Photo-inactivation mechanism over Candida biofilms by natural anthraquinones

1.53 JULIANA MARIONI, BRESOLÍ-OBACH ROGER, PARAJE MARÍA G, NONELL SANTI, NÚÑEZ MONTOYA SUSANA C.

Santiago de Chile

Congreso; 25th Inter- American Photochemical Society (I-APS) Meeting.; 2016

American Photochemical Society (I-APS)

Two photosensitizing anthraquinones (AQs), isolatedfrom Heterophyllaea pustulata Hook f.(Rubiaceae), rubiadin (AQ1) and 1-methyl ether rubiadin (AQ2), showed antifungalactivity on Candida tropicalis biofilms under actinicirradiation. It was hypothesized that the photoinactivation mechanism involves theReactive Oxygen Species (ROS): superoxide anion (O2?-)and/or singlet molecular oxygen (1O2). In this work, weset out to identify specifically the reactive intermediates involved.IsolatedAQs were identified by their RMN spectra (94 % purity). Biofilm quantification was performedby the O´Toole & Kolter method on C.tropicalis NCPF 3111. AQs were tested at 56 µM in triplicate, under darknessand irradiation (15 min with Philips TL/03 actinic lampemitting in the range 380-480 nm). 1O2 phosphorescence was detected by means of a customizedPicoQuant Fluotime 200 system employing a 355 nm diode-pumped pulsed Nd:Yaglaser for excitation (FTSS355-Q, Crystal Laser, Berlin, Germany; 10 kHzrepetition rate, 5 mW, 0.5 µJ per pulse) and a Hamamatsu NIR photomultiplierassembly (H9170-45) for detection. Transient absorption experiments werecarried out by using a home-built nanosecond laser flash photolysis apparatus,provided with a nanosecond laser that excited the samples at 355 nm. AQs transientswere detected at 550 nm (radical anion) and 675 nm (triplet state). Inaddition, the scavengingeffect of sodium azide (1O2 quencher) and Tiron (O2?- quencher)on biofilm photoinactivation by each AQs was studied at two concentrations (100 and 500 mM).The supernatant of those experiments was used to measure O2?- production and Superoxide dismutase (SOD)activation by means of the Nitro-Blue Tetrazolium assay. Both AQsreduced biofilm biomass only under exposure to light: AQ1 achieved 63.5 ± 4.5%reduction (R%) and AQ2 47.0 ± 10.0 R%. In phosphate buffered saline (PBS), the 1O2quantum yield of AQ2 was 0.02, whereas for AQ1 was lower than 0.01. Bycomparing the intensity of the phosphorescence signals, it was observed thatAQ2 generated three-fold more 1O2 than AQ1 in thebiofilms incubated with deuterated PBS. None of the ROS precursors (tripletstate and radical anion) was observed in the biofilms. The antibiofilm activityof AQ1 could not be reversed by azide (100 and 500 mM), whereas Tiron (100 and500 mM) reversed it completely. For AQ2 the same effect was observed, howeverthe antibiofilm activity was only reversed at the highest concentration ofTiron (500 mM). For both AQs, production of O2?-led to a significantSOD activation, which was not modified in the presence of sodium azide, but decreased to basal values in the presence of Tiron.Inconclusion, AQ1 and AQ2 reduced the formation of C. tropicalis biofilms under the action of light, mainly by meansof a Type 1 photodynamic mechanism (O2?-generation). Productionof 1O2 was observed in biofilm incubated with deuteratedPBS, however its participation in biofilm photoinactivation is unclear.