title: The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.
abstract: BACKGROUND
  Candida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity.
  
  
  OBJECTIVE
  This study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect.
  
  
  METHODS
  Biofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods. The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method.
  
  
  RESULTS
  Biofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74).
  
  
  CONCLUSIONS
  Under irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation.
authors.affiliation: Instituto Multidisciplinario de Biología Vegetal (IMBIV) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. Haya de la Torre y Medina Allende, X5000HUA Córdoba, Argentina.
authors.forename: Juliana
authors.initials: J
authors.lastname: Marioni
authors.affiliation: Instituto Multidisciplinario de Biología Vegetal (IMBIV) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Cátedra de Microbiología, Facultad de Ciencias Exactas Físicas y Naturales. Universidad Nacional de Córdoba, Argentina. Av. Vélez Sarsfield 299, Córdoba, Argentina.
authors.forename: María Angel
authors.initials: MA
authors.lastname: da Silva
authors.affiliation: Instituto Multidisciplinario de Biología Vegetal (IMBIV) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. Haya de la Torre y Medina Allende, X5000HUA Córdoba, Argentina.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Cabrera
authors.affiliation: Instituto Multidisciplinario de Biología Vegetal (IMBIV) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina. Haya de la Torre y Medina Allende, X5000HUA Córdoba, Argentina.
authors.forename: Susana C Núñez
authors.initials: SC
authors.lastname: Montoya
authors.affiliation: Instituto Multidisciplinario de Biología Vegetal (IMBIV) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Cátedra de Microbiología, Facultad de Ciencias Exactas Físicas y Naturales. Universidad Nacional de Córdoba, Argentina. Av. Vélez Sarsfield 299, Córdoba, Argentina. Electronic address: gabrielaparaje@gmail.com.
authors.forename: María Gabriela
authors.initials: MG
authors.lastname: Paraje
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000880:Anthraquinones / Q000737:chemistry / Q000494:pharmacology* / Q000528:radiation effects; D000935:Antifungal Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects*; D041022:Candida tropicalis / Q000187:drug effects* / Q000502:physiology; D008027:Light; D008826:Microbial Sensitivity Tests; D018384:Oxidative Stress / Q000187:drug effects; D026361:Reactive Nitrogen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000880:Anthraquinones; D000935:Antifungal Agents; D000975:Antioxidants; D026361:Reactive Nitrogen Species; C079172:rubiadin 1-methyl ether; D000666:Amphotericin B; C072500:rubiadin; D013482:Superoxide Dismutase
keywords: Antifungal; Biofilms; Candida tropicalis; Heterophyllaea pustulata; Oxidative stress; Synergistic activity