| Dendrobium huoshanense C.Z.Tang et S.J.Cheng is a perennial plant of Orchidaceae family and has been consumed as a tonic with the potential of regulating gastrointestinal function.Polysaccharide is the main active ingredient of D.huoshanense.The cultivated D.huoshanense is one of the few Dendrobium plants approved for food raw materials.However,very little research has been conducted up to date on the structure and activity of polysaccharides from cultivated D.huoshanense.In the present study,the structure features of homogeneous polysaccharides from stem,root,leaf and flower of cultivated D.huoshanense(cDHPS,cDHPR,cDHPL and cDHPF)were systematically investigated by HPGPC,FT-IR,HPLC,GC-MS and NMR techniques;Then we employed MFC-bearing mice as an animal model to evaluate the anti-gastric cancer activity of cultivated D.huoshanense polysaccharides from the aspects of tumor apoptosis,angiogenesis and T lymphocyte immune response;Lastly,based on principal component analysis and multiple linear regression analysis,the relationship between chemical structure and anti-gastric cancer activity of cultivated D.huoshanense polysaccharides was explored.The main results were as follows:(1)The homogeneous polysaccharides were extracted from stem,root and leaf flower of cultivated D.huoshanense and the chemical structure characteristics of these four polysaccharides were elucidated.After water extraction,ethanol precipitation and?-amylase digestion as well as purification on anion-exchange chromatography and gel permeation chromatography,the homogeneous polysaccharides including cDHPS from stems,cDHPR from roots,cDHPL from leaves and cDHPF from flowers were obtained,respectively.The molecular weights of cDHPS,cDHPR,cDHPL and cDHPF were 2.59×105 Da,1.41×104 Da,2.09×105 Da and 4.78×105 Da and their monosaccharide compositions were D-Man:D-Glc=3.04:1.00,D-Man:D-Glc:D-Gal:L-Ara=2.38:1.00:8.49:5.23,D-Man:D-Glc:D-Gal=19.15:1.32:1.00 and D-Man:D-Glc:D-Gal=9.68:3.26:1.00,respectively.The glycosidic linkages of cDHPS were?4)-?-D-Glcp-(1?,?4)-?-D-Manp-(1? and?4)-3-O-acetyl-?-D-Manp-(1?;The glycosidic linkages of cDHPR were terminal ?-L-Araf,?4)-?-D-Manp-(1?,3,5)-?-L-Araf-(1?,?4,6)-?-D-Manp-(1?,?6)-?-D-Galp-(1?,?4)-?-D-Glcp-(1?and;The glycosidic linkages of cDHPL were-+4)-?-D-Glcp-(1?,?4)-?-D-Manp-(1?,?4)-3-O-acetyl-?-D-Manp-(1?,?3,6)-?-D-Manp-(1? and terminal ?-D-Galp;The glycosidic linkages of cDHPF were ?4)-?-D-Glcp-(1?,?4)-?-D-Manp-(1?,?3,6)-?-D-Manp-(1? and terminal ?-D-Galp.(2)The anti-gastric cancer activity of cDHPS,cDHPR,cDHPL and cDHPF were investigated by flow cytometry and qRT-PCR.Compared to cDHPL,cDHPF and cDHPR,orally administrated cDHPS showed the best anti-gastric cancer activity with the tumor inhibition rate of 74.19±0.83%.Anti-gastric cancer effect of cDHPS can be attributed to tumor apoptosis induced by up-regulating the expression of Bax,Caspase-9 and Caspase-3 gene and down-regulating the expression of Bcl-2 gene,tumor angiogenesis inhibition inducing by down-regulating the gene expression of VEGF and CD34 as well as the secrtion of VEGF,and the changed T-lymphocyte response induced by increasing the proportion of CD3+CD4+T cells and the ratio of CD4+T cells to CD8+T cells as well as decreasing the percentage of CD4+ CD25+ Foxp3+ Treg cells.(3)The relationship between structural characteristics and anti-gastric cancer activity of polysaccharides from cultivated D.huoshanense was established based on the partial enzymatic hydrolysis and deacetylation of polysaccharides.Through controlling the enzymatic hydrolysis time of the endo-?-1,4-mannanase,three fragments including fragments ?,? and ? were obtained from cDHPS.Meanwhile,the fragment? was obtained from cDHPS by removing the acetyl groups with alkali treatment.The relative molecular weights of fragments ?,?,? and ? were 1.08×105 Da,5.57×104 Da,5.76×103 Da and 2.58×05 Da,respectively.All these fragments consisted of D-Man and D-Glc(1.46:1.00 for fragment 1,1.63:1.00 for fragment ?,1.53:1.00 for fragment ? and 3.12:1.00 for fragment ?).The fragments ?,? and ? were composed of ?4)-3-O-acetyl-?-D-Manp-(1?,?4)-?-D-Glcp-(1? and?4)-?-D-Manp-(1? and while fragment ? was composed of ?4)-?-D-Glcp-(1? and?4)-?-D-Manp-(1?.The evaluation of anti-gastric cancer activity showed that the tumor inhibition rate of cDHPS and its different fragments was cDHPS>fragment I>fragment ?>fragment ?>fragment ?.Taking cDHPS,cDHPR,cDHPL and cDHPF as well as the fragments ?,?,? and ? from cDHPS as investigation objects,the chemical structure characteristics of polysaccharides and their anti-gastric cancer activity characteristics were correlated by principal component analysis and multiple linear regression analysis,and the obtained results revealed that acetyl group,relative molecular weight and glycosidic linkages of ?3,6)-?-D-Manp-(1? and?6)-?-D-Galp-(1? were the key chemical structure factor influencing anti-gastric cancer activity of polysaccharides from cultivated D.huoshanense. |
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