Eva Machová

Scavenging and antioxidant activities of immunomodulating polysaccharides isolated from Salvia officinalis L.

Crude polysaccharides, isolated from the aerial parts of sage (Salvia officinalis L.) by sequential extraction with water (A), hot ammonium oxalate (B), dimethyl sulfoxide (C), 1M (D) and 4M (E) potassium hydroxide solutions, and six ion-exchange fractions of A were examined for their ability to inhibit peroxidation of liposome lipid by hydroxyl radicals and to reduced DPPH radical content. The highest inhibition of liposome lipid peroxidation was found with crude polysaccharides A, B and D, antioxidant activities reached approximately 37%. The purified fractions A1 and A2 inhibited the liposome peroxidation to approximately 35%. However, the radical scavenging abilities of the most active crude polysaccharides A, B and C on DPPH radicals were found in the range 80-90%, while the most active purified fractions A3-A6 in three or fourfold doses achieved 75-92%. The least effective tested polysaccharides succeeded 20% inhibition using both methods.

Mitogenic activity of particulate yeast β-(1 → 3)-d-glucan and its water-soluble derivatives

Particulate beta-D-glucan was isolated from bakers yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a beta-(1-->3)-linked backbone with beta-(1-->6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (carboxymethyl-,carboxyethyl-,hydroxyethyl-,sulfoethyl-), and the beta-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (Mw) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the carboxymethyl derivatives of various DS, the preparation with DS = 0.75 exhibited the highest activity. Water-soluble carboxymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.

Site-specific in vivo targeting of magnetoliposomes using externally applied magnetic field.

Abstract Human serum albumin labeled with technetium-99m was encapsulated together with magnetite particles into phosphatidylcholine/cholesterol liposomes. In order to investigate the stability of this complex and its ability to be used for magnetic drug targeting, the in-vivo distribution after intravenous administration in rats was estimated. For in-vivo targeting an SmCo permanent magnet with intensity ~ 0.3 5 T was attached near the right kidney. Difference between the relative radioactivity in the magnetically targeted right kidney (25.92±5.84%) and non-targeted left kidney (0.93±0.05%) is sufficiently high for relevant clinical applications.

Candida albicans mannan–protein conjugate as vaccine candidate

Mannan-branched polysaccharide with alpha-(1-->6)-linked mannose residues in the linear backbone, is a major virulence and protective factor of yeast Candida albicans. Injected alone does not induce sufficient level of protective antibodies. In this study, we have conjugated mannan to protein carrier by simple one step reaction using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) activation reagent. Prepared mannan conjugate is immunogenic in rabbits and reinjection elicited a booster response with significant increase of serum IgG level. Anti-C. albicans effectiveness of immune serum is clearly demonstrated. Based on these results, the mannan conjugate synthesized by this scheme can be considered as actual vaccine candidate for clinical evaluation.

Effect of ultrasonicated carboxymethylglucan on cyclophosphamide induced mutagenicity

Carboxymethylglucan (CMG) with ultrasonically lowered molecular weight (0.89 x 10(5)) was administered either intraperitoneally, intravenously or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in mouse bone marrow was evaluated. Both parenteral (intraperitoneal and intravenous) and oral administration of CMG decreased the clastogenic effect of CP. The protective effect induced by intravenous and intraperitoneal administration was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg body weight. With the lower dose of CMG a 2-h interval was necessary between intravenous CMG administration and CP injection. Oral pretreatment of mice with CMG decreased statistically significantly the frequency of micronuclei in polychromatic erythrocytes of the bone marrow. The fact that ultrasonically depolymerized CMG was effective also on oral administration is indicative of the passage of smaller CMG molecules through the wall of the gastrointestinal tract.

Sonication of chitin–glucan, preparation of water-soluble fractions and characterization by HPLC

A water-insoluble chitin-glucan complex, isolated from the mycelium of Aspergillus niger, was swollen in various aqueous media and treated subsequently by high-energy sonication. The concentration of the resulting water-soluble polysaccharide fractions was dependent on the swelling medium, the amount of the chitin-glucan complex in the suspension, and on the time of sonication. The yields of water-soluble chitin-glucan were within the range 13.6 to 24.4% relative to the mass of the original chitin-glucan. The nitrogen content obtained for the samples of water-soluble chitin-glucan indicated a higher content of chitin (3.45% of nitrogen in high-molecular fraction) than in the original water-insoluble chitin-glucan sample (1.8%). The distribution of the molecular weights of the water-soluble fractions prepared was determined by high-performance liquid chromatography.

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.

Carboxymethylated glucan inhibits lipid peroxidation in liposomes.

Protective capabilities were studied of carboxymethylated (1→3)-β-ᴅ-glucan from Saccharomyces cerevisiae cell wall against lipid peroxidation in phosphatidylcholine liposomes in duced by OH· radicals produced with Fenton’s reagent (H2O2/Fe2+) and also by microwave radiation using absorption UV-VIS spectrophotometry. A significant decrease in the conjugated diene production, quantified as Klein oxidation index, was observed in the presence of a moderate amount of added glucan. Increase of the oxidation index was accompanied with enhanced carboxyfluorescein leakage as a result of liposome membrane destabilization. This process was markedly suppressed with glucan present in the liposome suspension. Therefore, glucan may be considered as a potent protector against microwave radiation-induced cell damage.

Antioxidant capacities of mannans and glucans are related to their susceptibility of free radical degradation.

Microbial and plant polysaccharides in nature are frequently exposed to oxidative burst. They may act as antioxidants buffering the radical attack. This paper presents antioxidant properties of prepared yeast mannans, commercial β-glucans as well as the chemically prepared carboxymethylated β-glucan (CM-glucan). The hydroxyl radical antioxidant assay and the DPPH radical-scavenging assay were used. Yeast mannans and β-glucans (1.6 mg mL(-1)) showed antioxidant capacities against OH(·) up to 14.1%, while CM-glucan was significantly higher antioxidant (65.4%). In the DPPH(·) assay, the antioxidant capacities of yeast mannans and β-glucans (1.0 mg mL(-1)) were lower and reached up to ~6.5%. All polysaccharides tested were effectively degraded by OH(·) and the presence of salicylate considerably inhibited their degradation. Measure of Fe(2+) chelation revealed less than 13.1% effectivity for all polysaccharides. In all antioxidant and degradation experiments the yeast mannans showed very similar results to commercial β-glucans. The antioxidant capacities of polysaccharides may be assessed by simple HPLC monitoring.