A New Xanthone O-Glycoside from the Mangrove Endophytic Fungus Phomopsis sp.

Article abstract of DOI:10.1007/s10600-013-0497-0

A new xanthone O-glycoside, 3-O-(6-O-α-L-arabinopyranosyl)-β-D- glucopyranosyl-1,4-dimethoxyxanthone (1), together with three known compounds, phomapyrone D (2), 2-methoxy-3,4-methylenedioxybenzophenone (3), and cyclo(D-6-Hyp-L-Phe) (4), was isolated from the mangrove endophytic fungus, Phomopsis sp. (ZH76). Their structures were determined by analysis of spectroscopic data. Compound 1 inhibited HEp-2 and HepG2 cells with IC ₅₀ values of 9 and 16 μmol/mL, respectively.

Full text of DOI:10.1007/s10600-013-0497-0

Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013 [Russian original No. 1, January–February, 2013]
A NEW XANTHONE O-GLYCOSIDE FROM THE MANGROVE
ENDOPHYTIC FUNGUS Phomopsis sp.
1
2
1*
Zhongjing Huang, Jianxiang Yang, Fuhou Lei,
UDC 547.972 + 945
3
3
Zhigang She, and Yongcheng Lin
A new xanthone O-glycoside, 3-O-(6-O-ꢀ-L-arabinopyranosyl)-ꢁ-D-glucopyranosyl-1,4-dimethoxyxanthone
(
1), together with three known compounds, phomapyrone D (2), 2-methoxy-3,4-methylenedioxybenzophenone
(
3), and cyclo(D-6-Hyp-L-Phe) (4), was isolated from the mangrove endophytic fungus, Phomopsis sp. (ZH76).
Their structures were determined by analysis of spectroscopic data. Compound 1 inhibited HEp-2 and HepG2
cells with IC50 values of 9 and 16 ꢂmol/mL, respectively.
Keywords: xanthone O-glycoside, mangrove endophytic fungus, structure elucidation, cytotoxic activity.
Mangrove endophytic fungi have received some attention, and some promising compounds with unique chemical
structures and potent activities have been isolated [1–5]. This paper reports the isolation, structural elucidation, and biological
activities of a new xanthone O-glycoside, 3-O-(6-O-ꢀ-L-arabinopyranosyl)-ꢁ-D-glucopyranosyl-1,4-dimethoxyxanthone (1),
together with three known compounds, phomapyrone D (2), 2-methoxy-3,4-methylenedioxybenzophenone (3), and
cyclo(D-6-Hyp-L-Phe) (4), from the mangrove endophytic fungus Phomopsis sp. ZH76 that was isolated from the stem of the
mangrove tree Excoecaria agallocha from Dong Zai, Hainan, China.
Compound 1 was obtained as a yellow amorphous solid. The molecular formula of C H O was established by
2
6 30 14
–
HR-FAB-MS (m/z 565.1552, calcd for [M – H] 565.1557) and the NMR spectral data (Table 1). The UV absorption bands at
32, 258, 299, and 376 nm and IR (KBr) absorption bands at 3468, 1658, 1605, 1589, and 1478 cm suggested the presence
–
1
2
of the xanthone skeleton [6]. On acid hydrolysis, 1 gave glucose and arabinose, suggesting that 1 was a xanthone glycoside.
1
The H NMR spectrum exhibited four aromatic protons at ꢃ 7.64 (d, J = 8.3 Hz), 7.78 (t, J = 8.3 Hz), 7.42 (t, J = 7.5 Hz),
and 8.09 (d, J = 7.8 Hz), and one aromatic proton at 6.87 (s), indicating that one aromatic ring was a 1,2-disubstituted benzene
1
ring and the other was a pentasubstituted benzene ring. The H NMR spectrum also showed the signals of one anomeric proton
of Glc at ꢃ 4.97 (d, J = 7.5 Hz), one anomeric proton ofAra at 4.17 (d, J = 6.5 Hz), and two aromatic methoxyl groups at 3.85 and
3
.81. The remaining proton signals upfield were attributable to two sugar moieties.
OMe O
O
O
OMe
O
O
1
8
5
9
a
8a
4b
H
9
O
O
O
N
O
3
6
'
HO 5''
4a
O
HN
O
1'
4
MeO
O
5
'
O
OMe
OH
OH
OH
1
''
O
3
''
HO 3'
4
1
2
3
OH
OH
1) College of Chemistry and Ecology Engineering, Guangxi University for Nationalities, 530006, Nanning, P. R. China,
e-mail: leifuhou@163.com; 2) Department of Chemistry and Engineering, Guilin Medical University, 541004, Guilin,
P. R. China; 3) College of Chemistry and Chemical Engineering, Sun Yat-Sen (Zhongshan) University, 510275, Guangzhou,
P. R. China. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2013, pp. 27–29. Original article submitted
September 24, 2011.
0
0
009-3130/13/4901-0027 2013 Springer Science+Business Media New York
27

1
13
TABLE 1. H NMR (500 MHz) and C NMR (125 MHz) Data for 1 (DMSO-d , ꢃ, ppm, J/Hz)
6
C atom
ꢃC (DEPT)
ꢃH
HMBC
1
2
3
4
156.8 (C)
96.3 (CH)
157.0 (C)
138.5 (C)
148.2 (C)
154.4 (C)
117.8 (CH)
134.6 (CH)
124.1 (CH)
125.6 (CH)
121.6 (C)
179.8 (C)
110.9 (C)
H-2, 1-OCH3
6.87 (s)
H-2, 1ꢄ
H-2, 4-OCH3
4
4
a
b
H-5, 6, 8
H-6, 7
H-5, 7, 8
H-5, 6, 8
H-6, 7
H-5, 7, 8
H-8
5
7.64 (d, J = 8.3)
7.78 (t, J = 8.3)
7.42 (t, J= 7.5)
8.09 (d, J = 7.8)
6
7
8
8
a
9
H-2
9
a
1
4
-OCH
-OCH3
3
56.3 (CH
3
)
3.81 (s)
3.85 (s)
4.97 (d, J = 7.5)
3.35 (m)
61.7 (CH3)
101.5 (CH)
73.4 (CH)
76.1 (CH)
69.9 (CH)
75.6 (CH)
H-2ꢄ, 3ꢄ, 5ꢄ
H-1ꢄ, 3ꢄ
H-1ꢄ, 2ꢄ, 4ꢄ
H-5ꢄ
H-1ꢄ, 4ꢄ, 6ꢄ
H-5ꢄ, 1ꢄꢄ
H-6ꢄ, 2ꢄꢄ, 3ꢄꢄ
H-1ꢄꢄ, 3ꢄ, 4ꢄꢄ
H-2ꢄꢄ, 4ꢄꢄ
H-2ꢄꢄ, 3ꢄꢄ, 5ꢄꢄ
H-1ꢄꢄ, 4ꢄꢄ
Glc-1ꢄ
2
3
4
5
6
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
3.37 (m)
3.23 (m)
3.62 (m)
6
8.3 (CH
2
)
3.96 (m), 3.55 (dd, J = 6.5, 11.0)
4.17 (d, J = 6.5)
3.46 (t, J = 8.0)
3.35 (m)
3.58 (m)
3.65 (dd, J = 4.0, 11.6), 3.30 (m)
1
03.4 (CH)
Ara-1ꢄꢄ
7
7
6
0.5 (CH)
2.7 (CH)
7.3 (CH)
2
3
4
5
ꢄꢄ
ꢄꢄ
ꢄꢄ
ꢄꢄ
6
4.8 (CH
2
)
In the HMBC spectrum cross-peaks from H-2 (ꢃH 6.87) to C-1, C-3, C-4, and C-9a revealed the presence of a
1
,3,4-trisubstituted xanthone. Two methoxyl groups (ꢃ 3.81 and 3.85) were placed at C-1 and C-4, respectively, as determined
H
by the HMBC cross-peaks of the former proton with C-1, and of the latter proton with C-4. The HMBC cross-peaks from H-1ꢄ
ꢃ 4.97) of Glc to C-3 (ꢃ 157.0) of the aglycon and from H-1ꢄꢄ (ꢃ 4.17) of Ara to C-6ꢄ (ꢃ 68.3) of Glc showed that the
(
H
C
H
C
glucosyl residue was linked to C-3 of the aglycon, and the arabinosyl residue was linked to the glucosyl moiety by a (1ꢄꢄꢅ6ꢄ)
linkage. The anomeric configuration of the glucosyl residue was deduced to be ꢁ from the J value (J = 7.5 Hz) of anomeric
1
3
proton, and that of the arabinosyl residue to be ꢀ by comparison of the C NMR data [7]. So, compound 1 was identified
as 3-O-(6-O-ꢀ-L-arabinopyranosyl)-ꢁ-D-glucopyranosyl-1,4-dimethoxyxanthone.
In addition, three known compounds, 2, 3, and 4, were identified as phomapyrone D, 2-methoxy-3,4-methylenedioxy-
benzophenone, and cyclo(D-6-Hyp-L-Phe), respectively, through the comparison of spectroscopic data with those in the literature
[
8–10].
Primary bioassays showed that 1 exhibited cytotoxicity against HEp-2 and HepG2 cells with IC50 values of 9 and
6 ꢂg/mL, respectively.
1
EXPERIMENTAL
General Methods. NMR data were recorded in DMSO-d or CDCl , using TMS as internal reference, on a Varian
6
3
1
13
INOVA 500 NB nuclear magnetic resonance spectrometer ( H, 500 MHz; C, 125 MHz). Mass spectra were recorded on a
Thermo VG ZAB-HS double-focussing mass spectrometer or a Thermo MAT95XP high-resolution mass spectrometer.
IR spectra were measured on a Bruker Equinox 55 spectrophotometer. UV spectra were obtained on a Shimadzu UV-2501PC
spectrophotometer. Optical rotations were measured on a Schmidt + Haensch Polartronic HH W5 polarimeter and were
uncorrected. Column chromatography was performed on silica gel (200–300 mesh, Qingdao, China), and TLC on precoated
GF254 plates (Qiangdao, China). ꢁ-D-glucose (Sigma) and ꢀ-L-arabinose (Sigma) were used as reference compounds.
2
8

Fungus Material and Culture Conditions. The fungus Phomopsis sp. (ZH76) was isolated from the stem of
Excoecaria agallocha of the mangrove tree Euphorbiaceae from Dong Sai of the South China Sea coast. Starter cultures were
maintained on cornmeal seawater agar. Phomopsis sp. (ZH76) was stored at Sun Yat-Sen (Zhongshan) University, Guangzhou,
P. R. China. Plugs of agar supporting mycelium growth were cut and transferred aseptically into a 250 mL Erlenmeyer flask
containing 100 mL of liquid medium (glucose 10 g/L, peptone 2 g/L, yeast extract 1 g/L, NaCl 25 g/L). The flask was
incubated at 30ꢆC on a rotary shaker for 6 days. The mycelium was aseptically transferred to 1000 mL Erlenmeyer flasks
containing culture liquid (600 mL) and incubated at room temperature for 30 days.
Extraction and Separation of Metabolites. All cultures (90 L) were separated into mycelium and filtrate. The
mycelium was dried in room temperature and then immersed in methanol (6 L) for 20 days. The methanol extract was evaporated
under reduced pressure to yield a brown gum (48 g). The gum was chromatographed repeatedly on silica gel using gradient
elution from petroleum ether–ethyl acetate (9:1, 8:2, 7:3, 5:5, and 3:7), providing fractions A, B, C, D, and E. Fraction B was
repeatedly purified by column chromatography on silica gel using petroleum ether–ethyl acetate (9:1, 8:2, 7:3, and 6:4), giving
fractions B , B , B , and B . Fraction B was subjected to column chromatography on silica gel eluting with petroleum
1
2
3
4
3
ether–ethyl acetate (8:2) to give compound 1 as a yellow amorphous solid (7 mg). Fraction C was purified by column
chromatography on silica gel with petroleum ether–ethyl acetate (8:2, 7:3) to afford compound 2 as an amorphous white solid
(
8 mg) and compound 3 as a colorless oil (5 mg), respectively. Fraction D was purified by column chromatography on silica
gel with petroleum ether–ethyl acetate (6:4) to give compound 4 as a white solid (10 mg).
-O-(6-O-ꢀ-L-Arabinopyranosyl)-ꢁ-D-glucopyranosyl-1,4-dimethoxyxanthone (1). Yellow amorphous solid.
ꢀ] –62.0ꢆ (c 0.41, CH OH). UV spectrum (CH OH, ꢇmax, nm) (log ꢈ): 341 (4.35), 298 (4.68), 277 (4.14), 243 (3.90).
3
2
5
[
D
3
3
–
1
IR spectrum (KBr, ꢉ, cm ): 3434, 2942, 2886, 1615, 1469, 1422, 1316.
–
–
–
Mass spectrum (FAB, m/z, I , %): 565 [M – H] (25), 433 [M – H – 132] (10), 271 [M – H – 132 – 162] (100).
rel
–
1
13
Mass spectrum (HR-FAB, m/z, I , %): 565.1557 [M – H] , (calcd for C H O , 565.1557). H, C NMR see Table 1.
rel
26 29 14
1
Phomapyrone D (2). Amorphous white solid. H NMR (500 MHz, CDCl , ꢃ, ppm, J/Hz): 7.14 (1H, s), 6.53 (1H, s),
3
13
3
.95 (3H, s), 2.35 (6H, s), 2.05 (3H, s). C NMR (125 MHz, CDCl , ꢃ, ppm): 199.7 (C), 165.2 (C), 164.5 (C), 158.2 (C), 139.8 (C),
3
1
26.5 (CH), 97.5 (CH), 56.7 (CH ), 33.0 (CH ), 13.9 (CH ), 9.4 (CH ).
3
3
3
3
1
2
-Methoxy-3,4-methylenedioxybenzophenone (3). Colorless oil. H NMR (500 MHz, CDCl , ꢃ, ppm, J/Hz): 7.77
3
(
3
2H, br.dd, J = 8.0, 1.6), 7.52 (1H, tt, J = 8.0, 1.6), 7.42 (2H, br.t, J = 8.0), 6.94 (1H, d, J = 8.0), 6.58 (1H, d, J = 8.0), 5.59 (2H, br.s),
1
3
.81 (3H, s). C NMR (125 MHz, CDCl , ꢃ, ppm): 195.0 (C), 151.2 (C), 141.8 (C), 138.3 (C), 136.8 (C), 132.6 (CH), 129.5
3
(
2 ꢊ CH), 128.0 (2 ꢊ CH), 126.1 (C), 124.3 (CH), 102.7 (CH), 101.5 (CH ), 60.0 (CH ).
2 3
1
Cyclo(D-6-Hyp-L-Phe) (4). White solid. H NMR (500 MHz, CDCl , ꢃ, ppm, J/Hz): 7.36 (2H, t, J = 7.3), 7.29 (1H,
3
t, J = 7.3), 7.23 (2H, d, J = 7.3), 5.79 (s), 4.25 (1H, dd, J = 9.9, 3.6), 3.65 (2H, m), 3.62 (1H, dd, J = 14.3, 3.6), 2.73 (1H, dd,
1
3
J = 14.3, 9.9), 2.32 (1H, m), 1.93 (1H, m), 1.86 (2H, m). C NMR (125 MHz, CDCl , ꢃ, ppm): 168.1 (C), 166.1 (C), 135.7
3
(
C), 129.3 (2 ꢊ CH), 129.2 (2 ꢊ CH), 127.6 (CH), 87.6 (C), 55.8 (CH), 45.4 (CH ), 41.0 (CH ), 36.8 (CH ), 19.9 (CH ).
2 2 2 2
Acidic Hydrolysis of Compound 1. Compound 1 (3 mg) in 50% MeOH (3 mL) containing 5% HCl was refluxed in
a boiling H O bath for 5 h. After cooling, the reaction mixture was poured into 10 mL of H O. The mixture was extracted with
2
2
ethyl acetate, and the aqueous residue was then checked by co-TLC together with authentic sugar ꢁ-D-glucose and ꢀ-L-arabinose
(
EtOAc–MeOH–H O–AcOH, 13:4:4:5). Acid hydrolysis of 1 gave ꢁ-D-glucose (R 0.46) and ꢀ-L-arabinose (R 0.53).
2
f
f
Bioasays. The cytotoxic assays were performed using the MTT assay method [11]. In the preliminary bioassay,
compound 1 inhibited cytotoxicity against HEp-2 and HepG2 cells with IC50 values of 9 and 16 ꢂg/mL, respectively.
ACKNOWLEDGMENT
This research was supported by a grant from Guangxi Key Laboratory of Chemistry and Engineering of Forest
Products, the Natural Science Foundation of Guangxi Province (0832081), the Science Foundation of Guangxi University for
Nationalities (200702YJ18), and the National Natural Science Foundation of China (20772162, 20572136).
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0