The Identification Of Structure And Characterization Of Immunoregulatory Polysaccharide And Antioxidative Peptides Isolated From Dendrobium Aphyllumin

As the second largest genera of the Orchidaceae family,Dendrobium genus is widely distributed throughout Asia,Europe,and Australia.In China,there are more than 78 species and 2 varieties,some of which show bioactivities such as immunomodulatory properties,antitumor activity,and antioxidative activity.In the current market,the most popular edible Dendrobium orchids,which improve functional bioactivities,are Dendrobium officinale and Dendrobium huoshanense.However,their weak reproductive ability and high cultivation standards increase the cost of Dendrobium products.Dendrobium aphyllum(Roxb.)C.E.C.Fisch with the advantages of strong adaptability,easier artificial planting,and high resistance to various pests and diseases,is much less expensive than the Dendrobium products described above.Although these advantages indicate the economic benefits of D.aphyllum,only few studies have examined this species in detail.In order to enrich the study and increase the value of D.aphyllum,in gernal one immuoregulatory polysaccharide and antioxidative peptide were isolated from of D.aphyllum respectively.Firstly,D.aphyllum crude polysaccharide(cDAP,38.15±0.20% yield)extract was generated.The D.aphyllum polysaccharide(DAP,Mw 471.586 k Da),purified by DEAE Sepharose and Sephadex-G200 Fast Flow,was composed of mannose(71.3%)and glucose(28.7%).Its backbone was ?-D-mannopyranose and ?-D-glucopyranose residues and its glucosidic bond was 1,4-linked.DAP treatment enhanced cytokine secretion,and pinocytic capacity of RAW 264.7 mouse macrophages.The receptors of DAP on RAW 264.7 cells were found to be complement receptor 3 and mannose receptor,indicating that the Akt/mTOR/MAPK and IKK/nuclear factor-?B pathways are involved in DAP-activated immunomodulation.In vitro salivary and gastrointestinal digestion and fermentation of DAP were investigated in this study.Salivary amylase showed no influence on DAP.Gastrointestinal digestion were able to destory the spatial stucture of DAP,in which intestinal digestion could breakdown the primary stucture of DAP and therefore resulted in the increasing production of reducing sugars.In terms of the fermentation pattern,the pH values dropped significantly due to the formation of six types of SCFAs;Three types of carbohydrates,including Dendrobium officinale polysaccharide,and ?-glucans from yeast and barley,were examined and their structures were found to mainly contain 1,4-linked-?-D-Glcp.Artificially simulated gastrointestinal digestion was conducted to characterize the changes of stucture,which indicated that high-molecular weight and complex spatial structures contributed to delayed monosaccharide release following exposure to digestive solution.Additionally,for the developed 7890A/7000 GC-TQ/MS-MS,the new analytical method was successfully used to analyze very low concentrations of monosaccharides in the simulated gastrointestinal digestive system.Antioxidative peptides DA-PE were extracted from Dendrobium aphyllum.The Lactobacillus strain was identified as Lactobacillus amylolyticus L6.Analysis of amino acid composition and HPLC-MS/MS analysis indicated high antioxidative potential of the low molecular weight DA-PE fractions(DA-P),which contained 21 different types of polypeptides,as determined through chemical antioxidative assay.The cellular antioxidant activity of DA-P in HepG2 cells was used and the resultsrevealed an EC50 of 2.88 ± 0.143 mg/m L and a CAA unit of 63.46 ± 2.11 ?M QE/100 g peptides.Human erythrocytes subjected to an 2,2'-azobis [2-methylpropionamidine] dihydrochloride(AAPH)-induced hemolysis assay using the DA-P quenched the intracellular antioxidant system by inhibiting the generation of reactive oxygen species and malondialdehyde and regulating activity of cellular antioxidative enzymes,including superoxide dismutase,catalase,and glutathione peroxidase.Moreover,DA-P treatment enhanced the secretion of cytokines(nitric oxide,interleukin-6,and tumor necrosis factor-?)in RAW 264.7 mouse macrophages.An antioxidative peptide extracted from DA-P was used as a substrate for artificial stimulated in vitro gastrointestinal digestion and human feces-mediated fermentation.Reducing power,DPPH free radical-scavenging effects and ABTS free radical-scavenging effects of the peptide were improved after in vitro digestion.During fermentation process,DA-P could decrease the pH value and stimulated the mechanism of intestinal microbiota.In Caco-2 cell model,after demonstrating the presence of tight junctions in Caco-2 monolayers,the absorption was 25.57 ± 0.016% and 19.7 ± 0.012% from the different sides.Free amino acids and LC-MS/MS analysis indicated the degradation and expulsion of components after the absorption of DA-P.