| Astragalus membranaceus increase its efficacy of Tonifying the middle and replenishing qi after honey roasting according to the theory of traditional Chinese medicine.Previous studies have shown the difference of pharmacodynamics between Astragalus membranaceus before and after roasting,which may bond to the change of chemical structure or content of the main components.Active polysaccharide is a very important water-soluble component in honey-processed Astragalus.It is not reported that whether the pharmacodynamics were improved due to chemical structure of polysaccharide was changed during honey-processed.This research preparaed and characterized honey-processed Astragalus membranaceus polysaccharide(HAPS).By evaluating the effect of HAPS and Astragalus membranaceus polysaccharide(APS)on DSS-induced colitis in mice,we explored the potential mechanism of its efficacy,and laid the foundation for the study of the processing mechanism of Astragalus.Methods: HAPS and APS were obtained by water extraction and ethanol precipitation,combining papain enzymatic hydrolysis with Sevage method to remove proteins,and then dialysis.HAPS3 a and APS3 a were purified by DEAE-52 column and HW-65 F column.The structures of HAPS3 a and APS3 a were characterized by chemical means(phenol sulfuric acid method,complete acid hydrolysis)and chromatography(HPLC-GFC-ELSD,UPLC/Q-TOF-MS),including sugar content,molecular weight,monosaccharide composition and proportion.This research established DSS-induced colitis model in mice,and given 200 mg/kg of HAPS,APS,HAPS3 a and APS3 a respectively.The anti-inflammatory activity was evaluated comprehensively from body weight change,colon length,colon tissue section and cytokine expression.The relative abundance of bacteria in feces of mice was determined by 16 S RNA sequencing combined with bioinformatics.The contents of acetic acid,propionic acid and butyric acid in feces of mice were determined by gas chromatography.UPLC/Q-TOF-MS was applied to detect the changes of serum metabolites in mice,and multivariate statistical analysis was used to identify potential biomarkers and analyze the related metabolic pathways.Results: The sugar contents of HAPS3 a and APS3 a were 89% and 87% respectively,and their molecular weights were 3,388 kDa and 2,781 kDa,respectively.The monosaccharide composition of HAPS3 a and APS3 a contain mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose/xylose and fucose,corresponding molar ratios were 0.16:3.26:0.12:0.73:0.43:6.67:12.93:0.49 and 0.17:3.88:0.20:4.92:0.72:3.62:12.88:0.40,respectively.The experimental results of anti-inflammatory activity showed that HAPS,APS,HAPS3 a and APS3 a interfered with DSS-induced colitis model in mice,which could slow down the decrease of body weight and spleen index,increase the length of colon,alleviate the damage of colon recess,reduce colon ulcer,and significantly reduce the expression of IL-1β.Among them,200 mg/kg HAPS3 a had better effect.HAPS3 a could decrease the ratio of abundance of Firmicutes to Bacteroides,and increase the relative abundance of Lactobacillus,but APS3 a decrease the relative abundance of Verrucomicrobia.Morover,HAPS3 a could increase the contents of total SCFA,acetic acid and propionic acid of colitis model in mice,APS3 a mainly increased the contents of total SCFA and butyric acid.Twenty-seven and twenty-five biomarkers of HAPS3 a and APS3 a which related to colitis in mice respectively.These biomarkers were mainly phosphatidylcholines,lysophosphatidic acid choline and arachidonic acid metabolic products.Through these potential biomarkers,we found that the metabolic pathways of HAPS3 a related to arachidonic acid metabolism,linoleic acid metabolism and glycerophosphatidic acid metabolism,APS3 a related to fatty acids,linoleic acid metabolism and glycerophosphatidic acid metabolism.Conclusion: In this study,millions of high molecular weight polysaccharides were isolated and purified from honey-processed Astragalus membranaceus and Astragalus membranaceus,and they are differente in molecular weight and monosaccharide composition.HAPS3 a and APS3 a may interfere with colitis mice by protecting intestinal mucosa,regulating the expression of cytokines and influence microbiota diversity.However,they effect different metabolic pathway in colitis mice.HAPS3 a mainly influence arachidonic acid metabolism and APS3 a influence fatty acid metabolism pathway.The differences in the chemical structure and anti-inflammatory activity of the active polysaccharides HAPS3 a and APS3 a may be related to the changes in the structure of polysaccharide of Astragalus after honey roasting. |
