Synthesis Of Lipo-aconitine Derivatives And Their Inhibition Effects And Preliminary Mechanism Against MCF-7 Cells Line

As the main toxic component in traditional Chinese medicine Chuanwu,Caowu,Fuzi,aconitine has shown a wide range of physiological activities such as anti-tumor analgesia and cardiotonic effect.However,its serious neurological and cardiotoxicity restricts the clinical application.Its efficacy enhancing and toxicity reducing is still one of the future research directions.Based on the results of previous studies,taking aconitine as a raw material,a series of 3-lipo,8-lipo-substituted aconitine long-chain fatty acid ester compounds were synthesized and evaluated their proliferation inhibition against human breast cancer MCF-7 cell line.Furthermore,the structure-activity relationship and the preliminary anti-breast cancer mechanism were studied.The main findings are as follows:Using aconitine as raw material,twelve 3-lipo,8-lipo-substituted aconitine were synthesized by acylation reaction and transesterification with caprylic acid,myristic acid,palmitic acid,stearic acid,oleic acid and linoleic acid respectively.Their proliferation inhibition against MCF-7 cells were determined in vitro by MTS method,the results showed that compared with aconitine,the inhibition of 8-lipo aconitine were significantly improved,with IC₅₀0 less than 20μM,among which compound G8 was the most significant one with IC₅₀0 of 2.27μM.Further structure-activity relationship analysis showed that their antitumor activity against MCF-7 cells in vitro was significantly correlated with the length of fatty acid long chains and the number of double bonds contained.When the carbon chain length was 14 carbon,the anticancer activity was the most significant,then with the increase of the carbon chain length in saturated fatty acids chain,the anticancer activity shows a downward trend,but the introduction of a double bond can significantly increase the activity.Then,taking aconitine linoleate（G12）as example,the toxicity of aconitine long-chain fatty acid ester compounds were evaluated by acute toxicity test and histopathological examination.The results revealed the significant toxicity reducing of8-lipo aconitine for the 7 days’survival of rats injected of high dose aconitine linoleate（G12）（100 mg/kg）,in contrast with the total death by the medium dose of aconitine（0.4mg/kg）in half an hour.Histopathological examination of heart,liver,spleen,lung,kidney and thymus showed only milder lesions in the lung and liver tissues in aconitine linoleate group（100 mg/kg）than in aconitine group（0.4 mg/kg）.All these results indicated that the introduction of fatty acid chain into aconitine could significantly reduce the toxicity of aconitine.Finally,the inhibition mechanism of 8-lipo aconitine against MCF-7 cells was studied.The release of LDH in the supernatant of cell culture fluid was used to evaluate the effect of aconitine linoleate（G12）on the membrane integrity of MCF-7 cells.The effect of aconitine linoleate（G12）on the cell cycle and apoptosis of MCF-7 cells was detected by flow cytometry.The effect of aconitine linoleate（G12）on the ultrastructure of MCF-7cells was observed by transmission electron microscopy.Combined with molecular docking technology,enzyme activity inhibition assay and Western blotting,the target of aconitine linoleate（G12）was determined.The results showed that lipo-aconitine may induce cell death by cell autophagy and cell necrosis rather than apoptosis.At the same time,it can destroy the cell membrane integrity of MCF-7 cells,cause organelle damage,interfere with DNA replication during cell proliferation,and arrest cell cycle in G0G1phase.In addition,lipo-aconitine（G12）is TOPO IIαinhibitor,which can inhibit the activity of TOPO IIα,as significant as etoposide at the high concentration;meanwhile,it is a selective expression inhibitor of TOPO IIα,which can significantly inhibit the expression of TOPO IIαprotein in MCF-7 cells for affecting cell proliferation.