Okinawa Mozuku Research Articles

Effects of Ingesting Fucoidan Derived from Cladosiphon okamuranus Tokida on Human NK Cells: A Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Pilot Study.

The aim of this study was to evaluate the effects of ingesting fucoidan derived from Okinawa mozuku (Cladosiphon okamuranus) on natural killer (NK) cell activity and to assess its safety in healthy adults via a randomized, double-blind, parallel-group, placebo-controlled pilot study. Subjects were randomly divided into two groups—a placebo group (ingesting citric acid, sucralose, and caramel beverages; n = 20; 45.5 ± 7.8 years (mean ± standard deviation)) and a fucoidan group (3.0 g/day from beverages; n = 20; 47.0 ± 7.6 years); after 12 weeks, blood, biochemical, and immunological tests were performed. Clinically adverse events were not observed in any of the tests during the study period. In addition, adverse events due to the test food were not observed. In the immunological tests, NK cell activity was significantly enhanced at 8 weeks in the fucoidan group, compared to before ingestion (0 weeks). In addition, a significantly enhanced NK cell activity was observed in male subjects at 8 weeks, compared with the placebo group. These results confirm that Okinawa mozuku-derived fucoidan enhances NK cell activity and suggest that it is a safe food material.

Comparative genomics of four strains of the edible brown alga, Cladosiphon okamuranus

BackgroundThe brown alga, Cladosiphon okamuranus (Okinawa mozuku), is one of the most important edible seaweeds, and it is cultivated for market primarily in Okinawa, Japan. Four strains, denominated S, K, O, and C, with distinctively different morphologies, have been cultivated commercially since the early 2000s. We previously reported a draft genome of the S-strain. To facilitate studies of seaweed biology for future aquaculture, we here decoded and analyzed genomes of the other three strains (K, O, and C).ResultsHere we improved the genome of the S-strain (ver. 2, 130 Mbp, 12,999 genes), and decoded the K-strain (135 Mbp, 12,511 genes), the O-strain (140 Mbp, 12,548 genes), and the C-strain (143 Mbp, 12,182 genes). Molecular phylogenies, using mitochondrial and nuclear genes, showed that the S-strain diverged first, followed by the K-strain, and most recently the C- and O-strains. Comparisons of genome architecture among the four strains document the frequent occurrence of inversions. In addition to gene acquisitions and losses, the S-, K-, O-, and C-strains possess 457, 344, 367, and 262 gene families unique to each strain, respectively. Comprehensive Blast searches showed that most genes have no sequence similarity to any entries in the non-redundant protein sequence database, although GO annotation suggested that they likely function in relation to molecular and biological processes and cellular components.ConclusionsOur study compares the genomes of four strains of C. okamuranus and examines their phylogenetic relationships. Due to global environmental changes, including temperature increases, acidification, and pollution, brown algal aquaculture is facing critical challenges. Genomic and phylogenetic information reported by the present research provides useful tools for isolation of novel strains.

Intestinal Microbiota and Immune Modulation in Zebrafish by Fucoidan From Okinawa Mozuku (Cladosiphon okamuranus).

Fucoidan represents fucose-rich sulfated polysaccharides derived from brown seaweeds, which exerts various biological activities applicable for functional foods and therapeutic agents. The objective of the present study was to investigate in vivo effects of fucoidan extracted from Okinawa mozuku (Cladosiphon okamuranus), common edible seaweed in Japan, on immune responses and microbiota composition in zebrafish. We treated larvae and adult zebrafish with Okinawa mozuku (OM) fucoidan by immersion (100 and 500 μg/mL, 3 days) and by feeding (3 weeks), respectively. The effect of OM fucoidan on immune responses in zebrafish larvae was evaluated by live imaging of neutrophils and macrophages as well as quantitative polymerase chain reaction of pro- and anti-inflammatory cytokine genes. Whole microbiota of zebrafish larvae and intestinal microbiota of adult zebrafish treated with OM fucoidan were analyzed by Illumina MiSeq pair-end sequencing of the V3–V4 region of 16S rRNA genes. Fucoidan treatment only slightly affected the composition of the larvae microbiota and the number of neutrophils and macrophages, while pro- and anti-inflammatory cytokine gene expression levels were upregulated in the larvae treated with 500 μg/mL OM fucoidan. In contrast, feeding of OM fucoidan clearly altered the intestinal microbiota composition of adult zebrafish, which was characterized by the emergence and predominance of multiple bacterial operational taxonomic units (OTUs) affiliated with Rhizobiaceae and Comamonadaceae at the expense of E. coli-related Enterobacteriaceae, the dominant OTUs throughout the studied samples. These changes were accompanied by decreased expression levels of pro-inflammatory cytokine il1b in the intestines of the adult zebrafish. Our current study provides the first insights into in vivo modulatory effects of fucoidan on microbiota and immune responses of unchallenged zebrafish, which underscores the potential of fucoidan to play a modulatory role in the diet–microbiota–host interplay.

Evaluation of the Immunomodulatory Effects of Fucoidan Derived from Cladosiphon Okamuranus Tokida in Mice.

Okinawa mozuku (Cladosiphon okamuranus Tokida) is an edible seaweed classified as brown algae and is a native species of the Ryukyu Islands in Japan. In recent years, the genomic decoding of Okinawa mozuku has been completed. Previous studies on the anti-inflammatory, antiviral, and antitumor properties of Okinawa mozuku have suggested that it affects the regulation of cellular and humoral immunity. The aim of the present study was to examine the immunoregulatory effect of fucoidan derived from Okinawa mozuku in mice. A product containing fucoidan (purity, 88.3%; molecular weight, 49.8 kDa) was developed from Okinawa mozuku and tested for its immunoregulatory effects in mice. The experimental animals were 8-week-old female BALB/c mice to which fucoidan (0, 102.5, 205.0, 410.0, and 1025.0 mg/kg) was administered orally continuously for six weeks. Immune cell proliferation, cytokine production, macrophage phagocytosis, and serum antibody concentration were measured. We found that immune cell proliferation, interleukin (IL)-2, macrophage phagocytes, and serum antibodies (IgM, -G, -A) increased significantly, but IL-4, -5, and IgE decreased significantly. These results indicated that fucoidan modulated cellular and humoral immunity.

Antithrombotic Effect of Oral Administration of Mozuku (Cladosiphon okamuranus, Brown Seaweed) Extract in Rat.

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.

Improvement of defecation in healthy individuals with infrequent bowel movements through the ingestion of dried Mozuku powder: a randomized, double-blind, parallel-group study

Background: Okinawa mozuku (Cladosiphon okamuranu) is a type of edible seaweed of the family Chordariaceae that typically contains the polysaccharide fucoidan as a functional ingredient. In Okinawa, raw mozuku is eaten as vinegared mozuku together with vinegar or as tempura (deep-fried in batter). Polysaccharides such as fucoidan are generally known to regulate intestinal function, which is why we have used Okinawa mozuku to investigate this intestinal regulatory effect.Methods: The study was designed as a randomized, double-blind, parallel group study. Dried Okinawa mozuku powder at a dose of 2.4 g/day (1.0 g/day of fucoidan) and a placebo not containing any dried Okinawa mozuku powder were each made into capsules and given to healthy men and women with infrequent weekly bowel movements (2–4 movements a week) to ingest for eight weeks. We then investigated changes in the defecation situation, blood tests, and adverse events. Results: In the group that ingested the capsules containing dried Okinawa mozuku powder, the number of days with a bowel movement significantly increased compared with the placebo group after four weeks of ingestion (p < 0.05). Furthermore, after eight weeks of ingestion, the same increasing trend was seen compared with the placebo group (p = 0.0964). The volume of stool also increased significantly in the dried Okinawa mozuku powder group after eight weeks compared with the placebo group. In terms of blood tests and adverse events, no adverse events occurred that were the result of the test food.Conclusions: Ingestion of Okinawa mozuku was found to have a regulatory effect on intestinal function by promoting defecation in healthy individuals with a tendency for constipation. This demonstrated that Okinawa mozuku is a functional food capable of making defecation smoother and increasing the volume of stool.Key Words: Okinawa mozuku, Cladosiphon okamuranus, fucoidan, dry powder, bowel movement, constipation

Detection of Fucoidan in Urine after Oral Intake of Traditional Japanese Seaweed, Okinawa mozuku (Cladosiphon okamuranus Tokida).

Seaweed has been considered an indigestible food. Fucoidan, which is found abundantly in seaweed, especially in Cladosiphon okamuranus (Okinawa mozuku), has a high molecular weight and has been long believed to be hardly absorbed in the human digestive system due to a lack of certain digestive enzymes. We previously reported that fucoidan can be detected in serum and urine after oral intake of purified fucoidan in humans and rats. However, it is unclear whether the fucoidan in mozuku can be absorbed after digestion of mozuku. Therefore, we attempted to detect fucoidan in urine before and after mozuku intake. We determined the fucoidan concentration in urine after oral intake of Okinawa mozuku and urinary fucoidan was detected in several volunteers. In conclusion, these results suggest that fucoidan in mozuku can be absorbed after ingestion of mozuku.

A draft genome of the brown alga, Cladosiphon okamuranus, S-strain: a platform for future studies of 'mozuku' biology.

The brown alga, Cladosiphon okamuranus (Okinawa mozuku), is economically one of the most important edible seaweeds, and is cultivated for market primarily in Okinawa, Japan. C. okamuranus constitutes a significant source of fucoidan, which has various physiological and biological activities. To facilitate studies of seaweed biology, we decoded the draft genome of C. okamuranus S-strain. The genome size of C. okamuranus was estimated as ∼140 Mbp, smaller than genomes of two other brown algae, Ectocarpus siliculosus and Saccharina japonica. Sequencing with ∼100× coverage yielded an assembly of 541 scaffolds with N50 = 416 kbp. Together with transcriptomic data, we estimated that the C. okamuranus genome contains 13,640 protein-coding genes, approximately 94% of which have been confirmed with corresponding mRNAs. Comparisons with the E. siliculosus genome identified a set of C. okamuranus genes that encode enzymes involved in biosynthetic pathways for sulfated fucans and alginate biosynthesis. In addition, we identified C. okamuranus genes for enzymes involved in phlorotannin biosynthesis. The present decoding of the Cladosiphon okamuranus genome provides a platform for future studies of mozuku biology.

Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography

In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60×8.0mm I.D., 30nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210nm) is from 0 to 100ng of fucoidan with the linear calibration line inserting to zero. A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10mM sodium azide (respiration inhibitor) and 0.05mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all. We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells.

Excitation Energy-Transfer Dynamics of Brown Algal Photosynthetic Antennas.

Fucoxanthin-chlorophyll-a/c protein (FCP) complexes from brown algae Cladosiphon okamuranus TOKIDA (Okinawa Mozuku in Japanese) contain the only species of carbonyl carotenoid, fucoxanthin, which exhibits spectral characteristics attributed to an intramolecular charge-transfer (ICT) property that arises in polar environments due to the presence of the carbonyl group in its polyene backbone. Here, we investigated the role of the ICT property of fucoxanthin in ultrafast energy transfer to chlorophyll-a/c in brown algal photosynthesis using femtosecond pump-probe spectroscopic measurements. The observed excited-state dynamics show that the ICT character of fucoxanthin in FCP extends its absorption band to longer wavelengths and enhances its electronic interaction with chlorophyll-a molecules, leading to efficient energy transfer from fucoxanthin to chlorophyll-a.

Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku).

A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L)kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.

Toxicological evaluation of fucoidan from Cladosiphon okamuranus.

Fucoidans are sulfated polysaccharides extracted from brown seaweeds. They have a wide spectrum of activity in biological systems. Besides their well-known anticoagulant and anti-thrombotic activity, fucoidans modulate inflammation, possess antiproliferative and anti-adhesive effects on cells, protect cells from viral infection, and interfere with mammalian fertilization. Fucoidans are now used as raw material for development of drugs and are also widely used as a health-promoting food component. However, studies on the toxicity of fucoidans from different brown algae are limited. The present study examined the acute toxicity of varying levels of fucoidan extracted from Okinawa mozuku, a brown alga (Cladosiphon okamuranus), in Wistar rats after oral administration. No significant toxicological changes were induced by fucoidan at a dose of 600 mg/kg of body weight/day. However, with concentrations at and above 1,200 mg/kg of body weight/day, clotting time was significantly prolonged. No other signs of toxicity were observed.

Fucoidan, a major component of brown seaweed, prohibits the growth of human cancer cell lines in vitro

Fucoidan, the general term for sulfated polysaccharides, is reported to engage in various biological activities having anti-tumor, anti-coagulation and anti-viral effects. Though it has been investigated, the mechanism of its anti-tumor effects remains elusive. The current study examined the anti-tumor effects of fucoidan extracted from Okinawa mozuku on 15 human cancer cell lines (6 hepatocellular carcinomas, 1 cholangiocarcinoma, 1 gallbladder cancer, 2 ovarian cancers, 1 hepatoblastoma, 1 neuroblastoma and 3 renal cancers) using an MTT assay. Changes in apoptosis and the cell cycle were analyzed by flow cytometry. The results revealed that cell proliferation was suppressed in 13 cell lines in a time- and/or dose-dependent manner; this suppression was marked in the hepatocellular carcinoma, cholangiocarcinoma and gallbladder carcinoma cell lines. In contrast, proliferation of the neuroblastoma and 1 of the 2 ovarian carcinoma cell lines was not affected. The ratio of apoptotic cells significantly increased in 5 of the 6 hepatocellular carcinoma cell lines, and the ratio of G2/M cells increased in the 3 hepatocellular cell lines examined. These observations indicate that fucoidan is a potential anti-tumor agent for the treatment of bile duct cancers, such as hepatocellular carcinoma, cholangiocarcinoma and gall-bladder carcinoma.

Proportion of Murine Cytotoxic T Cells is Increased by High Molecular-Weight Fucoidan Extracted from Okinawa mozuku (Cladosiphon okamuranus)

Male BALB/c mice were administered three fucoidans with different molecular weights prepared from Okinawa mozuku (Cladosiphon okamuranus). The proportion of CD8+ cells in the spleens of mice fed with high molecular-weight fucoidan significantly increased compared with those in mice fed a control diet. In addition, the CD4+/CD8+ ratio tended to decrease and the proportion of CD11b+ cells to increase. These results suggest that high molecular-weight fucoidan promotes an increase in the proportion of murine cytotoxic T cells.

食用海藻類のラット盲腸内フローラおよび血清脂質レベルに及ぼす影響

To investigate the dietary effects of six different edible marine algae on the intestinal microflora and the level of serum lipids, diets containing 1% and 5% of the algae were administered to male rats for one week. These algae increased fecal weight. Cecal weight was increased by the diets containing 5% kombu Laminaria sp., 5% wakame Undaria pinatifida, and 5% mozuku (okinawa mozuku Cladosiphon okamuranus). In the case of the 5% aosa (hitoegusa Monostroma nitidum) diet, cecal pH value was higher. Most of the algae suppressed cecal ammonia. These marine algae had similar effects on serum lipid, such as the suppression of levels of serum triglycerides and free fatty acids, though these algae had no effect on serum cholesterol levels. On the other hand, these marine algae showed different effects on cecal microflora. The ratio of bifidobacteria to total viable counts increased in rats fed diets containing mekabu (sporophyll of wakame).