| Hyaluronan(HA, Mr=105~106) is a large, linear polysaccharide, which was present in all types of mammalian extracelluar matrix. HA is widely used in medicine, clinical diagnosis, cosmetics and health food industries for its unique properties. Recently, HA has been shown to exert different effects depending on its molecular size. Hyaluronan oligosaccharides(o-HAs, Mr within 104) have been demonstrated to have immunological activity, activate endothelial cell and inhibit multidrug resistance to tumours, et al. o-HAs were mostly achieved by bovine testicular hyaluronidase(BTH) that degrades HA into o-HAs with glucosamine at the reducing end by cleaving the β-1,4-N-acethylhexosaminide bonds. However, the preparation of o-HAs was inefficiency due to the limited source of BTH, hydrolysis inspecificity and the complex product. In contrast, o-HAs can be efficiently prepared by leech hyaluronidase(LHase) that degrades HA into o-HAs with gluconic acid at the reducing end by cleaving the β-1,3-glucuronide bonds. However, LHase and o-HAs produced by LHase were rarely studied before. In this study, HA was hydrolysed by LHase. The hydrolysis process was further quantitative investigated and the major products were separated and purified. Moreover, the biological functions including immunological activity, angiogenesis and tumor multidrug resistance of the major products were studied.First of all, the method for simultaneous determination of o-HAs by HPLC was optimized in this paper. Based on optimisation of the mobile phase type, concentration and flow rate, YMC-Pack Polyamine II column(250 mm×4.6 mm, 5 μm) was used at 30°C and mobile phase was 0.1 mol/L NH4H2PO4 with a flow rate of 0.5 mL/min. Detection wavelength was 210 nm. HA4 and HA6 were efficiently qualitatively and quantitatively analysed using the method which was demostrated to have good sensitivity, precision recovery and high stability.The stability of LHase was studied before analysis of the characteristics of HA hydrolysis process. LHase kept high enzyme activity when incubated in distilled water at 38°C. The reaction products of a series of incubation times and enzyme activities were analysed by ESI-MS and HPLC. The results showed that the major products were HA4 and HA6, and the minimum product was the disaccharide(HA2). 0.42 g HA4 and 0.46 g HA6 were obtained from 1 g HA with an enzyme activity of 1.6×104 U/m L after 24 hours at 38°C. Besides, the yield of HA4 in the products was increased with enzyme activity increasing.The optimal ion-exchanger Q-Sepharose Fast Flow was screened from many kinds of ion-exchangers(640, DEAE, D730, D750, Dowex 1×2), which exchanged o-HAs effectively according to its high adsorption and desorption percentages(more than 80%). Impressively, After optimisation of the operation parameters(balanced pH, elution concentration, elution volume and elution flow rate), HA4 was completely separated with HA6 without nucleic acid, protein and other impurities by balanced(pH 8.0 Tris-HCl) Q FF column eluted by the linear eluent 0.2 mol/L NaCl solution for 8 column volumes at a flow rate 5 mL/min according to chromatograms of UV and ESI-MS. Moreover, the purity of HA4 and HA6 reached 90% after desalination by Sephadex G-10.In addition, the biological functions of HA4 and HA6 were studied. The effect of HA4 and HA6 in vitro on the promotion of the proliferation of spleen cells was detected by MTT method. The results showed that compared with o-HAs(average Mr of 4 k), HA4 and HA6 could effectively improve the proliferation of the spleen cells. Especially, the proliferation rates were 64.4% and 68.2%, repectively, when HA4 and HA6(320 μg/m L) were combined with ConA; The role of HA4 and HA6 in angiogenesis was evaluated by using Matrigel plug assay. HA4 and HA6 were demonstrated to show higher activity for promoting angiogenesis. Compared with the negative group, the relative vessel area(RVA) was increased by 53.5 and 49.1 times in the HA4 and HA6 groups(50 μ/mice), repectively. However, the RVA was increased by 9.91 times in the o-HAs(average Mr of 4 k) groups(2 mg/mice); The reverse effect of HA4 and HA6 on muli-drug resistance in MCF-7/ADM was explored using MTT method. The results showed that HA4 and HA6 abrogated partly drug resistance of MCF-7/ADM while o-HAs with high mole weight have no effect. Moreover, the biological functions of HA4 and HA6 produced by BTH were also compared. The results demonstrated that there is no significant difference observed between the major products HA4 and HA6 produced by LHase and BTH in immunological activity, angiogenesis and tumor multidrug resistance. |
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