Study On The Structure-function Relationship Of Polysaccharides From Different Parts Of Angelica Sinensis And Their Protective Mechanism Against Colitis

As one of the main active components of Angelica sinensis(Oliv.)Diels,the polysaccharide has various biological activities such as immunoregulatory,antiinflammatory,antioxidant and anti-tumor effects.The roots of A.sinensis with four different parts are often used separately in Traditional Chinese Medicine,including head,body,tail and the whole body due to various pharmacological activities and components.However,less attention was paid to the untraditionally-used plant parts of A.sinensis,such as aboveground parts.The present studies primarily compared the content and bioactivity differences of its fat-soluble components,such as volatile oil and ferulic acid.However,there is a lack of research on the differences of its watersoluble component,especially polysaccharide.Based on this,A.sinensis was divided into five different plant parts: head,body,tail,whole body,and the aboveground part.Their crude polysaccharides were extracted by water extraction and ethanol precipitation method,and further purified.The structures of purified polysaccharides were characterized and analyzed.Combined with the LPS-induced IPEC-J2 cell inflammation model in vitro,the structure-activity relationship of polysaccharides from different part of A.sinensis was preliminarily analyzed.Furthermore,a polysaccharide fraction with the highest bioactivity and low price was selected to explore its preventive and protective effects on DSS-induced ulcerative colitis in mice.The relationship between serum metabolites,inflammatory factors and intestinal microbiota were analyzed and a metabolite with the greatest significance was chose to verify its antiinflammatory effect both in vitro and in vivo.1.Extraction,separation and purification of five acidic polysaccharides from different plant parts of Angelica sinensisThe crude polysaccharides from different parts of A.sinensis were extracted by water extraction and ethanol precipitation method.They were further purified by DEAE chromatography and gel filtration,and five fractions were obtained including the acid polysaccharide from the head of A.sinensis(ASP-H-AP),the acid polysaccharide from the body of A.sinensis(ASP-B-AP),the acid polysaccharide from the tail of A.sinensis(ASP-T-AP),the acid polysaccharide from the whole body of A.sinensis(ASP-Hb-AP),and the acid polysaccharide from the aboveground parts of A.sinensis(ASP-Ag-AP)with the yield of 24.0%,36.7%,47.1%,25.9%,and 28.6%,respectively.The results showed that the five polysaccharide fractions were homogeneous and the yield of polysaccharides from different parts of A.sinensis were significantly various.The yield of ASP-T-AP was the highest while that of ASP-H-AP was the lowest.2.The comparison of structures and anti-inflammatory effects of ASPsThe comparison of ASPs from different root parts were performed,including chemical compositions determined by colorimetric analysis,monosaccharide compositions measured by high performance liquid chromatography(HPLC),glycosidic linkage units determined by methylation and gas chromatography-mass spectrometry(GC-MS),organic functional groups determined by FT-IR,weightaverage molecular weight(Mw)demarcated by gel permeation chromatography(GPC),and the viscosities and solubilities.The anti-inflammatory and protective effects from oxidative stress indued by lipopolysaccharide(LPS)on IPEC-J2 cells of APSs were compared in vitro.The results showed that the Mw of polysaccharides from different parts of A.sinensis varied greatly,and the trends of viscosity were consistent in the trends of Mw.At the same time,structural domains belonging to pectin polysaccharides were found in all five acidic polysaccharides,including arabinogalactan II(AG-II),homogalacturonan(HG)and Rhamnalacturonan I(RG I).As pectin polysaccharides,ASPs possessed great anti-inflammatory activities,and their differences could be resulted from the structural diversity.The order of activity is: ASP-H-AP> ASP-Ag-AP = ASP-Hb-AP > ASP-B-AP>ASP-T-AP.Due to the economical and widely available sources of the stems and leaves of A.sinensis,and their acidic polysaccharide component ASP-Ag-AP also with high anti-inflammatory activity,this fraction would be further studied on the preventive and protective effects on DSS-induced ulcerative colitis.3.A.sinensis aboveground part polysaccharide ameliorates colitis via modulating gut microbiota,serum metabolite and TLR4/My D88/NF-κB pathwayThe ulcerative colitis model was induced by adding DSS(2.5%,m/v)in drinking water for 7 days.The blank control group,model group,positive drug group(Mesalazine,96 mg/kg)and different dosages of ASP-Ag-AP(50,100 and 150 mg/kg)were designed.All the mice were sacrificed on day 14;the colon tissues were collected for H&E section,inflammatory factors determination(RT-q PCR,ELISA)and signing pathway detection(RT-q PCR);serum was gathered for inflammatory factors,MPO and LPS determination(ELISA);the cecum contents were collected for SCFAs analysis(GC).The results showed that ASP-Ag-AP was shown with the protective effect on DSS-induced ulcerative colitis in mice,and could inhibit the inflammatory factors IL-6,IL-1 β and TNF-α in serum and colon,reduce the increased LPS content in serum and the increased MPO content in colon.Using RT-q PCR verification,TLR4/My D88/NF-κB pathway were shown to be inhibited by ASP-Ag-AP,and the secretion of inflammatory factors were also reduced resulting in an alleviation of ulcerative colitis in mice.4.ASP-Ag-AP ameliorates colitis via modulating gut microbiota and serum metaboliteFollow the above experiment,serum was gathered for metabolites detection(LCMS);the faeces were collected for intestinal analysis(16s DNA determination).The relationship between metabolites,intestinal flora and inflammatory factors was analyzed by Spearman correlation to screen a potential biomarker of ulcerative colitis.Non-targeted metabolomics analysis indicated that colitis caused the disorder of serum metabolic profile in mice,and 27 metabolites were effectively recovered after oral prevention of ASP-Ag-AP.The 16 s r DNA sequencing analysis demonstrated that colitis disrupted the composition of intestinal microbiota,while ASP-Ag-AP could regulate the structure of gut bacteria,including increase the abundance beneficial bacteria,Lactobacillus,and reduce the harmful bacteria Bacteroides,Streptococcus,Faecalibacterium,Staphylococcus,Alistipe.The concentrations of SCFAs in the cecum of colitis mice were found be increased by ASP-Ag-AP.Finally,the Spearman correlation analysis among metabolites,bacteria and inflammatory factors was performed,and it is concluded that 5-methyl-DL-tryptophan(5-MT)had negative relationship with inflammatory factors and intestinal bacteria,Bacteroides,Streptococcus,Alistipes,thus 5-MT might be able to alleviate inflammation by modulating the gut microbiota.5.5-MT,the metabolite of ASP-Ag-AP,protected DSS-induced colitis and LPSinduced IPEC-J2 cell inflammationThe ulcerative colitis was induced by oral administration of 2.5% DSS for 7 days as shown above.Blank control group(normal saline),model group(normal saline),ASP-Ag-AP group(ASP-Ag-AP,150 mg/kg),and 5-MT group(5-MT,30 mg/kg)were designed.All the mice were sacrificed on day 14;the colon tissues were collected for H&E section,inflammatory factors determination(RT-q PCR,ELISA)and signaling pathway detection(RT-q PCR);the serum was gathered for inflammatory factors,MPO and LPS determination(ELISA);the faeces were collected for analysis of intestinal flora(16s RNA determination).IPEC-J2 cells inflammation induced by LPS were employed in in-vitro experiments,and the effects on inflammatory factors and signaling pathway were determined by RT-q PCR.The results showed that 5-MT and ASP-AgAP were shown to have anti-inflammatory effects both in vivo and in vitro,and could reduce inflammatory factors by inhibiting TLR4/My D88/NF-κB pathway.In addition,in vivo,5-MT can effectively alleviate the bacteria disorder caused by colitis,reduce the abundance of harmful bacteria Alistipes,Murbaculaceae and Alloprevotella,and increase the abundance of beneficial bacteria Akkermansia and Lactobacillaceae.To sum up,all ASPs are homogeneous pectin polysaccharides with potent antiinflammatory activity in vitro,and the activity varies from different structures.ASPAg-AP is one of the five active polysaccharides with the best activity in vitro and economical profits with high yield.Therefore,animal experiments were performed to explore its protective effect on DSS-induced colitis.It can be concluded that ASP-AgAP and its metabolite 5-MT can inhibit TLR4/My D88/NF-κB pathway and regulate intestinal microbiota to alleviate inflammation,providing a theoretical basis for its further development and utilization.