Preparation And Characterization Of Chitooligosaccharide Monomers

Chitooligosaccharide(COS) with the advanage of high water solubility is thehydrolysis products of chitosan,it has extensive physiological functions which play animportant role in anti-tumor，antimicrobial，anti-oxidant，regulating blood sugar，decreasing blood lipid and biomaterial and so on.Chitosan with high degree of deacetylation could be hydrolyzed by chitosanasewhich was prepared in our lab to COS,which was analysed by TLC,MALDI-TOF-MS.The hydrolysis product consisted of COS with degree of polymerization(DP)2~6mainly and a spot of COS with higher DP. Hydrolytic model of chitosanase wasanalysed simply by TLC,HPLC,MALDI-TOF-MS. Chitobiose,chitotriose,chito-tetraose could not be hydrolyzed by chitosanase, chitopentose was hydrolyzed slowlyinto chitobiose and chitotriose and chitohexaose were hydrolyzed to chitotriose. Asuccession of COS could be prepared by contorlling substrate with different degreesof acetylation(DA) and reaction time.Applications of COS in anti-tumor and antimicrobial had been reported,but theresearch of theory and application about COS monomers reported rarely. Thiscondition was confined to the complex process of COS monomers separation andtheir expensive price. In this study, COS monomers were separated by ion exchangechromatography.Strongly acidic macroporous polystyrene cation exchange resins wasselected as separating medium,which has the functions with ion exchange andmolecular sieve. The separating effect was perfect in the conditions. Column volume:600mL;elution volume:12L;gradient elution:0.6mol/L-1.2mol/L,flow velocity:300mL/h;packed volume:150mL.Purity quotient of COS monomers was analysed byTLC,HPLC,MALDI-TOF-MS. Results indicated that the purity quotients of Chito-biose chitotriose chitotetraose and chitopentose were up to95%, chitohexaosecontained impurity,but he purity quotient was also up to85%.The activity of chitotetraose,chitopentose,chitohexaose aganist human hepatomacarcinoma cells(HePG-2),Human lung carcinoma cells(NCI-H460),human gastriccarcinoma cells(MGC-803),human colorectal carcinoma cells(LOVO) wereinvestigated in this study. Results indicated that three kinds of COS in highconcentration could inhibit tumor cell,which had some connection with degree ofpolymerization. The inhibition rates of cell reproduction on HePG-2increased withthe increase of degree of polymerization,but the rates on MGC-803declined with theincrease of DP.That might be in connection with the receptor of tumor cells.The agar diffusion method was used to measure the zone of inhibition ofglucosamine,chitobiose,chitotriose,chitotetraose,chitopentose and chitohexaoseagainst Colibacillus, Staphylococcus aureus, Vibrio anguillarum and Aeromonashydrophila.The results showed that chitobiose had strong antibaterial effect againstColibacillus, Staphylococcus aureus and Aeromonas hydrophila.Chitotriose hadantibaterial effect against Colibacillus and Aeromonas hydrophila. Chitopentose hadlittle antibaterial effect against Aeromonas hydrophila. Among the groups of COSmonomers,chitobiose had the intensest antibaterial effect.With increase of DP,anti-baterial effect of COS monomers delined.The reason might be that the interstice ofcytoderm was entered easily by COS with low molecular weight. The metabolism ofcell was interfered by COS that entered into cells.