Preparation Of Inulin From Codonopsis Pilosula Nannf.var.modesta L.T.Shen And Its Effect On Intestinal Mucosal Immune Function

As one of the main active constituents of Codonopisis pilosula,the polysaccharide composition has a variety of biological activities,among which the most widely studied is immunomodulatory function.C.pilosula Nannf.var.modesta L.T.Shen is one of the three major sources of C.pilosula,with wide resources and good quality,but few researches on its polysaccharides.Therefore,this subject,combined in vivo and vitro experiments,used cyclophosphamide（CY）-induced immunosuppressed mice as an animal model to evaluate the modulation of immunity and gut microbiota of the polysaccharides from C.pilosula（CPP）.Then,the majority of CPP,the inulin-type fructan（CPPF）,were optimizated with its extraction process,and the structure characterization and the prebiotics activity in vitro were studied;and the immunosuppressed mice were used again to evaluate the enhancement of intestinal mucosal immunity and the regulation of gut microbiota,further elucidating the mechanism of this neutral component.1 Effect of CPP on the enhancement of intestinal mucosal immunity and the regulation of gut microbiota in immunosuppressed mice40 BALB/c mice,male and female,were randomly divided into 5 groups:Control group,cyclophosphamide+saline group（CY group）,cyclophosphamide+high（200 mg/kg/d,CY+CPP-H）,medium（100 mg/kg/d,CY+CPP-M）,and low（50 mg/kg/d,CY+CPP-L）dosage of CPP,8 mice each group.After intraperitoneal injection of CY for 3 days to establish an immunosuppressed mice,the CPP solution was oral administered for 7 days,and the spleen,thymus and liver index were observed.The serum was detected by enzyme-linked immunosorbent assay（ELISA）;the concentration of the antibody IgG and cytokines IL-2,IL-10,IFN-γ,and secretion of secretory immunoglobulin A（sIgA）in the ileum were tested.Lastly,the amount of Bifidobacteria,Lactobacillus,Enterococcus and Escherichia coli in cecal contents were accounted by identification medium culture method;the content of Short Chain Fatty Acids（SCFAs）were determined by gas chromatography（GC）.The results showed that compared with CY group,CPP significantly increased the spleen index of mice after intragastric administration,and the high dose group significantly increased the thymus and liver indices,significantly promoted IL-2,IL-10,IFN-γand IgG secretion.At the same time,CPP in the high and middle dose groups promoted the secretion of sIgA in the ileum,which also significantly increased the amount of Lactobacillus in the cecum and decreased the amount of E.coli.The acetic acid in the cecum was significantly higher than that in the model group and the control group after the administration of high dosage of CPP.There was no significant change in the acid and butyric acid content.The above results indicated that CPP could improve the immune function,especilally the intestinal mucosal immune,and regulated the intestinal flora by the promotion of probiotics;and combined with the metabolic characteristics of biomacromolecules,we inferred that the target organ of the active polysaccharide of C.pilosula was intestine.In the previous study,approximately 60⁷0%（w/w）of CPP was a neutral component,namely,inulin-type fructan,so it is inferred that this component is the main active component of the CPP,thus those studies including extraction process optimization,structural analysis and bioactivities in intestine were studied.2 The optimization of extraction process and structural elusidation of CPPFIn this study,the extraction process of inulin from C.pilosula Nannf.var.modesta L.T.Shen was optimized by hot water.On the single factor experiments,the extraction rate of inulin was used as reference index for these extract condition:the different extraction temperatures（50¹00°C）,material-solvent ratio（1:10¹:60）and extraction time（0.5 h³.0 h）.Then,the experimental design of response surface analysis method with three-factor and three-level was carried out based on the principle of central combination experimental design.All analysed data was used for the second-order polynomial model regression.In the end,the regression equation is obtained;finally,the process is optimized on the basis of the regression equation,and the verificated by repeating experiments with same extract condition.The results showed that the inulin yield increased significantly with the increase of temperature,solvent-medicine ratio and extraction time.According to the statistical analysis of response surface,the fitting equations were obtained,and the coefficients showed that the fitting degree was good.The best conditions for extracting the inulin from C.pilosula Nannf.var.modesta L.T.Shen were obtained by maximizing the fitting equation:the extract temperature was 100°C,and the ratio of solvent to material was 40 mL/g,extracted twice,2.5 h each time,the inulin yield was20.6%.The CPPF was separated and purified by a DEAE-Sepharose from CPP,and then free-dried.The gas chromatography（GC）,paper chromatography,gas chromatography-mass spectrometry（GC-MS）,the size-exclusion chromatography-multiangle laser scattering（SEC-MALLS）and nuclear magnetic resonance（NMR）were used to detect the structure characterization,including monosaccharide compositions,glycosidic bond compositions,and molecular weight（degree of polymerization,DP）.The results showed that the CPPF consisted of a large amount of fructose（Fru）and a small amount of glucose（Glc）,which is based onβ-（2→1）-Fruf,and terminal Glcp linked withα-D-（2→1）glycosidic bond,with the molecular weight of 2810 Da,DP ranged from 2 to17,an average of 6.3 The prebiotic activity in vitro of CPPFSix different species of Lactobacillus were used as experimental bacteria;and two types of commential inulin（P95s and Orafti?HP,with different DP）were used as positive control.The?A₆₀₀ and pH in the medium were analyzed to evaluate the degree of proliferation of Lactobacillus compared with the basal medium.The prebiotic activity of CPPF be equivalent to the utilization degree.The results indicated that CPPF could significantly promoted the proliferation of all species of Lactobacillus,reduced the pH of the medium,with potential prebiotic activity.By comparing the different degrees of polymerization,the activity of the prebiotics was found to be related to the DP and the species of Lactobacillus.4 The effect of CPPF on intestinal mucosal immune function and gut microbiota in immunosuppressed miceAccording to the results above,it could be inferred that CPPF is the active component of CPP.Therefore,the activity of enhancement on intestinal mucosal immunity and regulation of gut microbiota in immunosuppressed mice were studied using the same method of section 1,and then the preliminary mechanism was explored.Fifty C57BL/6 mice were randomly divided into 5 groups:Control group,cyclophosphamide+saline group（CY group）,cyclophosphamide+high（200 mg/kg/d,CY+CPFP-H）,medium（100 mg/kg/d,CY+CPPF-M）,and low（50 mg/kg/d,CY+CPPF-L）dosage of CPPF.After establishing the immunosuppressed model,the spleen,thymus and liver indices were observed after CPPF oral administration for 7 days.The cytokines IL-1βand TNF-αin serum,jejunum and ileum were detected;and the secretion of sIgA and mucin 2 in the ileum was analyzed.After that,the microbiota composition of cecal contents were analyzed by 16S rRNA gene（V3/V4）sequencing and the SCFAs were quantitatively detected using GC.The results showed that compared with the CY group,the spleen and thymus indices of the high and medium groups increased significantly,and the liver index of the high dosage of CPPF was extremely higher than that of the Control group,and promoted the secretion of serum IL-1β.At the same time,the high and medium and dosage of CPPF inhibited the expression of pro-inflammatory cytokines IL-1β,TNF-αgene and protein expression in the jejunum and ileum,except colon;and the high dosage of CPPF increased ileal sIgA and mucin 2 concentration.In addition,theα-diversity index in CY group was significantly reduced,and the microbial structure was changed.Whereas,CPPF increased the microbial abundance,expressed as the rise abundance in the order of Clostridiale including the Lachnospiraceae family（Blautia genus）and the Ruminococcceae family（Unidentified Ruminococcaceae genus）.While,the content of SCFAs in the cecum did not change significantly.The above results demonstrated that CPPF improved the function of intestinal mucosal immune,and modulated the gut microbiota;the main active site was the jejunum and ileum.In summary,the CPP could promote the secretion of serum cytokines and sIgA in the ileal mucosa;it also promoted the proliferation of Lactobacillus and balanced the disordered intestinal flora.Among them,the neutral component of the CPP was the active component with a large content.The optimized extraction process and multiple structural detection analysis indicated that it is inulin-type fructan,ie inulin.The inulin is a potential prebiotic,which can enhance the body’s immunity,mainly the intestinal mucosal immune function of the jejunum and ileum（including the decrease of IL-1β,TNF-αgene and protein expression,and promotion of sIgA and Mucin 2 secretion）,and regulate the composition of the intestinal flora.Finally,it is further explained that the CPPF is the main active component of CPP,especially on immune enhancement and gut microbiota modulation.