Two new glycosidal metabolites of endophytic fungus Penicillium sp. (NO.4) from Tapiscia sinensis

Article abstract of DOI:10.1515/znb-2015-0184

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 IC₅₀values ranging from 6.8 to 35.8 μg mL^-1and were found to be inactive against three other cancer cell lines MCF-7, Caski and Hep G-2.

Full text of DOI:10.1515/znb-2015-0184

Z. Naturforsch. 2016; 71(4)b: 283–286
Qiao Wan, Ziwei Feng, Xueshuang Li, Mengmeng Lv, Zhiyong Guo*, Zhangshuang Deng*
and Kun Zou
Two new glycosidal metabolites of endophytic
fungus Penicillium sp. (NO.4) from Tapiscia
sinensis
DOI 10.1515/znb-2015-0184
potential for chemical diversity of secondary metabo-
lites is still highly attractive for natural products chem-
ists. Examples included penicillipyrone A and B [1],
eutypoid B–E [2], sorbicillamine A–E [3], 4,25-dehydro-
miniolutelide B, 4,25-dehydro-22-deoxyminiolutelide B
[4], isominiolutelide A [4], herqueiazol, herqueioxazole,
herqueidiketal [5], penibruguieramine A [6] and polyke-
tide-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 struc-
ture elucidation and cytotoxic activities of compounds
1–6 are reported.
Received November 4, 2015; accepted January 26, 2016
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 antimi-
crobial 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.
Keywords: cytotoxicity; endophytic fungus; Penicillium
sp.; structural elucidation; Tapiscia sinensis Oliv.
2 Results and discussion
Compound 1 was obtained as colorless crystals. The
1 Introduction
molecular formula was determined as C22H22O11 accord-
ing to the pseudomolecular ion peak at m/zꢀꢁ=ꢁꢀ463.1244
+
Though Penicillium species are typical and ubiqui- (calcd. 463.1240 for C22H23O11, [M+H] ) and 485.1063 (calcd.
+
tous filamentous fungi in nature and have been sys- 485.1060 for C22H22O11Na, [M+Na] ) in the HRMS, indicat-
1
tematically studied for over 80 years, their fascinating ing 12 degrees of unsaturation. The H NMR spectrum of
compound 1 exhibited signals for one methoxyl group at
δHꢀꢁ=ꢁꢀ3.86 ppm (s, 3H), one methyl group at δHꢀꢁ=ꢁꢀ2.44 ppm
*Corresponding authors: Zhiyong Guo and Zhangshuang Deng,
(s, 3H) and five aromatic protons at δHꢀꢁ=ꢁꢀ7.86 (t, 8.0, 7.6,
Hubei Key Laboratory of Natural Products Research and
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 δHꢀꢁ=ꢁꢀ7.86 (t, 8.0, 7.6, 1H),
7.71 (d, 8.0, 1H) and 7.39 ppm (d, 7.6, 1H) revealed the pres-
ence of a 1,2,3-trisubstituted benzyl ring in compound 1,
Development, College of Biological and Pharmaceutical Sciences,
China Three Gorges University, No. 8 University Road,
Yichang, 443002, P.R. China, Fax: +86-717-6397478,
E-mail: zhiyongguoctgu@hotmail.com (Z. Guo);
d.zhangshuang@gmail.com (Z. Deng)
Qiao Wan, Ziwei Feng, Xueshuang Li, Mengmeng Lv and
Kun Zou: Hubei Key Laboratory of Natural Products Research and
Development, College of Biological and Pharmaceutical Sciences,
China Three Gorges University, Yichang, 443002, P.R. China
1
1
which was supported by H– H COSY correlations. More-
over, the cross-peak between δHꢀꢁ=ꢁꢀ7.16 (s, 1H) and 7.13 ppm
1
(s, 1H) in the 1H– H COSY spectra displayed the framework
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ꢀꢀꢀꢁ
284ꢀ ꢀQ. Wan et al.: Two new glycosidal metabolites of endophytic fungus Penicillium sp. (NO.4)
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).
Compound 2 was obtained as colorless crystals.
The HRMS exhibited the pseudomolecular ion peak at
+
m/zꢀꢁ=ꢁꢀ435.1293 (cacld. 435.1291 for C21H23O10, [M+H] ) and
+
457.1113 (cacld. 457.1111 for C21H22O10Na, [M+Na] ), imply-
1
13
ing a molecular formula of C21H22O10. The H and C NMR
spectroscopic data of compound 2 were very similar to
those of compound 1, containing two 1,3,5,6-tetrasubsti-
tuted phenyl rings and a monosaccharide residue. The
differences between compounds 1 and 2 were in carbox-
ylic 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 chro-
matography, HPLC analysis and analysis of the coupling
constant of the anomeric proton.
The configuration of the anomeric proton was deter-
mined 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 ano-
meric proton of β-d-galactopyranose to C-4′ of djalonen-
sone in the HMBC spectrum, and the cross-peak between
the proton at δHꢀꢁ=ꢁꢀ5.02 (d, 6.8, H-1″) of the glycoside and
the proton at δHꢀꢁ=ꢁꢀ7.31 (d, 2.0, H-3′) of djalonensone. Thus,
the structure of compound 2 was determined as 4′-O-β-d-
galactopyranosyl djalonensone.
Fig. 1:ꢀThe structures of compounds 1 and 2.
of a 1,3,5,6-tetrasubstituted phenyl ring in 1. The remain-
ing protons were distributed ranging from δHꢀꢁ=ꢁꢀ2.0 to
13
5.0 ppm. The C and DEPT NMR spectra of compound 1
revealed 22 carbon resonance signals, including 2 methyl
groups, 1 methene group, 10 methine groups and 9 qua-
1
ternary carbon signals. Comprehensive analysis of the H
and 13C NMR spectra showed that compound 1 possibly
contained a sugar residue, because of the presence of six
proton signals at δHꢀꢁ=ꢁꢀ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 δCꢀꢁ=ꢁꢀ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 δHꢀꢁ=ꢁꢀ4.98 ppm (d, 7.6, 1H, H′-1) of the glucopyra-
nose to the quaternary carbon at δCꢀꢁ=ꢁꢀ157.6 ppm (C-8) and
the cross-peak between the anomeric proton at δHꢀꢁ=ꢁꢀ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
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 pub-
lished 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 Sclero-
tiumrol fsii Sacc, were also evaluated. Disappointingly,
compounds 1–6 did not display antimicrobial activity.
Fig. 2:ꢀThe key HMBC and NOESY correlations of compounds 1 and 2.
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ꢁꢀꢀꢀ
Q. Wan et al.: Two new glycosidal metabolites of endophytic fungus Penicillium sp. (NO.4)ꢂ
ꢂ285
Table 1:ꢀ1H (400 MHz) and 13C (100 MHz) NMR data of compounds 1 and 2 in [D6]DMSO.
Compound 1
ꢁ
Compound 2
NO ꢃ
13C (DEPT) ꢃ
133.4 (C) ꢃ
119.3 (CH)ꢃ
134.8 (CH)ꢃ
122.8 (CH)ꢃ
154.4 (C) ꢃ
156.4 (C) ꢃ
111.6 (CH)ꢃ
147.6 (C) ꢃ
113.2 (CH)ꢃ
157.6 (C) ꢃ
110.3 (C) ꢃ
119.1 (C) ꢃ
174.4 (C) ꢃ
169.2 (C) ꢃ
21.9 (CH3) ꢃ
52.5 (CH3) ꢃ
102.3 (CH)ꢃ
73.5 (CH) ꢃ
77.1 (CH) ꢃ
69.7 (CH) ꢃ
77.5 (CH) ꢃ
60.7 (CH2) ꢃ
1H (J in Hz)
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
NO
1
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
13C (DEPT) ꢃ
1H (J in Hz)
–
–
–
6.69 (d, 2.0)
–
7.05 (d, 2.2)
–
1
2
3
4
ꢃ
ꢃ
ꢃ
ꢃ
–
138.3 (C)
98.8 (C)
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
7.71 (d, 8.0)
2
7.86 (t, 7.6, 8.0)
3
164.1 (C)
99.7 (CH)
166.2 (C)
7.39 (d, 7.6)
4
4a ꢃ
4b ꢃ
–
–
5
6
102.8 (CH) ꢃ
5
6
7
8
ꢃ
ꢃ
ꢃ
ꢃ
7.16 (s)
7
164.5 (C)
111.1 (C)
152.3 (C)
ꢃ
ꢃ
ꢃ
–
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
1-CH3
–
–
7.13 (s)
–
104.6 (CH) ꢃ
157.5 (C)
119.6 (CH) ꢃ
7.31 (d, 2.0)
–
7.03 (d, 2.2)
–
5.02 (d, 6.8)
3.39 (m)
3.61 (m)
3.20 (m)
3.42 (m)
3.46 (m), 3.75 (m)
2.81 (s)
3.93 (s)
8a ꢃ
8b ꢃ
–
ꢃ
–
9
ꢃ
–
137.3 (C)
97.7 (CH)
72.9 (CH)
71.4 (CH)
69.8 (CH)
74.1 (CH)
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
10 ꢃ
11 ꢃ
12 ꢃ
–
2.44 (s)
3.73 (s)
4.98 (d, 7.7)
3.38 (m)
3.30 (m)
3.20 (t, 9.2)
3.39 (m)
1′
2′
3′
4′
5′
6′
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
60.7 (CH2) ꢃ
24.9 (CH3) ꢃ
55.9 (CH3) ꢃ
ꢃ
5-OCH3 ꢃ
3.73 (m), 3.50 (m)ꢃ
ꢃ
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
4 Experimental section
4.1 General experimental procedures
UV spectra were obtained on a SCINCO Spectrometer; IR were placed on PDA plates in an incubator at 28 °C. After
spectra were recorded on a Nicoler Auatar Spectrometer the microbes on the potato dextrose agar (PDA) plates had
series FT360 spectrophotometer. 1D and 2D NMR spectra developed sufficiently, the growing fungi were repeatedly
were recorded on a Bruker Ultrashield-400 MHz NMR spec- purified by the streak method, until obtained as single
trometer(Switzerland). MassspectrawereobtainedonanAPI colonies. The pure single colonies were deposited on
4000 mass spectrometer (USA). Silica gel GF 254 (10–40 μm) slants at 4 °C. In total, 25 endophytic fungi were obtained
prepared for thin-layer chromatography (TLC) and silica from the leaves and twigs of T. sinensis; these fungi were
gel (200–300 mesh) for column chromatography (CC) were fermented in 200 mL potato dextrose broth (PDB) liquid
obtained from Qingdao Marine Chemical Factory (Qingdao, medium in 500 mL Erlenmeyer flasks. Penicillium sp.
P.R. China). Fractions were monitored by TLC, and the spots (NO.4) was selected for chemical investigation according
were visualized under an ultraviolet lamp at 254 nm and in to the results of bioactivity assays. Penicillium sp. (NO.4)
an iodine cylinder. Semi-preparative HPLC was performed was cultured in 40 L PDB liquid medium in 200 ꢁ×ꢁ 500 mL
on a Dionex Ultra-3000 (USA) using a Cosmosil C-18 column Erlenmeyer flasks on an electronic oscillator at 28 °C at
(10 μm ꢁ×ꢁ 20 mm ꢁ×ꢁ 250 mm and 5 μm ꢁ×ꢁ 4.6 mm ꢁ×ꢁ 250 mm).
120 rpm for 30 days.
4.3 Extraction and isolation
4.2 Isolation and fermentation of the
endophytic fungus
The broth and mycelium were separated using eight layers
Healthy leaves and twigs of T. sinensis Oliv. were collected of gauze. The broth (40 L) was extracted five times with
in the Shennongjia National Forest Park, washed with 20 L ethyl acetate, and the extract was condensed under
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ꢀꢀꢀꢁ
286ꢀ ꢀQ. Wan et al.: Two new glycosidal metabolites of endophytic fungus Penicillium sp. (NO.4)
vacuum to give 20 g residue. This was subjected to silica (HyClone) supplemented with 10 % fetal bovine serum
gel CC and eluted with light petroleum to acetone, then to (FBS) (HyClone). The cells were maintained in 5 % CO2
methanol. The subfractions were merged to six fractions at 37 °C. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
from TLC results. Fraction A (1.5 g) was further subjected 2H-tetrazolium bromide (MTT, Sigma) colorimetric assay
to C-18 reverse phase silica gel CC with gradient elution was used to evaluate cell proliferation in the presence of
from 20 % methanol-H2O to 80 % methanol-H2O; fraction the different chemicals. The cells were seeded in 96-well
A was fractionated into five subfractions. Subfractions A-4 culture plates and treated with either vehicle or desired
and A-5 were purified by repeated silica gel CC, eluting concentrations of the chemicals for a further 24 h. After
with light petroleum–acetone (4:1) and light petroleum– treatment, the cells were incubated at 37 °C with MTT (1
acetone (3:1) to yield compounds 1 (8 mg), 2 (10 mg), 3 (15 μL per well, 5 mg mL−1) for 4 h; cell growth response to
mg), 4 (10 mg), 5 (20 mg) and 6 (50 mg).
the chemicals was determined by measuring the absorb-
ance 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.
4.4 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). – 4.8 Antimicrobial assays
[α]D25ꢀꢁ=ꢁꢀ–85 (cꢀꢁ=ꢁꢀ0.06, CH3OH). – IR(film): υꢀꢁ=ꢁꢀ3430, 2956,
1
13
2925, 2880, 1721, 1634, 1528, 1460 cm−1. – H and C NMR Antimicrobial assays against plant-pathogenic Erwinia caro-
data (see Table 1). – HRMS ((+)-ESI): m/zꢀꢁ=ꢁꢀ463.1244 tovora sub sp. Carotovora and Sclerotiumrol fsii Sacc. were
+
(cacld. 463.1240 for C22H23O11, [M+H] ) and 485.1063 (cacld. carried out using the well-diffusion method. Amphotericin
+
485.1060 for C22H22O11Na, [M+Na] ).
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).
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]D25ꢀꢁ=ꢁꢀ–68 (cꢀꢁ=ꢁꢀ0.07, CH3OH). – IR(film): υꢀꢁ=ꢁꢀ3363,
2924, 1668 (Cꢁ=ꢁO), 1609, 1448, 1342 cm−1. – 1H and 13C NMR
data (see Table 1). – HRMS ((+)-ESI): m/z:ꢀꢁ=ꢁꢀ435.1293
References
[1] L. Liao, J.-H. Lee, M. You, T. J. Choi, W. Park, S. K. Lee, D.-C. Oh,
K.-B. Oh, J. Shin, J. Nat. Prod. 2014, 77, 406.
+
(cacld. 435.1291 for C21H23O10, [M+H] ) and 457.1113 (cacld.
+
[2] D. Schulz, B. Ohlendorf, H. Zinecker, R. Schmaljohann,
J. F. Imhoff, J. Nat. Prod. 2011, 74, 99.
457.1111 for C21H22O10Na, [M+Na] ).
[3] W. Guo, J. Peng, T. Zhu, Q. Gu, R. A. Keyzers, D. Li, J. Nat. Prod.
2013, 76, 2106.
4.6 Acid hydrolysis
[4] Y. Zhang, X.-M. Li, Z. Shang, C.-S. Li, N.-Y. Ji, B.-G. Wang, J. Nat.
Prod. 2012, 75, 1888.
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 reac-
tion mixtures were extracted with ethyl acetate, the water
phase collected and concentrated under vacuum for HPLC
analysis.
[5] E. Julianti, J.-H. Lee, L. Liao, W. Park, S. Park, D.-C. Oh, K.-B. Oh,
J. Shin, Org. Lett. 2013, 15, 1286.
[6] Z.-F. Zhou, T. Kurtán, X.-H. Yang, A. Mándi, M.-Y. Geng, B.-P. Ye,
O. Taglialatela-Scafati, Y.-W. Guo, Org. Lett. 2014, 16, 1390.
[7] M. Iida, T. Ooi, K. Kito, S. Yoshida, K. Kanoh, Y. Shizuri,
T. Kusumi, Org. Lett. 2008, 10, 845.
[8] M. Macías, A. Gamboa, M. Ulloa, R. A. Toscano, R. Mata,
Phytochemistry 2001, 58, 751.
[9] P. A. Onocha, D. A. Okorie, J. D. Conolly, D. S. Roycroft,
Phytochemistry 1995, 40, 1183.
4.7 Cytotoxicity tests
[10] D. Liang, H. Luo, Y.-F. Liu, Z.-Y. Hao, Y. Wang, C.-L. Zhang,
Q.-J. Zhang, R.-Y. Chen, D.-Q. Yu, Tetrahedron 2013, 69, 2093.
A549, Caski, Hep G-2 and MCF-7 cells were cultured in
Roswell Park Memorial Institute (RPMI) 1640 medium [11] H. Noguchi, U. Sankawa, Phytochemistry 1982, 21, 319.
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