Pyrrolizidine alkaloids from Liparis nervosa with antitumor activity by modulation of autophagy and apoptosis

Article abstract of DOI:10.1016/j.phytochem.2018.06.001

Seven pyrrolizidine alkaloids, nervosine X–XV and nervosine VII N-oxide, together with a reaction product, namely chloride-(N-chloromethyl nervosine VII), were isolated from Liparis nervosa. Their structures were elucidated by extensive spectroscopic analyses. Most of these compounds were investigated for their cytotoxicity in vitro against HCT116 human cancer cell line, and the results showed that chloride-(N-chloromethyl nervosine VII) induced tumor cell death in a dose-dependent manner. Furthermore, the mechanisms underlying its cytotoxicity were investigated, including apoptosis and autophagy. Apoptosis in HCT116 cells was associated with up-regulation of caspase-3 and -9 expressions by activation of the mitochondrial pathway. The autophagy inducing effect was associated with the regulation of autophagic markers, including LC3-II, p62, and Beclin 1. Mechanistic studies showed that JNK, ERK1/2, and p38 MAPKs signaling cascades play an important role in chloride-(N-chloromethyl nervosine VII) induced autophagy and apoptosis.

Full text of DOI:10.1016/j.phytochem.2018.06.001

Phytochemistry 153 (2018) 147–155
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Pyrrolizidine alkaloids from Liparis nervosa with antitumor activity by
modulation of autophagy and apoptosis
Lin Chena,b,1, Shuai Huanga,c,1, Chun Ying Li , Feng Gaoa,∗∗, Xian Li Zhoua,∗
c
a
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, Sichuan, PR China
School of Chemistry and Chemical Engineering, China West Normal University, Nanchong, 637002, Sichuan, PR China
Center for Molecular and Translational Medicine, Institute of Biomedical Sciences, Georgia State University, 511 Research Science Center, 157 Decatur St SE, Atlanta,
b
c
GA, 30303, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Liparis nervosa
Orchidaceae
Pyrrolizidine alkaloids
Cytotoxicity
Human colorectal cancer cell line
Autophagy
Seven pyrrolizidine alkaloids, nervosine X–XV and nervosine VII N-oxide, together with a reaction product,
namely chloride-(N-chloromethyl nervosine VII), were isolated from Liparis nervosa. Their structures were elu-
cidated by extensive spectroscopic analyses. Most of these compounds were investigated for their cytotoxicity in
vitro against HCT116 human cancer cell line, and the results showed that chloride-(N-chloromethyl nervosine
VII) induced tumor cell death in a dose-dependent manner. Furthermore, the mechanisms underlying its cyto-
toxicity were investigated, including apoptosis and autophagy. Apoptosis in HCT116 cells was associated with
up-regulation of caspase-3 and -9 expressions by activation of the mitochondrial pathway. The autophagy in-
ducing effect was associated with the regulation of autophagic markers, including LC3-II, p62, and Beclin 1.
Mechanistic studies showed that JNK, ERK1/2, and p38 MAPKs signaling cascades play an important role in
chloride-(N-chloromethyl nervosine VII) induced autophagy and apoptosis.
Apoptosis
InChIKey:
MCJLHCBGPXQWLR-GKLPOLMESA-N
1. Introduction
extract of the same specimen led to the isolation of seven previously
undescribed saturated PAs, nervosine X–XV (1–3, 6–8) and nervosine
The genus Liparis, belonging to the Orchidaceae family that is
VII N-oxide (4), together with a reaction product of nervosine VII (5)
(Fig. 1). Compounds 1–3 and 5–8 were investigated in this study for
their cytotoxicity in vitro against HCT116 human cancer cells, and we
found that compound 5 induced tumor cell death in a dose-dependent
manner.
Programmed cell death (PCD), a major cytotoxic mechanism of
antitumor agents including two classical forms, apoptosis (type I PCD)
and autophagy (type II PCD), along with the regulation of PCD is an
important target in cancer chemotherapy (Kim et al., 2016). Accord-
ingly, the underlying molecular mechanism of the antitumor activity of
compound 5, and how it modulates the crosstalk between autophagy
and apoptosis were investigated in HCT116 human colon cancer cells.
Here, we report the isolation and structural elucidation of eight pre-
viously undescribed PAs from the whole plant of L. nervosa, as well as
the investigation about the mechanisms by which compound 5 affects
apoptosis and autophagy in HCT116 cells.
comprised of more than 250 species, is mainly distributed in the tropic
and subtropic region (Editorial committee of Flora of China, 1999).
Liparis nervosa (Thunb.) Lindl, is a herbaceous plant, widely distributed
in China, and used for detoxicating and hemostatic functions (Hua
et al., 1999). Research on phytochemical constituents has been con-
ducted since decades and a series of pyrrolizidine alkaloids (PAs) has
been isolated (Nishikawa and Hirata, 1967, 1968). The PAs represent a
class of typical metabolites, and it is well known that the majority of
PAs cause serious diseases in domestic animals and humans through
liver bioactivation (Bush et al., 1993). However, saturated PAs are not
hepatotoxic and must be distinguished from the 1, 2-unsaturated pyr-
rolizidine alkaloids (Bush et al., 1993; Castells et al., 2014). Further
evidence showed that PAs are biologically active molecules with anti-
tumor, antibiosis, and antifeedant activities (Hartmann, 1999; Siciliano
et al., 2005; Liu et al., 2017). Previous investigations on chemical
constituents of L. nervosa in our group have reported eleven pyrrolizi-
dine alkaloids and sixteen nervogenic acid derivatives (Huang et al.,
2013a, 2013b, 2013c, 2016). Our continuing study on the ethanol
∗
Corresponding author. Natural Products Laboratory of School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, PR China.
Corresponding author. Natural Products Laboratory of School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, PR China.
∗
∗
E-mail addresses: gaof@swjtu.edu.cn (F. Gao), zhouxl@swjtu.edu.cn (X.L. Zhou).
Joint first author.
1
https://doi.org/10.1016/j.phytochem.2018.06.001
Received 26 January 2018; Received in revised form 18 May 2018; Accepted 2 June 2018
0031-9422/ © 2018 Elsevier Ltd. All rights reserved.

L. Chen et al.
Phytochemistry 153 (2018) 147–155
Fig. 1. Structures of compounds 1–8 isolated from Liparis nervosa.
2
. Results and discussion
40.4–42.0 in 13C NMR, while in laburnine, H-1 and H-8 were trans-
configuration, C-1 was resonated at δ 45.0–46.3, respectively (Huang
et al., 2013a, 2016). 1-hydroxymethyl pyrrolizidine moiety of 1 pre-
(41.3, C-1, d), H-1 and H-8 were a cis-
C
2.1. Structure elucidation and identification
sented similar properties [δ
C
Compound 1 was isolated as a white, amorphous powder, and as-
configurated according to the NOESY correlations] with lindelofidine
signed a molecular formula of C37
molecule at m/z 722.3754 [M
H
+
55NO13 based on the protonated
based on the NMR spectra. Moreover, 1-hydroxymethyl pyrrolizidine
+
H]
(C37
H
56NO13
,
calcd for
was obtained by alkaline hydrolysis of 1, whose molecular formula was
1
3
+
722.3752), as revealed by HR-ESI-MS (positive mode) and C NMR
deduced as C
(Fig. S89), and the optical rotation [α]
8
H
15NO (calcd. for [M+H] , 142.1232) by HR-ESI-MS
−
1
20
spectra. IR spectrum of 1 gave a broad peak at 3369 cm
the presence of hydroxy groups, a peak at 1716 cm , which was at-
indicating
D
+62.1 (c 0.213, EtOH) was in
good agreement with that of lindelofidine [α]
(Nishikawa and Hirata, 1967). From the above-mentioned evidence, the
−1
20
+75.0 (c 3.2, EtOH)
D
−1
tributed to a carbonyl group, and two peaks at 1600 and 772 cm
confirming the presence of an aromatic ring. In the 1H NMR of 1
substructure in 1 was estimated as lindelofidine. HMBC correlations
(
Table 1), resonances for four vinylic methyl groups at δ
H
1.76 (6H, s)
5.31 (2H, t,
from H-9 (δ
H
C
4.39 and 4.52) to C-7′ (δ 167.5), the carboxyl carbon of
and 1.78 (6H, s), two olefinic methine protons at δ
H
the nervogenic acid unit, indicating that an ester group is attached at C-
9.
J = 7.2 Hz) and two vinylic methylene groups at δ
H
3.59 (4H, d,
The remaining signals in the H and 13C NMR spectra of 1 could be
assigned to two D-glucose units, which was supported by acid hydrolysis
of 1, and GC analysis of 1-(trimethylsilyl) imidazole derivatives.
According to the coupling constants of the hydrogens attached to the
anomeric center at δ 4.81 (1H, d, J = 7.8 Hz) and 4.83 (1H, d,
J = 7.8 Hz), the two glucose units (Glc-I and Glc-II) were determined as
β-configuraion (Huang et al., 2013a). In the HMBC, the proton signals
1
J = 7.2 Hz) were readily distinguished, which indicated the existence of
two prenyl moieties in the molecule (Nishikawa and Hirata, 1967,
1
968; Huang et al., 2013a, 2016). Moreover, resonances reminiscent of
a tetra-substituted aromatic ring [δ 7.67 (2H, s); δ 127.1, 130.1 × 2,
37.5 × 2 and 158.1] and a carboxyl group (δ 167.5) in the H and
C NMR spectra, along with the key HMBC correlations (Fig. 2), δ H-
167.5), suggested the
H
C
1
1
C
1
3
H
2
′/6′ (δ
H
7.67) to C-1″/1‴ (δ
C
29.6), and C-7′ (δ
C
presence of a 4-hydroxy-3,5-bis-(3-methyl- 2-butenyl)-benzoic acid
moiety, previously described as a nervogenic acid unit (Nishikawa and
Hirata, 1967, 1968; Huang et al., 2013a, 2016).
Glc-I H-1″″ (δ
correlations with carbons C-4′ (δ
H
4.83) and Glc-II H-1′′′′′ (δ
H
4.81) showed long-range
82.8),
C
158.1) and Glc-I C-2″″ (δ
C
respectively. Based on the above data and analysis, the structure of
compound 1 was elucidated as 4-O-[β-D-glucopyranosyl-(1 → 2)-β-D-
glucopyranosyl] nervogenic acid ester of lindelofidine, named as ner-
vosine X.
Additionally, the substructure 1-hydroxymethyl pyrrolizidine was
deduced from the NMR data, which displayed a nitrogen-bearing me-
thine (δ
H
4.20; δ
C
70.2), one oxymethylene (δ
3.28, 3.47; δ
57.1)], three methylene [(δ 2.00, 2.13; δ
27.0) and (δ 1.95, 2.09; δ 26.9)] and one methine (δ
H
4.39, 4.52; δ
55.0) and (δ
27.1), (δ
C
64.2), two
nitrogen-bearing methylene [(δ
H
C
H
2.98,
1.93,
2.91;
Compound 2 was purified as a white, amorphous powder with the
3
2
.74; δ
.18; δ
C
H
C
H
molecular formula C39
H
57NO14 based on its HR-ESI-MS m/z 764.3849
58NO14, 764.3857). Comparison of the
([M + H]+, calcd for C39
H
C
H
C
H
δ
C
41.3), which were connected by COSY and confirmed by HMBC
spectral data of 2 with that of 1 indicated that it also possessed one
nervogenic acid, one 1-hydroxymethyl pyrrolizidine moiety, and two β-
D-glucose units. The 1-hydroxymethyl pyrrolizidine moiety of 2 in the
1D NMR spectroscopic data (Table 1) were similar to those of 1, sug-
gesting that this compound also possessed a lindelofidine group, which
was further supported by UPLC-HR-ESI-MS and TLC analysis of the
reaction mixture of alkaline hydrolysis of 2, it's molecular formula was
correlations. Eleven pyrrolizidine alkaloids have been isolated from L.
nervosa, and 1-hydroxymethyl pyrrolizidine moiety of those alkaloids
was defined as lindelofidine and laburnine (Nishikawa and Hirata,
1
967, 1968; Huang et al., 2013a, 2016). The main differences between
lindelofidine and laburnine were that, H-1 and H-8 of lindelofidine
were cis-configurated and C-1 was resonated in the range at δ
C
148

L. Chen et al.
Phytochemistry 153 (2018) 147–155
Table 1
NMR spectroscopic data of compounds 1 and 2.a
No.
1
2
No.
1
2
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
1
2
2.91 m
41.3 d
27.1 t
2.93 m
41.1 d
27.0 t
Glc-I
1″″
Glc-I
4.74 d (6.0)
α 2.13 m
β 2.00 m
α 3.47 m
β 3.28 m
α 3.74 m
β 2.98 m
α 2.18 m
β 1.93 m
α 2.09 m
β 1.95 m
4.20 m
α 2.18 m
β 2.04 m
α 3.53 m
β 3.31 m
α 3.82 m
β 3.02 m
α 2.18 m
β 1.94 m
α 2.12 m
β 1.98 m
4.26 m
4.83 d (7.8)
3.83 m
104.2 d
82.8 d
78.4 d
71.3 d
78.2 d
62.6 t
105.5 d
76.1 d
75.3 d
81.0 d
74.0 d
63.8 t
3
5
6
7
8
9
55.0 t
57.1 t
27.0 t
26.9 t
70.2 d
64.2 t
54.9 t
57.1 t
27.0 t
26.9 t
70.4 d
64.1 t
2″″
3.61 m
3″″
3.63 m
3.59 m
4″″
3.42 m
3.62 m
5″″
3.15 m
3.50 overlapped
4.38 d (10.8)
Glc-II
6″″
a 3.65 dd (5.4,12.0)
b 3.78 dd (2.1,12.0)
a 4.39 dd (8.4, 11.4)
a 4.30 dd (8.4, 10.8)
Glc-II
b 4.52 dd (6.6, 11.4)
b 4.51 dd (6.0, 10.8)
1
2
3
4
7
1
′
–
127.1 s
130.1 d
137.5 s
158.1 s
167.5 s
29.6 t
–
127.1 s
130.0 d
137.4 s
157.9 s
167.4 s
29.5 t
1″″′
2″″′
3″″′
4″″′
5″″′
6″″′
4.81 d (7.8)
3.30 m
3.49 m
3.32 m
3.29 m
a 3.67 dd (5.4,12.0)
b 3.81 dd (2.4,12.0)
–
–
105.4 d
76.1 d
78.1 d
71.7 d
78.1 d
63.0 t
4.33 d (7.8)
3.21 m
3.35 m
3.29 m
3.35 m
a 3.66 br.d (10.8)
b 3.90 dd (6.0, 10.8)
1.94 s
104.9 d
74.8 d
77.8 d
71.4 d
78.2 d
62.5 t
′/6′
′/5′
′
′
7.67 s
–
–
–
7.66 s
–
–
–
″/1‴
3.59 d (7.2)
3.50 overlapped
2″/2‴
3″/3‴
4″/4‴
5″/5‴
5.31 t (7.2)
–
1.76 s
1.78 s
124.0 d
134.1 s
18.3 q
26.0 q
5.25 t (7.2)
–
1.73 s
1.75 s
123.8 d
134.1 s
18.1 q
25.9 q
OCOCH
OCOCH
3
3
–
–
20.7 q
172.3 s
–
a
OD ( H NMR for 600 MHz,13C NMR for 150 MHz).
1
Measured in CD
3
deduced as C
lindelofidine. The main differences between 1 and 2 were the presence
of an additional acetyl group [δ 1.94 (3H, s); δ 20.7, 172.3] as in-
dicated by the molecular formula and the connectivity of two glucose
units. In HMBC (Fig. 2), H-6′′′′ of Glc-I (δ 4.38) was correlated with the
172.3, indicating that the
8
H
15NO, the retention time and Rf value were same as
its molecular formula was determined to be C37
H
55NO14 by HR-ESI-MS
and 13C NMR. Comparison of the spectral data of 3 (Table 2) with that
of nervosine III (Huang et al., 2013a), previously isolated from this
plant, indicated that they were similar in structure, with the main dif-
ference being that one isoprene chain was substituted by a hydroxyl
group and consisting in a 3-hydroxy-3-methyl-butenyl moiety based on
H
C
H
carbonyl carbon of the acetyl group at δ
C
acetyl group was assigned to Glc-I C-6′′′′. Two D-glucose units were
connected through a 1 → 4 linkage, which was deduced from the long-
H
the following analyses. The signals at δ 7.17 (1H, d, J = 16.2 Hz) and
6.34 (1H, d, J = 16.2 Hz) in the 1H NMR spectrum indicated the pre-
sence of a trans-double bonds. The two methyl groups of the prenyl
moieties were shifted highfield compared to nervosine III, indicating
that the double bond was shifted from the 2, 3-position to the 1, 2-
position. Moreover, the 13C NMR spectral data showed the presence of
range correlations between Glc-II H-1′′′′′ (δ
H
4.33) and Glc-I C-4′′′′ (δ
C
8
1.0) in HMBC spectrum. Accordingly, the structure of 2, nervosine XI,
was defined as 4-O-[β-D-glucopyranosyl-(1 → 4)-6-O-acetyl-β-D- gluco-
pyranosyl] nervogenic acid ester of lindelofidine.
Compound 3 was also isolated as a white amorphous powder, and
C
an oxygenated quaternary carbon at δ 71.8 (C-3‴) and the lack of an
Fig. 2. Key 1H- H COSY and HMBC correlations of compounds 1–8.
1
149

L. Chen et al.
Phytochemistry 153 (2018) 147–155
Table 2
Table 3
NMR spectroscopic data of compound 3.a
NMR spectroscopic data of compounds 4 and 5.
a
No.
3
No.
4
5
δ
H
(J in Hz)
δ
C
No.
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
1
2
2.96 m
41.2 d
27.0 t
1‴
2‴
7.17 d (16.2)
6.34 d (16.2)
123.0 d
140.8 d
1
2
3.23 m
39.3 d
27.5 t
3.08 m
41.3 d
27.7 t
α 2.19 m
β 2.04 m
α 3.52 overlapped
β 3.32 m
α 3.78 m
β 3.03 m
α 2.13 m
β 1.93 m
α 2.17 m
β1.99 m
α 2.38 m
α 2.30 m
β 2.18 m
α 3.78 m
β 3.78 m
α 4.10 m
β 3.51 m
α 2.19 m
β 2.19 m
α 2.19 m
β 2.19 m
4.45 m
a 4.36 dd (6.4, 10.8)
b 4.47 dd (6.0, 10.8)
5.33 br.s
–
7.60 s
–
β 2.00 m
3
5
6
7
55.1 t
57.3 t
27.0 t
26.9 t
3‴
4‴
5‴
–
71.8 s
29.8 q
29.9 q
3
5
6
7
α 3.90 m
69.0 t
70.3 t
26.4 t
26.0 t
64.1 t
65.7 t
27.2 t
25.4 t
β 3.62 m
1.42 s
1.42 s
Glc-I
α 3.78 m
β 3.70 m
α 2.30 m
β 2.10 m
α 2.26 m
β 2.01 m
8
9
4.25 m
70.6 d
64.2 t
1″″
2″″
4.72 d (7.8)
3.57 m
105.8 d
76.4 d
8
9
4.13 m
85.8 d
63.9 t
80.8 d
63.1 t
a 4.43 dd (6.0, 11.4)
b 4.52 dd (6.0, 11.4)
–
8.01 d (1.8)
–
a 4.29 dd (8.4, 10.8)
b 4.42 dd (6.0, 10.8)
–
–
7.59 s
–
–
–
3.33 d (7.2)
5.31 t (7.2)
–
1.72 s
1.77 s
1′
2′
3′
4′
127.2 s
126.7 d
132.8 s
157.4 s
3″″
4″″
5″″
6″″
3.60 m
3.67 m
3.23 m
a 3.74 dd (2.4, 12.0)
b 3.80 dd (3.6, 12.0)
Glc-II
4.43 d (7.8)
3.21 m
75.4 d
80.6 d
76.7 d
61.8 t
10
1′
2′/6′
3′/5′
4′
–
69.4 t
121.8 s
129.9 d
129.5 s
158.8 s
168.0 s
29.3 t
123.0 d
134.3 d
17.9 q
26.0 q
121.6 s
130.0 d
129.6 s
158.9 s
167.9 s
29.3 t
122.9 d
134.3 d
17.9 q
26.0 q
–
–
–
5
6
7
1
′
′
′
″
–
138.3 s
130.7 d
167.3 s
29.6 t
7′
7.70 d (1.8)
–
a 3.64 overlapped
b 3.52 overlapped
5.30 t (7.2)
–
1″′″
2″′″
3″′″
104.6 d
74.9 d
77.9 d
1″/1‴
2″/2‴
3″/3‴
4″/4‴
5″/5‴
3.32 d (7.2)
5.30 t (7.2)
–
1.72 s
1.77 s
3.23 m
2″
3″
4″
123.7 d
134.2 s
18.1 q
4″′″
5″′″
6″′″
3.30 m
3.34 m
a 3.64 overlapped
b 3.89 dd (1.8, 12.0)
71.4 d
78.2 d
62.5 t
a
Measured in CD OD ( H NMR for 600 MHz,13C NMR for 150 MHz).
1
1.73 s
3
5
″
1.77 s
25.9 q
Compound 5 was obtained as a white amorphous powder. The po-
+
a
OD ( H NMR for 600 MHz,13C NMR for 150 MHz).
1
sitive ion, C25
peak at m/z 446.2468 [M] (calcd for C25
the isotopic peak at m/z 448.2448 [M + 2] in the HR-ESI-MS spec-
trum. The characteristic NMR spectra signals of compound 5 (Table 3)
resembled those of nervosine VII, except for the presence of a N-
chloromethyl group. This was supported by the appearance of a new
H
37ClNO
, was deduced from the quasimolecular ion
3
Measured in CD
3
+
+
H
37ClNO
3
, 446.2462) and
+
olefinic quaternary carbon in 3, which indicated that there was an
additional OH connected to C-3‴. The two methyl groups were con-
nected to C-3‴ and two trans olefinic carbons were located at C-1‴ and
C-2‴ on the basis of the cross-peaks in the HMBC spectrum as shown in
Fig. 2. The 1-hydroxymethyl pyrrolizidine unit of 3 was deduced as
lindelofidine by its homologous NMR data with 1, combined with TLC
and UPLC-HR-ESI-MS analysis of the reaction mixture of alkaline hy-
drolysis of 3. Consequently, 3 was established as 3-(3-methyl-2-bu-
tenyl)-4-O-[β- D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl]-5-(3-hy-
droxy-3-methyl-butenyl) benzoate ester of lindelofidine, and named
nervosine XII.
methylene signal at δ
H
5.33 (2H, br.s) and δ
C
69.4 (Perez-Castorena
et al., 1998; Shi et al., 2010; Bowman et al., 1994), and C-3, C-5, and C-
8
appeared shifted downfield (Δδc 10.1, 9.8 and 14.6 ppm, respec-
tively), in relation to those of nervosine VII (Huang et al., 2013a). The
existence of an N-chloromethyl group at nitrogen atom was confirmed
by the cross-peaks between H-10 (δ
H
5.33) with C-3 (δ
C
64.1), C-5 (δ
C
6
5.7) and C-8 (δ 80.8) in the HMBC spectrum (Fig. 2). From the above
C
evidence, compound 5 was determined to be chloride-(N-chloromethyl
nervosine VII). The tertiary amine was strongly nucleophilic and related
chloromethyl derivatives were easily obtained from cyclized tertiary
amines in the presence of methylene chloride (Beniddir et al., 2013;
Bowman et al., 1994; Ndongo et al., 2017; Shi et al., 2010). These ar-
tifacts frequently occur during extraction and/or isolation process (Tan
et al., 2013). Caution is therefore needed when quaternary ammonium
derivatives are isolated. UPLC-HR-ESI-MS analysis of the crude extract
from L. nervosa did not show any hint of chlorinated metabolites (see
Supplementary Information, Fig. S54), and after purification with di-
chloromethane in silica gel, strong chloromethyl derivatives signal
were identified (see Supplementary Information, Fig. S55). Thus,
compound 5 could be an artifact formed during the purification process
Compound 4, a white amorphous powder, had a molecular formula
as deduced by HR-ESI-MS (m/z 414.2638 [M + H]+
of C25H
35NO
4
,
calcd for C25
H
4
36NO , 414.2644). Comparison of the 1D NMR chemical
shifts of 4 (Table 3) with those of nervosine VII (Huang et al., 2016), a
known compound isolated from this plant, implied that they shared the
same planar structure except for the presence of an additional oxygen
1
3
atom as indicated by the molecular formula. In the C NMR spectrum
(
(
Table 3), the signals of C-3, C-5, and C-8 were displaced downfield
Δδc15.0, 14.4 and 19.6 ppm, respectively), in relation to those of
nervosine VII (Huang et al., 2016). Therefore, the additional oxygen
atom must be attached to the nitrogen atom with a coordination bond.
The positive correlation was observed between H-1 and H-8 in NOESY
spectrum, indicating a cis-configuration for these protons. The proposed
structure was further supported by comparison of the NMR spectral
data of 4 with liparis alkaloid A, a known compound described in the
literature (Jiang et al., 2015), which also contained a unit of lindelo-
fidine N-oxide. The above-mentioned conclusion was also confirmed
with CH
2 2
Cl .
The molecular formula of 6 (C25
H
5
35NO ) was ascertained by HR-
ESI-MS (m/z 430.2601 [M + H]+. Analyses of NMR data of 6 (Table 4)
suggested that it also possessed a 1-hydroxymethyl pyrrolizidine
moiety, which was estimated as lindelofidine following comparison of
the pyrrolizidine part of 1 with NMR data, which was proved by al-
kaline hydrolysis of 6, TLC and UPLC-HR-ESI-MS analysis of the reac-
tion mixture. Furthermore, the NMR spectra exhibited characteristic
2
0
through comparing the optical rotation [α] +18.2 (c 0.552, MeOH)
D
2
0
with that of nervosine VII [α] +23.0 (c 0.860, CH
3
Cl) (Huang et al.,
4.29 and 4.42) to
C
85.8) indicated
D
2
016). Furthermore, HMBC correlations from H-9 (δ
C-7′ (δ 168.0), C-1 (δ 39.3), C-2 (δ 27.5) and C-8 (δ
that the nervogenic acid unit was attached to C-9 by an ester linkage
Fig. 2). Thus, structure 4 was defined as nervosine VII N-oxide.
H
C
C
C
signals for a tetra-substituted phenyl unit [δ
H
7.75 (1H, br.s), 7.89 (1H,
br.s); δ 123.4, 125.3, 126.3, 130.9, 132.8, 164.0], a carboxyl group
(
C
150

L. Chen et al.
Phytochemistry 153 (2018) 147–155
and UPLC-HR-ESI-MS analysis of the reaction mixture of its alkaline
Table 4
NMR spectroscopic data of compounds 6–8.
hydrolysis. The major differences were the absence of one oxygen-
No. 6a
7b
8a
bearing methine carbon at δ
C
73.3 and the presence of a methylene at
30.5, exhibiting that the hydroxyl group at C-3‴ had disappeared.
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
Complete structure assignments were obtained from exhaustive analysis
of the HMQC, HMBC and COSY data. The circular dichroism curve of 7
showed the same pattern as that reported for acremine N (Arnone et al.,
2009), and the absolute configuration of C-2‴ was identified as (2‴S)-
conformer, named nervosine XIV.
1
2
2.92 m
41.2 d
27.0 t
2.68 m
40.8 d
27.6 t
2.93 m
41.2 d
27.0 t
α 2.17 m
β 2.01 m
α 3.51 m
β 3.30 m
α 3.82 m
β 3.00 m
α 2.13 m
β 1.94 m
α 2.10 m
β 1.92 m
4.29 m
α 1.84 m
β 1.62 m
α 3.07 m
β 2.66 m
α 3.16 m
β 2.49 m
α 1.89 m
β 1.76 m
α 1.70 m
β 1.42 m
3.62 m
a 4.28 dd
(7.8, 10.4)
b 4.40 dd
(7.2, 10.4)
–
α 2.19 m
β 2.03 m
α 3.51 m
β 3.32 m
α 3.82 m
β 3.00 m
α 2.17 m
β 1.95 m
α 2.13 m
β 1.97 m
4.30 m
a 4.40 dd
(6.6, 10.8)
b 4.49 dd
(6.0, 10.8)
–
3
5
6
7
54.9 t
57.1 t
26.9 t
26.8 t
54.2 t
56.0 t
26.8 t
26.6 t
55.0 t
57.2 t
26.9 t
26.8 t
Compound 8 was obtained as a white amorphous powder. HR-ESI-
MS data provided the molecular formula C25
H
35NO
6
(m/z 446.2544 [M
+
+
H] , calcd for C25H36NO , 446.2543). The NMR spectrum (Table 4)
6
exhibited characteristic signals attributable to a carboxyl group, a lin-
delofidine moiety and a dihydrobenzofuran derivative bearing a hy-
droxyl-isopropyl residue at C-2‴ and a hydroxyl group at C-3‴. The
lindelofidine moiety was further supported by TLC and UPLC-HR-ESI-
MS analysis of the reaction mixture of alkaline hydrolysis of 8. Com-
parison of the NMR data of 8 with those of 6 indicated that the mod-
ification of one prenyl 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. This was confirmed by the presence of
8
9
70.3 d
64.0 t
66.4 d
65.4 t
70.4 d
64.1 t
a 4.36 br. d
(
8.4)
b 4.47 dd
5.4, 10.2)
(
1
2
3
4
5
6
7
1
2
3
4
5
2
3
4
5
6
′
′
′
′
′
′
′
″
″
″
″
″
‴
‴
‴
‴
‴
–
130.9 s
122.9 s
121.4 s
131.5 d
121.9 s
163.1 s
132.1 s
126.7 d
167.3 s
7.75 br.s
–
–
–
7.89 br.s
–
3.33 (d, 7.2)
5.30 (t, 7.2)
–
1.73 s
1.74 s
4.34 (d, 4.2)
5.34 (d, 4.2)
–
132.8 d 7.67 br.s
130.8 d 7.97 br.s
125.3 s
164.0 s
123.4 s
–
–
–
123.1 s
161.9 s
127.1 s
–
–
–
signals at δ
and δ 6.72 (lH, d, J = 16.2 Hz, H-2″) in the H NMR and at δ
"/5″), δ 71.6 (C-3″), δ 142.4 (C-2″), and δ 121.4 (C-1″) in the
H
H
1.38 (6H, s, H-4"/5″), δ 6.65 (lH, d, J = 16.2 Hz, H-1″),
1
H
C
30.0 (C-
126.3 d 7.67 br.s
124.4 d 7.90 br.s
13
4
C
C
C
C
166.6 s
28.8 t
–
166.7 s
28.4 t
–
NMR (Flores et al., 2008). The dihydrobenzofuran derivative was at-
tached to C-9 of lindelofidine moiety by the ester linkage base on the
HMBC spectrum as shown in Fig. 2. The coupling constant (4.2 Hz) of
H-2‴ and H-3‴ suggested a trans-configuration (Friedrich et al., 2005).
Because the circular dichroism curve of 8 showed the same pattern as
that reported for the prenylated dihydrobenzofuran derivatives (Xu
et al., 2016), the absolute configuration of C-2‴ and C-3‴ was identified
as (2‴S, 3‴S)-conformer, and named nervosine XV.
3.30 (d, 7.2)
6.65 (d, 16.2) 121.4 d
122.4 d 5.27 (t, 7.2)
121.5 d 6.72 (d, 16.2) 142.4 d
134.3 s
17.9 q
25.9 q
98.9 d
73.3 d
71.8 s
25.8 q
25.1 q
–
133.3 s
18.0 q
25.9 q
90.2 d
30.5 t
72.0 s
24.2 q
25.9 q
–
71.6 s
30.0 q
30.0 q
99.3 d
72.9 d
71.7 s
25.4 q
25.6 q
1.73 s
1.74 s
4.69 (d, 9.0)
3.19 (d, 9.0)
–
1.38 s
1.38 s
4.38 (d, 4.2)
5.35 (d, 4.2)
–
1.22 s
1.29 s
1.21 s
1.33 s
1.28 s
1.30 s
a
OD ( H NMR for 600 MHz,13C NMR for 150 MHz).
Measured in CDCl ( H NMR for 600 MHz, C NMR for 150 MHz).
3
1
2.2. Biological evaluation
Measured in CD
3
b
1
13
2.2.1. Growth inhibition effects against HCT116 cells
To evaluate the effects of the isolated pyrrolizidine alkaloids on the
growth of human cancer cells, the growth inhibitory potential of
compounds 1–3 and 5–8 were determined by MTT assay. Compounds
–3, 7 and 8 did not show obvious growth inhibitory effects on
HCT116 cell lines at concentrations up to 80 μM. Compound 6 showed
IC50 values of 60.04 ± 0.8 μM against HCT116 cells. Compound 5,
possessing a chloromethyl group connected to the N atom, exhibited
cytotoxicity in a dose dependent manner against HCT116 cell lines with
IC50 values of 32.88 ± 1.6 μM. Herein, adriamycin was used as the
positive control, with IC50 values of 3.5 ± 6.7 μM against
HCT116 cells.
resonance at δ
C
166.6, and a prenyl group [δ
.33 (2H, t, J = 7.2 Hz), 1.73 (3H, s) and 1.74 (3H, s); δ
8.8, 25.9, 17.9]. Based on the HMBC spectrum as shown in Fig. 2, the
H
5.30 (1H, t, J = 7.2 Hz),
3
2
C
134.3, 122.4,
1
prenyl group and the carboxyl units were located at C-3′ and C-1′, re-
spectively. In combination with the HMQC data, the remaining five
1
3
aliphatic signals in C NMR, two oxymethine at δ
oxygen-bearing quaternary carbon at δ 71.8, and two methyl groups at
25.8 and 25.1 were observed. These NMR data hinted of a dihy-
C
73.3 and 98.9, one
C
δ
C
drobenzofuran skeleton bearing a hydroxyl group at C-3‴ and a hy-
droxyl-isopropyl residue C-2‴ (Duan et al., 2008). In addition, corre-
lation between H-9 and the carboxyl carbon (C-7′) of the
dihydrobenzofuran derivative unit in HMBC spectrum indicated that
the dihydrobenzofuran derivative ester was located at C-9 of lindelo-
fidine. The relative configuration of positions 2‴ and 3‴ could be de-
duced from the vicinal coupling constants, which should be 7.0 Hz in
2
.2.2. Induction of apoptosis by compound 5 is associated with caspase-
mediated pathway
In general, apoptosis occurs via either the mitochondrial (intrinsic)
pathway with the activation of caspase-3 and -9 or the death receptor
(extrinsic) pathway with the activation of caspase-8. To further explore
the molecular mechanism involved in the compound 5-mediated
apoptosis of HCT116 cells, the effects of 5 on the activation of cleavage
forms of caspase-3, -8, and -9 were observed after 24 h treatment with 5
the case of a cis-configuration and 4.0–5.0 Hz in trans-derivatives
3
(
Friedrich et al., 2005). As a coupling constant of J2‴, 3‴ = 4.2 Hz was
observed in 6, the dihydrofuran ring had to be 2‴, 3‴-trans-con-
figurated. The absolute configuration of C-2‴ and C-3‴ was identified
as (2‴S, 3‴S)-conformer by CD experiment, indicating that it had the
same curve pattern as that reported for the prenylated dihy-
drobenzofuran derivatives (Xu et al., 2016). Accordingly, the structure
of compound 6 was determined as shown in Fig. 1, and accorded the
trivial name nervosine XIII.
Compound 7, a white amorphous powder, showed the molecular
formula of C25H35NO4, as established by the positive HR-ESI-MS ion at
m/z 414.2644 [M + H] . The NMR data of compound 7 and nervosine
III (Huang et al., 2013a) showed a close resemblance (Table 4). The 1-
hydroxymethyl pyrrolizidine unit was identified as lindelofidine by TLC
(
Fig. 3). A dose-dependent cleavage of procaspase-3 and -9 was ob-
served, and the activated caspase-3 also cleaved PARP, which is re-
quired for repairing DNA damage. Meanwhile, cytochrome c expression
was increased in a concentration-dependent manner. These findings
suggested that the induction of apoptotic cell death by 5 might occur
through a caspase-9 independent pathway.
+
2
.2.3. Compound 5 induces autophagy in HCT116 cells
The conversion of LC3-I to LC3-II, as detected by western blotting, is
commonly used as a marker to evaluate autophagy activity (Green and
151

L. Chen et al.
Phytochemistry 153 (2018) 147–155
Fig. 3. Effect of compound 5 on the expression of apoptosis-related proteins in
HCT116 cells. HCT116 cells were cultured with indicated concentrations for
Fig. 5. Effect of compound 5 on the mitogen-activated protein kinase (MAPK)
signaling in HCT116 cells. Cells were lysed after treatment with indicated
concentrations of compound 5 for 24 h. The protein expression levels of p-ERK,
ERK, p-p38, p38, p-JNK and JNK were determined by western blot analysis.
GAPDH was used as an internal standard.
24 h. The activation of caspase-8, caspase-9 and caspase-3, PARP, and cyto-
chrome c was determined by western blot analysis. GAPDH was used as an
internal standard.
Levine, 2014). Western blot analyses showed that compound 5 induced
accumulation of LC3-II in HCT116 cells (Fig. 4A), indicating that
compound 5 might affect autophagy in HCT116 cells. To further con-
firm compound 5-induced autophagy, the autophagy markers of LC3-II,
2
.2.5. Compound 5 induced the switch from autophagy to apoptosis in
HCT116 cells
To elucidate the relationship between autophagy and apoptosis in
p62 and Beclin
1 were detected by western blot analysis in
HCT116 cells, the time-dependent expression of autophagy- and apop-
tosis-specific proteins after treatment with 40 μM of compound 5 was
inspected. As a result, the induction of autophagy by 5, as indicated by
the expression of LC3-II and Beclin 1, was gradually increased and
reached a peak at 24 h. In contrast, the activation of apoptosis by 5, as
indicated by the levels of cleaved caspase-9 and cleaved PARP, con-
tinued increasing up to 48 h (Fig. 6). These data suggest that the in-
duction of autophagy and apoptosis by 5 is not associated with a suc-
cessive process, but rather a simultaneous approach in an early stage
and the prolonged treatment with 5 inducing the switch from autop-
hagy to apoptotic cell death.
HCT116 cells after 24 h treatment with 5 in a concentration-dependent
manner. Treatment with compound 5 up-regulated the expression le-
vels of LC3-II and Beclin 1, and down-regulated the expression of p62 in
HCT116 cells (Fig. 4B), indicating that compound 5 could induce au-
tophagy (Kim et al., 2016).
2.2.4. Effects of the MAPKs signaling pathways on 5-treated HCT116 cells
MAPKs signaling pathways consisting of ERK, p38 MAPK, and JNK
have been considered as chemotherapeutic targets for sensitizing
cancer cells to autophagy and apoptosis. To investigate the role of
MAPK signaling pathways in 5-induced autophagy and apoptosis, the
activation of ERK1/2, p38 MAPK, and JNK was determined by western
blot analysis. As shown in Fig. 5, compound 5 activated the phos-
phorylation of ERK1/2, p-p38 and p-JNK in a concentration-dependent
manner. These results support that 5-induced autophagy and apoptosis
in HCT116 cells is associated with activation of MAPKs signaling
pathways.
3. Conclusions
Investigation on the whole plant of L. nervosa resulted in the isola-
tion of seven previously undescribed pyrrolizidine alkaloids and a re-
action artefact. Their structures were elucidated by extensive spectro-
scopic analyses and chemical reactions. Most of these compounds were
investigated for their cytotoxicity in vitro against HCT116 human
cancer cell line by MTT assay, and chloride-(N-chloromethyl nervosine
VII) (5) was found to induce tumor cell death with IC50
Fig. 4. (A) Effect of compounds 1–3 and 5–8 on the induction of autophagy in HCT116 cells. HCT116 cells were cultured with 40 μM of compounds 1–3 or 5–8 for
4 h, and the protein expression levels of LC3-II were determined by western blot analysis. (B) HCT116 cells were cultured with the indicated concentrations of
compound 5 for 24 h. The protein expression levels of autophagy markers were determined by western blot analysis.
2
152

L. Chen et al.
Phytochemistry 153 (2018) 147–155
4
. Experimental
.1. General experimental procedures
Optical rotations were measured on a Perkin–Elmer 341 polari-
4
meter. 1D and 2D NMR spectra were recorded on a Bruker AV 600 NMR
spectrometer with TMS, and IR spectra on a Thermo Fisher Nicolet
−1
6
700 spectrometer (KBr discs, cm ). UV spectra were determined with
a Shimadzu UV-2450 spectrophotometer. Circular dichroism was ob-
tained on a Jasco J-720 spectropolarimeter. Silica gel (Qingdao
Haiyang Chemical Co., Ltd., China, 200–300 mesh), RP-18 silica gel
(
Merck, 40–60 μm), D-101 macroporous resin (Rohm & Haas) and
alumina (Qingdao Haiyang Chemical Co., Ltd., China, 100–200 mesh)
were used for column chromatography (CC). HPLC was carried out on a
Waters SymmetryShield™ RP-18 column (5 μm, 4.6 × 250 mm) with a
Waters 600 controller and Waters 2487 detector. The 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
Dragendorff's reagent or iodine. The HR-ESI-MS data were measured
using a Q-TOF micro mass spectrometer (Waters). The antibodies for p-
ERK1/2, ERK1/2, p-p38, p38, JNK, p-JNK, LC3-II, p62, Beclin 1, and
the secondary antibodies were purchased from Cell Signaling
Technology (Beverly, MA, USA).
Fig. 6. Effect of compound 5 on the modulation of crosstalk between autophagy
and apoptosis. HCT116 cells were cultured with 40 μM of compound 5 for 6, 12,
24, 36 and 48 h, and the protein expression levels of autophagy marker (LC3-II
and Beclin 1) and apoptosis-related proteins (cleaved caspase 9, cleaved PARP)
were determined by western blot analysis.
32.88 ± 1.6 μM. The chloromethyl group connected to the N atom and
hydroxy group on aromatic ring in compound 5 appear to be the im-
portant factors for potential antitumor activity. Furthermore, the study
exploring possible mechanisms showed that compound 5 induced cell
death by activating both apoptosis and autophagy in HCT116 cells.
Compound 5 was able to induce apoptosis by activating the mi-
tochondrial pathway, which was associated with mitochondrial release
of cytochrome c, which functions to activate caspase-9, and then pro-
moting the activation of caspase-3, leading to apoptosis. Also, we found
that MAPK signaling pathways, ERK, p38 MAPK, and JNK, were the
common upstream signals that simultaneously triggered apoptosis and
autophagy. The time-dependent expression of autophagy- and apop-
tosis-specific proteins after treatment with compound 5 indicated that
there was a switch from autophagy to apoptotic cell death. A schematic
diagram summarizing these observed effects of compound 5 is shown in
Fig. 7. Therefore, our findings highlight that compound 5 could be a
suitable candidate for the treatment of human colorectal cancer cells.
4.2. Plant material
The whole plants of Liparis nervosa (Thunb.) Lindl were collected in
July 2014 from Kuankuo town, Suiyang county, Zunyi city, Guizhou
Province, China (N28°14′35.48″E107°0′55.40″). The plant was identi-
fied by Professor Liang-Ke Song in School of Life Science and
Engineering, Southwest Jiaotong University, Sichuan, China, where a
voucher specimen is deposited (No. ZN361520140801).
4.3. Extraction and isolation
The air-dried and powdered whole plant of L. nervosa (14 kg) was
extracted with 95% EtOH at room temperature (50 L × 3, every 7 days)
to yield a dark extract (750 g). The extract was suspended in H O (2 L)
2
Fig. 7. Schematic representing the proposed molecular mechanism underlying the action of compound 5 in induction of apoptosis and autophagy.
153

L. Chen et al.
Phytochemistry 153 (2018) 147–155
and then partitioned with petroleum ether, EtOAc, and n-butanol, re-
spectively. The n-butanol extract (180 g) was dissolved in 10% EtOH-
209 (−1.38), 251 (+0.51) nm. IR (KBr) νmax 3368, 2972, 2927, 2796,
1711, 1631, 1605, 1465, 1440, 1377, 1345, 1311, 1284, 1224, 1182,
−
1
1
13
H
2
O, and subjected to a D101 macroporous resin column using the
stepwise elution of H O-EtOH (90:10, 60:40, 40:60, and 5:95, v/v) to
yield four fractions (Fr.A–Fr.D). Fr.B was separated by silica gel CC
using a gradient of CH Cl -MeOH (90:10-5:95, v/v) to afford five sub-
fractions (Fr.B1–Fr.B5). Fr.B1 was separated by silica gel CC and eluted
with petroleum ether-Me CO-Et N (10:1:0.1–1:1:0.1, v/v/v) to produce
compounds 2 (12 mg) and 3 (17 mg). Fr.B2 was subjected to silica gel
CC with EtOAc-MeOH-Et N (20:1:0.2–1:1:0.2, v/v/v) to give three sub-
fractions, Fr.B2-1–Fr.B2-3. Fr.B2-2 was subjected to ODS CC with
CH CN-H O (2:8, v/v) to produce compounds 1 (14 mg) and 4 (2 mg).
Fr.B4 was applied to silica gel CC and eluted with EtOA-MeOH-Et
1084, 1008, 997, 913, 770 cm
;
H and C NMR data, see Table 4;
H]+ (calcd for
36NO ,
5
2
HR-ESI-MS (m/z): 430.2601 [M
+
C
25
H
430.2593).
2
2
4.3.7. Nervosine XIV (7)
2
0
White amorphous powder; [α]
= +20.4 (c 0.450, CHCl ); UV
3
2
2
D
(MeOH) λmax (log ε), 269 (0.29), 246 (0.76) nm; CD (MeOH) λmax(Δε):
248 (+1.51) nm. IR (KBr) νmax 2958, 2923, 2852, 1743, 1711, 1605,
2
−1
1
13
1463, 1377, 1304, 1178, 1092, 962, 863, 770, 721 cm
NMR data, see Table 4; HR-ESI-MS (m/z): 414.2644 [M + H] (calcd
for C25 36NO , 414.2644).
;
H and
C
+
3
2
2
N
H
4
(
10:1:0.2, v/v/v), continually separated over an ODS column with
MeOH-H
2
O (5:95-90:10, v/v) to yield compound 6 (11 mg) and a
4.3.8. Nervosine XV (8)
White amorphous powder; [α]
20
mixture (15.5 mg), which was further isolated by preparative HPLC
with CH CN-H O (3:7, v/v) to afford compound 7 (19 mg). Fr.C was
subjected to neutral Al CC and silica gel CC in succession to yield
three subfractions, Fr.C1–Fr.C3. Fr.C1 was submitted to silica gel CC
and eluted with CH Cl -MeOH (40:1, v/v) to produce compound 5
18 mg). Fr.C2 was subjected to ODS CC and silica gel CC in succession
to yield compound 8 (28 mg).
= +10.4 (c 0.500, MeOH); UV
D
(MeOH) λmax (log ε), 279 (0.56), 206 (1.22) nm; CD (MeOH) λmax(Δε):
217 (−0.46) nm. IR (KBr) νmax 3403, 2973, 2927, 1706, 1655, 1599,
1513, 1451, 1376, 1353, 1283, 1208, 1177, 1061, 1025, 973, 914, 839,
3
2
2 3
O
−
1
1
13
811 cm
;
H and+ C NMR data, see Table 4; HR-ESI-MS (m/z):
2
2
(
6
446.2544 [M + H] (calcd for C25H36NO , 446.2543).
4.4. Acid hydrolysis of 1–3
4
.3.1. Nervosine X (1)
White amorphous powder; [α] = +19.8 (c 0.550, MeOH); UV
2
0
Compound 1 (about 2 mg) was heated in 1 N HCl-dioxane (1:1,
2 ml) at 85 °C for 3 h. The solution was diluted with H O (3 mL), neu-
tralized with 1 N NaOH, and then extracted with CHCl . The H O layer
was evaporated under a stream of nitrogen. The residue was dissolved
in 1-(trimethylsilyl) imidazole (0.1 ml) and pyridine (0.2 ml), and the
solution was stirred at 60 °C for 20 min. After drying, the residue was
D
(
2
1
MeOH) λmax (log ε), 244 (0.37), 208 (0.84) nm; IR (KBr) νmax 3369,
2
958, 2920, 2879, 2851, 1716, 1631, 1600, 1455, 1384, 1350, 1317,
220, 1182, 1077, 1042, 899, 772, 634 cm
3
2
−1
1
13
.
H and C NMR data,
+
see Table 1; HR-ESI-MS (m/z): 722.3754 [M + H] (calcd for
C H56NO13, 722.3752).
37
2 3 3
partitioned between H O and CHCl . The CHCl layer was analyzed by
GC using a L-CP-Chirasil-Val column (0.32 mm × 25 m). D-glucose was
confirmed by comparison with the retention time of the authentic
standards at 7.65 min. The same analysis was carried out on compounds
2–3 (Huang et al., 2013a).
4
.3.2. Nervosine XI (2)
White amorphous powder; [α] = −1.47 (c 0.950, MeOH); UV
2
0
D
(
2
1
MeOH) λmax (log ε), 244 (0.31), 208 (0.80) nm; IR (KBr) νmax 3389,
966, 2924, 2879, 2072, 1720, 1629, 1602, 1456, 1384, 1284, 1243,
181, 1072, 1039, 901, 757, 637 cm
−1
1
13
;
H and C NMR data, see
+
Table 1; HR-ESI-MS (m/z): 764.3849 [M + H] (calcd for C39
H
58NO14
,
4.5. Alkaline hydrolysis of 1
764.3857).
A solution of compound 1 (8 mg) in 0.5 mL of MeOH was refluxed
with 1 mol/L NaOH (0.5 mL) for 1 h. The reaction mixture was acidified
4
.3.3. Nervosine XII (3)
White amorphous powder; [α] = +1.0 (c 0.300, MeOH); UV
2
0
with 1 mol/L HCl and extracted with CHCl
solution was made alkaline with 1 mol/L NaOH and extracted with
CHCl (3 × 4 mL), evaporation of the organic phase yielded lindelofi-
3
(3 × 4 mL). The aqueous
D
(
2
1
MeOH) λmax (log ε), 240 (0.86), 202 (0.68) nm; IR (KBr) νmax 3370,
964, 2922, 2852, 1716, 1677, 1631, 1600, 1455, 1425, 1383, 1322,
3
−
1 1
275, 1201, 1183, 1132, 1072, 902, 836, 800, 770, 721, 638 cm ; H
dine (3.8 mg) (Huang et al., 2013a). The process was repeated for
compounds 2, 3, and 6–8 (about 2 mg). 1-hydroxymethylpyrrolizidine
and C NMR data, see Table 2; HR-ESI-MS (m/z): 738.3707 [M + H]+
1
3
(
calcd for C37
H56NO14, 738.3701).
3
was analyzed by TLC using CHCl :MeOH:diethylamine = 7:3:0.1 as
developing solvent and iodine for detection, 1–3 and 6–8 gave linde-
lofidine (Rf 0.15) (Fig. S96). UPLC-HR-ESI-MS analysis was carried out
on Waters ACQUITY UPLC/Xevo G2 QTOF mass spectrometer, flow
rate: 0.3 ml/min, at 40 °C, eluent with CH OH/H O (2:8), its retention
3 2
time was at 1.02 min (Fig. S90–Fig. S95).
4
.3.4. Nervosine VII N-oxide (4)
White amorphous powder; [α] = +18.2 (c 0.550, MeOH); UV
2
0
D
(
2
1
MeOH) λmax (log ε), 264 (1.63), 216 (2.22) nm; IR (KBr) νmax 3380,
968, 2915, 2861, 1709, 1631, 1602, 1452, 1376, 1350, 1317, 1285,
246, 1099, 983, 934, 906, 769, 628, 592 cm ; H and C NMR data,
−1
1
13
+
see Table 3; HR-ESI-MS (m/z): 414.2638 [M + H] (calcd for
36NO , 414.2644).
4.6. Cell culture
C
25
H
4
HCT 116 human colon cancer cell line was obtained from ATCC.
These cells were maintained at subconfluence in a 95% air and 5% CO
2
humidified atmosphere at 37 °C. RPMI medium supplemented with 10%
fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin
was used for routine subculturing.
4
.3.5. Chloride-(N-chloromethyl nervosine VII) (5)
20
White amorphous powder; [α] = −59.8 (c 0.400, MeOH); UV
D
(
2
1
MeOH) λmax (log ε), 264 (0.63), 215 (1.19) nm; IR (KBr) νmax 3365,
969, 2926, 2877, 2858, 1711, 1631, 1604, 1464, 1382, 1351, 1313,
−1
1
13
284, 1243, 1184, 1098, 1007, 985, 769 cm ; H and C NMR data,
+
see Table 3; HR-ESI-MS (m/z): 446.2468 [M]
C H37ClNO , 446.2462), 448.2448 [M+2] .
25 3
(calcd for
4.7. Cytotoxicity assay
+
+
The cytotoxicity of the compounds against HCT116 human colon
cell lines was evaluated by the MTT method as described in our pre-
vious paper (Huang et al., 2013a). Cells treated with DMSO (0.1% v/v)
were used as negative controls, whereas adriamycin (≥98%; Sigma
4
.3.6. Nervosine XIII (6)
White amorphous powder; [α] = −4.3 (c 1.400, MeOH); UV
2
0
D
(
MeOH) λmax (log ε), 265 (0.69), 208 (1.48) nm; CD (MeOH) λmax(Δε):
154

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New nervogenic acid derivatives from Liparis nervosa. Planta Med. 79, 281–287.
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X.L., 2016. Three new pyrrolizidine alkaloids derivatives from Liparis nervosa. Chin.
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L. Chen and S. Huang contributed equally.
Acknowledgements
This research was supported by grants from National Natural
Science Foundation of China (81402803), the project of Science and
Technology Bureau of Chengdu (2015-HM01-00041-SF) and the United
States of America National Institutes of Health (R01 HL128647 to
Chunying Li). We thank Xiaoqing Gan, Yuning Hou, Xiaonan Sun and
Yu Zhou for technical assistance.
Jiang, P., Liu, H.D., Xu, X.H., Liu, B., Zhang, D.M., Lai, X.W., Zhu, G.H., Xu, P., Li, B.,
2
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in the
online version, at http://dx.doi.org/10.1016/j.phytochem.2018.06.
001. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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Callosine, a 3-alkyl-substituted pyrrolizidine alkaloid from Senecio callosus. J. Nat.
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