title: Reduction of Candida tropicalis biofilm by photoactivation of a Heterophyllaea pustulata extract.
abstract: BACKGROUND
  Biofilm formation is an important problem, since this growth mode confers resistance to drugs usually used in therapeutics.
  
  
  OBJECTIVE
  In vitro antifungal activity of extracts obtained from Heterophyllaea pustulata Hook f. (Rubiaceae) were studied against Candida tropicalis biofilms, evaluating the effect of irradiation and the oxidative and nitrosative stresses as possible mechanisms of action.
  
  
  METHODS
  Hexane, benzene, ethyl acetate and ethanol extracts were evaluated at three concentrations (0.2, 0.1 and 0.05 mg/mL) over mature biofilm, under darkness and irradiation. After 48 h of incubation, biofilm quantitation was performed by the O'Toole and Kolter method. Reactive oxygen species (ROS) was measured by nitro-blue tetrazolium (NBT) reaction and reactive nitrogen intermediates (RNI) by the Griess reagent. Superoxide dismutase activation (SOD, NBT assay) and total antioxidant system (FRAP test) were studied.
  
  
  RESULTS
  Only the benzene extract at 0.2 mg/mL reduced the biofilms formation. The slight decrease achieved in darkness (17.06 ± 2.80% reduction) was increased by light action (39.31 ± 3.50% reduction), clearly observing a photostimulation. This great reduction was confirmed by confocal microscopy. In darkness, biofilm reduction was mediated by an increase in RNI, whereas under irradiation, the ROS action was most important. Although no SOD activation was observed, a strong stimulation of the total antioxidant system was detected. HPLC analysis established a high content of several anthraquinones in this extract.
  
  
  CONCLUSIONS
  Biofilm reduction by benzene extract was mainly mediated by oxidative stress triggered under light action, confirming a photodynamic sensitization, which could be attributed to its high content of photosensitizing anthraquinones.
authors.affiliation: a IMBIV-CONICET, Departamento De Farmacia, Facultad De Ciencias Químicas , Universidad Nacional De Córdoba , Córdoba , Argentina.|b IMBIV-CONICET , Cátedra De Microbiología, Facultad De Ciencias Exactas Físicas Y Naturales . Universidad Nacional De Córdoba , Córdoba , Argentina.
authors.forename: Juliana
authors.initials: J
authors.lastname: Marioni
authors.affiliation: b IMBIV-CONICET , Cátedra De Microbiología, Facultad De Ciencias Exactas Físicas Y Naturales . Universidad Nacional De Córdoba , Córdoba , Argentina.
authors.forename: Julio E
authors.initials: JE
authors.lastname: Arce
authors.affiliation: a IMBIV-CONICET, Departamento De Farmacia, Facultad De Ciencias Químicas , Universidad Nacional De Córdoba , Córdoba , Argentina.
authors.forename: José L
authors.initials: JL
authors.lastname: Cabrera
authors.affiliation: b IMBIV-CONICET , Cátedra De Microbiología, Facultad De Ciencias Exactas Físicas Y Naturales . Universidad Nacional De Córdoba , Córdoba , Argentina.
authors.forename: María G
authors.initials: MG
authors.lastname: Paraje
authors.affiliation: a IMBIV-CONICET, Departamento De Farmacia, Facultad De Ciencias Químicas , Universidad Nacional De Córdoba , Córdoba , Argentina.
authors.forename: Susana C
authors.initials: SC
authors.lastname: Núñez Montoya
mesh_terms: D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D041022:Candida tropicalis / Q000187:drug effects* / Q000254:growth & development; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010775:Photic Stimulation / Q000379:methods; D017319:Photosensitizing Agents / Q000302:isolation & purification / Q000494:pharmacology*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D019666:Rubiaceae
chemical_list: D000935:Antifungal Agents; D017319:Photosensitizing Agents; D010936:Plant Extracts; D017382:Reactive Oxygen Species
keywords: Antioxidant defence system; Candida biofilms; HPLC analysis; Rubiaceae; nitrosative stress; oxidative stress; photosensitization