Determination of Polysaccharides in Undaria pinnatifida by Ionic Liquid-Modified Silica Gel Size Exclusion Chromatography

Ionic liquid-modified silica particles with a large pore size were synthesized and used as the stationary phase for size exclusion chromatography coupled with a refractive index detector for the determination of polysaccharides, such as fucoidan, alginic acid, and laminarin from Undaria pinnatifida (seaweed). The molecular weight of polysaccharide was determined by a dextran standard curve (5–1100?kDa). The ionic liquid-modified silica column exhibited excellent size exclusion properties in separating laminarin from other components. The 1-butyl-3-methyl-imidazolium bis-(trifluoromethylsulfonyl)-amino silica column has superior resolution in laminarin separation than the other columns because the amino-group in ionic liquid provide π–π interactions due to aromaticity of the ring structure, which enhances the effect of the hydroxyl group in the target compound separation. The concentrations of polysaccharides were calculated using a standard linear equation to be 0.332–0.484?mg/g of fucoidan, 0.207–0.301?mg/g of alginic acid, and 0.154–0.297?mg/g of laminarin.     

Retention of Large Biological Molecules by Size-Exclusion Chromatography

Size-exclusion chromatography has been developed for the separation of large biological molecules including proteins, polymers, peptides, nucleic acids, and polysaccharide according to their molecular size. This study determined the retention factors for dextran (5, 25, 50, 270, 670, and 1100?kDa) and polysaccharides, such as fucoidan, alginic acid, and laminarin, in the size-exclusion chromatography stationary phase. In addition, the molecular weights and retention factor of three polysaccharides were calculated from the dextran standard curve. The largest retention factor was 4.26 using the size-exclusion chromatography columns (5?kDa dextran). The molecular weights of fucoidan, alginic acid, and laminarin were determined to be 250, 200, and 5 to 64?kDa, respectively.     

Magnetic graphene oxide modified by imidazole‐based ionic liquids for the magnetic‐based solid‐phase extraction of polysaccharides from brown alga

Magnetic graphene oxide was modified by four imidazole‐based ionic liquids to synthesize materials for the extraction of polysaccharides by magnetic solid‐phase extraction. Fucoidan and laminarin were chosen as the representative polysaccharides owing to their excellent pharmaceutical value and availability. Fourier transform infrared spectroscopy, field‐emission scanning electron microscopy, and thermogravimetric analysis were applied to characterize the synthesized materials. Single‐factor experiments showed that the extraction efficiency of polysaccharides was affected by the amount of ionic liquids for modification, solid–liquid ratio of brown alga and ethanol, the stirring time of brown alga and ionic liquid‐modified magnetic graphene oxide materials, and amount of 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide materials added to the brown alga sample solution. The results indicated that 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide possessed better extraction ability than graphene oxide, magnetic graphene oxide, and other three ionic‐liquid‐modified magnetic graphene oxide materials. The highest extraction recoveries of fucoidan and laminarin extracted by 1‐(3‐aminopropyl)imidazole chloride modified magnetic graphene oxide were 93.3 and 87.2%, respectively. In addition, solid materials could be separated and reused easily owing to their magnetic properties.     

Molecularly Imprinted Polymers Modified by Deep Eutectic Solvents and Ionic Liquids with Two Templates for the Simultaneous Solid-Phase Extraction of Fucoidan and Laminarin from Marine Kelp

Molecularly imprinted polymers modified by deep eutectic solvents and ionic liquids (ILs) were prepared as packing materials for the solid-phase extraction (SPE) of fucoidan and laminarin. The prepared materials were characterized by field emission scanning electron microscopy and Fourier transform infrared spectroscopy. The polymers modified by the deep eutectic solvent prepared by choline chloride and urea had the best extraction efficiencies for fucoidan and laminarin (95.5% and 87.6%, respectively) from marine kelp. The relative standard deviations for intraday and interday determination were less than 4.23%. The molecularly imprinted polymers modified by deep eutectic solvents and ILs showed outstanding applications for SPE and may offer novel sample pretreatment for other analytes.     

Magnetic molecularly imprinted polymers for recognition and enrichment of polysaccharides from seaweed

New magnetic molecularly imprinted polymers with two templates were fabricated for the recognition of polysaccharides (fucoidan and alginic acid) from seaweed by magnetic solid‐phase extraction, and the materials were modified by seven types of deep eutectic solvents. It was found that the deep eutectic solvents magnetic molecularly imprinted polymers showed stronger recognition and higher recoveries for fucoidan and alginic acid than magnetic molecularly imprinted polymers, and the deep eutectic solvents‐4‐magnetic molecularly imprinted polymers had the best effects. The practical recovery of the two polysaccharides (fucoidan and alginic acid) purified with deep eutectic solvents‐4‐magnetic molecular imprinted polymers in seaweed under the optimal conditions were 89.87, and 92.0%, respectively, and the actual amounts extracted were 20.6 and 18.7 μg/g, respectively. To sum up, the developed method proved to be a novel and promising method for the recognition of complex polysaccharide samples from seaweed.     

褐藻糖胶及其与胶原复合物的体外抗凝血活性研究

用FTIR、UV对从海带中提取的褐藻糖胶结构进行了分析。采用部分凝血活醇时间、凝血酶原时间和凝血醇时间作为评价标准，对其体外抗凝血活性作了初步的评价，并与肝素的抗凝血活性作比较。结果表明：褐藻糖胶具有类似肝素的多糖结构，具有较好的抗凝血活性，但低于肝素。将褐藻糖胶固定于胶原上，所得的胶原-褐藻糖胶复合物仍具有一定的抗凝血活性。     

Quantitative determination of fucoidan using polyion-sensitive membrane electrodes

The use of polyanion and polycation-sensitive membrane electrodes to detect five different preparations of fucoidan is described. Unlike linear polyanionic molecules previously measured with polymer membrane-based electrochemical sensors, fucoidans from marine brown algae are all highly branched, sulfated polysaccharides with varying charge densities and structures, depending on the species of seaweed, method of extraction used and extent of purification. When tridodecylmethylammonium (TDMA) was used as the ion-exchanger, a large, non-equilibrium EMF response was observed over a concentration range of 0.5–50 μg mL⁻¹ fucoidan. Fucoidan was also measured by titration with polycationic protamine, using a dinonylnaphthalene sulfonate (DNNS)-doped membrane electrode as the potentiometric endpoint detector. Potentiometric titration was used to determine the binding ratio between protamine and fucoidan at the neutralization endpoint for each fucoidan preparation. This binding ratio was then used to successfully determine the fucoidan content of commercially available nutritional supplements. Fucoidan was also measured in undiluted blood serum, demonstrating that this method may be applicable for measuring fucoidan for clinical applications.     

Fucoidan and Alginate from the Brown Algae Colpomenia sinuosa and Their Combination with Vitamin C Trigger Apoptosis in Colon Cancer

Brown seaweeds are producers of bioactive molecules which are known to inhibit oncogenic growth. Here, we investigated the antioxidant, cytotoxic, and apoptotic effects of two polysaccharides from the brown algae Colpomenia sinuosa, namely fucoidan and alginate, in a panel of cancer cell lines and evaluated their effects when combined with vitamin C. Fucoidan and alginate were isolated from brown algae and characterized by HPLC, FTIR, and NMR spectroscopy. The results indicated that highly sulfated fucoidans had higher antioxidant and cytotoxic effects than alginate. Human colon cancer cells were the most sensitive to the algal treatments, with fucoidan having an IC₅₀ value (618.9 µg/mL⁻¹) lower than that of alginate (690 µg/mL⁻¹). The production of reactive oxygen species was increased upon treatment of HCT-116 cells with fucoidan and alginate, which suggest that these compounds may trigger cell death via oxidative damage. The combination of fucoidan with vitamin C showed enhanced effects compared to treatment with fucoidan alone, as evidenced by the significant inhibitory effects on HCT-116 colon cancer cell viability. The combination of the algal polysaccharides with vitamin C caused enhanced degeneration in the nuclei of cells, as evidenced by DAPI staining and increased the subG1 population, suggesting the induction of cell death. Together, these results suggest that fucoidan and alginate from the brown algae C. sinuosa are promising anticancer compounds, particularly when used in combination with vitamin C.     

Anti-Inflammatory Potential of Fucoidan for Atherosclerosis: In Silico and In Vitro Studies in THP-1 Cells

Several diseases, including atherosclerosis, are characterized by inflammation, which is initiated by leukocyte migration to the inflamed lesion. Hence, genes implicated in the early stages of inflammation are potential therapeutic targets to effectively reduce atherogenesis. Algal-derived polysaccharides are one of the most promising sources for pharmaceutical application, although their mechanism of action is still poorly understood. The present study uses a computational method to anticipate the effect of fucoidan and alginate on interactions with adhesion molecules and chemokine, followed by an assessment of the cytotoxicity of the best-predicted bioactive compound for human monocytic THP-1 macrophages by lactate dehydrogenase and crystal violet assay. Moreover, an in vitro pharmacodynamics evaluation was performed. Molecular docking results indicate that fucoidan has a greater affinity for L-and E-selectin, monocyte chemoattractant protein 1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) as compared to alginate. Interestingly, there was no fucoidan cytotoxicity on THP-1 macrophages, even at 200 µg/mL for 24 h. The strong interaction between fucoidan and L-selectin in silico explained its ability to inhibit the THP-1 monocytes migration in vitro. MCP-1 and ICAM-1 expression levels in THP-1 macrophages treated with 50 µg/mL fucoidan for 24 h, followed by induction by IFN-γ, were shown to be significantly suppressed as eight- and four-fold changes, respectively, relative to cells treated only with IFN-γ. These results indicate that the electrostatic interaction of fucoidan improves its binding affinity to inflammatory markers in silico and reduces their expression in THP-1 cells in vitro, thus making fucoidan a good candidate to prevent inflammation.     

Polysaccharides of algae 68. Sulfated polysaccharides from the Kamchatka brown alga Laminaria bongardiana

Several fucoidan fractions were isolated from the biomass of the Kamchatka brown alga Laminaria bongardiana by hot water extraction followed by anion-exchange chromatography. Fucoidans were found to be composed of l-fucose, d-galactose, and sulfate as the major components, whereas d-xylose, d-mannose, d-glucuronic acid, and acetate were detected as the minor constituents. Highly sulfated fucoidan fractions F-2 and F-3 were solvolytically desulfated by heating in dimethyl sulfoxide in the presence of pyridine. The structures of native and desulfated polysaccharides were investigated by the methylation analysis and NMR spectroscopy. It was shown that F-2 contains fucan sulfate, the backbone of which is made of 1→3-linked α-l-fucopyranose residues with single α-l-Fucp branches at positions 2 and sulfate groups predominantly at positions 4. Sulfated fucoglucuronomannan, fucoglucuronan, and fucogalactan were detected in F-2 as concomitant polysaccharides. Fucan sulfate and sulfated fucogalactan were the major components of the fraction F-3. The anticoagulant properties of fucoidan fractions were assessed. It was demonstrated that the activity of the fraction F-3 is comparable with that of low-molecular-weight heparin (enoxaparin), whereas the activity of total fucoidan F and the fraction F-2 is ~2/3 and ~1/2, respectively, of the activity of F-3, which is in accordance with the lower sulfate content in these samples. Desulfated preparations F-2deS and F-3deS were completely devoid of anticoagulant activity.

     

Solid-Phase Colorimetric Method for the Quantification of Fucoidan

We described the simple, selective, and rapid method for determination of fucoidans using methylene blue staining of sulfated polysaccharides, immobilized into filter paper and consequent optic density (at A ₆₆₃?nm) measurement of the eluted dye from filter paper. This solid-phase method allows selective determination of 1?C20???g fucoidan in presence of potentially interfering compounds (alginic acid, DNA, salts, proteins, and detergents). Further, we demonstrated the alternative way of using image processing software for fucoidan quantification without extraction of methylene blue dye from stained spots of fucoidan?Cdye complex.     

Composition,structural characteristics,and antitumor properties of polysaccharides from the brown algae <Emphasis Type="Italic">Dictyopteris polypodioides</Emphasis> and <Emphasis Type="Italic">Sargassum</Emphasis> sp.

The polysaccharide compositions of the brown algae Dictyopteris polypodioides and Sargassum sp. from the Mediterranean Sea were determined. The principal polysaccharide of the studied algae (about 12% of the dry alga weight) was alginic acid. The content of water-soluble polysaccharides was low. The amount of fucoidan was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%. The monosaccharide compositions of fucoidans and neutral polysaccharides were investigated. Experiments on soft agar-agar models showed that fucoidans from D. polypodioides and Sargassum sp. exhibited antitumor activity against RPMI-7951 human melanoma cells.     

Chemical Species of Iodine in Some Seaweeds II. Iodine-Bound Biological Macromolecules

The distribution of iodine in various biological macromolecules in Sargassum kjellmanianum was studied using neutron activation analysis combined with chemical and biochemical separation techniques. The results indicate that iodine is mainly bound with protein, part of iodine with pigment and polyphenol, and little with polysaccharides, such as algin, fucoidan and cellulose. This result is significant for the mechanism of enriching iodine of algae and utilization of alga iodine.     

The significance of complex polysaccharides in personal care formulations

Complex polysaccharides have numerous pharmacological activities, such as antioxidant, antibacterial, antiviral, immunomodulating, anticoagulant and anticancer effects. In personal care products, these biopolymers are used as thickener and stabilizing agents or as moisturizing ingredients that could be beneficial to the skin. Various polysaccharides, such as glycosaminoglycans (GAGs), hyaluronic acid (HA), carrageenan, fucose, fucoidan, chitin, chitosan, etc., have applications in cosmeceutical industries. GAGs and HA incorporated in moisturizing creams strengthen the skin barrier by forming a protective layer which prevents transdermal water loss. Therefore, these polysaccharides have been formulated into potential cosmeceuticals and biopharmaceuticals.     

Use of specific polysaccharide‐immobilized monodisperse poly(glycidyl methacrylate) core–silica shell microspheres for affinity purification of lectins

Immobilization of polysaccharides (yeast mannan and gum arabic) on the macroporous poly(glycidyl methacrylate) monodisperse microspheres coated with silica (SiO₂)‐containing amino groups on the surface was used to prepare affinity sorbents for lectin purification. The efficiency of isolating mannose specific Pisum sativum lectin was demonstrated on sorbent with immobilized yeast mannan and that of galactose specific Glycine hispida lectin on sorbent with immobilized gum arabic. The microspheres with immobilized polysaccharides can be used for selecting an affinity sorbent for purification of other mannose‐ and galactose‐specific lectins. In contrast to yeast mannan, the gum arabic immobilized on the microspheres possesses much narrower specificity and is suitable for purification of only those galactose specific lectins which interact well with l ‐rhamnose or l ‐arabinose. The synthesized macroporous particles are capable of immobilizing 50 mg of polysaccharide per 1 g of the matrix, which is 10 times higher than the capacity of epoxy‐activated Sepharose 6B. That makes it possible to obtain the same lectin quantity using a column of 10 times smaller volume. Another advantage of novel affinity sorbents comparing corresponding Sepharose gels is the possibility of sorbent drying after use. Copyright © 2014 John Wiley & Sons, Ltd.     

Polysaccharides of Algae 71*. Polysaccharides of the Pacific brown alga <Emphasis Type="Italic">Alaria marginata</Emphasis>

The polysaccharide composition in sporophylls of the brown alga Alaria marginata enriched with laminaran and sulfated polysaccharides was studied. It was shown that laminaran molecules had an average degree of polymerization about 30 and consisted mainly of 3-linked β-D-glucopyranose residues, having no more than 10% of 1→6 linkages. The majority of chains (about 60%) were terminated at "reducing" end by mannitol residue. Alginic acid of sporophylls contained mannuronic (M) and guluronic (G) acids residues distributed along the linear polymer molecules as MM, MG, and GG blocks at a ratio of 4: 1: 1. Fucoidan was found to be composed of fucose, galactose, and sulfate as the major constituents, while xylose, mannose, glucuronic acid, and acetate were the minor components. It was shown that fucoidan contained two major components: fucan sulfate, molecules of which are built up of 3-linked fucopyranose residues with branches and sulfate groups at different positions, and fucogalactan, also containing chains of 3-linked fucopyranose residues with branches at positions 4 together with highly branched galactan chains terminated by fucose residues. The fucoidan contained also sulfated glucuronomannan and sulfated glucuronan as minor components.     

Seasonal variations of the composition,structural features,and antitumor properties of polysaccharides from <Emphasis Type="Italic">Padina pavonica</Emphasis> (Lebanon) as a function of composition

The variation of the polysaccharide composition of the brown alga Padina pavonica from the Mediterranean Sea was studied as a function of the collection season (April–July). It was shown that the principal polysaccharide (8.0-13.3% of dry alga weight) was alginic acid. Its content did not undergo significant changes from April to July. The content of water-soluble polysaccharides (laminarans and fucoidans) was low (<0.3% of dry alga weight). Water-soluble polysaccharides of the studied alga were practically pure fucoidan. The amount of laminarans was insignificant (<0.01% of dry alga weight). The content of fucoidans increased in alga samples from May through July. It was shown that P. pavonica is a promising source of alginic acids, which have a high capacity for gelation, and fucoidans, which exhibit antitumor action against RPMI-7951 human melanoma cells.     

Sorption of Anionic Polysaccharides and Bovine Serum Albumin on a Macroporous Glass

Concentration and time dependences of the sorption of alginic acid and -carrageenan on an MPS-1000 macroporous glass were studied. The character of pH effect on the sorption of these polysaccharides was established. For a number of sorbents prepared by the preliminary adsorption of polysaccharides on an MPS-1000 glass, the dependence of the sorption of bovine serum albumin (BSA) on the surface content of alginic acid and -carrageenan was studied. The model of adsorption in the silica–polysaccharide–protein system accounting for the interaction between polysaccharide ionic groups and active sites at the sorbent surface and protein molecules was proposed. Acid-base properties of silica modified with polysaccharides were investigated by the potentiometric titration, and the results supporting the validity of the proposed model were obtained.     

Fucoidan‐Mimetic Glycopolymers as Tools for Studying Molecular and Cellular Responses in Human Blood Platelets

The marine sulfated polysaccharide fucoidan displays superior ability to induce platelet aggregation compared to other sulfated polysaccharides. As such, it is an attractive tool for studying molecular and cellular responses in activated platelets. The heterogeneous structure, however, poses a problem in such applications. This study describes the synthesis of sulfated α‐l ‐fucoside‐pendant poly(methacryl amides) with homogeneous structures. By using both thiol‐mediated chain transfer and reversible addition‐fragmentation chain transfer polymerization techniques, glycopolymers with different chain lengths are obtained. These glycopolymers show platelet aggregation response and surface changes similar to those of fucoidan, and cause platelet activation through intracellular signaling as shown by extensive protein tyrosine phosphorylation. As the platelet activating properties of the glycopolymers strongly mimic those of fucoidan, this study concludes these fucoidan‐mimetic glycopolymers are unique tools for studying molecular and cellular responses in human blood platelets.

     

Polysaccharides of algae 65. Unusual polysaccharide composition of the pacific brown alga Punctaria plantaginea

Quantitative determination revealed the presence of storage glucan (6.0%), fucoidan (19.2%) and alginate (12.7%) in the biomass of the brown alga Punctaria plantaginea collected from the Sea of Japan. The polysaccharides were isolated from the alga by fractional extraction followed by additional purification procedures. Unlike the well-known laminarans the storage polysaccharide from P. plantaginea was shown to be a linear (1→6)-β-d-glucopyranan, which is new for brown alga. The content of guluronic acid (G) residues in the alginate molecules exceeded the content of mannuronic acid residues (M), M/G = 0.5. Poly-G and poly-MG blocks were isolated from the products of partial hydrolysis of alginic acid; however, a heterogeneous mixture of polysaccharide fragments was obtained instead of the expected poly-M fraction. Preliminary data suggests that fucoidan from this alga is a new for brown algae type of sulfated polysaccharide (xylofucan) with a main backbone built of α-l-fucopyranose residues. This chain contains multiple sulfate groups and single non-sulfated β-d-xylopyranose residues as substituents.