Study On Separation,purification,and Antioxidant Activity Of Protein From Metaplexis Japonica Makino Nutshell

ObjectiveOptimize the extraction process and study its antioxidant activity of the protein from Metaplexis Japonica Makino nutshell?MJNP?.MethodsMJNP was extracted by alkali treatment and acid precipitation method,regarding the protein extraction rate as the key dimension.The suitable range of the main four factors?the ratio of raw materia to water,solution pH,temperature and extraction time?affecting the protein extraction yield were selected through the single factor tests preliminary.Based on the results of single-factor tests,orthogonal experiments were followed.The extraction conditions of the highest protein extraction rate were determined at last.The extraction of crude protein was further purified by DEAE-52 cellulose column,Sephadex G-75 gel column and dialysis.Besides,SDS–PAGE?sodium dodecyl sulphate polyacrylamide gel electrophoresis?was ready to analyze the electrophoretic map of MJNP-A of which ability to scavenge free radicals?DPPH·,·OH?and reductive was detected by antioxidant activity of protein.Moreover,superoxide dismutase?SOD?in hepatoma cells was measured,and the effect of MJNP-A on the proliferation of hepatoma cells was studied by MTT finally.ResultsThe optimum extraction parameters were:ratio of solid to liquid of 1?20?g/m L?,solution pH 11.5,temperature of 60?and extraction time of 1.0 h.Validation experiments showed that the average extraction yield was 89.18%.The MJNP1 and MJNP2 were gained after the MJNP through the DEAE-52cellulose column.The MJNP-A was gained after MJNP1 passing through the Sephadex G-75 gel column.By analyzing the result of SDS-PAGE,The MJNP-A purified by DEAE-52 cellulose column and Sephadex G-75 gel column was monocompound and its molecular weight was about 45 kDa.The scavenging rate of MJNP-A to DPPH·and·OH was 52.45%?800?g/mL?and 31.57%?800?g/m L?respectively.In addition,the IC₅₀0 value of DPPH·and·OH was 644.84?g/m L and 1392.03?g/mL,respectively.The SOD experiment showed that the activity of SOD in the hepatoma cells was enhanced.The MTT experiment suggested that the plasma shell protein could inhibit the proliferation of hepatoma cells dose-dependently.ConclusionsMJNP extraction rate was improved by the extraction technology of single factor experiment and orthogonal experiments.Furthermore,the crude protein was effectively purified by DEAE-52 cellulose column and Sephadex G-75 gel column.These findings indicated that the MJNP-A could inhibit the proliferation of hepatoma cells.