Fucoidan from brown seaweed Tubinaria decurrens: Structure and structure - anticancer activity relationship.

The aims of this study are to determine the structure of a fucoidan from brown seaweed Turbinaria decurrens, to investigate its anticancer activity and structure-activity relationship. SEC-MALLS, IR, ESI-MS and NMR spectra analysis indicated that dominant structure of the fucoidan, with a Mw 122.6 KDa, has a backbone of (1 â†’ 3)- and (1 â†’ 4)-α-L-Fucp residues, branched at C-4, sulfate groups are attached at C-2, C-3 and C-4; branches are (1 â†’ 4)-ß-D-Galp residues and sulfated at C-2. The fucoidan was hydrolyzed by HCl aqueous solution to obtain hydrolyzed fucoidans. It is assumed that native and hydrolyzed fucoidans have a rod-like conformation in solution with cross-sectional radius of gyration (Rgc) ranged from 0.53 to 1.52 nm as estimated from SAXS measurements. The fucoidans show great anticancer activity against HT29 human colon cancer cell line with IC50 ranging from 5.41 ± 0.36 to 73.52 ± 2.54 µg/mL. Anticancer activity of the fucoidan could be significantly improved by lowering molecular weight, furthermore, fucoidan required small molecular weight, small molecular weight distribution and rod-like structure with a short branch length for high anticancer activity.

Structure, anticoagulant and cytotoxic activity of a sulfated polysaccharide from green seaweed Chaetomorpha linum.

In this article, chemical structure and conformation in an aqueous solution of a new sulfated polysaccharide, PCL, extracted from green seaweed Chaetomorpha linum were elucidated by SEC-MALL, IR, NMR and SAXS. The results indicated that the obtained polysaccharide is a sulfated arabinogalactan with a molecular weight of 223 kDa, and is mainly composed of →3,6)-α-D-Galp4S→ and →2)-α-L-Araf→ connecting together through 1→3 glycoside linkages. It has a broken rod-like conformation in solution with Rgc estimated as 0.43 nm from SAXS measurements. The polysaccharide exhibited a notable anticoagulant activity measured by the assays of activated partial thromboplastintime, thrombintime and prothrombine time as well as a significant cytotoxic activity against hepatocellular, human breast cancer, and cervical cancer cell lines.

Tunable structure of chimeric isomaltomegalosaccharides with double α-(1 → 4)-glucosyl chains enhances the solubility of water-insoluble bioactive compounds.

Isomaltomegalosaccharides with α-(1 â†’ 4) and α-(1 â†’ 6)-segments solubilize water-insoluble ligands since the former complexes with the ligand and the latter solubilizes the complex. Previously, we enzymatically synthesized isomaltomegalosaccharide with a single α-(1 â†’ 4)-segment at the reducing end (S-IMS) by dextran dextrinase (DDase), but the chain length [average degree of polymerization (DP) ≤ 9] was insufficient for strong encapsulation. We hypothesized that the conjugation of longer α-(1 â†’ 4)-segment afforded the promising function although DDase is incapable to do so. In this study, the cyclodextrin glucanotransferase-catalyzed coupling reaction of α-cyclodextrin to S-IMS synthesized a new α-(1 â†’ 4)-segment at the nonreducing end (N-4S) of S-IMS to form D-IMS [IMS harboring double α-(1 â†’ 4)-segments]. The length of N-4S was modulated by the ratio between α-cyclodextrin and S-IMS, generating N-4Ss with DPs of 7-50. Based on phase-solubility analysis, D-IMS-28.3/13/3 bearing amylose-like helical N-4S with DP of 28.3 displayed a water-soluble complex with aromatic drugs and curcumin. Small-angle X-ray scattering revealed the chain adapted to rigid in solution in which the radius of gyration was estimated to 2.4 nm. Furthermore, D-IMS with short N-4S solubilized flavonoids of less-soluble multifunctional substances. In our research, enzyme-generated functional biomaterials from DDase were developed to maximize the hydrophobic binding efficacy towards water-insoluble bioactive compounds.

Oxygen permeability of regenerated cellulose films with different water regains.

The effect of water regain on the oxygen permeability coefficient (OP) of regenerated cellulose film was investigated. The OP of the dry film was extremely low, which was classified as a "very high" performance gas barrier; however, the OP increased with increasing water regain, and reached to the OP similar to that of low-density polyethylene film, which was categorized as a "poor" gas barrier. The film thickness increased with increasing water regain, and edge-view small-angle X-ray scattering revealed widening of the space between microcrystals in the thickness direction. Oxygen molecules likely passed through the space between cellulose molecules, which was widened with increasing water regain. The viscoelastic measurements indicated that regenerated cellulose existed in a rubbery state under wet conditions. Overall, the OP of regenerated cellulose was increased because of the widening and micro-Brownian motion of cellulose main chains caused by water.

Fractionation and characterization of cell wall polysaccharides from the brown alga Cladosiphon okamuranus.

Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

Dissolution of cellulose into supercritical water and its dissolving state followed by structure formation from the solution system.

Cellulose was treated with supercritical water at 668 K and 25 MPa for 0.04 s in this study. The cellulose/water system was transparent at room temperature for a while after supercritical water treatment before a precipitate gradually appeared over several hours. The precipitation process was monitored by synchrotron X-ray scattering. The scattering functions of fractal systems and flat-like structures were utilized to explain the experimentally observed small-angle scattering profiles. Immediately after supercritical water treatment, the cellulose appeared to dissolve with a fractal dimension D of approximately 1, indicating that the cellulose molecules were rigid, followed by aggregation into a 5-nm-thick flat-like structure. The flat-like structure was determined to be similar to the molecular sheets observed during the early stages of precipitation in the cellulose/aqueous sodium hydroxide and cellulose/aqueous lithium hydroxide/urea systems. Resultant regenerated cellulose had high crystallinity, large crystal size, and a low degree of polymerization.

Relaxation phenomenon and swelling behavior of regenerated cellulose fibers affected by organic solvents.

Regenerated cellulose fibers are extremely sensitive to water. In particular, their mechanical properties are greatly affected by water. Recently, it was clarified that the glass transition temperatures of regenerated cellulose over 500 K can be shifted to room temperature, and small-angle X-ray scattering (SAXS) showed the maxima and shoulders on the equator line in the wet state. In this study, glass transition caused by organic solvents was observed, although the peak heights of tangent δ (tan δ) were low, which suggested the regions affected by organic solvents were small. SAXS showed the maxima and shoulders, suggesting that organic solvents decreased the density of the amorphous region, i.e., widened space between cellulose molecules, creating sufficient space for the micro-Brownian motion of cellulose main chains. However, alkanes with molecular weight larger than n-nonane did not show tan δ peaks, which suggests that the solvent is hard to enter between microfibrils.

Relaxation phenomenon and swelling behavior of regenerated cellulose fibers affected by water.

Regenerated cellulose fibers are extremely sensitive to water; particularly, the mechanical properties are greatly affected by water. We examined the effect of water on regenerated cellulose fibers in respect of the relaxation phenomenon and swelling behavior. The peaks and shoulder of mechanical loss tangent δ were observed at room temperature and water regains of 56-78%. At the same time, the storage modulus markedly decreased around these water regains. Small angle X-ray scattering showed the maxima and shoulders in the wet state, which suggested that water decreased the density of the amorphous region and made space for the movement of polymer segments. It is possible that the glass transition temperatures of 510-550 K shift to room temperature at specific water regains. It is reasonable to suppose that water can penetrate into the amorphous region, loosening the interactions between cellulose molecules and widening the region, and in consequence decreasing the glass transition temperature.

Observation of in vitro cellulose synthesis by bacterial cellulose synthase with time-resolved small angle X-ray scattering.

Cellulose synthase is the enzyme that produces cellulose in the living organisms like plant, and has two functions: polymerizing glucose residues (polymerization) and assembling these polymerized molecules into a crystalline microfibril with a "cellulose I" crystallographic structure (crystallization). Many studies, however, have shown that an in vitro reaction of cellulose synthase produces aggregates of a non-native crystallographic structure "cellulose II", despite the remaining polymerizing activity. This is partial denaturation or loss of crystallization function in cellulose synthase, which needs to be resolved to reconstitute its native activity. To this end, we aimed to clarify the process of cellulose II formation by bacterial cellulose synthase in vitro, using in situ small angle X-ray scattering (SAXS). An increase in scattering specific to synthesis was observed around two distinct regions of q (0.2-0.4 nm-1 and <0.1 nm-1) by time-resolved SAXS measurement. The scattering at higher q-region appears prior to lower-q scattering at beginning of the reaction, indicating the existence of smaller primitive aggregations at the initiation stage. This study demonstrates the use of in situ SAXS measurement to decipher the dynamics of biosynthesized cellulose chains, which is a remarkable example of polymer assembly in ambient conditions.

Local Structure of Ca2+ Alginate Hydrogels Gelled via Competitive Ligand Exchange and Measured by Small Angle X-Ray Scattering.

Alginates, being linear anionic co-polymers of 1,4-linked residues ß-d-ManA (M) and α-l-GulA (G), are widely applied as hydrogel biomaterials due to their favourable in vivo biocompatibility and convenient ionic crosslinking. The "egg-box" model is the prevailing description of the local structure of junction zones that form between the alginate chains and divalent cations, such as Ca2+, when ionic gelation occurs. In the present study we address to what extent signatures of lateral dimerization and further lateral association of junction zones also represent a valid model for the gelation of alginate using the recently reported method of competitive ligand exchange of chelated Ca2+ ions as a method for introducing gelling ions at constant pH. Small angle X-ray scattering with a q range from 0.1 to 3.3 nm-1 was employed to determine local structure in the hydrogel, using a custom-made fluid sample cell inserted in the X-ray beam. The scattering volume was intended to be localized to the contact zone between the two injected aqueous alginate solutions, and data was captured to resolve the kinetics of the structure formation at three different conditions of pH. The data show evolution of the local structure for the Ca2+ induced formation of junction zones in an alginate with 68% G residues, characterized by cross-sectional radii that could be accounted for by a two-component, broken rod like model. The evolution of the two component weight fractions apparently underpinned the connectivity, as reflected in the rheological data.

Structural characterization of enzymatically synthesized glucan dendrimers.

Glucan dendrimers (GDs) were synthesized by using a branching enzyme. Spherical GDs of different molecular weights were obtained and had a narrow particle size distribution. Small-angle X-ray scattering (SAXS) measurements showed that the radius of gyration of the GDs was 4.2-14.6 nm in aqueous solution. The SAXS profiles of GDs with small molecular weights exhibited scattering behavior similar to that described by the hyperbranched polymer model, and tended to resemble that of a sphere as the molecular weight increased.

Gelation and Structural Formation of Amylose by In Situ Neutralization as Observed by Small-Angle X-ray Scattering.

The gelation and structural formation of two types of amylose in alkaline solution by in situ neutralization was monitored with time-resolved small-angle X-ray scattering (tr-SAXS). Sharp increases of SAXS profile in lower angle region were observed after gelation. The results showed that aggregation of amylose chains led to a gel point with crystal growth. The aggregation appeared to function as a junction zone, and the aggregate structure depended on the molecular weight of amylose. A high-molecular-weight sample was fitted using a Debye-Bueche function, and a low-molecular-weight sample was fitted using a stretched exponential function.

Structure, conformation in aqueous solution and antimicrobial activity of ulvan extracted from green seaweed Ulva reticulata.

The aim of this study is to elucidate the structure and investigate the antimicrobial activity of an ulvan obtained by water extraction from green seaweed Ulva reticulata collected at Nha Trang sea of Vietnam by using IR, NMR, SEC-MALLS and SAXS methods. The ulvan is composed of rhamnose, galactose, xylose, manose and glucose (mole ratio Rha: Gal: Xyl: Man: Glu = 1:0.12:0.1:0.06:0.03), uronic acid (22.5%) and sulphate groups (17.6%). Chemically structural determination showed that the ulvan mainly composed of disaccharide [→4)ß-D-GlcA(1→4)α-L-Rha3S-(1→]. The results from SAXS indicated that ulvan under study has a rod-like bulky chain conformation. Ulvan from U. reticulata showed high antimicrobial activity, with inhibition zone diameter of 20 mm against Enterobacter cloace and 18 mm against Escherichia coli.

Local structure of Ca(2+) induced hydrogels of alginate-oligoguluronate blends determined by small-angle-X-ray scattering.

Short oligoguluronates, oligoG's, are reported to affect the ionotropic gelation of alginates both with respect to altered gelation kinetics and elastic properties of the resulting gels. The local structure of Ca(2+) induced changes in oligoguluronates and blends of oligoguluronates and alginates was determined by small angle X-ray scattering (SAXS). Calcium was introduced in the aqueous polysaccharide solutions by in situ release of Ca(2+) from Ca-EGTA. The scattering profiles of the Ca(2+)-induced structures in the alginate-oligoG blends were accounted for by a two-component broken rod-like model, also with an additional term representing structural inhomogeneity by a Debye-Bueche term. Adding oligoG to the alginate yields an increase in the largest cross-sectional radius in the region of fractional Ca(2+) saturation of α-l-GulA units from 0.5 to 1. The time-lapse characterization during the Ca-induced changes in the alginate-oligoG blends shows that oligoG delays the emergence of the more extensive laterally aggregated junction zones.

Primary structure, conformation in aqueous solution, and intestinal immunomodulating activity of fucoidan from two brown seaweed species Sargassum crassifolium and Padina australis.

We studied the structure of fucoidans extracted from two brown seaweed species, Sargassum crassifolium and Padina australis, and their intestinal immunomodulating activity via Peyer's patch cells of C3H/HeJ mice. ESI-MS analysis indicated that the dominant structure of both fucoidans has a backbone of α-(1→4)-linked and α-(1→3)-linked l-fucose residues and sulfate groups are attached at the C-2 and C-4 positions; branches of fucoidan from S. crassifolium are galactose residues with (1→4)- linkage and branching points are at C-4 of fucose, while fucoidan from P. australis, branches are sulfated galactose-fucose disaccharides and sulfated galactose monosaccharides attached to the main chain through (1→3)- or (1→4)- linkages. According to small angle X-ray scattering (SAXS) measurements, the two fucoidans have a branched structure. We simulated them with molecular models based on our proposed primary structure. These fucoidan samples have the ability to stimulate intestinal immunological activity via Peyer's patch cells.

Conformation and physical properties of cycloisomaltooligosaccharides in aqueous solution.

We studied the conformation and physical properties of cyclic and linear isomaltooligosaccharides in aqueous solution by intrinsic viscosity measurement, small angle X-ray scattering (SAXS) and molecular modeling. We used four cycloisomaltooligosaccharide samples (CIs) with degree of polymerization (DP) 7-10 (CI-7-CI-10) and five linear isomaltooligosaccharide samples (LIs) with DP 7-11 (LI-7-LI-11). The values of α in the Mark-Houwink-Sakurada equation [η]=KM(w)(α) for the CI and LI were determined to be 0.50 and 0.78, respectively. The radii of gyration (R(G)) of CI-7, CI-8, CI-9 and CI-10 determined from SAXS data were 6.7, 6.9, 7.5 and 8.3Å, respectively. The scattering profile of CI-9 compared with those obtained for molecular models indicated that CI molecular chains are less flexible than those for LIs and adopt a rather compact circular conformation.

Structure of fucoidan from brown seaweed Turbinaria ornata as studied by electrospray ionization mass spectrometry (ESIMS) and small angle X-ray scattering (SAXS) techniques.

The purpose of this study is to elucidate both the chemical and conformational structure of an unfractionated fucoidan extracted from brown seaweed Turbinaria ornata collected at Nha-trang bay, Vietnam. Electrospray ionization mass spectrometry (ESI-MS) was used for determining the chemical structure and small angle X-ray scattering (SAXS) provided conformational of the structure at the molecular level. The results showed that the fucoidan has a sulfate content of 25.6% and is mainly composed of fucose and galactose residues (Fuc:Gal ≈ 3:1). ESIMS analysis suggested that the fucoidan has a backbone of 3-linked α-l-Fucp residues with branches, →4)-Galp(1→ at C-4 of the fucan chain. Sulfate groups are attached mostly at C-2 and sometimes at C-4 of both fucose and galactose residues. A molecular model of the fucoidan was built based on obtained chemical structure and scattering curves estimated from molecular model and observed SAXS measurement were fitted. The results indicated that fucoidan under study has a rod-like bulky chain conformation.

Gamma-crosslinked collagen gel without fibrils: analysis of structure and heat stability.

This paper reports an analysis of the structure and heat stability of two different collagen gels: conventional collagen gel (neutral gel) and gel without collagen fibrils (acidic gel), previously reported. We performed differential scanning calorimetry (DSC), observations by scanning electron microscope (SEM), observations by atomic force microscope (AFM), and small angle X-ray scattering (SAXS). Collagen fibrils were clearly observed in the neutral gel but not in the acidic gel by both SEM and AFM. A clear endothermic peak was observed at 53-55 degrees Celsius, representing disassembly of collagens in collagen fibrils in the neutral gel but not in the acidic gel. Only a small broad endothermic peak, at 35-43 degrees Celsius, representing the deformation of the triple helical structure of collagen, was observed in the acidic gel. The SAXS pattern also suggested that the neutral gel had a more heterogeneous structure than the acidic gel. The experimental results described here are compatible with the model proposed in a previous paper, and indicate more clearly that the acidic gel has no collagen fibrils and has a different molecular assembly state of Type I collagen than the neutral gel.

Conformational folding of xyloglucan side chains in aqueous solution from molecular dynamics simulation.

Molecular dynamics simulation was carried out on xyloglucan with explicit water molecules to investigate the folding mechanism of side chains onto a main chain in aqueous solution. The model xyloglucan was composed of 12 beta-D-glucopyranoses as a main chain substituted with six galactoses and three xyloses as side chains. Two conditions were set for the ribbon-like main chain; one is restricted to be 'flat' and the other is without restriction. The free main chain of xyloglucan has a 'twisted' conformation as the major one. Conformational folding of side chains onto the main chain was analyzed with dihedral angles at each glycosidic linkage. In a 5-ns calculation, the xyloglucan has a tendency to contract in both the restricted and the free systems, but the mode of contraction is different. Side chains tend to stick onto the flat surface of the main chain in the restricted system, while they do not tightly do so in the free one; instead the main chain takes a twisted and sometimes embowed conformation. This result indicates that the main chain has greater attractive forces to bind side chains when it is flat, while it loses the ability as it is twisted.

Structural observation of complexes of FGF-2 and regioselectively desulfated heparin in aqueous solutions.

In order to clarify the mechanism of interaction between FGF-2 and heparin, the association structures between human FGF-2 and different kinds of regioselectively desulfated heparins were observed by small angle X-ray scattering. In the FGF-2-native heparin complex, the global FGF-2 molecules appeared to attach along heparin chain as strained unilaterally. The complexes with the 6-O-, or N-desulfated heparin seemed to have randomly associated structure as compared with above system. On the other hand, 2-O-desulfated heparin did not indicate the aggregation with FGF-2, indicating that the sulfate groups at O-2 of iduronate residues in heparin is most essential for association with FGF-2. These structural characteristics will be deeply related with signal transduction in the association with FGF-2 receptor.