| Objective:Laggera Pterodonta belongs to Laggera,Compositae.As the representative of ethnic plant medicine,it has a long medicinal history in Yunnan.Laggera Pterodonta and its compounds have a variety of pharmacological activities,especially in the wide range of antiviral effects.In this study,we aim to explore the antiviral effect of Laggera Pterodonta from the following three aspects:1.To screen out the specific anti DENY-? active extracts through the confirmation of the anti DENV-? pharmacodynamic effect of Laggera Pterodonta.2.To screen out the active derivatives of Laggera Pterodonta against influenza virus through the pharmacodynamic effect in vitro,and then determine the anti influenza virus spectrum.3.To screen out the active compounds of Laggera Pterodonta against coronavirus through the pharmacodynamic effect in vitro.The purpose of the above research is to screen out effective antiviral components from Laggera Pterodonta,its compounds and even from derivatives,so as to provide new scientific ideas and pharmacodynamic research basis for the development of new antiviral drugs.Methods:1.Effects of different samples of Laggera Pterodonta on DENV-?(1)At the cellular level,the antiviral activity of each sample was tested at 24h,48h and 72h by observing the cytopathic effect(CPE),using two modes,mixing with the virus solution to inoculate cells(direct mode)and the virus adsorbed,then added the samples(treatment mode)at the concentration of 5?g/mL and 2.5 ?g/mL.(2)Real-time quantitative polymerase chain reaction(qPCR)was used to detect the effect of the samples on DENV-? replication at different time(24h,48h,72h)in the direct mode and the treatment mode.2.Anti-influenza virus effect of the derivatives of pterodontic acidAt the cellular level,MTT colorimetric method was used to determine the median toxic concentration(TC50)of 24 derivatives of pterodontic acid.The CPE of these derivatives on influenza A virus was evaluated,and the active derivatives were screened out by selection index(SI),and then confirmed their anti influenza virus spectrum.3.The anti coronavirus effect of Laggera Pterodonta and sesquiterpenoidsAt the cellular level,MTT colorimetric method was used to determine TC50 of the dry extract powder and sesquiterpenes of Laggera Pterodonta.The anti coronavirus effect of the dry extract powder and sesquiterpenes was evaluated by the determination of CPE,and then the components with anti coronavirus activity were screened out by selection index.Results:1.Effects of different extracts of Laggera Pterodonta on DENY-?(1)At the cellular level,the final concentration of the samples was 5?g/mL in the direct mode,at 48h after inoculation,the degree of cytopathy of pterodontic acid and ribavirin was lower than that of the control group(P<0.01).At 72 hours after inoculation,the cytopathic degree of the essential oil,silica gel separation of the 10th fraction from the extract,silica gel separation of the 70th fraction from the extract,supercritical fluid extraction and ilicic acid was lower than that of the control group(P<0.01).When the final concentration of the extract was 2.5 ?g/mL,at 72h after inoculation,the cytopathic degree of the essential oil,silica gel separation of the 70th fraction from the extract and pterodontic acid was lower than that of the control group(P<0.05).The final concentration of the samples was 5?g/mL in the treatment mode,at 24h after treatment,silica gel separation of the 10th fraction from the extract,silica gel separation of the 70th fraction from the extract,and supercritical fluid extraction,the pathological changes of cells were lower than those of the control group(P<0.05),ilicic acid and ribavirin cells were lower than those of the control group(P<0.01).At 48h after treatment,silica gel separation of the 10th fraction from the extract,silica gel separation of the 70th fraction from the extract,ilicic acid,pterodontic acid and ribavirin,the degree of cytopathy was lower than that of the control group(P<0.01).At 72h after treatment,the degree of cytopathy of silica gel separation of the 10th fraction from the extract was lower than that of the control group(P<0.01),and the degree of cytopathy of the essential oil and pterodontic acid were lower than that of the control group(P<0.05).When the concentration was 2.5?g/mL,compared with the control group,at 72h after treatment,the degree of cytopathic changes of the silica gel separation 70th fraction from the extract,pterodontic acid,pterodondiol and ribavirin was lower(P<0.05).(2)The results of qPCR showed that at 24h after inoculation of the direct mode,silica gel separation of the 70th fraction from the extract and ribavirin significantly inhibited the replication of DENY-?(P<0.05).At 48h after inoculation,the essential oil,silica gel separation of the 10th fraction from the extract,supercritical fluid extraction,ilicic acid and ribavirin significantly inhibited the replication of DENY-?(P<0.05).At 72h after inoculation,silica gel separation of the 10th fraction from the extract,silica gel separation of the 70th fraction from the extract and ilicic acid significantly inhibited the replication of DENV-?(P<0.05).At 24h after treatment of the treatment mode,pterodondiol significantly inhibited the replication of DENV-?(P<0.01).At 48h after treatment,the results showed that all samples(except the silica gel separation of the 10th fraction from the extract)and ribavirin significantly inhibited DENV-? replication(P<0.05).At 72h after treatment,all the samples had no effect on virus replication.2.Anti-influenza virus effect of the derivatives of pterodontic acidThe results of MTT showed that the TC50 of 1-2,1-9 and 1-63 of the derivatives of pterodontic acid were 200?g/mL,459.5?g/mL and 23.7?g/mL,respectively.The IC50 were 187?g/mL,132.8?g/mL and 2.2?g/mL,respectively,and SI were 1.1,3.5 and 10.6,respectively.These three derivatives showed inhibitory effects on influenza A(H1N1)virus in vitro.In the aspect of confirmation of anti influenza virus spectrum,the derivatives 1-2,1-9 and 1-63 showed inhibitory effect on influenza A(H1N1)virus in vitro,but had no inhibitory effect on Aichi strain of influenza A virus and H9N2 strain of avian influenza A virus.3.The anti coronavirus effect of Laggera PterodontaThe results of MTT showed that the TC50 of dry extract powder of Laggera Pterodonta,pterodontic acid and pterodondiol were 3440?g/mL,47.3 ?g/mL and 455?g/mL,respectively.The IC50 of pterodontic acid was 3.9 ?g/mL,SI was 12.1,and it could inhibit the coronavirus HCoV-OC43.But the dry extract powder of Laggera Pterodonta and pterodondiol had no inhibitory effect on the coronavirus.Conclusion:In this study,the antiviral activity of ethnic plant medicine Laggera Pterodonta was evaluated from DENY-?,influenza virus to HCoV-OC43 respectively.The results showed that it has broad-spectrum antiviral effect in vitro,and its extracts,compounds and even derivatives have the potential to be developed into antiviral drugs. |
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