for permeation into the cell. 
Fraction F15a containing fucoxanthin was considered less cytotoxic than the crude extract (IC50 UV 26.12 and 25.22 g/mL) but was deemed to be phototoxic (MPE: 0.343 and 0.478). Fourtanier A., Moyal D., Seite S. UVA filters in sun-protection products: Regulatory and biological aspects. Rautenberger R.W.C., Bischof K. Acclimation to UV radiation and antioxidative defence in the endemic Antarctic brown macroalga. The ethics committees of Human Research Ethics Committee of Faculty of Pharmaceutical Sciences of Ribeiro Preto, So Paulo, Brazil approved the experimental procedures using primary human fibroblasts and keratinocytes from donated foreskins with informed consent from legal representatives and ethics approval conforming to the principles of the Declaration of Helsinki (CAAE n: 55438216.0.0000.5403). The formazan production was measured at 570 nm. 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. UV light, beta-carotene and human skinBeneficial and potentially harmful effects. 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. 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. Calculation considering the Area under the Curve (AUC) between samples irradiated or not in triplicates. 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. 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]. (2015) study. and transmitted securely. 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]. 
Tovar-Snchez A., Snchez-Quiles D., Basterretxea G., Bened J.L., Chisvert A., Salvador A., Moreno-Garrido I., Blasco J. Sunscreen products as emerging pollutants to coastal waters. 
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. Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands. In our study, we replaced RHE with reconstructed human skin (RHS) to confirm the ability of the latter to detect the phototoxicity potential. However, there is a lack of scientific information regarding its properties when applied onto human skin. 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. 
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]. [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. A test substance with an MPE > 0.1 and < 0.15 (PIF > 1 and <5) is predicted to be probably phototoxic [26]. Following this trend, natural and biocompatible UV filters have led to research on carotenoids that could be interesting in the development of new UV filters or UV boosters to increase the protection or performance of sunscreens [11]. under stirring, in a thermal blanket with controlled temperature (not exceeding 30 C). 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. and L.R.G. 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). 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. 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]. They identified the macroalgae according to the standard taxonomic methodology in the Phycology Session and deposited exsiccates in the Phycological Herbarium of the Botanical Institute of So Paulo (SP), Brazil. The material was frozen until the time for transportation to our laboratories in Brazil, at the Laboratory of Organic Chemistry of the Marine Environment-Support Center for Research in Natural and Synthetic Products, School of Pharmaceutical Sciences of Ribeiro Preto (LQOAM-NPPNS, FCFRP-USP). The authors declare no conflict of interest. It is important to mention that skin models are more permeable than human skin [46], which means that they hardly produce false-negative results, which is very important when predicting the toxic potential of unknown substances. The dose of 4 J/cm2 used was defined by many authors, including Chignell and Sik [38], after no significant difference in viability was observed between control cells and cells exposed to this dose higher than 20%, while an increase in the DCF photochemical reactions (ROS formation) was observed [38]. Liebel and co-workers [3] showed that VIS light induced the generation of high levels of free radicals in humans and of proinflammatory cytokines in vitro. 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). However, the sunscreen plus fucoxanthin showed acceptable photodegradation (Figure 2d). The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop. Addressing the tiered strategy to assess acute phototoxicity according to ICH recommendations [18], 3T3 NRU-PT is the first step of the biological assays and it is considered to be a standalone test for negative results due to its high sensitivity (100%) for the identification of absence of phototoxic potential [45]. Scale bars = 100 m, n = 3; three independent experiments. However, since the sunscreen alone only provoked a reduction in the UVA absorption by 4%, the effect of fucoxanthin on UVA region was considered to be the difference of 12.5%. (d) Fx in a sunscreen formulation vs. sunscreen alone (dashed line) irradiated or not with 27.5 J/cm2; n = 3, three independent experiments. 
We measured UVA radiation with the same radiometer mentioned before. Gaspar L.R., Kawakami C.M., Benevenuto C.G. Test No. Photostability studies based on the electromagnetic spectrum of the samples irradiated (yellow line) or not (blue line). 
Cellular esterases hydrolyze the probe that is cell-permeable to the non-fluorescent DCFH derivative [39]. In addition, the viable epidermis is made of keratinocytes, which are less sensitive than fibroblasts to xenobiotics and UV radiation. Alternatives for Dermal Toxicity Testing. RT ~3 min, pink line, Figure S3: Chromatogram of the elution of the subfraction analytical scale, analyzed in (a) the 450 nmabsorption region of fucoxanthin at a higher intensity (3 Au), Retention time (RT) = 3.5 min; (b) 650 nmthe area of the chlorophyll absorption bands at a lower intensity (0.025 Au), RT ~6.5 min. The RHS were incubated with the DCFH2-DA probe in PBS (50 M) in the dark for 45 min. 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]. Learn more Zhang P., Omaye S.T. Marionnet C., Pierrard C., Golebiewski C., Bernerd F. Diversity of biological effects induced by longwave UVA rays (UVA1) in reconstructed skin. During the expedition of 2011 (January 4th, managed by the project PROANTAR (Programa Antrtico Brasileiro), the researchers collected 69.00 grams of D. anceps (wet) at the Punta Plaza locationAntarctic Continent (Admiralty Bay 620414.5621003.5 S and 582015582760 W). Booster is a term that is currently used in the cosmetic field to define substances that, in small amounts, are capable of increasing the effectiveness of some other products [11] (i.e., increasing the effects of UV filters protecting the skin from sunlight-induced ROS production). 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. Next, 1.15 g of the extract was submitted to fractionation in a classical column, which resulted in 40 fractions. The use of skin models is physiologically relevant in drug development, since it provides better prediction of human skin safety [16], besides ethical and economic concerns. 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. The beneficial use of topical fucoxanthin should be further investigated in vivo in the concentration range of 0.010.5% (w/v). 
Where * means significantly different from untreated control irradiated (+UV) (p < 0.001) and ** significantly different from fucoxanthin at 0.1, 1, and 10 g/mL (p < 0.001). (b) Fr15 fraction containing fucoxanthin in solution irradiated or not with 27.5 J/cm2. After the non-phototoxic response that was observed in the RHS, the protective effect of fucoxanthin was evaluated by the detection of intracellular ROS immediately after UVA radiation using a probe 2,7-DCFH2-DA in keratinocytes HaCat. Zeng J., Zhang Y., Ruan J., Yang Z., Wang C., Hong Z., Zuo Z. 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). Untreated (blue bar non-irradiated and yellow bars irradiated); sunscreen and sunscreen plus 0.5% fucoxanthin (Fx). Accessibility RHE and RHS, due to the presence of a stratum corneum, appear to be capable of detecting known human dermal phototoxicants. Where * means significantly different from untreated irradiated (+UV) and from sunscreen treated models (p < 0.001). Fernndez E., Fajar L., Rodrguez G., Ccera M., Moner V., Barbosa-Barros L., Kamma-Lorger C.S., de la Maza A., Lpez O. The high-resolution molecular mass spectrum (electrospray ionization, ESI) corresponding to the molecular weight of fucoxanthin, 658.90 g/mol, was identified based on the fragment pattern at m/z 659.4241 and 681.3878 corresponding to [M + H]+ and [M + Na]+ in the fractions F15 and F15a. In addition, it increased by 72% of the total sunscreen UV absorption spectra, presenting UV-booster properties. Fucoxanthin showed photoinstability when being evaluated in an organic solvent. We then evaluated fucoxanthin that was isolated in isopropanol solution using the same UVA dose used to induce phototoxicity to RHS (6 J/cm2), in order to compare both of the experiments. Consequently, under adequate test conditions, a negative result in a 3D skin model indicates that the acute photoirritation potential of the formulation can be regarded as low. Palmariapalmata, Cichorium intybus, and Medicago sativa extracts in cosmetic formulations: An integrated approach of in vitro toxicity and in vivo acceptability studies. Reduction of absorbance after irradiation with different doses of UVA light expressed in percentage. The peak with the 47 min. We expressed the results as a percentage of the area of irradiated samples related to the area of non-irradiated samples. Fucoxanthin only showed phototoxic potential (MPE: 0.920 and 0.915) and no cytotoxic potential (IC50 not determined in the range of 6.4 to 100 g/mL) to fibroblasts 3T3 (Table 3). 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 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. Intracellular Reactive Oxygen Species generation in HaCat after UVA irradiation (4 J/cm2) using a fluorescent probe DCFH2-DA; untreated irradiated (+UV) and non-irradiated (UV); norfloxacinNor (control); quercetinQuer (control); fucoxanthinFx. 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]. Liebsch M., Spielmann H., Pape W., Krul C., Deguercy A., Eskes C. UV-induced effects. Twenty hours after application of the test substances, the skin models were rinsed with phosphate-buffered saline (PBS), dried with a sterile swab, and then transferred to fresh wells with the medium. 
(c) Fucoxanthin (Fx) in solution irradiated or not with 6 J/cm2. 
Chlorophyll type a is predominant in algae due to its central role in the conversion of photochemical energy, while the chlorophyll c efficiently participates in photosynthesis as an accessory pigment (similar to the role of chlorophyll b in plants or green algae) [14]. 
); rb.psu.prfcf@anasoh (H.M.D. Photodegradation and photosensitization of mycosporine-like amino acids. The .gov means its official. The second analytical column was Polar RP column (100 mm 3 mm, 5 m) that was used to define a method to purify the carotenoid (from chlorophyll a as a contaminant). Next, we incubated the plates with the probe 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA) (10 M) in the dark for 30 min. The material was freshly thawed and washed with distilled water under a vacuum filter. After irradiation, the dried film was resuspended in 1 mL of solvent, and the absorption spectrum of the solutions in the 280 to 700 nm range was analyzed. In 2018, the governor of Hawaii banned the in-state sale of sunscreens containing either oxybenzone or octinoxate, suspected to harm coral reefs [10]. Each beaker subjected to irradiation had a negative control that was sheltered from light. The results demonstrated that UVA radiation-induced ROS generation in the keratinocyte cell line (100%) when compared to the untreated control non-irradiated. Biesalski H.K., Obermueller-Jevic U.C. However, the number of UVA filters allowed in the USA is quite limited [1,2]. 
Fraction F15a containing fucoxanthin was considered less cytotoxic than the crude extract (IC50 UV 26.12 and 25.22 g/mL) but was deemed to be phototoxic (MPE: 0.343 and 0.478). Fourtanier A., Moyal D., Seite S. UVA filters in sun-protection products: Regulatory and biological aspects. Rautenberger R.W.C., Bischof K. Acclimation to UV radiation and antioxidative defence in the endemic Antarctic brown macroalga. The ethics committees of Human Research Ethics Committee of Faculty of Pharmaceutical Sciences of Ribeiro Preto, So Paulo, Brazil approved the experimental procedures using primary human fibroblasts and keratinocytes from donated foreskins with informed consent from legal representatives and ethics approval conforming to the principles of the Declaration of Helsinki (CAAE n: 55438216.0.0000.5403). The formazan production was measured at 570 nm. 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. UV light, beta-carotene and human skinBeneficial and potentially harmful effects. 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. 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. Calculation considering the Area under the Curve (AUC) between samples irradiated or not in triplicates. 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. 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]. (2015) study. and transmitted securely. 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]. Tovar-Snchez A., Snchez-Quiles D., Basterretxea G., Bened J.L., Chisvert A., Salvador A., Moreno-Garrido I., Blasco J. Sunscreen products as emerging pollutants to coastal waters. 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. Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands. In our study, we replaced RHE with reconstructed human skin (RHS) to confirm the ability of the latter to detect the phototoxicity potential. However, there is a lack of scientific information regarding its properties when applied onto human skin. 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. 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]. [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. A test substance with an MPE > 0.1 and < 0.15 (PIF > 1 and <5) is predicted to be probably phototoxic [26]. Following this trend, natural and biocompatible UV filters have led to research on carotenoids that could be interesting in the development of new UV filters or UV boosters to increase the protection or performance of sunscreens [11]. under stirring, in a thermal blanket with controlled temperature (not exceeding 30 C). 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. and L.R.G. 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). 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. 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]. They identified the macroalgae according to the standard taxonomic methodology in the Phycology Session and deposited exsiccates in the Phycological Herbarium of the Botanical Institute of So Paulo (SP), Brazil. The material was frozen until the time for transportation to our laboratories in Brazil, at the Laboratory of Organic Chemistry of the Marine Environment-Support Center for Research in Natural and Synthetic Products, School of Pharmaceutical Sciences of Ribeiro Preto (LQOAM-NPPNS, FCFRP-USP). The authors declare no conflict of interest. It is important to mention that skin models are more permeable than human skin [46], which means that they hardly produce false-negative results, which is very important when predicting the toxic potential of unknown substances. The dose of 4 J/cm2 used was defined by many authors, including Chignell and Sik [38], after no significant difference in viability was observed between control cells and cells exposed to this dose higher than 20%, while an increase in the DCF photochemical reactions (ROS formation) was observed [38]. Liebel and co-workers [3] showed that VIS light induced the generation of high levels of free radicals in humans and of proinflammatory cytokines in vitro. 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). However, the sunscreen plus fucoxanthin showed acceptable photodegradation (Figure 2d). The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop. Addressing the tiered strategy to assess acute phototoxicity according to ICH recommendations [18], 3T3 NRU-PT is the first step of the biological assays and it is considered to be a standalone test for negative results due to its high sensitivity (100%) for the identification of absence of phototoxic potential [45]. Scale bars = 100 m, n = 3; three independent experiments. However, since the sunscreen alone only provoked a reduction in the UVA absorption by 4%, the effect of fucoxanthin on UVA region was considered to be the difference of 12.5%. (d) Fx in a sunscreen formulation vs. sunscreen alone (dashed line) irradiated or not with 27.5 J/cm2; n = 3, three independent experiments. We measured UVA radiation with the same radiometer mentioned before. Gaspar L.R., Kawakami C.M., Benevenuto C.G. Test No. Photostability studies based on the electromagnetic spectrum of the samples irradiated (yellow line) or not (blue line). Cellular esterases hydrolyze the probe that is cell-permeable to the non-fluorescent DCFH derivative [39]. In addition, the viable epidermis is made of keratinocytes, which are less sensitive than fibroblasts to xenobiotics and UV radiation. Alternatives for Dermal Toxicity Testing. RT ~3 min, pink line, Figure S3: Chromatogram of the elution of the subfraction analytical scale, analyzed in (a) the 450 nmabsorption region of fucoxanthin at a higher intensity (3 Au), Retention time (RT) = 3.5 min; (b) 650 nmthe area of the chlorophyll absorption bands at a lower intensity (0.025 Au), RT ~6.5 min. The RHS were incubated with the DCFH2-DA probe in PBS (50 M) in the dark for 45 min. 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]. Learn more Zhang P., Omaye S.T. Marionnet C., Pierrard C., Golebiewski C., Bernerd F. Diversity of biological effects induced by longwave UVA rays (UVA1) in reconstructed skin. During the expedition of 2011 (January 4th, managed by the project PROANTAR (Programa Antrtico Brasileiro), the researchers collected 69.00 grams of D. anceps (wet) at the Punta Plaza locationAntarctic Continent (Admiralty Bay 620414.5621003.5 S and 582015582760 W). Booster is a term that is currently used in the cosmetic field to define substances that, in small amounts, are capable of increasing the effectiveness of some other products [11] (i.e., increasing the effects of UV filters protecting the skin from sunlight-induced ROS production). 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. Next, 1.15 g of the extract was submitted to fractionation in a classical column, which resulted in 40 fractions. The use of skin models is physiologically relevant in drug development, since it provides better prediction of human skin safety [16], besides ethical and economic concerns. 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. The beneficial use of topical fucoxanthin should be further investigated in vivo in the concentration range of 0.010.5% (w/v). Where * means significantly different from untreated control irradiated (+UV) (p < 0.001) and ** significantly different from fucoxanthin at 0.1, 1, and 10 g/mL (p < 0.001). (b) Fr15 fraction containing fucoxanthin in solution irradiated or not with 27.5 J/cm2. After the non-phototoxic response that was observed in the RHS, the protective effect of fucoxanthin was evaluated by the detection of intracellular ROS immediately after UVA radiation using a probe 2,7-DCFH2-DA in keratinocytes HaCat. Zeng J., Zhang Y., Ruan J., Yang Z., Wang C., Hong Z., Zuo Z. 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). Untreated (blue bar non-irradiated and yellow bars irradiated); sunscreen and sunscreen plus 0.5% fucoxanthin (Fx). Accessibility RHE and RHS, due to the presence of a stratum corneum, appear to be capable of detecting known human dermal phototoxicants. Where * means significantly different from untreated irradiated (+UV) and from sunscreen treated models (p < 0.001). Fernndez E., Fajar L., Rodrguez G., Ccera M., Moner V., Barbosa-Barros L., Kamma-Lorger C.S., de la Maza A., Lpez O. The high-resolution molecular mass spectrum (electrospray ionization, ESI) corresponding to the molecular weight of fucoxanthin, 658.90 g/mol, was identified based on the fragment pattern at m/z 659.4241 and 681.3878 corresponding to [M + H]+ and [M + Na]+ in the fractions F15 and F15a. In addition, it increased by 72% of the total sunscreen UV absorption spectra, presenting UV-booster properties. Fucoxanthin showed photoinstability when being evaluated in an organic solvent. We then evaluated fucoxanthin that was isolated in isopropanol solution using the same UVA dose used to induce phototoxicity to RHS (6 J/cm2), in order to compare both of the experiments. Consequently, under adequate test conditions, a negative result in a 3D skin model indicates that the acute photoirritation potential of the formulation can be regarded as low. Palmariapalmata, Cichorium intybus, and Medicago sativa extracts in cosmetic formulations: An integrated approach of in vitro toxicity and in vivo acceptability studies. Reduction of absorbance after irradiation with different doses of UVA light expressed in percentage. The peak with the 47 min. We expressed the results as a percentage of the area of irradiated samples related to the area of non-irradiated samples. Fucoxanthin only showed phototoxic potential (MPE: 0.920 and 0.915) and no cytotoxic potential (IC50 not determined in the range of 6.4 to 100 g/mL) to fibroblasts 3T3 (Table 3). 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 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. Intracellular Reactive Oxygen Species generation in HaCat after UVA irradiation (4 J/cm2) using a fluorescent probe DCFH2-DA; untreated irradiated (+UV) and non-irradiated (UV); norfloxacinNor (control); quercetinQuer (control); fucoxanthinFx. 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]. Liebsch M., Spielmann H., Pape W., Krul C., Deguercy A., Eskes C. UV-induced effects. Twenty hours after application of the test substances, the skin models were rinsed with phosphate-buffered saline (PBS), dried with a sterile swab, and then transferred to fresh wells with the medium. (c) Fucoxanthin (Fx) in solution irradiated or not with 6 J/cm2. Chlorophyll type a is predominant in algae due to its central role in the conversion of photochemical energy, while the chlorophyll c efficiently participates in photosynthesis as an accessory pigment (similar to the role of chlorophyll b in plants or green algae) [14]. ); rb.psu.prfcf@anasoh (H.M.D. Photodegradation and photosensitization of mycosporine-like amino acids. The .gov means its official. The second analytical column was Polar RP column (100 mm 3 mm, 5 m) that was used to define a method to purify the carotenoid (from chlorophyll a as a contaminant). Next, we incubated the plates with the probe 2,7-dichlorodihydrofluorescein diacetate (DCFH2-DA) (10 M) in the dark for 30 min. The material was freshly thawed and washed with distilled water under a vacuum filter. After irradiation, the dried film was resuspended in 1 mL of solvent, and the absorption spectrum of the solutions in the 280 to 700 nm range was analyzed. In 2018, the governor of Hawaii banned the in-state sale of sunscreens containing either oxybenzone or octinoxate, suspected to harm coral reefs [10]. Each beaker subjected to irradiation had a negative control that was sheltered from light. The results demonstrated that UVA radiation-induced ROS generation in the keratinocyte cell line (100%) when compared to the untreated control non-irradiated. Biesalski H.K., Obermueller-Jevic U.C. However, the number of UVA filters allowed in the USA is quite limited [1,2].