title: Antiviral activity of hydroalcoholic extract from Eupatorium perfoliatum L. against the attachment of influenza A virus.
abstract: BACKGROUND
  Aerial parts of Eupatorium perfoliatum have been traditionally used by American natives as a treatment for fever and infections. Also modern phytotherapy in Europe documents the use of hydroalcoholic extracts of this herbal material for the treatment of infections of the upper respiratory tract.
  
  
  OBJECTIVE
  The aim of the present study was to characterize the anti-influenza A virus (IAV) potential of extracts derived from the aerial parts of E. perfoliatum and to identify their antiviral mode of action and potential active fraction's compounds of the extract.
  
  
  METHODS
  The inhibitory effects of extracts obtained by different organic solvents with different polarities on the cytopathic effect induced by IAV replication was determined in a Madin-Darby Canine Kidney Epithelial (MDCK II) cell-based assay measuring cell viability by MTT stain (MTTIAV assay). Plaque reduction assays were used for investigation of antiviral activity. The mode of action was investigated by different incubation and treatment cycles as well as hemagglutination inhibition assays. Influence of the test extract on tumor necrosis factor (TNF-α) and epidermal growth factor (EGF)-induced cell signaling was analyzed in human lung epithelial (A549) cells. Analytical characterization of extract and fractions obtained from the extract was performed by UHPLC-MS.
  
  
  RESULTS
  Hydroalcoholic extracts from the aerial parts of E. perfoliatum were shown to inhibit growth of a clinical isolate of IAV(H1N1)pdm09 I1 and the influenza virus A/Puerto Rico/8/34 (PR8; H1N1) with a half-maximal inhibitory concentration (IC50) of 7µg/mL and 14µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 52 and 26, respectively. Extracts prepared with dichloromethane and methanol were inactive. At concentrations >1-10µg/mL of the hydroalcoholic extract plaque formation of IAV(H1N1)pdm09 was abrogated. The extract was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. The extract blocked attachment of IAV and interfered with virus-induced hemagglutination. TNF-α-induced signal transduction in A549 cells was not affected, while the EGF-induced signaling to phosphorylated ERK was slightly upregulated by the extract. Bioassay-guided fractionation and subsequent LC-MS analysis indicated that the antiviral activity might be due to polyphenolic compounds with higher molecular weights, which strongly interact with stationary phases of different chromatographic systems. These still unknown compounds with probably high molecular weight could not be isolated in the present study. A variety of different flavonoid glycosides and caffeoyl quinic acids obtained from E. perfoliatum did definitely not contribute to the antiviral effect of the extract and its respective fractions.
  
  
  CONCLUSIONS
  Hydroalcoholic extracts from the aerial parts of E. perfoliatum and its main active polyphenolic constituents protect cells from IAV infection by inhibiting viral attachment to the host cells. The extract appears to be a promising expansion of the currently available anti-influenza agents.
authors.affiliation: Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Corrensstraße 48, 48149 Münster, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Derksen
authors.affiliation: Institute of Medical Microbiology - Clinical Virology, University Hospital Münster, Von Stauffenberg-Straße 36, 48151 Münster, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Kühn
authors.affiliation: Institute of Medical Microbiology - Clinical Virology, University Hospital Münster, Von Stauffenberg-Straße 36, 48151 Münster, Germany.
authors.forename: Wali
authors.initials: W
authors.lastname: Hafezi
authors.affiliation: Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Corrensstraße 48, 48149 Münster, Germany.
authors.forename: Jandirk
authors.initials: J
authors.lastname: Sendker
authors.affiliation: Institute of Molecular Virology, University of Münster, Von-Esmarch-Straße 56, 48149 Münster, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Ehrhardt
authors.affiliation: Institute of Molecular Virology, University of Münster, Von-Esmarch-Straße 56, 48149 Münster, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Ludwig
authors.affiliation: Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Corrensstraße 48, 48149 Münster, Germany. Electronic address: ahensel@uni-muenster.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hensel
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000998:Antiviral Agents / Q000302:isolation & purification / Q000494:pharmacology* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D036602:Eupatorium / Q000737:chemistry*; D006385:Hemagglutination Inhibition Tests; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000254:growth & development / Q000472:pathogenicity; D020128:Inhibitory Concentration 50; D061985:Madin Darby Canine Kidney Cells; D000432:Methanol / Q000737:chemistry*; D008517:Phytotherapy; D035261:Plant Components, Aerial / Q000737:chemistry; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology* / Q000633:toxicity; D010946:Plants, Medicinal; D015398:Signal Transduction / Q000187:drug effects; D012997:Solvents / Q000737:chemistry*; D010948:Viral Plaque Assay; D053585:Virus Attachment / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D010936:Plant Extracts; D012997:Solvents; D000432:Methanol
keywords: Antiviral activity; Attachment; Eupatorium perfoliatum; Hemagglutinin; Influenza A virus; Polyphenol