|
The the mussels raw materials, the use of enzyme technology, ultrafiltration, gel chromatography, ion exchange chromatography and reverse phase high performance liquid chromatography separation and purification technology means mussel protein enzymatic hydrolysis process, the preparation process and the active peptide in vitro antitumor bioactive peptides function and molecular mechanisms of anti-prostate cancer activity. Using orthogonal experimental method in the study of enzymatic process optimization, the main research and processing the amount of enzyme enzymatic hydrolysis of mussel-versus-tumor effect and enzymatic activity of the solid-liquid ratio, pH value, hydrolysis temperature, hydrolysis time influence of the experimental results is as follows: The experiment used the alkaline protease (Alcalase), of three kinds of protease trypsin (Trypsin) and pepsin (Pepsin), when a degree of hydrolysis of the hydrolysates as evaluation indexes, the obtained protease The best hydrolysis conditions: by Alcalase: solid-liquid ratio of 1:4, the pH value of 10, plus the amount of enzyme to 2000U / g, a temperature of 50 ° C hydrolysis time for 8h; Trypsin: solid-liquid ratio of 1:4, pH 8.5, enzyme dosage of 1000U / g, a temperature of 45 ° C, hydrolysis time 8h; Pepsin: liquid ratio of 1:5, the pH value of 5, the amount of enzyme to 1500U / g and a temperature of 45 ° C. hydrolysis time of 8h; When the anti-tumor activity of the hydrolysates from Evaluation protease optimal conditions: Alcalase: solid to liquid ratio of 1:4, and the pH value of 10, plus the amount of enzyme 1500U / g, a temperature of 50 ℃, hydrolysis time of 8h; Trypsin: solid to liquid ratio of 1:4, the pH value of 8, plus the amount of enzyme to 1500U / g and a temperature of 40 ° C, hydrolysis time of 2 h; Pepsin: material liquid ratio of 1:4, the pH value of 5, the amount of enzyme to 1500U / g, a temperature of 55 ° C hydrolysis time for 8h. When the optimal hydrolysis conditions on the anti-tumor activity of three kinds of protease indicators mussel protein hydrolysis, the obtained hydrolyzate Trypsin hydrolyzate of the highest degree of hydrolysis, while the strongest anti-tumor activity of Alcalase hydrolyzate. In the active peptide in the manufacturing process, the first choice is the ultrafiltration method, by using interception molecular weight 10KDa, 5KDa 3 kDa ultrafiltration membrane, after ultrafiltration, the total four component i.e. MH1 (gt; 10KDa), MH2 ( 5-10KDa), MH3 (3-5KDa) and MH4 (lt; 3KDa), of which the strongest anti-tumor activity to the lowest molecular weight MH4 concentration of 0.5mg/mL, after 48h of DU-145 and PC- 3 cell proliferation inhibition rate of 33.17% and 30.24%, respectively. MH4 by a DEAE-SepharoseFF ion exchange chromatography, and to obtain a 4-component, i.e. MH4 MH4-2-3 and MH4 MH4 4, these four components have anti-tumor activity, but strongest MH4-2 activity, through a SephadexG-25 gel filtration chromatography to obtain three fractions, i.e. MH4-2-1, MH4-2-2 and MH4-2-3, to MH4-2- 1 anti-tumor activity of the strongest. This component ZORBAX SB C18 was purified by RP-HPLC column, the finally obtained high purity active peptide APMH, the molecular weight of the peptide to 391.2 Da good proliferation were the DU-145 and PC-3 cells inhibitory activity, and a concentration and time-dependent inhibitory activity of DU-145 cells than PC-3. HE staining showed that DU-145 and PC-3 cells after APMH role the vacuolar and nucleus concentrated typical features of apoptosis; Annexin V-FITC/PI flow cytometry technique double staining analysis results show , 24h after APMH role, DU-145 and PC-3 cell apoptosis, and with the increase of the concentration, the rate of apoptosis and necrosis rate increase; immunohistochemistry results showed that, APMH DU-145 and PC-3 for 48h, apoptosis negative regulatory factor Bcl-2 expression and a sharp fall, while a significant increase in apoptosis inducing pathways important protease of Caspase-3 and Caspase-9 expression levels. Accordingly, we can one of the APMH antitumor mechanism induced apoptosis, the molecular mechanism of the induction of apoptosis by Bcl-2 and raised the expression of Caspase-3 and Caspase-9.
|