Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities

Li, Yong-Chao; Yang, Jing; Zhao, Yuan-Zeng; Ma, Qin-Di; Liu, Ben-Guo; Sun, Jun-Liang

doi:10.3390/jof9030374

Open AccessArticle

Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities

¹

School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China

²

Henan International Joint Laboratory of Plant Genetic Improvement and Soil Remediation, Xinxiang 453003, China

³

School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China

^*

Author to whom correspondence should be addressed.

J. Fungi 2023, 9(3), 374; https://doi.org/10.3390/jof9030374

Submission received: 13 February 2023 / Revised: 15 March 2023 / Accepted: 17 March 2023 / Published: 19 March 2023

(This article belongs to the Special Issue Recent Advances in the Biodiversity, Biotechnology, and Biomedicine of Fungi)

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Abstract

:

Six new cytochalasans—namely, aspergicytochalasins A–F (1–6)—together with five known analogs were isolated and characterized from the endophytic fungus Aspergillus sp. from the medicinal plant Lonicera japonica. The structures of the new compounds were established by NMR and MS methods as well as single crystal X-ray diffractions. Compounds 3 and 4 showed weak antibacterial activities to Staphylococcus aureus, with MIC values of 128 and 64 μg/mL, respectively. Compounds 1, 3, 5 and 6 showed inhibitory activities on NO production, with IC₅₀ values less than 40 μM.

Keywords:

Aspergillus sp. LE2; endophytic fungus; isolation and structure elucidation; antibacterial activity; NO production inhibition

1. Introduction

Endophytic fungi have established special functions that can produce a number of bioactive metabolites due to the process of co-evolution with host plants in a long history; these metabolites are widely used in healthcare and pharmaceutical areas [1]. Therefore, the discovery of bioactive substances and even lead compounds from endophytic fungi has become one of the important contents in the study of natural products of fungi.

Lonicera japonica Thunb. is a famous medicinal plant with a wide range of pharmacological activities, including antibacterial, anti-inflammatory, antiviral and anticancer, which has been utilized in most East Asian countries [2]. Its dried bud or the initial blooming flower, called honeysuckle, is used as a tea for daily healthcare in China [3]. As a widely used medicinal herb, L. japonica has been extensively studied on its chemical composition, which has led to identification of a number of flavonoids, iridoids, phenolic acids and saponins with a wide range of biological activities [4,5,6,7,8,9,10,11]. However, the endophytic fungi system of L. japonica and the chemical components have not been studied. Several endophytic strains were isolated from the plant of L. japonica, which exhibited potential plant growth improvement [12,13,14,15]. Therefore, it is necessary to carry out a systematical study on the endophytic fungi of L. japonica and their metabolites.

In our study, sixteen fungal species were identified from both the stem and flower of L. japonica. One of the strains, identified as Aspergillus sp. LE2, exhibited certain antibacterial and anti-inflammatory activities; therefore, we carried out a chemical study on its secondary metabolites. In the current study, six previously undescribed cytochalasans—namely, aspergicytochalasins A–F (1–6)—together with five known analogs, were obtained from the cultural broth of Aspergillus sp. LE2 (Figure 1). The structures of the new compounds were identified. The new compounds were evaluated for their antibacterial activity against Escherichia coli and Staphylococcus aureus as well as for their anti-inflammatory activities by inhibiting nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages. Herein, the isolation, structure elucidation and their biological activities are reported.

2. Materials and Methods

2.1. General Experimental Procedures

To acquire the infrared (IR) spectra, we used a Shimadzu Fourier Transform Infrared spectrometer with KBr pellets. The ultraviolet (UV) spectra were acquired using a double beam spectrophotometer (UH5300). The optical rotations were acquired using a Rudolph Autopol IV polarimeter (Hackettstown, NJ, USA). High-resolution electrospray ionization mass spectra (HRESIMS) were obtained using an Agilent 6200 Q-TOF MS system or a Thermo Scientific Q Exactive Orbitrap MS system. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance III 600 MHz spectrometer (Bruker, Karlsruhe, Germany). Column chromatography (CC) was performed using a Sephadex LH-20 (GE Healthcare) with silica gel (80–100 and 200–300 mesh) and RP-18 gel (20–45 μm; FuJi). High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 μm; 9.4 mm × 150 mm or 21.2 mm × 150 mm).

2.2. Fungal Material

The fungus Aspergillus sp. LE2 was isolated from the fresh stems and flowers of Lonicera japonica Thunb. that were collected from Xinxiang, Henan Province, China. The fungal species was identified by ITS sequencing with the accession number KM051392.1. A voucher specimen (LJEF20210513-LE2) was deposited at the Department of Life Science and Technology, Henan Institute of Science and Technology.

2.3. Fermentation, Extraction and Isolation

The rice culture medium consisted of 5% yeast, 6% glucose, 0.15% pork peptone, 0.04% KH₂PO₄ and 0.06% MgSO₄. The initial pH was adjusted to 6.5.

This fungal strain was cultured in a potato dextrose agar medium at 24 °C for 7 days to be used as the seed. The seed was then transferred to a rice medium and incubated at 20 °C in a dark place for 30 days. The rice medium in each bottle (500 mL) contained rice (70 g) and water (70 mL). A total of 50 bottles were used in this work.

The solid rice culture broth (3.5 kg) was extracted five times with EtOAc to obtain a crude extract (92 g). The crude extract was separated by CC over silica gel (80–100 mesh) to obtain six fractions (A–F), as eluted with a solvent system of CHCl₃/MeOH (from 1:0 to 0:1 v/v). Fraction B (12 g) was separated by MPLC over RP-18 silica gel eluted with MeOH/H₂O (from 0:100 to 100:0) to obtain five subfractions, B1–B5. Fraction B2 (1.8 g) was separated by CC over silica gel (200–300 mesh) eluted with CHCl₃/acetone (10:1) to obtain six subfractions, B2₁–B2₆. Fraction B2₃ (320 mg) was purified by prep-HPLC eluted with CH₃CN/H₂O (from 20:80 to 60:40 for 30 min) to obtain compounds 4 (1.8 mg) and 5 (6.3 mg). Fraction B2₄ (400 mg) was purified by prep-HPLC eluted with CH₃CN/H₂O (from 20:80 to 60:40 for 30 min) to produce 2 (2.6 mg) and 6 (4.5 mg). Fraction B2₅ (900 mg) was further separated by CC over silica gel (200–300 mesh) eluted with petroleum ether/acetone (7:1) to obtain 9 (12 mg), 10 (7 mg) and a mixture. The mixture was dissolved in MeOH to obtain crystal 8 (330 mg). Fraction D (16 g) was fractionated by MPLC over RP-18 silica gel eluted with MeOH/H₂O (from 0:100 to 100:0) to obtain 12 subfractions (D1–D12). Fraction D3 (800 mg) was separated by CC over silica gel (200–300 mesh) eluted with CHCl₃/acetone (12:1) to obtain compound 6 (4.5 mg) and a mixture. The colorless crystal 7 (19 mg) was deposited from the mixture. Fraction D5 (1.2 g) was separated by CC (silica gel, 200–300 mesh) to produce compounds 3 (3.8 mg) and 1 (3.1 mg), as eluted with petroleum ether/acetone (about 8:1).

Aspergicytochalasin A (1): colorless crystals (MeOH), mp: 218–220 °C;

{[α]}_{25}^{D}

+ 76.4 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) 248 (3.01) and 210 (3.76) nm; IR (KBr) ν_max 3423, 1688, 1651, 1277, 1102, 1016, 974 and 703 cm⁻¹; ¹H- (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 1; HRESIMS m/z 502.25627 [M + Na]⁺ (calcd for C₂₉H₃₇NO₅Na⁺: 502.25639).

Aspergicytochalasin B (2): colorless oil;

{[α]}_{25}^{D}

+ 88.8 (c 0.64, MeOH); UV (MeOH) λ_max (log ε) 210 (3.85) nm; ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 1; HR-ESI-MS m/z 518.25122 [M + Na]⁺ (calcd for C₂₉H₃₇NO₆Na⁺: 518.25131).

Aspergicytochalasin C (3): colorless oil;

{[α]}_{25}^{D}

+ 39.3 (c 0.18, MeOH); UV (MeOH) λ_max (log ε) 250 (3.02) and 210 (3.76) nm; ¹H- (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 1; HRESIMS m/z 516.23566 [M + Na]⁺ (calcd for C₂₉H₃₅NO₆Na⁺: 516.23566).

Aspergicytochalasin D (4): colorless oil;

{[α]}_{25}^{D}

+ 25.6 (c 0.21, MeOH); ¹H- (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 2; HRESIMS m/z 518.25116 [M + Na]⁺ (calcd for C₂₉H₃₇NO₆Na⁺: 518.25131).

Aspergicytochalasin E (5): colorless oil;

{[α]}_{25}^{D}

+ 100.7 (c 0.23, MeOH); ¹H- (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 2; HRESIMS m/z 496.26947 [M + H]⁺ (calcd for C₂₉H₃₈NO₆⁺: 496.26936).

Aspergicytochalasin F (6): colorless oil;

{[α]}_{25}^{D}

− 34.0 (c 0.14, MeOH); ¹H- (600 MHz) and ¹³C-NMR (150 MHz) data (methanol-d₄), see Table 2; HRESIMS m/z 520.26679 [M + Na]⁺ (calcd for C₂₉H₃₉NO₆Na⁺: 520.26696).

X-ray Crystallographic Data for aspergicytochalasin A (1): C₂₉H₃₇NO₅·H₂O, M = 497.61. Cell constants of a = 9.8035(6) Å, b = 12.4869(8) Å, c = 11.0745(7) Å, α = 90.00°, β = 98.951(2)°, γ = 90.00°, V = 1339.18(15) Å³ and T = 150(2) K. Space group P1 21 1, Z = 2 and μ(CuKα) = 1.54178. Goodness of fit: 1.054. Flack parameter: 0.08(4). CCDC number: 2240408 (data are available at https://www.ccdc.cam.ac.uk, accessed on 12 February 2023). For details of the crystallographic data, see the Supplementary Materials.

2.4. Antibacterial Assay

Bacterial strains Escherichia coli ATCC25922 and Staphylococcus aureus subsp. aureus ATCC29213, purchased from the China General Microbiological Culture Collection Center, were cultured in Mueller Hinton broth (MHB) (Guangdong Huankai Microbial Sci. &Tech. Co., Ltd., Guangzhou, China) at 37 °C overnight with shaking at 200 rpm. The assay was carried out by following the method reported in the literature [16]. Penicillin G sodium salt (Biosharp) was used as the positive inhibitor control.

2.5. Nitric Oxide Production in RAW 264.7 Macrophages

This assay was carried out by following the method reported in the literature [17]. Murine monocytic RAW 264.7 macrophages and a RPMI-1640 medium were acquired from HyClone. Griess reagents, including reagent A and reagent B, were purchased from Sigma. The absorbance of samples was measured at 570 nm with a 2104 Envision multilabel plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). S-methylisothiourea sulfate (SMS) was used as a positive control.

3. Results and Discussion

Compound 1 was isolated as colorless crystals (MeOH). Its molecular formula was identified as C₂₉H₃₇NO₅ by HRESIMS (measured at m/z 502.25627 [M + Na]⁺; calcd for C₂₉H₃₇NO₅Na⁺: 502.25639), corresponding with 12 degrees of unsaturation. In the ¹H-NMR spectrum (Table 1), five olefinic protons at δ_H 7.14 (2H, d, J = 7.4 Hz, H-2ʹ and H-6ʹ), 7.26 (2H, t, J = 7.4 Hz, H-3ʹ and H-5ʹ) and 7.18 (1H, t, J = 7.4 Hz, H-4ʹ) revealed the presence of a mono-substituted phenyl group. Four olefinic protons at δ_H 5.86 (1H, dd, J = 15.0, 10.1 Hz, H-13), 5.29 (1H, m, H-14), 5.63 (1H, d, J = 15.5 Hz, H-22) and 7.02 (1H, m, H-21) indicated two E-form double bonds whereas the protons at δ_H 5.30 and 5.08 (each 1H, s, H₂-12) suggested one terminal double bond. In addition, two protons at δ_H 3.59 and 3.27 suggested the presence of one oxygenated methylene group. ¹³C-NMR displayed 29 carbon resonances (Table 1), which could be classified as 2 CH₃, 7 CH₂, 15 CH and 5 non-protonated carbons by the DEPT method and HSQC data. Of them, one phenyl group and three double bonds could be readily identified, whilst the oxygenated methylene group could be observed at δ_C 65.9 (t). A preliminary analysis of these data suggested that 1 should be a cytochalasan product with a structure identical to that of cytochalasin B [18], a known cytochalasan isolated in this study. A further analysis of the NMR data suggested that 1 should be an isomer of cytochalasin B, with the loss of the hydroxy group at C-20 and the presence of oxygenated CH₂ at δ_C 65.9 (t). The HMBC correlations from δ_H 3.59 and 3.27 (each 1H) with δ_C 42.4 (d, C-16), 37.5 (t, C-15) and 30.7 (t, C-17) revealed that the oxygenated CH₂ should be placed at C-24 (Figure 2). It was supported by the ¹H-¹H COSY data, as shown in Figure 2. A detailed analysis of the ¹H-¹H COSY and HMBC data suggested that the other parts of 1 were the same as those of cytochalasin B (Figure 2). In the ROESY spectrum, the cross peaks of H-4/H-5, H-4/H-8 and H-7/H-13 suggested that 1 should have the same stereochemistry as cytochalasin B (Figure 3). Fortunately, the single crystal X-ray diffraction not only established the planar structure, but also determined the absolute configuration of 1, as shown in Figure 4 (Flack parameter = 0.08(4); CCDC: 2240408). Finally, the structure of 1 was established and trivially named aspergicytochalasin A.

Compound 2 was isolated as a colorless oil. Its molecular formula was identified as C₂₉H₃₅NO₆ by HRESIMS (measured at m/z 518.25122 [M + Na]⁺; calcd for C₂₉H₃₇NO₆Na⁺: 518.25131). All the ¹H- and ¹³C-NMR data (Table 1) of 2 showed similarities to those of 1 and cytochalasin B. A further analysis of the NMR data suggested that 2 should be 24-hydroxy-cytochalasin, as supported by the HMBC correlations from δ_H 3.60 and 3.26 (each 1H, H₂-24) with δ_C 42.6 (d, C-16), 37.6 (t, C-15) and 31.1 (t, C-17) (Figure 2). It was further supported by the ¹H-¹H COSY data, as shown in Figure 2. Therefore, the structure of compound 2 was established and named aspergicytochalasin B.

Compound 3 was isolated as a colorless oil. Its molecular formula was identified as C₂₉H₃₅NO₆ by HRESIMS (measured at m/z 516.23566 [M + Na]⁺; calcd for C₂₉H₃₅NO₆Na⁺: 516.23566). The analysis of the ¹H- and ¹³C-NMR data (Table 1) suggested that 3 should also be a cytochalasan, possessing a structure with a high similarity to that of cytochalasin B. However, a carbonyl carbon at δ_C 205.2 (s, C-15) suggested that there was one keto group in 3. In the HMBC spectrum, the correlations from δ_H 0.99 (3H, d, J = 6.6 Hz, H₃-24), 2.68 (1H, dd, J = 12.7 and 6.6 Hz, H-16) and 6.32 (1H, d, J = 15.3 Hz, H-14) with δ_C 205.2 indicated that the keto group should be placed at C-15, which allowed H-14 to be a doublet in the ¹H-NMR spectrum (Figure 2). A comprehensive analysis of the HMBC, ¹H-¹H COSY and ROESY data suggested that the other parts of 3 were the same as those of cytochalasin B. Therefore, the structure of compound 3 was constructed and named aspergicytochalasin C.

Compound 4 was isolated as a colorless oil. The molecular formula of C₂₉H₃₇NO₆ was determined based on HRESIMS (measured at m/z 518.25116 [M + Na]⁺; calcd for C₂₉H₃₇NO₆Na⁺: 518.25131), corresponding with 12 degrees of unsaturation. Similarly, the ¹H- and ¹³C-NMR data (Table 2) of 4 displayed high similarities with those of cytochalasin B. The main difference was that one non-protonated carbon at δ_C 74.3 (s, C-5) appeared in 4. These data, as well as the analysis of the MS data, suggested that 4 should be a hydroxy product of cytochalasin B. In the HMBC spectrum, the correlations between δ_H 1.34 (3H, s, H₃-11) and δ_C 74.3 (s), 55.6 (d, C-4) and 152.6 (s, C-6) supported that the OH should be placed at C-5 (Figure 2). A detailed analysis of the ¹H-¹H COSY and HMBC data suggested that the other parts of 3 were the same as those of cytochalasin B (Figure 2). In the ROESY spectrum, cross peaks of H₃-11/H-3 and H₃-11/H-7 were observed (Figure 3), which indicated that the OH at C-5 should be β-oriented. Based on the same biogenetic pathway for cytochalasin B and 4, the absolute configuration of 4 was established, as shown in Figure 1. Finally, the structure of 4 was figured out and named aspergicytochalasin D.

Compound 5 was isolated as a colorless oil. According to HRESIMS (measured at m/z 496.26947 [M + H]⁺; calcd for C₂₉H₃₈NO₆⁺: 496.26936), the molecular formula was identified as C₂₉H₃₇NO₆, indicating 12 degrees of unsaturation. The ¹H- and ¹³C-NMR data (Table 2) of 5 were closely related to those of cytochalasin B. A further analysis of these data suggested that 4 should have one additional OH group at the phenyl group, with respect to that of cytochalasin B. Four aromatic protons at δ_H 6.53 (1H, d, J = 1.8 Hz, H-2ʹ), 6.57 (1H, dd, J = 7.8 and 1.8 Hz, H-4ʹ), 6.55 (1H, d, J = 7.8 Hz, H-6ʹ) and 7.03 (1H, t, J = 7.8 Hz, H-5ʹ) could readily identify a 1,3-disubstituted phenyl moiety. The HMBC correlations from H-2ʹ to C-4ʹ and C-6ʹ further supported that the OH should be placed at C-3ʹ in 5. A comprehensive analysis of the HMBC, ¹H-¹H COSY and ROESY data suggested that the other parts of 5 should be the same as those of cytochalasin B. Therefore, compound 5 was identified as 3ʹ-hydroxy-cytochalasin B and trivially named aspergicytochalasin E.

Compound 6 was isolated as a colorless oil. Its molecular formula was identified as C₂₉H₃₉NO₆ by HRESIMS (measured at m/z 520.26679 [M + Na]⁺; calcd for C₂₉H₃₉NO₆Na⁺: 520.26696), corresponding with 11 degrees of unsaturation. The ¹H- and ¹³C-NMR data (Table 2) of 6 were similar to those of cytochalasin B. An analysis of these data indicated that the double bond between C-6 and C-12 in cytochalasin B had disappeared, instead of a methyl group at C-12 (δ_H 1.18; δ_C 22.3) and one non-protonated carbon at C-6 (δ_C 77.4) in 6. It was supported by the key HMBC correlations from δ_H 1.18 (3H, s, H₃-12) with δ_C 77.4 (s, C-6), 38.1 (d, C-5) and 76.3 (d, C-7) as well as the HMBC correlations from H-5 and H-7 with C-6 (Figure 2). A comprehensive analysis of the HMBC and ¹H-¹H COSY data suggested that the other parts of 5 were the same as those of cytochalasin B (Figure 2). In the ROESY spectrum, the cross peaks of H₃-12/H-4 and H₃-12/H-5 suggested that the OH at C-6 should be α-oriented, with respect to that of cytochalasin B (Figure 3). Therefore, the structure of 6 was established as 6-hydroxy-cytochalasin B and trivially named aspergicytochalasin F.

The structural characteristics of compounds 1–6 showed that they were similar to cytochalasin B, which was one of a large number of compounds found in this study. These results indicated that compounds 1–6 were likely to be obtained by the oxidation of cytochalasin B via p450, so the absolute configurations of compounds 1–6 should be consistent with that of cytochalasin B. The single crystal X-ray diffraction data for 1 also supported this conclusion.

In addition to the new compounds, the five known cytochalasans were identified as cytochalasin A (7) [18], cytochalasin B (8) [18], dihydrocytochalasin B (9) [19], cytochalasin Z2 (10) [20] and cytochalasin Z4 (11) [18] by a comparison of the spectroscopic data with those reported in the literature.

Compounds 1–6 were evaluated for their antibacterial activities against Staphylococcus aureus. Compounds 3 and 4 showed certain inhibitory activities, with MIC values of 128 and 64 μg/mL, respectively. In addition, compounds 1–6 were evaluated for their anti-inflammatory activities by inhibiting NO production in LPS-induced RAW 264.7 macrophages. As a result, compounds 1, 3, 5 and 6 showed inhibitory activities on NO production, with IC₅₀ values less than 40 μM (Table 3).

4. Conclusions

In summary, a total of eleven cytochalasans, including six new ones, were isolated from the honeysuckle endophytic fungus Aspergillus sp. LE2. The new compounds exhibited antibacterial and anti-inflammatory activities. Cytochalasans are a class of important natural products with a rich structural diversity and biological activity [21,22,23,24,25,26]. At the same time, cytochalasans can also reflect the chemical relationship between microorganisms and hosts, and are an important bridge molecule in the study of medicinal plants and their functions. To the best of our knowledge, this is the first chemical study on honeysuckle endophytic fungi. Therefore, the new cytochalasans found in this study can provide certain information for further investigations on this plant and its endophytic fungal system.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/jof9030374/s1. NMR and mass spectra for 1–6 and X-ray crystallographic data for 1.

Author Contributions

Conceptualization, Y.-C.L.; methodology, J.Y., Q.-D.M., B.-G.L. and J.-L.S.; resources, Y.-Z.Z. and Y.-C.L.; data curation, J.Y., Q.-D.M., B.-G.L. and J.-L.S.; writing, Y.-C.L.; project administration, Y.-C.L.; funding acquisition, Y.-C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key R&D projects in Henan Province (No. 221111110300, granted to Y.C.L.); Scientific and technological project in Henan Province (No. 212102310876).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The supporting information of this study is available from the corresponding author (Y.-C.L.). The CIF of 1 is available free of charge at https://www.ccdc.cam.ac.uk.

Acknowledgments

The authors thank the Analytical & Measuring Centre, South-Central University for Nationalities, for the spectra measurements.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structures of chemicals 1−11.

Figure 2. Key 2D NMR correlations of 1–6.

Figure 3. Key ROESY correlations of 1, 4 and 6.

Figure 4. ORTEP diagram of 1 showing absolute configurations.

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR spectroscopic data for 1–3 in methanol-d₄.

No.	1		2		3
No.	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)
1	173.9, C		174.0, C		173.0, C
3	55.0, CH	3.36, m	54.8, CH	3.40, m	55.3, CH	3.40, td (6.1, 2.8)
4	48.9, CH	2.80, m	48.5, CH	2.85, dd (5.0, 2.4)	48.9, CH	2.84, m
5	32.9, CH	3.15, m	32.8, CH	3.21, m	33.1, CH	3.17, m
6	151.5, C		151.4, C		151.1, C
7	71.1, CH	3.80, d (11.1)	71.5, CH	3.80, d (10.8)	70.8, CH	4.02, d (11.1)
8	49.9, CH	3.30, dd (11.1, 10.1)	49.2, CH	3.40, dd (10.8, 10.1)	49.0, CH	3.47, dd (11.1, 9.9)
9	84.9, C		85.4, C		86.1, C
10	44.0, CH₂	2.84, m 2.84, m	44, CH₂	2.80, m	43.9, CH₂	2.86, m
11	14.3, CH₃	0.83, d (6.7)	14.1, CH₃	0.85, d (6.7)	14.3, CH₃	0.88, d (6.7)
12	114.3, CH₂	5.08, s 5.30, s	114.4, CH₂	5.28, s; 5.09, s	114.6, CH₂	5.35, s; 5.13, s
13	129.1, CH	5.86, dd (15.0, 10.1)	129.2, CH	5.88, dd (14.7, 10.1)	145, CH	7.09, dd (15.3, 9.9)
14	136.5, CH	5.29, m	136.5, CH	5.24, m	134.5, CH	6.32, d (15.3)
15	37.5, CH₂	2.40, m; 1.69, m	37.6, CH₂	2.38, m; 1.66, m	205.2, C
16	42.4, CH	1.35, m	42.6, CH	1.29, m	46.0, CH	2.68, dd (12.7, 6.6)
17	30.7, CH₂	1.60, m; 0.86, m	31.1, CH₂	1.53, m; 0.84, m	35.1, CH₂	1.75, m; 1.29, m
18	27.6, CH₂	1.70, m; 1.07, m	21.2, CH₂	1.47, m; 1.29, m	22.0, CH₂	1.48, m; 1.13, m
19	27.3, CH₂	1.81, m; 1.35, m	35.4, CH₂	1.92, m; 1.53, m	36.2, CH₂	1.74, m; 1.66, m
20	35.2, CH₂	2.37, m; 2.18, m	70.9, CH	4.46, m	71.5, CH	4.25, m
21	154.0, CH	7.02, m	154.4, CH	6.94, dd (15.6, 4.3)	153.1, CH	6.91, dd (15.7, 6.6)
22	122.0, CH	5.63, d (15.5)	119.6, CH	5.79, dd (15.6)	121.7, CH	5.84, d (15.7)
23	166.2, C		166.3, C		166.4, C
24	65.9, CH₂	3.59, dd (10.8, 3.6) 3.27, dd (10.8, 7.5)	65.9, CH₂	3.60, dd (10.9, 3.4); 3.26, dd (10.9, 7.4)	15.1, CH₃	0.99, d (6.6)
1′	138.5, C		138.2, C		138.4, C
2′,6′	130.9, CH	7.14, d (7.4)	131.0, CH	7.13, d (7.4)	131.0, CH	7.15, d (7.3)
3′,5′	129.6, CH	7.26, t (7.4)	129.6, CH	7.27, t (7.4)	129.6, CH	7.27, t (7.3)
4′	127.8, CH	7.18, t (7.4)	127.9, CH	7.19, t (7.4)	127.9, CH	7.23, t (7.3)

Table 2. ¹H (600 MHz) and ¹³C (150 MHz) NMR spectroscopic data for 4–6 in methanol-d₄.

No.	4		5		6
No.	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)	δ_C	δ_H (J in Hz)
1	173.3, C		174.0, C		173.7, C
3	57.2, CH	3.25, m	54.8, CH	3.32, m	55.0, CH	4.18, m
4	55.6, CH	2.86, d (4.2)	48.6, CH	2.81, dd (5.2, 2.6)	50.1, CH	2.58, m
5	74.3, C		32.9, CH	3.16, m	38.1, CH	2.27, m
6	152.6, C		151.5, C		77.4, C
7	69.2, CH	3.94, d (12.2)	71.5, CH	3.76, d (10.9)	76.3, CH	3.45, d (12.3)
8	51.3, CH	3.83, dd (12.2, 10.2)	49.3, CH	3.34, dd (10.9, 9.8)	49.4, CH	2.86, dd (12.3, 10.2)
9	85.2, C		85.4, C		86.7, C
10	44.0, CH₂	3.02, d (6.6)	44.1, CH₂	2.68, m	45.0, CH₂	2.79, dd (13.7, 7.9) 2.93, dd (13.7, 4.4)
11	26.5, CH₃	1.34, s	14.1, CH₃	0.84, d (6.5)	13.0, CH₃	0.99, d (7.1)
12	114.4, CH₂	5.51, s; 5.47, s	114.3, CH₂	5.24, s; 5.05, s	22.3, CH₃	1.18, s
13	128.8, CH	5.62, dd (14.2, 10.2)	128.7, CH	5.79, dd (15.0, 9.8)	128.0, CH	5.64, dd (14.4, 10.2)
14	137.1, CH	5.41, m	136.7, CH	5.20, m	136.5, CH	5.25, m
15	42.7, CH₂	2.19, m; 1.73, m	43.2, CH₂	2.06, m; 1.61, m	42.8, CH₂	2.14, m; 1.70, m
16	33.9, CH	1.46, m	34.5, CH	1.18, m	34.3, CH	1.35, m
17	35.7, CH₂	1.83, m; 0.65, m	36.6, CH₂	1.68, m; 0.53, m	36.1, CH₂	1.77, m; 0.62, m
18	21.8, CH₂	1.53, m; 1.42, m	21.5, CH₂	1.41, m; 1.23, m	21.7, CH₂	1.41, m
19	35.3, CH₂	1.85, m; 1.61, m	35.4, CH₂	1.87, m; 1.52, m	35.4, CH₂	1.86, m; 1.58, m
20	72.8, CH	4.29, m	71.2, CH	4.42, m	72.0, CH	4.34, m
21	153.7, CH	7.04, dd (15.6, 6.3)	154.6, CH	6.92, dd (15.7, 5.4)	154.1, CH	6.99, dd (15.6, 6.4)
22	121.2, CH	5.79, d (15.6)	119.4, CH	5.74, d (15.7)	120.3, CH	5.75, d (15.6)
23	166.5, C		166.4, C		166.3, C
24	20.7, CH₃	0.90, d (6.5)	20.7, CH₃	0.84, d (6.5)	20.7, CH₃	0.88, d (6.6)
1′	139.2, C		139.7, C		139.0, C
2′	130.5, CH	7.20, d (7.5)	117.8, CH	6.53, (1.8)	130.7, CH	7.19, d (7.4)
3′	129.8, CH	7.30, t (7.5)	158.5, C		129.6, CH	7.27, t (7.4)
4′	127.9, CH	7.22, t (7.5)	114.9, CH	6.57, dd (7.8, 1.8)	127.7, CH	7.19, t (7.4)
5′	129.8, CH	7.30, t (7.5)	130.5, CH	7.03, t (7.8)	129.6, CH	7.27, t (7.4)
6′	130.5, CH	7.20, d (7.5)	122.2, CH	6.55, d (7.8)	130.7, CH	7.19, d (7.4)

Table 3. NO production inhibitions of Compounds 1–6.

Entry	IC₅₀ (μM)
1	26.8 ± 2.69
2	>40
3	17.1 ± 1.27
4	>40
5	31.2 ± 1.02
6	12.7 ± 1.85
SMS	7.3 ± 0.34

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Li, Y.-C.; Yang, J.; Zhao, Y.-Z.; Ma, Q.-D.; Liu, B.-G.; Sun, J.-L. Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities. J. Fungi 2023, 9, 374. https://doi.org/10.3390/jof9030374

AMA Style

Li Y-C, Yang J, Zhao Y-Z, Ma Q-D, Liu B-G, Sun J-L. Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities. Journal of Fungi. 2023; 9(3):374. https://doi.org/10.3390/jof9030374

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Li, Yong-Chao, Jing Yang, Yuan-Zeng Zhao, Qin-Di Ma, Ben-Guo Liu, and Jun-Liang Sun. 2023. "Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities" Journal of Fungi 9, no. 3: 374. https://doi.org/10.3390/jof9030374

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Cytochalasans from the Endophytic Fungus Aspergillus sp. LE2: Their Structures, Antibacterial and NO Production Inhibitory Activities

Abstract

1. Introduction

2. Materials and Methods

2.1. General Experimental Procedures

2.2. Fungal Material

2.3. Fermentation, Extraction and Isolation

2.4. Antibacterial Assay

2.5. Nitric Oxide Production in RAW 264.7 Macrophages

3. Results and Discussion

4. Conclusions

Supplementary Materials

Author Contributions

Funding

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Acknowledgments

Conflicts of Interest

References

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