Pinosylvin and Sanguinarine Combination to Enhance Antifungal Activity against Candida albicans

Abstract

Candida albicans is one of the major sources of fungal infections that can lead to life-threatening systemic infections. However, effective control of C. albicans remains a great challenge. Herein, this study aimed at investigating the antifungal effect of a combination of two natural compounds, pinosylvin (PIN) and sanguinarine (SAN), against C. albicans. In order to investigate the antifungal effect and mechanism of the combination of PIN and SAN, antimicrobial assay, time-kill assay, biofilm formation assay, cell membrane integrity assay, and reactive oxygen species (ROS) assay were performed. The results showed that the combination of PIN and SAN was more effective against C. albicans than PIN or SAN alone. PIN and SAN could jointly inhibit biofilm formation and thus attenuate C. albicans adhesion and colonization ability. PIN mainly targeting the cell membrane while SAN mainly inducing the cell to produce large amounts of ROS. Besides, PIN promoted the entry of SAN into C. albicans. Finally, the hemolysis experiment demonstrated that the combination of PIN and SAN is biocompatible. Taken together, the combination of PIN and SAN enhanced the antifungal effect against C. albicans, which has a broad application prospect in the control of C. albicans.

Keywords: Candida albicans; antifungal; pinosylvin; sanguinarine; synergy effect.

Fig. 1. The interaction between PIN and SAN against C. albicans.

(A) The checkerboard plot of the antimicrobial activity of the combination of PIN and SAN on C. albicans. (B) Synergy distribution mapped to the dose-response surface by the Bliss independence model. White arrows point to the areas where the strongest synergies occur.

Fig. 2. Time-kill curves of C. albicans cells treated with PIN, SAN, the combination, and control.

(A) Treated with different concentrations of PIN. (B) Treated with different concentrations of SAN. (C) Treated with P-1MIC+S-1MIC and with PIN or SAN at equivalent concentrations.

Fig. 3. The morphology of C. albicans by SEM.

(A1, and A2) Untreated; (B1, and B2) Treated with P-2MIC; (C1, and C2) Treated with S-2MIC; (D1, and D2) Treated with P-1MIC+S-1MIC. The arrows indicate significant changes in C. albicans morphology.

Fig. 4. Biofilm formation of C. albicans cells after being treated by PIN, SAN, the combination, and control.

(A) The three-dimensional plot of the effects of different concentrations of PIN, SAN, and combinations on biofilms. (B) Comparison of the effects of PIN, SAN, and the combination on biofilms at equivalent concentrations. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

Fig. 5. Cell membrane damage status of C. albicans after treatment with PIN, SAN or the combination.

(A) The CLSM images of C. albicans stained by PI and SYTO 9. PI (red) staining shows the disruptive effect of the sample on cell membrane integrity. SYTO 9 (green) staining marks the number of cells. (B) The plot of the percentage of membrane-damaged cells. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

Fig. 6. The intracellular ROS production in C. albicans.

(A) The CLSM images of C. albicans stained by DCFH-DA, green fluorescence reflects intracellular ROS. (B) The total fluorescence intensity graph was obtained by ImageJ software analysis of the CLSM images. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

Fig. 7. Effect of different concentrations of PIN on endocytosis of SAN by C. albicans.

(A) Autofluorescence pictures of intracellular SAN (red) observed in C. albicans. (B) The total fluorescence intensity graph. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significant differences.

Fig. 8. Hemolysis rates of different concentrations of PIN, SAN and the combination to reflect their biocompatibility.