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Abstract

Two new xanthone derivatives, spinosusones A (1) and B (2), and a new tryptoquivaline analogue, asperdiazapinone G (3), together with nine known compounds (4-12) were isolated from the EtOAc extract of the marine-derived fungus Aspergillus sp. WHUF03110. The structures of 1-3 were determined by spectroscopic analysis, and comparison with literature data. Most of these isolated compounds were evaluated for their antimicrobial activities against ten Gram-negative and seven Gram-positive bacteria, Mycobacterium smegmatis ATCC 607, Candida albicans ATCC SC5314, and C. albicans YY-1-4. Compound 10 displayed strong antibacterial activity against five Gram-positive bacteria (Bacillus subtilis 168, Staphylococcus aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271) with MIC values ranging from 1.0 to 2.0 μg/mL, and displayed moderate antibacterial activity against four Gram-negative bacteria (Helicobacter pylori 26695, H. pylori G27, H. pylori 159, H. pylori 129) and M. smegmatis ATCC 607 with a MIC value of 8.0 μg/mL.

Keywords

marine-derived fungus xanthone derivative alkaloid antibacterial activity

Introduction

In recent years, marine-derived fungi have attracted much attention from natural medicinal chemists, and many secondary metabolites possessing intriguing structures and various activities have been reported.¹ Aspergillus is a chemically brilliant fungal genus with immense potential to produce a wide range of unique secondary metabolites including polyketides,^2–4 alkaloids,^5–7 peptides^8,9 and terpenoids.^10,11 Some of these metabolites exhibit intriguing biological properties, including antimicrobial,^5,7,11 pro-angiogenesis,⁶ and cytotoxic,^2,4,10 as well as enzyme-inhibiting activities.⁸ In a current study, Aspergillus sp. WHUF03110 was fermented through a solid fermentation methodology and the ethyl acetate extract of the strain showed antibacterial activity against Staphylococcus aureus and Escherichia coli. Continuing our efforts on the discovery of the chemical diversity and biological activities of natural products, chemical investigation was performed. Three new compounds, spinosusone A (1), spinosusone B (2) and asperdiazapinone G (3), together with nine known compounds, aszonalenin (4),¹² 6-hydroxyaszonalenin (5),¹³ brocaenol A (6),¹⁴ microsphaeropsone C (7),¹⁵ 2,2′-oxodibis(1,4)-di-tert-butylbenzene (8),¹⁶ sartorypyrone D (9),¹⁷ sartorypyrone A (10),¹⁸ aszonapyrones A (11)¹⁹ and ergosterol peroxide (12)²⁰ (Figure 1) were obtained from fractionation of the EtOAc extract prepared from a rice fermentation culture. The antimicrobial activities of the majority of the isolates were evaluated. Herein, we report the details of isolation, structure elucidation, and bioactivity screening of these metabolites from Aspergillus sp. WHUF03110.

Figure 1.

Structures of compounds (1-12) isolated from Aspergillus sp. WHUF03110.

Results and Discussion

Compound 1 was isolated as yellow amorphous solid. Its molecular formula was determined to be C₁₇H₁₄O₆ based on the HRESIMS signal at m/z 315.0861 [M + H]⁺ (calcd. for C₁₇H₁₅O₆, 315.0869). The IR spectrum indicated the presence of hydroxy (approximately 3400 cm⁻¹) and carbonyl groups (1736, 1646 cm⁻¹). The UV spectrum of 1 showed maximum absorption peaks at 206, 232, 258 and 286 nm. The ¹H NMR spectrum (Table 1) displayed four aromatic protons at δ_H 6.78 (d, J = 8.3 Hz, 1 H), 6.89 (d, J = 8.3 Hz, 1 H), 7.57 (t, J = 8.3 Hz, 1 H) and 7.36 (s, 1 H), indicating the presence of one 12,3-trisubstituted benzene ring and one pentasubstituted benzene ring, respectively. Signals at δ_H 2.46 (s, 3 H), 3.86 (s, 3 H) and 4.06 (s, 3 H) were attributed to one methyl and two methoxyl groups. A signal at δ_H 12.28 (s, 1 H) was attributed to one hydroxy group. The ¹³C NMR data of 1 revealed 17 carbon resonances of which two were of carbonyl carbons (δ_C 180.9, 167.7), twelve of aromatic carbons (δ_C 161.9, 156.0, 152.5, 152.0, 142.1, 137.0, 126.2, 121.0, 116.4, 110.8, 108.7, 106.9), two of methoxyl carbons (δ_C 53.2, 62.6) and one of a methyl carbon (δ_C 17.1). The ¹H NMR and ¹³C NMR data indicated a xanthone skeleton, which was similar to that of 2,8-dihydroxy-3-methyl-9-oxoxanthene-1-carboxylic acid methyl ester, previously isolated from Picea glauca.²¹ The main difference is an additional methoxyl signal (δ_H 3.86; δ_C 62.6) and the chemical shifts at δ_C 126.2 and 142.1 in 1, which may be attributed to a methoxyl group at C-2. The 1D NMR data were fully assigned by analysis of the HSQC and HMBC spectra (Table 1). The key HMBC correlations observed from δ_H 7.36 (H-4) to δ_C 116.4 (C-9a) and δ_C 152.0 (C-2), and from δ_H 3.86 (H-13) to δ_C 152.0 (C-2) confirmed that the methoxyl group (δ_C 62.6) was connected to C-2. The HMBC correlations (Figure 2) observed from δ_H 4.06 (H-12) to δ_C 167.7 (C-11) suggested that the methoxyl group (δ_C 53.2) was connected to C-11. The location of the methyl was determined by the correlations from δ_H 2.46 (H-14) to δ_C 152.0 (C-2), δ_C 142.1 (C-3), and δ_C 121.0 (C-4) in the HMBC spectrum. The hydroxy group at C-8 was confirmed by the HMBC correlations from δ_H 7.57 (H-6) to δ_C 161.9 (C-8) and 156.0 (C-10a), and from δ_H 12.28 (8-OH) to δ_C 108.7 (C-8a), δ_C 161.9 (C-8) and δ_C 110.8 (C-7). Hence, the structure of 1 was determined and named as spinosusone A.

Figure 2.

The key HMBC correlations of 1-3.

Table 1.

¹H and ¹³C NMR Data for Compounds 1 and 2 (600/150 MHz, in CDCl₃, δ ppm).

	1			2
No.	δ_H (J in Hz)	δ _C	No.	δ_H (J in Hz)	δ _C
1		126.2	1		126.1
2		152.0	2		151.8
3		142.1	3		142.2
4	7.36 (s, 1 H)	121.0	4	7.34 (s, 1H)	120.9
4a		152.5	4a		152.8
5	6.89 (d, 8.3, 1 H)	106.9	5	6.88 (d, J = 9.0 Hz, 1 H)	105.6
6	7.57 (t, 8.3, 1 H)	137.0	6	7.30 (d, J = 9.0 Hz, 1 H)	120.9
7	6.78 (d, 8.3, 1 H)	110.8	7		142.9
8		161.9	8		150.7
8a		108.7	8a		108.9
9		180.9	9		181.5
9a		116.4	9a		115.7
10a		156.0	10a		149.6
11		167.7	11		167.8
12	4.06 (s, 3 H)	53.2	12	4.06 (s, 3 H)	53.2
13	3.86 (s, 3 H)	62.6	13	3.85 (s, 3 H)	62.6
14	2.46 (s, 3 H)	17.1	14	2.46 (s, 3 H)	17.1
8-OH	12.28 (s, 1 H)		15	3.94 (s, 3 H)	57.3
			8-OH	12.44 (s, 1 H)

Compound 2 was isolated as yellow amorphous solid. Its molecular formula was determined to be C₁₈H₁₆O₇ based on the HRESIMS signal at m/z 367.0799 [M + Na]⁺ (calcd. for C₁₈H₁₆NaO₇, 367.0788). The UV spectrum of 2 showed maximum absorption peaks at 237 and 270 nm. The IR spectrum showed absorption bands for hydroxy (3438 cm⁻¹) and carbonyl (1734, 1651 cm⁻¹) groups. The ¹H NMR spectrum (Table 1) displayed one hydroxy group at δ_H 12.44 (s, 1 H), three aromatic protons at δ_H 6.88 (d, J = 9.0 Hz, 1 H), 7.30 (d, J = 9.0 Hz, 1 H) and 7.34 (s, 1 H), three methoxyl groups at δ_H 3.85 (s, 3 H), 3.94 (s, 3 H) and 4.06 (s, 3 H), and one methyl signal at δ_H 2.46 (s, 3 H). The ¹³C NMR data of 2 revealed 18 carbon resonances containing two carbonyl carbons (δ_C 181.5, 167.8), twelve aromatic carbons (δ_C 152.8, 151.8, 150.7, 149.6, 142.9, 142.2, 126.1, 120.9, 120.9, 115.7, 108.9, 105.6), three methoxyl carbons (δ_C 62.6, 57.3, 53.2) and one methyl carbon (δ_C 17.1). The 1D NMR data of 2 were similar to those of 1, and fully assigned by analyzing the HSQC and HMBC spectra (Table 1). The main difference is the presence in 2 of two doublet aromatic proton signals at δ_H 6.88 (d, J = 9.0 Hz, 1 H) and 7.30 (d, J = 9.0 Hz, 1 H), and an additional methoxyl signal (δ_H 3.94; δ_C 57.3). The HMBC correlations (Figure 2) observed from δ_H 6.88 (H-5) to δ_C 142.9 (C-7) and δ_C 108.9 (C-8a), and from δ_H 3.94 (H-15) to δ_C 142.9 (C-7) confirmed a 1,2,3,4-tetrasubstitued benzene ring and suggested that the methoxyl group (δ_C 57.3) was connected to C-7. The HMBC correlations observed from δ_H 12.44 (8-OH) to δ_C 108.9 (C-8a), δ_C 150.7 (C-8) and δ_C 142.9 (C-7), suggested that a hydroxy group was connected to C-8. On the basis of the above analysis, the structure of 2 was determined and named as spinosusone B.

Compound 3 was isolated as pink amorphous solid. Its molecular formula was determined to be C₂₃H₂₃N₃O₃ based on the HRESIMS signal at m/z 388.1669 [M-H]⁻ (calcd. for C₂₃H₂₂N₃O₃, 388.1661). The UV spectrum of 3 showed maximum absorption peaks at 238 and 283 nm. The IR spectrum showed absorption bands for hydroxy (3246 cm⁻¹) and carbonyl (1686, 1647 cm⁻¹) groups. The ¹H NMR spectrum displayed four aromatic protons at δ_H 7.92 (d, J = 8.0 Hz, 1 H), 7.49 (t, J = 8.0, 7.4 Hz, 1 H), 7.26 (t, J = 8.0, 7.4 Hz, 1 H), and 6.97 (d, J = 8.0 Hz, 1 H), indicating the presence of a 1,2-disubstituted benzene ring (Table 2). Three aromatic protons at δ_H 7.14 (d, J = 8.7 Hz, 1 H), 6.91 (d, J = 2.3 Hz 1 H), and 6.77 (dd, J = 8.7, 2.3 Hz, 1 H) indicated the presence of a 12,4-trisubstituted benzene ring. Signals at δ_H 1.80 (s, 3 H) and 1.75 (s, 3 H) were attributed to two methyl groups. The ¹³C NMR and HSQC data of 3 revealed the presence of 23 carbon resonances, including two amide carbonyl (δ_C 171.7, 168.6), sixteen aromatic carbons (δ_C 149.7, 136.5, 135.6, 133.3, 132.0, 131.7, 128.7, 128.1, 125.6, 125.5, 121.0, 120.0, 111.9, 110.8, 106.9, 103.1), and five aliphatic resonances (δ_C 52.3, 44.5, 25.8, 24.4, 18.2). The ¹H NMR and ¹³C NMR data of 3 were similar to those of the previously reported compound asperdiazapinone D,²² except for the signals of aromatic protons of an indole moiety. In the ¹H NMR spectrum, signals of an ABX system were observed at δ_H 7.14 (d, J = 8.7 Hz), 6.77 (dd, J = 8.7, 2.3 Hz) and 6.91 (d, J = 2.3 Hz), while the signals of asperdiazapinone D were at δ_H 7.34, 6.65 and 6.76. In the HMBC spectrum, the correlations from δ_H 6.77 (H-7) to δ_C 132.0 (C-9) and δ_C 103.1 (C-5), and from 7.14 (H-8) to δ_C 128.7 (C-4) and δ_C 149.7 (C-6) confirmed that the hydroxy group was located at C-6. The HMBC cross peaks (Figure 2) from δ_H 4.58 (H-22) to δ_C 128.1 (C-2) and δ_C 132.0 (C-9), and from δ_H 7.02 (H-2) to δ_C 132.0 (C-9) and δ_C 128.7 (C-4) indicated that the isoprenyl moiety was connected to the indole nitrogen. The absolute configuration of C-11 was assigned as R according to a negative Cotton effect at 250 nm and a positive Cotton effect at 300 nm in the CD spectrum.²³ In addition, the negative specific rotation of 3, $[α]_{D}^{20} - 72$ (c 0.05, MeOH), also supported the result of the CD spectrum.^22,24 Thus, the structure of compound 3 was determined and named as asperdiazapinone G.

Table 2.

¹H and ¹³C NMR Data for Compound 3 (600/150 MHz, in CDCl₃, δ ppm).

No.	δ_H (J in Hz)	δ _C
1
2	7.02 (s, 1 H)	128.1
3		106.9
4		128.7
5	6.91 (d, 2.3, 1 H)	103.1
6		149.7
7	6.77 (dd, 8.7, 2.3, 1 H)	111.9
8	7.14 (d, 8.7, 1 H)	110.8
9		132.0
10	3.14 (dd, 15.2, 8.3, 1 H) 3.39 (dd, 15.2, 5.5, 1 H)	24.4
11	4.08 (dt, 8.3, 5.5, 1 H)	52.3
12		171.7
13
14		135.6
15	6.97 (d, 8.0, 1 H)	121.0
16	7.49 (t, 8.0, 7.4, 1 H)	133.3
17	7.26 (t, 8.0, 7.4, 1 H)	125.5
18	7.92 (d, 8.0, 1 H)	131.7
19		125.6
20		168.6
22	4.58 (d, 6.6, 2 H)	44.5
23	5.34 (t, 6.6, 1 H)	120.0
24		136.5
25	1.75 (s, 3 H)	25.8
26	1.80 (s, 3 H)	18.2

Most of the compounds were evaluated for their antimicrobial activities against ten Gram-negative bacteria (H. pylori 26695, H. pylori G27, H. pylori 159, H. pylori 129, E. coli MG1655, P. aeruginosa PAO1, A. baumannii ATCC19606, K. neumoniae ATCC35657, S. typhimurium 14028 s, S. dysenteriae), seven Gram-positive bacteria (E. faecalis FA2-2, E. faecium ATCC19434, B. subtilis 168, S. aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271), M. smegmatis ATCC 607, C. albicans ATCC SC5314 and C. albicans YY-1-4. The results are shown in Table S1. Compound 10 displayed strong antibacterial activity against five Gram-positive bacteria (B. subtilis 168, S. aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271) with MIC values ranging from 1.0 to 2.0 μg/mL, and displayed moderate antibacterial activity against four Gram-negative bacteria (H. pylori 26695, H. pylori G27, H. pylori 159, H. pylori 129) and M. smegmatis ATCC 607 with a MIC value of 8.0 μg/mL.

Experimental

General

Optical rotations were measured using a JASCO P-1020 polarimeter (Jasco Tokyo Japan), and the CD spectrum on a JASCO J-815 instrument. The HRESIMS data were obtained on an Agilent 6210 TOF MS system (Agilent Technologies, Santa Clara, CA, USA), UV spectra, recorded in MeOH, on a Shimadzu UV spectrometer-1800 (Shimadzu Corp., Kyoto, Japan), and IR spectra (KBr) on a Nicolet 6700 FT-IR spectrometer (Thermo electric nicoli, United States). ¹H and ¹³C NMR spectra were obtained on a Bruker AVANCE III 600 MHZ spectrometer (600 MHz for ¹H NMR and 150 MHz for ¹³C NMR) with TMS as internal standard (Bruker company, Switzerland). Semi-preparative reversed-phase HPLC (PR-HPLC) was performed on an Agilent ZORBAC SB-C18 column (5 μm, 250 × 9.4 mm id) with Agilent 1260 separation. Silica gel GF254 for TLC, supplied by the Yantai Zhifu Huangwu Silicone Factory, Yantai, China, and Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden) were used. Compounds were detected on TLC either under UV light or by heating after spraying with a solution of 40 mL H₂SO₄ and 10 g Vanillin in 500 mL H₂O. The microorganism test strains were: Gram-negative Bacteria (Helicobacter pylor 26695, H. pylor G27, H. pylor 159, H. pylor 129, Escherichia coli MG1655, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii ATCC19606, Klebsiella pneumoniae ATCC35657, Serovar typhimurium 14028 s, Shigella dysenteriae); Gram-positive bacteria (Enterococcus faecalis FA2-2, Enterococcus faecium ATCC19434, Bacillus subtilis 168, Staphylococcus aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271); Mycobacterium smegmatis ATCC 607, Candida albicans ATCC SC5314 and C. albicans YY-1 to 4. All were provided by the Department of Pathogen Biology & Jiangsu Key Laboratory of Pathogen Biology, Nanjing Medical University, Nanjing, China.

Fungal Material

Strain WHUF03110 was isolated on GPY medium (glucose 20 g, peptone 10 g, yeast extract 10 g, sea salts 15 g, agar 20 g, ddH₂O 1 L, pH 7.5) from a mangrove soil sample collected from Yalong Bay, at Sanya, Hainan, China, in Dec, 2018. The strain was first selected by the strong antibacterial activities against Staphylococcus aureus and Escherichia coli of its crude 80% methanol extract of its agar plate culture on PDA (Potato Dextrose Water 25 g, agar 20 g, sea-salt 15 g, ddH₂O, 1 L, pH 6.0 ± 0.2). The fungus was identified as Aspergillus sp. based on its morphological characteristics and the internal transcribed spacer (ITS) sequence (626 bp), which has 99.65% similarity to Aspergillus spinosus NRRL 5034 (NCBI Reference Sequence: NR_137489.1); the sequenced data derived from the fungal strain have been deposited in GenBank (accession no. MZ661122). The strain is stored at −80°C in the School of Pharmaceutical Sciences, Wuhan University, China.

Fermentation

The fungus was cultured on seed A medium-agar plates at 28°C for 3 days. The seed A medium (soluble starch 15 g, glucose 5 g, peptone 5 g, yeast extract 5 g, [NH₄]₂SO₄ 0.5 g, K₂HPO₄ 0.5 g, NaCl 0.5 g, MgSO₄ 0.5 g, CaCO₃ 1 g, made up to 1 L with water, pH 7.5) was inoculated with strain WHUF03110 and incubated at 28 °C for 72 h on a rotating shaker (180 rpm). The mass scale fermentation of strain WHUF03110 was carried out using solid rice medium in 1000 mL flasks (rice 200 g, distilled water 200 mL) inoculated with 15 mL of seed solution. Flasks were statically fermented for 30 days at 26 °C in a solid rice medium.

Extraction and Isolation

The entire fermented cultures from 80 flasks were harvested and ultrasonically extracted with EtOAc, three times for 30 min each time. The EtOAc extract (57.0 g) was fractionated by silica gel (200-300 mesh) column chromatography, eluting with a light petroleum-EtOAc gradient system (10:1, 4:1, 1:1, v/v), and a dichloromethane-MeOH gradient system (10:1, 0:1, v/v) to afford nine fractions A-I. Fraction D (3.6 g) was separated on a silica gel column, eluting with light petroleum-EtOAc (15:1, 8:1, 2:1, v/v), to yield fractions D1-D8. Fraction D2 (1.8 g) was subjected to silica gel column chromatography eluting with light petroleum-EtOAc (15:1, 12:1,8:1, v/v) to give 6 subfractions (Fr. D2A-D2F). Fr. D2C (650.0 mg) was purified by HPLC eluting with MeOH-H₂O (65:35, v/v) to afford 1 (5.7 mg). Fraction D2A was submitted to HPLC eluting with MeOH-H₂O (99:1, v/v) to afford 8 (3.8 mg). Fr. D5 (100.0 mg) was purified by HPLC, eluting with MeOH-H₂O (55:45, v/v), to afford 12 (17.0 mg). Fr. D3 (640.0 mg) was subjected to silica gel column chromatography, eluting with light petroleum-EtOAc (15:1, 12:1, 8:1, v/v), to yield fractions D3-1-D3-9. Fr. D3-6 (56.0 mg) was purified by HPLC eluting with MeOH-H₂O (55: 45, v/v) to afford 7 (6.4 mg). Fr. H (2.2 g) was separated on a Sephadex LH-20 column, eluting with dichloromethane-MeOH (1:1, v/v), to yield fractions H1-H8. HPLC of Fr. H3 (20.0 mg), eluting with MeOH-H₂O (90:10, v/v), afforded 11 (15.0 mg). Fr. H5 (185.0 mg) was fractionated by HPLC, eluting with MeOH-H₂O (60:40, v/v), to obtain eight subfractions. Fr. H5G, by HPLC eluting with MeOH-H₂O (58:42, v/v), yielded 3 (2.5 mg) and 5 (16.0 mg). Fr. E (2.1 g) was separated on a silica gel column, eluting with a light petroleum-EtOAc gradient system (10:1, 5:1, 0:1, v/v), to yield fractions Fr. E1-E14. Fr. E5 (40.0 mg) was further purified by HPLC, eluting with MeOH-H₂O (68:32, v/v), to afford 2 (3.0 mg). Fr. E9 was separated by silica gel chromatography to afford 4 (80.0 mg). Fr. E10 (60.0 mg) was purified by HPLC, eluting with MeOH-H₂O (55:45, v/v), to afford 6 (4.2 mg). Fr. G (3.0 g) was separated on a silica gel column, eluting with light petroleum-EtOAc (10:1, 5:1, 2:1, v/v), to afford nine fractions G1-G10. Fr. G11 (28.0 mg), by HPLC eluting with MeOH-H₂O (80:20, v/v), afforded 9 (4.6 mg). HPLC of Fr. G10 (100.0 mg), eluting with MeOH-H₂O (86:14, v/v), afforded 10 (20.0 mg).

Spinosusone A (1): Yellow amorphous solid; UV (c 0.01, CH₃OH) λ_max (log ε) 206 (4.39), 232 (4.47), 258 (4.45), 286 (4.04) nm; IR (KBr) v_max 2951, 2923, 2850, 1736, 1646, 1604, 1474, 1231 cm⁻¹; HRESIMS m/z 315.0861 [M + H]⁺ (calcd. for C₁₇H₁₅O₆, 315.0869); ¹H and ¹³C NMR data, see Table 1.

Spinosusone B (2): Yellow amorphous solid; UV (c 0.05, CH₂Cl₂) λ_max (log ε) 237 (4.14), 270 (4.21) nm; IR (KBr) v_max 3438, 2957, 2922, 2851, 1734, 1651, 1606, 1462, 1274, 1231 cm⁻¹; HRESIMS m/z 367.0799 [M + Na]⁺ (calcd. for C₁₈H₁₆NaO₇, 367.0788); ¹H and ¹³C NMR data, see Table 1.

Asperdiazapinone G (3): Pink amorphous solid; $[α]_{D}^{20} - 72$ (c 0.05, MeOH); UV (c 0.01, MeOH) λ_max (log ε) 238 (5.03), 283 (4.75) nm; IR (KBr) v_max 3246, 2958, 2921, 2851, 1686, 1647, 1607, 1577, 1465, 1402, 1260 cm⁻¹; HRESIMS m/z 388.1669 [M-H]⁻ (calcd. for C₂₃H₂₂N₃O₃, 388.1661); ¹H and ¹³C NMR data, see Table 2.

Antimicrobial Assays

The antibacterial and antifungal assays were conducted in 96-well plates using a broth micro dilution method.²⁵ The specific experiment was as follows: the initial cultures for H. pylori strains were maintained on Brain Heart Infusion (BHI) medium, and other pathogenic strains were maintained on Luria Broth (LB) agar. A single bacterial colony was picked, and suspended in LB/BHI broth to approximately 10⁶ CFU/mL. Each test compound was dissolved in MeOH and serially diluted to 9 different concentrations (16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg/mL) using LB broth on a 96-well plate. An aliquot (10 μL) of bacterial suspension was then added to each well. Sterile water was used as the negative control. Amoxicillin, vancomycin, and amphotericin B were used as the positive control for H. pylori, S. aureus, C. albicans strains, respectively. Ampicillin was used as the positive control for the other strains. The plate was incubated at 37°C aerobically for 24 h. MIC values were defined as the minimum concentration of compound that inhibited visible bacterial growth.

Conclusions

In conclusion, twelve compounds, including three new compounds (1-3), and nine known compounds (4-12) were isolated by fractionation of the EtOAc extract prepared from a rice fermentation culture of Aspergillus sp. WHUF03110. All the structures were confirmed by detailed analysis of their spectroscopic data. Compound 10 displayed strong antibacterial activity against five Gram-positive bacteria (B. subtilis 168, S. aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271), with MIC values ranging from 1.0 to 2.0 μg/mL, and displayed moderate antibacterial activity against four Gram-negative bacteria (H. pylori 26695, H. pylori G27, H. pylori 159, H. pylori 129) and M. smegmatis ATCC 607 with MIC value of 8.0 μg/mL.

Footnotes

Acknowledgments

This research was funded by grants from National Key Research and Development Program of China. (Grant Numbers: 2018YFC0311000) and National Natural Science Foundation of China. (Grant Numbers: 81603255). We thank Jun-Nan Shen and De-Sheng Li for their contribution on strain isolation and crude extracts preparation.

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.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Key Research and Development Program of China (grant number 2018YFC0311000, 81603255).

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

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.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Huawei Lv

Supplemental material

Supplemental material for this article is available online.

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Three New Metabolites From the Marine-Derived Fungus Aspergillus sp. WHUF03110

Abstract

Keywords

Introduction

Results and Discussion

Experimental

General

Fungal Material

Fermentation

Extraction and Isolation

Antimicrobial Assays

Conclusions

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Trial Registration

ORCID iD

Supplemental material

References

Supplementary Material