Characterization And In Vitro Metabolism Of Polysaccharide From Atractylodes Macrocephala Koidz And Active Polysaccharides Fractions Of Sijunzi Decoction

An active polysaccharide from the rhizome of Atractylodes macrocephala Koidz（PAM）was identified to improve and adjust disordered intestinal flora.High-performance gel permeation chromatography（HPGPC）and gas chromatography-mass spectrometry（GC-MS）were employed to identify the components of PAM as rhamnose,glucose,mannose,xylose and galactose at a ratio of 0.03:0.25:0.15:0.41:0.15.PAM was found to be metabolized in gastrointestinal tract when incubated with artificial gastric and intestinal juices.Anaerobic incubation of PAM with intestinal flora confirmed that PAM promoted the ability of intestinal bacteria to digest reducing sugar.Based on the Shannon′s index and similarity coefficient index of enterobacterial repetitive intergenic consensus-PCR（ERIC-PCR）fingerprinting of the total intestinal bacteria DNA,we concluded that PAM can significantly improve disordered intestinal flora and may be used as an oral adjuvant to regulate intestinal flora.Sijunzi decoction（SJZD）is a classical herbal prescription for curing spleen deficiency in traditional Chinese medicine（TCM）.Polysaccharides are the active ingredients of SJZD for its activities;however,the molecular mechanism is not clear yet.We investigated the active polysaccharide fraction from SJZD to explore the interaction among polysaccharides,intestinal microbiota,and host immune system.Three polysaccharides components（S-1,S-2,S-3）were obtained from SJZD with DEAE-52 cellulose chromatographic column,the effects of which on the immune system（organic,cellular and molecular levels）and intestinal microbiota,as well their metabolites（short chain fatty acids）were investigated using reserpine-treated wistar rat models.The spleen and thymus index of rats which usually measured as the immune organs indicators were calculated.The level of IL-2 and INF-γin intestine endothelial lymphocytes（IEL）of rat were determined by ELISA.The ratio between CD4+and CD8+in IEL（by FCM）was calculated as the cellular indicator.Moreover,the changes of intestinal microbiota were assessed by 16S rDNA PCR-DGGE,and the short chain fatty acids（SCFA）of rat faeces were analyzed by GC.The rats treated with reserpine,with flora-metabolites-immune disorders,shows similar signs to spleen deficiency,polysaccharides components S-3 from SJZD showed significant mitigating effect on the reserpine-induced flora-metabolites-immune disorders of spleen deficiency symptoms.The results revealed that the spleen index,IL-2 and IFN-g levels of S-3 group were significantly decreased compared to the model group,and the CD4+/CD8+ratio displayed a significant increase in the S-3 group,indicating the immune enhancing effect of polysaccharide fraction S-3.Moreover,the disturbance of gut microbiota induced by reserpine was restored after administration of polysaccharide fraction S-3,indicated as the Shannon’s diversity index and similarity coefficient index both increased significantly in S-3 group.In SCFA analysis,the content of acetic acid,propionic acid and butyric acid in faeces of S-3group rats was also significantly increased after treated with S-3.All the results consistently indicated that polysaccharide fraction S-3 from SJZD could mitigate effect in the reserpine-induced rats and potentially treat spleen deficiency.The comprehensive screening strategy established in this study may provide a new idea to achieve further understanding of TCM.Response surface method（RSM）was used to optimize the separation conditions of polysaccharides from the Sijunzi decoction.A three-level,three-variable Box–Behnken design（BBD）was applied for experimental design and analyzed of the results to obtain the optimal processing parameters.RSM analysis indicated good correspondence between experimental and predicted values.The optimal condition for the yield of polysaccharide was elution mode 0-0.2 M,0.3 M,sample quality 300 mg,flow rate 0.5 ml/min.Purification and structural characterization of SJZD polysaccharide were investigated and its activities were analyzed for the first time in this work.Results indicated that a single component polysaccharide with a molecular weight of 135 KDa was obtained after hot water extraction with sequential sevage deproteinization,DEAE-52 and Sephadex G-150 size-exclusion chromatography.The FTIR analysis showed that it had characteristic absorptive peaks.The methylation and GC-MS analysis showed that it comprised of Rha,Ara,Xyl,Man,Gal and Glc in molar ratio of 0.35:0.37:1.4:0.31:3:0.8 and that it mainly contained 1,3-linked-glc,1-linked-gal and 1,6-linked-gal glycosidic bonds.1H NMR and ¹³C NMR spectra analysis indicated that S-3-1 has a backbone of a-Glcp（1→4）-β-Galp（1→6）and that the side branches containα-Manp,β-Glcp,and terminal a-Araf,Rha,Glc residuces.AFM was used to observe the morphology of S-3-1 from SJZD.The determination of the anti-complement activities showed that it exhibited significant inhibitory effects on the classical pathway of the complement system,with IC₅₀ values of 530μg/mL.The S-3-1 were able to promote release of tumor necrosis factor alpha（TNF-α）and nitric oxide production.To investigate of metabolism of polysaccharides which were incubated in artificial gastric juice and artificial intestinal juice,and intestinal bacteria at different time points,DNS and phenol-sulfuric acid method were used to measure reducing sugar content and total sugar content changes.Monosaccharide composition changes were measured by GC-MS method.The results showed that the reducing sugar was hydrolysised from the polysaccharide in artificial gastric juice and artificial intestinal juice with the extension of incubation time.At 2 h,the reducing sugar content reached a steady state.Indicating that the polysaccharide has been metabolised both in gastric and intestinal juice.Investigation of polysaccharide in human intestinal bacteria anaerobic incubation medium,the results shown that the reducing sugar consumption curve and total sugar consumption curves were consistent.We sepeculated that metabolism in artificial gastric juice may be affected by the acidic environment of the incubation medium in artificial gastric juice,and the metabolism in the intestinal fluid may be affected by the role of the enzyme in the artificial intestinal juice.To Investigate the polysaccharides effect on metabolism of intestinal microflora and its structure,16S rDNA DGGE-PCR technology and illumina high-throughput sequencing technology were used to detect bacterial structure,GC method was used to detect intestinal microbial metabolism of fatty acids,pH changes was measured with pH meter.The results showed that,the intestinal bacteria metabolism of the acetic acid was promoted by polysaccharide;the pH in anaerobic Incubation fluid was decreased.Through the intestinal microflora study found that polysaccharide digested by gastric and intestinal,there is a better effect on the structure of intestinal flora.This can increase the growth of intestinal probiotics,such as faecalibacterium,bifidobacterium,megamona and parabacteroides.At the same time,with increasing of the metabolism of beneficial bacteria,such as acetic acid,propionic acid,butyric acid,the intestinal acidic environment were also improved.In addition to provide energy for the body,we speculated that polysaccharides could enhance the intestinal immune through metabolic acid of butyric stimulate the intestinal endothelial lymphocytes.