Comparative Assessment of APTT Reagents for Evaluating Anticoagulant Sensitivity of Fucosylated Glycosaminoglycans (FGs) Derived from Sea Cucumbers.

Fucosylated glycosaminoglycans (FGs) derived from sea cucumbers exhibit potent intrinsic Xase (iXase) inhibition, anticoagulation, and antithrombosis. Plasma activated partial thromboplastin time (APTT), a widely used screening test worldwide, is crucial for evaluating anticoagulant efficacy. However, the applicability of these commercially available APTT reagents for assessing anticoagulation of FGs remains unreported. In this study, we investigated the disparity between ellagic acid and colloidal silica APTT reagents in evaluating anticoagulation of dHG-5 and dHLFG-4, two depolymerized FGs, and elucidated the underlying rationale. The results demonstrated that dHG-5 and dHLFG-4 exhibited heightened sensitivity to the ellagic acid APTT reagent both in vitro and in vivo, and did not significantly affect the activation of APTT reagents for plasma. In addition, both ellagic acid and colloidal silica APTT reagents inhibited the anti-iXase of dHG-5 and dHLFG-4, and the inhibition of the ellagic acid APTT reagent was less pronounced compared to the colloidal silica APTT reagent. These findings suggest that the reduced impact of the ellagic acid APTT reagent on the anti-iXase activity of dHG-5 and dHLFG-4 is responsible for the increased sensitivity in plasma APTT analysis. This study offers valuable insights into the characteristics of two APTT reagents applied for assessing the anticoagulant activity of FG-related compounds.

High-molecular-weight fucosylated glycosaminoglycan induces human platelet aggregation depending on αIIbß3 and platelet secretion.

Fucosylated glycosaminoglycan (FG) from sea cucumbers has been reported to have anticoagulant effects via targeting intrinsic tenase. However, FG from natural source also potentially poses risks due to its FXIIa activation and platelet aggregating effects. Here, we found that the effect of FG on human platelet aggregation depended on both the sulfation pattern and chain length. FGs with higher content of Fuc2S4S and larger molecular weight (≥14 kD) had stronger activity. Both platelet aggregation and P-selectin expression induced by TaFG (an FG from Thelenota ananas) were decreased as the molecular weight reduced. Ticagrelor, aspirin and wortmannin completely blocked the secretion (ADP) but only partially blocked the aggregation induced by TaFG. Tirofiban an αIIbß3 antagonist however potently inhibited both the secretion and aggregation, with IC50 of 6.01 ± 1.1.97 nM. Furthermore, TaFG could bind to human αIIbß3 with high affinity, and the affinities of two FGs were paralleled with their activity in platelet aggregation or activation. These results indicated that αIIbß3 played an important role in TaFG-induced platelet aggregation which was mediated by PI3K, and that platelet secretion was required for the amplification of aggregation.

Pharmacokinetics and Pharmacodynamics of a Depolymerized Glycosaminoglycan from Holothuria fuscopunctata, a Novel Anticoagulant Candidate, in Rats by Bioanalytical Methods.

dHG-5 (Mw 5.3 kD) is a depolymerized glycosaminoglycan from sea cucumber Holothuria fuscopunctata. As a selective inhibitor of intrinsic Xase (iXase), preclinical study showed it was a promising anticoagulant candidate without obvious bleeding risk. In this work, two bioanalytical methods based on the anti-iXase and activated partial thromboplastin time (APTT) prolongation activities were established and validated to determine dHG-5 concentrations in plasma and urine samples. After single subcutaneous administration of dHG-5 at 5, 9, and 16.2 mg/kg to rats, the time to peak concentration (Tmax) was at about 1 h, and the peak concentration (Cmax) was 2.70, 6.50, and 10.11 µg/mL, respectively. The plasma elimination half-life(T1/2ß) was also about 1 h and dHG-5 could be almost completely absorbed after s.c. administration. Additionally, the pharmacodynamics of dHG-5 was positively correlated with its pharmacokinetics, as determined by rat plasma APTT and anti-iXase method, respectively. dHG-5 was mainly excreted by urine as the unchanged parent drug and about 60% was excreted within 48 h. The results suggested that dHG-5 could be almost completely absorbed after subcutaneous injection and the pharmacokinetics of dHG-5 are predictable. Studying pharmacokinetics of dHG-5 could provide valuable information for future clinical studies.

The Toxicology of Native Fucosylated Glycosaminoglycans and the Safety of Their Depolymerized Products as Anticoagulants.

Fucosylated glycosaminoglycan (FG) from sea cucumber is a potent anticoagulant by inhibiting intrinsic coagulation tenase (iXase). However, high-molecular-weight FGs can activate platelets and plasma contact system, and induce hypotension in rats, which limits its application. Herein, we found that FG from T. ananas (TaFG) and FG from H. fuscopunctata (HfFG) at 4.0 mg/kg (i.v.) could cause significant cardiovascular and respiratory dysfunction in rats, even lethality, while their depolymerized products had no obvious side effects. After injection, native FG increased rat plasma kallikrein activity and levels of the vasoactive peptide bradykinin (BK), consistent with their contact activation activity, which was assumed to be the cause of hypotension in rats. However, the hemodynamic effects of native FG cannot be prevented by the BK receptor antagonist. Further study showed that native FG induced in vivo procoagulation, thrombocytopenia, and pulmonary embolism. Additionally, its lethal effect could be prevented by anticoagulant combined with antiplatelet drugs. In summary, the acute toxicity of native FG is mainly ascribed to pulmonary microvessel embolism due to platelet aggregation and contact activation-mediated coagulation, while depolymerized FG is a safe anticoagulant candidate by selectively targeting iXase.

Characterization of the Hydrolysis Kinetics of Fucosylated Glycosaminoglycan in Mild Acid and Structures of the Resulting Oligosaccharides.

: Mild acid hydrolysis is a common method for the structure analysis of fucosylated glycosaminoglycan (FG). In this work, the effects of acid hydrolysis on the structure of FG from S. variegatus (SvFG) and the reaction characteristic were systemically studied. The degree of defucosylation (DF) and molecular weights (Mw) of partial fucosylated glycosaminoglycans (pFs) were monitored by 1H NMR and size-exclusion chromatography, respectively. The kinetic plots of DF, degree of desulfation (DS) from fucose branches, and degree of hydrolysis (DH) of the backbone are exponentially increased with time, indicating that acid hydrolysis of SvFG followed a first-order kinetics. The kinetic rate constants kDF, kDS, and kDH were determined to be 0.0223 h-1, 0.0041 h-1, and 0.0005 h-1, respectively. The structure of the released sulfated fucose branches (FucS) from SvFG and HfFG (FG from H. fuscopunctata) was characterized by 1D/2D NMR spectroscopy, suggesting the presence of six types of fucose: α/ß Fuc2S4S, Fuc3S4S, Fuc3S, Fuc4S, Fuc2S, and Fuc. The Fuc3S4S was more susceptible to acid than Fuc2S4S, and that the sulfate ester in position of O-2 and O-3 than in O-4 of fucose. The structure characteristic of pF18 indicated the cleavage of backbone glycosidic bonds. The APTT prolonged activity reduced with the decrease of the DF and Mw of the pFs, and became insignificant when its DF was 87% with Mw of 3.5 kDa.

Precise Structure and Anticoagulant Activity of Fucosylated Glycosaminoglycan from Apostichopus japonicus: Analysis of Its Depolymerized Fragments.

Apostichopus japonicus is one of the most economically important species in sea cucumber aquaculture in China. Fucosylated glycosaminoglycan from A. japonicus (AjFG) has shown multiple pharmacological activities. However, results from studies on the structure of AjFG are still controversial. In this study, the deaminative depolymerization method that is glycosidic bond-selective was used to prepare the depolymerized products from AjFG (dAjFG), and then a series of purified oligosaccharide fragments such as tri-, hexa-, nona-, and dodecasaccharides were obtained from dAjFG by gel permeation chromatography. The 1D/2D NMR and ESI-MS spectrometry analyses showed that these oligosaccharides had the structural formula of l-FucS-α1,3-d-GlcA-ß1,3-{d-GalNAc4S6S-ß1,4-[l-FucS-α1,3-]d-GlcA-ß1,3-}n-d-anTal-diol4S6S (n = 0, 1, 2, 3; FucS represents Fuc2S4S, Fuc3S4S, or Fuc4S). Thus, the unambiguous structure of native AjFG can be rationally deduced: it had the backbone of {-4-d-GlcA-ß1,3-d-GalNAc4S6S-ß1-}n, which is similar to chondroitin sulfate E, and each d-GlcA residue in the backbone was branched with a l-FucS monosaccharide at O-3. Bioactivity assays confirmed that dAjFG and nonasaccharides and dodecasaccharides from AjFG had potent anticoagulant activity by intrinsic FXase inhibition while avoiding side effects such as FXII activation and platelet aggregation.

Physicochemical Characteristics and Anticoagulant Activities of the Polysaccharides from Sea Cucumber Pattalus mollis.

Sulfated polysaccharides from sea cucumbers possess distinct chemical structure and various biological activities. Herein, three types of polysaccharides were isolated and purified from Pattalus mollis, and their structures and bioactivities were analyzed. The fucosylated glycosaminoglycan (PmFG) had a CS-like backbone composed of the repeating units of {-4-d-GlcA-ß-1,3-d-GalNAc4S6S-ß-1-}, and branches of a sulfated α-l-Fuc (including Fuc2S4S, Fuc3S4S and Fuc4S with a molar ratio of 2:2.5:1) linked to O-3 of each d-GlcA. The fucan sulfate (PmFS) had a backbone consisting of a repetitively linked unit {-4-l-Fuc2S-α-1-}, and interestingly, every trisaccharide unit in its backbone was branched with a sulfated α-l-Fuc (Fuc4S or Fuc3S with a molar ratio of 4:1). Apart from the sulfated polysaccharides, two neutral glycans (PmNG-1 & -2) differing in molecular weight were also obtained and their structures were similar to animal glycogen. Anticoagulant assays indicated that PmFG and PmFS possessed strong APTT prolonging and intrinsic factor Xase inhibition activities, and the sulfated α-l-Fuc branches might contribute to the anticoagulant and anti-FXase activities of both PmFG and PmFS.

Anti-HIV-1 activity and structure-activity-relationship study of a fucosylated glycosaminoglycan from an echinoderm by targeting the conserved CD4 induced epitope.

BACKGROUND: Fucosylated glycosaminoglycan (FG) is a novel glycosaminoglycan with a chondroitin sulfate-like backbone and fucose sulfate branches. The aim of this study is to investigate the mechanism and structure-activity relationships (SAR) of FG for combating HIV-1 infection. METHODS: Anti-HIV activities of FGs were assessed by a cytopathic effect assay and an HIV-1 p24 detection assay. The biomolecule interactions were explored via biolayer interferometry technology. The SAR was established by comparing its anti-HIV-1 activities, conserved CD4 induced (CD4i) epitope-dependent interactions and anticoagulant activities. RESULTS: FG efficiently and selectively inhibited the X4- and R5X4-tropic HIV-1 infections in C8166 cells with little cytotoxicity against C8166 cells and PBMCs. Our data indicated that FG bound to gp120 with nanomolar affinity and may interact with CD4i of gp120. Additionally, the CD4i binding affinity of FG was higher than that of dextran sulfate. SAR studies suggested that the unique sulfated fucose branches account for the anti-HIV-1 activity. The molecular size and present carboxyl groups of FG may also play important roles in various activities. Notably, several FG derivatives showed higher anti-HIV-1 activities and much lower anticoagulant activities than those of heparin. CONCLUSIONS: FG exhibits strong activity against X4- and R5X4-tropic HIV-1 infections. The mechanism may be related to targeting CD4i of gp120, which results in inhibition of HIV-1 entry. The carboxyl group substituted derivatives of FG (8.5-12.8kDa), might display high anti-HIV-1 activity and low anticoagulant activity. GENERAL SIGNIFICANCE: Our data supports further the investigation of FG derivatives as novel HIV-1 entry inhibitors targeting CD4i.

Structure and pharmacokinetics/pharmacodynamics of the anticoagulant tetradecasaccharide oHG-14 as an intrinsic tenase inhibitor.

The intrinsic tenase complex (iXase) is an attractive antithrombotic target to treat or prevent pathological thrombosis with negligible bleeding risk. Fucosylated glycosaminoglycan (FG) is a promising anticoagulant by inhibiting iXase. A depolymerized FG (dHG-5) as an anticoagulant has been approved for clinical trials. Given that dHG-5 is a multi-component drug candidate consisting of a homologous series of oligosaccharides, it is difficult to predict a clear pharmacokinetics. Here, as a major oligosaccharide component, the tetradecasaccharide (oHG-14) was purified from dHG-5 and its structure was defined as L-Fuc3S4S-α(1,3)-L-Δ4,5GlcA-α(1,3)-{D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ß(1,3)-}3-D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ol. oHG-14 showed potent iXase inhibitory activity in vitro and antithrombotic effect in vivo comparable to dHG-5. After single subcutaneous administration of oHG-14 at 8, 14.4 and 32.4 mg/kg to rats, the absolute bioavailability was 71.6 %-80.9 % determined by the validated bioanalytical methods. The maximum concentration (Cmax) was 3.73, 8.07, and 11.95 µg/mL, respectively, and the time reaching Cmax (Tmax) was about 1 h. oHG-14 was mainly excreted by kidney as the parent compound with the elimination kinetics of first-order linear model. Anticoagulant activity of oHG-14 was positively correlated with its concentration in rat plasma. The pharmacokinetics/pharmacodynamics (PK/PD) of oHG-14 is similar to that of dHG-5. This study could provide supportive data for the clinical trial of dHG-5 and further development of pure oligosaccharide as an antithrombotic drug candidate.

The glycosidic bond cleavage and desulfation investigation of fucosylated glycosaminoglycan during mild acid hydrolysis through structural analysis of the resulting oligosaccharides.

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 H2SO4 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 ß-D-GlcA-(1,3)-D-GalNAc and ß-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 ß-D-GalNAc-(1,4)-D-GlcA was more susceptible to acid than that of ß-D-GlcA-(1,3)-D-GalNAc during mild acid hydrolysis. Moreover, the sulfate ester groups in GalNAc4S6S 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.

Peroxidative depolymerization of fucosylated glycosaminoglycan: Bond-cleavage pattern and activities of oligosaccharides.

Peroxidative depolymerization is often used to elucidate the structure and structure-activity relationship of fucosylated glycosaminoglycan (FG), while the selectivity of bond cleavage and structural characteristics of the resulting fragments remain to be confirmed. Here, the FG from Stichopus variegatus (SvFG) was depolymerized by H2O2, and a series of yielded mono- and oligo-saccharides were purified. Almost all the non-reducing ends of oligosaccharides were d-GalNAc4S6S, suggesting that GlcA-ß1,3-GalNAc4S6S linkage was preferentially cleaved. The model reactions showed the glycosidic bond of uronate was more susceptible than those of N-acetyl hexosamine and fucose, which should be due to bond energy of the anomeric CH. The reducing ends of oligosaccharides include C4-C6 saccharic acid and GalNAc or GalNAcA, which should be derived from the oxidation of the reducing end. A hexasaccharide with tartaric acid exhibited increased anti-iXase activity, suggesting the oxidation of reducing end did not impair the anti-iXase activity of FG-derived oligosaccharides.

Deaminative-cleaved S. monotuberculatus fucosylated glycosaminoglycan: Structural elucidation and anticoagulant activity.

Stichopus monotuberculatus is a tropical sea cucumber species and used as a folk medicine and tonic food. In this study, a fucosylated glycosaminoglycan (SmFG), the depolymerized SmFG (dSmFG) and its oligosaccharide fractions were prepared. The SmFG and its depolymerized products were comprised of a chondroitin-sulfate-E backbone, and various sulfated fucose side chains, including an unusual disaccharide side chain connected to the C-3 position of D-glucuronic acid (GlcA) or GlcA-ol. A peeling reaction occurred during the deaminative depolymerization process. The dSmFG and its fractions showed strong anticoagulant activity by selectively inhibiting intrinsic tenase complex, and had no anti-factor IIa, Xa and VIIa activity. The anticoagulant activity reduced with the decrease of molecular weight, and the unusual branch and novel reducing end may enhance the anticoagulant activity. These findings can provide significant information for development and utilization of depolymerized products from SmFG in food and pharmaceutical industries.

Structural characterization and anticoagulant analysis of the novel branched fucosylated glycosaminoglycan from sea cucumber Holothuria nobilis.

Glycosaminoglycan HnFG was extracted from sea cucumber Holothuria nobilis. Its chemical structure was characterized by analyzing the physicochemical properties, oligosaccharides from its mild acid hydrolysates and depolymerized products. The disaccharide d-GalNAc4S6S-α1,2-l-Fuc3S-ol found in its mild acid hydrolysates provided a clue for the presence of a unique disaccharide-branch in HnFG. Furthermore, it was confirmed by a series of oligosaccharides from the low-molecular weight HnFG prepared by ß-eliminative depolymerization. Combining with the analysis of its peroxide depolymerized products, the precise structure of HnFG was determined: A chondroitin sulfate E (CS-E)-like backbone branched with sulfated monofucoses (~67%) and disaccharides d-GalNAcS-α1,2-l-Fuc3S (~33%) at O-3 position of each GlcUA. This is the first report on the novel branches in glycosaminoglycan. Biologically, the native and depolymerized HnFG showed potent activities in prolonging the activated partial thrombin time (APTT) and inhibiting intrinsic coagulation Xase (iXase), whereas the oligosaccharides (degree of polymerization ≤6) had no obvious effects.

Structural characterization of oligosaccharides from free radical depolymerized fucosylated glycosaminoglycan and suggested mechanism of depolymerization.

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 H2O2 and a series of oligosaccharides were purified and their structures were elucidated. For non-reducing ends of the trisaccharides were intact GalNAc4S6S, the cleavage site should mainly be the ß(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 CC bond of GlcA at the reducing ends. In addition, glycosidic linkages in D-GalNAc-ß(1,4)-D-GlcA and L-FucS-α(1,3)-D-GlcA located at the reducing end could be cleaved, and the released GalNAc4S6S were oxidized to N-acetylgalactosaminic acid.

Low-molecular-weight fucosylated glycosaminoglycan and its oligosaccharides from sea cucumber as novel anticoagulants: A review.

Antithrombotic drugs have some side effects, such as risk of serious bleeding. Development of antithrombotic drugs that inhibit components of the intrinsic coagulation pathway and have a low risk of causing bleeding has recently been a focus of research. Fucosylated glycosaminoglycan (FG), also named as fucosylated chondroitin sulfate (FCS), has potent anticoagulant activity and inhibits intrinsic factor tenase (FXase) complex. Low-molecular-weight FG (LFG) and its oligosaccharides show characteristics of anticoagulant and antithrombotic activities with negligible side effects, such as activation of human FXII, induction of platelet aggregation, and especially, the risk of serious bleeding. They are potential new anticoagulant drugs and have been extensively studied in recent years. This review presents recent findings regarding the preparation, structural analysis, pharmacological activity, and structure-activity relationships of LFG and its derived oligosaccharides, so as to provide a reference for the development of new anticoagulants with low side effects.

A new fucosylated glycosaminoglycan containing disaccharide branches from Acaudina molpadioides: Unusual structure and anti-intrinsic tenase activity.

A fucosylated glycosaminoglycan (AmFG) was extracted from the sea cucumber Acaudina molpadioides. And a series of oligosaccharides were purified from the size-homogeneous fractions, which were prepared from the ß-eliminative depolymerized AmFG. According to "bottom-up" strategy, the precise structure of AmFG was elucidated by analyzing the structures of these purified oligosaccharides, combining with NMR analysis of its free-radical depolymerized product. It contained a CS-E-like backbone, and each GlcUA was branched with a mono- or di-sulfated fucose (Fuc) at O-3. Intriguingly, besides two types of monosaccharide branches, Fuc2S4S (60 %) and Fuc4S (25 %), that were common in FG, AmFG also contained an unusual disaccharide branch GalNAc-α1,2-Fuc3S4S (15 %); this is the first report of such a structure in a glycosaminoglycan. Biological assays indicated that native AmFG and its oligosaccharides had potent anticoagulant and intrinsic tenase (iXase) inhibitory activities in a chain length-dependent manner. For these oligosaccharides, octasaccharide was the minimum structural fragment for potent anti-iXase activity, and the disaccharide branch might enhance this activity.

NMR characterization and anticoagulant activity of the oligosaccharides from the fucosylated glycosaminoglycan isolated from Holothuria coluber.

A glycosaminoglycan was isolated from the sea cucumber Holothuria coluber (HcFG). A series of oligosaccharide fragments (dp range 3-11) were prepared from its ß-eliminative depolymerized product (dHcFG). Extensive NMR characterization of the oligosaccharides indicated the d-GlcA-ß1,3-d-GalNAc4S6S repeating disaccharide backbone was substituted by monosaccharide branches comprising of Fuc2S4S, Fuc3S4S and Fuc4S, linked to O-3 of d-GlcA. For the prevailing Fuc3S4S at nonreducing end of dHcFG, the ß-eliminative depolymerization process of HcFG was compared with those of the FGs from Actinopyga miliaris (AmFG, branched with Fuc3S4S) and Stichopus variegatus (SvFG, branched with Fuc2S4S). The result suggested that d-GlcA substituted with Fuc3S4S was more susceptible to depolymerization than that with Fuc2S4S. It might be due to the larger steric hindrance effects from Fuc2S4S 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.

Precise structures and anti-intrinsic tenase complex activity of three fucosylated glycosaminoglycans and their fragments.

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 ß-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-ß(1→3)-D-GalNAc4S6S-ß(1}n-. The structures differed in sulfation types of FucS, namely, most of FucS in AmFG was Fuc3S4S, but the FucS in SvFG was Fuc2S4S, while the FucS in HaFG was Fuc3S4S, Fuc2S4S and Fuc4S. 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 Fuc2S4S had more potent anti-iXase activity.

Structural analysis and anticoagulant activities of two sulfated polysaccharides from the sea cucumber Holothuria coluber.

Sulfated polysaccharides such as fucosylated glycosaminoglycan and fucan sulfate from echinoderm possess complex chemical structure and various biological activities. The two sulfated polysaccharides were purified from the low-value sea cucumber Holothuria coluber. Their physicochemical properties and chemical structures were analyzed and characterized by chemical and instrumental methods. Structural analysis clarified that the sea cucumber fucosylated glycosaminoglycan contains a chondroitin sulfate-like backbone and fucosyl branches with four various sulfation patterns. The fucan sulfate with molecular weight of 64.6 kDa comprises a central core of regular α(1 → 4)-linked tetrasaccharide repeating units, each of which is linked by a 4-O-sulfated fucose residue. Anticoagulant assays indicated that these sulfated polysaccharides possessed strong APTT prolonging activities and intrinsic factor Xase inhibitory activities, both of which decreased with the reduction of their molecular weights. Our results expand knowledge on the structural types of sulfated polysaccharides from sea cucumbers and further illustrate their functionality.

Digestibility of fucosylated glycosaminoglycan from sea cucumber and its effects on digestive enzymes under simulated salivary and gastrointestinal conditions.

The digestibility of fucosylated glycosaminoglycan (FG) and its effects on digestive enzymes were investigated using an in vitro digestion model. Results showed that the molecular weight and the reducing sugar content of FG were not significantly changed, and no free monosaccharides released from FG were detected after the salivary, gastric and intestinal digestion, indicating that both the backbone and the sulfated fucose branches of FG are resistant to be cleaved in the saliva and gastrointestinal tract environments. Furthermore, FG can dose-dependently inhibit digestive enzymes such as α-amylase, pepsin and pancreatic lipase in different degrees under the simulated digestion conditions due to the sulfate and carboxyl groups. These physiological effects of FG may help control the postprandial glucose concentration and have the potential in the prevention or treatment of reflux disease and obesity. The findings may provide information on the digestibility and beneficial physiological effects of FG as a potential natural product to promote human health.