The Anti-Virulence Effect of Vismia guianensis against Candida albicans and Candida glabrata

In folk medicine, Vismia guianensis is used to treat skin diseases and mycoses in the Amazon region. We evaluated the anti-Candida activity of the hydroalcoholic extract from the leaves of Vismia guianensis (EHVG). HPLC-PDA and FIA-ESI-IT-MSn were used to chemically characterize EHVG. The anti-Candida activity was determined in vitro by the minimum inhibitory concentrations (MIC) against Candida glabrata (ATCC-2001); Candida albicans (ATCC-90028, ATCC-14053, and ATCC-SC5314), and C. albicans clinical isolates. EHVG effects on adhesion, growth, and biofilm formation were also determined. Molecular docking was used to predict targets for EHVG compounds. The main compounds identified included anthraquinone, vismione D, kaempferol, quercetin, and vitexin. EHVG was fungicidal against all tested strains. C. albicans ATCC 14053 and C. glabrata ATCC 2001 were the most sensitive strains, as the extract inhibited their virulence factors. In silico analysis indicated that vismione D presented the best antifungal activity, since it was the most effective in inhibiting CaCYP51, and may act as anti-inflammatory and antioxidant agent, according to the online PASS prediction. Overall, the data demonstrate that EHVG has an anti-Candida effect by inhibiting virulence factors of the fungi. This activity may be related to its vismione D content, indicating this compound may represent a new perspective for treating diseases caused by Candida sp.


Introduction
Candidemia has become one of the most common invasive Candida bloodstream infections. The high global incidence of candidiasis can be explained by the increase in the number of susceptible hosts, such as patients submitted to immunosuppressive

Characterization of the Plant Material and Extract
We determined the particle sizes of dried and powdered leaves to characterize the plant material and the yield after extraction. Figure 1 shows the particle size of the leaves and the product obtained from V. guianensis, which was classified as a semi-fine powder, with a large portion being retained in mesh M60 (34.09%), followed by mesh M80 (20.73%).
We determined the particle sizes of dried and powdered leaves to characterize the plant material and the yield after extraction. Figure 1 shows the particle size of the leaves and the product obtained from V. guianensis, which was classified as a semi-fine powder, with a large portion being retained in mesh M60 (34.09%), followed by mesh M80 (20.73%).  Table 1 shows the results related to the ash content and the total amount of residue from V. guianensis. The different hydromodules used to prepare the hydroethanolic extract of the V. guianensis leaves (EHVG) presented similar yields, but different ash content and total residues. The hydromodule 1:10 showed the best results, with 97% organic matter, and, therefore, it was chosen for chemical analysis and experiments.  15 13 a : X ± SD = mean ± standard deviation.

Chemical Profile of EHVG
To determine the chemical profile of EHVG, different hydromodules were evaluated qualitatively by HPLC-PDA at 254 nm. Figure 2 shows the chromatographic profile of the hydromodule 1:10, which qualitatively exhibited the best extraction profile in terms of peak area.  Table 1 shows the results related to the ash content and the total amount of residue from V. guianensis. The different hydromodules used to prepare the hydroethanolic extract of the V. guianensis leaves (EHVG) presented similar yields, but different ash content and total residues. The hydromodule 1:10 showed the best results, with 97% organic matter, and, therefore, it was chosen for chemical analysis and experiments.

Chemical Profile of EHVG
To determine the chemical profile of EHVG, different hydromodules were evaluated qualitatively by HPLC-PDA at 254 nm. Figure 2 shows the chromatographic profile of the hydromodule 1:10, which qualitatively exhibited the best extraction profile in terms of peak area.        Table 2. It is possible to observe a predominance of anthraquinones, flavonoids, and Vismione D.   Table 2. It is possible to observe a predominance of anthraquinones, flavonoids, and Vismione D.  Figure 4 shows the structures of the compounds identified in the leaf extract of V. guianensis (EHVG), according to the spectrum shown in Figure 3 and the compounds presented in Table 2. Herein, it is important to highlight, the structures of Vismione D, anthraquinone F, and kaempferol, which are important markers of the genus Vismia.
14 285 Kaempferol Figure 4 shows the structures of the compounds identified in the leaf extract of V. guianensis (EHVG), according to the spectrum shown in Figure 3 and the compounds presented in Table 2 Figure 4. Chemical structures of the compounds identified in the Vismia guianensis leaf extract.

EHVG Showed Low Toxicity Using Different Assays
To define the best concentrations of the extract of Vismia guianensis (EHVG) for the antifungal assays, its hemolytic and cytotoxic activities were evaluated. The extract showed low toxicity, inducing less than 10% cell death and hemolysis ( Figure 5A,B) at concentrations ≤ 5 mg/mL. Cell viability was greater than 80% at concentrations ≤ 10 mg/mL (Supplementary Materials: Figure S1). To define the best concentrations of the extract of Vismia guianensis (EHVG) for antifungal assays, its hemolytic and cytotoxic activities were evaluated. The ext showed low toxicity, inducing less than 10% cell death and hemolysis ( Figure 5A,B concentrations ≤ 5 mg/mL. Cell viability was greater than 80% at concentrations ≤ mg/mL).

Antifungal Activity of EHVG against C. albicans and C. glabrata
EHVG showed antifungal potential and fungicidal activity against all tested stra (Table 3). Against C. albicans isolates, EHVG MIC values ranged from 3.125-6.25 mg/ MFC from 3.125-12.5 mg/mL, and the CFM/MIC ratio ranged from 1-2. Agains glabrata, the MIC was 3.125 mg/mL, and the MFC was 6.25 mg/mL. MIC values were u as a parameter for selecting two strains for the subsequent assays: C. albicans (SC5314) C. glabrata (ATCC 2001), both of which are of clinical interest. 2.4. Antifungal Activity of EHVG against C. albicans and C. glabrata EHVG showed antifungal potential and fungicidal activity against all tested strains (Table 3). Against C. albicans isolates, EHVG MIC values ranged from 3.125-6.25 mg/mL, MFC from 3.125-12.5 mg/mL, and the CFM/MIC ratio ranged from 1-2. Against C. glabrata, the MIC was 3.125 mg/mL, and the MFC was 6.25 mg/mL. MIC values were used as a parameter for selecting two strains for the subsequent assays: C. albicans (SC5314) and C. glabrata (ATCC 2001), both of which are of clinical interest.  Figure 6 shows the fungicidal activity of EHVG. EHVG inhibited the growth of C. albicans and C. glabrata when tested at two different concentrations. Figure 6A shows the results for C. albicans SC5314, after 36 (2× MIC = 12.5 mg/mL) and 48 h (1× MIC = 6.25 mg/mL). Figure 6B shows similar results against C. glabrata (ATCC 2001), in a shorter period, for both concentrations, since the inhibition was observed after 24 and 36 h at concentrations of 2× (6.25 mg/mL) and 1× the MIC (3.125 mg/mL), respectively.  Figure 7A shows that EHVG was more effective than Amphotericin B in inhibiting C. glabrata (ATCC 2001) adhesion at the lowest   Figure 7A shows that EHVG was more effective than Amphotericin B in inhibiting C. glabrata (ATCC 2001) adhesion at the lowest tested concentration (1/2× MIC). At the highest concentration (1× MIC), all treatments had similar inhibitory effects in comparison with the untreated control. Figure 7B,C demonstrate that EHVG effects were equivalent to those of reference drugs (FLU and Ampho B), regardless of the concentration used, against the two different C. albicans strains.

Introduction
Candidemia has become one of the most common invasive Candida bloodstream infections. The high global incidence of candidiasis can be explained by the increase in the number of susceptible hosts, such as patients submitted to immunosuppressive treatments, long-term use of broad-spectrum antibiotics, use of catheters and probes, and hematopoietic transplants, amongst others. In addition, fungal resistance to antimicrobials has also increased [1,2].
Candidiasis is the fourth most common cause of nosocomial infections in the world, and C. albicans is the most frequent causative agent of medical relevance, due to its prevalence in both healthy hosts and those with underlying conditions or immune impairment. C. albicans is one of the most prevalent fungal species in the human microbiota, since it asymptomatically colonizes healthy individuals. This microorganism is, therefore, easily detected in the oral mucosa, gastrointestinal tract, urogenital tract, and skin of humans from birth [3]. Treatment with EHVG inhibited biofilm formation with an efficacy similar to that of Fluconazole or Amphotericin B, even at sub-inhibitory concentrations (1/4 MIC and 1/2 MIC). For Candida albicans SC5314, the reduction was more significant than that observed with Amphotericin B (Figure 9). MIC). For Candida albicans SC5314, the reduction was more significant than that observed with Amphotericin B (Figure 9).  Figure 10 shows the reduction in biofilm biomass in the groups treated with EHVG for 48 and 72 h, with the same intensity as that observed for Amphotericin B and sometimes higher than that for Fluconazole. This confirms the data obtained from the MTT assay.  Figure 10 shows the reduction in biofilm biomass in the groups treated with EHVG for 48 and 72 h, with the same intensity as that observed for Amphotericin B and sometimes higher than that for Fluconazole. This confirms the data obtained from the MTT assay.

Introduction
Candidemia has become one of the most common invasive Candida bloodstream infections. The high global incidence of candidiasis can be explained by the increase in the number of susceptible hosts, such as patients submitted to immunosuppressive treatments, long-term use of broad-spectrum antibiotics, use of catheters and probes, and hematopoietic transplants, amongst others. In addition, fungal resistance to antimicrobials has also increased [1,2].
Candidiasis is the fourth most common cause of nosocomial infections in the world, and C. albicans is the most frequent causative agent of medical relevance, due to its prevalence in both healthy hosts and those with underlying conditions or immune impairment. C. albicans is one of the most prevalent fungal species in the human microbiota, since it asymptomatically colonizes healthy individuals. This microorganism is, therefore, easily detected in the oral mucosa, gastrointestinal tract, urogenital tract, and skin of humans from birth [3].

In Silico Biological Activity and Toxicity for the Compounds Identified in EHVG
The PASS online tool evaluated the antifungal, anti-inflammatory, and antioxidant activities of the five selected compounds detected in EHVG, including catechin, kaempferol, anthraquinone, and Vismione D, in comparison to those of Fluconazole and Amphotericin. The compounds showed greater Pa than Pi as antifungal, considering Pa values higher than Pi (>0.3) due to the strong molecular potency. They also exhibited a high probability of gut permeability, and no violation to Lipinski's rules.
Vismione D showed the highest predictive antifungal and also showed high values as anti-inflammatory agent when compared to other compounds found in EHVG (Table 4). Kaempferol and quercetin showed the highest predictive value as anti-inflammatory. Quercetin and catechin showed the highest values as antioxidant compounds, and an-thraquinone, fluconazole, and amphotericin B had no antioxidant predictive activities with Pa values lower than 0.3. Table 4. PASS prediction of commercial drugs and three selected compounds detected in the Vismia guianensis extract, considering the Potential activity (Pa) higher than potential inactivity (Pi), as well as the antifungal, antioxidant, and anti-inflammatory activities. Pa (probability "to be active") estimates the chance that the studied compound belongs to the sub-class of active compounds. b : Pi (probability "to be inactive") estimates the chance that the studied compound belongs to the sub-class of inactive compounds.

Compounds Present in EHVG, Especially Vismione D, Interact with CaCYP51
The following compounds identified in EHVG were used for molecular docking: anthraquinone F, catechin, kaempferol, and vismione D. Figure 11 illustrates the molecular interaction between vismione D and the enzyme CaCYP51 of C. albicans. Analysis of the vismione D + CaCYP51 complex is characterized by the formation of hydrogen bonds between the ligand and the enzyme residues Tyr118, Ser378, and Met508, as well as hydrophobic contact with residues Leu121, Phe228, Pro230, Phe233, Gly303, Ile304, Gly307, Gly308, Thr311, Leu376, Phe380, and Ser508, including interactions with the heme group.  Pa (probability "to be active") estimates the chance that the studied compound belongs to the subclass of active compounds. b : Pi (probability "to be inactive") estimates the chance that the studied compound belongs to the sub-class of inactive compounds.

Compounds Present in EHVG, Especially Vismione D, Interact with CaCYP51
The following compounds identified in EHVG were used for molecular docking: anthraquinone F, catechin, kaempferol, and vismione D. Figure 11 illustrates the molecular interaction between vismione D and the enzyme CaCYP51 of C. albicans. Analysis of the vismione D + CaCYP51 complex is characterized by the formation of hydrogen bonds between the ligand and the enzyme residues Tyr118, Ser378, and Met508, as well as hydrophobic contact with residues Leu121, Phe228, Pro230, Phe233, Gly303, Ile304, Gly307, Gly308, Thr311, Leu376, Phe380, and Ser508, including interactions with the heme group.  (Table 5).  Figure 12 shows the spatial conformation obtained for the molecular docking analysis of vismione D and CaCYP51 structures. Several binding sites could be observed, which favor a possible action of the compound on the enzyme. Molecular docking analysis indicated that vismione D presents the highest affinity for the fungal enzyme CaCYP51, with a binding free energy (∆Gbind) of 10.96 kcal/mol and an inhibition constant (Ki) of 0.009 µM, when compared to the reference drugs posaconazole and fluconazole. Anthraquinone provided results similar to those of posaconazole, and the results for catechin and kaempferol were similar to those of fluconazole (Table 5).  Figure 12 shows the spatial conformation obtained for the molecular docking analysis of vismione D and CaCYP51 structures. Several binding sites could be observed, which favor a possible action of the compound on the enzyme.

Discussion
V. guianensis is a native plant found in different regions of Brazil, especially in the Amazon region, where its leaf extracts are used to treat skin inflammatory and infectious skin diseases [13]. Little is known about its mechanisms of action. Thus, our aim was to investigate its anti-Candida activity from the hydroethanolic extract prepared with a vegetal part (EHVG), considering that plants are still the main source of bioactive compounds and that there is a growing resistance to commercial antifungal drugs.
For the extract preparation, the particle size of the leaves and the product obtained were evaluated and classified as a semi-fine powder, with a large part retained in mesh M60, followed by mesh M80. It is important to highlight that the particle size of the dry plant material is important for the extract preparation, since the more homogenous the extraction is, the better, resulting in an increased yield and collection of chemical components, maximizing biological activities [19].
The preparation of EHVG with different hydromodules resulted in a similar yield, but different dry and total residues after evaluation of the ash content. The hydromodule 1:10 showed the best result for the chemical analysis, as it exhibited the best qualitative profile due to the more intense peaks and larger and better-defined areas, to confirm the presence of secondary metabolites in the extract. For this reason, it was used for antifungal assays.
Direct flow injection analysis of EHVG with ionization in negative mode confirmed the presence of phenolic compounds, including anthraquinones, catechins, epicatechins, kaempferol, vismione D, and flavonoids. The presence of these compounds was also previously reported by Seo et al. [20], who isolated five benzophenones, vismiaguianones A-E, and two benzocoumarins, vismiaguianins from chloroformic root extract during the investigation of the antitumoral effects of V. guianensis. In 2004, Politi et al. [14] described the presence of anthraquinones, vismiones, flavonoids, xanthones, and benzophenones in n-hexane, as well as dichloromethane, when investigating leaf methanol extracts in comparison with those from roots. Later, Hussain et al. [21], in a review related to the genus Vismia, described the presence of the same compounds which we found, and discussed anthraquinones and vismione as important chemical markers of this group.
According to Costa et al. [22], antifungal activities may be related to the presence of mono-and sesquiterpene metabolites, components also identified in the present study. This previous study demonstrated that such metabolites could promote cell membrane rupture, causing fungal death as well as impairments of membrane synthesis, cellular respiration, and spore germination. Flavonoids may also cause rupture of the microbial membrane due to their lipophilic nature [23]. Other V. guianensis chemical components, such as humulene epoxide II (not reported in this study) isolated from the oil of leaves, exhibited antifungal activity against several Candida species, including C. albicans and C. glabrata [22]. EHVG exhibited low toxicity using different techniques and different concentrations. The decision to investigate this by different assays was based on previous observations on the MTT assay; neutral red and crystal violet staining assays provided complementary data for cell viability [24].
The antifungal activity of EHVG was evaluated by time-kill assays, using the C. albicans (SC5314) and C. glabrata (ATCC 2001) strains. These strains were chosen due to their clinical interest [22,25,26]. EHVG showed a fungicidal effect against both strains, inhibiting all Candida strains over 48 h, especially for C. glabrata after the first 12 h.
As EHVG was antimicrobial against C. albicans and C. glabrata, we investigated its effects on Candida virulence (adhesion and biofilm formation). The extract impaired the adhesion of all tested strains with similar efficacy to that observed for reference drugs such as fluconazole. This is an important action, considering that adhesion is the first step of a successful fungal colonization and pathogenesis [1,7].
EHVG exerted an inhibitory concentration-dependent effect on early biofilm formation by C. albicans (ATCC90028) and C. glabrata (ATCC2001). The observed inhibitory effects were equivalent to Amphotericin B, but no differences were observed after incubation of sub-inhibitory concentrations of EHVG. The extract also exerted inhibitory activity on mature biofilms produced by C. albicans (ATCC 90028) and C. glabrata (ATCC 2001), with similar performance to those of amphotericin B and fluconazole when tested at 2× MIC. For C. albicans (SC5314), EHVG showed better results than fluconazole.
The biofilm acts as a protective barrier for the microorganism, rendering their cells more resistant to the immune system, as well as to the effects of antifungal therapies. Biofilms arising from Candida infections are difficult to treat and, depending on the biofilm phase, higher concentrations of antifungal compounds are necessary in comparison to planktonic cells [27]. In addition, biofilm formation may vary among Candida strains in response to different antifungals, contributing to increased virulence [5,7]. EHVG exhibited anti-Candida activity, reducing both adhesion and biofilm formation by all tested strains.
In order to better understanding the underlying mechanisms of EHVG anti-Candida activities, molecular modeling was used to identify the probable targets of EHVG compounds. We found that all tested compounds exhibited favorable interactions with CaCYP51 (cytochrome P450 lanosterol 14a-demethylase). However, vismione D was the most potent inhibitor of the enzyme, with a docking score of −10.96kcal/mol. Vismione D also exhibited the highest affinity for CaCYP51, whilst presenting the lowest inhibition constant and high negative binding free energy, with values even greater than those of posaconazole and fluconazole, used herein as reference drugs. These findings indicate vismione D as the most promising anti-Candida compound of EHVG. It is important to note that the other tested compounds were also predicted to interact with CaCYP51, allowing us to conclude that EHVG activity may be due to a phytocomplex present in the EHVG, in which vismione D may be the most effective.
Binding free energy is a parameter used to indicate how spontaneously the interaction between a molecule and a biological target occurs. Negative binding free energies indicate that these interactions favor the formation of ligand-receptor complexes [28].
Intermolecular interactions, such as hydrogen bonds with amino acid residues (Tyr-118, Ser-378 and Met-508), hydrophobic interactions, and interactions with the heme group, confer the ability to stabilize a given ligand at the binding site, forming ligand-receptor complexes. In the present study, vismione D was predicted to interact with practically the same amino acids as posaconazole, a commercially available fungicide, but more spontaneously and with a greater interaction potential. In addition, the spatial conformation obtained indicated a more linear structure of vismione D contributing to the molecular interactions.
Values lower than 2Å indicate that the docking protocol is valid, i.e., shows similarity with the experimental structure [28]. The CaCYP51 enzyme plays a key role in the synthesis of ergosterol from its precursor, lanosterol [29,30]. Blockade of this enzyme thus inhibits the process necessary for Candida survival [31]. Based on this, it is reasonable to propose that EHVG's anti-Candida activities are due to its ability to affect ergosterol synthesis.
It is important to emphasize the relevance of phytocomplexes present in EHVG for the development of new antifungals with, possibly, a broader spectrum of action than traditional agents [22], since the extract compounds may act additively as antifungals. The in silico absorption, distribution, metabolism, excretion (ADME), and toxicity modeling is an important tool for rational drug design for studying interactions of ligands with biological targets at the atomic scale. As shown by the in silico results, the evaluated compounds, including vismione D, presented good predictive values with high gut absorption, as well as accordance to the Lipinski's rule, an important characteristic for drug-likeness [32,33].
The in silico absorption, distribution, metabolism, excretion (ADME), and toxicity (T) modeling is an important tool for rational drug design and to study the interactions of ligands with the biological targets in atomic scale, especially if we consider the several restrictions for animal use in the world.
The molecular docking findings suggest that the compounds present in EHVG meet Lipinski's rules, which indicates that they might act as effective drug candidates with a low risk level for oral use. Furthermore, the ADMET predictor confirmed the in vitro results related to the low toxicity as an important characteristic to show the plant compounds as a good medicinal product [32,33].
Additionally, based on PASS prediction simulation, it is possible to propose other biological activities for vismione D, since it showed a higher value of Pa (potential activity) [34] as an antifungal compound, and good values as an anti-inflammatory and antioxidant compound.
To the best of our knowledge, there are no available data evaluating the in silico activities of compounds from V. guianensis extract (EHVG) or any other species of the genus Vismia. This highlights the importance of this analysis for elucidating the possible mechanisms underlying the antifungal action of this species.

Preparation of the Extract
The dried leaves were ground in an electrical grinder. The powder obtained was used to prepare the extract and the different hydromodules The extract yield was calculated as % of the total biomass of the plant material. The dependence of the extract yield on the hydromodule was studied to determine the best extraction process, and the choice of 1:10 was based on the highest extract yield and best chemical profile.
The extract was obtained by maceration for 7 days, protected from light. After this period, the solvent was evaporated in a rotary evaporator, and then the material was lyophilized and stored in a refrigerator for subsequent assessment.

Particle Size Analysis of the Leaves
For characterization of the plant material, two samples of 100 g of dried leaves were placed in a mesh strainer, with mesh sizes No. 16,20,40,60,80, and 120, and openings of 1190, 850, 425, 250, 180, and 125 µm, respectively, according to Brazilian normalization rules. This lasted for 20 min, with the rheostat being adjusted to intensity 7. The samples were removed and weighed for yield calculation [35].

Ash Content and Extract Yield
Total ash content was quantified as previously described [35]. Samples of the V. guianensis leaf powder (3 g), in duplicate, were placed in crucibles, stabilized for 30 min in a desiccator, and heated separately in a muffle furnace (Magnus Ltd.a. ® ) at an initial temperature of 200 • C, with increments of 200 • C every 2 h, until a temperature of 600 • C was reached. The samples were then removed, transferred to a desiccator, maintained at room temperature, and weighed for calculation of the ash volume. At the end of this process, the yield was calculated by placing 1 mL of the extract on Petri dishes and incubated at 37 • C until completely dried. Individual samples were tested in triplicate, and the yield was calculated considering the dry weight of the extract.

Analysis of EHVG by HPLC-PDA
The extract was analyzed by HPLC-PDA. A cleaning step was performed to remove any contaminants. Sample solution (30 mg/mL, 1 mL) was run through solid phase extraction (SPE) using Phenomenex Strata C18 cartridges (500.0 mg stationary phase), which had previously been activated with 5.0 mL MeOH and then equilibrated with 5.0 mL MeOH:H 2 O (1:1, v/v). The sample was then filtered through a 0.22 µm PTFE filter, and subsequently dried. The dried sample was dissolved at a concentration of 10.0 mg/mL in HPLC-grade methanol solvent. An aliquot of 10.0 µL was injected directly into the HPLC-PDA. The system used was a Shimadzu model HPLC system (Shimadzu Corp., Kyoto, Japan), consisting of a solvent injection module with a binary pump and PDA detector (SPA-20A. The column used was Luna 5.0 µm C18 100 0 A (150.0 µm × 4.6 µm). The elution solvents were A (2% acetic acid in water) and B (2% acetic acid in methanol). The sample was eluted according to the following gradient: 5% to 60% B in 60 min. The flow rate was 1 mL/min. The data were processed using the LC Solution software (Shimadzu) [36] Full-scan mass spectra were recorded in the range of 100-1000 m/z. Multistage fragmentations (ESI-MS n ) were obtained by the collision-induced dissociation (CID) method, using helium for ion activation. The different compounds of EHVG were identified, based on the comparison of UV spectra and characteristic fragmentations, to those from the data in the literature [30].  Table 6). The strains were obtained from the collection of the Laboratory of Applied Microbiology, Universidade CEUMA, São Luís, MA, Brazil. All Candida isolates were identified by multiplex PCR. The identification was performed as previously described [37], using the combination of eight different species-specific primers in a single PCR tube, by combining two yeast-specific fragments from the ITS1 and ITS2 regions and species-specific primers for C. albicans and C. glabrata. This method was chosen as it allows for the identification of clinical isolates with high specificity, and has the potential to discriminate individual Candida species in polyfungal infections. For the experiments, the isolates were subcultured on Sabouraud-dextrose agar (SDA) with chloramphenicol for 24 h at 37 • C.

Inoculum Preparation
The different strains were activated on SDA with chloramphenicol by seeding and incubation for 24 h at 35 • C. The colonies were resuspended in 5 mL of 0.85% sterile saline (0.145 mol/L; 8.5 g/L NaCl). The resulting suspension was homogenized in a Vortex ® for 15 s. The cell density was adjusted in a spectrophotometer (Global Trade Technology), at a wavelength of 530 nm, by the addition of saline to obtain an absorbance corresponding to 0.5 McFarland standards.

Determination of Minimum Inhibitory Concentration (MIC)
Antifungal susceptibility tests were standardized following previously established guidelines (M27-A3, [38]), and according to Ostrosky et al. [39]. For the test, 100 µL RPMI 1640 medium (with glutamine and phenol red, no bicarbonate) buffered with MOPS [3-(N-morpholine) propanesulfonic acid] was added to the wells of a 96-well sterile microtiter plate, which contained a cell suspension of Candida spp. (1 × 10 6 CFU/mL). Next, 100 µL EHVG at a concentration of 50 mg/mL was added to the first column, followed by serial dilutions up to 0.095 mg/mL. Amphotericin B (0.0313-16 µg/mL) and Fluconazole (0.125-256 µg/mL) (Sigma-Aldrich, São Paulo, Brazil) were used as positive controls. RPMI 1640 medium, without extract or an antifungal agent, served as the growth control. The Candida spp. inoculum plus EHVG or antifungal agent was incubated for 24 h at 37 • C.
The result was analyzed visually and read in a microplate reader at 540 nm. The MIC was defined as the lowest concentration of the extract or antifungal agent at which no growth was visible or detected. The test was carried out in triplicate in two different experiments.

Determination of Minimum Fungicidal Concentration (MFC)
The MFC was determined based on the MIC results. A 10 µL aliquot of the wells corresponding to up to 4× the MIC was seeded onto SDA plates with chloramphenicol. The plates were incubated for 24-48 h at 37 • C, and the MFC was defined as the lowest concentration of the EHVG and antifungal agents that inhibited fungal growth. All tests were carried out in triplicate. The MFC/MIC ratio was calculated in order to determine whether the extract had fungistatic (MFC/MIC ≥ 4) or fungicidal activity (MFC/MIC ≤ 4) [40].

Time-Kill Assay
C. albicans ATCC 2001 and C. albicans SC 5314 were used to establish the time-kill curves according to [26], with modifications. The inoculum was diluted in RPMI 1640 medium to a final concentration of 5 × 10 3 CFU/mL, and added to 96-well plates. EHVG was used at concentrations corresponding to the MIC and 2× MIC, using 1% DMSO as vehicle. Amphotericin B plus inoculum was used as a positive control, and RPMI 1640 plus inoculum as a negative control. The plates were incubated for 24 h at 37 • C, and the number of CFUs was determined.
Growth kinetics were evaluated after different periods of incubation: 0, 3, 6, 12, 24, 36, and 48 h. Serial dilutions were prepared for each sample (10 −1 to 10 −4 ), and 10 µL was transferred to Petri dishes containing SDA with chloramphenicol. After colony counting, the following formula was applied: CFU/mL = number of counted colonies × 10 n /q, where n corresponds to the absolute value of the chosen dilution and q to the volume (mL) of each dilution seeded on the plates [41]. The assay was carried out in triplicate in two different experiments.

Adhesion Assay
Candida spp. was activated as described in item 4.5.2, washed with PBS (3 mL), and centrifuged twice at 2060× g for 5 min. After the last wash, the inoculum was resuspended in 5 mL PBS, read at 530 nm, and adjusted to 1 × 10 7 CFU/mL [42].
For adhesion assessment, 100 µL PBS and 100 µL EHVG, at concentrations corresponding to the MIC and 1 /2 MIC, were added to each Candida strain. Serial dilutions were prepared, and the samples were incubated for 90 min at 37 • C. The supernatant was then discarded, and the plates were washed twice with PBS. In the last wash, PBS was discarded completely, and the cells were resuspended in 100 µL PBS. The microdroplet technique was used to seed dilutions of 10 −2 to 10 −4 on SDA plates with chloramphenicol, which were incubated for 24-48 h at 37 • C. The assay was carried out in triplicate in two different experiments.

Effect of EHVG on Biofilm Formation
The experiments were carried out as described previously [24], with modifications. Sub-inhibitory concentrations of EHVG (200 µL), corresponding to 1 /4 MIC and 1 /2 MIC, were used to assess the extract's interference with biofilm formation. The plates were incubated for 24 h at 37 • C. After this period, the supernatant was removed, and the pellet was washed twice with PBS. The number of CFUs was determined, and the metabolic activity was evaluated by the MTT assay, or by biomass formation using crystal violet staining. For the MTT (3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide) assay, a concentration of 5 mg/mL was added, and the samples were read in a microplate reader at 570 nm. For crystal violet staining, 0.2 mg/mL of the dye was added, and the samples were read at 595 nm, as described by Zago et al. [41].
For the quantification of CFU, dilutions of 10 −2 to 10 −4 were seeded on SDA plates with chloramphenicol by the microdroplet technique, and the plates were incubated for 24-48 h at 37 • C. For CFU quantification, the formula described in item 2.5.6 was applied.
The assay was carried out in triplicate in two different experiments.
The effect of the extract on the mature biofilm was analyzed following the same procedure as described above, and the microplates were incubated for 48 h at 37 • C. The culture medium was changed after 24 h. The supernatant was aspirated, and the biofilm was washed twice with PBS. Concentrations of EHVG (200 µL) corresponding to 2×-and 4× the MIC were used. The plates were incubated at 37 • C for an additional 24 h. After this period, the plates were washed twice with 100 µL PBS, and the number of CFUs, the metabolic activity (MTT assay), and the biomass (crystal violet staining) were analyzed [43] (Seneviratne et al., 2016). For CFU quantification, dilutions of 10 −2 to 10 −4 were seeded on SDA plates with chloramphenicol by the microdroplet technique, and the plates were incubated for 24-48 h at 37 • C. The number of CFUs was determined by applying the formula proposed previously [41]. In all experiments, biofilms without extract were used as negative controls, and biofilms with Amphotericin B and Fluconazole were used as positive controls.

Determination of EHVG Cytotoxicity by the MTT Assay
The cytotoxicity of the extracts against murine macrophages (RAW 264.7; APABCAM, Rio de Janeiro, Brazil) was evaluated. The cells were maintained in cell culture flasks (TPP) in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen, New York, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NE, USA), and then incubated for 1 h at 37 • C in the presence of 5% CO 2 . After reaching sub-confluence, the cells were detached with a cell scraper (TPP) and centrifuged at 2000 rpm for 5 min.
The supernatant was discarded, and the cells were, again, resuspended in RPMI medium and transferred to microtiter plates (100 µL, 1 × 10 6 /well). The plates were incubated for 4 h in the presence of 5% CO 2 for adherence of the cells. After this period, increasing concentrations of EHVG (0.5 to 50 mg/mL) were added, and the plates were incubated for 24 h at 37 • C in the presence of 5% CO 2 . As growth controls, the cells were incubated with RPMI without extract and 1% DMSO as vehicle.
After incubation, the supernatant was removed, and adherent cells were evaluated by the addition of 10 µL MTT solution (5 mg/mL) (Sigma, St. Louis, MO, USA) to the wells in the dark. The plates were incubated for 4 h at 37 • C. The supernatant was removed, and formazan was extracted by the addition of 100 µL sodium dodecyl sulfate (SDS) and incubation overnight. Absorbance was read at 570 nm after 24 h of incubation.

The Hemolytic Assay
Defibrinated sheep blood (EB FARMA, Rio de Janeiro, Brazil) was used in this study. Red blood cells were isolated by centrifugation at 290× g (5810R Centrifuge, Eppendorf) for 10 min at 4 • C. After the removal of plasma, the red blood cells were washed three times with PBS (pH 7.4) and immediately resuspended in 2% (v/v) of the same buffer. To evaluate the hemolytic activity of the extracts and fractions, 100-µL aliquots of the red blood cell solution were added to flat-bottom 96-well microplates (Kasvi, Italy), together with serial dilutions of the extract and fractions (0.05 to 50 mg/mL). Total hemolysis was achieved with 1% Triton X-100 (Sigma-Aldrich), and PBS was used as a negative control. DMSO (1%, vehicle) was also included as a control. After incubation for 60 min at room temperature, the cells were centrifuged at 300× g for 10 min, and absorbance was measured in the supernatant at 540 nm [11]. Relative hemolytic activity was expressed in relation to Triton X-100, and was calculated using the following formula: relative hemolytic activity (%) = [(As − Ab) × 100]/(Ac − Ab), where Ac is the absorbance of the control (blank, without extract), As is the absorbance in the presence of the extract, and Ab is the absorbance in the presence of Triton X-100. The assays were carried out in quadruplicate on three different experiments.

Evaluation of Cell Viability by the Neutral Red Assay
Neutral red (Vetec/Sigma Aldrich, São Paulo, Brazil) was prepared in PBS (20 µg/mL), and 100 µL/well of this solution was placed in the wells that contained RAW cells (100 µL/1 × 10 6 ) treated with different concentrations of EHVG, starting at 50 mg/mL (25, 12.5, 6.25, 3.125, 1.76, 0.78, and 0.39 mg/mL). After 2 h of incubation while protected from light, the plates were centrifuged, and the supernatant was discarded. Next, ethanol P.A. (100 µL) was added to the wells. The plates were shaken on an orbital shaker for 15 min. Absorbance was then determined at 570 nm, and the values obtained were converted into percentage of cell viability [44].

Ligands and Target Preparations
The GaussView 5.0.8 program was used to illustrate the three-dimensional (3D) structure of the compounds Vismione D, anthraquinone, kaempferol, and catechin, identified by HPLC-UV/Vis and FIA-ESI-IT-MS in the crude extract of V. guianensis leaves. The geometrical and vibrational properties were calculated in vacuum by the density functional theory combined with the 6-311++G** (d, p) basis set, using Gaussian 09. The 3D structure of 14-alpha-demethylase from C. albicans (CaCYP51) was obtained from the Protein Data Bank (PDB) (# 5FSA), then resolved by X-ray crystallography with a resolution of 2.86 Å.

Molecular Docking
The structures of CaCPY51 and of the ligands were prepared for the molecular docking calculations using the AutoDock Tools (ADT), version 1.5.6. The structure of CaCYP51 was considered rigid, while each ligand was considered flexible. Gasteiger partial charges were calculated after the addition of all hydrogens. Next, the apolar hydrogens of CaCYP51 and of the ligands were subsequently fused. A cubic box of 120 × 120 × 120 points, with spacing of 0.35 Å, was generated throughout the portion that corresponded to the active site of the macromolecule.
The grid box was centered on the heme group of CaCPY51. For molecular docking, a global search was performed using Lamarckian Genetic Algorithms (AGL), and a local search (LS) using pseudo-Solis and Wets. Each ligand was submitted to 100 independent simulations of ligand binding, and the remaining fit parameters were set to default values. The initial coordinates of the interactions between CaCYP51 and the compounds present in EHVG were chosen using the criterion of clustering low-energy conformations, combined with visual inspection [37]. Molecular docking was performed using AutoDock 4.2.

Pharmacokinetics and Toxicity Measurement
The SwissADME online method was used to assess the pharmacokinetic properties (ADME) of the compounds. Lipinski's five rule was determined based on the following parameters: molecular weight not more than 500 Dalton; H-bond donors ≤ 5; H-bond acceptors ≤ 10; molar refractivity ranging from 40 to 130; and lipophilicity positive druglike properties of any compound [45]. In addition, the online tool admetSAR (http:// lmmd.ecust.edu.cn/admetsar2, accessed on 28 September 2022) was used to calculate the toxicological properties of all the compounds.

Pass Prediction
The PASS online tool (http://www.pharmaexpert.ru/passonline/predict.php, accessed on 28 September 2022) was used to determine the other potential biological activities of the selected compounds. The potential biological activity considered the Pa and Pi values to range from 0.000 to 1.000 [46,47], and Pa values higher than the Pi values were considered to have the following characteristics: Pa < 0.7 suggests high drug activities; 0.5 < Pa < 0.7 shows moderate therapeutic potentials; and Pa < 0.5 shows poor pharmaceutical activity [48,49].

Statistical Analysis
The results were expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used, followed by Dunn's post-test for multiple comparisons and the Student t-test for comparison between two groups. All analyses were performed using the GraphPad Prism 8.0 software. The significance adopted was 5% (p < 0.05).

Conclusions
EHVG has important anti-Candida activity, being able to attenuate the growth and virulence factors of Candida ATCC strains and clinical isolates of interest. This anti-Candida effect may be due to the presence of vismione D, which may act synergistically to other compounds. This inhibits the Candida CaCYP51 enzyme, and, therefore, impairs the biosynthesis of ergosterol.
Overall, the results suggest that EHVG is a promising anti-Candida alternative that can be used as a reference to generate a lead compound for the purpose of antifungal drug discovery.