Polysaccharides of Algae: 55. Polysaccharide Composition of Several Brown Algae from Kamchatka

A new spectrophotometric procedure was developed for the quantitative determination of fucoidan and alginic acid and used for their analysis in extracts from biomass of 17 species of brown algae collected from coastal waters of the Kamchatka peninsula. In addition, neutral monosaccharides and mannitol were determined in the hydrolysis products of the alga biomass samples. The polysaccharide composition was shown to substantially depend on the algal species. The alginic acid content was maximal in the Alaria marginatablades; all the other representatives of the order Laminariales also are useful sources of this polysaccharide. At the same time, the fucoidan content is rather low in Laminariales. The highest content of fucoidan was found in Saundersella simplex, but Chordaria flagelliformisand Fucus evanescensare more practical fucoidan sources; the available supplies and the sugar composition make the latter alga the most suitable for the complex processing to prepare mannitol, fucoidan, and alginic acid.     

Algae polysaccharides. 55. Polysaccharide composition of some brown Kamchatka algae]

A new spectrophotometric procedure was developed for the quantitative determination of fucoidan and alginic acid and used for their analysis in extracts from biomass of 17 species of brown algae collected in coastal waters of the Kamchatka peninsula. In addition, neutral monosaccharides and mannitol were determined in the hydrolysis products of the alga biomass samples. The polysaccharide composition was shown to substantially depend on the algal species. The alginic acid content was maximal in the Alaria marginata blades; all the other representatives of the order Laminariales are also useful sources of the polysaccharides. At the same time, the fucoidan content is rather low in Laminariales. The highest content of fucoidan was found in Saundersella simplex, but Chordaria flagelliformis and Fucus evanescens are more practical fucoidan sources; the available supplies and the sugar composition make the latter alga the most suitable for the complex processing to prepare mannitol, fucoidan, and alginic acid.     

Seasonal variations in the fucoidan content of brown algae from Peter the Great Bay,Sea of Japan

The seasonal dynamics in the level of fucose-containing sulfated polysaccharides (fucoidans) have been studied in three brown alga species from Peter the Great Bay, Sea of Japan: Saccharina japonica, Sargassum pallidum, and Stephanocystis crassipes. In the latter two species, seasonal variations in the fucoidan content have been analyzed for the first time. During the year, the level of fucoidan in S. japonica varied from 0.98 to 4.19% dry weight; in S. crassipes, from 2.50 to 5.23%; in S. pallidum, from 1.86 to 6.31%. The maximum amount of this polysaccharide is accumulated in algae during reproduction: in August–October for S. japonica; in June and July for S. crassipes; and in July for S. pallidum. No significant correlation has been found between the level of fucoidan in algal tissues and the annual dynamics in sea water temperature, salinity, and concentration of biogenic elements. The obtained data can be helpful in determining the time to harvest algae for fucoidan extraction.     

Structural characteristics and biological activity of Fucoidans from the brown algae Alaria sp. and Saccharina japonica of different reproductive status

Structural characteristics and the antitumor activity of fucoidans isolated from vegetative and reproductive tissue of the brown algae Alaria sp. and Saccharina japonica were studied. The reproductive status of the brown algae affected the yield of fucoidans and their structural characteristics. The fucoidan yield was 5.7% (w/w on the basis of the dried algae weight) for fertile and 3.8% for sterile Alaria sp. and 1.42 and 0.71% for fertile and sterile S. japonica, respectively. The fucoidans from fertile Alaria sp. and S. japonica had a slightly higher degree of sulfation and a somewhat more homogeneous monosaccharide composition, with predominate amounts of fucose and galactose, than those isolated from sterile algae tissue. The fucoidans from both the sterile and fertile brown algae tissue tested possessed selective cytotoxicity towards human breast cancer (T-47D) and melanoma (RPMI-7951) cell lines, but not to normal mouse epidermal cells (JB6 Cl41), and effectively inhibited the proliferation and colony formation of the breast cancer and melanoma cell lines. The fucoidans from reproductive tissue of brown algae possessed higher antitumor activity than those from vegetative plants.     

Sulfated polysaccharides from brown seaweeds Saccharina japonica and Undaria pinnatifida: isolation, structural characteristics, and antitumor activity

During the last decade brown seaweeds attracted much attention as a source of polysaccharides, namely laminarans, alginic acids, and sulfated polysaccharides—fucoidans, with various structures and biological activities.In this study, sulfated polysaccharides were isolated from brown seaweeds Saccharina japonica (formerly named Laminaria) and Undaria pinnatifida and their antitumor activity was tested against human breast cancer T-47D and melanoma SK-MEL-28 cell lines.The sulfated polysaccharide form S. japonica was highly branched partially acetylated sulfated galactofucan, built up of (1→3)-α-l-fucose residues. The sulfated polysaccharide from U. pinnatifida was partially acetylated highly sulfated galactofucan consisting of (1→3)- or (1→3);(1→4)-α-l-fucose residues.Fucoidans from S. japonica and U. pinnatifida distinctly inhibited proliferation and colony formation in both breast cancer and melanoma cell lines in a dose-dependent manner. These results indicated that the use of sulfated polysaccharides from brown seaweeds S. japonica and U. pinnatifida might be a potential approach for cancer treatment.     

Tandem 5S ribosomal RNA genes and the spacer region sequences of three Japanese Laminaria species

The organization of 5S ribosomal RNA genes (rDNA) was examined for threeJapanese Laminaria species, L. japonica, L.religiosa and L. ochotensis. The linkage of 5SrDNA with the 18S-5.8S-25S rDNAs unit known in the brown algaScytosiphon lomentaria could not be detected inLaminaria. Instead, the tandem repeats of 5S rDNA were notassociated with the 18S-5.8S-25S rDNAs unit. The nucleotide sequence of 5S rDNAwas completely identical among these three species and its length was 118bp. However, a difference of nucleotide arrangement was detectedinthe spacer region of the tandemly repeated 5S rDNAs. Several nucleotideinsertion / deletions and substitutions were confirmed between differentindividuals of L. japonica, which were collected from notonly disjunct localities, but also the same locality. The lengths of the spacerregion of L. japonica, L. religiosaand L. ochotensis were 247–252 bp, 232bp and 252 bp, respectively.     

The composition and structural characteristics of polysaccharides of the brown alga Eisenia bicyclis

The present study examines the polysaccharide composition of the brown alga Eisenia bicyclis. The major polysaccharide of E. bicyclis is shown to be sodium alginate (15.8% of the dry defatted alga weight). The yields of laminaran and fucoidan were 1.4% and 1.3% of the dry defatted alga weight, respectively. The alga contains laminaran (1,3;1,6-β-D-glucan) that has a unique structure, a high content of 1,6-linked D-glucose residues (the ratio of 1,3: 1,6 linkage is 1.5: 1), and an unusually high molecular weight (19–27 kDa). The fucoidan fractions isolated from E. bicyclis are sulfated and acetylated heteropolysaccharides that differ in their sulfate content and monosaccharide composition.     

Extraction optimization and analysis of monosaccharide composition of fucoidan from Saccharina japonica by capillary zone electrophoresis

To optimize the extraction process of fucoidan from Saccharina japonica, an orthogonal test with four factors and three levels was designed in the present experiment. The results showed that the highest extraction yield was obtained under the following conditions: extraction temperature, 90 °C; extraction time, 1.5 h; number of extractions, 3; and pH of the extraction solution, 2.5. Furthermore, a capillary zone electrophoresis method using a UV detector was established for the analysis of the monosaccharide composition of fucoidan via 1-phenyl-3-methyl-5-pyrazolone derivatization. The results showed that fucoidans found in the three different samples all consisted of xylose, rhamnose, fucose, mannose, galactose, and glucuronic acid, while the mole ratios of monosaccharide components revealed differently. Moreover, fucose and galactose were found to be the most abundant monosaccharides in the samples. The above results could be further used for in-depth study on the mechanism of biological activity of fucoidan from S. japonica.     

Polysaccharides from mycelium of the fungus <Emphasis Type="Italic">Cunninghamella japonica</Emphasis>

Preliminary data on the polysaccharide composition of mycelium of the fungus Cunninghamella japonica (synonymous with C. echinulata) grown by the method of submerged cultivation were obtained. Mild acidic hydrolysis of mycelium resulted in the formation of glucose, mannose, and galactose; while the treatment with acid under drastic conditions afforded glucosamine as a product of hydrolysis of chitin and chitosan, their total content was about 35%. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water, 2% aqueous NaOH, and 10% AcOH; their monosaccharide composition was characterized. The yield of chitosan extracted with AcOH was insignificant. Additional purification of the fraction obtained after extraction with alkali afforded polysaccharide which was a linear (1 → 3)-α-D-glucopyranan according to the data of NMR spectroscopy and the chemical methods of structural analysis. The presence of this polysaccharide, as well as a low content of chitosan and polyuronides, distinguishes the studied strain C. japonica from most of the known Mucorales.     

Monthly changes in the content and monosaccharide composition of fucoidan from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Laminariales,Phaeophyta)

Crude fucoidan was extracted from the brown alga Undaria pinnatifida collected monthly from April to last July in Peter the Great Bay (Japan Sea, Russia). The amount of crude fucoidan rose markedly from April to June–July (from 3.2 to 16.0% dry weight) as the plant matures. An analysis of the monosaccharide composition of the fucoidan extracted showed that the alga synthesized polysaccharides with various structures which were dependent on the algae age. In juvenile plants collected in April–May, this was represented by sulfated manno-galactofucan containing up to 19–28 mol% of mannose and about 20 mol% of galactose, whereas in matured plants collected in June–July, the polysaccharide was represented by a sulfated galactofucan containing more than 38 mol% galactose. It is postulated that the production of sori causes a subsequent effect on fucoidan synthesis and leads to an enhanced of crude fucoidan content and an increased molar concentration of galactose. Crude fucoidan content in sporophylls increased 5 times, and galactose content in this polysaccharide rose s1.6 times with sori formation. The structural characteristics of the fucoidan extracted from sporophylls of Undaria collected in July were also studied. The fractionation of crude fucoidan on DEAE-Sephadex A-25 gave two fractions, F1 and F2 in equal quantities. F1 was characterized as manno-galactofucan sulfate and F2 was galactofucan sulfate. The molecular weights of both fractions were in a range of 30–80 kDa. Analysis of fucoidan structure using ESI-FTICR mass spectrometry showed the presence of mixed oligosaccharides consisting of fucose and galactose. Presumably, the polysaccharide molecules contain blocks built up of successively linked residues of fucose and galactose. These blocks are built from two to five or more residues of monosaccharides. According to IR-spectroscopy data, the main portion of sulfates is located at C2; in addition, sulfate esters are also present at C4 on the fucose and C3 and C6 of the galactose units.     

Effects of Alginic Acid from a Brown Alga Laminaria cichorioideson the Physical Activity of Experimental Animals

We studied the possibility of stimulating physical activity in experimental animals using alginic acid, a polysaccharide isolated from a brown alga Laminaria cichorioides(family Laminaraceae). It is shown that the alginic acid increases working capacity, prevents fatigue, and activates restoration processes after muscular activity.     

Polysaccharides composition from tropical brown seaweeds

Polysaccharides composition of the tropical brown seaweeds Turbinaria turbinata, Sargassum filipendula, Dictyota caribaea and Padina perindusiata collected at Yucatan Peninsula (Mexico) was determined in this study. Crude fucoidan extracted with HCl and alginate extracted with a hot alkali solution were characterized in terms of their molecular weight, sulfate content, uronic acid, total carbohydrate and neutral sugar components. Low molecular weight sulfated‐fucoidan was the major component in all species studied. Fucoidan from T. turbinata and from D. caribaea were characterized as a homofucan, with fucose as the neutral sugar. Fucoidan from S. filipendula was composed of a galactofucan, and fucoidan from P. perindusiata was characterized as a heterofucan consisting of fucose, glucose and galactose. The Fourier transform infrared (FT‐IR) spectra of fucoidan extracted from species studied indicated that the majority of sulfate groups are located at C‐4 and to a lesser extent at C‐2 and/or C‐3 of the fucopyranose residues. This could be advantageous since several therapeutic effects have been reported for fucoidans with similar characteristics. FT‐IR spectra from D. caribaea and P. perindusiata revealed the presence of O‐acetyl groups in crude fucoidan, which could be potentially utilized as an immune stimulant. Molecular weight of alginate varied between 595 and 1301 kDa with similar uronic acid content in all species. Alginate M : G ratio inferred from FT‐IR spectra suggests a high content of G‐block in all species. Potential applications of these polysaccharides are discussed.     

Monthly fluctuations in the content and monosaccharide composition of fucoidan from Undaria pinnatifida sporophylls from northern Patagonia

The invasive brown seaweed Undaria pinnatifida was first recorded in 1992 in Golfo Nuevo, northern Patagonia, Argentina (hereafter Undaria). Like other brown seaweeds, Undaria synthesizes fucoidans, a unique class of sulfated polysaccharides, which display an array of biological activities and have important commercial value. In this work we have measured the content, sulfate, and monosaccharide composition of fucoidans in Undaria sporophylls from Golfo Nuevo. These results were analyzed in relation to harvest month and development stage of algal thalli. Samples were collected between November 2015 and March 2016 and classified according to morphological traits into previously defined development stages. Acid extraction (0.01 M HCl, pH 2) was carried out at room temperature and at 70 °C. Average fucoidan content was 18.1% dry wt. and slightly increased with month progression and in senescent individuals. Average sulfate content in fucoidan extracts was 20.3% dry wt. decreasing with month and development. Predominant sugars were fucose and galactose averaging a total of 91 mol% of neutral sugars. This study confirms that sporophyll fucoidans from Patagonian Undaria are sulfated galactofucans. Fucoidan content significantly increases from 14.5 to 19.2% dry wt. with sporophyte development. Slight increments with month progression are not statistically significant. Molar proportion of neutral sugars is constant between maturity stages and varies slightly with month progression. Sulfate content of fucoidan decreased significantly with development and month progression. Pooled with previous reports about abundance and seasonality of Undaria in Patagonia, our results suggest that sporophylls could be harvested for fucoidan production at least during 5 months between November and March.

     

Polar Constituents of Brown Seaweed Colpomenia peregrina (Sauv.) Hamel from the Black Sea

GC-MS of trimethylsilyl derivatives of the compounds present in the butanolic extract of biomass of brown seaweed Colpomenia peregrina from the Black Sea aided in identification of 24 components, including aliphatic hydroxy and keto and aromatic acids, glycerol, mannitol, floridoside, and monosaccharides. The polysaccharide composition of the biomass was also studied, with high sodium alginate and laminaran contents and a comparatively low level of fucoidan being revealed. The polysaccharides were isolated from the biomass by fractional extraction and purified by precipitation or ion exchange chromatography. The structures of alginic acid and laminaran were deduced from ¹³C NMR spectra and confirmed, in the case of laminaran, by methylation analysis. The sodium alginate was shown to contain more guluronic (G) than mannuronic acid (M) residues, the M/G ratio being 0.48. Laminaran was demonstrated to be a -glucan with 1 3 linkages in its backbone and 1 6 linkages in its branching points, which is characteristic of brown algae. Fucoidan turned out to be a complex heteropolysaccharide containing, in addition to fucose and sulfate, other neutral monosaccharides and uronic acids.     

Mitochondrial DNA sequence variation of four <Emphasis Type="Italic">Saccharina</Emphasis> species (Laminariales,Phaeophyceae) growing in Japan

The complete mitochondrial DNA (mtDNA) was sequenced for four Saccharina species including three varieties of Saccharina japonica in Hokkaido: S. japonica; S. japonica var. religiosa; S. japonica var. ochotensis; S. japonica var. diabolica; Saccharina longipedalis; Saccharina angustata; and Saccharina coriacea. Furthermore, the structure and the sequence were compared among them. The total nucleotide length was 37,500–37,657 bp. All mtDNAs were mapped and no differences in the organization of the coding region were found. From the total alignment of S. japonica including the three varieties and S. longipedalis, nucleotide substitutions were detected at 68 sites, and a nucleotide insertion/deletion was detected at one site on rps19 for the whole genome. Variable regions useful for varieties distinction of S. japonica were trnI, trnM, rps19, ORF41, and seven spacers. The pairwise distance between S. japonica, S. angustata, and S. coraiacea was 0.000–0.116. Sequencing comparisons of rps7, rps11, rpl5, and tatC were thought to be useful tools for discrimination and phylogenetic analysis of Saccharina species having similar morphologies.     

陕甘花楸果实多糖的结构表征及抗氧化活性研究

陕甘花楸(Sorbus koehneana)是我国西北地区特有的灌木之一,主要被用于观赏和制作家具,但对其有效成分的研究却鲜见报道,从而限制了陕甘花楸产业的进一步开发和利用。该研究以陕甘花楸果实为原料,经石油醚脱脂后,采用超声辅助水提醇沉法提取、Sevag法脱蛋白,得到了较纯的花楸果实多糖(SSP),并对其进行结构表征和抗氧化活性研究。结果表明:(1)经苯酚-硫酸法测得多糖纯度为65.8%;FT-IR检测官能团,发现在3 420 cm~(-1)、2 929 cm~(-1)和1 733 cm~(-1)处存在多糖的典型吸收峰;用SEC-LLS测得重均分子量(Mw)为1.739×105,数均分子量(Mn)为5.052×104,多分散系数为3.443,表明分子量分布较为均一;经三氟乙酸酸解、糖腈衍生化等处理及气相-质谱联用法测定SSP的单糖组成,表明SSP由甘露糖、葡萄糖和未知单糖等3种单糖组成,摩尔比为2.2∶1.4∶6.4。(2)体外抗氧化活性实验表明:SSP具有很好的DPPH清除活性、超氧阴离子清除活性以及较强的还原力;当SSP浓度为2 mg·mL-1时,SSP对DPPH自由基的清除能力相当于BHT的96%,对超氧阴离子自由基清除能力为Vc的76.07%,还原能力等价于Vc的92%。以上表明该多糖可以用于抗衰老和抗炎等方面,是一种优良的天然抗氧化剂,为花楸资源的进一步开发利用提供了更为广阔的前景。     

Electrospray ionization mass spectrometry of oligosaccharides derived from fucoidan of Ascophyllum nodosum

Algal fucoidan is a complex sulfated polysaccharide whose structural characterization requires powerful spectroscopic methodologies. While most of the structural investigations reported so far have been performed using NMR as the main spectroscopic method, we report herein data obtained by negative electrospray ionization mass spectrometry. MS analysis has been carried out on oligosaccharides obtained by partial hydrolysis of fucoidan from the brown algae Ascophyllum nodosum. Oligosaccharide mixtures were fractionated by size exclusion chromatography, which allowed the analysis of oligomers ranging from monosaccharide to pentasaccharide. Monosaccharides were detected as monosulfated as well as disulfated forms. Besides, part of the oligosaccharides exhibited a high content of sulfate, evidencing that fucoidan contains disulfated fucosyl units. Fragmentation experiments yielded characteristic fragment ions indicating that the fucose units are mainly 2-O-sulfated. This study demonstrates that highly sulfated oligosaccharides from fucoidan can be analyzed by ESIMS which gives additional information about the structure of this highly complex polysaccharide.     

Isolation and Culture of a Marine Bacterium Degrading the Sulfated Fucans from Marine Brown Algae

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an α-l-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of α-1→3-l-fucopyranose-2-sulfate-α-1→4-l-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.     

Allium ampeloprasum var. babingtonii (Alliaceae): an isoclonal plant found across a range of habitats in S.W. England

Despite the widespread distribution and common habitat dominance of clonal plant species the mechanisms for their ecological success often remain uncertain. Allium ampeloprasum var. babingtonii is a sterile plant that reproduces with physiologically independent asexual propagules. The variety is found mainly in the S.W. of England over a wide habitat range including sea cliffs and open woodlands and displays an autumn-spring phenology in common with its close Mediterranean relatives. The role of population genetic structure in the remarkable habitat distribution of the variety is not known. Genetic variation in A. ampeloprasum var. babingtonii was analysed using RAPD markers in single individuals of var. babingtonii from 16 distinct populations and five further individuals from a single population. This analysis revealed no polymorphism suggesting that all sampled individuals are part of a single clone. We offer the conjecture that in the absence of genetic variation an ecological release, due to temporal separation from competing species, may contribute to the ability of var. babingtonii to exploit heterogeneous environmental conditions.     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.