The Beneficial Effect of Equisetum giganteum L. against Candida Biofilm Formation: New Approaches to Denture Stomatitis

Equisetum giganteum L. (E. giganteum), Equisetaceae, commonly called “giant horsetail,” is an endemic plant of Central and South America and is used in traditional medicine as diuretic and hemostatic in urinary disorders and in inflammatory conditions among other applications. The chemical composition of the extract EtOH 70% of E. giganteum has shown a clear presence of phenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol, in addition to styrylpyrones. E. giganteum, mainly at the highest concentrations, showed antimicrobial activity against the relevant microorganisms tested: Escherichia coli, Staphylococcus aureus, and Candida albicans. It also demonstrated antiadherent activity on C. albicans biofilms in an experimental model that is similar to dentures. Moreover, all concentrations tested showed anti-inflammatory activity. The extract did not show cytotoxicity in contact with human cells. These properties might qualify E. giganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis.


Introduction
Denture stomatitis (DS) is an inflammatory disease that affects the oral mucosa underlying a removable dental prosthesis, both partial and total. DS is considered a very common disease affecting denture wearers, especially those with maxillary complete dentures [1,2]. Due to the strong relationship between DS and Candida fungus, the use of proper hygiene aids by the denture wearer is fundamental, by physical means, such as brushing, and, if necessary, chemical, and immersion of dentures in disinfectant solutions. Local or systemic antifungal therapy may still be an option in some cases of DS [3]; however, one should know the predisposing factors related to the user and eliminate them or change them whenever possible. There is no sense in using drug therapy, if it is not associated with awareness and change in the factors that contribute to DS.
Currently, treatments directed to DS include topical antifungal therapy, such as several formulations based on nystatin, systemic antifungal medication such as azoles, attention to oral hygiene, denture disinfection procedures, removal of the denture overnight, and replacement of older dentures [4].
Continued use of topical antifungal agents, nystatin suspensions [5], and disinfectants, such as sodium hypochlorite (NaOCl), glutaraldehyde, and chlorhexidine, can induce changes in the properties of the resin surface, such as roughness, hardness, and wettability, which can contribute to fungal adhesion [5][6][7]. The continued use of systemic antifungal therapy can also lead to serious adverse effects, such as 2 Evidence-Based Complementary and Alternative Medicine hepatotoxicity and nephrotoxicity, and microbial resistance [4]. Moreover, after completion of antifungal therapy, there is a rapid recurrence of the DS, because the denture base resin serves as a reservoir for the fungus, and local or systemic antifungal therapies are incapable of eliminating the microorganisms present in denture bases, leading to the need of a further treatment [8].
Following this reasoning, the search for a better knowledge of antimicrobial action of medicinal plants has increased exponentially, and herbal products have proven to be an alternative to synthetic chemicals and can play an important role in the treatment of DS [9]. Studies using herbal medicines to combat dental plaque microorganisms or oral biofilm presenting Candida have revealed the efficacy of these agents as antimicrobial and antiadherent medications for the prevention of dental biofilm and treatment of candidiasis [10].
Equisetum giganteum L. (E. giganteum) is an endemic plant of Central and South America and is used as diuretic and hemostatic in urinary disorders and in inflammatory conditions in traditional medicine. Besides E. giganteum is commonly used as a substitute for Equisetum arvense; this drug is extensively commercialized for the purpose of beneficial role in the field of Medicine and Nutrition as a dietary supplement [11]. Although this plant has antimicrobial effect [12], until now there are no studies of E. giganteum against Candida albicans (C. albicans).
Propolis is a resinous substance that has notable antifungal, antibacterial, antioxidant, anti-inflammatory, and immunomodulatory properties. In Odontology, the propolis has been associated mainly with the process of healing [13][14][15][16][17]. Considering their antifungal and anti-inflammatory activities, the propolis could be an alternative for the local treatment of DS [9,18,19].
Antiatherogenic [20], antitumor, antioxidant [21], and virucidal [22] effects have been reported for Punica granatum (P. granatum). The findings of studies, including some that have related the inhibition of adherence, suggest that oral bacteria and C. albicans are sensitive to the extract of P. granatum [23][24][25]. In a clinical study, negativity of yeasts on lesions of DS in most subjects was observed after gel application of P. granatum. It could be concluded that P. granatum extract may be used as a topical antifungal drug for the treatment of this type of candidosis [9,26].
Therefore, in the present study, we evaluated the antimicrobial and anti-inflammatory potential of E. giganteum extract on C. albicans, as well as its influence on fungal adherence to the surface of heat-polymerized acrylic resin. The cytotoxic potential of the extract on human palatal epithelial cells and human monocytes was also assessed. This was done with a view to the future combination of the E. giganteum as a therapeutic/preventive alternative, for topical application, or inclusion in soft denture lining materials on inner surface of dentures, as a new alternative to the standard treatment in DS.   . Bacteria were grown and tested in brainheart infusion broth (BHI) (Difco, Sparks, MD, USA).

Antimicrobial
Assay. In vitro antimicrobial action was analyzed by the broth microdilution method with the objective of obtaining the minimum inhibitory concentration (MIC) of the E. giganteum extract. Bacterial and fungal suspensions (1 × 10 5 cells/mL) were, respectively, inoculated into BHI and Sabouraud broth using 96-well plates. Wells containing each inoculum with the 1% NaOCl solution (CTRL/NaOCl) and culture medium (Sabouraud or BHI broth) (CTRL/Medium) served as the positive and negative control, respectively.
According to the different treatments, the resin specimens were divided into five groups: E50, E25, and E16 (50, 25, and 16mg/mL) of E. giganteum extract; CTRL/PBS, sterile phosphate buffered saline (pH 7.2); or CTRL/NaOCl-1% NaOCl solution. The treatments were performed in 6-well plates, at room temperature for 10 min. Sequentially, the specimens were incubated for 90 min at 37 ∘ C at 75 rpm (Fanem, Guarulhos, Brazil) with fungal suspension (1 × 10 7 cells/mL), washed with PBS, and incubated in RPMI-1640 (GIBCO, Grand Island, NY) at 37 ∘ C for 12 h at 75 rpm. Three specimens for group were stained by LIVE/DEAD BacLight Bacterial Viability Kit solution (Molecular Probes, Eugene, OR, USA) [29] and incubated in the dark for 15 min at room temperature.
Subsequently, the specimens were conditioned in the confocal laser scanning microscopy (Leica Microsystems GmbH, Mannheim, Germany), from which digital images were obtained. The viable cells were visualized in green, while dead cells were visualized in red. With the use of BioImage Software program, the biofilm mass was quantified including both viable and nonviable cells and expressed as m 3 . The more the biofilm mass on the specimens decreased, the higher the antiadherent activity was.

Cell
Culture. HPEC were cultivated as previously described by Klingbeil et al. [30] and human monocytes were obtained from thirty-milliliter volumes of peripheral blood, which were drawn from apparently healthy five volunteers. After obtaining peripheral blood mononuclear cells by cell separation using Histopaque 1077 gradients, according to the manufacturer's instructions (Sigma-Aldrich Company), monocytes were counted using neutral red (0.02%) and incubated in 96-well culture plates by 2 h at 37 ∘ C in 5% CO 2 . Following, nonadherent cells were removed by aspiration. Monocytes were cultivated in RPMI-1640 (GIBCO, Grand Island, NY), supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). The medium was replaced every other day until the cells grew to about 70% confluence. The experimental protocol used in this study was approved by the Committee on Ethics in Human Experimentation, of Bauru School of Dentistry, University of São Paulo (grant number 6706412.2.0000.5417, November 8, 2012), and written informed consent was obtained from all patients. This study was performed in accordance with the Declaration of Helsinki.
2.6.1. Cell Viability Assay. The different extracts were evaluated regarding cytotoxicity by using MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) colorimetric assay (Sigma Chemical Co., St. Louis, USA). Briefly, monocytes (2 × 10 5 cells/well) suspended in 200 uL of medium were plated into 96-well tissue culture plates for 24 h. Thereafter, 200 uL of fresh medium containing various concentrations of E. giganteum extracts (50,25,16,8, and 4 mg/mL) was added to each well and incubated for time intervals of 1, 12, and 24 hours and compared with control cells (untreated and water). After these periods, the plates were washed twice in PBS and 200 uL of fresh medium mixed with 10 uL of 5 mg/mL MTT dye in PBS was added to the wells for 4 h in a humidified 5% CO 2 atmosphere at 37 ∘ C. After the removal of MTT solution, 150 uL of dimethyl sulphoxide (DMSO-LGCBiotecnologia, São Paulo, Brazil) was added and mixed to dissolve the dye crystals. The absorbance was measured at 500 nm by means of a multiplate reader (Synergy Mx Monochromator-Based, Biotek, Washington, DC, USA). The same assay was performed with human palatal epithelial cells (HPEC) (10 4 cells/well) for 12 and 24 hours. (ROS). Endogenous amounts of ROS in human monocytes were determined using the fluorescent probe 2 ,7dichlorofluorescin diacetate (Cell Rox Deep Red Reagent; Life Technologies, Grand Island, NY, USA). Monocytes (2 × 10 5 cells/well) were in vitro incubated with E. giganteum extract (50,25,16,8, and 4 mg/mL) for 1 hour at 37 ∘ C in 5% CO 2 atmosphere. After, they were stimulated with LPS at 5 g/mL or C. albicans SC5314 (1 × 10 7 cells/mL) for three hours and incubated with 5 M of Cell Rox at 37 ∘ C in 5% CO 2 atmosphere for 30 min. The fluorescence intensities (FIs) of the cells were measured using the multiplate reader at 640/665 nm, at 37 ∘ C.

Statistical
Analysis. The data are expressed as the mean ± standard deviation of at least three independent experiments, in triplicate. The data were analyzed, where appropriate, by means of the one-way ANOVA, two-way ANOVA, or Kruskal-Wallis Test followed by the Tukey and Dunnett Test using the STATISTICA software program (version 12.0; StatSoft Inc., Tulsa, OK, USA). We considered < 0.05 as a level of statistically significant difference.

Compounds Isolated from Equisetum giganteum L.
The constituents present in the hydroethanolic extract were identified by in RP-UHPLC, UV-vis, and MS/MS n spectra analysis and comparison with data from the literature [11]. In total, 13 constituents were identified in the 70% EtOH extract (Table 1). Figure 1 shows the structures of styrylpyrones and flavonoids identified.
The UHPLC-PAD-ESI-MS n analysis of the hydroethanolic extract highlighted UV spectrum characteristic of styrylpyrones with bands at 253-258 and 355-373 nm [31] and flavonoids at 240-290 nm related to A-ring and at 300-390 nm related to B-ring [32]. Diagnostics precursor ions at m/z 379, 301, and 285 confirm the presence of styrylpyrones and flavonoid glucosides derivatives of quercetin and kaempferol, respectively. The signals of mass fragments at m/z 178 and 192 were related to presence of caffeic and ferulic acid derivatives. The neutral losses of 162 mass units allowed the identification of hexoses (sophoroside or glucoside).
In Figure 2, the UPLC-PAD chromatogram of the E. giganteum 70% EtOH extract is presented.

Antimicrobial
Activity of E. giganteum. The E. giganteum extract (E50, E25, E16, E8, and E4) showed antimicrobial activity against all strains studied (Figure 3). The strain most sensitive to the E. giganteum extract was S. aureus.  At the highest concentration of the extract (50 mg/mL), the results were similar to 1% NaOCl for all strains tested. Even at its lowest concentrations, the extract still showed a microbicidal action.

Antibiofilm Effect of E. giganteum against C. albicans SC5314.
The specimens pretreated with sterile phosphate buffered saline (CTRL/PBS) showed an expressive presence of biofilm mass of C. albicans on the acrylic resin specimen surface, thereby revealing biofilms at the stage of proliferation and filamentation (Figure 4(d)). As expected, this biofilm was mainly formed of viable (green) cells (Table 2). In contrast with PBS, 1% sodium hypochlorite pretreatments removed the majority of biofilm (Figure 4(e)), leaving only a few viable cells; but most of the cells were nonviable ( Table 2). The surface of resin specimens pretreated with the different concentrations of E. giganteum extract (50, 25, and 16 mg/mL) revealed that increasing the concentration of the extract improved its antiadherent activity, because biofilm formation decreased with an increase in amount of herbal extract used (Figures 4(a), 4(b), and 4(c)). As shown for 1% NaOCl, E50 significantly reduced the biofilm mass    on the specimen surfaces, although scarce viable cells can still be noted (Figure 4(a)). All groups of the E. giganteum extract presented lower numbers of remaining fungal cells in comparison with control (PBS), showing marked antiadherent activity of the E. giganteum extracts against C. albicans biofilms. At 25 and 16 mg/mL, the values of biofilm mass were similar to those obtained for PBS; however, they differed statistically from both control (NaOCl) and E50. No statistical differences were observed between E50 and CTRL/NaOCl with respect to the total number of remaining nonviable cells.

ROS Production in Monocytes Treated with E. giganteum
Extract. The results revealed that all the extract concentrations (50,25,16,8, and 4 mg/mL) were able to decrease the levels of ROS production by cells stimulated with LPS or C. albicans, returning to baseline levels. Values for cells treated with E. giganteum extract, which were not stimulated with LPS or C. albicans, remained at baseline levels ( Figure 5).

Cytotoxicity Test.
Both monocytes and HPEC remained viable in the presence of E. giganteum extract; but, at the lowest concentration (E4) for 12 hours on monocytes, there were significant differences between the cells treated with extract and untreated cells. In addition, at highest concentrations of E. giganteum, the percentage of cell viability was higher than it was for untreated cells, suggesting an increased cell metabolism (Figures 6(a) and 6(b)).

Discussion
C. albicans is the predominant yeast species isolated from patients with DS, due to its greater capacity to form biofilms on surfaces [33][34][35][36], including denture acrylic surfaces. Therefore, decontamination of dentures is a fundamental aspect of effective oral hygiene, in order to prevent the denture stomatitis. Typically, research on Candida biofilms has evaluated the efficacy of different disinfectant solutions such as 1 and 2% NaOCl and 2% glutaraldehyde on heatpolymerized acrylic resin [37].
Considering that various disinfectants affect the physical properties of resin surface of denture [37][38][39][40][41][42][43][44], alternative therapies, such as phytotherapeutic agents, with antimicrobial potential and ability to eliminate or prevent the adhesion of C. albicans to the inner surface of the denture, without damaging the mucosa or dentures, may be of extreme interest to removable denture wearers, particularly those with maxillary complete dentures [45,46].
In the present study, the crude extract of E. giganteum showed a potent antimicrobial effect on C. albicans and on fungus adherence to heat-polymerized acrylic resin. Furthermore, the plant extract demonstrated a potential in vitro antiinflammatory effect on monocytes, while it had no cytotoxic effect in vitro on monocytes and HPEC. Our study is the first to verify the beneficial effect of E. giganteum against Candida biofilm formation on surfaces, and its biocompatibility.
Although E. giganteum does not have bacteriostatic potential, the antimicrobial action was evident in our findings, even at the lowest concentration tested (4 mg/mL). This Relative fluorescence unit (rfu) was shown not only against C. albicans, but also against the S. aureus and E. coli bacteria used as parameters for comparison in our study. At a concentration of 50 mg/mL, by means of antimicrobial assay based on the broth microdilution method, the herbal extract showed antimicrobial action similar to 1% sodium hypochlorite, an agent widely applied for immersion of dentures during treatment of DS.
We emphasize the values of antimicrobial action obtained in our study, which were higher than those reported in a previous study [12] that assessed the same herbal extract against Gram-negative and Gram-positive bacteria and obtained no activity against Escherichia coli (E. coli). In our study, another aspect that highlights the significant antimicrobial activity of E. giganteum is that this phytotherapeutic agent was effective against a clinical strain of E. coli (O1124), which usually presents more resistance and higher pathogenicity than standard strains when they are isolated from patients [47]. However, even at low concentrations of the extract (8 and 4 mg/mL), we observed that its antimicrobial activity against this strain of E. coli ranges from 65 to 54% of death.
The chemical composition of the extract EtOH 70% of E. giganteum has shown a clear presence of phenolic compounds derived from caffeic and ferulic acids and flavonoid heterosides derived from quercitin and kaempferol, in addition to styrylpyrones (Table 1; Figure 1). This herbal extract produces bioactive compounds such as flavonoids that are responsible for its anti-inflammatory and antioxidant effects, in addition to having analgesic, antimicrobial, and immunomodulatory properties [48,49]. Therefore, the flavonoids present in the composition of E. giganteum probably are the constituents responsible for the microbicidal effects observed here, because they have the ability to inactivate microbial adhesion proteins and transport proteins and to rupture the microbial membrane.
Furthermore, in order to simulate the resinous portion of removable dentures that have been contaminated by C. albicans, as frequently it occurs with dentures under the conditions of the intraoral environment, we developed contaminated specimens made of denture base material. The resin surface pretreated with extract of E. giganteum at all concentrations showed a significant reduction in the biofilm mass of viable C. albicans cells in comparison with the untreated resin. In other words, we can assert that a brief 10-minute treatment of the resin surface with the extracts was able to significantly reduce the capacity of C. albicans to adhere to the surface. We emphasize that, at 50 mg/mL, the treatment with E. giganteum resulted in the lowest biofilm mass found, when compared with any of the other concentrations. Interestingly, after these pretreatments, the resin surface showed dispersed fungus in both its filamentous and yeast forms, especially at the highest concentration of the extract. As observed in our control samples, confluent biofilms presenting predominantly filamentous forms are developed on the resin surface. Taken together, we believe that the herbal extract was able to interfere in the expression of fungal virulence factors, in addition to having an antiadherent effect on the microorganism.
One possible hypothesis for explaining the effect of biofilm removal by E. giganteum extract could be based on biofilm pH. Since the most of biofilms are formed at neutral pH, the conditions having very high or very low pH values can provide changes on microbial metabolism and surface properties, interfering in their adhesion process to surfaces [50]. Hypochlorite solutions, for example, having alkaline pH (pH > 11), can increase the electrostatic repulsion between the yeast cells and the surface of the resin material, dissolving the biofilm cells [51]. Thus, the acidic pH (pH = 5.48) of the hydroalcoholic extract of E. giganteum, associated with antimicrobial activity, especially in higher concentrations, could explain their important antiadherent effect on C. albicans biofilms; however, further studies are required about the mechanisms involved.
In addition to the beneficial results with respect to the antimicrobial and antiadherent activities, all concentrations of E. giganteum extract demonstrated suppression of reactive oxygen species in human monocytes stimulated in vitro by LPS or C. albicans. Previous studies have also shown reduced levels of ROS resulting from the action of E. arvense and have attributed this effect to the presence of flavonoids [52]. In fact, flavonoids are able to eliminate ROS and RNS by donation of electron and hydrogen (H + ) from the hydroxyl groups present in their composition, stabilizing these free radicals [49,53] or suppressing the formation of ROS by inhibiting the enzymes involved in generating them [49,54]. The presence of phenolic acids was also detected in the E. giganteum extract. This group of substances is characterized by their antioxidant properties, since they were able to reduce the levels of reactive oxygen species due to trapping free radicals directly or scavenging them through a series of combined reactions with antioxidant enzymes [49,52,53,55]. Other inflammatory mediators should be evaluated for a more accurate conclusion and to imply that E. giganteum extract could negatively modulate monocytemediated inflammatory responses.
Another interesting finding was that the extract did not alter the viability of monocytes and HPEC for up to 24 hours. This has important clinical repercussions, since these cells are closely related to the inner surface of the denture, in denture wearers. The oral epithelial cells are the first cells to interact with C. albicans during the establishment of DS.
In summary, our results suggest that the phytotherapeutic E. giganteum extract has the following properties: antimicrobial against C. albicans, S. aureus, and E. coli; antiadherence 8 Evidence-Based Complementary and Alternative Medicine of C. albicans to heat-cured acrylic resin specimens; and antiinflammatory effects on human monocytes activated by C. albicans. In addition, the phytotherapeutic extract did not compromise the viability of human monocytes or human palatal epithelial cells. These properties might qualify E. giganteum extract to be a promising alternative for the topic treatment and prevention of oral candidiasis and denture stomatitis.