Synthetic-biological approach for production of neoxanthin in Escherichia coli.

Carotenoids are isoprenoid pigments produced typically in plants, algae, and part of bacteria and fungi. Violaxanthin, neoxanthin, and lutein are xanthophylls biosynthesized specifically in land plants and part of algae. Nowadays, it is feasible to produce violaxanthin and lutein in Escherichia coli by pathway engineering, whereas there is no report to synthesize neoxanthin in E. coli. So far, several genes have been reported to code for neoxanthin synthases, e.g., NSY (NXS), ABA4 and VDL, which were assigned to catalyze a reaction for forming neoxanthin from violaxanthin. However, neither gene of these was common in plants or algae that biosynthesize neoxanthin, nor was confirmed by the E. coli complementation system. This study showed that the algal VDL gene (PtVDL1) was functional in recombinant E. coli cells accumulating violaxanthin to produce neoxanthin, whereas the E. coli cells failed to generate neoxanthin, when the NSY or ABA4 gene was introduced there instead of VDL. This result notes that VDL is one of veritable neoxanthin synthase genes.

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.

Production of Carotenoids from Cultivated Seaweed.

Cladosiphon (C.) okamuranus, a brown alga endemic to the Nansei Islands, Japan, has been conventionally ingested as food. Nowadays, it is a major aquatic product of the Okinawa Prefecture with an annual production of around 20,000 tons. The life cycle of C. okamuranus comprises the macroscopic sporophyte (algal body) generation and the microscopic gametophyte generation. The germlings in the latter generation can proliferate when floating in seawater. This floating form has been exploited in techniques involved in the commercial production of C. okamuranus seedlings.Brown algae contain fucoxanthin, a carbonyl carotenoid known to have anticancer, anti-obesity, and antidiabetic effect in addition to the anti-oxidation effect. We found that the fucoxanthin content of cultivated floating form of C. okamuranus discoid germlings becomes up to 50 times that of the mature alga. Since the discoid germlings repeatedly grow like microorganisms, although they are large algae, they are utilized to produce fucoxanthin. We optimize the culture conditions by changing the temperature, light intensity, photoperiod, light wavelength, and nutrient salt conditions for optimal fucoxanthin productivity. The cultivation has been successful to industrial plant scale, culminating in the use of 1 ton of cultivating medium.In brown algal cells, fucoxanthin is primarily found bound to the photosynthetic pigment-protein complexes known as fucoxanthin-chlorophyll protein (FCP). Consequently cultivated floating form of C. okamuranus also shows high content of FCP. Isolation and characterization of pigments bound to the FCP were determined precisely, and ultrafast spectroscopies were applied to elucidate the photosynthetic function of fucoxanthin bound to the pigment-protein complexes. This cultivation method has also been applied to the other edible brown algae. We found that the optimal cultivation conditions as well as the yields of fucoxanthin and FCP highly depend on the species.The floating form cultivation was also applied to a large-sized edible green alga, Codium intricatum, which is uniquely producing a carbonyl carotenoid, siphonaxanthin. This has several anti-disease effects and is also a primal photosynthetic pigment which is found bound to photosynthetic antenna complex usually called siphonaxanthin-chlorophyll protein (SCP). We are working on the improvement of productivity, scale-up of production, and development of cultivation technology of new macro algae.

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.

Are Helicobacter pylori Infection and Fucoidan Consumption Associated with Fucoidan Absorption?

We examined the associations of Helicobacter pylori and mozuku consumption with fucoidan absorption. Overall, 259 Japanese volunteers consumed 3 g fucoidan, and their urine samples were collected to measure fucoidan values and H. pylori titers before and 3, 6, and 9 h after fucoidan ingestion. Compared to the basal levels (3.7 ± 3.4 ng/mL), the urinary fucoidan values significantly increased 3, 6, and 9 h (15.3 ± 18.8, 24.4 ± 35.1, and 24.2 ± 35.2 ng/mL, respectively) after fucoidan ingestion. The basal fucoidan levels were significantly lower in H. pylori-negative subjects who rarely ate mozuku than in those who regularly consumed it. Regarding the ΔMax fucoidan value (highest value - basal value) in H. pylori-positive subjects who ate mozuku at least once a month, those aged ≥40 years exhibited significantly lower values than <40 years old. Among subjects ≥40 years old who regularly consumed mozuku, the ΔMax fucoidan value was significantly lower in H. pylori-positive subjects than in H. pylori-negative ones. In H. pylori-positive subjects who ate mozuku at least once monthly, basal fucoidan values displayed positive correlations with H. pylori titers and ΔMax fucoidan values in subjects <40 years old. No correlations were found in H. pylori-positive subjects who ate mozuku once every 2-3 months or less. Thus, fucoidan absorption is associated with H. pylori infection and frequency of mozuku consumption.

Activation of NK cells in male cancer survivors by fucoidan extracted from Cladosiphon okamuranus.

Cancer survivors are highly motivated to seek information about the use of dietary supplements and complementary nutritional therapies to improve their quality of life. Fucoidan, a sulfated polysaccharide extracted from brown marine alga, exhibits a wide range of bioactivities, including anticancer activity. As natural killer (NK) cells serve an important role in defenses against tumor cells, the present study examined the effects of fucoidan extracted from Cladosiphon Okamuranus on NK cell activity in cancer survivors. A prospective, open-label clinical study was conducted on cancer survivors treated with fucoidan via oral administration; 11 cancer survivors with a performance status of 0 or 1 participated and consumed 3 g of fucoidan for 6 months. No significant changes were observed in the mean activities of NK cells in total subjects following the ingestion of fucoidan. An analysis of each sex revealed that NK cell activity was significantly increased by the ingestion of fucoidan in male, yet not female subjects. Serum fucoidan levels were markedly increased following the ingestion of fucoidan and the peak levels ranged between 30 and 198 ng/ml. Tumor markers remained within the reference range during the trial period in subjects, in whom primary tumors were eradicated by treatment. The basal values of tumor markers were elevated in three cases; tumor markers were increased in two cases and decreased in one by the ingestion of fucoidan. These findings suggest that fucoidan enhances the activation of NK cells in male cancer survivors.

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.

Absorption Study of Mozuku Fucoidan in Japanese Volunteers.

We performed an oral administration study of fucoidan in 396 Japanese volunteers and investigated significant factors concerning the absorption of fucoidan. Urine samples were collected at 0, 3, 6, and 9 h after ingestion of 3 g of fucoidan. Fucoidan was detected in urine after ingestion in 385 out of 396 subjects. The maximum value (mean ± standard deviation (SD)) of urinary fucoidan was 332.3 ± 357.6 µg/gCr in subjects living in Okinawa prefecture, compared with 240.1 ± 302.4 µg/gCr in subjects living outside Okinawa. Compared with the estimated urinary excretion of fucoidan by place of residence, those of subjects living in Okinawa prefecture were significantly higher than those living outside Okinawa prefecture (p < 0.01). In addition, subjects living in Okinawa prefecture consumed significantly greater amounts of mozuku compared with those living outside Okinawa prefecture (p < 0.01). Multiple regression analysis showed that having Okinawa prefecture as a place of residence was a significant factor (p < 0.01) contributing to the estimated urinary excretion of fucoidan. Because the habit of eating mozuku was significantly higher (p < 0.01) in subjects living in Okinawa prefecture than in those living outside Okinawa prefecture, the habit of eating mozuku was speculated to be a factor in the absorption of fucoidan.

Strategies to enhance the excitation energy-transfer efficiency in a light-harvesting system using the intra-molecular charge transfer character of carotenoids.

Fucoxanthin is a carotenoid that is mainly found in light-harvesting complexes from brown algae and diatoms. Due to the presence of a carbonyl group attached to polyene chains in polar environments, excitation produces an excited intra-molecular charge transfer. This intra-molecular charge transfer state plays a key role in the highly efficient (∼95%) energy-transfer from fucoxanthin to chlorophyll a in the light-harvesting complexes from brown algae. In purple bacterial light-harvesting systems the efficiency of excitation energy-transfer from carotenoids to bacteriochlorophylls depends on the extent of conjugation of the carotenoids. In this study we were successful, for the first time, in incorporating fucoxanthin into a light-harvesting complex 1 from the purple photosynthetic bacterium, Rhodospirillum rubrum G9+ (a carotenoidless strain). Femtosecond pump-probe spectroscopy was applied to this reconstituted light-harvesting complex in order to determine the efficiency of excitation energy-transfer from fucoxanthin to bacteriochlorophyll a when they are bound to the light-harvesting 1 apo-proteins.

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.

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

INTRODUCTION: In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. 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. RESULTS: 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. CONCLUSION: We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells.

Intestinal absorption of fucoidan extracted from the brown seaweed, Cladosiphon okamuranus.

The aim of this study was to examine the absorption of fucoidan through the intestinal tract. Fucoidan (0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL) was added to Transwell inserts containing Caco-2 cells. The transport of fucoidan across Caco-2 cells increased in a dose-dependent manner up to 1.0 mg/mL. It reached a maximum after 1 h and then rapidly decreased. In another experiment, rats were fed standard chow containing 2% fucoidan for one or two weeks. Immunohistochemical staining revealed that fucoidan accumulated in jejunal epithelial cells, mononuclear cells in the jejunal lamina propria and sinusoidal non-parenchymal cells in the liver. Since we previously speculated that nitrosamine may enhance the intestinal absorption of fucoidan, its absorption was estimated in rats administered N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water. Rats were fed 0.2% fucoidan chow (BBN + 0.2% fucoidan rats), 2% fucoidan chow (BBN + 2% fucoidan rats) and standard chow for eight weeks. The uptake of fucoidan through the intestinal tract seemed to be low, but was measurable by our ELISA method. Fucoidan-positive cells were abundant in the small intestinal mucosa of BBN + 2% fucoidan rats. Most fucoidan-positive cells also stained positive for ED1, suggesting that fucoidan was incorporated into intestinal macrophages. The uptake of fucoidan by Kupffer cells was observed in the livers of BBN + 2% fucoidan rats. In conclusion, the absorption of fucoidan through the small intestine was demonstrated both in vivo and in vitro.

Light-dependent conformational change of neoxanthin in a siphonous green alga, Codium intricatum, revealed by Raman spectroscopy.

Siphonous green algae, a type of deep-sea green algae, appear olive drab and utilize blue-green light for photosynthesis. A siphonous green alga, Codium (C.) intricatum, was isolated from Okinawa prefecture in Japan, and a clonal algal culture in filamentous form was established. The major light-harvesting antenna was analogous to the trimeric LHCII found in higher plants, but the C. intricatum complex contained an unusual carbonyl carotenoid siphonaxanthin. Culture conditions were optimized to achieve high siphonaxanthin content in intact lyophilized filamentous bodies. Interestingly, the carotenoid composition was different when cultured under high irradiance: all-trans neoxanthin was accumulated in addition to the normal 9'-cis form in whole cell extract. Resonance Raman spectra of intact filamentous bodies, cultured under high- and low-light conditions, confirmed the accumulation of all-trans neoxanthin under high irradiance conditions. A plausible function of the presence of all-trans neoxanthin will be discussed in relation to the regulation against high light stress.

Characterization of the intramolecular transfer state of marine carotenoid fucoxanthin by femtosecond pump-probe spectroscopy.

Fucoxanthin, containing a carbonyl group in conjugation with its polyene backbone, is a naturally occurring pigment in marine organisms and is essential to the photosynthetic light-harvesting function in brown alga and diatom. Fucoxanthin exhibits optical characteristics attributed to an intramolecular charge transfer (ICT) state that arises in polar environments due to the presence of the carbonyl group. In this study, we report the spectroscopic properties of fucoxanthin in methanol (polar and protic solvent) observed by femtosecond pump-probe measurements in the near-infrared region, where transient absorption associated with the optically allowed S2 (1(1)B u (+) ) state and stimulated emission from the strongly coupled S1/ICT state were observed following one-photon excitation to the S2 state. The results showed that the amplitude of the stimulated emission of the S1/ICT state increased with decreasing excitation energy, demonstrating that the fucoxanthin form associated with the lower energy of the steady-state absorption exhibits stronger ICT character.

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 ~56 kDa 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.

The pigment stoichiometry in a chlorophyll a/c type photosynthetic antenna.

The trimeric fucoxanthin-chlorophyll a/c protein (FCP) was purified from a Japanese brown alga, Cladosiphon okamuranus TOKIDA. Its pigment stoichiometry was determined to be chlorophyll (Chl) a:Chl c (1):Chl c (2):fucoxanthin = 4.6:1.1:1.0:5.5 by a combination of binary HPLC and (1)H NMR spectroscopy. No violaxanthin found bound to the FCP. The ratio of Chl c/Chl a in this FCP is amongst the highest so far reported.

Ultrafast excited state dynamics of fucoxanthin: excitation energy dependent intramolecular charge transfer dynamics.

Carotenoids containing a carbonyl group in conjugation with their polyene backbone are naturally-occurring pigments in marine organisms and are essential to the photosynthetic light-harvesting function in aquatic algae. These carotenoids exhibit spectral characteristics attributed to an intramolecular charge transfer (ICT) state that arise in polar solvents due to the presence of the carbonyl group. Here, we report the spectroscopic properties of the carbonyl carotenoid fucoxanthin in polar (methanol) and nonpolar (cyclohexane) solvents studied by steady-state absorption and femtosecond pump-probe measurements. Transient absorption associated with the optically forbidden S(1) (2(1)A) state and/or the ICT state were observed following one-photon excitation to the optically allowed S(2) (1(1)B) state in methanol. The transient absorption measurements carried out in methanol showed that the ratio of the ICT-to-S(1) state formation increased with decreasing excitation energy. We also showed that the ICT character was clearly visible in the steady-state absorption in methanol based on a Franck-Condon analysis. The results suggest that two spectroscopic forms of fucoxanthin, blue and red, exist in the polar environment.

Attenuation of N-nitrosodiethylamine-induced liver fibrosis by high-molecular-weight fucoidan derived from Cladosiphon okamuranus.

BACKGROUND AND AIM: Liver fibrosis is closely associated with the progression of various chronic liver diseases. Fucoidan exhibits different biological properties such as anti-inflammatory, anti-oxidant and anti-fibrotic activities. The aim of this study was to determine whether oral fucoidan administration inhibits N-nitrosodiethylamine (DEN)-induced liver fibrosis. METHODS: Liver fibrosis was induced in rats by injecting DEN (50 mg/kg). Rats were given 2% of crude fucoidan solution or 2% of high-molecular-weight (HMW) fucoidan solution. They were divided into a crude fucoidan group, an HMW fucoidan group, a DEN alone group, a DEN + crude fucoidan group, a DEN + HMW fucoidan group and a control group. RESULTS: Liver fibrosis and hepatic hydroxyproline levels were significantly more decreased in the DEN + HMW fucoidan group than in the DEN-alone group. Anti-fibrogenesis was unremarkable in the DEN + crude fucoidan group. Hepatic messenger RNA levels and immunohistochemistry of transforming growth factor beta 1 were markedly increased by DEN. This increase was attenuated by HMW fucoidan. Hepatic chemokine ligand 12 expression was increased by DEN. This increase was suppressed by HMW fucoidan. HMW fucoidan significantly decreased the DEN-induced malondialdehyde levels. Also, fucoidan markedly increased metallothionein expression in the liver. Fucoidan was clearly observed in the liver by immunohistochemical staining in HMW fucoidan-treated rats, while it was faintly stained in the livers of crude fucoidan-treated rats. CONCLUSION: These findings suggest that the HMW fucoidan treatment causes anti-fibrogenesis in DEN-induced liver cirrhosis through the downregulation of transforming growth factor beta 1 and chemokine ligand 12 expressions, and that scavenging lipid peroxidation is well-incorporated in the liver.

Development of a fucoidan-specific antibody and measurement of fucoidan in serum and urine by sandwich ELISA.

Fucoidan exhibits various biological properties. We raised a novel antibody against fucoidan extracted from Cladosiphon okamuranus and developed a sandwich ELISA method to measure fucoidan. The fucoidan antibody was specific and did not cross-react with other polysulfated polysaccharides. Fucoidan recovery from serum and urine by ELISA was 86-113%. Intra- and inter-assay CVs were 1.5-13.4%. Assay linearity was maintained after 3-fold dilution of each sample with phosphate-buffer saline (PBS). In the serum and urine of healthy volunteers (n=10), fucoidan was not detected before administration, and the levels markedly increased 6 and 9 h after oral administration. The molecular weight of the serum fucoidan determined by HPLC gel filtration remained unchanged, whereas that of urine fucoidan was significantly reduced. This is the first ELISA method of measuring serum and urine fucoidan levels after oral administration. The method is simple, reliable, and practical for the analysis of samples, especially urine samples.

Inhibitory effect of fucoidan on Huh7 hepatoma cells through downregulation of CXCL12.

The aim of this study is to assess whether fucoidan modulates the expression of chemokine ligand 12 (CXCL12)/chemokine receptor 4 (CXCR4) and exerts antitumor activity toward Huh7 hepatoma cells. According to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fucoidan inhibited the growth of Huh7 cells and HepG2 cells in a dose-dependent manner, with a 50% inhibition of cell growth (IC50) of 2.0 and 4.0 mg/ml, respectively. alpha-fetoprotein levels in medium collected from fucoidan-treated cells were significantly decreased in Huh7 cells but not in HepG2 cells. Western blotting revealed that the amount of alpha-fetoprotein was decreased by 1.0 mg/ml of fucoidan in Huh7 cells, whereas it was unchanged in HepG2 cells. In Huh7 cells, CXCL12 mRNA expression was significantly downregulated by 1.0 mg/ml of fucoidan, whereas CXCR4 mRNA expression was unchanged by fucoidan. CXCL12 and CXCR4 mRNA were barely expressed in HepG2 cells. In addition, 1.0 mg/ml of fucoidan mildly arrested the cell cycle and induced apoptosis in Huh7 cells. The findings suggest that fucoidan exhibits antitumor activity toward Huh7 cells through the downregulation of CXCL12 expression.