Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to ß-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Suppressive effects of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on the production of NO and ROS in LPS-stimulated RAW264.7 cells.

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

Therapeutic effects of an orally administered edible seaweed-derived polysaccharide preparation, ascophyllan HS, on a Streptococcus pneumoniae infection mouse model.

Ascophyllan HS is a commercially available preparation of the edible brown alga Ascophyllum nodosum containing ascophyllan, a sulfated polysaccharide with diverse beneficial biological activities. In this study, the effects of ascophyllan HS were evaluated in a severe intranasal Streptococcus pneumoniae infection mouse model. The control untreated mice started to die on day 7 and 80% had died by day 14 post-infection. Continuous oral administration of ascophyllan HS before and after bacterial infection resulted in a remarkable increase in survival rate, with 90% of the low (167 mg/kg body weight/day) and 100% of the high (500 mg/kg body weight/day) dose ascophyllan HS-treated mice surviving at day 14 post-infection. Histopathological observation of the lungs of the infected mice revealed the induction of typical pneumonia features in the alveolar spaces of the untreated control mice, such as extensive infiltration of inflammatory cells, edema, and fibrin deposition. In contrast, notable levels of lung injuries or alterations were not observed in the ascophyllan HS-treated mice, and only a minor lesion was observed in one mouse. Furthermore, bacterial burdens in the lungs were significantly reduced in the ascophyllan HS-treated mice as compared to the control mice at day 4 post-infection. Significantly higher levels of IL-12 were detected in the serum of ascophyllan HS-treated mice than that of control mice measured at the end of the infection experiment (day 14). These results suggest that orally administered ascophyllan HS exerts a therapeutic effect on S. pneumoniae infection by activating the host defense systems. This is the first report of the therapeutic effect of an orally administered seaweed polysaccharide preparation on S. pneumoniae infection. Our findings suggest that ascophyllan HS has the potential to be developed as nutraceuticals and pharmaceuticals applicable for humans as well as a safe and promising therapeutic agent against S. pneumoniae infection.

Ascophyllan Induces Activation of Natural Killer Cells in Mice In Vivo and In Vitro.

Natural marine polysaccharides have demonstrated immune stimulatory effects in both mice and humans. Our previous study compared the ability of ascophyllan and fucoidan to activate human and mouse dendritic cells (DCs). In this study, we further examined the effect of ascophyllan on the activation of mouse natural killer (NK) cells in vivo and in vitro and compared it to that of fucoidan, a well-studied natural marine polysaccharide. Specifically, administration of ascophyllan to C57BL/6 mice increased the number of NK cells in the spleen when compared to the number in PBS-treated mice. Moreover, the number of IFN-γ-producing NK cells and expression of CD69 were markedly upregulated by ascophyllan treatment. Ascophyllan treatment also induced IFN-γ production and CD69 upregulation in isolated NK cells, but did not promote cell proliferation. Finally, ascophyllan treatment increased the cytotoxicity of NK cells against Yac-1 cells. The effects of ascophyllan on NK cell activation were considerably stronger than those of fucoidan. These data demonstrated that ascophyllan promotes NK cell activation both in mice and in vitro, and its stimulatory effect on NK cells is stronger than that of fucoidan.

Activation of Human Dendritic Cells by Ascophyllan Purified from Ascophyllum nodosum.

In our previous study, we showed that ascophyllan purified from Ascophyllum nodosum treatment promotes mouse dendritic cell (DC) activation in vivo, further induces an antigen-specific immune response and has anticancer effects in mice. However, the effect of ascophyllan has not been studied in human immune cells, specifically in terms of activation of human monocyte-derived DCs (MDDCs) and human peripheral blood DCs (PBDCs). We found that the treatment with ascophyllan induced morphological changes in MDDCs and upregulated co-stimulatory molecules and major histocompatibility complex class I (MHC I) and MHC II expression. In addition, pro-inflammatory cytokine levels in culture medium was also dramatically increased following ascophyllan treatment of MDDCs. Moreover, ascophyllan promoted phosphorylation of ERK, p38 and JNK signaling pathways, and inhibition of p38 almost completely suppressed the ascophyllan-induced activation of MDDCs. Finally, treatment with ascophyllan induced activation of BDCA1 and BDCA3 PBDCs. Thus, these data suggest that ascophyllan could be used as an immune stimulator in humans.

Suppressive effect of ascophyllan HS on postprandial blood sugar level through the inhibition of α-glucosidase and stimulation of glucagon-like peptide-1 (GLP-1) secretion.

A sulfated polysaccharide ascophyllan inhibited α-glucosidase in a concentration dependent manner, and >90% activity was inhibited at 1.0 mg/mL. The inhibitory activity was much higher than that of acarbose. No significant inhibitory effect of ascophyllan on α-amylase was observed up to 10.0 mg/mL. Ascophyllan HS, a commercially available ascophyllan preparation showed even higher inhibitory effect on α-glucosidase than ascophyllan. Interestingly, ascophyllan and ascophyllan HS induced the secretion of glucagon-like peptide-1 (GLP-1) from human intestinal NCI-H716 cell line in a concentration dependent manner (10-100 ng/mL). The oral glucose tolerance tests revealed that after continuous 8-week ingestion of ascophyllan HS at 100 mg/day, the glucose area under the curve values of the ascophyllan HS ingested group were significantly lower than placebo ingested group. Serum glycosylated hemoglobin (HbA1c) level in ascophyllan HS ingested group tended to decrease after 8-week ingestion, whereas no significant change was observed in placebo ingested group. This is the first report indicating that ascophyllan can induce the secretion of GLP-1 from human intestinal cell line (NCI-H716), besides the potent inhibitory effect on α-glucosidase. Furthermore, clinical trial suggested that ascophyllan HS may be a practically applicable blood glucose controlling agent in humans.

Acidic polysaccharides isolated from marine algae inhibit the early step of viral infection.

We examined the effects of various acidic polysaccharides isolated from marine algae on the infection and replication of human immunodeficiency virus type-1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type-1 (HTLV-1). It was found that sulfated fucan polysaccharides, ascophyllan, and two fucoidans derived from different sources significantly inhibited the early step of HIV-1 (R9 and JR-FL) infection, while they did not affect the late step. The alginate oligomer consisted of uronic acids and sulfated-galactan porphyran showed no significant inhibitory effects. In addition, ascophyllan and two fucoidans inhibited the early step of HBV infection in a dose-dependent manner. Furthermore, these polysaccharides inhibited the early step of HCV infection but had no inhibitory effects on HTLV-1 replication. To further examine the specificity of these polysaccharides in viral infections, we used vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection. Ascophyllan, the two fucoidans, and alginate oligomer also potently inhibited VSV-G-pseudotyped HIV-1 infection in HeLa cells. Taken together, these results suggest that the acidic polysaccharides used in this study are capable of inhibiting the early step of viral infections depending on the polysaccharides but not in a strict species-specific manner.

Ascophyllan functions as an adjuvant to promote anti-cancer effect by dendritic cell activation.

Our previous study demonstrated that ascophyllan, a sulfated polysaccharide purified from brown alga, has immune-activating effects. In this study, we evaluated ascophyllan as an adjuvant for its therapeutic and preventive effect on tumor in a mouse melanoma model. Ascophyllan induced migration of DCs to spleen and tumor-draining lymph node (drLN) in a mouse B16 melanoma model. Moreover, ascophyllan induced activation of dendritic cells (DCs), and promoted IFN-γ- and TNF-α-producing Th1 immune responses in tumor-bearing mice. In addition, treatment with a combination of ascophyllan and ovalbumin (OVA) in the tumor-bearing mice promoted proliferation of OVA-specific CD4 and CD8 T cells and migration of those cells into the tumor, consequently inhibiting the tumor growth. Immunization with the combination of ascophyllan and OVA caused enhanced OVA-specific antibody production and memory T cell responses compared to OVA immunization alone, and almost completely prevented B16-OVA tumor growth upon subsequent tumor challenge. Finally, the combination of ascophyllan and OVA prevented B16-OVA tumor invasion and metastasis into the liver. Thus, these results demonstrate that ascophyllan can function as an adjuvant to induce DC activation, antigen specific CTL activation, Th1 immune response and antibody production, and hence may be useful as a therapeutic and preventive tumor vaccine.

Anti-metastatic effects of the sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on B16 melanoma.

We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells.

Ascophyllan purified from Ascophyllum nodosum induces Th1 and Tc1 immune responses by promoting dendritic cell maturation.

Marine-derived sulfated polysaccharides have been shown to possess certain anti-virus, anti-tumor, anti-inflammatory and anti-coagulant activities. However, the in vivo immunomodulatory effects of marine-derived pure compounds have been less well characterized. In this study, we investigated the effect of ascophyllan, a sulfated polysaccharide purified from Ascophyllum nodosum, on the maturation of mouse dendritic cells (DCs) in vitro and in vivo. Ascophyllan induced up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in bone marrow-derived DCs (BMDCs). Moreover, in vivo administration of ascophyllan promotes up-regulation of CD40, CD80, CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen cDCs. Interestingly, ascophyllan induced a higher degree of co-stimulatory molecule up-regulation and pro-inflammatory cytokine production than fucoidan, a marine-derived polysaccharide with well-defined effect for promoting DC maturation. Ascophyllan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in the presence of DCs in an IL-12-dependent manner. Finally, myeloid differentiation primary response 88 (MyD88) signaling pathway was essential for DC maturation induced by ascophyllan. Taken together, these results demonstrate that ascophyllan induces DC maturation, and consequently enhances Th1 and Tc1 responses in vivo. This knowledge could facilitate the development of novel therapeutic strategies to combat infectious diseases and cancer.

Inhibitory effect of orally-administered sulfated polysaccharide ascophyllan isolated from ascophyllum nodosum on the growth of sarcoma-180 solid tumor in mice.

We evaluated the antitumor activity of crude extract and ascophyllan prepared from Ascophyllum nodosum in sarcoma-180 solid tumor-bearing mice with continuous intraperitoneal (i.p.) administration at a dose of 50 mg/kg body weight/day or oral administration at a dose of 500 mg/kg body weight/day. Ascophyllan and crude extract administered via the oral route showed greater antitumor effects than via i.p. route, and the tumor sizes in mice treated with ascopyllan and crude extract were reduced by a mean of 68.7±6.8% and 42.4±24.8% by the oral route, and 41.4±16.1% and 13.6±20.6% by i.p. route compared to control mice. Splenic natural killer cell activity in the mice treated with ascophyllan and crude extract by i.p. route was significantly enhanced, while only a slight increase of this activity was observed in orally-treated mice. Furthermore, increase in spleen weight of tumor-bearing mice was slightly suppressed by oral administration of ascophyllan, whereas i.p. administration resulted in further enlargement. Analysis of serum cytokines revealed that oral treatment with ascophyllan resulted in significant increase of tumor necrosis factor-α and interleukin-12 levels. Since ascophyllan showed no direct cytotoxic effect on sarcoma-180 cells, orally-administered ascophyllan is suggested to exhibit its antitumor activity through the activation of the host immune system.

Importance of sulfate groups for the macrophage-stimulating activities of ascophyllan isolated from the brown alga Ascophyllum nodosum.

To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.

In vitro antioxidant activities of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum.

Antioxidant activities of sulfated polysaccharide ascophyllan from Ascophyllum nodosum was investigated in vitro by various assays, and compared with those of fucoidan. A chemiluminescence (CL) analysis using a luminol analog, L-012, showed that ascophyllan scavenges superoxide, and the activity is greater than fucoidan. However, in the presence of 10µg/ml of ascophyllan or 10µg/ml and 100µg/ml of fucoidan, slightly enhanced CL-responses were observed. Since EDTA-treatment resulted in disappearance of the enhancement effects, it was suggested that metal ions especially iron ions in the polysaccharides might be involved in this phenomenon. In fact, metal element analysis revealed that ascophyllan and fucoidan inherently contain iron and other metal elements. EDTA-treatment resulted in significant increase in Fe(2+)-chelating activities of these polysaccharides. In an electron spin resonance (ESR)-spin trapping analysis in which direct UV-radiation to hydrogen peroxide was used as a source of hydroxyl radical, ascophyllan and fucoidan showed potent hydroxyl radical scavenging activity with similar extent. Reducing power of ascophyllan was stronger than that of fucoidan. Our results indicate that ascophyllan can exhibit direct and potent antioxidant activity.

Stimulatory effect of the sulfated polysaccharide ascophyllan on the respiratory burst in RAW264.7 macrophages.

In this study, we investigated the bioactivity of ascophyllan in terms of reactive oxygen species (ROS) generation. In RAW264.7 cells, we found that ascophyllan induced ROS generation in a concentration-dependent manner, but with bell-shaped profile. Immunoblot analysis demonstrated that ascophyllan promoted the translocation of cytosolic subunits (p67(phox) and p47(phox)) of NADPH oxidase to the plasma membrane. Among mitogen activated protein (MAP) kinase inhibitors tested, JNK inhibitor showed the most potent inhibitory effect on ascophyllan-induced ROS production. Consistently, significant level of phosphorylated JNK MAP kinase was detected in ascophyllan-treated RAW264.7 cells. Our findings suggest for the first time that ascophyllan can stimulate macrophages to produce ROS through the activations of NADPH oxidase and JNK MAP kinase.

Immunostimulatory activities of the sulfated polysaccharide ascophyllan from Ascophyllum nodosum in in vivo and in vitro systems.

Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.

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.

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.

Effects of sulfated fucan, ascophyllan, from the brown Alga Ascophyllum nodosum on various cell lines: a comparative study on ascophyllan and fucoidan.

The effects of fucose-containing sulfated polysaccharides, ascophyllan and fucoidan, isolated from the brown alga Ascophyllum nodosum, on the growth of various cell lines (MDCK, Vero, PtK(1), CHO, HeLa, and XC) were investigated. In a colony formation assay, ascophyllan and fucoidan showed potent cytotoxic effects on Vero and XC cells, while other cell lines were relatively resistant to these polysaccharides. Almost no significant effects of these polysaccharides were observed in the cell lines tested using the Alamar blue cytotoxicity assay over 48 h with varying initial cell densities (2500-20,000 cells/well) in growth medium. Interestingly, a significant growth promoting effect of ascophyllan on MDCK cells was observed, whereas treatment with fucoidan showed growth suppressive effects on this cell line under the same experimental conditions. These results suggest that ascophyllan is distinguishable from fucoidan in terms of their bioactivities. This is the first report of the growth promoting effects of a sulfated fucan on a mammalian cell line under normal growth conditions.