New Sesterterpenoid from the Marine Fungus Penicillium oxalicum M893

Abstract

Objectives

The aim of the project was the isolation, structural elucidation, and antimicrobial and antifungal activities of compounds from the culture broth of the marine fungus Penicillium oxalicum M893 (isolated from the marine brown alga Spatoglossum sp.).

Methods

Combined chromatographic techniques were used to isolate antibacterial and antifungal compounds from the MeOH extract Penicillium oxalicum M893. The structures were elucidated by analyses of high-resolution electrospray ionization mass spectral and nuclear magnetic resonance data. The antimicrobial and antifungal effects of compounds were evaluated using the dilution turbidimetric broth method.

Results

One new sesterterpenoid, oxaliterpenoid (1), and six known compounds, aspergillusidone C (2), nidulin (3), emeguisin B (4), aspergillusether A (5), aspergillusether J (6), and guisinol (7) were isolated from the methanol extract of the culture broth of Penicillium oxalicum M893. All compounds showed potent antibacterial activities against Gram-(+) bacteria, E. faecalis (ATCC299212), S. aureus (ATCC25923), and B. cereus (ATCC14579), and the yeast Candida albicans (ATCC10231), with MIC values ranging from 2 to 128 µg/mL.

Graphical Abstract

This is a visual representation of the abstract.

Keywords

sesterterpenoid oxaliterpenoid marine fungi antibacterial antifungal

Introduction

Marine fungi represent potential sources of structurally novel and diverse natural products, with approximately 38% (22,000) bioactive microbial-derived compounds from only 5% of discovered fungal species.^1,2 Those compounds have shown a broad spectrum of biological properties, including antimicrobial, antiviral, anti-neuritis, antioxidant, and antitumor activities.^3,4 Among these fungi, the Penicillium genus is one of the most widespread genera in the marine environment. The genus includes over 354 species, which have been intensively exploited due to their chemical diversity and associated biological activities.^5-7 They are well known to produce various classes of bioactive compounds, such as alkaloids, polyketides, terpenoids, and lipopeptides.^8,9 As a part of our ongoing search for secondary metabolites from marine-derived fungus, the methanol (MeOH) extract of Penicillium oxalicum M893 fermentation exhibited antimicrobial activity against three Gram-(+) bacteria (Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus) and one yeast strain (Candida albicans) with MIC values of 32, 128, 64, 16 µg/mL, respectively. Thus, the MeOH extract of M. oxalicum M893 has been further studied to identify the active compounds. Herein, we report the isolation, structural elucidation, and antibacterial and antifungal effects of one new sesterterpenoid and six known compounds from the fermentation broth of P. oxalicum M893 isolated from the marine brown alga Spatoglossum sp., collected at Phu Quy, Binh Thuan, Vietnam.

Results and Discussion

Compound 1 was isolated as white needles. Its HR-ESI mass spectrum showed a pseudo-molecular ion [M + H]⁺ at m/z 357.3141 (calcd. 357.3157 for C₂₅H₄₁O), leading to the molecular formula of C₂₅H₄₀O. The ¹H-NMR spectrum of 1 exhibited proton signals of six methyl groups at δ_H 0.71 (3H, d, J = 7.0 Hz), 0.85 (3H, d, J = 7.0 Hz), 0.91 (3H, d, J = 7.0 Hz), 0.94 (3H, s), 1.03 (3H, d, J = 7.0 Hz), and 1.11 (3H, s), and one oxygenated methine group at δ_H 3.68 (1H, m) (Table 1). Detailed analyses of the ¹³C- NMR and DEPT spectra of 1, along with the support of the HSQC spectrum, revealed signals of twenty-five carbons, including six methyls at δ_C 13.1, 14.9, 17.2, 17.7, 21.6, and 26.3, six methylenes at δ_C 22.2, 29.6, 31.1, 36.9, 38.4, and 39.6, nine methines at δ_C 30.1, 34.1, 48.2, 49.4, 50.5, 50.7, 55.6, 63.6, and 80.8, and four quaternary carbons at δ_C 30.8, 42.7, 54.1, and 60.8. Chemical shifts at δ_H 3.68 and δ_C 80.8 suggested the presence of a methine carbon bonding with the hydroxy group. Analysis of ¹H- and ¹³C-NMR spectroscopic data of 1 indicated its structure to be a sesterterpenoid possessing a hexacyclic (5/5/5/5/3/5) carbon skeleton, similar to those of niduterpenoids A and B, which have been reported from the fungus Aspergillus nidulans.¹⁰ The HMBC correlations between H₃-19 (δ_H 1.11)/H₂-2 (δ_H 1.54 and 1.65)/H₂-6 (δ_H 1.71)/H-17 (δ_H 1.79) and C-1 (δ_C 42.7)/C-5 (δ_C 30.8)/C-18 (δ_C 34.1) indicated the presence of a cyclopropane ring (E ring). Moreover, the HMBC correlations between H₃-21 (δ_H 0.85)/H₃-22 (δ_H 0.71) and C-4 (δ_C 50.7)/C-20 (δ_C 30.1), between H₃-19 (δ_H 1.11) and C-1 (δ_C 42.7)/C-2 (δ_C 38.4), between H₂-2 (δ_H 1.54 and 1.65)/H₂-3 (δ_H 1.15 and 1.36)/H-4 (δ_H 1.88) and C-1 and the COSY correlations of H₂-2/H₃-3/H-4/H-20 (δ_H 1.80)/H₃-21 (δ_H 0.85); H-20 (δ_H 1.80)/H₃-22 (δ_H 0.71) indicated the positions of methyl and isopropyl groups at C-1 and C-4 of a cyclopentane ring (F ring), respectively. The HMBC correlations between H₃-23 (δ_H 1.03) and C-10 (δ_C 80.8)/C-11 (δ_C 48.2)/C-12 (δ_C 63.6) and between H₃-24 (δ_H 0.94) and C-8 (δ_C 60.8)/C-14 (δ_C 36.9)/C-15 (δ_C 54.1)/C-16 (δ_C 50.5) indicated the positions of two methyl group at C-11 and C-15 and a hydroxy group at C-10. In addition, COSY correlations of H₂-9 (δ_H 1.54 and 1.90)/H-10 (δ_H 3.68)/H-11 (δ_H 1.34)/[H₃-23 (δ_H 1.03)]/H-12 (δ_H 1.43)/H₂-13 (δ_H 1.24 and 1.63)/H₂-14 (δ_H 1.22 and 1.61) suggested the presence of a pentalene skeleton (A and B rings). The HMBC correlations between H₃-25 (δ_H 0.91) and C-15 (δ_C 54.1)/C-16 (δ_C 50.5)/C-17 (δ_C 49.4) suggested the location of the methyl group at C-16. The second pentalene skeleton (C and D rings) was confirmed by HMBC correlations between H-7 (δ_H 1.93) and C-5 (δ_C 30.8)/C-12 (δ_C 63.6)/C-15(δ_C 54.1)/C-16 (δ_C 50.5)/C-18 (δ_C 34.1) and also COSY correlations of H₂-6 (δ_H 1.71)/H-7 (δ_H 1.93)/H-17 (δ_H 1.79)/H-16 (δ_H 1.37)/H₃-25 (δ_H 0.91). The HMBC correlations between H₂-6 (δ_H 1.71) and C-1 (δ_C 42.7)/ C-4 (δ_C 50.7)/C-5 (δ_C 30.8)/ C-8 (δ_C 60.8)/C-17 (δ_C 49.4)/C-18 (δ_C 34.1) and H-18 (δ_H 0.79) and C-1 (δ_C 42.7)/C-2 (δ_C 38.4)/C-4 (δ_C 50.7)/C-6 (δ_C 29.6) established a spiro[4.4]nonane skeleton of the D and F rings. Based on the above evidence, the constitutional structure of 1 was established as shown in Figure 1.

Figure 1.

The key HMBC, COSY, and NOESY correlations of compound 1.

Table 1.

¹H- and ¹³C-NMR Spectroscopic Data for Compound 1 in CDCl₃.

C	δ _C	Type	δ_H (mult., in Hz)
1	42.7	C	-
2	38.4	CH₂	1.54 (m)/1.65 (m)
3	22.2	CH₂	1.15 (m)/1.36 (m)
4	50.7	CH	1.88 (m)
5	30.8	C	-
6	29.6	CH₂	1.71 (m)
7	55.6	CH	1.93 (m)
8	60.8	C	-
9	39.6	CH₂	1.54 (m)/1.90 (m)
10	80.8	CH	3.68 (m)
11	48.2	CH	1.34 (m)
12	63.6	CH	1.43 (m)
13	31.1	CH₂	1.24 (m)/1.63 (m)
14	36.9	CH₂	1.22 (m)/1.61 (m)
15	54.1	C	-
16	50.5	CH	1.37 (m)
17	49.4	CH	1.79 (m)
18	34.1	CH	0.79 (br s)
19	14.9	CH₃	1.11 (s)
20	30.1	CH	1.80 (m)
21	21.6	CH₃	0.85 (d, 7.0)
22	17.2	CH₃	0.71 (d, 7.0)
23	17.7	CH₃	1.03 (d, 7.0)
24	26.3	CH₃	0.94 (s)
25	13.1	CH₃	0.91 (d, 7.0)

The relative configuration of 1 was determined by the NOESY experiment. The NOE cross-peaks of H-7 (δ_H 1.93)/H-12 (δ_H 1.43), H-12 (δ_H 1.43)/H₃-23 (δ_H 1.03), and H-17 (δ_H 1.79)/H₃-19 (δ_H 1.11) suggested that they were oriented on the same side and assigned as α-positions. As a result, a cis-fused configuration for both A/B and C/D ring junctions of 1 was determined. Similarly, the B/C ring junctions were also assigned as cis-fused the observation of NOE correlations between H₃-24 (δ_H 0.94) and H_β-9 (δ_H 1.54)/H-16 (δ_H 1.37). In addition, NOESY interactions of H-16 (δ_H 1.37)/ H-18 (δ_H 0.79), and H-18 (δ_H 0.79)/H-4 (δ_H 1.88) proved that they were β-oriented. In addition, a NOE correlation between H-10 (δ_H 3.68) and H₃-23 (δ_H 1.03) was also observed, indicating the configuration of the hydroxy group at C-10 to be β. Thus, the relative configuration of 1 was established, and the new compound was named as oxaliterpenoid.

The known compounds were identified to be aspergillusidone C (2),¹¹ nidulin (3),¹¹ emeguisin B (4),¹¹ aspergillusether A (5),¹² aspergillusether J (6),¹¹ and guisinol (7) (Figure 2).¹³ Their structures were identified by comparing their spectroscopic data with those reported in the literature.

Figure 2.

Chemical structures of compounds 1–7.

All compounds were evaluated for their antibacterial effects against Gram-(+) bacteria, Enterococcus faecalis (ATCC299212), Staphylococcus aureus (ATCC25923), and Bacillus cereus (ATCC14579), Gram-(–) bacteria, (Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Salmonella enterica (ATCC13076), and antifungal effect against Candida albicans (ATCC10231) (Table 2). Streptomycin and cycloheximide were used as positive controls. All compounds showed potent antibacterial activities against the Gram-(+) bacteria E. faecalis (ATCC299212), S. aureus (ATCC25923), and B. cereus (ATCC14579), and the yeast Candida albicans (ATCC10231), with MIC values ranging from 2 to 128 µg/mL. In previous studies, compounds from Penicillium sp. have been reported to exhibit an antibacterial effect.¹⁴ The results suggested that compounds 1–7 could be potential antimicrobial agents against Gram-(+) bacteria.

Table 2.

Antibacterial and Antifungal Activities of Compounds 1–7.

Compounds	MIC (μg/mL)
	Gram-(+)			Gram-(–)			Yeast
	E. faecalis	S. aureus	B. cereus	E. coli	P. aeruginosa	S. enterica	C. albicans
1	128	32	32	32	-	-	32
2	2	2	2	-	-	2	2
3	2	16	2	2	2	-	4
4	16	32	16	-	-	128	8
5	16	64	32	32	64	-	32
6	8	8	4	256	-	-	32
7	4	4	4	-	-	4	16
Streptomycin	256	256	128	32	256	128
Cyclohexamide							32

(-) MIC >256 µg/mL.

Material and Methods

General

See Supplemental Material.

Isolation and Taxonomic Identification

The fungal strain P. oxalicum M893 was isolated from the marine brown algalSpatoglossum sp., collected at a depth of 10 m, on the coast of Phu Quy, Binh Thuan, Vietnam in April 2021. (Spatoglossum sp. was identified by Prof. Do Cong Thung, Institute of Marine Environment and Resources, VAST); a voucher specimen (SS2104) was deposited at the Institute of Marine Environment and Resources, VAST.

The taxonomy of P. oxalicum M893 was identified by using 18S rRNA gene sequence analysis and compared with fungal 18S rRNA sequences in the GenBank database by NBCI Blast program. Its 18S rRNA gene sequence was registered on the GenBank database with the accession number OP522350.

Fermentation

P. oxalicum M893 was activated and inoculated into 1 L of PDA broth medium (comprising: 12 g/L potato extract, 8 g/L dextrose, 2 g/L soluble starch, 12 g/L instant ocean salt in 1.0 L of distilled water) to prepare the seed cultures. After 7 days of incubation at 30 ^oC with shaking of 150 rpm, the culture broth was used to inoculate into 20 flasks containing 2.5 L of PDA broth medium, pH 7.0 (comprising: 30 g/L potato extract, 20 g/L dextrose, 5 g/L soluble starch, 30 g/L instant ocean salt). The fermentation was conducted at 30 °C with a shaking speed of 100 rpm and harvested on the 14^th day.

Extraction and Purification of Compounds

The fermentation broth of P. oxalicum M893 (50 L) was passed through a XAD-16 column (XAD-16, 10 kg). The column was washed with distilled water (70 L) and then eluted with MeOH (80 L). The MeOH solution was concentrated under reduced pressure to obtain the MeOH extract (20.6 g). This was chromatographed by silica gel column chromatography (CC) eluting with a gradient solvent of CH₂Cl₂/acetone (100:1, 50:1, 20:1, 10:1, v/v) to give four fractions (F1-F4). Fraction F1 (600 mg) was separated on a silica gel column eluting with n-hexane/EtOAc 5:1, providing four fractions, F1.1-F1.4. Fraction F1.1 (100 mg) was subjected to Sephedex LH-20 CC (100% MeOH) to yield compound 1 (4.5 mg). Fraction F1.2 (120 mg) was further purified by HPLC (J'sphere H80 250 mm length × 20 mm ID) eluting with ACN in H₂O 95% (a flow rate of 3 mL/min) to yield compounds 5 (5 mg, R_t= 20 min), 6 (26 mg, R_t= 21 min), and 7 (15 mg, R_t= 18 min). Fraction F1.3 (90 mg) was separated by HPLC (J'sphere H80 250 mm length × 20 mm ID) eluting with ACN in H₂O 95% (a flow rate of 3 mL/min) to yield compounds 2 (5 mg, R_t= 17 min), 3 (3 mg, R_t= 14 min), and 4 (5 mg, R_t= 19 min).

Oxaliterpenoid (1)

White amorphous powder; $[α]_{D}^{25}$ : −20.0 (c 0.04, MeOH); HR-ESI-MS: m/z 357.3141; [M + H]⁺ (Calcd. for C₂₅H₄₁O: 357.3157); IR (KBr) ν_max 3341, 2950, 1466, 1198, 1172, 997, 666 cm^–1; ¹H- and ¹³C-NMR data (CDCl₃): see Table 1.

Antimicrobial Assays

See Supporting Information.¹⁵

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231191636 - Supplemental material for New Sesterterpenoid from the Marine Fungus Penicillium oxalicum M893

Supplemental material, sj-docx-1-npx-10.1177_1934578X231191636 for New Sesterterpenoid from the Marine Fungus Penicillium oxalicum M893 by Thi Hoang Anh Nguyen, Thi Quynh Do, Thuy Linh Nguyen and Hong Minh Le Thi, Mai Anh Nguyen, Brian T Murphy, Thanh Xuan Dam, Doan Thi Mai Huong, Pham Van Cuong in Natural Product Communications

Footnotes

Acknowledgements

This research is funded by Vietnam Academy of Science and Technology (Grant No: VAST.TĐ.DLB.03/20-22) and University of Illinois Campus Research Board grant (5D43 TW010530-05).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical approval is not applicable to this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Doan Thi Mai Huong

Pham Van Cuong

Supplemental Material

Supplemental material for this article is available online.

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