Purification And Structure Research Of Anticomplementary Polysaccharides From Lasiosphaera Fenzlii Reich

The complement system plays an important role in immune system against infection and the clearance of antigen-antibody complexes from the blood. However,when activated inappropriately, the complement system could cause a variety of diseases such as system lupus erythematosus, rheumatoid arthritis, and acute respiratory distress syndrome. With increasing in our understanding of complement activation that involved in the diversified pathological progresses, the inhibition of complement activation in these diseases or surgical applications would likely have significant benefits. Numerous naturally occurring agents have been found to have anti-complementary effects, and they offer a prospect of inexpensive and non-toxic strategies for the therapeutic inhibition of complement. By the bioassay guided isolation, homogeneous polysaccharides was obtained, and structural investigation and anti-complement activity test were analysis.The purification of homogeneous polysaccharides:extracted by ethanol, the Lasiosphaera Fenzlii Reich was extracted by 0.02mol/L NaOH. The residue was precipated by alcohol, remove proteins and then dialysised. The products (the classical passway IC50=20.10ug/ml) purified by DEAE-cellulose, eluded by 0-2mol/L sodium chloride to 5 parts.The parts eluded by water,0.8 and 2mol/L sodium chloride had no anticomplement activity, the parts eluded by 0.4mol/L sodium chloride(the classical passway ICso=77.88 ug/ml)1.2 mol/Lsodium chloride(the classical passway ICso=83.24 ug/ml)had activity.They were separated by SephacrylTM-300 and SephacrylTM-200. Eventually four homogeneous polysaccharides were separated from the Lasiosphaera Fenzlii Reich named TFP-1, TFP-2, TFP-3, TFP-4.The anticomplement activity of homogeneous polysaccharides and the research of mechanism:the IC50 for TFP-1, TFP-2, TFP-3, TFP-4 of classical passway were 181.7 ±14.45 ug/ml,132.7±13.70 ug/ml,78.81±3.21 ug/ml,136.1±11.60 ug/ml respectively. The value of alternative passway were 162.45±23.90 ug/ml,142.76±20.70 ug/ml, 136.23±10.60 ug/ml,139.1±11.60 ug/ml. From the result, those four polysaccharides shown great anticomplement activity. But compared with the crude polysaccharides, the homogeneous polysaccharide’s anticomplement activity decrease obviously.Preliminary mechanism studies indicated that the homogeneous polysaccharide inhibited the activation of the complement system by interacting with Clq, C2, C3, C4, C5, and C9.The structure research of the homogeneous polysaccharides:TFP-1 was brown flocculent loose material.The molecular weight about more than 500kDa.Total sugar content is96.94%, contains trace amounts of uronic acid and protein. Acid hydrolysis completely get three kinds of monosaccharide composition.Sugar residues mole ratio of Glc, Man, Gal was 6.30:1.03:2.07. Methylation results showed Terminal Glcp,1,3-linked Galp,1,2-linked Manp,1,3-linked Glcp,1,6-linked Glcp,1,2,3,6-linked Glcp with a molar ratio of 28.48:10.31:26.69:25.86:8.21:8.25. TFP-2 was brown flocculent loose material.The molecular weight more than 500kDa. Total sugar content is 97.24%, also contains trace amounts of uronic acid and protein. Acid hydrolysis completely get three kinds of monosaccharide composition. Sugar residues molar ratio of Glc:Man:Gal was 8.68:0.69:0.63. Methylation results showed Terminal Glcp,1,3-linked Galp,1,2-linked Manp,1,3-linked Glcp,1,6-linked Glcp,1,2,3,6-linked Glcp with a molar ratio of 14.42: 6.27:9.71:30.85:34.03:8.75. TFP-3 was tan flocculent loose material,The molecular weight about 600kDa,Total sugar content is 95.34%, about 9.50% uronic acid and 2.85% protein were determined. Acid hydrolysis completely got three kinds of monosaccharide composition. The result of the monosaccharide composition analysis showed that TFP-3 contained glucose, galactose, mannose, glucuronic acid in molar ratio of 6.46:1.25:1.29:1. Methylation results showed 1,3-linked Galp、1,2-linked Manp、Terminal Glcp、 1,3,6-linked Glcp、1,2,3,6-linked Glcp and 1,4-linked GlcpNAC with a molar ratio of 0.132:0.120:0.402:0.261:0.075. combining the result of 1H-NMR,13C-NMR and HMBC, HMQC, TOCSY, the linked type of TFP-3 is that 1,3,6-linked Glcp,1,2,3,6-linked Glcp and 1,2- linked Manp act as main chain while Terminal Glcp、Terminal Glcp A, 1,3-linked Galp as lateral chain. TFP-4 was blue flocculent loose material.The molecular weight about 1000kDa.Total sugar content is 56.94%, contains 47.95% uronic acid and 34.89% protein. Acid hydrolysis completely got three kinds of monosaccharide composition.Sugar residues mole ratio of Glc, Man, Gal was 5.886:1.222:2.894. Because of the complicated component, the methlation was failure.