Isolation And Purification,structure Identification And Immune Activity Study Of Polysaccharides From Agrocybe Aegerita(AA-P) And Hygrophorus Olivaceoalbus(HO-P)

In recent years,with the improvement of people’s living standards and the deepening understanding of the disease,the search for non-toxic and low side effects of natural products in treatment has attracted extensive attention.As an immune regulator,polysaccharides in natural products play an important regulatory role in the immune system by activating immune cells and promoting cytokine production.In this study,Agrocybe aegerita purchased from Nanchong City,Sichuan Province and Hygrophorus olivaceoalbus collected from Yajiang County,Ganzi Prefecture,Sichuan Province were used as research materials to explore the primary structure and in vitro immune activity of the two fungal polysaccharides,so as to provide a theoretical basis for the application of fungal polysaccharides in life science and clinical medicine.In this study,morphological identification and molecular biology techniques to identify fungi.The crude polysaccharides of Agrocybe aegerita and Hygrophorus olivaceoalbus were extracted by hot water extraction and ethanol precipitation,the two crude polysaccharides were separated and purified by Sevage method and DEAE-52cellulose column chromatography to obtain the purified Agrocybe aegerita polysaccharide and Hygrophorus olivaceoalbus polysaccharide,which were named AA-P and HO-P respectively.Using High-performance gel permeation chromatography,Fourier transform infrared,High performance liquid chromatography,Gas Chromatography-mass spectrum and Nuclear magnetic resonance were used to identify the structure of AA-P and HO-P.Cell Counting Kit-8 and flow cytometry analysis were used to explore the effects of AA-P and HO-P on T cells,B cells and RAW264.7 cells effects of cell proliferation and cell cycle.Enzyme linked immunosorbent assay was used to investigate the effects of AA-P and HO-P on cytokine secretion by T cells,B cells and RAW264.7 cells.The research results of this paper are as follows:（1）The morphological identification results showed that the strain purchased from a supermarket in Nanchong,Sichuan Province was Agrocybe aegerita.By combining the morphological identification results with ITS sequence analysis results,it was determined that the wild strain collected from Yajiang County,Ganzi Prefecture,Sichuan Province was Hygrophorus olivaceoalbus.（2）Structural analysis of Polysaccharides:The results of HPGPC showed that the weight average molecular weight of Agrocybe aegerita polysaccharide was 21,062 Da,and the weight average molecular weight of Hygrophorus olivaceoalbus polysaccharide was 24,481 Da.Both polysaccharides were homogeneous polysaccharides.The primary structure of polysaccharide was analyzed by FT-IR,HPLC,GC-MS and NMR.The results showed that Agrocybe aegerita polysaccharid was composed of Arabinose,Glucose and Galactose in the ratio of 1:2:2,its primary structure was based on（1→4）-Arap,（1→4,6）-Glup and（1→6）-Galp.One（1→4,6）-Glup was connected to Glup 4-O,and the Glup 1-O and 4-O were connect to a→1)Glup respectively.Hygrophorus olivaceoalbus polysaccharid was composed of Mannose,Glucose and Galactose in a ratio of 1:2:1,its primary structure was based on（1→2,6）-Galp,（1→4）-Glup and（1→4,6）-D-Manp.One（1→2,6）-Galp was connected to two Galactose 2-O,the Galp 2-O was connect to one→1)Glup,one（1→4,6）-D-Manp was connected to Manp 4-O,the Manp 1-O and4-O were connect to one→1)Glup respectively,and another Manp 4-O on the main chain was connect to one→1)Glup.In conclusion,the results showed that AA-P showed a more linear structure,while HO-P had larger molecular weight and more branches.（3）Study on in vitro immune activity of Polysaccharides:In the concentration range of 1.25-10μg/mL,Agrocybe aegerita polysaccharide and Hygrophorus olivaceoalbus polysaccharide can stimulate T cells,B cells and RAW.2647 cells proliferation.When the concentration of AA-P was 10μg/mL,the stimulation effect on T cells and B cells was the best（P<0.01）,and the maximum proliferation rates were 49.12%and 32.47%,respectively.When the concentration of AA-P was 5μg/mL,the stimulation effect on RAW264.7 cells was the best（P<0.01）,and the proliferation rate was 99.90%.When the concentration of HO-P was 5μg/mL,the stimulation effect on T cells and RAW264.7cells was the best（P<0.01）,and the maximum proliferation rates were 35.80%and38.59%,respectively.When the concentration of HO-P was 2.5μg/mL,the stimulation effect on B cells was better（P<0.01）,and the maximum proliferation rate was 48.61%.In conclusion,the AA-P has a better stimulation effect on RAW264.7 cells,and the stimulation effect on RAW264.7 cells is much higher than that of HO-P at the same concentration.Based on the research results of proliferation experiment,the concentration with the best proliferation effect was selected to further explore the effects of Polysaccharide on T cells,B cells and RAW264.7 effect of cell cycle.The results showed that both Agrocybe aegerita polysaccharide and Hygrophorus olivaceoalbus polysaccharide could reduce the percentage of cells in the G₀/G₁ phase of the three immune cells.When AA-P was 10μg/mL,the percentage of T cells in G₂/M phase was significantly increased,while that of B cells in S phase was significantly increased.When HO-P was 2.5μg/mL,the percentage of B cells increased significantly in S and G₂/M phases.When HO-P was 5μg/mL,the percentage of RAW264.7 cells in G₂/M phase was significantly increased.Using the optimal concentration to stimulate the proliferation of three kinds of immune cells,the effect of AA-P and HO-P on the secretion of cytokines by T cells,B cells and RAW264.7 cells was further explored.The results showed that when the concentration of AA-P was 10μg/mL and the concentration of HO-P was 5μg/mL,AA-P could stimulate T cells to secrete TNF-α,but HO-P could not stimulate T cells to secrete TNF-α.When the concentration of AA-P was 10μg/mL and the concentration of HO-P was 2.5μg/mL,both polysaccharides could promote the secretion of Ig A,Ig D,Ig E,Ig G and Ig M from B cells.When the concentrations of AA-P and HO-P are both 5μg/mL,both can stimulate RAW264.7 cells to secrete TNF-α,but have no statistical significance on IL-1β.In this paper,two novel homogeneous polysaccharides,Agrocybe aegerita polysaccharide and Hygrophorus olivaceoalbus polysaccharide,were studied.The primary structures of the two polysaccharides were analyzed and their in vitro immune activities were measured,which provided a theoretical basis for the development of edible fungi value and clinical application of Agrocybe aegerita polysaccharide and Hygrophorus olivaceoalbus polysaccharide.