Correlation evaluation of antioxidant properties on the monosaccharide components and glycosyl linkages of polysaccharide with different measuring methods

Abstract

The relationships of antioxidant properties (AOPs), measured by four conventional in vitro methods, with monosaccharides and glycosyl linkages in the polysaccharide, were evaluated using multiple linear regression analysis with minor modifications. Polysaccharides extracted from culture broth filtrates of Lentinula edodes were used as model samples for evaluation. Results indicate that the composition of monosaccharides and the type of glycosyl linkage modulates the AOPs of the polysaccharides. The AOPs of the polysaccharides were dependent on the ratios of different monosaccharides in the composition. Among the monosaccharides, rhamnose was the most significant determinant factor associated with AOPs. The glycosyl linkages of the monosaccharides also affected the anti-oxidation characteristics of the polysaccharides. Specifically, the arabinose 1 → 4 and mannose 1 → 2 linkages of the side-chain were significantly related to the reducing power, whereas the glucose 1 → 6 linkage and arabinose 1 → 4 linkages were related to the scavenging on DPPH⁻ radicals.

Introduction

Superoxide and hydroxyl radicals are potent oxidants that can react with all biological molecules, such as DNA, proteins, lipids, and carbohydrates; meanwhile, induced oxidative stress can mediate a wide variety of pathological effects (Chattopadhyay et al., 2010, Kardošová and Machová, 2006, Tsai et al., 2007). Normally, living cells have the ability of self-protection against oxidative damage through several defense mechanisms, such as the enzymatic conversion of reactive oxygen species (ROS) into less toxic substances and detoxification by reaction with antioxidants (Tsai et al., 2007). The discovery of polysaccharides, a new antioxidant active agent that can be extracted from plants or fungi, have pushed many researchers to explore many clinically serviceable dietary supplements, as they offer advantages in the prevention of human diseases (Li et al., 2006a, Li et al., 2006b, Tsai et al., 2007, Tseng et al., 2008).

Previously, polysaccharides extracted from mushrooms have been shown to be effective and non-toxic, and offers the ability to scavenge free radicals (Liu, Ooi, & Chang, 1997). Among various mushrooms, the shiitake mushroom, Lentinula edodes, which is widely cultivated in Korea, Russia, Taiwan, China, and Japan (Campbell and Slee, 1987, Fox et al., 1994), have been intensively studied for their immune-modulating, antimicrobial, antioxidant, and anti-atherogenic activities (Hobbs, 2000). Liu et al. (1997) reported that proteins in polysaccharide extracts contribute directly in free radical scavenging activities. Particularly, the sulfated, acetylated, and phosphorylated derivatives, as well as the water extract from polysaccharides, could exhibit significant antioxidant properties (AOPs) in in vitro experiments (Kardošová and Machová, 2006, Zhang et al., 2003). Recently, Chen, Xie, Nie, Li, and Wang (2008) reported that a water-soluble protein-bound polysaccharide extracted from the fruiting bodies of Ganoderma atrum containing mannose (Man), galactose (Gal), and glucose (Glu) in a molar ratio of 1:1.28:4.91, with an average molecular weight about 1013 kDa, exhibits strong AOP. Li and Zhou (Li, Li, & Zhou, 2007) also showed the multiple AOPs of polysaccharides extracted from Lycium barbarum fruits (e.g., rhamnose (Rha), xylose (Xyl), arabinose (Ara), fucose (Fuc), Glu, and Gal). Bučková, Labuda, Šandula, Križková, Štěpánek, and Ďuračková (2002) found that AOP and protection against DNA damage by chelation of transition metals of polysaccharides was in the order of mannan (Candida krusei), extracellular glucomannan (Candida utilis), mannan (Candida albicans), and glucomannan (C. utilis).

In general, the AOPs of polysaccharides are influenced by chemical characteristics like molecular weight, degree of branching (DB), types of monosaccharides, intermolecular associations of polysaccharides, glycosidic branching, and modification of polysaccharides. However, the detailed mechanisms of antioxidant effects reflected by monosaccharide composition and glycosyl linkage in polysaccharides remain largely unexplored, even if the vital roles of glucans and glycans have been well-established (Liu et al., 1997).

In previous papers, the monosaccharide composition, molecular weight, and structural linkage of polysaccharides extracted from 10 regional L. edodes were investigated and compared for genetic and structural similarities and differences (Lo et al., 2007a, Lo et al., 2007b). All polysaccharides isolated from these mushrooms exhibited similar molecular weight ranging between 1 × 10⁴ Da and 3 × 10⁶ Da. A major form of polysaccharide linkage with a backbone of (1 → 4)-glucan and side chains of (1 → 6)-glucan was identified. Ara (1 → 4) and Man (1 → 2) linkages also existed in all polysaccharides. Based on composition analysis, the heterogeneous monosaccharides contained Ara, Xyl, Man, Gal, Glu, Fuc, and Rha. Structural analysis did not indicate presence of proteins and absence of uronic acids. The only differences in these linkages manifested in their monosaccharide compositions, leading to different degrees of backbone and branch formations.

Most of the extracts exhibited significant AOPs. All of the previously mentioned studies clearly demonstrate the AOPs of polysaccharides in fungi. In the present work, the AOPs of the extracted polysaccharides were assayed by using conventional methods; that is, by measuring conjugated diene, reducing power ability, scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, and chelating ability on ferrous ions.

Numerous methods have recently applied mathematics and statistics in the classification, summary, and/or simplification of complex data, which consequently have assisted in the interpretation of the relationships between structural characteristics and bio-functions of polysaccharides (Xu & Hagler, 2002). Meanwhile, regression analyses have been broadly used to predict sources or structure–function relationships in biological sciences (Lo et al., 2007b, Pytelaa and Klimešova, 2011).

The aim of this study is to utilize this method to evaluate the relationship between in vitro AOP and chemical characteristics (i.e., monosaccharide ratio and glycosyl linkage) in polysaccharides by using conventional measurement methods. This study is the first to conduct research on the relationship between AOPs and monosaccharide components (i.e., Ara, Xyl, Man, Gal, Glu, Fuc, and Rha) of the L15 polysaccharide.