Study On The Quality Control Method And Biological Activity Of Venom From Vespa Bicolor Fabricius

Vespa bicolor venom(VBV)is the secretion of the venom glands of Vespa bicolor Fabricius.Objective: In order to establish the quality control method of VBV and improve the quality control level.At the same time,the biological activity research of VBV is carried out to clarify its potential medicinal value and promote the further development and utilization of this medicinal resource.Methods:(1)Twenty-two batches of VBV from summer and autumn in Guangdong,Guangxi,and Guizhou were collected as the research objects,and the character description,SDS-PAGE gel electrophoresis identification,HPLC fingerprint analysis,and index content determination were carried out.(2)Combined LC-MS/MS,NMR,and MALDI-TOF-MS detection methods to identify the components of VBV.(3)The antibacterial activity,antioxidant activity,anti-inflammatory activity,and tumor cytotoxicity of VBV were extensively screened.(4)Research on the anti-liver tumor activity of VBV alone and in combination with the chemotherapeutic drug cisplatin(DDP).The effects of VBV alone and in combination on the viability,proliferation,migration,invasion,and apoptosis of tumor cells were investigated in vitro by the CCK-8 method,cell proliferation assay,cell scratch experiment,transwell invasion assay,and cell apoptosis assay,respectively.(5)Perform subcutaneous tumor-bearing experiments in mice to further clarify the in vivo anti-liver tumor activity of VBV alone and combination with DDP.Results:(1)The typical appearance,solubility,p H,and UV absorption spectra of VBV was described.For the first time,a gel electrophoresis method with 6 characteristic protein bands and an HPLC fingerprint analysis method with 15 common peaks were established.In addition,the content of the 5-hydroxytryptamine(5-HT)and mastoparan-like peptide(Vb-MLP 12a)were determined in the range of3.23%-6.14% and 32.54%～51.27%,respectively.(2)Identified 6 protein bands in the electropherogram.Proteins 1 to 4 were hyaluronidase A,phospholipase A1(two isoforms),and antigen 5 respectively.Proteins 5and 6 were two unreported proteins.Proteins 5 and 6 are two unreported proteins,which may belong to the exclusive proteins of VBV.Two main common peaks in the fingerprint were identified,and common peaks 5and 10 were 5-HT and Vb-MLP 12 a,respectively.(3)In the antibacterial activity experiment,it was found that VBV has strong inhibitory and killing activities against the anaerobic bacteria P.acnes.Minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)are both 12.5 μg/m L;The half-maximal inhibitory concentration(IC50)of VBV on DPPH and ABTS free radicals were 0.178 mg/m L and1.41 mg/m L,respectively.And at a low concentration(0.0625 mg/m L),it has a significant scavenging effect on hydroxyl radicals,but the concentration dependence is weak.In the study of anti-inflammatory activity,VBV showed significant anti-inflammatory activity,and Vb-MLP 12 a had a significant activity of promoting the proliferation of macrophage RAW264.7.In the study of tumor cytotoxicity,VBV showed a broad-spectrum anti-tumor activity,and the hepatocellular carcinoma cell Hep G2 was more sensitive,with an IC50 of 42 μg/m L.(4)In the study of anti-tumor activity in vitro,it was found that VBV(15μg/m L～50 μg/m L)had significant anti-proliferation,anti-invasion,and apoptosis-inducing activities on Hep G2 cells,and at a low concentration(15 μg/m L),it had a significant pro-proliferation activity on normal hepatocyte L-02.In addition,the combination with DDP can significantly enhance the antitumor activity of DDP,and the IC50 is enhanced from18.7 μg/m L to 6.1 μg/m L.When the DDP concentration was lower than 4μg/m L,the combined administration had a significant protective effect on L-02 cells.(5)In vivo tumor-bearing experiments,it was found that the tumor inhibition rate of VBV(0.5 mg/kg,4 d/time)was comparable to that of DDP(1 mg/kg,2 d/time).And after combined use(VBV 0.5mg/kg + DDP 1 mg/kg,4 d/time),the tumor inhibition rate was significantly increased by about double.In addition,the application of VBV can significantly improve the immune capacity of tumor-bearing mice,especially the thymus,and the central immune function of tumor-bearing mice can be restored to normal levels after administration of VBV.Conclusion: A quality control method including VBV authenticity identification and VBV quality evaluation is established,which greatly improves the quality control level of VBV and provides a reference for the quality control research of others.At the same time,VBV under this quality control has a wide range of biological activities such as antibacterial,anti-inflammatory,antioxidant,and antitumor.In addition,VBV can inhibit the proliferation and migration of Hep G2 cells,induce the apoptosis of Hep G2 cells,improve the immune function of tumor-bearing mice,and combined with DDP can significantly improve the therapeutic effect of liver tumors.