Citrus hystrix DC leaves powder obtained from Khaolaor Company (Samut Prakan, Thailand) was macerated in three different solvents sequentially, as previously described (Buakaew et al., 2021a,b). Crude hexane, crude ethyl acetate, and crude ethanol extracts were obtained. Citronellol, the active compound was identified from C. hystrix leaves extract (Buakaew et al., 2021a,b). For in vitro experiments, citronellol was purchased from Sigma-Aldrich Company (St. Louis, MO, United States). They were stored at 4°C for future experiments.

C. albicans ATCC 90028 was used as the model. Yeast inoculum was prepared from 24 h colonies on Sabouraud dextrose agar (SDA: HiMedia Laboratories, Mumbai, India). The cell suspension was adjusted in different broth media using a spectrophotometer (A₆₀₀nm) at a final concentration of 10⁶ cells/ml for cytotoxicity and biofilm formation assays. For proteomic analysis and other methods, yeast cells were adjusted to 10⁷ cells/ml for final concentration.

A broth microdilution test was carried out to determine the minimal inhibitory concentration (MIC) value of all crude extracts and β-citronellol according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A2 with some modifications. In brief, 100 μl of 2-fold serial dilution of crude extracts (0.20–100 mg/ml) and β-citronellol (2–1,024 μg/ml) in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, United States) were added to a 96-well plate. The suspension of C. albicans was adjusted at 530 nm to match the turbidity of 0.5 McFarland standard. Then, 10 μl of yeast cell suspension was added to each well of pre-diluted crude extracts and β-citronellol to reach the final concentration of 0.5–1 × 10³ cells/ml. The cells were then incubated at 35 ± 2°C for 48 h. After incubation, 100 μl of resazurin (0.02 mg/ml) was added and further incubated for 3 h at 35 ± 2°C. A change in color was observed and wells with no change in color (the blue color of resazurin remained unchanged) were evaluated as MIC values. While the spread plate technique was used to establish the minimum fungicidal concentration (MFC). Ten microliter of cell suspension was spread on SDA and incubated at 35 ± 2°C for 48 h. The MFC was determined based on the concentration in the absence of colony growth.

To determine the effect of β-citronellol on yeast growth, the cells were incubated in 100 μl of Sabouraud dextrose broth (SDB: HiMedia Laboratories, Mumbai, India) containing different concentrations of β-citronellol in a 96-well plate. The plate was incubated at 35 ± 2°C for 48 h in a multidetector microplate reader. The turbidity was measured at OD₆₀₀nm every 2 h.

To prepare C. albicans treated with β-citronellol for proteomic analysis, the yeast cells were incubated with β-citronellol (128 μg/ml) in SDB for 24 h. The treated cells were washed twice with 1× phosphate-buffered saline (PBS) pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄). After that, the cells were resuspended with lysis buffer [1% SDS, 5 mM dithiothreitol (DTT) in 0.1× PBS pH 7.4 (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na₂HPO₄, and 0.147 mM KH₂PO₄) with 1× protease inhibitor cocktail], sonicated for 30 s and rested on ice for 30 s × 5 rounds. Then, the cells were centrifuged at 12,000 RPM, 4°C for 20 min. Proteins extracted were precipitated using ice-cold acetone and stored at −20°C for 16 h. After precipitation, the protein pellet was reconstituted in 0.25% RapidGest SF (Waters, United Kingdom) in 10 mM ammonium bicarbonate. The protein concentrations were determined by the bicinchoninic acid assay kit (Pierce, New York, NY, United States), using bovine serum albumin as the standard. Then, 25 μg of protein were subjected to gel-free based digestion. Reduction of sulfhydryl bonds was performed using 5 mM DTT, with incubation at 80°C for 20 min. Alkylation of sulfhydryl groups was performed using 25 mM iodoacetamide (IAA) at room temperature for 30 min in the dark. The solution was cleaned-up using a 7 kDa molecular weight cut-off, 0.5 ml (Zeba™ Spin Desalting Columns, Thermo Fisher, United States). Proteolytic digestion using 500 ng of sequencing grade trypsin (Promega, Germany) was added to the protein solution and incubated at 37°C for 3 h. The peptides were resolubilized in 0.1% formic acid/LC–MS water before transfer to a TruView LC-MS vial (Waters, United Kingdom).

To investigate the protein expression profiles of C. albicans, the peptide mixtures were analyzed in data-dependent mode on tandem mass spectrometers using an Orbitrap HF hybrid mass spectrometer combined with an UltiMate 3000 LC system. Briefly, the peptides were first desalted on line on a reverse-phase C18 PepMap 100 trapping column, before being resolved onto a C18 PepMapTM 100 capillary column with a 150-min gradient of CH₃CN, 0.1% formic acid at a flow rate of 300 nl/min. Peptides were analyzed by applying a data-dependent Top15 method consisting of a scan cycle initiated by a full scan of peptide ions in the Orbitrap analyzer, followed by high-energy collisional dissociation and MS/MS scans of the 15 most abundant precursor ions. Full scan mass spectra were acquired from m/z 400–1,600 with an AGC target set at 3 × 10⁶ ions and resolution of 60,000. The MS/MS scan was initiated when the ACG target reached 10⁵ ions. Ion selection was performed by applying a dynamic exclusion window of 12 s.

Raw files were analyzed by Proteome Discoverer software version 2.4 (Thermo Scientific) using the SEQUEST, Percolator and Minora algorithms. The LC-MS spectrum was matched against the UniProt C. albicans reference proteome (UP000000559; downloaded 08/08/2021; 6,035 entries). For protein identification and quantification, the setting parameters were as follows: a maximum of two trypsin missed cleavages were allowed with a precursor mass tolerance of 5 ppm and fragment mass tolerance of 0.01 Da. Carbamidomethylating +57.021 Da (cysteine) and oxidation +15.995 Da (methionine) were chosen as static modifications and oxidation +15.995 Da (methionine) were chosen as dynamic modifications. The false discovery rate (FDR) for peptide and protein identification was set to 0.05 in both cases. Normalization of the relative protein abundance ratio was performed by the total peptide amount for each LC-run (across all runs; n = 6) by the normalization algorithm of Proteome Discoverer software. To assemble the differentially expressed protein list, multiple consensus workflows were used within the Proteome Discoverer software to assemble the PSMs into peptide groups, protein database matches and finally non-redundant protein groups using the principle of strict parsimony as defined by the vendor software defaults. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD030379 and 10.6019/PXD030379.

To determine the effect of β-citronellol on biofilm formation, C. albicans biofilm formation assay was performed according to the protocol previously described (Gulati et al., 2018). Briefly, 48 h of C. albicans colonies on SDA were adjusted in RPMI-1640 to obtain 10⁶ CFU/ml. Then, 100 μl of yeast was added to a flat-bottom cell culture 96-well plate (Wuxi NEST Biotechnology Co., Ltd., Jiangsu, China) and incubated at 35 ± 2°C for 1.5 h with constant shaking at 250 RPM. The plate was washed once with PBS to remove non-adherent cells. To determine the effects of active compounds, 100 μl of different concentrations of citronellol were added and incubated at 35 ± 2°C for 24 h. The cells treated with 2 μg/ml of amphotericin B were served as positive control. After incubation, biofilm from C. albicans was rinsed three times with PBS to eliminate non-adherent cells. The plate was air-dried for 45 min at room temperature. Then, yeast biofilm was stained with 0.4% crystal violet for 45 min and washed three times with PBS. To measure the absorbance, 200 μl of 95% ethanol was added to destain crystal violet for 45 min and 100 μl of each well was transferred to a new plate. The absorbance was measured at OD₅₉₅nm using a microplate reader.

To determine the production of reactive oxygen species (ROS), cell were treated with β-citronellol and the level of ROS was measured using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA; Invitrogen, Waltham, MA, United States) following the manufacturer’s protocol. C. albicans (10⁶ cells/ml) was incubated at 35 ± 2°C for 4 h with different concentrations of β-citronellol in SDB. Yeast cells treated with 2 mM hydrogen peroxide (H₂O₂) in SDB was used as positive control in this experiment. After incubation, cells were collected by centrifuging at 12,000 RPM for 5 min and washed once with PBS. Cells were incubated with 10 μM H₂DCFDA in PBS at 35 ± 2°C for 30 min in the dark. Then, cells were washed once with PBS and resuspended in the same buffer. One hundred microliter of cell suspensions were transferred to a 96-well fluorescence plate. The fluorescence intensity was measured at excitation/emission at 485/535 nm.

To determine the effect of β-citronellol on yeast membrane potential, DiBAC₄(3) [Bis-(1,3-dibutylbarbituric acid) trimethine oxonol; Invitrogen, Waltham, MA, United States] was used to determine the cell membrane potential as described by Yang et al. (2019). Briefly, yeast cells (10⁶ cells/ml) were prepared in SDB, then incubated with diluted β-citronellol at 35 ± 2°C for 4 h. For positive control group, yeast cells were treated with 2 μg/ml of amphotericin B. The cells were harvested by centrifuging at 12,000 RPM for 5 min and washed once with PBS. After that, yeast cells were incubated at 35 ± 2°C in the dark with 20 μg/ml DiBAC₄(3) in PBS. After incubation, the cells were centrifuged and resuspended in PBS and transferred to a 96-well fluorescence plate. Fluorescence intensities were measured with a fluorescence spectrophotometer at excitation/emission wavelengths at 492/518 nm.

To determine the effect of β-citronellol on inducing programmed cell death by apoptosis, protoplast preparation was conducted following a previously described protocol (Lone et al., 2020) and protoplast buffer preparation (Khan et al., 2014). Briefly, cells exposed with various concentrations of β-citronellol (64, 128, and 256 μg/ml) for 6 h were washed twice with protoplast buffer-I (1 M sorbitol, 30 mM DTT, 50 mM Tris base, and 10 mM MgCl₂), then incubated in the same buffer at 25°C for 15 min, followed by centrifugation at 12,000 RPM for 5 min. The pellets were resuspended with protoplast buffer-II (1 M sorbitol, 1 mM DTT, 50 mM Tris base, and 10 mM MgCl₂) supplemented with lyticase enzyme (≥1 μg/ml; Sigma-Aldrich, MO, United States) and incubated at 25°C for 1 h. Then, the cells were centrifuged to remove buffer-II, followed by incubation in protoplast buffer-III (1 M sorbitol, 50 mM Tris base, and 10 mM MgCl₂) at 25°C for 20 min. After incubation, cells were centrifuged, and supernatants were discarded and washed once with PBS. To examine the apoptosis phenotype, the Muse™ Annexin V & Dead Cell Kit (Merck, Darmstadt, Germany) was used. The protoplasts were resuspended in SDB with 1% fetal bovine serum (FBS) and 100 μl of protoplasts were mixed with 100 μl of the Muse™ Annexin V & Dead Cell Kit. The mixture was incubated at room temperature for 20 min in the dark. After that, yeast cells were analyzed using Muse™ Cell Analyzer following the manufacturer’s protocol.

To study the effect of β-citronellol on C. albicans morphology, tested yeast cells was observed using Scanning Electron Microscopy (SEM) according to a previously described protocol (Fischer et al., 2012). The cells were incubated with various concentrations of β-citronellol at 35 ± 2°C for 4 h, and then the suspension was centrifuged at 12,000 RPM for 5 min. The pellets were resuspended in PBS and 50 μl of cells were dropped on a glass slide and incubated at room temperature for 30 min. After that, 0.5 ml of primary fixative (4% paraformaldehyde) was added, followed by incubation at room temperature for 1 h. The cells were washed three times (2 min each), then post-fixed with 1% OsO₄ at room temperature for 1 h. The post-fixed cells were washed once with PBS and dehydrated using 25, 50, 75, 95, and 100% ethanol. Then, the cells were dehydrated using 1:1 hexamethyldisilazane (HMDS): ethanol for 15 min, followed by 100% HMDS for 15 min. The excess volume of solution was wiped away, and the cells were dried at room temperature for 4 h. Then, the cells were gold-covered by cathodic spraying, and the morphology of yeast cells was observed 5,000× under Apreo 2 SEM (Thermo Scientific) in high vacuum mode at 5 kV.

Statistical analyses of all data were performed using GraphPad Prism version 8.01 (GraphPad Software, San Diego, CA, United States). A one-way analysis of variance (ANOVA) was used to determine the variables significance, with comparison of means by Tukey’s range test. All tests were performed in triplicate, and data were expressed as mean ± standard error. The p-value ≤0.05, was considered statistically significant. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, and ^****p ≤ 0.0001.

The purpose of this experiment was to determine the MIC and MFC values for all crude C. hystrix DC. leaf extracts prepared using our extraction protocol and to investigate the relationship between these values and the amount of β-citronellol in each extract. The yeast cells were treated with different concentrations of crude extracts (0.2–100 mg/ml) and β-citronellol (2–1,024 μg/ml) at 35 ± 2°C for 48 h. In crude extracts group, the extract from ethanol had the lowest MIC value of 50 mg/ml, while the MIC values from n-hexane and ethyl acetate were equal to or greater than 100 mg/ml. However, the MFC of all three crude extracts was greater than 100 mg/ml. This result was related to the higher relative mass percentage of β-citronellol in ethanolic extract than n-hexane found in our previous studies (Ho et al., 2020; Buakaew et al., 2021a,b). The MIC and MFC values for β-citronellol treatment were 128 and 256 g/ml, respectively, as shown in Table 1. Based on these findings, β-citronellol was chosen as the candidate compound for further investigation.

To evaluate the influence of β-citronellol on the growth curve of yeast cells, the growth inhibitory effect of various concentrations of β-citronellol (32–256 μg/ml) on yeast cells is shown in Figure 1. Compared to the control, the β-citronellol treated condition showed significantly delayed cell growth with decreasing absorbance at 600 nm in a dose-dependent manner. The MIC and MFC values of β-citronellol on C. albicans ATCC 90028 were 128 and 256 μg/ml, respectively.

To investigate the protein expression profiles of C. albicans, yeast cells were treated with 128 μg/ml of β-citronellol for 24 h. Proteomics data were obtained using LC-MS/MS, while post-sample analysis was performed to generate high-certainty and high-specificity protein lists. Mass deviation showed that 94.93% of identified peptide groups had ≤10 ppm. For digestion specificity, 91.44% of all identified peptides had no miscleavage site, while 8.35% had a miscleavage site. In this study, the false discovery rate was set to <0.05. Overall, the total number of identified proteins was 1,326 from the control and treatment groups. As shown in Figure 2A, proteins commonly found among the two groups numbered 1,199 and unique proteins identified in the treatment and control groups numbered 59 and 68, respectively. Different expression proteins in each group were clustered hierarchically, as represented in the heatmap (Figure 2B). The columns show relative protein expression, while rows present significantly expressed proteins with maximum distance = 1.0.

Based on statistical significance (p < 0.05) with differential expression ≥1.5-fold from the treatment group compared to the control, 126 significant altered proteins were functionally classified using PANTHER-GO Slim Molecular Function analysis tool. Results showed that altered proteins were mainly involved in catalytic activity (41.8%), binding (39.1%), molecular function regulator (6.4%), structural molecule activity (5.5%), transporter activity (3.6%), translation regulator activity (2.7%), and molecular adaptor activity (0.9%; Figure 3A). Protein-protein interactions in both up-regulated and down-regulated proteins were analyzed using the STRING v.11 database with a confidence score of 0.4 (Figure 3B and Table 2). Several pathways involved in the mode of action on killing C. albicans based on the 126 significant proteins altered in response to β-citronellol treatment. Among 46 downregulated and 80 upregulated proteins, the yeast cells exposed to β-citronellol suppress 10 interesting proteins, as shown in Table 3. These proteins include cell wall-associated proteins, such as cell wall protein RTB1 (Rbt1p; encoded by the gene RBT1), agglutinin-like protein 2 (Als2p; encoded by the gene ALS2), and 1,3-beta-glucanosyltransferase PGA4 (Pga4p; encoded by the gene PGA4). In addition, the treatment downregulates proteins involved in cellular response to oxidative stress, such as superoxide dismutase [Cu-Zn] (Sod1p; encoded by the gene SOD1), glutathione S-transferase (Gst2p; encoded by the gene GST2), and stress protein DDR48 (Ddr48p; encoded by the gene DDR48). Furthermore, the expression of ATP synthesis related proteins was decreased, such as ATP synthase subunit gamma (Atp3p; encoded by the gene ATP3), ATP synthase subunit d, mitochondrial (Atp7p; encoded by the gene ATP7), cytochrome c oxidase subunit 1 (Cox1p; encoded by the gene COX1), and cytochrome b (Cobp; encoded by the gene COB). In contrast, β-citronellol treatment upregulated the expression of proteins involved in various pathways, including heat shock proteins, such as heat shock protein SSA2 (Ssa2p), Hsp30p, and Hsp90 cochaperone (Sti1p), and ribosomal proteins, such as 40S ribosomal protein S27 (Rps27Ap), ribosomal protein P1B (Rpp1Bp), 60S ribosomal protein L13 (Rpl13p), ribosomal protein L37 (Rpl37Bp), and ribosomal 40S subunit protein S15 (Rps15p).

The aim of this experiment was to determine the effect of β-citronellol on yeast biofilm formation. Biofilm formation of C. albicans was altered after β-citronellol treatment, as shown in Figure 4. The lowest concentration (32 μg/ml) of β-citronellol significantly suppressed the formation of yeast biofilm compared to the control. The inhibitory effect was higher when concentration increased, and the result at the highest concentration (256 μg/ml) was comparable to amphotericin B treatment.

In this experiment, the effect of β-citronellol on induced ROS production was investigated. Production of ROS as a key factor in apoptosis was evaluated using an H₂DCFDA probe. Results are shown in Figure 5. C. albicans treated with β-citronellol for 4 h showed an increase of ROS production by upregulation of green fluorescence intensity compared to the control group. ROS production increased in a dose-dependent manner.

The effect of β-citronellol on yeast membrane potential was determined using a DiBAC₄(3) probe. Fluorescence dye enters cells through depolarized membrane binding to intracellular proteins or membrane, resulting in a fluorescence signal. As shown in Figure 6, red fluorescence in β-citronellol treated groups significantly increased in a dose-dependent manner compared to the control, indicating the alteration effect of β-citronellol on the cell membrane.

The goal of this study was to determine the apoptosis profile of yeast cells after being exposed to β-citronellol. The apoptosis-inducing effect of β-citronellol is shown in Figure 7. After incubating the cells at concentrations of 64, 128, and 256 μg/ml for 6 h, the rate of early apoptosis increased to 20.75, 47.20, and 26.60%, respectively compared to the control. For late apoptosis/dead positive cells, the rate demonstrated a dose-dependent manner ranging from 0.20, 1.25% and maximum at 16.35% in β-citronellol treatment at 64, 128, and 256 μg/ml, respectively. The rate of dead or necrotic cells was positive for 0.20 and 0.05% in 256 μg/ml of β-citronellol and amphotericin B treated conditions, respectively. Results showed that 6 h β-citronellol treatment caused C. albicans programmed cell death, primarily activating the early to late apoptosis phases.

Morphological and structural changes of C. albicans under β-citronellol treatment were observed by SEM, with results shown in Figure 8. The yeast cells had spherical shapes and smooth surfaces (A). After 24 h of β-citronellol treatment, the cells exhibited wrinkled surfaces with irregular shapes (B–D), this result could indicate damage to the cell wall and/or cell membrane. The wrinkles increased as the concentration of β-citronellol increased.