Elsevier

Carbohydrate Polymers

Volume 61, Issue 1, 4 July 2005, Pages 18-28
Carbohydrate Polymers

Antioxidant properties of yeast (1→3)-β-d-glucan studied by electron paramagnetic resonance spectroscopy and its activity in the adjuvant arthritis

https://doi.org/10.1016/j.carbpol.2005.02.010Get rights and content

Abstract

Radical-scavenging activity of the water-soluble derivative obtained from cell wall of the baker's yeast Saccharomyces cerevisiae was investigated using the technique of electron paramagnetic resonance. The experiments involved a study of the scavenging activity of carboxymethyl (1→3)-β-d-glucan (CMG) towards the radicals formed in the thermally initiated decomposition of potassium persulfate, hydrogen peroxide, or 2,2′-azo-bis(2-amidinopropane)-dihydrochloride in aqueous solutions using spin trapping as an indicative technique. In the absence of glucan, high intensity spectra of generated free radicals in the form of their adducts with 5,5-dimethylpyrroline-N-oxide were observed.

Addition of CMG resulted in concentration-dependent substantial decrease of spectral intensities of adducts as a result of competition of CMG in the scavenging of reactive radicals formed.

In the in vivo experiments involving administration of CMG to rats with experimentally induced adjuvant arthritis (AA) a substantial decline of the level of plasmatic carbonyls, a parameter indicating oxidative tissue damage during the progress of arthritic diseases, was observed. We assume that radical-scavenging properties of CMG can be responsible for its antioxidant activity in the AA model, suggesting possible application of the yeast glucan derivatives in the treatment of arthritis.

Introduction

In the last decade, much support has been obtained for the assumption that many pathogenic processes including rheumatoid arthritis and osteoarthritis, neoplastic and mitochondrial diseases, as well as ageing and death of cells and complete tissues may be initiated by damaging action of free radical species, especially reactive oxygen species (ROS) (Basu et al., 1999, Halliwell and Gutteridge, 1989). For this reason, growing attention of researchers and physicians has been paid to the group of natural substances, known as antioxidants, which are able to protect living organisms from the attack of reactive radical species, and in this way to decrease the risk of several diseases (Babincová and Sourivong, 2001, Wang et al., 1996).

Although most often applied and investigated natural antioxidants are ascorbic acid (vitamin C) and α-tocopherol (vitamin E), recently increased attention has been paid to the antioxidants of polysaccharide origin (Bobek et al., 1997, Xie et al., 2001). Among the representatives of this class of antioxidants are (1→3)-β-d-linked glucose polymers that occur as a primary component in the cell walls of fungi and several bacteria or they are secreted extracellularly by various fungi (Bohn and BeMiller, 1995, Kogan, 2000). Due to their ability to modulate the immune system of the host, glucans belong to the group of substances known as biological response modifiers. (1→3)-β-d-glucans have been reported to have stimulating effects on the defense mechanisms of the living organisms by enhancing the host resistance to viral, bacterial, fungal, and parasitic infections, as well as to neoplastic and other pathogenic conditions (Tzianabos et al., 1995, Williams, 1997, Williams et al., 1996). Some indications have been obtained that protective and immunomodulating properties of (1→3)-β-d-glucan may in part be ascribed to its ability to exert free radical scavenging properties. It has been demonstrated that (1→3)-β-d-glucans prepared from yeast or mushrooms are able to protect blood macrophages from X- or γ-ray irradiation, to restore bone marrow production, to increase hemopoiesis, and to protect animals from the lethal effect of the γ-ray irradiation (Hofer et al., 1995, Patchen et al., 1987, Patchen and MacVittie, 1983).

Using the prepared water-soluble carboxymethylated (1→3)-β-d-glucan (CMG), Chorvatovičová (1991) described its inhibitory effect on the micronucleus frequencies induced in mice by γ-ray irradiation and later demonstrated the ability of this compound to suppress the mutagenic and toxic activity of cyclophosphamide in mice (Chorvatovičová, Machová, & Šandula, 1996). It was suggested that the observed protective activity of CMG as well as the described protective effect of sulfoethylated (1→3)-β-d-glucan (SEG) against mutagenic action of hexavalent chromium (Chorvatovičová, Kovačíková, Šandula, & Navarová, 1993) could be attributed to the free radical scavenging activity of the used glucan derivatives. Recently, using the same compounds, Križková et al. (2003) described pronounced antioxidant effect of CMG and SEG in the Trolox equivalent antioxidant capacity (TEAC) assay and demonstrated that, due to the observed antioxidant activity, these two glucan derivatives were able to inhibit significantly Euglena gracilis chloroplast DNA damaging action of ofloxacin, which is known to be associated with the generation of reactive radical species.

Previously, we have studied the ability of CMG prepared from the cell walls of baker's yeast Saccharomyces cerevisiae to inhibit lipid peroxidation in liposomes induced by hydroxyl radicals produced using Fenton's reagent (H2O2/Fe2+) or using microwave radiation. The liposome model is an ideal approximation to the lipid bilayers in the cell membranes and therefore the protective efficiency of glucan in such a model can imply its possible application as antioxidant in protection of living cells. Our results showed that CMG exerted a potent protective effect against lipid peroxidation induced in both experimental models, comparable to that of the well-known antioxidants, α-tocopherol and d-mannitol (Babincová et al., 2002, Babincová et al., 1999). More recently, we demonstrated a protective effect of CMG and another water-soluble derivative, sulfoethyl glucan, against oxidative DNA lesions in hamster lung cells, using comet assay. On the basis of the observations made we suggested that the glucans exhibited their protective effect against oxidative DNA damage as a consequence of scavenging both radical dotOH radicals and singlet oxygen (Slameňová et al., 2003).

Despite all the indications that CMG acts as free radical scavenger and antioxidant, no direct evidence of trapping of free radicals by glucan has ever been produced. Thus, in this paper we attempted to demonstrate directly the free radical dotOH radical scavenging activity of CMG by using the EPR technique.

Another goal of our work was to investigate whether the antioxidant activity of CMG could lead to beneficial effects in animals with induced adjuvant arthritis, an experimental model of human rheumatoid arthritis (RA), a severe disease of joints, in which pathogenesis an important role is played by reactive oxygen species and various nitrogen oxides (Bauerová and Bezek, 1999, Jaswal et al., 2003, Ostrakhovitch and Afanas'ev, 2001).

Section snippets

Reagents

All chemicals used were of analytical purity grade and distilled water was used throughout all procedures. 5,5-Dimethylpyrroline-N-oxide (DMPO) was purchased from Aldrich Chemical Company (Milwaukee, WI); K2S2O8 and d-mannitol were from Merck GmbH (Darmstadt, Germany); H2O2 from Lachema (Brno, Czech Republic); 2,2′-azo-bis(2-amidinopropane)-dihydrochloride (AAPH) from Polysciencies, Inc. (Warrington, PA). Cyclosporin A (CsA) (Consupren®, Ivax-CR, Opava, Czech Republic) was used in the

EPR experiments

Previously, we have demonstrated antioxidant properties of CMG in the lipid peroxidation model induced by radical dotOH radicals produced by Fenton's reagent and microwave irradiation (Babincová et al., 2002, Babincová et al., 1999). In these experiments, CMG proved to be a potent antioxidant preventing oxidative damage of liposomes. Its antioxidant activity revealed at relatively low concentration (30 μM) was intermediate between that of α-tocopherol and d-mannitol, which were however used at much higher

Conclusions

By means of EPR spin trapping technique and using several types of free radical initiators, we observed high radical scavenging activity of carboxymethyl (1→3)-β-d-glucan derivative. These properties of yeast polysaccharide have been directly demonstrated for the first time and provide a possible explanation for its antioxidant activity observed in vitro and in vivo in various experimental models. Additional data on antioxidant activity of carboxymethyl (1→3)-β-d-glucan have been obtained in

Acknowledgements

The authors are indebted to the Slovak grant agencies APVT [Grants 20-005702 (V.B.) and 51-020802 (K.B., L.Š.)] and VEGA [Grants 2/4143/04 (G.K.), 2/2049/22 (K.B., L.Š.), and 1/0053/03 (A.S., M.P., and V.B.)] for the financial support. Technical assistance of Ms V. Dytrichová, Ms Š. Hájovská, Ms D. Komendová, Ms M. Proftová, and Ms B. Uhliariková is gratefully acknowledged.

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