The Effect Of Molecular Weight And Molecular Modification On Immunoregulatory Activity Of Dendrobium Officinale Polysaccharide

Dendrobium officinale belongs to the perennial adnascent herbaceous plant of Dendrobium Orchidaceae,and was listed as top grade among Dendrobium species.Dendrobium officinale is a kind of “drug homologous food” with a variety of medicinal values.A large number of studies have shown that Dendrobium officinale polysaccharide has various functions,such as anti-tumor,immuno-stimulating activities,antioxidant,promote the health of intestine.With the progress of research,it was found that the biological activity of polysaccharides was related to its structure.Currently,there are a lot of researches on the structure and bioactivity of Dendrobium officinale polysaccharides.However,there are few studies on its structure-activity relationship.Therefore,in this study,the Dendrobium officinale polysaccharides with different molecular weight and modified method were obtained by gradient alcohol precipitation,enzymatic hydrolysis,sulfation,carboxymethylation and deacetylation.The immunoregulatory activity of Dendrobium officinale polysaccharide was studied at the whole,cell and molecular level in vitro and in vivo by the modern molecular biology method,and the relationship between structure and immunological activity was discussed.The main research contents were summarized as follows:1.Study on preparation and physicochemical properties of different Dendrobium officinale polysaccharides.The results showed that DOP was neutral sugar with molecular weight of 262.4 Kda.With the increase of ethanol concentration,the molecular weight and the intrinsic viscosity of F30,F40,F50 and F50 S were decreased while Mw/Mn increased gradually.All components were homogeneous polysaccharide,and were mainly composed by mannose and glucose with the molar ratio of mannose to glucose at 5.16:1,3.67:1,4.16:1,5.62:1 and 5.86:1,respectively,in which F50 S component also contains a small amount of galactose.The results of IR spectroscopy showed that SDOP,DADOP and CMDOP which prepared by sulfation,deacetylation and carboxymethylation of Dendrobium officinale polysaccharide were successfully prepared with the degree of substitution at 1.13,0.07 and 0.52,respectively.And the acetyl substitution of DOP and HDOP that hydrolyzed by DOP were 0.29 and 0.79,respectively.SDOP,DADOP and HDOP were single peak,while CMDOP had three peaks,indicating that sulfation,deacetylation and mannanase enzymatic hydrolysis had no significant effect on the homogeneity of polysaccharides,while carboxymethylation had a great effect.SDOP,CMDOP and HDOP molecular weight were decreased,while DADOP molecular weight was increased,and the intrinsic viscosity of all samples were decreased.2.To assess the immunoregulatory activity of different Dendrobium officinale polysaccharides in vitro,murine macrophage cell line RAW264.7 was used as cell model.The difference of immunoregulatory activity between different components were also compared,and discussed the relationship of structure and activity.The results showed that all samples,besides CMDOP,had significant effect on the proliferation,phagocytosis,NO release and TNF-? and IL-1? secretion of RAW264.7 macrophages,indicating that they all had good immunoregulatory activity.Compared with DOP,the immunoregulatory activity of F30,F40 and F50 were weaker,while F50 S was stronger.It may be due to the F50 S has lower molecular weight and intrinsic viscosity;the F30 has higher molecular weight and intrinsic viscosity which was inhibit the immunoregulatory active;the F40 and F50,although the molecular weight is lower than DOP,but the intrinsic viscosity and ratio of Man/Glc were different,resulting in their immunoregulatory activity were different.Sulfation and Deacetylation can enhance the immunoregulatory activity of DOP,the mannanase enzymatic hydrolysis had no significant change,while the carboxymethylation significantly inhibited the immunoregulatory activity of DOP.3.To verify the immunoregulatory activity of different Dendrobium officinale polysaccharides in vivo,immunosuppressive mice was used as animal model,and its regulation on intestinal health was also studied.The results showed that both DOP and F50 S could significantly increase the thymus index,promote the secretion of serum and spleen cytokines,upregulate the gene expression of T-bet and Gata-3 and their ratio in spleen,regulate the balance of Th1/Th2 shift to Th1 type immune.Moreover,the fecal moisture content,pH value and SCFA concentration level in intestinal were also significantly increased,indicating that DOP and F50 S could had positive effect on the immune function and intestinal health of cyclophosphamide-induced immunosuppressive mice.F30,F40,F50,SDOP and DADOP had some positive effect on improving the cyclophosphamide-induced mice immunosuppressive and intestinal health.Compared with the previous in vitro results,we could found that the immune activity of F30,F40,F50 in vivo were consistent in vitro,and all weaker than DOP,indicating that Dendrobium officinale polysaccharides immune activity were related to molecular weight,intrinsic viscosity,the ratio of composition,and was effected together by a variety of factors.While F50 S,SDOP and DADOP were different in vivo and in vitro which could be due to the different ways.The digestion,absorbtion and hydrolysis of different polysaccharides in gastrointestinal were different,and result in the different microenvironment of intestinal,than led to the difference of immune response and showed different immunoregulatory activity.