Introduction
Rugulopteryx okamurae is a species of brown macroalgae native to the Pacific Ocean that has become highly invasive along the southwestern coasts of Europe. Its proliferation has led to significant economic impacts on the fishing sector, as well as serious ecological consequences, threatening the biodiversity of marine ecosystems. To mitigate the effects associated with the accumulation of algal biomass, current research is exploring its valorization through applications such as compost production, the development of bioplastic materials, and its use as a source of bioactive compounds (Barcellos et al., 2023). However, information on the functionality of bioactive compounds derived from this macroalgal species remains limited. This study aimed to characterize two lipophilic extracts obtained from R. okamurae using ethanol at two different concentrations and to evaluate their antioxidant and immunomodulatory properties. For this purpose, lipid extracts ROLT100 and ROLT50 were obtained using ethanol and ethanol:water (1:1, v/v), respectively. Lipid composition as well as in vitro antioxidant and immunomodulatory activities were subsequently assessed.
Material and methods
The brown seaweed R. okamurae was collected in spring from Faro de Punta Carnero (Algeciras). Algal biomass was washed, lyophlised and milled. Thereafter, 5 g of algal biomass powder were incubated in 50 ml of two different solvents: ethanol and an ethanol:water mixture (1:1, v/v), yielding a more hydrophobic (ROLT100) and a more hydrophilic (ROLT50) fraction, respectively. At the end of the extraction, the suspensions were filtered with a strainer and lyophilized. Lipid content was determined as described previously by C��rdoba-Granados et al. 2024. Macrophages isolation was performed as described by Ponce et al. 2024 from head kidney of Dicentrarchus labrax adults (average weight 369.23 21.89 g) provided by Cupibar S.L. (C��diz, Spain). After 24h of incubation with the extracts (5, 50 and 100 µg ml-1) cells were collected, cell viability was determined by trypan blue assay, total RNA was isolated using the RNeasy �� Mini Kit (Qiagen) and expression analyses were performed by RT-qPCR. The selected genes related to the immune system were lysozyme g (lyg), interleukin 1b (il1b), interleukin 6 (il6), interleukin 10 (il10) and tumor necrosis factor alpha (tnf).
Results and discussion
The yield of the lyophilized ROLT100 and ROLT50 extracts obtained from R. okamurae was 13.48 0.09 and 17.82 0.26% of the algal dry weight, respectively. The total lipid as well as the neutral and glycolipid content were higher in the ROLT100 extract than in the ROLT 50 extract (Table 1). In the same way, ROLT100 showed a greater total phenolic content and enhanced antioxidant activity, as evidenced by higher DPPH and ABTS radical scavenging activities. Cell viability decreased significantly when macrophages were incubated with both extracts at concentrations of 50 and 100 µg ml-1 with respect to the controls. Therefore, subsequent gene expression analyses were conducted in macrophages incubated with ROLT100 and ROLT50 at a non-cytotoxic concentration of 5 µg ml-1. A significant coordinated decrease in the mRNA levels of lyg, i1lb, il6, il10 and tnf was observed in macrophages treated with ROLT100 compared to the controls. In contrast, macrophages incubated with ROLT50 showed a significant downregulation of lyg, while the expression levels of il10 and tnf were significantly upregulated relative to the controls. The differential gene expression patterns observed may be partially related to the presence of diterpenoids, including dilkamural-type compunds, previously described in R. okamurae, which have been reported to display anti-inflammatory activities in macrophages (Cuevas et al., 2021). The coordinated decreased in cytokine-related gene expression observed in macrophages treated with ROLT100 is consistent with a higher content of terpenes. Overall, the results indicate that both lipid extracts have antioxidant activity and can modulate macrophage immune responses, although the cytotoxic effects observed at higher concentrations highlight the importance of dose selection when considering their potential bioactive applications.
Table 1
Lipid content (mg g-1 dry weight of the extract) in the extracts obtained from R. okamurae. Values are expressed as average and standard deviation (n = 3).
Total lipids
Neutral fraction
Glycolipid fraction
Fucoxantine
Fucosterol
Terpenes
SQDG
DGDG
MGDG
ROLT100
874.56 18.14
136.55 2.09
77.93 2.07
165.52 9.23
33.93 1.78
20.52 3.81
62.34 1.68
ROLT50
427.25 30.31
70.39 7.46
28.04 3.85
110.46 17.00
30.19 5.74
14.24 1.68
28.61 2.49
SQDG: sulfoquinovosyldiacylglycerols; DGDG: digalactosyldiacylglycerol; MGDG: monogalactosyldiacylglycerol;
Acknowledgment
This work was supported by the project PR.FEMPA.DIP2023A.001 co-financed at 70% by the European Maritime, Fisheries and Aquaculture Fund.
References
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