Isolation and Purification of Alcohol-Extracted Protein from Eupolyphaga Sinensis Walker and Its in vitro Anti-Hepatocellular Carcinoma and Anti-Hepatic Fibrosis Activities

  OBJECTIVE  In vitro cell experiments were used to evaluate the anti-hepatocarcinoma and anti-hepatic fibrosis activities of the macromolecular proteins isolated from the alcohol extract of Eupolyphaga sinensis Walker, and gradually focused on the effective parts and components.

  METHODS  The protein fractions were recovered from the alcohol extracts of Eupolyphaga sinensis Walker by salting out, and the crude protein products of Eupolyphaga sinensis Walker were divided into three different protein sites EEPA, EEPB and EEPC, according to their molecular weight by ultrafiltration. Based on the activity of inhibiting the proliferation of abnormal liver cells, the best part was screened by cell experiments. Then, a purified protein (PC3-Ⅲ) was isolated from EEPC by DEAE anion chromatography and Sephadex G-100 gel chromatography, and identified by LC-MS/MS. Finally, cell experiments were performed to evaluate the in vitro anti-hepatocarcinoma and anti-hepatic fibrosis activities of EEPC and PC3-Ⅲ.

  RESULTS  Among EEPA, EEPB, and EEPC, the protein site EEPC with the largest molecular weight showed the most potent inhibitory activity against abnormal liver cell proliferation. The molecular weight of PC3-Ⅲ was about 10 kDa. LC-MS/MS identification showed that the coverage rate of PC3-Ⅲ to the homologous protein A0A0M3STY1 reached 35%. The peptide sequences obtained by checking the reference were QYSINFISAR, CNGDSCVCTFR and SNNFR. In vitro experiments suggested that PC3-Ⅲ not only exerted anti-hepatoma by inhibiting tumor cell growth, inducing tumor cell apoptosis, and inhibiting tumor cell migration, but also inhibited the proliferation of activated hepatic stellate cells and delayed the process of liver fibrosis. The efficacy was comparable to that of EEPC.

  CONCLUSION  This study confirms the anti-tumor and anti-liver fibrosis activities of the macromolecular protein of Eupolyphaga sinensis Walker and isolates the homogeneous protein product PC3-Ⅲ, laying a foundation for the advanced understanding of the material basis for the efficacy of Eupolyphaga sinensis Walker and the research and development of related products.