Studies On The Preparation And Anti-prostate Cancer Activity Of Peptide From Mussel Hydrolysate

In this paper,studies on preparation of antitumor activity peptide by use of enzymatic hydrolysis of mussel protein, ultrafiltration, DEAE Sepharose FF ion-exchange, Sephadex G-25 gel chromatography and reversed-phase HPLC method were carried out,and antitumor mechanism of this peptide were also done.Studies of enzymatic hydrolysis were mainly on the effect of each factor in solid-liquid ratio, PH, hydrolyzed temperature, time and enzyme addition on degree of hydrolysis and antitumor activity of hydrolysates. Mussel was hydrolysate with three commercial proteinases including Alcalase, Trypsin and Pepsin. The hydrolysis conditions of three kinds of enzymes were investigated by orthogonal test. The experimental results that the optimal hydrolysis conditions of three enzymes which based on the evaluation index of degree of hydrolysis were: solid-liquid ration 1:4, pH10, enzyme dosage 2000U/g, 50℃, time 8h for Alcalase; solid-liquid ration 1:4, pH 8.5, enzyme dosage 1000U/g, 45℃, time 8h for Trypsin; solid-liquid ration 1:4, pH 5, enzyme dosage 1500U/g, 45℃,time 8h for Pepsin, respectively. The optimal hydrolysis condition of three enzymes on the basis of the index of antitumor activity of hydrolysates were: solid-liquid ration 1:4, pH 10, enzyme dosage 1500U/g, 50℃,time 8h for Alcalase; solid-liquid ration 1:4, pH 8, enzyme dosage 1500U/g, 40℃, time 2h for Trypsin; solid-liquid ration 1:4, pH 5, enzyme dosage 1500U/g, 55℃, time 8h for Pepsin, respectively. Mussel was respectively hydrolysated with three enzymes in the optimal conditions on the basis of antitumor activity. The hydrolysate of Trypsin had the highest degree of hydrolysis, while the hydrolysate of Alcalase had the best activity of antitumor.Ultrafiltration was first used to purify the active peptide in the preparing technology. 10KDa, 5KDa and 3KDa molecular weight cut-off membranes were used to fractionate the mussel hydrolysate, and four ranges of molecular weight fractions (MH1,>10KDa; MH2,5KDa-10KDa; MH3,3KDa-5KDa; MH4,<3KDa) were obtained. MH4 which had the lowest molecular weight showed the highest activity of antitumor. Teatment of the DU-145 and PC-3 cells with MH4 at 0.5mg/ml for 48h inhibited the proliferation of DU-145 and PC-3 cells by 33.17% and 30.24%. MH4 was further fractionated into four portions (MH4-1, MH4-2, MH4-3 and MH4-4) by DEAE Sepharos FF ion exchange. MH4-2 had the highest antitumor activity and was further fractionated into three fractions (MH4-2-1, MH4-2-2 and MH4-2-3) by Sephadex G-25 gel filtration chromatography. MH4-2-2 which showed the best actitumor activity was furified with RP-HPLC on a zorbax C18 column, and a peptide (APMH) was finally obtained. APMH was identified its molecular weight as 391.2Da. MTT assay showed that APMH inhibited proliferation of DU-145 and PC-3 cells in both concentration-dependent and time-dependent manners.Cell apoptosis morphological feature was observed under HE staining when DU-145 and PC-3 cells were treated with APMH for 24h and 48h. Flow cytometry analysis with Annexin V-FITC and PI double staining showed that APMH induced early apoptosis and cellular necrosis in concentration-dependent manner on both DU-145 and PC-3 cells. When treatment with APMH for 48h, the expression of Bcl-2 was significantly down-regulated, whereas the expressions of caspase-3 and caspase-9 were all up-regulated.