Effect of red seaweed sulfated galactans on initial steps of complement activation in vitro.

The research described here presents data on the effect of galactans of red algae, carrageenans (λ/µ/ν-, κ-, κ/ß-, and ι/κ-types), and agar on complement system activation in normal human serum. The experiments were based on well surfaces coated with triggering agents for binding initiating complement components -C3 and C4. The sulfated galactans inhibited C3 binding to lipopolysaccharide with direct dependence on the sulfation degree of polysaccharides. Sulfation degree was also important in carrageenans' capacity to reduce C4 binding to mannan. However, C4 binding to antibodies was considerably activated by carrageenans, especially with 3,6-anhydrogalactose. The gelling carrageenans were able to block antigen binding centers of total serum IgM and with more intensity than non-gelling. No structural characteristics mattered in ameliorating C5 cleavage by plasmin in extrinsic protease complement activation, but λ/µ/ν- and κ/ß-carrageenans almost completely inhibited C5 cleavage. Thus, galactans participated in cell surface biology by imitating surface glycans in inhibition of C3 binding and mannose binding lectin, but as to the tthe heclassical pathway these substances stimulated complement, probably due to their structure based on carrabiose.

Effect of carrageenans on some lipid metabolism components in vitro.

The research described here focused on the effect of sulfated red algal polysaccharides (κ-, κ/ß-, ι/κ-carrageenan) individually and in combination with lipopolysaccharide (LPS) on the synthesis of prostaglandin E2 (PGE2) and cytokines (interleukin [IL]-1ß and IL-6) in whole blood model in vitro. The results demonstrated that, at high concentrations, carrageenans have substantial ability to modulate PGE2 synthesis and stimulate IL-1ß and IL-6 synthesis. A low degree of sulfate and high molecular weight were a prerequisite for the ability of carrageenans to modulate PGE2 synthesis. Further, we investigated the ability of the carrageenans alone and in combination with casein to affect bile salt permeability through an artificial membrane imitating the gastrointestinal barrier. The least sulfated κ/ß-carrageenan could retain bile salt permeation the most but less efficiently than cholestyramine. The polysaccharides did not affect pancreatic lipase activity. Our data confirm a possible mechanism of the cholesterol-reducing properties of carrageenan.

Morphology, electrokinetic characteristics and the effect on biofilm formation of carrageenan:chitosan polyelectrolyte complexes.

Carrageenan:chitosan (CG:CH) polyelectrolyte complexes (PEC) were obtained and the effect of the initial components ratio on formation was studied by dynamic light scattering, atomic force microscopy (AFM) and electrokinetic measurements. Positively charged PEC particles (average ζ-potential 40.2 mV) were formed, provided that the polycation was present in excess in the complex and was stabilized by chitosan amino groups. According to the AFM data, chitosan was located on the surface of the carrageenan fibers. In PEC where carrageenan prevailed, the number of unbound sulfate groups decreased when the chitosan content increased, this resulted in a decrease in the PEC negative surface charge (from -92.4 to -55.6 mV). In this case, AFM showed that chitosan was incorporated into the network structure of carrageenan and breaks it at a CG:CH ratio of 1:0.5 w/w. Complexes with a high content of kappa-CG inhibited biofilm formation by Gram negative and Gram positive microorganisms.

Effect of carrageenans alone and in combination with casein or lipopolysaccharide on human epithelial intestinal HT-29 cells.

The research described here was focused on the effect on human intestinal epithelial cell monolayers of sulfated red algal polysaccharides (κ-, λ-, and κ/ß-carrageenans) alone and in combination with casein or lipopolysaccharide (LPS). HT-29 cells were investigated under normal and stress conditions; stress was induced by exposure to ethanol. Cell viability was monitored with a real-time system. The change in binding properties of negatively sulfated red algal polysaccharides assessed by the measurement of free carrageenans in mixtures with casein or McCoy's 5 A culture medium by means of toluidine blue O. Low sulfate content and the presence of 3,6-anhydogalactose are prerequisites for the recovery of ethanol-exposed HT-29 cells by carrageenans. Analysis of carrageenan binding ability confirmed that casein and LPS should affect carrageenan activity. Whether the combined action of the mucin-containing layer and carrageenans or the action of carrageenans alone was responsible for enhanced cell viability under stress conditions induced by ethanol is a subject for further research. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2843-2850, 2017.

Influence of carrageenan on cytokine production and cellular activity of mouse peritoneal macrophages and its effect on experimental endotoxemia.

The in vivo effect of κ/ß-carrageenan isolated from the red alga Tichocarpus crinitus on cytokine synthesis and cellular activity of murine peritoneal macrophages and also the protective effect of polysaccharides in LPS-induced endotoxemia in mice was studied. It was established that κ/ß-carrageenan given orally at a dose of 100 mg/kg stimulates the induction of anti-inflammatory cytokines (IL-10) in mouse blood cells by more than 2.5-fold compared with control, with no effect on pro-inflammatory cytokine (TNF-α) production. Pretreating mice with carrageenan once a day before injecting LPS increased the levels of IL-10 by 2.5-fold and reduced TNF-α production by 2-fold compared with control. So, κ/ß-carrageenan alone and in combination with LPS enhanced the cellular activity and mobility of peritoneal macrophages by increasing cell adhesion and migration compared with control. LPS activated cells intensively, sometimes resulting in their destruction by necrosis; carrageenan pretreatment reduced the excessive inflammatory cell activation caused by LPS. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1549-1557, 2017.

Carrageenans effect on neutrophils alone and in combination with LPS in vitro.

Influence of sulfated red algal polysaccharides (κ-, λ-, and κ/ß-carrageenans) and degraded derivative of κ/ß-carrageenan on neutrophils/monocytes activation alone and in combination with lipopolysaccharide was investigated by means of determination of reactive oxygen species production, latex microparticles engulfment, total and extracellular myeloperoxidase induction and the analysis of silhouette and contour two-dimensional images of flattened cells. Carrageenans alone can activate neutrophils with much less potency than lipopolysaccharide (LPS) and the sulfation degree of carrageenans stipulates high activity in this role. On the other hand, carrageenans especially with low contents of sulfate groups are able to interfere with LPS in vitro resulting in reducing inter- and intracellular activation of neutrophils killing mechanisms. Further research is necessary to relate these findings to actions on the whole animal or human in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1603-1609, 2016.

Influence of structural features of carrageenan on the formation of polyelectrolyte complexes with chitosan.

The polyelectrolyte complexes (PEC) of carrageenans (CG)-κ-, κ/ß-, λ-and x-CG with chitosan were obtained. The formation of PEC was detected by Fourier-transform infrared (FTIR) spectroscopy and by centrifugation in a Percoll gradient. The influence of the structural peculiarities of CG on its interaction with chitosan was studied. The results of centrifugation showed that x-CG with a high degree of sulphation (SD) was completely bound to chitosan, unlike low SD κ-CG and κ/ß-CG. Binding constant values showed there was a high affinity of CG for chitosan. CG with flexible macromolecule conformation and high SD exhibited the greatest binding affinity for chitosan. The full-atomic 3D-structures of the PEC κ-CG: chitosan in solution have been obtained by the experiments in silico for the first time. The amino groups of chitosan make the largest contribution to the energy of the complex formation by means of hydrogen and ionic bonds. The most probable complexes have stoichiometries of 1:1 and 1:1.5.

The supramolecular structure of LPS-chitosan complexes of varied composition in relation to their biological activity.

The complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5 (E-LPS) and Yersinia pseudotuberculosis 1B 598 (Y-LPS) of various weight compositions were investigated using quasi-elastic light scattering, ζ-potential distribution assay and atomic force microscopy. The alteration of ζ-potential of E-LPS-Ch complexes from negative to positive values depending on Ch content was detected. The Y-LPS-Ch complexes had similar positive ζ-potentials regardless of Ch content. The transformation of the supramolecular structure of E-LPS after binding with to Ch was revealed. Screening of E-LPS and Y-LPS particles by Ch in the complexes with high polycation was detected. The ability of LPS-Ch complex to induce biosynthesis of TNF-α and reactive oxygen species in stimulated human mononuclear cells was studied. A significant decrease in activity complexes compared to that of the initial LPS was observed only for E-LPS-Ch complexes.

Soluble chitosan-carrageenan polyelectrolyte complexes and their gastroprotective activity.

The soluble polyelectrolyte complexes (PEC) κ-carrageenan (κ-CG):chitosan was obtained. Binding constant value (2.11 × 10(7)mol(-1)) showed high affinity of κ-CG to chitosan. The complex formation of κ-CG:chitosan 1:10 and 10:1 w/w was shown by centrifugation in a Percoll gradient. Using atomic force microscopy we showed that the supramolecular structure of the complexes is different from each other and from the macromolecular structure of the initial polysaccharides. The gastroprotective and anti-ulcerogenic effect of κ-CG, chitosan and their complexes was investigated on the model of stomach ulcers induced by indometacin in rats. PEC κ-CG:chitosan have gastroprotective properties which depend on their composition. Complex κ-CG:chitosan 1:10 w/w possesses higher gastroprotective activity than the complex 10:1 w/w. These results suggest that the gastroprotective effect of complexes can be associated with their protective layer on the surface of the mucous membrane of a stomach, which avoids a direct contact with the ulcerogenic agent.

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus.

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.

Atomic force microscopy imaging of carrageenans from red algae of Gigartinaceae and Tichocarpaceae families.

In the present article, the atomic force microscopy was applied to investigate macromolecular structures of various carrageenan types including hybrid polysaccharides (κ-, κ/ß-, κ/ι-, λ-, and X-carrageenans) depending on polysaccharide concentration. The structures dependence on a polysaccharide concentration also was focused. κ-Carrageenan forms both single and two stranded structures at a low concentration. At high concentrations κ-, κ/ß-, and κ/ι-carrageenans form fibrous network-like structures by a side-by-side association type at the same time for κ/ι-carrageenan end-to-end association type also was found. Comparably to κ-carrageenan, κ/ß-carrageenan network was more open with coarser fibers while κ/ι-carrageenan structure is characterized with a more flexible network. Honeycombed structures due to end-to-end and side-by-side association types were observed for X-carrageenan, while λ-carrageenan formed honeycombed structures only at high concentrations. In order to investigate topographical parameters of the carrageenans macromolecular structure a new method of the autocorrelation function analysis was used for the first time.

Correlation between influence of polysaccharides on hydrolase activity and their antiviral effect in tobacco leaves.

The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-ß-D-glucan, fucoidan, and κ/ß-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

In vitro and ex vivo studies of antioxidant activity of carrageenans, sulfated polysaccharides from red algae.

Antioxidant properties of structurally different sulfated polysaccharides (carrageenans) were studied in vitro and ex vivo. Ferric reducing antioxidant activity of carrageenans and their inhibitory effects on hydroxyl radicals and superoxide anion radicals were demonstrated in vitro. Activity of carrageenans depends on the polysaccharide structure. Carrageenans stimulate catalytic activity of SOD from donor erythrocyte.

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes.

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.

Determination of binding constants of lipopolysaccharides of different structure with chitosan.

The interaction of endotoxins--lipopolysaccharides (LPS) different in degree of the O-specific chain polymerization--with 20- and 130-kD chitosan was studied using the competitive binding of LPS with the complex of chitosan-anionic dye (tropaeolin 000-2) and the direct binding of (125)I-labeled LPS with chitosan immobilized on Sepharose 4B. The interaction of 20-kD chitosan with LPS was non-cooperative, and immobilization of the polycation on Sepharose resulted in its binding to (125)I-labeled LPS with a positive cooperativity. The interaction of LPS possessing a long O-specific chain with 130-kD chitosan was characterized by negative cooperativity. Binding constants of LPS with the polycation and the number of binding sites per amino group of chitosan were determined. The interaction affinity and stoichiometry of the LPS-chitosan complexes significantly depend on the LPS structure and concentration in the reaction mixture. The increase in the length of carbohydrate chains of LPS results in increase in the binding constants and decrease in the bound endotoxin amount.

Comparative study of carrageenans from reproductive and sterile forms of Tichocarpus crinitus (Gmel.) Rupr (Rhodophyta, Tichocarpaceae).

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.

Interaction of bacterial endotoxins with chitosan. Effect of endotoxin structure, chitosan molecular mass, and ionic strength of the solution on the formation of the complex.

The interaction of endotoxins of different structure (lipopolysaccharides (LPS) and lipopolysaccharide-protein complexes (LPPC)) with chitosan has been studied. It was shown that the mechanism of interaction is rather complicated and depends on the macromolecular organization of endotoxin as well as on the degree of polymerization of the chitosan. Chitosan with molecular mass of 20 kD reveals higher affinity to LPS than chitosan with molecular mass of 140 kD. Endotoxins with long O-specific chains can bind completely with chitosan with the formation of LPS-chitosan and LPPC-chitosan complexes with weight ratios between the original components of 1:1 and 1:5. When endotoxins with higher degree of hydrophobicity and short O-specific chains were mixed with chitosan, a part of the LPS remained unbound. The stability of the complexes formed depends on ionic strength. It was shown that, in addition to electrostatic forces, other types of forces take part in the formation of the complexes. A decrease in acute toxicity of various LPSs is observed on their binding with chitosans.

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan.

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.

Studies on a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis. 1 isolation and characterization.

A comparative study of various procedures of a lipopolysaccharide-protein complex (LPPC) from Yersinia pseudotuberculosis was carried out. The materials obtained were fractionated by molecular-sieve chromatography on Sepharose 2B resulting in highly aggregated complexes with antigen activity. LPPC aggregates dissociated in the presence of sodium dodecylsulphate (SDS) and urea. The chemical composition and serologic properties of fractions obtained are under consideration. The protein component of the complex consists of two major polypeptides (molecular weights--45,000 and 20,000) and some minor ones. The LPS component appeared to give 2--3 narrow bands in gel under conditions of SDS-polyacrylamide gel electrophoresis. It is suggested that such fractionation is caused by LPS association-dissociation in the course of electrophoresis.