1. Introduction
Infections caused by species of the genus
Candida are opportunistic and more severe in immunocompromised, hospitalized individuals, using invasive devices and with comorbidities [
1]. Superficial candidiasis affects the oral and vaginal mucosa, skin and nails; and factors external to the individual, such as climatic conditions in tropical and subtropical regions, and factors inherent to it, such as local humidity, use of immunosuppressive or antibacterial drugs and some comorbidities, such as diabetes, facilitate the development of the disease [
2]. In superficial infections, the most frequent
Candida species is
C. albicans; however, in recent years,
Candida non-
albicans species have shown relevance among the causative agents of vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC), including the species
C. glabrata,
C. krusei and
C. tropicalis [
2,
3,
4].
The azole antifungals have been one of the therapeutic options for the treatment of superficial mycoses since the 1960s–1970s [
5,
6]. In this sense, topical formulations containing azoles are attractive for VVC and RVVC due to the lower incidence of adverse effects compared to the same drug class for oral use and systemic action [
3]. Clotrimazole has a cure rate of between 73% and 100% of infections, similar to other topical antifungals such as nystatin [
6,
7].
In the last decades, the report of new
Candida species and in vitro resistant isolates to traditional antifungals has been an incentive for the search and development of new ways of managing these infections [
8]. Resistance is a result of multiple factors that include structural changes in the drug target and the ability of
Candida spp. to form biofilms [
9,
10,
11]. In this sense, the community structure and firm adherence between the microorganisms of the biofilm allow a barrier condition that makes penetration of drugs difficult and consequently reduces the effectiveness of the treatment [
8,
10,
11].
Essential oils are plant-derived products with potential activity against microorganisms, attributable to the complex mixture of chemotypes [
12,
13,
14]. Recently, combinations of multiple agents have optimized antifungal activity against clinically relevant fungi. Thus, a new therapeutic approach combining conventional antifungal drugs, such as clotrimazole, and natural products with antifungal activity may have the potential for clinical use [
15,
16,
17,
18].
The in vivo screening of compounds with proven in vitro antimicrobial action is one of the necessary steps within the current safety context to identify the toxicity of new anti-infective agents [
19]. In this context, in vivo studies using alternative animal models such as
Drosophila melanogaster,
Galleria mellonella and
Caenorhabditis elegans have been proposed to assess the preliminary toxicity of new health products [
20,
21]. Thus, the free-living nematode
C. elegans can be an alternative predictive model option, being of low cost, fast cultivation and not very complex laboratory handling, and lending itself to the evaluation and screening of acute toxicity for use in animals, including humans, and contamination of the environment [
11,
19,
22]. In this sense, the evident anti-
Candida action of isolated essential oils could mean they present lower inhibitory values when combined with other essential oils or antifungal substances, such as clotrimazole, and its acute toxic repercussions.
Thus, in this study, the in vitro inhibitory activity of the essential oils of Cupressus sempervirens, Citrus limon, Litsea cubeba and Melaleuca alternifolia, alone and in combination, and associated with clotrimazole, against Candida species biofilms were analyzed. Furthermore, the in vivo toxicity of these essential oils against C. elegans was also evaluated.
4. Discussion
EO have been extensively studied nowadays and can be a complementary alternative for the treatment of infections caused by Candida species, especially mucocutaneous infections. This study investigated the activity of the EO of C. limon, C. sempervirens, L. cubeba and M. alternifolia, alone and in combination with each other and with clotrimazole, on four species of the genus Candida, to determine in vitro the MIC, MBIC and MBEC; in addition to this, an in vivo toxicity assessment for the nematode C. elegans was performed.
The EO extracted from plants of the studied species are products that have a complex chemical composition and may have more than 20 identified compounds [
12,
13,
14,
15,
18]. Our study used EO that presented limonene (65.6%), α-pinene (52.4%), terpinen-4-ol (41%) and geranyl acetate (42%) as the main component, respectively, for
C. limon,
C. sempervirens,
M. alternifolia and
L. cubeba. These constituents are like those described for EO of these plants in other studies [
15,
18,
29,
35,
36,
37,
38,
39,
40,
41]. Terpene derivatives, a class that includes the mentioned constituents, are closely related to the antimicrobial biological action of these EO, as already demonstrated for
Candida spp. in other studies [
13,
15,
16,
41,
42,
43].
In the present study, the MIC varied according to the isolate and according to the EO, but the EO of
C. sempervirens and
L. cubeba presented the lowest MIC (500 µg/mL) for the same species (
C. krusei SV 03), while
M. alternifolia and clotrimazole combined (62.5–0.25 µg/mL) inhibited
C. albicans ATCC 90028 at lower concentrations than in isolation. In this sense, the ranges of results for the EO showed the effectiveness of plant-derived products in inhibiting microorganisms [
42,
43], a significant finding for the genus, given the recognized adaptive antifungal arsenal associated with
C. albicans and the intrinsic fluconazole resistance of
C. krusei [
9,
20,
44].
This variability of MIC can be observed in the literature [
9,
18] and is due to the characteristics of each isolate, which may be related to virulence factors and the origin of the isolate (blood, feces, respiratory tract or environment). In addition to this, storage and the constant activation and reactivation of cells, which occur in repeated cultures, may have generated adaptive changes in the phenotypic profile of the reference strains [
45].
The MFC were, on average, 2 × MIC for most isolates and EO, but for some, it was not possible to make this determination, as the values were greater than 4000 µg/mL, that is, greater than the limits of concentrations tested. Still, MIC and MFC of 2000 and 1000 µg/mL, respectively, were observed for
C. albicans ATCC 90028 when evaluated for the EO of
C. limon. This fact may be explained if the growth curve of cells in contact with this EO is evaluated, as it is possible that the fungicidal effect occurs through mechanisms that involve the depletion of some essential intracellular constituent for growth, such as ergosterol reserves, associated with other mechanisms of enzymatic inhibition, or action on the membrane or cell wall [
46] which is time-dependent, but other assays need to be performed to elucidate this finding.
The anti-
Candida activity of EO may be a direct result of the interaction of the various chemical components present and the association of different mechanisms, which may explain the fungistatic and fungicidal effects. The characteristics common to EO, such as lipophilicity and ability to cause damage to vital structures, membrane, and cell wall, result in increased membrane permeability and release of intracellular contents, with consequent death of
Candida spp. cells [
13,
37].
The fungistatic action of clotrimazole at low concentrations is due to structural changes in ergosterol; at high concentrations, it has a fungicidal effect [
6]. Thus, it can be assumed that the potentiation of the effects, demonstrated by the synergism observed in the association of clotrimazole with different proportions of EO, is the result of the multiplicity of mechanisms resulting from the various constituents of the EO, leading to the fungicidal effect [
47]. The activity of EO against biofilm [
10,
13,
17,
25,
26,
48,
49,
50,
51] is another factor that contributes to the need for studies that evaluate the combination of other drugs and a greater number of EO [
26,
47,
51,
52].
The application of a product with simultaneous inhibition of microbial growth and biofilm is advantageous since it allows for more efficient satisfactory results in different structures of Candida spp. The present study demonstrated that there was inhibition of biofilm formation and a reduction in the viability of the cells of previously formed biofilm, with MIC up to five times lower for the synergistic combination when compared to the same MIC found for the drugs evaluated alone. This study is the first, to our knowledge, to present a synergistic relationship of EO–EO and EO–clotrimazole on Candida spp., evaluating their action on biofilms.
The initial assessment of a substance, such as toxicity and antifungal activity, is a preliminary step in the design of new drug and health product candidates [
32]. Our study sought to evaluate the safety of EO and clotrimazole alone, as well as in combinations, exposed for 24 h to the in vivo model
C. elegans. It was observed that more than 80% of
C. elegans larvae survived at concentrations of 500 µg/mL for three of the evaluated EO. For
C. sempervirens, 80% of the larvae survived at the concentration of 4000 µg/mL, and for clotrimazole, the survival of 100% of the larvae was observed at all concentrations.
Among the EO evaluated, the biological activity of the EO of
C. limon and
M. alternifolia is better known when compared with those of
L. cubeba and
C. sempervirens [
18]. As observed in
Table 4, the LC
50 was not determined for the EO of
C. sempervirens (LC
50: >4000 µg/mL), suggesting that it is the least toxic for
C. elegans larvae among the four evaluated. Our study demonstrated that lethal toxicity of
L. cubeba EO against
C. elegans larvae was at 2000 µg/mL; however, lower concentrations such as 0.120–0.525 mg/mL (120–525 µg/mL) were found previously for the nematode
Bursaphelenchus xylophilus [
37]. In our study, we found lower toxicity of
C. sempervirens EO alone; however, it was moderate and high for other combinations (OE–OE and OE–clotrimazole). Some studies have provided other models for assessing toxicity by evaluating different cell cultures, showing that in vitro inhibitory concentrations (IC
50) for MCF-7 and MDA-MB-231 mammary tumor cells were lower than 34.5 and 65.2 μg/mL, respectively [
53], and that
C. sempervirens is lethal at higher concentrations in human promyelocytic leukemia strains (HL-60 and NB4) (LC
50: 333.79 to 365.41 µg/mL) [
38] and in experimental animal Ehrlich ascitic carcinoma (LC
50: 372.43 µg/mL) [
38]. In the larvae of
Culex quinquefasciatus, a non-vertebrate model and vector of filariasis, the LC
50 was 16.1 μg/mL after 24 h of exposure [
39].
The complexity of factors intrinsic to EO, such as the variability and concentration of chemotypes, which can vary in the same plant species according to the part of the plant used for extraction, region of cultivation and stage of development, may be, in part, responsible for the different results obtained in the same toxicity model used. In different models, this variability of constituents can be even greater, as can be seen in some studies [
12,
15,
18,
41,
49,
53]. Thus, it is suggested that toxicity is evaluated in different models to obtain evidence of greater safety and definition of the best drug concentrations that may have biological action and an absence or reduction of damage.
Our study focused on the preliminary assessment of EO–EO and EO–clotrimazole combinations, using concentration ranges applied predictively to planktonic cells and subsequently to biofilm and C. elegans after 24 h. Therefore, the totality of combinations that the checkerboard provides for the biofilm was not explored, nor was the influence of different exposure times of the substances for inhibition, eradication and toxicity. Our study used evaluation in the C. elegans model; therefore, it is important to evaluate correlation with the results in other models for a better understanding of the mechanism related to toxicity, including the use of EO in biocompatible pharmaceutical applications in nanosystems to improve aspects of physicochemical and biological agents against Candida spp.
The complexity of the composition of EO allows wide use in alternative and complementary medicine. The exploration of antimicrobial activity may enable new strategies and therapeutic alternatives for infectious diseases, especially mucocutaneous ones, where topical application is possible. The association of EO makes it possible for some constituents, even though they are not in the majority, to interact, enhancing or evidencing biological effects and reducing toxicity. In this context, studies still need to be carried out to determine the practical relevance of the combinations, better concentrations of each one of them, and the economic and market viability, in addition to the advantages over existing products.