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- An Insight into Sargassum muticum Cytoprotective Mechanisms against Oxidative Stress on a Human Cell In Vitro ModelPublication . Pinteus, Susete; Lemos, Marco; Silva, Joana; Alves, Celso; Neugebauer, Agnieszka; Freitas, Rafaela; Duarte, Adriana; Pedrosa, RuiSargassum muticum is a brown seaweed with strong potential to be used as a functional food ingredient, mainly due to its antioxidant properties. It is widely used in traditional oriental medicine for the treatment of numerous diseases. Nevertheless, few studies have been conducted to add scientific evidence on its effects as well as on the mechanisms of action involved. In this work, the human cell line MCF-7 was used as an in vitro cellular model to evaluate the capability of Sargassum muticum enriched fractions to protect cells on an oxidative stress condition. The concentration of the bioactive compounds was obtained by vacuum liquid chromatography applied on methanol (M) and 1:1 methanol:dichloromethane (MD) crude extracts, resulting in seven enriched fractions from the M extraction (MF2-MF8), and eight fractions from the MD extraction (MDF1-MDF8). All fractions were tested for cytotoxic properties on MCF-7 cells and the nontoxic ones were tested for their capacity to blunt the damaging effects of hydrogen peroxide-induced oxidative stress. The nontoxic effects were also confirmed in 3T3 fibroblast cells as a nontumor cell line. The antioxidant potential of each fraction, as well as changes in the cell's real-time hydrogen peroxide production, in the mitochondrial membrane potential, and in Caspase-9 activity were evaluated. The results suggest that the protective effects evidenced by S. muticum can be related with the inhibition of hydrogen peroxide production and the inhibition of Caspase-9 activity.
- Antioxidant and neuroprotective potential of the brown seaweed Bifurcaria bifurcata in an in vitro Parkinson’s Disease ModelPublication . Silva, Joana; Alves, Celso; Freitas, Rafaela; Martins, Alice; Pinteus, Susete; Ribeiro, Joana; Gaspar, Helena; Alfonso, Amparo; Pedrosa, RuiBifurcaria bifurcata is a marine brown seaweed mainly found on the Atlantic coast. Herein, we report the antioxidant and neuroprotective activities of seven fractions (F1–F7) obtained by normal phase chromatography from the B. bifurcata dichloromethane extract, as well as of its two major isolated diterpenes. Total phenolic content of fractions was determined by the Folin–Ciocalteu method, while antioxidant activity was evaluated by the DPPH, ORAC, and FRAP assays. Neuroprotective effects were evaluated in a neurotoxic model induced by 6-hydroxydopamine (6-OHDA) in a human neuroblastoma cell line (SH-SY5Y), while the mechanisms associated to neuroprotection were investigated by the determination of mitochondrial membrane potential, H2O2 production, Caspase-3 activity, and by observation of DNA fragmentation. Fractions F4 and F5 exhibited the best neuroprotective and antioxidant activities, respectively. F4 fraction prevented changes in mitochondrial potential, and induced a reduction of H2O2 levels production and an increase in cell viability, suggesting that it may contain multi-target compounds acting on different pathways. Hence, this fraction was subjected to purification steps, affording the known diterpenes eleganolone and eleganonal. Both compounds exhibited antioxidant potential, being interesting candidates for further neuroprotective studies.
- Medusa polyps adherence inhibition: A novel experimental model for antifouling assaysPublication . Pinteus, Susete; Lemos, Marco F.L.; Freitas, Rafaela; Duarte, Inês M.; Alves, Celso; Silva, Joana; Marques, Sónia C.; Pedrosa, RuiAlthough in the last decades significant advances have been made to improve antifouling formulations, the main current options continue to be highly toxic tomarine environment, leading to an urgent need for new safer alternatives. For anti-adherence studies, barnacles and mussels are commonly the first choice for experimental purposes. However, the use of these organisms involves a series of laborious and time-consuming stages. In the present work, a new approach for testing antifouling formulations was developed under known formulations and novel proposed options. Due to their high resilience, ability of surviving in hostile environments and high abundance in different ecosystems, medusa polyps present themselves as prospect candidates for antifouling protocols. Thus, a complete protocol to test antifouling formulations using polyps is presented, while the antifouling properties of two invasive seaweeds, Asparagopsis armata and Sargassum muticum, were evaluated within this new test model framework. The use of medusa polyps as model to test antifouling substances revealed to be a reliable alternative to the conventional organisms, presenting several advantages since the protocol is less laborious, less time-consuming and reproductive. The results also show that the seaweeds A. armata and S. muticum produce compounds with anti-adherence properties being therefore potential candidates for the development of new greener antifouling formulations.
- Dermocosmetic potential of the macroalgae Fucus spiralisPublication . Freitas, Rafaela Patrícia Freire; Pedrosa, Rui Filipe Pinto; Alves, Celso Miguel da Maia; Martins, Alice Isabel MendesA pele é o maior órgão do corpo humano estando sujeita a fatores intrínsecos e extrínsecos que influenciam negativamente a sua homeostasia, particularmente a exposição à radiação ultravioleta. A contínua exposição da pele a agentes nocivos pode despoletar o stress oxidativo que está associado não só ao envelhecimento cutâneo, mas também ao desenvolvimento de patologias dérmicas mais severas, sendo de extrema importância o seu cuidado e proteção. Com a progressiva consciencialização dos consumidores sobre a importância da pele e o impacto de alguns ingredientes sintéticos na saúde, existe uma procura crescente por formulações de origem natural. Neste contexto, o objetivo da presente dissertação consistiu na avaliação do potencial cosmético de onze frações obtidas da alga Fucus spiralis. As frações foram obtidas através de três metodologias de extração distintas, usando diferentes solventes permitidos na área cosmética, tempos e temperaturas de extração. O potencial mocosmético das frações foi determinado através da avaliação das suas propriedades antioxidantes (quantificação total de polifenóis, capacidade de redução do radical 2,2-difenil-1-picril-hidrazilo (DPPH), capacidade de redução do ião ferro (III) (FRAP), e capacidade de absorção dos radicais de oxigénio (ORAC)), da capacidade de inibir enzimas envolvidas no envelhecimento da pele (colagenase, elastase e hialuronidase), da capacidade de influenciar o crescimento de microrganismos associados à microbiota da pele (Staphylococcus epidermis, Cutibacterium acnes e Malassezia furfur) e da capacidade citoprotetora e fotoprotetora numa condição de stress oxidativo induzida por peróxido de hidrogénio (H2O2) e por UVB nas células HaCaT, respetivamente. A citotoxicidade das frações foi avaliada em fibroblastos (3T3) e queratinócitos (HaCaT). Todas as frações foram caracterizadas por espectroscopia UV-Vis. A fração biologicamente mais ativa foi analisada por HPLC-DAD e LC-MS, numa tentativa de identificar os compostos responsáveis pelas bioatividades demonstradas. Todas as frações demonstraram ter propriedades antioxidantes, no entanto, as frações F3, F5 e F10 destacaram-se pela sua elevada capacidade de estabilizar radicais livres nos ensaios efetuados. No ensaio de DPPH, a fração F10 destacou-se com a maior capacidade de redução deste radical (EC50 de 38,55 μg/ mL), seguida das frações F3 (157,60 μg/ mL) e F5 (244,70 μg/ mL). Relativamente aos ensaios FRAP e ORAC, F10 demonstrou a maior capacidade antioxidante exibindo um valor de 2.378,20 ± 14,40 Eq FeSO4/ g extrato e 16.322,80 ± 576,20 μmol Eq Trolox/ g extrato, respetivamente. Já as frações F3 e F5 apresentaram valores mais baixos quando comparados com a fração F10 no ensaio FRAP (F3 - 1821,76 ± 37,82 μM Eq FeSO4/ g extrato e F5 - 1180,28 ± 117,00 μM Eq FeSO4/ g extrato) e no ensaio ORAC (F3 - 3557,94 ± 466,60 μmol Eq Trolox/ g extrato e F5 - 4273,78 ± 181,80 μmol Eq Trolox/ g extrato). Estas frações foram as que exibiram maior conteúdo em polifenóis (F3 – 362,06 ± 12,10 mg Eq P/ g extrato; F5 – 309,47 ± 6,30 mg Eq P/ g extrato e F10 – 1677,90 ± 24,13 mg Eq P/ g extrato). No efeito antienzimático, a fração F10 demonstrou a maior capacidade inibitória para as enzimas colagenase (IC50 = 0,04 μg/ mL) e elastase (IC50 = 3,01 μg/ mL), não tendo um efeito notório contra a hialuronidase. Por sua vez a fração F4 exibiu capacidade para inibir a atividade de todas as enzimas, nomeadamente a colagenase (IC50 = 40,40 μg/ mL), a elastase (IC50 = 67,78 μg/ mL) e a hialuronidase (IC50 = 61,10 μg/ mL). Na citotoxicidade, a fração F10 apresenta efeitos citotóxicos nas células 3T3 à concentração de 1000 μg/ mL, no entanto, quando testada a 600 μg/ mL não demonstrou qualquer efeito na viabilidade celular dos fibroblastos e nas células HaCaT não exibiu qualquer citotoxicidade. Quanto às restantes frações, apresentaram baixa citotoxicidade (viabilidade acima dos 65%) nas células 3T3 e nenhum efeito citotóxico nas células HaCaT. No que diz respeito ao efeito fotoprotetor, as frações F7 e F10 inibiram ativamente a produção de espécies reativas de oxigénio quando as células HaCaT foram tratadas com H2O2 (F7 - IC50 = 99,50 μg/ mL e F10 - IC50 = 41,30 μg/ mL) e quando expostas a UVB (F7 - IC50 = 100,30 μg/ mL e F10 - IC50 = 31,30 μg/ mL). Relativamente aos efeitos das frações no crescimento dos microrganismos referidos, a fração F2 inibiu o crescimento de C. acnes (IC50 de 905,60 μg/ mL) e F7 inibiu o crescimento de M. furfur (IC50 de 1018,00 μg/ mL). No entanto, a fração F10 estimulou o crescimento de S. epidermidis. Por fim, a caraterização química por LC-MS da fração mais promissora, F10, revelou a presença de vários compostos fenólicos do grupo dos florotaninos. Em suma, em face dos resultados obtidos, e numa perspetiva geral, as diferentes frações de Fucus spiralis demonstram potencial cosmético, destacando-se a fração F10 pelas bioatividades evidenciadas. Esta fração poderá vir a ser explorada para aplicações cosméticas, através da sua inclusão em formulações com atividade antioxidante, antienzimática e fotoprotetora, aliada à capacidade de manutenção da microbiota da pele.
- Highlighting the biological potential of the brown seaweed Fucus spiralis for skin applicationsPublication . Freitas, Rafaela; Martins, Alice; Silva, Joana; Alves, Celso; Pinteus, Susete; Alves, Joana; Teodoro, Fernando; Ribeiro, Helena Margarida; Gonçalves, Lídia; Petrovski, Zeljko; Branco, Luís; Pedrosa, RuiSkin aging is a biological process influenced by intrinsic and extrinsic factors. The last ones, mainly exposure to UV radiation, increases reactive oxygen species (ROS) production leading to a loss of extracellular matrix, also enhanced by enzymatic degradation of matrix supporting molecules. Thus, and with the growing demand for eco-friendly skin products, natural compounds extracted from brown seaweeds revealed to be good candidates due to their broad range of bioactivities, especially as antioxidants. The aim of this study was to assess the dermo-cosmetic potential of di erent fractions obtained from the brown seaweed Fucus spiralis. For this purpose, in vitro antioxidant (Total Phenolic Content (TPC), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, Ferric Reducing Antioxidant Power (FRAP), Oxygen Radical Absorbance Capacity (ORAC)), anti-enzymatic (collagenase, elastase and hyaluronidase), antimicrobial, anti-inflammatory (NO production) and photoprotective (ROS production) capacities were evaluated. Although nearly all fractions evidenced antioxidant e ects, fraction F10 demonstrated the highest antioxidant ability (EC50 of 38.5 g/mL, DPPH assay), and exhibited a strong e ect as an inhibitor of collagenase (0.037 g/mL) and elastase (3.0 g/mL). Moreover, this fraction was also the most potent on reducing ROS production promoted by H2O2 (IC50 of 41.3 g/mL) and by UVB (IC50 of 31.3 g/mL). These bioactivities can be attributed to its high content of phlorotannins, as evaluated by LC-MS analysis, reinforcing the potential of F. spiralis for further dermatological applications.
- Bromoditerpenes from the red seaweed Sphaerococcus coronopifolius as potential cytotoxic agents and proteasome inhibitors and related mechanisms of actionPublication . Alves, Celso; Silva, Joana; Pintéus, Susete; Guedes, Romina A.; Guedes, Rita C.; Alvariño, Rebeca; Freitas, Rafaela; Goettert, Márcia I.; Gaspar, Helena; Alfonso, Amparo; Alpoím, Maria C.; Botana, Luis M.; Pedrosa, RuiSeaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 μM. 12Rhydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.