Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection

Abstract

Background: Vaginal candidiasis is a frequent and common distressing disease affecting up to 75% of the women of fertile age; most of these women have recurrent episodes. Essential oils from aromatic plants have been shown to have antimicrobial and antifungal activities. This study was aimed at assessing the anti-fungal activity of essential oil from Mentha suaveolens (EOMS) in an experimental infection of vaginal candidiasis.

Methods: The in vitro and in vivo activity of EOMS was assessed. The in vitro activity was evaluated under standard CLSI methods, and the in vivo analysis was carried out by exploiting a novel, non-invasive model of vaginal candidiasis in mice based on an in vivo imaging technique. Differences between essential oil treated and saline treated mice were evaluated by the non-parametric Mann-Whitney U-test. Viable count data from a time kill assay and yeast and hyphae survival test were compared using the Student's t-test (two-tailed).

Results: Our main findings were: i) EOMS shows potent candidastatic and candidacidal activity in an in vitro experimental system; ii) EOMS gives a degree of protection against vaginal candidiasis in an in vivo experimental system.

Conclusions: This study shows for the first time that the essential oil of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal in vitro, and has a degree of anticandidal activity in a model of vaginal infection, as demonstrated in an in vivo monitoring imaging system. We conclude that our findings lay the ground for further, more extensive investigations to identify the active EOMS component(s), promising in the therapeutically problematic setting of chronic vaginal candidiasis in humans.

Figure 1

Time kill curve of EOMS and TTO against Candida albicans gLUC59. Cells were untreated (control) or treated with 0.39, 0.78, 1.56, or 3.12 g/L of EOMS or treated with 3.12, 6.25, 12.5 or 25 g/L of TTO for different time periods (0, 0.5, 1, 2, 4, 6, 8, 24 and 48 hours). After incubation survival cells were determined by cultivation on Sabouraud dextrose agar plates at 37°C for 48 h. Results are expressed as CFU/ml and indicated as mean ± SEM of triplicate samples. Data are representative of one of three independent experiments. The statistical significance was evaluated with the Student's t-test (two-tailed). *P-values of < 0.05 were considered significant.

Figure 2

Effect of essential oil on yeast and hyphae survival. gLUC59 Candida albicans yeast cells (A) and preformed hyphae (B) were treated with different concentrations of essential oils (EOMS, TTO and JO) for 24 h. After incubation 10 μl of coelenterazine substrate (1 mg/ml) was added to each well and samples were read using a luminometer. Results are expressed as percentage of yeast or hyphae survival and indicated as mean ± SD of triplicate samples are from one of three experiments with similar results. The statistical significance was evaluated with the Student's t-test (two-tailed). *P-values of < 0.05 were considered significant. The effect on preformed hyphae was microscopically examined after 24 h of treatment with essential oil (C). Original magnification of 20x or 40x is indicated in the micrographs. The results are representative of one of three independent experiments.

Figure 3

Cytotoxicity of essential oils on mammalian cells. Monomac 6 and L929 cells were treated with different concentrations of essential oils for 2 h. The cytotoxicity was tested by the determination of the cell ATP level by a bioluminescent method. Results, expressed as Relative luciferase activity (RLUC), represent the mean ± SD of three different experiments. The statistical significance was evaluated with the Student's t-test (two-tailed). *P-values of < 0.05 were considered significant.

Figure 4

In vivo imaging of mice vaginally infected with Candida albicans and treated with essential oils. The vaginal lumen of mice under pseudoestrus condition were infected for 2 consecutive days with 10 μl of a 10⁹ cell/ml suspension of Candida albicans gLUC59 (first three mice of each group) or control strain (fourth mouse of each group) and treated with the different essential oils 2 h before the first challenge and then every two days. After 5, 7, 9, 15 and 21 days post-infection mice were treated intravaginally with 10 μg of coelenterazine and imaged in the IVIS-200TM imaging system under anaesthesia with 2.5% isoflurane. Total photon emission from vaginal areas within the images (Region Of Interest, ROI) of each mouse was quantified with Living ImageR software package. Data are from one of three experiments with similar results.

Figure 5

Measurement of Candida albicans load in mice treated with different essential oils. The vaginal lumen of mice under pseudoestrus condition were infected with Candida albicans gLUC59 and then treated with the essential oils 2 h before the first challenge and then every two days. After 5, 7, 9, 15 and 21 days post-infection 6 mice per group were anaesthetized, imaged in the IVIS-200TM imaging system, and the vaginal lumen was thoroughly washed with 150 μl of saline using a mechanical pipette. The fungal burden of vaginal lavage fluids was determined by evaluating the colony forming units (CFU) assay. For CFU assay 50 μl of the lavage fluids were diluted and seeded in YPD agar plus chloramphenicol. Results were reported and the statistical significance was evaluated with the non-parametric Mann-Whitney U-test. *P < 0.05 (essential oil treated mice versus saline treated mice).