New nervogenic acid derivatives from liparis nervosa

Article abstract of DOI:10.1055/s-0032-1328109

Ten new nervogenic acid derivatives (1-4, 6-11) and one known compound (5) have been isolated from Liparis nervosa. Their structures were determined using extensive spectroscopic analysis, including 1D and 2D NMR experiments. Compounds 3, 4, 9, 10, and 11

Full text of DOI:10.1055/s-0032-1328109

Original Papers 281
New Nervogenic Acid Derivatives from Liparis nervosa
Shuai Huang1,2, Xian-li Zhou , Cui-juan Wang , Hong-yan Wang , You-song Wang , Lian-hai Shan , Jie Weng
1
1
3
1
1
2
Authors
1
Affiliations
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, Peopleʼs Republic of China
Key Laboratory of Advanced Technology of Materials, Ministry of Education, School of Material Science and Engineering,
Southwest Jiaotong University, Chengdu, Sichuan, Peopleʼs Republic of China
2
3
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou, Peopleʼs Republic of China
Key words
Abstract
pounds 3, 4, 9, 10, and 11 were evaluated for their
cytotoxicity against A549, H460, Hela, MCF-7, Ca-
co2, and HepG2 human cancer cell lines.
"
l Orchidaceae
!
"
l Liparis nervosa
Ten new nervogenic acid derivatives (1–4, 6–11)
and one known compound (5) have been isolated
from Liparis nervosa. Their structures were deter-
mined using extensive spectroscopic analysis, in-
cluding 1D and 2D NMR experiments. Com-
"
l benzamide acid derivatives
"
l nervogenic acid derivatives
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
"
l cytotoxicity
Introduction
!
Materials and Methods
!
Liparis nervosa (Thunb.) Lindl, a member of the
Orchidaceae family, is an herbaceous plant that is
widely distributed in China. This plant usually
grows in broad-leaved forests, particularly on
moist slopes at altitudes from 850 to 1000 m. Li-
paris nervosa historically has been used in folk
medicine as an antipyretic, detoxicating, and he-
mostatic agent [1]. The chemical components of
this plant have been investigated by Kunisuke
Nishkawa, leading to the identification of a new
pyrrolizidine alkaloid, nervosine [2,3]. However,
the nervogenic acid derivatives and their bioac-
tivities have not been studied in detail yet, which
prompted us to undertake a systematic study of
this plant. Here we report on the isolation and
structural elucidation of new nervogenic acid de-
General experimental procedures
Optical rotations were measured on a Perkin-El-
mer 341 polarimeter. 1D and 2D NMR spectra
were recorded on a Bruker AV 400 or a Varian
Unity INOVA 400/54 NMR spectrometer
1
13
(400 MHz for H and 100 MHz for C) with TMS
as an internal standard. IR spectra were obtained
with a Thermo Fisher Nicolet 6700 spectrometer,
−1
KBr pellets in cm . UV spectra were determined
with a Shimadzu UV-2450 spectrophotometer.
HR‑ESI‑MS were measured using a Q‑TOF micro
mass spectrometer (Waters). GC analyses were
performed using a Hewlett Packard GC6890 in-
strument on an Agilent HP-5 column (0.25 mm,
30 m, i.d., 0.25 µm). Silica gel (Qingdao Haiyang
Chemical Co., Ltd., 200–300 mesh), Sephadex LH-
20 (Pharmacia Co.), RP-18 silica gel (Merck, 40–
60 µm), and D-101 macroporous resin (Rohm &
Haas) were used for column chromatography
(CC). Semipreparative HPLC was carried out on a
Waters Symmetry Prep™ C-18 column (7 µm,
∅ 19.0 × 300 mm) with a Waters 600 controller
and Waters 2487 detector. TLC plates were pre-
coated with silica gel GF254 (Qingdao Haiyang
Chemical Co., Ltd.) and visualized under a UV
lamp at 254 nm or by spraying 5% vanillin-H SO
received
revised
accepted
June 7, 2012
Dec. 6, 2012
Dec. 10, 2012
Bibliography
DOI http://dx.doi.org/
0.1055/s-0032-1328109
Published online January 15,
013
Planta Med 2013; 79: 281–287
Georg Thieme Verlag KG
"
1
rivatives (1–4, 6–11) (l Fig. 1) by means of 1D
and 2D NMR experiments. In addition, one known
compound was isolated and identified as methyl
2
3,5-bis (3-methyl-2-butenyl)-4-O-(β-D-glucopyr-
©
anosyl) benzoate (5) by comparison of its spectro-
scopic data with those reported in the literature
Stuttgart · New York ·
ISSN 0032‑0943
[4]. To the best of our knowledge, the cytotoxic ac-
Correspondence
Prof. Dr. Xian-li Zhou
tivity of the nervogenic acid derivatives has not
been studied yet, and we have here investigated
part of the isolated compounds for their cytotox-
icity in vitro against several human cancer cell
lines.
Natural Products Laboratory of
the School of Life Science and
Engineering
Southwest Jiaotong University
Chengdu 610031, Sichuan
Peopleʼs Republic of China
Phone: + 862887600185
Fax: + 862887600185
xxbiochem@163.com
2
4
(w/v) or by iodine.
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

2
82 Original Papers
Fig. 1 Structures of compounds 1–11 isolated
from Liparis nervosa.
Plant material
to yield six fractions (FIII-1–FIII-6), among which FIII-3 (400 mg)
was further purified by an RP-18 silica gel column (∅ 2.0 ×
The whole plant of L. nervosa was collected in Chongqing, China
in July 2007. The plant was identified by Professor Liangke Song
in the School of Life Science and Engineering, Southwest Jiaotong
University, Sichuan Province, China, where a voucher specimen
was deposited (No. Z36150701).
45 cm) with MeOH:H O (45:55) as the mobile phase to yield
2
compounds 6 (7 mg), 7 (10 mg), and 11 (16 mg), respectively.
Meanwhile, the EtOAc extract (25.0 g) was subjected to RP-18 sil-
ica gel column (∅ 7.0 × 60 cm) and eluted with MeOH:H O (0:1–
2
1
:0) to yield five fractions (F1–1-F1–5). F1–3 (2.0 g) was purified us-
Extraction and isolation
ing a Sephadex LH-20 column (∅ 1.5 × 60 cm, MeOH) first and
The shade dried L. nervosa (5.0 kg) was powdered and extracted
with 95% EtOH (3 × 20 L) at room temperature for 3 days. After
the solvent was removed by evaporation, the ethanol extract
then by an HPLC system using MeOH:H O (62:38) to afford com-
pounds 1 (6 mg) and 2 (4 mg). F1–5 (1.7 g) was subjected to a silica
2
gel column (∅ 3.5 × 45 cm) and eluted with EtOAc:MeOH:H O
2
(300.0 g) was recovered. The extract was then suspended in
(120:1:1) to yield compounds 3 (25 mg), 4 (22 mg), and 5
water (1 L) at 50°C and adjusted to pH 2.8 with HCl. The aqueous
solution was then extracted with CHCl3 (500 mL × 3) and EtOAc
(25 mg).
(
(
500 mL × 3) to obtain the CHCl3 (82.0 g) and EtOAc extracts
25.0 g), respectively. The pH of the aqueous layer was adjusted
Isolated compounds
{6-O-[(4-oxo-4H-pyran-3-yloxy)-O-β‑D-glucopyranosyl]}-4-hy-
to 9.4 with aqueous ammonia solution and then extracted with
EtOAc (500 mL × 3) and n-butanol (500 mL × 3) to obtain the
crude alkaline EtOAc extract (10.0 g) and the n-butanol extract
droxy-3,5-bis(3-methyl-2-butenyl) benzoate (1): Amorphous sol-
20
D
id, purity: 96% (HPLC); [α] − 14.4 (c 0.305, MeOH); UV (MeOH)
λmax (log ε), 223 (3.27), 261 (3.46) nm; IR (KBr) νmax 3401, 2970,
(
50.0 g), respectively.
The n-butanol extract (50.0 g) was subjected to a column
∅ 9.5 × 60 cm) using D101 resin and eluting with H O, 30%,
2925, 1711, 1642, 1607, 1439, 1384, 1310, 1284, 1241, 1194,
−1
1
13
"
1072, 875, 842, 769 cm ; H and C NMR data, see l Tables 1
and 3. HR‑ESI‑MS at m/z 553.2064 [M + Na]+ (calcd. for
C28H34O10Na, 553.2050).
(
6
2
0%, and 95% EtOH sequentially, to afford four fractions (I, II, III,
and IV).
{1-O-[6-acetyl-O-β‑D-glucopyranosyl]}-4-hydroxy-3,5-bis(3-
methyl-2-butenyl) benzoate (2): Amorphous solid, purity: 95%
Fraction II (6.5 g) was separated by RP-18 silica gel column CC
20
(
∅ 4.0 × 45 cm) using MeOH and H O as the mobile phase with a
(HPLC); [α]
D
− 6.2 (c 0.225, MeOH); UV (MeOH) λmax (log ε) 226
2
gradient from 2% to 100% to afford fractions FII-1-FII-6 based on
TLC analysis. FII-2 (400 mg) was subjected to another silica gel
(2.72), 267 (2.44) nm; IR (KBr) νmax 3411, 2972, 2926, 1724, 1607,
−
1 1
1438, 1375, 1308, 1282, 1247, 1186, 1077, 875, 769 cm ; H and
1
3
"
column (∅ 1.5 × 30 cm) and eluted with CHCl :MeOH:EtOAc
C NMR data, see l Tables 1 and 3. HR‑ESI‑MS at m/z 477.2132
3
−
(
12:1:1) to yield compound 10 (42 mg). FII-3 (1.5 g) was sub-
jected to a silica gel column (∅ 3.5 × 45 cm) and eluted with
CHCl :MeOH:EtOAc (25:1:1) to yield six fractions (F
[M – H] (calcd. for C25H33O , 477.2125).
9
Methyl 3,5-bis(3-methyl-2-butenyl)-4-O-(α‑L-arabinopyranosyl)
2
0
–
II-3–1
benzoate (3): Amorphous solid, purity: 96% (HPLC); [α]
D
+ 26.4
3
FII-3–6). FII-3–2 was subjected to a Sephadex LH-20 column
(c 0.700, MeOH); UV (MeOH) λmax (log ε) 238 (2.64) nm; IR
(KBr) νmax 3400, 2973, 2927, 1715, 1649, 1604, 1435, 1384,
(
(
∅ 1.5 × 60 cm) and eluted with MeOH to yield compound 9
43 mg). FII-3–3 was first purified by column chromatography us-
−1
1
13
1313, 1265, 1238, 1082, 1018, 951, 912, 772 cm ; H and
C
"
ing a Sephadex LH-20 column (∅ 1.5 × 60 cm) and eluted with
MeOH. It was further purified by a preparative HPLC, eluted with
NMR data, see l Tables 1 and 3. HR‑ESI‑MS at m/z 443.2038 [M
+
+ Na] (calcd. for C23H32O Na, 443.2046).
7
MeOH:H O (58:42) at a flow rate of 8 mL/min to afford com-
pound 8 (6 mg).
Methyl 3,5-bis(3-methyl-2-butenyl)-4-O-[β‑D-glucopyranosyl(1 →
2)-α‑L-arabinopyranosyl] benzoate (4): White amorphous pow-
2
20
Fraction III (9.0 g) was subjected to
a
silica gel column
D
der, purity: 97% (HPLC); [α] − 6.0 (c 0.300, MeOH); UV (MeOH)
(
∅ 5.0 × 50 cm) and eluted with CHCl :EtOAc:MeOH (10:10:1)
λmax (log ε) 243 (3.44) nm; IR (KBr) νmax 3377, 2926, 2875, 1722,
3
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

Original Papers 283
Table 1 1H NMR data of compounds 1–6.
H
1a, b
2a, b
3a, c
4a, d
5c
6b
2
6
7.64 s
7.73 s
7.67 s
7.68 s
7.64 s
7.67 s
7.64 s
7.73 s
7.67 s
7.68 s
7.64 s
7.67 s
1
2
4
5
6
7
9
1
′
3.34 d (7.2)
5.31 m
1.72 s
3.34 d (7.2)
5.32 m
1.73 s
3.45 d (7.2)
5.21 t (7.2)
1.67 s
3.55 d (7.6)
5.29 t (7.6)
1.74 s
3.46 d (7.2)
5.19 t (7.2)
1.63 s
3.32 d (7.1)
5.30 t (7.1)
1.75 s
′
′
′
1.76 s
1.76 s
1.71 s
1.77 s
1.68 s
1.76 s
′
3.34 d (7.2)
5.31 m
1.72 s
3.34 d (7.2)
5.32 m
1.73 s
3.45 d (7.2)
5.21 t (7.2)
1.67 s
3.55 d (7.6)
5.29 t (7.6)
1.74 s
3.46 d (7.2)
5.19 t (7.2)
1.63 s
3.32 d (7.1)
5.30 t (7.1)
1.75 s
′
′
0′
1.76 s
1.76 s
1.71 s
1.77 s
1.68 s
1.76 s
Glc
Glc
Ara
Ara
Glc
Glc
1
2
3
4
5
″
″
″
″
″
4.79 d (7.2)
3.49 m
3.49 m
3.47 m
3.74 m
5.65 d (8.0)
3.48 m
3.48 m
3.45 m
3.64 m
4.54 d (7.6)
4.02 br.t (8.8)
3.69 m
4.75 d (7.2)
4.15 dd (7.2, 8.8)
3.81 m
4.66 d (7.6)
3.62–3.79 m
3.62–3.79 m
3.62–3.79 m
3.15 m
4.72 d (7.6)
3.53 m
3.43 m
3.93 m
3.91 m
3.43 m
a 3.34 br.d (12.4)
b 3.93 br.d (12.4)
–
a 3.41 ov
b 3.93 ov
–
3.15 m
6
″
a 4.42 dd (6.4, 12.0)
b 4.63 dd (2.0, 12.0)
a 4.22 dd (5.6, 12.0)
b 4.37 dd (2.0, 12.0)
a 3.49 dd (6.0,12.0)
b 3.70 ov
a 3.65 dd (5.6, 12.0)
b 3.79 dd (2.0, 12.0)
Glc
1
2
3
4
5
6
′″
′″
′″
′″
′″
′″
–
–
–
–
–
–
–
–
–
–
–
–
–
4.72 d (8.0)
3.37 m
–
–
–
–
–
–
–
–
–
–
–
–
8.17 d (0.6)
–
3.43 m
–
3.88 m
6.46 d (5.6)
3.35 m
7.97 dd (5.6, 0.6)
a 3.69 dd (5.2,12.0)
b 3.80 ov
–
-
-
OCOCH3
OCH3
–
–
2.05 s
–
–
–
–
–
3.86 s
3.88 s
3.86 s
a
Data are based on DEPT, HSQC, and HMBC experiments; 1H NMR (400 MHz, δ, J in Hz in parentheses).b Data in CD3OD; c data in CDCl3; d data in CDCl3+ CD3OD; ov means over-
lapped
1
604, 1436, 1383, 1319, 1284, 1224, 1082, 1039, 1017, 956, 896,
(log ε) 239 (3.82) nm; IR (KBr) νmax 3363, 2968, 2924, 1660,
−
1
1
13
"
772 cm ; H and C NMR data, see l Tables 1 and 3. HR‑ESI‑MS
1611, 1604, 1579, 1423, 1384, 1265, 1199, 1162, 1075, 1041,
+
−1
1
13
"
at m/z 605.2574 [M + Na] (calcd. for C29H43O12Na, 605.2574).
Methyl
925, 899, 774 cm ; H and C NMR data, see l Tables 2 and 3.
+
3,5-bis(3-methyl-2-butenyl)-4-O-(β‑D-glucopyranosyl)
HR‑ESI‑MS at m/z 476.2262 [M + Na] (calcd. for C23H35NO Na,
8
benzoate (5): White amorphous powder, purity: 90% (HPLC);
476.2260).
20
[α]
D
− 6.0 (c 0.450, MeOH); UV (MeOH) λmax (log ε) 240 (3.46)
3-(3-Hydroxy-3-methylbutyl)-4-O-[β‑D-glucopyranosyl-(1 → 2)-
nm; IR (KBr) νmax 3390, 2923, 2853, 1722, 1601, 1456, 1435,
β‑D-glucopyranosyl]-5-(3-methyl-2-butenyl)
White amorphous powder, purity: 97% (HPLC); [α]
benzamide
(9):
20
1
377, 1323, 1284, 1237, 1082, 1196, 1180, 1068, 1010, 903,
D
− 20.8 (c
−
1
1
13
"
773 cm ; H and C NMR data, see l Tables 1 and 3. HR‑ESI‑MS
0.125, MeOH); UV (MeOH) λmax (log ε) 241 (4.22) nm; IR (KBr)
νmax 3364, 2970, 2925, 1660, 1610, 1579, 1423, 1384, 1261,
+
at m/z 473.2140 [M + Na] (calcd. for C28H34O Na, 473.2151).
8
−1
1
13
3
,5-bis(3-Methyl-2-butenyl)-4-O-(β‑D-glucopyranosyl)
benza-
− 5.3 (c
1200, 1159, 1078, 1035, 950, 909, 778 cm ; H and C NMR data,
2
0
"
+
mide (6): Amorphous solid, purity: 96% (HPLC); [α]
D
see l Tables 2 and 3. HR‑ESI‑MS at m/z 638.2766 [M + Na] (calcd.
for C29H45NO13Na, 638.2789).
0.150, MeOH); UV (MeOH) λmax (log ε) 240 (3.52) nm; IR (KBr)
νmax 3419, 2923, 2953, 1659, 1648, 1456, 1398, 1384, 1261,
3-(3-Hydroxy-3-methylbutyl)-4-O-[β‑D-glucopyranosyl-(1 → 2)-
−
1
1
13
"
1
195, 1162, 1077, 1034, 776 cm ; H and C NMR data, see l Ta-
α‑L-arabinopyranosyl]-5-(3-methyl-2-butenyl) benzamide (10):
+
20
bles 1 and 3. HR‑ESI‑MS at m/z 436.2324 [M + H] (calcd. for
Amorphous solid, purity: 95% (HPLC); [α]
D
+ 23.0 (c 0.300,
C23H34NO , 436.2335).
MeOH); UV (MeOH) λmax (log ε) 238 (3.31) nm; IR (KBr) νmax
7
3
-[(1E)-(3-Hydroxy-3-methyl-1-butenyl)-4-O-[β‑D-glucopyrano-
3392, 2971, 2929, 1716, 1602, 1459, 1383, 1371, 1311, 1272,
−
1
1
13
syl-(1 → 2)-α‑L-arabinopyranosyl]-5-(3-methyl-2-butenyl) benza-
1190, 1078, 1031, 948, 912, 772 cm ; H and C NMR data, see
2
0
"
+
mide (7): Amorphous solid, purity: 97% (HPLC); [α]
.350, MeOH); UV (MeOH) λmax (log ε) 236 (2.65) nm; IR (KBr)
νmax 3364, 2969, 2925, 1660, 1613, 1579, 1423, 1384, 1265,
D
+ 4.4 (c
l Tables 2 and 3. HR‑ESI‑MS at m/z 608.2668 [M + Na] (calcd.
for C28H43NO12Na, 608.2683).
0
1-(Ethoxy)-3-[(3-hydroxy-3-methylbutyl)-4-O-[β‑D-glucopy-
−
1
1
13
"
1
200, 1070, 1018, 902, 775 cm ; H and C NMR data, see l Ta-
ranosyl-(1 → 2)-α‑L-Arabinopyranosyl]]-5-(3-methyl-2-butenyl)
+
20
bles 2 and 3. HR‑ESI‑MS at m/z 606.2529 [M + Na] (calcd. for
benzoate (11): White amorphous solid, purity: 94% (HPLC); [α]
D
C28H41NO12Na, 606.2526).
− 22.7 (c 0.600, MeOH); UV (MeOH) λmax (log ε) 236 (3.02) nm; IR
(KBr) νmax 3415, 3338, 2970, 2971, 2877, 1638, 1610, 1579, 1420,
1384, 1297, 1259, 1238, 1194, 1100, 1070, 1017, 929, 894,
3-(3-Hydroxy-3-methylbutyl)-4-O-(β‑D-glucopyranosyl)-5-(3-
methyl-2-butenyl) benzamide (8): White amorphous powder, pu-
2
0
rity: 96% (HPLC); [α]
D
+ 9.4 (c 0.900, MeOH); UV (MeOH) λmax
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

2
84 Original Papers
Table 2 1H NMR data of compounds 7–11.
H
7a, b
8a, b
9a, c
10a, c
11a, b
2
6
7.88 d (2.0)
7.57 d (2.0)
7.27 d (16.2)
6.42 d (16.2)
1.42 s
7.58 d (2.3)
7.54 d (2.3)
3.13 m
7.60 br.s
7.51 br.s
2.86 m
7.58 br.s
7.50 br.s
2.82 m
1.77 br.t (7.6)
1.29 s
7.72 d (2.0)
7.65 d (2.0)
2.90 m
1
2
4
5
6
7
9
1
′
′
1.80 m
1.79 br.t (8.4)
1.30 s
1.79 m
′
1.27 s
1.28 s
′
1.42 s
1.27 s
1.30 s
1.29 s
1.28 s
′
3.55 d (7.2)
5.32 t (7.2)
1.74 s
3.59 d (7.2)
5.30 t (7.2)
1.75 s
3.55 ov
5.29 br.s
1.71 s
3.52 ov
5.28 br.s
1.70 s
3.54 d (7.2)
5.30 t (7.2)
1.74 s
′
′
0′
1.74 s
1.75 s
1.71 s
1.70 s
1.76 s
Ara
Glc
Glc-I
Ara
Ara
1
2
3
4
5
″
″
″
″
″
4.74 d (7.2)
4.14 dd (7.2, 8.6)
3.84 m
4.72 d (7.6)
3.53 m
4.95 d (7.2)
4.01 br.t (8.0)
3.80 m
4.88 ov
4.24 br.t (8.0)
3.99 m
3.99 m
a 3.46 ov
b 3.88 ov
–
4.70 d (7.6)
4.16 dd (6.8, 8.0)
3.87 m
3.43 m
3.86 m
3.43 m
3.56 m
3.88 m
a 3.39 ov
b 3.88 dd (2.8, 8.4)
–
3.14 m
3.26 m
a 3.40 ov
b 3.88 ov
–
6
″
a 3.67 dd (5.6,12.0)
a 3.73 ov
b 3.88 br.d (12.0)
Glc-II
b 3.76 dd (2.0,12.0)
Glc
–
–
–
–
–
–
–
Glc
Glc
1
2
3
4
5
6
′″
′″
′″
′″
′″
′″
4.76 d (7.8)
5.00 d (8.0)
3.44 m
4.90 ov
3.39 m
3.46 m
3.52 m
3.46 m
a 3.75 ov
b 3.85 ov
–
4.80 d (6.4)
3.28 m
3.35 m
3.40 m
3.76 m
3.40 m
3.35 m
3.42 m
3.37 m
3.30 m
3.58 m
3.31 m
a 3.67 dd (5.0, 11.3)
a 3.77 ov
b 3.81 brd (8.8)
–
a 3.70 dd (5.7, 12.0)
b 3.82 dd (2.0, 12.0)
4.32 q (7.2)
1.37 t (7.2)
b 3.77 dd (2.2, 11.3)
-
-
OCH2CH3
OCH2CH3
–
–
–
–
–
–
a
Data are based on DEPT, HSQC, and HMBC experiments; 1H NMR (400 MHz, δ, J in Hz in parentheses). b Data in CD3OD; c data in D2O; ov means overlapped
−
1
1
13
"
7
75 cm ; H and C NMR data, see l Tables 2 and 3. HR‑ESI‑MS
Cell culture and cytotoxicity assay
+
at m/z 637.2824 [M + Na] (calcd. for C30H46O13Na, 637.2836).
A549 (non-small cell lung cancer, NSCLC), H460 (human lung ad-
enocarcinoma), Hela (human cervical cancer), MCF-7 (human
breast cancer), Caco2 (human colon cancer), HepG2 (human he-
patoblastoma) cell lines were obtained from ATCC. The cells were
maintained in a growth medium containing RPMI 1640 or Dul-
beccoʼs MEM with glutamate supplemented with 10% fetal bo-
vine serum (FBS) and antibiotics. All cells were cultured at 37°C
Acid hydrolysis
Compound 1 (2 mg) was heated in 1 M HCl-dioxane (1:1, 4 mL) at
0°C for 4 h. After cooling, the solution was diluted with H O
3 mL), neutralized with 1 M NaOH and then extracted with
8
(
2
CHCl3 (3 × 3 mL). The aqueous layer was concentrated under a
stream of nitrogen. The residue was then dissolved in anhydrous
pyridine (0.1 mL), followed by the addition of 0.1 M L-cysteine
methyl ester hydrochloride (0.2 mL). The resulting solution was
stirred at 60°C for 1 h, followed by the addition of trimethysilyla-
tion reagent HMDS-TMCS-pyridine (hexamethyldisilazane-tri-
methylchlorosilane-pyridine, 3:1:9). It was then stirred at 60°C
for additional 30 min. The thiazolidine derivatives were analyzed
by GC for sugar identification. D-glucose and L-arabinose were
confirmed by comparison with the retention time of the authen-
tic standards at 11.81 min and 9.04 min, respectively. The same
analysis was carried out on compounds 2–11 [5].
with 5% CO (v/v). All cell culture media supplements for the cell
2
culture were purchased from Invitrogen. Treatments with xeno-
biotics were carried out in growth medium minus serum. Cells
treated with DMSO (0.1% v/v) were used as negative controls,
while adriamycin (Sigma–Aldrich) was used as the positive con-
4
trol. Cells were seeded in a 96-well plate at 1 × 10 cells per well.
After 24 h, cells were treated with compounds 3, 4, 9, 10, and 11
at different concentrations, respectively, in a medium minus FBS
for 24 h and then compared to the untreated cells. Cell viability
was determined with an MTS Cell Proliferating Assay Kit (Prome-
ga) [6,7].
HPLC analysis
Supporting information
HPLC analyses were carried out at 40°C on a Waters Symmetry-
Shield™ RP-18 column (5 µm, 4.6 × 250 mm, detection at UV
The 1D, 2D NMR, UV, and HR‑ESI‑MS spectra of compounds 1–11
as well as the data of cell viability of compounds 3, 4, 9, 10, and 11
on different cell lines are available as Supporting Information.
254 nm) with water and methanol as the mobile phases with a
linear gradient from 5 to 95% of methanol and 40 min eluting
time. The flow rate was kept constant at 1 mL/min.
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

Original Papers 285
Table 3 13C NMR data of compounds 1–11.
C
1a, b
2a, b
3a, c
4a, d
5c
6b
7a, b
8a, b
9a, e
10a, e
11a, b
1
2
3
4
5
6
7
122.0 s
130.1 d
129.5 s
158.7 s
129.5 s
130.1 d
168.3 s
29.4 t
121.5 s
130.5 d
129.6 s
159.0 s
129.6 s
130.5 d
167.1 s
29.4 t
126.7 s
129.3 d
135.6 s
155.9 s
135.6 s
129.3 d
167.0 s
28.6 t
125.5 s
128.3 d
135.2 s
155.9 s
135.2 s
128.3 d
166.9 s
27.7 t
121.8 d
132.3 s
16.7 q
24.5 q
27.7 t
121.8 d
132.3 s
16.7 q
24.5 q
Ara
126.9 s
129.3 d
135.5 s
155.6 s
135.5 s
129.3 d
166.9 s
29.4 t
131.1 s
128.3 d
137.1 s
156.7 s
137.1 s
128.3 d
172.6 s
29.8 t
131.1 s
125.0 d
132.2 s
156.1 s
138.2 s
129.0 d
172.2 s
123.2 d
140.9 d
72.1 s
30.1 q
30.0 q
29.9 t
123.8 d
133.7 s
18.2 q
26.0 q
Ara
131.1 s
128.2 d
138.2 s
156.8 s
137.1 s
128.4 d
172.4 s
26.0 t
132.2 s
129.8 d
139.8 s
157.0 s
138.7 s
130.0 d
174.6 s
26.9 t
45.8 t
74.1 s
30.1 q
30.7 q
31.1 t
124.8 d
136.9 s
20.0 q
27.5 q
Glc-I
132.1 s
130.0 d
139.6 s
157.5 s
138.5 s
130.0 d
174.6 s
27.3 t
46.0 t
74.1 s
30.4 q
30.4 q
31.1 t
124.5 d
137.1 s
19.9 q
27.5 q
Ara
127.7 s
130.0 d
138.3 s
158.2 s
137.2 s
129.9 d
168.1 s
26.2 t
45.4 t
71.4 s
29.5 q
29.2 q
29.7 t
123.9 d
134.1 s
18.2 q
26.0 q
Ara
1
2
3
4
5
6
7
8
9
1
′
′
123.0 d
134.2 s
17.9 q
26.0 q
29.4 t
123.0 d
134.1 s
17.9 q
25.9 q
29.4 t
122.4 d
133.4 s
18.0 q
25.7 q
28.6 t
122.4 d
133.7 s
17.9 q
25.7 q
29.4 t
124.2 d
133.7 s
18.2 q
25.9 q
29.8 t
45.4 t
′
71.6 s
′
29.7 q
28.9 q
29.7 t
′
′
′
123.0 d
134.2 s
17.9 q
26.0 q
Glc
123.0 d
134.1 s
17.9 q
25.9 q
Glc
122.4 d
133.4 s
18.0 q
25.7 q
Ara
122.4 d
133.7 s
17.9 q
25.7 q
Glc
124.2 d
133.7 s
18.2 q
25.9 q
Glc
124.2 d
133.7 s
18.2 q
25.9 q
Glc
′
′
0′
1
2
3
4
5
6
″
″
″
″
″
″
103.2 d
74.7 d
77.0 d
71.5 d
75.9 d
64.5 t
95.8 d
74.0 d
78.0 d
71.3 d
76.1 d
64.5 t
104.9 d
72.2 d
73.4 d
68.5 d
66.6 t
102.8 d
78.8 d
72.0 d
67.2 d
65.2 t
–
104.0 d
74.1 d
76.3 d
69.3 d
75.5 d
61.2 t
105.9 d
75.7 d
78.0 d
71.7 d
78.2 d
62.8 t
105.8 d
81.3 d
74.1 d
69.5 d
67.2 t
–
105.9 d
75.8 d
78.0 d
71.4 d
78.2 d
62.4 t
105.0 d
81.8 d
79.1 d
71.7 d
78.6 d
63.3 t
Glc-II
105.5 d
80.6 d
74.5 d
70.0 d
67.7 t
–
105.6 d
81.1 d
73.7 d
69.7 d
66.7 t
–
–
Glc
Glc
Glc
Glc
1
2
3
4
5
6
″′
″′
″′
″′
″′
″′
–
–
–
103.3 d
73.7 d
75.9 d
69.5 d
75.8 d
60.1 t
–
–
–
–
–
–
–
–
–
–
–
–
–
104.8 d
76.0 d
77.9 d
71.4 d
78.2 d
62.6 t
–
–
–
–
–
–
–
–
–
–
–
–
104.6 d
76.4 d
78.5 d
72.2 d
78.4 d
62.3 t
–
105.2 d
76.3 d
78.3 d
72.0 d
78.4 d
63.1 t
–
104.6 d
75.7 d
78.0 d
71.6 d
78.1 d
62.6 t
–
146.2 d
–
–
–
148.0 s
–
–
–
175.9 s
–
–
–
117.0 d
–
–
–
157.8 d
–
–
–
-
-
-
-
-
OCOCH3
OCOCH3
OCH3
–
–
–
–
–
172.8 s
–
–
20.6 q
–
–
–
–
–
–
–
–
–
–
52.0 q
51.0 q
–
52.0 q
–
–
–
–
OCH2CH3
OCH2CH3
–
–
–
–
–
–
–
62.0 t
14.6 q
–
–
–
–
a
Data are based on DEPT, HSQC, and HMBC experiments; 13C NMR (100 MHz, δ). b Data in CD3OD: c data in CDCl3; d data in CDCl3+ CD3OD; e data in D2O
Results and Discussion
aglycone moiety as 4-hydroxy-3,5-bis(3-methyl-2-butenyl)-ben-
zoic acid, previously described as a nervogenic acid unit [9,10].
The H NMR spectrum showed three unsaturated protons for the
!
1
Compound 1 was obtained as an amorphous solid. Its molecular
formula was determined as C28H34O10 by HR‑ESI‑MS at m/z
ABX pattern [δ 8.17 (1H, d, J = 0.6 Hz, H-2′″), δ 6.46 (1H, d,
J = 5.6 Hz, H-5′″), and δ 7.97(1H, dd, J = 5.6, 0.6 Hz, H-6′″)]. Fur-
5
53.2064 [M + Na]+ (calcd. for C28H34O10Na, 553.2050). The IR
−1
13
spectrum indicated the presence of hydroxyl groups (3401 cm ),
thermore, the C‑NMR and DEPTspectra of 1 revealed the signals
−
1
a carbonyl group (1711 cm ), and an aromatic ring (1607 and
of five unsaturated carbons (175.9, 157.8, 148.0, 146.2, and
117.0), consistent with those in a pyromeconic acid unit [11].
Next, the proton resonances of the sugar units were observed.
The sugar residue was identified as β-D-glucose by gas chroma-
tography of the hydrolyzed product and by the coupling constant
of its anomeric proton δ 4.79 (1H, d, J = 7.2 Hz). In the HMBC spec-
trum, the long-range correlation between Glc H-1″ [δ 4.79 (d,
J = 7.2 Hz)] and C-3′″ (δ 148.0) of the pyromeconic acid unit indi-
cated that the sugar moiety was located at C-3′″.
−
1
1
"
7
69 cm ). The H NMR spectrum (l Table 1) displayed a singlet
at δ 7.64 (2H, s) integrating for two protons, supporting the pres-
ence of an aromatic ring. In addition, signals due to two prenyl
groups were present, namely, δ 5.31 (2H, m), δ 3.34 (4H, d,
J = 7.2 Hz), and two singlets at δ 1.72 (6H, s) and δ 1.76 (6H, s) [8].
Further information was obtained from 2D NMR experiments.
"
HMBC (l Fig. 2) was observed from H-2 and H-6 (δ 7.64) with
the carbon resonance at δC 168.3 (C-7), indicating that the car-
boxyl group was attached to C-1. More HMBC correlations were
observed from the same proton signal to the resonances at δC
Furthermore, long-range correlations between Glc H-6″ [δ 4.42
(dd, J = 6.4, 12.0 Hz), 4.63 (dd, J = 2.0, 12.0 Hz)] and C-7 of the
nervogenic acid unit were observed.
158.7 (C-4) and 129.5 (C-3, C-5), supporting the presence of a
benzoic acid derivative. The HMBC correlations observed from
H-1′ and H-6′ (δ 3.34) with C-2, C-3, C-4, C-5, C-6, C-2′, C-3′, C-7′,
C-8′ and H-2, and H-6 (δ 7.64) with C-1′ revealed the linkage of
prenyl units to C-3 and C-5. These data led us to establish the
Based on the above data and analysis, the structure of compound
1 was determined to be {6-O-[(4-oxo-4H-pyran-3-yloxy)-O-β-D-
glucopyranosyl]}-4-hydroxy-3,5-bis(3-methyl-2-butenyl) ben-
zoate.
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

2
86 Original Papers
Fig. 2 Key HMBC correlations of compounds 1, 2,
and 7.
The molecular formula of compound 2 was determined as
C25H34O9 by HR‑ESI‑MS at m/z 477.2132 [M – H] (calcd. for
drolyzed product and by the coupling constant of its anomeric
protons, Glc H-1″ [δ 4.66 (1H, d, J = 7.6 Hz)]. By analysis of the
HMQC, HMBC, and COSY data, compound 5 was determined to
be methyl 3,5-bis(3-methyl-2-butenyl)-4-O-(β-D-glucopyrano-
syl) benzoate [4].
The molecular formula of 6 was determined as C23H33NO7 by
HR‑ESI‑MS at m/z 436.2324 [M + H]+ (calcd. for C23H34NO7,
436.2335). The NMR spectroscopic data of 6 were comparable to
those of 5, meaning 6 was also a nervogenic acid derivative with
one β-D-glucopyranoside unit. Comparison of NMR data to
nervogenic acid indicated that the -COOH group at C-7 was re-
−
"
C25H33O , 477.2125). From the NMR spectra of 2 (l Tables 1 and
9
3
), the presence of a nervogenic acid unit as a part of the struc-
ture was evident. Furthermore, one β-D-glucopyranoside unit
and one acetyl unit were confirmed by NMR spectra. Complete
structure assignments were obtained from exhaustive analysis
1
1
of the HMQC, HMBC, and H- H COSY data. The HMBC experi-
ment showed long-range correlations between the anomeric
proton signal of Glc at δ 5.65 (1H, d, J = 8.0 Hz) with C-7, indicat-
"
ing that the Glc unit was linked as an ester linkage (l Fig. 2).
Moreover, the acetyl unit was located at C-6″ of the Glc by the
correlations between Glc H-6″ [δ 4.22 (dd, J = 5.6, 12.0 Hz), 4.37
placed by a CONH group. Therefore, the structure of 6 was deter-
mined to be 3,5-bis(3-methyl-2-butenyl)-4-O-(β-D-glucopyran-
osyl) benzamide.
2
(
(
dd, J = 2.0, 12.0 Hz)] and the carboxyl carbon of the acetyl unit
δ 172.8). Thus, structure 2 was determined to be {1-O-[6-ace-
HR‑ESI‑MS of compound 7 gave a molecular ion at m/z 606.2529
+
tyl-O-β-D-glucopyranosyl]}-4-hydroxy-3,5-bis(3-methyl-2-bu-
tenyl) benzoate.
Compound 3 was isolated as an amorphous solid. Its molecular
formula was deduced as C23H32O7 by HR‑ESI‑MS at m/z
[M + Na] (calcd. for C28H41NO12Na, 606.2526), corresponding to
the molecular formula C28H41NO12. The NMR and HR‑ESI‑MS da-
ta of 7 implied that compound 7 should be a benzamide deriva-
tive. Comparison of the NMR data of 6 with those of 7 indicated
that the modification of one isoprene chain was the most notable
difference, revealing the presence of a chain with a trans double
bond, two methyl groups, and a tertiary hydroxyl group. The
+
1
4
43.2038 [M + Na] (calcd. for C23H32O Na, 443.2046). The H
7
1
3
"
and C NMR signals (l Tables 1 and 3) were assigned by a com-
bination of DEPT, HMQC, and HMBC spectra which included sig-
nals due to one nervogenic acid unit, one α-arabinopyranosyl
unit, and one methoxy (δ 3.86, 52.0) unit. The HMBC experiment
showed the long-range correlations from the methoxy unit (δ
"
HMBC experiment (l Fig. 2) showed as the most relevant con-
nectivities on this side chain those of the two vinylic protons at
δ 6.42 (H-2′, d, J = 16.2 Hz) and 7.27 (H-1′, d, J = 16.2 Hz) with δ
132.2 (C-3) and a correlation of the signals at δ 1.42 (Me-4′, Me-
5′) with δ 72.1(C-3′) [8]. The sugar residues were identified as β-
D-glucopyranoside and α-L- arabinopyranosyl by gas chromatog-
raphy of the hydrolyzed product and by the coupling constant of
their anomeric protons, Ara H-1″ [δ 4.74 (1H, d, J = 7.2 Hz)] and
Glc H-1′″ [δ 4.76 (1H, d, J = 7.8 Hz)]. Long-range correlations in
HMBC experiment were observed between Ara H-1″ with C-4 (δ
156.1) and Glc H-1′″ with Ara C-2″ (δ 81.3). By analysis of the
HMQC, HMBC, and COSY data, the structure of compound 7 was
elucidated to be 3-[(1E)-3-hydroxy-3-methyl-1-butenyl-4-O-[β-
D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl]]-5-(3-methyl-
2-butenyl) benzamide.
3
.86, 52.0) to C-7, and the Ara H-1″ [δ 4.54 (1H, d, J = 7.6 Hz)] to
C-4 (δ 155.9). Thus, structure 3 was determined to be methyl
,5-bis(3-methyl-2-butenyl)-4-O-(α-L-arabinopyranosyl) ben-
3
zoate.
Compound 4 was obtained as a white amorphous powder. Its
molecular formula was determined as C29H42NO12 by HR‑ESI‑MS
+
at m/z 605.2574 [M + Na] (calcd. for C29H42NO12Na, 605.2574). In
the comparison of NMR data of 4 with those of 3, the main differ-
ence was the presence of one β-D-glucopyranoside unit. The sug-
ar residues were identified as α-L-arabinopyranosyl and β-D-glu-
copyranoside by gas chromatography of the hydrolyzed product
and by the coupling constant of their anomeric protons, Ara H-
1
″ [δ 4.75 (1H, d, J = 7.2 Hz)] and Glc H-1′″ [δ 4.72 (1H, d,
The molecular formula of compound 8 was determined as
+
J = 8.0 Hz)]. In the HMBC experiment, long-range correlations
were observed from Ara H-1″ with C-4, Glc H-1′″ with Ara C-2″,
and the methoxy unit (δ 3.88, 51.0) to C-7. These data supported
the structure of 4 as methyl 3,5-bis(3-methyl-2-butenyl)-4-O-[β-
D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl] benzoate.
Compound 5 was also obtained as an amorphous solid. Its molec-
ular formula was established as C H34O8 by analysis of HR‑E-
C23H35NO8 by HR‑ESI‑MS at m/z 476.2262 [M + Na] (calcd. for
1
3
"
C23H35NO Na, 476.2260). The HR‑ESI‑MS and C NMR (l Table
8
3) spectra revealed that compound 8 was also a benzamide deriv-
ative. The major differences between compounds 8 and 6 were
the absence of the double bond and the presence of the quater-
1
nary hydroxy group at one of the prenyl side chains. H‑NMR
"
spectrum of 8 (l Table 2) indicated the presence of two aromatic
2
4
SI‑MS at m/z 473.2140 [M + Na]+ (calcd. for C24H34O Na,
protons at δ 7.58 (1H, d, J = 2.3 Hz) and 7.54 (1H, d, J = 2.3 Hz),
characteristic of a 1,3,4,5-tetrasubstituted aromatic ring. More-
over, one 3-hydroxy-3-methylbutyl moiety was confirmed by
8
473.2151). Comparison of the NMR data of compounds 5 and 3
"
(
l Tables 1 and 3) indicated that 5 possessed the same aglycone
1
as 3 but differed in the sugar part. The sugar residue was identi-
fied as β-D-glucopyranoside by gas chromatography of the hy-
the presence of signals in the H NMR spectrum at δ 1.27 (6H, s),
1
3
1.80 (2H, m), 3.13 (2H, m) and signals in the C NMR spectrum at
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

Original Papers 287
δ 26.0 (C-1′), 45.4 (C-2′), 71.6 (C-3′), 29.7 (C-4′), and 28.9 (C-5′)
12].
dividually for their in vitro cytotoxicities against A549, H460, He-
La, MCF-7, Caco2, and HepG2 human cell lines by the MTS meth-
od, as described previously in the literature [6,7]. None of these
compounds showed significant inhibitory activity against the tu-
mor cells used (IC50 > 10 µM, n = 3).
[
The linkage position of the isoprene side chain to the aromatic
ring was determined by the HMBC experiment, in which correla-
tions between the signals of H-2′/C-3 and H-1′/C-2, C-3, C-4, C-2′,
C-3′ were observed. Moreover, the long-range correlation in the
HMBC experiment was observed from Glc H-1″ [δ 4.72 (d,
J = 7.6 Hz)] with C-4 (δ 156.8). Therefore, 8 was elucidated as 3-
Acknowledgements
!
[(3-hydroxy-3-methylbutyl)-4-O-(β-D-glucopyranosyl)]-5-(3-
methyl-2-butenyl) benzamide.
The molecular formula of compound 9 was determined as
C29H45NO13 (HR‑ESI‑MS). The H (l Table 2) and C NMR data
This work was financially supported by the National Natural
Science Foundation of China (21142004, 31171695) and the
Fundamental Research Funds for Central Universities
(SWJTU2010ZT09, SWJTU12CX048).
1
"
13
"
(
l Table 3) of 9 showed close structural similarity to the aglycone
moiety of compound 8, indicating that they had the same agly-
cone moiety. Complete assignment of the glycosidic protons and
carbons along with an acid hydrolysis experiment indicated the
presence of two β-D-glucopyranosyl units. Their sequencing pat-
terns were obtained from the HMBC spectrum. Long-range corre-
lations in the HMBC experiment were observed from Glc-I H-1″
Conflict of Interest
!
All the authors have no conflict of interest to declare.
[
(
δ 4.95 (d, J = 7.2 Hz)] with C-4 (δ 157.0) and Glc-II H-1′″ [δ 5.00
d, J = 8.0 Hz)] with Glc-I C-2″ (δ 81.8). Thus, structure 9 was as-
References
1 Hua YX, Liu SF, Yang ZQ. The Chinese herbal, Vol. 12. Shanghai: Shang-
hai Science and Technology Publishing House; 1999: 997
signed to be 3-(3-hydroxy-3-methylbutyl)-4-O-[β-D-glucopy-
ranosyl-(1 → 2)-β-D-glucopyranosyl]-5-(3-methyl-2-butenyl)
benzamide.
The molecular formula of 10 was determined as C28H43NO12 by
HR‑ESI‑MS at m/z 608.2668 [M
2
3
Nishikawa K, Hirata Y. Chemotaxonomical alkaloid studies. I. Structure
of nervosine. Tetrahedron Lett 1967; 27: 2591–2596
Nishikawa K, Miyamura M, Hirata Y. Chemotaxonomical alkaloid stud-
ies structures of Liparis alkaloids. Tetrahedron 1969; 25: 2723–2741
Na]+ (C28H43NO12Na,
4 Abe F, Yamauchi T. Glycosyl nervogenic acid esters of carbohydrates
from Anodendron affine (Anodendron. VI). Chem Pharm Bull 1985; 33:
+
6
(
08.2683). Comparison of the NMR data of compounds 10 and 9
2
712–2720
"
l Tables 2 and 3) indicated that 10 possessed the same aglycone
5
6
Tang L, Jiang Y, Chang HT, Zhao MB, Tu PF, Cui JR, Wang RQ. Triterpene
saponins from the leaves of Ilex kudingcha. J Nat Prod 2005; 68: 1169–
1174
Chou TC, Motzer RJ, Tong Y, Bosl GJ. Computerized quantitation of syner-
gism and antagonism of taxol, topetecan, and cisplatin against human
teratocarcinoma cell growth: a rational approach to clinical protocol
design. J Natl Cancer Inst 1994; 86: 1517–1524
moiety as 9. After hydrolysis, sugars were identified as D-glucose
and L-arabinose, in a ratio of 1:1, based on the GC analysis of
their chiral derivatives. Their sequencing was identified as the
same as in compound 4 by the HMBC experiment. On the basis
of these spectral data, the structure of 10 was established as 3-
(
3-hydroxy-3-methylbutyl)-4-O-[β-D-glucopyranosyl-(1 → 2)-α-
7 Buttke TM, McCubrey JA, Owen TC. Use of an aqueous soluble tetrazo-
lium/formazan assay to measure viability and proliferation of lympho-
kine-dependent cell lines. J Immunol Methods 1993; 157: 233–240
L-arabinopyranosyl]-5-(3-methyl-2-butenyl) benzamide.
The molecular formula of compound 11 was determined as
C30H46O13 by HR‑ESI‑MS at m/z 637.2824 [M + Na] (calcd. for
C30H46O13Na, 637.2836). It was found to have a similar structure
to compound 10 by comparison of their NMR data (l Tables 2
and 3). The main difference observed was that the -NH group at
C-7 was replaced by a carboxyethyl group [δH 1.37 (3H, t,
J = 7.2 Hz), 4.32 (2H, q, J = 7.2 Hz), and δ 14.6 and 62.0]. The posi-
tion of the carboxyethyl group was unambiguously defined by
the HMBC experiment. Structure 11 was therefore determined
to be 1-(ethoxy)-3-[(3-hydroxy-3-methylbutyl)-4-O-[β-D-gluco-
pyranosyl-(1 → 2)-α-L-arabinosyl]-5-(3-methyl-2-butenyl) ben-
zoate.
8
Flores N, Jiménez IA, Giménez A, Ruiz G, Gutiérrez D, Bourdy G, Bazzocchi
IL. Benzoic acid derivatives from Piper species and their antiparasitic
activity. J Nat Prod 2008; 71: 1538–1543
+
"
9 Šlapetová T, Šmejkal K, Innocenti G, DallʼAcqua S, Heilmann J, Babula P,
Hamzová E, Žemličk M. Glycosylated nervogenic acid derivatives from
Liparis condylobulbon (Reichb.f.) leaves. Carbohydr Res 2009; 344:
2
1
770–1774
C
1
1
0 Orjala J, Erdelmeier CAJ, Wright AD, Rali T, Sticher O. Five new preny-
lated p-hydroxybenzoic acid derivatives with antimicrobial and mol-
luscicidal activity from Piper aduncum leaves. Planta Med 1993; 59:
5
46–551
1 Hashidoko Y. Pyromeconic acid and its glucosidic derivatives from
leaves of Erigeron annuus, and the siderophile activity of pyromeconic
acid. Biosci Biotechnol Biochem 1995; 59: 886–890
In order to evaluate the biological activitiy of these newly identi-
fied compounds isolated from the whole plant of L. nervosa for
future applications, compounds 3, 4, 9, 10, and 11 were tested in-
12 Kuo WL, Huang YL, Shen CC, Shieh BJ, Chen CC. Prenylated benzoic acids
and phenanthrenes from Liparis nakaharai. J Chin Chem Soc 2007; 54:
1
359–1362
Huang S et al. New Nervogenic Acid… Planta Med 2013; 79: 281–287

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