Browsing by Author "Cunha, Marta"
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- 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.
- 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
- Peptide fraction identification by SE-HPLC and LC-MS/MS analysis of the body mucus from Portugal coastal fish Halobatrachus didactylusPublication . Cunha, Marta; 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 pathogens damaging. 1,2These include antimicrobial peptides secreted in the mucus, which play an essential role in defense against microbial pathogens since these belong to the innate immune system2,3. In this study, two adult Halobatrachus didactylus individuals were captured from the wild in Sesimbra. Then, mucus collection was performed by scraping the dorsal-lateral body of the fish with a sponge. Our objective was the identification of new peptides with bioactive potential in mucus samples by chromatography analysis. Size exclusion high-performance liquid chromatography (SE-HPLC) analysis performed on mucus samples from the two individuals revealed a similar profile with an intense highlight peak which resulted in a distribution of about 775 Dalton. With interest in that peak, the two mucus samples were pooled for fractionation by SEC. The resulting fraction was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the most probable peptide sequences. Identification from databases did not provide reliable results, indicating a lack of information on the matrix analyzed. We resorted to de novo sequencing with good results using PEAKS Studio software. Five identified peptides were selected according to their bioactivities predicted in silico. Furthermore, the five identified peptides were synthesized, and the molecular size was validated by SE-HPLC analysis. Overall, this chromatographic approach enabled the identification of promising peptides, which bioactivities will be evaluated in vitro in future work.