title: Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus.
abstract: BACKGROUND
  Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns.
  
  
  OBJECTIVE
  This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling.
  
  
  METHODS
  A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation.
  
  
  RESULTS
  Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone.
  
  
  CONCLUSIONS
  HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.
authors.affiliation: a Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy , University of Helsinki , Helsinki , Finland ;|b Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry , University of Helsinki , Helsinki , Finland ;
authors.forename: Päivi
authors.initials: P
authors.lastname: Järvinen
authors.affiliation: a Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy , University of Helsinki , Helsinki , Finland ;
authors.forename: Susanna
authors.initials: S
authors.lastname: Nybond
authors.affiliation: c School of Pharmaceutical Sciences, EPGL , University of Geneva, University of Lausanne , Geneva , Switzerland ;
authors.forename: Laurence
authors.initials: L
authors.lastname: Marcourt
authors.affiliation: c School of Pharmaceutical Sciences, EPGL , University of Geneva, University of Lausanne , Geneva , Switzerland ;
authors.forename: Emerson
authors.initials: E
authors.lastname: Ferreira Queiroz
authors.affiliation: c School of Pharmaceutical Sciences, EPGL , University of Geneva, University of Lausanne , Geneva , Switzerland ;
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Wolfender
authors.affiliation: b Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry , University of Helsinki , Helsinki , Finland ;
authors.forename: Aila
authors.initials: A
authors.lastname: Mettälä
authors.affiliation: d Department of Chemistry and Bioengineering , Tampere University of Technology , Tampere , Finland ;
authors.forename: Matti
authors.initials: M
authors.lastname: Karp
authors.affiliation: e Pharmaceutical Biology, Division of Pharmaceutical Biosciences, Faculty of Pharmacy , University of Helsinki , Helsinki , Finland.
authors.forename: Heikki
authors.initials: H
authors.lastname: Vuorela
authors.affiliation: e Pharmaceutical Biology, Division of Pharmaceutical Biosciences, Faculty of Pharmacy , University of Helsinki , Helsinki , Finland.
authors.forename: Pia
authors.initials: P
authors.lastname: Vuorela
authors.affiliation: b Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry , University of Helsinki , Helsinki , Finland ;
authors.forename: Annele
authors.initials: A
authors.lastname: Hatakka
authors.affiliation: a Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy , University of Helsinki , Helsinki , Finland ;
authors.forename: Päivi
authors.initials: P
authors.lastname: Tammela
mesh_terms: D000890:Anti-Infective Agents / Q000302:isolation & purification / Q000494:pharmacology*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D045424:Complex Mixtures / Q000302:isolation & purification / Q000494:pharmacology*; D048168:Escherichia coli K12 / Q000187:drug effects / Q000254:growth & development; D059627:Liquid Phase Microextraction; D008826:Microbial Sensitivity Tests; D055494:Pycnoporus / Q000737:chemistry / Q000254:growth & development
chemical_list: D000890:Anti-Infective Agents; D045424:Complex Mixtures
keywords: Bioluminescent bacterial strain; Escherichia coli; Gram-negative bacteria; phlebiarubrone