The Molecular Structure,Conformation And Immunomodulatory Activity Of Glucans From Floccularia Luteovirens

The Floccularia luteovirens is an important biological resource and featured product in the Qinghai Tibet Plateau area.It was listed as one of the most valuable edible fungi with the most development potential in the plateau area and has been traditionally used as Chinese herbs.Recently,it has also been listed as one of the“national drug and food homologous resources”.The edible and medicinal values of Floccularia luteovirens have gone through a long history of verification,showing good antiviral,anti-inflammatory,and immune regulatory effects.The bioactive effects have been closely associated with the existing polysaccharides.However,to our best knowledge,the relationships between the structural and functional activity of polysaccharides from Floccularia luteovirens have not been systematically reported.In this study,the Floccularia luteovirens polysaccharide was extracted and fractionated into three fractions according to Mw differences.Their proposed structures,conformational properties and immunomodulatory effects,in vivo and in vitro,were investigated.The structural and immunomodulatory relationships of the Floccularia luteovirens polysaccharides were partially established.This study provides a theoretical basis for the value-added application of Floccularia luteovirens polysaccharides in functional foods and pharmaceutical areas.The detailed sub-conclusions were as follows.(1)Three high molecular weight polysaccharide fractions of Floccularia luteovirens were prepared by hot water extraction combined with sequential ethanol fractionation(15Et%,20 Et%,25 Et%)(yield:2.40±0.3%,Mw:1.96×10~6g/mol),ALP-2(yield:3.67±0.5%;Mw:2.15×10~7g/mol)and ALP-3(yield:3.03%,Mw:8.07×10~6g/mol).The total sugar contents measured by the phenol sulfuric acid method were 89.2%,90.40%and88.3%,respectively.Rheological analysis indicated that ALP-1 showed much higher apparent viscosity and was easier to form a gel when compared to ALP-2 and ALP-3.However,when compared with lentinan with a similar structure,the apparent viscosity of ALP-1 was relatively low,and the gelling property is slightly weak.(2)The molecular structure of ALPs was characterized by partial molecular degradation,methylation analysis,1D&2D nuclear magnetic resonance spectroscopy,combined with FT-IR.The results showed that ALP-1,ALP-2 and ALP-3 were all homogenous comprising only glucose.ALP-1 mainly contained three sugar residues,namely,T-,1,3-and 1,3,6-Glcp,with a molar ratio close to 1:2:1.ALP-2 and ALP-3contain T-Glcp,4-Glcp and 4,6-Glcp sugar residues,with molar ratios of 1:11:1 and 1:4:1,respectively.Through 1D and 2D NMR,the proposed structure of ALP-1 was obtained as follows:β-1,3-Glcp is connected as the main chain,andβ-T-Glcp was connected at the C6position of every three sugar residues along the main chain.The proposed structures of ALP-2 and ALP-3 were as follows:α-1,4-Glcp is connected as the main chain,andα-T-Glcp is connected at the C6 position of every twelve and five sugar residues along the backbone for ALP-2 and ALP-3,respectively.HPSEC-MALL-RI-UV analysis indicating that ALP-1 existed in a rigid and triple helix conformation with ALP-2 and ALP-3 fractions showed typical spherical conformation in an aqueous solution.(3)Through in vitro digestion analysis,β-Glucan(ALP-1)were unchanged whileα-Glucan(ALP-2 and ALP-3)were gradually degraded into oligosaccharide fragments.The results from cell culture showed that ALP-1,ALP-2 and ALP-3 all could effectively activate RAW-blue cells,and improve cell proliferation,phagocytosis and cytokine secretion in RAW 264.7(p<0.05).However,the immunomodulatory effects were greatly reduced when ALP-2 and ALP-3 were degraded by the enzymes through the GI tract.In vitro fermentation of ALP-1,ALP-2,ALP-3,ALP-2-D3 and ALP-3-D3 by faecal inoculum from pigs indicated that they all could be utilized by gut microbiota the p H value were decreased by 2.30,3.55,4.33,1.75 and 1.36,respectively,in the system.Meanwhile,the total SCFAs continuously increased within 24 h for ALP-1 group and 12 h for ALP-2-D3.16S r RNA showed that ALP-1,ALP-2 and ALP-3-D3 could effectively decrease the value of Firmicutes/Bacteroides and the diversity of Escherichia Shigella.(4)The immunomodulatory effects of glucans were also determined using the animal trial.The results showed that intraperitoneal injection for ALP-2 at the dose of 1 mg/kg body weight and gavage for ALP-1 at the dose of 10 mg/kg both could effectively restore the cyclophosphamide(Cy)-induced decrease in thymus indexes in mice and the damaged intestinal barrier.They also increased cytokine and immunoglobulin levels,promoted the production of SCFAs,increased the abundance of Bacteroidetes and Muribaculaceae,and decreased the proportion of Firmicutes and Helicobacter in the microbial community.In addition,the recovery effects of ALP-1 on body weight,intestinal SIg A and lysozyme content,and SCFAs production were better than that of ALP-2.