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Fucan sulfates from echinoderm possess characteristic structures and various biological activities. Herein, comprehensive methods including enzymolysis, ion-exchange chromatography and size exclusion chromatography lead to the purification of five fucan sulfates (FSI, FSII, FSIII, FSIV, FSV) from the sea cucumber Pattalus mollis. Chemical composition analysis showed that they were all composed of l -fucose. Their sulfate content was determined by a conductimetric method. The molecular weight (Mw) of FSI, FSII, FSIII, FSIV and FSV were measured as 238.3 kDa, 81.0 kDa, 82.0 kDa, 23.2 kDa and 6.12 kDa, respectively. Detailed NMR spectroscopic analysis revealed that the structural sequence of FSI and FSII was →3)- l -Fuc S -α(1→, where Fuc S were Fuc 2S4S (10%), Fuc 2S (44%), Fuc 0S (10%), Fuc 4S (36%), that of FSIII was →4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 → 4)- l -Fuc 0S/3S -(α1→, where Fuc 0S and Fuc 3S were in equal molar, and that FSIV was →4)- l -Fuc 2S3S -(α1 → 4)- l -Fuc 2S3S -(α1 → 4)- l -Fuc 2S -(α1→4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 →. This is the first report that such a diversity of fucan sulfates were obtained from the same sea cucumber species. Biological activity showed that FSI, FSII, FSIII and FSIV exhibited potent anticoagulant by prolonging the APTT. Among them, FSII, FSIII and FSIV showed the similar potency, while FSI owned the strongest. Structure-activity relationships analysis showed that molecular weight and sulfation degree should be the crucial factors for the activity. • Five fucan sulfates were purified from sea cucumber Pattalus mollis. • The physicochemical properties of four fucan sulfates were characterized. • Structural unit of FSI and FSIII was →3)- l -Fuc S -α(1→ and →4)- l -Fuc 2S -(α1→4)- l -Fuc 2S -(α1→4)- l -Fuc 0S/3S -(α1→, respectively. • FSIV was composed of an α1,4-linked hexafucose repeating unit. • FSI exhibit strong inhibition of the intrinsic coagulation pathway. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides from sea cucumber possess unique chemical structure and various biological activities. In this study, four sulfated polysaccharides were purified from the body wall of Holothuria fuscopunctata by anion exchange chromatography and chemical depolymerization. They were identified as sulfated fucan (SFI, SFII), fucosylated chondroitin sulfate (FCS) and sulfated aminoglycan (AG) by physicochemical and structural analyses. The Mw of SFI, SFII, FCS and AG were 470.6, 36.8, 42.6, 39.6 kDa and the sulfate content was 21.40%, 35.86%, 33.70%, 35.70%, respectively. Their primary structures were clarified both by monosaccharide composition and 1D/2D NMR spectroscopy analysis. As a result, the repeating sequences of FCS and SFII were →4)-[L-Fuc 3S4S -(α1 → 3)]-D-GlcA-(β1 → 3)-D-GalNAc 4S6S -(β1 → and →4-L-Fuc-(3SO 3 −)-α1→, respectively. The primary structure of SFI was →3)-L-Fuc 2S4S -(α1 → 4)-L-Fuc-(α1 → 3)-L-Fuc 2S -(α1 → 4)-L-Fuc-(α1→. The sulfated AG was composed of four types of monosaccharides. Their anticoagulant activities were further evaluated in vitro. FCS and AG showed potent anticoagulant activity and intrinsic factor Xase inhibition activity. These results expand the knowledge on the structure types of sulfated polysaccharides from sea cucumber and further illustration of their functionality. • Four sulfated polysaccharides were purified from the sea cucumber H. fuscopunctata. • The physicochemical properties of these polysaccharides were characterized. • The depolymerization methods could facilitate to separate sulfated polysaccharides. • The anticoagulant activities of these polysaccharides were evaluated. [ABSTRACT FROM AUTHOR]

• A new glycosaminoglycan (LF-GAG) was obtained from the slug Limacus flavus. • Physicochemical properties of LF-GAG were analyzed. • Four oligosaccharides were obtained by deacetylation-deamination and purification. • The precise structure of LF-GAG was determined. • Structure and heparinase inhibitory activity relationships were discussed. A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N -acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA 2R -(α1,4)-D-GlcNAc-(α1-, in which R was −OH (˜80%) or –OSO 3 − (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC 50 , 0.10 μM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity. [ABSTRACT FROM AUTHOR]

Fucoidan or fucan sulfate, a sulfated polysaccharide from algae or echinoderm mainly containing fucoses, possesses complex chemical structure and various biological activities. Herein, three fucan sulfates consisted of distinctive simplest repeating units were isolated from Holothuria fuscopunctata, Thelenota ananas and Stichopus horrens . The structural sequences of these fucan sulfates from H. fuscopunctata, T. ananas and S. horrens are →4-α- l -Fuc p -(3 S O 3 − )-1→, →4-α- l -Fuc p -(2 S O 3 − )-1→ and →3-α- l -Fuc p -(2 S O 3 − )-1→, respectively, revealing the existence of the highly regular homogeneous fucan sulfates and their structural diversity in the sea cucumber species for the first time. Pharmacological assays indicated their specific sulfation pattern and position of the glycosidic linkage may contribute to the anticoagulant action. Further mechanism analysis suggested that these fucan sulfates may exhibit strong inhibition of the intrinsic coagulation pathway by targeting the intrinsic coagulation factor Xase. Our results provide novel information to enrich knowledge on structural types of fucan sulfate and to illustrate its functionality. [ABSTRACT FROM AUTHOR]

The slug Vaginulus alte is used as folk medicine in China, but the structure and activities of its galactan components remain to be clarified. Here, the galactan from V. alte (VAG) was purified. The Mw of VAG was determined as ~28.8 kDa. Chemical composition analysis showed that VAG was composed of d -galactose (75 %) and l -galactose (25 %). To elucidate its precise structure, a series of disaccharides and trisaccharides were purified from mild acid hydrolyzed VAG and their structures were characterized by 1D/2D NMR spectroscopy. Based on methylation analysis and structural analysis of oligosaccharides, VAG was elucidated as a highly branched polysaccharide and mainly composed of (1 → 6)- or (1 → 3)-linked β- d -galactose, and distinct (1 → 2)-linked α- l -galactose. The investigation of probiotic effects in vitro revealed that VAG could promote the growth of B. thetaiotaomicron and B. ovatus , while had no effect on the growth of L. acidophilus , L. rhamnosus , B. longum subsp. infantis and B. animalis subsp. lactis , but dVAG-3 with Mw ~1.0 kDa could promote the growth of L. acidophilus. These results will provide insights into specific structures and functions of polysaccharides from the V. alte. • A highly branched galactan VAG was obtained from the slug Vaginulus alte. • Structures of oligosaccharides released from VAG were identified. • VAG was consisted of (1 → 3)-, (1 → 6)-linked β- d -galactose and (1 → 2)-linked α- l -galactose. • VAG could promote the growth of B. thetaiotaomicron and B. ovatus. • dVAG-3 with Mw of 1.0 kDa could promote the growth of L. acidophilus. [ABSTRACT FROM AUTHOR]

A fucan sulfate was isolated and purified from the sea cucumber Holothuria albiventer by papain enzymolysis, alkaline hydrolysis and ion-exchange chromatography. The water-soluble polysaccharide had high molecular weight and contained fucose and sulfate in a molar ratio of about 1:0.83. Methylation analysis of the native polysaccharide indicated that its glycosidic linkages and sulfate substituents might be at O- 3 or O -3, 4 or O -2, 3, or O -2, 3, 4 positions. FT-IR and 2D NMR spectroscopies further revealed that the fucan sulfate is characteristically composed of a regular α (1 → 3) linked hexasaccharide repeating unit which is substituted with sulfate esters in a distinctive pattern. Anticoagulant properties of the fucan sulfate and its depolymerized product were assessed in vitro in comparison with a low-molecular-weight heparin. The fucan sulfate exhibits strong APTT and TT prolonging activities and intrinsic factor Xase inhibitory activity, and its molecular size seemed to be required for these activities. [ABSTRACT FROM AUTHOR]

Edible snails have been widely used as a health food and medicine in many countries. A unique glycosaminoglycan (AF-GAG) was purified from Achatina fulica . Its structure was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, analysis of monosaccharide composition, and 1D/2D nuclear magnetic resonance spectroscopy. Chemical composition analysis indicated that AF-GAG is composed of iduronic acid (IdoA) and N -acetyl-glucosamine (GlcNAc) and its average molecular weight is 118 kDa. Structural analysis clarified that the uronic acid unit in glycosaminoglycan (GAG) is the fully epimerized and the sequence of AF-GAG is →4)-α-GlcNAc (1 → 4)-α-IdoA2S (1→. Although its structure with a uniform repeating disaccharide is similar to those of heparin and heparan sulfate, this GAG is structurally highly regular and homogeneous. Anticoagulant activity assays indicated that AF-GAG exhibits no anticoagulant activities, but considering its structural characteristic, other bioactivities such as heparanase inhibition may be worthy of further study. [ABSTRACT FROM AUTHOR]

Edible snails have been widely used as a health food and medicine in many countries. In our study, a water-soluble polysaccharide (AF-1) was isolated and purified from Achatina fulica by papain enzymolysis, alcohol precipitation and strong anion exchange chromatography. Structureof the polysaccharide was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, high performance liquid chromatography, analysis of monosaccharide composition, methylation analysis, and nuclear magnetic resonance (NMR) spectroscopy ( 1 H, 13 C, COSY, TOCSY, NOESY, HSQC and HMBC). Chemical composition analysis indicated that AF-1 is composed of glucose (Glc) and its average molecular weight is 1710 kDa. Structural analysis suggested that AF-1 is mainly consisted of a linear repeating backbone of (1 → 4) linked α- d -Glc p residues with one branch, α- d -Glc p , attached to the main chain by (1 → 6) glycosidic bonds at every five main-chain units. Further studies on biological activities of the polysaccharide are currently in progress. [ABSTRACT FROM AUTHOR]

Mild acid hydrolysis is a common method to study the chemical structure of fucosylated glycosaminoglycan (FG). It was generally considered that the fucose branches α-L-FucS-(1, of FG could be hydrolyzed selectively in mild acid. This report focused on the selectivity of glycosidic bond cleavage and extensive desulfation characteristics of the backbone during mild acid hydrolysis. The hydrolyzed product of native SvFG (dfSvFG) was prepared by mild acid hydrolysis in 0.1 M H 2 SO 4 at 100 °C for 2 h. A series of oligosaccharides were purified by GPC and SAX-HPLC from dfSvFG, then they were analyzed by HPGPC, 1D/2D NMR and ESI-Q-TOF-MS. The precise structure of these oligosaccharides was elucidated to be trisaccharides, tetrasaccharides and pentasaccharides, indicating SvFG branches hydrolyzed basically and its' backbone composed of repeating β-D-GlcA-(1,3)-D-GalNAc and β-D-GalNAc-(1,4)-D-GlcA unit. The prevalent presence of the GlcA residues at the non-reducing terminal of these oligosaccharides, suggesting the glycosidic bond of β-D-GalNAc-(1,4)-D-GlcA was more susceptible to acid than that of β-D-GlcA-(1,3)-D-GalNAc during mild acid hydrolysis. Moreover, the sulfate ester groups in GalNAc 4S6S unit could also be hydrolyzed by acid, and it at position C-4 was more susceptible to hydrolysis than that at C-6. This extensive degradation and desulfation of the backbone should be taken into consideration when mild acid hydrolysis was used in elucidating the exact structure or structure-activity relationship of native FG. [Display omitted] • Structures of oligosaccharides from mild acid hydrolysis of SvFG were analyzed. • The β-D-GalNAc-(1,4)-D-GlcA glycosidic bond was preferentially cleaved. • Sulfate ester at C-4 in GalNAc 4S6S was more susceptible to acid than that at C-6. • Desulfation should be accounted when using mild acid hydrolysis for FG structure study. [ABSTRACT FROM AUTHOR]

Abstract: Fucosylated chondroitin sulfate (FuCS) is a glycosaminoglycan from sea cucumber, made up of alternating β-d-glucuronic acid and N-acetyl-β-d-galactosamine units. The β-d-glucuronic acid residues have branches of sulfated fucose, while the fucose branches may have distinguishable patterns and proportions of sulfate substitution. The structure of the sulfated fucose branches of the fucosylated chondroitin sulfate from the sea cucumber Thelenata ananas has been characterized by mild acid hydrolysis and NMR technology. The results showed that the fucose residues are made up of 3-,4-mono-O-sulfated fucose and 2,4-di-O-sulfated fucose with about 25:22:53, respectively. The sulfated fucose branches are essential for the anticoagulant action of FuCS, and this potent effect is possibly related to the occurrence of 2,4-di-O-sulfated fucose units. Furthermore, these branches could constitute the structural requirement for the binding of the glycosaminoglycan to coagulant enzymes, such as thrombin by HCII, factor X by the intrinsic tenase complex. [Copyright &y& Elsevier]

Free radical depolymerization is a common method in structural analysis of polysaccharides, the major challenge is the analysis of the cleavage site and characterization of newly formed ends in this reaction. Here, a fucosylated glycosaminoglycan from H. fuscopunctata (HfFG) was depolymerized by H 2 O 2 and a series of oligosaccharides were purified and their structures were elucidated. For non-reducing ends of the trisaccharides were intact GalNAc 4S6S , the cleavage site should mainly be the β(1,3) linkages between GlcA and GalNAc in the backbone of FG. Meanwhile, the reducing ends of the disaccharides and trisaccharides were almost dicarboxylic acid derivatives of GlcA, possibly arising from oxidative breaking of the C C bond of GlcA at the reducing ends. In addition, glycosidic linkages in D-GalNAc-β(1,4)-D-GlcA and L-FucS-α(1,3)-D-GlcA located at the reducing end could be cleaved, and the released GalNAc 4S6S were oxidized to N -acetylgalactosaminic acid. • Structures of oligosaccharides from free radical depolymerized HfFG were determined. • The preferential cleavage site in FG during depolymerization was identified. • Oligosaccharides with dicarboxylic acid as the reducing end were found. • The possible mechanism of free radical depolymerization of FG was proposed. [ABSTRACT FROM AUTHOR]

• Oligosaccharide fractions H1 – H4 from depolymerized HcFG was purified and characterized. • HcFG was deduced as -{→4)-[ l -FucS-α(1→3)]- d -GlcA-β(1→3)- d -GalNAc 4S6S - β(1}n-. • FG branched with Fuc 3S4S was susceptible to β -eliminative depolymerization. • Anticoagulant activities of native and depolymerized HcFG were assessed. A glycosaminoglycan was isolated from the sea cucumber Holothuria coluber (HcFG). A series of oligosaccharide fragments (dp range 3–11) were prepared from its β -eliminative depolymerized product (dHcFG). Extensive NMR characterization of the oligosaccharides indicated the d -GlcA- β 1,3- d -GalNAc 4S6S repeating disaccharide backbone was substituted by monosaccharide branches comprising of Fuc 2S4S , Fuc 3S4S and Fuc 4S , linked to O-3 of d -GlcA. For the prevailing Fuc 3S4S at nonreducing end of dHcFG, the β- eliminative depolymerization process of HcFG was compared with those of the FGs from Actinopyga miliaris (AmFG, branched with Fuc 3S4S) and Stichopus variegatus (SvFG, branched with Fuc 2S4S). The result suggested that d -GlcA substituted with Fuc 3S4S was more susceptible to depolymerization than that with Fuc 2S4S. It might be due to the larger steric hindrance effects from Fuc 2S4S on the esterification of GlcA. Biological assays confirmed that the minimum chain length (dp8), regardless of the Fuc branch types, was required for the potent anti-iXase and anticoagulant activities in FG fragments. [ABSTRACT FROM AUTHOR]

• Three FGs were isolated from three species of sea cucumbers. • The structures of A1 - A4 , H1 - H4 and S1 - S4 from depolymerized FGs were determined. • Three FGs were deduced as -{→4)-[ L -FucS-α(1→3)]- D -GlcA-β(1→3)- D - GalNAc 4S6S -β(1}n-. • The effects of fucose types on anti-intrinsic tenase activity were discussed. Fucosylated glycosaminoglycan (FG), a glycosaminoglycan derivative containing distinct sulfated fucose (FucS) branches, displays potent anticoagulant activity by inhibiting the intrinsic tenase complex (iXase). Herein, AmFG, SvFG and HaFG from three species of sea cucumbers were isolated and depolymerized by β-eliminative cleavage. Three series of fragments, A1 - A4 , S1 - S4 and H1 - H4 , were purified from the depolymerized FGs. Based on structural analysis of these fragments, three FGs were deduced as -{→4)-[ L -FucS-α(1→3)]- D -GlcA-β(1→3)- D -GalNAc 4S6S -β(1} n -. The structures differed in sulfation types of FucS, namely, most of FucS in AmFG was Fuc 3S4S , but the FucS in SvFG was Fuc 2S4S , while the FucS in HaFG was Fuc 3S4S , Fuc 2S4S and Fuc 4S. However, all FucS branches attached to C -3 of GlcA as monosaccharides. Anticoagulant and anti-iXase assays showed the octasaccharide is the minimum fragment for potent anticoagulant activity via anti-iXase irrespective of FucS types. Among FG fragments with same degree of polymerization, oligosaccharides containing Fuc 2S4S had more potent anti-iXase activity. [ABSTRACT FROM AUTHOR]