Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate

A new method for the chemical modification of chitosan sulfate was used to prepare N-propanoyl-, N-hexanoyl- and N,O-quaternary substituted chitosan sulfate. Structural analysis by elemental analysis, FTIR, 13C NMR, and 1H NMR spectroscopy, and gel-permeation chromatography showed that these methods could conveniently be used for the introduction of functional groups. The influences of the acyl or quaternary groups on the anticoagulant activity of the polysaccharides were studied with respect to activated partial thromboplastin time (APTT) thrombin time (TT), and prothrombin time (PT). The propanoyl and hexanoyl groups increased the APTT activity, and the propanoyl groups also increased the TT anticoagulant activity slightly, while the N,O-quaternary chitosan sulfate showed only a slight TT coagulant activity.     

Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan

Synthesis of chitosan sulfates with low molecular weight (M_v 9000–35,000 Da) was carried out by sulfation of low molecular weight chitosan (M_v 10,000–50,000 Da). The oleum was used as sulfating agent and dimethylfornamide as medium. The chitosans were prepared by enzymatic and acidic hydrolysis of initial high molecular weight chitosan as well as by extrusion solid-state deacetylation of chitin. As was shown by FT-IR and NMR-methods and elemental analysis, the sulfation occurred at C-6 and C-3 positions and substitution degree is 1.10–1.63. The molecular weight sulfated chitosan was determined by viscometric method and the Mark–Houwink equation [η]=10⁻⁵ 4.97 M^0.77. Study of anticoagulant activity showed that chitosan sulfates with lowered molecular weight demonstrated a regular increase of anti-Xa activity like heparins.     

Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution

The kinetics and mechanisms of depolymerization of aqueous chitosan and alginate solutions at elevated temperatures have been investigated. Chitosan salts of different degree of acetylation (F_A), type of counterions (-glutamate, -chloride) and degree of purity were studied. One commercially available highly purified sodium alginate sample with high content of guluronic acid (G) was also studied. Furthermore, the influence of oxygen, H⁺ and OH⁻ ions on the initial depolymerization rates was investigated. Depolymerization kinetics was followed by measuring the time courses of the apparent viscosity and the intrinsic viscosity. The initial rate constants for depolymerization were determined from the intrinsic viscosity data converted to a quantity proportional to the fraction of bonds broken. The activation energies of the chitosan chloride and chitosan glutamate solutions with pH close to 5 and the same degree of acetylation, F_A = 0.14, were determined from the initial rate constants to be 76 ± 13 kJ/mol and 80 ± 11 kJ/mol, respectively.The results reported herein suggest that the stability of aqueous chitosan and alginate solutions at pH values 5–8 will be influenced by oxidative–reductive depolymerization (ORD) as the primary mechanism as long as transition metal ions are presented in the samples. Acid – and alkaline depolymerization will be the primary mechanisms for highly purified samples.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Sulfonation of papain-treated chitosan and its mechanism for anticoagulant activity

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, ¹³C NMR, and ¹H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.     

Anticoagulant activity of fucosylated chondroitin sulfate isolated from Cucumaria syracusana

Fucosylated chondroitin sulfate (FCScs) isolated from sea cucumber Cucumaria syracusana was characterized by Fourier Transform InfraRed spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR) spectroscopy and high performance size exclusion chromatograph, a multi-angle laser light scattering detector, a viscometer and a differential refractive index (dRI) detector (HPSEC-MALLS-dRI). The anticoagulant activities of FCScs were studied by the classical clotting time assays and the purified systems containing thrombin and antithrombin or heparin cofactor II. The effect on thrombin generation was investigated using calibrated automated thrombography (CAT). The results obtained showed that the FCS with high sulfate content 31 % and relatively low average molecular weight of 36.3 kDa was isolated from C. syracusana in amount of ∼ 35.6 mg/g dry body wall. Structural analysis of this polysaccharide revealed the presence backbone structure of chondroitin sulfate chain branched by two types of fucose 2,4-O-di and 3,4-O-disulfated residues in respective ratios of 57.5 and 42.5 %. The FCScs exhibited a high anticoagulant activity mediated essentially by heparin cofactor II (HCII) and to lesser extent by antithrombin (AT) with IC₅₀ values of 0.05 μg/mL and 0.09 μg/mL, respectively. Furthermore, the results of CAT assay showed that the velocity index decreases 3-times at 50 μg/mL in comparison with normal plasma. The overall results showed high anticoagulant activity attributed to the high sulfate content and abundance of disulfated fucose branches of FCScs which made it a promising candidate of anticoagulation drug.     

Separation,purification, anticoagulant activity and preliminary structural characterization of two sulfated polysaccharides from sea cucumber Acaudina molpadioidea and Holothuria nobilis

In this study, we isolated two fucosylated polysaccharide sulfates (ACP and HOP) from sea cucumber Acaudina molpadioidea and Holothuria nobilis, with an average molecular weight of 90.8 and 135.8 kDa, respectively. We investigated and compared their anticoagulant activities through anticoagulant assay. Our data showed that both polysaccharides possessed good anticoagulant activity, but HOP's activity was higher than that of ACP. Due to the different anticoagulant activities of ACP and HOP, we compared the preliminary structural characterizations of these two sulfated polysaccharides, and found that both ACP and HOP consisted of β-d-glucuronic acid, β-d-N-acetyl-galactosamine, α-l-fucose and sulfate groups. ACP and HOP had almost identical ratios of glucuronic acid, N-acetyl-galactosamine and fucose. However, the sulfate contents and sulfation patterns of fucose residues of ACP and HOP were obviously different. There were 4-O-sulfated fucose, 3,4-O-disulfated fucose and 2,4-O-disulfated fucose in ACP, but only 3-O-sulfated fucose and 2,4-O-disulfated fucose were present in HOP. Therefore, their distinct anticoagulant activities might be due to the different sulfate contents and sulfation patterns of their fucose residues.     

Solvolytic depolymerization of chondroitin and dermatan sulfates

It is essential to establish a library of glycosaminoglycan oligosaccharides from the chondroitin and dermatan sulfates to investigate their biological functions and structure-activity relationships (SARs). There are several approaches to obtain oligosaccharides using chemical and enzymatic degradation procedures; however, purification of each resulting oligosaccharide is complicated because of the diversity of sulfonation patterns present in these oligosaccharides. We have developed a new method for the solvolytic degradation for chondroitin and dermatan sulfates to obtain an oligosaccharide mixture that can be easily purified into chondro/dermato oligosaccharides for characterization by both ¹H NMR and MALDI-TOFMS. These oligosaccharides have a methyl-esterified uronate residue and a methyl 2-acetamido-2-deoxy-d-galactofuranoside at the nonreducing and reducing ends, respectively. All other internal repeating disaccharide units were desulfonated, but maintained their core carbohydrate structures.     

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2^T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into l-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

     

Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation

Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, ¹H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways.     

A structural basis for depolymerization of alginate by polysaccharide lyase family-7

Alginate lyases depolymerize alginate, a heteropolysaccharide consisting of alpha-L-guluronate and beta-D-mannuronate, through a beta-elimination reaction. Their structure/function relationships are expected to provide information valuable to future industrial alginate processing and drug design for Pseudomonas aeruginosa alginate biofilm-dependent infection, but much remains unknown. Here, we present the crystal structure at 1.0 A resolution and the results of mutational analysis of Sphingomonas sp. A1 alginate lyase A1-II', which is grouped into the polysaccharide lyase (PL) family-7. The overall structure of A1-II' uses a beta-sandwich fold, and it has a large active cleft covered by two short flexible loops. Comparison with other family PL-7 structures indicated that loop opening is necessary for substrate binding when the catalytic reaction is initiated. In contrast to the disorder in many side-chains on the protein surface, the three adjacent beta-strands at the center of the active cleft are well ordered. This results from hydrogen bond networks and stacking-like associations identical with those in other family PL-7 structures. Disruption of these interactions by site-directed mutagenesis (R146A, E148A, R150A, Q189A, and K280A) makes the protein insoluble or greatly decreases its activity. The A1-II' structure includes two sulfate ions in the active cleft. Ammonium sulfate was a potent inhibitor with a Ki of 2.5 mM, indicating that our structure represents a model of the inhibitory state. Results of mutational analysis and continuous hydrogen bond networks suggest that Arg146, Gln189, His191, and Tyr284 form an active center. Tyr284OH appears particularly crucial to the catalytic reaction, which is supported by sulfate ion binding and the proximity to the C5 and O4 atoms of subsite +1 in the model obtained by energy minimization calculations using tri-mannuronate. The structural basis shown by this study is similar in many respects to that of the family PL-5 enzymes.     

Anticoagulant activity of a sulfated chitosan

Chitin prepared from the shells of rice-field crabs (Somanniathelphusa dugasti) was converted into chitosan with a degree of acetylation of 0.21 and then sulfated with chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions to give 87% of water-soluble sulfated chitosan with degree of substitution (d.s) of 2.13. 1H NMR revealed the sulfate substitution at C-2, C-3 and C-6. Gel filtration on Sepharose CL-6B of the sulfated chitosan gave three fractions with average molecular weights of 7.1, 3.5, and 1.9 x 10(4). The three sulfated chitosan preparations showed strong anticoagulant activities, with the same mechanism of action observed for standard therapeutic heparin.     

樟芝固态发酵产物抗氧化性分析

本文对樟芝谷物固态发酵产物抗氧化性能及活性成分进行了研究.在所选谷物中,樟芝青稞固态发酵产物的乙醇提取物总抗氧化性最好,较未发酵青稞提高了4.02倍.通过无水乙醇50℃水浴振荡提取80 min,其总抗氧化性达到了769.60 U/g.对其抗氧化性能分析发现,樟芝青稞固态发酵乙醇提取物为6 mg/L时,对DP P H自由基、羟基自由基以及超氧阴离子的去除率分别为91.9%、51.2%、61.3%,对铁离子的螯合能力为79.5%.相对于未发酵谷物,大米、小米、玉米以及青稞的樟芝发酵产物中总酚含量均有显著的提升,其中青稞乙醇提取和水提取物中总酚含量分别提高了2.36倍和4.23倍.通过HP LC分析可知,樟芝固态发酵产物含有丰富的活性化合物,包括马来酸衍生物(Antrodin)以及泛醌类衍生物(Antroquinonol),且各组分含量较为均衡;而樟芝液态发酵菌丝体乙醇提取物中主要活性成分为马来酸衍生物,不含有泛醌类化合物.     

N-Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin

The deacetylation and depolymerization reactions of chitin/chitosan from three crustacean species (Paralomis granulosa, Lithodes antarcticus and Palinurus vulgaris) were evaluated under the same conditions. The average molecular weight and the mole fraction of N-acetylated units were the parameters studied in the resulting chitosans. During the N-deacetylation process P. granulosa, L. antarcticus and P. vulgaris follow a pseudo-first order kinetics and their apparent rate constants are very similar. However, the degradation rate of chitosan in the first 45 min of this process is higher for P. vulgaris. The depolymerization process follows a pseudo-first order kinetics for the three species, but in the first 9 min P. vulgaris shows a slightly lower depolymerization rate. Hence, depending on the ash contents, crystallinity and the physicochemical characteristics of chitin from these sources, the obtained chitosans show different qualities.     

Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M_n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.     

Current state on the enzymatic synthesis of glycosaminoglycans

Glycosaminoglycans (GAGs) are linear anionic polysaccharides, and most of them show a specific sulfation pattern. GAGs have been studied for decades, and still, new biological functions are discovered. Hyaluronic acid and heparin are sold for medical or cosmetic applications. With increased market and applications, the production of GAGs stays in the focus of research groups and the industry. Common industrial GAG production relies on the extraction of animal tissue. Contamination, high dispersity, and uncontrolled sulfation pattern are still obstacles to this process. Tailored production strategies for the chemoenzymatic synthesis have been developed to address these obstacles. In recent years, enzyme cascades, including uridine-5′-diphosphate sugar syntheses, were established to obtain defined polymer size and dispersity, as well as defined sulfation patterns. Nevertheless, the complex synthesis of GAGs is still a challenging research field.     

Low molecular weight chitosans--preparation by depolymerization with Aspergillus niger pectinase,and characterization

The viscosity of a chitosan solution was rapidly lowered in the presence of pectinase from Aspergillus niger at pH 3.0 and 37 degrees C. The low molecular weight chitosans (LMWC) had a molecular weight in the range 20,000-5000 Da. Circular dichroism spectra showed a decrease in the segment of acetylated glucosamine units, whereas X-ray diffraction and CP-MAS 13C NMR indicated higher crystallinity and polymorphism in LMWC. The latter on thermal drying resulted in structural alterations, and yielded an insoluble product. FT-IR and X-ray diffraction showed no evidence of either Schiff's base linkage or any annealed polymorph. CP-MAS 13C NMR showed marked changes in the chain conformations of LMWC, which are believed to be responsible for its loss of solubility and functionality.