The antibiofilm effects of the dichloromethane fraction of Sideroxylon obtusifolium T. D. Penn on Candida spp.Abstract
Objective To analyze the phytochemical profile of the dichloromethane fraction (FDIC) of the leaves of Sideroxylon obtusifolium T. D. Penn and determine its antifungal and antibiofilm activity. Materials and Methods: Phytochemical characterization was performed by qualitative and quantitative screening and Nuclear Magnetic Resonance. Antimicrobial activity was determined by Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). Antifungal activity on the growth kinetics (Time-Kill Curve) of Candida albicans was measured. Finally, the prescription and metabolism of C. albicans and Candida glabrata monospecies and mixed biofilms treated with FDIC were determined. Data were analyzed by one-way ANOVA and Tukey's post-test (α = 0.05). Results: Phytochemical characterization suggested a greater presence of saponins and phenolic compounds. FDIC showed MIC values between 250-1000 µg/mL and MFC values between 1000 and >1000 µg/mL. FDIC showed fungicidal activity for 24 h against C. albicans and disrupted the biofilm formation of Candida spp. Conclusion: FDIC showed renewed antifungal activity against adhesion and biofilm formation of Candida spp.
Keywords:
biofilm; Candida; oral candidiasis; Sideroxylon
Resumo
Objetivo Analisar o perfil fitoquímico da fração diclorometano (FDIC) das folhas de Sideroxylon obtusifolium T. D. Penn e determinar sua atividade antifúngica e antibiofilme Materiais e Métodos: A caracterização fitoquímica foi realizada por triagem qualitativa e quantitativa e Ressonância Magnética Nuclear. A atividade antimicrobiana foi determinada pela Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM). A atividade antifúngica na cinética de crescimento (Curva Time-Kill) de Candida albicans foi medida. Por fim, foi determinada a viabilidade e o metabolismo das monoespécies C. albicans e Candida glabrata e dos biofilmes mistos tratados com FDIC. Os dados foram analisados pelo teste ANOVA unidirecional e pós-teste de Tukey (α = 0,05). Resultados: A caracterização fitoquímica sugeriu maior presença de saponinas e compostos fenólicos. A FDIC apresentou valores de CIM entre 250-1000 µg/mL e valores de CFM entre 1000 e >1000 µg/mL. A FDIC apresentou atividade fungicida por 24 h contra C. albicans e rompeu a formação de biofilme de Candida spp. Conclusão: A FDIC apresentou atividade antifúngica reduzindo a aderência e formação de biofilme de Candida spp.
Palavras-chave:
biofilme; Candida; candidíase oral; Sideroxylon
1. Introduction
Oral candidiasis is an opportunistic fungal infection caused mainly by Candida. albicans, but more recent evidence has shown an important role of non-albicans species in the onset and progression of the disease such as C. glabrata, C. guillermondii, C. krusei, C. lusitaniae, C. parapsilosis, C. pseudotropicalis, C. stellatoidea and C. tropicalis (Hellstein and Marek, 2019). Oral candidiasis can develop in different clinical types in susceptible individuals, such as newborns, patients with oral cancer, those undergoing immunosuppressive therapy, and denture users (Pereira et al., 2016).
Yeast cell adhesion to host tissues is the first step of biofilm development and infection. This process depends on the composition of the cell walls as well as the characteristics of the adhesion surface (Araújo et al., 2017). The ability to form biofilms is one of the major virulence factors of C. albicans (Teodoro et al., 2018) which limits the penetration of molecules through the extracellular matrix, and provides resistance to phagocytic cell attacks (Souza et al., 2018).
Three large families group the most commonly used antifungal drugs: polyenes (amphotericin B and nystatin), echinocandins (anidulafungin, caspofungin and micafungin) and azoles. Azoles constitute the most extensive group being divided into imidazoles (clotrimazole, miconazole, ketoconazole, etc.) and triazoles (fluconazole, isavuconazole, itraconazole, posaconazole and voriconazole) (Quindós et al., 2019). While these drugs are effective, some patients have experienced adverse effects, such as allergic reactions, antagonistic interactions with other drugs, and gastrointestinal effects such as vomiting, nausea, and diarrhea (Lyu et al., 2016). Moreover, microbial resistance rates have been increasing alarmingly worldwide (Li et al., 2018). Therefore, there has been a quest for effective therapeutic alternatives for microbial control (Oliveira et al., 2013; Silva et al., 2012). In this context, the use of plant raw materials, especially those that contain bioactive compounds with antimicrobial activity, becomes relevant. Sideroxylon obtusifolium TD Penn, a medicinal plant native to the Brazilian Caatinga biome, of the Sapotaceae family, popularly known as “quixabeira”, “sapotiaba”, “sacutiaba” or “rompe-gibão” (Araújo-Neto et al., 2010; Gomes et al., 2021), appears as a possible promising alternative in the scenario of fungal infections.
The leaves and barks of S. obtusifolium are used in the form of infusions for their anti-inflammatory properties to treat gastritis, colic, kidney issues, duodenal ulcer, heartburn, and cardiovascular conditions (Aquino et al., 2016). Previous studies report the therapeutic potential of the methanolic extract of S. obtusifolium leaves through the modulating action of antibacterial activity and anti-inflammatory activity. The methanolic fraction stimulated human keratinocyte cells and improved wound healing by modulating inflammatory mediators in burns, in addition to showing potential antifungal promise against the species C. albicans, C. glabrata and C. tropicalis (Aquino et al., 2016; Souza et al., 2021; Aquino et al., 2019; Souza et al., 2021; Aquino et al., 2019; Silva et al., 2017). Anthocyanins from “quixaba” have antioxidant activity, and the dichloromethane and n-butanol fractions of S. obtusifolium extract demonstrated antifungal activity in initial analyses against C. albicans, with potential for bioprospecting phytocompounds for the treatment of periodontal fungal diseases caused by this microorganism. (Figueiredo and Lima, 2015; Pereira et al., 2016; Sampaio et al., 2017).
Thus, we determined the phytochemical composition of the dichloromethane fraction of S. obtusifolium T. D. Penn and its inhibitory effects on the adhesion capacity and biofilm development of Candida spp.
2. Materials and Methods
2.1. Extract preparation and fractionation
Sideroxylon obtusifolium T.D. Penn leaves were harvested in April 2017 in the city of Campina Grande, PB, Brazil (7º 22’ 25” S, 35º 59’ 32” W). S. obtusifolium T.D. voucher specimens (JPB 57.985) were deposited in the Prof. Lauro Pires Xavier Herbarium at the Dept. of Systematics and Ecology of the Federal University of Paraíba, João Pessoa, Brazil. This study was registered in the National System for Management of Genetic Heritage and Associated Traditional Knowledge, Ministry of Environment (accession number A39F2C5).
S. obtusifolium leaves were dried in a circulating-air oven (FANEM - Model 330/5) at 40°C for 14 days and ground in a knife mill (SOLAB - Model SL 30). A hydroethanolic extract (7:3, v/v) was produced by mixing 200 g of the dried-ground plant with 1 L of solvent through maceration. The solvent of the hydroethanolic extract was eliminated in a rotary evaporator at 40°C at 70 rpm. Subsequently, the hydroetahnolic extract was lyophilized (Lyophilizer LS 3000 Terroni®) at -20°C to -40°C (LHE).
The LHE was dissolved in a solution of 50 mL of methanol:water (7:3, v/v) and partitioned in a separation funnel using the following solvents in an increasing degree of polarity: hexane, dichloromethane, ethyl acetate, and n-butyl alcohol. The final portion corresponded to the aqueous phase. The dichloromethane fraction of the extract was subjected to a rotary evaporator at 40°C (70 rpm), lyophilized as previously described (Lyophilizer LS 3000 Terroni®) and stored under refrigeration.
2.2. Phytochemical characterization
2.2.1. Qualitative screening
The dichloromethane fraction of S. obtusifolium extract was first screened for the presence of saponins, polysaccharides, tannins, and total phenolics, flavonoids, alkaloids, steroids, and triterpenes, according to the literature (Matos, 2009).
2.2.2. Quantitative screening
The content of total polyphenols (Chandra and Gonzalez, 2004), total flavonoids (Meda et al., 2005) and condensed tannins (Makkar and Becker, 1993) in the dichloromethane fraction of the extract was measured in triplicate in a microplate reader (Biochrom, EZ Reader 400 Microplate Reader).
2.2.3. 1H Nuclear magnetic resonance (NMR) spectroscopic analysis
The 1H NMR spectra were obtained on a Bruker Ascend spectrometer at 400 MHz. The samples were solubilized in chloroform (CDCl3) and the solvent was used as an internal reference.
2.3. Determination of the antifungal activity
2.3.1. Microorganisms
The following American Type Culture Collection (ATCC) strains were used: C. albicans (ATCC 10231), C. glabrata (ATCC 90030), C. krusei (ATCC 34135), C. tropicalis (ATCC 750), and C. parapsilosis (ATCC 22019). Moreover, clinical isolates of C. albicans (A4, A5, and A6) were also used, which were stored in the Pathology Laboratory at the State University of Paraíba School of Dentistry.
2.3.2. Reactivation of microorganisms and inocula preparation
Both the reference strains and clinical isolates were reactivated in Sabouraud Dextrose agar media (SDA, Kasvi, Paraná, Brazil) and incubated at 37ºC for 24 h in an anaerobic atmosphere. The cell concentration was determined by a visible spectrophotometer (Model GT 7220 BioPet Technologies, Monte Alto, Brazil) at 530 nm (abs 0.08–0.1) to a final concentration of 2.5 x 103 CFU/mL in the antimicrobial assays23.
2.3.3. Minimum inhibitory and fungicidal concentrations (MIC/MFC)
The MIC and MFC values of the fraction against Candida spp. were determined (CLSI, 2008). The fraction was dissolved in 40% ethyl alcohol to 4,000 μg/mL. Then, 100 µL of Sabouraud Dextrose broth (SDB) was added, followed by 100 µL of the fraction, which was serially diluted (1,000 to 7.8 μg/mL). Lastly, 100 µL of the yeast suspension was added. Nystatin and fluconazole were used as positive controls (64 to 0.5 μg/mL). A growth control (untreated suspension), a vehicle control (40% ethanol), and a sterility control (culture media only) were included. The MIC was defined as the lowest concentration of the fraction capable of inhibiting visible microbial growth, which was confirmed by the resazurin dye.
To determine the MFC, a 10 μL aliquot from each well with concentrations equal to or greater than MIC was subcultured onto Sabouraud Dextrose agar plates and incubated at 37 °C for 24 hours. The MFC was defined as the lowest concentration of the subculture that inhibited visible growth. The tests were carried out in triplicate in three independent experiments.
The MFC/MIC ratio was calculated to determine whether the fraction had a fungistatic (MFC/MIC ≥4) or fungicidal (MFC/MIC <4) activity (Siddiqui et al., 2013).
2.3.4. Effect on the growth kinetics of Candida albicans
In a 96-well flat-bottom microdilution plate, 100 µL of SDB was added to each well, followed by the dichloromethane fraction at MIC, 2xMIC, and 4xMIC (500 μg/mL, 1000 μg/mL, and 2000 μg/mL, respectively). Lastly, 100 µL of C. albicans inoculum (2.5 x 103 CFU/mL) was added. The plate was incubated at 37ºC for 24 h in an anaerobic atmosphere. Aliquots of 10 µL were seeded onto Petri dishes containing SDA at intervals of 0 (initial), 1, 2, 3, 4, 6, 8, 12, and 24 h. The number of viable microorganisms (CFU/mL) was counted for each concentration and timepoint. Nystatin was used as a positive control at MIC, 2xMIC, and 4xMIC (2 μg/mL, 4 μg/mL, and 8 μg/mL, respectively). A vehicle control (40% ethanol), a growth control, and a control for sterility of the culture media were included (Castro et al., 2013). The assays were carried out in triplicate in three independent experiments.
2.3.5. Effects on adherence and biofilm formation
The adherence assay was performed in 96-well microplates containing 100 µL of yeast suspensions, 100 µL of nutrient broth (Sabouraud Dextrose supplemented with 4% sucrose), and 100 µL of the fraction or nystatin at MIC, 2xMIC, and 4xMIC. After 24 h, the liquid content was removed, and the adhered biofilm formed at the bottom of the plates was resuspended in 200 µL of saline solution.
Biofilm formation assays were performed in 96-well microplates containing 100 µL of SDB supplemented with 4% sucrose and 100 µL of the yeast suspension. The plates were incubated for 48 h at 37ºC in aerobiosis for biofilm growth and maturation. Then, the culture media were removed and 100 µL of nutrient broth (Sabouraud Dextrose supplemented with 4% sucrose), 100 µL of the fraction or nystatin at MIC, 2xMIC, and 4xMIC were added. The plates were incubated again for another 24 h at 37ºC in aerobiosis. The liquid content was removed and the biofilm formed at the bottom of the plates was collected in 200 µL of saline solution and analyzed as further described (Almeida et al., 2018).
2.3.6. Quantification of viable cells
The biofilm suspension was subjected to serial dilutions (10-1 to 10-5) and an aliquot (10 μL) of each dilution was seeded onto Sabouraud Dextrose agar. The plates were incubated at 37 °C for 24 hours. The CFUs were counted and the results were reported as CFU/mL. The assay was performed in triplicate in three independent experiments (Almeida et al., 2018).
2.3.7. Metabolic activity
The metabolic activity of treated cells was determined by the MTT method [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium] (Sigma-Aldrich - thiazolyl bromide-tetrazolium blue), as previously described. An aliquot of 180 µL of the solution containing residual biofilm cells was centrifuged (KASVI, K14-0602) at 1500 rpm for 5 min. The supernatant was discarded and 200 μL of MTT solution was added (0.5 mg/mL). Cells were incubated for 3 h at 37 °C. The supernatant was discarded and 200 μL of acid isopropanol (95: 5 v/v isopropyl alcohol: hydrochloric acid) was added. Cells were incubated again at 37 °C for 15 min. The absorbance was read in a microplate reader (Biochrom, EZ Reader 400 Microplate Reader) at 490 nm. The assay was performed in triplicate in three independent experiments (Almeida et al., 2018).
2.3.8. Ethical considerations
This study was previously approved by the Research Ethics Committee at the State University of Paraíba (UEPB), under protocol number CAAE 16618919.4.0000.5187. This study complies with the Resolution 466/12 of the National Health Council (CNS/MS), which regulates ethics in studies involving human beings in Brazil.
2.3.9. Data analysis
The growth kinetics and biofilm data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test in the Statistical Package for the Social Sciences program (SPSS for Windows, version 25.0, IBM Corp., Armonk, NY, USA), considering a 5% significance level.
3. Results
3.1. Phytochemical analysis
3.1.1. Qualitative screening
As shown in Table 1, different classes of chemical compounds were tentatively identified in the dichloromethane fraction of S. obtusifolium, such as saponins, tannins, and total phenolics, alkaloids, and steroids/triterpenes.
3.1.2. Quantitative screening
Table 2 shows the concentration of total polyphenols, total flavonoids, and condensed tannins detected in the dichloromethane fraction of S. obtusifolium leaf extract.
Quantification of total polyphenols, total flavonoids, and condensed tannins in the dichloromethane fraction of the hydroethanolic extract of S. obtusifolium.
3.1.3. Nuclear magnetic resonance (NMR) spectroscopic analysis
The 1H NMR spectrum (400 MHz, CDCl3) of the dichloromethane fraction showed a signal envelope between δH 2.5 and 0.75 ppm in addition to multiplets between δH 5.33 and 5.10, suggestive of the presence of methyl and methylenic carbons and oxymetinic hydrogens (Kojima et al., 1990). Other signals were identified in the region of δH 4.1 and 3.2 ppm, suggestive of the presence of osidic units in the sample (Figure 1) (Kasai et al., 1987).
1H NMR spectrum of the dichloromethane fraction of S. obtusifolium (400 MHz, CDCl3). The analysis of these signals suggests the presence of triterpenoid nuclei linked to the sugar molecule (s) in the sample, which are indicative of saponins.
3.2. Antifungal activity
3.2.1. Minimum inhibitory and fungicidal concentrations (MIC/MFC)
Table 3 shows the MIC and MFC values of the dichloromethane fraction against reference and clinical Candida spp strains. The fraction showed MIC values ranging from 250 μg/mL to 1000 μg/mL and MFC values of 1000 μg/mL and higher, which is suggestive of fungistatic activity. While all strains were susceptible, C. glabrata (ATCC 90030) and the combined culture of C. albicans (ATCC 10231) and C. glabrata (ATCC 90030) showed greater sensitivity to the fraction. As expected, the tested strains were highly susceptible to the positive controls (nystatin and fluconazole).
MIC and MFC values of the dichloromethane fraction of S. obtusifolium leaf extract and positive controls against Candida spp strains. The values are expressed in µg/mL.
3.2.2. Time-killing effects of the fraction on Candida albicans
At all tested concentrations, treatment with the fraction and nystatin reduced the average number of CFU/mL when compared to the untreated growth control (GC) (P < 0.05) (Figure 2).
Effects of the dichloromethane fraction of S. obtusifolium leaf extract and nystatin on the growth kinetics of C. albicans (ATCC 10231) at (2a) MIC, (2b) 2xMIC, and (2c) 4xMIC. The results were expressed as the average number of microorganisms (CFUmL) exposed to the treatments over time. Note: * Indicates a significant difference between the fraction and nystatin (P < 0.05); # indicates a significant difference between each treatment and the untreated growth control (P < 0.05) by one-way ANOVA and Tukey's posthoc test.
Interestingly, treatment with the fraction at MIC (500 μg/mL) and 2xMIC (1000 μg/mL) and nystatin at 2xMIC (4 μg/mL) and 4xMIC (8 μg/mL) showed a similar killing behavior against C. albicans up to 12 h. After that period, both the fraction and nystatin no longer inhibited C. albicans growth. At 4xMIC (2000 μg/mL), the treatment with the dichloromethane fraction reduced significantly and continuously any yeast growth for up to 24 h (P < 0.05) (Figure 2c).
3.2.3. Effects against C. albicans adherence and biofilm development
The treatment with the dichloromethane fraction at 4xMIC (2000 μg/mL) significantly reduced the adherence and the number of viable C. albicans biofilm cells (ATCC 10231) (Figure 3), with no significant difference in anti-adherent activity compared to 2xMIC (1000 μg/mL) and in antibiofilm activity between MIC (500 μg/mL) and 2xMIC (1000 μg/mL) (P > 0.05).
Metabolic activity (3a) and cell viability (3b) of C. albicans cells after exposure to different concentrations of the DICF of S. obtusifolium and to Nystatin (4MIC). Note: * Indicates significant difference between the substances and growth control (GC).
The dichloromethane fraction also inhibited adherence and biofilm formation in a mixed suspension of C. albicans (ATCC 10231) + C. glabrata (ATCC 90030) at 1000 μg/mL (Figure 4), with no statistical difference in anti-adherent activity (P > 0.05) when compared to the concentration of 500 μg/mL and in antibiofilm activity between concentrations of 250 μg/mL, 500 μg/mL, and 1000 μg/mL (P > 0.05).
Metabolic activity (4a) and cell viability (4b) of a mixed culture of C. albicans (ATCC 10231) and C. glabrata (ATCC 90030) after exposure to different concentrations of the DICF of S. obtusifolium and to Nystatin (4MIC). Note: * Indicates significant difference between the substances and growth control (GC).
The treatment with nystatin at 4xMIC (4 μg/mL) significantly reduced the number of viable microorganisms and the metabolic activity of C. glabrata (ATCC 90030), with no significant difference compared to the treatment with the fraction at 4xMIC (1000 μg/mL) (Figure 5). No significant difference between the concentrations of 250 μg/mL and 500 μg/mL was observed (P > 0.05).
Metabolic activity (5a) and cell viability (5b) of C. glabrata cells after exposure to different concentrations of the DICF of S. obtusifolium and to Nystatin (4MIC). Note: * Indicates significant difference between the substances and growth control (GC).
4. Discussion
In this study, we characterized chemically the dichloromethane fraction of S. obtusifolium and determined its inhibitory effects on the adhesion capacity and biofilm development of clinically relevant Candida spp strains.
The 1H NMR spectra suggested the presence of terpenic structures. The association of this finding with the suggestive presence of osidic units in the sample is indicative of the presence of saponins. These are steroids or triterpenes linked to sugars widely distributed in plant species with remarkable antimicrobial activity (Rahman et al., 2017). The active compounds found in the dichloromethane fraction of S. obtusifolium are consistent with those reported in other studies (Araújo-Neto et al., 2010; Aquino et al., 2016; Sampaio et al., 2017; Pereira et al., 2016).
Studies have shown that terpenes, including triterpene saponins, have antifungal and antibiofilm activity (Sajjadi et al., 2016; Soberón et al., 2017) and that phenolic compounds such as flavonoids and tannins also have anti-Candida activity (Araújo et al., 2017; Sampaio et al., 2017). According to a previously established parameter (Holetz et al., 2002), our findings indicated that the active fraction of S. obtusifolium showed mostly moderate antifungal activity (MIC values of 100-500 µg/mL) against Candida spp. While these findings confirm those of other studies showing that the dichloromethane fraction of S. obtusifolium has antimicrobial activity against C. albicans, different MIC values were found in our study as compared to those studies (Araújo et al., 2017; Sampaio et al., 2017). Regional differences regarding the season and location of harvest (Reis et al., 2011) as well as the proportion of chemical substances present may result in samples with varying MIC and MCF values (Almeida et al., 2016).
In our study, C. glabrata was the most susceptible strain to the treatment with the dichloromethane fraction of S. obtusifolium. C. glabrata has been associated with resistance to azoles and is considered one of the pathogens responsible for difficult-to-treat fungal infections (Miranda-Cadena et al., 2018; Quindós, 2014). In addition, after the widespread use of immunosuppressive agents and treatment with broad-spectrum antibiotics, the number of systemic and mucosal infections caused by C. glabrata increased considerably (Silva et al., 2012).
The fungistatic properties of the fraction against most of the tested strains can favor the control of infection without causing an imbalance in the normal oral microbiome, which usually occurs with fungicidal substances (Almeida et al., 2018). In addition, the success of antifungal therapy requires maintenance of the biofilm at levels consistent with oral health (Marsh, 2010).
This is the first study in the literature reporting the effects of the dichloromethane fraction of S. obtusifolium on the growth kinetics of yeast strains. The fraction showed fungicidal activity at 4xMIC (2000 µg/mL) against C. albicans cells over 24 h. In addition, both nystatin (4 µg/mL and 8 µg/mL) and the fraction (500 µg/mL and 1000 µg/mL) were similarly effective in inhibiting fungal growth over 12 h.
The dichloromethane fraction significantly reduced C. albicans and C. glabrata adhesion and biofilm formation at the concentration of 2000 µg/mL and 1000 µg/mL, respectively. The molecules present in the fraction may act through cell breakdown – affecting metabolism - and inhibition of adherence. The reduction in the metabolic activity caused may have occurred due to the intracellular stress caused by the action of the fraction at higher concentrations (Almeida et al., 2018). Our data indicated that the treatment with the fraction at 4xMIC (2000 µg/mL and 1000 µg/mL) effectively reduced metabolism and cell viability in C. albicans and C. glabrata monospecies biofilms and in a combined culture between them.) The MIC values of antifungal substances against Candida biofilms can be up to 1000-fold higher than those that are effective against planktonic cells (Souza et al., 2018).
In our study, nystatin inhibited C. albicans adhesion and biofilm formation to a lesser extent as compared to other strains. This can be explained by the fact that nystatin has good antibiofilm activity against C. albicans only when used in high concentrations (Pierce et al., 2013). In contrast, nystatin significantly reduced the adherence and biofilm formation of C. glabrata cultures at the concentration of 4 µg/mL. Nystatin is known to complex with ergosterol in the cell membrane, increasing permeability and leading to cell lysis (Pereira et al., 2016).
When co-cultured with C. glabrata, C. albicans biofilms were more susceptible to the action of the dichloromethane fraction. Some studies have been developed to understand the mechanisms of interaction between Candida spp. The reduction of CFU/mL of C. albicans cells grown in mixed biofilms can be attributed to competitive interactions between Candida spp for adhesion sites during the early phases of biofilm formation, which attenuated the pathogenicity of C. albicans in animal models (Rossoni et al., 2015).
Our data further indicated that inhibition of C. albicans and C. albicans + C. glabrata biofilm formation was not causally related to the impaired ability of the strains to adhere to biotic and abiotic surfaces. Although the dichloromethane fraction had a significant inhibitory effect on yeast adherence, it presented a greater inhibitory potential on biofilm formation at the lowest tested concentration. This fact suggests that cell adhesion was likely not a major requirement for biofilm development when cells were exposed to the fraction of S. obtusifolium leaf extract (Gondim et al., 2018).
The secondary metabolites (saponins, flavonoids, tannins) present in the fraction are likely responsible for its anti-adherent and antibiofilm properties. The bioactive compounds found in S. obtusifolium leaves complex with ergosterol in the cell membrane, creating pores, ionic imbalance, and the loss of membrane integrity (Pereira et al., 2016).
To date, there are no studies in the literature testing the antifungal activity of the dichloromethane fraction of S. obtusifolium leaf extract against Candida non-albicans spp. Our findings may provide relevant insights into the complex pathogenicity of fungal infections and the modulatory behavior of bioactive fractions on yeast growth and development. However, the present work has the limitations of an initial in vitro study, requiring additional analyses, such as cytotoxicity tests, investigations into the mechanisms of direct action of the fraction against microorganisms, as well as toxicity and antifungal activity tests in vivo to continue the results presented here on the potential of this promising plant species.
The dichloromethane fraction of S. obtusifolium extract showed antifungal activity against Candida spp and reduced the adherence and metabolic activity of Candida biofilms. While the fraction showed mostly fungistatic activity, it had a fungicidal effect on Candida albicans over 24 hours. These effects may be attributed to the secondary metabolites present in the fraction, such as saponins and phenolic compounds. Further toxicological and antioxidant assessments are needed to validate the use of this fraction as an alternative antifungal therapy in dental care.
Acknowledgements
This study was funded by the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) and the Foundation for Research Support in the State of Paraíba (FAPESQ, Brazil). The article originated from a thesis.
Data Availability Statement
The entire data set that supports the results of this study was published in the article itself.
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Editor:
Jairo Lizandro Schmitt
