Abstract
Two new glycosides, 8-O-β-d-glucopyranosyl-6-methyl-1-carboxylate methyl ester xanthone (1) and 4′-O-β-d-galactopyranosyl djalonensone (2), together with four known compounds, 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate methyl ester (3), cassionllin (4), djalonensone (5) and alternariol (6), were isolated from the endophytic fungus Penicillium sp. (NO.4) of Tapiscia sinensis Oliv. The structures of compounds 1–6 were elucidated by the analysis of 1D and 2D NMR and HRMS. The cytotoxic activities of these compounds were evaluated against four cancer cell lines, as well as antimicrobial activities against two plant-pathogenic microbes. Compounds 1–6 showed moderate cytotoxicity against the A549 cancer cell line with IC50 values ranging from 6.8 to 35.8 μg mL−1 and were found to be inactive against three other cancer cell lines MCF-7, Caski and Hep G-2.
1 Introduction
Though Penicillium species are typical and ubiquitous filamentous fungi in nature and have been systematically studied for over 80 years, their fascinating potential for chemical diversity of secondary metabolites is still highly attractive for natural products chemists. Examples included penicillipyrone A and B [1], eutypoid B–E [2], sorbicillamine A–E [3], 4,25-dehydrominiolutelide B, 4,25-dehydro-22-deoxyminiolutelide B [4], isominiolutelide A [4], herqueiazol, herqueioxazole, herqueidiketal [5], penibruguieramine A [6] and polyketide-terpenoid hybrids [7]. Our recent studies revealed that the ethyl acetate extract of the culture broth of the plant-associated fungus Penicillium sp. (NO.4) isolated from Tapiscia sinensis Oliv. showed significant in vitro cytotoxicity against two cancer cell lines, A549 and Hep G-2, with the IC50 at 30.5 and 35.6 μg mL−1. Subsequently, the chemical investigation of the culture broth extract led to the isolation of six metabolites including two new compounds 1 and 2 (see Fig. 1). In this paper, the structure elucidation and cytotoxic activities of compounds 1–6 are reported.
The structures of compounds 1 and 2.
2 Results and discussion
Compound 1 was obtained as colorless crystals. The molecular formula was determined as C22H22O11 according to the pseudomolecular ion peak at m/z=463.1244 (calcd. 463.1240 for C22H23O11, [M+H]+) and 485.1063 (calcd. 485.1060 for C22H22O11Na, [M+Na]+) in the HRMS, indicating 12 degrees of unsaturation. The 1H NMR spectrum of compound 1 exhibited signals for one methoxyl group at δH=3.86 ppm (s, 3H), one methyl group at δH=2.44 ppm (s, 3H) and five aromatic protons at δH=7.86 (t, 8.0, 7.6, 1H), 7.71 (d, 8.0, 1H), 7.39 (d, 7.6, 1H), 7.16 (s, 1H) and 7.13 ppm (s, 1H). In the downfield region, the coupling constants of the three protons at δH=7.86 (t, 8.0, 7.6, 1H), 7.71 (d, 8.0, 1H) and 7.39 ppm (d, 7.6, 1H) revealed the presence of a 1,2,3-trisubstituted benzyl ring in compound 1, which was supported by 1H–1H COSY correlations. Moreover, the cross-peak between δH=7.16 (s, 1H) and 7.13 ppm (s, 1H) in the 1H–1H COSY spectra displayed the framework of a 1,3,5,6-tetrasubstituted phenyl ring in 1. The remaining protons were distributed ranging from δH=2.0 to 5.0 ppm. The 13C and DEPT NMR spectra of compound 1 revealed 22 carbon resonance signals, including 2 methyl groups, 1 methene group, 10 methine groups and 9 quaternary carbon signals. Comprehensive analysis of the 1H and 13C NMR spectra showed that compound 1 possibly contained a sugar residue, because of the presence of six proton signals at δH=3.20 (t, 6.5, 1H), 3.30 (m, 1H), 3.38 (m, 1H), 3.39 (m, 1H), 3.50 (m, 1H) and 3.73 ppm (m, 1H) and six carbon signals at δC=102.3, 77.5, 77.1, 73.5, 69.7 and 60.7 ppm. Extensive analysis of the 2D NMR spectra and comparison of the NMR data with 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate methyl ester [8] allowed the elucidation of the structure of compound 1 as 8-O-β-d-glucopyranosyl-6-methyl-1-carboxylate methyl ester xanthone (1). The monosaccharide residue was attached to the 8-OH group, which was confirmed by heteronuclear multiple-bond correlation (HMBC) from the anomeric proton at δH=4.98 ppm (d, 7.6, 1H, H′-1) of the glucopyranose to the quaternary carbon at δC=157.6 ppm (C-8) and the cross-peak between the anomeric proton at δH=4.98 (d, 7.6, 1H, H′-1) and 7.12 ppm (s, 1H, H-7) in the nuclear Overhauser effect spectroscopy (NOESY) spectrum (see Fig. 2). The monosaccharide residue was identified as a glucopyranose by acid hydrolysis and high-performance liquid chromatography (HPLC) analysis. The configuration of the anomeric proton was determined as β-d through the coupling constant of 7.6 Hz (see Table 1). Thus, compound 1 was established as 8-O-β-d-glucopyranosyl-6-methyl-1-carboxylate methyl ester xanthone (1).
The key HMBC and NOESY correlations of compounds 1 and 2.
1H (400 MHz) and 13C (100 MHz) NMR data of compounds 1 and 2 in [D6]DMSO.
| Compound 1 | Compound 2 | ||||
|---|---|---|---|---|---|
| NO | 13C (DEPT) | 1H (J in Hz) | NO | 13C (DEPT) | 1H (J in Hz) |
| 1 | 133.4 (C) | – | 1 | 138.3 (C) | – |
| 2 | 119.3 (CH) | 7.71 (d, 8.0) | 2 | 98.8 (C) | – |
| 3 | 134.8 (CH) | 7.86 (t, 7.6, 8.0) | 3 | 164.1 (C) | – |
| 4 | 122.8 (CH) | 7.39 (d, 7.6) | 4 | 99.7 (CH) | 6.69 (d, 2.0) |
| 4a | 154.4 (C) | – | 5 | 166.2 (C) | – |
| 4b | 156.4 (C) | – | 6 | 102.8 (CH) | 7.05 (d, 2.2) |
| 5 | 111.6 (CH) | 7.16 (s) | 7 | 164.5 (C) | – |
| 6 | 147.6 (C) | – | 1′ | 111.1 (C) | – |
| 7 | 113.2 (CH) | 7.13 (s) | 2′ | 152.3 (C) | – |
| 8 | 157.6 (C) | – | 3′ | 104.6 (CH) | 7.31 (d, 2.0) |
| 8a | 110.3 (C) | – | 4′ | 157.5 (C) | – |
| 8b | 119.1 (C) | – | 5′ | 119.6 (CH) | 7.03 (d, 2.2) |
| 9 | 174.4 (C) | – | 6′ | 137.3 (C) | – |
| 10 | 169.2 (C) | – | 1″ | 97.7 (CH) | 5.02 (d, 6.8) |
| 11 | 21.9 (CH3) | 2.44 (s) | 2″ | 72.9 (CH) | 3.39 (m) |
| 12 | 52.5 (CH3) | 3.73 (s) | 3″ | 71.4 (CH) | 3.61 (m) |
| 1′ | 102.3 (CH) | 4.98 (d, 7.7) | 4″ | 69.8 (CH) | 3.20 (m) |
| 2′ | 73.5 (CH) | 3.38 (m) | 5″ | 74.1 (CH) | 3.42 (m) |
| 3′ | 77.1 (CH) | 3.30 (m) | 6″ | 60.7 (CH2) | 3.46 (m), 3.75 (m) |
| 4′ | 69.7 (CH) | 3.20 (t, 9.2) | 1-CH3 | 24.9 (CH3) | 2.81 (s) |
| 5′ | 77.5 (CH) | 3.39 (m) | 5-OCH3 | 55.9 (CH3) | 3.93 (s) |
| 6′ | 60.7 (CH2) | 3.73 (m), 3.50 (m) | |||
Compound 2 was obtained as colorless crystals. The HRMS exhibited the pseudomolecular ion peak at m/z=435.1293 (cacld. 435.1291 for C21H23O10, [M+H]+) and 457.1113 (cacld. 457.1111 for C21H22O10Na, [M+Na]+), implying a molecular formula of C21H22O10. The 1H and 13C NMR spectroscopic data of compound 2 were very similar to those of compound 1, containing two 1,3,5,6-tetrasubstituted phenyl rings and a monosaccharide residue. The differences between compounds 1 and 2 were in carboxylic groups and monosaccharide residues. Comparing the NMR data with djalonensone [9] and lysilactone A [10] and detailed analysis of the HMBC correlations of 2, the basic framework was deduced as djalonensone and the monosaccharide residue was determined to be β-d-galactopyranose by acid hydrolysis, paper thin layer chromatography, HPLC analysis and analysis of the coupling constant of the anomeric proton.
The configuration of the anomeric proton was determined as β-d by the coupling constant of 6.8 Hz. The β-d-galactopyranose unit was linked to the 4′-OH group, which was confirmed by the correlation from the anomeric proton of β-d-galactopyranose to C-4′ of djalonensone in the HMBC spectrum, and the cross-peak between the proton at δH=5.02 (d, 6.8, H-1″) of the glycoside and the proton at δH=7.31 (d, 2.0, H-3′) of djalonensone. Thus, the structure of compound 2 was determined as 4′-O-β-d-galactopyranosyl djalonensone.
Compounds 3–6 were verified as 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester [8] (3), cassionllin [11] (4), djalonensone [9] (5) and alternariol [9] (6) by exhaustive comparison of the NMR data with published results.
3 Conclusions
Compounds 1–6 were evaluated for cytotoxicity against four cancer cell lines A549, Caski, Hep G-2 and MCF-7 in vitro using the MTT method. Compounds 1–6 exhibited moderate cytotoxicity against A549 with IC50 values of 6.8, 7.2, 17.2, 20.5, 26.6 and 35.8 μg mL−1, respectively, but were inactive against the other three cancer cell lines Caski, Hep G-2 and MCF-7. The antimicrobial activities against two plant-pathogenic microbes, Erwinia carotovora sub sp. Carotovora (Jones 1901) Berseyetal (1923) and Sclerotiumrol fsii Sacc, were also evaluated. Disappointingly, compounds 1–6 did not display antimicrobial activity.
4 Experimental section
4.1 General experimental procedures
UV spectra were obtained on a SCINCO Spectrometer; IR spectra were recorded on a Nicoler Auatar Spectrometer series FT360 spectrophotometer. 1D and 2D NMR spectra were recorded on a Bruker Ultrashield-400 MHz NMR spectrometer (Switzerland). Mass spectra were obtained on an API 4000 mass spectrometer (USA). Silica gel GF 254 (10–40 μm) prepared for thin-layer chromatography (TLC) and silica gel (200–300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, P.R. China). Fractions were monitored by TLC, and the spots were visualized under an ultraviolet lamp at 254 nm and in an iodine cylinder. Semi-preparative HPLC was performed on a Dionex Ultra-3000 (USA) using a Cosmosil C-18 column (10 μm × 20 mm × 250 mm and 5 μm × 4.6 mm × 250 mm).
4.2 Isolation and fermentation of the endophytic fungus
Healthy leaves and twigs of T. sinensis Oliv. were collected in the Shennongjia National Forest Park, washed with running water, then immersed in 5–10 % NaOCl solution for 5 min and 75 % ethanol solution for 3 min, and finally washed three times with aseptic water; the water was removed by sterile filter papers. The sterilized leaves and twigs were cut into small pieces of 0.5 × 0.5 cm2, and these were placed on PDA plates in an incubator at 28 °C. After the microbes on the potato dextrose agar (PDA) plates had developed sufficiently, the growing fungi were repeatedly purified by the streak method, until obtained as single colonies. The pure single colonies were deposited on slants at 4 °C. In total, 25 endophytic fungi were obtained from the leaves and twigs of T. sinensis; these fungi were fermented in 200 mL potato dextrose broth (PDB) liquid medium in 500 mL Erlenmeyer flasks. Penicillium sp. (NO.4) was selected for chemical investigation according to the results of bioactivity assays. Penicillium sp. (NO.4) was cultured in 40 L PDB liquid medium in 200 × 500 mL Erlenmeyer flasks on an electronic oscillator at 28 °C at 120 rpm for 30 days.
4.3 Extraction and isolation
The broth and mycelium were separated using eight layers of gauze. The broth (40 L) was extracted five times with 20 L ethyl acetate, and the extract was condensed under vacuum to give 20 g residue. This was subjected to silica gel CC and eluted with light petroleum to acetone, then to methanol. The subfractions were merged to six fractions from TLC results. Fraction A (1.5 g) was further subjected to C-18 reverse phase silica gel CC with gradient elution from 20 % methanol-H2O to 80 % methanol-H2O; fraction A was fractionated into five subfractions. Subfractions A-4 and A-5 were purified by repeated silica gel CC, eluting with light petroleum–acetone (4:1) and light petroleum–acetone (3:1) to yield compounds 1 (8 mg), 2 (10 mg), 3 (15 mg), 4 (10 mg), 5 (20 mg) and 6 (50 mg).
4.4 8-O-β-d-glucopyranosyl-6-methyl-1-carboxylate methyl ester xanthone (1)
Colorless crystals, M.p. 166–168 °C. – UV/Vis (CH3OH): λmax (lg εmax)=205 nm (2.4), 254 nm (3.9), 287 nm (3.5). – [α]D25=–85 (c=0.06, CH3OH). – IR(film): υ=3430, 2956, 2925, 2880, 1721, 1634, 1528, 1460 cm−1. – 1H and 13C NMR data (see Table 1). – HRMS ((+)-ESI): m/z=463.1244 (cacld. 463.1240 for C22H23O11, [M+H]+) and 485.1063 (cacld. 485.1060 for C22H22O11Na, [M+Na]+).
4.5 4′-O-β-d-galactopyranosyl djalonensone (2)
Colorless crystals, M.p. 156–157 °C. – UV/Vis (CH3OH): λmax (lg εmax)=208 nm (2.3), 255 nm (4.1), 287 nm (3.8), 330 nm (2.6). – [α]D25=–68 (c=0.07, CH3OH). – IR(film): υ=3363, 2924, 1668 (C=O), 1609, 1448, 1342 cm−1. – 1H and 13C NMR data (see Table 1). – HRMS ((+)-ESI): m/z:=435.1293 (cacld. 435.1291 for C21H23O10, [M+H]+) and 457.1113 (cacld. 457.1111 for C21H22O10Na, [M+Na]+).
4.6 Acid hydrolysis
Compounds 1 (2 mg) and 2 (2 mg) were added separately to 10 mL of a 2 m HCl solution in 25 mL flasks. These were then heated with stirring at 90 °C for 2 h. Finally, the reaction mixtures were extracted with ethyl acetate, the water phase collected and concentrated under vacuum for HPLC analysis.
4.7 Cytotoxicity tests
A549, Caski, Hep G-2 and MCF-7 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone) supplemented with 10 % fetal bovine serum (FBS) (HyClone). The cells were maintained in 5 % CO2 at 37 °C. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) colorimetric assay was used to evaluate cell proliferation in the presence of the different chemicals. The cells were seeded in 96-well culture plates and treated with either vehicle or desired concentrations of the chemicals for a further 24 h. After treatment, the cells were incubated at 37 °C with MTT (1 μL per well, 5 mg mL−1) for 4 h; cell growth response to the chemicals was determined by measuring the absorbance at 570 nm with a plate reader. Three replicates were used for each treatment. In the anticancer activity in vitro experiment, mitomycin was used as positive control for the anticancer assay.
4.8 Antimicrobial assays
Antimicrobial assays against plant-pathogenic Erwinia carotovora sub sp. Carotovora and Sclerotiumrol fsii Sacc. were carried out using the well-diffusion method. Amphotericin B was used as positive control for the antifungal assay.
Acknowledgments
This work was financial supported by the Natural Science Foundation of China (21002058 and 21272137).
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