Carvacrol (>99%, CAS: 499-75-2) was obtained from Weikeqi Biotechnology Co., Ltd. (Sichuan, China). Its chemical structure was shown in Figure 1. A stock carvacrol (494 mg/ml) was made by dissolving this compound in dimethyl sulfoxide (DMSO). The three strains of C. albicans used in this study were SC5314, ATCC18804, and 07318, and each was cultured at 30°C in yeast extract peptone dextrose (YPD) medium.

Microdilution methods were used to determine the minimal inhibitory concentration (MIC). Specifically, aliquots of 100 μl of C. albicans cell suspensions (1 × 10⁵ cells/ml) with different carvacrol concentrations were transferred into a series of 96-well plates. After a 48-h incubation at 30°C, the MIC of carvacrol against SC5314 strain was examined (Tian et al., 2017; Yun and Lee, 2017). This experiment was conducted in triplicate.

Overnight cultures were diluted in YPD medium to an OD₆₀₀ value of 0.1. After reaching the mid-exponential phase, C. albicans cells cultures were subjected to 3-h treatments with 247 and 494 μg/ml carvacrol. The treated cells were then collected, and about 10³ cells were coated onto plates of YPD agar. Following a 24-h aerobic incubation at 30°C, the numbers of colony forming units (CFUs) were determined. Percentage survival estimates were detected relative to the untreated control cells, and each experiment was conducted independently in triplicate.

Analysis of cell membrane integrity was conducted according to a modified version of the method described by Li et al. (2016). Specifically, after C. albicans cells were treated with carvacrol, they were collected, and stained for 30 min at 4°C with 10 mg/ml propidium iodide (PI) in darkness. Then, cell membrane integrity was assayed using a FACSCalibur flow cytometer (Becton Dickinson, United States).

DiBAC₄(3)(bis-(1,3-dibarbituric acid)-trimethine oxanol) was used as a membrane potential molecular probe to assess changes in plasma membrane potential as previously described (Li et al., 2016). In brief, C. albicans cells were treated, harvested, and washed thrice with phosphate-buffered saline (PBS). Next, the C. albicans cell suspension was incubated with 20 mg/ml DiBAC₄(3) at 37°C for 30 min in darkness. Depolarized C. albicans cells were examined by flow cytometry, and the percentage of depolarized cells was recorded.

The CaspACE FITCVAD-FMK in situ marker (Promega, Madison, WI, USA) was utilized to detect metacaspase activation (Tian et al., 2017). Specifically, carvacrol-treated C. albicans cells were collected and washed with PBS. Then, cells staining was conducted with CaspACE FITC-VAD-FMK at a 5 mM final concentration at 37°C for 20 min in darkness. Next, the stained cells were again washed with PBS and assayed using flow cytometry.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was utilized to examine C. albicans DNA fragmentation (Jia et al., 2018). Specifically, treated C. albicans were harvested, washed with PBS, and fixed for 30 min in 3.6% paraformaldehyde followed by a 2-min permeabilization on ice. Next, the cells were again washed before being stained with an in situ cell death detection kit at 37°C for 1 h in darkness. DNA fragmentation was finally assessed by flow cytometry.

To measure C. albicans reactive oxygen species (ROS) levels, the fluorescent probe 2′, 7′–dichlorofluorescein diacetate (DCFH-DA) was utilized. DCFH-DA can be cleaved by intracellular esterase into the membrane-impermeable agent DCFH, which ROS can then oxidize into its fluorescent derivative, DCF. Thus, the fluorescence intensity of DCF is an indicator of the total ROS levels (Jia et al., 2019). Accordingly, carvacrol-treated C. albicans cells were gathered, washed with YPD medium one time, and resuspended in 0.5 ml of YPD medium containing 10 mM DCFH-DA. Following a 30-min incubation in the dark, the fluorescence intensity of C. albicans cells was assessed using flow cytometry.

MitoSOX Red mitochondrial superoxide indicator was utilized to determine the mitochondrial ROS levels (Zhou et al., 2019). Specifically, carvacrol-treated C. albicans cells were collected, washed, stained with MitoSOX Red mitochondrial superoxide indicator at a final concentration of 5 μM, and incubated for 20 min at 37°C. After staining, cells were harvested and assayed using a flow cytometer.

To examine the mitochondrial transmembrane potential, 5,5′,6,6′-tetrachloro-1,1′,3, 3′-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) was applied (Jia et al., 2019). Carvacrol-treated C. albicans cells were gathered by centrifugation, twice washed with PBS, and then incubated for 20 min with 2.5 μg/ml JC-1 at 37°C in darkness. After the stained cells were washed again with PBS, they were assayed using a FACSCalibur flow cytometer. The mitochondrial membrane potential was then estimated as the ratio of the fluorescence intensity of JC-1 aggregates (FL2) to that of its monomer (FL1).

Rhod-2 AM and Fluo-3 AM were, respectively, used to examine the mitochondrial and cytosolic calcium levels (Tian et al., 2017; Jia et al., 2018). C. albicans cells were treated with 247 and 494 μg/ml carvacrol. Next, the carvacrol-treated C. albicans cells were centrifuged, collected, washed twice with Hank's balanced salt solution (HBSS), and resuspended in 500 μl of HBSS buffer. To assay mitochondrial calcium levels, the washed C. albicans cells were stained with 4 mM Rhod-2 AM for 30 min at 37 °C in the dark. To measure cytosolic calcium levels, cells were stained with 2 mM Fluo-3 AM for 40 min at 30°C in the dark. Next, the stained cells were washed again, resuspended in 600 μl of HBSS, and incubated for another 20 min at 30°C. The fluorescence intensities of Rhod-2 AM and Fluo-3 AM were then immediately detected with a flow cytometer.

Whether carvacrol can clear C. albicans infections was determined by infecting female Institute of Cancer Research (ICR) mice (25–30 g) with 100 μl of a 5 × 10⁶ C. albicans cells/ml normal saline suspension via tail vein injection. One hour post-infection, 200 μl of normal saline, 16 mg/kg carvacrol, and 32 mg/kg carvacrol were, respectively, administered via oral-gastric (OG) gavage, and this was repeated daily on days 2–5. Each group included six mice. Mice survival was assessed on day 10. Mice were sacrificed on the fourth day after infection in order to assess the fungal burdens in their kidneys, which were then homogenized. Fungal loads were examined by plating dilutions of the resulting homogenate onto YPD agar plates supplemented with 50 μg/ml ampicillin and 100 μg/ml streptomycin followed by a 2-days incubation at 30°C. To evaluate the toxicity of carvacrol, ICR female mice (25–30 g) were administrated with carvacrol at doses of 16 and 32 mg/kg body weight once daily for 5 days by OG gavage with each group comprising six mice, and the survival of mice was monitored over the course of 10 days. SPSS (Version 17.0) was used to conduct data analyses, with the Kaplan–Meier logrank test used to examine the statistical differences between groups, and significant P-values noted by asterisks.

To examine the effect of carvacrol on RAW264.7 cell viability, RAW264.7 cells (10⁵ cells per well) were incubated at 37°C and 5% CO₂ in 96-well plates for 24 h to facilitate cell attachment and spreading. Then cells were treated with 0.15625, 0.3125, 0.625, 1.25, or 2.5 μg/ml carvacrol for 24 h. Next, a CCK8 assay was conducted to assess cell viability.

To detect the anti-inflammatory activity of carvacrol, RAW264.7 cells were pretreated with carvacrol for 24 h before being stimulated with 1 μg/ml lipopolysaccharide (LPS) for 4 h. Next, cells were harvested, and their total RNA was extracted. Then, RT-PCR was used to determine the mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Table 1 showed the primers utilized in this study.

In this study, each experiment was independently conducted at least in triplicate. Data were expressed as mean ± standard deviation (SD) value, and were analyzed using SPSS version 17.0. All data generated did not deviate from a normal distribution. Thus, Duncan's Multiple Range tests following one-way analysis of variance were conducted to make comparison among three or more groups, and two-tailed unpaired Student's t-tests were used to make comparison between two experimental groups. Statistically significance was indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001).

The antifungal action of carvacrol against C. albicans was investigated. The MIC value of carvacrol against SC5314 strain was 247 μg/ml. To examine the effect of carvacrol on C. albicans survival, the CFUs assays were conducted after treatment with this compound for 3 h. The survival percentage was significantly decreased after carvacrol treatment (42.6 ± 2.7 and 21.4 ± 1.8% under 247 and 494 μg/ml carvacrol treatments, respectively) compared with the control (100 ± 6.2%; P < 0.05; and P < 0.01, respectively; Figure 1), indicating that carvacrol caused C. albicans cell death. To better demonstrate the antifungal activity of carvacrol against C. albicans, two clinical strains, ATCC18804 and 07318, were treated with carvacrol. Consistent with the above results, similar effects were observed in these clinical strains (data not shown).

Previous studies have reported that carvacrol can interfere with ergosterol biosynthesis, which ultimately affects plasma membrane integrity (Chami et al., 2005; Ahmad et al., 2011). To confirm the effect of carvacrol on C. albicans cell membranes, cell membrane integrity and potential were, respectively, analyzed using PI and DiBAC₄(3) staining. As shown in Figure 2A, PI staining analysis revealed a remarkably intense fluorescence signal in 247 μg/ml carvacrol-treated cells (18 ± 2%) compared with control cells (0.15 ± 0.08%, P < 0.05; Figure 2A), indicating that carvacrol disrupted C. albicans cell membrane integrity. More cells were stained by DiBAC₄(3) after 247 μg/ml carvacrol treatment (38.97 ± 2.27%) compared with the control cells (1.51 ± 0.5%, P < 0.05; Figure 2B). To further assess the results, ATCC18804 and 07318 plasma membranes were also examined. In line with SC5314 observation, carvacrol treatment increased cell membrane permeability and depolarization in the two clinical strains (data not shown). These data together revealed that carvacrol affected both cell membrane integrity and potential in C. albicans cells.

To further examine the mode of C. albicans cell death due to carvacrol, apoptosis-related parameters, including DNA fragmentation and metacaspase activation, were analyzed. As shown in Figure 3A, carvacrol treatment significantly increased the percentage of TUNEL-positive cells (29.1 ± 0.82 vs. 4.12 ± 0.48%, P < 0.05; Figure 3A), indicating that DNA fragmentation occurred in carvacrol-treated C. albicans cells. Moreover, metacaspase activity was also remarkably increased in C. albicans cells following carvacrol treatment (68.97 ± 1 vs. 1.72 ± 0.21%, P < 0.05; Figure 3B). These results demonstrate that carvacrol triggered C. albicans apoptosis. To confirm this finding, DNA fragmentation and metacaspase activity were also determined in the aforementioned clinical strains. As expected, the percentage of TUNEL-positive cells and metacaspase activity levels were obviously elevated in both clinical strains following carvacrol treatment (data not shown). Collectively, these results indicated that the antifungal action of carvacrol was realized by inducing apoptosis.

As is known, ROS play a key role in the induction and regulation of apoptotic processes. To examine the effect of carvacrol on ROS levels, the ROS indicator DCFDH-DA was utilized. Carvacrol addition significantly elevated ROS levels (18.67 ± 1.27%) compared with the control (1.55 ± 0.15%, P < 0.05; Figure 4A). Mitochondria are a major source of ROS production. Therefore, mitochondrial ROS levels were analyzed using MitoSOX Red, a mitochondrial superoxide indicator. Carvacrol treatment remarkably increased the ROS levels of mitochondria (75.68 ± 5.75% and vs. 2.74 ± 2.15%, P < 0.01; Figure 4B). Mitochondrial ROS production is associated with mitochondrial function. Thus, mitochondrial transmembrane potential was evaluated. As expected, mitochondria membrane potential was significantly decreased after treatment with carvacrol (1.52 ± 0.1 and vs. 3.56 ± 0.62%, P < 0.05; Figure 4C). Moreover, mitochondrial morphology was remarkably affected by carvacrol (Figure 4D). In addition, we also validated these results in the two clinical strains examined, and similar effects were observed in the carvacrol-treated clinical strains (data not shown). Together, these results indicate that carvacrol treatment disturbed mitochondrial functions.

Calcium is known to be essential in apoptotic processes (Pinton et al., 2008). Thus, Rhod-2 AM and Fluo-3 AM were, respectively, utilized to measure mitochondrial and cytosolic and calcium levels. In contrast to the control (2.57 ± 0.56%), there were significantly more Rhod-2 AM-positive carvacrol-treated C. albicans cells (10.68 ± 3%, P < 0.05; Figure 5B). Moreover, the percentage of Fluo-3 AM-positive cells was notably increased in carvacrol-treated cells (26.4 ± 1.87) compared with control cells (6.04 ± 0.22%, P < 0.05; Figure 5A). The two clinical strains responded similarly to SC5314 in terms of calcium changes following carvacrol treatment (data not shown). Thus, carvacrol treatment led to accumulation of both mitochondrial and cytosolic calcium levels in C. albicans cells.

Previous studies have reported that calcium can induce calcineurin activation, which induces calcineurin-mediated BAD dephosphorylation and, in turn, activation of the caspase-3 apoptotic cascade, ultimately thereby inducing apoptosis (Wang et al., 1999; Saito et al., 2000; Springer et al., 2000). Another study has also demonstrated that Ca²⁺ and its downstream calcineurin/Crz1p/CaMCA1 pathway are involved in H₂O₂-induced C. albicans apoptosis (Lu et al., 2010). Our above findings have demonstrated that calcium levels were increased after carvacrol treatment. Therefore, we anticipated that calcineurin might be involved in carvacrol-induced C. albicans apoptosis. To confirm this assumption, a calcineurin inhibitor, cyclosporine A (CsA), was applied to pretreat C. albicans for 2 h. Supplementation with CsA significantly rescued C. albicans growth (0.147 ± 0.006 vs. 0.088 ± 0.001, P < 0.05; Figure 6A), and notably inhibited metacaspase activation (30.45 ± 3.1 vs. 58.4 ± 4.26%, P < 0.05; Figure 6B), indicating that carvacrol predisposed C. albicans cells to apoptosis via calcineurin activation. To further substantiate that carvacrol induced C. albicans apoptosis via Ca²⁺/calcineurin pathway, the calcineurin mutant cmp1Δ/Δ, and calcium-scavenger BAPTA were used to observe metacaspase activity. As expected, BAPTA addition and CMP1 deletion significantly decreased carvacrol-mediated metacaspase activity (32.55 ± 2.5% for carvacrol treament plus CMP1 deletion and 37.15 ± 2% for carvacrol treament plus BAPTA addition vs. 55.44 ± 2.5% for carvacrol treatment only, P < 0.05; Figure 6C).

To assess the effect of carvacrol on in vivo C. albicans virulence, this compound was administrated in a mouse model of systemic candidiasis via oral-gastric (OG) gavage. Mice in the infection group died within 10 days, and 50 and 83.3% of mice treated with 16 and 32 mg/kg carvacrol, respectively, survived the entire experiment (^*P < 0.05 and ^**P < 0.01) (Figure 7A), indicating that carvacrol could be used to treat C. albicans infection. Evaluation of fungal burdens showed that carvacrol administration obviously decreased CFUs in kidney samples from the carvacrol-treated group compared with the control (infection) group (Figure 7B). All uninfected mice survived the entire experiment after administration with carvacrol alone (Figure 7C), indicating that this phytomolecule was non-toxic at doses of 32 mg/kg or less.

Previous studies have reported that carvacrol can modulate the inflammatory response (Du et al., 2016). To elucidate whether carvacrol eliminates C. albicans through regulating immunity in addition to its direct antifungal efficacy, we will first find a carvacrol concentration which was effective to promote macrophage viability and proliferation through observing its effects on RAW264.7 macrophage. A low concentration of carvacrol significantly promoted cell viability and proliferation (Figure 7D). Moreover, addition of carvacrol at a low concentration notably down regulated LPS-induced mRNA transcript levels of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β (Figure 7E). Taken together, these results indicated that carvacrol diminished C. albicans infection through both antifungal and immunomodulation activities.