Separation And Identification Of Nucleoside Compounds From Cordyceps Militaris And Its Inhibitory Effect On Human Hepatocellular Carcinomas HepG2 Cells

Hepatic carcinoma is one of the common malignant tumors,due to its high incidence,insidious onset,toxic and side effects of advanced chemotherapy drugs,the cure rate of patients is generally low and poor after recovery.Therefore,the separation and extraction of safe and efficient active ingredients from natural products for the development of new products for hepatic carcinoma prevention,treatment,and post-healing conditioning has become a hot research topic at home and abroad.Cordyceps militaris(L.)Link is a fruiting body formed by Cordyceps fungus infecting insect larvae or insect pupa.Studies have shown that Cordyceps militaris extract(CME)has broad-spectrum antitumor activity,and the efficacy is closely related to its rich nucleoside compounds.In this paper,Cordyceps militaris was used as the raw material,and the orthogonal experiment method was used to optimize the extraction process of nucleoside compounds,four monomeric compounds were separated and purified by preparative liquid phase method,and then ultra-high phase liquid chromatography-triple quadrupole flight Time mass spectrometry(UPLC/Q-TOF/MS),nuclear magnetic resonance spectroscopy(NMR)were used to identify the structural of unknown compounds.Using HepG2 cells as amodel,the antitumor activity of compound I(PTUh),compound II(PTGh)and cordycepin was compared,and the antitumor mechanism of PTUh and PTGh was preliminarily inferred.The research results of this paper were as follows:(1)Taking the extraction rate of cordycepin as an indicator,the extraction condition was optimized by orthogonal experiment.Considering the stability of the extraction solution comprehensively,the best extraction process of nucleoside compounds in Cordyceps militaris was determined as follows:extracted with 1%acetic acid aqueous solution,the material-liquid ratio was 1:30,the extraction temperature was 40?,the extraction time was 40 min,the extraction rate of cordycepin obtained by this extraction process was 87.33%±3.83%.(2)Using two liquid chromatography columns,C18 and C18-HCE,to compare the separation effects of the crude extract of Cordyceps militaris.The results showed that the C18 column had poor selectivity and resolution,but it could effectively retain cordycepin and adenosine,and could be used for one-dimensional preparation;the C18-HCE column has high resolution for the six compounds in the crude extract,ans coule be used for two-dimensional preparation to separate and prepare nucleoside compounds other than cordycepin and adenosine.(3)Using high-efficiency preparative liquid phase method,one-dimensional preparation was used to separate cordycepin and adenosine from crude extract of Cordyceps militaris,and two-dimensional preparation was used to separate PTUh and PTGh,the chromatographic purity was 98.11%,90.49%,96.34%and 98.33%.UPLC/Q-TOF/MS and NMR were used to identify the structure of PTUh and PTGh,and it was confirmed that both were amino acids and their derived nucleoside compounds.The Sci Finder database comparison confirmed that PTGh was a new compound.(4)MTT colorimetric method and cell scratch test were used to analyze the effects of cordycepin,PTUh and PTGh on the relative viability and lateral migration ability of HepG2 cells.The results showed that all three could effectively reduce the viability of HepG2 cells,inhibit cell proliferation,and showed a time-dose dependence;72 hours after administration,the half maximal inhibitory concentration of cordycepin,PTUh and PTGh on HepG2 cells(IC₅₀)were 0.24 m M,0.09 m M,and 0.51 m M,respectively;within the same time range,all three could inhibit the lateral migration of HepG2 cells,of which cordycepin had the best inhibitory effect,followed by PTUh and PTGh.(5)Flow cytometry was used to measure apoptosis and cell cycle of HepG2 cells.The results showed that after 48 hours of administration,0.2 m M cordycepin,0.2 m M PTUh and 0.2 m M PTGh increased the total apoptosis rate of cells from 4.75%to29.81%,29.15%,13.13%(p<0.01)significantly;cordycepin,PTUh and PTGh could delay the cell cycle,thereby effectively inhibit the proliferation of HepG2 cells,PTGh could cause G0/G1 phase arrest of the cell cycle,and cordycepin and PTUh G2/M phase block.(6)The results of mitochondrial membrane potential(MMP)and quantitative real-time polymerase chain reaction(q RT-PCR)test showed that cordycepin and PTUh could reduce the mitochondrial membrane of HepG2 cells(p<0.01),significantly,in a dose-dependent manner,and the effect of PTUh was better than cordycepin;PTGh had little effect on the mitochondrial membrane potential of HepG2 cells;cordycepin,PTUh and PTGh could increase the expression levels of Fas,Cyt C,p53,caspase-9,caspase-8,and caspase-3 genes obviously,and increase the ratio of Bax and Bcl-2 gene expression,and promote HepG2 cell apoptosis.In summary,using C18 and C18-HCE two preparative chromatographic columns,using high-performance liquid preparation method could simultaneously prepare a variety of nucleoside compounds in Cordyceps militaris,and one-dimensional preparation could obtain high purity cordycepin.PTUh and PTGh isolated and purified from Cordyceps militaris have been confirmed by in vitro experiments,and they could inhibit the proliferation of HepG2 cells,especially PTUh,which could promote the apoptosis of HepG2 cells,the ability was equivalent to cordycepin.According to the experimental results,the specific anti-tumor mechanism of PTUh and PTGh was inducing apoptosis of tumor cells,blocking cell cycle at G2/M and G0/G1 phase respectively,ans regulating the expression of genes related to endogenous mitochondrial pathway and exogenous death receptor pathway.The results of this paper provided a theoretical basis for the application and development of Cordyceps militaris in the prevention,treatment and post-healing conditioning of hepatic carcinoma.