Studies On Structure Of Polysaccharides From Dendrobium Officinale And Bioactivities Of Its Sulfated Derivatives

Objective: Aims of this project were to investigate structure characteristics of polysaccharides from Dendrobium officinale Kimura et.Migo and bioactivities of its sulfated derivatives,and lay the theoretical and material foundation for further studies involving bioactivity material basis,relative quality control based on polysaccharides and innovative drug development based on Dendrobium polysaccharides and their derivatives.Methods: In this study,crude polysaccharides were obtained by boiling water-extracted and Na OH-extracted combined with ethanol precipitated methods.Then the crude polysaccharides were purified by α-amylase,anion-exchange and gel permeation chromatograph columns to obtain homogeneous polysaccharides.Chemical and physical methods were employed to elucidate the structure features of homogeneous polysaccharides,including monosaccharide analysis,partial hydrolysis,methylation analysis,IR and NMR analysis etc.Dendrobium officinale polysaccharides were sulfated by chlorosulfonic acid-pyridine(1: 3)method.The anti-angiogenesis bioactivity of polysaccharides was evaluated by tube formation assay and wound healing assay of HMEC-1 cells,and MTT assay was employed to evaluate the anti-tumor bioactivity as well as the cytotoxicity of polysaccharides and its derivatives on HMEC-1 and LO2 cells.Results:(1).304.0 g water-extracted(designated DOW,yield 20.3%)and 14.0 g alkali-extracted(designated DOA,yield 0.9%)crude polysaccharides were obtained from dried stems of Dendrobium officinale.(2).Two homogeneous polysaccharides(designated 5A and 5B)were isolated from DOW with an average molecular weight of 1.6 × 104 Da and 8.2 × 103 Da,respectively.The result of monosaccharides composition analysis indicated that 5A was consisted of glucose(Glc),mannose(Man),arabinose(Ara),galactose(Gal)and xylose(Gal)in a molar ratio of 55.7: 39.7: 1.4: 0.6: 2.6.Results of structure analysis suggested that 5A contained a backbone of 1,4-α-D-Glcp and 1,4-β-D-Manp with branches of terminal(T)-Xyl and T-Ara and T-D-Glcp with α-and β-configuration attached to the C-6 position of 1,4-α-D-Glcp.5B was composed of Glc,Man and Xyl with a molar ratio of 91.9: 6.5: 1.6.According to the structure analysis result,the backbone of 5B was consisted of 1,4-α-D-Glcp and a trace amount of 1,4-β-D-Manp with T-α-D-Glcp as branch attached at C-6 position of 1,4-α-D-Glcp.(3).Two homogeneous acidic heteroxylans designated S32 and S33S1 were purified from DOA.Their molecular weights(Mw)were 3.7 × 104 Da and 6.9 × 103 Da,respectively.The result of monosaccharides composition analysis showed that S32 was composed of Ara,Xyl,Glc,Gal,Rha and 4-methoxy-glucuronic acid(4-MGA)in a molar ratio of 8.9: 62.7: 8.5: 3.7: 3.9: 12.3.S33S1 contained Xyl,Ara,Glc and 4-MGA in a molar ratio of 6.1: 79.8: 1.2: 12.9.The structure analysis results indicated that both S32 and S33S1 contained a backbone of 1,4-β-D-Xylp with branches substituted to C-2 position of the backbone.The branches of S32 were composed of T-4-MeO-α-D-GlcAp,1,4-α-D-Glcp,1,3-α-L-Rhap,T-α-L-Araf,T-β-D-Galp and T-β-D-Xylp.S33S1 showed a simpler structure with less branches consisted of T-4-MeO-α-D-GlcAp,T-β-D-Xylp and T-α-L-Araf.(4).5B and S32 were modified by chlorosulfonic acid-pyridine method to achieve the sulfated derivatives(S5B and S32S),with an average Mw of 1.8 × 104 Da and 5.4 × 104 Da,respectively.According to the result of BaCl2-gelatin method,the substitution degrees(DS)of S5 B and S32 S were 0.92 and 0.90,respectively.The structure analysis of S5 B indicated that sulfuric group was substituted at C-6 position of 1,4-α-D-Glcp and T-α-D-Glcp,while the main substitution sites of S32 were determined to be C-2 or C-3 of 1,4-β-D-Xylp,C-3 of partial 1,2,4-β-D-Xylp,C-3 of T-4-MGA and C-6 of 1,4-α-D-Glcp.(5).The results of bioactivity tests suggested that native polysaccharides 5B and S32 had no significant effects on tube formation of HMEC-1 cell,while sulfated derivatives S5 B and S32 S could inhibit the tube formation and the migration of HMEC-1 cells at lower concentration(5.95 μM,0.29 μM),without significant cytotoxicity on HMEC-1 and LO2 cells even at 1 mg/m L(47.62 μM,18.52 μM).Conclusion:(1).Four kinds of homogeneous Dendrobium officinale polysaccharides were extracted,purified and characterized,while 5A and 5B were purified from water-extracted crude polysaccharide,S32 and S33S1 were from alkali-extracted crude polysaccharide.(2).5A and 5B were mannoglucan with a backbone contained 1,4-α-D-Glcp and 1,4-β-D-Manp.S32 and S33S1 were acidic heteroxylan with a backbone of 1,4-β-D-Xylp.(3).Sulfated polysaccharides S5 B and S32 S have anti-angiogensis ability,and the sulfuric group was essential to the bioactivities.