The Separation And Purification Of Konjac Mannatide And Its Anti-tumor Activity

Mannatide is a novel biological response modifier and biological immune enhancer developed by Chinese scientists, and its function includes regulating immune response and enhancing the body’s anti-tumor ability. Currently, the clinical application of mannatide mainly extracted from the culture medium of a-hemolytic streptococcus strain, which is from a single source. Therefore, the exploration of other sources and preparation methods of mannatide has become a hot research. China is the most productive country of konjac powder in the world, while konjac flying powder as a by-product during the production of fine powder, having large output, which is at the rate of 30%-40% of fine powder. For a long time, there are few studies and applications of konjac flying powder, most of them were abandoned or used as animal fed, and inappropriate diapose will cause the environment pollution. Literature and previous research showed that konjac flying powder contained abundant protein and gulcomannan, was considered a good source of preparing konjac mannatide.Konjac fiying powder was selected as the raw material in this research. Konjac protein was extracted from konjac fiying powder by alkali-solution and acid-isolation method, and then was used to prepare konjac peptide by enzymatic hydrolysis. In order to remove the impurity peptides from the enzymolysis solution, konjac polypeptide was purified by ammonium sulfate precipitation, and after the column chromatography of DEAE-52 cellulose, Sephadex G-75 and ConA-Sepharose 4B, konjac mannatide was separated and purified from it, and studied its anti-tumor activity. Experiment results can be concluded as follows:1. Konjac protein was extracted from konjac fiying powder by alkali-solution and acid-isolation method, and then was used to prepare konjac peptide by enzymatic hydrolysis. The yield of polypeptide was 13.68%, and the degree of hydrolysis was 10.32%. Based on above, the extraction process of konjac glycopeptide by ammonium sulfate precipitation was studied. The glycopeptide extraction yield was taken as an index, and through the single factor and orthogonal experiments, the extraction process of konjac glycopeptide was optimized. The results showed that the addition of ammonium sulfate had significant influence on the yield of glycopeptide extraction compared with pH and precipitation time under the experimental conditions; and the optimal conditions were confirmed as follows:70% degree of ammonium sulfate saturation, pH 4.5, 10h of precipitation time. At the optimal conditions, the glycopeptide extraction yield could reach 17.21%.2. The separation and purification process of konjac mannatide was studied, konjac mannatide was separated initially by DEAE-52 cellulose column chromatography, and obtained one common peak. After the optimization on elution conditions, the optimal conditions for DEAE-52 cellulose separation were confirmed as follows:distilled water and 0.05,0.1,0.2mol/L NaCl were selected as eluent,2mL of sample amount,0.8mL/min of elution flow rate. Then, sephadex G-75 column chromatography was used to ulteriorly separate konjac mannatide, and obtained one common peak. The optimal flow rate of Sephadex G-75 was 1.0mL/min. Finally, konjac mannatide was purifed by ConA-Sepharose 4B column chromatography. The adsorption rate was taken as an index, after the optimization on the effect of different ion strength and pH to adsorption rate, the optimal conditions for adsorption was confirmed as follows: 0.15mol/L of NaCl concentration, pH 7.5; Based on the desorption rate as an index, the optimal concentration of a-D-methylglucoside was 0.2mol/L.3. Qualitative analysis of konjac mannatide was studied by HPLC, the results showed that konjac mannatide had the similar amino acid composition with the standard mannatide; the monosaccharide of konjac mannatide were mannose and glucose. By SDS-PAGE method, the molecular weight of konjac mannatide was about 20.1-42.7ku. The concentration of konjac mannatide was 79.51ug/mL4. The Preliminary anti-tumor activity of konjac mannatide was detected by MTT test. The results showed that konjac mannatide can inhibit the proliferation of colon cancer cells HCT-116 and hepatoma cells HepG2, especially to hepatoma cells HepG2, and with dose dependence at the range of 1-100μmol/L. The date analysis showed half-inhibit concentration of konjac mannatide to colon cancer cells HCT-116 and hepatoma cells HepG2 were respectively 140.86μ.mol/L and 57.63μmol/L according to SPSS 22.0 software.