Browsing by Author "Almada, Frederico"
Now showing 1 - 10 of 12
Results Per Page
Sort Options
- 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 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.
- 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.
- Exploring bioactivities and peptide content of body mucus from the lusitanian toadfish Halobatrachus didactylusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Marques, Rita; Neto, José; Almada, Frederico; Gonçalves, David; Pintado, ManuelaIdentifying bioactive molecules from marine organisms is still vastly understudied. Fish remain an untapped source of bioactive molecules, even when considering species whose toxicity to other fish species has been noticed before. We assessed potential applications of crude body mucus of the Lusitanian toadfish (Halobratachus didactylus) and characterized its peptide fraction composition. Mucus samples from three individuals (two wild and one captive) revealed potential antioxidant, antihypertensive, and antimicrobial activities. For antioxidant activity, the best results of 2371 ± 97 µmol Trolox Equivalent/g protein for ORAC and 154 ± 6 µmol Trolox Equivalent/g protein for ABTS were obtained. For antihypertensive activity, the relevant inhibitory activity of ACE resulted in IC50 of 60 ± 7 µg protein/mL. Antimicrobial activity was also identified against the pathogenic bacteria Escherichia coli and Listeria monocytogenes. The peptide profile of the crude body mucus was obtained through size exclusion chromatography, with a conspicuous peak at ca. 800 Da. LC-MS/MS allowed the detection of the most probable peptide sequences of this dominant peptide. This is the first study where the bioactive potential of mucus from the Lusitanian toadfish is demonstrated. Peptides with such properties can be applied in the food and pharmaceutical industries.
- First bioactive characterization of the skin mucus from Portugal coastal fish halobatrachus didactylusPublication . Cunha, Marta; Coscueta, Ezequiel R.; Brassesco, Maria Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe marine environment has several promising features for the discovery of new molecules. The oceans have half of the planet’s biodiversity and are a harsh environment for aquatic organisms challenging them to have a set of bioactive molecules to survive [1]. It is well known the mucus secreted by fish epidermis works as a defense barrier against harmful elements from the external environment [2]. Therefore, this study aimed to explore some bioactive properties such as antimicrobial activity (agar drop diffusion method), antioxidant activity (ABTS and ORAC scavenging assays) and ACE inhibitory activity (iACE) exerted by the mucus collected scraping the Halobatrachus didactylus skin, from the Tagus estuary. Direct drop application did not show inhibitory activity on the growth of E. coli and S. aureus. The protein contentin the mucus, determined by the bicinchoninic acid methodology, was 13260 ± 342 μg BSA/mL. The antioxidant activity resulted in 1.49 ± 0.04 μmol TE/mL for ABTS and 5.47 ± 0.8 μmol TE/mL for ORAC. iACE resulted in an IC50 of 60 ± 7 μg protein/mL. Also, a peptidic profile of the mucus was obtained through size exclusion chromatography showing a shortchain peptide profile (> 3000 Da) as general distribution, which is consistent with the observed bioactivities. In conclusion, fish mucus peptide fraction showed potential as an antioxidant and even more so as an antihypertensive, but not as an antimicrobial. Furthermore, more studies are needed to discover the key molecules behind these bioactivities.
- First bioactive characterization of the skin mucus from Portugal coastal fish Halobatrachus didactylusPublication . Cunha, Marta; Coscueta, Ezequiel R.; Brassesco, Maria Emilia; Almada, Frederico; Gonçalves, David; Pintado, Manuela
- Methods for the collection of fish mucus: a systematic reviewPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe aquatic environment holds a vast source of organisms that provide numerous opportunities to bioprospect new molecules. Notably, fish are producers of an epidermal mucus that offers protection against pathogens, making it a promising source of bioactive molecules. This source of molecules, however, has yet to be thoroughly explored, and particularly, optimization of methods for collection and study is needed. This review concentrates on the methods of mucus collection employed to secure high-quality samples, enabling the extraction and characterization of molecules with bioactive potential. A comprehensive search was conducted, and publications were selected based on the following criteria: (i) the mucus has been collected from the external body of the fish, not involving dissection or damage; (ii) mucus crude extracts have undergone a chemical or genetic characterization; (iii) mucus was used in bioactivity assays (e.g., antimicrobial or immune-related). Scraping, bagging, and absorption are the primary methods for collecting fish mucus. They were assessed based on fish handling, sample volume, and processing, including anesthesia and starvation. Scraping with a soft tool, such as cotton balls or sponges, proved most effective and minimized contamination, dilution, and injury risk. This review aids future studies of mucus composition and properties.
- NanoLC-MS/MS sequencing and in silico analysis of bioactive peptides from Lusitanian toadfish halobatrachus didactylus body mucusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe mucus covering a fish's body works as a protective barrier and contains molecules, such as antimicrobial peptides, that defend against pathogens. In this study, two adult Halobatrachus didactylus were captured from the wild in Sesimbra, and mucus was collected by scraping the dorsal-lateral body with a sponge. Our objective was to identify new bioactive peptides in mucus samples via chromatography. SE-HPLC revealed a prominent peak at 775 Dalton. The mucus samples were pooled, fractionated, and analyzed by nanoLC-MS/MS. Database identification was unreliable, so de novo sequencing with PEAKS Studio was used, resulting in five peptides selected for their predicted bioactivities.
- NanoLC-MS/MS sequencing and in silico analysis of bioactive peptides from Lusitanian Toadfish Halobatrachus didactylus body mucusPublication . Cunha, Marta Fernandez; Coscueta, Ezequiel R.; Brassesco, María Emilia; Almada, Frederico; Gonçalves, David; Pintado, ManuelaThe mucus covers the fish's body, working as a protective barrier. Besides physical protection, mucus provides molecules that protect the fish from damaging pathogens [1,2]. Has been reported that antimicrobial peptides are secreted in the mucus, which play an essential role in defense against microbial pathogens since these belong to the innate immune system [2,3].