielmann et al. Obermüller-Jevic et al. Except for the plain oil.10 12.70 2. F29. Surfactant system composed by surfactant A: Steareth-2 and surfactant B: Ceteareth-20. Formulation Buriti oil (%) Water (%) C. Nevertheless. (1999) observed that beta-carotene strongly enhances the stress response in UVA-irradiated skin fibroblasts. Furthermore. F29.0 – – – – – – – – – – – 1.. 1998) due to the high correlation with in vivo tests. especially in the pre-treatment condition.1 – – – – – – – – – 1. except for the formulations F51S and F37M.VE.Pc F37Nc F37N. when compared to the emulsion without these components. Considering that the surfactants caused an initial oxidative damage. as has been previously observed (Merwald et al. HaCat keratinocytes showed to be less sensitive to photo-irritant compounds when compared to 3T3 fibroblasts.VE. 2008). Surfactant system composed by surfactant A: Sorbitan Monooleate and surfactant B: PEG-40 castor oil.VE in the case of HaCaT cell line.Pc a b c 10 10 5 5 5 5 5 5 5 5 80 79 80 79 80 79 90 89 90 89 4. where all surfactants and emulsion had their cytotoxicity evaluated. 2004). prooxidant activity was observed at higher levels and was reported as adverse effects (Eichler et al. Control plates that remained in the dark. leads to a reduction in intracellular glutathione levels.VE. However. F51LC.0 – 1.30 0. F37N. also presented slightly higher viability values when compared to those that were previously treated with the emulsions.30 2. higher reduction on cell viability than UVA/UVB radiation. F51S. These results were more evident in the assays where UVA and UVB radiation were employed (Table 3).P. According to the literature.VE. Another possibility is that the antioxidants added to the emulsions combined with carotenoids present in Buriti oil might have exerted a prooxidant effect in the cells submitted to UV radiation. F37M. the treatments with F37N. F37N. F37N. The phototoxic damages caused to the cells by the emulsions. like cosmetic’s safety assays. resulting in an increase in hydrogen peroxide concentration.90 2. cellular viability did not varied when compared to control values.1 0.90 2. F37N.. authors hypothesized that the 1O2 quencher activity of beta-carotene in UVA-irradiated fibroblasts was not effective and that the observed prooxidant potential of beta-carotene could also have enhanced inflammatory responses to UVA-induced oxidative stress such as production of pro-inflammatory cytokines.VE. higher viability for keratinocytes compared to fibroblasts. it would be interesting to use in future 3D skin models to demonstrate the effects of emulsions due to their more realistic behaviour. cells defenses were reduced even before they were exposed to UV.72 Table 1 Concentration of emulsion’s components.10 2.53 12.. which presented higher viability values in pre-treatment when compared to the post-treatment (Table 3).VEb F37Mc F37M.90 2.P and Buriti oil not only did not increase cytotoxicity. F51S and F51S. F51LC. which is responsible for part of the oxidative stress that causes damages to the cells (Hirama et al. The assays performed with UVA/UVB irradiation showed a reduction in cell viability and the pre and post-treatment with F29. these effects showed to be dose-dependent. Consequently. The same pattern was observed for the HaCat keratinocytes.VE. F51S. F51LC.70 Sodium polyacrylate (%) Vitamin E acetate (%) Panthenol (%) F29Ra F29R. all other samples analyzed resulted in lower values in fibroblasts and significant differences were observed for emulsions F29. Significant differences in viability values between the treatments were only found for F29 and F29. F37M. justifying the lower viability values found for the pre-treatment condition. That would explain the fact that the emulsions with a-tocopherol.0 – 1. showing once more. when the red dye was applied after 3 h of post-treatment.P. 2002) and also could be related to the . F37M. UVA radiation for 60 min induced in general.70 2. keratinocytes are preferred since they are the first cells exposed to the products and to the sun light. Nevertheless.. Analysis showed that when the red dye was applied immediately after the post-treatment. F37M. 2005).VE increased phototoxicity (Table 3).10 12. since their cytotoxic effect could be enough to make more sensitive-labile the cells prior to UV exposure. F51S. Significant differences were found in HaCaT cells posttreated with the F29. the oxidative stress caused by the radiation (Chen et al.P. the exposure of the cell membrane to non-ionic surfactants. 2004.90 2. F51LC. The results of the NRU of the control plate showed that the formulations and the Buriti oil caused a small decrease in HaCaT cell viability (Table 2) when compared to the controls cells without product. Interesting. / Food and Chemical Toxicology 48 (2010) 70–75 Surfactant system (%) Surfactant A Surfactant B 5. F51LC. 2006). for some specific purposes. However.0 – 1.. 2007). determined by induction of heme oxygenase-1 (HO-1). F51S.70 2. significant differences in viability values were only found for the plain oil (Table 3).. led to lower viability values. there is a significant decrease on 3T3 cell viability after treatment with F29.P when compared to the values of the cells pre-treated with the same emulsions. could be attributed to the surfactants used in their production. These results could be partially explained by the use of a surfactant (Ceteareth-20) that was considered cytotoxic in a moderate grade in a previous study (Zanatta et al. Zanatta et al.VE.30 2. Viability values were significantly higher when 3T3 fibroblasts were post-treated with formulations F29.P compared to pre-treated cells.P and Buriti oil increased cell viability of irradiated cells. passed through all steps except the radiation. We have observed that in general. However.P and F37M.47 2.10 12.P and F37M.VEa F51Sb F51S.. F51LC.0 Surfactant system composed by surfactant A: Steareth-2 and surfactant B: Ceteareth-5.

6 102.8 ± 3.8 ± 6. / Food and Chemical Toxicology 48 (2010) 70–75 Table 2 Cellular viability values of non-irradiated control plates of 3T3 and HaCat pre and post-treated with Buriti oil emulsions.0 81.3 94.3 23.b 78.2a.8 ± 3.0 76.7 ± 4.VE F51S F51S.2 ± 8.5c 37.6 ± 6.6 ± 2.5b 33.1 ± 4. b Indicates significant difference in comparison to the 3T3 fibroblasts in the same treatment condition.2 89.b 51.1 ± 0.7 69.6 ± 2.5b 72.6 ± 9.5 ± 2.8 ± 3.2 ± 5.6 ± 3.7 ± 11.7 98.4b 68. at significance level of 95%.8 ± 4.5 ± 5.9 ± 4.63 92.b 77.2 ± 4.4 ± 1.6 ± 3.6 40.5 ± 2.9 67.1 ± 4. interactions of the carotenoids and the a-tocopherol (Black and Gerguis.9 44.9 ± 4.1 ± 4.5 59.9a.7 ± 0.8 62.9 ± 4.3 ± 2.9 ± 4.6 81. the accumulation of these oxidation products associated to excess of antioxidants (carotenoids and the a-tocopherol) leads to chain reaction due to a prooxidant activity.9 69.5 52.5 ± 5.8 ± 1.5 ± 3.6 18.VE F51S.9 Irradiated 3T3 controls (%) UVA irradiated HaCat cells (%) Pre-treatment 44.1 ± 8. tumor promotion by activation of intracellular signaling and also the generation of free radicals and related oxidants that contributes to carcinogenesis (Merwald et al.9 87.9 40.9 ± 4.8 95.6 75.5 105.0b 25.P F37M.5 ± 2.1 106. 6 ± 4.4 ± 25.1 37.5 ± 3.1 ± 6. however.6 22 ± 2.2 ± 3.3 100.0 ± 4.8 ± 19.0 ± 3. Table 4 Cellular viability values obtained by the NRU in 3T3 and HaCat pre.9 ± 5.3 ± 17.0 ± 6.9 101.P Buriti oil 39.2 113.8 ± 7.3a 22.b 51.2 ± 2.7 68.3 79.2 23.2 ± 3.7 82.7 ± 1.4 ±.2 ± 4.9 ± 20.9 53.2 ± 13 66.6 ± 1.VE F51LC.4 73.0 66.7 73 F29.7 ± 1. Formulation tested Non-irradiated 3T3 cells (%) F29 F51LC F51S F37N F37M Buriti oil Non-irradiated HaCat cells (%) F29 F51LC F51S F37N F37M Buriti oil Pre-treatment 46.7 ± 4.8 96.4 92.6 94. Formulation tested UVA irradiated 3T3 cells (%) Pre-treatment F29 F29.P F37 M.5 93.2 30.VE F51S.8 91.1 ± 1.4 72.8a.2 ± 3.0 99.3 ± 12.7 69.7 42.6 Irradiated HaCat controls (%) Results are expressed as mean ± standard error of triplicates of three independent experiments.8 44. c Indicates that it is significantly higher than viability of the irradiated control wells.9 ± 20.3 ± 3.3 ± 11. at significance level of 95%.VE F51S F51S.2 ± 1.5 97.8 68.6c 84.3a 67.9 96.P Buriti oil 16.0 89.2 101.1b 80.5 ± 0.6 Post-treatment 57.2c 57.4 70.6 ± 7.2 ± 8.2 84. They can be rapidly reduced by other cellular reductants.4 ± 8.2 103.5 18.6 ± 7.8 98.7 ± 3.8 ± 15.5a.6 ± 5.6b.2 ±.2 Irradiated 3T3 controls (%) UVA/UVB irradiated HaCat cells (%) Pre-treatment 45.4 ± 27.8 ± 4.9 ± 17.2 95.1 ± 5.6 ± 7.5 99.5 ± 18.6 ± 4.5 Formulation tested F29.VE F37N F37N.b 54.3 87.8 ± 1.9 ± 9.0 ± 2.0 86.9 ± 3.5 ± 6.6 Table 3 Cellular viability values obtained by the NRU assay in 3T3 and HaCat cell lines pre and post-treated with Buriti oil emulsions and exposed for 30 min of UVA and UVB irradiation.9 ± 2.0 ± 1.1 ± 20.1 ± 1.VE F37N F37N.3 ± 1.5 62.9 ± 1. c Indicates significant difference in comparison to the formulation without active ingredient.6 ± 7.4b 79.5 ± 3. there are the induction of DNA photodamage that leads to mutation in critical genes.2 93.9 ± 16.0 100.0 ± 17.7 ± 29.2 ± 0.5 ± 5.5 ± 6.2c 92.6b 29.6 40. at significance level of 95%.3 78.9 ± 23.7 ± 5.d 89.P F37M F37M.8 ± 16.8 ± 19.7 Post-treatment 25.7 ± 3.4 67.9 84.6 98.3.9 ± 9.8 ± 8.1 ± 0.5 ± 2.7b 48.8 ± 2.9 ± 4.2 ± 3.8 ± 6.9 ± 4.4 89.6 ± 13.4 59.3 61.1 88.0 ± 23.6a 84.7 ± 4.4 91.2 87. a Indicates significant difference in comparison to the pre-treatment condition.3 60.3b 32.VE F51LC F51LC.4 ± 15.b 75.2 73.7 ± 2.6 85.8 80. at significance level of 95%.9 ± 3.8 107.1 ± 2.F.0 90.1 ± 5.9.3 ± 7.6 64.1 100.3 96. at significance level of 95%.4 ± 4.4 55.3 65.1 ± 38.VE F37N.5 ±.2a 71.4 ± 5.0 Post-treatment 53.5 86. d Indicates that it is significantly higher than viability of the irradiated control wells.5a 72.3.7 ± 3. Zanatta et al.9 72.P 93.8 ± 5.3 ± 8. Percentage of viability related to control cells non-irradiated. at significance level of 95%.3a 23.9 ± 4.8 ± 2.9 ± 2.2 45. 2005).2 ± 2.5b.5 ± 2.3 ± 9. tocopheroxyl radicals are formed.0 ± 5.2 ± 11.2 ± 2.1 62. Percentage of viability related to control cells non-irradiated.2 57.6 ± 23.and post-treated with Buriti oil emulsions exposed to UVA radiation for 60 min.1 ± 7.9 89.2 ± 13.1 16.6 101.6 66.1 ± 26.4 96.2 74.8a.7 102.0 77. a Indicates significant difference in comparison to the pre-treatment condition.8 99.5 38.8 81.c Post-treatment 77.3c 92. Formulation tested UVA/UVB irradiated 3T3 cells (%) Pre-treatment F29 F29. b Indicates significant difference in comparison to the 3T3 fibroblasts in the same treatment condition.4 ± 5.8 Post-treatment 64.2 81.6a.0 71.5c 18. 2003).VE F37N.6 ± 14.7 20.8 40.3 ± 7.4 ± 3.0 88.0 91.8b. at significance level of 95%.P F37M F37M.5a.7 81.5a 66. Among the effects of UVB.3 77.8 ± 5.8 ± 19.4 ± 0.9 84.6 69.4 ± 8. Initially our purpose was to verify if the antioxidant properties of the Buriti oil emulsions were able to diminish or even retard .5 94.0 ± 9.1 78.7 94.2b 57.8a.1 ± 2.5 20.6 ± 6.3 Irradiated HaCat controls (%) Results are expressed as mean ± standard error of triplicates of three independent experiments.7 106.9 100.2 ± 4.0 ± 5.VE F51LC F51LC.8 65.1b 68.4 62..4 ± 3.4 ± 24.7 ± 10.8 ± 5.7 ± 1.6 ± 7.P Pre-treatment 31.3 93.4 ± 1.2 71.9 ± 19.C.1 ± 3.2 ± 19.8 ± 10.4 82.0b 44.6 ± 8.0 69.b 74.9 60.7 ± 8.4 74.VE F51LC.5 ± 7.6 ± 21. When the UVB photons reach the cells.c 87.2 ± 11.6 ± 10.c 83.7 ± 7.3 ± 2.0 Post-treatment 61.9 24.

as surfactant system. Oyama. Pharm. T.. Horikawa. Ultraviolet A radiation-induced apoptosis.C. Alcantara. Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Combe. 1998.M. H. 101..M.. H. / Food and Chemical Toxicology 48 (2010) 70–75 the damages caused by the UV radiation.. Photostability and efficacy studies of topical formulations containing UV-filters combination and vitamins A.. produced with Steareth-2 associated to Ceteareth-5 or Ceteareth-20. Nutr. T.. Fukunaga.C. 36. Der-Petrossian.H. H. because the panthenol is precursor of intracellular antioxidants. F. Huang.. A. J. Cabral. 79. A. the glutathione (Slyshenkov et al..B. 2006.. Francz... and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis. bcarotene and lutein protecting against UVB irradiation in human fibroblasts. containing or not a-tocopherol. 2003. C. J. U. I. Sladowski. K. F37M. Da Rocha. Technol. The keratinocytes showed higher viability values when they were pre and post-treated with F37N. Gaspar. Toxicol. 2000. Ueda. Klosner. Lovell. 298–308.. Runge. Biesalski.... W. Frank. without having prooxidant activity (Slyshenkov et al. J. 2007. Gerberick. Holzhutter.. Scaletta. due to its indirect antioxidant activity.. G. Bito.. 55–59.N. J. Burlando. M. P. The damages caused by these emulsions can be attributed to a prooxidant activity.. Modulation of dietary vitamins E and C fails to ameliorate b-carotene exacerbation of UV carcinogenesis in mice.P.. Cardiol. Spain). M. Oliveira. 2002.S. 343. Int. Nishizaki... Obermüller-Jevic. vitamin E. 31. FEBS Lett. Rallis. M. C... 2004. Fang. Mandawgade. H. Garcia-Quiroz. A. 302–309. 2004. H... M. This might be explained because panthenol is not chemically considered an antioxidant compound but a precursor of an intracellular antioxidant. Skin Therapy Lett. T. Parodi.. 2004. J. G. Conclusions The Buriti oil emulsions.P (Tables 3 and 4). Eichler.. 6. Photochem. S. Guingand. Lin. Campos. Antioxidants used in skin care formulations.F. Bassi. Pape. Flaccus.. L. P.. V. Matsumura. Redox Rep. Krutmann. F37NP.. did not caused phototoxic damages to the cells (especially keratinocytes). Applegate.W. Lo.P and the plain oil. 2003. Rocha-Filho.J.. J. Potthast. Csato. Slyshenkov. 2002. Mason. U.V. Balls. A. S. J..G. Silva. 59. Based on this. Divergent optimum levels of lycopene. Photobiol.I.. Res. Toxicol. R.. namely F37N.. Kimball. Cancer. J.. 4. Toxicology 199. Jarvi.. Bourgeois. Technol. Photochem.. the results showed us that the emulsions containing high levels of a combination of antioxidants (carotenoids associated to a-tocopherol) did not protect the cells but contributed to undesirable phototoxic effects. 24.. P. Y. N. B. S.. T.T. Fromageot. Dymkowska. R. Gautier. Photochem. Ferret. Merwald. P. M. 93–101. Mortensen. Dispers. Lockwood. Volante. F37M. Wojtczak.. F37M.. P. P. R. 42. Y. Using the singlet oxygen scavenging property of carotenoid in photodynamic molecular beacons to minimize photodamage to non-targeted cells.C. Scavenging of benzylperoxyl radicals by carotenoids.. Stefflova. Photobiol... Toxicol. H.. J.. Free Radic. O. V. Doctorate Thesis.M.P and with the pure Buriti oil. F37M.. H.Y. In addition. Sci..A. Liebsch. 2007. 149.N. Costa.N. De Morais. Am.. Desolle.A. Spielmann.-G.. Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes. Y. J.. Biol.. McNulty... K.. assessment. 2009.. Demetzos. 2003..C. 177.. 26. 45. L. Photobiol.C. Budiyanto. O. Harmacol.. 169–172.K... J.. Int.F. J. M.. 1293–1303. Melø. Dermatol. Pharm. On the other hand. b-carotene. Br... P.V. Ichihashi.. 137–143. carotenoids. UVA-induced oxidative damage and cytotoxicity depend on the mode of exposure. Photoprotective potential of lycopene. Nakao.. Universitat de Barcelona.. vitamin C and carnosic acid in UVA irradiated human skin fibroblasts. 75. Technol. Brantom. F. B: Biol. O. Enhancement of the UVA induction of heme oxygenase-1 expression by betacarotene in human skin fibroblasts.R. especially those containing panthenol. In Vitro 20. Morita.. Kobayashi... The emulsions prepared with Sorbitan Monooelate and PEG-40 castor oil.. Phytosterols: applications and recovery methods. Patravale. W. P... 98. R.. although no significant differences were found. 211–216. Clothier. UV-induced skin damage.. References Black. M. Naqvi. 2005.. Dupuis. not only the formulations F37N.. minerals and botanical ingredients as modulators of environmental and chronological skin damage. 2005.H. An evaluation of three in vitro cytotoxicity assays...A. C. 2007. Moraes. J.74 C.D. the Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection formulations and especially in after sun formulations... H. 2008. 2004). 2004). W.. Development of SLNs from natural lipids: application to topical delivery of tretinoin.J. Toxicol. Physical and chemical analysis of dielectric properties and differential scanning calorimetry techniques on Buriti oil. Dijoux. Lett. Durand.K.P.R. Bioresour. K.. 1986. 363. the viability values of the non-treated cells that were irradiated with UVA and UVA plus UVB. 2008. which were able to defend the cells against the oxidative stress generated by UVA and UVB radiation. H. revealed that radiation by itself caused more damage to 3T3 fibroblasts and HaCat keratinocytes in comparison to the cells that were treated with the emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil. F37N. Kokesch.. Stahl.A. Iwase. Pharmacological activity of natural lipids on a skin barrier disruption model. F. and relevance to photoprotection. That effect was possible. Santos. 197–207. 212–216. On the contrary. M. Zheng. Bogdan Allemann. Biologic activity of carotenoids related to distinct membrane physicochemical interactions. as well as the plain oil were able to reduce damages caused by UV radiation..S. Krämer. Zanatta et al.. FEBS Lett.D. Offord..P. Baumann. Food Chem.. Fernandes. Y. C.. Merzlyak.. L.. Comparison of the irritation potentials of Boswellia serrata gum resin and of acetyl-11-keto-beta-boswellic acid by in vitro cytotoxicity tests on human skin-derived cell lines. S. 195. Stelling. Topical vitamins. Clothier. A. D.. 2335–2350. Appl. K. Instrum. Free Radic. Toxic effects of ultraviolet radiation on the skin. Sies. Sci. Toxicology 189.. modelling. Riddell. Jacob. 480–489. Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay. Hönigsmann. C...B.. Based on these results. Oka. C. but also had time to induce glutathione’s synthesis during incubation period after irradiation. added to the phototoxic effect of the surfactant system in the formulations. K. Pantothenic acid and pantothenol increase biosynthesis of glutathione by boosting cell energetics. M. J. 2007. but also were able to reduce part of the damages caused by the UV radiation.R. A.. Wilson. F37NP. 13.M. 2008. 503–506. O.. Maurer. M. 681–691. Tsuru. 569. Hix. L. P... 1311–1317.. 132–138. Miotto... 2003. A. Bot. A.. Y. D. H. S. Y. S. J.. Hirama. 21–39.. 2008. E. 349–359. M. J. Ananthaswamy.. W. Molecules 14. J. 243–249.. 1999. Res. Med. C and E. Bioactive carotenoids: potent antioxidants and regulators of gene expression. T.. 2002. F37M and F37. Gerguis. 181–191.M. S. W.. 2000.S. A. were not capable to avoid or reduce the damages caused by UVA and UVB radiation. M.. Acknowledgements This work was funded by CAPES (Brazil) and Project 501 (Fundació Bosch i Gimpera. G. 144–149. L. A. In conclusion.. A. 36–45. The international EU/COLIPA in vitro phototoxicity . Z. R... Trautinger. Ishida. 469–471. Conflict of interest The authors declare that there are no conflicts of interest. Umebayashi.M. Balls. De Silva.W.. Tatsuishi... G. Bertram. A.F. tocopherols and phytosterols characterization in north/northeast fruits in Brazil. 9.. Moreira. 319. Fatty acids. Their effectiveness against UVA/UVB radiation should be reevaluated in association with chemical UV filters to investigate if the antioxidants can provide increased photoprotection when compared just with the chemical filters..S. due to the excess of antioxidant. 20–29. Pfannennbecker. 32. Oechovitch. 540–554. M. J....B. Chen. Attainment of emulsions of liquid crystal from Marigold oil using required HBL method. 460. B. G. Hatziantoniou. Pharmacol. Effect of anthocyanins. 2008. C. firstly because the surfactants present in those formulations were less harmful to the cells and secondly. Okano. Methods Enzymol. 181–189.. 2004. we postulated that not only panthenol did not act as a prooxidant... 5–9. Sci. viability values were higher when the cells were post-treated with the emulsions containing panthenol in comparison to those that were treated with formulations without this compound (Tables 3 and 4). Chiu. Avanti. P. Papaioannou. Exp.

/ Food and Chemical Toxicology 48 (2010) 70–75 validation study: results of phase II (blind trial). T. J. Appl. Polysorbate 80 increases the susceptibility to oxidative stress in rat thymocytes.C. J. Formation and stability of nano-emulsions. A. A. Solar UV irradiation and dermal photoaging. Skin Physiol. M. Razi-Wolf.. A. Pharm. Ugartondo. 75 Tatsuishi.. Esquena. K. Hrelia. T. Cantelli-Forti. Sci. Toxicol. Y. 623–629. 2001. C. Food Chem. Carotenoids and protection against solar UV radiation. 305–327. 303–318... 46–59. 26–30. P. C.A. L. Scharffetter-Kochanek.. 81. Fiori. Schuller. Part 1: the 3T3 NRU phototoxicity test. Biotechnol. I.. Nishimura.. Carotenoids and flavonoids contribute to nutritional protection against skin damage from sunlight..F. Photochem. Hirama. H. Solans.. Kanada. P... J. M. H.. Yamaguchi. W. Mitjans..... Photochem. Wlaschek.. Adv. Naderi. M. Tadros. 41–51.. Ma... Y. Mol. Alternative methods for eye and skin irritation tests: and overview. 2004.. In Vitro 12. Toxicology 199. Stahl. 2008... Photobiol... Tarozzi. J.A. P. H. Toxicol. 15. Z. G.. K. Stahl. 137–143. Rocha-Filho.. Colloid Interf.P. V. W.. Iwase. 37.. 20. J..P. W. Mitjans.. Tantcheva-Poor. L. 2002. Oyama. Vinardell. M... Andrisano. 2004. Sies. Zanatta et al. Kobayashi. Vinardell. 2007. V.F.. Hrelia.. 291–296.. Angeloni. . 2776– 2781. Marchesi. M. C.. B 63.. Schneider. 2005. Sies. J. 46. 97. Protective effects of cyanidin-3-O-b-glucopyranoside against UVA-induced oxidative stress in human keratinocytes. M. Izquierdo.. Low cytotoxicity of creams and lotions formulated with Buriti oil (Mauritia flexuosa) assessed by the neutral red release test. Photobiol. Zanatta. A. Skin Pharmacol. Sci. 2008.