Elsevier

Process Biochemistry

Volume 45, Issue 6, June 2010, Pages 874-879
Process Biochemistry

Purification, structural analysis and hydroxyl radical-scavenging capacity of a polysaccharide from the fruiting bodies of Russula virescens

https://doi.org/10.1016/j.procbio.2010.02.007Get rights and content

Abstract

In this study, one water-soluble polysaccharide (RVP) was isolated from the fruiting bodies of Russula virescens. Its molecular weight (Mw) was ∼3.9 ×104 Da, as determined by high-performance size-exclusion chromatography (HPSEC). The RVP was composed of d-galactose and d-mannose in a ratio of 2:1, as detected by gas chromatography (GC). The structural features of RVP were investigated by partial hydrolysis with acid, periodate oxidation and Smith degradation, acetylation, methylation analysis, and NMR spectroscopy (1H, 13C). The results revealed that RVP had a backbone consisting of (1  6)-linked-α-d-galactopyranosyl and (1  2, 6)-linked-α-d-galactopyranosyl residues that terminated in a single non-reducing terminal (1  )-α-d-mannopyranosyl residue at the O-2 position of each (1  2, 6)-linked-α-d-galactopyranosyl residues along the main chain in the ratio of 1:1:1. Preliminary, in vitro antioxidant activity tests showed that RVP could dose-dependently enhance the scavenging effect on hydroxyl radicals at concentrations from 20 to 160 μg/ml. These results indicated that RVP was mainly composed of water-soluble galactomannan, and it had a significant effect on scavenging of hydroxyl radicals in vitro.

Introduction

Today, increasing attention is being placed on polysaccharides by biochemical and nutritional researchers, due to their various biological activities that could be applied to healthcare foods or medicine, especially antioxidant, immunostimulatory, and antitumor effects [1], [2], [3], [4]. Overmuch production of reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals, superoxide, and nitric oxide was proved to play key roles in tissue damage and loss of function in many tissues and organs. Thus, it is essential to develop and utilize effective and natural antioxidants so that they can protect the human body from free radicals and retard the progress of many chronic diseases [5], [6], [7]. Published data indicate that plant polysaccharides in general have strong antioxidant activities and can be explored as novel potential antioxidants [4], [8], [9].

Russula virescens is a wild, edible fungus that grows on the roots of pine trees distributed throughout China. Sun et al. had identified one alkali-soluble (1  3)-β-d-glucan from R. virescens by 0.5 M NaOH aqueous solutions, and evaluated the antitumor activity of it and its sulfated derivatives [10]. There was also report that the wild R. virescens had distinct effects on the regulation of blood lipids and anti-oxidation [11]. However, to date, no investigation has been carried out on the structural feature of water-soluble polysaccharides from R. virescens. Since, structure and functions are intimately related, structural identification of the polysaccharides is necessary to better effectively explore its structure–activity relationship.

Therefore, the aim of this work was mainly to report on the extraction and purification of a water-soluble polysaccharide from the fruiting bodies of R. virescens by ion exchange chromatography and gel filtration chromatography on an ÄTKA explore 100 purification system, and elucidate its structural characterization by a combination of chemical and instrumental analyses. In addition, the hydroxyl radical-scavenging activity of this polysaccharide was also evaluated.

Section snippets

Chemicals

DEAE Sepharose Fast Flow, Sepharose 6 Fast Flow, and Sephadex G-25 were purchased from Amersham (Sweden). T-series dextran, dimethyl sulfoxide (DMSO), standard sugars, deoxyribose, trichloride ferric, ethylene diamine tetra-acetic acid (EDTA), H2O2, ascorbate acid, and thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. (St. Louis, MO). All of other reagents were analytical grade from Peking Chemical Co. (Peking, China).

General methods

UV–vis absorption spectra were recorded with a UV–vis

Isolation, purification and structural analysis of polysaccharides

The RVP showed a single, symmetrically sharp peak, indicating its homogeneity on HPSEC (Fig. 2). According to the retention time, its molecular weight was estimated to be 3.9 × 104 Da, and it was shown that [α]D20=+28.1 (c 2 mg/ml, H2O). It had a negative response to the Bradford test and no absorption at 280 or 260 nm in the UV spectrum, indicating the absence of protein and nucleic acid. Total carbohydrate content was determined to be 93.4%. The RVP was composed of d-galactose and d-mannose as

Conclusions

We had successfully isolated and characterized the structure of a polysaccharide isolated from the fruiting bodies of R. virescens. Its structural features included a backbone consisting of (1  6)-linked-α-d-galactopyranosyl and (1  2, 6)-linked-α-d-galactopyranosyl residues that terminated in (1  )-α-d-mannopyranosyl residues in the ratio of 1:1:1. Preliminary, in vitro antioxidant activity tests showed that RVP could dose-dependently enhance scavenging effects on hydroxyl radicals.

Acknowledgement

This work was financially supported by the Natural Science Foundation for Young Scientists of Heilongjiang Province, China (Grant No. QC2009C106).

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