Polyphenol-rich fraction from Ecklonia cava (a brown alga) processing by-product reduces LPS-induced inflammation in vitro and in vivo in a zebrafish model

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  • ABSTRACT

    Ecklonia cava is a common edible brown algae that is plentiful in Jeju Island of Republic of Korea. Polyphenols from E. cava have strong anti-inflammatory activity. However, a large number of the by-products from E. cava processing are discarded. In the present study, to utilize these by-products, we assessed the anti-inflammatory activity of the polyphenol-rich fraction (PRF) from E. cava processing by-product (EPB) in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. Four compounds, namely eckol, eckstolonol, dieckol, and phlorofucofuroeckol-A, were isolated and identified from PRF. We found that PRF suppressed the production of nitric oxide (NO), inducible nitric oxide synthase, and cyclooxygenase-2 in the LPS-induced cells. Furthermore, the protective effect of PRF was investigated in vivo in LPS-stimulated inflammation zebrafish model. PRF had a protective effect against LPS-stimulated toxicity in zebrafish embryos. In addition, PRF inhibited LPS-stimulated reactive oxygen species and NO generation. According to the results, PRF isolated from EPB could be used as a beneficial anti-inflammatory agent, instead of discard.


  • KEYWORD

    anti-inflammation , by-product , Ecklonia cava , polyphenol , seaweeds

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  • [Fig. 1.] High-performance liquid chromatography (HPLC) chromatogram and HPLC-diode array detection / electrospray ionization mass spectrometryspectra of ethyl acetate fraction from Ecklonia cava processing by-product.
    High-performance liquid chromatography (HPLC) chromatogram and HPLC-diode array detection / electrospray ionization mass spectrometryspectra of ethyl acetate fraction from Ecklonia cava processing by-product.
  • [Fig. 2.] Nitric oxide (NO) production and cytotoxicity by Ecklonia cava processing by-product (EPB) and polyphenol-rich fraction (PRF) on lipopolysaccharide (LPS)-induced RAW264.7 cells. The production of nitric oxide was assayed in the culture medium of cells incubated with LPS (1 μg mL-1) for after 24 h in the presence of EPB and PRF (25, 50, and 100 μg mL-1). Cytotoxicity was determined by lactate dehydrogenase (LDH) assay. ■, % NO production; □, cytotoxicity. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-eValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
    Nitric oxide (NO) production and cytotoxicity by Ecklonia cava processing by-product (EPB) and polyphenol-rich fraction (PRF) on lipopolysaccharide (LPS)-induced RAW264.7 cells. The production of nitric oxide was assayed in the culture medium of cells incubated with LPS (1 μg mL-1) for after 24 h in the presence of EPB and PRF (25, 50, and 100 μg mL-1). Cytotoxicity was determined by lactate dehydrogenase (LDH) assay. ■, % NO production; □, cytotoxicity. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-eValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 3.] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression by polyphenol-rich fraction (PRF) in RAW264.7 cells. Cells (1 × 105 cells mL-1) were pre-incubated for 16 h, and the cells were stimulated with lipopolysaccharide (LPS; 1 μg mL-1) in the presence of PRF (25, 50, and 100 μg mL-1) for 24 h. iNOS and COX-2 protein level were determined using a western blot analysis. Equal protein loading was confirmed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-eValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
    Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression by polyphenol-rich fraction (PRF) in RAW264.7 cells. Cells (1 × 105 cells mL-1) were pre-incubated for 16 h, and the cells were stimulated with lipopolysaccharide (LPS; 1 μg mL-1) in the presence of PRF (25, 50, and 100 μg mL-1) for 24 h. iNOS and COX-2 protein level were determined using a western blot analysis. Equal protein loading was confirmed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-eValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 4.] Toxicity of polyphenol-rich fraction (PRF) or lipopolysaccharide (LPS) in zebrafish embryos. Survival rates were assessed after treatment with LPS or co-treatment with PRF. The embryos were stimulated with 10 μg mL-1 LPS and co-treated with PRF (25, 50, and 100 μg mL-1). Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-cValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
    Toxicity of polyphenol-rich fraction (PRF) or lipopolysaccharide (LPS) in zebrafish embryos. Survival rates were assessed after treatment with LPS or co-treatment with PRF. The embryos were stimulated with 10 μg mL-1 LPS and co-treated with PRF (25, 50, and 100 μg mL-1). Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-cValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
  • [Fig. 5.] Reactive oxygen species (ROS) and nitric oxide (NO) production as well as cell death in zebrafish. The zebrafish embryos were exposed to lipopolysaccharide (LPS) (10 μg mL-1) and treated with polyphenol-rich fraction (PRF; 25, 50, and 100 μg mL-1). (A) ROS level. (B) NO level. (C) Cell death. Assessments were measured by image analysis and fluorescence microscope. The fluorescence intensity of individual zebrafish was quantified using image J program. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-dValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.
    Reactive oxygen species (ROS) and nitric oxide (NO) production as well as cell death in zebrafish. The zebrafish embryos were exposed to lipopolysaccharide (LPS) (10 μg mL-1) and treated with polyphenol-rich fraction (PRF; 25, 50, and 100 μg mL-1). (A) ROS level. (B) NO level. (C) Cell death. Assessments were measured by image analysis and fluorescence microscope. The fluorescence intensity of individual zebrafish was quantified using image J program. Experiments were performed in triplicate and the data are expressed as the mean ± standard error. a-dValues with different alphabets are significantly different p < 0.05 as analyzed by Duncan’s multiple range test.