| Flammulina velutipes is rich in polysaccharides, approximately 35% of total Zn in Flammulina velutipes is combined with polysaccharide. In recent years, foreign and domestic researchers have carried out preliminary analysis on Zn polysaccharides, but most studies stay in the aspects of bioavailability and antioxidant and total content of Zn. Only a few studies about the biological activity, monosaccharide composition and structural analysis of Zn polysaccharides in Flammulina velutipes were reported. However, the study on the structure of Zn polysaccharides is very important since the biological function of any particular element greatly depend on its chemical form. It is related closely to the activity of the immune function. Therefore, it is laid a good foundation to analysis its primary strcture, study its antioxidant activity, develop a new functional products and expand its application scope. The main results are concluded as follows.1. The single factor and orthogonal experiment was applied to optimize the water extraction condition of Zn polysaccharide in Flammulina velutipes. The content of Zn was determined by flame atomic absorption spectrometry (FAAS). The results showed that the optimized conditions for the extraction of Zn were as followed:extraction temperature 75 ?, extraction time 120 min and ratio of material to water was 1:30. About 34.43 ug Zn was extracted from per gram dried Flammulina velutipes under the optimal conditions.2. Four different species of Zn, namely the suspended, soluble, inorganic, organic species, protein and the polysaccharides fraction were separated through dissoluting, extracting and using 0.45?m membrane filter and D101 macroporous resin. The extraction rate of Zn was 76.34%. In the soluble form, about 43.36% and 23.90% of total Zn was combined with organic and inorganic compound, respectively. Furthermore,14.86%, 10.64% of Zn was combined with polysaccharide and protein.3. Crude Zn polysaccharides were obtained after hot water extraction, deproteininzation and alcohol precipitation. The extraction yeild of crude sample was 6.67%. The content of polysaccharides and Zn was 72.37% and 140 ?g/g. The crude sample was separated through DEAE cellulose-52 and Sephadex G-100 gel chromatography. Two fractions named as CFl, CF2 were obtained. The qualitative experiment and ultraviolet spectra indicated that CF1, CF2 didn't contain starch, phenolic compounds and amino acids and other impurities. Two components were both high purity with high performance liquid chromatography (HPLC). The molecular weight was 890 KDa,1244 KDa.The yields of the extracts were 20.97% and 12.25%, the content of Zn was 191 ?g,429 ?g per gram dried Flammulina velutipes. The results further proved that the two components of high purity. This provided a good basis for further study and comparison about the structural characteristics and activity analysis of the different Zn polysaccharide components.4. The results of monosaccharide composition analysis showed that CF1 was composed of mannose, glucose, glucuronic acid, xylose, galactose, fucose six monosaccharide, with molar ration of 7.15:77.28:1.77:0.89:9.20:3.66. CF2 was composed of mannose, glucuronic acid, rhamnose, glucose, galacturonic acid, arabinose, galactose and fucose eight monosaccharide, with molar ration of 9.45:0.82:1.10:13.08:9.66:2.40:0.89: 62.49.5. Zn polysaccharide was studied by infrared spectrum, NMR, congo red and X-ray experiments. The results showed that the presence of furanose ring in CF1, pyranose ring in CF2. CF1 and CF2 were linked with ?-glycosidic bond, ?-D-glucose and ?-D-mannose existed in them, a-D-xylopyranose existed in CF1. The absorption peak at 1640 cm-1moved to low wavenumber indicates that Zn was bonded with -NH2, the red-shifts of the absorption peak between 1100-1010 cm'1 showed that Zn formed a chelated structure with polysaccharide.They were both triple helical structures and non-crystal structure, and Zn didn't exist in Single crystal.6. The results of Antioxidant activity in vitro of Zn polysaccharides in Flammulina velutipes showed that crude sample and CF1, CF2 sample had a moderate DPPH radical scavenging activity, OH·radical scavenging activity and O2-radical scavenging activity, strong scavenging capacity of H2O2 and weak reducing power. The scavenging activity was increased with the increasing concentration of sample. The order of the antioxidant effect of Zn polysaccharides was crude sample> CF2> CFl. The results indicated the content of Zn and structure of polysaccharides was related to the antioxidant activity of Zn polysaccharides of Flammulina velutipes. |
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