Isolation, Purification And Structural Analysis Of A Polysaccharide From Hirsutella Sinensis

Cordyceps is one of Precious traditional Chinese medicine,its real anamorpHis Hirsutella sinensis. Hirsutella sinensis has many active substances, Among them,Cordyceps Polysaccharide is one of the most important and abundant component. Thestudy shows it has many activities.Hirsutella sinensis was used as raw material. By means of water extraction andethanol precipitation and washing with organic solvents to obtain crudepolysaccharides.DNS method was used to detect the amount of reducing sugar which producedby α-amylase, beta-amylase and isoamylase at different times and which reflected theeffect of removing starch. The results show that beta-amylase can remove starchentirely at30℃, pH4.8,25:4(w/w) within6h and α-amylase at57℃, pH5.8,25:1(w/w)within6h.Crude polysaccharides was segregate by anion-exchange chromatograpHy（2.6×50cm）. The first is that through gradient elution of Tris-Hcl buffer, getting twosets of polysaccharides——HST-1and HST-2. Next, HST-1is eluted with Boric acid-borate buffer gradient elution. Finally,We got HSP-A1from HST-1at elution(0%Nacl)and HSP-B1from HST-1(0~15%Nacl).Through the research of GPC, we selected NaAc elution at0mol/L、0.005mol/L、0.01mol/L and0.02mol/L,we got polysaccharides segregated well at0.005mol/l NaAc,and SepHadex G-100was selected for gel chromatograpHyaccording to the molecular weight of HSP-A1and HSP-B1with0.005mol/L NaAc.Gel chromatography experiment was divided into three parts.The first part was using2.8×50cm column to purificate HSP-A1and HSP-B1twice respectively, we gotHSN-A3which contained much polysaccharide was separated from HSP-A1andHSN-B1from HSP-B1.The second part was using1.8×100cm column to purificateHSN-A3and HSN-B1,the result showed that HSN-A3and HSN-B1produced only one symmetrical sugar peak respectively.It illustrated that the purity of the twosamples are already high.The third part was using HPLC at25℃，TSK-GEL,G4000SW(7．5mmx300mm)，0.005mol/LNaAc elution，0.5mL/min，detected by RI to purificated samples further and detected the purity of the samples.The result was that we got two single and symmetrical peak HSH-3and HSH-4which had reached a certain requirement.HPLC and GPC2000sftware analysis show that the molecular weight of HSH-2was1.2×104Da and HSH-4was5.3kDa, HPLC,IR and Varian400NMRsepectroscopy HSH-4was mainly consisted of Man, Glc, Gal, with a ratio of1.27:6.62:1.00, and α-pyranose present in the HSH-4, and HSH-4had a backbone ofα-1,3-glucosyl residues and α-1,6-glucosyl residues.