Purification Of Sodium Hyaluronate With Alkaline Treatment And Ultrafiltration Method

The crude sodium hyaluronate(SH) was treated by an aqueous Na OH solution, and the solution was ultrafiltrated to obtain purified SH(medical grade). The structure of purified SH was characterized with Fourier transform infrared spectroscopy(FTIR), its intrinsic viscosity and molecular weight were determined with dilute solution viscometry, its thermal stability was characterized with thermogravimetric analysis(TG), its content of glucuronic acid(Glcua) was measured with UV spectrophotometry, its content of protein was measured with koumas blue method, its content of nucleic acid was measured with UV spectrophotometry, and its cytotoxicity against HEK293 was evaluated with MTT cell counting and microscopic observation. The effects of temperature, time and Na OH concentration for alkaline treatment of crude SH on the content of Glcua, protein and nucleic acid, the intrinsic viscosity and molecular weight, and the yield of purified SH were researched with orthogonal design and single factor experiments. The cell counting principle was applied to study the r emoval of bacteria added in crude SH with alkaline treatment and ultrafiltration processes. The traditional TCID50 half death and fluorescence quantitative PCR(q PCR) methods were applied to investigate the effect of removal of pathogens added in crude SH with alkaline treatment and ultrafiltration processes.Results showed that temperature for alkaline treatment had great effect on the viscosity average molecular weight(VAMW) of purified SH. We got purified SH with high VAMW at about 60 ?,and its VAMW and yield decreased as temperature increased. While temperature had little effect on protein content and DNA content of SH, this may imply that protein and DNA degradated completely after alkaline treatment. And the concentration of NaOH for alkaline treatment contributed a lot to the Glcua content. When using 0.2 wt.% of NaOH solution(70 ?, 90 min), we obtained purified SH with Glc UA content of 46.17%, VAMW of 1.49×106, protein content of 0.04%, DNA OD value of 0.63, yield of 40.75%, and with increasing the concentration of Na OH, its Glcua content and yield decreased significantly. Still, we found that the time for alkaline treatment mainly affected the protein and DNA content, as with increasing time, the protein and DNA degradated more sufficiently and filtered by ultrafiltration. And pathogen detection showed that alkaline treatment followed by ultrafiltration removes pathogenic bacteria and virus almost completely. Hence, we proposed that the optimal condition of alkaline treatment of SH would be that the temperature was 60 ?, the time was 60 min, the concentration of NaOH was 0.2 wt.%, and followed by ultrafiltration we got SH with high purity. Thus we provided a simplified purification method, to reduce the complexcity of current methods, and this may facilitate the medical application of pure SH greatly.