Evaluation The Preparation And Function Of Hyaluronic Acid From Cockscomb

Used comb as material to study the technologies of extraction and purification and degradation of hyaluronic acid, then optimized conditions.To study the hyaluronic acid yield as the index, compared double reagent and enzymatic method of extracting effect, and used the Box-Behnken central composite design and response surface method analysis of the establishment of the second regression model. Determined the best process conditions for double enzymatic method:enzyme dosage is0.004g, enzymolysis temperature46℃, enzymolysis time2.3h and the content of hyaluronic acid was10.36%. The optimum conditions of the reagent method extraction of hyaluronic acid were as follows: material liquid than is1:177, ethanol concentration is90%, the incubation time was25.7h, hyaluronic acid of cockscomb in yield of27.37%.To study the purification test as the index, through compared the anion resin and solvent precipitation method to purity hyaluronic acid in comb, determined the best purification conditions of hyaluronic acid by dynamic adsorption, and analysis was as follows:The rate of adsorption amount of liquid and resin volume is4:1(g/mL); the adsorption time is45min;the adsorption velocity is1.0mL/min; the eluant was0.6mol/LNaCl solution; the amount of eluant and resin volume ratio of4:1(g/mL). Selected CTAB as the best solvent of precipitation method, determined the optimum conditions of solvent precipitation method purification HA by single factors and Box-Behnken central composite design and response surface method experiments were as follows:CTAB concentration is11.90%, pH value of9.0, HA coarse extraction liquid material and CTAB solution fluid volume ratio of1:3.70(g/mL).The content of hyaluronic acid in cockscomb was precipitated with the methods of column chromatography and solvent precipitation by dual-beam UV spectrophotometry, which respectively was40.05%and82.29%. This showed that the solvent precipitation method as the best method. Significantly improve the purity of hyaluronic acid in the extract.Through qualitative test after extraction of the tested material, extraction material for hyaluronic acid.It was proved that the material of extraction contented HA. Detected the substances of purification by Column chromatography through UV and HPLC, And the results was showed that the peak positions of purification was as similar as standard HA. The stracture of purification was as similar as standard hyaluronic acid through IR. Turning out that the extraction substance from the cockscomb was hyaluronic acid.Determination of molecular weight degradation application method of reducing sugar of hyaluronic acid, through the single factor experiment and response surface method analysis establishes two regression modle.the degradation time, the free radical content, degradation of pH, factors of the degradation temperature and ratio of material to liquid. Optimized combination to determine the optimum process conditions of hyaluronic acid degradation study for. Degradation time of5min, the concentration of H2O2was1.67mol/L. the ratio of material to liquid (Vc:H2O2) for1.413,8.0for pH, temperature is25℃degree of degradation is the best. Degradation of hyaluronic acid molecular minimum weight was1791.Research the antioxidant activity of hyaluronic acid from cockscomb. The result indicated that the hyaluronic acid had the strong elimination ability to the free radical. Total antioxidant ability was18.97U/mL, the maximum inhibition rate to hydroxy free radical was50.69%,the maximum ability to Anti-superoxide anion was42.87U/L, the alkyl free radical is41.22%and Anti-liposome was50.69%.