Structure Characterization,Antitumor And Immunomodulatory Activity Of Polysaccharides Co-Fermented With Morus Alba L. And Phellinus Igniarius

Objective In this paper,a strain of Phellinus igniarius isolated and identified in this laboratory was used as a material and Morus alba L.was used as the fermentation substrate.The polysaccharide components were extracted from the end-point of fermentation broth to study its structural characteristics,anti-tumor activity,immunomodulatory activity and the molecular mechanism.It provides a theoretical basis and experimental basis for anti-tumor and regulating immunity of traditional Chinese medicine polysaccharides,which has guiding significance for clinical application.Methods Using hot water leaching,ethanol precipitation,Sevag deproteinization,and AB-8 macroporous resin decolorization,crude polysaccharides were obtained from mulberry leaves and mulberry co-fermentation broth.The homopolysaccharide MPFP2-1 was isolated by DEAE-cellulose anion exchange column chromatography and Shephadex G-100 column chromatography.The structure of MPFP2-1 was determined by HPLC,FT-IR and NMR.Characterization;MTT assay was used to detect the proliferation of mouse macrophage RAW264.7 by MPFP2-1,and Griess and ELISA were used to detect the content of NO and TNF-αin the supernatant of MPFP2-1 treated cells,in order to further investigate its immunity.Regulatory mechanisms were used to detect the expression of NF-κB pathway-related proteins by Western Blot.H22 hepatoma-bearing mice models were successfully established and randomly divided into model group,positive control group and treatment group,and they were intragastrically administered to study their anti-tumor and immunomodulatory activities in vivo.Results The monosaccharide composition and molar ratio of the homoleptic polysaccharide MPFP2-1 was L-rhamnose:D-glucose=1.0:5.4.High-performance gel permeation chromatography coupled with evaporative light detector analysis showed that the molecular weight of MPFP2-1 was 5.03×10⁴Da;The results of the spectrum show that MPFP2-1 has a special absorption peak of polysaccharides,¹H-NMR and¹³C-NMR NMR spectra show that the two sugars are linked by-β-1,6-glycosidic bonds,and the results are consistent with the results of monosaccharide composition.The results of MTT assay showed that MPFP2-1 had no cytotoxicity to macrophages in the concentration range of 25-200μg/m L;Griess and ELISA results showed that MPFP2-1 could significantly promote the secretion of NO and TNF-αby macrophages,Western Blot results Further shows that MPFP2-1 can significantly increase the phosphorylation of NF-κB,and the results are consistent with the ELISA results.H22 tumor-bearing mice experimental results show that MPFP2-1 can improve the H22 tumor-bearing mice immune organ index,serum cytokine levels,changes in splenic lymphocytes CD4+T/CD8+T,inhibit tumor growth.Conclusion The Morus alba L.and Phellinus igniarius co-fermentative glycosaminoglycan MPFP2-1 is a non-starch polysaccharide,which is composed of L-rhamnose and D-glucose with-β-1,6-glycosidic bonds to form a homogeneous polysaccharide with a molecular weight of 5.03×10⁴Da.MPFP2-1 promotes the secretion of NO and TNF-αby macrophages through the NF-κB signaling pathway.In addition,MPFP2-1 can also increase the immune function of H22 tumor-bearing mice,thereby inhibiting tumor growth and improving quality of life.