Schinus terebinthifolius (Brazilian Peppertree) extract used as antifungal to control Candida spp. in planktonic cultures and biofilms

Objective: The use of medicinal plants may be an alternative method for the control of Candida spp. responsible for human infections. This study evaluated the antifungal effect of Schinus terebinthifolius extract (Brazilian Peppertree) on C. albicans, C. dubliniensis, C. glabrata, and C. krusei planktonic cultures and biofilms. Material and Methods: Minimum inhibitory concentration (MIC) and minimum fungal concentration (MFC) of the plant extract were determined by the broth microdilution method. Biofilms formed in microplate wells were exposed to the extract for 5 min (50, 100 and 200 mg/mL) or 24 h (25, 50 and 100 mg/mL). After determination of colony-forming units per milliliter (CFU/mL), the data were analyzed by one-way ANOVA and Tukey’s Test (P ≤ 0.05). Results: Different MIC (mg/mL) were found, such as 0.39 (C. dubliniensis), 1.56 (C. albicans), and 3.13 (C. glabrata and C. krusei). Besides, MFC (mg/mL) of 0.78 (C. dubliniensis) and 3.13 (C. albicans, C. glabrata and C. krusei) were also observed. Regarding the biofilms, significant reductions (log10) were found after 5 min and 24 h exposure to the plant extract, compared to the control group. However, C. dubliniensis was significantly affected only in 24 h treatment. Conclusion: S. terebinthifolius extract presented a significant antifungal effect on C. albicans, C. dubliniensis, C. glabrata, and C. Krusei both in planktonic cultures and biofilms. RESUMO

Candida pathogenicity is associated with its capacity to form hyphae, which can favour the invasion of the host's tissues, formation of biofilm on biotic and abiotic surfaces and production of hydrolytic enzymes such as proteases and phospholipases [4].
There are several antifungal agents commercially available for treatment of candidiasis, such as nystatin, amphotericin B, clotimazole, miconazole, itraconazole, fluconazole and ketoconazole. However, these drugs may produce adverse effects such as bitter taste, allergic reactions and resistant cell selection [6].
S. terebinthifolius (Brazilian Peppertree), Anacardiaceae family, is a native species from the South and Central America, but it can also be found in tropical and subtropical regions of the United States and Africa. In Brazil, this plant species is distributed throughout the country's eastern coast, from northern to southern region [7]. Antimicrobial, anti-inflammatory and antifungal activities of this plant have been reported [7,8].
The search for therapeutic applications using medicinal plants and their derivatives has been increasing around the world. The use of products obtained from these plants such as extracts, dyes, essential oils and phytocompounds may be an important alternative for control of strains resistant to the antimicrobial drugs available, as well as for treatment of infections caused by Candida spp. Therefore, this study evaluated the antifungal effect of S. terebinthifolius extract on C. albicans, C. dubliniensis, C. glabrata and C. krusei both in planktonic cultures and biofilms.

Antifungal activity on planktonic cultures
For determination of MIC, the broth microdilution method was used according to Clinical and Laboratory Standards Institute (CLSI) [9,10]. Fungal suspensions were prepared in sterile saline solution (0.9% NaCl) from a culture incubated at 37 o C for 24 h and standardised to 10 6 CFU/mL in a spectrophotometer (Micronal, São Paulo, Brazil). From this suspension were performed two dilutions, one of 1:50 and other of 1:20, in order to obtain a concentration of approximately 5 x 10² to 2.5 x 10³ CFU/mL. The assay was performed in microplates with addition of 100 μL/well of culture medium and 100 μL of extract only in the first well from where 10 serial dilutions were obtained, then 100 μL/well of standardised yeast suspension were added. Negative control composed by inoculum and culture medium and positive control constituted only by culture medium were added. The culture medium used was RPMI 1640 (Himedia) with glutamine, without bicarbonate and phenol red indicator, buffered to pH 7,0±0,1 with MOPS [3-(N-morpholino) propanesulfonic acid] (Sigma-Aldrich, St. Louis, USA). After 24 h incubation, MIC was determined in the first well that showed no turbidity. For determination of MFC, 100 μL of MIC and adjacent concentrations were seeded in Sabouraud dextrose (SD -Himedia) agar. After 48 h incubation at 37°C the MFC was determined on the plaque with no colonial growth.

Antifungal activity on biofilms
Monomicrobial biofilms were formed in 96-well plates (TPP, Trasadingen, Switzerland). Initially, the yeasts were cultured in SD agar and then in yeast nitrogen base (YNB -Himedia) supplemented with 100 mM of glucose at 37 o C for 24 h each one. Posteriorly, the fungal suspension was centrifuged (358 g/10 min) and the supernatant disregarded and the pellet suspended in sterile saline solution. After another centrifugation, the suspension was standardised to 10 7 CFU/mL in spectrophotometer (Micronal, São Paulo, Brazil). After, 200 μL/well of this suspension was added in the microplate and followed to incubation (37 o C) under shaking (75 rpm) for 90 min, for adherence of the biofilms. The supernatant was discarded and YNB broth was added. After 48 h, the biofilms were exposure to the extract for 5 min at 50, 100 and 200 mg/mL; or, for 24 h at 25, 50 and 100 mg/mL. Saline solution (0.9% NaCl) and culture medium were used as controls on the treatments of 5 min and 24 h, respectively. The biofilm was washed three times with sterile saline solution before being disaggregated by ultrasonic homogenizer (Sonopuls HD 2200 -Bandelin Eletronic, Berlin, Germany) with power of 25% for 30 s. The suspension was serially diluted and 20 μL were added to SD agar in single drops in triplicate. After 48 h incubation, the concentration of CFU/mL was determined by calculating the mean CFU of each drop multiplied by 50 and dilution factor used and the values were converted in log 10 . Three assays were performed on an independent basis, with four repetitions each, totalising 12 for each experimental group.

Statistical analysis
Results were analysed by ANOVA and Tukey's Test (P ≤ 0.05), using GraphPad Prism software version 5.0.
Regarding the biofilms, this plant extract at 50, 100, and 200 mg/mL produced significant reductions of CFU/mL after 5 min exposure (Figure 1) in relation to the control group, except C. dubliniensis, when applied the concentrations of 100 and 50 mg/mL. After 24 h exposure to the extract at 25, 50 and 100 mg/mL, significant reductions of all fungal species were observed ( Figure 2).   [13] demonstrated that the dye extracted from S. terebinthifolius presented effective action against C. tropicalis, with MIC and MFC values of 625 μg/mL. Martínez et al. [14] conducted a study with different concentrations of S. terebinthifolius alcoholic extract and found no microbial growth inhibition in Gram-positive and Gramnegative bacteria and C. albicans at the lowest concentration used (10%), but an inhibitory effect was observed at higher concentrations (50% and 100%). In addition, Gomes et al. [15] reported that lectin, a compound extracted from S. terebinthifolius, had an effect on C. albicans (MIC = 6.5 μg/mL; MCF = 26 μg/mL). Johann et al. [16] demonstrated that S. terebinthifolius leaf extract contains saponins, flavonoids, triterpenes, steroids and tannins, all capable of affecting the development of fungal specimens. Studies on plant compounds are necessary to find out bioactive molecules and elucidate their action mechanisms, thus allowing their addition to new drugs.
In this study was also shown the effect of S. terebinthifolius extract on Candida spp. biofilms. Nevertheless, higher concentrations were needed to achieve an effective control of biofilms compared to those used on planktonic forms. In fact, it was reported that biofilms can be more resistant than planktonic cells [17]. C. albicans biofilm exposed to S. terebinthifolius extract presented significant reductions of CFU/ mL. However, it was found that concentrations of 100 and 200 mg/mL were more effective than 50 mg/mL in 5 min exposure. Similar reductions of these biofilms were observed after 24 h exposure to the extract at concentrations of 25, 50 and 100 mg/mL. Alves et al. [11] also reported that the dye extracted from S. terebinthifolius showed antibiofilm effect on C. albicans demonstrating that the MIC values (1x, 2x and 4x) of this product affected significantly this fungal species, causing significant reductions of CFU/mL after 60 min exposure; however they were similar to controls after 120 and 180 min exposures. Barbieri et al. [8] reported inhibitory effect on the initial formation of C. albicans biofilm. According to the authors, this effect can be measured by the presence of some compounds of the plant such as alkaloids, phenols and terpenes. Therefore, it can suggest that this product may be potentially used for both prevention and treatment of infections associated with Candida biofilm formation.
Non-albicans Candida biofilms were also significantly affected such as C. dubliniensis, C. glabrata and C. krusei after 24 h exposure to different concentrations of S. terebinthifolius. There were also significant reductions in CFU/mL after 5 min exposure, except with C. dubliniensis at 50 and 100 mg/mL. These results are important because non-albicans Candida are present in different candidoses such as oropharyngeal candidiasis in HIVinfected individuals [18], invasive candidiasis in intensive care units [19] and vulvovaginal candidiasis [20], including strains resistant to most conventional antifungal like fluconazole and itraconazole.
Similarly to C. albicans, C. dubliniensis can be considered as human commensal yeast and thus can cause opportunistic infections [21]. However, some differences regarding adherence to oral epithelium [22] and filamentation of biofilm cells [23] were related. C. krusei was cited as a pathogen involved in cases of systemic infections, mainly in patients with acquired immune deficiency syndrome [24]. This specie can also inhibit the development of C. albicans in an interspecific association [25].

CONCLUSION
The antifungal potential of the S. terebinthifolius extract suggests opportunities to study this plant more closely so that other possible biological effects (i.e. antibiotic, antitumor and anti-inflammatory) can be explored, including application to cytotoxicity tests to ensure its use in possible formulations, broadening the antifungal therapeutic scope. Thus, it was demonstrated that S. terebinthifolius extract to control Candida spp. in planktonic cultures and biofilms