Research on Secondary Metabolites From the Soft Coral-Associated Fungus Aspergillu s sydowii SCSIO41203 and Their Bioactivities

Abstract

Objective: As part of our ongoing search for bioactive secondary metabolites, the Beibu Gulf soft coral-derived fungus Aspergillus sydowii SCSIO41203 was investigated, which led to the isolation of 15 compounds. Methods: These compounds were purified by chromatographic methods, including normal phase silica gel column chromatography, octadecylsilyl column chromatography, Sephadex LH-20, and high-performance liquid chromatography. Their structures were elucidated by nuclear magnetic resonance, and mass spectrometry and compared their data with those related metabolites in the literature. Results: The structures of 1 to 15 were identified as expansol F (1), expansol C (2), expansol D (3), (Z)-5-(hydroxymethyl)-2-(6′-methylhept-2′-en-2′-yl)phenol (4), (E)-5-(hydroxymethyl)-2-(6′-methylhept-2′-en-2′-yl)phenol (5), sydonol (6), (-)-(7R,10S)-10-hydroxysydowic acid (7), (-)-(7R,10R)-iso-10-hydroxysydowic acid (8), sydowic acid (9), 1-hydroxyboivinianin A (10), (+)-sydonic acid (11), 3-hydroxydiorcinol (12), cordyol C (13), (R)-mevalonolactone (14), and 4-methyl-5,6-dihydropyren-2-one (15). These isolated compounds were evaluated for antibacterial, cytotoxic, and enzyme-inhibitory activities. Among them, compounds 1 to 3 and 13 showed moderate acetylcholinesterase inhibitory activity, with IC₅₀ values of 3.9, 33.8, 11.5, and 19.8 μM, respectively, with the positive control (tacrine) of 0.5 μM.

Introduction

The South China Sea is the largest and deepest portion of China offshore. As a significant resource plateau for marine drug research and development, it is also the richest distribution of tropical marine ecosystems, such as coral reefs and mangroves, which contain rich biological resources, including microorganisms. Due to the demand for resource conservation and the characteristics of microorganisms, such as easy metabolic regulation, more and more attention has been paid to marine co-epiphytes in recent years, hoping to find active metabolites with more novel structures and more potential development. ¹

Although heteropterpenes are widespread natural products produced by microorganisms, fungal heteropterpenes have attracted extensive attention due to their remarkable biological activities and complex and diverse structures.^2,3 Several star molecules with application potential were screened. For example, mycophenolic acid produced by Penicillium sp. can effectively noncompetitively inhibit inosine monophosphate dehydrogenase, and its derivatives have been developed into current immunosuppressive drugs.^4,5 In recent years, a series of aromatic myrrh sesquiterpenoids from a number of fungi strains have been obtained by Chinese researchers. These structures show antibacterial, antioxidant, and cytotoxic activities, which are valuable for further study.^6–10 In addition, acetylcholinesterase (AChE) is one of the key enzymes involved in Alzheimer's disease (AD). ¹¹ The compounds inhibiting AChE are thought to have promising anti-AD activities worth further research.

In this study, 15 known compounds, including 11 aromatic myrrh sesquiterpenoids, were isolated from the rice solid culture of Aspergillus sydowii SCSIO41203 gathered from a soft coral from the South China Sea. Four compounds showed significant anti-AChE activity.

Results and Discussion

Investigation of bioactive secondary metabolites from the coral-derived fungi, Aspergillus sydowii SCSIO41203, resulted in the isolation of fifteen compounds, including 11 aromatic myrrh-type sesquiterpenoids, which greatly enriched the structural types of heteroterpenoids metabolites from coral-derived fungi of the South China Sea (Figure 1).

Figure 1.

The chemical structures of compounds 1 to 15 from Aspergillus sydowii SCSIO41203.

It's reported that aromatized myrrh-type sesquiterpenoids have shown good antioxidant, antibacterial, and antitumor activities. For example, the compound expansol C (2) showed cytotoxic activity against human acute promyeloid leukemia cells (HL-60). ⁹ Compound 1-hydroxyboivinianin A (10) showed strong inhibition against V harveyi with MIC of 19.4 μM. ¹² Expansol F (1) exhibited antifouling activity against Bugula neritina with LC₅₀ of 6.8 μM. ¹² To further explore the potential of these structures, we screened for their inhibitory activities of pancreatic lipase and AChE, antitumor, and antibacterial activities. We found that many compounds showed significant inhibitory activity of AChE. All the compounds were tested at the concentration of 50.0 μg/mL. Compounds 5 and 12 showed weak inhibitory activities with inhibition rates of 11.5% and 21.0%, respectively, and compounds 1 to 3 and 13 showed strong inhibitory activities with inhibition rates of 86.5%, 98.0%, 99.0%, and 99.1%, respectively. In further investigation, the IC₅₀ values of AChE inhibition of compounds 1 to 3 and 13 were determined to be 3.9, 33.8, 11.5, and 19.8 μM, respectively, with the positive control (tacrine) of 0.5 μM. These compounds showed no pancreatic lipase and cytotoxic activity at 50.0 μg/mL. Except for compound 13, which showed inhibition against Staphylococcus aureus, other compounds showed no antibacterial activity.

Subsequently, molecular docking analysis was conducted to investigate the binding modes between active compounds and AChE. Generally, the docking score and the glide score are the same, according to the official website of Schrodinger. We use the docking score to assess the docking of our ligands to make people understand molecular docking easily. Compounds 1 to 3 and 13 appeared to interact with AChE protein (PDB ID: 4EY7) perfectly with the docking scores from −7.313 to −10.914 (Table 1) (the positive ligand E20 with the docking score −12.482, and tacrine with the docking score −9.965). As shown in Figure 2, phenolic hydroxyl groups of 1 (the docking score −10.914) formed 4 hydrogen bonds with the active site residues SER 293, TYR 72, and ASP 74. Additionally, the aromatic ring of 1 formed a π–π stacking interaction with TYR 341. Phenolic hydroxyl groups of 2 (the docking score −8.843) formed a hydrogen bond with the active site residue TYR 124. Additionally, the aromatic ring of 2 formed 2 π–π stacking interactions with TYR 341 and TYR 337 (Figure 2). Phenolic hydroxyl groups of 3 (the docking score −8.645) formed 4 hydrogen bonds with the active site residues TYR 341, ASP 74, and TYR 124. Additionally, the aromatic ring of 3 formed 2 π–π stacking interactions with TYR 341, TYR124, and TYR 337 (Figure 2).

Figure 2.

The molecular docking analysis of compounds 1 to 3.

Table 1.

The Molecular Docking Analysis of Compounds 1 to 3 and 13.

Compound	IC50 (μM)	Docking Score	Glide Score
1	3.9	−10.914	−10.914
2	33.8	−8.843	−8.843
3	11.5	−8.645	−8.645
13	19.8	−7.313	−7.327
The positive ligand E20	—	−12.482	−12.482
Tacrine	0.5	−9.965	−9.965

As shown in Figure 3, phenolic hydroxyl groups of tacrine (the docking score −9.965) formed one hydrogen bond with the active site residues ASP74. Additionally, the aromatic ring of tacrine formed a π–π stacking interaction with TYR 341 and TYR124. There are also 2 pi-cation interactions with TYR341 and TYR337 (Figure 3).

Figure 3.

The molecular docking analysis of tacrine.

Based on our results, the following cytotoxic structure–activity relationships can be revealed. Regarding these compounds with 3 aromatic rings, such as compounds 1 to 3, the cytotoxic effects depend on the presence/absence of the aromatic ring. These aromatic rings formed π–π stacking interactions with active site residues. Additionally, phenolic hydroxyl groups are also necessary to form hydrogen bonds with active site residues. In summary, molecular docking is a computational chemical method designed to predict the binding pattern and affinity between proteins and small molecules, and the results can be regarded as a reference to further screening for active lead compounds.

Materials and Methods

General Experimental Procedures

Refer to Supplemental Material.

Fungal Material

To ensure the reproducibility of the experimental process and the traceability of the results, the fermentation and metabolic extraction processes were operated by the standard unified procedure of the laboratory. The fungal strain SCSIO41203 was isolated from soft coral BH4, collected in Beihai, Guangxi Province, China, in November 2018. It was identified as Aspergillus sydowii SCSIO41203 based on the sequence analysis of the rDNA's internal spacer (ITS) regions (GenBank accession no. MH398045.1) and morphological analysis. A voucher specimen was deposited in the CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

Fermentation and Extraction

Refer to Supplemental Material.

Isolation and Purification

Refer to Supplemental Material.

Bioactivity Assay

All compounds 1 to 15 were screened for inhibitory effects of antitumor, antibacterial activity, pancreatic lipase, and AChE. Screening of pancreatic lipase and AChE activities was conducted according to the literature.^13–15 In order to screen compounds with inhibitory activity, a mixture of compounds and AChE react at 30 °C; if the compounds inhibited AChE, which can decrease the amount of substrate degradation, corresponding with the reaction of 5-5″-Dithio-bis(2-nitrobenzoic acid) generating less yellow compound, namely at 405 nm lower absorption value. The cytotoxic against A549 of these compounds was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide method. The antibacterial activity against Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (MRSA, clinical strain), Enterococcus faecalis (ATCC 29212), and Klebsiella pneumonia (ATCC 13883) were screened by disk diffusion method according to the literature. ¹⁶

Nuclear Magnetic Resonance Spectroscopic Data of Compounds 1 to 15

Refer to Supplemental Material.

Molecular Docking Analysis

The Schrödinger 2017-1 suite (Schrödinger Inc ) was employed to perform the docking analysis. The crystal structure of AChE (PDB code: 4EY7) ¹⁷ obtained from the Protein Data Bank was used as a starting model with all of the waters and the N-linked glycosylated saccharides removed. It was constructed following the Protein Prepare Wizard workflow in Maestro 11-1. The prepared ligands were then flexibly docked into the receptor using the induced-fit module with the default parameters. The figures were generated using PyMol molecular graphics software (Schrödinger 2017-1, Schrödinger Inc).

Conclusion

In total, 15 compounds were isolated from the Beibu Gulf soft coral-derived fungus Aspergillus sydowii SCSIO41203. Compounds 1 to 3 and 13 exhibited moderate AChE inhibitory activity, with IC₅₀ values of 3.9, 33.8, 11.5, and 19.8 μM, respectively. Moreover, molecular docking analysis was conducted to investigate the binding modes between active compounds and AChE. Compounds 1 to 3 and 13 appeared to interact with AChE protein (PDB ID: 4EY7) perfectly, with the docking scores from −7.313 to −10.914. As one of the essential sources of drug lead compounds, aromatic myrrh sesquiterpenoids not only have rich chemical diversity but also have a broad spectrum of biological activities. Further studies on their activity screening and mechanism of action can be conducted to explore these compounds’ medicinal potential. This study enriched the structural diversity of sesquiterpenoids derived from coepiphytic fungi derived from corals in the South China Sea. Further revealed the AChE inhibitory activity of aromatic myrrh sesquiterpenoids. This work also provided some potential molecules for the development of AChE inhibitors.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231213220 - Supplemental material for Research on Secondary Metabolites From the Soft Coral-Associated Fungus Aspergillus sydowii SCSIO41203 and Their Bioactivities

Supplemental material, sj-docx-1-npx-10.1177_1934578X231213220 for Research on Secondary Metabolites From the Soft Coral-Associated Fungus Aspergillus sydowii SCSIO41203 and Their Bioactivities by Ying Chen, Bin Yang and Juan Liu in Natural Product Communications