Study On Properties, Fractionation, And Immune Activity Of Chitooligosaccharides

This research work was concerned with fractionation, and immune activity sieving of chitooligosaccharides(COS). Their structure, quantitative analysis , immune activity ,and mechanism were studied,also their physicochemical and functional properties.The results of the physicochemical and functional properties showed that the COS with average molecular weight of 1389 and DD 91%, possessed good solubility in water from pH1 to pH13. But it showed a little decrease above pH 5.1 . The COS solubility decreased with ethanol concentration and it presented positive ion characteristic at pH<9.7 . At temperature <80℃ and pH 2⁵.5, COS solution was stable as confirmed by DSC analysis. COS had excellent water absorption , water holding capacity and can significantly resist starch aging.Fractionation and isolation of chitooligosaccharides were studied by applying membrane separation, ethanol stepwise sedimentation and column chromatography. The isolated component with higher immune activity was sieved by combination of the proliferation of splenocytes in vitro , phagocytization of PMΦ and HC₅₀ experiment in vivo. Firstly, NF retentate COS (NFR-COS) was sieved from UF retentate COS (UFR-COS) and NFR-COS . Secondly, 87.5% ethanol supernatant (COS-87.5SU)was sieved among 75% ethanol sedimentation (COS-75SE), 87.5% ethanol sedimentation (COS-87.5SE), 87.5% ethanol supernatant (COS-87.5SU) from NFR-COS whose molecular weight was 766 Da .Thirdly, COS-87.5SU were flowed through ion-exchange resin CM-Sephadex C-25. The elution was done by 0.01M pH4.8 buffer solution and 0—1.2N NaCl. Six components (COS1, COS2, COS3, COS4, COS5, COS6) were isolated. Component with high immune activity (COS6) and component with low immune activity (COS4 and COS5) were sieved by the above activity sieving method. Two monomers (COS61 and COS62) were got by HPLC . COS62 with highest immune activity were sieved by the proliferation of splenocytes.HPLC column and mobile phase for COS analysis were optimized using GAH/(GLcN)₂/ (GLcN)₃/(GLcN)₄/(GLcN)₅/(GLcN)₆ as mixed standards. HPLC analysis showed that 1) there was good linear relationship between logarithm of retention time(Rt) and degree of polymer(DP), so Rt of amino-sugars could be calculated from DP and vice versa;2)there was rather good linear relationship between DP and minus logarithm of relative quantitativecorrective factor(f') , so quantitative corrective factor(fi) of other chitooligosaccharides couldbe calculated from f_s of standard GAH (benchmark) . The qualitative and quantative analyticmethod were validated by COS6 and found that it had rather good assurance. So various kind of COS monomer could be qualitatively and quantatively determined without standards or only with cheap GAH/NAG standard.The composition and structure of the two monomers (COS61 and COS62) isolated from COS6 were identified by LC/ESI-MS. COS62 was an hexamer ( GLcN-GLcN-GLcN-GLcN -GLcN-GLcN) and represented 93.11%. COS61 was a pentamer (GLcN-GLcN-GLcN -GLcN-GLcN) and represented 6.89%.The two components (COS4 and COS5) with low immune activity were also identified by LC/ESI-MS. The component COS4 was 68.61 %of a trimer (GLcN-GLcN-GLcNAc) and 31.39% of a tetramer (GLcN-GLcN-GLcN-GLcN). The component of COS5 was 59.84% of a tetramer (GLcN-GLcN-GLcN-GLcN )and 40.16 % of a pentamer (GLcN-GLcN-GLcN-GLcN-GLcN).Besides, nonspecific immune activity, cell immune and humoral immune activity mechanism, cell cycle and cell factor of COS6 with known composition and structure were studied. It showed that COS6 could promote the increase of spleen index ,thymus index, phagoiytic activity of peritoneal macrophage(PMO) and proliferation rate of splenocytes and HC50 in normal mice(NM) and immunodeficiency mice (IDM). It also could resist decreasing immune organs' weight, weakening phagoiytic activity of PMO, killing splenocytes by cyclophosphamide(Cy) and reducing the production of HC50 due to Cy. Thus COS6 promoted specific immune activity, nonspecific immune activity, cell immune and humoral immune activity. COS6 was mitosis source of splenocytes and it could cooperate with karyokinesis matrix. It indicated that COS6 could promote the dividing and proliferation of spleen cell of mice by cell cycle of NM using cell instrument. It could repair cell dying induced by Cy used in spleen cell cycle change experiment on IDM.Effects of (GLcN)5 and (GLcN)6 in vivo and vitro on gene transcription and translation level of IL-1, TNF-a , IL-2* IFN-y were studied, respectively by methods of relatively quantitive RT-PCR and ELISA. It showed that the mRNA expression and release of IL-K TNF-cu IFN- could be promoted more significantly by (GLcN)6 than those by (GLcN)s. Effects of (GLcN)5 and (GLcN>6 in vivo and vitro on gene expression of CR3 on the surface of PMO and splenocyte were also studied by RT-PCR. It showed that the mRNA expression of CR3 could be promoted more significantly by (GLcN)6 than those by (GLcN)5 This maybe concerned with that (GLcN)6 binded more sites of CR3 than (GLcN)s? led to change conformation of CR3 and (GLcN)6 possesed more affinity with CR3 than (GLcN)s. After (GLcN)5 and (GLcN)6 had been bound with CR3, signal transfer had been started through membrane and then leaded to stimulate gene expression of cytokines and CR3, thus immunomodulating ability for body were promoted. There was maybe one of mechanisms for elucidation of immunity of COS as well as the structure-activity relationship.