FOOD SCIENCE ›› 2019, Vol. 40 ›› Issue (11): 167-174.doi: 10.7506/spkx1002-6630-20180421-281

• Nutrition & Hygiene • Previous Articles     Next Articles

Enzymatic Preparation of Peptide from Cyclina sinensis Proteins and Its Inhibitory Activity toward Prostate Cancer DU-145 Cells

ZHANG Yaru1, YAN Haiqiang2, YANG Zuisu1, YU Fangmiao1, DING Guofang1,3, GONG Jianfang2,*   

  1. 1. Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food Science and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China; 2. College of Donghai Science and Technology, Zhejiang Ocean University, Zhoushan 316004, China; 3. Marine Fisheries Research Institute of Zhejiang, Zhoushan 316022, China
  • Online:2019-06-15 Published:2019-06-28

Abstract: The purpose of this study was to investigate the enzymatic preparation of peptide from Cyclina sinensis proteins and to evaluate its activity against prostate cancer DU-145 cells. Different proteases were screened to increase the amino nitrogen content of hydrolysates and the hydrolysis conditions were optimized using orthogonal array experiments. An active peptide was purified from the hydrolysates through ultrafiltration, gel chromatography and preparative high performance liquid chromatography. The amino acid sequence of the purified peptide was identified as Ile-Leu-Tyr-Met-Pro. The antiproliferative activity of this peptide against DU-145 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and the morphologic change of DU-145 cells was observed by using an inverted microscope, Hoechst 33258 staining and transmission electron microscopy. A fluorescence method was used to detect the generation of intracellular reactive oxygen species (ROS). Cell apoptosis was evaluated by determination of mitochondrial membrane potential using flow cytometry. Furthermore, the expression of nm23H1 was detected by an immunohistochemical method. Our results showed that papain was the optimal protease for hydrolyzing Cyclina sinensis proteins and the optimum hydrolysis conditions were determined as follows: solid-to-solvent ratio 1:4, pH 7.0, enzyme dosage 1 500 U/g, temperature 45 ℃ and hydrolysis time 4 h. The purified peptide had a significant inhibitory effect on DU-145 cells in a time- and dosedependent manner. The treated cells exhibited apoptotic morphological characteristics. The concentration of the peptide was positively correlated with the generation of ROS in cells. Flow cytometry revealed that the percentage of cells with decreased mitochondrial membrane potential was increased from 4.22% to 25.07%. Immunohistochemical results indicated that the expression of nm23H1 was increased. Therefore, the enzymatic peptide from Cyclina sinensis could significantly inhibit the proliferation of DU-145 cells by inducing apoptosis.

Key words: Cyclina sinensis, enzymatic hydrolysis, prostate cancer, cell apoptosis

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