Secondary Metabolites From the Grasshopper-Derived Entomopathogenic Fungus Aspergillus Tamarii NL3 and Their Biological Activities

Abstract

Chromatographic purification of the ethyl acetate extract of the entomopathogenic fungus Aspergillus tamarii NL3 culture broth led to the isolation of griseofulvin (1), isogriseofulvin (2), cytochalasin J (3), solamargine (4), and solasonine (5). The chemical structures of these compounds were identified by HRESIMS and NMR spectra, as well as by comparison with literature data. This is the first report on the isolation of 1-4 from the Aspergillus genus and compound 5 from A tamarii. Compounds 1 and 2 exhibited antifungal activity against the reference fungi with MICs of 16 to 128 µg/mL. Compound 3 displayed weak antimicrobial activity against most of the tested microorganisms with MICs ≥ 128 µg/mL, except for Bacillus subtilis and Aspergillus niger with MICs of 64 µg/mL. Compounds 4 and 5 showed activity against a wide spectrum of the reference microorganisms with MICs of 16 to 128 µg/mL but showed stronger antifungal than antibacterial activity. Furthermore, all isolates exhibited weak ABTS and DPPH scavenging activities with scavenging rates of 18.92% to 32.64% and 24.62% to 31.06%, respectively, at the concentration of 100 µg/mL.

Introduction

Fungi are one of the most important sources of bioactive components for drug discovery and development. Literature surveys indicate that about 15 600 bioactive compounds are derived from fungi. The discovery rate of fungal-derived new compounds has increased significantly in the past 2 decades. ¹ Aspergillus is a diverse genus with 339 accepted species; many of them are considered as decomposers of organic materials and cause destructive rots in agricultural products. ² The genus Aspergillus is rich in alkaloids, terpenes, steroids, and polyketones. ³

Aspergillus tamarii has produced structurally diverse compounds with a wide range of bioactivities, including an antibacterial pentacyclic indole alkaloid, ⁴ anti-inflammatory dipyrrolobenzoquinones, ⁵ antibiotic and cytotoxic cyclic peptides, ⁶ butenolides, ⁷ and anti-phytopathogenic indolyl diketopiperazines. ⁸ In a continuing search for new antimicrobial and antioxidant agents, we report herein the isolation and structural determination of 5 secondary metabolites from the ethyl acetate (EtOAc) extract of the entomopathogenic fungus A tamarii NL3 culture broth. All isolates were further evaluated for their antimicrobial activity against 6 reference bacterial strains (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Bacillus subtilis) and 4 reference fungal strains (Candida albicans, Aspergillus niger, Fusarium oxysporum, and Pythium ultimum), as well as their 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities.

Results and Discussion

Five products were obtained from the culture broth of the entomopathogenic fungus Aspergillus tamarii NL3. Their chemical structures (Figure 1) were determined as griseofulvin (1), ⁹ isogriseofulvin (2), ¹⁰ cytochalasin J (3), ¹¹ solamargine (4), ¹² and solasonine (5), ¹³ by NMR spectroscopic and HRESIMS techniques. This is the first time that compounds 1-4 have been isolated from the genus Aspergillus and compound 5 from A tamarii.

Figure 1.

The structures of 1-5 isolated from Aspergillus tamarii.

The obtained compounds 1-5 were tested for antimicrobial activity against 6 reference bacterial strains (E coli, S enterica, P aeruginosa, E faecalis, S aureus, and B subtilis) and 4 reference fungal strains (C albicans, A niger, F oxysporum, and P ultimum). As shown in Table 1, 1 and 2 showed weak effects against the reference bacteria with MICs > 128 µg/mL, but exhibited more potent antifungal activity against the tested fungi with MICs of 16 to 128 µg/mL. Compound 3 displayed weak effects against almost all the tested microorganisms with MICs ≥ 128 µg/mL, except for B subtilis and A niger with MICs of 64 µg/mL. Interestingly, compounds 4 and 5 inhibited a wide spectrum of the reference microorganisms with MICs of 16 to 128 µg/mL. Compounds 4 and 5 showed stronger antifungal activity than antibacterial activity. It is noteworthy to mention here that the spirosolane alkaloid glycosides (4 and 5) exhibited antibacterial activity stronger than that of a common antifungal drug, griseofulvin (1).

Table 1.

Antimicrobial Activities of Isolated Compounds 1-5.

Tested microorganisms	MIC (µg/mL)
	1	2	3	4	5	Cip	Mic
Bacteria
E scherichia coli	>128	>128	128	64	64	0.5	-
S almonella enterica	>128	>128	>128	64	128	2	-
P seudomonas aeruginosa	>128	>128	>128	64	128	1	-
E nterococcus faecalis	>128	>128	>128	128	64	2	-
Staphylococcus aureus	>128	>128	>128	32	128	2	-
Bacillus subtilis	>128	>128	64	32	64	4	-
Fungi
Candida albicans	64	128	>128	64	64	-	16
Aspergillus niger	32	64	64	32	64	-	8
Fusarium oxysporum	16	64	128	16	16	-	16
Rhizoctonia solani	16	64	128	16	16	-	8

Abbreviations: Cip, ciprofloxacin; Mic, miconazole.

The radical scavenging ability of these compounds on ABTS and DPPH radicals was evaluated (Table 2). Unfortunately, all isolated compounds (1-5) exhibited weak scavenging effects on ABTS and DPPH radicals, with scavenging rates of 18.92% to 32.64% and 24.62% to 31.06%, respectively, at the concentration of 100 µg/mL.

Table 2.

Antioxidant Activities of Isolated Compounds 1-5.^a

Compound	Scavenging rate (%) at 100 µg/mL
	DPPH	ABTS
1	27.12	23.04
2	24.62	18.92
3	25.07	20.71
4	31.06	32.64
5	29.89	31.32
Ascorbic acid (20 µg/mL)	92.35	92.28

^a

Ascorbic acid was used as a positive control.

Conclusion

Five known compounds, griseofulvin (1), isogriseofulvin (2), cytochalasin J (3), solamargine (4), and solasonine (5), were purified from the ethyl acetate portion of the culture broth of the entomopathogenic fungus A tamarii NL3. Except for solasonine (5), the isolated compounds are reported for the first time in the Aspergillus genus. Griseofulvin (1) and isogriseofulvin (2) exhibited antifungal effects against the reference fungi with MICs of 16 to 128 µg/mL, while cytochalasin J (3) inhibited B subtilis and A niger with MICs of 64 µg/mL. The 2 glycoalkaloids (4 and 5) inhibited a wide spectrum of the reference microorganisms with MICs of 16 to 128 µg/mL. Collectively, the current study provides a new insight that these natural products may be useful candidates for further antibiotic studies.

Materials and Methods

General Experimental Procedures

The HRESIMS were obtained from a MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255,748 Germany). All NMR spectra were recorded on either a Bruker AM500 FT-NMR spectrometer (500 MHz for ¹H-NMR and 125 MHz for ¹³C-NMR) or a Bruker Avance NEO600 spectrometer (600 MHz for ¹H-NMR and 150 MHz for ¹³C-NMR). IR spectra were obtained from KBr pellets on an IR Prestige-21 spectrometer (Shimadzu). Optical rotations were measured on a JASCO P-2000 polarimeter. The stationary phases for column chromatography (CC) included silica gel (Kieselgel 60, 70-230, 230-400 mesh, Merck), reverse phase resins (150 µm, Fuji Silysia Chemical Ltd), and Sephadex LH-20 (Pharmacia Co.). For thin layer chromatography (TLC), precoated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck) were used. Compounds were detected on TLC either under UV light or by heating after spraying with 10% H₂SO₄ in C₂H₅OH (vol/vol).

Fungal Materials

Aspergillus tamarii NL3 was isolated from the grasshopper Oxya chinensis collected from rice fields in Thua Thien Hue province, Vietnam, in July 2020. The strain NL3 was identified as A tamarii using the ITS gene sequence with primers ITS4 and ITS5. ¹⁴ The ITS gene sequence of the NL3 strain showed 100% identity to A tamarii L1 (accession number MT340979) in the NCBI GeneBank database.

The fungus was cultivated in 125 mL Potato Dextrose Broth (PDB, Himedia) in a shaking incubator at 25 °C for 72 h. Following that, the culture was then inoculated in a 5 L fermentor (BioFlo 120, Eppendorf) containing 2.5 L Glucose Peptone Yeast Broth (GPYB, Himedia) at an inoculum of 5% (vol/vol). The fermentation was performed at 25 °C for 72 h with an agitation speed of 150 rpm. Afterward, the culture was moved to a 100 L bioreactor (BioFlo 610, Eppendorf) containing 50 L GPYB at an inoculum of 5% (vol/vol). The bioreactor was maintained at 25 °C for 15 days at an agitation speed of 150 rpm.

Extraction and Isolation

The culture solution of A tamarri (50 L) was extracted with EtOAc (50 L × 3 times). The EtOAc residue (APE, 16.3 g) was separated by RP-18 CC using an increasing ratio of methanol (MeOH) (10%-100%) in water as mobile phases to obtain six fractions, APE1-APE6. Fraction APE5 (2.1 g) was separated by silica gel CC using CH₂Cl₂/EtOAc (10/1, vol/vol) as the mobile phase to yield 5 subfractions, APE5.1-APE5.5. Compound 1 (12 mg) was obtained from subfraction APE5.2 (182 mg) by RP-18 CC eluting with MeOH/water (2/1, vol/vol). Subfraction APE5.3 (110 mg) was purified by RP-18 CC, eluting with MeOH/water (2/1, vol/vol) to yield 2 (7 mg). Subfraction APE5.5 (153 mg) was separated by RP-18 CC, eluting with MeOH/water (1.5/1, vol/vol), to afford a smaller fraction, APE5.5A. Further purification of this (68 mg) by Sephadex C,C eluting with MeOH/water (1/1, vol/vol), yielded 3 (4 mg). Additionally, the fraction APE3 (1.6 g) was separated by silica gel CC by eluting with CH₂Cl₂/acetone/water (1/5/0.3, vol/vol/vol) to obtain 3 subfractions, APE3.1-APE3.3. Compound 4 (9 mg) was obtained from subfraction APE3.3 (114 mg) by purifying on a Sephadex column eluting with MeOH/water (1/1, vol/vol). Finally, subfraction APE3.1 (310 mg) was separated by silica gel CC by eluting with ethyl acetate/MeOH/water (4/1/0.2, vol/vol/vol) to yield 5 (6 mg).

Antimicrobial Assay

Antimicrobial activity of the isolated compounds was evaluated against reference microorganisms obtained from Mientrung Institute for Scientific Research, Thua Thien Hue province, Vietnam, including Gram-negative bacteria (ie, E coli ATCC 25922, S enterica ATCC 13076, P aeruginosa ATCC 27853), Gram-positive bacteria (ie, E faecalis ATCC 29212, S aureus ATCC 25923, and B subtilis ATCC 6633), and fungi (C albicans ATCC 10231, A niger MISR 11215, F oxysporum MISR 20415, and Rhizoctonia solani MISR 11115). Minimum inhibitory concentrations (MICs) of the compounds against the reference microorganisms were determined using the microdilution method, as described by Dat et al. ¹⁵ The antibiotics ciprofloxacin and miconazole were used as positive controls.

Antioxidant Assay

The ABTS free radical scavenging activity of each sample was determined using the method described by Pellegrini et al. ¹⁶ A quantity of 0.1 mL sample (concentrations ranging from 25 to 100 µg/mL) was mixed with 3.9 mL of ABTS (7 mM) solution, and then the absorbance was measured at 734 nm. Ascorbic acid was employed as a positive control. The scavenging capability toward ABTS was calculated as follows:

$$\mathrm{Scavenging}\mathrm{rate}(\text{\%})=[1\text{–}\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}/\mathrm{O}{\mathrm{D}}_{\mathrm{Blank}}]\times 100$$

The DPPH free radical scavenging effect of the sample was measured using the method described by Gopi et al, with some minor modifications. ¹⁷ One milliliter of the sample (concentrations ranging from 25 to 100 µg/mL) was added to 1 mL of 100 µM DPPH in ethanol. The reaction was homogenized for 1 min and then incubated at room temperature for 30 min. Then, its optical density was measured at 517 nm. Ascorbic acid was employed as a positive control and ethanol as a blank sample. The DPPH free radical scavenging capability was calculated as follows:

$$\mathrm{Scavenging}\mathrm{rate}(\text{\%})=[1\text{–}\mathrm{O}{\mathrm{D}}_{\mathrm{sample}}/\mathrm{O}{\mathrm{D}}_{\mathrm{Blank}}]\times 100$$

Supplemental Material

sj-doc-1-npx-10.1177_1934578X221141548 - Supplemental material for Secondary Metabolites From the Grasshopper-Derived Entomopathogenic Fungus Aspergillus Tamarii NL3 and Their Biological Activities

Supplemental material, sj-doc-1-npx-10.1177_1934578X221141548 for Secondary Metabolites From the Grasshopper-Derived Entomopathogenic Fungus Aspergillus Tamarii NL3 and Their Biological Activities by Dat Ton That Huu, Ha Tran Phuong, Phan Thi Diem Tran, Bakeo Souvannalath, Hieu Le Trung, Duc Viet Ho and Cuong Le Canh Viet in Natural Product Communications