| ObjectiveTo isolate and purify the active compound against the influenza virus infection from the Traditional Chinese Medicine Laggera pterodonta(DC.) Benth, and to analyse the chemical composition, which will definite the effective substance. Furthermore, the antiviral spectrum, efficacy strength and the influence to the host signaling pathway and cytokine, chemokine induced by influenza virus will be analyzed. Then, the antiviral mechanism will be clarified. This will supply the scientific evidence for its clinical using.MethodsIsolating and purifying the various extracts or fractions from the Laggera pterodonta(DC.) Benth by using different polar solvent, inclueding Methanol and ethanol. The cytotoxicity of extracts were measuring with MTT assay, the antiviral ef fection and spectrum were testing with cytopathic effect assay (CPE asasy) in vitro. The component analysis of effective fraction was measuring with UHPLC-QTOF MS assay. The effect of fractions to the host signaling pathway and cytokine induced by influenza virus were studied by western-blot and Quantitative real-time RT-PCR assay, and also, the bio-plex suspension chip technology were used to verify the influence to the cytokine from the level of protein. The mice model of influenza virus infection was applieded to evaluate the potency in vivo.Results(一) the anti-influenza virus activity of the ethanol extracts from Laggera pterodonta (DC.) BenthThe results showed that the extracts can inhibit influenza virus infection in the model of giving drug before infection and after infection, the 50% inhibiting concentration(IC50) were 0.31mg/mL,0.73mg/mL, the selective index (SI) were 9.9,4.21 repectively. The results of mice model in vivo showed that the ethanol extract can decrease the lung index and alleviate the lung inflammation of influenza virus infection.(二) the anti-influenza virus activity of C8 and its antiviral mechanism, the chemical composition.Base on these results, several fractions were obtained from the methanol extract elution by Petroleum ether, acetic ether, Butyl alcohol, and H2O. The antiviral effects of C5-C10 were screen,C5 and C8 were efficacious against influenza virus infection, the IC50 were 68.2μg/mL,25μg/mL. Because the C8 was more potency, then antiviral spectrum of C8 were done, and the results showed that, the IC50 against the 2009 new H1N1, seasonal H1N1, FluB, H6N2, H7N3, H9N2 were 50μg/mL,19.9μg/mL,50μg/mL,84.8μg/mL,80.2μg/mL,91.4μg/mL. The antiviral mechanisms results showed that C8 can effectively inhibit the NF-κB activation at the concentration 10μg/mL,20μg/mL induced by TNF-α and influenza virus in HEK293 cell(stabletransfection pNF-κB-Luc plasmid), and also, the western-blot showed that the NF-KB Phosphorylation were inhibited by C8 at the concentration lOμg/mL,20μg/mL,40μg/mL, completely inhibited at the concentration 80μg/mL, but ineffective to ERK. The component analysis of C8 by UHPLC-MS displayed that it contained sixty-three compounds. The active ingredient of C8 were Eudesmane sesquiterpenes, Glycoside and Flavone compounds. The molecular weight (MW) of peak 23 was 222, the molecular formula(MF) was C15H26O, it was identified as Laggerol; Isointermedeol; ent-7(11)-selinen-4-ol;7-Epi-β-eudesmol;7-epi-γ-eudesmol, the structure was mainly Eudesmane SesquiterPenols; peak 28+29, the MWof 28 was 234, the MF was C15H22O2 and the structure was costic acid/isocostic acid;Pterodontic acid, belonging to SesquiterPene acid; the MW of peak 29 was 452, the MF was C24H36O8, which is the isomer with peak 26(4-0-acetyl-cuauthemone-3-0-[2-methyl-2-hydroxy-3-acetoxybutyrate]pterodonoic acid), is SesquiterPene acid.(三) the chemical composition of Fr 14 and its anti-influenza virus mechanismThe component analysis of Laggera pterodonta (DC.) Benth were analyzed. Base on the Laggera chemical constituent of Dictionary of Natural Products and the reference, the database of the Laggera chemical constituent was established. The chemical composition of and the chromatographic peaks of Laggera pterodonta (DC.) Benth analyzed by UHPLC-QTOF MS were identified.138 compounds were obtained. The fractions were analyzed by UHPLC-QTOF MS, and the similar elution was merged, the MCI different parts were weight. The results diaplayed that FrO4 to FrO7 were cyclic alco, the main ingredients were peak 18,19,20, and identified as 2-dicaffeoylquinic acid. SesquiterPene acid were accumulation in Fr12 to Fr14, the peak of main compounds were 99,100, the [M+H]+ was 235.1693 and 527.3339, The MF were C15H2202 and C32H46O6 respectively, the structure of peak 9 was the isomer of costic acid、2α-Acetoxycostic acid, isocostic acid, Pterodontic acid, the structure of peak 10was unknown。 Flavones was focus on Frl6to Fr17, the main ingredients was peak102, the [M+H]+ was 375.1074, and the MF was C19H1808, the structure was identified as the isomer of chrysosplenetin B, casticin. The other fractions such as peak 121, the M+H]+was 389.1231, The MW was C20H208, and the structure was the isomer of artemitin. Furthermore, the MF of compounds signed with *、# and $ in Fig 4and Fig 6 were C24H23NO4、C23H21NO3、C21H22O8, the weight analysis of solid matter results demonstrated that SesquiterPene acid. (Fr12-Fr14) and the Flavones (Fr16-Fr17) were the main compounds.Twenty-three fractions were isolated, the antiviral results showed that only the Fr14 and Fr16 were effective, the IC50 were 79.4μg/mL,25μg/mL, The component analysis displayed that the Fr14 were sesquiterpenes compounds, Fr16 were flavonoid compounds. Fr14 had a wide antiviral spectum, The antiviral mechanisms results showed it acted on the early phase of influenza virus replication by the time course assay, Fr14 can effectively inhibit the NF-KB, COX-2,P38 activation at the concentration of 150μg/mL,100μg/mL and 50μg/mL, especially at the 150μg/mL, but ineffective to ERK. Frl4 can effectively inhibited the TNF-α, IP-10, IFN-α, MIG, IL-8, MIP-1α over expression at the concentration of 50μg/mL,150μg/mL after influenza virus infection of 4 h, inhibit the IP-10, IL-6, MCP-1 over expression at the concentration of 50μg/mL and 150μg/mL, inhibited the MIG over expression at the 150μg/mL after the infection of 8h, inhibit the IP-10 at the concentration of 50μg/mL and 150μg/mL, inhibit the MIG, IL-6 at the 150μg/mL after the infection Of 16h. The bio-plex suspension chip technology results showed that Frl4 can effectively inhibit the TNF-a, IL-8, IL-6, MCP-1, IP-10, RANTES over expression at the concentration of 150μg/mL,100μg/mL and 50μg/mL from the protein level.ConclusionThe definite anti-influenza virus infections were verified and the effective substances were sesquiterpenes compound. The effective fractions were also the active fractions. The mechanisms demonstrated that it had a wide antiviral spectrum, effectiveness against different influenza virus including human and avain influenza virus. Besides, the host signaling pathway and cytokine induced by influenza virus were inhibited. All the results indicated that the Laggera pterodonta(DC.) Benth will have a good prospect to be developed as an anti-influenza virus drugs. |
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