Isolation and Structure Elucidation of Secondary Metabolites From an Endophytic Fungus Annulohypoxylon ilanense

Abstract

Fractionation of the EtOAc-soluble fraction of the liquid fermentation of an endophytic fungus Annulohypoxylon ilanense associated with the wood of medicinal plant Cinnamomum species resulted in the isolation of 1 new α-pyrone, ilanpyrone (1), along with 3 previously identified compounds, 2 to 4. Their structures were elucidated by means of spectroscopic and mass-spectrometric analyses, especially 1D and 2D nuclear magnetic resonance (NMR) spectroscopy as well as high-resolution electrospray ionization mass spectrometry. The isolated compounds were tested for their cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines using the MTT assay. Among them, compounds 1 and 4 were found to have moderate-to-weak cytotoxic effects against these 3 cell lines in vitro.

Microorganisms are potentially exploitable for biotechnological processes, aiming the development of new antibiotics, probiotics, enzymes, and polymers with pharmaceutical and industrial applications. Endophytes were regarded as such a kind of special biotope microorganisms by residing in the tissues of living plants or animals without causing any apparent symptoms. ^{1 -4} Most reports on endophytes have shown the diversity and potential of fungi as a prospect for bioactive compounds. ^{5 -8} In previous research done by our group members for secondary metabolites from the endophyte, 3 new and 22 known compounds were isolated with biological activities from a BuOH-soluble extract of the endophytic fungus Annulohypoxylon ilanense (family Xylariaceae). ⁹ In our further investigation on the EtOAc-soluble extract of the same plant endophyte, 1 new α-pyrone, ilanpyrone (1), together with 3 known compounds were characterized. The isolation and structure elucidation of the new compound, and an assessment of their cytotoxic activity, are described herein.

Compound 1 had a molecular formula C₁₂H₁₈O₄, as established by high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 249.1099, ([M+Na]⁺) and ¹H and ¹³C nuclear magnetic resonance (NMR) data (see the section “Experimental”), indicating 4 indices of hydrogen deficiency. The UV spectrum absorption at 286 nm, and a strong IR absorption at 1660 cm^–1, as well as the observation of the characteristic carbon resonances [δ_C 97.7 (C-3), 164.1 (C-6), 170.0 (C-2) and 174.0 (C-4)] in the ¹³C NMR spectrum, revealed the presence of one α-pyrone ring in 1. ¹⁰ The ¹H NMR and COSY data of 1 confirmed the presence of 1 olefinic proton at δ_H5.90 (1H, s, H-5), 1 vinyl methyl at δ_H 1.83 (3H, s, Me-13), a pair of coupled methylene H-atoms at δ_H1.47 (1H, m, H-8b), 1.79 (1H, m, H-8a) and 2.43/2.51 (each 1 H, m, H₂-7), 2 methyl doublets at δ_H0.93 (3H, d, J = 6.6 Hz, H₃-11) and 1.12 (3H, d, J = 6.6 Hz, H₃-12), 1 quartet of doublets oxymethine at δ_H3.68 (1H, qd, J = 6.6, 4.2 Hz, H-10), and 1 methine at δ_H1.47 (1H, m, H-9). In the ¹³C and DEPT spectra of 1, there were 12 carbon signals in total, including 1 sp² methyl at δ_C 8.4, 2 sp³ methyls at δ_C 14.5 and 20.0, 2 sp³ methylenes at δ_C 31.3 and 32.3, 2 sp³ methines at δ_C 40.3 and 71.5, 1 olefinic methine at δ_C 104.2, and 4 quaternary sp² carbon resonances signals, including one C=O group. Comparison of ¹H and ¹³C NMR data of 1 with those of violapyrone B in literature ¹⁰ revealed that 1 had the same ring C-atom skeleton as a 3,4,6-trisubstituted α-pyrone moiety. The NMR data of 1 were similar to those of violapyrone B, except that a 4-hydroxy-3-methylpentyl side chain in 1 replaced a heptyl group in the C-6 position in violapyrone B. ¹⁰ The position of the 4-hydroxy-3-methylpentyl group at C-6 was readily determined by HMBC correlations from H-7 (δ_H 2.43) to C-3 (δ_C 104.2), combined with the nuclear Overhauser effect spectroscopy (NOESY) association of H-5/H₂-7 to support the alkyl chain. Thus, the gross structure of 1 was deduced as shown in Figure 1. The absolute configuration at C-10 cannot be confirmed by application of Mosher ester method due to insufficient amounts of compound 1. In addition, the absolute configuration at C-9 could not be determined due to insufficient data. An effort to determine the relative configuration at C-9 and 10 in 1, we compared the [α]_D value and NMR chemical shifts data with those of one similar analog 2-(4-hydroxy-3,4-dimethylbutyl)-4-pyrone (1a), ¹¹ and also showed a laevorotatory optical activity with $[\alpha {]}_D^{25}$ : −15.2 (c 2.56, CHCl₃). The laevorotatory optical activity of 1 ( $[\alpha {]}_D^{25}$ : −25.2 (c 0.02, MeOH)) indicated C-9 and 10 in rel-(R,R)-configuration, and the NMR chemical shifts of C-9 and C-10 of 1 were consistent with those of 1a. ¹⁰ Therefore, the relative configurations at C-9 and C-10 of the new α-pyrone 1 were temporarily deduced to be the (9R*,10R*) configuration. ¹⁰ Accordingly, compound 1 was determined to be 4-hydroxy-6-((3R*,4R*)-4-hydroxy-3-methyl-pentyl)-3-methyl-2H-pyran-2-one, named ilanpyrone. The other known isolates, ie ergosterol (2), ¹² 2,3-dihydroxypropyl palmitate (3), ¹³ and methyl asterrate (4), ¹⁴ were readily identified by comparison with literature data.

Figure 1

Chemical structure of 1 and its similar analog 1a ¹⁰ and important COSY (H▬H), HMBC (H→C) and NOESY (H↔H) correlations of 1.

Compounds 1 to 4 were evaluated for their cytotoxicity in vitro against MCF-7 (human breast adenocarcinoma), NCI-H460 (nonsmall-cell lung cancer), and SF-268 (glioblastoma cells) with actinomycin D as positive control. In cell proliferation activity, compound 1 displayed moderate cytotoxic effects against MCF-7 cancer cell line with IC₅₀ value of 4.79 µM. Compound 4 exhibited weak cytotoxicity against MCF-7, NCI-H460, and SF-268 cancer cell lines with IC₅₀ values of 8.56, 7.44, and 5.46 µM, respectively (Table 1). Compounds 2 and 3 exhibited no cytotoxicity. The α-pyrone skeleton, 4-hydroxy-6-(4-hydroxy-3-methylpentyl)-3-methyl-2H-pyran-2-one, as shown in ilanpyrone (1) is common in natural products, and several related fungal metabolites have been reported. Dothideopyrone F, isolated from a culture of the endolichenic fungus Dothideomycetes sp. possess moderate anti-inflammatory effects in LPS-stimulated BV2 microglial cell. ¹⁵ Nodulisporipyrones A-D produced by Nodulisporium sp., having antifungal activity against the filamentous fungus (A. niger R330). ¹⁶

Table 1

The Cytotoxicity of Compounds 1 to 4 in Four Cancer Cells.

Compounds	IC50 (μM)
	MCF-7	NCI-H460	SF-268
1	4.79	> 100	> 100
2	> 100	> 100	> 100
3	> 100	> 100	> 100
4	8.56	7.44	5.46
Actinomycin D a	0.13	0.07	0.12

a

Positive control.

Experimental

General

CC: silica gel 60; TLC: silica gel 60 F₂₅₄ precoated plates; IR Mattson Genesis II spectrophotometer; NMR: 600 MHz VNMRS; ESIMS: VG-Biotech Quatro-5022 mass spectrometer; HRESIMS: Bruker APEX II mass spectrometer.

Isolation and Fermentation of the Fungal Strain

The fungus was isolated by potato dextrose agar medium from the wood of plant Cinnamomum sp, which was collected from Ta-tung, Chi-lan-shan, I-lan County, Taiwan. Both a traditional morphological assessment and internal transcribed spaces sequencing were performed to characterize it as A. ilanense. The strain is preserved with the Bioresource Collection and Research Center of the Food Industry Research and Development Institute, with the ID number 34015. ⁹ The fermentation was performed as described previously. ⁹

Extraction and Separation

The rice containing the mycelium (1 kg) was lyophilized and extracted 3 times with 95% EtOH at room temperature. The extract was concentrated under reduced pressure, and the ethanol syrup extract was partitioned with H₂O/EtOAc 1:1 to afford the EtOAc-soluble fraction (Fraction A, 3.5 g) and the H₂O-soluble fraction. Fraction A was subjected to fractionation over silica gel CC (90 g SiO₂, 70 to 230 mesh, eluted with n-hexane-EtOAc (10:1→5:1→2:1→1:1→0:1) to give 7 fractions (Fractions A1-A7). Fraction A1 (250 mg) was chromatographed over silica gel CC (9 g SiO₂, 70-230 mesh), eluting with a gradient step-wise of EtOAc 10%, 30%, 50%, 70%, 90% and 100% in n-hexane to provide 6 subfractions: A1-1-A1-6. Subfraction A1-3 (25 mg) was subjected to CC (SiO₂, n-hexane-EtOAc 5:1→1:1) to produce ergosterol (2) (3.5 mg). Fraction A2 (175 mg) was chromatographed over silica gel, eluting with n-hexane–EtOAc (15:1→0:1) to obtain 5 subfractions (A2-1-A2-5). Subfraction A2-1 (16 mg) was chromatographed over silica gel, eluting with n-hexane-EtOAc (15:1→5:1) to obtain 2,3-dihydroxypropyl palmitate (3) (3.6 mg). Fraction A5 (212 mg) was applied to silica gel CC (6 g SiO₂, 70-230 mesh), eluting with CH₂Cl₂–acetone (20:1→5:1), to obtain 13 subfractions: A5-1-A5-13. Subfraction A5-3 was repeatedly purified by preparative thin-layer chromatography (TLC) (CH₂Cl₂-MeOH, 50:1) to afford methyl asterrate (4) (2.9 mg). Fraction A6 (128 mg) was subjected to Sephadex LH-20 (in MeOH) to obtain 5 subfractions: A6-1-A6-5. Subfraction A6-4 (40 mg) was applied to a RP-C18 column (1.2 g), eluting with acetonitrile–H₂O (1:1) to give 8 fractions (A6-4-1-A6-4-8). Subfraction A6-4-6 (30 mg) was applied to a silica gel column (1 g, 230-400 mesh), eluting with a gradient of CH₂Cl₂-MeOH (10:1) to obtain 3 subfractions (A6-4-6-1-A6-4-6-3). Subfraction A6-4-6-3 (5 mg) was further purified by preparative TLC (CH₂Cl₂-acetone, 10:1) to afford ilanpyrone (1) (1.3 mg).

Biological Assay

A cytotoxicity assay was performed as described previously. ¹⁷ Each cell was seeded in each well containing 100 mL of Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal calf serum. Cell cultures were maintained at 37°C under a humidified atmosphere of 5% CO₂. Compounds at least 8 different concentrations were added to replicate wells and treated cells for 72 hours, respectively, with actinomycin D as a positive reference. The number of viable cells was estimated using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma) reduction assay ^18,19 and the experiment was performed as manufacturer recommended (Promega, Madison, WI, USA). DMSO 0.1% (final concentration) were used as vehicle control. The experiments were repeated 3 times. Cell viability was expressed as percentage of cell proliferation in comparison to the negative control.

Ilanpyrone

Colorless oil.

$[\alpha {]}_D^{25}$ : −25.2 (c 0.02, MeOH).

IR (neat): ν_max 3410 (OH), 1660 (C = O) cm^–1.

UV/Vis λ_max (MeOH) nm (log ε): 286 (2.92).

¹H NMR (600 MHz, CD₃OD): δ_H0.93 (3H, d, J = 6.6 Hz, H₃-11), 1.12 (3H, d, J = 6.6 Hz, H₃-12), 1.47 (2H, m, H-8b and H-9), 1.79 (1H, m, H-8a), 1.83 (3H, s, H₃-13), 2.43/2.51 (each 1 H, m, H₂-7), 3.68 (1H, qd, J = 6.6, 4.2 Hz, H-10), 5.90 (1H, s, H-5).

¹³C NMR (150 MHz, CD₃OD): δ_C8.4 (C-13), 14.5 (C-11), 20.0 (C-12), 31.3 (C-8), 32.3 (C-7), 40.3 (C-9), 71.5 (C-10), 97.7 (C-3), 104.2 (C-5), 164.1 (C-6), 170.0 (C-2), 174.0 (C-4).

HR-ESIMS (positive-ion mode) m/z 249.1099 [M+Na]⁺ (calcd. 249.1098 for C₁₂H₁₈O₄Na); ESIMS (positive-ion mode) m/z 249 [M+Na]⁺.

Supplemental Material

Supplementary data - Supplemental material for Isolation and Structure Elucidation of Secondary Metabolites From an Endophytic Fungus Annulohypoxylon ilanense

Supplemental material, Supplementary data, for Isolation and Structure Elucidation of Secondary Metabolites From an Endophytic Fungus Annulohypoxylon ilanense by Ming-Jen Cheng, Shuen-Shin Yang, Ming-Der Wu, Hsun-Hsuo Chang, Yueh-Hsiung Kuo, Sung-Yuan Hsieh, Jih-Jung Chen and Ho-Cheng Wu in Natural Product Communications