| The frequent mutation of influenza(abbreviated as flu)virus has caused a series of unexpected severe flu outbreak,mostly with a higher rate of pathogenic and lethal,bringing heavy burden and harm to human society,which makes its outbreak become serious threat to public health.After influenza virus infected the host,not only the replication and proliferation of the virus will cause direct damage to the tissue,but also infection flu virus will induce excessive immune and inflammatory response,causing secondary damage to the host.At present,drugs commonly-used with effect against influenza and inflammatory have stuck into the difficulty of drug resistance and side effects.In the long-term clinical application,it has been found that Chinese medicine has a good therapeutic effect on influenza virus,which means discovering feasible new anti-viral drugs from traditional Chinese medicine becomes valuable in current research.The traditional Chinese medicine Laggera pterodonta(DC.)Benth.commonly used in China as a folk herbal medicine,with ethnic and local characteristics,with its proven preventive effect against colds in basic or clinical studies in the past,.of which active ingredients have not yet been fully identified.In the early stage of its activity-tracking study,we have found that total flavonoids,sesquiterpenoids and other components of Laggera pterodonta(DC.)Benth.have inhibitory activity against influenza virus and inflammation-inducing activity.Objective1.To clarify the anti-influenza and inflammation-modulating effects of the total flavonoids of Laggera pterodonta through in vivo experiments,and then to explore the anti-influenza and inflammatory-inducing effects of the monomer compounds obtained from the total flavonoids of odorantum flavonoids.After influenza virus infected the host,we explored the role of flavonoids on the regulation of the related signaling pathways and changes in cytokines and chemokines.Further,we attempted to elucidate the pharmacological mechanisms of flamingoflavonoids in the treatment of influenza.2.According to previous activity tracking study results,it has been confirmed that the Pterodontic acid has a definite anti-influenza effect.In this study,a combination of Pterodontic acid and different chemical drugs was used.In order to find out if the combination with chemical drugs that can produce synergistic effects with Laggera pterodonta.It has been explored to provide a more precise service to clinical treatment.Methods1.A number of flavonoid compounds were isolated from the total flavonoids of total flavonoids in Laggera pterodonta(DC.)Benth by using ultra performance liquid chromatography(HPLC)and their structures were identified.2.In vivo anti-influenza virus and its efficacy in mediated inflammation experiments to determine the anti-influenza and anti-inflammatory effects of total flavonoids.The total flavonoid drugs were divided into high and low dose groups(200mg/kg · d,100mg/kg/d).After treatment with the positive drug oseltamivir phosphate(60 mg/kg · day)for 1 day,influenza-infected mice were continued to be treated for 5 days,and body weight changes,death protection,and lung viral drop were observed.Degree,lung pathological changes and other indicators clear the body of the total flavonoids anti-influenza effect in vivo.3.For the isolated flavonoids,the cytotoxicity of each compound and the inhibitory effect on influenza virus in vitro were respectively observed by MTT method and cytopathic inhibition method(CPE,Cytopathic Effect).4.Western blot was used to detect the effect of flavonoids on host cell signaling pathways associated with influenza virus infection,including p38/MAPK,NF-KB,STAT-3.Real-time fluorescence quantitative PCR technique was used to detect the effect of flavonoids on influenza virus-induced cytokine and chemokine messenger RNA expression levels.5.The sesquiterpenes of Laggera pterodonta has been proven effective against virus.In order to explore its application by studying the treatment effect to different subtypes of influenza virus in vitro with the combination of sesquiterpenes of Laggera pterodonta and different types of antiviral drugs.Results1.Six flavonoid monomer compounds were isolated from total flavonoids,and their respective structures were identified as 5,6-dihydroxy-3,7,4'-trimethoxyflavone and vitexin.5,4'-dihydroxy-6,7,-dimethoxyflavone,5-hydroxy-3,6,7,3',4'-pentamethoxyflavone,5,4'-dihydroxy-3,6,7-trime-thoxyflavone and 5,4'-dihydroxy-3,5,7,4'-tetramethoxyflavone.2.The result of total flavonoids from styrylammonium efficacy in vivo:The total flavonoids of styrax pentaphyllum has certain inflammatory regulation effects on mice infected with influenza A virus FM1 strain,and the drug group(200,100 mg/kg · d)can Effectively reduce the lung index of influenza virus-infected mice compared with the virus group;lung pathological results show that the high dose of total flavonoids silyantil high-dose treatment group(200mg/kg · d)lung tissue pathological inflammatory changes than the virus The group had a certain degree of relief P<0.05),and the total f lavone-treated group also could regulate IL-6,IP-10 and other inflammatory factors in bronchoalveolar lavage fluid of influenza-infected mice(P<0.05).However,the stinking flavonoids-treated group did not effectively inhibit the weight loss of mice caused by viral infection,and there was no significant death protection in influenza-infected mice,suggesting that the drug directly inhibited the replication and replication of influenza virus in mice.Not significant(P>0.05).3.The half-toxicity concentration of several flavonoids isolated from the total flavonoids of stinkyil in MDCK cells was in the range of 8-13 ?g/mL in vitro.In vitro inhibition of influenza virus observation showed that the isolated compounds had no obvious inhibitory effect on the influenza virus of various subtypes.Combined with their selection index SI,they could all be considered to be ineffective in inhibiting the virus.4.The flavonoid monomer compounds can inhibit the Chemokine ligand 5(CCL-5),monocyte chemoattractant protein1(MCP-1),and gamma interferon-inducing protein 10(IP-10),macrophage inflammatory protein 1(MIP-1?),chemokine CXCL9(also known as MIG),tumor necrosis factor ?(TNF-?),interleukin 6(IL-6),leukocyte Interleukin-8(IL-8),which are induced by A/H1N1 influenza virus in different concentrations(P<0.05).Phlegm Flavanone Compound 5-hydroxy-3,6,7,3,4'-pentamethoxy flavonoids and flavonoids 5,4'-dihydroxy-3,6,7-trimethoxyflavone.The phosphory-lation of p38/MAPK,STAT3,NF-?B and other signal pathways were inhibited by 3 ?g/mL,10 ?g/mL,and 30?g/mL concentrations.5.The Pterodontic acid combined with oseltamivir,ribavirin,and amantadine acted on influenza A(H1N1,H3N2,and influenza B)-infected MDCK cells in vitro,with a range of synergy values between-50 and 50.There was an additive effect between each other,and no antagonistic effect was found.ConclusionThe total flavonoids from the Laggera pterodonta(DC.)Benth have a good regulatory effect on the host inflammatory response induced by infecting of influenza virus.The material basis of the Laggera pterodonta active effects maybe is the flavonoids.What' s more,the flavonoids in Laggera pterodonta(DC.)Benth plays an effective role in regulated influenza-induced inflammation related signaling pathways and inflammatory factors.And,showing that it can play a role in the regulation of immune inflammation in the treatment of host infected influenza virus.The sesquiterpenes of Laggera pterodonta has shown an additive effect in the in vitro combination study.In the future,we are still hope to explore the synergistic effect of the drug. |
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