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.

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.

[Cytokine-inducing and anti-inflammatory activity of chitosan and its low-molecular derivative].

A low-molecular derivative of the polysaccharide (5 kDa) was obtained and its cytokine-inducing and anti-inflammatory activity was studied by free radical depolymerization of chitosan (110 kDa). It was shown that high-molecular chitosan in vitro inhibited the synthesis of anti-inflammatory cytokine, the tumor necrosis factor alpha induced by endotoxin. In the case of peroral introduction to experimental animals, high- and low-molecular chitosans stimulated synthesis of the anti-inflammatory cytokine IL-10 in the blood serum of mice; in this case, the activity of the high-molecular derivative was two times higher as compared with the initial polysaccharide. With peroral introduction, the initial polysaccharide (50 mg/kg) and its derivative inhibited the development of chemically induced inflammation of experimental animals' large intestines, which was manifested as a decrease in the affected area and the degree of damage to the large intestine wall, as well as a two-fold reduction of myeloperoxidase activity. According to morphological and biochemical characteristics, the effect of chitosans was similar to that of a hormone anti-inflammatory drug, prednisolone.

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.

Antitumor activity of cucumarioside A2-2.

BACKGROUND: The cytotoxic activity of sea cucumber glycosides against different types of cells and cell lines, including human tumor cell lines, has been studied for many years. However, the molecular mechanism(s) of the antitumor action of triterpene glycosides on cancer cells remain unclear. This article reports a continuation of investigations of triterpene glycoside cucumarioside A2-2 isolated from the Far-Eastern sea cucumber Cucumaria japonica. It describes a study of glycoside anticancer activity in vivo and glycoside interaction with mouse Ehrlich carcinoma cells in vitro. METHODS: The cytotoxicity of cucumarioside A2-2 and its effect on apoptosis, the cell cycle, DNA biosynthesis and p53 activity, and glycoside anticancer action against Ehrlich carcinoma cells were studied. RESULTS: Cucumarioside A2-2 influences tumor cell viability at micromolar concentrations. The EC50 for glycoside estimated by nonspecific esterase assay and MTT assay was 2.1 and 2.7 µM, respectively. Cucumarioside A2-2 at a subcytotoxic range of concentrations exhibits a cytostatic effect by blocking cell proliferation and DNA biosynthesis in the S phase. It may induce apoptosis in tumor cells in a caspase-dependent way, bypassing the activation of the p53-dependent segment. CONCLUSION: The anticancer and proapoptotic properties of cucumarioside A2-2 may be due to direct interaction of the glycoside with tumor cells. The in vivo anticancer effect of cucumarioside A2-2 may be associated with the ability of the drug to arrest the cell cycle in the synthetic phase and induce programmed tumor cell death.

[Effect of chitosan on the cell ultrastructure and activity of hydrolases in tobacco leaves].

Effect of chitosan on the mesophyll cell ultrastucture and activity of hydrolases in leaves of tobacco cv. Samsun was studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased) and, at the same time, caused some activation of lytic compartment expressed in the stimulation of the formation of dictyosomes, smooth ER elements and cytoplasmic vacuoles, which are all prominent constituents of this compartment. In biochemical experiments, it was established that chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) in the leaves as compared to untreated leaves. In some cells chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm and so on) that can be classified as a result of programmed cell death development.

[Electron microscopic study of the chitosan effect on the intracellular accumulation and the state of tobacco mosaic virus particles in tobacco leaves].

Influence of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in the mesophyll cells of Nicotiana tabacum L. var Samsun leaves in early period of infection development (3 days after infection of leaves) has been studied. The virus accumulated in the cells of the leaves treated for 24 h before infection with chitosan to a lesser degree than in the control cells. The chitosan affected the formation of TMV-specific granular and tubular inclusions which are known to consist of the viral replicase components. Three days after infection of the leaves treated with the chitosan, a typical sign of the infection development was the predominant formation of granular inclusions which are known to appear at the early stages of TMV replication. The infected cells of the leaves untreated with chitosan contained mainly tubular inclusions which had been shown previously to be formed from granular ones at the last stages of the infection process. This indicates that chitosan treatment of the leaves leads to a delay of the development of infection. In phosphotungstic acid-stained suspensions obtained from the infected leaves, abnormal (swollen and "thin") TMV particles were observed along with normal ones. The appearance of abnormal virus particles seems to be caused by virus-induced activation of intracellular lytic processes. The most lytic activity in the infected cells as well as the highest number of abnormal viral particles was observed under the chitosan action. Therefore, it appears that chitosan-mediated stimulation of lytic processes causing destruction of TMV particles may be one of the protective mechanisms limiting virus accumulation in cells.

[Chitosan antiviral activity: dependence on structure and depolymerization method].

Enzymatic (the action of lysozyme) and chemical (hydrogen peroxide) hydrolysis of chitosans with various degree ofacetylation (DA)--25, 17, and 1.5%--was performed. Purification and fractioning of the hydrolysis products were performed using dialysis, ultrafiltration, and gel-penetrating chromatography Low-molecular (LM) derivatives of the polysaccharide with molecular masses from 17 to 2 kDa were obtained. The study of their antiviral activity against the tobacco mosaic virus (TMV) showed that these samples inhibited the formation of local necroses induced by the virus for 50-90%. The antiviral activity of the LM chitosans significantly increased with the lowering of their polymerization degree. Furthermore, the products of the enzymatic hydrolysis possessed higher activity than the chitosan samples obtained as a result of chemical hydrolysis. It was revealed that the exhibition of the antiviral activity weakly depended on the degree of acetylation of the samples.

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.

Modification of biological activity of lipopolysaccharide in the complex with chitosan.

In the complex with chitosan, lipopolysaccharide partially lost its ability to induce lymphokines tumor necrosis factor and interleukin-8, but retained immunostimulating properties and increased phagocytic function of macrophages by improving digestion of bacteria.

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.

[Comparative study of the physico-chemical properties of chitosans with varying degree of polymerization in neutral aqueous solutions]. / Sravnitel'noe izuchenie fiziko-khimicheskikh svoistv khitozanov razlichnoi stepeni polimerizatsii v neitral'nykh vodnykh rastvorakh.

The physicochemical properties of chitosan samples with high (130 kD) and low (30 kD) molecular masses in neutral aqueous solutions (pH 6.0) were studied by the methods of high-speed and equilibrium sedimentation, viscosimetry, and NMR and UV spectroscopies. Differences in the hydrodynamic characteristics of the samples were revealed. It was found that low-molecular-weight chitosan represents flexible linear macromolecules which undergo conformational changes upon temperature increase. The high-molecular-weight chitosan forms more rigid asymmetric structures whose conformation does not vary significantly with temperature increase. It was found that the high-molecular-weight chitosan has a higher constant of binding to the anionic dye tropeoline 000-II, which can be explained by different conformations of their macromolecules in solution.