Phytochemistry Letters 10 (2014) 7679

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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Bioactive alkaloids from Palhinhaea cernua

Dong-Bo Zhang a, Jian-Jun Chen a, Li Zhang a, Qiu-Yan Song a, Kun Gao a,b,*
a
b

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, PR China

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 23 May 2014
Received in revised form 30 July 2014
Accepted 5 August 2014
Available online 19 August 2014

In screening for acetylcholinesterase (AChE) inhibitors and cytotoxic constituents from medicinal plants,
a bioactivity-guided isolation of the alkaloidal extract of the whole plant of Palhinhaea cernua led to the
isolation of six lycopodium alkaloids. Their structures were elucidated by spectroscopic methods (IR, MS
and NMR). The absolute conguration of 1 was established by computational methods. To our best
knowledge, compound 1 was a rare cernuane-type alkaloid bearing an oxygen bridge between C-12 and
C-15 in Lycopodiaceae. Among these alkaloids, compound 5 was the most active against AChE with IC50
value of 1.9 mM, and the new compound 1 showed weak inhibitory activity with IC50 value of 102 mM.
Moreover, the new compound 1 and the known one 3 inhibited A549 cell line with IC50 values of 12.6 and
18.4 mM, respectively. Meanwhile, preliminary structureactivity relationships were discussed in this
study.
2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords:
Palhinhaea cernua
Cernuane alkaloid
Acetylcholinesterase
Cytotoxicity
Structureactivity relationships

1. Introduction
Palhinhaea cernua (Lycopodiaceae) is mainly used to heal
contusions, scald and rheumatism in traditional medicine and is
broadly distributed in Eastern and Southern China (Zhang et al.,
1997). Previous phytochemical investigations on P. cernua have
resulted in the isolation of various secondary metabolites, mainly
those of lycopodium alkaloids (Zhao et al., 2010; Dong et al., 2013)
and serratene triterpenoids (Yan et al., 2012). In addition, pcoumaroylated apigenin glycosides, dillenetin, rhamnazin, aonocerin, b-sitosterol and (E)-2-hydroxy-5-methoxycinnamic acid
were isolated from an ethanol extract of the whole plant of P.
cernua (Jiao et al., 2006). However, the biological activities of the
compounds of this plant have not been reported adequately. In our
continuing search for active compounds from medicinal plants, the
alkaloidal extract of the whole plant of P. cernua showed
remarkable AChE inhibitory activity and moderate cytotoxicity.
The bio-guided chromatographic fractionation of the alkaloidal
extract led to the isolation of one new compound (1) and ve
known lycopodium alkaloids (26). Reported herein are the
isolation, structural elucidation, AChE inhibitory activity and

* Corresponding author at: State Key Laboratory of Applied Organic Chemistry,

College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou
730000, PR China. Tel.: +86 0931 8912592; fax: +86 931 8912582.
E-mail address: npchem@lzu.edu.cn (K. Gao).

cytotoxicity of these compounds. Furthermore, preliminary

structureactivity relationships were discussed.
2. Results and discussion
2.1. Phytochemical investigation
Compound 1, obtained as a white powder, had a molecular
formula of C16H24N2O2 by the HRESIMS ion peak at m/z 277.1915
[M+H] + (calcd for C16H25N2O2, 277.1911) with six degrees of
unsaturation. The IR absorption at 1616 cm1 gave a suggested the
presence of amidic carbonyl. In agreement with its molecular
formula, all the sixteen carbon signals were observed in the 13C
NMR spectrum (Table 1), and were further classied by DEPT
experiments into one methyl, eight methylenes, ve methines, and
two quaternary carbons. Among these, four methines (dC 48.3,
53.0, 62.2, 66.3) were ascribed to those carbons bearing a nitrogen
atom. One methine at dC 74.8 (C-12) and one quaternary carbon at
dC 80.7 (C-15) were accounted for those carbons attached to an
oxygen atom. The 1H NMR spectrum of 1 displayed one singlet
methyl group at dH 1.36 (3H, s, H-16), one oxymethine group at dH
4.01 (1H, br d, J = 5.8 Hz, H-12), four N-methine groups at dH 3.35
(1H, br s), 3.56 (1H, m), 3.62 (1H, m) and 5.57 (1H, dd, J = 11.2,
5.3 Hz). Comparison of the 1H and 13C NMR data (Table 1) of 1 with
those of lycocernuine (2) indicated that the two structures are
closely related. The main differences were the presence of
one oxygenated quaternary carbon (dC 80.7) in place of a methine

http://dx.doi.org/10.1016/j.phytol.2014.08.008
1874-3900/ 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

D.-B. Zhang et al. / Phytochemistry Letters 10 (2014) 7679

Table 1
1
H (400 MHz),
Position

13

C (150 MHz) and DEPT NMR data of compounds 1 and 2 in CDCl3, and some HMBC correlations of 1.
1

dC (DEPT)
1
2a
2b
3a
3b
4a
4b
5
6a
6b
7
8a
8b
9
10a
10b
11a
11b
12
13
14a
14b
15
16
a

77

168.8 (C)
32.8 (CH2)
20.0 (CH2)
30.7 (CH2)
53.0 (CH)
39.1 (CH2)
48.3 (CH)
43.0 (CH2)
66.3 (CH)
13.9 (CH2)
24.8 (CH2)
74.8 (CH)
62.2 (CH)
44.2 (CH2)
80.7 (C)
25.7 (CH3)

dH (J in Hz)
2.37
2.46
1.62
1.85
1.56
1.96
3.56
1.38
1.64
3.62
1.39
1.71
5.57
1.38
2.24
1.99
2.26
4.01
3.35
1.78
1.84

(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(dd, 11.2, 5.3)
(m)
(m)
(m)
(m)
(br d, 5.8)
(br s)
(m)
(m)

1.36 (s)

HMBC

dC (DEPT)

1, 3, 4

168.5 (C)
33.1 (CH2)

1, 2, 4, 5

19.2 (CH2)

2, 3, 5, 6

30.5 (CH2)

3, 4, 6, 7, 9
4, 5,7, 8

51.0 (CH)
41.6 (CH2)

5, 6, 9, 15
6, 14, 15, 16

49.1 (CH)
41.7 (CH2)

1, 5, 7, 10, 13
9, 11, 12

67.2 (CH)
15.9 (CH2)

9, 10, 12, 13

33.7 (CH2)

10, 14, 15
7, 12, 15
8, 12, 15, 16

71.0 (CH)
58.6 (CH)
37.9 (CH2)

8, 14, 15

26.3 (CH)
23.0 (CH3)

dH (J in Hz)
2.33
2.42
1.60
1.81
1.47
1.94
3.53
1.16
1.75
3.51
0.83
1.69
5.45
1.22
2.33
1.91
1.98
3.80
2.96
1.48
1.79
2.31
0.87

(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(m)
(dd, 10.8, 5.6)
(m)
(m)
(m)
(m)
(br s)
(br d, 6.8)
(m)
(m)
(m)
(d, 6.8)

HMBC correlations from proton to carbon.

(dC 26.3) in 13C NMR, meanwhile the presence of one singlet methyl
group (dH 1.36) instead of a doublet methyl group (dH 0.87) in 1H
NMR. These ndings showed that oxygenated quaternary carbon
(dC 80.7) might be localized at C-15 in 1. From the six degrees of
unsaturation of compound 1, apart from one carbonyl and four
rings, the remaining one-degree of unsaturation was assumed to
due to an oxygen ring that must be formed between C-12 (dC 74.8)
and C-15 (dC 80.7). Moreover, the HMBC cross peaks of C-15 with
H-12, H-7, H-13 and H-16; H-12 with C-14 and C-10 conrmed the
inference (Fig. 1). Consequently, the gross structure of 1 was
deduced to be as shown in Fig. 2.
The relative conguration of compound 1 was deduced from
NOE difference experiment. In biogenetic consideration of
ceruane-type alkaloid derivatives isolated from Lycopodiaceae

Fig. 1. Key HMBC correlations (H!C) and 1H1H COSY of 1 (

).

family, the protons at C-9 and C-13 both possess b-orientation

(Morita et al., 2004). Irradiation of H-13 enhanced the signals of H9 (3.88%), H-12 (2.39%), and H-16(2.26%) which therefore the
epoxy group at C-12 and C-15 was suggested to be an aorientation. The H-5 and H-7 were also a-oriented because of the
NOE correlation of H-5 with H-7. Accordingly, compound 1 was
assigned as 12a, 15a-epoxycernuine (Fig. 2).
The absolute conguration of 1 was established by computational methods. Recently, Gaussian package has become a popular
method to predict the optical rotation in chiral rigid compounds
(Liu et al., 2006; Stephens et al., 2008). For the assignment of
absolute conguration, the optical rotation values for
(5S,7S,9S,12R,13R,15S)-1 and its enantiomer were calculated and
were further compared with the experimental data for 1. The
geometries of the molecules were optimized with Gaussian 09
package (Frisch et al., 2009) at B3LYP/6-31G (d) computational
level. The minimum nature of the structure was conrmed by
frequency calculations at the same computational level. These
geometries were then used to evaluate the optical rotation at
B3LYP/6-311++G (d, p) computational level. All optical rotation
calculations were performed using PCM solvation model and the
chloroform was specied as the solvent which was in agreement
with the experiment condition. The calculated optical rotation for
(5S,7S,9S,12R,13R,15S)-1 was 11.998 and that for its enantiomer
was +19.598. The former was close to the experimental value of
208 for 1 (Fig. 2), which strongly suggested that the absolute

Fig. 2. The structures of compounds 16.

78

D.-B. Zhang et al. / Phytochemistry Letters 10 (2014) 7679

conguration of 1 was 5S/7S/9S/12R/13R/15S. Finally, the structure

of 1 was elucidated and named as Palhinua A.
In addition to the new compound 1, ve other known
compounds were isolated and identied as lycocernuine (2) and
cernuine N-oxide (3) (Morita et al., 2004); huperzinine (4) and Ndemethylhuperzinine (5) (Shen and Chen, 1994); b-obcurine (6)
(Nakashima et al., 1975) by comparison of their physical and
spectroscopic data with those reported in the literatures.
Lycopodium alkaloids are a group of chemically and pharmacologically interesting secondary metabolites. Many of them
continue to be the hot points because of complex polycyclic
structures and biological activities. For example, the well-known
huperzine A, potent selective AChE inhibitor, has been studied well
so far (Liu et al., 1986). During a search for active compounds from
medicinal plants, one new and ve known lycopodium alkaloids
were obtained. Among these, compounds 13 possessed a rare
cernuane-type skeleton consisting of a fused tetracyclic ring
system containing two nitrogen atoms, with a methyl group at C15 (Braekman et al., 1974). To the best of our knowledge, the new
compound 1 was the rst reported example of cernuane-type
alkaloid with the epoxy group at C-12 and C-15.
2.2. AChE inhibiting activity and cytotoxicity
Initially, methanol crude extract of the whole plant of P. cernua
showed signicant AChE inhibitory activity and moderate
cytotoxicity. The alkaloidal extract was found more active than
the methanol extract. Compounds 16 were tested for inhibitory
activity against AChE by Ellmans method in 96-well microplates
(Ellman et al., 1961; Orhan et al., 2004). Huperzine A was used as
the positive control with an IC50 value of 74 nmol. Within alkaloids
tested, compound 5 showed the most potent inhibition against
AChE with IC50 value of 1.9 mM which was consistent with
previous report (Zhang et al., 2014), and the new compound 1
exhibited weak inhibitory activity against AChE with IC50 value of
102 mM.
Moreover, the isolated alkaloids (16) were evaluated for in
vitro cytotoxicity by MTT assay using paclitaxel (IC50
2.26  103 mM) as a positive control. The results showed that
compound 1 and 3 inhibited A549 cell line with IC50 values of 12.6
and 18.4 mM respectively, while the rest alkaloids showed no or
weak cytotoxicity. The observed cytotoxicity of compound 1, 2, 3,
indicated that the presence of the epoxy group at C-12 and C-15
and an N-oxide group might be important in the cytotoxicity of
cernuane-type alkaloids.
3. Materials and methods
3.1. Plant material
The whole plant of P. cernua were collected in Tuncang County
of Hainan Province, China, in July 2009, and identied by Qiongxin
Zhong of Hainan Normal University. The voucher specimen (No.
2009019) was deposited in the State Key Laboratory of Applied
Organic Chemistry, Lanzhou University, China.
3.2. General
Optical rotations were measured on a Perkin-Elmer 341
polarimeter. IR spectra were recorded on a 170SX FT-IR instrument
using KBr discs over the range of 4004000 cm1. UV spectra were
measured using a Shimadzu UV-260 spectrophotometer. NMR
spectra were recorded on a Bruker AM-400 NMR spectrometer
using TMS as an internal standard. High-resolution electro spray
ionization mass spectra (HRESIMS) were measured on a Bruker
Daltonics APEX II 47e spectrometer. Column chromatography was

performed on silica gel (200300 mesh, Qingdao Marine Chemical

Inc., Qingdao, Peoples Republic of China) and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Sweden). Fractions were monitored by
TLC, which were visualized by heating the silica gel GF254 plates
(1040 mm, Qingdao Marine Chemical Inc., Qingdao, China) after
being sprayed with 5% H2SO4 in EtOH.
3.3. Extraction and isolation
An air-dried and powdered sample (2 kg) of the whole plant of
P. cernua was extracted with 95% MeOH (7 d  3 times). The
extract was partitioned between EtOAc and 2% HCl solution. The
acidic water-soluble materials, adjusted to pH 910 with 10%
ammonia solution, were extracted with CHCl3 to give an alkaloidal
extract (3.1 g). The extract was subjected to MCI gel column
chromatography (H2O/MeOH, 1:0 to 0:1) to afford eight crude
fractions (Fr. 18). Fr.7 (1.3 g) was subjected to a silica gel column
(EtOAc/MeOH/Et2NH, 20: 1: 0.02) to give 1 (3.2 mg) and 2
(489.3 mg). Fr.5 (0.9 g) was chromatographed on a silica gel
column (CHCl3/EtOAc/MeOH/Et2NH, 5:5:1:0.01) to yield 4
(20.0 mg), 5 (2.6 mg) and 6 (10.1 mg). Compound 3 (2.2 mg)
was obtained from Fr.3 (0.5 g) after purication by a Sephadex LH20 column (CHCl3/MeOH, 1:1), and nally applied to silica gel
column
chromatography
(CHCl3/EtOAc/MeOH/Et2NH,
2:2:1:0.005).
3.4. Characteristic data of compound 1
Palhinua A (1): white powder; [a]D25 208 (CHCl3, c 1.0); IR
(KBr): nmax 2938, 2869, 1616, 1449, 1374 cm1; UV (MeOH) lmax
202 nm; HRESIMS m/z: 277.1915 [M+H] + (calcd. for 277.1911); 1H
NMR, 13C NMR, and DEPT data see Table 1.
3.5. Assay of AChE inhibition
The six alkaloids were tested for AChE inhibitory activities by
the modied Ellmans method in 96-well microplates (Ellman
et al., 1961; Orhan et al., 2004). Briey, 140 mL of 0.1 M sodium
phosphate buffer (pH = 8.0), 20 mL sample solution and 15 mL
enzyme solution were mixed and incubated at 4 8C for 20 min.
10 mL of 0.01 M DTNB was added and the reaction was then started
by adding 10 mL of 0.075 M ATCI. After incubating the reaction
solution at 37 8C for 20 min, the optical densities were measured in
a 96-well plate reader at 405 nm immediately. A blank positive
control was set up by adding 20 mL huperinze A (0.100 mg/mL in
phosphate buffered saline) instead of 20 mL sample solution.
Blanks were set up by adding 20 mL buffer solutions instead of
20 mL sample solution. Experiment control was set up by adding
15 mL buffer solutions instead of 15 mL enzyme solution in order to
deduct the sample background. All reactions were carried out
thrice. The inhibition rate (%) was calculated by the following
equation (Table 2):
Table 2
AChE inhibiting activity and cytotoxicity of compounds 16.
Compound

1
2
3
4
5
6
a
b

AChE inhibiting
activity

Cytotoxicity against
A549 cell line

IC50 (mM)a

IC50 (mM)a

102
b
b
b
1.9
b

12.6
>50
18.4
b
b
b

IC50 represents the 50% inhibitory concentration.

No or very weak activity.

D.-B. Zhang et al. / Phytochemistry Letters 10 (2014) 7679

Inhibition% Blank  Blankpositivecontrol

79

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 Experiment  Experimentcontrol=
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The concentration of test samples that inhibited the hydrolysis
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3.6. Cytotoxicity assay
The isolated alkaloids (16) were evaluated for their cytotoxicity by the MTT assay. Generally, Cells (1  105) were incubated
with compounds in triplicate in a 96-well plate for the indicated
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Acknowledgements
The research work was nancially supported by National
Natural Science Foundation of China (Nos. 31270396 and
21002046).
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.phytol.2014.08.008.

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