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- Response surface methodology in the optimization of extraction conditions for Gracilaria gracilis extracts for use in thermoplastic food coatingsPublication . Reboleira, João; Andrade, Mariana A.; Sanches-Silva, Ana; Ganhão, Rui; Mendes, Susana; Vilarinho, Fernanda; Félix, Rafael; Pinheiro, Joaquina; Bernardino, Susana M.Incorporation of antioxidant agents in edible films and packages often relies in the usage of essential oils and other concentrated hydrophobic liquids, with reliable increases in antimicrobial and antioxidant activities of the overall composite, and consequent improvements in product shelf life. These oils are often obtained from plant sources and the usage of organic solvents in their extraction has been criticized as to whether it constitutes a health hazard for consumers [1, 2, 3]. The use of water-soluble antioxidant agents is less common, due to lower activities and extraction yields. As such, using commonly available seaweeds as the source for both the main polymers and the supplementing antioxidant/antimicrobial agents in bioactive films can potentially reduce production costs and create a safer, more sustainable product [1]. Hydroethanolic extracts of commercially available red macroalgae Gracilaria gracilis were evaluated for their antioxidant potential and phenolic content, as part of the preliminary assays for the selection of algal biomass for the enrichment of thermoplastic films. The extracts were obtained through use of solid-liquid extractions, over which yield, DPPH radical reduction capacity, total phenolic content, and FRAP activity assays were measured [4, 5]. Solid to liquid ratio (SLR), extraction time, and ethanol to water ratio were selected as independent variables with experimental ranges and configurations obtained using a Box-Behnken design with three factors, resulting in 15 experimental conditions. Extraction duplicates were used [4]. Response surface methodology was then used to estimate the effect of each extraction condition on the tested bioactivities. Bioactivities were significantly (p<0.05) and positively affected by the presence of ethanol in the extraction solvent, while extraction yield was reduced. Lower solid to liquid ratios (higher solvent proportions) resulted in higher yields, but had no significant (p<0.05) impact on bioactivities. Time was not a relevant factor in any of the measured variables, and as such will be minimized in any further work. 100% ethanol with minimum extraction time (10 minutes) and 1g to 5mL SLR maximize antioxidant activities per gram of dry extract. Maximum ethanolic yields were obtained with a theoretical extraction of 100 minutes and a 1g to 25mL SLR. Aqueous extractions displayed similar results in terms of optimum conditions, but with overall lower bioactivities and higher yields, very likely due to high amounts of soluble polysaccharides. While the high antioxidant activities from the ethanolic extracts encourage the selection of this solvent for extraction of bioactives, low yields may make them later unfeasible. Future studies over the compatibility of these extracts in the polymeric matrix of the films, as well as further optimization of the extraction process will be necessary before the definitive choice for bioactive origin and processing is made.
- Asparagopsis armata exudate cocktail: the quest for the mechanisms of toxic action of an invasive seaweed on marine invertebratesPublication . Silva, Carla; Simões, Tiago; Félix, Rafael; A.M.V.M., Soares; Barata, Carlos; Novais, Sara C.; Lemos, Marco F.L.The seaweed Asparagopsis armata exhibits a strong invasive behavior, producing halogenated compounds with effective biological effects. This study addresses the biochemical responses to sublethal concentrations of A. armata exudate on the marine snail Gibbula umbilicalis whole body and the shrimp Palaemon elegans eyes and hepatopancreas. Antioxidant defenses superoxide dismutase (SOD) and glutathione-S-transferase (GST), oxidative damage endpoints lipid peroxidation (LPO) and DNA damage, the neuronal parameter acetylcholinesterase (AChE), and the fatty acid profile were evaluated. Results revealed different metabolic responses in both species. Despite previous studies indicating that the exudate affected G. umbilicalis’ survival and behavior, this does not seem to result from oxidative stress or neurotoxicity. For P. elegans, the inhibition of AChE and the decrease of antioxidant capacity is concomitant with the increase of LPO, suggesting neurotoxicity and oxidative stress as contributor mechanisms of toxicity for this species. Fatty acid profile changes were more pronounced for P. elegans with a general increase in polyunsaturated fatty acids (PUFAs) with the exudate exposure, which commonly means a defense mechanism protecting from membrane disruption. Nonetheless, the omega-3 PUFAs arachidonic acid (ARA) and docosapentaenoic acid (DPA) increased in both invertebrates, indicating a common regulation mechanism of inflammation and immunity responses
- A biorefinery approach to the biomass of the seaweed Undaria pinnatifida (Harvey Suringar, 1873): obtaining phlorotannins-enriched extracts for wound healingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using lowcost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 +- 10.04 mmol mg-1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 +-1.13mMeq. Fe II mg-1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 ug mL-1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Stress response markers in the blood of São Tomé green sea turtles (Chelonia mydas) and their relation with accumulated metal levelsPublication . Morão, Inês F.C.; Lemos, Marco F.L.; Félix, Rafael; Vieira, Sara; Barata, Carlos; Novais, Sara C.Metals are persistent worldwide being harmful for diverse organisms and having complex and combined effects with other contaminants in the environment. Sea turtles accumulate these contaminants being considered good bioindicator species for marine pollution. However, very little is known on how this is affecting these charismatic animals. Sâo Tomé and Príncipe archipelago harbours important green sea turtle (Chelonia mydas) nesting and feeding grounds. The main goal of this study was to determine metal and metalloid accumulation in the blood of females C. mydas nesting in São Tomé Island, and evaluate the possible impacts of this contamination by addressing molecular stress responses. Gene expression analysis was performed in blood targeting genes involved in detoxification/sequestration and metal transport (mt, mtf and fer), and in antioxidant and oxidative stress responses (cat, sod, gr, tdx, txrd, selp and gclc). Micronuclei analysis in blood was also addressed as a biomarker of genotoxicity. Present results showed significant correlations between different gene expressions with the metals evaluated. The best GLM models and significant relationships were found for mt expression, for which 78% of the variability was attributed to metal levels (Al, Cu, Fe, Hg, Pb and Zn), followed by micronuclei count (65% - Cr, Cu, Fe, Hg, Mn and Zn), tdx expression (52% - Cd, Fe, Mn, Pb and Se), and cat expression (52% - As, Fe, Se and Cd x Hg). Overall, this study demonstrates that these green sea turtles are trying to adapt to the oxidative stress and damage produced by metals through the increased expression of antioxidants and other protectors, which raises concerns about the impacts on these endangered organisms’ fitness. Furthermore, promising biomarker candidates associated to metal stress were identified in this species that may be used in future biomonitoring studies using C. mydas’ blood, allowing for a temporal follow-up of the organisms.
- Industry-friendly hydroethanolic extraction protocols for Grateloupia turuturu UV-shielding and antioxidant compoundsPublication . Félix, Rafael; Carmona, Ana M.; Félix, Carina; Novais, Sara C.; Lemos, Marco F.L.Grateloupia turuturu is an invasive macroalga on the Iberian coast, known to produce bioactive compounds with di erent cosmeceutical bioactivities, namelyUVshielding and antioxidants. The goal of this study was to optimize the extraction procedure of main bioactivities of this species with cosmetic potential, using Response Surface Methodology. Two Box–Behnken designs were used to evaluate the e ect of ethanol concentration (0–50%), liquid-solid ratio, time, pH, and temperature on yield, UV absorbance, and antioxidant activity. Both optimizations showed a similar trend: aqueous extracts have higher yields and extracts performed with ethanol as part of the solvent have higher activities concerning UV absorbance and antioxidant activity. For all the extracts an absorption peak between 320 and 340 nm was observed. This data now allows further studies by narrowing the extracts worthful of characterization. The development of industry-friendly extraction methods allows the valorization of this invasive species, contributing for the potential creation of natural and eco-friendly products by the cosmetic industry while contributing to the restoration of affected environments.
- Marine macroalgae, a source of natural inhibitors of Fungal PhytopathogensPublication . Vicente, Tânia F. L.; Lemos, Marco F.L.; Valentão, Patrícia; Félix, Rafael; Félix, CarinaFungal phytopathogens are a growing problem all over the world; their propagation causes significant crop losses, affecting the quality of fruits and vegetables, diminishing the availability of food, leading to the loss of billions of euros every year. To control fungal diseases, the use of synthetic chemical fungicides is widely applied; these substances are, however, environmentally damaging. Marine algae, one of the richest marine sources of compounds possessing a wide range of bioactivities, present an eco-friendly alternative in the search for diverse compounds with industrial applications. The synthesis of such bioactive compounds has been recognized as part of microalgal responsiveness to stress conditions, resulting in the production of polyphenols, polysaccharides, lipophilic compounds, and terpenoids, including halogenated compounds, already described as antimicrobial agents. Furthermore, many studies, in vitro or in planta, have demonstrated the inhibitory activity of these compounds with respect to fungal phytopathogens. This review aims to gather the maximum of information addressing macroalgae extracts with potential inhibition against fungal phytopathogens, including the best inhibitory results, while presenting some already reported mechanisms of action.
- Cosmeceutical potential of grateloupia turuturu: using low-cost extraction methodologies to obtain added-value extractsPublication . Félix, Carina; Félix, Rafael; Carmona, Ana M.; Januário, Adriana P.; Dias, Pedro D.M.; Vicente, Tânia F. L.; Silva, Joana; Alves, Celso; Pedrosa, Rui; Novais, Sara C.; Lemos, Marco F.L.The invasive macroalga Grateloupia turuturu is known to contain a diversity of bioactive compounds with different potentialities. Among them are compounds with relevant bioactivities for cosmetics. Considering this, this study aimed to screen bioactivities with cosmeceutical potential, namely, antioxidant, UV absorbance, anti-enzymatic, antimicrobial, and anti-inflammatory activities, as well as photoprotection potential. Extractions with higher concentrations of ethanol resulted in extracts with higher antioxidant activities, while for the anti-enzymatic activity, high inhibition percentages were obtained for elastase and hyaluronidase with almost all extracts. Regarding the antimicrobial activity, all extracts showed to be active against E. coli, S. aureus, and C. albicans. Extracts produced with higher percentages of ethanol were more effective against E. coli and with lower percentages against the other two microorganisms. Several concentrations of each extract were found to be safe for fibroblasts, but no photoprotection capacity was observed. However, one of the aqueous extracts was responsible for reducing around 40% of the nitric oxide production on macrophages, showing its anti-inflammatory potential. This work highlights G. turuturu’s potential in the cosmeceutical field, contributing to the further development of natural formulations for skin protection.
- Seaweed as a natural source against phytopathogenic bacteriaPublication . Vicente, Tânia F.L.; Félix, Carina; Félix, Rafael; Valentão, Patrícia; Lemos, Marco F.L.Plant bacterial pathogens can be devastating and compromise entire crops of fruit and vegetables worldwide. The consequences of bacterial plant infections represent not only relevant economical losses, but also the reduction of food availability. Synthetic bactericides have been the most used tool to control bacterial diseases, representing an expensive investment for the producers, since cyclic applications are usually necessary, and are a potential threat to the environment. The development of greener methodologies is of paramount importance, and some options are already available in the market, usually related to genetic manipulation or plant community modulation, as in the case of biocontrol. Seaweeds are one of the richest sources of bioactive compounds, already being used in different industries such as cosmetics, food, medicine, pharmaceutical investigation, and agriculture, among others. They also arise as an eco-friendly alternative to synthetic bactericides. Several studies have already demonstrated their inhibitory activity over relevant bacterial phytopathogens, some of these compounds are known for their eliciting ability to trigger priming defense mechanisms. The present work aims to gather the available information regarding seaweed extracts/compounds with antibacterial activity and eliciting potential to control bacterial phytopathogens, highlighting the extracts from brown algae with protective properties against microbial attack.
- Multifunctional Gelatin/Chitosan Electrospun Wound Dressing Dopped with Undaria pinnatifida Phlorotannin-Enriched Extract for Skin RegenerationPublication . Ferreira, Carolina A. M.; Januário, Adriana P.; Félix, Rafael; Alves, Nuno; Lemos, Marco F.L.; Dias, Juliana R.The similarities of electrospun fibers with the skin extracellular matrix (ECM) make them promising structures for advanced wound dressings. Moreover, infection and resistance in wounds are a major health concern that may be reduced with antibacterial wound dressings. In this work, a multifunctional wound dressing was developed based on gelatin/chitosan hybrid fibers dopped with phlorotannin-enrich extract from the seaweed Undaria pinnatifida. The intrinsic electrospun structure properties combined with the antimicrobial and anti-inflammatory properties of phlorotannin-enrich extract will enhance the wound healing process. Electrospun meshes were produced by incorporating 1 or 2 wt% of extract, and the structure without extract was used as a control. Physico-chemical, mechanical, and biological properties were evaluated for all conditions. Results demonstrated that all developed samples presented a homogenous fiber deposition with the average diameters closer to the native ECM fibrils, and high porosities (~90%) that will be crucial to control the wound moist environment. According to the tensile test assays, the incorporation of phlorotannin-enriched extract enhances the elastic performance of the samples. Additionally, the extract incorporation made the structure stable over time since its in vitro degradation rates decreased under enzymatic medium. Extract release profile demonstrated a longstanding delivery (up to 160 days), reaching a maximum value of ~98% over time. Moreover, the preliminary antimicrobial results confirm the mesh’s antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In terms of biological characterization, no condition presented cytotoxicity effects on hDNF cells, allowing their adhesion and proliferation over 14 days, except the condition of 2 wt% after 7 days. Overall, the electrospun structure comprising phlorotannins-enriched extract is a promising bioactive structure with potential to be used as a drug delivery system for skin regeneration by reducing the bacterial infection in the wound bed.
- Red seaweed-derived compounds as a potential new approach for acne vulgaris carePublication . Januário, Adriana P.; Félix, Rafael; Félix, Carina; Reboleira, João; Valentão, Patrícia; Lemos, Marco F.L.Acne vulgaris (AV) is a chronic skin disease of the pilosebaceous unit affecting both adolescents and adults. Its pathophysiology includes processes of inflammation, increased keratinization, sebum production, hormonal dysregulation, and bacterial Cutibacterium acnes proliferation. Common AV has been treated with antibiotics since the 1960s, but strain resistance has emerged and is of paramount concern. Macroalgae are known producers of substances with bioactive properties, including anti-viral, antibacterial, antioxidant, and anti-inflammatory properties, among several others. In particular, red algae are rich in bioactive compounds such as polysaccharides, phenolic compounds, lipids, sterols, alkaloids, and terpenoids, conferring them antioxidant, antimicrobial, and anti-inflammatory activities, among others. Thus, the exploration of compounds from marine resources can be an appealing approach to discover new treatment options against AV. The aim of this work is to provide an overview of the current knowledge of the potentialities of red macroalgae in the treatment of AV by reviewing the main therapeutic targets of this disease, and then the existence of compounds or extracts with bioactive properties against them.
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