The potent activity of sulfated polysaccharide, ascophyllan, isolated from Ascophyllum nodosum to induce nitric oxide and cytokine production from mouse macrophage RAW264.7 cells: Comparison between ascophyllan and fucoidan

Abstract

Ascophyllan isolated from the brown alga Ascophyllum nodosum is a fucose-containing sulfated polysaccharide, which has similar but distinct characteristic monosaccharide composition and entire chemical structure to fucoidan. In this study, we examined the effects of ascophyllan, fucoidan isolated from A. nodosum (A-fucoidan), and fucoidan from Sigma (S-fucoidan) as a representative fucoidan derived from other source (Fucus vesiculosus) on mouse macrophage cell line RAW264.7 cells. No significant cytotoxic effects of ascophyllan and A-fucoidan on RAW264.7 cells were observed up to 1000μg/ml, while S-fucoidan showed cytotoxic effect in a concentration-dependent manner. Ascophyllan induced extremely higher level of nitric oxide (NO) production from RAW264.7 cells than those induced by fucoidans over the concentration range tested (0-200μg/ml). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that expression level of inducible NO synthase (iNOS) in ascophyllan-treated RAW264.7 cells was much higher than the levels detected in the cells treated with fucoidans. Furthermore, the activities of ascophyllan to induce the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from RAW264.7 cells were also greater than those induced by fucoidans especially at lower concentration range (3.1-50μg/ml). The activities of ascophyllan to induce NO and cytokine production in mouse peritoneal macrophages were also stronger than those of fucoidans. Electrophoretic mobility shift assay (EMSA) using infrared dye labeled nuclear factor-kappa B (NF-κB) and AP-1 consensus sequences suggested that ascophyllan can strongly activate these transcription factors. Marked increase in the nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also observed in ascophyllan-treated RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors and western blot analysis suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase are mainly involved in ascophyllan-induced NO production.

Reevaluation of bactericidal, cytotoxic, and macrophage-stimulating activities of commercially available Fucus vesiculosus fucoidan

“…Besides some structural and bioactive similarities between fucoidan and ascophyllan (Heinzelmann et al 1998, Nakayasu et al 2009), our previous study demonstrated that ascophyllan showed a growth-promoting activity on MDCK cells, while fucoidan was rather toxic to this cell line (Jiang et al 2010). During the course of our comparative studies on the cellular level biological activities of sulfated polysaccharides from different seaweed species, we found that F. vesiculosus fucoidan, which is the only commercially available one, was highly cytotoxic to RAW264.7 cells, whereas ascophyllan and fucoidan isolated from A. nodosum showed no significant cytotoxicity on RAW264.7 cells up to 1,000 μg mL -1 (Jiang et al 2011). Fucoidans isolated from several algal species including commercially available fucoidan have been reported to possess numerous biological activities such as anticoagulant (Pereira et al 1999), antithrombotic activities (Kusaykin et al 2008), antiviral (Karmakar et al 2010, Sinha et al 2010, antitumor, anti-inflammatory (Cumashi et al 2007, Croci et al 2011), immuno-modulatory (Raghavendran et al 2011, and apoptosis-inducing activities (Jin et al 2010.…”

“…To estimate NO level in RAW264.7 cells, nitrite, a stable reaction product of NO with molecular oxygen, was measured by Griess assay as described previously (Jiang et al 2011). In brief, adherent RAW264.7 cells in 96-well plates (3 × 10 4 cells well -l ) were incubated with varying concentrations of test samples for 24 h in the growth medium at 37°C, and then the nitrite levels in the culture medium were measured. )…”

“…were treated with varying concentrations of test samples in the growth medium at 37°C. After 24 h incubation, the levels of TNF-α in the culture supernatants of treated cells were measured by sandwich ELISA with two antibodies to two different epitopes on TNF-α molecule as described previously (Jiang et al 2011). The TNF-α concentrations were estimated from a reference to a standard curve for serial three-fold dilution of murine recombinant TNF-α.…”

“…We have previously reported that F-fucoidan, but not A-fucoidan and ascophyllan, showed cytotoxic effect on RAW264.7 cells (Jiang et al 2011). To investigate the involvement of the methanol-extractable agents of F-fucoidan in the cytotoxicity of F-fucoidan, we examined the Table 2.…”

“…To investigate the involvement of the methanol-extractable agents of F-fucoidan in the cytotoxicity of F-fucoidan, we examined the Table 2. Chemical composition analysis of the methanol-extract of Fucoidans including commercially available F. vesiculosus fucoidan are known to induce secretion of NO and cytokines from RAW264.7 cells (Jiang et al 2011). We investigated whether or not the methanol-extract is involved in such activities of F-fucoidan.…”