After the period of incubation, we irradiated one plate with 4 J/cm2 of UVA radiation (sun simulator), while the other one we kept in dark box. 
Alternatives for Dermal Toxicity Testing. The positive phototoxic control 3% ketoprofen was correctly classified, since it showed a reduction of approximately 41% in cell viability when compared to the non-irradiated tissue, which is higher than the cut off value of 30% reduction that was determined for the assay (Figure 3). Thus, although Rodrguez-Luna et al. In this test, fibroblasts of the Balb 3T3 clone A31 that were provided by Banco de Clulas do Rio de Janeiro, BCRJ code 0047 (Rio de Janeiro, Brazil) cultured on two 96 well microtiter plates were pre-incubated with eight different concentrations of the test chemical (6.8100 g/mL) for one hour. 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, the viable epidermis is made of keratinocytes, which are less sensitive than fibroblasts to xenobiotics and UV radiation. fucoxanthin While the other carotenoids have virtually no scavenging ability against ROS, fucoxanthin donates electrons as a part of its mechanism of capturing free radicals [12]. In the presence of hydrogen peroxide, hydroxyl radicals, carbonate, and nitrite, DCFH is oxidized to the highly fluorescent DCF, which can be monitored by several fluorescence-based techniques derivative [39,40]. In the present study, fucoxanthin was, for the first time, evaluated in vitro by the toxicological and photoprotective perspective of its use in the human skin using 3D RHS. The skin models were irradiated (sun simulator mentioned before) with 6 J/cm2, the dose that was recommended by Kandarova and Liebsch [28], as necessary to produce a phototoxic response in the positive controls without damage to the tissue and the one used to pre-validate the test in 1999 by European Centre for the Validation of Alternative Methods (ECVAM) [29]. Santos A.J., Miranda M.S., Esteves da Silva J.C. We repeated the photostability assay applying 0.5% (w/v) of pure fucoxanthin in a sunscreen formulation, formulation 3 (F3), based on the photostability studies of Freitas et al. 
By their coloring feature, other pigments, such as any xanthophyll common to brown algae, such as cis-fucoxanthin, diadinoxanthin, diatoxanthine, and -carotene, could be present in the alga extract in addition to fucoxanthin [12]. 
We used a mass of 69.00 g (wet weight) to obtain the extract. On the 10th day of tissue cultivation, the skin models were exposed to fucoxanthin solubilized in c12-c15 alkyl benzoate (0.5%, v/v), which is a vehicle that is commonly used to solubilize lipophilic chemicals in cosmetics [5]. 
The .gov means its official. kombu tororo diet eating healthy japanese slism seaweed shredded benefits (2015) study. Zeng J., Zhang Y., Ruan J., Yang Z., Wang C., Hong Z., Zuo Z. Rodrguez-Luna A., vila-Romn J., Gonzlez-Rodrguez M.L., Czar M.J., Rabasco A.M., Motilva V., Talero E. Fucoxanthin-Containing Cream Prevents Epidermal Hyperplasia and UVB-Induced Skin Erythema in Mice. RHE and RHS, due to the presence of a stratum corneum, appear to be capable of detecting known human dermal phototoxicants. After analyzing the chemical UVA/VIS photoinstability of fucoxanthin, with degradation being over the acceptable range of 20% after a 6 J/cm2 UVA dose, we added fucoxanthin at 0.5% to a sunscreen formulation to assess photostability at a 27.5 J/cm2 UVA dose. Since fucoxanthin contains an alene bond, a conjugated carbonyl group, one 5,6-monoepoxide, and an acetyl group, there is a biological potential that is associated with this structure of the molecule when orally administered [13]. Absorption spectra of (a) The alga D. anceps crude extract; isopropanol solution, 100 g/mL; (b) The fractions obtained from this crude extract presented different spectrum profiles; F = fraction, numbers ordered by elution (nonpolar to polar a indicates the second day of the extraction. The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop. The second instrument was Shimadzu chromatograph (Shimadzu, Kyoto, Japan), Prominence model, CBM-20th controller, SPD-20th detector UV/VIS, with two pumps (LC-6AD), an FCR-10th automatic collector DGU-20A5 degasser and LC-Solution Single Software, a semi-preparative column Synergi Polar-RP (250 mm 10 mm, 4 mm), and a semi-preparative Polar-RP guard column (10 10 mm, 4 mm), both being from Phenomenex (Torrance, CA, USA). Following the same mechanism of the probe DCFH2-DA, but this time with a sunscreen containing or not 0.5% of fucoxanthin applied in RHS tissues [33,34], the results demonstrated that the UVA radiation increased ROS generation in the untreated RHS control irradiated when compared to the untreated and not irradiated control (p > 0.05), (Figure 5 and Figure 6a,b). energy plus extract acai berry clarity constituent aging demonstrated increased mental benefits provide memory anti support been We selected fractions that yielded higher weight to have enough mass for all of the following tests; firstly, the selected fractions were screened in terms of their UV spectra. Concerning brown algae, the typical absorptions in the regions of 400 nm and 660 nm (areas of blue and red, respectively) are due to the presence of carotenoids and chlorophylls, especially chlorophyll a, which is common to all photosynthetic organisms having a maximum absorption at 420 and 660 nm, respectively. 
The reduction in ROS production was probably due to its antioxidant properties under anoxic conditions, well described by other studies using in chemico (DPPH; ABTS) [14] and in vitro methods (HepG2, HaCat, PC12) [47]. Whitehead K., Hedges J.I. The RHS were incubated with the DCFH2-DA probe in PBS (50 M) in the dark for 45 min. weight loss healthy skincare fucoxanthin supplement With this lower dose, we observed no degradation in the UVB region, 35% in the UVA region, and 21% in the VIS region; the depletion of absorption of the isolated compound was not in the range considered to be photostable (Figure 2c) [2,4,37]. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (, GUID:2C58C507-D41F-486B-A4DF-89E29C1A0AF2, antioxidant, fucoxanthin, phototoxicity, photostability, reconstructed human skin. Alves G.A.D., Oliveira de Souza R., Ghislain Rogez H.L., Masaki H., Fonseca M.J.V. Untreated (blue bar non-irradiated and yellow bars irradiated); sunscreen and sunscreen plus 0.5% fucoxanthin (Fx). additives thistle 
Where * means significantly different from untreated irradiated (+UV) and from sunscreen treated models (p < 0.001). We obtained the dry extract (1.29 g) from fresh wet alga material (69.00 g), with a yield of 2.31%. Antioxidant and prooxidant roles for beta-carotene, alpha-tocopherol and ascorbic acid in human lung cells. The marine carotenoid fucoxanthin that was isolated in this study from the alga D. anceps was the main carotenoid present in this brown alga [41]. 
Photostability studies based on the electromagnetic spectrum of the samples irradiated (yellow line) or not (blue line). Maia-Campos P.M.B.G., Benevenuto C.G., Calixto L.S., Melo M.O., Pereira K.C., Gaspar L.R. Hashimoto T., Ozaki Y., Taminato M., Das S.K., Mizuno M., Yoshimura K., Maoka T., Kanazawa K. The distribution and accumulation of fucoxanthin and its metabolites after oral administration in mice. Nevertheless, RHE does not present the dermal component, which is essential for many epidermal characteristics and proper skin functionalities, including the improvement of barrier function [19]. The experimental data was analyzed statistically by analysis of variance (ANOVA), a parametric test, followed by Tukeys test. for permeation into the cell. 
These carotenoids also have a maximum absorption peak around 448 nm, as is the case for chlorophylls (b), (c), and (a), which are the main chlorophyll pigments in algae [14]. Pennacchi P.C., de Almeida M.E., Gomes O.L., Faio-Flores F., de Arajo Crepaldi M.C., Dos Santos M.F., de Moraes Barros S.B., Maria-Engler S.S. Glycated Reconstructed Human Skin as a Platform to Study the Pathogenesis of Skin Aging. Licensee MDPI, Basel, Switzerland. [35], different structures of chlorophyll c are responsible for its intense absorption in the region of 530 nm; also, a peak in the 680 nm region (Figure 1a) could be related to chlorophyll a. Liebsch M., Spielmann H., Pape W., Krul C., Deguercy A., Eskes C. UV-induced effects. Furthermore, treatment with fucoxanthin at 100 g/mL induced the maximal reduction of ROS production, which was statistically different from the other studied concentrations (0.1, 1, and 10 g/mL) (p < 0.001). The final sunscreen formulation was yellow-colored, but it was not able to stain the skin. For the D. anceps extract and Fr15, we applied a cumulative dose of 27.5 J/cm2. It absorbs in the UV/VIS region, which is interesting for photoprotection and presents promising antioxidant properties that are observed in mice reported in the literature [12]. Accessibility The formazan production was measured at 570 nm. bladderwrack capsules We concentrated the organic D. anceps extract in a rotary evaporator under low pressure (Bchi R-300, Buchi Labotechnik, Flawil, Switzerland) and subjected it to a classical chromatographic column30 cm with stationary phase Silica Gel 4063 m/ASTM Macherey-Nagel (Merck, Darmstadt, Germany), with a polarity gradient using n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) (JT Baker, Port of Spain, Trinidad Y Tobago) to fractionate the extract. In the efficacy assay on monolayers, fucoxanthin not only did not harm the HaCat keratinocytes, but also protected the cells from UVA-induced ROS formation in a concentration-dependent manner (maximal effect at 0.01%) (Figure 4). Freitas J.V., Gaspar L.R. 
Proceedings of International Scientific Conference, Alternatives to Animal Testing. * Fucoxanthin rich fraction, ** dose defined as enough to induce phototoxic responses, NE: Non-evaluated (sunscreen with absence of absorbance in the VIS range). 
We measured UVA radiation with the same radiometer mentioned before, while we kept the non-irradiated plates in a dark box. According to the OECD Test Guideline 432, a substance is predicted to be phototoxic if MPE is higher than 0.15 or the PIF is higher than 5. retention time eluted the substance of interest that was monitored by absorption ( max ~ 450 nm) (Supplementary Figure S1); chlorophyll, (a; c) together with fucoxanthin. D. anceps extract presented absorption in the region of interest for photoprotection (280400 nm), especially in the UVA and in the visible range (Figure 1a). That dose is recommended as being similar to 66 min. bgg 
Zhang H., Tang Y., Zhang Y., Zhang S., Qu J., Wang X., Kong R., Han C., Liu Z. Fucoxanthin: A Promising Medicinal and Nutritional Ingredient. After Hawaii, Florida and Key West followed this trend. Riccioni G., DOrazio N., Franceschelli S., Speranza L. Marine carotenoids and cardiovascular risk markers. 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. before exposure to a UVA dose of 27.5 J/cm2. Optimizing the spectral absorption profile of sunscreens. Besides the evaluation of fucoxanthin photosafety, we evaluated the photoprotective potential of this molecule by its photostability under different irradiation exposure and when used in a sunscreen formulation. Therefore, the combined phototoxicity assays (monolayer and RHS) suggest that fucoxanthin would not be phototoxic to the human skin at 0.5% due to the reduced bioavailability through the stratum corneum and in the stratified epidermis. We used the area under the curve (AUC), which is the integral of the absorption spectrum of the samples in the UVB (280320 nm), UVA (320400 nm), and VIS (400700 nm) ranges using the integration function of the MicroCal OriginPro Software (8 SRO, OriginLab Corporation, Northampton, MA, USA) to calculate the photostability [24]. However, this effect was not observed for fucoxanthin 0.1 g/mL, a dose that provided a reduction of 12%, which was not considered statistically significant when compared to the control (p > 0.05). The following are available online at https://www.mdpi.com/2076-3921/9/4/328/s1, Figure S1: Chromatogram of the elution semi-preparative scalecarotenoid isolation, Figure S2: Chromatogram of the semi-preparative scale subfraction, analyzed in 450 nmabsorption region of fucoxanthin at a higher intensity (Au 450), Retention Time (RT) = 5.75 min black line, 650 nmregion of the bands absorption of chlorophyll a lower intensity. Conceptualization, R.S.N.T., L.R.G., H.M.D. On the other hand, the sunscreen containing fucoxanthin 0.5% (w/v) induced a significant reduction of ROS generation after UVA irradiation when compared to the untreated control irradiated (+UV) and when compared to the sunscreen without fucoxanthin (Figure 5 and Figure 6a,d). The treatment with fucoxanthin at 1, 10, and 100 g/mL induced a significant reduction of ROS production of about 17%, 15%, and 65%, respectively, when compared to the untreated irradiated control (p < 0.001) (Figure 4). Reconstructed human skin after UVA irradiation (10 J/cm2) using a fluorescent probe DCFH2-DA. 
Cambon M., Issachar N., Castelli D., Robert C. An in vivo method to assess the photostability of UV filters in a sunscreen. Concerning ultraviolet (UV) damage, at the beginning of this century, not many compounds were available for the protection against UVA (320400 nm) radiation and, in response to the growing concern regarding the additional damage that is caused by this radiation, various UVA filters are now available for formulations worldwide. n = 1 or 2 independent experiments, -: values not determined in the studied concentration range. Thus, the objective of the present study was to assess the photoprotective and phototoxicity potential of fucoxanthin based on its UV/VIS absorption, photostability, phototoxicity in 3T3 mouse fibroblast culture vs. full-thickness RHS, and its ability to inhibit reactive oxygen species formation that is induced by UVA on HaCaT keratinocytes. The complete assignments of the 1H and 13C NMR spectra of fucoxanthin revealed signals that were assignable to polyene containing acetyl, conjugated ketone, olefinic methyl, two quaternary germinal oxygen methyls, two quaternaries geminal dimethyl, and allene groups. However, -Carotene is able to protect the collagen structure from infrared light in the skin [53] and it is used in cosmetic formulations for aged skin and actinic keratosis [51], which, for instance, could also suggest some skin benefits from the topical use of fucoxanthin when tested in vivo, due to the similarities of their molecular structures. Additionally, we evaluated the maximal antioxidant potential of this molecule onto immortalized human keratinocytes (HaCat) cells, determining the ideal concentration range that is to be used for this effect. This research was funded by call N 64/2013, MCTI/CNPq/FNDCT-Ao Transversal-Programa Antrtico Brasileiro-PROANTAR, from the Brazilian Government, R.S.N.T. Based on the historical data that was produced in our laboratory, we defined this dose as a dose high enough to elicit a phototoxic response in positive controls and as a dose that did not produce interference higher than 20% in cell viability, as recommended by the Organisation for Economic Co-operation and Development (OECD) [26]. 8600 Rockville Pike The material was filtered and extracted two more times while using ultrasound equipment for 15 minutes in the third procedure. 
On the other hand, the antioxidant quercetin (10 g/mL) induced a reduction of ROS generation of around 46% after UVA irradiation (Figure 4). DOrazio N., Gemello E., Gammone M.A., de Girolamo M., Ficoneri C., Riccioni G. Fucoxantin: A treasure from the sea. However, the sunscreen plus fucoxanthin showed acceptable photodegradation (Figure 2d). 
This suggests that fucoxanthin can also protect viable epidermis against UVA-induced ROS production, which is in agreement with the fucoxanthin protection observed in HaCat keratinocytes. They were kept submerged for 24 h. Next, the culture was maintained at the air-liquid interface for 12 days to allow for keratinocytes differentiation [27]. In addition, it showed a significant inhibition of ROS formation at 0.01% (p < 0.001), in HaCat, and in a sunscreen at 0.5% (w/v) (p < 0.001), in RHS. 
kelp seaweeds Furthermore, we also observed a significant reduction in the ROS production in RHS (complex 3D tissue) treated and irradiated with sunscreen plus fucoxanthin that we did not observe in the RHS that was treated with sunscreen alone (Figure 5 and Figure 6). fucoidan 
Alternative in vitro methods validated for preclinical trials are being used to predict the safety and efficacy of unknown natural compounds instead of using animal models to evaluate the potential risk of a test chemical. and L.R.G. To isolate the carotenoid, we employed high-performance liquid chromatography (HPLC), analytical, and semi-preparative analyses while using two different types of equipment with three different columns. We are thankful to Ribeiro Preto State Hospital and to the Maria de Ftima Galli Sorita, Ana Laura Ferreira Marsico Dalto, and Alexandra Longo Camperoni for the partnership providing the foreskin. Damiani E., Baschong W., Greci L. UV-Filter combinations under UV-A exposure: Concomitant quantification of over-all spectral stability and molecular integrity. For the identification of fucoxanthin, the highest colored mass fractions were subjected to Electrospray Ionization Mass Spectrometry (ESI-MS) techniques (amaZon SL, Bruker, Bil-lerica, MA, USA). We expressed the results as a percentage of the area of irradiated samples related to the area of non-irradiated samples. However, its topical properties and safety for the human skin are still unknown. lymphoma bcl fucoidan photoprotective abbreviations sod Federal government websites often end in .gov or .mil. When a positive result is obtained, i.e. kelp composition In the previous study of our group, commercial fucoxanthin at the same concentration, presented an absence of irritation, absence of morphological changes in the histology, and no significant dysregulation on homeostasis, metabolism, and in the inflammatory genes studied after following the OECD protocol for skin irritation [49]. carnitine fucoxanthin 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. We applied a cumulative dose of 6 J/cm2 to determine the photostability of fucoxanthin in isopropanol (the dose recommended for phototoxicity studies and similar to 14 min. The positive control norfloxacin (100 g/mL) induced an increase of ROS production of around 25% in comparison to the untreated irradiated control (Figure 4). government site. sampler week try sample RHS viability was measured by the end of the experiment of phototoxicity. It has been reported that this carotenoid shows intense antioxidant activity, as well as anti-inflammatory, anti-obesity, anti-diabetic, anti-tumor, antihypertensive, and anticancer properties [13,14]. The results were normalized to area/pixels and expressed in a percentage of fluorescence intensity in comparison to the irradiated and non-irradiated untreated controls. We exposed other constructs in every batch to fucoxanthin, but not to MTT, to evaluate the ability of fucoxanthin to stain the constructs under test conditions. wakame The images were quantified using Image J software [33,34]. fucoxanthin induce fucus inhibit duodenal carcinoma substances inhibits induzieren Diffey B.L. Our study corroborates those reported before [48], which employed the HaCat cell line and hairless mice to study the UVB protective effects of commercial all-trans-fucoxanthin (Sigma). The reduction of mitochondrial dehydrogenase activity was assessed via the decreased formazan production following incubation with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, Cotia, Brazil). 432: In vitro 3T3 NRU Phototoxicity Test, OECD Guidelines for the Testing of Chemicals. ; investigation, R.S.N.T., C.M.K., K.d.C.P. FOIA In this study, the sunscreen formulation that contains a combination of two UV filters considered to be photo unstable (avobenzone and ethylhexyl methoxycinnamate) was studied in order to assess the fucoxanthin capabilities of alteration in the photostability of this photo unstable combination. Liebsch M., Traue D., Barrabas C., Spielmann H., Gerberick G.F., Cruse L., Diembeck W., Pfannenbecker U., Spieker J., Holzhutter H.-G., et al. saccharina ingredient loss fat natural fucoxanthin japonica extract powder bulk of exposure to sunlight at midday (6.94 mW/cm2) on a typical September sunny day in the Ribeiro Preto, Brazillatitude 211039 south and longitude 474837 west [4,23]. 
Tavares R.S.N., Maria-Engler S.S., Colepicolo P., Debonsi H.M., Schfer-Korting M., Marx U., Gaspar L.R., Zoschke C. Skin Irritation Testing beyond Tissue Viability: Fucoxanthin Effects on Inflammation, Homeostasis, and Metabolism. This movement has stimulated the urgent research on alternatives and possibly eco-friendly photoprotective compounds [10]. Photostability evaluation of five UV-filters, trans-resveratrol and beta-carotene in sunscreens.
Alternatives for Dermal Toxicity Testing. The positive phototoxic control 3% ketoprofen was correctly classified, since it showed a reduction of approximately 41% in cell viability when compared to the non-irradiated tissue, which is higher than the cut off value of 30% reduction that was determined for the assay (Figure 3). Thus, although Rodrguez-Luna et al. In this test, fibroblasts of the Balb 3T3 clone A31 that were provided by Banco de Clulas do Rio de Janeiro, BCRJ code 0047 (Rio de Janeiro, Brazil) cultured on two 96 well microtiter plates were pre-incubated with eight different concentrations of the test chemical (6.8100 g/mL) for one hour. 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, the viable epidermis is made of keratinocytes, which are less sensitive than fibroblasts to xenobiotics and UV radiation. fucoxanthin While the other carotenoids have virtually no scavenging ability against ROS, fucoxanthin donates electrons as a part of its mechanism of capturing free radicals [12]. In the presence of hydrogen peroxide, hydroxyl radicals, carbonate, and nitrite, DCFH is oxidized to the highly fluorescent DCF, which can be monitored by several fluorescence-based techniques derivative [39,40]. In the present study, fucoxanthin was, for the first time, evaluated in vitro by the toxicological and photoprotective perspective of its use in the human skin using 3D RHS. The skin models were irradiated (sun simulator mentioned before) with 6 J/cm2, the dose that was recommended by Kandarova and Liebsch [28], as necessary to produce a phototoxic response in the positive controls without damage to the tissue and the one used to pre-validate the test in 1999 by European Centre for the Validation of Alternative Methods (ECVAM) [29]. Santos A.J., Miranda M.S., Esteves da Silva J.C. We repeated the photostability assay applying 0.5% (w/v) of pure fucoxanthin in a sunscreen formulation, formulation 3 (F3), based on the photostability studies of Freitas et al. 
By their coloring feature, other pigments, such as any xanthophyll common to brown algae, such as cis-fucoxanthin, diadinoxanthin, diatoxanthine, and -carotene, could be present in the alga extract in addition to fucoxanthin [12]. We used a mass of 69.00 g (wet weight) to obtain the extract. On the 10th day of tissue cultivation, the skin models were exposed to fucoxanthin solubilized in c12-c15 alkyl benzoate (0.5%, v/v), which is a vehicle that is commonly used to solubilize lipophilic chemicals in cosmetics [5]. The .gov means its official. kombu tororo diet eating healthy japanese slism seaweed shredded benefits (2015) study. Zeng J., Zhang Y., Ruan J., Yang Z., Wang C., Hong Z., Zuo Z. Rodrguez-Luna A., vila-Romn J., Gonzlez-Rodrguez M.L., Czar M.J., Rabasco A.M., Motilva V., Talero E. Fucoxanthin-Containing Cream Prevents Epidermal Hyperplasia and UVB-Induced Skin Erythema in Mice. RHE and RHS, due to the presence of a stratum corneum, appear to be capable of detecting known human dermal phototoxicants. After analyzing the chemical UVA/VIS photoinstability of fucoxanthin, with degradation being over the acceptable range of 20% after a 6 J/cm2 UVA dose, we added fucoxanthin at 0.5% to a sunscreen formulation to assess photostability at a 27.5 J/cm2 UVA dose. Since fucoxanthin contains an alene bond, a conjugated carbonyl group, one 5,6-monoepoxide, and an acetyl group, there is a biological potential that is associated with this structure of the molecule when orally administered [13]. Absorption spectra of (a) The alga D. anceps crude extract; isopropanol solution, 100 g/mL; (b) The fractions obtained from this crude extract presented different spectrum profiles; F = fraction, numbers ordered by elution (nonpolar to polar a indicates the second day of the extraction. The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop. The second instrument was Shimadzu chromatograph (Shimadzu, Kyoto, Japan), Prominence model, CBM-20th controller, SPD-20th detector UV/VIS, with two pumps (LC-6AD), an FCR-10th automatic collector DGU-20A5 degasser and LC-Solution Single Software, a semi-preparative column Synergi Polar-RP (250 mm 10 mm, 4 mm), and a semi-preparative Polar-RP guard column (10 10 mm, 4 mm), both being from Phenomenex (Torrance, CA, USA). Following the same mechanism of the probe DCFH2-DA, but this time with a sunscreen containing or not 0.5% of fucoxanthin applied in RHS tissues [33,34], the results demonstrated that the UVA radiation increased ROS generation in the untreated RHS control irradiated when compared to the untreated and not irradiated control (p > 0.05), (Figure 5 and Figure 6a,b). energy plus extract acai berry clarity constituent aging demonstrated increased mental benefits provide memory anti support been We selected fractions that yielded higher weight to have enough mass for all of the following tests; firstly, the selected fractions were screened in terms of their UV spectra. Concerning brown algae, the typical absorptions in the regions of 400 nm and 660 nm (areas of blue and red, respectively) are due to the presence of carotenoids and chlorophylls, especially chlorophyll a, which is common to all photosynthetic organisms having a maximum absorption at 420 and 660 nm, respectively. 
The reduction in ROS production was probably due to its antioxidant properties under anoxic conditions, well described by other studies using in chemico (DPPH; ABTS) [14] and in vitro methods (HepG2, HaCat, PC12) [47]. Whitehead K., Hedges J.I. The RHS were incubated with the DCFH2-DA probe in PBS (50 M) in the dark for 45 min. weight loss healthy skincare fucoxanthin supplement With this lower dose, we observed no degradation in the UVB region, 35% in the UVA region, and 21% in the VIS region; the depletion of absorption of the isolated compound was not in the range considered to be photostable (Figure 2c) [2,4,37]. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (, GUID:2C58C507-D41F-486B-A4DF-89E29C1A0AF2, antioxidant, fucoxanthin, phototoxicity, photostability, reconstructed human skin. Alves G.A.D., Oliveira de Souza R., Ghislain Rogez H.L., Masaki H., Fonseca M.J.V. Untreated (blue bar non-irradiated and yellow bars irradiated); sunscreen and sunscreen plus 0.5% fucoxanthin (Fx). additives thistle 
Where * means significantly different from untreated irradiated (+UV) and from sunscreen treated models (p < 0.001). We obtained the dry extract (1.29 g) from fresh wet alga material (69.00 g), with a yield of 2.31%. Antioxidant and prooxidant roles for beta-carotene, alpha-tocopherol and ascorbic acid in human lung cells. The marine carotenoid fucoxanthin that was isolated in this study from the alga D. anceps was the main carotenoid present in this brown alga [41]. Photostability studies based on the electromagnetic spectrum of the samples irradiated (yellow line) or not (blue line). Maia-Campos P.M.B.G., Benevenuto C.G., Calixto L.S., Melo M.O., Pereira K.C., Gaspar L.R. Hashimoto T., Ozaki Y., Taminato M., Das S.K., Mizuno M., Yoshimura K., Maoka T., Kanazawa K. The distribution and accumulation of fucoxanthin and its metabolites after oral administration in mice. Nevertheless, RHE does not present the dermal component, which is essential for many epidermal characteristics and proper skin functionalities, including the improvement of barrier function [19]. The experimental data was analyzed statistically by analysis of variance (ANOVA), a parametric test, followed by Tukeys test. for permeation into the cell. These carotenoids also have a maximum absorption peak around 448 nm, as is the case for chlorophylls (b), (c), and (a), which are the main chlorophyll pigments in algae [14]. Pennacchi P.C., de Almeida M.E., Gomes O.L., Faio-Flores F., de Arajo Crepaldi M.C., Dos Santos M.F., de Moraes Barros S.B., Maria-Engler S.S. Glycated Reconstructed Human Skin as a Platform to Study the Pathogenesis of Skin Aging. Licensee MDPI, Basel, Switzerland. [35], different structures of chlorophyll c are responsible for its intense absorption in the region of 530 nm; also, a peak in the 680 nm region (Figure 1a) could be related to chlorophyll a. Liebsch M., Spielmann H., Pape W., Krul C., Deguercy A., Eskes C. UV-induced effects. Furthermore, treatment with fucoxanthin at 100 g/mL induced the maximal reduction of ROS production, which was statistically different from the other studied concentrations (0.1, 1, and 10 g/mL) (p < 0.001). The final sunscreen formulation was yellow-colored, but it was not able to stain the skin. For the D. anceps extract and Fr15, we applied a cumulative dose of 27.5 J/cm2. It absorbs in the UV/VIS region, which is interesting for photoprotection and presents promising antioxidant properties that are observed in mice reported in the literature [12]. Accessibility The formazan production was measured at 570 nm. bladderwrack capsules We concentrated the organic D. anceps extract in a rotary evaporator under low pressure (Bchi R-300, Buchi Labotechnik, Flawil, Switzerland) and subjected it to a classical chromatographic column30 cm with stationary phase Silica Gel 4063 m/ASTM Macherey-Nagel (Merck, Darmstadt, Germany), with a polarity gradient using n-hexane, ethyl acetate (EtOAc), and methanol (MeOH) (JT Baker, Port of Spain, Trinidad Y Tobago) to fractionate the extract. In the efficacy assay on monolayers, fucoxanthin not only did not harm the HaCat keratinocytes, but also protected the cells from UVA-induced ROS formation in a concentration-dependent manner (maximal effect at 0.01%) (Figure 4). Freitas J.V., Gaspar L.R. 
Proceedings of International Scientific Conference, Alternatives to Animal Testing. * Fucoxanthin rich fraction, ** dose defined as enough to induce phototoxic responses, NE: Non-evaluated (sunscreen with absence of absorbance in the VIS range). We measured UVA radiation with the same radiometer mentioned before, while we kept the non-irradiated plates in a dark box. According to the OECD Test Guideline 432, a substance is predicted to be phototoxic if MPE is higher than 0.15 or the PIF is higher than 5. retention time eluted the substance of interest that was monitored by absorption ( max ~ 450 nm) (Supplementary Figure S1); chlorophyll, (a; c) together with fucoxanthin. D. anceps extract presented absorption in the region of interest for photoprotection (280400 nm), especially in the UVA and in the visible range (Figure 1a). That dose is recommended as being similar to 66 min. bgg Zhang H., Tang Y., Zhang Y., Zhang S., Qu J., Wang X., Kong R., Han C., Liu Z. Fucoxanthin: A Promising Medicinal and Nutritional Ingredient. After Hawaii, Florida and Key West followed this trend. Riccioni G., DOrazio N., Franceschelli S., Speranza L. Marine carotenoids and cardiovascular risk markers. 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. before exposure to a UVA dose of 27.5 J/cm2. Optimizing the spectral absorption profile of sunscreens. Besides the evaluation of fucoxanthin photosafety, we evaluated the photoprotective potential of this molecule by its photostability under different irradiation exposure and when used in a sunscreen formulation. Therefore, the combined phototoxicity assays (monolayer and RHS) suggest that fucoxanthin would not be phototoxic to the human skin at 0.5% due to the reduced bioavailability through the stratum corneum and in the stratified epidermis. We used the area under the curve (AUC), which is the integral of the absorption spectrum of the samples in the UVB (280320 nm), UVA (320400 nm), and VIS (400700 nm) ranges using the integration function of the MicroCal OriginPro Software (8 SRO, OriginLab Corporation, Northampton, MA, USA) to calculate the photostability [24]. However, this effect was not observed for fucoxanthin 0.1 g/mL, a dose that provided a reduction of 12%, which was not considered statistically significant when compared to the control (p > 0.05). The following are available online at https://www.mdpi.com/2076-3921/9/4/328/s1, Figure S1: Chromatogram of the elution semi-preparative scalecarotenoid isolation, Figure S2: Chromatogram of the semi-preparative scale subfraction, analyzed in 450 nmabsorption region of fucoxanthin at a higher intensity (Au 450), Retention Time (RT) = 5.75 min black line, 650 nmregion of the bands absorption of chlorophyll a lower intensity. Conceptualization, R.S.N.T., L.R.G., H.M.D. On the other hand, the sunscreen containing fucoxanthin 0.5% (w/v) induced a significant reduction of ROS generation after UVA irradiation when compared to the untreated control irradiated (+UV) and when compared to the sunscreen without fucoxanthin (Figure 5 and Figure 6a,d). The treatment with fucoxanthin at 1, 10, and 100 g/mL induced a significant reduction of ROS production of about 17%, 15%, and 65%, respectively, when compared to the untreated irradiated control (p < 0.001) (Figure 4). Reconstructed human skin after UVA irradiation (10 J/cm2) using a fluorescent probe DCFH2-DA. Cambon M., Issachar N., Castelli D., Robert C. An in vivo method to assess the photostability of UV filters in a sunscreen. Concerning ultraviolet (UV) damage, at the beginning of this century, not many compounds were available for the protection against UVA (320400 nm) radiation and, in response to the growing concern regarding the additional damage that is caused by this radiation, various UVA filters are now available for formulations worldwide. n = 1 or 2 independent experiments, -: values not determined in the studied concentration range. Thus, the objective of the present study was to assess the photoprotective and phototoxicity potential of fucoxanthin based on its UV/VIS absorption, photostability, phototoxicity in 3T3 mouse fibroblast culture vs. full-thickness RHS, and its ability to inhibit reactive oxygen species formation that is induced by UVA on HaCaT keratinocytes. The complete assignments of the 1H and 13C NMR spectra of fucoxanthin revealed signals that were assignable to polyene containing acetyl, conjugated ketone, olefinic methyl, two quaternary germinal oxygen methyls, two quaternaries geminal dimethyl, and allene groups. However, -Carotene is able to protect the collagen structure from infrared light in the skin [53] and it is used in cosmetic formulations for aged skin and actinic keratosis [51], which, for instance, could also suggest some skin benefits from the topical use of fucoxanthin when tested in vivo, due to the similarities of their molecular structures. Additionally, we evaluated the maximal antioxidant potential of this molecule onto immortalized human keratinocytes (HaCat) cells, determining the ideal concentration range that is to be used for this effect. This research was funded by call N 64/2013, MCTI/CNPq/FNDCT-Ao Transversal-Programa Antrtico Brasileiro-PROANTAR, from the Brazilian Government, R.S.N.T. Based on the historical data that was produced in our laboratory, we defined this dose as a dose high enough to elicit a phototoxic response in positive controls and as a dose that did not produce interference higher than 20% in cell viability, as recommended by the Organisation for Economic Co-operation and Development (OECD) [26]. 8600 Rockville Pike The material was filtered and extracted two more times while using ultrasound equipment for 15 minutes in the third procedure. On the other hand, the antioxidant quercetin (10 g/mL) induced a reduction of ROS generation of around 46% after UVA irradiation (Figure 4). DOrazio N., Gemello E., Gammone M.A., de Girolamo M., Ficoneri C., Riccioni G. Fucoxantin: A treasure from the sea. However, the sunscreen plus fucoxanthin showed acceptable photodegradation (Figure 2d). This suggests that fucoxanthin can also protect viable epidermis against UVA-induced ROS production, which is in agreement with the fucoxanthin protection observed in HaCat keratinocytes. They were kept submerged for 24 h. Next, the culture was maintained at the air-liquid interface for 12 days to allow for keratinocytes differentiation [27]. In addition, it showed a significant inhibition of ROS formation at 0.01% (p < 0.001), in HaCat, and in a sunscreen at 0.5% (w/v) (p < 0.001), in RHS. kelp seaweeds Furthermore, we also observed a significant reduction in the ROS production in RHS (complex 3D tissue) treated and irradiated with sunscreen plus fucoxanthin that we did not observe in the RHS that was treated with sunscreen alone (Figure 5 and Figure 6). fucoidan Alternative in vitro methods validated for preclinical trials are being used to predict the safety and efficacy of unknown natural compounds instead of using animal models to evaluate the potential risk of a test chemical. and L.R.G. To isolate the carotenoid, we employed high-performance liquid chromatography (HPLC), analytical, and semi-preparative analyses while using two different types of equipment with three different columns. We are thankful to Ribeiro Preto State Hospital and to the Maria de Ftima Galli Sorita, Ana Laura Ferreira Marsico Dalto, and Alexandra Longo Camperoni for the partnership providing the foreskin. Damiani E., Baschong W., Greci L. UV-Filter combinations under UV-A exposure: Concomitant quantification of over-all spectral stability and molecular integrity. For the identification of fucoxanthin, the highest colored mass fractions were subjected to Electrospray Ionization Mass Spectrometry (ESI-MS) techniques (amaZon SL, Bruker, Bil-lerica, MA, USA). We expressed the results as a percentage of the area of irradiated samples related to the area of non-irradiated samples. However, its topical properties and safety for the human skin are still unknown. lymphoma bcl fucoidan photoprotective abbreviations sod Federal government websites often end in .gov or .mil. When a positive result is obtained, i.e. kelp composition In the previous study of our group, commercial fucoxanthin at the same concentration, presented an absence of irritation, absence of morphological changes in the histology, and no significant dysregulation on homeostasis, metabolism, and in the inflammatory genes studied after following the OECD protocol for skin irritation [49]. carnitine fucoxanthin 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. We applied a cumulative dose of 6 J/cm2 to determine the photostability of fucoxanthin in isopropanol (the dose recommended for phototoxicity studies and similar to 14 min. The positive control norfloxacin (100 g/mL) induced an increase of ROS production of around 25% in comparison to the untreated irradiated control (Figure 4). government site. sampler week try sample RHS viability was measured by the end of the experiment of phototoxicity. It has been reported that this carotenoid shows intense antioxidant activity, as well as anti-inflammatory, anti-obesity, anti-diabetic, anti-tumor, antihypertensive, and anticancer properties [13,14]. The results were normalized to area/pixels and expressed in a percentage of fluorescence intensity in comparison to the irradiated and non-irradiated untreated controls. We exposed other constructs in every batch to fucoxanthin, but not to MTT, to evaluate the ability of fucoxanthin to stain the constructs under test conditions. wakame The images were quantified using Image J software [33,34]. fucoxanthin induce fucus inhibit duodenal carcinoma substances inhibits induzieren Diffey B.L. Our study corroborates those reported before [48], which employed the HaCat cell line and hairless mice to study the UVB protective effects of commercial all-trans-fucoxanthin (Sigma). The reduction of mitochondrial dehydrogenase activity was assessed via the decreased formazan production following incubation with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, Cotia, Brazil). 432: In vitro 3T3 NRU Phototoxicity Test, OECD Guidelines for the Testing of Chemicals. ; investigation, R.S.N.T., C.M.K., K.d.C.P. FOIA In this study, the sunscreen formulation that contains a combination of two UV filters considered to be photo unstable (avobenzone and ethylhexyl methoxycinnamate) was studied in order to assess the fucoxanthin capabilities of alteration in the photostability of this photo unstable combination. Liebsch M., Traue D., Barrabas C., Spielmann H., Gerberick G.F., Cruse L., Diembeck W., Pfannenbecker U., Spieker J., Holzhutter H.-G., et al. saccharina ingredient loss fat natural fucoxanthin japonica extract powder bulk of exposure to sunlight at midday (6.94 mW/cm2) on a typical September sunny day in the Ribeiro Preto, Brazillatitude 211039 south and longitude 474837 west [4,23]. 
Tavares R.S.N., Maria-Engler S.S., Colepicolo P., Debonsi H.M., Schfer-Korting M., Marx U., Gaspar L.R., Zoschke C. Skin Irritation Testing beyond Tissue Viability: Fucoxanthin Effects on Inflammation, Homeostasis, and Metabolism. This movement has stimulated the urgent research on alternatives and possibly eco-friendly photoprotective compounds [10]. Photostability evaluation of five UV-filters, trans-resveratrol and beta-carotene in sunscreens.