Menthol Inhibits Candida albicans Growth by Affecting the Membrane Integrity Followed by Apoptosis

Inclusion of Candida albicans in the list of pathogens with potential drug resistance threat in recent years has compelled scientists to explore novel and potent antifungal agents. In this study, we have evaluated anti-Candida potential of menthol against different growth forms and synergistic potential with fluconazole. Menthol inhibited planktonic growth of all the isolates completely at ≤3.58 mM and killed 99.9% inoculum at MIC, indicating that menthol is fungicidal. Menthol inhibited hyphal form growth completely at 0.62 mM. It has inhibited developing a biofilm by 79% at 3.58 mM, exhibiting excellent activity against recalcitrant biofilms. FIC index values of 0.182 and 0.093 indicate excellent synergistic activity between fluconazole and menthol against planktonic and biofilm growth, respectively. Menthol enhanced rate of OxPhos among 22% cells; arrested 71% cells at G2-M phase of cell cycle and induced apoptosis in 15% cells. Thus, menthol exhibits excellent anti-Candida activity against differentially susceptible isolates as well as various growth and morphological forms of C. albicans. Menthol affects membrane integrity thereby inducing oxidative stress followed by cell cycle arrest and apoptosis. Considering the excellent anti-Candida potential and as it is Generally Recognized as Safe by the Food and Drug Administration, it may find use in antifungal chemotherapy, alone or in combination.


Introduction
e Candida species is the fourth most common pathogen responsible for life-threatening invasive candidiasis including candidemia and nosocomial infections in the form of biofilms exhibits very high mortality (40-50%) [1,2]. It is considered as difficult-to-treat infections especially among immunocompromised individuals [2,3]. Morphophysiological plasticity enables C. albicans cells to respond differentially towards host defense mechanisms and chemotherapeutic agents [2,3]. Candida species is included in the list of pathogen with the potential drug resistant threat by CDC, recently in consultation with other institutions, considering its current and projected impact in clinics and on economy [4].
us, considering the promise plantderived agents has shown against various infectious and noninfectious diseases, plant-derived anti-Candida agents are being explored on highest priority worldwide [5].
Menthol, a cyclic monoterpene alcohol, is one of the major active ingredients of peppermint (Mentha canadensis L) [6]. Peppermint plants are being used in traditional systems of medicine worldwide from centuries, long before identification of menthol in eighteenth century and were reported to exhibit various biological activities like antimicrobial, anticancer, immunomodulatory, antiinflammatory, antiviral, etc. [7,8]. Menthol was isolated and identified by Gambius (1771). It exists in L (+) or (−) forms naturally. It is one of the most frequently used flavoring agent in pharmaceuticals, cosmetics, pesticides, liqueurs, toothpastes, shampoo, tobacco, etc. [6,8]. Menthol exhibits various biological activities like it binds and activate TRPM8 (transient receptor potential cation channel) that leads to increase in calcium concentration and induce cold response, causes the vasodilation of cutaneous microvasculature and thus is being used as a very good enhancer of drug uptake in various topically applied analgesics [9]. Insecticidal activity of peppermint oil against Culex quinquefasciatus, Aedes aegypti, and Anopheles tessellatus was ascribed to menthol [10]. Derivatization of menthol into menthyl cinnamate and menthyl chloroacetate significantly enhanced the mosquitocidal activity of menthol [10].
Antimicrobial (antibacterial and antifungal) activity of menthol as well as synergistic activity with antibiotics, like oxacillin, norfloxacin, or erythromycin, against bacterial and fungal pathogens were reported in several recent studies [8]. However, the mechanism of antimicrobial activity is not understood. Trombetta et al. suggested that membrane solubilizing activity of monoterpene could be associated with antibacterial activities. Menthol exhibited excellent antifungal activity against various plant and human pathogenic fungi at very low concentrations [11,12]. Anticandida activity of menthol is reported recently and antifungal activity was found to be isomer-dependent, i.e., (+)-menthol and (−)-menthol were most effective ones exhibiting MIC 1.50 mM [13]. However, the mechanism of antifungal activity is not known. e present study is aimed at evaluating anti-Candida activity of menthol against different growth forms viz. planktonic, biofilm, and hyphae. We have also made an attempt to understand mechanism of anti-Candida activity by cell cycle, MMP, real-time qPCR, and apoptosis assay. Our results provide an insight into the mechanism of anti-Candida activity of menthol.

Candida albicans Isolates and Strains.
Fourteen clinical isolates and two strains of Candida albicans, differentially susceptible to fluconazole (FLZ) (Resistant-N � 6, S-DD-N � 02 and susceptible-N � 8) used in this study were isolated from sputum, blood, cerebrospinal fluid (CSF), ascitic fluid, pus and stool [14]. Clinical isolates were received from Swami Ramanand Teerth Culture Collection (SRTCC) of School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded (MS) India. A fluconazole-resistant strain of C. albicans ATCC 10231 [15,16], and a susceptible strain ATCC 90028 were procured from the Microbial Type Culture Collection, Institute of Microbial Technology (IMTECH), Chandigarh (India), and included as a quality control in this study. All the cultures were maintained on yeast extract peptone dextrose (YPD) agar slants at 4°C [16].

Inhibition of Planktonic Growth of Candida albicans
2.3.1. Inoculum Preparation. C. albicans cells grown in YPD for 24 h at 30°C were harvested, washed with sterile distilled water, and resuspended in phosphate buffered saline (PBS). Cell density was adjusted to 2 × 10 3 cells/ml, aseptically and used in following experiments.

Broth Microdilution Assay.
Anti-Candida activity of menthol was tested with different concentration (from 0.22 mM to 28.6 mM) against fourteen clinical isolates and two standard strains of C. albicans differentially susceptible to fluconazole (Resistant-N � 6, S-DD-N � 02 and susceptible-N � 8) by using a broth microdilution assay as mentioned previously with slight modification as per Wayne and akre et al. [16][17][18]. e lowest concentration required for complete growth inhibition (no visible growth) was determined as minimum inhibitory concentration (MIC) [18]. Triplicates were used for each concentration and the experiment was repeated thrice. 5 µl cultures from these wells were inoculated on YPD agar plates and number of colonies appeared on the agar plates after 48 h of incubation at 35°C were counted. Lowest concentration required for killing of 99.9% inoculums was considered as MFCs [16,17].

Time Kill Assay.
Time-dependent killing of C. albicans (ATCC 10231) inoculums by menthol at MFC was studied as described previously by Zore et al. and akre et al. [14,16,19]. In brief, 500 µl of C. albicans inoculum (prepared in YPD broth medium) containing 3.58 mM (MFC) of menthol was aliquoted in 1.5 ml vials. Vials were incubated at 35°C. Cells were harvested at different time intervals (0, 5, 10, 20, 40, 80, and 160 min), washed to remove any drug carryover and resuspended in 100 µl of PBS. 50 µl of these cells were inoculated on YPD agar plates and number of colonies appeared were counted after 48 h of incubation and compared with control plates. Triplicate vials were harvested after each time interval, vials without menthol were used as controls, and the experiment was repeated thrice. (ATCC 10231). Effect of menthol (28.6 to 0.44 mM) on seruminduced morphogenesis of C. albicans (ATCC 10231) was studied by using a microtiter plate-based morphological assay as done previously by Zore et al. and akre et al. [14,16,19]. In brief, 100 µl of inoculum (2 × 10 5 cells/ml) prepared in YPD broth-containing horse serum (20%) was distributed in each wells of a 96 well microtiter plate. Menthol (28.6 to 0.44 mM) was added to each well as a doubling dilutions and incubated at 37°C for 90 min. Cells of different morphological types (budded, unbudded, hyphae, and pseudohyphae) were counted after incubation microscopically using hemocytometer. Percentage inhibition of hyphae induction was calculated by comparing with control. Wells without menthol were used as controls. Each concentration was tested in triplicates and the experiment was repeated thrice. Triplicates were used for each concentration and experiment was repeated three times. MTT assay was performed as described previously by akre et al. [16,20].

Synergistic Assay.
e effect of menthol on fluconazole susceptibility in planktonically growing C. albicans (ATCC 10231) cells was studied by a microdilution checkerboard assay as done previously akre et al. and Odds [16,17,22]. Concentrations of menthol and fluconazole used were 0.22 to 7.16 mM and 2 to 128 mg/L, respectively. Similarly, the synergistic activity of fluconazole (≥256-8 µg/ml) and menthol (0.22 to 7.16 mM) was evaluated against development and maturation of C. albicans biofilms as done previously. Fractional inhibitory concentrations (FICs) and FIC index values were calculated for menthol as per Odds [22]. FIC is inhibitory concentration in combinations divided by the concentration that has same effect when used individually while FIC index values are sum of the FICs. e FIC index ( FIC) value shows the type of interaction between two compounds viz. value 0.5 to 4.0 shows additive interaction while a value less than 0.5 indicates synergistic interaction [22].

Mitochondrial Membrane Potential Assay.
Effect of menthol on mitochondrial membrane potential was evaluated using MitoTracker ® Red dye and Alexa Fluor ® 488 annexin V (Component A) in a flow cytometer as done previously by akre et al. [20]. In brief, C. albicans cells (5 × 10 6 cells/ml) grown for 24 h at 30°C were exposed to menthol (MIC50) for 6 h. After incubation, 1 μl of the MitoTracker ® Red dye (10 mM) was added to each well and incubated for 30 minutes at 37°C in an atmosphere of 5% CO 2 . Cells were washed with PBS and resuspended into 100 μl of 1X annexin-binding buffer. 5 μl of Alexa Fluor ® 488 annexin V (Component A) was added into each well and incubated for 15 min at room temperature. Finally, 400 μl 1X annexin-binding buffer was added in each well and stained cells were analyzed using a flow cytometer by measuring the fluorescence emission at 530 nm and 585 nm.

Cell Cycle Assay. Starved (1 h) cells of C. albicans
(2 × 10 3 cells/ml) were exposed to subtoxic concentration of menthol (1 mM) for 24 h at 30°C and used for cell cycle analysis using Attune flow cytometry (Invitrogen, USA) as described previously by akre et al. and Zore et al. [17,19]. In brief, inoculum of C. albicans treated with menthol (1 mM) for 24 h in YPD broth. After incubation, cells were harvested and washed twice using 1 ml of 50 mM Tris pH 7.8. ese cells were treated with 10 µg of RNase A in 500 µl of 50 mM Tris and incubated for 2 h at 37°C, followed by addition of protease (5 mg/ml pepsin in 0.05 M HCl) for 1 h at 25°C. ereafter, cells were harvested and resuspended in 500 µl FACS assay buffer (200 mM Tris/HCl pH 7.5; 200 mM NaCl; 78 mM MgCl 2 ). Finally, 15 µl of propidium iodide (1 mg/ml) was added and incubated for 30 min, and the cell cycle was monitored using flow cytometry.
2.5.6. Apoptosis Assay. Menthol (MIC50)-induced apoptosis in C. albicans cells was assessed after 24 h of incubation using Annexin V/Alexa Fluor ® 568 conjugate/PI binding and flow cytometry analysis as described previously by akre et al. [17]. Samples were used in triplicates and the experiment was repeated thrice.

Inhibition of Planktonic and Hyphal Form Growth.
Antifungal potential of menthol against different morphological (planktonic and hyphal) and growth (biofilm) forms of C. albicans isolates and strains (n � 16) is evaluated in present study. Among the morphological forms, the hyphal form was found more sensitive compared to planktonic growth. Menthol inhibited planktonic growth of all the clinical isolates and strains differentially susceptible to fluconazole (Resistant-N � 6, S-DD-N � 02 and susceptible-N � 8) tested in this study at or below 3.58 mM ( Table 2). Among the sixteen isolates and strains tested, eleven required 3.58 mM menthol for complete growth inhibition, while five were inhibited completely only at 1.79 mM ( Table 2).
Hyphal form growth was more sensitive to menthol than that of planktonic growth. Menthol inhibited serum (20%)induced hyphae induction completely at 0.62 mM concentration (Figures1(a)-1(c) and 2). To confirm whether menthol is fungistatic or fungicidal, MFC (minimum fungicidal concentration) assay was carried out. It showed that menthol was fungicidal at MIC for all the isolates and strains tested in this study (Table 2). To determine the time required for exerting fungicidal effect, a kill curve analysis of menthol at MFC was performed. Results of the kill curve analysis showed time-dependent killing of inoculums as MFC of menthol killed 99.9% inoculums within 160 min of exposure ( Figure 3).

Inhibition of Biofilm Growth.
Considering the excellent activity of menthol against planktonic and hyphal form growth, we have evaluated effect of menthol onstage biofilm growth at different stages, i.e., a developing and mature biofilm. e MIC of planktonic growth (3.58 mM) inhibited developing and mature biofilm by 79% and 55%, respectively ( Figure 4, Table 2). e developing biofilm was found to be more sensitive as 7.16 mM menthol caused 84% inhibition compared to 70% in case of a mature biofilm (Figure 4, Table 2). Doubling the concentration to 14.32 mM could achieve inhibition of 87% in case of developing and 75% in case of mature of biofilm growth and further increase in concentration could not increase the % inhibition, significantly ( Figure 4, Table 2). Our result suggests that menthol exhibit excellent activity against C. albicans biofilm.  (Table 2). e FIC index value of 0.182 indicates excellent synergistic activity of menthol with fluconazole against planktonic growth (Table 2). Similarly, menthol (1.79 mM) brought down MIC 90 of a developing biofilm and MIC 70 of a mature biofilm to 8 mg/L from 256 mg/L fluconazole (Table 2). e FIC index value of 0.093 indicates excellent synergy between fluconazole and menthol against recalcitrant biofilm growth of a fluconazole-resistant strain of C. albicans ATCC 10231 ( Table 2).

Mitochondrial Membrane Potential Assay.
Menthol is known to modulate membrane permeability and induce membrane damage stress. We have evaluated the effect of menthol on C. albicans mitochondrial membrane using a mitochondrial membrane potential assay. Our results showed that menthol (1 mM) at subtoxic concentration enhanced oxidative phosphorylation by 22.4% compared to 4% in control ( Figure 5).

Cell Cycle and Apoptosis Assay.
Membrane damage stress is reported to exert oxidative stress followed by cell cycle arrest and apoptosis. In this study, our cell cycle analysis suggests that at subtoxic concentration (1 mM), menthol arrests 71% cells in G2-M phase, compared to 57% in control (Table 3). It indicates an apoptosis-inducing potential. We have confirmed apoptosis inducing potential of menthol using apoptosis assay based on Annexin V Alexa Fluor ® 568 conjugate/PI binding and flow cytometry.

Real-Time qPCR Analysis of Selected Genes.
e expression analysis of three genes viz., KRE9, RPL11, and CDC37 was carried out using real-time qPCR to support our study. Out of the three, two were upregulated while one was downregulated as compared to control. e expression pattern of three genes is shown in (Figure 7).

Discussion
Menthol is a cyclic monoterpene alcohol found in peppermint (Mentha canadensis L) and exists in L (+) or (−) forms naturally. It is a major active principle and thus is reported to be associated with most of the biological activities of peppermint like antimicrobial, anticancer, immunomodulatory, anti-inflammatory, antiviral, etc., to cite a few [6][7][8]. Monoterpene molecules, in addition to multifarious biological activities, were reported to enhance membrane permeability and transdermal uptake of several drugs [23]. Menthol is considered as a better and safer enhancer of transdermal drug uptake than other monoterpene molecules as it does not induce convulsions in humans in response to overdose and thus being used as an adjuvant [23,24]. ough membrane destabilizing activity of monoterpenes is hypothesized to cause depolarization and inhibition of microbial growth, the mechanism of anti-Candida activity of menthol is not understood fully.

Evidence-Based Complementary and Alternative Medicine
Menthol demonstrated differential activity against different morphological and growth forms of C. albicans in our study. Menthol inhibited planktonic growth and hyphae induction completely at 3.58 and 0.62 mM concentrations, respectively, while 7.16 mM concentration could inhibit growth of a developing biofilm by 84% and mature biofilm by 70%, only (Table 2, Figure 4). e differential activity could be associated with membrane destabilizing activity of menthol. Membrane, in addition to encapsulating the cellular components, also plays a very important role in cellular signaling. Signaling pathways (MAPK, cAMP-PKA, and HOG1) regulate morphophysiology of C. albicans cells in response to environmental cues [3,25]. Membrane properties are highly dynamic and reported to depend on    morphological and growth forms of C. albicans [26]. Terpene-induced membrane integrity damage is reported to induce oxidative stress, enhance fluconazole uptake, and affect signaling pathways regulating hyphae induction, previously [14,16,17,19]. us, membrane destabilizing activity of menthol could be affecting signaling pathways, thereby inhibiting hyphae induction in C. albicans at very low concentration. e difference in efficacy of menthol could be due to the difference in bioavailability of menthol under planktonic and biofilm form growth [27]. Biofilms are made up of adherent microbial cells colonizing living surfaces or tissues embedded in an extracellular matrix [28]. It is a highly structured growth form composed of yeast, pseudohyphae, and hyphal cells and thus exhibits distinct properties, making biofilms recalcitrant to antifungal agents as well as Values shown were percentages of each quadrant. * p < 0.05, in comparison to control. Evidence-Based Complementary and Alternative Medicine immune responses [29]. Menthol inhibited biofilm growth significantly, i.e., more than 70%. Extracellular matrix, metabolic plasticity, and upregulation of drug efflux pumps are believed to confer resilience toward antifungal agents and immune responses [30,31]. e β-glucan present in extracellular matrix is reported to protect C. albicans cells from neutrophil attacks [32]. FIC index values suggest that menthol exhibits excellent synergistic activity with fluconazole against planktonic (0.182) and biofilm growth (0.093). Membrane-destabilizing activity of menthol could enhance bioavailability of fluconazole in a fluconazoleresistant strain used in our study and reduce MIC of  Figure 7: Expression analysis of selected genes of Candida albicans using real-time qPCR (KRE9, RPL11 and CDC37) in response to menthol. mRNA copy was normalized initially to GAPDH levels and later to the untreated control mRNA copy number. Data are expressed as mRNA copies in cells, where significance referrers to the difference between untreated control and treated cells (n � 3, * * p < 0.01, * p < 0.05). Bar indicates mean, and error bar indicates SD.
fluconazole against both planktonic and biofilm growth. As menthol is reported to enhance membrane permeability, we conclude that synergistic activity of menthol with fluconazole is a result of membrane destabilizing activity of menthol. Menthol is known for increasing membrane permeability and drug uptake; thus, it is being used as an adjuvants [8]. us, menthol may find use in antifungal chemotherapy alone or as an adjuvant and reduce side effects associated with higher or prolonged use of chemotherapeutic agents especially against drug-resistant isolates and difficult-to-treat infections like biofilms [33]. Membranedestabilizing activity of monoterpenes is also reported to induce leakage of intracellular components and inhibit microbial growth [11].
In addition to permeability, terpene-induced cell wall and membrane integrity damage is reported to exert oxidative stress in microorganisms including C. albicans [11,14,17,19]. Our real time qPCR analysis revealed that KRE9 was significantly upregulated in response to menthol compared to control (Figure 7). KRE9 gene encodes an Oglycoprotein involved in assembly of 1-6, beta glucan in the cell wall and was reported to be upregulated in response to limonene-induced cell wall and membrane damage [17]. Upregulation of KRE9 suggests menthol-induced cell wall and membrane damage. It was further supported by our MMP assay wherein, menthol modulated membrane potential ( Figure 5). Modulation of membrane potential by menthol is well documented; wherein, it was reported to accelerate inactivation of L-type (high-threshold) Ca 2+ currents (activated from a holding potential of −80 mV to positive potentials above −20 mV). Menthol inhibits Ca 2+ influx through the low voltage-activated Ca 2+ channel, and enhances the inactivation of the L-type (high voltage-activated) Ca 2+ channel [34,35]. Our MMP assay confirms increased membrane potential in C. albicans.
As terpene-induced membrane and cell wall damage is reported to generate ROS and thus oxidative stress that often induce DNA damage in C. albicans [17,19]. Upregulation of RPL11 in response to menthol indicates nucleolar stress. RPL11 is an indicator of nucleolar stress caused by DNA damage induced by oxidative stress. Our earlier studies have shown that oxidative stress caused by membrane and cell wall damage in C. albicans by limonene and piperine exerts nucleolar stress followed by cell cycle arrest and apoptosis [17,20]. Menthol downregulated CDC37 an essential chaperone reported to be involved in cell cycle regulation in our RT-qPCR analysis. CDC37 is reported to be indispensable for cell growth as heterozygous mutants were susceptible to chemicals while homozygous null mutants were inviable. us, downregulation of CDC37 by menthol confirms cell cycle arrest in C. albicans. It was further confirmed using cell cycle and apoptosis assay; wherein, menthol arrested C. albicans cells at G2-M phase and induced apoptosis.
In general, our data suggest that membrane-destabilizing activity of menthol is associated with differential inhibition of C. albicans growth (planktonic, biofilm, and hyphae) as membrane properties varies according to morphological forms. Modulation of mitochondrial membrane potential, overexpression of KRE9 (cell wall and membrane damage), RPL11 (nucleolar stress), and downregulation of CDC37 (cell cycle) genes confirm membrane damage stress inducing ROS followed by nucleolar stress, cell cycle arrest, and apoptosis. Demonstration of cell cycle arrest and apoptosis by flow cytometry-based assays further confirms that menthol inhibit C. albicans growth by destabilizing membrane causing oxidative stress that leads to nucleolar stress followed cell cycle arrest and apoptosis."

Conclusion
Our data suggest that menthol exhibits considerable anti-Candida activity against various growth forms alone or in combination with fluconazole. Based on our findings, we conclude that menthol-induced membrane and cell wall destabilization exert oxidative stress (ROS), leading to DNA damage followed by cell cycle arrest and apoptosis in C. albicans. Considering the excellent anti-Candida potential and as it is Generally Recognized as Safe (GRAS) by the Food and Drugs Administration (FDA), toxicity concern may not arise and thus menthol may find use in antifungal chemotherapy alone or as an adjuvant.

Data Availability
All our data were included in our MS in tables and figures.

Conflicts of Interest
e authors declare that they have no conflicts of interest.