| Opuntia Milpa Alta is the edibable Opuntia ficus indica variety belonging to the Cactaceae family.The objectives of the current project were to extract,isolate and purify the polysaccharides with anticoagulant activity from O.ficus indica,and to study the structure and functional properties of the bioactive polysaccharide.Chemical composition analysis revealed that Opuntia Milpa Alta contained 0.73%rude protein,0.17%crude fat,0.11%reducing sugar,0.86%polysaccharide,0.88%ash,95.7% moisture,0.77%crude fiber.Size distribution and features in FT-IR were compared between polysaccharides by aqueous extraction and polysaccharides by homogenize-assisted aqueous extraction,which indicated no significant.The intrinsic viscosity of polysaccharides by homogenize-assisted aqueous extraction reduced 34%.Hence,homogenize-assisted aqueous extraction was used to obtain the crude polysaccharides and the extraction procedure was optimized using Box-Behnken design and Surface Response Methodology(RSM).The obtained optimal parameters of the extraction procedure of CP were:temperature 86.1℃, time 3.61 h,and water to solid ratio 3.72,CP yield 0.694%.WSP1,WSP2a & WSP3 fraction were obtained from the crude polysaccharides(CP) following DEAE-Sepharose CL-6B and Sepharose CL-6B.The three fractions were identified by gel filtration chromatography on Sepharose CL-6B column and HPSEC as homogeneous in molecular weight,their molecular weights were 2.32x106Da,1.26x106 Da,and 7.92x106 Da,respectively.WSP2a consisted mainly of arabinose,galactose,xylose,rhamnose,along with low amount of mannose and glucose,was regard as a rhalnnogalacturonan confirmed by proportion of monosaccharide and FT-IR spectra.The degree of esterification(DE) value of WSP2a was 41.9%based on a FT-IR method.Structure characterization of WSP2a was carried out using partial acid hydrolysis and methylation analysis,combined with GC-MS and 1D & 2D NMR spectroscopy techniques. The results indicated that the configuration of sugar constituents were mainlyα-D-GalpA,β-L-Rhap,α-D-Galp,α-L-Arap andα-L-Xylf.The backbone of WSP2a-28H was found to consisting of a→4)-β-D-GalpA(1→2,4)-α-L-Rhap(1→.The galactose side-chain are 1,4-1inked and are attached mainly to C-4 of the rhamnose units in the backbone.The other side-chain consisting of two 1,6-β-linked galactosyl residues,attached to the backbone by the linkages of 1,4-D-Galp via the O-4 position of the rhamnose residues.T-L-Arafor T-L-Xylf were situated as the nonreducing terminals,which were linked to the O-3 position of the 1,6-D-Galp units.Through AFM observations of CP and WSP2a,it was found that CP and WSP2a have strand-like structure,with diameter about 150~-200 nm,10-40 nm,respectively,average length of CP and WSP2a was respectively about 0.7-1.5 gm,0.05-0.7 gm.Actived partial thromboplastin time(APTT),Prothrombin time(PT) amd Thrombin time (TT) were chosen as screening targets for Anticoagulation activity of CP,WSP1,WSP2a,and WSP3 in vitro,and APTT of WSP2a was prolonged remarkably comparing with normal saline. APTT and TT of WSP2 were prolonged while PT did not prolonged in vivo.The results showed that WSP2 with anticoagulant activity by way of intrinsic pathway and common pathway.Compared with positive control,WSP2 in combination with fibrinogen and ATⅢ, but did not like heparin and prompted AT thrombinⅢrapidly,resulted in fibrinogen inverting Thrombin slowly.
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