In Vitro and In Vivo Inhibitory Activity of Limonene against Different Isolates of Candida spp.

Commensal yeast from the genus Candida is part of the healthy human microbiota. In some cases, Candida spp. dysbiosis can result in candidiasis, the symptoms of which may vary from mild localized rashes to severe disseminated infections. The most prevalent treatments against candidiasis involve fluconazole, itraconazole, miconazole, and caspofungin. Moreover, amphotericin B associated with prolonged azole administration is utilized to control severe cases. Currently, numerous guidelines recommend echinocandins to treat invasive candidiasis. However, resistance to these antifungal drugs has increased dramatically over recent years. Considering this situation, new therapeutic alternatives should be studied to control candidiasis, which has become a major medical concern. Limonene belongs to the group of terpene molecules, known for their pharmacological properties. In this study, we evaluated in vitro the limonene concentration capable of inhibiting the growth of yeast from the genus Candida susceptible or resistant to antifungal drugs and its capacity to induce fungal damage. In addition, intravaginal fungal infection assays using a murine model infected by Candida albicans were carried out and the fungal burden, histopathology, and scanning electron microscopy were evaluated. All of our results suggest that limonene may play a protective role against the infection process by yeast from the genus Candida.


Introduction
Candidiasis, caused by the opportunistic fungus Candida albicans, is one of the most prevalent infections that affect humans and can display severe morbidity in immunosuppressed individuals [1]. In fact, hospitalized patients infected with HIV/AIDS (human immunodeficiency virus/Acquired immunodeficiency syndrome) or chronic obstructive pulmonary disease or undergoing chemotherapy treatments and transplant patients represent the most relevant groups at risk of developing Oropharyngeal Candidiasis (OPC) [2,3].
Isogenic female BALB/c mice (six animals per group) that were six to eight weeks old were housed in polypropylene cages under Specific-Pathogen-Free (SPF) conditions with a sawdust substrate. Food and water were provided ad libitum and all mice environments were sterilized. Animals used in this study were bred at the University of São Paulo animal facility. All experiments involving animals were conducted and approved by the Ethics Committee of the University of São Paulo (No. 042-127-02) and were conducted in accordance with international recommendations.
To set up the vulvovaginal candidiasis model, it was necessary to induce the female mice to a pseudo-oestrus phase by the subcutaneous administration of 0.5 mg of 17 beta-estradiol valerate (Sigma Chemicals, St Louis, MO, USA) dissolved in sesame oil (Sigma Chemicals, St Louis, MO, USA) three days before vaginal infection, as described by Hamad et al. [24].

Antimicrobial Compounds
(R)-(+)-limonene was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments. To carry out in vivo assays, we prepared a 10% (308.28 µM) limonene topical formulation, adding the compound to commercial neutral cream (10% Wax self-nonionic emulsifier, 2% mineral oil, 5% propylene glycol, and 84% distilled water). Fluconazole (Pfizer Inc., New York, NY, USA) was dissolved in PBS solution to obtain a concentration of 10 mg/kg and then mixed with the same neutral cream described above.

Conditions of Limonene Exposure for Growth Inhibition Assays
Limonene was added to a 30 mL suspension of C. albicans and non-albicans yeast cultures (OD 600 = 0.4) at final concentrations of 25, 50, 250, and 500 µM (1% DMSO concentration). Cultures were then incubated under the conditions described above, for 12 h, and fungal growth was monitored hourly, through OD 600 measurements. All experiments were performed in triplicate.

MTT Assay
The viability of C. albicans (1 × 10 6 ) after treatment with limonene was tested in vitro by cultivating cells in increasing concentrations of limonene (0 µM, 25 µM, 50 µM, 100 µM, 250 µM, 500 µM, and 600 µM) for 8 and 17 h. The cell viability was determined by measuring the cleavage of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Aldrich, St. Louis, MO, USA). After incubation with limonene, 50 µL of MTT solution (5 mg/mL) was added and cultures were further incubated, at 37 • C, for 5 h. Next, samples were centrifuged at 10.000 rpm for two minutes, the MTT solution was carefully removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to solubilize the produced formazan, prior to absorbance measurements at 570 nm. Assays were performed in triplicate, and results are expressed as the mean percent reduction of cells, compared to untreated controls.

Intravaginal Infection of BALB/c Mice
The C. albicans ATCC 90028 virulent strain was grown in brain heart infusion (Hi-Media Laboratories, Mumbai, India), at 37 • C, for 24 h, under 200 rpm agitation. Afterward, yeasts were centrifugated, washed in PBS (pH 7.2), and analyzed to determine the cell viability. Six animals per group were inoculated intravaginally with a maximum volume of 10 µL of 3 × 10 5 yeast cells, as described by Muñoz et al. (2017) [25].

Assay for Colony-Forming Units (CFUs)
BALB/c female mice were infected intravaginally with the virulent C. albicans strain ATCC 90028 (3 × 10 5 yeast cells) and submitted to limonene or fluconazole treatment 24 h post-infection. Treatment was continuous for seven days, every 24 h. On the 8th day, mice were sacrificed, and the vaginal canal was removed, weighed, and homogenized in PBS. A 100 µL sample of this suspension was plated on solid brain heart infusion medium (BHI), supplemented with 10 IU/mL streptomycin/penicillin (Cultilab, São Paulo, Brazil), and 500 µg/mL cycloheximide (Sigma, St. Louis, MO, USA). The petri dishes were then incubated at 37 • C, for 24 h, and colonies were counted (1 colony = 1 CFU).

Histopathology
The vaginal canal was removed and fixed in 10% neutral buffered formalin for subsequent paraffin embedding. Sections (5 mm thick) were stained with silver (Grocott stain) and examined microscopically at 100× magnification (Optiphot-2, Nikon, Tokyo, Japan).

Transmission Electron Microscopy (TEM)
C. albicans ATCC 90028 yeasts untreated (control group) or treated with 500 µM limonene for 24 h at 37 • C, in RPMI 1640 and buffered with 0.16 M MOPS were harvested in log phase and fixed at room temperature, for two hours, with 2.5% glutaraldehyde and 4% paraformaldehyde in PBS. After this process, yeasts were incubated in 1% osmium for 2 h, dehydrated, and embedded in araldite-Epon, as previously described [26]. Ultrathin sections were collected on grids stained with alcoholic 1% uranyl acetate and lead citrate. The grids were analyzed using a Jeol 100 CX (Tokyo, Japan) transmission electron microscope.

Scanning Electron Microscopy (SEM)
Vaginal canals from untreated or limonene-treated mice were excised and then fixed in 2.5% glutaraldehyde, for 1 h, at room temperature. Afterwards, samples were transferred to a critical point drier (BAL-TEC CPD-030-Electron Microscopy Sciences, Buffalo Grove, IL, USA). Sample assembly was conducted as previously described [26] and observed using an EM Jeol 100 CX transmission electron microscope.

Statistical Analysis
Statistical analysis was conducted using GraphPad Prism, version 7.05 software (GraphPad Software, San Diego, CA, USA). Results were expressed as means ± the standard deviations (SD) of the indicated numbers of animals or experiments. Statistical comparisons were conducted by analysis of variance (one-way ANOVA) followed by nonparametric Tukey's test. p Values of ≤0.05 and ≤0.01 indicated statistical significance. Conditions of limonene exposure for the growth inhibition assay were performed in triplicate, and the graphs show the average values and their respective standard deviations. Figure 1 shows the under-curve area values of the growth patterns of the different strains exposed or not exposed to the limonene treatment for 12 h. The one-way ANOVA was carried out followed by a nonparametric test.

Evaluation of the Antifungal Activity of Limonene In Vitro
The time-dependent growth of C. albicans ATCC 90028, exposed to different concentrations of limonene (0 µM, 25 µM, 50 µM, 250 µM, and 500 µM), for a period of 12 h, showed inhibition at concentrations of 250 and 500 µM ( Figure 1). Moreover, we observed a significant decrease in the cell viability of C. albicans when yeasts were previously treated with limonene, at 500 µM and 600 µM concentrations, for a period of 8 h ( Figure 2). The calculated EC50% was 444 ± 35 µM.   Limonene antifungal activity using 250 and 500 µM was analyzed in ATCC strains of C. albicans, C. krusei, C. glabrata, C. parapsilosis, and in 10 additional C. albicans clinical isolates. In the case of the three non-albicans species, a significant growth decrease was observed compared to the control group that did not receive limonene treatment. The clinical strains (n = 10) analyzed showed a less marked, but statistically significant, growth decrease, as shown in Figure 3.
Limonene antifungal activity using 250 and 500 µM was analyzed in ATCC strains of C. albicans, C. krusei, C. glabrata, C. parapsilosis, and in 10 additional C. albicans clinical isolates. In the case of the three non-albicans species, a significant growth decrease was observed compared to the control group that did not receive limonene treatment. The clinical strains (n = 10) analyzed showed a less marked, but statistically significant, growth decrease, as shown in Figure 3.

Treatment with Limonene Reduces the Fungal Burden in Mice with Vaginal Candidiasis
The number of CFUs in the vaginal canal of mice that received 10% (308.28 µM) limonene was significantly reduced compared to the control (infected but not treated) group. Moreover, we observed a decrease in the number of CFUs in the limonene-treated animals, when compared with the group of animals treated with fluconazole ( Figure 4).

Treatment with Limonene Reduces the Fungal Burden in Mice with Vaginal Candidiasis
The number of CFUs in the vaginal canal of mice that received 10% (308.28 µM) limonene was significantly reduced compared to the control (infected but not treated) group. Moreover, we observed a decrease in the number of CFUs in the limonene-treated animals, when compared with the group of animals treated with fluconazole ( Figure 4).

Histopathology of the Vaginal canal in Treated, Intravaginally Infected, BALB/c Mice
The vaginal canal of the untreated control animals showed multiple yeast-like cells ( Figure  5A). In contrast, the animals that received treatment with 10% (308.28 µM) limonene displayed significantly reduced numbers of yeast cells and large areas of preserved vaginal tissue ( Figure 5B).

Histopathology of the Vaginal canal in Treated, Intravaginally Infected, BALB/c Mice
The vaginal canal of the untreated control animals showed multiple yeast-like cells ( Figure 5A). In contrast, the animals that received treatment with 10% (308.28 µM) limonene displayed significantly reduced numbers of yeast cells and large areas of preserved vaginal tissue ( Figure 5B).

Transmission Electron Microscopy (TEM)
C. albicans ATCC 90028 cells treated with different concentrations of limonene displayed a marked decrease in cell density, as fewer cells were detected within the microscope's field when compared to the untreated controls. A total of 48% of the yeasts treated with 500 µM of limonene showed morphological modifications such as cell wall disruption and significant intracellular damage when compared to the untreated control group ( Figure 6).

Transmission Electron Microscopy (TEM)
C. albicans ATCC 90028 cells treated with different concentrations of limonene displayed a marked decrease in cell density, as fewer cells were detected within the microscope's field when compared to the untreated controls. A total of 48% of the yeasts treated with 500 µM of limonene showed morphological modifications such as cell wall disruption and significant intracellular damage when compared to the untreated control group ( Figure 6).

Scanning Electron Microscopy (SEM)
Yeast cells could be easily detected by SEM in the vaginal canal of animals inoculated with C. albicans without limonene treatment ( Figure 7A), but not in the vaginal canal of animals infected and treated with 500 µM of limonene. However, we observed some epithelial irritation signs, as evidenced by the presence of squamous cells ( Figure 7B).

Scanning Electron Microscopy (SEM)
Yeast cells could be easily detected by SEM in the vaginal canal of animals inoculated with C. albicans without limonene treatment ( Figure 7A), but not in the vaginal canal of animals infected and treated with 500 µM of limonene. However, we observed some epithelial irritation signs, as evidenced by the presence of squamous cells ( Figure 7B).

Discussions
In the present work, we evaluated the antifungal activity of R-(+)-limonene against clinical and ATCC isolates of Candida spp. susceptible or resistant to antifungal drugs. R-(+)-limonene is largely used in food, beverage, and cosmetic industries and is classified as a low-toxicity additive [27]. The efficacy of limonene as an anti-diabetic, anticarcinogenic, and antimicrobial agent has also been

Discussion
In the present work, we evaluated the antifungal activity of R-(+)-limonene against clinical and ATCC isolates of Candida spp. susceptible or resistant to antifungal drugs. R-(+)-limonene is largely used in food, beverage, and cosmetic industries and is classified as a low-toxicity additive [27]. The efficacy of limonene as an anti-diabetic, anticarcinogenic, and antimicrobial agent has also been evaluated with relative success [27].
In vivo and preclinical models identified that the oral administration of R-(+)-limonene was absorbed from the rat gastrointestinal tract by diffusion (reviewed by Chandrakala et al., 2018) and also by humans with little toxicity [28]. These data are important considering the small amount of R-(+)-limonene used here in the intravaginal treatment compared with the oral treatment. Future studies may be carried out to analyze the possible absorption of R-(+)-limonene by the vaginal mucous.
To define the best concentration of R-(+)-limonene able to interfere with the growth of yeast cells, a kinetic analysis was performed and our results confirmed that limonene at concentrations ≥500 µM can inhibit the growth of C. albicans, C. krusei, C. glabrata, and C. parapsilosis in vitro, reinforcing previous observations made by Freire and collaborators (2017), who originally evaluated the antimicrobial properties of limonene-containing essential oils, derived from Mentha piperita L., Origanum vulgare, and Zingiber officinalepara [29]. Interestingly, all these oils were capable of exerting a fungicidal effect on Candida strains (obtained from dental prostheses), inducing morphological alterations in yeast cells and inhibiting pseudohyphae formation [29]. Similarly, Leite and coworkers (2014) detected that C. albicans yeasts treated with citrus essential oils, containing several substances structurally related to limonene, also displayed morphological alterations and decreased chlamydoconidia production, which directly affected the pathogenicity of the treated yeasts [30].
In addition to inhibiting growth, we assessed the ability to interfere with the viability of yeast. We observed that the viability of the yeasts treated with limonene in concentrations ≥500 µM was significantly reduced. Previous studies described that the use of this monoterpene in concentrations between 0.6 to 5 mM induces apoptosis in C. albicans, due to the damage caused to both the cell wall and membrane. In that study, the authors claimed that cell death was likely due to oxidative stress produced by limonene, which leads to DNA damage, alterations of the cell cycle, and, finally, yeast apoptosis [31].
Mondello and collaborators (2003) employed a rat vulvovaginal candidiasis model to demonstrate that the essential oils of Malaleuca alternifolia (composed of a complex mixture of monoterpenes) were protective against C. albicans strains either susceptible or resistant to fluconazole and/or itraconazole [32]. Cases of recurrent vulvovaginal candidiasis, mainly caused by C. albicans, have drawn particular attention from the medical community over the past years, given their high prevalence and challenging treatment. It has been described that the difficulty in treating this mycosis is directly associated with a variety of factors, including the presence of drug-resistant Candida strains (mostly non-albicans species), overall predisposition of patients to infection, use of antibiotics, uncontrolled diabetes, estrogen use, pregnancy, a host's genetic factors, and alterations in the vaginal microbiota, among others [33][34][35].
To the best of our knowledge, the present work represents the first example of a successful R-(+)-limonene-based treatment of vulvovaginal candidiasis in mice. Moreover, our studies lead us to verify that limonene also exerts a protective role against C. albicans infections, since animals that were submitted to intravaginal limonene treatments showed a significant decrease in the fungal burden when compared to untreated animals.
Transmission electron microscopy studies performed on Candida yeasts showed damage to the cell wall, as well as to intracellular structures, including nuclear alterations, such as the condensation of genetic material and specific changes in mitochondria. Moreover, yeasts treated with limonene also displayed dramatic structural changes in organelles, accompanied by cell wall rupture in most of the observed fields. These alterations may have derived from the action of limonene on the yeast genetic material, as described by Thakre and collaborators (2018). Recent work has also demonstrated that monoterpenes can interfere with the permeability and fluidity of plasma membranes and can inhibit efflux pumps, possibly creating permanent membrane pores, resulting in cytoplasmic leakage and irreversible cellular damage [36].
Finally, scanning electron microscopy analyses showed distinct alterations in the appearance of the vulvovaginal canal epithelium, possibly due to an irritation process. This observation conflicts with observations made by Arruda and coworkers (2009), who carried out cytotoxic assays using R-(+)-limonene at concentrations reaching up to 1.012 µM, which resulted in no observable cytotoxic effect on monkey epithelial cells (LLCMK2) or human cells (HEK-293). Another important aspect is that R-(+)-limonene did not induce mutagenic, carcinogenic, or nephrotoxic effects in humans [37]. In mice, the oral LD50 for limonene was reported to be 5.6 g/kg body weight in males and 6.6 g/kg body weight in females [37].
Since vulvovaginal candidiasis is an infection that affects up to 70% of all women, at least once during their reproductive years, and given the fact that approximately 10% of these cases may turn into recurrent vulvovaginal candidiasis, it is important to search for new therapeutic alternatives to treat this pathology [33]. In this sense, the present study strongly suggests that topical R-(+)-limonene has significant therapeutic benefits in experimental vulvovaginal candidiasis.