UHPLC-HRMS-Based Untargeted Lipidomics Reveal Mechanism of Antifungal Activity of Carvacrol against Aspergillus flavus

Abstract

Aspergillus flavus is a common contaminant in grain, oil and their products. Its metabolite aflatoxin B₁ (AFB₁) has been proved to be highly carcinogenic. Therefore, it is of great importance to find possible antifungal substances to inhibit the growth and toxin production of Aspergillus flavus. Carvacrol (CV) was reported as a potent antifungal monoterpene derived from plants. In this paper, the antifungal effects and mechanism of CV on Aspergillus flavus were investigated. CV was shown good inhibition on the growth of Aspergillus flavus and the production of AFB₁. CV used in concentrations ranging from 0, 50, 100 and 200 μg/mL inhibited the germination of spores, mycelia growth and AFB₁ production dose-dependently. To explore the antifungal mechanism of CV on Aspergillus flavus, we also detected the ergosterol content of Aspergillus flavus mycelia, employed Scanning Electron Microscopy (SEM) to observe mycelia morphology and utilized Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS) to explore the lipidome profiles of Aspergillus flavus. The results showed that the production of ergosterol of mycelia was reduced as the CV treatment concentration increased. SEM photographs demonstrated a rough surface and a reduction in the thickness of hyphae in Aspergillus flavus treated with CV (200 µg/mL). In positive ion mode, 21 lipids of Aspergillus flavus mycelium were downregulated, and 11 lipids were upregulated after treatment with 200-µg/mL CV. In negative ion mode, nine lipids of Aspergillus flavus mycelium were downregulated, and seven lipids upregulated after treatment with 200-µg/mL CV. In addition, the analysis of different lipid metabolic pathways between the control and 200-µg/mL CV-treated groups demonstrated that glycerophospholipid metabolism was the most enriched pathway related to CV treatment.

1. Introduction

As a pathogenic fungus, Aspergillus flavus Link (A. flavus) frequently contaminates grains, oil and nuts, resulting in a significant reduction of nutritional quality and a serious loss of food commodities [1,2]. Since A. flavus could produce hazardous secondary metabolite aflatoxins (AFTs), it also causes contamination of food commodities during storage and processing and endangers human health [3]. AFTs mainly includes aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂) [4]. Among them, AFB₁ is considered to be the most mutagenic and teratogenic and is well-documented to be a pathogenic agent of hepatocellular carcinoma [5]. Therefore, it is of great importance to control aflatoxin contamination.

Using synthetic preservatives and fungicides was an effective way to inhibit the growth of A. flavus and the production of AFTs. However, the application of synthetic antifungal agents had led to a series of problems, such as drug resistances, and was harmful to human health [6,7]. Consequently, investigators had been searching for new natural products to control A. flavus reproduction and to reduce AFTs contamination.

EOs are natural aromatic oily liquids obtained from flowers, leaves, roots, bark, fruits, seeds and resin [8]. A number of studies had reported the essential oils (EOs) were effective substances with antifungal activity and had been kept under Generally Recognized as Safe (GRAS) category by United States Food and Drug Administration [9]. Previous reports demonstrated that EOs performed antifungal activity by the way of destruction of fungal plasma membrane of fungus [10,11]. Oxidative stress, membrane permeability and amino acid metabolism were significantly changed after perilla frutescens essential oil-treated A. flavus [12]. The essential oil from dill (Anethum graveolens L.) caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity [13]. Thymol has a variety of antifungal mechanism of action, such as disruption of cell membranes, inhibition of efflux pumps, alteration of mycelial morphology and production of ROS and nitric oxide [14].

As one kind of EO compounds, Carvacrol (CV, C₁₀H₁₄O), 5-isopropyl-2-methylphenol, is a liquid phenolic monoterpenoid found in herb like oregano (Origanum vulgare), pepperwort (Lepidium flavum), wild bergamot (Citrus aurantium var. Bergamia Loisel) and other plants [15,16]. Hsin-Bai Yin [17] investigated the effect of CV on contaminated poultry feed and discovered that CV can reduce AFTs production in broth culture and chicken feed by at least 60% when compared to control group, downregulated the expression of main genes (aflC, nor1 and norA) associated with AFT synthesis in the molds. Sharifi et al. [18] presented that the antimicrobial potential bioactivities of CV were high due to the presence of a free hydroxyl group, hydrophobicity and a phenol moiety. For a more comprehensive analysis of the antifungal mechanism of CV, a lipidomics analysis of A. flavus may provide important information.

Lipidomics, which aimed to study the lipid of specific tissues or organisms at the system-wide level, was widely used in food science for a variety of purposes [19]. Lipids are one of the main classes of compounds in biological systems and perform important physiological tasks. Their hydrophobicity allows them to form cell membranes that constitute a boundary against the hydrophilic environment of the cells [20]. As the important components of biological membranes, lipids (e.g., phospholipids, sphingolipids and phytosterol) are crucial for their pathogenicity and could be a potential target to inhibit fungal growth [21,22]. In recent years, some studies on the effect of EOs on the lipid profile of A. flavus were reported. Helal et al. found that mycelia fumigated with Cymbopogon citratus L. essential oil reduced the total lipid content, reduced the saturated fatty acid and increased the unsaturated fatty acid compared to samples without EO treatment [23]. Paeonol acting on A. flavus reduced the total lipid content, induced lipid peroxidation and altered the glycerol content [24].

As of today, the underlying antifungal mechanism of CV on A. flavus is still far from unequivocal. Therefore, in the present study, we evaluated the inhibitory of CV on the growth and toxin production of A. flavus. The UPLC-HRMS-based untargeted lipidomics approach was utilized for lipid profile analysis in A. flavus. A multivariate statistical analysis was used to analyze significant lipids and metabolic pathways to clarify alterations in lipid metabolic pathways of A. flavus treated by CV. Our work mainly explored the potential anti-A. flavus mechanism of CV so as to provide a theoretical reference for the use of CV for control of A. flavus.

2. Materials and Methods

2.1. Materials

The strain A. flavus 127-1 was isolated from the soil of the principal peanut regions (Liaoning Province, China) and preserved in our laboratory, which was a medium aflatoxin producer. CV was purchased from Jiangxi Cedar natural medicinal oil Co., Ltd. (Ji an, China). All other chemicals were analytical grade. Formic acid and ammonium formate were obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Deionized water was purified by a Milli-Q system (Millipore-Co., Ltd., Milford, MA, USA) and was used to prepare all aqueous solutions. Dichloran Glycerol-18 Agar medium (DG-18) and Liquid Sabourand medium were purchased from Haibo Biotechnology Co., Ltd. (Shanghai, China).

2.2. Fungal Culture

The strain was cultured in DG 18 Agar medium (Haibo Biotechnology Co., Ltd.) (Qingdao, China) at a constant temperature and humidity (28 °C, 75%) for 5 days. The A. flavus spores were washed off by 0.1% Tween-80 solution, and A. flavus spore suspension with 2 × 10⁸ spores/mL was obtained.

2.3. Inhibitory Effect of CV on A. flavus Growth and Toxin Production

2.3.1. Inhibitory Effect of CV on Spore Germination

Twenty microliters of A. flavus spore suspension (2 × 10⁸ spores/mL) were inoculated into 20-mL liquid sabourand medium with different concentrations of CV (0, 50, 100 and 200 μg/mL) and cultured at 28 °C for 8 h. Then, the spore germination was observed by a light microscope. When the length of the germination tube was longer than the diameter of the spore, the spore was considered as “germinated”.

2.3.2. Inhibitory Effect of CV on A. flavus Mycelia Growth

The antifungal activity of CV was estimated by the contact agar phase effects on the mycelia growth of A. flavus. Primarily, 500 µL of CV was well-dissolved in 4.5 mL of 0.1% Tween-80. Different amounts of CV solution were aseptically transferred into disposable plastic petri dishes containing 20 mL of preheated DG18 agar under sterile conditions, respectively. The sample without CV treatment was used as the control. After the DG 18 agar medium was colloid, 5 µL of A. flavus spore suspension was added at each culture plate. A. flavus was continuously cultured at 28 °C and 75% humidity for 5 days, and the colony diameter was measured by the crossing method every day [25].

2.3.3. Inhibitory Effect of CV on Mycelia Dry Weight

A series of volumes of CV was pipetted aseptically into a conical flask containing 50 mL of the liquid sabourand medium to procure the required concentrations of CV 50, 100 and 200 μg/mL. The sample without CV treatment was used as the control. One hundred microliters of A. flavus spore suspension (2 × 10⁸ spores/mL) were cultured at each conical flask. After incubating at 28 °C and a speed of 200 r/min for 5 days, the A. flavus mycelia solution was obtained. Then, the mycelia were collected, lyophilized and weighed.

2.3.4. Inhibition Effect of CV on AFB₁ Production

One hundred microliters of A. flavus mycelia solution (as described in Section 2.3.3) was added to 900-µL methanol, vortex-mixed for 2 min and centrifuged at 10,000× g r/min for 10 min. The supernatant was used as the AFB₁ sample. The AFB₁ samples were analyzed by an Agilent HPLC 1100 system equipped with a reverse-phase column Sycronis C18 (150 mm × 4.6 mm, 5 μm) at 35 °C and detected by a fluorescence detector (excitation and emission wavelengths 360 nm and 440 nm, respectively). The mobile phase conditions were as below: 40% methanol and 60% water, 0.8 mL/min flow rate and isocratic elution. The content of AFB₁ was calculated by the standard curve y = 1.7841x − 6.024 (concentration range 6.25–400 μg/L).

2.3.5. Scanning Electron Microscopy (SEM) Analysis

Mycelia were cultured as described in Section 2.3.3; 5-mm × 10-mm segments were filtered from mycelia and washed twice by 0.1-M phosphate-buffered saline (PBS, pH 7.2). The samples were fixed by 2.5% glutaraldehyde solution for a fixed 24 h and washed three times in 0.2-M phosphate-buffered saline (PBS, pH 7.4) for 15 min each time. The segments were dehydrated twice in graded ethanol in a series (15–100%) over a period of time for 20 min each time, then air-dried, fixed on the radio with a conductive adhesive, sprayed with gold for 30 s and, finally, observed by SEM. Samples were fixed by 2.5% glutaraldehyde solution for 24 h and washed three times in 0.2-M phosphate-buffered saline (PBS, pH 7.4) for 15 min each time. Sections were dehydrated twice (15–100%) in graded ethanol in series for 20 min each time, then air dried, fixed on radio with conductive gel, sprayed with gold for 30 s and, finally, observed by SEM.

2.4. Antifungal Mechanism of CV

2.4.1. The Content of Ergosterol in Mycelia Plasma Membrane

The content of ergosterol in an A. flavus mycelia plasma membrane was determined as Tian et al. [13] described. A. flavus spores (100 μL, 2 × 10⁸ spores/mL) were inoculated into 50-mL liquid sabourand medium with different concentrations of CV (0, 50, 100 and 200 µg/mL) and cultured at 28 °C with a speed of 200 r/min for 2 days. Then, A. flavus mycelia were filtered and collected. Five milliliters of 25% alcoholic potassium hydroxide solution were added to each sample, vortex-mixed for 2 min and then incubated at 85 °C for 4 h. Two milliliters of sterile distilled water and 5-mL n-heptane were then added, followed by vortex mixing for 2 min. The n-heptane layer (upper layer) was collected and scanned in the range of 230–300 nm by UV spectrophotometry. The presence of ergosterol (at 282 nm) and 24(28) dehydroergosterol (at 230 and 282 nm) in mycelium led to a characteristic curve. The ergosterol amount was calculated as a percentage of the wet weight of the cells and was based on the absorbance and wet weight of the initial pellet. Samples without CV treatment were considered as the control group. The ergosterol amount was calculated by a formula as follows:

$$(\%) \mathrm{ergosterol} =\left(A282/290\right)/\mathrm{net} \mathrm{wet} \mathrm{weight} \mathrm{of} \mathrm{mycelia}-(\%) 24 \left(28\right) \mathrm{dehydroergosterol}$$

(1)

$$\%24 \left(28\right) \mathrm{dehydroergosterol} = \left(A230/518\right)/\mathrm{net} \mathrm{wet} \mathrm{weight} \mathrm{of} \mathrm{mycelia}$$

(2)

where A282 and A230 are the absorbance of the n-heptane layer in 282 nm and 230 nm, respectively. The E-values are 290 and 518 (in percentages per cm) determined for crystalline ergosterol and 24 (28) dehydroergosterol, respectively.

2.4.2. Lipid Extraction from A. flavus Mycelia

Firstly, 50-mg dried A. flavus mycelia (as described in Section 2.3.3) was dissolved in 2-mL anhydrous alcohol. The lipid in A. flavus mycelia was extracted by ultrasonic extraction for 20 min. The mixture was centrifuged at 4500× g r/min for 10 min. Subsequently, the supernatant was pipetted into a Ostro 96-well plate, which was activated by 200-μL methanol. The lipid in the supernatant was adsorbed on the plate. Then, 2-mL chloroform/methanol/triethylamine (4.5/4.5/1, v/v/v) was added to elute the lipid. The collected lipids were dried by nitrogen blowing and dissolved in 200 µL of chloroform/methanol (2/1, v/v). Finally, the lipid in A. flavus mycelia was analyzed by the UPLC-HRMS system. Before the lipidomics analysis, the quality control samples (QC) were mixed equally from equal amounts of samples from each group (control group, 100 and 200 μg/mL). QC samples were used to balance the mass accuracy before injection and correct for peak intensity or retention time, and then, they were employed to evaluate the system stability during the experiment. Five QC samples were determined at the beginning of each run. One QC sample was determined after every 6 analyzed samples were measured. QC samples were used for filtering lipids during data processing, and in order to better show the relationship between the changes in the control and treatment groups, so quality control samples were not presented in the paper.

2.4.3. Lipid Detection by UPLC-HRMS

The analysis of the mycelial lipids was achieved by the Dionex high-performance liquid chromatography system with tandem Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). An ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) provided by Waters (Milford, MA, USA) was used for the separation of the target compounds. The temperature of the column was maintained at 40 °C, and the injection volume was 1 μL. Separations under a gradient elution at 0.2 mL/min based on acetonitrile/H₂O (6:4, v/v) + 0.1% formic acid + 10-mmol/L ammonium formate (solvent A) and isopropanol/acetonitrile (9:1, v/v) + 0.1% formic acid + 10-mmol/L ammonium formate (solvent B) was as follows: 0–4 min 15% B, 5 min 48% B, 17 min 70% B, 20 min 82% B, 24 min 99% B and 24.2–30 min 15% B.

The following parameters were used for MS. The source parameters were as below: ion source type, ESI source; scan mode, positive (+)/negative (−) ion mode; spray voltage, 3500 v (+)/3000 v (−); ion transfer tube temperature, 320 °C and gas heater temperature, 320 °C. Both sheath gas and auxiliary gas were nitrogen, with the sheath gas flow 40 arb, auxiliary gas flow 5 arb and sweep gas flow 0 arb. The main parameters of the full scan were as follows: MS1, Orbitrap resolution, 240,000; scanning range, m/z 150–2000; RF lens (%), 60; AGC target, 1.0e6, MS2 and MS3, activation type, HCD; HCD collision energy (%), 35 ± 5; Orbitrap resolution, 30,000; AGC target, 5.0 × 10⁴ [26].

2.4.4. Lipidomics Analysis of A. flavus Mycelia

The raw data obtained from UPLC-HRMS were collected by Xcalibur 4.0 software (Thermo Scientific, Waltham, MA, USA). Lipid identification used LipidSearch 4.0 software (Thermo Scientific) (Shanghai, China), which contained 18 lipid species and over 1,500,000 fragment ions to match the experiment data [27,28].

The adductions included +H⁺, +NH₄⁺ and +2H⁺ for the positive mode and H⁺, +CHOO^- and −2H⁺ for the negative mode. Subsequently, the lipid molecules in the extracted data with missing values >50% were deleted. The missing values that were not eliminated were filled with 1/5 minimum values. After that, the data were processed with quality controls (QCs), and data with relative standard deviations (RSD) > 30% were eliminated, and the peak area was normalized. Preprocessed data were imported to Metaboanalyst 5.0 for the multivariate statistical analysis, including a principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Metaboanalyst 5.0, a web-based platform with rich compound databases and path libraries, has become widely used for comprehensive metabolomics data analysis, interpretation and multi-omics data integration of a wide range of species [29]. The significantly differential lipids between the control and CV treatment groups were selected using the protocol p < 0.05, fold change (FC) > 2.0 (upregulated) or 0.5 (downregulated) and variable importance in projection (VIP) > 1.0 [30]. The significantly different lipids (p < 0.05, VIP > 1) between the control group and 200-µg/mL CV-treated group were imported into a “pathway analysis” panel of Metaboanalyst 5.0 for the metabolic pathway analysis. The lipid metabolic pathway analysis was performed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.

3. Results and Discussion

3.1. Antifungal Activity of CV

It can be seen from Figure 1A that spore germination of A. flavus was inhibited effectively by CV. Following 8 h, in the experimental group, with the increase of CV concentration, the percentage of A. flavus spore germination decreased. Specifically, the percent germination of 50-, 100- and 200-µg/mL CV-treated spores were 84.0% and 26.7% and 11.3%, respectively.

The inhibitory effect of CV at different concentrations on the mycelia growth of A. flavus is displayed in Figure 1B,C. In general, all tested concentrations of CV exhibited inhibition of mycelia growth, and the inhibition degree was concentration-dependent. After 5 days, compared to the mycelia diameter of the control (6.4 cm), a significant inhibition effect of CV on mycelia diameters at 50-µg/mL, 100-µg/mL and 200-µg/mL concentrations were found at 5.8 cm, 5.3 cm and 2.1 cm, respectively. As can be seen from the data, CV had a good inhibitory effect on the growth of A. flavus.

The antifungal effects of presenting different concentrations of CV on the dry mycelia weigh of A. flavus cultured for 5 days are displayed in Figure 1D. The concentration of CV was negatively correlated with the dry weight of mycelia. Compared to the control group (0.68 g), the dry weight of A. flavus mycelia treated with CV of 50 and 100 µg/mL decreased to 0.34 and 0.24 g, respectively. At the concentration of 200 µg/mL, the dry weight of mycelia was only 0.19 g.

The inhibitory effect of CV at different concentrations on the AFB₁ is displayed in Figure 1E. CV had a significant inhibitory effect on AFB₁ production. The AFB₁ amount of the control group was 248.19 µg/l. After treated with 50-, 100- and 200-µg/mL CV, the amount of AFB₁ were 84.58 µg/L, 25.78 µg/L and 0 µg/L, respectively.

3.2. CV Destroyed the Integrity of A. flavus Cell Membrane

The fungal cell membrane can absorb nutrients, exchange substances and energy with the surroundings and maintain cell viability [31]. Some essential oils may cause cell necrosis by damaging the cell membrane of A. flavus [8]. Since the growth and toxin production of A. flavus was significantly inhibited by CV, we wondered whether CV damaged the integrity of A. flavus cell membrane. Hence, we investigated the cell morphology using SEM.

An intact morphology (the control) with striated linear, smooth hyphae is shown in the SEM image of A. flavus mycelia (Figure 2A). However, after being incubated with CV, a slightly shrunken appearance was observed. The deformation can be seen, including a rough and wrinkled surface, in the mycelia morphology following the 200-μg/mL treatment (Figure 2D). The reason for the rough and wrinkled surface of the A. flavus mycelia was the rupture of the membrane integrity and loss of the cytoplasmic contents [32].

3.3. Ergosterol Content of Mycelium

Ergosterol is a key component of the fungal cell membrane, which is an important substance to maintain membrane integrity and normal physiological function of fungal cells. It is also considered as a marker for estimating fungal biomass in different matrices [33]. The ergosterol content in the plasma membrane of A. flavus was determined, and the inhibition result of CV on the synthesis of ergosterol can be seen in Figure 3. The total ergosterol content was determined at 0-, 50-, 100- and 200-μg/mL concentrations of CV with values of 0.341 ± 0.001%, 0.230 ± 0.000%, 0.210 ± 0.041% and 0.074 ± 0.006%, respectively. Furthermore, the ergosterol contents of A. flavus mycelium after 100-μg/mL and 200-μg/mL CV treatment were significantly different from the control. The reduction of ergosterol content in A. flavus following treatment with different concentrations of CV suggested that the inhibitory effect of CV on the ergosterol content in the A. flavus cell membrane was shown in a dose-dependent manner.

3.4. UPLC-HRMS Lipid Metabolite Profiling of A. flavus

3.4.1. Multivariate Analysis

Lipid extracts of 100-μg/mL and 200-μg/mL CV-treated A. flavus mycelia and the control group were analyzed in positive and negative ion modes using a UPLC-HRMS. A total of 369 lipid molecules in positive ionization mode and 368 lipid molecules in negative ionization mode were detected. The subclasses of the defined lipids of A. flavus could be classified into ceramides (Cer), monoglycosylceramide (CerG1), diglycosylceramide (CerG2), triglycosyl-ceramide (CerG3), ceramides phosphate (CerP), cardiolipin (CL), cyclic phosphatidic acid (cPA), dimethylphosphatidylethanolamine (dMePE), diglyceride (DG), digalactosyldiacylglycerol (DGDG), digalactosylmonoacylglycerol (DGMG), fatty acid (FA), monoglyceride (MG), monogalactosyldiacyiglycerol (MGDG), monogalactosyl monooctanoyl glycerol (MGMG), lysodimethylplosphatidylethanolamine (LdMePE), lysophosphatidic acid (LPA), lysophosphatidylcholines (LPC), lysophosphatidylethanol (LPEt), phosphatidylethanolamines (LPE), lysophosphatidylmethanol (LPMe), lyso-lysophosphatidylinositol (LPI), phosphatidylglycerols (LPG), lipopolysaccharide (LPS), phosphatidic acid (PA), platelet-activating factor (PAF), phosphatidylcholines (PC), phosphatidylethanolamine (PE), phosphatidylethanol (PEt), phosphatidylinositols (PI), phosphatidylinositrol (PIP, PIP2), phosphatidylglycerols (PG), phosphatidylmethanol (PMe), phosphatidylserine (PS), sphingomyelin (SM), sphingoshine (So), sphingoshine phosphate (SoP), sulfoquinovosyldiacylglycerol (SQDG), sulfoquinovosylmonoacylglycerol (SQMG), triglyceride (TG), (O-acyl)-1-hydroxy fatty acid (OAHFA) and sphingomyphlin (phytosphingosine) (phSM).

As shown in Figure 4A,B, the explained variance by the first two principal components in the control group (red dots), 100 μg/mL (green dots) and 200 μg/mL group (blue dots) were 24.5% (PC1) and 12.7% (PC2) in the positive mode and 21.1% (PC1) and 11.7% (PC2) in the negative mode. Furthermore, the blurred boundary among the areas occupied groups can be seen in the negative mode. In order to obtain clearer separations, a supervised pattern recognition method, OPLS-DA, was used to discover significant discriminants among the samples. In the OPLS-DA score plots, the control, 100- and 200-μg/mL CV-treated groups were clearly distinguished in positive and negative modes. To ensure that the calculated models were reliable, 100 times the permutation was performed with the model parameters in positive mode (R²Y = 0.997, Q² = 0.858) and negative mode (R²Y = 0.999, Q² = 0.706), showing a more predictive performance and goodness-of-fit results (Figure 4C,D). Both positive and negative mode datasets presented clear segregation among the three groups, showing that the lipid metabolism of A. flavus was affected by CV significantly.

Based on the OPLS-DA models (Figure 4C,D), the 100-μg/mL CV-treated group was closer to the control group when compared to the 200-μg/mL CV-treated group, indicating that lipid metabolism was dependent on CV concentration. Variable important in projection (VIP) scores from the OPLS-DA models were calculated. Lipids with VIP > 1 and p-values < 0.05 were regarded as significantly different metabolites. A total of 88 differential lipid metabolites were selected according to their VIP and p-values, which are shown in

Table 1. Significantly altered lipid metabolites among the control and the CV-treated groups by the ANOVA test analysis with Fisher’s post hot analysis and OPLS-DA analysis.

No.	Extract Mass	Compound	p Value	VIP
1	(M + H)+	PC (16:2/18:2)	2.07 × 10−3	1.039
2	(M+ H)+	PE (20:4/21:2)	2.08 × 10−5	1.056
3	(M + NH4)+	TG (18:1/24:0/24:0)	2.50 × 10−3	1.090
4	(M + H)+	PC (16:0/22:6)	3.47 × 10−5	1.113
5	(M + H)+	PS (30:0/18:3)	8.21 × 10−4	1.125
6	(M + NH4)+	DG (10:0e/22:6)	6.62 × 10−6	1.158
7	(M + NH4)+	DG (18:1/18:2)	5.94 × 10−5	1.207
8	(M + NH4)+	TG (16:0/24:0/24:0)	8.55 × 10−5	1.284
9	(M + H)+	PE (18:2/18:1)	5.00 × 10−3	1.292
10	(M + H)+	PMe (10:0e/23:3)	1.58 × 10−6	1.312
11	(M + NH4)+	DG (18:2/18:2)	9.25 × 10−8	1.327
12	(M + NH4)+	DG (20:0/21:0)	8.58 × 10−6	1.544
13	(M + H)+	PE (18:3/18:2)	1.33 × 10−5	1.552
14	(M + H)+	DG (9:0/21:5)	1.63 × 10−5	1.586
15	(M + NH4)+	TG (16:0/18:2/24:0)	1.91 × 10−3	1.711
16	(M + NH4)+	TG (17:0/18:2/24:0)	6.75 × 10−3	1.799
17	(M + NH4)+	LdMePE (20:1p)	2.05 × 10−4	1.800
18	(M + NH4)+	TG (18:0/18:1/18:1)	3.95 × 10−3	1.840
19	(M + NH4)+	TG (18:1/18:2/18:2)	4.26 × 10−3	1.842
20	(M + NH4)+	TG (18:2/18:2/23:0)	4.26 × 10−3	1.891
21	(M + H)+	PS (18:2/18:2)	6.80 × 10−4	1.902
22	(M + NH4)+	TG (18:1/18:2/24:0)	1.11 × 10−3	1.906
23	(M + H)+	CerP (d22:1/32:1)	2.29 × 10−5	1.989
24	(M + NH4)+	TG (16:0/18:1/24:0)	8.24 × 10−4	1.991
25	(M + NH4)+	TG (18:0/17:0/18:2)	2.17 × 10−4	1.999
26	(M + H)+	MG (26:6)	2.93 × 10−8	2.052
27	(M + NH4)+	TG (16:0/17:0/18:1)	5.77 × 10−4	2.052
28	(M + NH4)+	TG (17:0/18:2/18:2)	2.41 × 10−4	2.121
29	(M + NH4)+	TG (18:0/18:1/24:0)	1.23 × 10−6	2.155
30	(M + H)+	PE (18:2/15:0)	3.34 × 10−4	2.169
31	(M + NH4)+	TG (26:0/18:0/18:1)	3.03 × 10−5	2.201
32	(M + NH4)+	TG (18:2/18:2/18:2)	3.98 × 10−6	2.217
33	(M + H)+	DG (4:0/20:2)	1.28 × 10−7	2.309
34	(M + NH4)+	TG (18:3/18:2/18:3)	2.06 × 10−13	2.396
35	(M + NH4)+	TG (18:0/18:0/18:1)	1.44 × 10−5	2.406
36	(M + NH4)+	TG (16:0/16:1/21:4)	1.99 × 10−6	2.464
37	(M + NH4)+	TG (18:2/18:2/16:2)	2.17 × 10−13	2.528
38	(M + NH4)+	TG (24:3/12:0/21:4)	1.65 × 10−9	2.545
39	(M + NH4)+	TG (18:0/17:0/18:1)	2.56 × 10−8	2.580
40	(M + NH4)+	TG (18:2/17:2/18:2)	3.32 × 10−12	2.591
41	(M + NH4)+	TG (18:2/18:2/18:3)	6.14 × 10−12	2.593
42	(M + NH4)+	TG (15:0/18:2/18:3)	3.01 × 10−10	2.618
43	(M + NH4)+	TG (25:0/18:2/18:2)	9.93 × 10−11	2.648
44	(M − H)−	CerG1 (d17:0/15:0)	1.52 × 10−3	1.846
45	(M − H)−	DGDG (13:0/15:1)	6.76 × 10−3	1.807
46	(M − H)−	dMePE (14:0/18:3)	1.44 × 10−7	1.998
47	(M − H)−	dMePE (15:0/16:1)	5.01 × 10−5	2.298
48	(M − H)−	dMePE (16:2/18:3)	1.32 × 10−6	1.448
49	(M − H)−	dMePE (18:0p/22:5)	1.24 × 10−9	2.185
50	(M − H)−	dMePE (18:2/13:0)	4.55 × 10−5	2.527
51	(M − H)−	dMePE (18:2/15:0)	1.80 × 10−3	2.147
52	(M − H)−	dMePE (18:2/16:1)	3.16 × 10−3	1.296
53	(M − H)−	dMePE (24:3/8:0)	3.62 × 10−4	1.879
54	(M − H)−	dMePE (25:3/8:0)	1.35 × 10−3	2.266
55	(M − H)−	dMePE (27:5/23:4)	6.32 × 10−4	1.637
56	(M − H)−	LdMePE (38:4)	5.74 × 10−9	2.504
57	(M − H)−	LPE (16:0)	1.76 × 10−6	2.445
58	(M − H)−	LPE (18:2)	2.46 × 10−5	2.148
59	(M − H)−	MGDG (12:0/18:2)	5.26 × 10−3	1.991
60	(M + HCOO)−	MGDG (12:0/23:1)	5.76 × 10−3	1.733
61	(M − H)−	MGDG (16:1/18:5)	1.07 × 10−4	2.429
62	(M − H)−	MGDG (16:1/25:9)	6.67 × 10−3	1.692
63	(M − H)−	PA (16:0/18:1)	6.60 × 10−3	2.003
64	(M − H)−	PA (18:2/18:2)	4.41 × 10−3	1.819
65	(M − H)−	PA (27:6/20:5)	6.51 × 10−3	1.692
66	(M + HCOO)−	PC (16:2/18:2)	1.14 × 10−3	1.389
67	(M + HCOO)−	PC (18:1/18:1)	4.01 × 10−3	1.349
68	(M + HCOO)−	PC (18:3/18:3)	6.06 × 10−5	1.080
69	(M + HCOO)−	PC (8:0p/10:0)	1.57 × 10−3	1.491
70	(M − H)−	PE (15:0/18:1)	8.70 × 10−4	1.834
71	(M − H)−	PE (17:0/18:2)	2.09 × 10−3	2.126
72	(M − H)−	PE (18:2/15:0)	8.34 × 10−5	2.490
73	(M − H)−	PE (18:2/18:1)	3.34 × 10−3	1.291
74	(M − H)−	PE (18:3/18:2)	9.03 × 10−7	1.495
75	(M − H)−	PEt (16:0/16:1)	5.94 × 10−3	2.018
76	(M − H)−	PEt (24:6/21:6)	5.53 × 10−3	1.838
77	(M − H)−	PEt (26:4/8:0)	4.74 × 10−3	1.556
78	(M − H)−	PI (16:0/18:1)	3.30 × 10−3	1.993
79	(M − H)−	PI (16:0/18:2)	4.81 × 10−3	1.818
80	(M − H)−	PI (18:2/18:2)	6.06 × 10−3	1.339
81	(M − H)−	PMe (16:0/17:1)	6.89 × 10−3	2.002
82	(M − H)−	PMe (17:1/18:2)	5.26 × 10−3	1.991
83	(M − H)−	PMe (18:2/17:2)	4.13 × 10−3	1.318
84	(M − H)−	PMe (4:0/8:0)	4.88 × 10−6	1.341
85	(M − H)−	PS (18:2/18:2)	1.10 × 10−4	2.442
86	(M+HCOO)−	SM (d14:0/17:1)	1.28 × 10−5	1.114
87	(M − H)−	SQDG (27:4/27:4)	4.22 × 10−6	1.561
88	(M+HCOO)−	SQMG (7:1)	4.31 × 10−3	1.921

. In addition, glycerolipids (in the positive ion mode) and glycerophospholipids (in the negative ion mode) were the main significant differential lipids between the control group and CV-treated groups.

3.4.2. Univariate Statistical Analysis

To better understand the possible antifungal mechanism of CV to A. flavus, metabolic differences between the control and CV-treated groups (volcano plots) were investigated to recognize the lipids that showed significant differences (p < 0.05, FC > 2).

Compared with the control group, four lipids downregulated while 22 lipids upregulated for the 100-μg/mL CV treated group (Figure 5A), and 21 lipids downregulated while 11 lipids upregulated for the 200-μg/mL CV-treated group in the positive ion mode (Figure 5B). The significantly differential lipids are supplied in Supplementary Materials Table S1. In addition, in the negative ion mode, three lipids were downregulated with eight lipids upregulated (100-μg/mL CV-treated, Figure 5C) and nine lipids downregulated with seven lipids upregulated (200-μg/mL CV-treated, Figure 5D) in comparison with the control. The significantly differential lipids were supplied in Supplementary Materials Table S2. Furthermore, TG (18:2/17:2/18:2), TG (18:3/18:2/18:3), TG (18:2/18:2/16:2), TG (18:2/18:2/18:3), TG (25:0/18:2/18:2), TG (15:0/18:2/18:3), TG (24:3/12:0/21:4), TG (16:0/16:1/21:4), dMePE (18:2/13:0) and PE (18:2/15:0) are significantly downregulated with increasing CV concentrations in Supplementary Materials Table S3.

3.4.3. Hierarchical Clustering Analysis

To observe the discrimination trend in more detail and evaluate the rationality of differential metabolites, a hierarchical clustering heat map of up- and downregulated significantly differential lipids in A. flavus treated by CV were performed to show the overall trend of the 25 top significant ion features. From Figure 6A, we can find that most of the significant features expressed a downregulated trend after being 200-μg/mL CV-treated compared with the control group and 100-μg/mL CV-treated, principally for triglycerides. A study in yeast showed that TG was a novel lifespan-promoting factor, stored more TG in yeast cells and had a significantly extended lifespan than those cells depleted of TG [34]. Thus, treatment with 200-μg/mL CV may not be conducive to mycelial life extension. From Figure 6B, in the negative mode, specific lipid classes (e.g., PI, DGDG, MGDG and SQMG) were upregulated with 200-μg/mL CV treatment. PIs have physiological regulatory functions in membrane trafficking, membrane cytoskeletal interactions and ion channel and transporter activity [21], and the upregulation of PIs and glycolipids 200-μg/mL CV-treated may trigger the relevant signaling pathways.

3.4.4. Analysis of Metabolic Pathways

The metabolic pathway analysis was of great significance in revealing the antifungal mechanism of CV and understanding the metabolic process alteration of A. flavus after CV treatment. The significantly different lipids (p < 0.05, VIP > 1) between the control group and 200-µg/mL CV-treated group were imported into the pathway analysis panel of Metaboanalyst 5.0 for the metabolic pathway analysis.

As shown in Figure 7A and

Table 2. Results from the ingenuity pathway analysis with Metaboanalyst in the 200-μg/mL group vs. control group.

Pathway Name	Total 1	Hits 2	Raw p	−log10 (p)	Holm Adjust p 3	FDR 4	Impact 5
Glycerophospholipid metabolism	32	2	0.012	1.924	0.86	0.43	0.229
Glycerolipid metabolism	14	2	0.002	2.642	0.17	0.17	0.020
Glycosylphosphatidylinositol (GPI)-anchor biosynthesis	14	1	0.077	1.113	1	1	0.004
Phosphatidylinositol signaling system	26	1	0.139	0.856	1	1	0

Total ¹ means the number of compounds in the pathway. Hits ² is the actually matched number from the user uploaded lipids. Holm adjust p ³ is the p-value adjusted by the Holm–Bonferroni method. FDR ⁴ is the p-value adjusted using the false discovery rate. Impact ⁵ is the pathway impact value calculated from the pathway topology analysis.

, four metabolic pathways of A. flavus were affected by CV at 200 μg/mL, including glycerophospholipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis (GPI-B) and glycerolipid metabolism and phosphatidylinositol signaling. Among these pathways, only one distinctly altered pathway, glycerophospholipid metabolism, was filtered based on specific criteria (raw p < 0.05 and impact value > 0.1). The biosynthesis of glycerolipids and phospholipids in A. flavus is shown in Figure 7B. The glycerolipids (TG and DG) and phospholipids (PC, PE and PS) were significantly downregulated, whereas PI and PG were upregulated. The abundance of the lipid metabolites and total lipids in the selected pathways indicated that the CV treatment had a potent inhibition of the growth of A. flavus.

4. Discussion

CV is one of the potent monoterpenes that can be utilized to control fungal species [37]. In the present study, we determined the effectiveness of CV on inhibiting the growth of A. flavus both in the agar and liquid phases. In our study, spore germination, mycelia growth and aflatoxin production of A. flavus were significantly inhibited by CV. Similar results for the antifungal effect of CV were also found in Fusarium oxysporum, Neocosmospora solani and Microdochium nivale, and CV at a 0.2–0.4-mg/mL concentration also inhibited the spore germination in a dose-dependent manner [37]. In addition, CV also made the mycelia of A. flavus shrink and form a rough surface. Our results confirmed that CV was able to damage the cell membrane and reduced the ergosterol content of A. flavus. Cell disruption in the cell membrane and imbalance in cell permeability may lead to the loss or reduction of the ergosterol content [38]. Similar results were found for the ergosterol content of A. flavus treated with 0.6-μL/mL Cuminum cyminum L. seed essential oil [39]. Furthermore, Xu et al. found that nine differentially expressed genes involved in ergosterol synthesis were downregulated after exposure of A. flavus mycelia to 0.25 μL/mL of cuminaldehyde [8].

Lipids have long been believed to have a structural role in the biomembrane and a role in energy storage [40]. Therefore, in order to investigate the antifungal mechanism of CV from a new perspective, lipidomic technology based on UPLC-HRMS was used to detect and analyze the metabolites of A. flavus after CV treatment. Our data suggested that CV concentrations at 100 μg/mL or greater showed evident mycelia growth and AFB₁ production inhibition compared to the control group. In order to explore lipid metabolism changes caused by different concentrations of CV, 100 and 200 μg/mL were chosen for further study. The inhibitory mechanism of CV against A. flavus was studied using a lipidomic metabolism analysis.

Most of the significant differential lipids of A. flavus mycelia were downregulated after 200-μg/mL CV treatment. Li et al. investigated the antifungal efficacy of paeonol on A. flavus; they found that the lipid content and glycero content of mycelia treated with paeonol at the minimum inhibitory concentration (MIC) was remarkably reduced by 46.21% and 64.68% [25]. Similar results of the total lipids content reduction were also found in α-phellandrene, nonanal-treated P. cyclopium [41] and Mentha piperita essential oil-treated Fusarium sporotrichioides [42]. The mechanism of action resulting in the reduction of the lipid content was that α-phellandrene oil and nonanal had the ability to penetrate the lipid structure of the cell and disrupt the integrity of the cell membrane [43]. Mentha piperita essential oil was capable of acting on the cell membrane structure and disrupting the cell membrane integrity. The result of reduction of the glycerol content was similar with pyrrolnitrin-treated Neurospora crassa, which could be the inhibition of respiration of Neurospora crassa by pyrrolizidine [44].

It was found that significant differential metabolites were involved in four metabolic pathways in the A. flavus mycelia. The glycerophospholipids metabolic pathway was a potential target metabolic pathway for CV intervention. Glycerophospholipids were key components of the cellular lipid bilayer, and their fatty acid compositions have important effects on membrane characteristics, such as membrane fluidity, transport triacylglycerols and cholesterol in the body and the formation of lipid rafts [43]. The glycerophospholipid bilayer was an integral cell membrane structure that played an important role in protecting cellular components from external influences and maintaining internal cellular biological functions without interference [37]. At the same time, glycerophospholipid metabolism was essential to the cell membrane dynamics [45]. Thus, the amounts of some kinds of glycerophospholipids induced by external factors may affect the overall cell metabolism. Moreover, the lipids associated with cell membrane homeostasis-related pathways, such as glycerophospholipid metabolism, were evidentially downregulated upon 200-μg/mL CV treatment.

The decrease of the abundance of most of the glycerolipids and phospholipids of A. flavus indicated the inhibition of the lipid metabolism by CV. Neutral lipids in the fungi primarily included TGs, which were primarily responsible for the storage of free fatty acids, sterols and DG [21]. The general pattern of neutral lipid turnover in many fungi manifests itself as a mechanism of resistance to chemical fungicides [46,47,48]. After treatment with 200-μg/mL CV, the abundance of neutral lipid (TG) was downregulated. Similar results were found for the decreased neutral lipids in Aspergillus parasiticus spear treated with propolis [49]. Phospholipids, which are the most abundant lipids in cells, account for 40–60% of the total lipids in eukaryotic cells [50]. After treatment with 200-μg/mL CV, the abundance of PC, PE and PS was significantly downregulated, whereas PI and PG were upregulated, indicating a blocking in the pathway of other phospholipids fractions from PI and PG.

5. Conclusions

In this study, we discovered that CV had a high potential to reduce spore germination, mycelia growth, aflatoxin production and the ergosterol content of A. flavus. In addition, CV also made the mycelia of A. flavus shrink and form a rough surface. Most of the significant differential lipids of A. flavus mycelia were downregulated after 200-μg/mL CV treatment. In addition, the analysis of different lipid metabolic pathways between the control and 200-μg/mL CV-treated groups showed a significant enrichment of lipids in the glycerophospholipid metabolic pathway. This work deeply elucidated the lipidomics changes contributed to the prevention of A. flavus growth and the promotion of cell apoptosis, which is promising for providing novel molecular targets for the prevention of A. flavus contamination. These results provide a new perspective for the antifungal mechanisms of CV and promote its application as an antifungal agent.