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

Abstract

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.

Keywords: CaCYP51; Candida albicans; Candida glabrata; Vismia guianensis; antifungals; lacre; vismione D.

Figure 1

Percentage of different particles sizes in the powder of dry Vismia guianensis leaves after grinding and sifting using different mesh sizes.

Figure 2

Chromatogram of the hydroalcoholic extracts of Vismia guianensis obtained by HPLC with UV detection (HPLC–PDA) for the hydromodule 1:10 with defined peaks and areas.

Figure 3

First-order spectrum of direct flow injection analysis (FIA-ESI-IT-MS) obtained in the negative mode for EHVG. (▲) Fragments of the chemical compounds identified.

Figure 4

Chemical structures of the compounds identified in the Vismia guianensis leaf extract.

Figure 5

Different concentrations of the hydroalcoholic leaf extract of Vismia guianensis (EHVG) showed low toxicity when evaluated by the hemolysis (A) and MTT assay (B). Triton was the positive control for hemolysis (A) and medium was the negative control in the cytotoxic assay. Data represent the mean ± standard deviation of individual samples tested in quadruplicate. (*) p < 0.05 compared to the control; (#) p < 0.05 compared with other concentrations.

Figure 6

Effects of EHVG on time-kill curves. The effects of EHVG against Candida albicans SC5314 (A) and Candida glabrata ATCC 2001 (B) were tested at two different concentrations corresponding to the MIC or 2× MIC, then compared to untreated cultures (Control) or with cultures treated with Amphotericin B (AMPHO B; MIC—0.5 µg/mL and 2× MIC—1 µg/mL).

Figure 7

EHVG inhibited the adhesion of Candida albicans ATCC 90,028 (A), C. albicans SC5314 (B), and C. glabrata ATCC 2001 (C) in cultures treated with concentrations of ½ MIC and MIC when compared to untreated samples (control). The EHVG-treated cultures were also compared to those treated with the reference drugs Amphotericin B (Ampho B) or Fluconazole (Flu). (*) p < 0.05 differs from untreated control; (#) p < 0.05 differs from Amphotericin B.

Figure 8

EHVG inhibited the formation of early (A,C,E) and mature (B,D,F) biofilms. The CFU/mL values were established in cultures of Candida albicans (ATCC 90028, A,B; SC5314, C,D) and C. glabrata (ATCC 2001, E,F) after 48 h and 72 h. The samples treated with different concentrations of EHVG (1/4 and 1/2× MIC for early biofilms, and 2× and 4× MIC for mature biofilms) were compared to cultures treated with Amphotericin B (Ampho B) or fluconazole (Flu), and with the untreated control. (*) p < 0.05 in comparison to control; (@) in comparison to Fluconazole, and (&) p < 0.05 Fluconazole compared to Amphotericin B.

Figure 9

Percentage of inhibition on EHVG-treated early (A,C,E) and mature biofilms (B,D,F) of Candida albicans ATCC 90,028 (A,B), C. albicans SC5314 (C,D) and C. glabrata ATCC 2001 (E,F) after 48 and 72 h, evaluated by MTT assay. Samples were treated with EHVG (1/4 and 1/2 for early biofilms, and 2× and 4× MIC for mature biofilms) and compared to untreated controls or the reference drugs Amphotericin B (Ampho B) and Fluconazole (Flu). (*) p < 0.05 compared to control. (#) p < 0.05 compared to Amphotericin B and (@) p <0.05 compared to fluconazole.

Figure 10

Reductions in biofilm biomass stained with crystal violet (CV) of Candida spp. (ATCC 90028, A,B), (ATCC 2001, C,D) and (SC5314, E,F) after 48 and 72 h. The samples were treated with EHVG and compared to untreated controls or to the reference drugs Amphotericin B (AMHO B) and Fluconazole (FLU). (*) p < 0.05 differs from control; (#) p < 0.05 differs from Amphotericin B and (@) p <0.05 compared to fluconazole.

Figure 11

Schematic representation of the interactions between vismione D and CaCYP51, identified by molecular docking. It is possible to observe the formation of hydrogen bonds and hydrophobic contacts between the ligand and the enzyme residues, including interactions with the heme group. The figure was obtained with PoseView.

Figure 12

Spatial conformation showing several common binding sites between the CaCYP51 enzyme (PDB: 5FSA) and Vismione D (blue) or the antifungal posaconazole (green) through molecular docking. The image was obtained with USCF Chimera.