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- Mecanismos de neurotoxicidade induzidos pela Dopamina e pela 6–OH-dopamina num modelo celular humano: efeito da presença de extratos de algas com elevada capacidade antioxidantePublication . Silva, Joana Rita Martins da; Pedrosa, Rui Filipe PintoO aumento da esperança média de vida tem elevado a prevalência de doenças neurodegenerativas, como é o caso da doença de Parkinson. Nos últimos anos a procura de novas soluções terapêuticas, assim como a minimização dos efeitos dos tratamentos atualmente utilizados tem promovido a procura de novas soluções. Deste modo, o objetivo deste trabalho consistiu no estudo dos mecanismos moleculares de neurotoxicidade induzidos pela dopamina (DA) e 6-hidroxidopamina (6-OHDA) num modelo celular do neuroblastoma humano (SH-SY5Y), bem como na avaliação do potencial neuroprotetor de extratos de algas com elevada capacidade antioxidante. O efeito neurotóxico da DA e 6-OHDA, assim como o efeito protetor dos extratos das algas com maior atividade antioxidante (Sargassum muticum, Saccorhiza polyschides, Padina pavonica, Codium tomentosum, Ulva compressa) foi avaliado através da viabilidade celular das células SH-SY5Y utilizando o método de MTT. De modo a compreender os efeitos induzidos na viabilidade celular pela DA e 6-OHDA procedeu-se ao estudo da atividade da caspase-3, alterações do potencial mitocondrial e quantificação de H2O2. Os resultados demonstraram um claro efeito dependente da concentração da DA (30-3000μM) e 6-OHDA (10-1000μM) na viabilidade celular das células SH-SY5Y, bem como do tempo de exposição (6-48h). No que diz respeito a prevenção do efeito neurotóxico da DA (1000μM (56,41±5,05% de células viáveis); 24h) e 6-OHDA (100μM (66,76±3,24% de células viáveis);24h) pelos extratos das algas (1mg/mL; 24h) verificou-se que os extratos que apresentaram um efeito preventivo mais marcado pertencem as algas Sargassum muticum (82,37±6,41% de células viáveis e 115,8±8,53% de células viáveis, após tratamento com DA e 6-OHDA, respetivamente), Saccorhiza polyschides (89,26±8,62% de células viáveis e 106,51±4,26% de células viáveis, após tratamento com DA e 6-OHDA, respetivamente) e Codium tomentosum (81,28±3,68% de células viáveis e 103,17±7,25% de células viáveis, após tratamento com DA e 6-OHDA, respetivamente). A morte celular induzida pela DA e pela 6-OHDA foi acompanhada pelo aumento da atividade da caspase-3 quando comparado com o controlo (DA - 66,46±1,49fluorescência (u.a)/mg de proteína/minuto; 6-OHDA - 22,56±1,71fluorescência (u.a)/mg de proteína/minuto; controlo – 4,8 ±0,48fluorescência (u.a)/mg de proteína/minuto), pela presença de elevadas quantidades de peróxido de hidrogénio (H2O2) (363,81±28,58 % do controlo e 214,26 ± 8,46 % do controlo, após tratamento com DA e 6-OHDA, respetivamente) e pela despolarização da membrana mitocondrial (162,3±2,34 % do controlo e 144,7±2,87 % do controlo, após tratamento com DA e 6-OHDA, respetivamente). Por sua vez, durante o tratamento com extratos das algas (1mg/mL) na presença de DA e 6-OHDA verificou-se uma inibição da atividade da caspase-3 induzida pelas algas Sargassum muticum (2,53±2,49fluorescência (u.a)/mg de proteína/minuto e 4,52±1,36 fluorescência (u.a)/mg de proteína/minuto, após tratamento com DA e 6-OHDA, respetivamente), Saccorhiza polyschides (4,71±0,70fluorescência (u.a.)/mg de proteína/minuto e 2,73±1,11 fluorescência (u.a.)/mg de proteína/minuto, após tratamento com DA e 6-OHDA, respetivamente) e Codium tomentosum (17,05±1,72fluorescência (u.a.)/mg de proteína/minuto e 2,58±1,77fluorescência (u.a)/mg de proteína/minuto, após tratamento com DA e 6-OHDA, respetivamente). De igual modo verificou-se uma diminuição da produção de H2O2 pelas células SH-SY5Y na presença dos extratos das algas Sargassum muticum (132,58 ± 10,68% controlo), Saccorhiza polyschides (150,54 ± 23,54% controlo) e Codium tomentosum (54,074 ± 6,66% do controlo), quando expostas a 6-OHDA, contudo não se verificou o mesmo efeito na presença de DA. Relativamente ao potencial mitocondrial observou-se uma inibição da despolarização mitocondrial induzida pela DA e 6-OHDA nas células SH-SY5Y pela presença dos extratos das algas Sargassum muticum (135,7±2,97% controlo e 49,3±1,17% controlo, após tratamento com DA e 6-OHDA, respetivamente), Saccorhiza polyschides (126,7±5,46% controlo e 94,3±1,70% controlo, após tratamento com DA e 6-OHDA, respetivamente). Os resultados obtidos demonstraram o potencial citoprotetor dos extratos de algas sobre efeitos neurotóxicos induzidos pela DA e 6-OHDA no modelo celular SH-SY5Y. O efeito protetor é mediado pela diminuição da condição de stress oxidativo, com redução da produção de H2O2, diminuição da atividade da caspase-3 e prevenção da alteração do potencial mitocondrial induzido pela DA e 6-OHDA. Conclui-se que os extratos de algas produzem moléculas bioativas com elevado potencial antioxidante, podendo ser uma fonte promissora de novos compostos neuroprotetores com aplicação terapêutica para doenças neurodegenerativas como a doença de Parkinson.
- 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.
- The biotechnological and seafood potential of Stichopus regalisPublication . Santos, Rita; Dias, Simaura; Pinteus, Susete; Silva, Joana; Alves, Celso; Tecelão, Carla; Pombo, Ana; Pedrosa, RuiSea cucumbers are highly used not only for the production of the bêche-de-mer, but also recent studies have been showing the high potential of these marine invertebrates on the pharmaceutical industry, as they are rich in bioactive compounds with important functions. Stichopus regalis extracts (1 mg∙mL−1) were used in antioxidant, antimicrobial and antitumor assays. The lipid profile was also evaluated. No significant antioxidant activity was detected in both methanolic and dichloromethane extracts. The methanolic fraction showed the highest antimicrobial potential against Candida albicans with an IC50 of 475.4 μg∙mL−1. In the antitumor assays, the dichloromethane fraction showed a high potential for both cell lines, as revealed by the MTT method. The total fat content was 3.63% ± 0.11% and the fatty acid profile revealed the highest amount in C16:0 (9.43% ± 0.77%), C18:0 (12.43% ± 0.83%), C18:1 ω7 (5.63% ± 0.33%), EPA (12.49% ± 0.15%), DHA (7.35% ± 0.02%), ARA (19.29% ± 0.14%) and a ω3/ω6 ratio of 1.078. These findings led us to suggest the potential use of S. regalis as a new source of bioactive compounds with pharmacological potential and its nutritional benefits for human health.
- Antimicrobial and antileukemic effects: in vitro activity of Calyptranthes grandifolia aqueous leaf extractPublication . Majolo, Fernanda; Bitencourt, Shanna; Monteiro, Bruna Wissmann; Haute, Gabriela Viegas; Alves, Celso; Silva, Joana; Pinteus, Susete; Santos, Roberto Christ Vianna; Torquato, Heron Fernandes Vieira; Paredes-Gamero, Edgar Julian; Oliveira, Jarbas Rodrigues; Souza, Claucia Fernanda Volken De; Pedrosa, Rui; Laufer, Stefan; Goettert, Márcia InêsNatural products are still a promising source of bioactive molecules. Food and Drug Administration data showed that approximately 49% of the approved molecules originate naturally or chemicallyresemble these substances, of which more than 70% are being used in anticancer therapy. It is noteworthy that at present there are no scientific studies to prove the effectiveness and safety of a number of plants used in folk medicine such as in the case of Calyptranthes grandifolia O. Berg (Myrtaceae) originally from South America. The aim of the present study was to determine the biological potential and toxicological effects of the aqueous leaf extract of C. grandifolia. The main detected phytoconstituents were condensed tannins and flavonoids and a high quantity of polyphenols. Regarding the antimicrobial potential, the extract exerted inhibitory activity against Pseudomonas aeruginosa. The results also revealed the extract induced DNA damage in a concentration-dependent manner in RAW 264.7 cells. In addition, C. grandifolia produced cytotoxicity in leukemia cell lines (HL60 and Kasumi-1) without affecting isolated human lymphocytes but significantly inhibited JAK3 and p38α enzyme activity. Taken together, these findings add important information on the biological and toxicological effects of C. grandifolia, indicating that aqueous extract may be a source of natural antimicrobial and antileukemic constituents.
- Sulfated polysaccharides from macroalgae: A simple roadmap for chemical characterizationPublication . Martins, Alice; Alves, Celso; Silva, Joana; Pinteus, Susete; Gaspar, Helena; Pedrosa, RuiThe marine environment presents itself as a treasure chest, full of a vast diversity of organisms yet to be explored. Among these organisms, macroalgae stand out as a major source of natural products due to their nature as primary producers and relevance in the sustainability of marine ecosystems. Sulfated polysaccharides (SPs) are a group of polymers biosynthesized by macroalgae, making up part of their cell wall composition. Such compounds are characterized by the presence of sulfate groups and a great structural diversity among the different classes of macroalgae, providing interesting biotechnological and therapeutical applications. However, due to the high complexity of these macromolecules, their chemical characterization is a huge challenge, driving the use of complementary physicochemical techniques to achieve an accurate structural elucidation. This review compiles the reports (2016–2021) of state-of-the-art methodologies used in the chemical characterization of macroalgae SPs aiming to provide, in a simple way, a key tool for researchers focused on the structural elucidation of these important marine macromolecules.
- Gelidiales are not just agar: Revealing the antimicrobial potential of Gelidium corneum for skin disordersPublication . Matias, Margarida; Pinteus, Susete; Martins, Alice; Silva, Joana; Alves, Celso; Mouga, Teresa; Gaspar, Helena; Pedrosa, RuiIn recent decades, seaweeds have proven to be an excellent source of bioactive molecules. Presently, the seaweed Gelidium corneum is harvested in a small area of the Portuguese coast exclusively for agar extraction. The aim of this work was to fully disclosure Gelidium corneum as a sustainable source of antimicrobial ingredients for new dermatological formulations, highlighting its potential to be explored in a circular economy context. For this purpose, after a green sequential extraction, these seaweed fractions (F1–F5) were chemically characterized (1H NMR) and evaluated for their antimicrobial potential against taphylococcus aureus, Staphylococcus epidermidis and Cutibacterium acnes. The most active fractions were also evaluated for their effects on membrane potential, membrane integrity and DNA damage. Fractions F2 and F3 displayed the best results, with IC50 values of 16.1 (7.27–23.02) g/mL and 51.04 (43.36–59.74) g/mL against C. acnes, respectively, and 53.29 (48.75–57.91) g/mL and 102.80 (87.15–122.30) g/mL against S. epidermidis, respectively. The antimicrobial effects of both fractions seem to be related to membrane hyperpolarization and DNA damage. This dual mechanism of action may provide therapeutic advantages for the treatment of skin dysbiosis-related diseases.
- Antiulcerogenic potential of the ethanolic extract of Ceiba speciosa (A. St.-Hil.) Ravenna evaluated by in vitro and in vivo studiesPublication . Dörr, Juliana Andréa; Majolo, Fernanda; Bortoluzzi, Luísa; Vargas, Evelin Zen de; Silva, Joana; Pasini, Manoela; Stoll, Stefani Natali; Rosa, Rafael Lopes da; Figueira, Mariana Moreira; Fronza, Marcio; Beys-da-Silva, Walter O.; Martins, Alice; Gaspar, Helena; Pedrosa, Rui; Laufer, StefanGastrointestinal diseases, such as peptic ulcers, are caused by a damage in the gastric mucosa provoked by several factors. This stomach injury is regulated by many inflammatory mediators and is commonly treated with proton-pump inhibitors, histamine H2 receptor blockers and antacids. However, various medicinal plants have demonstrated positive effects on gastric ulcer treatment, including plants of the Ceiba genus. The aim of this study was to evaluate the antiulcer and anti-inflammatory activities of the stem bark ethanolic extract of Ceiba speciosa (A. St.-Hil.) Ravenna. We performed a preliminary quantification of phenolic compounds by high-performance liquid chromatography-diode array detection (HPLC-DAD), followed by the prospection of other chemical groups through nuclear magnetic resonance (NMR) spectroscopy. A set of in vitro assays was used to evaluate the extract potential regarding its antioxidant activity (DPPH: 19.83 +- 0.34 ug/mL; TPC: 307.20 +- 6.20 mg GAE/g of extract), effects on cell viability and on the release of TNF-α in whole human blood. Additionally, in vivo assays were performed to evaluate the leukocyte accumulation and total protein quantification in carrageenan-induced air pouch, as well as the antiulcerogenic effect of the extract on an ethanol-induced ulcer in rats. The extract contains flavonoids and phenolic compounds, as well as sugars and quinic acid derivatives exhibiting potent antioxidant activity and low toxicity. The extract reduced the release of TNF-α in human blood and inhibited the activity of p38α (1.66 ug/mL), JAK3 (5.25 ug/mL), and JNK3 (8.34 ug/mL). Moreover, it reduced the leukocyte recruitment on the pouch exudate and the formation of edema, reverting the effects caused by carrageenan. The extract presented a significant prevention of ulcer formation and a higher reduction than the reference drug, Omeprazole. Therefore, C. speciosa extract has demonstrated relevant therapeutic potential for the treatment of gastric diseases, deserving the continuation of further studies to unveil the mechanisms of action of plant bioactive ingredients.
- Antioxidant and antimicrobial potential of the Bifurcaria bifurcata epiphytic bacteriaPublication . Horta, André; Pinteus, Susete; Alves, Celso; Fino, Nádia; Silva, Joana; Fernandez, Sara; Rodrigues, Américo; Pedrosa, RuiSurface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.
- Mitigating the negative impacts of marine invasive species – Sargassum muticum - a key seaweed for skincare products developmentPublication . Susano, Patrícia; Silva, Joana; Alves, Celso; Martins, Alice; Pinteus, Susete; Gaspar, Helena; Goettert, Márcia Inês; Pedrosa, RuiSargassum muticum is a highly invasive species, threatening marine biodiversity worldwide. One strategy to reduce marine invaders’ impacts is to promote their use as valuable biomass for new products development. On the other hand, there is a rising conscience of natural compounds importance as health promoters. The present work was designed to sustainably use the marine invasive seaweed S. muticum collected off the Portuguese shore for novel skincare products. The antioxidant, anti-enzymatic (collagenase, elastase, hyaluronidase, tyrosinase), antimicrobial (Staphylococcus epidermidis, Cutibacterium acnes, Malassezia furfur), photoprotective, and antiinflammatory properties of five fractions (F1–F5) obtained by a sequential extraction of S. muticum were evaluated. The diethyl ether fraction (F2) demonstrated the most promising results, with the highest antioxidant and photoprotective capacity, reducing reactive oxygen species (ROS) production promoted by UVA and UVB radiation in 3T3 cells. On the other hand, the ethyl acetate fraction (F3) exhibited the highest anti-enzymatic capacity, inhibiting the activities of collagenase, hyaluronidase and tyrosinase (IC50 of 97.5, 23.7 and 72.3 μg/mL, respectively). Moreover, fractions from S. muticum showed anti-inflammatory potential by reducing tumor necrosis factor – α and interleukin-6 release. A chemical screening by 1H NMR of S. muticum fractions evidenced signals that can be attributed to the presence of different chemical classes, including lipids, pigments, amino acids, polyphenols, and sugars, being related to the observed multitarget properties. This work highlights a strategic valorisation of S. muticum as a source of treasured ingredients for skincare applications.
- Lymphocyte genotoxicity and protective effect of Calyptranthes tricona (Myrtaceae) against H 2 O 2-induced cell death in MCF-7 cellsPublication . Kich, Débora Mara; Bitencourt, Shanna; Caye, Bruna; Faleiro, Dalana; Alves, Celso; Silva, Joana; Pinteus, Susete; Mergener, Michelle; Majolo, Fernanda; Boligon, Aline Augusti; Santos, Roberto Christ Vianna; Pedrosa, Rui; Souza, Claucia Fernanda Volken de; Goettert, Márcia InêsCalyptranthes tricona is a species (Myrtaceae) native to South Brazil. Plants belonging to this family are folkloric used for analgesia, inflammation, and infectious diseases. However, little is known about the toxic potential of C. tricona. The present study aimed to evaluate the antioxidant activity of C. tricona ethanol and hexane leaf extracts, as well as verify their effect on human lymphocytes and MCF-7 cells. The extracts were subjected to preliminary phytochemical screening, antioxidant activity using DPPH and ORAC methods. Genotoxic and mutagenic effects in cultured human lymphocytes were assessed using the comet assay and the micronucleus assay, respectively. In addition, cell viability by MTT assay and fluorometric analysis of mitochondrial potential and caspases-9 activity were performed in order to verify the possible effects of both extracts on H2O2-induced cell death of MCF-7 cells. Our findings revealed that the phenol content and the antioxidant activity were only present in the ethanol extract. Also, the phytochemical screening presented steroids, triterpenoids, condensed tannins, and flavones as the main compounds. However, both extracts were capable of inducing concentration-dependent DNA damage in human lymphocytes. When treating MCF-7 cells with the extracts, both of them inhibited MCF-7 cell death in response to oxidative stress through a decrease of mitochondrial depolarization and caspases-9 activity. Thus, our results need to be considered in future in vitro and in vivo studies of C. tricona effects. In the meanwhile, we recommend caution in the acute/chronic use of this homemade preparation for medicinal purpose.