The Research Of Tussah Pupa Extracted Optimally By Response Surface Method And Its Effected On SMMC-7721 Hepatoma Carcinoma Cell

Objective Polysaccharides are one of the important macromolecules in the organism.They are linked by more than 10 monosaccharides through glycosidic linkages.The polysaccharide was extracted from Tussah pupa by microwave method,and the polysaccharide was further isolated and purified by response surface methodology.The effect of polysaccharide on the apoptosis of tumor cells was investigated.Methods Microwave method was used to extract polysaccharides from Tussah pupa under weak alkaline condition.On the basis of single factor experiment,the optimum extraction technology of polysaccharide from Tussah pupa was optimized by response surface methodology using polysaccharide yield as an index.Sevag method was used to remove the protein from the polysaccharide extract,the effects of H2O2 volume fraction on the decolorization rate of polysaccharides were investigated by removing the pigment from crude polysaccharide with hydrogen peroxide.and the content of polysaccharide in the Tussah pupa was determined by phenol-sulfuric acid method.In this study,the separation of molecular weight of 5000 Da dialysis bag to remove small molecules.The DEAE-52 weak anion exchange chromatography was used to purify polysaccharides.Sephadex G-150 gel chromatography The PSP1 active component,which has a value-added inhibitory effect on tumor cells,is further purified.CCK-8 method was used to detect the inhibitory effect of PSP1 on the growth of SMMC-7721 human hepatocellular carcinoma cells.Western blot was used to detect the expression of apoptosis-related proteins(Bax,Bcl-2 and P53).Detection of P50 and P65 protein localization by laser confocal immunofluorescence.Results The microwave extraction technology of polysaccharide was optimized by response surface methodology.The optimum extraction conditions were microwave time 23 min,microwave extraction power 516 W,ratio of material to liquid was 1: 28.Under the optimum extraction conditions,the yield of polysaccharide was 4.72%,and the polysaccharide was white powder.The polysaccharide purity was 71.8 %.The optimum conditions for the decolorization of H2O2 volume fraction 25 %,the decolorization rate of polysaccharide was 75.31 %,and the retention rate of polysaccharide was 68.47 %.DEAE-52 weak anion exchange chromatography,with deionized water and different concentrations of sodium chloride gradient elution,get a total of three polysaccharide components,namely PSP1,PSP2,PSP3.Three polysaccharides acted on SMMC-7721 cells,The results showed that PSP1 polysaccharide had a significant inhibitory effect on SMMC-7721 hepatocarcinoma cells.Sephadex G-150 gel was used to purify the PSP1 active component,the elution curve was symmetrical,indicating that the PSP1 active component was relatively homogeneous and the purity was high.The results of CCK-8 showed that the polysaccharide extract of Tussah pupa had inhibitory effect on the proliferation of SMMC-7721 cells in a time and concentration-dependent manner(P<0.01),and the half inhibitory concentration(50 % Inflammatory concentration,IC50)were 6.606,4.114 and 2.569 mg/ml,respectively.The results of Western blot showed that the expression of Bax and P53 protein in SMMC-7721 cells increased gradually with the increase of polysaccharide concentration,and the expression of Bcl-2 protein decreased gradually.The results showed that polysaccharide PSP1 could inhibit the growth of tumor cells by inhibiting the migration of P65 / P50 protein from the cytoplasm to the nucleus in the NF-?B pathway.Conclusion Polysaccharide extracted from Tussah chrysalis was isolated PSP1,PSP2,PSP3.acting on SMMC-7721 cells.The results showed that PSP1 could inhibit the expression of Bax and P53 protein and increase the expression of Bcl-2 protein in a time-dependent and concentration-dependent manner.Inhibition of tumor cell growth by inhibiting the migration of P65 /P50 from the cytoplasm to the nucleus in the NF-?B pathway.The results showed that the inhibitory effect of polysaccharide PSP1 on the proliferation of tumor cells could provide a basis for the future development of anticancer drugs and the adjuvant therapy of cancer.