Polyketides and Meroterpenes from the Marine-Derived Fungi Aspergillus unguis 158SC-067 and A. flocculosus 01NT-1.1.5 and Their Cytotoxic and Antioxidant Activities

Abstract

Ten secondary metabolites, including a new grifolin analog, grifolin B (1); a new homovalencic acid derivative, 12-hydroxyhomovalencic acid (7); and a compound isolated from a natural source for the first time (9), along with seven known compounds, grifolin (2), averantin (3), 7-chloroaverantin (4), 1′-O-methylaverantin (5), 7-hydroxy-2-(2-hydroxypropyl)-5-pentylchromone (6), homovalencic acid (8), and bekeleylactone E (10), were isolated from two fungal strains. The structures of 1–10 were identified by detailed analysis and comparison of their spectroscopic data with literature values. Compounds 9 and 10 showed moderate cytotoxic activity against a panel of cancer cell lines (PC-3, HCT-15, MDA-MB-231, ACHN, NCI-H23, NUGC-3), with the GI₅₀ values ranging from 1.1 µM to 3.6 µM, whereas 1 displayed a weak 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity without cytotoxicity against all tested cell lines.

1. Introduction

Marine habitats have been acknowledged as prolific sources of new chemical entities with various worthwhile pharmacological activities [1]. Over the past decade, more than 1000 new marine natural products have been reported annually [2]. Whereas the discovery of new compounds from tunicates, cnidarians, and sponges is diminishing, there is a remarkable increase in the number of new substances isolated from marine-derived bacteria and fungi [2]. According to the latest statistics, new natural products (NPs) reported from marine-derived fungi accounted for almost half (47%) of the total new marine NPs reported in 2019 [2].

The genus Aspergillus is one of the most ubiquitous genera of filamentous fungi, and they are the major contributor to marine-derived fungal natural products [2,3]. A great number of secondary metabolites with structural diversity, such as polyketides, alkaloids, terpenes, steroids, and peptides, have been isolated from this genus, and many of them display potent biological activities [2].

As part of our ongoing program to investigate marine-derived fungi as an underexplored source of new natural products, we focused our attention on Aspergillus unguis 158SC-067 and A. flocculosus 01NT-1.1.5 strains, which showed good antimicrobial activity in the preliminary screening. Our previous studies on the EtOAc extract of A. flocculosus 01NT-1.1.5 grown on rice medium led to the isolation of fungal metabolites having antimicrobial properties and the suppression of RANKL-induced osteoclastogenesis activities [4,5]. To further study the secondary metabolites from marine-derived fungi, the “one strain many compounds” (OSMAC) strategy was applied by changing the culture medium from rice medium to Bennett’s broth medium. Interestingly, the ¹H NMR spectra of the crude extracts from A. unguis 158SC-067 and A. flocculosus 01NT-1.1.5 grown in Bennett’s broth medium showed some unique peaks in aromatic and olefinic regions, which did not appear or were much smaller when cultured in the rice medium. Therefore, the extracts from two strains were chemically investigated. As a result, two new phenolic compounds (1 and 7), together with eight known compounds (2–6 and 8–10), were isolated (Figure 1). Herein, we report the isolation, structure determination, and bioactivities of these compounds.

2. Results and Discussion

Compound 1 was isolated as a brown solid, and its molecular formula was deduced as C₁₉H₂₆O₄ by HRESIMS data (m/z 341.1728 [M + Na]⁺, calculated for C₁₉H₂₆O₄Na 341.1724), requiring seven degrees of unsaturation. The ¹H NMR spectrum revealed signals of two aromatic protons at δ_H 6.12 (2H, s, H-4 and H-6); two olefinic protons at δ_H 5.21 (t, J = 7.0, H-2′) and 5.12 (t, J = 7.0, H-6′); ten methylene protons at δ_H 3.24 (d, J = 7.1, H₂-1′), 2.26 (m, H₂-9′), 2.20 (m, H₂-8′), 2.07 (dd, J = 7.3, 14.6, H₂-5′), and 1.96 (t, J = 7.4, H₂-4′); and three methyl groups at δ_H 2.13 (s, H₃-7), 1.74 (s, H₃-12′), and 1.57 (s, H₃-11′) (

Table 1. ¹H and ¹³C NMR spectroscopic data for 1 and 7.

Compound	1		7
Position	δH (Mult, J in Hz)	δC, Type	Position	δH (Mult, J in Hz)	δC, Type
1, 3		156.9, C	1		176.0, C
2		113.3, C	2	3.52, s	41.1, CH2
4, 6	6.12, s	108.5, CH	3		128.2, C
5		137.2, C	4, 8	7.18, d (8.5)	131.3, CH
7	2.13, s	21.3, CH3	5, 7	6.86, d (8.6)	115.7, CH
1′	3.24, d (7.1)	22.9, CH2	6		159.2, C
2′	5.21, t (7.0)	125.2, CH	9	4.60, d (6.3)	65.6, CH2
3′		134.2, C	10	5.71, td (1.2, 6.3)	121.1, CH
4′	1.96, t (7.4)	40.7, CH2	11		140.8, C
5′	2.07, dd (7.3, 14.6)	27.5, CH2	12	3.98, s	67.8, CH2
6′	5.12, t (7.0)	126.0, CH	13	1.74, s	14.0, CH3
7′		134.6, C
8′	2.20, m	35.9, CH2
9′	2.26, m	34.2, CH2
10′		177.9, C
11′	1.57, s	16.0, CH3
12′	1.74, s	16.2, CH3

¹H and ¹³C NMR spectra were recorded in CD₃OD at 600 MHz and 150 MHz, respectively.

). The ¹³C NMR spectrum, in combination with the gHSQC NMR spectrum, displayed nineteen resonances belonging to a carboxyl carbon at δ_C 177.9 (C-10′); six non-protonated sp² carbons at δ_C 156.9 (C-1 and C-3), 137.2 (C-5), 134.6 (C-7′), 134.2 (C-3′), and 113.3 (C-2); four protonated sp² carbons at δ_C 126.0 (C-6′), 125.2 (C-2′), and 108.5 (C-4 and C-6); five sp³ methylene carbons at δ_C 40.7 (C-4′), 35.9 (C-8′), 34.2 (C-9′), 27.5 (C-5′), and 22.9 (C-1′); and three methyls at δ_C 21.3 (C-7), 16.2 (C-12′), and 16.0 (C-11′). One carboxyl and ten sp² carbons were accounted for six out of seven degrees of unsaturation, indicating that 1 possesses a monocyclic skeleton.

The gross structure of 1 was identified by a detailed analysis of ¹H-¹H COSY and HMBC data. The structure of a symmetrical 1,2,3,5-tetrasubstituted benzene ring was identified by the HMBC cross peaks from H-4 to C-2, C-3, and C-6, and from H-6 to C-2, C-4, and C-5 (Figure 2). A methyl group attached to C-5 of the benzene ring was confirmed by the HMBC correlations from H₃-7 to C-4, C-5, and C-6, and those of H-4/C-7 and H-6/C-7. The side chain was determined as a 4,8-dimethyldeca-4,8-dienoic acid by the COSY correlations from H₂-1′/H-2′, H₂-4′/H₂-5′, H₂-5′/H-6′, and H₂-8′/H₂-9′; as well as the HMBC cross peaks from H₃-12′ to C-2′, C-3′, C-4′; from H₃-11′ to C-6′, C-7′, C-8′; and from H₂-8′ to C-6′ and C-10′. The side chain connected to the ring at C-2 was supported by the HMBC cross peaks from H₂-1′ to C-1, C-2, and C-3.

The NOESY correlations from H-2′ to H₂-4′, H₃-12′ to H₂-5′, and no observed correlation from H-2′ to H₃-12′ confirmed the geometry of ∆^2′ as 2′E. Similarly, ∆^6′ was deduced as 6′E as shown in Figure 2. Thus, 1 is a new derivative of the co-isolated compound, grifolin (2) [6], and named grifolin B (Figure 1).

Compound 7 was isolated as a yellowish powder with a molecular formula of C₁₃H₁₆O₄ based on its HRESIMS data (m/z 259.0945 [M + Na]⁺, calculated for C₁₃H₁₆O₄Na 259.0946), requiring six degrees of unsaturation. The ¹H NMR spectrum revealed the presence of seven signals, which were classified into two pairs of magnetically symmetrical protons at δ_H 7.18 (d, J = 8.5, H-4 and H-8) and 6.86 (d, J = 8.6, H-5 and H-7); an olefinic proton at δ_H 5.71 (td, J = 1.2, 6.3, H-10); two oxygenated sp³ methylenes at δ_H 4.60 (d, J = 6.3, H₂-9) and 3.98 (s, H₂-12); a sp³ methylene at δ_H 3.52 (s, H₂-2); and a methyl group at δ_H 1.74 (s, H₃-13). The ¹³C and gHSQC NMR spectra revealed the presence of thirteen carbon signals belonging to a carboxyl carbon at δ_C 176.0 (C-1); three non-protonated sp² carbons at δ_C 159.2 (C-6), 140.8 (C-11), and 128.2 (C-3); two pairs of magnetically symmetrical carbons at δ_C 131.3 (C-4 and C-8) and 115.7 (C-5 and C-7); a protonated sp² carbon at δ_C 121.1 (C-10); two oxygenated sp³ methylene carbons at δ_C 67.8 (C-12) and 65.6 (C-9); a sp³ methylene at δ_C 41.1 (C-2); and a methyl at δ_C 14.0 (C-13).

The structure of a symmetrical 1,4-disubstituted benzene ring was determined by the COSY correlations from H-4 to H-5 and from H-7 to H-8, and the HMBC correlations from H-4 to C-6 and C-8, from H-5 to C-3 and C-7, from H-7 to C-3 and C-5, and from H-8 to C-4 and C-6 (Figure 2). A carboxy methyl group attached to the benzene ring at C-3 was supported by the HMBC correlations from H₂-2 to C-1, C-3, C-4, and C-8. The HMBC correlation from H₂-9 to C-6 supported that a prenyl unit was attached to C-4 via an ether linkage. The fact that CH₂-12 bears a hydroxy group was evidenced by the chemical shift values of H₂-12 (δ_H 3.98) and C-12 (δ_C 67.8) as well as the molecular formula. The geometry of the double bond between C-10 and C-11 was determined as 10E by the strong NOESY correlation from H-10 to H₂-12 (Figure S14). Thus, 7 is a new derivative of the co-isolated compound, homovalencic acid (8) [7], and named 12-hydroxyhomovalencic acid.

The previously described compounds were identified as grifolin (2) [6], averantin (3) [8], 7-chloroaverantin (4) [8], 1′-O-methylaverantin (5) [8], 7-hydroxy-2-(2-hydroxypropyl)-5-pentylchromone (6) [9], homovalencic acid (8) [7], (5R,6S,16R,3E)-5,6-dihydroxy-16-methyloxacyclohexadec-3-en-2-one (9) [10], and bekeleylactone E (10) [11] by comparison of their spectroscopic data and the signs of optical rotation with those reported in the literature. It is noteworthy that 9 was isolated for the first time from natural source in this study, and its spectroscopic data were identical to those reported for a synthetic analog by Stierle et al. (Figures S15–S18) [10].

Since some of the previously reported compounds isolated in this work have been shown to possess cytotoxic activity [8,12], 1, 7, 9, and 10 were evaluated for their cytotoxicity against six cancer cell lines, HCT-15 (colon), NUGC-3 (stomach), NCI-H23 (lung) ACHN (renal), PC-3 (prostate), and MDA-MB-231 (breast), which are the most common cancer types in Korea. However, only 9 and 10 showed moderate cytotoxic activity against all of the tested cell lines, with GI₅₀ values ranging from 1.1 μM to 3.6 μM (

Table 2. Growth Inhibition (GI₅₀, μM) of 9 and 10 against human cancer cell lines.

Cell Lines	9	10	Adr.
PC-3	2.7	3.6	0.17
HCT-15	3.0	2.8	0.12
MDA-MB-231	2.4	3.1	0.16
ACHN	3.4	3.1	0.16
NCI-H23	1.1	1.2	0.13
NUGC-3	2.7	2.6	0.16

Adr. Adriamycin as a positive control. GI₅₀ values are the concentration corresponding to 50% growth inhibition.

). Additionally, 1 and 7 were screened for their DPPH radical scavenging activity. Compound 1 showed a weak DPPH radical scavenging activity with an IC₅₀ value of 86.4 µM, whereas 7 showed neither cytotoxic nor DPPH radical scavenging activity.

3. Materials and Methods

3.1. General Experimental Procedures

High-resolution ESIMS data were measured with a hybrid ion-trap time-of-flight mass spectrometer (Shimadzu LC/MS-IT-TOF, Kyoto, Japan). IR spectra were obtained on a JASCO FT/IR-4100 spectrophotometer (JASCO Corporation, Tokyo, Japan). The 1D and 2D NMR spectra were recorded by a Bruker 600 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). HPLC was performed using a semi-preparative ODS column (YMC-Triart C18, 250 × 10 mm i.d, 5 µm) and an analytical ODS column (YMC-Triart C18, 250 × 4.6 mm i.d, 5 µm) (YMC Corporation, Kyoto, Japan). UV spectra were measured with a Shimadzu UV-1650PC spectrophotometer in 1 mm quartz cells (Shimadzu Corporation, Kyoto, Japan). All the reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), and the organic solvents and water were distilled prior to use. Cancer cell lines were obtained from Japanese Cancer Research Resources Bank (JCRB) (NUGC-3, gastric adenocarcinoma, JCRB Cell Bank/Cat. # JCRB0822) and American Type Culture Collection (ATCC) (PC-3, prostate adenocarcinoma, ATCC/Cat. # CRL-1435; MDA-MB-231, breast adenocarcinoma, ATCC/Cat. # HTB-26; ACHN, renal adenocarcinoma, ATCC/Cat. # CRL-1611; NCI-H23, lung adenocarcinoma, ATCC/Cat. # CRL-5800; HCT-15, colorectal adenocarcinoma, ATCC/Cat. # CCL-225).

3.2. Fungal Material, Fermentation and Isolation of Secondary Metabolites

3.2.1. Fungal Material, Fermentation, and Isolation of 1–6 from Aspergillus unguis 158SC-067

The strain Aspergillus unguis 158SC-067 was isolated from a seawater sample collected at the depth of 30 m near the Socheongcho Ocean Research Station, Korea, in August 2015. The fungus was identified as Aspergillus unguis on the basis of DNA amplification and ITS gene sequencing (GenBank accession number MZ489151). The strain was deposited in the Microbial Culture Collection, KIOST, with the name of Aspergillus sp. 158SC-067 under the curatorship of Hee Jae Shin.

The seed and mass cultures were conducted in Bennett’s medium (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, natural sea salts 3.2%, and agar 1.7% for agar medium). At first, the fungus was grown on Bennett’s agar medium in a Petri dish under static condition for 7 days. Agar plugs were cut into small pieces and aseptically transferred into a 500 mL conical flask containing 300 mL of Bennett’s broth medium and placed on a rotary shaker (140 rpm) at 28 °C for 7 days for the seed culture. An aliquot (0.1% v/v) from the seed culture was inoculated into 2.0 L flasks, each containing 1.0 L of the medium, and cultured under the same conditions as described for the seed culture for 14 days. In total, 20 flasks were prepared for the mass production.

After cultivation, the culture broth and mycelium were separated by filtration. The broth was extracted with EtOAc (20 L, twice). The EtOAc layer was evaporated under reduced pressure at 37 °C to yield a broth extract (1.0 g). Afterward, the extract was separated into 10 fractions (fractions 1b–10b) by vacuum liquid chromatography on an ODS column using a stepwise elution with 100 mL each of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% MeOH in H₂O and 100% MeOH. Compound 1 (3.0 mg) was isolated from fraction 7b by a semipreparative HPLC (YMC-PackODS-A, 250 × 10 mm i.d., 5 µm, flow rate 2.0 mL/min) with an isocratic elution of 60% MeOH in H₂O for 40.0 min.

The mycelium was extracted with EtOAc (3.0 L, three times) and the EtOAc solution was evaporated under reduced pressure to yield a mycelium extract (2.0 g). The extract was fractionated into 10 fractions (fraction 1m–10m) by the same procedure described for the broth extract. Compounds 2 (1.0 mg, t_R = 54 min), 3 (10.0 mg t_R = 64 min), and 4 (1.0 mg, t_R = 92 min) were purified from fraction 9m by a semipreparative HPLC (YMC-PackODS-A, 250 × 10 mm i.d., 5 μm, flow rate 2.0 mL/min) with an isocratic elution of 80% MeOH in H₂O. Fraction 10m was subjected to a semipreparative HPLC (YMC-PackODS-A, 250 × 10 mm i.d., 5 μm, flow rate 2.0 mL/min) with an isocratic elution of 90% MeOH in H₂O to obtain compound 5 (2.0 mg, t_R = 70 min). Compound 6 (1.5 mg) was isolated from fraction 7m by an analytical HPLC (YMC-PackODS-A, 250 × 4.6 mm i.d., 5 μm, flow rate 0.8 mL/min) with an isocratic elution of 60% MeOH in H₂O for 38 min.

3.2.2. Fungal Material, Fermentation, and Isolation of 7–10 from Aspergillus flocculosus 01NT-1.1.5

Aspergillus flocculosus 01NT-1.1.5 was isolated from a Stylissa sp. sponge as previously described [4]. Based on NMR-guided isolation, the ¹H NMR spectrum of the crude extract from the culture broth of A. flocculosus 01NT-1.1.5 showed some interesting peaks in olefinic and aromatic regions. Therefore, the broth extract was selected for further study. The culture broth was extracted with EtOAc, and the organic extract was fractionated into 15 fractions as described previously [13]. Compound 7 (10.0 mg) was purified from fraction 8 by a semipreparative HPLC (YMC-PackODS-A, 250 × 10 mm i.d., 5 μm, flow rate 2.5 mL/min) with an isocratic elution of 25% MeCN in H₂O for 28.0 min. Compound 8 (10.0 mg) was isolated from fraction 10 by an analytical HPLC (YMC-PackODS-A, 250 × 4.6 mm i.d., 5 μm, flow rate 1.0 mL/min) with an isocratic elution of 50% MeCN in H₂O for 15 min. Fraction 12 was subjected to an analytical HPLC (YMC-PackODS-A, 250 × 4.6 mm i.d., 5 μm, flow rate 1.0 mL/min) with an isocratic elution of 50% MeCN in H₂O to yield 9 (3.0 mg, t_R = 20.0 min) and 10 (3.0 mg, t_R = 27 min).

Grifolin B (1): brown solid, UV (MeOH) λ_max (log ε) 204 (4.15), 228 (3.73), 277 (3.12) nm; IR ν_max 3678, 2987, 1706, 1452, 1058 cm⁻¹; HRESIMS m/z 341.1728 [M + Na]⁺, calculated for C₁₉H₂₆O₄Na 341.1724); ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz), see Table 1.

12-Hydroxyhomovalencic acid (7): yellowish powder, UV (MeOH) λ_max (log ε) 203 (4.23), 227 (3.81), 276 (3.15) nm; IR ν_max 3373, 2925, 1705, 1509, 1224, 1176 cm⁻¹; HRESIMS m/z 259.0945 [M + Na]⁺, calculated for 259.0946, C₁₃H₁₆O₄Na; ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz), see Table 1.

3.3. Cytotoxicity Test by SRB Assay

Cytotoxicity Test by SRB Assay has been described previously [14].

3.4. DPPH Radical Scavenging Assay

DPPH radical scavenging assay was performed according to the previously described method with minor modification [3,15]. The samples and a positive control, ascorbic acid, were dissolved in DMSO with final concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL. DPPH was dissolved in anhydrous ethanol (EtOH) with a concentration of 0.04 mg/mL. Tested samples (50 µL) were added to 50 µL of fresh DPPH, then kept in room temperature in the dark for 30 min. The optical density (OD) was measured by an AMR-100 microplate reader (Hangzhou Allsheng Instruments, Hangzhou, China) at 517 nm. The EtOH and DMSO were used as a blank and negative control, respectively. The IC₅₀ values were determined by the software of GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) [3].

4. Conclusions

In summary, on the basis of the OSMAC strategy, ten secondary metabolites, including two new phenolic derivatives (1 and 7), and a substance isolated from a natural source for the first time (9), together with seven known compounds (2–6, 8, and 10), were isolated from two fungal strains of the genus Aspergillus. Compounds 9 and 10 showed moderate cytotoxic activity, while 1 exhibited a weak DPPH radical scavenging activity without cytotoxicity. To the best of our knowledge, the known compounds (2–6) were isolated from A. unguis for the first time. Moreover, we also found that A. flocculosus 01NT-1.1.5 produces various chemical constituents in different culture media [4,13]. This study expanded the chemical and biological diversity of natural products isolated from marine-derived fungi. The results indicate that marine-derived fungi, particularly the Aspergillus genus, could be a promising source to search for bioactive natural products with unique structures for discovery of new anti-cancer drugs.