Study On Preparation Of Low-molecular-weight Hyaluronan Oligomers By Microwave And Its Biological Activity

Hyaluronic acid(HA),a nonbranched polysaccharide,is composed of regularly altemating units of N-acetyl glucosamine and D-glucuronic acid linked byβ-(1→4)andβ-(1→3)glycosidic bonds.The average molecular weight((?)_W)range of natural HA is within 100 kDa～10 000 kDa,and the molar ratio of the two monosaccharides in the molecule is 1:1.HA is generally prepared by extraction from animal tissues or by bacteria fermentation.Many works have been done on the study of the structure,properties and physiological functions of HA,and HA is used extensively in clinic.Studies showed that some biological activities of HA have close relationship to its molecular weight, High molecular weight HA has different biological activities from low molecular weight HA(LMWHA)and they even have opposite biological activities.Studies of LMWHA gradually become a hotspot in domestic and abroad.In the paper,the preparation method,physical and chemical properties,molecular structure analysis and angiogenesis effects of MW-HA were studied.1 Studies on the preparing procedure of LMWHAThe preparing procedures of LMWHA by microwave degradation and the major factors affecting the preparation of MW-HA were studied in this paper.The optimum degradation condition was determined as 105℃,pH3.2 Studies on the characteristics and structure of LMWHAThe characteristics and structure of LMWHA obtained by three different degradation methods were studied.Their UV,IR and ¹³C-NMR are very similar, suggesting that the monosaccharide constituents of LMWHA are the same as natural HA.3 Studies on the angiogenesis activity of LMWHAThe angiogenesis promoting effect of LMWHA on human umbilical vein vascular endothelial cell(ECV304)and chicken embryo chorioallantoic membrane (CAM)were studied.We investigated the effect of LMWHA on proliferation of human umbilical vein endothelial cells(ECV304)with MTT method,the result showed that LMWHA(150-600μg/mL)could promote the growth of EVC304.The angiogenesis activity of LMWHA studied by using CAM model demonstrated that comparing with the blank group,the LMWHA groups and the bFGF group showed obviously increased number of blood vessels(P＜0.05).In the LMWHA groups,the thickness and density of the blood vessels increased with the increasing LMWHA dosage,indicating that the angiogenesis activity of LMWHA was dose dependent. The angiogenesis promoting activity of the three LMWHA obtained by different degradation methods was:ED-HA＞MW-HA＞CH-HA,and there was no significant difference between ED-HA group and MW-HA group.4 Conclusion(1)The preparing procedure of LMWHA by microwave degradation was studied; the optimum condition of the MW-HA preparation was obtained.(2)Products of MW-HA with molecular weight lower than 10 kDa were obtained, and the analyzed physical and chemical parameters showed no change compared to undegradated.HA.(3)The structure changes of MW-HA,ED-HA and CH-HA before and after degradation were analyzed according to the spectra of UV,IR and ¹³C-NMR and the results showed that when the molecular weight of MW-HA and CH-HA are less than 8 kDa,both of them showed some degree structure change.The spectra analysis showed that:MW-HA have less structure change comparing with the same molecular weight CH-HA,which may due to the uniformity and fast speed of microwave heating; MW-HA and CH-HA possessed the same structure change mode,indicating that the mechanism of MW-HA degradation was hydrolization of glycosidic band under acid condition.(4)Angiogenesis activity of LMWHA was investigated.The results showed that the angiogenesis promoting activity of the three LMWHAs obtained by different degradation methods was:ED-HA＞MW-HA＞CH-HA,and there was no significant difference between each two LMWHA groups.