Genomics- and Transcriptomics-Guided Discovery of Clavatols from Arctic Fungi Penicillium sp. MYA5

Clavatols exhibit a wide range of biological activities due to their diverse structures. A genome mining strategy identified an A5cla cluster from Penicillium sp. MYA5, derived from the Arctic plant Dryas octopetala, is responsible for clavatol biosynthesis. Seven clavatols, including one new clavatol derivate named penicophenone F (1) and six known clavatols (2–7), were isolated from Penicillium sp. MYA5 using a transcriptome mining strategy. These structures were elucidated by comprehensive spectroscopic analysis. Antibacterial, aldose reductase inhibition, and siderophore-producing ability assays were conducted on compounds 1–7. Compounds 1 and 2 demonstrated inhibitory effects on the ALR2 enzyme with inhibition rates of 75.3% and 71.6% at a concentration of 10 μM, respectively. Compound 6 exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli with MIC values of 4.0 μg/mL and 4.0 μg/mL, respectively. Additionally, compounds 1, 5, and 6 also showed potential iron-binding ability.


Introduction
Iron is an indispensable element for plant growth and development.Iron mainly exists in the form of insoluble substances such as iron oxide or iron hydroxide, which is difficult to be directly absorbed by plants [1].The endophytic fungi in the vast majority of plants can synthesize siderophores, facilitating increased iron absorption by the host from the surrounding environment [2][3][4][5][6][7].Some endophytes residing within plants cultivated in extreme environments enhanced host plant resistance to nutrient deficiencies and cold conditions by producing siderophores [8][9][10].Siderophores, which function as iron chelators secreted by microorganisms, can be classified into four groups based on their functional groups, including catecholates, hydroxamates, carboxylates, and phenolates [11][12][13].
Clavatols, a family of aromatic polyketides with phenolic groups, play a pivotal role in microorganisms against adverse environmental changes.Clavatols showed inhibitory effects against various plant pathogenic fungi, such as Staphylococcus greyi, Dictyostelium tylosporium, and Mycosphaerella solanacearum [14].Furthermore, clavatol derivatives revealed structure diversity and a variety of biological activities, such as antiviral [15] and inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B [16].
The development and popularity of sequencing technology have led to a new trend in the genome-directed discovery of specified secondary metabolites.Through our investigation focusing on siderophores, it was elucidated that Penicillium sp.MYA5 possesses the capacity to produce siderophores (Figure S1).Genome sequencing and mining of secondary metabolism biosynthesis gene clusters in Penicillium sp.MYA5 revealed gene clusters related to clavatols.Eight clavatols, including one new clavatol derivate, penicophenone F (1), and seven known clavatols (2-7), were isolated from Penicillium sp.MYA5 based on transcriptome mining strategy (Figure 1).The structures of all the compounds were unambiguously elucidated by extensive spectroscopic analysis, complemented by literature comparisons.This study contributes to the comprehensive understanding of clavatols, encompassing their discovery, isolation, purification, structural characterization, and bioactivity evaluation.
Mar. Drugs 2024, 22, x FOR PEER REVIEW 2 of the capacity to produce siderophores (Figure S1).Genome sequencing and mining of s ondary metabolism biosynthesis gene clusters in Penicillium sp.MYA5 revealed gene clu ters related to clavatols.Eight clavatols, including one new clavatol derivate, penicoph none F (1), and seven known clavatols (2-7), were isolated from Penicillium sp.MY based on transcriptome mining strategy (Figure 1).The structures of all the compoun were unambiguously elucidated by extensive spectroscopic analysis, complemented literature comparisons.This study contributes to the comprehensive understanding clavatols, encompassing their discovery, isolation, purification, structural characteriz tion, and bioactivity evaluation.

Based on Genomics and Transcriptomics to Discovery Clavatols.
The strain Penicillium sp.MYA5 underwent genome sequencing using the Illumi HiSeq 2000 platform.The complete genome was composed of 32,800,485 bp, with an a erage GC content of 47.97%.Genome annotation resulted in the detection of 11,248 p tein-coding sequences, 235 tRNA genes, and 55 rRNA operons.
For the determination of the evolutionary relationship of Penicillium sp.MYA5 w other Penicillium strains, a whole-genome core SNP-based phylogenetic tree was co structed and phylogenetic analysis revealed that Penicillium sp.MYA5 was closely relat to Peniciilium isariiforme (Figure S2).
Secondary metabolite biosynthetic gene clusters (BGCs) in the draft genome of Pe cillium sp.MYA5 were predicted using the antiSMASH version 6.1.1 [17].The results vealed that Penicillium sp.MYA5 could synthesize abundant secondary metabolites, whi might be an important source of novel bioactive compounds.Forty-six completely quenced biosynthetic gene clusters were predicted in the Penicillium sp.MYA5 genom and contained seven polyketide synthases (PKSs), twenty non-ribosomal peptide synth tases (NRPSs), twelve hybrid NRPS-PKSs, five terpenes, two indoles, and others.The d tails of the location of all the sequenced biosynthetic gene clusters are shown in Table S Aromatic polyketones were synthesized by non-reducing polyketide synthase (N PKS) in fungi and the Penicillium sp.MYA5 genome contained three NR-PKS-containi clusters.We identified a unique NR-PKS cluster 3.1 (A5cla) as a candidate biosynthe gene cluster.In order to postulate putative cluster products from the identified cluster, w conducted BLAST analyses of all the clusters across the genomes present in the GenBa archive.This identified cluster 3.1 (A5cla) with high homology to cluster cla in Penicilliu crustosum [18] (Tables S3), for which the biosynthetic product had been experimenta

Based on Genomics and Transcriptomics to Discovery Clavatols
The strain Penicillium sp.MYA5 underwent genome sequencing using the Illumina HiSeq 2000 platform.The complete genome was composed of 32,800,485 bp, with an average GC content of 47.97%.Genome annotation resulted in the detection of 11,248 proteincoding sequences, 235 tRNA genes, and 55 rRNA operons.
For the determination of the evolutionary relationship of Penicillium sp.MYA5 with other Penicillium strains, a whole-genome core SNP-based phylogenetic tree was constructed and phylogenetic analysis revealed that Penicillium sp.MYA5 was closely related to Peniciilium isariiforme (Figure S2).
Secondary metabolite biosynthetic gene clusters (BGCs) in the draft genome of Penicillium sp.MYA5 were predicted using the antiSMASH version 6.1.1 [17].The results revealed that Penicillium sp.MYA5 could synthesize abundant secondary metabolites, which might be an important source of novel bioactive compounds.Forty-six completely sequenced biosynthetic gene clusters were predicted in the Penicillium sp.MYA5 genome and contained seven polyketide synthases (PKSs), twenty non-ribosomal peptide synthetases (NRPSs), twelve hybrid NRPS-PKSs, five terpenes, two indoles, and others.The details of the location of all the sequenced biosynthetic gene clusters are shown in Table S2.
Aromatic polyketones were synthesized by non-reducing polyketide synthase (NR-PKS) in fungi and the Penicillium sp.MYA5 genome contained three NR-PKS-containing clusters.We identified a unique NR-PKS cluster 3.1 (A5cla) as a candidate biosynthetic gene cluster.In order to postulate putative cluster products from the identified cluster, we conducted BLAST analyses of all the clusters across the genomes present in the GenBank archive.This identified cluster 3.1 (A5cla) with high homology to cluster cla in Penicillium crustosum [18] (Table S3), for which the biosynthetic product had been experimentally determined as clavatols, allowing us to predict clavatols that are produced by Penicillium sp.MYA5.The A5cla cluster comprised eleven ORFs, encoding one NR-PKS (gene03823), two HR-PKSs (gene03826 and gene03827), one Enoyl-CoA isomerase (gene03820), one cytochrome P450 oxidase (gene03828), one Fe (II) oxygenase (gene03821), one ABC-type transporter (gene03824), one transcription factor (gene03818), and three hypothetical proteins (gene03819, gene03822, and gene03825) (Figure 2).
Mar. Drugs 2024, 22, x FOR PEER REVIEW 3 of 12 determined as clavatols, allowing us to predict clavatols that are produced by Penicillium sp.MYA5.The A5cla cluster comprised eleven ORFs, encoding one NR-PKS (gene03823), two HR-PKSs (gene03826 and gene03827), one Enoyl-CoA isomerase (gene03820), one cytochrome P450 oxidase (gene03828), one Fe (II) oxygenase (gene03821), one ABC-type transporter (gene03824), one transcription factor (gene03818), and three hypothetical proteins (gene03819, gene03822, and gene03825) (Figure 2).We performed GO enrichment analysis using A5cla to identify its involvement with secondary metabolic processes.We searched the genes of each GO term whose function related to transferase activity, acyltransferase activity, oxidoreductase activity, modified amino acid binding, etc.There were two genes (gene03823 and gene03827) under each of the GO terms "secondary metabolite biosynthetic process" and "secondary metabolic process" (Figure 3) and four genes (gene03821, gene03823, gene03824, and gene03828) under "oxidoreductase activity", "transferase activity", and "binding" (Figure 3).In this study, by annotating the gene function through GO analyses, we identified two key genes and four modification genes.We performed GO enrichment analysis using A5cla to identify its involvement with secondary metabolic processes.We searched the genes of each GO term whose function related to transferase activity, acyltransferase activity, oxidoreductase activity, modified amino acid binding, etc.There were two genes (gene03823 and gene03827) under each of the GO terms "secondary metabolite biosynthetic process" and "secondary metabolic process" (Figure 3) and four genes (gene03821, gene03823, gene03824, and gene03828) under "oxidoreductase activity", "transferase activity", and "binding" (Figure 3).In this study, by annotating the gene function through GO analyses, we identified two key genes and four modification genes.However, cluster A5cla still differed from the identified cluster cla, suggesting tha further research on Penicillium sp.MYA5 would undoubtedly be worthwhile.
To investigate the optimum cultivation conditions of secondary metabolite accumu lation in Penicillium sp.MYA5, we selected PDA medium for 45 days and KSB medium fo 45 days of fermentation of crude extract for transcriptome analysis.The expression pat terns of the eleven genes in cluster A5cla were analyzed using clustering analysis (Figur 4A).The figure shows that gene03818-gene03824 and gene03828 exhibited higher expres sion levels under PDA culture conditions for 45 days, while gene03825 and gene0382 demonstrated the highest expression levels under KSB culture conditions for 45 days.Ad ditionally, quantitative real-time polymerase chain reaction (RT-qPCR) was employed t However, cluster A5cla still differed from the identified cluster cla, suggesting that further research on Penicillium sp.MYA5 would undoubtedly be worthwhile.
To investigate the optimum cultivation conditions of secondary metabolite accumulation in Penicillium sp.MYA5, we selected PDA medium for 45 days and KSB medium for 45 days of fermentation of crude extract for transcriptome analysis.The expression patterns of the eleven genes in cluster A5cla were analyzed using clustering analysis (Figure 4A).The figure shows that gene03818-gene03824 and gene03828 exhibited higher expression levels under PDA culture conditions for 45 days, while gene03825 and gene03826 demonstrated the highest expression levels under KSB culture conditions for 45 days.Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) was employed to determine the expression profiles of target gene clusters under two different culture conditions.The results showed that the A5cla cluster exhibited higher gene expression under PDA culture conditions for 45 days compared to KSB culture conditions for 45 days (Figure 4B).The strains cultured in Penicillium sp.MYA5 on PDA for 45 days exhibited high expression of gene03819, gene03823, gene03824, and gene03828 and low expression of gene03825 and gene03826 (Figure 4B).Considering the expression profiles of A5cla under different culture conditions, we ultimately selected the PDA culture for large-scale fermentation for 45 days.

Structural Characterization of Compounds 1-7
Compound 1 was isolated as a yellow amorphous solid.Its molecular formula was determined to be C13H18O5 by HRESIMS (m/z 277.1048 [M + Na] + , calculated for C13H18O5Na 277.1046), indicating five degrees of unsaturation.The IR spectrum exhibited major absorption bands at 3331 cm −1 , 2954 cm −1 , 2922 cm −1 , 2852 cm −1 , and 1449 cm −1 , corresponding to hydroxyl and aromatic rings, respectively [19].The 1 H NMR spectrum of 1 revealed signals attributed to two methyl singlets at δH 2.18 and 2.55 and one aromatic proton singlet at δH 7.52 (Table 1).The 13 C NMR spectrum of 1 revealed one ketone carbonyl at δC 203.2, two oxygenated quaternary carbons at δC 161.1 and 161.5, three sp 2 hybridized carbons at δC 112.4,112.9, and 116.7, and two oxygenated methylenes at δC 61.8 and 61.8 (Table 1).A comparison of the 1 H and 13 C NMR data of 1 with those of the known compound 2 revealed that they shared the same phenol skeleton [20], which was further confirmed by the HMBC from H-9 to C-3, C-4, and C-2, from H-2 to C-7, C-1, C-6, C-4, and C-3, and from H-8 to C-1 and C-7, as shown in Figure 5.The COSY spectrum revealed the presence of one isolated spin system: C-10-C-11-C-12-C-13 (Figure 5).Ultimately, one new clavatol derivate, penicophenone F (1), and seven known clavatols (2-7) were identified from the crude extract of Penicillium sp.MYA5 after 45 days of PDA growth.

Structural Characterization of Compounds 1-7
Compound 1 was isolated as a yellow amorphous solid.Its molecular formula was determined to be C 13 H 18 O 5 by HRESIMS (m/z 277.1048 [M + Na] + , calculated for C 13 H 18 O 5 Na 277.1046), indicating five degrees of unsaturation.The IR spectrum exhibited major absorption bands at 3331 cm −1 , 2954 cm −1 , 2922 cm −1 , 2852 cm −1 , and 1449 cm −1 , corresponding to hydroxyl and aromatic rings, respectively [19].The 1 H NMR spectrum of 1 revealed signals attributed to two methyl singlets at δ H 2.18 and 2.55 and one aromatic proton singlet at δ H 7.52 (Table 1).The 13 C NMR spectrum of 1 revealed one ketone carbonyl at δ C 203.2, two oxygenated quaternary carbons at δ C 161.1 and 161.5, three sp 2 hybridized carbons at δ C 112.4, 112.9, and 116.7, and two oxygenated methylenes at δ C 61.8 and 61.8 (Table 1).A comparison of the 1 H and 13 C NMR data of 1 with those of the known compound 2 revealed that they shared the same phenol skeleton [20], which was further confirmed by the HMBC from H-9 to C-3, C-4, and C-2, from H-2 to C-7, C-1, C-6, C-4, and C-3, and from H-8 to C-1 and C-7, as shown in Figure 5.The COSY spectrum revealed the presence of one isolated spin system: C-10-C-11-C-12-C-13 (Figure 5).HMBC correlations from H-11 to C-5, from H-10 to C-5, and from H-10 to C-6 provided evidence of the direct linkage between C-10 and C-5.Furthermore, two hydroxy groups were attached to C-6 (δ C 161.1) and C-4 (δ C 161.5) based on the molecular formula, respectively.

Biological Activity Assay
Compounds 1-7 were evaluated for antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Vibrio vulnificus, and Vibrio parahaemolyticus (Table 2).Compound 6 exhibited antibacterial activity against S. aureus and E. coli with MIC values of 4.0 µg/mL for both, whereas compounds 1-5 and 7 exhibited different levels of antibacterial activity against different microbes, as shown in Table 2.

Biological Activity Assay
Compounds 1-7 were evaluated for antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Vibrio vulnificus, and Vibrio parahaemolyticus (Table 2).Compound 6 exhibited antibacterial activity against S. aureus and E. coli with MIC values of 4.0 µg/mL for both, whereas compounds 1-5 and 7 exhibited different levels of antibacterial activity against different microbes, as shown in Table 2.In the current study, an in vitro ALR2 inhibition assay demonstrated that compounds 1 and 2, with inhibition rates of 75.3% and 71.6% at a concentration of 10 µM, respectively, displayed superior inhibitory activities compared with other compounds (Table 3) with epalrestat as the positive control.Compounds 1 and 2, therefore, represented the potential drug candidates for the treatment of diabetic complications.The Fe(III)-binding properties of compounds 1-7 were assessed by the chrome azurol S (CAS) assay.Compounds 1, 5, and 6 showed binding activity (Table 4).Compounds 6 and 7 displayed a purple color when mixed with the CAS assay solution (Figure 6h,i), and compound 1 displayed a red color when mixed with the CAS assay solution (Figure 6c).Structurally, the compounds 1, 5, and 6 belong to the phenolates family.For this type of siderophore, the phenolic moiety and the number of this group are both very important in the interaction of these compounds with iron [23].
TLC analyses were conducted on pre-coated silica gel GF254 plates, and spots were visualized under UV light (254 nm) or by heating after spraying with anisaldehyde-H 2 SO 4 reagent.

Biological Material and Bioinformatics Analysis
The strain Penicillium sp.MYA5 was isolated from the Arctic Svalbard Islands (E 10 • 35 • , N 74 • 81 • ).The rDNA-ITS sequence of the strain was sequenced, and an ITS (SUB14112691) phylogenetic tree was constructed to identify the fungal strain as Penicillium sp.The strain has been deposited at the Chinese Typical Culture Preservation Center (CCTCC NO: SF2021062) and the Laboratory of Marine Biomedicine and Polar Medicine, Naval Specialty Medical Center, PLA Naval Medical University, Shanghai, People's Republic of China.
The fungal strain underwent genome sequencing using the Illumina Hiseq 2000 platform at Shanghai Meiji Biomedical Technology, and its genomic data were stored at NCBI (JAYRCP010000000).To analyze the presence of biosynthetic gene clusters on the scaffolds, we employed antiSMASH https://fungismash.secondarymetabolites.org/#!/start (accessed on 15 May 2023) and 2ndFind https://biosyn.nih.go.jp/2ndfind/ (accessed on 16 May 2023).Gene prediction for fungi was performed using Maker v2.31.9, while rRNAs and tRNAs contained in the genome were predicted using Barrnap v0.8 and tRNAscan-SE v2.0, respectively.The gene annotation results were used to analyze the expression differences and gene ontology (GO) https://www.blast2go.com/(accessed on 19 August 2023) and predict the protein-coding frames (CDS).GO enrichment analyses were performed.Verification was also conducted by Blast analysis https://blast.ncbi.nlm.nih.gov/Blast.cgi(accessed on 23 September 2023) by manually comparing the homologous gene/protein sequences in the GenBank database.

Fermentation, Extraction, and Isolation
The fungus Penicillium sp.MYA5 was cultured on potato dextrose agar medium at 28 • C for 45 days.After cultivation, the culture underwent extraction by immersion in a methanol-dichloromethane mixture (1:1) for 24 h, followed by ultrasonication for 30 min to disrupt the cells.The resulting mixture was filtered through eight layers of gauze to obtain the filtrate.Subsequently, the culture underwent three rounds of ultrasonic extraction, and the combined filtrates were concentrated under reduced pressure using a rotary evaporator until all the organic solvents were removed.An appropriate amount of water suspension was added, followed by three additional extractions with an equal volume of ethyl acetate.The ethyl acetate layer was collected and further concentrated under reduced pressure using a rotary evaporator until dry, yielding 17.38 g of the fermented extract.The total fermented extract was then subjected to silica gel (200-300 mesh) column chromatography and separated into nine fractions (Fr.A-Fr.I) using a step gradient elution of petroleum ether and ethyl acetate.

Analysis of Transcriptomic Data and RT-qPCR
Penicillium sp.MYA5 underwent culturing under two distinct conditions: potato dextrose agar (PDA) for 45 days and KSB solid medium (containing glucose 12.36 g/L, tryptone 1.05 g/L, beef extract 6.08 g/L, manganese sulfate monohydrate 0.246 g/L, agar 20 g/L) for 45 days.The fermented crude extract was then collected.Sequencing was conducted using the Illumina Novaseq 6000 platform by Shanghai Meiji Biomedical Tech-nology.To validate the RNA-seq data, RT-qPCR was performed following established protocols [24].The primers utilized for RT-qPCR analysis are detailed in Table S1, with the 16S rDNA-coding gene employed as the internal control.

In Vitro ALR2 Enzyme Inhibitory Activity Assay
100 µL of reaction mixture was composed of 20 µL of buffer (100 mM sodium phosphate, pH 6.2), 30 µL of enzyme extract, 20 µL of substrate (10 mM), 20 µL of cofactor (0.1 mM of NADPH), and 10 µL of test compound (1 mM) [25,26].The reaction mixture without cofactor was incubated at 32 • C for 10 min, then the enzymatic reaction was initiated with the addition of NADPH and monitored for 5 min.DL-glyceraldehyde was used as the substrate for the ALR2 assays.
The compounds were dissolved in 100% DMSO and diluted with deionized water, keeping the DMSO concentration equal to 0.1% in the assay.The compounds were initially tested for percent inhibition at a concentration of 10 µM.ALR2 activity was determined by monitoring the change in absorbance at 340 nm caused by decreasing the NADPH [27].Epalrestat served as the positive control.

Antibacterial Assay
The antimicrobial activities of compounds 1-7 against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Vibrio vulnificus, and Vibrio parahaemolyticus were assessed using the broth dilution method [28,29].Levofloxacin hydrochloride served as the positive control.

Chrome Azurol S Assay
An assay solution was prepared to measure the iron-binding activity of the compounds [30].Hexadecyltrimethylammonium bromide (CTAB) (21.9 mg) was diluted in 25 mL of H 2 O at 35 • C.An iron(III) chloride solution (1.5 mL, 1.0 mM) (prepared by dissolving FeCl 3 •6H 2 O in a 10 mM aqueous HCl solution) and 7.5 mL of a 2.0 mM aqueous chrome azurol S (CAS) solution were added to this solution.In a separate container, 9.76 g of 2-(N-morpholino) ethanesulfonic acid (MES) was dissolved in 50 mL of H 2 O, and the pH of this solution was adjusted to 5.6 with a 50% KOH solution.Then, the previous CTAB-CAS-Fe(III) solution was mixed with the MES buffer solution slowly, and we filled the solution with water to obtain 100 mL.The modified CAS assay solution (100 µL) was loaded into each well of a 96-well plate, which was then mixed with 100 µL of solution of compounds diluted in H 2 O.The final concentrations of each compound were 10 µg/mL.The color changes were observed by visual inspection after incubation at 37 • C for 1 h.Then, the corresponding absorbance changes were measured on a microplate reader at 630 nm.The saturated EDTA solution (which reacts with CAS detection solution in red) was used as the positive control, and the solvent dissolving the sample was mixed with CAS detection solution in the same volume as the blank control.The color changed to red, orange red, and purple, indicating that the compounds could bind with iron.

Conclusions and Discussion
In summary, genomic and transcriptomic approaches were employed to activate the expression of the A5cla cluster in Penicillium sp.MYA5.One new clavatol derivate, penicophenone F (1), and seven known clavatols (2-7) were isolated, and the structures of all the compounds were elucidated through comprehensive spectroscopic analysis.The moderate inhibitory activities of compounds 1 and 2 against ALR2 enzyme were demonstrated.Compounds 1, 5, and 6 showed potential antibiotic activity and ironbinding ability.
Siderophores produced by endophytic fungi played a pivotal role in supporting plant growth.It was conjectured that clavatols extracted from Penicillium sp.MYA5 might similarly enhance the survival and development of Dryas octopetala in iron-deficient polar environments.The synthetic biogenesis cluster of hydroxylate-type siderophores was annotated in Penicillium sp.MYA5.Hydroxamate-type siderophores constituted the primary class of siderophores synthesized by endophytic fungi [31].The correlation analysis of the A5cla cluster and the hydroxylate-type siderophore biosynthesis cluster revealed that the genes contained in the two clusters were significantly correlated (Figure S12).It was hypothesized that clavatols might act synergistically with other forms of siderophores to facilitate the normal growth and development of hosts in extreme environments.Overall, this work underscored the utility of integrating genomic and transcriptomic approaches as a systematic approach to expedite the discovery of bioactive compounds.

Figure 3 .
Figure 3. GO enrichment analysis of the cluster A5cla.

Figure 4 .
Figure 4. Transcriptomic analyses of cluster A5cla for different culture conditions.(A) Cluster heatmap of metabolite abundance based on hierarchical cluster analysis; (B) The quantitative realtime PCR (qPCR) results of several genes.

Figure 4 .
Figure 4. Transcriptomic analyses of cluster A5cla for different culture conditions.(A) Cluster heatmap of metabolite abundance based on hierarchical cluster analysis; (B) The quantitative real-time PCR (qPCR) results of several genes.
a Positive control.'NA' stands for no inhibitory effect at greater than 64 µg/mL.
a Positive control.