Introduction
Seaweeds are a source of bioactive compounds, including proteins, amino acids, polysaccharides fatty acids, vitamins, pigments and polyphenols with antioxidant, antimicrobial and antiviral functions. In recent years, there has been a growing interest in the research of polysaccharides of marine origin obtained from macroalgae being the most investigated for their potential applications in the fields of biomedicine and biotechnology: fucoidan, alginate and laminarin, present in brown macroalgae; ulvan obtained from green macroalgae; agar and carrageenan from red macroalgae (Salehi et al., 2019). Recently, the role of ulvan extracted from Ulva ohnoi as immunonutrient in Solea senegalensis, a species of great interest in aquaculture, has been reported (Ponce et al., 2024). Fucoidan is formed mainly by L-fucose and sulfate groups, although monosaccharides such as xylose, mannose, galactose, rhamnose and glucose, uronic acids and proteins may also be part of its structure. Like other macroalgae polysaccharides, fucoidan has a broad spectrum of biological properties, including antioxidant, immunomodulatory, antimicrobial, antiviral, anticancer and anticoagulant properties (Luthuli et al., 2019). The aim of this study was to characterize a sulphated polysaccharides extract obtained from the brown macroalgae Rugulopteryx okamurae and evaluate its antioxidant and immune activity. For this reason, sulphated polysaccharides extract was obtained using the hot water method and isolated by precipitation with ethanol. Chemical characterization and in vitro antioxidant and immunomodulatory activity was evaluated. Finally, the antimicrobial activity against Photobacterium damselae subsp. piscicida, an important fish pathogen, was determined.
Material and methods
R. okamurae from Faro de Punta Carnero (Algeciras) was collected in spring. After washed, lyophilised, and milled, algal powder was mixed with ethanol 80% and stirred overnight at room temperature. The solution was centrifuged, the sediment was dried at 45ºC for 24 h and thereafter, it was treated with hot water (70ºC) for 2 h. The extract was centrifuged, and this process was repeated twice. The supernatant was collected and precipitated with ethanol overnight at 4ºC. The precipitated extract was recovered by centrifugation and freeze-dried. Total protein concentration was determined using the Bradford reagent following the manufacturer´s instructions for the 96 well plate assay. Sulfate, L-fucose, fucoidan and uronic acid content were determined as described previously by (January et al., 2019). Total phenolic assay was performed by the Folin- Ciocalteu method. For in vitro assays, macrophages from head kidney of 9 S. senegalensis juveniles obtained from facilities of IFAPA Centro El Toruño were extracted and cultured as described by Secombes and Fletcher, (1992). After 24 h of incubation with the sulphated polysaccharides extract (0.05, 0.5 y 1 mg ml-1), cells were collected. Total RNA was isolated using the RNeasy® Mini Kit (Qiagen) and expression analyses were performed by RT-qPCR.
Results and discussion
The yield of lyophilised sulphated polysaccharides extract obtained from R. okamurae was 3.16 ± 1.31% of the algal dry weight. High levels of L-fucose, a high sulphate content and low levels of contaminants such as uronic acid and protein are considered characteristics of good quality fucoidan (Ale et al., 2011). The sulphated polysaccharides extract obtained in this study contained 13.33 ± 2.92%, 5.34 ± 0.93% and 11.45 ± 2.14% of sulphate, L-fucose and fucoidan, respectively. Moreover, protein and uronic acid content was 2.34 ± 0.20 and 15.70 ± 0.81%, respectively. The protein content was lower than 3% indicating the purity of the sulphated polysaccharides extract. The main sugars were fucose (23.55 ± 0.03%), mannose (21.14 ± 1.0%) and xylose (15.17 ± 0.91) followed by galactose (13.78 ± 1.44%), glucuronic acid (10.19 ± 0.74%), glucose (8.58 ± 0.9) and rhamnose (8.01 ± 0.93%).
The results obtained in this study indicate that the sulphated polysaccharides extract has antioxidant activity (Table 1). The polyphenol content of macroalgae contributes significantly to their antioxidant and immunomodulatory properties. Therefore, these results could be explained by the higher content of phenolic compounds associated with the sulphated polysaccharides extract. To investigate the effects of the sulphated polysaccharides extract on the defence system, the expression level of a panel of genes related to the immune system was evaluated in Senegalese sole macrophages. The selected genes were as follows: lysozyme g (lyg), interleukin 1b (il1b), interleukin 6 (il6), interleukin 10 (il10), complement C3 (c3), hepcidin (hamp) and transferrin (tf). A significant coordinated increase in the mRNA levels of il1b, il6, il10, hamp and tf were observed in macrophages incubated with the sulphated polysaccharides extract at all concentrations assayed (0.05, 0.5 and 1 mg ml-1) with respect to the control groups whereas no significant differences were observed in c3 expression levels. All these results together with the antibacterial activity of the sulphated polysaccharides extract will be presented and discussed in depth during the oral presentation.
Acknowledgements
This work was supported by the project PR.FEMPA.DIP2023A.001 co-financed at 70% by the European Maritime, Fisheries and Aquaculture Fund.
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