Antagonistic Activity of Oroxylin A against Fusarium graminearum and Its Inhibitory Effect on Zearalenone Production

Fusarium graminearum produces zearalenone (ZEA), a mycotoxin that is widely found in food and feed products and is toxic to humans and livestock. Piper sarmentosum extract (PSE) inhibits F. graminearum, and Oroxylin A appears to be a major antifungal compound in PSE. The aim of this study is to quantify the Oroxylin A content in PSE using UPLC-QTOF-MS/MS, and to investigate the antagonistic activity of Oroxylin A against F. graminearum and its inhibitory effect on ZEA production. The results indicate that Oroxylin A inhibits both fungal growth and ZEA production in a dose-dependent manner. Oroxylin A treatment downregulated the mRNA expression of zearalenone biosynthesis protein 1 (ZEB1) and zearalenone biosynthesis protein 2 (ZEB2). The metabolomics analysis of F. graminearum mycelia indicated that the level of ribose 5-phosphate (R5P) deceased (p < 0.05) after Oroxylin A treatment (64–128 ng/mL). Moreover, as the Oroxylin A treatment content increased from 64 to 128 ng/mL, the levels of cis-aconitate (p < 0.05) and fumarate (p < 0.01) were upregulated successively. A correlation analysis further showed that the decreased R5P level was positively correlated with ZEB1 and ZEB2 expression, while the increased cis-aconitate and fumarate levels were negatively correlated with ZEB1 and ZEB2 expression. These findings demonstrate the potential of Oroxylin A as a natural agent to control toxigenic fungi and their mycotoxin.


Introduction
Zearalenone (ZEA) is a mycotoxin that is mainly produced by Fusarium species, including F. graminearum, which is found to contaminate a wide range of foods and feeds [1,2].ZEA has a high thermal stability, so it is difficult to degrade during the processing, storage, or handling of foods and feeds, constituting a considerable threat to human and animal health [3,4].After ingestion, ZEA and its reduced metabolites can bind to and activate estrogen receptors because of their structural similarity to estrogens, which, in turn, causes reproductive dysfunction in female animals such as abortion and false estrus, as well as immunotoxicity and advanced puberty [5].
Toxins 2023, 15, 535 2 of 14 In recent years, many synthetic antifungal agents have been applied to inhibit the growth of F. graminearum and other fungi, but the use of synthetic antifungal agents has resulted in adverse effects on the environment, the safety of agricultural products, and human and livestock health [6].Therefore, there is a need to find new and less harmful antifungal and mycotoxin controlling agents; fortunately, multiple active ingredients in plant extracts provide excellent options as alternatives to synthetic antifungal agents due to their good antifungal activities and bio-degrading nature and because they are not toxic to the environment [7][8][9][10].
P. sarmentosum is an edible herb from the Piperaceae (pepper) family; it is widely distributed throughout tropical and sub-tropical regions of the world, and it is commonly used in cooking.It has antioxidant [11], anti-inflammatory [12], and antimicrobial activities [13], and these beneficial health effects have been primarily associated with alkaloids and phenolic compounds [12,14,15].In our previous study, P. sarmentosum extract (PSE) exerted antifungal activity against F. graminearum at doses of 1 and 2 mg/mL, and 17 compounds were tentatively identified as the main antifungal constituents of PSE [16].Among the 17 compounds, Oroxylin A was the only flavonoid, and this was the first report of Oroxylin A in P. sarmentosum.Studies have shown that Oroxylin A has a broad spectrum of pharmacological effects including anti-inflammatory, anti-cancer, and anti-bacterial effects, and so on [17,18].Meanwhile, it was suggested that Oroxylin A could be functional in multiple signaling pathways and could be a promising and effective compound in the development of new additives, nutraceuticals, and therapeutic agents [18,19].However, the antifungal activity of Oroxylin A against F. graminearum has not been extensively studied.
In the past, we merely performed the tentative identification of Oroxylin A in PSE through the mzCloud database (https://www.mzcloud.org,accessed on 30 May 2021) [16].Therefore, in the present study, UPLC-QTOF-MS was used for further qualitative and quantitative validation of Oroxylin A in PSE based on the MS and MS 2 fragmentation patterns of an Oroxylin A reference standard in order to enable the determination of the suitable dosages of Oroxylin A for subsequent experiments.In addition, the antifungal and anti-mycotoxin properties of Oroxylin A against F. graminearum were assessed.S1 and Figure S1) as well as the comparison with standard Oroxylin A in this study.

Qualitative and Quantitative Analyses of
A calibration graph was plotted based on a linear regression analysis of the integrated peak areas (y) versus the concentrations (x, ng/mL) of Oroxylin A in the standard solution (regression equation, y = 571.80x− 10575.42;linear range, 20-500 ng/mL; R 2 = 0.9937), which was used to determine the Oroxylin A content of PSE as 64 ng/mg in PSE.
A previous study [16] found that 1 and 2 mg/mL of PSE had antifungal activities against F. graminearum and that Oroxylin A is one of the main antifungal compounds of PSE.In this study, 1 and 2 mg/mL of PSE were found to contain 64 ng and 128 ng Oroxylin A, respectively; therefore, the three test concentrations of Oroxylin A used here were 0 (T0; control), 64 (T1), and 128 ng/mL (T2).A calibration graph was plotted based on a linear regression analysis of the integrated peak areas (y) versus the concentrations (x, ng/mL) of Oroxylin A in the standard solution (regression equation, y = 571.80x− 10575.42;linear range, 20-500 ng/mL; R 2 = 0.9937), which was used to determine the Oroxylin A content of PSE as 64 ng/mg in PSE.
A previous study [16] found that 1 and 2 mg/mL of PSE had antifungal activities against F. graminearum and that Oroxylin A is one of the main antifungal compounds of PSE.In this study, 1 and 2 mg/mL of PSE were found to contain 64 ng and 128 ng Oroxylin

Antifungal Activity of Oroxylin A against F. graminearum
Oroxylin A contents of 64 and 128 ng/mL both inhibited the growth of F. graminearum on agar plates 3 and 5 days after inoculation (Figure 2A,B), and the inhibition was quantified (Figure 2C).An Oroxylin A content of 64 ng/mL (T1) inhibited growth by 22.4 and 15.0% after 3 and 5 days of incubation, respectively, and 128 ng/mL (T2) of Oroxylin A inhibited growth by 43.4 and 20.1% after 3 and 5 days, respectively.The T2 inhibition was higher than that of T1 (p < 0.05).
Oroxylin A contents of 64 and 128 ng/mL both inhibited the growth of F. gramin on agar plates 3 and 5 days after inoculation (Figure 2A,B), and the inhibitio quantified (Figure 2C).An Oroxylin A content of 64 ng/mL (T1) inhibited growth b and 15.0% after 3 and 5 days of incubation, respectively, and 128 ng/mL (T2) of Or A inhibited growth by 43.4 and 20.1% after 3 and 5 days, respectively.The T2 inh was higher than that of T1 (p < 0.05).

Anti-Mycotoxin Activity of Oroxylin A against F. graminearum
The anti-mycotoxin activity of Oroxylin A against F. graminearum was determined; there was a downward trend in the ZEA production with an increasing Oroxylin A concentration (Figure 3A), and an Oroxylin A content of 128 ng/mL significantly inhibited ZEA production compared with T0 (p < 0.05).
To help elucidate the mechanism of ZEA production inhibition by Oroxylin A, the mRNA expressions of two key genes (ZEB1 and ZEB2) that regulate ZEA synthesis were assessed.Oroxylin A at both treatment concentrations markedly and similarly reduced ZEB1 expression (p < 0.001) (Figure 3B), whereas it reduced ZEB2 expression less strongly, but in a dose-dependent manner (p < 0.01).

Untargeted Metabolomic Analysis of Oroxylin A-Treated F. graminearum
The global metabolomic profiling of F. graminearum mycelia detected 201 metabolite features across all experimental groups.To examine the metabolic differences in F. graminearum resulting from the T0, T1, and T2 treatments, the metabolomic data were analyzed using unsupervised PCA; the score plot revealed that PC1 accounted for 23.7% of the total variation and PC2 accounted for 14.3% of the total variation (Figure 4A).However, the separation between the three treatments was poor, and this model was unable to clearly distinguish between the treatments.To help elucidate the mechanism of ZEA production inhibition by Oroxylin A, the mRNA expressions of two key genes (ZEB1 and ZEB2) that regulate ZEA synthesis were assessed.Oroxylin A at both treatment concentrations markedly and similarly reduced ZEB1 expression (p < 0.001) (Figure 3B), whereas it reduced ZEB2 expression less strongly, but in a dose-dependent manner (p < 0.01).

Untargeted Metabolomic Analysis of Oroxylin A-Treated F. graminearum
The global metabolomic profiling of F. graminearum mycelia detected 201 metabolite features across all experimental groups.To examine the metabolic differences in F. graminearum resulting from the T0, T1, and T2 treatments, the metabolomic data were analyzed using unsupervised PCA; the score plot revealed that PC1 accounted for 23.7% of the total variation and PC2 accounted for 14.3% of the total variation (Figure 4A).However, the separation between the three treatments was poor, and this model was unable to clearly distinguish between the treatments.Supervised PLS-DA was applied to the data (Figure 4B), which resulted in a clear separation between the groups and a marked dose-response relationship, with the T0/T2 separation being approximately double that of the T0/T1 separation.This also indicates a dose-response relationship in the metabolic changes induced by Oroxylin A treatment.
Among all the metabolites detected in the fungal mycelium, there were nine differential metabolites between T1 and T0, and eight differential metabolites between T2 and T0 (Table S2).Out of these, cis-aconitate and fumarate are two important intermediate metabolites of the TCA cycle.It was noticed that compared with T0, the cis-aconitate concentration (p < 0.05) in T1 and that of fumarate (p < 0.01) in T2 both increased (Figure 5A).In addition, the concentration of ribose-5-phosphate (R5P) decreased in both T1 and T2 (p < 0.05); this was the only differential metabolite that was common to both treatments (Figure 5B).Supervised PLS-DA was applied to the data (Figure 4B), which resulted in a clear separation between the groups and a marked dose-response relationship, with the T0/T2 differential metabolites between T1 and T0, and eight differential metabolites between T2 and T0 (Table S2).Out of these, cis-aconitate and fumarate are two important intermediate metabolites of the TCA cycle.It was noticed that compared with T0, the cis-aconitate concentration (p < 0.05) in T1 and that of fumarate (p < 0.01) in T2 both increased (Figure 5A).In addition, the concentration of ribose-5-phosphate (R5P) decreased in both T1 and T2 (p < 0.05); this was the only differential metabolite that was common to both treatments (Figure 5B).

Correlation Analysis between Differential Metabolites and Expression Changes of ZEB1 and ZEB2 Genes
Pearson's correlation analysis (Figure 6) showed significant correlations between most of the differential metabolites and the ZEB1 and/or ZEB2 mRNA levels.Seven metabolites correlated with both ZEB1 and ZEB2 (p < 0.05), namely aminoadipic acid, cis-aconitate, p-aminobenzoate, and R5P in T1 (Figure 6A), and spermine, fumarate, and L-homocysteic acid in T2 (Figure 6B).

Correlation Analysis between Differential Metabolites and Expression Changes of ZEB1 and ZEB2 Genes
Pearson's correlation analysis (Figure 6) showed significant correlations between most of the differential metabolites and the ZEB1 and/or ZEB2 mRNA levels.Seven metabolites correlated with both ZEB1 and ZEB2 (p < 0.05), namely aminoadipic acid, cisaconitate, p-aminobenzoate, and R5P in T1 (Figure 6A), and spermine, fumarate, and Lhomocysteic acid in T2 (Figure 6B).

Discussion
In our previous study, we found that P. sarmentosum extract (PSE) inhibited F. graminearum growth at concentrations of 1 and 2 mg/mL, and Oroxylin A was tentatively identified as a major contributor to F. graminearum inhibition in PSE [16].In this study, the Oroxylin A of the PSE was further qualified and quantified based on UPLC-QTOF-MS/MS analysis by using the authentic standard.The analyses revealed a very low Oroxylin A content of 64 ng/mg in PSE.Subsequently, the in vitro antifungal activity evaluation demonstrated that Oroxylin A showed effective inhibitory activity against F. graminearum at concentrations of both 64 and 128 ng/mL, and the inhibition was dose-dependent.Moreover, we also found that pure Oroxylin A treatment inhibited ZEA production and downregulated the expressions of ZEB1 and ZEB2.It was known that ZEA is a polyketide mycotoxin produced by F. graminearum and its biosynthesis is closely related to the expressions of two genes (ZEB1 and ZEB2) [20].This may suggest that ZEB1 and ZEB2 act as effector sites for Oroxylin A and are involved in regulating ZEA production.

Discussion
In our previous study, we found that P. sarmentosum extract (PSE) inhibited F. graminearum growth at concentrations of 1 and 2 mg/mL, and Oroxylin A was tentatively identified as a major contributor to F. graminearum inhibition in PSE [16].In this study, the Oroxylin A of the PSE was further qualified and quantified based on UPLC-QTOF-MS/MS analysis by using the authentic standard.The analyses revealed a very low Oroxylin A content of 64 ng/mg in PSE.Subsequently, the in vitro antifungal activity evaluation demonstrated that Oroxylin A showed effective inhibitory activity against F. graminearum at concentrations of both 64 and 128 ng/mL, and the inhibition was dose-dependent.Moreover, we also found that pure Oroxylin A treatment inhibited ZEA production and downregulated the expressions of ZEB1 and ZEB2.It was known that ZEA is a polyketide mycotoxin produced by F. graminearum and its biosynthesis is closely related to the expressions of two genes (ZEB1 and ZEB2) [20].This may suggest that ZEB1 and ZEB2 act as effector sites for Oroxylin A and are involved in regulating ZEA production.
Previous work has revealed that Oroxylin A is a methoxyflavone, belongs to the class of phenolic compounds, and displays diverse biological activities including antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory activities [21], while the antimicrobial activities of phenolic compounds result from their hydrophobic characteristic.And this chemical characteristic permits them to cross the lipid layers of the cell membrane and interact with cell compounds, which could lead to a deformation in the cell structure and functionality, ultimately causing cell death [6].It was also reported that the phenolic hydroxyl group at the C-7 position of Oroxylin A is essential for antimicrobial activity [22], and the presence of a methoxyl (-OCH 3 ) group at C-6 contributes to its ability to bind to intracellular membrane structures, which may improve its bioavailability and antimicrobial activity [23,24].It appears that the inhibitory effects of Oroxylin A against F. graminearum are related to its chemical structure.
Metabolically, there are some similarities and differences of the differential metabolites in the groups treated with various concentrations of Oroxylin A (T1 and T2) compared to T0.This seems to imply that the different concentrations of Oroxylin A could have resulted in different fine-tuning levels of the inhibition mechanism on F. graminearum, which, in turn, could have led to different levels of metabolites [25].Notably, R5P was found be the only shared differential metabolite between both treatment concentrations (T1 and T2).It was reported that R5P is not only an important product of the pentose phosphate pathway [26], but can also be converted to phosphoribosyl pyrophosphate (PRPP) [27].Speculatively, Oroxylin A suppresses the R5P concentration by possibly inhibiting the pentose phosphate pathway and, consequently, purine metabolism, by decreasing the supply of PRPP, which is required for ATP generation and purine nucleotide biosynthesis in F. graminearum (Figure 5B) [28,29].Alternatively, a downregulated pentose phosphate pathway may result in decreased NADPH generation and increased oxidative stress, which imbalances the redox system in F. graminearum, thereby retarding its growth and development [30,31].
Most importantly, Oroxylin-A-treated F. graminearum had increased concentrations of cis-aconitate (T1) or fumarate (T2); both are important metabolic cycling products in the TCA cycle (Figure 5A) and were negatively correlated with ZEB1 and ZEB2 expressions.Therefore, the accumulation of cis-aconitate or fumarate suggests that the mitochondrial TCA cycle in F. graminearum might be inhibited, which would decrease ATP production, induce mitochondrial dysfunction, and reduce macromolecule (lipids, nucleotides, and proteins) biosynthesis.These metabolic imbalances would impair F. graminearum growth and ZEA production or even induce fungal death [32,33].Moreover, the increased itaconic acid level in both T1 (FC = 1.75; p = 0.008) and T2 (FC = 1.51; p = 0.287) suggests that Oroxylin A may increase the decarboxylation of the TCA cycle's intermediate cis-aconitate into itaconic acid, which is an important anti-bacterial compound [33].

Conclusions
To sum up, Oroxylin A effectively suppressed the growth of F. graminearum and ZEA production, and we suggest that Oroxylin A could be used as a promising antifungal and mycotoxin controlling agent.

Fungal Culture
The pathogenic ZEA-producing fungus, the F. graminearum strain (AF 2015031), isolated from diseased maize stems, was from the China Center for Type Culture Collection (CCTCC, Wuhan, China).Fungal cultures were maintained on PDA medium and were incubated in the dark at 28 • C, as described previously [16].

Preparation of Oroxylin A Standard Solution and PSE
Oroxylin A standard was made with up to 1 mg/mL stock solution in methanol.The stock solution was further diluted with methanol to produce a dilution series (20,50,100,200, and 500 ng/mL), and was used to prepare calibration curves.PSE was prepared as described previously [16] and made up to 10 mg/mL in DMSO, then diluted to a final concentration of 1 mg/mL with methanol for quantitative analysis of Oroxylin A.

UPLC-QTOF-MS/MS Analysis
Oroxylin A in PSE was quantified via UPLC-QTOF-MS/MS.Peak area was extracted and integrated after peak alignment and retention time correction.Calibration curves (five points) were obtained using external standard calibrations for Oroxylin A by injecting the standard solution in the concentration range of 20 ng/mL to 500 ng/mL.Calibration curves were established by plotting the peak area (y) versus concentration (x) of each analyte.
Samples were analyzed in the electrospray ionization (ESI)-negative mode.Data acquisition was carried out in full scan mode (m/z range from 70 to 1000) combined with information-dependent acquisition (IDA) mode.For IDA analysis, a collision energy of 30 eV was used to fragment the parent ions.The drying gas temperature was set at 450 • C (ESI-), and the ion spray voltage was −4500 V (ESI-).Atomization gas pressure, auxiliary heating gas pressure, and curtain gas pressure in negative ionization mode were set at 55, 55, and 35 psi, respectively.The instrument was mass calibrated automatically by infusing the Sciex Negative Calibration Solution (part no.4460134) for negative mode after every six samples injected.One QC sample and one blank vial were run after every 10 samples.

Experimental Design
This study consisted of two experiments.Experiment I was performed with antifungal assays, and Experiment II was performed with a mycotoxin inhibition assay.For Experiment I, PDA was used as the growth medium for fungal culture, and for Experiment II, PDB liquid culture medium was used for fungal growth and mycotoxin production.Experiments I and II were both performed with 0 (control), 64, and 128 ng/mL Oroxylin A and designated as T0, T1, and T2, respectively.Three replicates of Experiment I and five replicates of Experiment II were performed.The Oroxylin A concentrations in T1 was equivalent to 1 mg of PSE, and that of T2 was equivalent to 2 mg of PSE, calculated based on the quantitation of Oroxylin A in PSE.

Antifungal Activity of Oroxylin A against F. graminearum
The antifungal activity of Oroxylin A against F. graminearum was determined in vitro by measuring the linear growth rate of mycelium as reported previously [16].Briefly, plugs of fungal growth on agar (4 mm diameter, from 7-day-old cultures) were inoculated onto plates and incubated at 28 • C for up to 5 days.The colony diameter of each plate was measured with the original mycelial plug diameter (4 mm) subtracted from the measurement after 3 and 5 days of inoculation, respectively, and the growth inhibition rate was calculated as follows: inhibition rate (%) = (Dc − Dt)/ Dc × 100%, where Dc represents the control (T0) colony diameter and Dt represents the colony diameter of the Oroxylin A treatments (T1 or T2).The results are presented as the mean ± standard error of the mean (SEM).

Anti-Mycotoxin Activity of Oroxylin A against F. graminearum
The anti-mycotoxin activity of Oroxylin A in liquid cultures was determined as described previously [34] with minor modifications.In brief, a stock solution of Oroxylin A (1 mg/mL in DMSO) was prepared; then, an appropriate volume was added to a 100 mL centrifuge tube containing PDB (50 mL) to prepare media T1 and T2.Subsequently, fungal spore suspension (5 µL, 1 × 10 6 spores/mL) from a 7-day-old culture was inoculated into tubes of T0, T1, and T2 media and inoculated with shaking (140-160 rpm) at 28 • C for 14 days.
After 14 days, the fungal biomass was separated from the culture medium with Whatman No. 1 filter paper, and the ZEA concentration in the filter was determined.The fungal mycelium was washed twice with sterile distilled water, transferred to cryotubes, frozen in liquid nitrogen, and stored at −80 • C until needed for RNA and metabolite extraction.

UPLC Analysis of ZEA
Before the UPLC analysis, the ZEA in the growth medium was extracted and cleaned up using an IAC-SEP ® ZEA-specific immunoaffinity column (Clover Technology Group, Beijing, China) according to the manufacturer's instructions.The resulting sample was transferred to an autosampler vial, and 100 µL was injected into the UPLC column for analysis.
The ZEA content was tested following the Chinese standard method GB 5009.209-2016[35].Quantitative analysis was performed on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) consisting of an Ultimate 3000 RS autosampler, pump, column oven, and fluorescence detector, and controlled by Chromeleon 7.2 software.Separation was performed on a Zorbax Eclipse XDB-C18 RRHD column (100 × 2.1 mm, 1.8 µm; Agilent).The isocratic mobile phase was acetonitrile/water/methanol (46:46:8, v/v) with a flow rate of 1.0 mL/min.The fluorescence detection excitation wavelength was 274 nm, and the emission wavelength was 440 nm.
The ZEA was identified and quantified via comparison with the retention time and the chromatographic peak area of an external standard.The standard curve was obtained by preparing standard solutions at six ZEA concentrations (5,10,50,100,200, and 500 ng/mL), and each concentration was analyzed in triplicate.The resulting regression equation for ZEA was as follows: y = 2912.3x− 6081.6 (R 2 = 0.9998).
mRNA Expression Analysis of ZEA Biosynthesis Genes via RT-PCR The total RNA was extracted using Trizol reagent (Tiangen, Beijing, China), according to the manufacturer's protocol.Quality and quantity of RNA were measured via RNA 6000 Nano LabChip kit using a Bioanalyzer (Agilent).The cDNA was synthesized using RevertAid Reverse Transcriptase following the manufacturer's protocol (Thermo Fisher).Quantitative RT-PCR was performed in an ABI Q1 PCR system (Thermo Fisher) using PerfectStart Green qPCR SuperMix (TransGen, Beijing, China).In brief, the 10 µL reaction mixture consisted of 2 × SYBR Green RT-PCR reaction mix (5 µL), forward primer (10 nM; 0.3 µL), reverse primer (10 nM; 0.3 µL), cDNA (2 µL), and nuclease-free water (PCR grade; 2.4 µL).The temperature program comprised 10 min of starting template denaturation at 94 • C for 1 cycle, followed by 40 cycles of PCR at 95 • C for 5 s, 60 • C for 15 s, and 72 • C for 10 s.The primer sequences used were as described previously [36] and are listed in Table S3.The synthesized primer sequences were obtained from Sangon Biotech (Shanghai, China).Elongation factor 1-α (EF1-α) was used as a house-keeping gene.Each reaction was completed with a melting curve analysis to ascertain that only the expected amplification products were generated.PCR data were analyzed using the 2 −∆∆CT method to calculate the relative expression levels of the target gene.

Metabolomic Analysis
Fungal mycelia were extracted and analyzed as described previously [11].Briefly, aliquots of mycelium (100 mg) were extracted with ice-cold methanol, and after centrifugation at 20,000× g for 10 min at 4 • C, the supernatant was collected, dried in an SPD121P SpeedVac vacuum concentrator (Thermo Fisher), and resuspended in acetonitrile.Analysis was performed via liquid chromatography/tandem mass spectrometry on a Triple TOF TM Oroxylin A in Piper Sarmentosum Extract (PSE) Oroxylin A was characterized in PSE using UPLC-QTOF-MS/MS.The extracted ion chromatogram (EIC) and MS/MS fragmentation patterns of Oroxylin A in PSE are shown in Figure 1A,B, respectively.Oroxylin A (C 16 H 12 O 5 ) had the following characteristics: retention time (Rt), 3.11 min; [M-H] − /m/z, observed value of 283.0351, calculated value of 283.0606, and mass error of −9.0 ppm; and MS 2 fragment ion, 268.0136 [M-H-CH 3 ] − .The identification of the observed compound as Oroxylin A was confirmed (Figure 1B) by the [M-H] − precursor ion at m/z 283.0351 [C 16 H 12 O 5 ] and CID of [M-H] − (loss of CH 3 ; 15 Da) to generate a fragment ion at m/z 268.0136 [C 15 H 9 O 5 ], which is consistent with our previous MS-based qualitative analysis of Oroxylin A (Table

Figure 3 .
Figure 3. Inhibitory effect of Oroxylin A on (A) zearalenone (ZEA) production, and (B) the mRNA expression of ZEA-biosynthesis-related genes (ZEB1 and ZEB2) of F. graminearum in liquid culture.All data are expressed as the mean ± SEM.Significance: * p < 0.05 and *** p < 0.001.

Figure 3 .
Figure 3. Inhibitory effect of Oroxylin A on (A) zearalenone (ZEA) production, and (B) the mRNA expression of ZEA-biosynthesis-related genes (ZEB1 and ZEB2) of F. graminearum in liquid culture.All data are expressed as the mean ± SEM.Significance: * p < 0.05 and *** p < 0.001.

Figure 6 .
Figure 6.Pearson's correlation analysis of differential metabolites and ZEA-biosynthesis-related genes between T1 and T0 (A), and between T2 and T0 (B).Each column represents a gene, each row represents a metabolite, and each square represents a correlation coefficient between the corresponding gene and metabolite.The color scale indicates the correlation strength, red indicates a positive correlation, and blue indicates a negative correlation.Significance: * p < 0.05.

Figure 6 .
Figure 6.Pearson's correlation analysis of differential metabolites and ZEA-biosynthesis-related genes between T1 and T0 (A), and between T2 and T0 (B).Each column represents a gene, each row represents a metabolite, and each square represents a correlation coefficient between the corresponding gene and metabolite.The color scale indicates the correlation strength, red indicates a positive correlation, and blue indicates a negative correlation.Significance: * p < 0.05.