New Cytotoxic Steroid Produced by the Soil-Derived Fungus Aspergillus flavus JDW-1

Abstract

One new steroid 1, together with seven known compounds 2 to 8, were discovered in the extract of a soil-derived fungus Aspergillus flavus JDW-1. The structure, including the absolute stereochemistry of new compound 1, was established by interpretation of extensive nuclear magnetic resonance spectroscopic data and further confirmed by X-ray crystallographic analysis. The cytotoxicity of 1 against the A-549, Hela, HCT-116, MGC-803, and HO-8910 cell lines was evaluated, which showed cytotoxicity with the half-maximal inhibitory concentration (IC₅₀) values as 5.00 to 22.38 μM. Compounds 2 to 8 exhibited moderate radical scavenging activity against DPPH with IC₅₀ values ranging from 4.7 to 28.5 μM.

The filamentous fungus Aspergillus flavus is one of the most important species in the Aspergillus genus and is distributed worldwide as a prevalent aflatoxin-producing food and feed contaminant. ¹ Meanwhile, it can also produce a wide variety of natural products, such as stilbenoids, ² diketopiperazine, ³ lactones, ⁴ and steroids ⁵ . As a widespread class of natural compounds occurring in animals, plants, and microorganism, steroids comprise a wide repertoire of structurally related natural compounds with important functions such as physiological regulators, hormones, and pro-vitamins. ^6-8 In our search for bioactive compounds from soil-derived fungus, the strain of JDW-1 was isolated from the rhizosphere soil of economic plant Cinnamomum bodinieri Levl. Its extract exhibited cytotoxicity against the P388 cells in preactive screening. Investigation of the secondary metabolites led to the isolation of a new steroid 1, helvolic acid (2), ⁹ and known sesquiterpene derivatives 3 to 8 (Figure 1, NMR data showed in Supplemental Material). ^10-12 In this paper, we described the isolation, structure elucidation, and bioactivity of those compounds.

Figure 1

The structures of Compounds 1 to 8.

Compound 1 was obtained as an amorphous powder. The molecular formula was determined as C₂₈H₄₄O₄ on the basis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis, indicating 7° of unsaturation. The infrared (IR) spectrum showed the presence of hydroxy (3422 cm⁻¹) and carbonyl (1674 cm⁻¹) groups. Analysis of the nuclear magnetic resonance (NMR) data (Table 1) of 1 revealed the presence of 5 methyls, 9 methylenes, 8 methines (2 oxygenated and 1 olefinic), and 6 quaternary carbons (1 oxygenated, 2 olefinic, and 1 carbonyl). Based on the degrees of unsaturation, it could be indicating that 1 should have a steroid core skeleton.

Table 1

¹H (600 MHz, CDCl₃) and ¹³C (150 MHz, CDCl₃) NMR Data for Compound 1.

C/H	δ H (J in Hz)	δ C
1	2.46 m; 2.30 m	37.4 (CH2)
2	2.86 m; 1.60 m	38.6 (CH2)
3		211.2 (C)
4	2.19 m; 2.27 m	43.6 (CH2)
5	1.60 m	52.6 (CH)
6	1.94 m	39.5 (CH2)
7	5.56 s	119.2 (CH)
8		133.0 (C)
9	1.77 d (9.9)	61.6 (CH)
10		34.5 (C)
11	3.98 m	68.9 (CH)
12	1.43 m; 2.30 m	50.6 (CH2)
13		43.0 (C)
14	1.96 m	61.6 (CH)
15	4.20 m	69.1 (CH)
16	1.41 m; 1.23 m	34.4 (CH2)
17	1.92, m	42.5 (CH)
18	1.14, s	11.4 (CH3)
19	0.63 s	13.3 (CH3)
20	1.43 m	35.1 (CH)
21	0.98 d (6.6)	17.8 (CH3)
22	1.89 m; 1.60 m	29.0 (CH2)
23	2.27 m; 1.93 m	26.7 (CH2)
24		155.6 (C)
25		72.8 (C)
26	1.35 s	28.5 (CH3)
27	1.35 s	28.6 (CH3)
28	5.09 s; 4.75 s	106.2 (CH2)
OH-11	4.39 d (5.5)
OH-15	4.37 d (6.6)
OH-24	4.44 s

NMR, nuclear magnetic resonance.

Spectra were recorded at 600 MHz for ¹H NMR using dimethyl sulfoxide as solvent and tetramethylsilane (TMS) as internal standard.

The planar structure of 1 was determined by the analysis of the two-dimensional NMR data (Figure 2). The COSY correlations (H₂-2/H₂-1, H₂-4/H-5/H₂-6/H-7) and the heteronuclear multiple bond correlation (HMBC) correlations between H₂-1, H₂-2, H₂-4/C-3 suggested the connectivity of C-1 to C-7. The HMBC correlations between H-9/C-7, C-8, C-10; H₂-12/C-9, C-11, C-13, C-14; H-14/C-7, C-8; and H-16/C-13, C-17 with the COSY correlations (H-14/H-15/H₂-16/H-17) led to the establishment of core ergostane skeleton. ¹³ The HMBC correlations between H₂-28/C-23, C-24, C-25 and H₃-26/C-24, C-25, C-27 together with the COSY correlations (H₂-16/H-17/H-20/H₂-22/H₂-23) suggested that the side chain was located at C-17. Additional correlations from H₃-18 C-9, C-10; H₃-19/C-13, C-14 and H₃-21/C-17, C-20 indicated the location of Me-18 on C-10, Me-19 on C-13, and Me-21 on C-20, respectively. These observed data demonstrated that 1 was a new ecdysteroid.

Figure 2

Selected key ¹H-¹H COSY and HMBC correlations of 1.

The relative configuration and absolute configuration of 1 were determined using X-ray. Crystals of 1 were obtained, and the absolute configurations (5S,9R,10S,11R,13R,14R,15S,17R,20R) were based on X-ray diffraction analysis (Flack parameter –0.08) using Cu-Kα radiation (Figure 3) and assigned by comparison with published data, ^14,15 which was established as a new steroid.

Figure 3

The Oak Ridge Thermal Ellipsoid Plot of single-crystal X-ray data of 1.

The cytotoxicities of Compounds 1 and 2 were evaluated against A-549, Hela, HCT-116, MGC-803, and HO-8910 cell lines. Compound 1 exhibited various activities with the IC₅₀ values 5.0 to 22.4 µM (Table 2), respectively. The radical scavenging activities of Compounds 3 to 8 were evaluated against DPPH with IC₅₀ values ranging from 4.7 to 28.5 µM (Table 3).

Table 2

The Cytotoxicity of Compounds 1 and 2.

	A-549	Hela	HCT-116	MGC-803	HO-8910
1	13.1	5.0	10.1	12.7	22.4
2	>100	>100	>100	>100	6.7
ADM	0.5	0.6	0.04	0.2	0.8

^aADM = doxorubicin (positive control).

Table 3

The DPPH Radical Scavenging Activities of Compounds 3 to 8.

	IC50 (μM)		IC50 (μM)
3	28.5	6	4.7
4	25.2	7	6.3
5	5.9	8	5.7
AA	15.5

AA, ascorbic acid (positive control); IC₅₀, half-maximal inhibitory concentration.

Steroids, a widespread class of natural compounds occurring in animals, plants, and fungi, had shown great therapeutic value for a broad array of pathologies. ¹⁶ Natural steroids had been showing notable cytotoxic activities, and were potential lead compounds and probed to disclose new mechanisms of anticancer. ^17,18 In our research, the compound 1 was reported the first time of the activity to Hela cell line with theIC₅₀ 5.00 µM, which might be as the potential anticancer lead compound.

Experimental

General

Colum chromatography: silica gel and silica gel GF254 (SiO₂; 200-300 mesh, 10-40 µm, Qingdao Marine Chemical Inc., China). Sephadex LH-20 (GE Healthcare, Sweden). Semi-prep: high-performance liquid chromatography (HPLC) was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µm). UV spectra were recorded on Waters 2487. IR spectra were taken on a NICOLET NEXUS 470 spectrophotometer in KBr discs. ¹H- and ¹³C-NMR, DEPT, and 2D NMR spectra: JEOL Eclipse-600 spectrometer. High-resolution electrospray ionization mass spectrometry data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer.

Strain Identification

Fungal identification was performed based on sequencing of the ITS region. The nucleotide BLAST search result showed that the sequence was the most similar to the sequence of Aspergillus flavus.

Extraction and Isolation

The media contained yeast powder, peptone, glucose, and malt extract dissolved in 1 L water with pH 7. The flasks were cultured with standing at 28°C for 30 days. All extracts were evaporated under reduced pressure and concentrated to give a crude extract (20.0 g). The extract was separated by ODS in the gradient elution step of MeOH-H₂O. Fraction 7 was further separated on a Sephadex LH-20 column to provide eight fractions. MPLC was applied to Fraction 7 to 7 to exclude the impurities, and isolated by HPLC employing isocratic elution with CH₃CN-H₂O (65:35; v/v) to obtain Compounds 1 and 2 (12.5 and 15.8 mg), and with MeOH-H₂O (70:30; v/v) to obtain Compounds 3 and 4 (14.0 and 13.0 mg). Fraction 8 to6 was applied isocratic elution with MeOH-H₂O (75:25, v/v) to yield Compounds 5 to 8 (9.0, 10.0, 15.3, and 7.3 mg).

Biological Assay

The new compound was evaluated for its cytotoxicity against A-549 and Hela cell lines by the MTT method , ¹⁹ and HCT-116, MGC-803, and HO-8910 cell lines by the SRB method. ²⁰ The new compound 1 showed moderate cytotoxicity against all the five cell lines with the IC₅₀ values as 5.00 to 22.38 μM, respectively (Table 2). In the DPPH scavenging assay, samples to be tested were dissolved in MeOH and the solution (160 µL) was dispensed into wells of a 96-well microtiter tray. Forty microliters of the DPPH solution in MeOH was added to each well. The mixture was shaken and left to stand for 30 minutes. After the reaction, absorbance was measured at 510 nm, and Compounds 3 to 8 were evaluated for low radical scavenging activity against DPPH with IC₅₀ values in the range 4.7 to 28.5 µM, respectively (Table 3).

X-Ray Crystallographic Analysis of Compound 1

Single-crystal X-ray diffraction data were collected on an Agilent Gemini Ultra diffractometer with CuKα radiation (λ = 1.54184 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. Carbon, oxygen, and nitrogen atoms were refined anisotropically. Hydrogen atoms were either refined freely with isotropic displacement parameters or positioned with an idealized geometry and refined riding on their parent C atoms. Crystals suitable for X-ray diffraction were obtained by slow evaporation of a solution in MeOH-H₂O. Crystallographic data (excluding structure factors) for 1 have been deposited with the Cambridge Crystallographic Data Centre: CCDC reference numbers 922 839 (for Compound 1). The data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

Compound 1

Amorphous powder.

[α] $25D$ : +51 (c 0.1, MeOH).

UV (MeOH) λ _max: (log ε) 210 (2.32) nm.

IR (KBr) ν _max 3422, 2955, 1674 cm^–1.

¹H NMR (600 MHz, CDCl₃): Table 1.

¹³C NMR (150 MHz, CDCl₃): Table 1.

HRESIMS: m/z [M + H]⁺ m/z 445.3314 (calcd for C₂₈H₄₄O₄ + H, 445.3312).

Crystallographic Data

Monoclinic, C₂₈H₄₄O₄, space group P2₁ with a = 10.8171(4) Å, b = 6.2921(3) Å, c = 15.0930(5) Å, α = 90°, β = 96°, γ = 90°, V = 1021.46(7) ų, Z = 2, T = 291(2) K, D_c = 1.360 g/cm³, μ = 0.781 mm⁻¹, and F (000) =440. Crystal size: 0.32 × 0.2 × 0.2 mm³. Independent reflections: 3227 with R _int = 0.0228. The final agreement factors are R ₁ = 0.0476 and w R ₂ = 0.1282 [I > 2σ (I)]. The absolute configuration was determined by Flack parameter x = –0.08(19).