Study On The Preparation And Biological Activities Of Oligosaccharides Of Hyaluronan

Hyaluronic acid (HA), a nonbranched polysaccharide, is composed of regularly alternating units of N-acetyl glucosamine and D-glucuronic acid linked by β-(1→4) andβ-(1→3) glycosidic bonds. The weight average molecular weight (M|-_w) range ofnatural HA is within 1 × 10⁵～1×10⁷D, and the molar ratio of the two monosaccharides in the molecular structure is 1:1. HA is generally prepared by extraction from animal tissues or by bacteria fermentation.Till now a lot of work has been done on the study of the structure, properties and physiological functions of HA, and at present HA is used extensively in clinic. TheM|-_wused now is above 1 × 10⁵D, while oligosaccharides of hyaluronan (o-HA) is lessthan 1 × 10⁴ D with one to twenty repeats (2-mers to 40-mers). O-HA and HA have different biological activities and they even have opposite activities. In the paper, the preparation method, purification technologies and biological activities of o-HA were studied.1. Preparation methods and purification technologies of o-HAThe optimal reaction conditions of HA hydrolysis catalyzed by hyaluronidase (HAase) were studied by orthogonal desigen. It was found that the hydrolysis reaction is affected by temperature, pH, substrate concentration, the amount of HAase used and reaction time. The optimal reaction condition is as follows: 10 g/L of HA, 1.5 × 10⁵U/L of HAase, pH 5.0, reaction temperature 50°C. It is important that well-characterized and highly purifyied o-HA is used in biological and structural studies, so we established a method for the purifyication of o-HA of defined molecular weight using Bio-gel P6 chromatography following digestion of HA with HAase. These preparations were characterized by a combination of ultraviolet spectrometry (UV), infrared spectra (IR), mass spectrometry (MS) and confirmed they are o-HAs composed of 4 saccharide residues (o-HA4), 6 saccharide residues (o-HA6), 8 saccharide residues (o-HA8) and 10 saccharide residues(o-HA10).2. Angiogenesis promoting effect of o-HA on chicken embryo chorioallantoic membrane and cultured microvascular endothelial cellsThe angiogenesis promoting effect of o-HAs on chicken embryo chorioallantoic membrane (CAM) was sdudied and found O-HA6, o-HA8, o-HA10 could promote angiogenesis in CAM. The effect of o-HAs on proliferation of human umbilical vein endothelial cells (HUVEC) was invesgated by MTT method, the result showed that o-HA6, o-HA8, o-HA10 (18.75-150μg/mL) could proliferate HUVEC.3. The reverse effect of o-HAs on muli-drug resistance in K562/A02The reverse effect and mechanism of o-HAs on muli-drug resistance in K562/A02 were explored using adriamycin (ADR) as model a drug. 50% inhibition concentration (IC₅₀) of ADR on K562/A02 was determined by MTT method, intracellular ADR concentrations were determined by flow cytometry. The results showed that the IC₅₀ of ADR decreasead from 61.6μg/mL to 30.17, 30.04, 32.29, 33.33 μg/mL after 100 μg/mL of O-HA4, o-HA6, o-HA8, o-HA10 were added respectively. O-HAs of 100 μg/mL could also enchance ADR accumulation in K562/A02 cell by 3.90, 3.92, 3.76 and 3.39 folds.The main achievements obtained in the study are as follows:1.On the basis of single factor investation, the optimized conditions of HA hydrolysis catalyzed by hyaluronidase was obtained by orthogonal desigen as: 10g/L of HA concentration, 1.5×10⁵U/L of HAase, pH 5.0, reaction temperature 50℃. O-HAs composed of 4 saccharide residues (o-HA4), 6 saccharide residues( o-HA6), 8 saccharide residues( o-HA8) and 10 saccharide residues(o-HA10) were prepared using Bio-gel P6 chromatography.2. Angiogenesis promoting effect of o-HAs was compared, 0-HA6, 0-HA8, o-HA10 (18.75-150 μg/mL ) could proliferate HUVEC and they could promote angiogenesis in CAM.3. O-HAs can reverse muli-drug resistance in K562/A02 was found for the first time.The study has laid the foundation for the development of o-HAs as new drugs.