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      Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

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          Abstract

          An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

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          Author and article information

          Journal
          Phytochemistry
          Phytochemistry
          1873-3700
          0031-9422
          Sep 2014
          : 105
          Affiliations
          [1 ] School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
          [2 ] Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne, Switzerland.
          [3 ] School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. Electronic address: emerson.ferreira@unige.ch.
          [4 ] University of Airlangga, Fak Pascasarjana, 60286 Surabaya, Indonesia.
          [5 ] Swiss Federal Research Station Agroscope Changins Wädenswil ACW, Route de Duiller 50, P.O. Box 1012, 1260 Nyon, Switzerland.
          Article
          S0031-9422(14)00255-6
          10.1016/j.phytochem.2014.06.004
          24984572
          d9d9fbfe-8f77-422c-8578-e02536822fa0
          Copyright © 2014 Elsevier Ltd. All rights reserved.

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