Studies On The Structural Identification And Immune Activity Of Polysaccharide From Canthar-ellus Cibarius Fr. And Agaricus Rusiophyllus Lasch

Canthar-ellus cibarius Fr.is also called Huangsi-jun or Xing-jun.It is a natural wild edible fungus of basidiomycetes.Agaricus rusiophyllus Lasch belongs to the natural wild edible fungi of Tricholomataceae.Its name is due to its deep brown color,its cap,bacteria and stipe are all white.The two kinds of fungi are very representative of wild edible fungi in Xiaojin County,Sichuan.They are famous for their delicious taste and rich nutrition.However,people only pay attention to their edible value at present,and the medicinal value of two kinds of polysaccharides has not been effectively exploited and utilized.Therefore,we hope to study the medicinal value of two kinds of polysaccharides.The polysaccharide of Canthar-ellus cibarius Fr.and the polysaccharide of Agaricus rusiophyllus Lasch were extracted by hot water extraction and ethanol precipitation.The polysaccharide of Canthar-ellus cibarius Fr.named CC-1 based on the Latin name of the Canthar-ellus cibarius Fr..In the same way,we named polysaccharides of Agaricus rusiophyllus Lasch as Ar L-1.The CC-1 was separated and purified by DEAE cellulose column chromatography.The ArL-1 was purified by DEAE-FF gel column chromatography.The relative molecular weight of two polysaccharideswasidentifiedbyhighperformancegelpermeation chromatography?HPGPC?.Fourier transform Infrared Spectroscopy?FT-IR?,Nuclear Magnetic Resonance Spectroscopy?NMR?and Gas Chromatography-Mass Spectrum?GC-MS?were used to identify the structure of the polysaccharides.Finally,the immune activity of the polysaccharides was identified by cell culture in vitro.Through the research of extraction,separation,purification,structure identification and biological activity of CC-1 and ArL-1,this paper provided a theoretical basis for the research and development of the economic and medicinal value of polysaccharides.It provided the theoretical support and scientific basis for the fungal polysaccharides drug products,provided the premise for the immunomodulatory activity,anti-tumor and its action mechanism,and provided the scientific basis for the development of the multi-carbohydrate targeted immune regulation.The main results are as follows:?1?The two kinds of polysaccharides were purified in different ways.The DEAE cellulose column chromatography was suitable for the purification of Canthar-ellus cibarius Fr.Polysaccharide?CC-1?,and the DEAE-FF gel column chromatography was better for the purification of Agaricus rusiophyllus Lasch polysaccharide?Ar L-1?.The elution curves of the two polysaccharides in different NaCl segments showed that CC-1 had only one elution peak in distilled water,while Ar L-1 had two elution peaks in distilled water and 0.05mol/L NaCl,respectively.It shows that the optimal elution solution of CC-1 is distilled water,while the optimal eluant of ArL-1 is distilled water and 0.05mol/L Tris-NaCl.?2?The result of High performance gel permeation chromatography?HPGPC?showed that the molecular weight curves of the two polysaccharides is a single peak,indicating that the two polysaccharides were homogeneous and the purification effect was better.But the Mw,Mn,Mp of CC-1 and ArL-1 are different.The Mw of ArL-1 is much larger,which is up to 1682458 Da,which indicates that the polysaccharide chain of Ar L-1 has more monosaccharides.?3?The FT-IR,¹H-NMR,¹³C-NMR and GC-MS analysis of the two polysaccharides showed that the two extracts contained the characteristic peaks of polysaccharides and the characteristic fingerprint peaks of polysaccharides,which indicated that the two extracts were polysaccharides.CC-1 is composed of glucose and xylose with the ratio of 5:1,which was most likely to be connected with?1?4?-linked-?-D-glucosepyranose and?6?1?-linked-?-D-xylopyranose revealing that?1?4?-linked-D-glucosepyranose possible form the backbone structure.Residues of branch structure were terminated with?-D-xylopyranose resides.The polysaccharide Ar L-1 was composed of glucose and galactose with a molar mass of 3:2.The result inferred that the structure was to connect the?1?6?-linked-D-galactose possible form the backbone structure and galactose?3?6?–linked-D-glucosepyranose-1-protein as cyclic monomer.?4?After the analysis of two kinds of polysaccharide structure,they were used to stimulate three kinds of different immune cells respectively,and proliferation effect of three immune cells immune cells was tested.First,the proliferation test of Raw264.7cell in vitro was carried out.The results showed that CC-1 reached the best proliferation effect at 10?g/mL,and the proliferation rate was 36.46%.When the concentration of Ar L-1 was 5?g/m L,the optimal proliferation level reached to 93.09%.The two kinds of polysaccharides had a proliferation effect on Raji cells,and the proliferation effect of CC-1 was more significant.When the concentration of CC-1reached 20?g/mL,the best proliferation effect was 85.09%.The optimum proliferation concentration of ArL-1 was 5?g/mL,and the proliferation rate was 66.15%.The maximum proliferation rate of the two polysaccharides was higher than of the positive control group.Finally,we also studied the proliferation effect of two polysaccharides on Jurkat cells.The results showed that the optimal concentration of the two polysaccharides against Jurkat cells was 5?g/mL,and there was no cytotoxicity to three kinds of immune cells.In summary,we studied the specific structures of two polysaccharides,that is,the structure of CC-1 was?1?4?-linked-?-D-glucosepyranose and?6?1?-linked-?-D-xylopyranose revealing that?1?4?-linked-D-glucosepyranose possible form the backbone structure.Residues of branch structure were terminated with?-D-xylopyranose resides.And the structure of ArL-1 was?1?6?-linked-D-galactose possible form the backbone structure and galactose?3?6?–linked-D-glucosepyranose-1-protein as cyclic monomer.Secondly,we found that the structures of the two polysaccharides are different,which make many properties different.For example,the molecular weight,color and solubility of the two polysaccharides are different.there are also obvious differences in their ¹H-NMR spectra and ¹³C-NMR spectra.These differences are related to connection mode,the ligation group of polysaccharides and so on.We also preliminarily determined the immunological activity of the two polysaccharides.The two kinds of polysaccharides could promote the proliferation of three kinds of immune cells,and it can be seen that CC-1 has the best effect on the proliferation of Raji cells in the three kinds of cells.The maximum proliferation rate was 85%,while ArL-1 had the best effect on Raw264.7,and the maximum proliferation rate was 93%.The determination of the specific structure of the polysaccharides laid a foundation for the determination of the active sites and the functional sites.The specific structure of protein-containing polysaccharide ArL-1 was analyzed for the first time,which is of great value in the study of polysaccharides,which provides a prerequisite for immunomodulatory activity,anti-tumor and its mechanism,provides theoretical support and scientific basis for the future bacterioglycan biopharmaceutical products.