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To evaluate the safety and the synergistic effects of tea tree, lavender, eucalyptus and tangerine essential oils in combination on the skin using in vitro, ex vivo and clinical studies.The phototoxicity was predicted using 3T3 neutral red uptake phototoxicity test (OECD TG 432). Skin penetration was evaluated by confocal Raman microspectroscopy using direct application of essential oils to pig ears. For the clinical studies, 40 participants were enrolled and randomized in three groups: (1) lavender, eucalyptus and tangerine, (2) the same essential oils plus melaleuca and (3) placebo group. The skin was evaluated by noninvasive techniques before and after a 90-day period of topical use.The essential oils were non-phototoxic, but the tangerine oil showed dose-dependent cytotoxicity (IC50: 33.1 µg/ml), presenting 35% of penetration in the viable epidermis. On the contrary, 17.7 µg/ml in combination was applied per day in the clinical study and the penetration rate for the combinations (10%, 1.77 µg/ml achieving the viable epidermis) guaranteed the safety, since in the clinical study, the application of the four essential oils improved skin barrier and morphologic skin characteristics, as well as increased skin hydration and decreased sebum levels, with no unwanted effects reported.All essential oils studied were considered non-cytotoxic or non-phototoxic separately except tangerine, which present a dose-dependent cytotoxicity. Finally, the essential oils in combination in an appropriate amount were safe and effective in the improvement of the hydrolipidic balance and morphological properties of the skin.évaluer la sécurité d’emploi et les effets synergiques des associations d’huiles essentielles d’arbre à thé, de lavande, d’eucalyptus et de mandarine sur la peau à l’aide d’études in vitro, ex vivo et cliniques. MÉTHODES: la phototoxicité a été prédite avec le test de phototoxicité de fixation du rouge neutre 3T3 (OCDE TG 432). La pénétration cutanée a été évaluée par microspectroscopie confocale de Raman grâce à l’application directe d’huiles essentielles sur les oreilles de cochons. Pour les études cliniques, 40 participants ont été inclus et randomisés dans trois groupes : (1) lavande, eucalyptus et mandarine, (2) les mêmes huiles essentielles plus melaleuca et (3) un groupe placebo. La peau a été évaluée par des techniques non invasives avant et après une période d’utilisation topique de 90 jours. RÉSULTATS: les huiles essentielles se sont avérées non phototoxiques, mais l’huile de mandarine a montré une cytotoxicité dose-dépendante (CI 50 : 33,1 µg/ml), représentant 35 % de pénétration dans l’épiderme viable. À l’inverse, dans l’étude clinique, une quantité de 17,7 µg/ml par jour en association a été appliquée, et le taux de pénétration des associations (10 %, soit 1,77 µg/ml atteignant l’épiderme viable) a garanti la sécurité d’emploi, puisque dans l’étude clinique, l’application des quatre huiles essentielles a amélioré la barrière cutanée et les caractéristiques morphologiques de la peau, et a entraîné une augmentation de l’hydratation cutanée et une diminution des taux de sébum, sans signalement d’effets indésirables.chacune des huiles essentielles étudiées a été considérée comme non cytotoxique ou non phototoxique, à l’exception de la mandarine, qui présente une cytotoxicité dose-dépendante. Enfin, l’association d’huiles essentielles en quantité appropriée a démontré sa sécurité d’emploi et son efficacité dans l’amélioration de l’équilibre hydrolipidique et des propriétés morphologiques de la peau.

Background/Aims: The selection of suitable raw materials in the cosmetic research and development is a key point, not only in order to obtain the expected results but also to avoid undesirable side...

Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability

The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen's Egg Test - Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as "severe irritants", it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as "no prediction can be made". However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.

A challenge for cosmetic and dermatologic products is to develop new high-performance and safer anti-aging products based on new compounds to enhance the stability of retinyl palmitate combined with broad-spectrum UV-filters. Consequently, the aim of this work was to evaluate the effects of three often used avobenzone photostabilizers-ethylhexyl methoxycrylene (EHMCR), tris(tetramethylhydroxypiperidinol) citrate (TTMHP) and tris-biphenyl triazine (TBPT)-on the photostability and phototoxicity of the combination of avobenzone (AVO), octyl methoxycinnamate (OMC) and retinyl palmitate (RP). The photostability studies were performed by the exposure of formulations to UVA radiation. The phototoxicity was evaluated by the 3T3 neutral red uptake phototoxic assay (OECD TG 432). The addition of EHMCR, TBPT, and TTMHP in the formulations, with/or without RP, improved the photostability of AVO and RP, but EHMCR was the most effective in stabilizing RP. In the phototoxicity assay, the combinations AVO-OMC containing or not RP showed phototoxic potential. EHMCR and TTMHP reduced the phototoxicity of the combination AVO-OMC, whereas EHMCR also decreased the phototoxicity of the combination containing RP. Therefore, EHMCR might be used to the photostabilization of formulations of AVO-OMC with/or not RP, while TTMHP can be added to this photounstable UV-filter combination.

UV light catalyzes the ozone formation from air pollutants, like nitrogen oxides. Since ozone reacts with cutaneous sebum lipids to peroxides and, thus, promotes inflammation, tumorigenesis, and aging, even broad-spectrum sunscreens cannot properly protect skin. Meanwhile, xanthophylls, like fucoxanthin, proved their antioxidant and cytoprotective functions, but the safety of their topical application in human cell-based models remains unknown. Aiming for a more detailed insight into the cutaneous fucoxanthin toxicity, we assessed the tissue viability according to OECD test guideline no. 439 as well as changes in inflammation (IL-1α, IL-6, IL-8), homeostasis (EGFR, HSPB1) and metabolism (NAT1). First, we proved the suitability of our 24-well-based reconstructed human skin for irritation testing. Next, we dissolved 0.5% fucoxanthin either in alkyl benzoate or in ethanol and applied both solutions onto the tissue surface. None of the solutions decreased RHS viability below 50%. In contrast, fucoxanthin ameliorated the detrimental effects of ethanol and reduced the gene expression of pro-inflammatory interleukins 6 and 8, while increasing NAT1 gene expression. In conclusion, we developed an organ-on-a-chip compatible RHS, being suitable for skin irritation testing beyond tissue viability assessment. Fucoxanthin proved to be non-irritant in RHS and already showed first skin protective effects following topical application.

Fucoxanthin possesses a well-described antioxidant activity that might be useful for human skin photoprotection. However, there is a lack of scientific information regarding its properties when applied onto human skin. Thus, the objective of the present study was to assess the photoprotective and phototoxicity potential of fucoxanthin based on its ultraviolet (UVB 280&ndash, 320 nm, UVA 320&ndash, 400 nm) and visible (VIS 400&ndash, 700 nm) absorption, photostability, phototoxicity in 3T3 mouse fibroblast culture vs. full-thickness reconstructed human skin (RHS), and its ability to inhibit reactive oxygen species formation that is induced by UVA on HaCaT keratinocytes. Later, we evaluated the antioxidant properties of the sunscreen formulation plus 0.5% fucoxanthin onto RHS to confirm its bioavailability and antioxidant potential through the skin layers. The compound was isolated from the alga Desmarestia anceps. Fucoxanthin, despite presenting chemical photo-instability (dose 6 J/cm2: 35% UVA and 21% VIS absorbance reduction), showed acceptable photodegradation (dose 27.5 J/cm2: 5.8% UVB and 12.5% UVA absorbance reduction) when it was added to a sunscreen at 0.5% (w/v). In addition, it increased by 72% of the total sunscreen UV absorption spectra, presenting UV-booster properties. Fucoxanthin presented phototoxic potential in 3T3 fibroblasts (mean photo effect 0.917), but it was non-phototoxic in the RHS model due to barrier function that was provided by the stratum corneum. In addition, it showed a significant inhibition of ROS formation at 0.01% (p &lt, 0.001), in HaCat, and in a sunscreen at 0.5% (w/v) (p &lt, 0.001), in RHS. In conclusion, in vitro results showed fucoxanthin protective potential to the skin that might contribute to improving the photoprotective potential of sunscreens in vivo.

Leishmaniasis, malaria, and neosporosis are parasitic diseases that affect humans and animals, causing public health problems and billions in economic losses. Despite the advances in the development of new drugs, the severe side effects of available leishmaniasis treatments, the Plasmodium spp. resistance to antimalarial drugs, and the lack of a specific treatment against neosporosis lead us to the search for new anti-protozoan molecules from underexplored sources such as the Antarctic marine environment. Herein, we describe for the first time the chemical profile of Desmarestia antarctica crude extract and fractions using GC-MS and LC-MS/MS (molecular networking) approaches, and evaluate their antiparasitic activity against Leishmania amazonensis, Neospora caninum, and multi-drug-resistant Plasmodium falciparum. Furthermore, the cytotoxicity in 3T3 BALB/c fibroblasts and Vero cells was evaluated. D. antarctica fraction E ( IC50 of 53.8±4.4 μg mL− 1 and selectivity index of 3.3) exhibited anti-promastigote activity and was fourfold more selective to L. amazonensis rather than to the host cells. D. antarctica fraction D (IC50 of 1.6±1.3 μg mL− 1 and selectivity index of 27.8), D. antarctica fraction F (IC50 of 3.1±2.1 μg mL− 1 and selectivity index of 23.1), and D. antarctica fraction H (IC50 of 3.1±2.0 μg mL− 1 and selectivity index of 12.9) presented the highest antiparasitic effects against N. caninum with no cytotoxic effects. Also, D. antarctica fraction D presented a significant antiplasmodial inhibitory effect (IC50 of 19.1±3.9 μg mL− 1 and selectivity index of 6.0). GC-MS analysis indicated palmitic acid, myristic acid, fucosterol, phthalic acid, di(2-methylbutyl) ester, loliolide, and neophytadiene as the main components in the active fractions. In addition, this is the first report of a biological screening of macroalgae secondary metabolites against N. caninum parasites.

Natural products, or secondary metabolites, obtained from fungal species associated with marine algae have been widely used in sunscreens due to their antioxidant activity and protective potential against solar radiation. The endophytic fungus isolated from Bostrychia radicans algae collected in the Rio Escuro mangrove, São Paulo State, Brazil, Annulohypoxylon stygium (Xylariaceae family) was studied to evaluate the photoprotective potential of its metabolites. The Annulohypoxylon genus can produce secondary metabolites with interesting cytotoxic, antibacterial and antioxidant properties and was never isolated before from a marine alga or had its metabolites studied for UV protection. The fungal culture (code As) extracted with dichloromethane: methanol (2:1) yielded 9 fractions (Asa to Asi) which were submitted to different chromatographic methodologies to obtain pure compounds, and to spectroscopic methodologies to elucidate their structures. Also, a screening was conducted to evaluate the qualitative production of the metabolites, besides the absorption in the UVA/UVB range, their photostability and phototoxicity potential using the 3T3 NRU phototoxicity test (OECD TG 432). This study led to the isolation of a novel compound, 3-benzylidene-2-methylhexahydropyrrolo [1,2-α] pyrazine-1,4-dione (1), from fractions Ase3 and Asf3; Ase1 was identified as 1-(1,3-Benzodioxol-5-yl)-1,2-propanediol (2), two metabolites were isolated as diastereomers (1S,2R)-1-phenyl-1,2-propanediol (3) from Asd2 and (1R,2R)-1-phenyl-1,2-propanediol (4) from Asd3, and Ase1 and 1,3-benzodioxole-5-methanol (5) from Asc1. The results obtained showed a great potential source of new molecules to be used as UVB filters in sunscreens, since substances 1-2 presented UVB absorption, had no phototoxic potential and were considered photostable. In conclusion, these compounds can be considered as a potential new class of molecules for photoprotection, since their photosafety and non-cytotoxicity were predicted using in vitro methods for topical use. Meanwhile, further efficacy assays shall be conducted for the establishment of their Sun Protection Factor (SPF). Also, this work provided new information concerning the metabolic profile of A. stygium, since it was possible to obtain two enantiomer compounds (3) and (4). One of them belonged to the same skeleton, but with a methylenedioxy moiety, showing the richest enzymatic pattern for this microorganism.

This chapter presents some considerations in order to foster debate and to place the issue of animal research in a context of the growing social, scientific and ethical debate. It shows that the timing is appropriate before the widespread introduction of a context that emphasized the use of animals for dealing with protection of human health and where alternative methods are brought up as real possibilities of scientific research. The chapter provides a description of the current international practices regarding the acceptance of alternative methods to animal testing and their situation. In fact, the process of validation of alternative methods is aimed at assessing the optimization, reproducibility and relevance of the method in order to be submitted to the regulatory agency and, become officially available for toxicological purposes. Optimization of existing systems is an important part of the strategy to accelerate the implementation of a mostly animal-free safety science, in addition to the more time-demanding development of entirely new methods.

Made available in DSpace on 2019-10-04T12:36:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Leishmaniasis is considered a neglected disease and affects billions around the world, currently presenting few therapeutic options, which makes the development of new antileishmanial drugs urgent. Secondary metabolites from marine and terrestrial organisms are important sources of new chemical entities. Herein, the potential activity of crude extracts and fractions from the Antarctic macroalga Iridaea cordata (Turner) Bory de Saint-Vincent was studied against promastigote and intracellular amastigotes forms of Leishmania amazonensis. The cytotoxicity of the active fractions was evaluated on macrophages and 3T3 BALB/c fibroblasts. The chemical profile of volatile substances was analyzed using the GC-MS technique. The fractions IC-FE (IC50-AMA = 23.6 +/- 3.4 mu g mL(-1); SI > 11) and IC-FF (IC50-AMA = 12.4 +/- 1.2 mu g mL(-1); SI > 24) showed promising activity against amastigotes and higher selectivity to the parasite rather than to the mammalian host cells, when compared to the reference drug amphotericin B (IC50-AMA = 5.9 +/- 0.3 mu g mL(-1); SI = 3.9). Their estimated LD50 in rodents are also higher than that in amphotericin B (LD50 IC-FE = 594.5 +/- 25.87 mg kg(-1); LD50 IC-FE = 580.1 +/- 11.84 mg kg(-1); LD50 amphoter = 172.20 +/- 2.40 mg kg(-1)). The chemical profile of these fractions showed the presence of phthalates, esters, ketones, fatty acids, and carboxylic acids, which might be contributing alone or synergistically to the observed antileishmanial activity. Consequently, I. cordata might be used to identify new antileishmanial compounds. Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Ribeirao Preto, SP, Brazil Univ Estadual Paulista, Dept Anal Clin, Fac Ciencias Farmaceut, BR-14800903 Araraquara, SP, Brazil Univ Sao Paulo, Inst Quim, BR-05508000 Sao Paulo, SP, Brazil Univ Estadual Paulista, Dept Anal Clin, Fac Ciencias Farmaceut, BR-14800903 Araraquara, SP, Brazil FAPESP: 2017/03552-5 FAPESP: 2016/06931-4 CNPq: 407588/2013-2

Considering the global trend in the search for alternative natural compounds with antioxidant and sun protection factor (SPF) boosting properties, bacterial carotenoids represent an opportunity for exploring pigments of natural origin which possess high antioxidant activity, lower toxicity, no residues, and no environmental risk and are readily decomposable. In this work, three pigmented bacteria from the Antarctic continent, named Arthrobacter agilis 50cyt, Zobellia laminarie 465, and Arthrobacter psychrochitiniphilus 366, were able to withstand UV-B and UV-C radiation. The pigments were extracted and tested for UV absorption, antioxidant capacity, photostability, and phototoxicity profile in murine fibroblasts (3T3 NRU PT–OECD TG 432) to evaluate their further potential use as UV filters. Furthermore, the pigments were identified by ultra-high-performance liquid chromatography–photodiode array detector–mass spectrometry (UPLC-PDA-MS/MS). The results showed that all pigments presented a very high antioxidant activity and good stability under exposure to UV light. However, except for a fraction of the A. agilis 50cyt pigment, they were shown to be phototoxic. A total of 18 different carotenoids were identified from 23 that were separated on a C18 column. The C50 carotenes bacterioruberin and decaprenoxanthin (including its variations) were confirmed for A. agilis 50cyt and A. psychrochitiniphilus 366, respectively. All-trans-bacterioruberin was identified as the pigment that did not express phototoxic activity in the 3T3 NRU PT assay (MPE

Currently most of sunscreens provide effective protection in the full UV range but lack VIS protection. The addition of effective antioxidants to sunscreens might afford suitable UV-VIS protection. Apigenin (API), chrysin (CRI) and beta-carotene (BTC) have shown potential for UV-VIS protection. This paper reports a photosafety and efficacy screening of such antioxidants through evaluation of the photostability, photoreactivity and phototoxicity as well as UVA/UVB ratio and critical wavelength. The assessment of the photostability, photoreactivity and phototoxicity of API, CRI and BTC, isolated and combined (CMB) was performed by HPLC, ROS assay and 3T3 NRU phototoxicity test, respectively. The phototoxicity test was also performed for CMB plus bemotrizinol (BMZ). The in vitro evaluation of the UVA protection was assessed by the determination of the UVA/UVB ratio and the critical wavelength. The antioxidants API, CRI, BTC and CMB were stable under UVA/VIS and VIS light. However weak photoreactivity after UVA/VIS irradiation was observed for API, CRI and CMB in the ROS assay. In the 3T3 NRU phototoxicity test, phototoxic potential was observed for CRI, BTC, CMB and CMB+BMZ after UVA/VIS exposure, and for BTC and CMB after VIS exposure. BMZ reduced the phototoxic potential of CMB in the VIS range. In the in vitro evaluation of UVA protection API, CRI, BTC, CMB and CMB+BMZ presented ultra UVA protection (UVA/UVB ratio>0.9) and exhibited critical wavelength close to or above 370nm. In conclusion, the use of API, CRI, BTC and their CMB aiming skin photoprotection could be considered safer in the VIS range. Furthermore, API presented the best performance in the photosafety screening among the studied antioxidants, since it was photostable and non-phototoxic in UVA/VIS and photostable, non-photoreactive and non-phototoxic in VIS range.

Skin model cultivation under static conditions limits the observation of the toxicity to this single organ. Biology-inspired microphysiological systems associating skin with a liver in the same circulating medium provide a more comprehensive insight into systemic substance toxicity; however, its advantages or limitations for topical substance toxicity remain unknown. Herein, we performed topical (OECD test guideline no. 439) and systemic administration of terbinafine in reconstructed human skin (RHS) vs. a RHS plus liver model cultured in TissUse' HUMIMIC Chip2 (Chip2). Aiming for a more detailed insight into the cutaneous substance irritancy/toxicity, we assessed more than the MTT cell viability: lactate dehydrogenase (LDH), lactate and glucose levels, as well as inherent gene expressions. Sodium dodecyl sulfate (SDS) was the topical irritant positive control. We confirmed SDS irritancy in both static RHS and Chip2 culture by the damage in the morphology, reduction in the lactate production and lower glucose consumption. In the static RHS, the SDS-treated tissues also released significantly high LDH (82%; p 0.05) and significantly lower IL-6 release (p 0.05), corroborating with the other metabolic levels. In both static RHS and Chip2 conditions, we confirmed absence of irritancy or systemic toxicity by LDH, glucose or lactate levels for topical 1% and 5% terbinafine and systemic 0.1% terbinafine treatment. However, topical 5% terbinafine treatment in the Chip2 upregulated IL-1α in the RHS, unbalanced apoptotic and proliferative cell ratios in the liver and significantly increased its expression of CYP1A2 and 3A4 enzymes (p 0.05), proving that it has passed the RHS barrier promoting a liver impact. Systemic 0.1% terbinafine treatment in the Chip2 increased RHS expression of EGFR, increased apoptotic cells in the liver, downregulated liver albumin expression and upregulated CYP2C9 significantly (p 0.05), acting as an effective hepatotoxic terbinafine control. The combination of the RHS and liver model in the Chip2 allowed a more sensitive assessment of skin and hepatic effects caused by chemicals able to pass the skin (5% terbinafine and SDS) and after systemic 0.1% terbinafine application. The present study opens up a more complex approach based on the microphysiological system to assess more than a skin irritation process.

Botanical and marine extracts have been used in cosmetic products as natural antioxidants or UV-absorbers to reduce the use of organic UV filters. However, despite the high UV exposure of Antarctic macroalgae that induces the production of photoprotective compounds, the use of marine macroalgae extracts for photoprotection has been little explored. Thus our objective was to assess the photoprotective potential and the toxicity of extracts from two red macroalgae Curdiea racovitzae and Iridaea cordata. Chemical characterization was based on the qualitative and quantitative analyses of mycosporine-like amino acids (MAAs) by high performance liquid chromatography coupled to mass spectrometry. Photoprotective properties were assessed by UV spectrophotometry and photostability assay. Antioxidant capacity was studied by DPPH and superoxide radical scavenging assays. UVB and UVA photoprotection was assessed in keratinocytes by cell viability analysis after UVB irradiation and quantification of intracellular reactive oxygen species (ROS) induced by UVA, respectively. Toxicity was estimated by cytotoxicity, phototoxicity and HET-CAM (Hen's egg test-chorioallantoic membrane) assays. Red algae extracts had high concentrations of MAAs, mainly consisted of palythine. The extracts absorbed in the UVB and UVA-II and were photostable. The C. racovitzae extract showed antioxidant activity against superoxide radicals. In addition, the C. racovitzae (1.0 and 0.5 mg/mL) and I. cordata (1.0 mg/mL) extracts had an UVB photoprotection similar to benzophenone-3, which was dependent on the total MAAs concentration. Besides, the C. racovitzae extract decreased ROS generation by 31 and 24% at 100 and 50 μg/mL, respectively. Furthermore, it was more effective in reducing ROS generation than quercetin and Helioguard 365®. Finally, both extracts were considered to be non-cytotoxic, non-phototoxic and non-irritant. Therefore, the C. racovitzae extract is a good candidate to be added to anti-aging and sunscreen formulations in order to protect against oxidative stress and to improve the absorption spectra of UV filters.

An iridescent yellow pigmented bacterium isolated from the Antarctic continent, named Cellulophaga fucicola strain 416, was found to be able to tolerate UV-B radiation. Its crude pigment extract was tested for antioxidant capacity, UV light stability and phototoxicity profile against murine fibroblast lines. The pigments were further isolated and chemically identified by ultra-high-performance liquid chromatography with photodiode array and mass spectrometry detectors. The results showed that the pigment extract presented weak stability under exposure to UV light, a phototoxic profile in the 3t3 Neutral Red Uptake test and a very high antioxidant activity, suggesting that it could be used as food and feed colourants. Zeaxanthin and two isomers of zeaxanthin, β-cryptoxanthin and β-carotene, were identified using a C18 column. These five carotenoids were the major pigments isolated from C. fucicola 416. In conclusion, the identification of pigments produced by the bacterial strain under study may help us understand how bacteria thrive in high UV and cold environments, and opens avenues for further biotechnological application towards a more sustainable and environmentally friendly way of pigment exploitation.

The aim of this study was to assess the photoprotective effects of cosmetic formulations containing UV filters, red algae, Porphyra umbilicalis, extracts and combinations of the extract with vitamins and Ginkgo biloba through the use of in vivo preclinical studies. For this study, 4 groups of 4 hairless mice each were treated with topical formulations applied on the dorsum for 5 days as follows: group 1 - control (no treatment); group 2 - application of the formulation F (sunscreen formulation containing only UV filters); group 3 - application of the formulation FA (sunscreen formulation with red algae extract); and group 4 - application of the formulation FVGA (sunscreen formulation with red algae extract, G. biloba and vitamins A, C and E). The effects of these formulations were evaluated by determining the transepidermal water loss (TEWL) and erythema index. Apoptosis was detected by immunohistochemical staining with anti-p53 and anti-caspase-3 antibodies. The results showed that the formulations protected the skin from erythema when exposed to UV radiation. The group that received the formulation FVGA presented a greater TEWL than did the other groups, suggesting that this formulation was involved in cell renewal. Immunohistochemical analysis showed that UV radiation caused an increase in the expression of p53 and active caspase-3, confirming that the damage caused by UV radiation exposure led to apoptosis. The application of all formulations studied resulted in a statistically significant reduction in the expression of p53 and caspase-3, with a more pronounced effect observed following treatment with FA. In conclusion, extracts from the red algae P. umbilicalis could be considered effective ingredients to be used in sunscreen formulations. The combination of vitamins A, E, C and G. biloba along with red algae extracts can improve significantly the performance of the sunscreens, preventing UV-induced DNA damage and inflammation. Thus, they should be considered an interesting combination for an effective photoprotective formulation with anti-aging properties.

Cutaneous permeation is a critical parameter when topical application of sunscreens containing antioxidants is considered. The aim of this study was to evaluate the cutaneous penetration of most marketed UV-filters combined with trans-resveratrol (RES) and beta-carotene (BTC) since few studies report skin penetration when such compounds are applied. Formulations containing octocrylene, octyl methoxycinnamate, avobenzone and bemotrizinole were prepared and supplemented or not with BTC, or with RES, or with both compounds in combination. Penetration studies were performed using Franz vertical diffusion cells and porcine ear skin as the biological membrane. The quantification of UV-filters and antioxidants in the stratum corneum (SC), viable epidermis plus dermis and receptor fluid was performed by HPLC. Results suggested that UV-filters and antioxidants did not permeate the skin but were retained for 12h post application. About 90% and 80%, respectively, of the total penetrated amount of UV-filters and antioxidants was found in the SC. Interestingly, it was observed that BTC, alone or combined with RES, reduced the skin retention of UV-filters on average by 63%. In conclusion, this study demonstrated that the combination of antioxidants and UV-filters in sunscreens is advantageous for cutaneous penetration, since BTC and BTC+RES improved sunscreen safety by reducing delivery of the four UV-filters in the study into SC and viable epidermis.

This study aimed to assess the phototoxic potential of combined UV-filters and retinyl palmitate (RP) in the presence or not of bemotrizinol (BMTZ), employing photostability and in vitro and in vivo phototoxicity assays. The formulations tested contained octocrylene (OCT), octyl methoxycinnamate (OMC), benzophenone-3 (BZP-3) and RP (photostable) or octocrylene (OCT), octyl methoxycinnamate (OMC), avobenzone (AVO) and RP (less photostable). Both formulations were supplemented with bemotrizinol. Photostability was evaluated by exposing, or not, formulations spread on a glass plate to UVA/UVB irradiation. The resulting products were quantified by HPLC analysis. In vitro phototoxicity of UV-filters and combinations were evaluated using 3T3 viable monolayer fibroblast cultures submitted, or not, to irradiation according to OECD TG 432. In vivo photoallergy and photoxicity were assessed by clinical studies (photopatch test). Photostability assays showed that UV-filter bemotrizinol was a better photostabilizer for RP/benzophenone-3 than for RP/avobenzone. The in vitro phototoxicity of the combination RP/avobenzone was reduced by bemotrizinol. Clinical studies did not indicate phototoxic or photoallergenic potentials in all formulations tested. It is concluded that the 3T3 NRU phototoxicity test may be considered a supplementary assay in formulation developments, since it can detect chemically unstable and potentially phototoxic combinations. However, extrapolation of in vitro positive results to human photopatch tests may be performed only to a limited extent.

Photo-induced effects are based on the photosensitization processes by which a photosensitizer, i.e., a molecule absorbing nontoxic dose of ultraviolet (UV) or visible (VIS) radiation (chromophores), interacts with other molecules leading eventually to an adverse effect. The assessment of photo-induced toxicity is required when chemical preparations are intended to be used on sunlight-exposed skin, i.e., products topically applied to the skin or systemic drugs. It includes evaluation of phototoxic effects and of photoallergy, which will be discussed hereafter. Moreover, the recommended tiered phototoxic strategy, including nonbiological and biological assays, will be reported together with the ICH (International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) recommendations. In this photosafety strategy, UV/VIS spectral analysis should be employed as the first screening to indicate a potential phototoxic risk by measuring the molar extinction coefficient (MEC), followed by the ROS (reactive oxygen species) assay. The biological assays are then carried out to evaluate the phototoxic potential of those materials presenting potential phototoxic risk and include the 3T3 neutral red uptake phototoxicity test (3T3 NRU-PT) followed, if a positive result is obtained, by the human 3D reconstructed skin model phototoxicity test (H3D-PT). The last step is the confirmatory photopatch test to be applied to a group of volunteers and to be performed using the first non-phototoxic concentration determined in the H3D-PT using a margin of safety factor of 10. Finally, some epidemiological data regarding photoallergy will be presented.

Benzophenone-3 (BP-3) is a UV filter with absorption at the UVB and UVA wavelengths which has not been extensively studied in experiments involving its absorbing effects and toxicity. We synthetized four BP-3 derivatives and characterized their photoprotective potential by UV absorption and photodegradation, their phototoxicity potential by 3T3 Neutral Red Uptake (3T3 NRU PT) and their photoreactivity by the reactive oxygen species (ROS) assay. The UV absorption, photodegradation, phototoxicity and photoreactivity of the four BP-3 derivatives (BP-3 carbonate, BP-3 carbazole, BP-3 phenylamine and BP-3 methoxy-phenylamine) were evaluated and compared to those of BP-3. Results showed that all derivatives were photostable, except BP-3 carbonate, which did not absorb in the UVA range. BP-3 phenylamine and BP-3 methoxy-phenylamine were considered non-phototoxic and weakly photoreactive in the ROS assay, while the carbazole derivative was considered phototoxic and non-photoreactive due to its rigid structure. The UV spectra of BP-3 carbonate, BP-3 phenylamine and BP-3 methoxy-phenylamine showed the influence of hydrogen bonding on their UV absorption. Based on these results, we concluded that BP-3 phenylamine and BP-3 methoxy-phenylamine could be promising UVA filters.

The aim of the present study was to investigate the photochemical behavior of DHHB and its photostabilizing effect on avobenzone (AVO) in different sunscreen formulations. The formulations were subjected to photostability studies by HPLC and spectrophotometry. In vitro phototoxicity was assessed using 3T3 fibroblast cultures. The mechanism of interaction between DHHB and AVO was investigated by steady state and time-resolved fluorescence spectroscopy. All formulations provided ultra-protection against UVA radiation. HPLC results demonstrated that DHHB did not present a photostabilizing effect on AVO. Fluorescence spectroscopy showed that AVO and DHHB interact by a static quenching mechanism and DHHB did not affect the AVO excited state lifetime. In addition, the energy transfer by Forster mechanism (FRET), which is the most often mechanism responsible for singlet-singlet quenching, is unlikely in this work. These results suggest why DHHB did not work as a photostabilizer on AVO singlet excited state. Phototoxicity results demonstrated that combinations containing DHHB (C2) did not show a phototoxic potential. Finally, although DHHB was considered to be photostable for all formulations studied (F2 and F3) it did not increase the photostability of AVO (F3). Thus, we suggested that formulations containing DHHB (F2) should be considered more advantageous than formulations containing AVO and AVO/DHHB (F1 and F3 respectively).

The aim of this study was to evaluate the in vitro skin phototoxicity of cosmetic formulations containing photounstable and photostable UV-filters and vitamin A palmitate, assessed by two in vitro techniques: 3T3 Neutral Red Uptake Phototoxicity Test and Human 3-D Skin Model In Vitro Phototoxicity Test. For this, four different formulations containing vitamin A palmitate and different UV-filters combinations, two of them considered photostable and two of them considered photounstable, were prepared. Solutions of each UV-filter and vitamin under study and solutions of four different combinations under study were also prepared. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (3T3-NRU-PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D-PT). Avobenzone presented a pronounced phototoxicity and vitamin A presented a tendency to a weak phototoxic potential. A synergistic effect of vitamin A palmitate on the phototoxicity of combinations containing avobenzone was observed. H3D-PT results did not confirm the positive 3T3-NRU-PT results. However, despite the four formulations studied did not present any acute phototoxicity potential, the combination 2 containing octyl methoxycinnamate (OMC), avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) presented an indication of phototoxicity that should be better investigated in terms of the frequency of photoallergic or chronic phototoxicity in humans, once these tests are scientifically validated only to detect phototoxic potential with the aim of preventing phototoxic reactions in the general population, and positive results cannot predict the exact incidence of phototoxic reactions in humans.

Quercetin is a flavonoid with high antioxidant activity, conceivably useful for pharmaceutical and cosmetic applications. Additionally, its molecular structure is potentially suited to stabilize emulsions. In this work the interaction of quercetin with cationic and nonionic surfactants in a mixed-surfactant bioactive nanoemulsion was investigated. The high-loaded quercetin nanoemulsion (prepared with 0.5% w/w and entrapment efficiency of 99.8%), prepared by the sub-PIT method, was assessed in terms of thermal/photostability and safety for cosmetic applications. It proved to be highly stable to chemical degradation under ambient or sub-ambient storage conditions and very photostable to UV artificial radiation. Results of UV absorption/emission spectroscopy, differential scanning calorimetry and confocal fluorescence microscopy studies showed that in the nanoemulsion: (i) quercetin appears, mainly in the form of a neutral non-emissive 5OH hydrogen bonded tautomer with a minor population of both the emissive 3OH hydrogen bonded tautomer and the mono-anionic form; (ii) quercetin is very stable under UV radiation. (iii) quercetin interacts with the surfactants and is predominantly located at the oil/water interface of the nanodroplets. In contrast, when present in a coarse emulsion quercetin is preferentially dispersed in the aqueous phase. The nanoemulsion showed an excellent biocompatibility with skin, a low eye irritancy potential (HET-CAM assay). This favorable safety profile coupled with the high antioxidant content and thermal/photophysical stability, suggests a large potential of the quercetin nanoemulsion studied in this work for skin care applications.

Chemically stable ester derivatives of vitamins A, C and E have become a focus of interest for their role in the satisfactory results in skin aging treatments. Accordingly, the aim of this study was to evaluate the physical and chemical stability of a cosmetic formulation containing 1% retinyl palmitate, ascorbyl tetraisopalmitate and tocopheryl acetate, alone or in combination. In the studies of physical stability, a Brookfield rheometer was used to determine rheological behavior of formulations containing the vitamins. Chemical stability was determined by HPLC on a Shimadzu system with UV detection. Results showed that formulations had pseudoplastic behavior and that vitamins did not alter their apparent viscosity and thixotropy. In the chemical stability studies, first-order reaction equations were used for determinations of the shelf-life of vitamins derivatives considering a remaining concentration of 85%. Combined vitamins in a single formulation had a slightly lower degradation rate as compared to different preparations containing only one of the vitamins. Considering that many cosmetic formulations contain vitamin combinations it is suggested that the present study may contribute to the development of more stable formulations containing liposoluble vitamins.

The beneficial photoprotective effects of topical formulations containing combined extracts of Ginkgo biloba (GB) and green tea (GT) have not been investigated. The present study aims to assess the photoprotective effects of topical formulations containing GB and GT combined or not by applications on the dorsal skin of hairless mice prior to UVA/B irradiation. After 20 h, skin barrier damage (TEWL), erythema, histological alterations and sunburn cell formation were evaluated. The results showed that only extract supplemented formulations protected the skin against the UV-induced damage. Formulations containing GB provided total protection of the skin barrier function avoiding UV radiation damage such as TEWL and erythema and were more effective than those containing GT. However, it is suggested that formulations containing combined GB and GT may provide substantial photoprotective effects since different aspects of skin damage were influenced by each extract. Copyright © 2011 John Wiley & Sons, Ltd.

Vitamin A palmitate has been used in cosmetics; however, studies report that this substance shows photoreactivity that can lead to loss of safety and efficacy. On the other hand, photostabilizers have been used to increase sunscreen photostability and consequently their safety and effectiveness. Thus, this study aimed to evaluate the influence of photostabilizers on the photoprotective effects of a cosmetic formulation containing UV-filters and vitamin A palmitate. The formulation containing UV-filters was supplemented with vitamin A palmitate and the photostabilizers diethylhexyl 2,6-naphthalate (DEHN), bumetrizole and benzotriazolyl dodecyl p-cresol (BTDC). Hairless mice were treated daily by topical applications and irradiated (UVA/B). Erythema index, transepidermal water loss, histological/histometric analysis and number of sunburn cells (SBC) were evaluated. The results showed that all formulations protected from UV-induced enhancement of erythema and SBC but there was no difference among them. The formulation with no stabilizers reduced viable epidermis thickness due to atrophy induced by UV radiation. Thus, it can be concluded that the presence of photostabilizers influenced the effects of formulations containing UV-filters and vitamin A palmitate, which could be seen by histological and histometric analysis. Furthermore, the formulations containing the stabilizers DEHN and BTDC showed better protective effects on hairless mice skin.

Green tea (Camellia sinensis) and Ginkgo biloba extracts in cosmetic formulations have been suggested to protect the skin against UV-induced damage and skin ageing. Thus, it is very important to assess the human skin penetration of their major flavonoids to verify if they penetrate and remain in the skin to exert their proposed effects. The aim of this study was to evaluate the human skin penetration of epigallocatechin-3-gallate (EGCG) and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations. This study was conducted with fresh dermatomed human Caucasian skin from abdominal surgery mounted on static Franz diffusion cells. Skin samples were mounted between two diffusion half-cells and 10 mg/cm2 of formulations supplemented with 6% of green tea or G. biloba extract were applied on the skin surface. The receptor fluid was removed after 6 and 24 h and analyzed by high-performance liquid chromatography for the quantification of the flavonoids. The stratum corneum was removed by tape stripping and immersed in methanol and the epidermis was mechanically separated from the dermis and triturated in methanol to extract EGCG and quercetin. The results showed that the flavonoids under study penetrated into the skin, without reaching the receptor fluid. The majority of EGCG was quantified in the stratum corneum (0.87 μg/cm2), which was statistically higher than the EGCG concentrations found in viable epidermis (0.54 μg/cm2) and in the dermis (0.38 μg/cm2). The majority of quercetin was quantified in the viable epidermis (0.23 μg/cm2), which was statistically higher than the EGCG concentration found in the stratum corneum layer (0.17 μg/cm2). Finally, it can be concluded that EGCG and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations presented good skin penetration and retention, which can favor their skin effects.

Saccharomyces cerevisiae extract (SCE) is used in cosmetics since it can act in oxidative stress and improve skin conditions. This study investigated dermatological effects of cosmetic formulations containing SCE and/or vitamins A, C and E. The formulation studied was supplemented or not (F1: vehicle) with vitamins A, C and E esters (F2) or with SCE (F3) or with the combination of vitamins and SCE (F4). Formulations were patch tested on back skin of volunteers. For efficacy studies, formulations were applied on volunteers and transepidermal water loss (TEWL), skin moisture (SM), skin microrelief (SMR) and free radicals protection were analysed after 3h, 15 and 30 days of application. Volunteers were also asked about efficacy perception. It was observed that F4 provoked a slight erythema in one volunteer. All formulations enhanced forearm SM. Only F3 and F4 presented long term effects on SMR and showed higher texture values; F3 had the highest brightness values. Our results suggest that vitamins and SCE showed effects in SM and SMR. Only formulations containing SC had long term effects in the improvement of SMR. Thus, these kinds of evaluations are very important in cosmetics development to evaluate the best risk and benefit correlation.

Background/purpose: Vitamins C and its derivatives, mainly due to their antioxidant properties, are being used in cosmetic products to protect and to reduce the signs of ageing. However, there are no studies comparing the effects of vitamin C [ascorbic acid (AA)] and its derivatives, magnesium ascorbyl phosphate (MAP) and ascorbyl tetra-isopalmitate (ATIP), when vehiculated in topical formulations, mainly using objective measurements, which are an important tool in clinical efficacy studies. Thus, the objective of this study was to determine the in vitro antioxidant activity of AA and its derivatives, MAP and ATIP, as well as their in vivo efficacy on human skin, when vehiculated in topical formulations. Methods: The study of antioxidant activity in vitro was performed with an aqueous and a lipid system. The in vivo methodology consisted of the application of these formulations on human volunteers' forearm skin and the analysis of the skin conditions after 4-week period daily applications in terms of transepidermal water loss (TEWL), stratum corneum moisture content and viscoelasticity using a Tewameter®, Corneometer® and Cutometer®, respectively. Results: In vitro experiments demonstrated that in an aqueous system, AA had the best antioxidant potential, and MAP was more effective than ATIP, whereas in the lipid system ATIP was more effective than MAP. In in vivo studies, all formulations enhanced stratum corneum moisture content after a 4-week period daily applications when compared with baseline values; however, only the formulation containing AA caused alterations in TEWL values. The formulations containing MAP caused alterations in the viscoelastic-to-elastic ratio, which suggested its action in the deeper layers of the skin. Conclusion: AA and its derivates presented an in vitro antioxidant activity but AA had the best antioxidant effect. In in vivo efficacy studies, only the formulation containing AA caused alterations in TEWL values and the formulation containing MAP caused alterations in the viscoelastic-to-elastic ratio. This way, vitamin C derivatives did not present the same effects of AA on human skin; however, MAP showed other significant effect-improving skin hydration, which is very important for the normal cutaneous metabolism and also to prevent skin alterations and early ageing.

It is already known that the photostability of a sunscreen is important for its performance on human skin. On the other hand, there are many formulations besides sunscreens containing combinations of UV-filters and daily use active substances with other claims like hydration and anti-aging effects. Vitamins A, C and E are frequently added in these kinds of products and it is not known if the UV-filters have some influence on the hydration and anti-aging effects of these vitamins on the skin as well as on their stability mainly when photounstable UV-filters like avobenzone and octyl methoxycinnamate are present in the formulation. Thus, the aim of this study was to evaluate the influence of two different UV-filters combinations, a photostable and a photounstable one, on the photostability as well as on the efficacy of a formulation containing vitamin A, C and E derivatives. The formulations that were investigated contained or not (vehicle: formulation 1) a combination of 0.6 % (w/w) vitamin A palmitate (1,700,000 UI/g), 2 % (w/w) vitamin E acetate and 2% (w/w) ascorbyl tetraisopalmitate (formulation 2) supplemented with a photounstable UV filter combination octyl methoxycinnamate (OMC), avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) (formulation 3) or with a photostable UV filter combination OMC, benzophenone-3 (BP-3) and octocrylene (OC) (formulation 4). In the photostability studies, all formulations were spread onto a glass plate and exposed to UVA/UVB irradiation. The filter components and vitamins were quantified by HPLC analysis with detection at 325 and 235 nm and by spectrophotometry. To simulate the effects of these formulations daily use, all of them (formulations 1-4) were applied on the dorsum of hairless mice, which were submitted to a controlled light-dark cycle (and were not irradiated), once a day for 5 days. Transepidermal water loss (TEWL), water content of the stratum corneum and viscoelastic properties of the skin were analyzed by using different non-invasive Biophysics Techniques in order to evaluate hydration and anti-aging effects of these formulations as well as erythema to assess skin irritation. Histopathology, viable epidermal thickness as well as the number of epidermal cell layers were also evaluated. It was observed that both UV filters combinations (photounstable one containing OMC, AVB and MBC and photostable one containing OMC, BP-3 and OC) enhanced vitamin A photostability and F4 was more photostable than F3, in terms of vitamin A. In vivo efficacy studies showed that F2, F3 and F4 enhanced the viable epidermal thickness, the number of epidermal cell layers, TEWL and Uv/Ue parameter, when compared to the vehicle, which can suggest that they enhanced viable epidermis hydration and acted in cell renewal. However formulation 2 (containing only vitamins), which was the most photounstable formulation, provoked an irritation on hairless mouse skin, and consequently it cannot be considered as safe as the other formulations. It can be concluded that both UV filters combinations did not influence the hydration and anti-aging effects of the formulations containing the vitamins under study and reduced the skin irritation observed when the vitamins were present in the formulation. In addition, the photostable UV-filters combination had the highest recovery of vitamin A in the photostability studies. Finally, it could be suggested that the presence of UV-filters can be considered interesting for the reduction of skin irritation and the most suitable formulation was the one containing the combinations of vitamins A, C and E with photostable UV-filters.

Efficient UV-absorbing molecules are designed to protect against UV-light exposure. However, the development of photostable sunscreens is important to preserve the photoprotective efficacy and to prevent the formation of reactive oxygen species (ROS) and photodegradation products, which can promote phototoxic or photoallergic contact dermatitis. The aim of this study was to evaluate the effects of mangiferin and naringenin on the photostability and phototoxicity of sunscreens containing avobenzone. Cosmetic sunscreen formulations containing octocrylene (OCT), octyl methoxycinnamate (OMC) and avobenzone (AVO) were prepared and supplemented or not with mangiferin, naringenin, or with both compounds in combination. For photostability studies, samples of the formulations were spread onto glass plates, exposed to UVA radiation and then analyzed by high performance liquid chromatography (HPLC) to determine UV filters and the antioxidants recovery. The phototoxicity of the UV filters and antioxidants was evaluated using 3T3 fibroblast cultures that were subjected (or not) to irradiation according to OECD TG 432. The photostability studies demonstrated that AVO and naringenin showed the highest photodegradation when present in formulation FN (containing octocrylene, avobenzone, octyl methoxycinnamate and naringenin). The addition of mangiferin to this combination (FMN) resulted in an improved photostability of both substances compared to FN. The in vitro phototoxicity test showed that only avobenzone was considered phototoxic. The combination containing AVO/naringenin exhibited phototoxic potential; however, this was reduced by the addition of mangiferin (combination CMN). The results of this study are promising because it was demonstrated that mangiferin could increase the photostability and reduce the phototoxic potential of the combination of naringenin and AVO.

Retinoids and hydroxy acids have been widely used due to their effects in the regulation of growth and in the differentiation of epithelial cells. However, besides their similar indication, they have different mechanisms of action and thus they may have different effects on the skin; in addition, since the topical formulation efficiency depends on vehicle characteristics, the ingredients of the formulation could alter their effects. Thus the objective of this study was to compare the effects of retinoic acid (RA) and glycolic acid (GA) treatment on the hairless mouse epidermis thickness and horny layer renewal when added in gel, gel cream, or cream formulations. For this, gel, gel cream, and cream formulations (with or without 6% GA or 0.05% RA) were applied in the dorsum of hairless mice, once a day for seven days. After that, the skin was analyzed by histopathologic, morphometric, and stereologic techniques. It was observed that the effects of RA occurred independently from the vehicle, while GA had better results when added in the gel cream and cream. Retinoic acid was more effective when compared to glycolic acid, mainly in the cell renewal and the exfoliation process because it decreased the horny layer thickness.

Background/purpose: The polysaccharide-rich composition of Aloe vera extracts (Aloe barbadensis Miller), often used in cosmetic formulations, may impart moisturizing properties to the product. The aim of this study was to evaluate the effect of cosmetic formulations containing different concentrations of freeze-dried Aloe vera extract on skin hydration, after a single and a 1- and 2-week period of application, by using skin bioengineering techniques. Methods: Stable formulations containing 5% (w/w) of a trilaureth-4 phosphate-based blend were supplemented with 0.10%, 0.25% or 0.50% (w/w) of freeze-dried Aloe vera extract and applied to the volar forearm of 20 female subjects. Skin conditions in terms of the water content of the stratum corneum and of transepidermal water loss (TEWL) (CorneometerTM CM 825 and TewameterTM TM 210) were analysed before and after a single and 1- and 2-week period of daily application. Results: After a single application, only formulations supplemented with 0.25% and 0.50% (w/w) of Aloe vera extract increased the water content of the stratum corneum, while after the 2-week period application, all formulations containing the extract (0.10%, 0.25% and 0.50%) had the same effect, in both cases as compared with the vehicle. TEWL was not modified after a single and after 1- and 2-week period of application, when compared with the vehicle. Conclusion: Our results show that freeze-dried Aloe vera extract is a natural effective ingredient for improving skin hydration, possibly through a humectant mechanism. Consequently, it may be used in moisturizing cosmetic formulations and also as a complement in the treatment of dry skin.

A produção de espécies reativas de oxigênio (EROs) por leucócitos polimorfonucleares (LPMNs) durante a fagocitose é essencial para a defesa do organismo contra microrganismos invasores. Entretanto, em algumas doenças ocorre a deposição de imunocomplexos, o que leva à produção e liberação excessiva dessas EROs, e conseqüente lesão nos tecidos hospedeiro. Neste trabalho avaliamos o efeito de substâncias naturais (quercetina e 7-aliloxicumarina) sobre a produção de EROs por LPMNs de coelho, estimulados com imunocomplexos de imunoglobulina G (ICIgG), empregando o ensaio de quimioluminescência (QL) dependente de luminol (QL lum) e de QL dependente de lucigenina (QL luc). A interação entre ICIgG e LPMNs, mediada pelo receptor de membrana Fcgama, foi observada por microscopia eletrônica de transmissão, pela intensa marcação dos ICIgG com partículas de ouro coloidal. Os resultados obtidos indicaram que as substâncias analisadas inibiram a QL lum e a QL luc e que tais efeitos não parecem estar relacionados com a inibição da fagocitose, conforme observado no ensaio por microscopia eletrônica.The generation of reactive oxygen species (EROs) by polymorphonuclear leukocytes (LPMNs) during phagocytosis plays a key role in the organism defense against invading microorganisms. However, in some diseases the immune complexes deposition occurs, that causes an increase in the production and release of EROs. Consequently, the host tissue is damaged. In this work we studied the effect of natural compounds (quercetin and 7-allyloxycoumarin) on EROs generation by rabbit (LPMNs) stimulated with immunecomplexes (IC) of chicken egg albumin (antigen) and class G immuneglobulins (IgG) - ICIgG. The EROs production was measured by chemiluminescence assay (QL) enhanced by luminol (QL lum) or by lucigenin (QL luc). The interaction between ICIgG (conjugated with colloidal gold) e LPMNs, mediated by Fcgamma membrane receptor, was observed by transmission electron microscopy. The results indicated that the studied compounds inhibited QL lum and QL luc. They also suggest that such effects are not related to phagocytosis inhibition, as observed by the electron microscopy assay.

A new trend in cosmetic formulations is the use of biotechnological raw materials as the polysaccharides from Klebsiella pneumoniae, which are supposed to enhance cell renewal, improve skin hydration and micro-relief. Botanical extracts of Myrtus communis leaves contain different sugars, which may provide the same benefits. Thus, the objective of this study was to evaluate through objective and subjective analysis the immediate and long-term effects of cosmetic formulations containing polysaccharides biotechnologically-originated and / or the ones contained in Myrtus communis extracts. Three polysaccharide-based and placebo formulations were applied on the forearm skin of 40 volunteers. Skin hydration, transepidermal water loss (TEWL), viscoelasticity and skin micro-relief measurements were made before and 2 hours after a single application and after 15 and 30 day-periods of daily applications. Answers to a questionnaire about perceptions of formulation cosmetic features constituted the subjective analysis. All polysaccharide-based formulations enhanced skin hydration. Formulations with isolated or combined active substances improved skin barrier function as compared to placebo, in the short and long term studies. Formulations containing Myrtus communis extracts had the highest acceptance. Results suggest that daily use of formulations containing these substances is important for protection of the skin barrier function.Uma nova tendência em formulações cosméticas é a utilização de matérias-primas biotecnológicas como os polissacarídeos de Klebsiella pneumoniae, que pode aumentar a renovação celular e melhor a hidratação e micro-relevo da pele. Por outro lado, o extrato vegetal de Myrtus communis contém diferentes polissacarídeos, que também podem proporcionar benefícios à pele. Assim, o objetivo do estudo foi a avaliação dos efeitos imediatos e em longo prazo, de formulações cosméticas contendo polissacarídeos obtidos por processo biotecnológico e/ou de extrato de M. communis por meio de análises objetivas e subjetivas. Três formulações contendo os polissacarídeos e um placebo foram aplicadas na pele dos antebraços de 40 voluntários. As medidas foram realizadas antes e após 2 horas da aplicação das formulações e após 15 e 30 dias de aplicações diárias em termos de hidratação da pele, perda transepidérmica de água (TEWL), viscoelasticidade e micro-relevo da pele. Para a análise subjetiva, os voluntários responderam um questionário a fim de obter-se informações sobre a percepção relativa à qualidade de cosméticos. Todas as formulações provocaram aumento da hidratação cutânea. As formulações que continham os polissacarídeos melhoraram a função barreira da pele, em curto e em longo prazo. A formulação contendo extrato de M. communis apresentou maior aceitação. Os resultados sugerem que o uso diário dos polissacarídeos avaliados é importante na proteção da função barreira da pele.

The present study aimed to evaluate the photoprotective effects of cosmetic formulations containing a dispersion of liposome with magnesium ascorbyl phosphate (MAP), alpha-lipoic acid (ALA) and kinetin, as well as their effects on the hydration and viscoelastic skin properties. The photoprotection was determined in vitro (antioxidant activity) and in vivo on UV-irradiated hairless mouse skin. The hydration effects were performed with the application of the formulations under study on the forearm of human volunteers and skin conditions were analyzed before and after a single application and daily applications during 4 weeks in terms of transepidermal water loss (TEWL), skin moisture and viscoelastic properties. The raw material under study possessed free-radical scavenging activity and the formulation with it protected hairless mouse skin barrier function against UV damage. After 4 weeks of application on human skin, the formulation under study enhanced stratum corneum skin moisture and also showed hydration effects in deeper layers of the skin. Thus, it can be concluded that the cosmetic formulation containing a dispersion of liposome with MAP, ALA and kinetin under study showed photoprotective effects in skin barrier function as well as pronounced hydration effects on human skin, which suggests that this dispersion has potential antiaging effects.

Liposoluble vitamin C derivatives, such as tetra-isopalmitoyl ascorbic acid (IPAA), are often used in dermocosmetic products due to their higher stability than vitamin C free form as well as its proposed effects in skin; however, there are no studies analyzing IPAA stability or its in vivo effects when present in dermocosmetic formulations. Thus, this study aimed to evaluate chemical stability and pre-clinical and clinical efficacy of dermocosmetic formulations containing IPAA in skin hydration and microrelief. Chemical stability of the formulations added with 1% IPAA was evaluated by heat stress during 35 days by HPLC. For pre-clinical evaluation, experimental formulations were topically applied on hairless skin mice during 5 days and animal skins were analyzed by non-invasive biophysic techniques (water content of stratum corneum, TEWL, viscoelasticity, and microrelief) and by histopathological studies. For clinical efficacy tests, the formulations were topically applied to the forearm and face of human volunteers, and 3h and 15 days after applications, the skins were evaluated by the same non-invasive techniques mentioned before. Results showed that formulations containing IPAA had medium stability and had pronounced moisturizing effects on stratum corneum and on viable epidermis. These formulations also improved skin microrelief especially in relation to skin smoothness and roughness.

The beneficial photoprotective effects of topical formulations containing combined extracts of Ginkgo biloba (GB) and green tea (GT) have not been investigated. The present study aims to assess the photoprotective effects of topical formulations containing GB and GT combined or not by applications on the dorsal skin of hairless mice prior to UVA/B irradiation. After 20 h, skin barrier damage (TEWL), erythema, histological alterations and sunburn cell formation were evaluated. The results showed that only extract supplemented formulations protected the skin against the UV-induced damage. Formulations containing GB provided total protection of the skin barrier function avoiding UV radiation damage such as TEWL and erythema and were more effective than those containing GT. However, it is suggested that formulations containing combined GB and GT may provide substantial photoprotective effects since different aspects of skin damage were influenced by each extract.

Vitamin A palmitate has been used in cosmetics; however, studies report that this substance shows photoreactivity that can lead to loss of safety and efficacy. On the other hand, photostabilizers have been used to increase sunscreen photostability and consequently their safety and effectiveness. Thus, this study aimed to evaluate the influence of photostabilizers on the photoprotective effects of a cosmetic formulation containing UV-filters and vitamin A palmitate. The formulation containing UV-filters was supplemented with vitamin A palmitate and the photostabilizers diethylhexyl 2,6-naphthalate (DEHN), bumetrizole and benzotriazolyl dodecyl p-cresol (BTDC). Hairless mice were treated daily by topical applications and irradiated (UVA/B). Erythema index, transepidermal water loss, histological/histometric analysis and number of sunburn cells (SBC) were evaluated. The results showed that all formulations protected from UV-induced enhancement of erythema and SBC but there was no difference among them. The formulation with no stabilizers reduced viable epidermis thickness due to atrophy induced by UV radiation. Thus, it can be concluded that the presence of photostabilizers influenced the effects of formulations containing UV-filters and vitamin A palmitate, which could be seen by histological and histometric analysis. Furthermore, the formulations containing the stabilizers DEHN and BTDC showed better protective effects on hairless mice skin.

This paper reports a simple and reliable HPLC method to evaluate the influence of two currently available photostabilizers on cosmetic formulations containing combined UV-filters and vitamins A and E. Vitamins and UV-filters, widely encountered in products of daily use have to be routinely evaluated since photoinstability can lead to reductions in their efficacy and safety. UV-irradiated formulation samples were submitted to a procedure that included a reliable, precise and specific HPLC method employing a C18 column and detection at 325 and 235 nm. Methanol, isopropanol and water were the mobile phases in gradient elution. The method precision was between 0.28 and 5.07. The photostabilizers studied [diethylhexyl 2,6-naphthalate (DEHN) and benzotriazolyl dodecyl p-cresol (BTDC)], influenced the stability of octyl methoxycinnamate (OMC) associated with vitamins A and E. BTDC was considered the best photostabilizer to vitamins and OMC when the UV-filters were combined with both vitamins A and E.