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Roque Lino Felgueiras Barreto, Maria do Carmo
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- Constructing ethanol-derived bioactive extracts using the brown seaweed Zonaria tournefortii (J.V.Lamouroux) Montagne performed with Timatic extractor by means of response surface methodology (RSM)Publication . Nunes, N.; Valente, S.; Ferraz, S.; Barreto, Maria Carmo; Carvalho, M.A.A. Pinheiro deUnderstanding the biochemical and antioxidant composition and capacity of a target biomass is the first step to its selectivity as functional food which can enhance the ability to promote health by reducing the risk of chronic diseases. The main purpose of this work was to employ response surface methodology (RSM) to determine the effect of the independent variables, % of ethanol (50–96%), time of sonication (0–20 min), and number of extraction cycles (6–18 cycles) in the primary extract of brown seaweed Zonaria tournefortii, studying the yield variation of some bioactive compounds, assessing the potential of these bioextracts to integrate as a natural additives or supplements in the functional food industry. The extractions were performed employing “Green Chemistry” techniques executed with the Timatic extractor, which applied pressurized ethanol solution at a maximum pressure of 8.5 bar through milled dehydrated biomass in the extraction vessel. Several parameters were assessed in the primary bioactive extract which included extract yield (11.56–28.49 g (100 g)−1 dw), total chlorophyll content (0.14–1.42 g (100 g)−1 dw), total carotenoid content (0.35–0.80 g (100 g)−1 dw), total fucoxanthin content (0.04–0.13 g (100 g)−1 dw), total phenolic content (3.58–5.84 g (100 g)−1 dw), total flavonoid content (0.22–4.70 g (100 g)−1 dw), DPPH (56.05–76.45%), and reducing activity (3.83–6.04 g (100 g)−1 dw). A second objective was to determine the suitability of the residue for subsequent extraction of valuable compounds such as fucoidan (4.87 to 6.59 g (100 g)−1 dw) and cellulose (18.88 to 20.27 g (100 g)−1 dw), implementing the first step to a biorefinery strategy, using a cascade approach.
- Nutraceutical potential of Asparagopsis taxiformis (Delile) Trevisan extracts and assessment of a downstream purification strategyPublication . Nunes, N.; Valente, S.; Ferraz, S.; Barreto, Maria Carmo; Carvalho, M. A. A. Pinheiro deThe main goal of the present work was to determine the nutraceutical potential of Asparagopsis taxiformis D. extracts from Madeira Archipelago south coast. Extraction methodologies consisted either/or in 72 hours stirring, at room temperature (M1), or 6 cycles of Soxhlet extraction (M2), both with re-extraction. Solvents used were distilled water, ethanol, methanol and ethyl acetate. M1 allowed to obtain the highest values for extraction yield (31.65 g.100g 1 dw) using water, whereas iodine content (3.37 g.100g 1 dw), TPC (1.71 g GAE.100g 1 dw) and chlorophyll a (45.96 mg.100g 1 dw) were obtained using ethanol, and TCC (36.23 mg.100g 1 dw) with methanol. Extracts that showed higher reduction activity in M1 were derived from ethanol extraction (1,908 mg AAE.100g 1 dw). Water and ethanol were the best solvents for higher DPPH scavenging activity in M2, both with same result (IC50 1.37 mg.mL 1 ). The lowest value of IC50 for chelating activity (1.57 mg.mL 1 ) was determined in M1, using ethyl acetate. The remaining residue was used to obtain other products, i.e. lipid extraction (M1, 2.05 g.100g 1 dw), carrageenans (M2, 21.18 g.100g 1 dw) and cellulose (M1, 23.81 g.100g 1 dw) with subsequent FTIR ATR analysis. Our results show that A. taxiformis is a valuable source of bioactive compounds. The M1 extraction methodology using ethanol is the most effective solvent to produce an iodine rich bioactive extract with potential of being used as a nutraceutical supplement. Also, we have demonstrated a possible downstream strategy that could be implemented for multiple compound extraction from A. taxiformis residue. This has a vital importance for future feasibility, when using this biomass as an industrial feedstock for multiple products production. Statistical analysis, using SPSS 24.0, was also performed and important correlations were found between assays and methods.
- Evaluation of fucoxanthin contents in seaweed biomass by vortex-assisted solid-liquid microextraction using high-performance liquid chromatography with photodiode array detectionPublication . Nunes, N.; Leça, João M.; Pereira, Ana C.; Pereira, Vanda; Ferraz, S.; Barreto, Maria Carmo; Marques, José C.; Carvalho, M.A.A. Pinheiro deFucoxanthin is considered an important marine bioactive compound with biological properties with promising effects, namely on health. A simple and efficient analytical methodology is proposed for its quantification in seaweed biomass by using vortex-assisted solid-liquid microextraction (VASLME) followed by reversed phase high-performance liquid chromatography (RP-HPLC) photodiode array detection (PDA) analysis. This microextraction uses reduced quantities of sample (25 mg) and solvent (300 μL of ethanol) to efficiently extract this high-valued xanthophyll, in a vortex time of 15 min. These extraction parameters were optimized performing a Central Composite Design (CCD) analysis, running 32 individual experiments. In turn, the method validation was assessed. The linearity of the method was confirmed (R2 = 0.99998) in a concentration range from 12 to 3600 μg·g−1 dw. Also, good sensitivity and accuracy results were observed through the LOD (3.33 μg·g−1 ), LOQ (10.09 μg·g−1 ) and recovery (varied from 95 to 97%) assessments. Good precision was also verified, with intra-day variation within 2.0–3.3%, and inter-day within 1.0–3.8%. Matrix effect was also evaluated and an acceptable variation of 3.4% was found. The method applicability was confirmed by the analysis of 22 seaweed biomass samples and fucoxanthin content was found to vary from about 10 to 853 μg·g−1 dw. This method demonstrated a good performance and can be successfully implemented for a rapid, reliable and accurate screening of fucoxanthin in seaweed biomass.
- Nutraceutical potential of Asparagopsis taxiformis (Delile) Trevisan extracts and assessment of a downstream purification strategyPublication . Nunes, Nuno; Valente, S.; Ferraz, Sónia; Barreto, Maria Carmo; Carvalho, M .A. A. Pinheiro deThe main goal of the present work was to determine the nutraceutical potential of Asparagopsis taxiformis D. extracts from Madeira Archipelago south coast. Extraction methodologies consisted either/or in 72 hours stirring, at room temperature (M1), or 6 cycles of Soxhlet extraction (M2), both with re-extraction. Solvents used were distilled water, ethanol, methanol and ethyl acetate. M1 allowed to obtain the highest values for extraction yield (31.65 g.100g 1 dw) using water, whereas iodine content (3.37 g.100g 1 dw), TPC (1.71 g GAE.100g 1 dw) and chlorophyll a (45.96 mg.100g 1 dw) were obtained using ethanol, and TCC (36.23 mg.100g 1 dw) with methanol. Extracts that showed higher reduction activity in M1 were derived from ethanol extraction (1,908 mg AAE.100g 1 dw). Water and ethanol were the best solvents for higher DPPH scavenging activity in M2, both with same result (IC50 1.37 mg.mL 1 ). The lowest value of IC50 for chelating activity (1.57 mg.mL 1 ) was determined in M1, using ethyl acetate. The remaining residue was used to obtain other products, i.e. lipid extraction (M1, 2.05 g.100g 1 dw), carrageenans (M2, 21.18 g.100g 1 dw) and cellulose (M1, 23.81 g.100g 1 dw) with subsequent FTIR ATR analysis. Our results show that A. taxiformis is a valuable source of bioactive compounds. The M1 extraction methodology using ethanol is the most effective solvent to produce an iodine rich bioactive extract with potential of being used as a nutraceutical supplement. Also, we have demonstrated a possible downstream strategy that could be implemented for multiple compound extraction from A. taxiformis residue. This has a vital importance for future feasibility, when using this biomass as an industrial feedstock for multiple products production. Statistical analysis, using SPSS 24.0, was also performed and important correlations were found between assays and methods.
- Biochemical composition, nutritional value, and antioxidant properties of seven seaweed species from the Madeira ArchipelagoPublication . Nunes, N.; Ferraz, S.; Valente, S.; Barreto, Maria Carmo; Carvalho, M. A. A. Pinheiro deBiochemical composition and antioxidant activity were assessed in seven seaweeds from Madeira Archipelago, namely, the chlorophyte (Ulva lactuca), the rhodophytes (Asparagopsis taxiformis, Chondrus crispus, Galaxaura rugosa, Grateloupia lanceola and Nemalion elminthoides), and the phaeophyte (Zonaria tournefortii). Seaweed mineral content varied from 16.60 to 84.16 g (100 g)-1 dry weight (dw). Organic matter, composed by fiber and matrix polysaccharides (8.33 to 54.04 g (100 g)-1 dw), starch (1.95 to 25.41 g (100 g)-1 dw), protein (2.80 to 17.55 g (100 g)-1 dw), and fat (1.46 to 12.04 g (100 g)-1 dw), was also determined. Asparagopsis taxiformis was found to have substantial quantities of protein, fat, fiber, and matricial polysaccharides, compared to the other analyzed seaweeds. Analysis of antioxidant components included the measurement of chlorophyll a (28.81 to 244.3 g (100 g)-1 dw), total carotenoids (0 to 297.8 g (100 g)-1 dw), total phenolic compounds (0 to 2154 mg GAE (100 g)-1 dw), and total flavonols (7.27 to 604.8 mg QE (100 g)-1 dw). Zonaria tournefortii was found to possess the highest contents of chlorophyll a, total carotenoids, total phenolic content (TPC), and antioxidant activity, determined through ferric reduction antioxidant potential (FRAP), ferrous ion chelating (FIC), free radical-scavenging assay (FRSA), and β-carotene bleaching (β-CB). Statistical analysis showed 38 significant correlations between various biochemical and antioxidant parameters or activity and determined that fat content showed the highest number of correlations. Overall, this study gives a better understanding of Madeira autochthonous seaweeds in their potential of being introduced as a raw material for nutrient supplementation in various food products or to produce functional foods using seaweed natural properties.
- Fatty acid composition, TLC screening, ATR-FTIR analysis, anti-cholinesterase activity, and in vitro cytotoxicity to A549 tumor cell line of extracts of 3 macroalgae collected in MadeiraPublication . Nunes, N.; Rosa, G. P.; Ferraz, S.; Barreto, Maria Carmo; Carvalho, M. A. A. Pinheiro deThree macroalgae collected at Madeira Island were included in this study to determine their potential for drug, nutraceutical, food, or supplement application. Fatty acid content was higher in Zonaria tournefortii (12.32 mg g−1 dw) with 16.58% of PUFAs, eicosapentaenoic acid (C20:5ω3), and arachidonic acid (20:4ω6) having concentrations of 2.59 and 1.17%, respectively. The anti-thrombogenic and anti-atherogenicity potential was higher for Z. tournefortii due to relevant fatty acids in the biochemical composition this macroalgae. Lipid classes were assessed in the lipid extract and neutral lipids (NL) were in higher yield in Asparagopsis taxiformis (51.16%) and lower in Z. tournefortii (26.96%). The glycolipids (GL) were between 36.03 and 16.11% in Z. tournefortii and Ulva lactuca. Phospholipids (PL) fraction varied from 35.91 and 31.60% in A. taxiformis and Z. tournefortii. TLC screening identified that U. lactuca contains phytol and cholesterol in its NL, digalactosyldiacylglycerol in its GL, and cardiolipin and L-α-phosphatidylcholine in its PL. Zonaria tournefortii contains phytol and cholesterol in its NL classes, and the PL classes contain L-α-phosphatidylethanolamine and 1-(3-sn-phosphatidyl)-rac-glycerol. The macroalgae A. taxiformis revealed cholesterol in its NL fraction and the same phospholipids as Z. tournefortii in its PL fraction. ATRFTIR analysis enabled a “fingerprint” spectra and important sulfation absorption bands were identified, revealing the functional polysaccharides within these macroalgae. Anti-cholinesterasic activity was assessed in A. taxiformis, with a low IC50 for AChE (8.92 ± 0.43 μg mL−1 ) and BuChE (13.96 ± 0.32 μg mL−1 ), demonstrating dual inhibitory activity, justifying the interest to identify the active principle which may be the scaffold of a novel drug.
- Validation of a spectrophotometric methodology for a rapid iodine analysis in algae and seaweed castsPublication . Nunes, N.; Valente, S.; Ferraz, S.; Barreto, Maria Carmo; Carvalho, M.A.A. Pinheiro deIodine plays an important role in human metabolism and its deficiency is particularly harmful in pregnancy and childhood. It remains a major public health concern in many countries, especially in Portugal. The main purpose of this work was to develop a validated spectrophotometric analysis for a fast and reliable iodine quantification in algal samples. Absorbance was determined at 410 nm demonstrating a good linearity (R2 ≈ 1.0) in the range of 0–0.06 mg I/100 g. LOD and LOQ were 1.7 × 10−3 and 5.0 × 10−3 mg I/100 g, respectively. Accuracy was determined using recovery and varied between 101 and 118%. For precision analysis, an intra-day test performance (RSD = 8.7%) and a repeatability assay (RSD = 3.8%) were performed. Matrix effect assessment demonstrated that this had a negligible effect (3.2%) in the iodine quantification. The spectrophotometric method was externally validated, for iodine quantification in algal samples, by INSA certified laboratory. The correlation coefficient between external iodine quantification and our work was R2 ≈ 0.9, showing a good correlation. Applicability was assessed in 25 macroalgae species (5 green, 9 red and 11 brown), 12 seaweed casts, collected in Canary Islands and 1 microalga (Isochrysis galbana) provided by ITC (Instituto Tecnologico de Canarias).
- Pharmacological and cosmeceutical potential of Seaweed Beach-Casts of MacaronesiaPublication . Zárate, Rafael; Portillo, Eduardo; Teixidó, Sílvia; Carvalho, Miguel A. A. Pinheiro de; Nunes, Nuno; Ferraz, Sónia; Seca, Ana M. L.; Rosa, Gonçalo P.; Barreto, Maria CarmoSeaweed beach-casts are a seasonal phenomenon that regularly deposits tons of algae biomass on beaches, which are usually disposed of in landfills. The present work aimed to contribute to the valorization of this biomass by studying bioactivities that reveal its potential in the pharmaceutical and/or cosmeceutical industries. Methanol and ethanol extracts and fractions from 15 beach-casts biomass were tested for a range of bioactivities. Eight of the most active samples exhibit IC50 values between 11.38 µg/mL and 19.28 µg/mL and selectivity indexes higher than 3.8, against NCI-H1299 (lung cancer) and T47D (breast cancer). Concerning antiaging potential, all the extracts tested presented UV protection, with SPF values above 30, like the standard sunscreen. Regarding aging-related enzymes, 24 samples were tyrosinase inhibitors exhibiting IC50 values from 9.01 µg/mL to 200.09 µg/mL. Eight extracts inhibited collagenase (IC50 < 248.76 µg/mL), two of them more actively than positive control EDTA (IC50 = 59.26 µg/mL). In addition to UV protection, the cumulative effect of antityrosinase and anticollagenase activities shown by the samples suggest that they may play a significant role in preventing skin photoaging. The results obtained demonstrate the high potential of beach-cast seaweed biomass as sources of cosmetic bioproducts with antiaging effects, and of selective cytotoxic metabolites.
- Biochemical study of attached macroalgae from the Madeira Archipelago and beach-cast macroalgae from the Canary Islands: multivariate analysis to determine bioresource potentialPublication . Nunes, Nuno; Valente, Sofia; Ferraz, Sónia; Barreto, Maria Carmo; Carvalho, Miguel Â. A. Pinheiro deFifteen attached macroalgae from the Madeira Archipelago, comprising three green, three red and nine brown algal species, as well as two beach-cast macroalgal samples, collected along the north shore of Gran Canaria, were assessed for their biochemical properties. The anal ysis included the determination of total minerals, total carbohydrates, protein, lipids, chlorophyll a, total carote noids, total phenolic content, fucoxanthin and phycobilins (allophycocyanin, phycocyanin and phycoerythrin). The results showed a high variability of biochemical composi tion, allowing for the targetting of specific bioresources for particular purposes, including functional foods. This work provides the foundation for a biorefinery strategy implementation plan, for which specific macroalgae may be tar geted for valuable and beneficial compounds
- Nutraceutical potential of Asparagopsis taxiformis (Delile) Trevisan extracts and assessment of a downstream purification strategyPublication . Nunes, N.; Valente, S.; Ferraz, S.; Barreto, Maria Carmo; Carvalho, M. A. A. Pinheiro deThe main goal of the present work was to determine the nutraceutical potential of Asparagopsis taxiformis D. extracts from Madeira Archipelago south coast. Extraction methodologies consisted either/or in 72 hours stirring, at room temperature (M1), or 6 cycles of Soxhlet extraction (M2), both with re-extraction. Solvents used were distilled water, ethanol, methanol and ethyl acetate. M1 allowed to obtain the highest values for extraction yield (31.65 g.100g 1 dw) using water, whereas iodine content (3.37 g.100g 1 dw), TPC (1.71 g GAE.100g 1 dw) and chlorophyll a (45.96 mg.100g 1 dw) were obtained using ethanol, and TCC (36.23 mg.100g 1 dw) with methanol. Extracts that showed higher reduction activity in M1 were derived from ethanol extraction (1,908 mg AAE.100g 1 dw). Water and ethanol were the best solvents for higher DPPH scavenging activity in M2, both with same result (IC50 1.37 mg.mL 1 ). The lowest value of IC50 for chelating activity (1.57 mg.mL 1 ) was determined in M1, using ethyl acetate. The remaining residue was used to obtain other products, i.e. lipid extraction (M1, 2.05 g.100g 1 dw), carrageenans (M2, 21.18 g.100g 1 dw) and cellulose (M1, 23.81 g.100g 1 dw) with subsequent FTIR ATR analysis. Our results show that A. taxiformis is a valuable source of bioactive compounds. The M1 extraction methodology using ethanol is the most effective solvent to produce an iodine rich bioactive extract with potential of being used as a nutraceutical supplement. Also, we have demonstrated a possible downstream strategy that could be implemented for multiple compound extraction from A. taxiformis residue. This has a vital importance for future feasibility, when using this biomass as an industrial feedstock for multiple products production. Statistical analysis, using SPSS 24.0, was also performed and important correlations were found between assays and methods.