Browsing by Author "Coscueta, Ezequiel R."
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- Applications in medicine: joint healthPublication . Coscueta, Ezequiel R.; Brassesco, María Emilia; Batista, Patrícia; Borges, Sandra; Pintado, ManuelaRheumatic and joint diseases are among the most widespread pathologies associated with pain and cause a great impact in society across the globe. Given the continuing rise in life expectancy, their prevalence is destined to grow. Osteoarthritis, a degenerative joint disease, is on its way to becoming the fourth leading cause of disability worldwide by 2020. Accompanying osteoarthritis is rheumatoid arthritis, which is a chronic systemic disease that often causes pain and deformity. The joint function involves several processes and understanding the mechanism of action of the joint disease process (proliferation, degradation, regeneration, and infection) is important for the diagnostic, prophylaxis, and treatment. These mechanisms are associated with active peptides that are involved in almost all physiological processes, including cell differentiation, proliferation, inflammatory processes, and immune regulation. Therefore the study of bioactive peptides as biological markers and therapeutic targets for the diagnosis, prevention, and therapy of joint diseases has a very important research ahead. In this context, the present chapter will highlight the importance of bioactive peptides in joint health, underline peptides natural sources, their mechanisms of action, and their potential applications on joint diseases.
- Assessment of collagen content in fish skin: development of a flow analysis method for hydroxyproline determinationPublication . Melo, Maria M. P.; Mesquita, Raquel B. R.; Coscueta, Ezequiel R.; Pintado, Manuela E.; Rangel, António O. S. S.This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L −1, with a limit of detection (LOD) of 2.6 mg L −1 and a limit of quantification (LOQ) of 23.8 mg L −1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).
- Bioaccessibility of novel bioactive peptides from the body mucus of the Lusitanian Toadfish Halobatrachus didactylus using an in vitro digestion modelPublication . Fernandez Cunha, Marta; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe bioprospection of marine resources for drug discovery is receiving increasing attention (1). Adverse marine environmental conditions lead organisms to develop a collection of bioactive molecules for survival (1). Mucus, acting as a first line of defense against pathogens (2), is known to protect fish from the surrounding environment. Our previous studies have already demonstrated the bioactive potential of body mucus from the Lusitanian toadfish Halobatrachus didactylus. We performed LC-MS/MS to identify potential peptides within the mucus peptide fraction, selecting them based on in silico predictions of their bioactivities. It was important to assess the capacity of our bioactive peptides to resist the gastrointestinal tract and cross the intestinal epithelial barrier, thereby confirming their possible applicability as health potentiators (3). In this study, two peptides coded HdLPN (sequence PFPGPLPN) and HdVLPN (sequence VYPFPGPLPN) were submitted to an in vitro digestion model using the protocol INFOGEST 2.0. The digested content from the dialysis process after gastrointestinal simulation, both permeate and retentate using 3.5 kDa membranes, were evaluated in vitro for their antioxidant activity through ORAC assay, and the permeate for antihypertensive potential through inhibition of angiotensin-converting enzyme (iACE). The antioxidant activity of the two digested peptides, in both permeate and retentate forms, was comparable to the control (which utilized water in place of peptides); similar results were obtained for their antihypertensive activity in the permeate form. The results for both bioactivities showed no significant differences when comparing the digested peptides' retentate and permeate forms with control. This suggests that the enzymatic hydrolysis occurring during digestion degrades the bioactivity of these peptides, as they exhibited antioxidant activity prior to digestion (HdLPN 0.20±0.02 µmol Eq. Trolox/ mg peptide and HdVLPN 1.51±0.07 µmol Eq. Trolox/ mg peptide), and there was no subsequent activation or potentiation related to antihypertensive activity, which was also absent before digestion. This research primarily focused on evaluating how digestion affects bioactive peptides derived from the mucus of H. didactylus. However, comprehensive analysis, including mass spectrometry, is essential to fully understand the impact of digestion on the hydrolysis of these peptides. A forthcoming study could explore biocompatible materials for safe delivery methods of peptides, e.g. encapsulation, enabling them to withstand gastrointestinal digestion and effectively reach target organs to exert their intended bioactivity (3).
- Bioaccessibility of novel bioactive peptides from the body mucus of the Lusitanian Toadfish Halobatrachus didactylus using an in vitro digestion modelPublication . Fernandez Cunha, Marta; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe bioprospection of marine resources for drug discovery is receiving increasing attention (1). Adverse marine environmental conditions lead organisms to develop a collection of bioactive molecules for survival (1). Mucus, acting as a first line of defense against pathogens (2), is known to protect fish from the surrounding environment. Our previous studies have already demonstrated the bioactive potential of body mucus from the Lusitanian toadfish Halobatrachus didactylus. We performed LC-MS/MS to identify potential peptides within the mucus peptide fraction, selecting them based on in silico predictions of their bioactivities. It was important to assess the capacity of our bioactive peptides to resist the gastrointestinal tract and cross the intestinal epithelial barrier, thereby confirming their possible applicability as health potentiators (3). In this study, two peptides coded HdLPN (sequence PFPGPLPN) and HdVLPN (sequence VYPFPGPLPN) were submitted to an in vitro digestion model using the protocol INFOGEST 2.0. The digested content from the dialysis process after gastrointestinal simulation, both permeate and retentate using 3.5 kDa membranes, were evaluated in vitro for their antioxidant activity through ORAC assay, and the permeate for antihypertensive potential through inhibition of angiotensin-converting enzyme (iACE). The antioxidant activity of the two digested peptides, in both permeate and retentate forms, was comparable to the control (which utilized water in place of peptides); similar results were obtained for their antihypertensive activity in the permeate form. The results for both bioactivities showed no significant differences when comparing the digested peptides' retentate and permeate forms with control. This suggests that the enzymatic hydrolysis occurring during digestion degrades the bioactivity of these peptides, as they exhibited antioxidant activity prior to digestion (HdLPN 0.20±0.02 µmol Eq. Trolox/ mg peptide and HdVLPN 1.51±0.07 µmol Eq. Trolox/ mg peptide), and there was no subsequent activation or potentiation related to antihypertensive activity, which was also absent before digestion. This research primarily focused on evaluating how digestion affects bioactive peptides derived from the mucus of H. didactylus. However, comprehensive analysis, including mass spectrometry, is essential to fully understand the impact of digestion on the hydrolysis of these peptides. A forthcoming study could explore biocompatible materials for safe delivery methods of peptides, e.g. encapsulation, enabling them to withstand gastrointestinal digestion and effectively reach target organs to exert their intended bioactivity (3).
- Bioactive extracts from brewer's spent grainPublication . Bonifácio-Lopes, Teresa; Boas, Ana Vilas; Coscueta, Ezequiel R.; Costa, Eduardo M.; Silva, Sara; Campos, Débora; Teixeira, José A.; Pintado, ManuelaIn this study antioxidant-rich extracts from brewer’s spent grain (BSG) extracted with solid-to-liquid extraction using different solvents water and ethanol and their mixtures at two ratios (80% ethanol:water (v/v) and 60% ethanol:water (v/v)) were characterized. Nutritional composition was evaluated for the extracts and for the solid residues obtained after extraction. Additionally, the extracts were analyzed for the total phenolic content and individual phenolic compounds and related biological properties including antioxidant capacity (ABTS; ORAC and DNA protection), antihypertensive capacity, antibacterial activity and antibiofilm capacity. Safety was also demonstrated through genotoxicity and cytotoxicity tests. The results obtained showed that while all the extracts exhibited high antioxidant capacity (except ethanolic extract), the highest values were obtained for the 60% ethanol:water extract. The identification of phenolic compounds using HPLC showed that catechin and vanillin were the main compounds identified with the highest concentration being obtained for 60% ethanol:water extraction. In the biological activity assays water and hydroethanolic extracts showed to be multifunctional (antioxidant and antihypertensive capacity, antibacterial and antibiofilm activity), the 80% ethanol:water presented better results in some assays. All were non-genotoxic, but the cytotoxicity was dependent on the extract concentration, with complete safe application for all up to 1 mg/mL. Therefore, this study shows the potential of a viable green solvent based and a low cost extraction recovery method of bioactive compounds from brewer’s spent grain.
- Bioactive hydrolysates from chlorella vulgaris: optimal process and bioactive propertiesPublication . Cunha, Sara A.; Coscueta, Ezequiel R.; Nova, Paulo; Silva, Joana Laranjeira; Pintado, Maria ManuelaMicroalgae have been described as a source of bioactive compounds, such as peptides. Microalgae are easy to produce, making them a sustainable resource for extracting active ingredients for industrial applications. Several microalgae species have interesting protein content, such as Chlorella vulgaris with around 52.2% of protein, making it promising for peptide hydrolysate production. Therefore, this work focused on the production of water-soluble hydrolysates rich in proteins/peptides from the microalgae C. vulgaris and studied bioactive properties. For that, a design of experiments (DOE) was performed to establish the optimal conditions to produce hydrolysates with higher levels of protein, as well as antioxidant and antihypertensive properties. Four experimental factors were considered (cellulase percentage, protease percentage, hydrolysis temperature, and hydrolysis duration) for three responses (protein content, antioxidant activity, and antihypertensive activity). The optimal conditions determined by the DOE allowed producing a scaled-up hydrolysate with 45% protein, with antioxidant activity, measured by oxygen radical absorbance capacity assay, of 1035 µmol TE/g protein, IC50 for angiotensin-converting enzyme inhibition activity of 286 µg protein/mL, and α-glucosidase inhibition of 31% (30 mg hydrolysate/mL). The obtained hydrolysates can be used as functional ingredients for food and nutraceuticals due to their antioxidant, antihypertensive, and antidiabetic potential. Moreover, the antioxidant potential of the extracts may be relevant for the cosmetic industry, especially in antiaging formulations.
- Bioactive potential and chromatographic characterization of body mucus from Portugal coastal fish halobatrachus didactylusPublication . Cunha, Marta; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe body mucus in fish provides a stable physical or chemical barrier against invading pathogens. In mucus, antimicrobial peptides are secreted as a response to immunestimulation. Studies have shown antimicrobial activity against multidrugresistant pathogens and low toxicity to eukaryotic cells. Previously, body mucus from five captive Halobatrachus didactylus individuals was collected. We aim to explore the mucus studying molecules with bioactive potential. Size exclusion highperformance liquid chromatography (SEHPLC) analyses were performed on the five body mucus samples showing a similar molecular size distribution with a maximum peak of ca. 800 Da. These five mucus samples were pooled to assess the following bioactivities: antioxidant (ABTS and ORAC), antimicrobial (minimal inhibitory concentration), andcytotoxicity (Caco2 and HaCaT human cell lines). The protein content in the mucus, determined by the bicinchoninic acid methodology, was 16836 ± 1020 µg BSA/mL. The antioxidant activity resulted in 268 ± 11 µmol TE/g mucus protein for ABTS and 306 ± 11 µmol TE/g mucus protein for ORAC. The antibacterial activity was assessed against five pathogenic bacteria: Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, within minimal inhibitory concentrations of 421 to 105 µg mucus protein/mL. Moreover, the mucus showed noncytotoxic for Caco2 cells in concentrations between 196 to 25 µg mucus protein/mL, while it showed cytotoxicity for HaCaT cells. In the future, liquid chromatographytandem mass spectrometry (LCMS/MS) analysis will be performed to determine the molecules behind these bioactivities, namely antimicrobial peptides.
- Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysisPublication . Coscueta, Ezequiel R.; Amorim, Maria M.; Voss, Glenise B.; Nerli, Bibiana B.; Picó, Guillermo A.; Pintado, M. E.Enzymatic hydrolysis of soybean meal protein isolate (SPI) obtained under two temperature conditions with Corolase PP was studied, assessing the impact of hydrolysis on potential antioxidant and antihypertensive activities. The protein was isolated from soybean meal under controlled conditions of time and temperature (70 C, 1 h; 90 C, 30 min). Degree of hydrolysis assessed the progress of hydrolysis at different sampling times. For hydrolysates the antioxidant and angiotensin-converting-enzyme (ACE) inhibitory activities were measured. As observed, the DH was increasing until reaching 20% at 10 h with disappearance of globular proteins and generation of low molecular weight peptides (less than 3 kDa). A significant increase in antioxidant and ACE inhibitory capacities was observed. Five main peptides were identified, which may explain through their sequences the bioactive properties analyzed. Through this study was possible to obtain for the first time with Corolase PP soy hydrolysates with potential antioxidant and ACE inhibitory activities, which can be used to obtain new added value functional ingredients from soy meal.
- Bioactivity and bioaccessibility of bioactive compounds in gastrointestinal digestion of tomato bagasse extractsPublication . Coelho, Marta; Oliveira, Carla; Coscueta, Ezequiel R.; Fernandes, João; Pereira, Ricardo N.; Teixeira, José A.; Rodrigues, António Sebastião; Pintado, Manuela E.A nutrient-rich diet is a key to improving the chemical signals, such as antioxidants, which modulate pathogens’ resistance in the gut and prevent diseases. A current industrial problem is the generation of undervalued by-products, such as tomato bagasse, which are rich in bioactive compounds and of commercial interest (carotenoids and phenolic compounds). This work analyzed the effect of gastrointestinal digestion on the bioactivity and bioaccessibility of carotenoids and phenolic compounds from tomato bagasse extracts. Thus, the extraction by ohmic heating (OH) technology was compared with conventional (organic solvents). The results showed that the main phenolic compounds identified by UPLC-qTOF-MS were p-coumaric acid, naringenin, and luteolin. A higher recovery index for total phenolic compounds throughout the gastrointestinal digestion was observed for OH while for carotenoids, a strong reduction after stomach conditions was observed for both extracts. Furthermore, colon-available fraction exhibited a prebiotic effect upon different Bifidobacterium and Lactobacillus, but a strain-dependent and more accentuated effect on OH. Thus, the extraction technology highly influenced bioaccessibility, with OH demonstrating a positive impact on the recovery of bioactive compounds and related health benefits, such as antioxidant, anti-hypertensive, prebiotic, and anti-inflammatory properties. Of these properties, the last is demonstrated here for the first time.
- Bioprospecting bioactive peptides in halobatrachus didactylus body mucus: from in silico insights to essential in vitro validationPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, Maria ManuelaFish body mucus plays a protective role, especially in Halobatrachus didactylus, which inhabits intertidal zones vulnerable to anthropogenic contaminants. In silico predicted bioactive peptides were identified in its body mucus, namely, EDNSELGQETPTLR (HdKTLR), DPPNPKNL (HdKNL), PAPPPPPP (HdPPP), VYPFPGPLPN (HdVLPN), and PFPGPLPN (HdLPN). These peptides were studied in vitro for bioactivities and aggregation behavior under different ionic strengths and pH values. Size exclusion chromatography revealed significant peptide aggregation at 344 mM and 700 mM ionic strengths at pH 7.0, decreasing at pH 3.0 and pH 5.0. Although none exhibited antimicrobial properties, they inhibited Pseudomonas aeruginosa biofilm formation. Notably, HdVLPN demonstrated potential antioxidant activity (ORAC: 1.560 μmol TE/μmol of peptide; ABTS: 1.755 μmol TE/μmol of peptide) as well as HdLPN (ORAC: 0.195 μmol TE/μmol of peptide; ABTS: 0.128 μmol TE/μmol of peptide). Antioxidant activity decreased at pH 5.0 and pH 3.0. Interactions between the peptides and mucus synergistically enhanced antioxidant effects. HdVLPN and HdLPN were non-toxic to Caco-2 and HaCaT cells at 100 μg of peptide/mL. HdPPP showed potential antihypertensive and antidiabetic effects, with IC50 values of 557 μg of peptide/mL for ACE inhibition and 1700 μg of peptide/mL for α-glucosidase inhibition. This study highlights the importance of validating peptide bioactivities in vitro, considering their native environment (mucus), and bioprospecting novel bioactive molecules while promoting species conservation.