Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production in RAW264.7 macrophages

Article abstract of DOI:10.1016/j.phytochem.2013.03.009

Six pyrrolizidine alkaloids were isolated from the whole herb of Liparis nervosa together with two previously known ones. Their structures were elucidated by extensive spectroscopic analyses and chemical reactions. The cytotoxicity of the isolates was eva

Full text of DOI:10.1016/j.phytochem.2013.03.009

Phytochemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against
LPS-induced NO production in RAW264.7 macrophages
a
a
a
a
Shuai Huang a,b, Xian-li Zhou a, , Cui-juan Wang , You-song Wang , Feng Xiao , Lian-hai Shan ,
⇑
Zhi-yun Guo a, Jie Weng b
a School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, PR China
b Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031,
Sichuan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six pyrrolizidine alkaloids were isolated from the whole herb of Liparis nervosa together with two previ-
ously known ones. Their structures were elucidated by extensive spectroscopic analyses and chemical
reactions. The cytotoxicity of the isolates was evaluated against A549, HepG2, and MCF-7 human cancer
cell lines; however, no significant growth inhibition was observed. All compounds were evaluated for the
inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, and most significantly
Received 19 May 2012
Received in revised form 10 October 2012
Accepted 4 March 2013
Available online xxxx
inhibited NO production with IC50 values in the range of 2.16–38.25
lM.
Keywords:
Liparis nervosa
Ó 2013 Elsevier Ltd. All rights reserved.
Orchidaceae
Pyrrolizidine alkaloids
Nervosine
Nervogenic acid derivatives
NO production inhibitor
1. Introduction
2. Results and discussion
Liparis nervosa Thunb.Lindl belonging to the Orchidaceae family,
is a herbaceous plant widely distributed in China. It usually grows
in broad-leaved forests on slopes under moist conditions at the
altitude of 850–1000 m. L. nervosa has been used in folk medicine
for detoxicating and hemostatic functions. Howerer its chemical
constituents and their bioactivities have not been studied in detail
yet; other than investigations leading K to identification of nervo-
sine (Nishikawa and Hirata, 1967; Nishikawa et al., 1969). Here,
the isolation and structural determination of six new pyrrolizidine
alkaloids, nervosine I–VI, along with two known compounds of the
same structural class (Fig. 1) are reported. The cytotoxicity of the
isolated compounds against several cancer cell lines and their
inhibitory activity against LPS-induced nitric oxide (NO) produc-
tion in vitro by RAW264.7 cells are discussed herein. In addition,
an attempt to provide a structure–activity relationship was also
undertaken.
2.1. Structure elucidation and identification
The crude alkaloid fraction of L. nervosa was repeatedly sub-
jected to silica gel column and Sephadex LH-20 column chroma-
tography to afford six new pyrrolizidine alkaloids (1, 2, 5, 6, 7,
and 8), along with two known pyrrolizidine alkaloids (3 and 4).
Compound 1 was obtained as an amorphous solid. Its molecular
formula was determined as C30H43NO7 by HR-ESI-MS at m/z
530.3116 [M+H]+ (calcd. for C30H44NO7, 530.3118), whereas its IR
spectrum of
1 indicated the presence of hydroxy groups
(3387 cmꢀ1) and carbonyl group (1716 cmꢀ1) and an aromatic ring
(1600 and 773 cmꢀ1) respectively, In the 1H NMR spectrum (Ta-
ble 1), a singlet at d 7.67 (2H, s) was indicators of the presence of
an aromatic ring. Also, signals [d 5.23 (2H, t, J = 7.0 Hz), d 3.49
(4H, d, J = 7.0 Hz), and two singlets at d 1.70 (6H, s) and d 1.73
(6H, s) integrating together for 12 protons] in the 1H NMR spec-
trum established the existence of two prenyl groups (Flores
et al., 2008). HMBC correlations Fig. 2 were also observed from
H-20 and H-60 (d 7.67) with the carbon resonance at d 166.3 (C-
70), suggestions that the carbonyl group was attached to C-1. More
HMBC correlations Fig. 2 were observed from the same proton sig-
nals to the resonances at d 156.2 (C-40) and d 135.8 (C-30, C-50), sup-
porting the presence of a benzoic acid derivative. The HMBC
⇑
Corresponding author. Tel.:/fax: +86 28 8760 0185.
E-mail address: xxbiochem@163.com (X.-l. Zhou).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.03.009
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

2
S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
Fig. 1. Structures of compounds 1–8 isolated from Liparis nervosa.
Table 1
1H and 13C NMR Spectroscopic Dataa for Compounds 1–4.
No.
1b
2b
3c
4c
dH
dC
dH
dC
dH
dC
dH
dC
1
2
2.75 m
40.4
27.1
2.21 m
45.0
30.5
2.75 m
41.8
27.4
2.25 m
46.3
31.2
a
1.93 m
a
2.05 m
a
1.96 m
a
2.11 m
b 1.65 m
b 1.70 m
b 1.70 m
b 1.81 m
3
5
6
7
a
3.21 m
b 2.75 m
3.38 m
b 2.55 m
1.93 m
b 1.83 m
1.83 m
53.8
55.7
26.4
26.4
a
3.30 m
b 2.60 m
3.10 m
b 2.60 m
1.91 m
b 1.70 m
2.02 m
54.4
54.7
25.9
31.7
a
3.17 m
b 2.92 m
3.39 m
b 2.73 m
2.03 m
b 1.86 m
1.86 m
54.8
56.8
27.3
27.2
a
3.24 m
b 2.72 m
2.99 m
b 2.72 m
1.95 m
b 1.72 m
2.05 m
55.3
55.6
26.6
32.8
a
a
a
a
a
a
a
a
a
a
a
a
b 1.57 m
b 1.63 m
b 1.64 m
b 1.67 m
8
9
3.77 m
a 4.27 dd (8.4, 11.2)
66.5
64.6
3.42 m
a 4.17 dd (8.3, 10.9)
68.7
66.9
3.75 m
a 4.30 dd (8.8, 11.2)
68.4
65.4
3.44 m
a 4.19 dd (8.2, 10.9)
69.9
67.9
b 4.39 dd (6.8, 11.2)
—
7.67 s
—
—
—
3.49 d (7.0)
5.23 t (7.0)
—
1.70 s
1.73 s
b 4.38 dd (5.9, 10.9)
—
7.68 s
—
—
—
3.48 d (7.0)
5.24 t (7.0)
—
1.70 s
1.73 s
b 4.44 dd (6.4, 11.2)
—
7.66 s
—
—
b 4.41 dd (5.6, 10.9)
—
7.67 s
—
—
—
10
126.4
129.4
135.8
156.2
166.3
28.7
122.5
133.3
18.0
126.3
129.3
135.7
156.1
166.2
28.7
122.4
133.3
18.0
127.2
130.0
137.2
158.1
167.6
29.5
123.9
134.1
18.2
127.3
130.0
137.3
158.0
167.9
29.6
123.9
134.1
18.1
20/60
30/50
40
70
—
100/1000
200/2000
300/3000
400/4000
500/5000
3.52 d (7.2)
5.29 t (7.2)
—
1.74 s
1.76 s
Glc
3.54 d (7.1)
5.30 t (7.1)
1.74 s
1.77 s
Glc
25.8
25.8
26.0
26.0
Ara
Ara
10000
20000
30000
40000
50000
4.57 d (7.6)
3.94 m
3.66 m
105.1
72.2
73.4
68.3
66.6
4.56 d (7.6)
3.94 m
3.66 m
104.8
72.4
73.3
68.2
66.5
4.72 d (7.6)
3.55 m
3.42 m
3.40 m
3.15 m
105.9
75.7
77.8
71.6
78.2
4.70 d (7.7)
3.53 m
3.43 m
3.38 m
3.15 m
105.9
75.7
77.9
71.7
78.2
3.91 m
3.92 m
a 3.38 br.d (12.0)
b 3.97 dd (2.6, 12.0)
—
a 3.39 br.d (12.0)
b 3.97 br.d (12.0)
—
60000
—
—
a 3.65 dd (5.6, 12.0)
b 3.77 dd (2.0, 12.0)
62.7
a 3.66 dd (5.6, 12.0)
b 3.77 dd (2.0, 12.0)
62.8
a
b
c
Data are based on DEPT, HSQC, and HMBC experiments. 1H NMR (400 MHz, d, J in Hz in parentheses), 13C NMR (100 MHz, d).
Data in CDCl3.
Data in CD3OD.
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
3
Fig. 2. Key HMBC correlations for 1, 2 and 5–8.
correlations observed from H-100/1000 (d 3.49) with C-20, C-30, C-40,
C-50, C-60, C-200/2000, C-300/3000 and H-20, H-60 (d 7.67) with C-100/1000
demonstrated linkage of two prenyl units to C-30 and C-50, these
data let us to establish the moiety as 4-hydroxy-3, 5-bis-
showing a correlation between Ara H-10000 and C-40. Thus, structure
1 was confirmed as 4-O-( -arabinopyranosyl) nervogenic acid
a
-L
ester of lindelofidine, named nervosine I.
Compound 2, an amorphous solid, had a HR-ESI-MS [M+H]+ ion
at m/z 530.3115, suggesting the same molecular formula of
(3-methyl-2-butenyl)-benzoic acid, previously described as
nervogenic acid unit (Slapetova et al., 2009).
a
C
30H44NO7 (calcd. 530.3118) as for compound 1. Its NMR spectro-
scopic data were comparable to those of 1, which means that it
also possessed one nervogenic acid, one -arabinose and one 1-
A further unit was established by analysis of the 13C NMR and
DEPT spectra these displayed eight carbon signals comprising
one oxymethylene carbon (d 64.6, C-9), one nitrogen-bearing
methine carbon (d 66.5, C-8), two nitrogen-bearing methylene car-
bons (d 55.7 and 53.8, C-3 and -5), three methylene carbons (d 27.1,
26.4 and 26.4, C-2, -6 and -7) and one methine (d 40.4, C-1) respec-
tively. Further analyses of the COSY, HMQC, and HMBC spectra sug-
gested that 1 consisted of a 1-hydroxymethyl pyrrolizidine moiety
(Table 1). The 1-hydroxymethyl pyrrolizidine moiety was esti-
mated as lindelofidine by comparison of the NMR spectroscopic
data with the literature (Koulman et al., 2008). This being con-
firmed further by NOE experiments. The NOE cross-peaks observed
between H-1 and H-8, indicated a cis-configuration for these pro-
tons. Full structure elucidation was achieved by alkaline hydrolysis
of 1, which gave the necine base, whose molecular formula was
determined to be C8H15NO (calcd. for [M+H]+, 142.1232) by HR-
a
hydroxymethylpyrrolizidine unit. The main difference between
those two compounds was in the absolute configuration of 1-
hydroxymethylpyrrolizidine unit. In the 1H NMR spectrum of 2,
the signal of H-1 was shifted upfield about d 0.54. In the 13C
D
NMR spectrum, the signals of C-1, C-2, C-7, C-8 and C-9 were
shifted downfield about d 4.6, 3.4, 5.3, 2.2 and 2.2 respectively.
D
The alkaloid base was estimated as laburnine by comparison of
the NMR spectroscopic data of the pyrrolizidine part of 2 with lit-
erature values (Mohanraj et al.,1982; Ravi and Lakshmanan, 2000)
and the positive NOEs between the protons at H-9/H-8, H-1/H-7
a,
indicating the -orientation of H-1, the b-orientation of H-8. Con-
a
firmatory evidence was provided by alkaline hydrolysis. The necine
base was comfirmed by HR-ESI-MS which gave the molecular for-
mula of C8H15NO (calcd. for [M+H]+, C8H16NO, 142.1232), and the
ESI-MS. Moreover its and the optical rotation ½a D20
ꢁ
+ 74.5 (c 0.600,
optical rotation ½a D20
ꢁ
+ 15.3 (c 0.025, EtOH) was again in good
EtOH) was in good agreement with that of lindelofidine,
agreement with that of laburnine, ½a D20
ꢁ
+ 14.6 (c 3.2, EtOH) (Gali-
½
a 2D0
ꢁ
+ 75 (c 0.103, EtOH) (Nishikawa and Hirata, 1967). In addition,
novsky et al., 1949). Complete structure assignments were ob-
tained from exhaustive analysis of the HMQC, HMBC and COSY
data. The correlations between Ara H-10000 to C-40 and H-9 to C-70
can be observed in the HMBC experiment Fig. 2. Therefore, struc-
HMBC correlations from H-9 to the carbonyl carbon (C-70) and to
C-1, C-2, and C-8 indicated the position of the ester attachment
at C-9.
Further signals in the 1H and 13C NMR spectra of 1 could be as-
ture 2 was elucidated to be 4-O-(a-L-arabinopyranosyl) nervogenic
signed to a sugar unit, which was identified as
a-
L-arabinose by gas
acid ester of laburnine, named nervosine II.
chromatography of the hydrolyzed product, and its anomeric pro-
ton resonance at d 4.57 (H-10000, 1H, d, J = 7.6 Hz) and carbon signals
at d 105.1 (d), 72.2 (d), 73.4 (d), 68.3 (d) and 66.6 (t). The connec-
tivity of the sugar unit was established from the HMBC spectrum
Compound 3, isolated as an amorphous solid, and its molecular
formula, C31H45NO8 was established from a protonated molecular
ion [M+H]+ at m/z 560.3229 (calcd. for C31H46NO8, 560.3223).
It was found to have a similar structure to compound 1 by
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

4
S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
comparison of their NMR data spectroscopic (Table 1). The main
difference observed was in the -arabinopyranose unit being
substituted by a b- -glucopyranoside unit. The mecine moiety
C-40 (d 158.1) and Glc H-100000 (d 4.54, J = 7.8 Hz) with Glc C-40000(d
80.9) (Table 2). Complete structure assignments were obtained
from exhaustive analysis of the HMQC, HMBC and COSY data. Thus,
a
-L
D
was identified as lindelofidine by TLC and HPLC analysis of the
reaction mixture of alkaline hydrolysis of 3, Rf and its retention
time was the same as lindelofidine. Thus, structure 3 was deter-
mined as paludosine, a nervogenic acid ester of lindelofidine. Spec-
troscopic NMR data of compound 3 (Table 1), is provided herein, as
thus was not given in the literature (Lindstrom and Luning, 1971).
Compound 4 was obtained as an amorphous solid. Its molecular
formula was deduced as C31H45NO8 by HR-ESI-MS at m/z 560.3222
[M+H]+ (calcd. for C31H46NO8, 560.3223). By TLC and HPLC analysis
of the reaction mixture of alkaline hydrolysis of 4, the necine moi-
ety was again determined to be laburnine. Comparison of the NMR
(Table 1) spectic of compounds 4 and 2 established that 4 was a
nervogenic acid ester of laburnine, the main difference begins the
structure 5 was defined as the 4-O-[b-
b- -glucopyranosyl] nervogenic acid ester of lindelofidine, which
was given the trival name nervosine III.
D-glucopyranosyl-(1 ? 4)-
D
Compound 6 was shown to possess a molecular formula of C36-
H53NO12 (HR-ESI-MS, [M+H]+, m/z 692.3639). The 1-hydroxymeth-
ylpyrrolizidine unit was identified as lindelofidine by TLC and HPLC
analysis of the reaction mixture of its alkaline hydrolysis. Its 1H
and 13C NMR spectroscopic data (Table 2) were similar to those
of compound 1. The difference was confined to the presence of
the signals assigned to the glucopyranoside unit. Long-range corre-
lations in HMBC experiment Fig. 2 were observed from Ara H-10000 (d
4.74, J = 8.0 Hz) with C-40 (d 158.2) and Glc H-100000 (d 4.77,
J = 7.8 Hz) with Ara C-20000 (d 80.7). By analysis of the HMQC, HMBC
a-
L
-arabinopyranose unit was replaced by a b-
D
-glucopyranoside
and COSY data, compound 6 was elucidated to be 4-O-[b-
D-gluco-
unit. Thus, structure 4 was determined as auriculine (Lindstrom
and Luning, 1971), and again the NMR spectroscopic data is pro-
vided, as it was not reported previously in the literature.
pyranosyl-(1?2)- -arabinopyranosyl] nervogenic acid ester of
a-L
lindelofidine, named nervosine IV, which is the diastereoisomer
of grandifoline, a compound previously isolated from Malaxis gran-
difolia (Lindstrom et al., 1971).
The molecular formula of compound 5 was determined as
C
37H55NO13 by HR-ESI-MS at m/z 722.3765 [M+H]+ (calcd. for
The molecular formula of compound
7 was established
C37H56NO13, 722.3752). The 1-hydroxymethylpyrrolizidine unit
was identified as lindelofidine by TLC and HPLC analysis of the
reaction mixture of alkaline hydrolysis of 5. The difference with
compound 3 was confined to the presence of the signals ascrib-
abled to the cellobiose unit. Long-range correlations in the HMBC
experiment were observed from Glc H-10000(d 4.74, J = 7.6 Hz) with
C
30H45NO8 from its HR-ESI-MS (m/z 548.3221 for [M+H]+). Alkaline
hydrolysis of 7, TLC and HPLC analysis of the reaction mixture, 1-
hydroxymethylpyrrolizidine unit was determined to be lindelofi-
dine. The 1H NMR spectrum (Table 3) exhibited signals for two
aromatic protons at d 7.67 (2H, s), characteristic of a 1,3,4,5-tetra-
substituted aromatic ring. The structure of compound 7 was
Table 2
1H and 13C NMR Spectroscopic Dataa for Compounds 5 and 6.
No.
5b
No.
6c
dH
dC
No.
dH
dC
dH
dC
No.
dH
dC
1
2
2.83 m
41.4
27.2
Glc
4.54 d (7.8)
1
2
2.74 m
42.0
27.8
Glc
4.77 d (7.8)
a
2.05 m
100000
200000
300000
400000
500000
600000
104.6
74.9
77.9
71.4
78.2
62.5
a
1.88 m
100000
200000
300000
400000
500000
600000
105.4
75.8
77.9
71.5
78.0
62.9
b 1.83 m
b 1.70 m
3
5
6
7
8
9
a
3.37 m
b 3.16 m
3.62 m
b 2.87 m
2.10 m
b 1.90 m
2.03 m
54.9
57.0
27.1
27.0
69.6
64.7
3.26 m
3.35 m
3.31 m
3.35 m
3
5
6
7
8
9
a
3.30 m
b 2.74 m
3.22 m
b 2.58 m
1.96 m
b 1.81 m
1.82 m
54.9
56.8
27.6
27.6
67.5
66.1
3.37 m
3.35 m
3.42 m
3.44 m
a
a
a
a
a
a
b 1.84 m
4.03 m
b 1.57 m
3.56 m
b 3.68 dd (5.6, 12.0)
a 3.90 dd (2.0, 12.0)
a 3.58 overlapped
b 3.71 dd (4.8, 12.0)
a 4.35 dd (8.4, 11.2)
a 4.27 dd (8.4, 11.0)
b 4.49 dd (6.5, 11.2)
—
7.67 s
—
—
b 4.45 dd (6.8, 11.0)
—
7.68 s
—
—
—
3.56 d (7.2)
5.31 t (7.2)
—
1.74 s
1.76 s
10
127.1
130.1
137.3
158.1
167.5
29.5
123.9
134.1
18.1
10
127.4
130.1
137.2
158.2
167.9
30.0
123.8
134.3
18.6
20/60
30/50
40
20/60
30/50
40
70
—
70
100/1000
200/2000
300/3000
400/4000
500/5000
3.54 d (7.0)
5.29 t (7.0)
—
1.74 s
1.76 s
Glc
100/1000
200/2000
300/3000
400/4000
500/5000
25.6
26.4
Ara
10000
20000
30000
40000
50000
4.74 d (7.6)
3.58 m
3.61 m
3.68 m
3.29 m
105.8
76.3
75.4
80.9
76.7
10000
20000
30000
40000
50000
4.74 d (8.0)
4.18 dd (6.8, 8.0)
3.82 m
3.91 m
a 3.38 overlapped
b 3.85 dd (2.8, 8.8)
104.9
80.7
73.9
69.1
67.0
60000
a 3.77 dd (2.6, 12.0)
b 3.82 dd (3.7, 12.0)
62.0
a
b
c
Data are based on DEPT, HSQC, and HMBC experiments. 1H NMR (400 MHz, d, J in Hz in parentheses), 13C NMR (100 MHz, d).
Data in CD3OD.
Data in CDCl3 + CD3OD.
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
5
Table 3
1H and 13C NMR Spectroscopic Dataa for Compounds 7 and 8.
No.
7b
No.
8b
dH
dC
No.
dH
dC
dH
dC
1
2
2.73 m
40.7
27.4
Ara
4.64 d (7.6)
1
2
2.78 m
40.5
27.3
a
1.90 m
10000
20000
30000
40000
50000
60000
104.7
72.4
73.5
68.5
66.7
104.7
a
1.88 m
b 1.61 m
b 1.57 m
3
5
6
7
a
3.11 m
b 2.68 m
3.24 m
b 2.52 m
1.93 m
b 1.79 m
1.82 m
54.2
56.0
26.7
26.6
4.07 br.t (9.2)
3.68 m
3
5
6
7
a
3.22 m
b 2.80 m
3.42 m
b 2.56 m
1.97 m
b 1.86 m
1.84 m
54.0
56.0
26.6
26.5
a
a
a
3.92 m
a
a
a 3.38 br.d (12.0)
b 3.93 br.d (12.0)
4.64 d (7.6)
a
b 1.48 m
3.67 m
a 4.27 dd (8.1, 11.0)
b 1.51 m
3.83 m
a 4.27 dd (8.0, 11.0)
8
9
66.4
65.4
8
9
66.8
64.6
b 4.35 dd (7.2, 11.0)
—
7.67 s
—
—
—
7.67 s
—
b 4.36 dd (7.1, 11.0)
—
7.65 (d, 2.0)
—
—
—
7.50 (d, 2.0)
—
3.27 (d, 7.3)
10
20
30
40
50
60
70
100
126.7
129.6
136.9
155.9
136.1
129.6
166.4
24.6
10
20
30
40
50
60
70
100
121.7
131.2
129.5
155.2
120.6
126.1
166.6
28.3
3.09 overlapped
2.69 overlapped
1.82 overlapped1.70 overlapped
—
1.24 s
200
44.3
71.3
28.3
30.5
28.8
122.7
133.4
18.1
25.9
200
5.22 (t, 7.3)
—
1.73 s
122.1
132.8
18.1
300
300
400
500
1000
2000
3000
4000
5000
400
500
1000
2000
3000
4000
5000
1.24 s
1.73 s
26.0
3.53 br.d (7.0)
5.21 t (7.0)
—
1.69 s
1.72 s
6.35 (d, 10.0)
5.65 (d, 10.0)
—
122.2
131.0
77.1
1.44 s
28.4
1.44 s
28.4
a
Data are based on DEPT, HSQC, and HMBC experiments. 1H NMR (400 MHz, d, J in Hz in parentheses), 13C NMR (100 MHz, d).
Data in CDCl3.
b
elucidated by physical methods, showing it to be related to com-
pound 1, with the absence of the double bond at one of the prenyl
side-chains and the presence of the quaternary-hydroxy group
on the side-chain being the major difference. Moreover, the 3-
hydroxy-3-methylbutyl moiety was confirmed by the presence of
which indicated
8
to have
a
2,2-dimethyl-6-carbonyl-8-
prenylchromene unit. (Moreira et al., 1998). The 1-hydroxymethyl-
pyrrolizidine unit was confirmed by TLC and HPLC analysis of the
reaction mixture of alkaline hydrolysis of 8. Thus, compound 8
was elucidated as 2,2-dimethyl-6-carbonyl-8-prenylchromene
ester of lindelofidine, and named nervosine VI.
13
signals in the C NMR spectrum at d 28.3 (C-400), d 30.5 (C-500), d
24.6 (C-100), d 44.3 (C-200), and d 71.3 (C-300) (Kuo et al., 2007). In
the HMBC experiment Fig. 2, correlations between the resonance
of H-200/C-30 and H-100/ C-20, C-30, C-40, C-200, C-300 were observed,
indicating the linkage of the 3-hydroxy-3-methylbutyl moiety at
C-30 of the aromatic ring. Finally, complete assignment of the NMR
spectroscopic data was made by analysis of 2D NMR experiments.
HMBC correlations were observed from Ara H-10000 (d 4.64 , J =
7.6 Hz) with C-40 (d 155.9) which established the structure of 7 as
2.2. Biological evaluation
Compounds 1–8 were evaluated for their cytotoxicity against
human cancer cell lines including MCF-7, A549 and HepG2 by
MTT method (Tao et al., 2007). However, all compounds had no
3-[(3-hydroxy-3-methylbutyl)-4-O-(a-L-arabinopyranosyl)]-5-(3-methyl-
Table 4
Effects of compounds 1–8a on nitric oxide (NO) production in LPS-stimulated RAW
264.7 cells and cytotoxicity to RAW 264.7 Cell viability (n = 3).
2-butenyl) benzoate ester of lindelofidine, named nervosine V.
Compound 8 was assigned the molecular formula of C25H33NO3
as established from HR-ESI-MS at m/z 396.2545 [M+H]+ (calcd. for
Compounds IC50 of inhibitory effects
M)
IC50 of cytotoxicity activity
(lM)
C
25H34NO3, 396.2539). In the 1H NMR spectrum (Table 3), com-
(l
pound 8 showed signals of two aromatic protons [d 7.65 (1H, d,
J = 2.0 Hz), d 7.50 (1H, d, J = 2.0 Hz)] , two cis-coupled olefin protons
[d 6.35 (1H, d, J = 10.0 Hz), d 5.65 (1H, d, J = 10.0 Hz)], one prenyl
group [d 5.22 (1H, t, J = 7.3 Hz), d 3.27 (2H, d, J = 7.3 Hz), d 1.73
(6H, s)] and two quaternary methyl groups d 1.44 (6H, s), respec-
tively. Analysis of the 13C NMR spectrum of 8 (Table 3) indicated
resonances due to one carbonyl carbon d 166.6, one oxygenated
aromatic carbon (d 155.2), five carbons for a prenyl group (d
132.8, 122.1, 28.3, 26.0, 18.1), two olefinic carbons (d 131.0,
122.2), one oxygen-bearing carbon (d 77.1), respectively. The loca-
tion of these groups was decided on the basis of the 2D NMR data,
1
2
3
4
5
6
7
8
38.25 3.71
38.14 2.54
9.94 0.32
10.87 1.21
7.92 1.83
7.74 0.67
2.16 0.57
29.33 2.39
2.91 0.73
>100
>100
>100
>100
>100
>100
>100
37.0
1400Wb
>100
a
The purity of the compounds used in the assay was more than 97% by HPLC
analysis.
b
1400W: N-(3-(Aminomethyl)benzyl)acetamidine, positive control.
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

6
S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
obvious inhibitory activity against the tumor cells used (IC50 > 10 -
Life Science and Engineering, Southwest Jiaotong University, Sich-
uan Province, China, where a voucher specimen is deposited (No.
Z36150701).
lM, n = 3).
In this study, all the alkaloids were tested on their cytotoxic
activities on RAW264.7 macrophages. Most were not toxic, and
these were then tested for inhibitory activities against LPS-induced
NO production in this cell line under the concentration range from
4.3. Extraction and Isolation
2.5 to 40
suggested that all of these compounds significantly inhibited NO pro-
duction. Interestingly, the occurrence of the b- -glucopyranoside unit
in compounds (3, 4, 5, 6) clearly enhances the inhibitory effects
lM. The IC50 values in the range of 2.16–38.25 lM (Table 4)
L. nervosa whole plant tissue (5.0 kg) was extracted with EtOH–
H2O (20 L ꢂ 3, 95:5 V/V) times at room temperature with each
soaking process lasting a week. After removal of the solvent by
evaporation, the EtOH extract (300 g) was recovered. Then its res-
idue was suspended in H2O (1 L) at 50 °C and adjusted to pH 2.8
with HCl, and extracted with CHCl3 (500 mL ꢂ 3), EtOAc
(500 mL ꢂ 3). The pH of aqueous layer was adjusted to 9.4 with
aqueous ammonia solution and extracted with EtOAc (500 mL ꢂ
3). Removal of the solvent by evaporation in vacuo gave the crude
alkaloid fraction (10 g).
D
on NO production compared to compounds (1, 2) in which
a-L-
arabinopyranose unit appear at C-40. Also, compound 7 showed
stronger inhibitory effect than the other compounds, this unfore-
seen result being because of the quaternary-hydroxy group in
place of the double bond on the side-chain. In conclusion, the
b-D-glucopyranoside unit and the quaternary-hydroxy group
on the side-chain in the nervogenic acid unit appear to be the
important factors for potential anti-inflammatory activities.
The latter was subjected to CC on silica gel, eluted with CH2Cl2:
CH3OH (1:0 ? 0:1), to give six fractions: A (200 mg), B (344 mg), C
(243 mg), D (229 mg), E (1.1 g), and F (300 mg).
3. Conclusions
Fraction B (344 mg) was submitted to CC on silica gel eluting
with CH2Cl2:MeOH:NH4OH (10:1:0.1) to yield compounds
(8 mg) and 8 (7 mg) .
Fraction C (243 mg) was subjected to CC on silica gel, eluted
with CH2Cl2:MeOH:NH4OH (10:1:0.1) to yield compounds
7
Six new pyrrolizidine alkaloids I-VI (1, 2, 5, 6, 7, and 8) and two
known pyrrolizidine alkaloids (3 and 4) were isolated from the
EtOH extract of the whole plant of L. nervosa. Their structures were
elucidated by extensive spectroscopic examinations. In a cytotox-
ity assay, all tested compounds displayed no cytotoxicity against
three cancer cell lines (A549, MCF7 and HepG2). The NO produc-
tion and cell growth inhibitory activities were assayed using
RAW 264.7 macrophage cells in vitro, where the obtained IC50
values demonstrated significant inhibitory activities for all the
pyrrolizidine alkaloids for NO production furthermore a struc-
ture–activity relationship analysis was proposed. Therefore, the
inhibitory activity of these pyrrolizidine alkaloids is not due to
their cytotoxicity but to their ability for NO suppression. These
findings many provide significant insight for the future design of
anti-inflammatory agents.
1
(44 mg) and 2 (25 mg) .
Fraction E (1.1 g) was subjected to CC on silica gel, eluted with
EtOAc:MeOH:NH4OH (12:1:0.5 ? 1:1:0.5) to get four fractions (E1-
E4). Fraction E1 was subjected to Sephadex LH-20 CC (MeOH) to
yield compound 5 (7 mg); Fraction E2 was purified using silica
gel CC eluted with EtOAc:MeOH:NH4OH (7:1:0.5) to obtain com-
pounds 3 (42 mg), 4 (11 mg) and 6 (124 mg).
4.3.1. Nervosine I (1)
Amorphous solid; ½a D20
ꢁ
= + 18.3 (c 0.350 MeOH); UV (MeOH)
kmax (log e), 242 (3.97) nm; IR (KBr) mmax 3387, 2970, 2967, 2918,
2858, 1716, 1600, 1453, 1384, 1317, 1284, 1219, 1182, 1075,
1014, 950, 774, 651 cmꢀ1; for 1H and 13C NMR spectroscopic data
see Table 1. HR-ESI-MS at m/z 530.3116 [M+H]+ (calcd. for
4. Experimental
C30H44NO7, 530.3118).
4.1. General experimental procedure
Optical rotations were measured on a Perkin-Elmer 341 polar-
imeter. NMR spectra, including 1H–1H COSY, HMQC, HMBC, and
NOESY experiments, were recorded on Varian Unity INOVA 400/
54 NMR or a Bruker AV 400 spectrometers with TMS as an internal
standard, d in ppm, J in Hz. IR spectra were obtained with a
ThermoFisher Nicolet 6700 spectrometer, KBr pellets in cmꢀ1. UV
spectra were determined with a Shimadzu UV-2450 spectropho-
tometer. HR-ESI-MS were measured using a Q-TOF micro mass
spectrometer (Waters, USA). Silica gel (Qingdao Haiyang Chemical
Co., Ltd., China, 200–300 mesh), and Sephadex LH-20 (Pharmacia
Co.) were used for column chromatography (CC). HPLC was carried
4.3.2. Nervosine II (2)
Amorphous solid; ½a D20
ꢁ
= 0 (c 0.250 MeOH); UV (MeOH) kmax
(log
e), 241 (3.59), 216 (3.10) nm; IR (KBr) mmax 3415, 2964,
2922, 2854, 1719, 1631, 1602, 1436, 1319, 1287, 1220, 1181,
1083, 1027, 951, 774, 651 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Table 1. HR-ESI-MS at m/z 530.3115 [M+H]+ (calcd. for
C30H44NO7, 530.3118).
4.3.3. Paludosine (3)
Amorphous solid; ½a D20
ꢁ
= + 9.1 (c 0.550 MeOH); UV (MeOH) kmax
(loge), 244 (3.91) nm; IR (KBr) mmax 3399, 2963, 2923, 2729, 1716,
out on
a lM,
Waters SymmetryShieldTM RP-18 column (5
1601, 1455, 1384, 1316, 1283, 1220, 1182, 1099, 1070, 1014, 901,
772, 634 cmꢀ1; for 1H and 13C NMR spectroscopic data, see Table 1.
HR-ESI-MS at m/z 560.3229 [M+H]+ (calcd. for C31H46NO8,
560.3223).
4.6 ꢂ 250 mm) with a Waters 600 controller and Waters 2487
detector. TLC plates were precoated with silica gel GF254 (Qingdao
Haiyang Chemical Co., Ltd., China) and visualized under a UV
lamp at 254 nm or by spraying the Dragendorff’s reagent or by
iodine. GC analyses were performed using a Hewlett Packard
GC6890 instrument on a L-CP-Chirasil-Val column (0.32 mm ꢂ
25 m).
4.3.4. Auriculine (4)
Amorphous solid; ½a D20
ꢁ
= -20.5 (c 0.020 MeOH); UV (MeOH) kmax
(log e), 244 (3.97) nm; IR (KBr) mmax 3499, 2964, 2918, 2728, 1716,
4.2. Plant material
1601, 1453, 1567, 1454, 1383, 1322, 1284, 1221, 1184, 1069, 1016,
901, 772, 635, 567 cmꢀ1; for 1H and 13C NMR spectroscopic data,
see Table 1. HR-ESI-MS at m/z 560.3222 [M+H]+ (calcd. for
L. nervosa whole plant were collected from Chongqing in China
in July 2007. With it identified by Prof. Liangke Song in School of
C31H46NO8, 560.3223).
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
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S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
7
4.3.5. Nervosine III (5)
the solution was diluted with H2O (3 mL), neutralized with 2 N
NH4OH, and then extracted with CHCl3 (3 ꢂ 3 mL). The H2O layer
was checked for identification of sugar moieties. Sugars were ana-
lyzed by TLC by comparison with authentic samples using CH3
OH:EtOAc:HCOOH = 8:2:0.1 as developing solvent and aniline
Amorphous solid; ½a D20
ꢁ
= + 4.3 (c 0.035 MeOH); UV (MeOH) kmax
(log e), 239 (4.04) nm; IR (KBr) mmax 3399, 2967, 2923, 2730, 1716,
1600, 1456, 1383, 1316, 1285, 1229, 1185, 1073, 1044, 901, 771,
640, 612, 570 cmꢀ1; for 1H and 13C NMR spectroscopic data, see Ta-
ble 2. HR-ESI-MS at m/z 722.3765 [M+H]+ (calcd. for C37H56NO13
,
phthalate for detection, 1–7 gave D-glucose (Rf 0.26) and L-arabi-
722.3752).
nose (Rf 0.33), respectively. The H2O layer was evaporated under
a stream of N2. The residue was dissolved in 1-(trimethylsilyl)
imidazole (0.1 ml) and pyridine (0.2 ml), and the solution was stir-
red at 60 °C for 20 min. After drying, the residue was partitioned
between H2O and CHCl3. The CHCl3 layer was analyzed by GC using
a L-CP-Chirasil-Val column (0.32 mm ꢂ 25 m). Temperatures of
both the injector and detector were 200 °C. A temperature gradient
system was used for the oven, starting at 60 °C for 2 min
and increasing up to 260 °C at a rate of 15 °C/min. Peaks of
the hydrolysate were detected by comparison with retention
4.3.6. Nervosine IV (6)
Amorphous solid; ½a D20
ꢁ
= + 12.0 (c 0.035 MeOH); UV (MeOH)
kmax (log e), 245 (4.12) nm; IR (KBr) mmax 3397, 2965, 2915, 1716,
1601, 1454, 1384, 1316, 1284, 1219, 1220, 1183, 1079, 1032,
1016, 956, 773, 654, 597 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Table 2. HR-ESI-MS at m/z 692.3639 [M+H]+ (calcd. for
C36H54NO12, 692.3646).
4.3.7. Nervosine V (7)
times of authentic samples of D-glucose (11.08 min), L-arabinose
(9.04 min) (Sigma Aldrich), after treatment with 1-(trimethylsilyl)
imidazole in pyridine. Absolute configurations of the sugars were
Amorphous solid; ½a D20
ꢁ
= + 19.2 (c 0.025 MeOH); UV (MeOH)
kmax (loge), 245 (3.15), 220 (3.09) nm; IR (KBr) mmax 3396, 2962,
2923, 2854, 1716, 1601, 1457, 1382, 1314, 1277, 1217, 1184,
1076, 1015, 951, 773, 650 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Table 3. HR-ESI-MS at m/z 548.3221 [M+H]+ (calcd. for
C30H46NO8, 548.3223).
identified as
D-glucose, L-arabinose.
4.6. Biological evaluation
4.6.1. Cell lines and cell culture and Cytotoxicity assay
MCF-7 (human breast adenocarcinoma cell line), A549 (human
lung adenocarcinoma epithelial cell line, NSCLC), and HepG2 (hu-
man hepatocellular liver carcinoma cell line) were obtained from
ATCC. Proliferation inhibition of compounds 1– 8 against MCF-7,
A549 and HepG2 cells was measured by the MTT method (Tao
et al., 2007).
Cells were plated in 96-well plates 24 h before treatment and
continuously exposed to different concentrations of compounds
for 72 h. DMSO (0.1% v/v) were used as negative controls.
4.3.8. Nervosine VI (8)
Amorphous solid; ½a D20
ꢁ
= + 17.7 (c 0.030 MeOH); UV (MeOH)
kmax (log e), 245 (3.27), 213 (2.7) nm; IR (KBr) mmax 3409, 2969,
2925, 1713, 1640, 1602, 1462, 1376, 1310, 1286, 1194, 1124,
1004, 1049, 989, 954, 930, 769 cmꢀ1; for 1H and 13C NMR spectro-
scopic data, see Table 3. HR-ESI-MS at m/z 396.2545 [M+H]+ (calcd.
for C25H34NO3, 396.2539).
4.3.9. Lindelofidine
½
a 2D0
= + 74.5° (c 0.600, EtOH); UV (MeOH) kmax (log e), 213
ꢁ
(2.17); HR-ESI-MS at m/z 142.1233 [M+H]+ (calcd. for C8H16NO,
4.6.2. Assay for inhibition ability against LPS-induced NO production
and cytotoxicity testing
142.1232).
Those two experiments were performed as previously described
(Qin et al., 2009; Qin et al., 2010; Denizot and Lang, 1986). Briefly,
the inhibitory activity of the isolated compounds toward NO pro-
duction by RAW 264.7 cells was determined using Griess reagent.
RAW264.7 cells (ATCC) were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% heat-inactivated
FCS in a humidified 5% CO2/95% air atmosphere at 37 °C. RAW264.7
cells were seeded in 96-well plates at 5 ꢂ 104 cells/well for NO pro-
duction. The plates were pretreated with various concentrations of
samples for 30 min and then incubated for 24 h with or without
4.3.10. Laburnine
½
a 2D0
= + 15.3° (c 0.025, EtOH); UV (MeOH) kmax (loge), 213
ꢁ
(2.20); HR-ESI-MS at m/z 142.1234 [M+H]+ (calcd. for C8H16NO,
142.1232).
4.4. Alkaline Hydrolysis (Ikeda et al., 2005)
A solution of compound 1 (19 mg) in MeOH (0.5 mL) was heated
with 1 N NaOH (0.5 mL) until reflux began, thus being maintained
for 1 h. After cooling, the reaction mixture was acidified with 1 N
HCl and extracted with CHCl3 (3 ꢂ 4 mL). The aqueous solution
was then made alkaline with 1 N NaOH and extracted with CHCl3
(3 ꢂ 4 mL), following which evaporation of the organic phase
yielded lindelofidine (2.1 mg). The process was repeated for com-
pound 2 (5 mg) to afford laburnine (0.25 mg), and then for com-
pounds 3–8 (about 2 mg). 1-hydroxymethylpyrrolizidine was
analyzed by TLC using CHCl3:MeOH:diethylamine = 7:3:0.1 as
developing solvent and iodine for detection, 1–8 gave laburnine
(Rf 0.13) and lindelofidine (Rf 0.15), respectively. HPLC analysis
was carried out on a Waters SymmetryShieldTM RP-18 column
1 lg/mL of LPS (Escherichia coli, serotype O111: B4, Sigma). The ni-
trite concentration in the culture supernatant was measured by the
Griess reaction. Cell viability was measured by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
(Sigma–Aldrich). The absorbance at 540 nm was read using a micro-
plate reader (POLARstar).
Acknowledgments
This research work was financially supported by the New Cen-
tury Talents Scheme of the Ministry of Education (NCET-08-0820),
the Fundamental Research Funds for Central Universities
(SWJTU2010ZT09) and the National Natural Science Foundation
of China (21142004).
[5
l
M, 4.6 ꢂ 250 mm, flow rate: 1 ml/min, at 40 °C, eluent with
CH3CN/H2O/H3PO4(35/65/0.1), detection at UV 204 nm] with a
Waters 600 controller and Waters 2487 detector, by comparison
with lindelofidine and laburnine.
Appendix A. Supplementary data
4.5. Acid Hydrolysis (Tang et al., 2005)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
03.009.
A solution of each compound (about 2 mg) in 1 N HCl-dioxane
(1:1, 1.5 ml) was heated at 90 °C for 4 h, respectively. After cooling,
Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009

8
S. Huang et al. / Phytochemistry xxx (2013) xxx–xxx
Mohanraj, S., Subramanian, P.S., Herz, W., 1982. Minor alkaloids of Heliotropium
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Please cite this article in press as: Huang, S., et al. Pyrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPS-induced NO production
in RAW264.7 macrophages. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.03.009