Antifungal activity of a gel containing Thymus vulgaris essential oil against Candida species commonly involved in vulvovaginal candidosis

Abstract

Previous reports of the antifungal potential of Thymus vulgaris L. (Lamiaceae) essential oil against Candida spp. suggests that this natural product may play a valuable role in the management of vulvovaginal candidosis. A polycarbophil-based gel, containing Thymus vulgaris essential oil (1%, w/w), was formulated and its antifungal activity was evaluated in vitro against four strains of Candida commonly involved in vulvovaginal candidosis. Results show that the gel allows the essential oil to maintain its activity at pH values near those observed in the healthy vagina. Also, the lack of significant antifungal activity of the polycarbophil-based gel seems to be advantageous when considering its use as a placebo formulation. Further in vivo studies are needed in order to evaluate the potential of this drug formulation in the management of vulvovaginal candidosis.

Keywords::

• Candida spp
• drug formulation
• polycarbophil
• Thymus vulgaris L.
• vulvovaginal candidosis

Introduction

Vulvovaginal candidosis (VVC) is a frequent gynecological problem, affecting women worldwide (Sobel, 2007). It is estimated that three out of four women will experience at least one episode of VVC during their lifetime, and that 20–30% of all symptomatic vulvovaginitis are caused by Candida species. Although uncomplicated VVC, which represents most cases, is easily treatable with conventional oral or local azole therapy, complicated forms of this disease (severe, chronic or recurrent VVC) are more difficult to manage (Pappas et al., 2004). These last cases are becoming more frequent, being commonly associated with azole-resistant strains of Candida albicans and, particularly, C. glabrata and C. krusei (Spinillo et al., 1997). The increasing rate of VVC cases that do not respond to conventional therapies has led to the investigation of several alternative regimens, namely those using plant products (Pauli, 2006).

Thyme (Thymus spp.) is a widely available Lamiaceae, native of meridional Europe, with its essential oil (approximately 1.0 to 2.5% of total dry plant weight) being of particular interest. Several traditional uses of this essential oil have been reported, namely as antispasmodic, antiseptic, and expectorant in respiratory diseases or as a topic antiseptic and rubefacient (Cunha et al., 2003). Several essential oils of the genus Thymus, particularly Thymus vulgaris L. essential oil (TVEO), have previously shown in vitro activity against Candida spp. usually involved in VVC (Pina-Vaz et al., 2004). TVEO is mainly composed of two phenolic monoterpenes, carvacrol and/or thymol, with these constituents being responsible for antifungal activity. Thus, this natural product may play a significant role in the quest for alternative therapies, namely in the treatment of non-albicans and azole-resistant cases.

Even if a substance is shown to have pharmacological activity, its therapeutic efficacy depends on the adequate choice of drug delivery system and its correct formulation. This fact seems particularly important when vaginal drug administration is considered (das Neves et al., 2008). Previous reports found that using different drug formulations or drug delivery systems may influence the antimicrobial activity of incorporated active substances (Ahmad et al., 2005; Chang et al., 2002; Thorgeirsdottir et al., 2005). In the last years, hydrophilic gels have been widely used as vaginal drug delivery systems, showing the ability of incorporating virtually any type of drug substance. Indeed, some studies indicate that these systems are, from a pharmaceutical point of view, adequate for essential oils (Cal, 2006; Maccioni et al., 2002). Also, hydrophilic gels are easy to produce, inexpensive, generally recognized as safe, and widely accepted or even preferred by patients (das Neves & Bahia, 2006).

The objective of this work was to produce a polycarbophil-based gel containing TVEO (1%, w/w) (TVEO gel) to be administered in the vagina, and to evaluate its in vitro antifungal activity against four strains of Candida spp., with different azole-resistance patterns, commonly involved in VVC.

Materials and methods

Materials

TVEO (carvacrol type) was purchased from Segredo da Planta (Produtos Naturais e Biológicos, Lisbon, Portugal). The composition of the sample used in this work was previously assessed by gas chromatography, as reported by Pina-Vaz et al. (2004). Its six major components were carvacrol (70.3%), p-cymene (11.7%), γ-terpinene (3.2%), E-caryophyllene (2.9%), linalool (2.2%), and myrcene (1.7%). Polycarbophil (Noveon^® AA-1) was a kind gift from Noveon (Cleveland, USA). All other materials were of analytical grade or equivalent.

Preparation of TVEO gel

The composition (Table 1) and preparation of TVEO gel has been previously reported (das Neves et al., 2007). In brief, polycarbophil was dispersed in the mixture of hydrochloric acid (1 mM) and lactic acid by mechanical stirring. Following a 24-h rest period at 2–8°C, the mixture of propylene glycol, triacetin, and TVEO was incorporated by gentle stirring. Triethanolamine was added to achieve pH 4.2 ± 0.1. Also, the same gel was obtained without adding the essential oil (gel base). Both gels were then transferred to glass containers and allowed to rest for 48 h at 20°C, away from light, prior to the antifungal studies.

Table 1.  Composition of polycarbophil-based gel containing Thymus vulgaris L. essential oil (1%, w/w) (TVEO gel).

Ingredients	% (w/w)	Functions
TVEO	1	Antifungal drug
Polycarbophil	3	Gelling agent and mucoadhesive
Triacetin	1	Fixative
Lactic acid 90% (v/v)	1	Acidifier
Propylene glycol	15	Moisturizer and solvent
Hydrochloric acid 1 mM	75	Acidifier and solvent
Triethanolamine 70% (v/v)	2.3	Neutralizing agent
Purified water	e.q. 100	Solvent

Candida spp. strains

Three clinical strains isolated from recurrent cases of VVC (C. albicans H37, C. glabrata H16 and C. krusei H9) and one strain from the American Type Culture Collection (C. albicans ATCC 10231), presenting different resistance patterns to fluconazole, were utilized.

In vitro antifungal studies

Minimal inhibitory concentration (MIC) of TVEO, TVEO gel and the gel base were determined by a macrodilution broth method based on the CLSI (formerly NCCLS) M27-A2 protocol (NCCLS, 2002). Since the developed gel possesses high buffer properties, the method was performed at pH 5.5 for both the TVEO and the TVEO gel. Also, values of MIC were determined at pH 7 for TVEO, according to the CLSI protocol M27-A2, and compared with those obtained at pH 5.5. All experiments were performed in triplicate.

Results and discussion

Antifungal activity of TVEO at both pH values and TVEO gel are presented in Table 2. These results are comparable to those previously reported by Pina-Vaz et al. (2004), confirming the considerable antifungal activity of TVEO. According to Aligiannis et al. (2001), it is possible to say that essential oils are potent inhibitors of fungal growth for MIC values below 1.27 mg/mL. In a more conservative classification, Duarte and colleagues (2005) consider that only essential oils with MICs below 0.5 mg/mL can be regarded as potent antifungal products. In accordance with these two groups of investigators and the obtained MIC results, it is therefore possible to say that TVEO is a potent antifungal product.

Table 2.  MICs of TVEO, TVEO gel, and gel base for tested Candida strains.

Strains	TVEO gel^a	Gel base^a	TVEO^a	TVEO^b	Flu
C. albicans ATCC 10231	0.32	> 2.56^c	0.32	0.32	S
C. albicans H37	0.32	> 2.56^c	0.32	0.32	R
C. glabrata H16	0.32	> 2.56^c	0.32	0.32	R
C. krusei H9	0.32	> 2.56^c	0.32	0.32	R

All MICs expressed in mg/mL (w/v) of TVEO and determined at ^apH 5.5 or ^bpH 7.0. MICs for gel base ^c expressed as virtual concentration of TVEO. Phenotype to fluconazole (Flu) determined following the rules of the CLSI M27-A2 protocol.

MICs obtained at pH 5.5 and 7.0 show that the acidic moiety does not seem to influence the TVEO activity against selected Candida strains. The maintenance of antifungal activity of TVEO for pH values in close proximity to those observed in the healthy human vagina (4.0-5.0) (Boskey et al., 2001) and during VVC suggests that this essential oil has the potential to be effective in vivo.

Obtained values indicate that the incorporation of the TVEO in the gel base did not influence significantly its activity. Nonetheless, it may be expected that this polycarbophil-based gel may be advantageous in vivo, due to its pH-buffering and moisturizing properties (Robinson & Bologna, 1994). Also, it seems possible to state that the amount of TVEO in the gel (1%, w/w) is enough to achieve significant amounts of this substance in the vagina, in order to manage VVC. Considering the amounts of vaginal semisolid preparations normally administered (2.5 to 3.5 mL) and the quantity of fluid usually present at this organ (about 0.75 mL) (das Neves et al., 2008), we estimate that concentrations approximately 24 times higher than the determined MICs can be achieved in vivo. The gel base did not present antifungal activity in the range of concentrations tested, allowing normal fungal growth as compared to negative controls. This fact is significant when considering the utilization of the gel base as a placebo formulation in clinical experimentation.

In conclusion, TVEO showed promising antifungal activity against Candida spp. commonly involved in VVC, which may justify its use in clinical practice. The utilization of this essential oil may be particularly interesting when azole-resistant strains of Candida albicans or non-albicans species are present. Proposed TVEO gel seems to be adequate in order to administer TVEO in the vagina, demonstrating ability to maintain the antifungal activity of this natural product. Also, the gel base showed to be ineffective against tested Candida spp., being capable of playing an important role in the upcoming clinical trials as a placebo formulation.

Acknowledgments

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.