Two new compounds from an endophytic fungus Pestalotiopsis heterocornis

Article abstract of DOI:10.1080/10286020.2011.621422

Two new compounds, 7-hydroxy-5-methoxy-4,6-dimethyl-7-O-α-L- rhamnosyl-phthalide (2) and 7-hydroxy-5-methoxy-4,6-dimethyl-7-O-β-D- glucopyranosyl-phthalide (3), along with one known and related metabolite 7-hydroxy-5-methoxy-4,6- dimethylphthalide (1) were isolated from the EtOAc extract of fermentation broth of an endophytic fungus Pestalotiopsis heterocornis. The structures of these compounds were elucidated on the basis of spectroscopic methods (UV, IR, HR-ESI-MS, 1D NMR, and 2D NMR).

Full text of DOI:10.1080/10286020.2011.621422

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Two new compounds from an
endophytic fungus Pestalotiopsis
heterocornis
a
a
a
Jian-Guang Xing , Hui-Ying Deng & Du-Qiang Luo
a
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of
Ministry of Education, College of Life Science, Hebei University ,
Baoding, 071002, China
Published online: 25 Nov 2011.
To cite this article: Jian-Guang Xing , Hui-Ying Deng & Du-Qiang Luo (2011) Two new compounds
from an endophytic fungus Pestalotiopsis heterocornis , Journal of Asian Natural Products Research,
13:12, 1069-1073, DOI: 10.1080/10286020.2011.621422
To link to this article: http://dx.doi.org/10.1080/10286020.2011.621422
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Journal of Asian Natural Products Research
Vol. 13, No. 12, December 2011, 1069–1073
Two new compounds from an endophytic fungus Pestalotiopsis
heterocornis
Jian-Guang Xing, Hui-Ying Deng and Du-Qiang Luo*
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College
of Life Science, Hebei University, Baoding 071002, China
(
Received 6 June 2011; final version received 4 September 2011)
Two new compounds, 7-hydroxy-5-methoxy-4,6-dimethyl-7-O-a-L-rhamnosyl-phtha-
lide (2) and 7-hydroxy-5-methoxy-4,6-dimethyl-7-O-b-D-glucopyranosyl-phthalide
(
3), along with one known and related metabolite 7-hydroxy-5-methoxy-4,6-
dimethylphthalide (1) were isolated from the EtOAc extract of fermentation broth of
an endophytic fungus Pestalotiopsis heterocornis. The structures of these compounds
were elucidated on the basis of spectroscopic methods (UV, IR, HR-ESI-MS, 1D NMR,
and 2D NMR).
Keywords: Pestalotiopsis heterocornis; endophytic fungus; phthalide derivatives
1
.
Introduction
we report the isolation and structural
elucidation of these compounds.
Endophytic fungi that inhabit normal
tissues of the host plants without causing
apparent pathogenic symptoms have been
demonstrated to be rich sources of
bioactive natural products [1–3]. As one
class of the most widely distributed
endophytic fungi, Pestalotiopsis spp. have
attracted much attention in recent years for
their ability to produce a variety of natural
products such as triterpenoids, polyketides,
meroterpenoids and so on [4–15]. Pesta-
lotiopsis heterocornis (L421) was isolated
from the stem of Bruguiera gymnorrhiza
collected in Dongzhai, Hainan Province of
China. Chemical investigation on the ethyl
acetate extract of the fermentation broth of
L421 afforded two new compounds 7-
hydroxy-5-methoxy-4,6-dimethyl-7-O-a-
L-rhamnosyl-phthalide (2) and 7-hydroxy-
2. Results and discussion
Compound 2 was obtained as a yellow oil,
and its molecular formula was determined
to be C H O by HR-ESI-MS at m/z
1
7 22 8
þ
3
77.1215 [M þ Na] . The IR spectrum
indicated the presence of hydroxyl
(
2
1
21
3374 cm ), ester carbonyl (1744 cm ),
21
and benzene ring (1605 and 1441cm ).
The absorption maxima in the UV spectrum
(216, 247, and 292 nm) also exhibited the
1
presence of a benzene ring. The H NMR
spectrum showed one methoxyl at d 3.79
H
(3H, s) and three methyls at d 2.27 (3H, s),
H
2.21 (3H, s), and 1.13 (3H, d, J ¼ 6.2 Hz).
In addition, five proton signals were found
in the range of d 3.5–6.0, which suggested
the existence of a sugar moiety. On acid
H
21
5-methoxy-4,6-dimethyl-7-O-b-D-gluco-
pyranosyl-phthalide (3), together with one
hydrolysis using 0.5 mol l HCl at 708C
for 3 h, rhamnose was detected in the water
2
D
2
known compound, 7-hydroxy-5-methoxy-
4
solution. The specific rotation (½aꢀ 2 64)
,6-dimethylphthalide (1; Figure 1). Here,
demonstrated its L-configuration. The
*Corresponding author. Email: duqiangluo@163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.621422
http://www.tandfonline.com

1
070
J.-G. Xing et al.
O
O
O
1
2
3
O
O
O
8
CH3
CH3
OH
CH3
7
4
O
HO
O
HO
HO
O
6
5
H C
O
OCH3
OCH3
3
OCH3
OH
H C
H3C
H C
HO
3
3
HO
OH
1
2
3
Figure 1. Structures of compounds 1–3.
anomeric proton signal at d 5.53 (d . 5)
H
methyls, a methylene, and a rhamnose
moiety located at C-5, C-4, C-6, C-2, and
C-7, respectively (Figure 2). Thus, the
structure of 2 was characterized as 7-
hydroxy-5-methoxy-4,6-dimethyl-7-O-a-
L-rhamnosyl-phthalide (Figure 1). It was a
new phthalide derivative, i.e. the rhamno-
side of known compound 1.
and the anomeric carbon signal at d 106.4
C
(
JC1–H1 ¼ 166 Hz) further suggested the
presence of a-L-rhamnose in the molecule.
Seventeen carbon signals were shown in the
1
3
C NMR spectrum, including the carbon
signals of hexa-substituted benzene ring at
dC 164.5, 154.9, 148.5, 126.6, 122.2, and
1
and an ester carbonyl at d 171.3. With the
13.2, an oxygenated methylene at d 69.8,
Compound 3 was obtained as a color-
less crystal, and its molecular formula was
determined to be C H O by HR-ESI-
C
C
1
3
help of the HSQC spectrum, the C NMR
spectrum showed one methoxyl carbon at
d 60.9, three methyls at d 18.0, 11.3, and
1
7 22 9
þ
MS at m/z 393.1156 [M þ Na] . Its IR
spectrum indicated the presence of
C
C
2
hydroxyl (3374 cm ), ester carbonyl
1
1
0.6, six carbon signals belonging to the a-
rhamnose moiety at d 106.4, 73.5, 72.4,
2
(1736 cm ), and benzene ring (1513 and
1
C
2
1
72.2, 72.0, and 18.0. The structure assem-
bly of 2 was mainly achieved using the
1452 cm ). The UV spectrum showed
absorption maxima at 212, 249, and
1
291 nm. The H NMR and C NMR
13
HMBC experiment. The HMBC corre-
lations between the methoxyl at d 3.79 and
spectral data of 3 were very similar to
those of 2 (Table 1), except that the methyl
H
C-5 at d 164.5; the methyl at d 2.21 and
C
H
0
C-3 at d 148.5, C-4 at d 122.2, and C-5 at
C
at d 18.0 (C-6 ) in 2 was replaced by the
C
C
0
d 164.5; the methyl at d 2.27 and C-5 at
C
methylene at dC 62.4 (C-6 ) in 3. This
difference indicated that a hydroxyl group
H
d 164.5, C-6 at d 126.6, and C-7 at d
C
C
C
0
1
dC 171.3 and C-3 at dC 148.5; and H-1
54.9; the methylene at d 5.21 and C-1 at
0
was attached at C-6 in 3 and in another
H
word, the rhamnosyl in compound 2 was
changed to glucosyl in compound 3. This
conclusion was supported by the result of
at d 5.53 (1H, d, J ¼ 1.2 Hz) and C-7 at
H
d
154.9 suggested a methoxyl, two
C
O
O
O
O
CH3
OH
CH3
O
O
O
HO
HO
H C
3
O
OCH3
OCH3
OH
HO
H C
H C
3
3
HO
OH
2
3
Figure 2. The key HMBC correlations of compounds 2 and 3.

Journal of Asian Natural Products Research
1071
1 13
Table 1. H (600 MHz) and C (150 MHz) NMR spectral data of 2 and 3 in MeOD and
pyridine-d , respectively.
5
2
3
Position
d
C
d
H
(J, Hz)
d
C
H
d (J, Hz)
1
2
3
4
5
6
7
8
171.3
69.8
170.2
69.5
5.21 (2H, s)
5.02 (2H, s)
148.5
122.2
164.5
126.6
154.9
113.2
60.9
11.3
10.6
106.4
72.0
72.4
146.5
120.8
163.0
126.9
153.6
112.5
59.8
10.9
10.8
106.0
75.9
78.7
5
4
6
1
2
3
4
5
6
-OCH
3
3.79 (3H, s)
2.21 (3H, s)
2.27 (3H, s)
5.53 (1H, d, 1.2)
4.48 (1H, dd, 3.3, 1.2)
3.89 (1H, dd, 9.5, 3.3)
3.52 (1H, dd, 9.5, 9.5)
4.00 (1H, m)
3.59 (3H, s)
1.94 (3H, s)
2.50 (3H, s)
5.98 (1H, d, 7.5)
4.40 (1H, m)
4.34 (1H, m)
4.31 (1H, m)
4.00 (1H, m)
4.44 (1H, m) 4.31(1H, m)
-CH
-CH
3
3
0
0
0
0
0
0
73.5
72.2
18.0
71.2
78.5
62.4
1.23 (3H, d, 6.2)
acid hydrolysis that D-glucose was
detected in the water solution of 3. The
coupling constant (J ¼ 7.5 Hz) of the
anomeric proton (dH 5.98) demonstrated
that the glucose was in b-orientation.
The HMBC correlation of H-1 at d 5.98
with C-7 at d 153.6 indicated that the
glucosyl moiety was linked as C-7 position
(Figure 2). Thus, the structure of com-
China). Optical rotations were measured
on a Perkin-Elmer 241 polarimeter
(Perkin-Elmer, Waltham, MA, USA). UV
spectra were obtained on a Shimadzu UV-
2
10 spectrometer (Shimadzu, Kyoto,
0
Japan). IR spectra were obtained on a
Perkin-Elmer 577 spectrometer (Perkin-
Elmer, Waltham, MA, USA). The NMR
spectral data were recorded on Bruker
pound 3 was established as 7-hydroxy-5-
methoxy-4,6-dimethyl-7-O-b-D-glucopyr-
anosyl-phthalide (Figure 1). It was also a
new phthalide derivative, i.e. the glucoside
of known compound 1.
1
AM-600 (600 MHz for H and 150 MHz
13
for C; Bruker, Faellanden, Switzerland)
with tetramethylsilane (TMS) as an
internal standard. The HR-ESI-MS
data were obtained on a Bruker apexultra
The known compound was readily
identified as 7-hydroxy-5-methoxy-4,
7.0 T spectrometer (Bruker, Karlsruhe,
Germany). HPLC was performed on
Waters series (717 Plus Autosampler, 600
Controller, 2489 UV/Visible Detector;
Waters, Milford, MA, USA) by using
SymmetryPrepTM C18 semi-preparative
column (7 mm, 7.8 mm £ 150 mm; Waters,
Milford, MA, USA). Column chromato-
graphy (CC) was performed on silica gel
(200–300 mesh; Yantai Zhi Fu Chemical
Co., Ltd., Yantai, China) and RP-18
6
-dimethylphthalide (1) by comparing its
NMR spectral data with those reported in
the literature [16].
3
.
Experimental
3
.1 General experimental procedures
Melting point (MP) was determined on an
X-4 micromelting point apparatus (Cany
Precision Instruments Co., Ltd., Shanghai,

1
072
J.-G. Xing et al.
(12 nm, S-50 um, YMC Co., Ltd., Kyoto,
Japan).
followed by 30–40% for 18 min; detection
2
1
at 254 nm; t ¼ 12 min; 2.0 ml min ) to
R
afford compound 2 (10 mg). Fraction 9
(1.6 g) was then subjected to silica gel
reverse-phase column chromatography,
3
.2 Strain isolation and cultivation
Strain L421 was isolated from the stems of
B. gymnorrhiza collected in Dongzhai,
Hainan Province of China. The strain L421
was identified as P. heterocornis by Prof.
Jing-Ze Zhang and has been deposited in
Key Laboratory of Medicinal Chemistry
and Molecular Diagnosis of Ministry of
Education of Hebei University.
eluted with H O–MeOH (100:0–0:100),
2
yielding 10 subfractions (1–10). Com-
pound 3 (15 mg) was obtained as a
colorless crystal from subfraction 2.
3.3.1 Compound 2
2
2
A yellow oil, ½aꢀ 2 64 (c ¼ 0.25,
D
The fungal strain was cultured on
slants of comprehensive potato dextrose
agar (CPDA) at 288C for 5 days. Agar
plugs were used to inoculate 1000-ml
Erlenmeyer flasks each containing 600 ml
of media (CPDA), and the final pH of the
media was adjusted to 6.5 before steriliza-
tion. Flask cultures were incubated at 288C
on a rotary shaker at 150 rpm for 5 days.
Fermentation was carried out in eighty
MeOH). UV (MeOH) l
(nm): 216,
max
21
247, and 292. IR (KBr) vmax (cm ): 3374,
13
1
1744, 1605, and 1441. For H and
C
NMR spectral data, see Table 1. HR-ESI-
þ
MS: m/z 377.1215 [M þ Na] (calculated
for C H O Na, 377.1207).
17 22 8
3
.3.2 Compound 3
A colorless crystal, MP 169–1718C.
2
2
D
5
1
00-ml Erlenmeyer flasks each containing
00 g of rice. Distilled H O (100 ml) was
½aꢀ þ 22.8 (c ¼ 0.1, MeOH). UV
2
(MeOH) l
max
(nm): 212, 249, and 291.
2
IR (KBr) vmax (cm ): 3374, 1736, 1513,
1
added to each flask, and the contents were
soaked overnight before autoclaving at 15
1 13
and 1452. For H and C NMR spectral
2
2
lb in for 30 min. After cooling to room
temperature, each flask was inoculated
with 10.0 ml of the spore inoculum and
incubated at 288C for 60 days.
data, see Table 1. HR-ESI-MS: m/z
þ
393.1156 [M þ Na] (calculated for
C H O Na, 393.1156).
17 22 9
Acknowledgements
3
.3 Extraction and isolation
This work was financially supported by
Hundred Excellent Innovation Talents from
the Universities and Colleges of Hebei
Province (SPRC008), the Key Project of
Chinese Ministry of Education and the Key
Applied Basic Research Programs of Hebei
Province (0996030917D) and National Natural
Science Foundation of China (31071701).
The fermented material was extracted with
EtOAc (8 £ 1.0 l), and the organic solvent
was evaporated to dryness under vacuum
to afford the crude extract (108.0 g). The
extract was applied on a silica gel column
chromatography, and eluted with pet-
roleum ether–EtOAc–MeOH gradient to
afford 10 fractions. Compound 1 (20 mg)
was obtained as a colorless crystal from
fraction 2 (230 mg). Fraction 6 (2.0 g) was
subjected to silica gel column chromato-
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