Identification and characterization of the peptides with calcium-binding capacity from tilapia (Oreochromis niloticus) skin gelatin enzymatic hydrolysates.

The aim of this study was to isolate and identify the peptides with calcium-binding capacity from the different tilapia skin gelatin enzymatic hydrolysates. The complex protease was selected and its hydrolysates were further separated using gel filtration chromatography (Sephadex G-25) and reverse phase high-performance liquid chromatography. Two purified peptides with strong calcium-binding capacity were identified as Tyr-Gly-Thr-Gly-Leu (YGTGL, 509.25 Da) and Leu-Val-Phe-Leu (LVFL, 490.32 Da). The calcium-binding capacities of YGTGL and LVFL reached 76.03 and 79.50 µg/mg, respectively. The structures of the complex of purified peptides and calcium (YGTGL-Ca and LVFL-Ca) were characterized by ultraviolet-visible spectroscopy (UV-VIS), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and mass spectrometry (LC-MS/MS). The results of UV-VIS, SEM, and XRD indicated that YGTGL-Ca and LVFL-Ca were formed as new compounds. The results of FTIR and LC-MS/MS indicated the nitrogen atom of the amino group and the oxygen atom of the carboxyl group in terminates of the peptides provided primary binding sites. Moreover, the hydrophobic amino acids in purified peptides could provide more chelating spaces. This study was of great significance for the development of calcium supplement foods. PRACTICAL APPLICATION: Compared with inorganic calcium and organic calcium, the bioactive gelatin peptide chelated calcium has the characteristics of high utilization rate, high solubility, and high absorption rate. The raw materials are extracted from the tilapia processed waste, which reduce the cost, make full use of resources, and improve the bioavailability. The tilapia skin gelatin peptide calcium chelate can be directly absorbed by the human body, and the absorption efficiency is high, further improving the resource utilization rate and having high economic benefits, which is a comprehensive supplement that can also be used as a functional food.

Induction effect of TTF1-NP on human hepatoma cell apoptosis through ERS-mediated pathway / 吉林大学学报(医学版)

Objective To explore the effects of different doses of 5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone nanoparticles (TTF1-NP)on the apoptosis of human hepatoma cells and human normal hepatocytes,and to explore their mechanisms through endoplasmic reticulum stress (ERS)-meditated apoptosis pathway. Methods The human hepatoma cell lines (HepG2,Hep3B and PLC/PRF/5)and human hepatocytes (Chang Liver)were used as cell model, and divided into vehicle, 5-Fu and TTF1-NP treated groups with the concentrations of 50, 100 and 200 μmol·L-1 respectively. The inhibitory effects of TTF1-NP on the cell growth were assessed using MTT assay and the best inhibitory one (HepG-2)was selected as the main research cell lines.Flow cytometry was used to detect the TTF1-NP-induced apoptosis;Western blotting and immunocytochemistry were used to determine the expressions of ERS key proteins.Finally,the expressions of key proteins were detected by Western blotting after using the ERS inhibitor 4-PBA.Results Compared with vehicle group,the inhibitory rates of growth of 4 kinds of human hepatoma cells in different concentrations of TTF1-NP groups were increased (P <0.05 or P <0.01);moreover,the inhibitory effects of TTF1-NP were in a time-and dose-dependent manner.Compared with vehicle group,the apoptotic rates of the cells in TTF1-NP groups were increased in a dose-dependent manner (P <0.05 or P < 0.01 );the expression levels of ERS key proteins GRP78 and caspase-4 were increased with the increasing of the concentration of TTF1-NP (P < 0.05 or P < 0.01).The expression levels of ERS key proteins GRP78 and caspase-4 induced by TTF1-NP were inhibited by ERS inhibitor 4-PBA (P < 0.05 or P < 0.01 ). Conclusion TTF1-NP can induce the apoptosis of HepG2 cells;ERS pathway plays a central role in TTF1-NP-induced apoptosis of HepG-2 cells.