Anti-Candida albicans effectiveness of citral and investigation of mode of action

Abstract

Context: Candidiasis is a mycosis caused by Candida species, which is of clinical importance due to the increase in resistant yeasts. Candida infection has been a serious health problem due to the inappropriate use of antibiotics. Therefore, it is necessary to study molecules with an antifungal action. Citral is a monoterpene with known pharmacological properties, including antimicrobial action.

Objective: The aim of this work was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral and the probable mode of action.

Materials and methods: The MIC of citral was determined by the broth microdilution method using Sabouraud dextrose medium. Additionally, the interference of citral in cell wall (sorbitol assay) and the binding of citral to ergosterol and cholesterol were studied, carried out by broth microdilution method.

Results: The MIC and MFC of citral were 512 and 1024 µg/mL, respectively. The MIC of amphotericin B was 1 µg/mL. The mechanism of action did not involve either the cell wall or ergosterol. However, the presence of cholesterol increased the MIC of citral to 1024 µg/mL, indicating there is some interaction between citral and cholesterol. Amphotericin B was used as the positive control, and it showed a high MIC in the presence of ergosterol (32 µg/mL), while in the presence of cholesterol MIC increased to 4 µg/mL.

Conclusion: Citral inhibits the growth of C. albicans. The probable mechanism of action did not involve the cell wall or ergosterol. Citral is able to interact with cholesterol. More studies are necessary to describe their effects completely.

Keywords::

• Ergosterol
• sorbitol
• cholesterol

Introduction

Disseminated candidiasis is associated with mortality in excess of 25%. Candida spp. is a normal commensal of the skin and gastrointestinal and genitourinary tracts, and Candida albicans is the most frequent species isolated from clinical specimens (Kett et al., 2011; Enoch et al., 2006). Among the Candida species, C. albicans is associated with bloodstream infections in 44% of cases in Latin America and 62% in Europe (Richardson & Lass-Flörl, 2008).

Prior surgery, acute renal failure, previous yeast colonization, neutropenia, antibacterial agents, parenteral nutrition, and central nervous catheters are associated with an increased risk of invasive candidiasis (Eggimann et al., 2003).

The incidence of invasive opportunistic mycoses occurs due to an expanding population of immunosuppressed patients, including patients with cancer, diabetes, AIDS, solid-organ transplant, hematopoietic stem cell transplant, premature neonates and patients recovering from major surgery (Nucci et al., 2010).

The resistance of microbes to antimicrobial agents has potentially serious implications for the management of infections (Sanglard & Odds, 2002). Existing antifungals can treat mucosal fungal infections, but very few treatments are available for invasive diseases. (Cohen, 1998). The polyenes cause serious host toxicity. The azoles are fungistatic and their prolonged use contributes to the development of drug resistance in C. albicans and other related species (Sanglard et al., 2003). Overexpression of efflux genes is the most common mechanism of resistance to azoles in Candida species, and has also been associated with cross-resistance within the class. Progressive loss of echinocandin activity has been observed in Candida species with prolonged exposure to echinocandin. The most common mechanism of echinocandin resistance is associated with mutation in the FSK1 gene, a gene that produces the FSK1 protein for β-1,3-d-glucan synthase (Kriengkauykiat et al., 2011). For these reasons, new drugs and other therapies are necessary for the treatment of infections caused by C. albicans.

Terpenes form structurally and functionally different classes. They are made by the combinations of several 5-carbon-base (C5) units called isoprene. The main terpenes are the monoterpenes (C10) and sesquiterpenes (C15). A terpene containing oxygen is called a terpenoid (Bakkali et al., 2008).

Citral is the name given to a natural mixture of two isomeric acyclic monoterpene aldehydes: geranial (trans-citral, citral A) and neural (cis-citral, citral B). This phytoconstituent is active against HSV-1 in vitro and it has anti-inflammatory action, where it blocks the NF-κB pathway (Adorjan & Buchbauer, 2010).

Garcia et al. (2008) demonstrated the activity of citral against the fungi Colletotrichum musae, Colletotrichum gloeosporioides and Fusarium subglutinans f.sp. ananas. Citral is active against methicillin-resistant Staphylocoocus aureus, Penicillium italicum and Rhizopus stolonifer (Saddiq & Khayyat, 2010).

The aim of this work was to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) and to investigate the mode of action of this phytoconstituent against C. albicans by sorbitol, ergosterol and cholesterol assays.

Materials and methods

Phytoconstituents and substances

The following substances used in this work were acquired commercially: citral, ergosterol, sorbitol, cholesterol and amphotericin B (all from Sigma-Aldrich®, Chemie, Germany). Furthermore, Sabouraud dextrose broth (SDB) and Sabouraud dextrose agar (SDA) were purchased from Difco (Detroit, MI, USA).

Cell strains used

The assays were performed with 13 strains of Candida albicans. The strains were isolated from blood (LM-13, LM-36, LM-39, LM-41, LM-45, LM-52, LM-57, LM-68, LM-77, and LM-86), urine (LM-70), respiratory tract (LM-67) and vaginal secretion (LM-84), and are part of the Collection of the Laboratory of Microbiology of Yeasts, College of Pharmacy, Federal University of Ceará (LMY/CF/UFC). The strains were streaked on potato agar (Himedia, Mumbai, India) and incubated at 37°C for 24 h. They were then plated on CHROMagar Candida (Himedia, Mumbai, India) to assess purity. Identification was done by micromorphology on rice agar Tween 80, germ tube production, fermentation and assimilation of carbohydrates as well as molecular tests (Menezes et al., 2009; Gomes et al., 2010; Vasconcelos-Júnior, 2011).

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)

Minimum inhibitory concentration (MIC) was determined by the microdilution method, according to NCCLS (2008), with some modifications. Cultures of Candida albicans were placed on Sabouraud dextrose and incubated for 24–72 h at 37°C. Colonies of this culture were suspended in sterile 0.85% NaCl, and the inoculum was standardized to 0.5 on the McFarland scale (1–5 × 10⁶ CFU/mL). Sabouraud broth containing citral at concentrations of 1024–1 μg/mL was added a 96-well plate. The plates were incubated at 37°C for 24–48 h, at which time fungal growth was observed (NCCLS, 2008).

To determine the MFC, 10 μL of each of the wells with no fungal growth were seeded on plates containing Sabouraud dextrose agar (SDA), which were then incubated at 37°C for 24–48 h. Negative control (without drugs) was performed to confirm cell viability (Ernst et al., 1999; Costa et al., 2008; Eloff, 1998). Sensitivity control to Tween 80 was performed at the same concentrations used to dissolve the products.

MFC was considered as the lowest concentration in a plate with SDA at which growth was less than 3 CFU. Afterwards, 20 μL of 0.5% TTC (triphenyl tetrazolium chloride) were added to each of 96-well for MIC determination, and the plate was incubated for 24 h. MIC was defined as the lowest citral concentration that inhibited visible growth of the microorganism, as also indicated by TTC by reading 96-well plates. Three independent experiments were performed on different occasions (Klepser, et al., 1998; Ernst et al., 1999; Costa et al., 2008).

Sorbitol assay

The MIC of citral was determined with Candida albicans (LM-13) using the broth microdilution method (NCCLS, 2002). Sorbitol was added to the culture medium to give a final concentration of 0.8 M. Following incubation at 25°C, the plates were read at 48 h and after 7 days (Frost et al., 1995). This assay was carried out in duplicate and the geometric mean values were calculated.

Effect of ergosterol and cholesterol on MIC of citral

This experiment was performed according to the method of Lunde and Kubo (2000) and Escalante et al. (2008), with some modifications. The MIC of citral against C. albicans was determined by the microdilution method (NCCLS, 2002), in the presence and absence of ergosterol and cholesterol at concentrations of 100, 200, and 400 μg/mL. This assay was carried out in duplicate and the geometric mean values were calculated. Amphotericin B was used as a control drug in the ergosterol assay.

Results

MIC and MFC were determined by the broth microdilution technique. The MIC of citral ranged between 256 and 512 μg/mL. The concentration of 512 μg/mL inhibited the growth of all of the strains tested, while 256 μg/mL inhibited approximately 50% of the strains tested (MIC₅₀). MFC in these microorganisms ranged between 256 and 1024 μg/mL, the latter being fungicidal for all strains tested. The MIC of the amphotericin B against C. albicans strains ranged between 0.5 and 4 µg/mL. The growth of most of the strains studied was inhibited by 0.5 µg/mL amphotericin B (Table 1).

Table 1.   Minimum inhibitory concentration (MIC) of citral and amphotericin B and minimum fungicidal concentration (MFC) in µg/mL of citral against C. albicans.

C. albicans strains	Control	Amphotericin B (µg/mL)	Citral (MIC) (µg/mL)	Citral (MFC) (µg/mL)
LM-13	+	1	256	512
LM-36	+	4	512	1024
LM-39	+	1	256	256
LM-41	+	2	512	512
LM-45	+	0.5	512	512
LM-52	+	0.5	512	512
LM-57	+	0.5	256	512
LM-67	+	0.5	512	512
LM-70	+	0.5	256	512
LM-77	+	1	256	512
LM-84	+	0.5	256	256
LM-86	+	0.5	256	256
LM-88	+	0.5	512	512

+, presence of growth; C. albicans, Candida albicans.

According to the above results, the strain LM-13 was chosen for the other tests, the MICs of citral and amphotericin B against this strain were 256 and 1 µg/mL, respectively. The sorbitol assay consists of determining of MIC in the presence and absence of 0.8 M sorbitol. The present work showed no difference between MICs of citral against strain LM-13 in the absence or presence of sorbitol, the MIC remained 256 µg/mL (Table 2).

Table 2.   Sorbitol assay with citral of different concentrations against Candida albicans (LM-13).

Concentration (µg/mL)	Citral in presence of sorbitol	Citral in absence of sorbitol
2048	−	−
1024	−	−
512	−	−
256	−	−
128	+	+
64	+	+
32	+	+
16	+	+
8	+	+
4	+	+
2	+	+
1	+	+

+, growth fungal; −, absence of growth fungal

No alterations in MIC of citral were observed in the presence of various ergosterol concentrations (100, 200, and 400 µg/mL). In the presence of various cholesterol concentrations (100, 200, and 400 µg/mL), the MIC of citral increased from 512 to 1024 μg/mL (Table 3). Table 4 shows an increase in MIC of amphotericin B value when ergosterol was added to the medium, where the MIC increased from 1 to 32 µg/mL with all concentrations of ergosterol tested. The presence of the cholesterol in the medium increased the MIC of amphotericin B to 4 μg/mL (Table 4).

Table 3.   Ergosterol and cholesterol assay with citral of different concentrations against C. albicans (LM-13).

Citral (µg/mL)	C. albicans LM-13				C. albicans LM-13
	Absence of ergosterol	Ergosterol (100 µg/mL)	Ergosterol (200 µg/mL)	Ergosterol (400 µg/mL)	Absence of cholesterol	Cholesterol (100 µg/mL)	Cholesterol (200 µg/mL)	Cholesterol (400 µg/mL)
2048	−	−	−	−	−	−	−	−
1024	−	−	−	−	−	−	−	−
512	−	−	−	−	−	+	+	+
256	+	+	+	+	+	+	+	+
128	+	+	+	+	+	+	+	+
64	+	+	+	+	+	+	+	+
32	+	+	+	+	+	+	+	+
16	+	+	+	+	+	+	+	+
8	+	+	+	+	+	+	+	+
4	+	+	+	+	+	+	+	+
2	+	+	+	+	+	+	+	+
1	+	+	+	+	+	+	+	+

+, growth fungal; −, absence of growth fungal, C. albicans, Candida albicans.

Table 4.   Ergosterol and cholesterol assay with amphoterin B of different concentrations against C. albicans (LM-13).

Amphotericin B (µg/mL)	C. albicans LM-13				C. albicans LM-13
	Absence of ergosterol	Ergosterol (100 µg/mL)	Ergosterol (200 µg/mL)	Ergosterol (400 µg/mL)	Absence of cholesterol	Cholesterol (100 µg/mL)	Cholesterol (200 µg/mL)	Cholesterol (400 µg/mL)
2048	−	−	−	−	−	−	−	−
1024	−	−	−	−	−	−		−
512	−	−	−	−	−	−	−	−
256	−	−	−	−	−	−	−	−
128	−	−	−	−	−	−	−	−
64	−	−	−	−	−	−	−	−
32	−	−	−	−	−	−	−	−
16	−	+	+	+	−	−	−	−
8	−	+	+	+	−	−	−	−
4	−	+	+	+	−	−	−	−
2	−	+	+	+	−	+	+	+
1	−	+	+	+	−	+	+	+

+, growth fungal; −, absence of growth fungal, C. albicans, Candida albicans.

Discussion

The high incidence of Candida albicans infections and emergence of resistance indicate the need to study new sources of antifungals, such as natural products and their phytoconstituents. Essential oils are important because of their various pharmacological activities including antifungal, antibacterial and antiparasitic properties (Bakkali et al., 2008; Rates, 2001).

The present work studied the activity of citral against Candida albicans and its mode of action. Citral concentration of 512 μg/mL inhibited the growth of all strains, and 1024 μg/mL was fungicidal to all strains (Table 1). The control consisted of the strains, medium and vehicle, where the last did not inhibit fungal growth.

It has been reported that the citral and the essential oil of Cymbopogon citratus, shows antifungal activity against C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and especially C. albicans (Silva Cde et al., 2008). Lemongrass oil is characterized for monoterpenes compounds, and citral is the major component present at levels of approximately, 65–85% (Ferreira & Fonteles, 1989). The activity of lemongrass oil is associated with the content of citral.

Lima et al. (2005) showed the antifungal action of citral against C. albicans, C. tropicalis, C. parapsilosis, C. stellatoidea, C. guilhermondii, C. krusei and Cryptococcus neoformans. Dellau et al. (2008) demonstrated that citral has antibiofilm activities against C. albicans.

Ferreira et al. (2009) demonstrated the antifungal action of citral against C. albicans, C. tropicalis, C. parapsilosis and C. glabrata isolated from patients with clinical suspicion of infection. These authors showed that citral inhibited the fungal growth and this action was more effective than miconazol.

In the present work, citral showed activity against C. albicans strains and these results confirm the results obtained by Silva Cde et al. (2008) and Ferreira et al. (2009).

Park et al. (2009) observed other actions of citral. This component was able to cause prominent hyphal degeneration, the separation of membrane from cell wall, and the disintegration of mitochondria in Trichophyton mentagrophytes.

In this context, it is important to investigate the mode of action of citral. In this work, we investigated the effect of citral on the cell wall by sorbitol assay and the binding of citral to ergosterol and cholesterol.

The sorbitol has an osmoprotector function and is essential for fungal growth, when fungi are in the presence of drugs that act on the cell wall (Frost et al., 1995). Sorbitol-protected cells can grow in the presence of an inhibitor of fungal cell wall synthesis, while fungi are growth-inhibited in its absence. This effect is detected by the great difference observed between the MICs obtained with and without sorbitol (Svetaz et al., 2007). The protection of growth with sorbitol is not limited to β-(1,3) glucan synthesis inhibitors but can also be applied to inhibitors of the synthesis of other cell wall polymers and of the mechanisms controlling cell wall synthesis. The sorbitol protection assay is a broad spectrum screen that can identify not only agents that directly affect cell wall synthesis and assembly but also regulatory mechanisms involved in this process, including signal transduction pathways such as protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) (Frost et al., 1995). In this work, the MIC of citral was not altered in the presence of sorbitol, suggesting that citral does not act by modifying the fungal cell wall, but probably by affecting another target.

Ergosterol is a sterol of the fungal cell, while cholesterol is a sterol of mammalian cells (Paquet et al., 2002). If the activity of the compound were due by binding to ergosterol, the exogenous ergosterol would prevent binding to ergosterol in the fungal membrane, and as a consequence, it would cause an increase in MIC in the presence of exogenous ergosterol with respect to the control assay (Escalante et al., 2008). The effect of ergosterol on the MIC of citral and amphotericin B was determined. In the presence of ergosterol, the MIC value of amphotericin B increased 32-times (Table 4); however, the same was not observed for the MIC of citral (Table 3). This indicates that this phytoconstituent does not bind to ergosterol.

According to the above findings, if the compound binds to cholesterol, it increases MIC in the presence of exogenous cholesterol. In the presence of cholesterol, the MIC value of citral increased to 1024 µg/mL (two-fold), and the MIC of amphotericin B increased four times, as shown in Tables 3 and 4. Amphotericin B has a strong affinity for ergosterol, while it has a weak affinity for cholesterol, and this is due to the double bond in the ergosterol molecule increasing the stability of interaction between amphotericin B and ergosterol (Baran et al., 2009).

It was demonstrated that a direct interaction can occur between sterol and a polyene antibiotic. The structure required for this effect is similar to that needed to prevent the biological action of polyenes, and the addition of sterols will prevent the antifungal activity of the polyenes (i.e., the presence of sterols increases the antifungal concentration required for growth inhibition). The interaction of the sterol and polyene reduces the effective concentration of the antibiotic and in this manner it increases the amount that must be used to inhibit growth or metabolic activity of the fungus (Lampen et al., 1960).

This work demonstrated that citral inhibits the growth of C. albicans. The probable mechanism of action does not involve either cell wall synthesis or ergosterol. It is important to know the mode of action of a drug, because this information can be used to increase the effectiveness of the substance. A drug interaction with cholesterol can result in toxic effects to mammalian cells; however, it is necessary to study its effect against human cells to confirm it. Considering that the incidence of both community-acquired and nosocomial fungal infections has significantly increased over the past few decades, the search for new alternatives for treatment of fungal diseases is important, and natural products, especially from plants, can be a source of new drugs.

Acknowledgement

The authors conducted the experiments at the Universidade Federal de Paraíba. Dr. A. Leyva helped with English editing of the manuscript.

Declaration of interest

The authors are grateful to CNPq for financial support. The authors report no conflicts of interest.