Optimization of citric acid treatment for the sequential extraction of fucoidan and alginate from Sargassum latifolium and their potential antioxidant and Fe(III) chelation properties

In this study, citric acid was applied as a safe organic acid for the treatment of the brown macroalga Sargassum latifolium to facilitate the sequential extraction of fucoidan and alginate without cross contamination. Box-Behnken experimental design was used to investigate the effects of citric acid concentration, temperature, and time on fucoidan yield, its fucose and sulfate contents, and molecular weight (MW), while the investigated responses of the sequentially extracted alginate were yield, mannuronic/guluronic acid ratio (M/G) and MW. Under the optimized conditions, the fucoidan yield, fucose content, sulfate content, and MW were 6.55%, 21.01%, 30.92% and 7.12 × 10³ Da, respectively, while alginate yield, M/G, and MW were 28.81%, 0.36, and 1.50 × 10⁵ Da, respectively. The optimized products showed good Fe(III) chelating properties and the iron contents were 509.82 mg g^?1 for fucoidan-Fe and 406.71 ± 5.18 mg g^?1 for alginate-Fe. Under simulated gastrointestinal conditions, fucoidan-Fe and alginate-Fe complexes released 46 and 57% iron, respectively, which are nutritionally relevant amounts. Both fucoidan and alginate showed potent antioxidant properties, but their Fe complexes exhibited a reduced activity. The results of the present study indicated that citric acid could support the basis for an economical biorefinery process for the sequential extraction of fucoidan and alginate. Additionally, these polysaccharides could be good candidates for the preparations of iron supplements.

     

羊栖菜褐藻糖胶抗凝血活性的研究

本文研究了羊栖菜褐藻糖胶的化学组成和抗凝血活性之间的关系。采用热水提取得羊栖菜粗多糖,CaCl2纯化得褐藻糖胶,DEAE Sepharose CL-6B柱层析与Sepharose CL-6B柱层析对褐藻糖胶进行分级,得到F1、F2、F31、F32和F33五个级分,均为岩藻糖、半乳糖和甘露糖等糖基组成的杂多糖,并含有硫酸酯和糖醛酸以及少量的蛋白质,相对分子质量范围2．5万～95万。采用活化部分凝血活酶时间(APTT)和凝血酶时间(TT)检测了这5个级分的抗凝血活性,结果显示,羊栖菜褐藻糖胶能显著延长APTT的凝血时间,而对TT的影响不明显。F1、F31和F32对APTT的影响比较显著,而F2、F33和羊栖菜粗多糖的影响较小。研究表明,羊栖菜褐藻糖胶主要是通过抑制内源凝血途径而达到抗凝血的效果,其抗凝血活性与褐藻糖胶的硫酸基含量成正相关,而与相对分子质量和糖醛酸含量无关。     

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.     

Structural Characterization and α‐Glucosidase Inhibitory and Antioxidant Activities of Fucoidans Extracted from Saccharina japonica

Two sulfated fucoidan fractions (Lj3 and Lj5) were extracted from Saccharina japonica and then subjected to acid hydrolysis to obtain Lj3h and Lj5h. Lj3h and Lj5h were characterized using IR, methylation analysis, and mass spectrometry. It was found that Lj3h and Lj5h were homogeneous low molecular weight fucoidans. Specifically, Lj3h was composed of the main chain of 1,3‐linked α‐L‐fucopyranose residues with sulfate at C‐2 and/or C‐4 and three different monosaccharides (galactose, glucose, mannose) branched at C‐2 and/or C‐4 of fucose residue. Lj5h contained backbones of alternating galactopyranose residues and fucopyranose residues attached via a 1→3 linkage (galactofucan) and 1→6 linked galactan. The sulfation pattern was mainly located at C2/C4 fucose or galactose residues and more branches occupied at C‐4 of fucose residue and C‐2, C‐3 or/and C‐6 of galactose residue. In vitro assay indicated that, among the four fucoidans tested, only Lj5 showed potent α‐glucosidase inhibitory activity with IC₅₀ of 153.27±22.89 μg/mL, and the two parent fucoidans, Lj3 and Lj5, showed better antioxidant activity than their derivatives. These findings highlight the structure and bioactivity diversity of Saccharina japonica‐derived fucoidans.     

Polar components of Black sea seaweed Colpomenia peregrina (Sauv.) Hamel

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 beta-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. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru     

裙带菜褐藻糖胶的分离纯化及其结构分析

裙带菜经水提法提取得到褐藻糖胶粗多糖,经DEAE-Sepharose FF离子交换层析和 Sepharose 4B层析后,得到Sl、S2两个单一组分,相对分子质量分别为550 808、38 335.基本结构及单糖组成分析表明,二者均含有岩藻糖、糖醛酸、硫酸基、半乳糖、甘露糖、葡萄糖、鼠李糖、木糖、阿拉伯糖,但含量差别较大,推测与二者的生理活性的差异有关.     

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.     

A comparison of the effect of fucoidan from alga <Emphasis Type="Italic">Fucus vesiculosus</Emphasis> and its fractions obtained by anion-exchange chromatography on HeLa G-63, Hep G2, and Chang liver cells

The aim of this study was to compare the effects of sulfated fucopolysaccharides isolated from Fucus vesiculosus on HeLa G-63, Hep G2, and Chang liver cells. Native fucoidan F3 and two fractions (F3-0.5 and F3-1) obtained by anion-exchange chromatography were analyzed using chemical methods and IR spectroscopy. It was demonstrated that F3 and F3-1 are characterized by a higher content of sulfates, location of sulfo groups mostly at the C4 atom of fucose residue, and low content of uronic acids inhibited cell proliferation. Human liver carcinoma Hep G2 appeared to be the most sensitive to fucoidan, whereas nonmalignant human Chang liver cells were the least sensitive.     

Sugar constituents of fucose-containing polysaccharides from various Japanese brown algae

Sugar constituents of the fucose-containing polysaccharides (FCPs) from 21 species of brown algae were analyzed. FCPs were extracted with hot water (100 °C, 4 h), separated by precipitation with 20% (v:v) ethanol in the presence of 0.05 M MgCl₂ to remove contaminating soluble alginate, and purified by DEAE-Sephadex column chromatography. The samples were hydrolyzed with HCI, and neutral sugar and uronic acid were separated by anion exchange chromatography. Their amounts were determined by gas-liquid chromatography. The neutral sugars in the FCPs from Ishige okamurae, Laminaria ochotensis, Myelophycus simplex, Padina arborescens and Sargassum thunbergii all contained arabinose, fucose, galactose, glucose, mannose, rhamnose and xylose residues. The FCPs from Ishige okamurae, Padina arborescens, Sargassum hemiphyllum, S. patents and S. sagamianum contained the four uronic acids, galacturonic acid, glucuronic acid, guluronic acid and mannuronic acid.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola I. Indications for a Polysaccharide Resembling Alginic Acid in Seven P. syringae Pathovars

Extracellular polysaccharides (EPS) produced by Pseudomonas syringae pv. phaseolicola are obviously composed of two main components: the long known levan consisting of fructose, and a mannuronan consisting mainly of mannuronic acid (manA), thus resembling alginic acid (alginate). The identification of manA was established by TLC utilizing different developing systems, and by cellulose acetate electrophoresis in different buffers. References were authentic uronic acids and hydrolyzed authentic alginate. A rough quantification of the “alginate” present in crude EPS was achieved with a selective colour reaction which largely excluded compounds other than uronic acids. Levan was only synthesized with sucrose as primary carbon source. When grown on several other sugars and related compounds “alginate” was the predominant component of the EPS. Additionally, rhamnose, fucose, glucose and amino sugars were found in some instances in hydrolysates of crude EPS, suggesting the release of lipopolysaccharides (LPS) from the bacterial cell walls during culture. Growth on carbon sources not related to sugars resulted in these “LPS” as the main constituent of EPS. After cultivation with sucrose, the “alginate” was restricted to the “slime” fraction of the EPS. In the “capsular” fraction, levan was predominating. A screening program revealed the capacity to synthesize the “alginate” in six additional P. syringae pathovars: pisi, lachrymans, aptata, tomato, syringae, and glycinea. All of the strains tested so far produced levan from sucrose, however, the “alginate” was formed not by all of them. There was a tendency that fresh isolates produced more “alginate” than strains subcultured for an extended time in vitro. This was also true for the total amount of EPS.     

Sulfated galactofucan from <Emphasis Type="Italic">Lobophora variegata</Emphasis>: Anticoagulant and anti-inflammatory properties

Sulfated polysaccharides (fucans and fucoidans) from brown algae show several biological activities, including anticoagulant and anti-inflammatory activities. We have extracted a sulfated heterofucan from the brown seaweed Lobophora variegata by proteolytic digestion, followed by acetone fractionation, molecular sieving, and ion-exchange chromatography. Chemical analyses and ¹³C-NMR and IR spectroscopy showed that this fucoidan is composed of fucose, galactose, and sulfate at molar ratios of 1:3:2. We compared the anticoagulant activity of L. variegata fucoidan with those of a commercial sulfated polysaccharide (also named fucoidan) from Fucus vesiculosus and heparin. The experimental inflammation models utilized in this work revealed that fucoidan from L. variegata inhibits leukocyte migration to the inflammation site. Ear swelling caused by croton oil was also inhibited when sulfated polysaccharides from F. vesiculosus and L. variegata were used. The precise mechanism of different action between homo-and heterofucans is not clear; nevertheless, the polysaccharides studied here may have therapeutic potential in inflammatory disorders. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 9, pp. 1265–1273.     

Phytochemical Findings Evidencing Botanical Origin of New Propolis Type from North‐West Argentina

Propolis samples from north‐west Argentina (Amaicha del Valle, Tucumán) were evaluated by palynology, FT‐IR spectra, and RP‐HPTLC. In addition, the volatile fraction was studied by HS‐SPME‐GC/MS. The botanical species most visited by Apis mellifera L. near the apiaries were collected and their RP‐HPTLC extracts profiles were compared with propolis samples. In addition, GC/MS was performed for volatile compounds from Zuccagnia punctata Cav. (Fabaceae). FT‐IR spectra and RP‐HPTLC fingerprints of propolis samples showed similar profiles. In RP‐HPTLC analyses, only Z. punctata presented a similar fingerprint to Amaicha propolis. The major volatile compounds present in both were trans‐linalool oxide (furanoid), 6‐camphenone, linalool, trans‐pinocarveol, p‐cymen‐8‐ol, and 2,3,6‐trimethylbenzaldehyde. Potential variations for the Amaicha del Valle propolis volatile fraction as consequence of propolis sample preparation were demonstrated.     

FT-IR Spectroscopy and Artificial Neural Network Identification of Fusarium Species

A rapid spectroscopic approach for whole‐organism fingerprinting of Fourier transform infrared (FT‐IR) spectroscopy was used to analyse 16 isolates from five closely related species of Fusarium: F. graminearum, F. moniliforme, F. nivale, F. semitectum and F. oxysporum. Principal components analysis and hierarchical cluster analysis were used to study the clusters in the data. On visual inspection of the clusters from both methods, the spectra were not differentiated into five separate clusters corresponding to species and these unsupervised methods failed to identify these fungal strains. When the data were trained by back propagation algorithm of artificial neural networks (ANNs) with principal components scores of spectra used as input modes, the strains were accurately predicted and recognized. The results in this study show that FT‐IR spectroscopy in combination with principal component artificial neural networks (PC‐ANNs) is well suited for identifying Fusarium spp. It would be advantageous to establish a comprehensive database of taxonomically well‐defined Fusarium species to aid the identification of unknown strains.     

Distribution of O-glycosylhydrolases in marine invertebrates. Enzymes of the marine mollusk Littorina kurila that catalyze fucoidan transformation

The distribution of O-glycosylhydrolases (fucoidan hydrolases, -D-mannosidases, -D-glucosidases, and -D-galactosidases) in 30 species of marine invertebrates occurring in the Sea of Japan was studied. It is shown that fucoidanases and glycosidases are widespread in the animals analyzed. Some molluscan, annelid, and echinoderm species can probably serve as objects for isolation and detailed study of the fucoidan-hydrolyzing enzymes. Fucoidan hydrolase, -L-fucosidase, and arylsulfatase from the marine mollusk Littorina kurila were isolated and described. It was found that -L-fucosidase and arylsulfatase hydrolyze synthetic substrates and cannot hydrolyze natural fucoidan, whereas fucoidan hydrolase cleaves fucoidan to produce sulfated oligosaccharides and fucose.     

The invasive brown seaweed Sargassum muticum as new resource for alginate in Morocco: Spectroscopic and rheological characterization

The Japanese brown seaweed Sargassum muticum, recently invaded several shorelines worldwide including the Atlantic coast of Morocco with large well‐established populations. Within the framework of a sustainable strategy to control this invasive seaweed, we report on extraction yield, spectroscopic characterization and rheological properties of alginate, a commercially valuable colloid, from harvested biomass of S. muticum. Extraction yield was about 25.6% on dry weight basis. Infrared spectroscopy analysis shows that the obtained Fourier transform infrared spectra of the extracted biopolymer exhibit strong similarities with that of the commercial alginate. Furthermore, Proton nuclear magnetic resonance spectroscopy revealed that S. muticum alginate has almost equal amounts of β‐D‐mannuronic acid (M; 49%) and α‐L‐guluronic acid (G; 51%) with an M/G ratio of 1.04 and a high content of heteropolymeric MG GM diads suggesting a sequence distribution of an alternated polymer type. Rheological measurements were performed at different sodium alginate concentrations, temperatures and shear rates. The hydrocolloid exhibited pseudoplastic behavior and showed shear thinning, particularly at high solution concentration and low temperature which is consistent with the rheological behavior reported for commercial alginates. Considering the abundance of S. muticum in the Northwestern Atlantic coast of Morocco and the quality of the extracted hydrogel, this invasive species could be considered as a potential source of alginates.     

Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica

Fucoidan, a group of sulfated heteropolysaccharide, was extracted from Laminaria japonica, an important economic alga species in China. Three sulfated polysaccharide fractions (F1, F2, and F3) were successfully isolated through anion-exchange column chromatography and had their antioxidant activities investigated employing various established in vitro systems, including superoxide and hydroxyl radical scavenging activity, chelating ability, and reducing power. Chemical analysis suggested that F1 and F3 were heteropolysaccharide in which galactose was the major component, while F2 was a typical fucoidan. All fractions possessed considerable antioxidant activity, and F1, F2 and F3 had stronger antioxidant ability than fucoidan in certain tests. The correlation between the sulfate content and scavenging superoxide radical ability was positive. Available data obtained with in vitro models suggested that the ratio of sulfate content/fucose was an effective indicator to antioxidant activity of the samples.     

VARIATIONS IN THE SUBSTITUTED 3‐LINKED MANNANS CLOSELY ASSOCIATED WITH THE SILICIFIED WALLS OF DIATOMS1

The polysaccharides from cleaned frustules of the diatoms Pinnularia viridis (Nitzsch) Ehrenberg, Craspedostauros australis Cox, Thalassiosira pseudonana Hasle et Heimdal, and Nitzschia navis‐varingica Lundholm et Moestrup were extracted with hot alkali that dissolved the silica and were characterized by constituent sugar and linkage analyses. The polysaccharides from P. viridis were investigated further by permethylation, partitioning according to solubility, desulfation, and CD₃I‐methylation. Yields of carbohydrate in the hot alkali extracts ranged from 0.9% to 1.8% w/w based on the dry weight of the silica. Mannose was the dominant sugar in the polysaccharides from all four species (54–69 mol% of constituent sugars), although 14 other monosaccharides, including neutral sugars (glucose, galactose, xylose, arabinose, rhamnose, fucose), acidic sugars (glucuronic acid, galacturonic acid, 2‐O‐methylglucuronic acid), and O‐methylated neutral sugars (2‐O‐methylrhamnose, 3‐O‐methylrhamnose, 2,3‐di‐O‐methylrhamnose, 3‐O‐methylxylose, 4‐O‐methylxylose) were also detected in varying proportions among the four samples. The polysaccharides were predominantly composed of a 3‐linked mannopyranose backbone with a prevalence of linkage and/or substitution at O‐2 of the 3‐linked mannopyranosyl residues, and they were polyanionic, bearing uronic acid residues and/or sulfate esters. There were, however, species‐specific differences in the degree and position of substitution on the mannan backbone, the type and substitution patterns of the anionic substituents, and the type and linkage patterns of sugars other than mannose. Although definitive functions for these polysaccharides in diatom biology remain uncertain, a possible role in biosilicification is 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.

     

Studies on by-products from the industrial extraction of alginate

The chemical composition of fucans isolated from leach-water, an industrial alginate extraction by-product, was investigated. Several fractions were obtained by anion exchange and gel permeation chromatography. They all contained fucose, but differed in the uronic acid, sulfate, xylose and galactose contents. They distributed as a continuum between uronic acid rich and sulfate poor to sulfate rich and uronic acid poor molecules. Two highly sulfated fractions were studied in particularly by chemical means (methylation, carboxy reduction, desulfation, controlled acid hydrolysis) and by¹³C nuclear magnetic resonance spectroscopy. One fraction consisted of a highly branched fucan (43.8% fucose) composed mostly of 1,2,3,4- and 1,2,4-linked fucose with some 1,4-,1,3,4- and 1,3-linkages and sulfate (23.9%) occurring on O₂ and/or O₃ and/or O₄. The other was composed mainly of fucose (31.6%), galactose (24.7%) and sulfate (23.7%). It consisted primarily of 1,6-, 1,4,6-, 1,3- and 1,3,6-linked galactose 6-and/or 4- and/or 3-sulfate on which are linked essentially terminal fucose or 1,4-linked with sulfate on O₂ and/or O₃ and/or O₄. None of these highly branched fractions contained sufficiently regular segments to yield series of homologous oligosaccharides on partial acid hydrolysis or interpretable¹³C NMR spectra.author for correspondence     

Important Role of β1-Integrin in Fucoidan-Induced Apoptosis via Caspase-8 Activation

Fucoidan induces apoptosis by activating caspase-8 in human MCF-7 breast cancer cells, but the detailed mechanism for this is not understood. We demonstrate here that fucoidan interacted with the cell surface, and silencing the β₁-integrin gene expression inhibited fucoidan-induced apoptosis accompanied by caspase-8 activation. Fucoidan induced formation of the β₁-integrin-caspase-8 complex. These data indicate that β₁-integrin is an important factor for the cell-surface binding of fucoidan and plays an important role in fucoidan-induced apoptosis. Fucoidan also induced recruitment of caspase-8 to the β₁-integrin intracellular domain, cleaved it into the activated protein by direct combination with β₁-integrin, and induced apoptosis via the caspase cascade in MCF-7 cells.