Mode of action of extract from a phototoxic plant on fungal biofilms

MARIONI J, ARCE MIRANDO JE, COMINI LR, CABRERA J, NÚÑEZ MONTOYA SC, PARAJE MG

Congreso; IX CONGRESO DE MICOLOGÍA GENERAL. SOCIEDAD ARGENTINA DE MICOLOGÍA; 2013

Comite Organizador del Evento

In recent years, different species from Candida no albicans are becoming the main pathogens causing nosocomial infections in our country, especially because of their ability to form biofilms. Heterophyllaea pustulata Hook. f. (Rubiaceae) is a phototoxic vegetal species from NW Argentina and Bolivia, popularly known as ??cegadera?. Previously, only the anthraquinones (AQs)-rich extracts have been shown to have in vitro antibacterial and antifungal effects against planktonic microorganisms, with a low acute toxicity in vivo. The aim of this study was to evaluate the activity of different polarity extracts from H. pustulata against biofilms formed by a Candida no albicans strain, and to investigate if any effects could be enhanced by light, quantifying the reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI) generation. Four extracts were obtained: hexane (Ext-H), benzene (Ext-B), ethyl acetate (Ext-AE), and ethanol (Ext-E) from the aerial parts of this plant. Biofilm quantification was performed by the O?Toole & Kolter method, using an ATCC strain of C. no albicans. Sensitivity to the extracts was determined following the protocols of the Clinical and Laboratory Standards Institute, at three concentrations (0.2, 0.1 and 0.05 mg/ml) in triplicate. The assay was carried out under two conditions: darkness and irradiation, simultaneously. The irradiation system comprised a 20W Phillips actinic lamp (380? 480 nm, 0.65 mW/cm2) with a maximum at 420 nm, placed inside a black box at 20 cm above the samples. The supernatant was separated by measuring the superoxide anion (O2?-) production by the reduction of the nitro-blue tetrazolium (NBT) reaction; the nitric oxide (NO) generation was evaluated by Griess reagent; and the total system antioxidant capability was determined through FRAP assay (Ferrous Reduction Antioxidant Potency). No extracts tested resulted active under darkness, while under irradiation Ext-B, AE y E showed the ability to decrease biofilms with the most active being Ext-E (biofilm reduction of 34.8±2.3 % at 0, 2 mg/ml). Furthermore, when the system was irradiated, O2?- and NO were generated. The total system´s antioxidant capability was higher with respect to darkness. We established that the ability of the extracts obtained from H. pustulata to inhibit the biofilm growth of ATCC C. no albicans was correlated with the irradiation effect that caused the generation of two reactive toxic species: O2?- and NO. These results encouraged us to evaluate the qualitative and quantitative bioactive extract composition, try to explain the observed effect. Since Candida biofilms are resistant to the antifungal agents used in medicine, the natural compounds found to be active against this form of growth, warrants the continuity of our research, in order to assess the effects of the pure compounds both isolated and purified from extracts of H. pustulata.