Investigation of the antifungal activity of carvacrol against strains of Cryptococcus neoformans

Abstract

Background: Cryptococcus neoformans is the etiologic agent of opportunistic systemic fungal infection cryptococcosis, which affects individuals with compromised immune systems. Thus, natural products research has become important, since monoterpenes such as carvacrol, a promising molecule in the search antifungal agents, have shown significant biological activity.

Objective: The study aimed to investigate the antifungal activity and mode of action of carvacrol against strains of C. neoformans.

Methods: The minimum inhibitory concentration (MIC) was determined by microdilution method. Minimum fungicidal concentration (MFC) was performed by seeding technique on solid media. Studying the mode of action was performed using broth microdilution.

Results: The MIC ranged from 25 to 81 μg/mL and the MFC ranged from 25 to 102 μg/mL. Carvacrol bonded to exogenous ergosterol and cholesterol.

Discussion: The results suggest that carvacrol has antifungal activity against C. neoformans and its mode of action is related to fungal membrane instability.

Conclusions: The phytoconstituent carvacrol may eventually become a drug; however, further studies are needed to elucidate its mechanism.

Keywords:

• Antifungal activity
• carvacrol
• ergosterol
• fungal membrane

Introduction

Cryptococcosis is a fungal infection that most commonly affects people with compromised immune systems. The etiological agent is Cryptococcus neoformans. The fungus appears as yeast surrounded by a capsule (Queiroz et al. 2008). Infection occurs by inhalation of infectious particles and can reach various parts of the body including the lungs, yet also displays tropism for the central nervous system, causing meningitis (Contin et al. 2011). According to Park et al. (2009), roughly one million cryptococcal meningitis cases are diagnosed worldwide and the disease is responsible for over 600 thousand deaths.

Treatment is performed with fungistatic/or fungicidal drugs (Nobre et al. 2002). Amphotericin B and flucytosine are used in combination, when treating meningitis caused by Cryptococcus spp. (Chen & Sorrell 2007). However, a high incidence of renal toxicity is one of the effects seen with the use of amphotericin B (Sara et al. 1990).

Limitations in the use of antifungals have provoked research for treatment alternatives. Phytochemicals isolated from essential oils are effective alternatives (Bakkali et al. 2008), helping to obtain new drugs that are used in the treatment of these infections and other diseases (Lima et al. 2005; Bezerra et al. 2013).

To this end, new compounds such as carvacrol, a phytoconstituent present in essential oils with biological properties, for example; oregano (Origanum vulgare), thyme (Tymus vulgaris), lipia (Lippia graveolens) rosemary, and pepper-rosmarin (Lipia sidoides) are under test (Pozzatti et al. 2006; Freitas et al. 2013). Carvacrol is a phenolic compound, and the presence of a hydroxyl group and a delocalized electron system may be responsible for its biological activity (Ultee et al. 2002).

The objective of this study was to determine carvacrol’s minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and to evaluate the interference of ergosterol and cholesterol (at MIC), in order to elucidate the possible mechanism of activity.

Material and methods

Test substances

The substances carvacrol, ergosterol, cholesterol and amphotericin B (used to perform the tests) were purchased from Sigma Aldrich® (Steinheim, Germany). Further, Sabouraud dextrose agar (SDA) and Sabouraud dextrose broth were purchased from Difco Laboratories Ltd (Le Pont de Claix, France).

Fungal strains

Various strains of Cryptococcus neoformans (LM-0310, FCF-119, ICB-59, FGF-102, ICB-2601, JM-10, LM-5, LM-120, LM-39, LM-22), belonging to the Mico-Tech of the Federal University of Paraíba, Mycology Laboratory, were used and provided by Professor Dr. Edeltrudes de Oliveira Lima.

Inoculum

The strains were maintained in SDA culture medium for 72 h at 37 °C. To prepare the inoculum, the fungal colonies were suspended in 5 mL of sterile saline (0.85%). The suspensions were then shaken with the aid of a vortex and adjusted to a 0.5 McFarland scale, which corresponds to 1–5 × 10⁶ CFU/mL (Cleeland & Squires 1991; Hadacek & Greger 2000).

Determination of MIC and MFC

The MIC was determined by the microdilution method in 96 well plates for each of the strains. A 100 μL of Sabouraud dextrose broth and carvacrol were distributed in all the wells of the plate. Through serial dilution at a ratio of two, concentrations from 1024 to 1 μg/mL were obtained, made in each plate itself, from column 1 to 11. The last column (12) was reserved for micro-organism growth control (Sabouraud dextrose broth, without the tested product). Control with a standard synthetic antifungal (amphotericin B) was realized. Then 10 μL of the fungal suspension was added to each well. The assay was performed in triplicate and subjected to incubation at 37 °C for 72 h. The readings were made by observing turbidity of the medium; indicating growth of the microorganism. Thus, the MIC was determined as the lowest concentration which could inhibit fungal growth (Cleeland & Squires 1991; Eloff 1998; Hadacek & Greger 2000; CLSI 2002).

To determine the MFC, 10 μL of the volume of each well with no microorganism growth (MIC, 2xMIC and 4xMIC) was seeded onto plates containing Sabouraud dextrose agar. The plates were then incubated at 37 °C temperature for 72 h. The reading was performed by counting the number of colony forming units (CFU), and the MFC was considered as the lowest concentration able to inhibit fungal growth up to a limit of three CFUs (Klepser et al. 1998; Ernst et al. 1999; Cantón et al. 2003; Costa et al. 2008).

Ergosterol and cholesterol assay

The assay was performed in accordance with Escalante et al. (2008). In this trial, two strains of C. neoformans LM-22 and FGF-102 were used. The carvacrol MIC determination was made through the microdilution method, comparing strains in the absence and presence of ergosterol and cholesterol in concentrations of 100, 200 and 400 μg/ml. As a positive control, amphotericin B was used, and the negative control consisted of culture medium and inoculum.

Results

The results of tests determining the minimum inhibitory concentration are shown in Table 1. The MIC of carvacrol ranged from 25 to 81 μg/mL. There was an increase in the positive control (culture medium and inoculum), and none in the sterility control (culture medium); these same were also used to validate the method of this study (Cavalcanti et al. 2001).

Table 1. MIC results for amphotericin B and carvacrol against C. neoformans strains.

Strains	Control (micro-organisms)	Amphotericin B (μg/mL)	Carvacrol (μg/mL)
LM-05	+	0.62	25
FCF-102	+	0.5	32
FCF-119	+	0.62	40
ICB-59	+	0.5	40
JM-10	+	1	51
LM-39	+	0.79	51
ICB-2601	+	0.62	51
LM-120	+	0.5	64
LM-0310	+	0.5	64
LM-22	+	1	81

+: Presence of fungal growth. The MIC was determined by calculating the geometric mean.

Amphotericin B (AB) was used for MIC determination as positive control. The MIC values ranged from 0.5 to 1 μg/ml (Table 1). All strains were sensitive to AB, given that Nguyen and Yu (1998) and Lozano-Chiu et al. (1998) have defined resistance to AB as being MIC values equal to or greater than 2 μg/mL.

After determining the MIC of carvacrol, minimum fungicidal concentration (MFC) was determined according to the method of Ernst et al. (1999). The results are shown in Table 2.

Table 2. MFC results of carvacrol against C. neoformans strains.

Strains	Control (micro-organism)	Carvacrol (μg/mL)
LM-05	+	25
FCF-102	+	32
FCF-119	+	40
JM-10	+	51
ICB-2601	+	51
LM-120	+	64
LM-0310	+	64
ICB-59	+	80
LM-22	+	81
LM-308	+	102

+: Presence of fungal growth. The MIC was determined by calculating the geometric mean.

The carvacrol MFC was considered as the lowest concentration that resulted in growth of three colonies (Ernst et al. 1999). We used the control of each strain to facilitate growth on solid medium. The MFC ranged from 25–102 mg/mL, for all strains, MFC was equal to MIC, except for the ICB-59 and LM-309 strains where MFC was 2× MIC.

To investigate the mode of action, carvacrol was tested in the presence (and absence) of ergosterol according to the methodology of Escalante et al. (2008), and also in the presence and absence of cholesterol (Lima et al. 2013), a sterol present in mammalian cells (Zacchino 2001). Ergosterol (sterol) is the lipid component of fungal membranes considered most important for permeability and fluidity (Thevissen et al. 2003). It may be observed that the MIC of carvacrol in concentrations of 100, 200, 400 μg/ml increased in the presence of ergosterol and cholesterol (Tables 3 and 4).

Table 3. Study of the effect of ergosterol on MIC against carvacrol C. neoformans LM-22, and C. neoformans FGF-102.

	C. neoformans LM-22				C. neoformans FCF-102
Carvacrol (μg/mL)	Absence of the ergosterol	Ergosterol 100 μg/mL	Ergosterol 200 μg/mL	Ergosterol 400 μg/mL	Absence of the ergosterol	Ergosterol 100 μg/mL	Ergosterol 200 μg/mL	Ergosterol 400 μg/mL
1024	–	–	–	–	–	–	–	–
512	–	–	–	+	–	–	–	–
256	–	–	–	+	–	–	–	–
128	–	+	+	+	–	+	+	+
64	–	+	+	+	–	+	+	+
32	+	+	+	+	–	+	+	+
16	+	+	+	+	–	+	+	+
8	+	+	+	+	+	+	+	+
4	+	+	+	+	+	+	+	+
2	+	+	+	+	+	+	+	+
1	+	+	+	+	+	+	+	+
0.5	+	+	+	+	+	+	+	+

+: Fungal growth; –: No fungal growth.

Table 4. Study of the effect of cholesterol on carvacrol’s MIC against C. neoformans LM-22 and C. neoformans FGF-102.

	C. neoformans LM-22				C. neoformans FCF-102
Carvacrol (μg/mL)	Absence of the Cholesterol	Cholesterol 100 μg/mL	Cholesterol 200 μg/mL	Cholesterol 400 μg/mL	Absence of the Cholesterol	Cholesterol 100 μg/mL	Cholesterol 200 μg/mL	Cholesterol 400 μg/mL
1024	–	–	–	+	–	–	–	+
512	–	–	–	+	–	–	–	+
256	–	+	+	+	–	–	–	+
128	–	+	+	+	–	+	+	+
64	–	+	+	+	–	+	+	+
32	+	+	+	+	–	+	+	+
16	+	+	+	+	–	+	+	+
8	+	+	+	+	+	+	+	+
4	+	+	+	+	+	+	+	+
2	+	+	+	+	+	+	+	+
1	+	+	+	+	+	+	+	+
0.5	+	+	+	+	+	+	+	+

+: Fungal growth; –: No fungal growth.

In the presence of ergosterol, the MIC increased by four times the concentration of 100 and 200 μg/mL, and eight times at the concentration of 400 μg/mL when tested against the LM-22 strain (Table 3). In the same test, when carried out against the FGF-102 strain, the MIC increased by eight times at all concentrations (100, 200, 400 μg/mL) when tested in the presence of ergosterol (Table 3). The MIC was determined by calculating the geometric mean.

In Table 4, it is observed that the carvacrol MIC changed in the presence of cholesterol. The strain LM-22 MIC increased to six times at concentrations of 100 and 200 μg/mL, the concentration of 400 μg/mL increased MIC by 10-fold (Table 4). Also in this test, against the FCF-102 strain we observed that the MIC increased by eight times when used at concentrations of 100 and 200 μg/mL, and the concentration of 400 μg/mL MIC increased the MIC by 14 times in the presence of cholesterol. The MIC was determined by calculating the geometric mean.

In this study, testing was of ergosterol also performed with AB as a positive control. This drug acts by binding to ergosterol, leading to increased permeability and ultimately, to cell death (Moreira 2005).

In the presence of ergosterol the AB MIC against the LM-22 strain, increased 10 times for the concentration of 100 μg/mL, and at concentrations of 200 and 400 μg/mL it increased 14-fold (Table 5). Also in this test, the AB MIC for the FCF-102 strain increased 10 times for the concentration of 100 μg/mL, and at concentrations of 200 and 400 μg/ml increased 12 times (Table 5). The MIC was determined by calculating the geometric mean.

Table 5. Study of the effect of ergosterol on the MIC of amphotericin B against C. neoformans LM-22 and C. neoformans FGF-102.

	C. neoformans LM-22				C. neoformans FCF-102
Amphotericin B (μg/mL)	Absence of the ergosterol	Ergosterol 100 μg/mL	Ergosterol 200 μg/mL	Ergosterol 400 μg/mL	Absence of the ergosterol	Ergosterol 100 μg/mL	Ergosterol 200 μg/mL	Ergosterol 400 μg/mL
64	–	–	+	+	–	–	–	–
32	–	–	+	+	–	–	–	–
16	–	+	+	+	–	–	+	+
8	–	+	+	+	–	+	+	+
4	–	+	+	+	–	+	+	+
2	–	+	+	+	–	+	+	+
1	–	+	+	+	–	+	+	+
0.5	+	+	+	+	–	+	+	+
0.25	+	+	+	+	+	+	+	+
0.125	+	+	+	+	+	+	+	+
0.062	+	+	+	+	+	+	+	+
0.031	+	+	+	+	+	+	+	+

+: Fungal growth; –: No fungal growth.

The effect of cholesterol on the MIC of AB was twice that of the antifungal’s MIC value for FGF-22 and LM-109 at all concentrations (Table 6). It is noteworthy that in conjunction with these experiments were held assays of the positive and negative controls which indicated that the strains were viable, and the medium sterile. The MIC was determined by calculating the geometric mean.

Table 6. Study of the effect of cholesterol on the MIC of amphotericin B against C. neoformans LM-22 and C. neoformans FGF-102.

	C. neoformans LM-22				C. neoformans FCF-102
Amphotericin B (μg/mL)	Absence of the Cholesterol	Cholesterol 100 μg/mL	Cholesterol 200 μg/mL	Cholesterol 400 μg/mL	Absence of the Cholesterol	Cholesterol 100 μg/mL	Cholesterol 200 μg/mL	Cholesterol 400 μg/mL
64	–	–	–	–	–	–	–	–
32	–	–	–	–	–	–	–	–
16	–	–	–	–	–	–	–	–
8	–	–	–	–	–	–	–	–
4	–	–	–	–	–	–	–	–
2	–	–	–	–	–	–	–	–
1	–	+	+	+	–	–	–	–
0.5	+	+	+	+	–	+	+	+
0.25	+	+	+	+	+	+	+	+
0.125	+	+	+	+	+	+	+	+
0.062	+	+	+	+	+	+	+	+
0.031	+	+	+	+	+	+	+	+

+: Fungal growth; –: No fungal growth.

Discussion

The MIC results obtained were lower and different than the results obtained by Lima et al. (2013), where, in evaluating the antifungal activity of carvacrol against the Candida albicans strain, MICs of 128 and 256 μg/mL were obtained. We show that this phytoconstituent has antifungal effect. Another study by Kaloustion et al. (2008) demonstrated that carvacrol had an MIC of 400 μg/mL for E. coli ATCC 10536, and S. aureus ATCC 65288 by broth dilution method.

Studies with essential oils rich in carvacrol demonstrate antimicrobial activity. Thus, Pozzatti et al. (2010) reported that the essential oil of Mexican oregano with carvacrol (58%) presented an MIC ≤800 μg/mL against species of the genus Candida. In another study, Carmo et al. (2008) determined the antifungal activity of oregano essential oil against Aspergillus species and found an MIC of between 20 and 80 μL/mL.

Comparison of this study with the findings of other authors, shows agreement, though certain factors may interfere with MIC values; such as culture conditions (incubation time, temperature and oxygen%), culture medium, concentration of the tested substances, and dispersion and emulsion of the agents used in oil-water emulsions (Hammer et al. 1999; Rios & Recio 2005). These factors may contribute to the difference in results and hamper comparison with other studies.

Lima et al. (2013) determined the MFC of carvacrol against strains of Candida albicans and obtained values between 256 and 512 μg/mL. In this study, the MFC of carvacrol was between 25 and 102 μg/mL; this being different from the value obtained by Lima et al. (2013). In another study by Manohar et al. (2001), the MFC of carvacrol against a standard strain of C. albicans (ATCC-48274) was 500 μg/mL. The concentrations of MIC and MFC determined in this study were low and could completely inhibit the strains, which shows that at the evaluated concentrations, carvacrol has fungicidal action. It is believed to carvacrol fungal growth inhibition occurs thru alteration of the cell wall/membrane functions (Lee et al. 1999).

Based on the results, the MIC of carvacrol increased proportionally to the concentration of ergosterol in the medium. This result suggests that the carvacrol may act by binding to exogenous ergosterol. Since ergosterol is a sterol present in fungal cells, carvacrol-ergosterol binding would result in increased permeability, causing the output of Ca²⁺, and K⁺ ions, radicals, and proteins (Bakkali et al. 2008) and thus destabilization of the membrane and inhibition of fungal growth, as shown in Figure 1. Cell membrane integrity is essential for survival of fungi as it is responsible for the cell function (Gray et al. 2012).

Figure 1. Representation of carvacrol’s mechanism with its possible link to ergosterol.

Hammer et al. (2004) evaluated the antifungal activity of M. alternifolia essential oil (rich in terpenoids) on Candida albicans, C. glabrata and Saccharomyces cerevisiae, finding that the essential oil at concentrations of 0.25 and 1.0% changed fluidity and permeability of the fungal membrane.

Gill and Holley (2006) investigated cell membrane rupturing in strains of L. monocytogenes, E. coli O157H7, and Lb. sake when exposed to bactericidal concentrations of aromatic compounds (carvacrol, eugenol and cinnamaldehyde). The results indicated that carvacrol caused rupture of the cytoplasmic membrane and increased its permeability.

However, carvacrol interacted with cholesterol; this result showed that the MICs were higher in the presence of cholesterol, as compared to medium containing ergosterol. The result suggests that the phytoconstituent might be toxic to human cells, since cholesterol is a component of animal cell plasma membranes.

Cyclic hydrocarbons, such as terpenes, cyclic alkanes, may be toxic. Such action is attributed to interactions between the lipophilic portions of these groups with the hydrophobic part of the membrane structure (Sikkema et al. 1995), the lipophilic portions of these essential oils tend to accumulate within the cytoplasmic membrane (Pozzatti et al. 2006).

The lipophilicity of a physicochemical characteristic of a compound depends on other characteristics as well, such as polarity, solubility and density (Leo et al. 1976). For information on the lipophilic character of carvacrol, Ultee et al. (2002) evaluated its partitioning behaviour in a mixture of octanol and water. Carvacrol had a preference for the octanol phase (being more hydrophobic) which resulted in a log Po/w 3.64. Given this information, carvacrol may have affinity to fat tissues, and concentrating there, may cause harmful effects (Ultee et al. 2002).

Wright and Shadnia (2008) found the co-existence of two toxicity mechanisms for phenols, one being nonspecific, and dependent on the lipophilicity, and the other, being the phenolic ability to form radicals by interacting with reactive oxygen species, generated in cells by oxidative stress. Thus, a toxicological study of the phytoconstituent is necessary to ensure its safety.

Yet, a study by Chami et al. (2004) evaluated carvacrol and eugenol activity against oral candidiasis in immune compromised mice. No acute toxicity at the doses used (160 and 380 mg/kg) was found. Categorizing these oils as promising agents for the treatment of oral candidiasis became possible. In another study, carried out by Cleff et al. (2008) evaluated the toxicity of 3% O. vulgare essential oil, orally and topically in rats. When used for 30 d, it caused no changes in organ mass, or in erythrocyte or leukocyte count; a demonstration of low toxicity.

Today, liposomes are well used in the development of technologies. These vesicles are of amphoteric nature which allow encapsulation of various hydrophilic and/or lipophilic molecules. When used as chemotherapeutic agents they provide greater efficacy, and reduced toxicity and dosage of the substance (Machado et al. 2007). Thus, liposomes might be a good alternative to serve the carvacrol and achieve efficient therapeutic application.

AB is a polyenic antibiotic and used for systemic antifungal therapy; it acts quickly and causes lethal damage to most endemic and opportunistic mycosis agents. Primary AB therapy indications are cases of cryptococcosis, aspergillosis, and disseminated Candida spp. infections due to triazole resistance (Ghannoum & Rice 1999).

AB molecules self-associate in high concentrations to form dimers or larger aggregates (Larabi et al. 2004). A study by Huang et al. (2002) found that AB as a monomer or as an aggregate may form channels in membranes containing ergosterol, but only the aggregated forms channels in membranes containing cholesterol.

Though AB has a greater affinity for ergosterol, many of its attributed toxic effects are the result of its ability to bind to cholesterol (Bolard et al. 1993; Huang et al. 2002). A higher incidence in renal toxicity is seen when using this antifungal agent, the main characteristics are: reduction of glomerular filtration, hypokalemia and hypomagnesemia (Sara et al. 1990). In a comparative study between amphotericin B and caspofungin, 24.8% of patients who were treated with AB (0.6–1.0 mg/kg/day) showed nephrotoxicity confirmed by laboratory findings (Mora-Duarte et al. 2002).

Another limiting factor in the treatment of fungal infections is fungal resistance to the action of the available drugs. Dalazen et al. (2011) demonstrated a high rate of AB resistance (96.6%) in clinical isolates of Candida spp. In the face of these considerations, the phytoconstituent carvacrol shows promise as an alternative for the treatment of fungal infections. Despite the interaction of carvacrol with cholesterol, toxicity studies to prove possible effects are necessary since the techniques developed in this study do not replace toxicity studies.

One way to optimize the use of carvacrol is by association with conventional drugs. These associations have been widely studied in in vitro methods; the “checkerboard” technique being widely used (Greco et al. 1995; Odds 2003). A carvacrol association study with AB to assess whether there is a synergistic effect is needed. Rosato et al. (2008) demonstrated the potential synergistic activity of essential oils from Melaleuca alternifolia, Origanum vulgare, Pelargonium graveolens and AB against species of Candida sp. It is noteworthy that the pharmacologically active compound present in the essential oil of oregano is carvacrol at 3–17% (Farmacopea Ufficiale Della Republica Italiana 1998).

The antifungal of the future should have a broad spectrum of activity, being safe and non- toxic to the host cells (Carrillo-Muñoz et al. 2006). Thus, in view of the results, the phytoconstituent carvacrol may eventually become a drug; however, further studies are needed to elucidate its mechanism and toxicity.

Conclusions

Under the conditions evaluated, the phytoconstituent carvacrol showed antifungal activity against Cryptococcus neoformans strains. It may act by binding to exogenous ergosterol and presented interaction with cholesterol, a sterol present in mammalian cells.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.