Correlation evaluation of antioxidant properties on the monosaccharide components and glycosyl linkages of polysaccharide with different measuring methods
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.
Section snippets
Chemicals and reagents
Linoleic acid, ascorbic acid, potassium ferricyanide, trichloroacetic acid, ferric chloride, butylated hydroxylanisole (BHA), DPPH, ferrozine, ethylenediamine-tetraacetic acid (EDTA), Ara, Xyl, Man, Gal, Glu, Fuc, Rha, dextrose, and cellulose were purchased from Sigma (St. Louis, MO, USA). Sodium phosphate buffer and citric acid were purchased from Merck (Darmstadt, Germany). Methanol (ACS) was purchased from Echo (Hsinchu, Taiwan). The methanolic solution containing 0.2 mM DPPH radicals was
Results
The relationships of AOPs, as measured by the four conventional in vitro methods (i.e., conjugated diene, reducing power, DPPH− radical-scavenging, and ferrous ions chelating activity methods) and structural characteristics of polysaccharides (i.e., monosaccharides and glycosyl linkages in the composition) were evaluated individually using MLRA.
Discussion
To the best of our knowledge, there have only been a few reports using objective statistical methods to conduct a comprehensive analysis of experimental data as a means of deducing polysaccharide structure–function relationships, particularly because data related to these are limited (Lo, Jiang, et al., 2007). The MLRA adopted in the current research allowed for the detection of differences in the major characteristic compositions (i.e., polysaccharide antioxidant competence) in each of the 10
Conclusion
The in vitro AOPs of polysaccharides extracted from the CBF of L. edodes are dependent on the composition and ratio of monosaccharide and the type of glycosidic side chain. This mechanism underscores the four different AOPs, which have been modulated by different characteristics of the polysaccharide structure (monosaccharide composition and glycosyl linkage). This study not only contributes to the understanding of the relationships between structures and AOPs, but also simplifies the problems
Acknowledgements
The authors thank the National Science Council of Taiwan for financial support under Grants of NSC 95-2113-M-009-025 and NSC-95-2113-M-005-0021-MY3 and 97-2116-M-259-001-MY3, and the Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.
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