| ObjectiveIngredient C8(Sesquiterpenoid mixture)isolated form Yunnan traditional drug Laggera pterodonta(DC.)Benth will be studied for further component analysis,and isolation of chemical monomer with antiviral activities.To study the inhibitory effect of chemical monomer on different subtypes of influenza virus,further study its blocking effect on different stages of replication cycle of influenza virus,and clarify the related host signaling pathway and cytokines caused by influenza virus infection.Finially,to clarify the mechanism of anti-influenza virus and anti-inflammation associated with influenza virus infection,and provide the basic research reference for the clinical application.MethodsThe components of C8(Sesquiterpenoid mixture)were analyzed by ultra performance liquid chromatography(HPLC),and the chemical monomers were further separated by Silica gel column chromatography.Isolated chemical monomers were screened by the cytopathology of different respiratory viruses and different subtypes of influenza virus in vitro.In addition,the chemical monomer having antivral activities was tested for its effective monomer to carry out prevention,treatment and direct effects.Furthermore,hemagglutination inhibition test,viral polymerase activity inhibition test,NF-?B activation test,immunofluorescence assay and neuraminidase activity inhibition test were employed to find out the drug target.Finally,the influence of drug on host was tested by real-time quantitative PCR in mRNA level and Bio-Plex suspension chip system at the protein level.Results(?)Isolation,identification and antiviral analysis of sesquiterpenoid monomers isolated form Laggera pterodonta(DC.)BenthC8 was subjected to silica gel column chromatography to separate mononers.Compound 1 was obtained according to iodized smoked color,and the compound 2 was purified by recrystallization of ethyl acetate.Content of two compounds were detected by ultra performance liquid chromatography and quadrupole mass spectrometry,and were 107.98 mg/g and 111.3 mg/g,respectively.Through the identification of the two compounds by nuclear magnetic resonance technology,it was found that the two compounds 1,2 were Pterodontic acid and Pterodondiol.The results showed that the maximum non-toxic concentrations of the two compounds were 278.9?g/mL and 100?g/mL in cytotoxicity test(MTT).In anti-respiratory virus efficacy screening test,Pterodontic acid showed antiviral activities on influenza A(H1N1)PR8 strain,influenza A(H1N1)pdm09 California04 strain and GIRD07 strain,and its half effective inhibition concentration Were 37.14?g/mL,21.75?g /mL and 9.47?g/mL respectively.While it didn't show any those inhibitory effects on influenza a virus(H3N2,H7N3,H6N3 and H9N2),respiratory syncytial virus,I type herpes simplex virus,enterovirus 71 and Coxsackie virus 16 at maximum non-toxic concentrations.In order to confirm the inhibitory effect of Pterodontic acid on influenza a virus,we used progeny virus reduction test to detect the number of progeny virus in the supernatant of the cells with different concentration of Pterodontic acid treatment.The data showed that Pterodontic acid had obvious inhibitory effect on the H1N1 influenza virus PR8 strain in the range of 25-100?g/mL compared to the virus control group(P <0.01).Pterodondiol didn't show antiviral activity against influenza virus and other respiratory and enteric viruses in vitro.(?)Study on anti-influenza virus mechanism of Pterodontic acidThe results showed that Pterodontic acid had no inhibitory effect on the virus particles and had no protective effect on the cells.Only in the treatment mode,Pterodontic acid could inhibit replication of virus,and its half effective concentration was 35.93?g/mL.In addition,Pterodontic acid was tested by a series of experiments for detecting its target on replication cycle of the virus.The hemagglutination inhibition test confirmed that the compound had no inhibition effect on virus attachment.In the inhibition test of neuraminidase activity,it was found that Pterodontic acid at a concentration of 668.1?g/mL had a mild inhibitory effect on the neuraminidase of influenza A H1N1 pdm09 virus California04 strain,indicating that it could inhibit the neuraminidase of the activity to prevent the releasing of virus.The detection for activity of the polymerase showed that the Pterodontic acid had a mild inhibition effect on the polymerase activity of influenza A(H1N1)virus in the range of 10-80?g/mL.Since activation of the NF-?B signaling pathway is critical for the replication of influenza viruses,inhibition experiments on the activation of this pathway was corried out.It was found that when the concentration of the Pterodontic acid was at 20-80?g/mL,either by the influenza virus or TNF-?-stimulated NF-?B pathway activation could be inhibited.Since the NF-?B pathway was inhibited,the virus could not produce nuclear export and was retained in the nucleus.Therefore,in order to further verify the inhibitory effect of the Pterodontic acid on NF-?B pathway activation,the influenza virus protein core stained experiment was conducted.In this experiment,virus-infected cells were found under the fluorescence microscope,and the virus nucleoprotein could not be output through the nucleus,which resulted in its retention in the nucleus.There was no way to produce the progeny virus,and this inhibiting concentration range is 25-100?g/mL in a dose-dependent manner.(?)Effect of Pterodontic acid on Cytokine Changes in Host immunol reponse induced by influenza virus infectionAlthough Pterodontic acid can only inhibit the influenza A H1N1 influenza virus with a mild inhibitory effect on virus' neuraminidase and polymerase,it can significantly inhibit NF-?B pathway activation caused by virus infection.Therefore,the cytokines expression associated with influenza virus infection had been detected.The results showed that the compound could significantly inhibit the mRNA of IP-10,MIP-1? and IL-6 induced by the infection of influenza a virus H1N1 PR8 strain at the concentration of 25-100?g / mL.The expression of IP-10 and MIP-1? induced by avian influenza A H9N2 virus infection was significantly inhibited at the concentration of 25-100 ?g/mL.High,100?g/mL concentration of Pterodontic acid can significantly inhibit the expression of CCL-5 and TNF-?.In order to further confirm its inhibitory effect on inflammatory factors,we used the bio-plex platform to detect the expression of related cytokines at the protein levels.The results showed that the Pterodontic acid could significantly inhibit the expression of A-H1N1 at 25-100?g / mL.The protein levels of IP-10,IL-6 and TNF-? was inhibited by Pterodontic acid at the concentration of 100?g/mL.The level of protein expression of IP-10 and IL-6 induced by avian influenza virus infection increased significantly inhibited by Pterodontic acid at 100?g/mL.ConclusionThe separation of two major components from sesquiterpene mixture isolated from Laggera pterodonta(DC.)Benth were Pterodontic acid and Pterodondiol.Pterodontic acid was identified that it had anti-influenza A(H1N1)virus effects achieved by inhibition Neuraminidase and viral polymerase activity,as well as the viral nucleoprotein output in vitro.In addition,the compound was also effective in inhibiting the increase in inflammatory cytokines caused by human or avian influenza virus infection.Pterodondiol had no inhibitory effect on influenza virus,respiratory syncytial virus and enterovirus.The results showed that the direct antiviral effect of Pterodontic acid was not strong and only effective against influenza A(H1N1),but it had a good inhibitory effect on the cytokines and chemokines of the host caused by viral infection.The effects of Pterodontic acid on cytokines and chemokines can be considered as immune response regulator to influenza virus infection for further research. |
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