Cytotoxic 7S-oxindole alkaloids from Gardneria multiflora

Article abstract of DOI:10.1016/j.phytol.2014.08.001

Seven new oxindole alkaloids, gardmutines A-F (1-6) and 18-hydroxy-chitosenine (7), along with 15 known alkaloids, were isolated from the aerial parts of Gardneria multiflora Makino. The structures of the alkaloids were established by spectroscopic methods. Alkaloids 1-6 are the first Gardneria alkaloids possessing a 7S configuration. Gardmutines D and E were cytotoxic to HeLa, MCF-7 breast, and SW-480 colon cancer cell lines.

Full text of DOI:10.1016/j.phytol.2014.08.001

Phytochemistry Letters 10 (2014) 55–59
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Cytotoxic 7S-oxindole alkaloids from Gardneria multiflora
Xiu-Hong Zhong a,b, Lei Xiao a, Qi Wang c, Bing-Jie Zhang a,b, Mei-Fen Bao a,
Xiang-Hai Cai a, , Lei Peng
*
d,**
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
China
b University of Chinese Academy of Sciences, Beijing 100049, China
c School of Public Health, Kunming Medical University, Kunming 650500, China
d College of Horticulture and Landscape, Yunnan Agricultural University, Kunming 650201, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Seven new oxindole alkaloids, gardmutines A–F (1–6) and 18-hydroxy-chitosenine (7), along with 15
known alkaloids, were isolated from the aerial parts of Gardneria multiflora Makino. The structures of the
alkaloids were established by spectroscopic methods. Alkaloids 1–6 are the first Gardneria alkaloids
possessing a 7S configuration. Gardmutines D and E were cytotoxic to HeLa, MCF-7 breast, and SW-480
colon cancer cell lines.
Received 18 May 2014
Received in revised form 16 July 2014
Accepted 1 August 2014
Available online 15 August 2014
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Gardneria multiflora
7S-oxindole alkaloids
Structure elucidation
Cytotoxicity
1. Introduction
results showed the presence of oxindole alkaloids with the 7S-
configuration, in addition to the 7R-isomers. This paper describes
Monoterpenoid indole alkaloids (MIAs) have been a focus of
natural products research (Bonjoch and Sole, 2000; O’Connor and
Maresh, 2006). Our previous studies have reported several novel
alkaloids from plants of the family Apocynaceae, e.g., E/Z-
alstoscholarine and scholarisine A. In addition, some of them
exhibit cytotoxic activities (Bao et al., 2013; Liu et al., 2012). Plants
of the genus Gardneria in the family Loganiaceae are also sources of
MIAs (Haginiwa et al., 1970; Jiang et al., 2012). As part of our search
for new and cytotoxic alkaloids, phytochemical research on this
genus of plants was performed. In the literatures (Jiang et al.,
2012), almost all reported oxindole alkaloids from the genus
Gardneria were assigned to C-7R configuration. On the other hand,
all of the MIAs from Gardneria shown no cytotoxicity against tumor
cells to date (Bonjoch and Sole, 2000). However, oxindole alkaloids
in nature usually possess C-7R and S configurations, such as in the
genera Uncaria and Gelsemium (Kitajima et al., 2010; Wang et al.,
2011). Therefore, although our previous research did not explore
the cytotoxic MIAs from G. ovate (Li et al., 2011), we performed
research on the congeneric species, G. multiflora. The present
the isolation and structural elucidation of new alkaloids (1–7) and
15 known alkaloids. The cytotoxicity of these compounds against
three human cancer cell lines was evaluated.
2. Results and discussion
The alkaloid fraction of G. multiflora was separated as described
in Section 3 to yield a total of 22 MIAs, including 7 new ones (1–7 in
Fig. 1). All of the products probably belong to the class of alkaloid,
as they were positive in a reaction with Dragendorff’s reagent.
The molecular formula C22H28N2O5 of alkaloid 1 was deter-
mined by the molecular ion peak at m/z 400.1992 [M]+ in the
HREIMS in combination with the 1H and 13C NMR and DEPT spectra
(Tables 1 and 2). Its UV and IR spectra showed absorption bands at
313 and 256 nm and bands at 3440, 1692, and 1633 cmꢀ1
,
respectively, which were consistent with those of monoterpenoid
oxindole alkaloids (Li et al., 2011). The 1H and 13C NMR and DEPT
spectra displayed a trisubstituted oxindole ring [dC 179.9 (s, C-2),
57.2 (s, C-7), 131.0 (s, C-8), 139.8 (s, C-9), 150.9 (s, C-10), 99.9 (d, C-
11), 140.4 (s, C-12), 124.2 (s, C-13); dH 6.65 (s, H-11)] (Yang et al.,
2008). In addition to the indole-ring signals, the 13C NMR and DEPT
spectra displayed 13 additional carbon signals, including a quaternary
*
Corresponding author. Tel.: +86 871 65223188.
Corresponding author.
**
carbon (
d
C 139.8), five methines (
dC 114.2, 67.7, 61.9, 47.9, 28.3), three
E-mail addresses: xhcai@mail.kib.ac.cn (X.-H. Cai), penglei69@126.com
(L. Peng).
methylenes (dC 66.3, 50.2, 28.0), three methoxyls (dC 62.0, 57.4, 57.2)
http://dx.doi.org/10.1016/j.phytol.2014.08.001
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

56
X.-H. Zhong et al. / Phytochemistry Letters 10 (2014) 55–59
Fig. 1. Alkaloids (1–7) isolated from G. multiflora.
and a methyl (dC 13.0). These NMR patterns indicated that 1 was
similar to alkaloid M (Sakm et al., 1977) with the exception of a methyl
methylene signal of C-6 at ab. dC 41.0 ꢁ 2 (Yang et al., 2008). The
ROESY correlations of dH 3.54 (H-21) with dH 5.20 (H-19) and of dH
2.74 (H-15) with dH 1.55 (H-18) established the E-orientation of the C-
19/20 double bond (Fig. 2). Similarly, correlations of H-16 (dH 2.23)
with H-5 (dH 3.05) placed H-16 in the b-orientation. All of the signals
of 1H and 13C NMR spectroscopic data were assigned by the HSQC,
HMBC, and ROESY spectra. Thus, 1 was named gardmutine A.
The 13C NMR and DEPT data of 2 were similar to those of 1 except
group (C-18) instead of
presumption was supported by the HMBC correlations (Fig. 2) of
the double bond proton H 5.20 (H-19) with C 28.3 (C-15), 50.2 (C-21)
and C 139.8 (C-20), and of H 1.55 (H-18) with C-20.
a hydroxymethyl in the latter. This
d
d
d
d
To date, almost all oxindole alkaloids from Gardneria have been
identified with the 7R-configuration (Jiang et al., 2012). However, a
special methylene signal at dC 42.7 was present in the 13C NMR
that a methylene signal at
one at C 13.0 (C-18) in 1. This indicated that alkaloid 2 and alkaloid M
differ in the configuration of C-7. Additionally, the molecular formula
22H28N2O6 of 2 from HREIMS m/z at 416.1956, 16 mass units higher
dC 56.2 in alkaloid 2 substituted the methyl
spectrum of 1, instead of at ab.
d
C 31.0 ꢁ 2 (C-6), as in the 7R-isomers
d
(Li et al., 2011). Further, the HMBC correlations from the methylene
proton signals at dH 2.70 (dd, J = 11.6, 6.4 Hz) and 1.81 (d, J = 11.6 Hz)
to dC 179.9 (C-2), 131.0 (C-8), 67.7 (C-3), and 47.9 (C-16) assigned the
methylene to C-6. This chemical shift difference reminded us that 1
should be a 7S isomer, such as alstonisine and its derivatives with the
C
than that of 1, further indicated that it was an 18-hydroxy derivative of
gardmutine A. This presumption was supported by the HMBC
correlations from dH 3.86 (2H, H-18) to dC 140.6 (C-20) and 119.6
Table 1
1H NMR spectroscopic data for alkaloids 1–7 (
d
in ppm and J in Hz)a.
dH (3)
Position
dH (1)
dH (2)
dH (4)
dH (5)
dH (6)
dH (7)
NH
3
9.01 (1H, br.s)
3.19 (br. d, 8.4)
3.05 (1H, d, 6.4)
10.17 (1H, br.s)
10.24 (1H, s)
10.15 (1H, br.s)
9.02 (1H, s)
9.47 (1H, br.s)
10.12 (1H, br.s)
3.15 (1H, br. d, 8.4) 3.12 (1H, br.d, 8.0) 3.06 (1H, d, 8.4)
3.08 (1H, br.d, 8.0) 2.91 (1H, br.d, 8.0) 3.10 (1H, br.d, 8.0)
5
2.82 (1H, d, 6.6)
3.02 (1H, m)
2.54 (1H, dd,
11.8, 6.6)
2.85 (1H, d, 7.8)
2.53 (1H, dd,
11.2, 7.8)
2.87 (1H, d, 7.8)
2.53 (1H, dd,
12.0, 7.8)
3.63 (1H, d, 7.6)
2.05 (1H, dd,
12.0, 7.6)
2.83 (1H, d, 7.6)
2.45 (1H, dd,
12.0, 7.6)
6
2.70 (dd, 11.6, 6.4) 2.52 (1H, dd,
11.2, 6.6)
1.81 (1H, d, 11.6)
/
1.66 (1H, d, 11.8)
1.69 (1H, d, 11.8)
/
1.66 (1H, d, 11.2) 1.66 (1H, d, 12.0)
2.31 (1H, d, 12.0)
7.27 (1H, d, 8.0)
6.55 (1H, d, 8.0)
/
1.98 (1H, d, 12.0)
/
9
/
/
/
10
11
14
/
/
/
/
/
/
6.65 (1H s
2.52 (1H, br.d,
11.2)
6.61 (1H, s)
6.63 (1H, s)
2.37 (1H, d, 11.6)
6.60 (1H, s)
6.61 (1H, s)
6.60 (1H, s)
2.48 (1H, d, 11.6)
2.36 (1H, d, 11.6)
2.35 (1H, d, 11.6) 2.37 (1H, d, 11.6)
2.90 (1H, d, 11.4)
1.38 (1H, dd,
11.2, 8.4)
2.74 (1H, br.s)
2.23 (1H, m)
3.43 (1H, m)
1.32 (1H, dd,
11.6, 8.4)
1.34 (1H, dd,
11.6, 8.0)
1.32 (1H, dd,
11.6, 8.4)
1.31 (1H, dd,
11.6, 8.0)
1.21 (1H, dd,
11.4, 8.0)
1.15 (1H, dd,
11.6, 8.0)
15
16
17
2.65 (1H, s)
2.07 (1H, m)
3.32 (1H, m)
2.18 (1H, m)
2.18 (1H, m)
3.73 (1H, m)
3.24 (1H, m)
3.80 (2H, m)
2.12 (1H, s)
1.97 (1H, m)
3.25 (1H, m)
3.17 (1H, m)
3.83 (2H, m)
2.19 (1H, s)
2.00 (1H, m)
3.26 (1H, m)
3.19 (1H, m)
3.76 (2H, m)
2.20 (1H, s)
2.11 (1H, m)
3.90 (1H, m)
3.83 (1H, m)
4.00 (1H, m)
3.64 (1H, m)
5.25 (1H, t, 5.0)
3.80 (2H, m)
/
2.13 (1H, s)
/
3.62 (1H, m)
3.83 (1H, m)
3.86 (2H, m)
18
1.55 (3H, d, 5.0)
3.86 (2H, m)
19
5.20 (1H, q, 5.0)
3.54 (2H, m)
3.82 (3H, s)
3.80 (3H, s)
3.69 (3H, s)
/
5.30 (1H, t, 6.4)
3.43 (2H, m)
3.75 (3H, s)
3.74 (3H, s)
3.57 (3H, s)
/
5.15 (1H, t, 5.0)
3.50 (2H, m)
3.75 (3H, s)
3.74 (3H, s)
3.59 (3H, s)
3.18 (3H, s)
5.11 (1H, t, 6.0)
3.49 (2H, m)
3.75 (3H, s)
3.74 (3H, s)
3.59 (3H, s)
5.10 (1H, t, 5.0)
3.53 (2H, m)
3.73 (3H, s)
3.73 (3H, s)
3.59 (3H, s)
3.16 (3H, s)
5.10 (1H, t, 6.0)
3.52 (2H, m)
3.75 (3H, s)
3.74 (3H, s)
3.62(3H, s)
21
9-OCH3
10/11-OCH3
12-OCH3
18-OCH3
3.81 (3H, s)
3.76 (3H, s)
a
Compounds 1 and 6 in acetone-d6, 2, 3, 4, 5 and 7 in DMSO.

X.-H. Zhong et al. / Phytochemistry Letters 10 (2014) 55–59
57
Table 2
13C NMR spectroscopic data for alkaloids 1–7a.
Position
dC (1)
dC (2)
dC (3)
dC (4)
dC (5)
dC (6)
dC (7)
2
179.9 s
67.7 d
61.9 d
42.7 t
179.5 s
65.7 d
59.7 d
41.2 t
179.7 s
65.3 d
60.3 d
41.3 t
179.5 s
65.5 d
60.2 d
41.3 t
179.5 s
65.4 d
60.2 d
41.3 t
180.3 s
66.8 d
58.4 d
38.0 t
179.1 s
67.3 d
65.1 d
32.6 t
3
5
6
7
57.2 s
58.4 s
130.2 s
138.8 s
148.9 s
98.7 d
140.3 s
122.9 s
25.9 t
58.5 s
130.3 s
138.9 s
149.0 s
98.7 d
140.2 s
122.9 s
27.1 t
58.5 s
130.3 s
138.8 s
148.9 s
98.7 d
140.3 s
122. s
27.1 t
58.4 s
130.2 s
138.8, s
148.9 s
98.7 d
140.3 s
122.9 s
27.0 t
55.3 s
57.7 s
129.7 s
138.8 s
149.1 s
98.8 d
140.8 s
122.9 s
23.2 t
8
131.0 s
139.8 s
150.9 s
99.9 d
140.4 s
124.2 s
28.0 t
133.3 s
120.1 d
106.5 d
153.3 s
133.3 s
134.5 s
23.7 t
9
10
11
12
13
14
15
28.3 d
47.9 d
66.3 t
26.9 d
45.6 s
63.8 t
33.9 t
33.7 d
45.9 d
64.5 t
33.8 d
45.7 d
64.5 t
34.5 d
44.4 d
61.5 t
39.6 d
74.9 s
63.7 t
16
42.9 d
72.7 t
17
18
13.0 q
114.2 d
139.8 s
50.2 t
56.2 t
67.4 t
56.8 t
67.2 t
58.4 t
57.0 t
19
119.6 d
140.6 s
48.3 t
116.4 d
142.7 s
46.0 t
120.1 d
139.6 s
45.9 t
116.0 d
143.1 s
46.0 t
118.3 d
146.3 s
47.1 t
120.1 d
140.8 s
46.3 t
20
21
9-OCH3
10/11-OCH3
12-OCH3
18-OCH3
57.2 q
57.4 q
62.0 CH3
56.5 q
56.5 q
61.0 q
56.5 q
56.5 q
61.1 q
57.2 q
56.4 q
56.4 q
61.0 q
56.4 q
56.4 q
61.0 q
57.0 q
56.4 q
56.4 q
61.1 q
56.3 q
61.1 q
a
Compounds 1 and 6 in acetone-d6, 2, 3, 4, 5 and 7 in DMSO.
(C-19). Analysis of the HMBC and HSQC spectroscopic data of 2 further
confirmed that its molecule skeleton was identical to that of 1. The
Alkaloid 4 had the same molecular formula C22H28N2O6 as 2 as
determined by HREIMS. The 1H, 13C NMR and DEPT spectra
displayed were similar to those of 2 except for a downfield methine
ROESY correlations of H-18/H-15 (
H 2.82) indicated the C-19/C-20 double bond E-configuration and the
H-16 -orientation. Therefore, 2 was named gardmutine B.
dH 2.65) and H-16 (dH 2.07 m)/H-5
(d
signal of dC 33.7 instead of dC 26.9 (C-15) in 2. The HMBC and HSQC
b
correlations supported that the alkaloids 4 and 2 (Supporting
Information) possessed identical plane structures. The 1H, 13C NMR
and DEPT spectra of 5 showed 23 carbon signals with an additional
methoxy (dC 57.0 and dH 3.16) compared to 4. The HMBC
correlation of 5 between H-19 (dH 5.10) with this methoxy signal
placed it connected with C-18. However, the ROESY correlations of
H-19 to H-15 in 4 and 5 indicated the Z-configuration of the C-19/
20 double bond. Thus, 4 and 5 were named gardmutines D and E,
respectively.
Alkaloid 3 was assigned the molecular formula C29H40N2O11 by
HREIMS. The 1H, 13C NMR and DEPT spectra displayed a sugar unit
(glucose) on the basis of an anomeric methine [dH 4.09 (d,
J = 7.8 Hz) and dC 103.3 d], a methylene group (dC 61.1), and four
other methine signals between dC 70 and 77. The J values of the
anomeric proton of the sugar moieties revealed the configuration
of the glucosyl residue. The identification of the sugar residue was
continued by hydrolysis with 10% HCl to afford
was confirmed by comparison with authentic samples and the
determination of their optical rotation values ([
20 = +25.58)
D-glucose, which
The 1H NMR spectral data of 6 showed a bis-substituted indole
ring A [dH 7.27 (d, J = 8.0 Hz) and 6.55 (d, J = 8.0 Hz)]. Additionally,
a]
D
(Eskander et al., 2005). Apart from the sugar unit, the 1H, 13C NMR
and DEPT spectra of 3 showed 23 carbon signals with an additional
methoxyl (dC 57.2 and dH 3.18) compared to 2. The HMBC
correlation from dH 5.15 (H-19) with this methoxy signal
determined that it is connected with C-18. The glycosyl position
was unambiguously determined to be at C-17 from the correlation
its molecular formula C21H26N2O5 determined by HREIMS
indicated the absence of a methoxyl group compared with 4.
The 13C NMR and DEPT spectral data of 6 were similar to those of 4
with the exception of the indole-ring signals (Table 2). The HMBC
correlation of
methoxyls at C-11 and C-12. Correlations of
19) with H 2.20 (1H, m, H-15) and of H 4.00 (1H, H-18b) with dH
3.80 (1H, d, J = 17.0 Hz, H-21b) in the ROESY spectrum indicated
d
H 7.27 with
d
C 55.3 (C-7) placed the two neighboring
d
H 5.25 (t, J = 5.0 Hz, H-
of the anomeric proton with C-17 (
d
C 72.7) in the HMBC spectrum.
d
d
The compound 3 was named gardmutine C.
Fig. 2. Key HMBC and ROESY correlations of 1.

58
X.-H. Zhong et al. / Phytochemistry Letters 10 (2014) 55–59
Table 3
Anhui University of Traditional Medicine. A voucher specimen (No.
Liu20120720) has been deposited at Kunming Institute of Botany,
Chinese Academy of Sciences.
Cytotoxicity data of the alkaloids (IC50
,
m
M).
MCF-7
Compd.
HeLa
SW480
4
4.52 ꢁ 0.42
8.10 ꢁ 0.36
1.67ꢁ 0.21
13.63 ꢁ 0.45
1.37 ꢁ 0.10
3.01 ꢁ 0.14
19.63 ꢁ 1.59
3.3. Extraction and isolation
5
2.52 ꢁ 0.12
Cisplatin
11.84 ꢁ 1.04
The dried whole-plant of G. multiflora (10 kg) was extracted
three times with 90% MeOH (25 L each). The extract was
partitioned between EtOAc and a 0.5% HCl solution three times.
The acidic water layer, adjusted to pH 9–10 with a 10% ammonia
solution, was extracted with EtOAc to give an alkaloidal extract
(34 g). The total alkaloid was subjected to a silica gel column
(CHCl3/acetone, 10:1 to 1:1) to give the five fractions I–V. Fraction I
(0.5 g) was separated by silica gel CC (petroleum ether/Me2CO,
4:1–1:1), then by HPLC, eluted with MeOH/H2O (60–75%), to give
vallesiachotamine (35.7 mg). Fraction II (1.2 g) was separated by
silica gel CC (petroleum ether/Me2CO, 2:1 to 1:1), to give a mixture
A. Fraction III (4.7 g) was subjected to a C18 silica gel column and
eluted with MeOH/H2O (2:5–4:5, v/v) to yield fraction III-1,
fraction III-2 and the mixture of compounds B. A and B were further
purified by RP-18 CC (MeOH/H2O, from 3:20 to 3:10, v/v) to yield
cantleyine (260.1 mg). Chitosenine (222.1 mg) was crystallized in
methanol from fraction III-1. Fraction III-1 (107.5 mg) was further
purified by HPLC (MeOH/H2O, from 50% to 60%). Fraction III-2
(1.2 g) was separated by a C18 column (MeOH/H2O, 2:5–13:20, v/
v) to afford fraction III-2-1 (19 mg) and fraction III-2-2 (27 mg).
Fraction III-2-1 and fraction III-2-2 were further purified by HPLC
(MeOH/H2O, from 52% to 63%) to yield 18-demethoxygardner-
amine (6.4 mg) and 18-demethoxygardfloramine (8.7 mg), respec-
tively. Fraction IV (19 g) was subjected to RP-18 (MeOH/H2O, from
1:5 to 4:5, v/v), yielding three subfractions (IV-1–IV-3). Fraction
IV-1 (5.4 g) was applied to MPLC (MeOH/H2O, 1:9-7:20, v/v) to
offer fraction IV-1-1 and fraction IV-1-2. Fraction IV-1-1 was
further purified by HPLC to yield 7 (22.4 mg), 4 (8.2 mg), Alkaloid M
that the C-19/C-20 double bond had the Z-configuration. Thus, 6
was named gardmutine F.
The 13C NMR and DEPT data of 7 were closed to those of
chitosenine with the exception of an additional downfield
methylene at dC 57.0 and the absence of a methyl signal at dC
13.0 (C-18) in chitosenine (Aimi et al., 1978). The ROESY
correlations of H-19 (dH 5.10) to H-15 (dH 2.13) in 7 indicated
the Z-configuration of the C-19/20 double bond. Thus, 7 was named
18-hydroxy-chitosenine.
Other known alkaloids were identified, such as vallesiachota-
mine (Djerassi et al., 1966), cantleyine (Massiot et al., 1992),
chitosenine (Sakai et al., 1975), 18-demethoxygardneramine (Aimi
et al., 1978), 18-demethoxygardfloramine (Sakai et al., 1987),
gardfloramine (Sakai et al., 1987), 18-demethylgardneramine
(Aimi et al., 1978), gardneramine (Aimi et al., 1978), N4-oxide
gardneramine (Sakm et al., 1977), Alkaloid M (Sakm et al., 1977),
Alkaloid N (Sakm et al., 1977), Alkaloid I (Sakm et al., 1977),
Alkaloid J (Sakm et al., 1977), bemethoxygardmultine (Sakai et al.,
1982), and gardmultine (Sakm et al., 1977) by comparison of their
NMR spectroscopic data with that in the literature. Alkaloids 1–6
are the first Gardneria alkaloids possessing the 7S-configuration.
The cytotoxicities of all of the alkaloids against three human cancer
cell lines were evaluated by using the MTT method. Only alkaloids
4 and 5 exhibited inhibitory effects against these three cell lines.
These are the first Gardneria alkaloids discovered with moderate
cytotoxicity (Table 3).
(10.5 mg), and
combined and purified by HPLC (MeOH/H2O: 50–70%) to yield
18-demethylgardneramine (5.8 mg), Alkaloid (3.7 mg),
2 (6.6 mg). Fractions IV-1-2 and IV-2 were
3. Experimental
N
3
3.1. General experimental procedures
(11.5 mg), 5 (21.7 mg), Alkaloid J (3.9 mg), Alkaloid I (39.6 mg),
and 1 (6.4 mg). Fraction IV-3 (6.2 g) was subjected to a C18 silica
gel column and eluted with MeOH/H2O (1:4–3:5, v/v) to give
fraction IV-3-1 (1.37 g) and fraction IV-3-2 (0.11 g). Fraction IV-3-1
was further purified by HPLC (MeOH/H2O: 55–75%) to yield
gardneramine (8.6 mg) and 6 (11.5 mg). Fraction IV-3-2 was
purified by HPLC (MeOH/H2O: 60–80%) to yield gardfloramine
(27.9 mg) and gardneramine (6.5 mg). Fraction V (3.9 g) was
subjected to RP-18 (MeOH/H2O, from 1:5 to 13:20, v/v), affording
to 2 subfractions (V-1–V-2). Subfraction V-1 and fraction V-2 were
further purified by HPLC (MeOH/H2O, from 70% to 80%) to yield
bemethoxygardmultine (2.5 mg) and gardmultine (3.3 mg), re-
Optical rotations were measured with an Horiba SEPA-300
polarimeter. UV spectra were obtained using a Shimadzu UV-
2401A spectrometer. IR spectra were obtained with a Bruker FT-IR
Tensor 27 spectrometer using KBr pellets. 1D and 2D NMR
spectroscopic data were run on a Bruker AVANCE III-600, DRX-500,
and AM-400 MHz spectrometer with TMS as an internal standard.
ESI–MS spectra were measured on a Bruker HTC/Esquire spec-
trometer, and HREIMS was recorded on a Waters Auto Premier
P776 spectrometer. Column chromatography (CC) was performed
on silica gel (200–300 mesh, Qingdao Marine Chemical, Ltd.,
Qingdao, People’s Republic of China), RP-18 gel (20–45
m
m, Fuji
spectively.
17
Silysia Chemical Ltd., Japan). Fractions were monitored by TLC (GF
254, Qingdao Haiyang Chemical Co., Ltd., Qingdao), and spots were
visualized by Dragendorff’s reagent. Medium-pressure liquid
chromatography was employed using a Buchi pump system coupled
with a C18-silica gel-packed glass column (15 mm ꢂ 230 mm and
26 mm ꢂ 460 mm, respectively). HPLC was performed using a
Waters 1525EF pumps coupled with an Xbridge/Sunfire analytical,
semipreparative, or preparative C18 column (150 mm ꢂ 4.6 mm,
150 mm ꢂ 10 mm, and 250 mm ꢂ 19 mm, respectively). The HPLC
system employed a Waters 2998 photodiode array detector and a
Waters fraction collector III.
Gardmutine A (1): White powder; [
a
]
ꢀ18.2 (c 0.14, MeOH);
D
UV (MeOH) lmax (log
e
) 313 (2.73), 256 (2.94), 203 (3.56) nm; IR
(KBr) nmax 3440, 2925, 1692, 1633, 1497, 1245 cmꢀ1
;
1H
(400 MHz) and 13C NMR (100 MHz) data (acetone-d6), see Tables
1 and 2, respectively; HREIMS m/z 400.1992 (calcd for C22H28N2O5
[M]+., 400.1998, error: 1.5 ppm).
17
Gardmutine B (2): White powder; [
UV (MeOH) lmax (log
(KBr)
a
]
ꢀ23.4 (c 0.12, MeOH);
D
e
) 313 (2.88), 253 (3.11), 203 (3.75) nm; IR
n
max 3435, 2930, 1690, 1633, 1498, 1247 cmꢀ1; 1H (400 MHz)
and 13C NMR (100 MHz) data (DMSO), see Tables 1 and 2,
respectively; positive ESIMS m/z 417 [M + H]+, HREIMS m/z
416.1956 (calcd for C22H28N2O6 [M]+., 416.1947, error: 2.2 ppm).
17
D
3.2. Plant material
Gardmutine C (3): Colorless oil; [
(MeOH) max (log
max 3440, 2925, 1703, 1640, 1489, 1244 cmꢀ1; 1H (600 MHz) and
13C NMR (150 MHz) data (DMSO), H 4.09 (1H, d, J = 7.8 Hz, H-10),
a
]
ꢀ32.4 (c 0.13, MeOH); UV
l
e) 313 (2.91), 252 (3.10), 203 (3.78) nm; IR (KBr)
The whole herbs of G. multiflora were collected from Yellow Mount,
Anhui Province, P.R. China and authenticated by Prof. Shou-Jin Liu,
n
d

X.-H. Zhong et al. / Phytochemistry Letters 10 (2014) 55–59
59
3.70 (2H, overlap, H-60), 3.11 (1H, overlap, H-50) 3.08 (1H, overlap,
H-30), 3.06 (1H, overlap, H-40), 2.96 (1H, m, H-20), C 103.3 (d, C-10),
76.9 (d, C-50), 76.8 (d, C-30), 73.5 (d, C-20), 70.0 (d, C-40), 61.1 (t, C-
60), others see Tables 1 and 2, respectively; positive ESIMS m/z 593
[M + H]+, HREIMS m/z 592.2617 (calcd for C29H40N2O11 [M]+.,
0.0624, 0.32, 1.6, 8, and 40
m
M in triplicate for 48 h, with cisplatin
d
(Sigma, USA) as a positive control. After compound treatment, the
cell viability was detected (Table 3), and the cell-growth curve
was graphed. The IC50 value was calculated by Reed and Muench’s
method.
592.2632, error: 2.5 ppm).
17
D
Gardmutine D (4): Colorless oil; [
a]
ꢀ24.7 (c 0.11, MeOH); UV
Acknowledgements
(MeOH) max (log e) 313 (3.03), 253 (3.24), 203 (3.89) nm; IR (KBr)
l
n
max 3440, 2925, 1688, 1639, 1498, 1247 cmꢀ1; 1H (400 MHz) and
This project was supported in part by the National Natural
Science Foundation of China (21172225, 31370377), the Young
Academic and Technical Leader Raising Foundation of Yunnan
Province (No. 2010CI049), and XiBuZhiGuang Project of Chinese
Academy of Sciences. We are grateful to Prof. Dr. Shou-Jing Liu,
Anhui University of Traditional Medicine, for the collection and
identification of samples.
13C NMR (100 MHz) data (DMSO), see Tables 1 and 2, respectively;
positive ESIMS m/z 417 [M + H]+, HREIMS m/z 416.1939 (calcd for
C
22H28N2O6 [M]+., 416.1947, error: 1.9 ppm).
17
Gardmutine E (5): White powder; [
a]
ꢀ17.5 (c 0.14, MeOH);
D
UV (MeOH) lmax (log
e
) 312 (2.88), 252 (3.14), 204 (3.74) nm; IR
(KBr) nmax 3425, 2927, 1711, 1699, 1642, 1498, 1245 cmꢀ1 1H
;
(400 MHz) and 13C NMR (100 MHz) data (DMSO), see Tables 1 and
2, respectively; positive ESIMS m/z 431 [M + H]+, HREIMS m/z
430.2115 (calcd for C23H30N2O6 [M]+., 430.2104, error: 2.6 ppm).
References
17
Gardmutine F (6): White powder; [
a
]
ꢀ53.2 (c 0.11, MeOH);
D
UV (MeOH) lmax (log
e
) 221 (3.79), 203 (3.72) nm; IR (KBr) nmax
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17
D
18-Hydroxy-chitosenine (7): White powder; [
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