Study On Isolation, Purification, Structure And Biological Activity Of The Polysaccharides From Herba Asari

This paper studied on the isolation, fractionation, physicochemical properties, structural analysis and immunological activity of polysaccharides from Herba Asari. Crude polysaccharide was extracted by hot water and precipated by alcohol. And then, was fractionated by anion-exchange and gel chromatography. Further, the structural features were determined by chemical methods such as periodate oxidation, Smith degradation, methylation and spectroscopy methods including infrared spectrum, GC-MS, ¹³C-NMR. Finally, the immunological activity of Herba Asari polysaccharide was assayed in vivo.The Herba Asari was first cut into small pieces, followed by extraction with hot water. The yield of crude polysaccharide from Herba Asari was 9.6%. The crude polysaccharide was composed of Glc, GalUA and trace of Xyl, Gal and Ara. The content of carbohydrate was 51.9% determined with phenol-sulfuric acid method. The content of uronic acid was 17.4% determined with m-hydroxydiphenyl method. The content of protein was 0.9% measured with Bradford method and the iodine coloration method was used to determined the content of starch (24.17%). The specific rotation was [α]_D²⁰= +168.8.After being deproteinated by Sevag method, the polysaccharides were fractionated by DEAE-cellulose and Sepharose CL-6B gel chromatography to obtain 17 fractions HA, HA1,HA1-1,HA1-2,HA1-3,HA1-4,HA1-3a,HA1-3b,HA1-3c,HA1-3d,HA1-3e,HA2,HA2-1,HA2-2,HAS,HAM and HAN.HAS was a homogeneous polysaccharide identified by high-performance liquid chromatography (HPLC) and its molecular weight was 110000 Da. Sugar analysis showed it was composed of Ara (50.1%), Gal (28.7%) and GalUA (21.2%).The structure of HAS was determined by composition analysis, molecular weight analysis, periodate oxidation, Smith degradation, methylation, infrared spectrum and nuclear magnetic resonance (NMR) analysis. The results showed that HAS was consitituted byα-(1→3)-D-Araf,β-(1→4)-D-Galp andα-(1→4)-D-GalpA . The branching rate of HAS is low. There is one branch every 15 sugar residues on the average.The bioassay showed that Herba Asari polysaccharide had potent stimulating effects on proliferation of both T and B lymphocytes in vivo.