Structural Characterization Of Fermented Astragalus Membranaceus Polysaccharides And Its Improvement Effect On Intestinal Mucosal Immunity In Mice

After the fermentation of Astragalus membranaceus by FGM(isolated in chicken cecal contents),the extraction yield of polysaccharides was significantly improved,and it has better biological activities such as promoting growth,repairing liver damage and promoting dendritic cell maturation,but the regulatory effect of fermented Astragalus membranaceus polysaccharides(FAPS)on intestinal mucosal immunity in immunosuppressed mice has not been reported.In this thesis,the ultrasonic-assisted extraction process and structure characterization of FAPS were studied.The immunoprotective effect of FAPS on intestinal mucosal immunity in mice was analyzed.The aim was to provide theoretical support for the further development and application of FAPS.1.In order to utilize polysaccharides extracted from fermented Astragalus membranaceus more efficiently,ultrasonic-assisted extraction method was used to analyze the yield of crude fermented Astragalus membranaceus polysaccharides without deproteinization(CFAPS)by single-factor test,Box-Behnken and Design-Expert software.The model equations of extraction time,ratio of water to material and extraction power,the contribution of each factor to CFAPS yield was ratio of water to material,extraction time and extraction power,and interaction between the first and third factor are most significant.The optimum extraction process parameters are extraction temperature 50?,extraction time 60 min,ratio of water to material 8 mL/g and extraction power 480 W,the yield is 7.35±0.08%,which is higher than that of traditional hot water extraction method(6.95±0.24%).The results showed that the application of ultrasound-assisted extraction can increase the yield of polysaccharide while shortening the extraction time.2.In order to investigate the structural characterization and antioxidant activity of FAPS,by different chemical methods,the total carbohydrate content(86.34±1.19%)and sulfate radical content(1.36±0.05%)of FAPS were slightly higher than the total carbohydrate content(77.33±2.60%)and sulfate radical content(1.1910.03%)of Astragalus membranaceus polysaccharides(APS),while the proteins content(6.38±0.62%)of FAPS was slightly lower than that(7.01±0.94%)of APS.Both have no reducing sugar and polyphenol.The molar ratio of the main monosaccharide of APS was determined by high performance liquid chromatography(HPLC)to be Glc:Gal:Ara=27.92:5.20:2.86,and that of FAPS was Glc:Gal:Ara=23.45:2.50:3.16.The average molecular weight of FAPS is lower than that of APS by high performance gel permeation chromatography(HPGPC),especially in the high molecular weight fraction.Both of them contain pyranose and ?-glycosidic linkages by fourier transform infrared spectroscopy(FT-IR).The diameter and height of particles of FAPS are smaller than that of APS by atomic force microscopy(AFM),and neither of them contains triple helix structure by Congo red test.FAPS showed better DPPH and hydroxyl radical scavenging activity by antioxidation activity test.The results showed that there was no significant difference in the structural characterization between FAPS and APS,and the improvement of antioxidant activity of FAPS may be related to the decrease of average molecular weight.3.To investigate the effect of FAPS on intestinal mucosal immunity in immunosuppressed mice,blood routine,tissue section,ELISA,RT-PCR and other methods were used.It was found that intraperitoneal injection of 60 mg/kg cyclophosphamide(CY)for 3 days was effective on establishment of intestinal mucosal immunosuppression model,oral administration of 400 and 600 mg/kg FAPS for 10 days can restore body weight,immune organ index,white blood cell count and intestinal tissue morphology,and promote SIgA secretion by regulate the gene expression of SIgA a-chain and plgR,the serum levels of IgA,IgG,IgM and IL-2,IFN-y were increased,and even restore the total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)activity of the intestine.The results showed that medium and high doses of FAPS could improve intestinal mucosal immune function in immunosuppressed mice.4.To investigate the effect of FAPS on intestinal epithelial barrier function in immunosuppressed mice,intraperitoneal injection of 60 mg/kg CY for 3 days significantly reduced the expression of relevant protein of tight junctions(TJ)and adheren junctions(AJ)in small intestine by RT-PCR,Western blotting and tissue immunofluorescence assay,oral administration of 400 and 600 mg/kg FAPS for 10 days can promote gene expression of E-cadherin,Occludin,ZO-1,ZO-2,ZO-3,Cingulin and Claudin-2,and can also promote protein expression of ZO-1,E-cadherin,Claudin-2 and Claudin-7.The results showed that medium and high doses of FAPS have the effect of protecting and maintaining the expression and integrity of TJ/AJ proteins,and improve the intestinal mucosal damage caused by CY by regulating the intestinal epithelial cell barrier function.