Reduction ofCandida tropicalisbiofilm by photoactivation of aHeterophyllaea pustulataextract

Abstract

Context: 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. Materials and 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. Discussion and conclusion: 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. ARTICLE HISTORY

On the mechanism of Candida tropicalis biofilm reduction by the combined action of naturally-occurring anthraquinones and blue light

“…They were unequivocally identified by their spectroscopic/spectrometric data ( 1 H NMR, 13 C NMR, IR, UV–Vis, MS) [10]. The purity was 93.6% ± 0.1% for 1 and 93.8 ± 0.1% for 2 , as established by HPLC analysis [13] (Fig 2 in S1 File). …”

“…An actinic Phillips 20 W lamp (380–480 nm, emission maximum at 420 nm, 0.65 mW cm -2 ) was used in photo-induced antibiofilm studies, which was placed 20 cm above the samples inside a black box [13,14]. …”

“…Biofilm formation was achieved following the previously published procedure [13,14,20], which is an adaptation from the method of O’Toole & Kolter (1998) [21] using flat-bottomed 96-well microplates (Greiner Bio-One, Germany). In this assay, we used the C .…”

“…In this assay, we used the C . tropicalis NCPF 3111 strain previously classified as a strong biofilm producer [13,22]. A strain suspension (1 × 10 7 cells/mL) in SDB was inoculated in pre-treated microplates with 50% (v/v) Fetal Bovine Serum [19], and then were incubated 90 min at 37°C.…”

“…tropicalis biofilms. The concentration necessary to inhibit the biofilm (Biofilm Inhibitory Concentration) [13] was 0.2 mg/mL. Meanwhile, both purified AQs, 1 and 2 , showed greater antibiofilm effect than the photoactive extract under irradiation, which was mediated by a strong redox imbalance.…”