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- 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.
- Avaliação do potencial biotecnológico da alga Asparagopsis armata e das suas bactérias epífitas: citotoxicidade e atividade antimicrobianaPublication . Horta, André Gustavo Cavadas da; Pedrosa, Rui Filipe PintoOs organismos marinhos têm-se revelado como fonte de compostos bioativos com elevado potencial. As algas têm demonstrado que produzem moléculas pelo seu metabolismo secundário com aplicação farmacológica, entre outras. Contudo, as bactérias epífitas das macroalgas podem ter um papel importante na produção das moléculas com potencial biotecnológico encontrado nas macroalgas. O estudo das bactérias associadas a macroalgas são escassos e isto é ainda mais evidente no caso das algas da costa de Portugal. Neste trabalho, foi avaliado o potencial antimicrobiano e antitumoral da alga vermelha Asparagopsis armata, bem como das suas bactérias epífitas, de modo a perceber se o potencial biotecnológico da A. armata depende das suas bactérias epífitas. A avaliação da capacidade antimicrobiana foi realizada nos microrganismos Escherichia coli, Salmonella enteritidis, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Saccharomyces cerevisiae e Candida albicans. A avaliação da capacidade antitumoral foi realizada em células do carcinoma hepatocelular humano (HepG-2) e células provenientes do carcinoma mamário (MCF-7).
- Bioaccessibility of antioxidants and fatty acids from Fucus spiralisPublication . Francisco, João; Horta, André; Pedrosa, Rui; Afonso, Cláudia; Cardoso, Carlos; Bandarra, Narcisa M.; Gil, Maria MFucus spiralis is an edible brown seaweed (SW) found in the Portuguese Coast. It has been reported to have high antioxidant activity, which may elicit a potential use for the food industry. However, little information is available on how the SW behaves during the digestive process and how the freeze-drying process might affect the bioaccessibility of the di erent compounds. Therefore, antioxidant activity, total polyphenols, lipid, and fatty acid contents were measured before and after in vitro simulation of the human digestive process, both in fresh and freeze-dry SW. F. spiralis had a lipid content of 3.49 +- 0.3% of dry weight (DW), which is a usual amount described for this SW genus. The total lipid bioaccessibility was 12.1 +- 0.1%. The major omega-3 fatty acid detected was eicosapentaenoic acid, 7.5 +- 0.1%, with a bioaccessibility percentage of 13.0 +- 1.0%. Four different methods—total phenolic content (TPC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and 1,1-diphenyl-2-picryl-hydrazyl (DPPH)—were used to assess the antioxidant activity of F. spiralis. The bioaccessibility of the antioxidants studied, ranged between 42.7% and 59.5%, except the bioaccessibility of polyphenols in freeze-dried SW (23.0% +- 1.0%), suggesting that the freeze-drying process reduces the bioaccessibility of these compounds.
- Antioxidant and Cytoprotective Activities of Fucus spiralis Seaweed on a Human Cell in Vitro ModelPublication . Pinteus, Susete; Silva, Joana; Alves, Celso; Horta, André Gustavo Cavadas da; Thomas, Olivier P.; Pedrosa, RuiAntioxidants play an important role as Reactive Oxygen Species (ROS) chelating agents and, therefore, the screening for potent antioxidants from natural sources as potential protective agents is of great relevance. The main aim of this study was to obtain antioxidant-enriched fractions from the common seaweed Fucus spiralis and evaluate their activity and efficiency in protecting human cells (MCF-7 cells) on an oxidative stress condition induced by H₂O₂. Five fractions, F1-F5, were obtained by reversed-phase vacuum liquid chromatography. F3, F4 and F5 revealed the highest phlorotannin content, also showing the strongest antioxidant effects. The cell death induced by H₂O₂ was reduced by all fractions following the potency order F4 > F2 > F3 > F5 > F1. Only fraction F4 completely inhibited the H₂O₂ effect. To understand the possible mechanisms of action of these fractions, the cellular production of H₂O₂, the mitochondrial membrane potential and the caspase 9 activity were studied. Fractions F3 and F4 presented the highest reduction on H₂O₂ cell production. All fractions decreased both caspase-9 activity and cell membrane depolarization (except F1). Taken all together, the edible F. spiralis reveal that they provide protection against oxidative stress induced by H₂O₂ on the human MCF-7 cellular model, probably acting as upstream blockers of apoptosis.
- Antitumor and antimicrobial potential of bromoditerpenes isolated from the red alga, Sphaerococcus coronopifoliusPublication . Rodrigues, Daniel; Alves, Celso; Horta, André; Pinteus, Susete; Silva, Joana; Culioli, Gérald; Thomas, Olivier P.; Pedrosa, RuiCancer and infectious diseases continue to be a major public health problem, and new drugs are necessary. As marine organisms are well known to provide a wide range of original compounds, the aim of this study was to investigate the bioactivity of the main constituents of the cosmopolitan red alga, Sphaerococcus coronopifolius. The structure of several bromoditerpenes was determined by extensive spectroscopic analysis and comparison with literature data. Five molecules were isolated and characterized which include a new brominated diterpene belonging to the rare dactylomelane family and named sphaerodactylomelol (1), along with four already known sphaerane bromoditerpenes (2–5). Antitumor activity was assessed by cytotoxicity and anti-proliferative assays on an in vitro model of human hepatocellular carcinoma (HepG-2 cells). Antimicrobial activity was evaluated against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Compound 4 exhibited the highest antimicrobial activity against S. aureus (IC50 6.35 μM) and compound 5 the highest anti-proliferative activity on HepG-2 cells (IC50 42.9 μM). The new diterpene, sphaerodactylomelol (1), induced inhibition of cell proliferation (IC50 280 μM) and cytotoxicity (IC50 720 μM) on HepG-2 cells and showed antimicrobial activity against S. aureus (IC50 96.3 μM).
- Algae from Portuguese Coast Presented High Cytotoxicity and Antiproliferative Effects on an In vitro Model of Human Colorectal CancerPublication . Alves, Celso; Pinteus, Susete; Rodrigues, Ana; Horta, André; Pedrosa, RuiBackground: The marine environment has shown to be an interesting source of new antitumor agents, representing an important tool in cancer research. Objective: The aim of this study was to evaluate the antitumor activities of 12 algae from Peniche coast (Portugal) on an in vitro model of human colorectal cancer (Caco‑2 cells). Materials and Methods: The antitumor potential was accessed by evaluating Caco‑2 cell’s viability and proliferation through the 3‑[4, 5‑dimethylthiazol‑2‑yl]‑2, 5‑diphenyl tetrazolium bromide and calcein‑AM methods. Results: The dichloromethane extracts of Asparagopsis armata and Sphaerococcus coronopifolius induced the highest decrease on cell’s viability (1 mg/mL; 24 h), 98.96% ± 0.39% and 98.08% ± 0.89%, respectively, followed by the methanolic extracts of S. coronopifolius (96.47% ± 1.26%) and A. armata (92.68% ± 1.17%). Regarding cell proliferation, the highest decrease of Caco‑2 cell’s proliferation (1 mg/mL; 24 h) was induced by the dichloromethane extract of A. armata (100% ± 0.48%), S. coronopifolius (99.04 ± 0.51%), and Plocamium cartilagineum (95.05% ± 1.19%). The highest potency was shown by the dichloromethane extract of S. coronopifolius in both, cytotoxicity and antiproliferative tests, with an IC50 of 21.3 and 36.5 µg/mL, respectively. Conclusion: The extracts of A. armata and S. coronopifolius are promising sources of new bioactive molecules with application in cancer therapeutics.
- Cytoprotective effect of seaweeds with high antioxidant activity from the Peniche coast (Portugal)Publication . Pinteus, Susete; Silva, Joana; Alves, Celso; Horta, André; Fino, Nádia; Rodrigues, Ana Inês; Mendes, Susana; Pedrosa, RuiScreening of antioxidant potential of dichloromethane and methanolic extracts of twenty-seven seaweeds from the Peniche coast was performed by: total phenolic contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Seaweeds revealing the highest antioxidant activity were screened for cytoprotective potential in MCF-7 cells, including the mitochondrial membrane potential analysis and the caspase-9 activity. High correlation was found between TPC of seaweed extracts and their scavenging capacity on DPPH and peroxyl radicals. The highest antioxidant activity was displayed by the methanolic fraction of brown seaweeds belonging to Fucales, however Ulva compressa presented the highest cytoprotective effect by blunting the apoptosis process. These results suggest that high antioxidant activity may not be directly related with high cytoprotective potential. Thus, seaweeds reveal to be a promising source of compounds with potential against oxidative stress.
- Antifungal and Antioxidant Properties of Chitosan Polymers Obtained from Nontraditional Polybius henslowii SourcesPublication . Avelelas, Francisco; Horta, André; Pinto, Luís F.V.; Marques, Sónia Cotrim; Nunes, Paulo; Pedrosa, Rui; Leandro, Sérgio MiguelChitin was extracted from Polybius henslowii, a swimming crab, captured in large quantities throughout the Portuguese coast by purse seine vessels as bycatch. After standard chitin extraction procedures, water-soluble chitosan products were obtained via two di erent methods: (1) N-acetylation with the addition of acetic anhydride and (2) a reaction with hydrogen peroxide. The chemical structure and molecular weight of chitosan derivatives, water-soluble chitosan (WSC) and chitooligosaccharides (COS), were confirmed by Fourier Transform Infrared Spectroscopy (FT-IR) and gel permeation chromatography (GPC).Antioxidant and metal chelation activities were evaluated, and the growth inhibition capacity was tested on four phytopatogens. The chitooligosaccharides from pereopods (pCOS) and shell body parts (sCOS) inhibited all fungal species tested, particularly Cryphonectria parasitica with 84.7% and 85.5%, respectively. Both radical scavenging and antifungal activities proved to be dose-dependent. Chitooligosaccharides with a low molecular weight (2.7, 7.4, and 10.4 Kg mol1) showed the highest activity among all properties tested. These results suggested that chitosan derivatives from P. henslowii raw material could potentially be used against phytopathogens or as ingredient in cosmetics and other products related to oxidative stress.
- Study of the seasonal variations of the fatty acid profiles of selected macroalgaePublication . Pereira, Tatiana; Horta, André; Barroso, Sonia; Mendes, Susana; Gil, Maria M.Due to the high consumption of fat-rich processed foods, efforts are being done to reduce their saturated fat (SFA) contents and replace it with polyunsaturated fatty acids (PUFA), creating a necessity to find alternative PUFA sources. Macroalgae, being a promising natural source of healthy food, may be such an alternative. The fatty acid (FA) profile of Fucus spiralis, Bifurcaria bifurcata, Ulva lactuca, and Saccorhiza polyschides were determined through direct transesterification and their seasonal variation was studied. F. spiralis showed the highest FA content overall, B. bifurcata presented the higher PUFA amounts, and U. lactuca and S. polyschides the higher SFA. The production of FA was shown to be influenced by the seasons. Spring and summer seemed to induce the FA production in F. spiralis and B. bifurcata while in U. lactuca the same was verified in winter. U. lactuca presented a !6/!3 ratio between 0.59 and 1.38 while B. bifurcata presented a ratio around 1.31. The study on the seasonal variations of the macroalgal FA profile can be helpful to understand the best season to yield FA of interest, such as ALA, EPA, and DHA. It may also provide valuable information on the best culturing conditions for the production of desired FAs.
- Bioaccessibility of target essential elements and contaminants from Fucus spiralisPublication . Francisco, João; Cardoso, Carlos; Bandarra, Narcisa; Brito, Pedro; Horta, André; Pedrosa, Rui; Gil, Maria M.; Delgado, Inês Margarida; Castanheira, Isabel; Afonso, CláudiaFucus spiralis is an edible brown seaweed (SW) found in the Portuguese Coast that is still undervalued and not very well studied. However, SW are known to be nutritious and healthy foods, in part, for their potential to supply mineral nutrients to the body. Nevertheless, with the increasing levels of pollution in the coastal areas, SW can also accumulate some contaminants. Therefore, to expand the knowledge about this marine resource, the contents of Mg, Mn, Co, I, Cd, Pb, As and Hg in fresh and freeze-dried Fucus spiralis were determined before and after in vitro human digestion simulation. Magnesium was the major element with 11.86 mg/g dry weigh (DW), but it must be highlighted that I in this SW presented concentration values of approximately 135 μg/g DW. Concerning contaminants, F. spiralis seems to have low levels of Cd, Pb, and Hg. The elements’ bioaccessibility was always high, ranging between 45.8% (I – Fresh SW) and 88.5% (Hg – FD SW). The freeze-drying process seemed to enhance element bioaccessibility.