Preparation Of Polysaccharides From Polygonati Rhizoma And Its Effects On Intestine And Lung In Aged Mice

Polygonati rhizoma polysaccharide is one of the main active substances of Polygonati Rhizoma,which has a wide range of pharmacological activities.The 《 Chinese Pharmacopoeia》 2020 edition has three sources of medicinal P.rhizoma,Polygonatum sibiricum Red.,P.cyrtonema Hua,and P.kingianum.The Pharmacopoeia uses total polysaccharides to control the quality of P.rhizoma,but the differences in polysaccharide composition and activity of the three types of Polygonatum have not been systematically studied.In this experiment,the polysaccharide content and component differences of the three medicinal P.rhizoma were compared by the same extraction,separation,and purification methods;and the antioxidant and anti-inflammatory activities of the purified polysaccharides of different varieties in vitro were compared.PSP-NP-Ι was chosen to do the further study for its best antioxidant effect and best yield among three polysaccharides.Then P.sibiricum polysaccharide PSP-NP-Ι effect on intestinal and lung function in vivo was studied using aged mice as model,and its mechanism and structure was analyzed.This research provided a reference for the quality evaluation of P.rhizoma,layed the foundation for the further development and utilization of P.rhizoma polysaccharides,provided a natural medicine for the treatment of intestinal and lung diseases,and provided a basis for the structure-activity relationship of P.rhizoma polysaccharides.1 Extraction,separation,purification and component comparison of three different varieties of P.rhizomaAfter removing impurities by 95% ethanol and 50% ethanol,the crude polysaccharides of three varieties of P.rhizoma were extracted by water extraction,and the contents of polysaccharides,proteins,and polyphenols in three crude polysaccharides were determined by the phenol sulfuric acid method,the Coomassie blue method,and the fulvic phenol method.The crude polysaccharides were separated and purified by DEAE-agarose gel and agarose gel 6FF chromatography columns,then purified neutral polysaccharides and acid polysaccharides was obtained,which were precisely weight and the yield was calculated.The results showed that a crude polysaccharide from P.sibiricum was obtained,and named PSP,showing a loose flocculent shape,which was identified composing with 65.83%polysaccharide,20.24% poluphenol;a crude polysaccharide from P.cyrtonema was obtained,and named PCP,showing a loose foam shape,which was identified composing with 74.02%polysaccharide,24.83% poluphenol;a crude polysaccharide from P.kingianum was obtained,and named PKP,dense plate-like structure,which was identified composing with 37.47%polysaccharide,25.47% poluphenol.PSP was most consist of neutral polysaccharides accounting for 70.50%.P.cyrtonema crude polysaccharides were mainly acid polysaccharides,accounting for 64.67%.The content S of neutral and acid polysaccharides of P.kingianum is not much different,neutral polysaccharides accounting for 41.75% and acid polysaccharides 58.25%.After further purification,one neutral(PSP-NP-Ι)and acidic(PSP-Ι-Ι)polysaccharide was obtained from PSP.One neutral(PCP-NP-Ι)and acidic(PCP-Ι-Ι)polysaccharide was obtained from PCP.One neutral(PKP-NP-Ι)and acidic(PKP-Ι-Ι)polysaccharide was obtained from PKP.2 Antioxidant and anti-inflammatory activities of different P.rhizoma purified polysaccharides on porcine small intestinal epithelial cells in vitroThe effects of 4 purified polysaccharides on the gene expression of SOD1 and IL-6 in pig small intestinal epithelial cells were detected by qRT-PCR.The results showed that PSP-NP-Ι had a certain inhibitory effect on the expression of IL-6 gene,and significantly increased the expression of SOD1 gene(P <0.01).PSP-Ι-Ιpossessed no significant effect on IL-6 gene(P >0.05),but significantly raised the expression of SOD1 gene(P <0.01).PCP-Ι-Ι significantly reduced the expression of IL-6 gene in cells(P <0.01),but suppressed the expression of SOD1 gene in cells(P <0.05);PKP-Ι-Ιsignificantly reduced the expression of IL-6 gene in cells(P <0.01),however,there was no significant change in SOD1 gene expression(P >0.05).3 Effects of P.sibiricum polysaccharides PSP-NP-Ι on intestinal and lung of aged miceSixty 10-month-old male SPF C57 BL / 6Cnc mice were divided into 4 groups of 16 mice each.Dosing groups were separated as high,medium,and low-dose groups,and the polysaccharide PSP-NP-Ι was orally administered,the dose was 200 mg/kg/d,100 mg/kg/d,50 mg/kg/d,and the control group was given saline.Samples were taken 45 days after the administration to observe its effects on different intestinal segments and lungs of aged mice.1.Effect on organ index and histopathology: Observed the changes of lung and brain organ indexes;HE staining was used to observe the pathological changes of jejunum,ileum,colon and lung tissues,and pathological damage score was assessed.2.Effect on the mucosal barrier: Counted goblet cells in different intestinal segments by AB-PAS staining;ELISA was used to determine the LPS content in serum;qRT-PCR analysis was performed to detect Mucin2,tight junction protein ZO-1,Occludin gene expression in different intestinal segments and lung.3.Effect on immune indicators: The effect of sIgA content in different intestinal tissues and lungs was determined by ELISA.4.Effect on oxidative stress indicators:The content of carbonyl protein content in different intestinal tissues and lungs was determined by ELISA.5.Effect on intestinal microbe: Preliminarily analyzed intestinal microbe changes through bacterial identification culturing of cecum contents,and detection of short-chain fatty acid content in cecum contents by gas chromatography.6.Effects on brain-gut peptides: The gene expression of neuropeptide P(SP),vasoactive intestinal peptide(VIP),and calcitonin-related gene peptide(CGRP)in different intestinal segments and lungs were analyzed by qRT-PCR analysis.7.Effect on inflammatory cytokines: Gene expression of interleukins 1β,4,6,10,12 A,12B,17,23,and tumor necrosis factor α(TNF-α)and transforming growth factor β(TGF-β)in different intestinal segments and lungs were detected by qRT-PCR analysis.The results showed that: 1.Effects on organ index and histopathology: PSP-NP-Ι lowdose group significantly increased lung organ index(P <0.05);pathological changes of jejunum,ileum,colon,and lung tissue significantly improved after administration(P <0.05).2.Effect on the mucosal barrier: jejunal goblet cells were significantly increased after drug administration(P <0.01),while ileal goblet cells increased as well;The serum LPS content was significantly reduced(P <0.05),and in a dose dependence manner;PSP-NP-Ι could significantly increase the expression of Muc2 gene in the jejunum,ileum and lung tissues(P<0.01),but no significant change in the colon(P> 0.05).The administration group significantly increased the expression of ZO-1 gene in the jejunum,ileum and lung(P <0.01).Occludin gene expression was significantly up-regulated in the lung and different intestinal segments(P <0.01).3.Impact on immune indicators: PSP-NP-Ι can significantly increase the sIgA content in the jejunum,ileum and lung(P <0.01),and the effects of the medium and low dose groups are significantly better than those of the high dose group.4.Effects on oxidative stress indicators: PSP-NP-Ι significantly reduced the content of carbonylated proteins in the jejunum,colon,and lungs(P <0.01).5.Effect on intestinal microbe: PSPNP-Ι can significantly increase the number of Bifidobacteria(P <0.05),and showed a dosedependent manner;it could increase the number of Lactobacillus,abd reduce the number of Escherichia coli.The contents of acetic acid,propionic acid,and butyric acid were significantly higher compared with the control group.6.Effect on brain-gut peptides: SP and VIP gene expression in different parts of intestine and lung were significantly inhibited(P<0.05).And PSP-NP-Ι had a significant upregulation effect on CGRP gene expression(P<0.01).7.Effect on inflammatory cytokines: PSP-NP-Ι had significant inhibitory effects on gene expression of pro-inflammatory factors including IL-12 A,IL-12 B,IL-23,IL-17,IL-6,IL-1β and TNF-α expression(P <0.05),but the activity in different parts is significantly different;PSP-NP-Ι could significantly promoted the gene expression of anti-inflammatory factors including IL-4,IL-10 and TGF-β(P <0.05),and the gene expression in different parts of different doses had significant differences.4 Structure determination of P.sibiricum polysaccharide PSP-NP-ΙFourier infrared spectroscopy(FT-IR)was used to analyze the functional groups contained in PSP-NP-Ι;its molecular weight was measured by gel column chromatography;monosaccharide compositions were determined by gas chromatography(GC);Chromatography(GC-MS)and nuclear magnetic resonance(NMR)detection was sued to study the glycosidic bond composition and connection type,then clarify the basic structure of PSP-NP-Ι by the information obtained above.The results showed that PSP-NP-Ι contained O-H,C-H,C=O and C-O,with a molecular weight of 43.0 k Da,and consist of a large amount of galactose,mannose and a small amount of glucose in a ratio of 68.7:24.3:7.0.It’s a neutral heteropolysaccharide composed of 1,4-β-D-Gal;1,4,6-β-D-Gal;T-α-D-Man;1,4-α-D-Glc and T-α-D-Glc.In summary,this experiment systematically compared the yield and composition of polysaccharides from three different varieties.And compared their anti-inflammatory and antioxidant activities in vitro.Obtained the PSP-NP-Ι with the best antioxidant activity and highest yield,which could change the pathological changes of the intestine and lung tissue in old mice,could improve the intestinal permeability by increasing the number of goblet cells and the expression of mucin and tight junction protein genes,increase sIgA content in intestine and lung,reduce intestinal and lung carbonyl protein content,and could increase the quantity of Bifidobacterium and increase short-chain fatty acid content in caecum,and also regulate the movement and inflammation in intestine and lung tissue by affecting the gene expression of the neuropeptides and inflammatory factor.PSP-NP-Ι is a neutral heteropolysaccharide composed of 1,4-β-D-Gal;1,4,6-β-D-Gal;T-α-D-Man;1,4-α-DGlc and T-α-D-Glc with a molecular weight of 43.0 kDa.