Optimization of Extraction Process from Tortoise Shell Protein by Response Surface Methodology and Its Proliferation Activity on MC3T3-E1 Cells

Objective:To optimize the extraction process of tortoise shell protein,screen the active constituent of tortoise shell,and explore the effect of tortoise shell on the proliferation of osteoblasts(MC3T3-E1). Methods:The contents of protein and the proliferation rate of MC3T3-E1 cells were used as the index to screen the extraction method of tortoise shell protein. The ratio of material to liquid,extraction temperature and time were used as independent variables,and the contents of tortoise shell protein was used as dependent variable to carry out single factor extraction research.According to the design of response surface test,the best extraction technology of tortoise shell protein was obtained.By ultrafiltration technology,the tortoise shell protein was divided into different constituent:Total extract(Component 1),M_W>10 kDa(Component 2),M_W:3~10 kDa(Component 3),M_W:1~3 kDa(Component 4),M_W<1 kDa(Component 5).CCK8 method was used to determine the effect of different concentrations(12.5,25,50,100,200 μg/mL)of tortoise shell protein and its different constituent on the proliferation rate of MC3T3-E1 cells. Results:The contents of tortoise shell protein peptide obtained by magnetic stirring method was the highest,and the osteogenic activity was the best.The best extraction condition of tortoise shell protein was at 30 ℃ and the ratio of material to liquid was 1:13 (g/mL)for 3 hours,under this condition,the contents of tortoise shell protein peptide was 15.16%. At the concentration of 200 μg/mL,the proliferation rate of fraction 1~5 to MC3T3-E1 cells was 20.58%,25.30%,30.24%,37.89% and 38.15%,respectively. Among them,fraction<1 kDa had the best proliferation activity.Conclusion:This study would provide a reference for the preparation of tortoise shell protein,and prove that tortoise shell protein and its different components have better proliferation promoting activity in MC3T3-E1 cells,in which the molecular weight sess than 1 kDa had the best effect on MC3T3-E1 cells proliferation.