Jozef Šandula

Protective effects of fungal (1→3)-β-d-glucan derivatives against oxidative DNA lesions in V79 hamster lung cells

beta-Glucans belong to the class of substances known as biological response modifiers with a broad range of activity. We have investigated two types of glucans: (1-->3)-beta-D glucan from the bakers yeast Saccharomyces cerevisiae and beta-glucan-chitin complex from the mycelium of filamentous fungus Aspergillus niger. Since these fibrillar beta-glucans are insoluble in water, their water-soluble derivatives--carboxymethyl glucan (CM-G), sulfoethyl glucan (SE-G), and carboxymethyl chitin-glucan (CM-CG) were prepared and tested. The aim of the present work was to investigate the protective effect of the prepared glucan derivatives against oxidative DNA damage induced by H2O2 and visible light-excited Methylene Blue in V79 hamster lung cells. The level of DNA damage (DNA strand breaks) was measured using the single cell gel electrophoresis, so called comet assay. Our findings demonstrate that all three tested glucans reduce oxidative DNA damage. The ability to reduce genotoxic activity increased in the order: CM-G<SE-G<CM-CG. We suggest that the analyzed glucans exhibit protective effects against oxidative damage to DNA as a consequence of scavenging of both *OH radicals and singlet oxygen.

Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro.

Antioxidative and antimutagenic effect of yeast cell wall mannans, in particular, extracellular glucomannan (EC-GM) and glucomannan (GM-C.u.) both from Candida utilis, mannan from Saccharomyces cerevisiae (M-S.c.) and mannan from Candida albicans (M-C.a.) was evaluated. Luminol-dependent photochemical method using trolox as a standard showed that EC-GM, GM-C.u., M-S.c. and M-C.a. have relatively good antioxidative properties. EC-GM exhibited the highest antioxidative activity, followed by GM-C.u. and M-S.c. M-C.a. showed the least antioxidative activity. These mannans were experimentally confirmed to exhibit different, statistically significant antimutagenic activity in reducing damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange (AO). We suggest that the antimutagenic effect of EC-GM, GM-C.u., M-S.c. and M-C.a. against ofloxacin is based on their ability to scavenge reactive oxygen radicals. With AO, the reduction of the chloroplast DNA lession could be a result of the absorptive capacity of the mannans. The important characteristics of mannans isolated from the yeast cell walls, such as good water solubility, relatively small molecular weight (15-30kDa), and antimutagenic effect exerted through different mode of action, appear to be a promising features for their prospective use as a natural protective (antimutagenic) agents.

Influence of the drying method on the physical properties and immunomodulatory activity of the particulate (1→3)-β-d-glucan from Saccharomyces cerevisiae

Abstract The particulate (1→3)-β- d -glucan isolated from Saccharomyces cerevisiae cell walls was recovered from the aqueous medium as water-insoluble particles by different drying methods: solvent exchange (GE), lyophilisation (GL), and spray drying (GS). The samples were characterised by optical microscopy, FT-IR spectroscopy, swelling capacity, and rheological behaviour of aqueous dispersions. The immunological activity of the glucan samples was examined using the assay for the mitogenic and comitogenic activities. The drying method affected the microstructure of the glucan particles leading to differences in their physical properties (particle size and shape, swelling capacity, interparticle hydrogen bonding) as well as in the flow and viscoelastic properties. In comparison to GL and GE, the GS particles preserved the ellipsoid shape of yeast cells and exhibited a very low extent of interparticle hydrogen bonds. The rather liquid-like GS dispersion showed a several times lower apparent viscosity than the gel-like GE and GL dispersions. The results suggest that the physical state of the variously dried particulate glucan samples influenced significantly also their immunomodulatory activity which was found to be about twice higher with GS than with the GL and GE samples. The results indicated that for application of the particulate (1→3)-β- d -glucan as immunomodulator/adjuvans in form of aqueous suspension it is important to use spray-dried preparations.

Detection of damage to DNA and antioxidative activity of yeast polysaccharides at the DNA-modified screen-printed electrode

A simple procedure for the voltammetric detection of the DNA damage and antioxidants protecting DNA from its damage using a disposable electrochemical DNA biosensor is reported. The carbon-based screen-printed electrode (SPE) modified by a surface layer of the calf thymus double stranded (ds) DNA was used as a working electrode in combination with a silver/silver chloride reference electrode and a separate platinum auxiliary electrode. The [Co(phen)(3)](3+) ion served as the dsDNA redox marker and the [Cu(phen)(2)](2+) and [Fe(EDTA)](-) complex compounds were used as the DNA cleavage agents under the reduction by a chemical reductant (ascorbic acid). Four yeast polysaccharides with different chemical structure were investigated as the antioxidants within the concentration range of 0.05-4 mg ml(-1) in the cleavage mixture. A remarkable antioxidative activity of polysaccharides in order mannan (Candida krusei)>extracellular glucomannan (Candida utilis)>mannan (Candida albicans)>glucomannan (C. utilis) was found which is in agreement with that refered to trolox (a structural derivative of alpha-tocopherol) and determined by photochemiluminescent method.

Extracellular polysaccharide-protein complexes produced by selected strains ofCandida albicans (Robin) Berkhout

Water-soluble extracellular polysaccharide-protein complexes of a molecular weight of about 200,000 were isolated from glucose-containing nutrient media after cultivation of slightly, moderately and highly virulent strains ofCandida albicans (Robin) Berkhout. Chemical, physico-chemical and immunological properties of these compounds were studied. The complexes contain 74–86% of mannose, 21–31% of glucose, 1–1.5% of glucosamine and 11–14% of proteins built from 17 aminoacids. The polysaccharideprotein complexes are immunologically active and differ from each other by their precipitation ability if added to specific antisera prepared against moderately highly virulentCandida albicans strains.

Increased efficiency of Lewis lung carcinoma chemotherapy with a macrophage stimulator—yeast carboxymethyl glucan

The efficiency of chemotherapy of Lewis lung carcinoma with cyclophosphamide was affected by administration of the water-soluble yeast polysaccharide derivative--carboxymethylated (1 --> 3)-beta-D-glucan (CMG)-a well-known macrophage stimulator. It was found that while cyclophosphamide showed 57% growth inhibition of the intramuscular tumor implants in comparison with the control group, its combined administration with CMG led to 75-90% inhibition. Similarly, increased inhibition of occurrence of lung metastases (up to 92-94%) was observed using the combined application of the two compounds. The stimulatory effect of CMG is not associated with the changed cellularity of peripheral blood, but is rather due to the obviously increased concentration of the intracellular inhibitor of cysteine proteases-stefin A and cystatin C in tumor tissue.

Ultrasonication: the way to achieve antimutagenic effect of carboxymethyl-chitin–glucan by oral administration

Carboxymethyl-chitin-glucan (CMCG) isolated from Aspergillus niger was ultrasonicated to decrease its molecular weight. Ultrasonicated CMCG with molecular weight 0.19 x 10(-5) was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in polychromatic erythrocytes of mouse bone marrow was evaluated. Both ways of CMCG administration significantly decreased the clastogenic effect of CP. The protective effect of CMCG was concentration dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b.wt. Ultrasonic depolymerization of high molecular CMCG resulted in its anticlastogenic effect against CP not only on intraperitoneal, but also on oral administration, achieved by decreasing its molecular weight. Ultrasonication proved to be an efficient way to obtain molecules of CMCG able to pass through the cell walls of the gastrointestinal tract.

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.

ULTRASONIC DEPOLYMERIZATION OF THE CHITIN-GLUCAN COMPLEX FROM ASPERGILLUS NIGER AND ANTIMUTAGENIC ACTIVITY OF ITS PRODUCT

Sonication was effective for the depolymerization of carboxymethylated chitin-glucan complex (CM-CG) isolated from the cell wall of Aspergillus niger. After 10 min of sonication and subsequent gel filtration, two samples (CM-CG(I) and CM CG(II)) with significantly distinct molecular weights (660 kDa and 19 kDa, respectively) and different nitrogen contents (3.02 and 1.69%) were obtained. CM-CG(II) with lower Mw was also effective against cyclophosphamide mutagenicity by oral administration in mice.

Effect of ultrasonic treatment on the molecular weight of carboxymethylated chitin-glucan complex from Aspergillus niger.

Different factors affecting the efficiency of the ultrasonic depolymerization of the high-molecular-weight carboxymethylated chitin-glucan prepared from the fungal mycelium of Aspergillus niger have been investigated. The influence of the following parameters was examined: concentration of the chitin-glucan complex, duration of ultrasonic irradiation, reaction temperature and volume of the ultrasonicated solution. The optimized conditions for the efficient ultrasonic depolymerization include: polysaccharide concentration--0.2 mg ml-1; volume of the sonicated solution--25 ml; duration of the sonication--10 min; and constant cooling of the sonicated sample in an ice-water bath.