Study On Structure And Bioactivities Of Polysaccarides From Lactarius Volemus Fr.

Lactarius volemus Fr.is a typical edible fungi,which belongs to Agaricales,Russlaceae,Lactarius,mainly distributed in Yunnan,Guizhou,Tibet and other places inChina.Lactarius volemus Fr.has strong antioxidant and antitumor activity,as well as significantly enhance the immune ability.However,there are few studies on the structure identification and biological activities of polysaccharides from Lactarius volemus Fr.In this study,we systematically studied the extraction,isolation,purification,structure identification and biological activities of polysaccharides from the Lactarius volemus Fr.The main results are as follows:Single factor screening and response surface analysis were used to analyze the best extraction technology conditions.Taking into account the limitations of the actual operation,the parameters will be modified.the ratio of material to liquid is 1:25,80 ℃,for 150 min.The results showed that the yield of the crude polysaccharides from the Lactarius volemus Fr.was 4.15%.Lactarius volemus Fr.polysaccharides extracted liquid obtained in the best extraction condition conducted the following steps: decoloring by resins,deproteinizing with Sevag method,precipitating by ethanol alcohol,concentrating and drying.The crude polysaccharide was isolated by DEAE-52 ion-exchange column chromatography,which was used to get the LVF-Ⅰ,LVF-Ⅱ,LVF-ⅡI,which accounts for 32.10%,18.33% and6.22% respectively.The LVF-Ⅰ and LVF-Ⅱ polysaccharides were identified as homogeneous polysaccharides by gel permeation chromatography(GPC)and Sephadex G-100 column chromatography.In addition,the molecular weight Mw were 12894 Da and 14404 Da respectively.LVF-Ⅲ was miscellaneous polysaccharides,and the Mw of LVF-Ⅲ was 9259 Da.It was found that LVF-Ⅰ,LVF-Ⅱ,LVF-Ⅲ were like white cotton,canary yellow cotton,yellowish cotton,respectively.The protein contents were 0.97%,4.66%,6.31%;total sugar contents followed by 70.03%,75.77% and 63.25%;uronic acid contents followed by 8.29%,13.36%,8.70%,respectively.Structure analysis: LVF-Ⅰ,LVF-Ⅱ,LVF consists of Glucuronic acid,fucose,mannose,Glucose,Galactose.The molar ratio of LVF-Ⅰ was 1.0:2.8:6.9:10.4:10.0,molarratio of corresponding monosaccharide LVF-Ⅱ was 1 : 1.1 : 4.4 : 24.4 : 4.7.The infrared spectra show that LVF-Ⅰ and LVF-Ⅱ were polysaccharides containingα-D-mannose Glucose.By periodate oxidation and Smith degradation Methylation and NMR analysis,LVF-Ⅰ contains 1→2,6 1→3 1→4 and 1→6 glycoside bonds,and 1→6glycoside bonds is the main chain.LVF-Ⅱ contains 1→3,1→2,6,1→4 and 1→6glycoside bonds,and 1→6 glycoside bonds is the main chain.The results of Congo red test and SEM showed that the conformation of LVF-Ⅰ and LVF-Ⅱ did not exist multiple spiral.In addition,they were irregular flakes,porous like clusters of different shapes,etc.The biological activity of polysaccharides: All the polysaccharide samples have certain antioxidant activity,including Fe reducing power,and ABTS+ free radical scavenging capacity,especially at high concetration.There was a positive correlation between the antioxidant activity and concentration of polysaccharide samples.And LVF polysaccharide shows higher antioxidant activity than the other polysaccharide samples.The purified polysaccharides from Lactarius volemus Fr.had strong inhibitory effects on MCF-7 and H1299,and they were positively correlated with the concentration.The antitumor activity of LVF-Ⅱ had the best effect,comparable to the positive control 5-Fu uracil.The polysaccharides of LVF alone in mouse spleen lymphocytes could promote cell proliferation,but the degree of the improvement was lower.However,when LPS and ConA synergistic effect on cells,they could evidently promote proliferation,but between them has no significant difference.In 125-500 μg/mL,phagocytosis of polysaccharides was stronger than the 1μg/mL LPS,and the role in setting the range of concentration significantly.Lactarius volemus Fr.polysaccharides could promote the secretion of NO,IL-6 and TNF-α,with the dose dependent effect.The expression of iNOS,IL-6 and TNF-α,mRNA gene in macrophages were detecte by RT-PCR.LVF-Ⅰ,LVF-Ⅱ,LVF-ⅡI could increase the expression of iNOS,IL-6 and TNF-α gene,which indicated that they could promote the production of immune factors and improve the cellular immune activity.In vitro digestion profiles of LVF-Ⅰ in a simulated gastrointestinal digestive system were investigated.Results showed that gastric juice could change the molecular weight of polysaccharides but cannot increase the content of reducing sugars.After digestion in simulated intestinal juice could change the molecular weight of polysaccharidesobviously.The content of reducing sugar content in digestive juice increased.This indicates that the simulated intestinal fluid destroyed a small amount of glycoside bondss of LVF-Ⅰ.