Study On The Active Ingredients Of Periplaneta Americana L. Against Hepatocellular Carcinoma And Analysis Of Heavy Metals And Harmful Elements

Objective:Periplaneta americana L.is the Periplaneta genus insect of the family Blattidea in Blattodea from Insecta.There are polypeptides,nucleosides and amino acids in Periplaneta americana.It has the functions of anti-tumor,promoting tissue repair and immune regulation.The preparation developed with Periplaneta americana as raw material has achieved good results in clinical adjuvant treatment of liver diseases.However,there are relatively few reports on the material basis and molecular mechanism of its pharmacodynamic components.In addition,with the increasing use of Periplaneta americana in medical and daily supplies,artificial breeding has become the main source of Periplaneta americana.At present,the heavy metal limit standards in various countries and regions are mainly based on plant medicinal materials,and there is no unified standard for heavy metal limit in animal medicinal materials such as Periplaneta americana.We study the screening of anti-hepatocellular carcinoma active ingredients from Periplaneta americana in vitro,chemical composition analysis and its mechanism of active ingredients,as well as to determine and analyze the content of heavy metals and harmful elements.Methods:1.To explore the active ingredients group and mechanism of Periplaneta americana against hepatocellular carcinoma by network pharmacology.The targets of composition in Periplaneta americana and hepatocellular carcinoma were obtained through literatures.2.The 95%ethanol extract of Periplaneta americana was prepared with water,30%,50%,70%and 90%ethanol eluted parts by HP-20 macroporous resin,respectively.Using Hep G2 as the target cell,the anti-hepatocellular carcinoma active ingredients were screened by MTT assay.3.To analyze the chemical components of the anti-hepatocellular carcinoma active ingredients of Periplaneta americana by an Ultra-high performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry（UPLC-Q-Orbitrap HRMS）method with the database,literature reports and related reference materials were used for identification.4.The expression of related proteins in the PI3K/Akt pathway was detected by Western blot to further study the molecular mechanism of the active ingredients.5.The contents of Cd,As,Pb,Hg and Cu in 13 batches of Periplaneta americana were determined by ICP-MS,and conduct analysis according to international and domestic heavy metal limit standards.Results:1.The network pharmacological results showed that PI3K/Akt,RAP1,TNF,FOXO,RAS,MAPK and Erb B were seven signaling pathways closely related to hepatocellular carcinoma,and Akt1,MAPK1,MAP2K1,EGFR and RAF1 were potential drug targets enriched in these pathways.Cyclo（Val-Tyr）,Cyclo（L-Tyr-L-Tyr）,Cyclo（L-Pro-L-Tyr）,Cyclo（Gly-Leu）and Cyclo（Trp-Asn）in Periplaneta americana are highly associated substances with potential drug targets.2.The ethanol extract of Periplaneta americana was enriched and separated by HP-20 macroporous resin,and the inhibition rate was increased from（17.71±0.16）%to（30.38±0.50）%with70%ethanol elution fraction（P<0.05 or P<0.01）.3.A total of 35 compounds were identified from the active ingredients by UPLC-Q-Orbitrap HRMS,including 10 amino acids,1 alkaloid,6 nucleosides and their bases,4 dipeptides and cyclodipeptides,8organic acids,2 isoflavones,and 4 others.4.The results of WB showed that compared with blank control group,the relative expression levels of PI3K and p-Akt proteins in active ingredients of Periplaneta american at 0.15624 mg·m L^-1 and 0.31250 mg·m L^-1groups were significantly decreased（P<0.05 or P<0.01）.There was no significant change in AKT protein expression level（P>0.05）.5.The results of ICP-MS showed that the contents of the five elements were Cd 0.009～0.979 mg·kg^-1,As 0.093～2.644mg·kg^-1,Pb 0.361～2.308 mg·kg^-1,Cu 10.265～77.360 mg·kg^-1,Hg not detected～0.044 mg·kg^-1.Conclusion:1.This study found that the peptides and PI3K/Akt signaling pathways might be the main component groups and molecular mechanism of Periplaneta americana anti-hepatocellular carcinoma by network pharmacology.MTT showed that 70%ethanol elution site was the main active ingredients of Periplaneta americana anti-hepatocellular carcinoma.UPLC-Q-Orbitrap HRMS and WB methods were used to study the chemical components and mechanism of the active ingredients of Periplaneta americana.It was found that the anti-hepatocellular carcinoma effect of Periplaneta americana was a combination of multiple components,which could reduce the relative expression of PI3K and p-Akt proteins and regulate the PI3K/Akt pathway to play an anti-hepatocellular carcinoma role.2.According to international and domestic standards,it is common for Cu to exceed the standard due to large difference in the set limits,and most standards do not require it.There are regulations on the limit of Pb,but the set value is relatively wide,and the limit can be appropriately lowered.The limits of Cd,As and Hg in the international standards have little difference compared with the domestic standards,and the unqualified rates are similar.