| Dendrobium officinale is a traditional and valuable Chinese herb,and polysaccharides are considered as its main active components which have been widely reported to possess a variety of bioactivities.However,due to the lack of detection methods,there is a relative lack of research on the in vivo pharmacokinetic characteristics of polysaccharides,which limits the exploitation of polysaccharides.Therefore,in this study,Dendrobium officinale polysaccharide(DOP)was characterized and labeled with two different fluorescein,and we further established the methodology to investigate the pharmacokinetic behavior and mechanism of DOP after oral administration by various means.The highly purified polysaccharide DOP was obtained by water extraction-alcohol precipitation,repeated freeze-thawing,microfiltration,and ultrafiltration.DOP was identified as a typical acetylated glucomannan with a molecular weight of 141.2 k Da,which was composed of glucose and mannose in the molar ratio of 1.00l: 4.44,and consisted of(1→4)-β-D-Glcp and(1→4)-β-D-Manp with some attached 2-O-acetylated groups.The absorption and distribution behavior of fluorescently labeled DOP after oral administration was investigated,and the results showed that DOP was stable in the in vitro simulated digestive fluid and exerted no significant toxicity on Caco-2 cells.After oral administration,DOP was hardly absorbed directly by the body in its prototype form;rather,it moved with the intestinal contents to the distal part of the intestine and degraded into a homogeneous oligosaccharide substance,where it remained for a long time and performed metabolic activities.Therefore,the in vivo metabolic behavior and mechanism of DOP will be further investigated.The specific metabolic mechanism of DOP in the intestine was analyzed by metagenomics combined with 16 S r RNA sequencing.The results showed that DOP could stimulate the expression of polysaccharide metabolism-related genes in the intestine of mice after oral administration,and then increase the synthesis of polysaccharide metabolismrelated enzymes such as glycoside hydrolases,and the absence of Bacteroides was found to prevent DOP from being metabolized into stable homogeneous oligosaccharide-like metabolites in the distal part of the intestine by establishing a mouse model for the targeted clearance of Bacteroides,suggesting that the in vivo metabolic behavior of DOP may be closely related to endo-β,1-4-mannanasee expressed mainly by Bacteroides.In vitro enzymatic digestion of DOP by three enzyme systems selected according to its structural characteristics further confirmed that endo-β,1-4-mannanase was able to degrade DOP into an oligosaccharide-like fragment(EDOP)similar to the in vivo metabolite,which was further employed to investigate whether EDOP is the core active domain of DOP by structural characterization and bioactivities comparison by the dextran sodium sulfateinduced ulcerative colitis model.The results showed that EDOP is a class of acetylated glucomannan-like oligosaccharide fractions with molecular weights in the 2000-3000 Da region,consisting of(1→4)-β-D-Glcp and(1→4)-β-D-Manp with some attached 2-Oacetylated groups,which are highly similar to the in vivo metabolites of DOP,and both DOP and EDOP could dramatically attenuate the clinical signs via blocking pro-inflammatory cytokines(TNF-α,IL-6,IL-1β,and their related m RNA),restoring the levels of short-chain fatty acids(SCFAs),activating the G-protein coupled receptors(GPRs)and modulating the gut microbiota.In addition,EDOP was labeled and the study of the absorption and metabolism characteristics of EDOP showed that DOP was most likely to be recognized by Bacteroides after oral administration and degraded into an oligosaccharide-like fraction similar to EDOP by endo-β,1-4-mannanase,part of which can be directly absorbed and utilized by intestinal tissues,while the other part is further metabolized by other gut microbiota into SCFAs or other metabolites and released in intestine. |
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