In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species

Abstract

Background: Recent investigations on the antifungal properties of essential oil of Melaleuca alternifolia Cheel (Tea Tree Oil, TTO) have been performed with reference to the treatment of vaginal candidiasis. However, there is a lack of in vivo data supporting in vitro results, especially regarding the antifungal properties of TTO constituents. Thus, the aim of our study was to investigate the in vitro and the in vivo anti-Candida activity of two critical bioactive constituents of TTO, terpinen-4-ol and 1,8-cineole.

Methods: Oophorectomized, pseudoestrus rats under estrogen treatment were used for experimental vaginal infection with azole (fluconazole, itraconazole) -susceptible or -resistant strains of C. albicans. All these strains were preliminarily tested for in vitro susceptibility to TTO, terpinen-4-ol and 1,8-cineole for their antifungal properties, using a modification of the CLSI (formerly NCCLS) reference M27-A2 broth micro-dilution method.

Results: In vitro minimal inhibitory concentrations (MIC90) values were 0.06% (volume/volume) for terpinen-4-ol and 4% (volume/volume) for 1,8-cineole, regardless of susceptibility or resistance of the strains to fluconazole and itraconazole. Fungicidal concentrations of terpinen-4-ol were equivalent to the candidastatic activity. In the rat vaginal infection model, terpinen-4-ol was as active as TTO in accelerating clearance from the vagina of all Candida strains examined.

Conclusion: Our data suggest that terpinen-4-ol is a likely mediator of the in vitro and in vivo activity of TTO. This is the first in vivo demonstration that terpinen-4-ol could control C. albicans vaginal infections. The purified compound holds promise for the treatment of vaginal candidiasis, and particularly the azole-resistant forms.

Figure 1

Vaginal infection outcome by a fluconazole-susceptible strain C. albicans (SA-40) in oophorectomized, oestradiol-treated rats inoculated intravaginally with TTO 5% v/v (open squares), 2.5% v/v (open triangles), 1% v/v (open circles), terpinen-4-ol 1% v/v (filled triangles), fluconazole 100 μg (filled circles), Tween-80 0.001% v/v (control; filled squares) at 1, 24 and 48 hr after intravaginal C. albicans challenge (10⁷ cells in 0.1 mL). Each curve represents the mean (± S.E.) of cfu of five rats. Data are from one of two independent experiments with similar results. At each time point considered, starting from day 2 to day 14, there was a statistically significant difference between the cfu of rats treated with fluconazole or TTO 5% or TTO 2.5% or terpinen-4-ol and those of the untreated animals.

Figure 2

Vaginal infection outcome by fluconazole-itraconazole resistant strain C. albicans (AIDS-68) in oophorectomized, oestradiol-treated rats inoculated intravaginally with TTO 5% v/v (open squares), 1% v/v (open circles), terpinen-4-ol 1% v/v (filled triangles), fluconazole 100 μg (filled circles), Tween-80 0.001% v/v (control; filled squares) at 1, 24 and 48 hr after C. albicans challenge (10⁷ cells in 0.1 mL). Each curve represents the mean (± S.E.) of the fungal cfu of five rats. At each time point (day1-day14) there was a statistically significant difference (P < 0.05, Student's t test, two tailed) between the untreated control and cfu of rats treated with TTO 5%, TTO 1% and terpinen-4-ol. At scattered day intervals, there was also a statistically significant difference (P < 0.05, Student's t test, two tailed) between cfu of rats treated with TTO 5% or terpinen-4-ol and TTO 1% (for example day 1).