Naphtho‐γ‐pyrones From Aspergillus foetidus, an Endophytic Fungus Isolated From Gardenia jasminoides J. Ellis

ABSTRACT

A new naphtho‐γ‐pyrone dimer, asperforone A (1), along with thirteen known compounds, was isolated from the endophytic fungus Aspergillus foetidus of Gardenia jasminoides J. Ellis. Their structures were elucidated through comprehensive spectroscopic analysis and by comparison with literature data. Aspernigrin A (11) showed notable acetylcholinesterase inhibitory activity (46.98% at 50 µM), while asperforone A (1) and asperpyrone D (9) exhibited antimicrobial activity against Plectosphaerella cucumerina (minimum inhibitory concentration = 32 µg/mL). This study extends the chemical profile of A. foetidus and underscores its potential for bioactive metabolite discovery.

A new naphtho‐γ‐pyrone dimer, asperforone A (1), and thirteen known compounds are isolated from the endophytic fungus Aspergillus foetidus. Asperforone A (1) and asperpyrone D (9) show antimicrobial activity against Plectosphaerella cucumerina (MIC = 32 µg/mL). This work expands the chemical profile of A. foetidus, highlighting its potential for bioactive metabolite discovery.

1. Introduction

Fungi of the genus Aspergillus are widely distributed in nature, including marine sediments, soil, and plants [1]. As a key focus in microbial research, Aspergillus species are renowned for their ability to produce diverse and biologically active secondary metabolites [2], particularly monomeric and dimeric naphtho‐γ‐pyrones [3, 4, 5]. Naturally occurring naphtho‐γ‐pyrones‐based metabolites have been reported from different ascomycetes, including A. tubingensis [6], A. niger [7], A. ochraceus [8], and A. carbonarius [9]. The species A. foetidus has emerged as a source of structurally unique naphtho‐γ‐pyrones. For example, A. foetidus KMM4694 has been found to produce nine naphtho‐γ‐pyrones, which affect the viability and colony formation of human drug‐resistant prostate cancer 22Rv1 cells [10]. In addition, this type of compound shows a wide range of biological activities, including antimicrobial [11], antioxidant [12], and anti‐inflammatory activities [13]. The structural variation of the specialized metabolites provides the basis for chemophenetic analysis, which exhibits a broad spectrum of pharmacological properties. Despite the recognized potential of naphtho‐γ‐pyrones, chemophenetic analyses and detailed data on potential biological activities remain limited for A. foetidus.

In this study, we investigated the fungal strain A. foetidus isolated from the rhizosphere of Gardenia jasminoides J. Ellis. The primary objectives were to isolate its secondary metabolites (Figure 1), assess their bioactivity, and employ a polyphasic approach integrating molecular, chemophenetic, and classical morphological data to characterize this fungal species.

FIGURE 1.

Chemical structures of compounds 1–14.

2. Results and Discussion

2.1. Strain Origin and Identification

Based on the analysis of internal transcribed spacer gene sequences of the mwg22 against NCBI databases, species were identified as closely related to Aspergillus sp. Through morphological characteristics and molecular identification, a phylogenetic tree was constructed using the homologous sequences in GenBank by the Maximum Likelihood method [14, 15]. The results showed that the ITS gene sequence of mwg22 had a similarity to A. foetidus CBS 121.28 (Figure S6), and they clustered in the same branch with a support rate of 100%.

2.2. Structural Elucidation

Compound 1 has a molecular formula of C₂₄H₁₉O₁₀ deduced from the positive high‐resolution electrospray ionization mass spectrometry (HR‐ESI‐MS) (m/z 467.0979 [M + H]⁺, calcd for 467.0973) and consistent with 16 degrees of unsaturation. The ultraviolet (UV) spectrum exhibited a characteristic absorption maximum at 218 nm, indicative of a conjugated diene system. The ¹H nuclear magnetic resonance (NMR), ¹³C NMR and heteronuclear single quantum coherence spectra of 1 revealed the presence of 24 carbon resonances corresponding to two methoxy groups [δ _H 3.68 (3H, s) and 3.91 (3H, s); δ _C 52.6 and 56.4], one methyls [δ _H 2.25 (3H, s); δ _C 20.3], one methylenes [δ _H 3.54 (2H, s); δ _C 29.2], four methines [δ _H 5.98 (1H, s), 6.37 (1H, d, J = 2.1 Hz), 6.41 (1H, d, J = 2.1 Hz) and 6.97 (1H, s); δ _C 107.8, 98.2, 100.1 and 136.0, respectively], and sixteen quaternary carbons (Table 1). Furthermore, the NMR spectrum in DMSO‐d₆ revealed two phenolic hydroxy protons [δ _H 10.43 (OH, s) and 15.12 (OH, s); δ _C 162.0 and 164.4]. Notably, the NMR signals of the aromatic protons [δ _H 6.37 (H‐7) and 6.41 (H‐9)], the conjugated enone system [δ _C 185.5 (C‐4) and 107.8 (C‐3)], and the markedly downfield‐shifted chelated hydroxy proton at δ _H 15.12 (5‐OH), collectively suggest the presence of a typically characteristic naphtho‐γ‐pyrone unit [16].

TABLE 1.

¹H and ¹³C nuclear magnetic resonance (NMR) data of compound 1 (δ in ppm, J in Hz).

Pos.	1 (in CD3OD)
	δ H (J in Hz)	δ C, type	Pos.	δ H (J in Hz)	δ C, type
2		169.9, C	1’		184.3, C
3	5.98 (s)	107.8, CH	2’	6.97 (s)	136.0, CH
4		185.5, C	3’		145.9, C
4a		103.7, C	4’		186.8, C
5		164.4, C	5’		116.2, C
5a		108.2, C	6’		156.6, C
6		162.0, C	7’	3.54 (s)	29.2, CH2
7	6.37 (d, 2.1)	98.2, CH	8’		173.0, C
8		162.8, C	CH3‐2	2.25 (s)	20.3, CH3
9	6.41 (d, 2.1)	100.1, CH	OCH3‐8	3.91 (s)	56.4, CH3
9a		140.9, C	OCH3‐8’	3.68 (s)	52.6, CH3
10		105.7, C	OH‐5	15.12 (s)
10a		151.5, C	OH‐6	10.43 (s)

Recorded‐OH in (CD₃)₂SO at 400 MHz.

To further confirm the above inference, the heteronuclear multiple bond correlation (HMBC) correlations of CH₃‐2 to C‐2 (δ _C 169.9) and C‐3, H‐3 to C‐4 and C‐4a (δ _C 103.7), H‐7 to C‐6 (δ _C 162.0) and C‐8 (δ _C 162.8), H‐9 to C‐5a (δ _C 108.2) and C‐8 (δ _C 162.8), as well as OCH₃‐8 to C‐8 were observed. Additionally, the hydroxy proton at 5‐OH (δ _H 15.12) showed interactions with C‐4a, C‐5 (δ _C 164.4), and C‐5a, along with that of 6‐OH (δ _H 10.43) correlated with both C‐6 and C‐7 (δ _C 98.2). This evidence proved that naphtha‐γ‐pyrone exists, which might closely resemble that of rubrofusarin [17]. Unlike rubrofusarin, the C‐10 position in compound 1 is a quaternary carbon, as the original hydrogen atom is substituted by a benzosemiquinone unit linked through a C‐3ʹ ‐C‐10 bond. The HMBC correlations of H‐2ʹ to C‐1ʹ, C‐3ʹ, C‐4ʹ, C‐6ʹ (δ _C 156.6) and notably to C‐10 (δ _C 105.7), confirmed the presence of a benzosemiquinone unit. The acetonyl side chain at C‐5ʹ was determined by the HMBC correlation from H‐7ʹ to C‐4ʹ, C‐5ʹ (δ _C 116.2), C‐6ʹ, and C‐8ʹ (δ _C 173.0), and OCH₃‐8ʹ to C‐8ʹ. Thus, the structure of 1 was determined and named asperforone A.(Figure 2)

FIGURE 2.

Key ¹H‐ ¹H correlated spectroscopy (COSY), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser effect spectroscopy correlation of 1.

The current phytochemical investigation of A. foetidus has led to the isolation of one unrevealed naphto‐γ‐pyrone, asperforone A (1), seven known naphtho‐γ‐pyrone monomers, rubrofusarin (2) [17], TMC‐256A1 (3) [18], rubrofusarin B (4) [3], fonsecin (5) [3], flavasperone (6) [19], oxisterigmatocystin C (7) [20], and RF‐3192C (10) [16], two naphtho‐γ‐pyrone dimers, rubasperone B (8) [21], and asperpyrone D (9) [6], and four other pyridone alkaloids, aspernigrin A (11) [22], aspernigrin B (12) [23], aspernigrin C (13) [22] and carbonarone B (14) [24].

To elucidate the chemotaxonomic relationships among closely related Aspergillus species, a network analysis (Figure 3) was performed [25]. The primary purpose of this analysis was to map the shared metabolic profile of A. foetidus against other species in the genus, thereby identifying potential characteristic chemical markers and evolutionary links. The network clearly illustrates that A. foetidus shares a significant number of metabolites, compounds 2–6 and 9–14 with A. niger, suggesting a close chemotaxonomic and phylogenetic relationship between A. niger and A. foetidus. Compounds 2 and 5–14 were isolated and identified for the first time from A. foetidus. Compounds 11–14 are pyridone alkaloids, which have previously been reported only in A. niger and A. carbonarius within the Aspergillus genus and have not been found in other fungal species. Thus, this type of metabolite can serve as a characteristic chemical marker for A. foetidus. Together with compounds 2–6 and 8–10 reported from A. tubingensis as well as compounds 2–6 and 14 obtained from A. carbonarius, the network collectively delineates a distinct, metabolically coherent subgroup within the genus.

FIGURE 3.

Compound‐species network. (The pink circles represent the compounds isolated from A. foetidus in this work. The blue boxes represent fungi containing these compounds, edges represent the isolation relationship.).

2.3. Assessment of Bioactivity

In the bioassay, all compounds 1–14 were evaluated for anti‐acetylcholinesterase activity. At a concentration of 50 µM, compound 11 showed acetylcholinesterase (AChE) inhibitory activity with an inhibition rate of 46.98%, while compound 4 exhibited moderate inhibition at 37.88%. Additionally, all the compounds were assessed for antimicrobial activity against five pathogenic microbial strains: Fusarium solani, Plectosphaerella cucumerina, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus, using a previously reported method. Among them, compounds 1 and 9 demonstrated moderate antimicrobial activity against P. cucumerina with minimum inhibitory concentrations of 32 µg/mL, respectively. Compound 11 also showed antimicrobial activity against B. subtilis with a minimum inhibitory concentration (MIC) of 32 µg/mL. (Table 2).

TABLE 2.

The antifungal activity of isolated compounds 1–14 (µg/mL).

Compounds	P. coumarin	F. solan	B. subtilis	E. coli	S. aureus
1	32	256	256	32	256
2	64	256	128	256	128
3	>256	>256	>256	256	>256
4	>256	>256	>256	256	>256
5	256	256	64	128	128
6	>256	>256	256	>256	>256
7	64	256	128	128	256
8	256	>256	>256	256	256
9	32	256	>256	128	128
10	128	>256	>256	>256	>256
11	256	256	32	128	>256
12	>256	>256	>256	256	>256
13	>256	>256	256	>256	256
14	256	>256	64	128	128
Nystatin a	8	8	—	—	—
Kanamycin b			16	4	2

a: Nystatin is positive control for antifungal activity against P. coumarin and F. solan. b: Kanamycin is positive control for antibacterial activity against B. subtilis, E. coli and S. aureus.

3. Conclusions

In conclusion, one new naphtho‐γ‐pyrone, asperforone A (1), along with thirteen known compounds (2–14), was isolated from the endophytic fungus A. foetidus. This profile, abundant in naphtho‐γ‐pyrones, reinforces the chemophenetic signature of the Aspergillus genus. Bioactivity evaluation revealed a novel finding. Asperforone A (1) exhibited moderate activity against the P. cucumerina (MIC = 32 µg/mL). The aspernigrin A (11) was identified as an AchE inhibitor (46.98% inhibition at 50 µM) and showed antimicrobial efficacy against B. subtilis (MIC = 32 µg/mL). Furthermore, based on reports, the potent anti‐HIV activity of aspernigrin C (13) (50% inhibitory concentration [IC₅₀] = 4.7 ± 0.4 µM) and its effectiveness against human erythroleukemic K562 cells for carbonarone B (14) (IC₅₀ = 27.8 µg/mL) are presented as promising antiviral leads. Similarly, the moderate antifungal activity of rubrofusarin B (4) against C. albicans further consolidates its status as a broad‐spectrum antimicrobial agent. This discovery contributes significantly to the elucidation of A. foetidus chemical diversity and suggests promising pharmacological potential.

4. Experimental

4.1. General Experimental Procedures

NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer or Bruker Avance III 600 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. HR‐ESI‐MS data were acquired on an Agilent 1290 UPLC/6545Q‐TOF‐MS spectrometer. High‐performance liquid chromatography (HPLC) analyses were performed on an Agilent 1260 instrument equipped with a Zorbax SB‐C 18 column (5 µm, 9.4 × 250 mm). Silica gels (200–300 and 300–400 mesh; Qingdao Haiyang Chemical Co., China) were used for column chromatography (CC). Fractions were monitored by thin‐layer chromatography (TLC) and visualized by spraying with 10% H₂SO₄ in ethanol, followed by heating.

4.2. Material, Identification, and Fermentation

Gardenia jasminoides J. Ellis was collected from Yunshan Botanical Garden in Kunming City, Yunnan Province, China, in October 2021 and identified by Professor Chen Yang from the School of Pharmacy, Guizhou Medical University (Figure S10). The plant material (No. YNU202110‐GJZZ‐1) has been deposited in the School of Chemical Science and Technology, Yunnan University, China. A. foetidus mwg22 was isolated from the root of Gardenia jasminoides J. Ellis. The strain (mwg22) was cultured on a potato dextrose agar (PDA) slant at 4°C and has been deposited at the School of Chemical Science and Technology, Yunnan University, China.

The colony morphology is silky, with a black‐brown mycelium. The edges are white, and a large number of spores are gathered in the middle. Genomic DNA sequence was extracted, and the ITS region was amplified using the universal primers ITS1 and ITS4 by polymerase chain reaction, then sequenced. A phylogenetic tree was constructed using the homologous sequences in GenBank by the Maximum Likelihood method in MEGA software, with 1000 bootstrap replicates. The organism was most closely related to A. foetidus (100% bootstrap support) [A. foetidus CBS 121.28].

The fermentation products from nine distinct solid culture media (potato, oat, rice, yam, sweet potato, taro, millet, corn, and peanut) were analyzed and compared after a 35‐day fermentation period. A comparative analysis by TLC and HPLC revealed considerable chemical diversity in the extracts from both taro and potato media. Furthermore, the potato medium demonstrated a clear yield advantage over the taro medium. Based on the chemical diversity and richness, the potato‐based solid medium was selected for large‐scale fermentation. A. foetidus mwg22 was added to the 20 kg fermentation culture medium (500 g potato in each seed bottle) that had been sterilized at 121°C for 30 min before, and it was then incubated at 28°C for a period of 35 days.

4.3. Extraction and Isolation

The solid culture was extracted with MeOH (3 × 25 L) at room temperature (25°C). The extract was filtered and evaporated under reduced pressure to completely remove methanol, obtaining an extract (56.4 g).

The extract (56.4 g) was subjected to column chromatography using silica gel with a gradient of petroleum ether and CHCl₃‐MeOH (100:0, 50:1, 10:1 and 3:1, ν/ν) to obtain five fractions (Fr. 1 ‐ Fr. 5). Fr. 2 (19.3 g) was separated by column chromatography with petroleum ether‐acetone (30:1 to 1:1, ν/ν), resulting in the isolation of Fr. 2.1 ‐ Fr. 2.3. Fr. 2.2 was subjected to silica gel with silica gel column chromatography by repeated gradient elution CHCl₃‐MeOH (100:1 to 20:1, ν/ν) to yield compounds 4 (0.9 g) and 8 (6.1 mg), and further purified through Sephadex LH‐20 CHCl₃‐MeOH (1:2, ν/ν) to yield compound 1 (5.0 mg). Compound 6 (1.4 g) was isolated from Fr. 2.1 through silica gel column chromatography using ether‐acetone (30:1 to 1:1, ν/ν), and compound 14 (5.0 mg) was obtained from Fr. 2.3 by means of Sephadex LH‐20 with MeOH. Fr. 3 (2.9 g) was successively chromatographed over a Sephadex LH‐20 (MeOH) and silica gel CHCl₃‐MeOH (60:1 to 30:1, ν/ν) to afford compounds 5 (18.0 mg), 11 (8.2 mg), and 13 (176.0 mg). Meanwhile, Compound 3 (1.2 g) precipitated during this process. Fr. 4 (15.6 g) was separated using silica gel CHCl₃‐MeOH (30:1 to 10:1, ν/ν) to obtain compounds 7 (7.2 mg), 9 (700.0 mg), 10 (20.0 mg), and 12 (3.6 mg), and furthermore, compound 2 (4.8 mg) was separated using Sephadex LH‐20 with MeOH.

4.3.1. Asperforone A (1)

Yellowish‐brown powder; m.p. 112‐119°C; UV (MeOH) λmax (logε): 218 (3.31) nm; ¹H NMR (400 MHz, CD₃OD) and ¹³C NMR (100 MHz, CD₃OD) see Table 1; HR‐ESI‐MS m/z: 467.0979 [M + H]⁺ (Calcd. for C₂₄H₁₉O₁₀, 467.0973).

4.4. Antimicrobial Activity Assay

An antimicrobial assay was performed against two fungal strains (F. solani and P. cucumerina) and three bacterial strains (B. subtilis, E. coli, and S. aureus) for compounds 1‐14. The inoculum was prepared in PDA medium (1 L water, 200 g potato, 20 g dextrose, and natural pH) to achieve a concentration of 1×10⁵ units/mL. Samples were dissolved in dimethyl sulfoxide (DMSO) (10.24 mg/mL), serially double‐diluted from 1 to 512 µg/mL in 96‐well plates, inoculated with microbial suspensions, and incubated at 28°C for 24 h [26]. Nystatin was used as a positive control for antifungal activity, while kanamycin served as the positive control for antibacterial activity.

Based on the literature references [27, 28, 29] and the positive control observed MIC values, the compounds were categorized as follows: excellent activity (MIC < 2 µg/mL), good activity (MIC = 4–8 µg/mL), moderate activity (MIC = 16–32 µg/mL), and weak or no activity (MIC > 32 µg/mL).

4.5. AChE Inhibitory Assay

The AChE inhibitory activity of the isolated compounds was evaluated using a modified spectrophotometric method based on Ellman's assay [30, 31]. AChE, acetylthiocholine iodide, and DTNB were dissolved in phosphate buffer (pH 8.0). Test compounds were dissolved in a 2% DMSO aqueous solution to a concentration of 1 mM. The assay was performed in a 96‐well plate. Tacrine was used as the positive control at a final concentration of 0.333 µM, and a blank group (NC) was used as the negative control. After incubation, the absorbance was measured at 405 nm using a microplate reader.

4.6. Compound‐Species Network

The data on the distribution of specialized metabolites across different Aspergillus species were manually curated from an extensive literature search in the SciFinder database. The columns for compound names and their corresponding fungal sources are stored in the Excel spreadsheet (Table S1), and the 48 sample entries (representing compound‐species pairs) from Excel were subsequently imported into Cytoscape (version 3.9.1, https://cytoscape.org) for network visualization and analysis. A compound‐species association network was constructed where nodes represent either a compound or a fungal species, and edges represent the isolation relationships. The visual style of the network was modified for clarity: nodes representing compounds isolated from A. foetidus in the present study are depicted as pink circles, while nodes representing fungi species are shown as blue boxes.

Author Contributions

Yi‐Nong Wang and Li Zhu have contributed equally to this work. Yi‐Nong Wang isolated and identified the compounds. Li Zhu wrote the manuscript. Jia‐Qi Liu experimented with the biological activity and identified the fungi. Liang Deng and Le Cai guided and supervised this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This work was supported by a grant from the Natural Science Foundation of China (No. 82160799) and the Yunnan Characteristic Plant Screening and R&D Service CXO Platform (2022YKZY001).

Supporting information

Supporting file 1: cbdv70891‐sup‐0001‐SuppMat.pdf

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.