Extraction, Isolation, Purification, Structural Elucidation And Antioxidant Activity Of Polysaccharides From The Fruting Bodies Of Ramaria Flava

In this paper, the extraction, antioxidant activity, purification, physical-chemical character research and sugar structure elucidation for polysaccharides from fruiting bodies of Ramaria flava were systematic studied.The extraction conditions for polysaccharides were optimized on the basis of Design-Expert software and response surface methodology. The optimum extraction conditions for Ramaria flava polysaccharides production were extraction temperature 72℃, the ratio of material to water 1:21 (g/g), and extraction time 15 min, while the extracting yield reached 15.99%. Three water-soluble crude polysaccharides, Ramaria flava Polysaccharide Fraction 30 (RFPF30), Ramaria flava Polysaccharide Fraction 60 (RFPF60), Ramaria flava Polysaccharide Fraction 80 (RFPF80) were obtained under the optimal condition. The antioxidant activities of RFPF30, RFPF60 and RFPF80 were evaluated on the basis of hydroxyl radical assay, superoxide radical assay, Fe2+-chelating assay and reducing power assay. Among these polysaccharides, RFPF60 showed better reducing power and chelating activity than others, it also revealed the highest inhibitory effects on superoxide radical and hydroxyl radical. The results demonstrated that all fractions exhibited antioxidant activities in a concentration-dependent manner, which indicated that polysaccharides extracted from Ramaria flava had potential use as functional food ingredients to alleviate oxidative stress. And we also found that the component with the smaller molecular weight and higher polysaccharide content, showed better antioxidant effect.RFPF60 was purified by Sephacryl S gel chromatography after DEAE Sepharose Fast Flow anion-exchange chromatography. Two novel water-soluble heteropolysaccharides RFPF1 and RFPF2A were obtained, then their chemical and physical properties were determined by a combination of chemical and instrumental analysis methods. UV scanning images at 280 nm and 260 nm showed that both of them didn’t contain protein or nucleic acid. IR spectra indicated that they all had sugar characteristic peaks. They were eluted as a single symmetrical narrow peak on HPLC, and the average molecular weight were 2.18×105 Da and 9.87×105 Da respectively.The sugar composition, the number of sugar residues and the form of glycosyl linkage of RFPF2A were confirmed by sugar analysis, methylation analysis and NMR spectroscopic methods. RFPF2A was mainly composed of D-mannose, D-glucose and D-galactose as the molar ratio of 0.26:1:0.15. Among them, the glucosyl residues are mainly 1, 6-linked units, the galactosyl residues are mainly 1,2,6-linked units, and the mancosyl residues are mainly 1,4-linked units. And we also found that glucosyl residues and galactosyl residues were β configuration, galactosyl residues existed as a configuration. While RFPF1 was composed of D-galactose, D-mannose and D-glucose, with a molar ratio of 1:0.28:0.47. The paper identified and researched the structure of polysaccharides from Ramaria flava systematically, which could provide some reference on ideas and methods for the study on relationship between polysaccharides’ bioactivity and struction.