Study On The Preparation And Biological Activities Of4,5-unsaturated Hyaluronate Disaccharide

Hyaluronan (Hyaluronic Acid, HA) is a high-molecular mass polysaccharide consisting of alternating D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) units linked by β-(1â†'4) and β-(1â†'3) glycosidic bonds. As a significant component of extracellular matrix which is also an important constituent of skin, HA has unique physicochemical property and biological activities. It is reported that HA plays an important role in maintaining tissue integrity, as well as in developing and remodeling tissues during inflammation, wound repair, and embryonic development.In1934, HA was first isolated from the vitreous humor of bovine eyes by American scientists Meyer and Palmer, HA has been widely used in medicine, cosmetic and food area as the research on structure, property and biological functions of HA going deeply. It has been reported that HA with different molecular weight has different even opposite effects. There have been more and more researches on degradation and degradation products of HA during recent years. Oligosaccharides of hyaluronan (o-HA) are HA fragments with the molecular weight less than104Da which consist of2-40monosaccharide resedues. and they are getting more attention because of the important role in wound healing, angiogenesis, immunoregulation and antineoplastic activity. The methods of preparing o-HA include chemical degradation. enzymatic degradation and biosynthesis among which enzymatic degradation is a more proper way because of its mild reaction condition, high degradation efficiency and so on. This research focuses on the preparation of unsaturated hyaluronan disaccharide (ADiHA) and it’s biological activities.1Preparation methods and structure elucidation of ΔDiHAA method of preparation of ΔDiHA using a hyaluronate lyase derived from Bacillus A50was investigated and then ΔDiHA was purified considering the degradation characteristics of this enzyme. After that, the chemical structure of ΔDiHA was confirmed by ultraviolet spectrum (UV), mass spectrum (MS),1H NMR spectrum and13C NMR spectrum, and then two assay methods of ADiHA, carbazole method and HPLC method, were discussed.2Cytotoxicity of ΔDiHAThe cytotoxicity of ΔDiHA was evaluated using mouse fibroblast cells L929through MTT test. The results showed that ΔDiHA didn’t affect the proliferation of L929cells and hadn’t change cellular morphology which means ΔDiHA has no cytotoxicity to L929cells.3Effects of ΔDiHA on proliferation of human vein enthothelial cells and human corneal endothelial cellsThe effects of ΔDiHA on the proliferation of human vein enthothelial cell (HUVEC) and human corneal endothelial cell (HCEC) was investigated with MTT test and the effects of ΔDiHA on HCEC cycle and apoptosis were also analyzed by flow cytometry (FCM). The results displayed ΔDiHA has a strong ability to stimulate the proliferation of HUVEC and can promote the proliferation of HCEC without changing cell cycle and apoptosis of HCEC.4Antioxidation activity of ΔDiHAThe effects of different concentrations H2O2on ROS level of HCEC and whether ΔDiHA has the ability to restrain the rising of intracellular ROS were assessed. The results show that intracellular ROS rises as the concentrations of H2O2rising in certain concentration range and certain amount of ΔDiHA in culture medium can restrain the rising of intracellular ROS. The study has initiatorily discussed the preparation and purification of ΔDiHA, evaluated its cytotoxicity, antioxidant activity and its effects on proliferation of HUVEC and HCEC, which will be reference for the application of ΔDiHA.