Two new sesquiterpenes from Artemisia sieversiana

Article abstract of DOI:10.1016/j.fitote.2014.05.007

Two new sesquiterpenes, together with 32 known compounds(3-34), were isolated from Artemisia sieversiana Ehrhart ex willd. and the compounds 3-21 were isolated from this plant for the first time. The new compounds were elucidated as 2α,9α-dihydroxymuurol-3(4)-en-12-oic acid (1) and 13α-methyl-(5αH,6αH,7αH,8αH)-austricin 8-O-β-D-glucopyranoside (2), respectively. The structural identification of these compounds was mainly achieved by spectroscopic methods including 1D and 2D NMR techniques, and the structure of compound 1 was confirmed by a single crystal X-ray diffraction experiment. Compounds 1-2 were evaluated for cytotoxic activity in vitro against MCF-7, NCI-H460 and Hep-G2 cell lines, respectively.

Full text of DOI:10.1016/j.fitote.2014.05.007

Fitoterapia 97 (2014) 43–49
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Two new sesquiterpenes from Artemisia sieversiana
a
b
b
Shi-Jun Liu a,c, Zhi-Xin Liao a,c, , Chao Liu , Lan-Ju Ji , Hong-Fa Sun
⁎
a
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, PR China
Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new sesquiterpenes, together with 32 known compounds(3–34), were isolated from
Artemisia sieversiana Ehrhart ex willd. and the compounds 3–21 were isolated from this plant for
the first time. The new compounds were elucidated as 2α,9α-dihydroxymuurol-3(4)-en-12-oic
acid (1) and 13α-methyl-(5αH,6αH,7αH,8αH)-austricin 8-O-β-D-glucopyranoside (2), respec-
tively. The structural identification of these compounds was mainly achieved by spectroscopic
methods including 1D and 2D NMR techniques, and the structure of compound 1 was confirmed
by a single crystal X-ray diffraction experiment. Compounds 1–2 were evaluated for cytotoxic
activity in vitro against MCF-7, NCI-H460 and Hep-G2 cell lines, respectively.
Received 26 March 2014
Accepted in revised form 12 May 2014
Available online 24 May 2014
Keywords:
Artemisia sieversiana
Compositae
Sesquiterpene
Lignan
© 2014 Elsevier B.V. All rights reserved.
Flavone
Cytotoxic activity
1. Introduction
effects against MCF-7, NCI-H460 and Hep-G2 cancer cells are
described in this paper.
The genus Artemisia belongs to the tribe Anthemideae, family
of Compositae, comprising more than 186 species native to China
[1]. Many species have been used as folk medicines in China.
Artemisia sieversiana Ehrhart ex willd. is an annual herb that is for
the most part abundantly distributed in Qinghai, Gansu, Ningxia,
Shaanxi and Sichuan provinces. It grows on the roadside,
wasteland, floodplains, steppe and forest edges in altitudes of
500–4500 m [1], and it has been used for detumescence,
hemostasis and relieving heat in Tibetan medicines.
However, there have been few reports[2] on phytochem-
ical investigations of this plant up to now. To ascertain its
chemical composition and medicinal value, the petroleum
ether-Et2O-MeOH (1:1:1) extract of A. sieversiana was
investigated. Herein, the isolation and structural elucidation
of the two new sesquiterpenes as well as their cytotoxic
2. Experimental
2.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer-343
spectropolarimeter. IR spectra were recorded on a NICOLET
IR200 FT-IR spectrophotometer. NMR spectra were scanned on
a Bruker Avance DRX-500 spectrometer at 500 MHz (1H) and
125 MHz (13C). HR-ESI-MS was carried out on an Agilent
Technologies 6224 TOF LC-MS apparatus. Column chromatog-
raphy (CC) was performed using silica gel (200–300 mesh,
Qingdao Marine Chemical, Inc.). MCI GEL CHP20p (75–150 μm,
Mitsubishi Kasei Corporation), or Sephadex LH-20 (20–100 μm,
Pharmacia) was also used for CC. TLC was conducted on silica
gel GF254 plates (10–40 μm; Qingdao Marine Chemical, Inc.).
Petroleum ether (30–60 °C), ethyl acetate and other reagents
were purchased from Nanjing Wanqing Reagent, Inc. Spots
were observed by UV light as well as by spraying with 10%
H2SO4–EtOH followed by heating. The cell lines (MCF-7,
NCI-H460 and Hep-G2) were purchased from the center cell
⁎
Corresponding author at: Department of Pharmaceutical Engineering, School
of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189,
PR China. Tel.: +86 25 52090620; fax: +86 25 52090618.
E-mail address: zxliao@seu.edu.cn (Z.-X. Liao).
http://dx.doi.org/10.1016/j.fitote.2014.05.007
0367-326X/© 2014 Elsevier B.V. All rights reserved.

44
S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
resources of Shanghai Institute of Life Science Chinese Academy
of Sciences. Tumor cells were incubated in an HF-212UV CO2
incubator and observed under an OLYMPUS CKX41 inverted
microscope. Optical density (OD) values were read under a
BIO-RAD Model 680 microplate reader. Cisplatin (HPLC N98%)
was purchased from the National Institutes for Food and Drug
Control. The purity of the two new sesquiterpenes (1: 99.84%;
2: 99.12%) was analyzed by HPLC (Agilent 1260: DEACA01043),
which was carried out on a column (Agilent ZORBAX SB-C18;
4.6 × 250 mm, 5 μm) using methanol and 0.1% glacial acetic
acid–water soln. (v/v 80/20) at a flow rate of 1 mL/min within
about 35 min.
gel (100 g, column: 2.5 × 100 cm) with a petroleum ether–
ethyl acetate gradient (v/v 8:1–2:1) to yield 28 (6.00 mg),
15 (105 mg), 23 (100 mg) and 4 (3.00 mg) while Fr.4.2 (10.6 g,
petroleum ether–ethyl acetate 2:1–1:1) was subjected to
separation repeated by silica gel (150 g, column: 3 × 100 cm)
column chromatography eluted with petroleum ether–ethyl
acetate (v/v 4:1–0:1) to yield 5 (200 mg), 11 (10.0 mg),
10 (305 mg) and 14 (4.00 mg). After that, a repeated separation
of the residue over Sephadex LH-20 with chloroform–methanol
(1:1) resulted in 6 (5.00 mg), 7 (3.00 mg), 8 (4.00 mg),
33 (3.00 mg) and 36 (6.00 mg). Concerning to Fr.5 (60.4 g,
petroleum ether-ethyl acetate 0:1), it was fractionated by
column chromatography over silica gel (600 g, column: 6 ×
100 cm) with the gradient system of increasing polarity with
chloroform–methanol (v/v 50:1–0:1). In this case, four frac-
tions (Fr.5.1–Fr.5.4, TCL data) were obtained. Compounds
19 (2.00 mg), 27 (2.00 mg), 25 (3.00 mg) and 24 (3.00 mg)
were subsequently eluted from Fr.5.1 (14.4 g, chloroform–
methanol 50:1–20:1) when eluted with a chloroform–methanol
of increasing polarity (v/v 50:1–10:1) by column chromatogra-
phy over silica gel (150 g, column: 4 × 100 cm), and com-
pounds 16 (4.00 mg), 1 (25.0 mg) and 17 (12.0 mg) were
precipitated successively from Fr.5.2 (20.6 g, chloroform–
methanol 10:1–8:1) with increasing polarity of chloroform–
methanol (v/v 12:1–7:1) by column chromatography over silica
gel (200 g, column: 5 × 100 cm). Fr.5.3 (13.8 g, chloroform–
methanol 6:1–4:1) was subjected to a silica gel column (150 g,
column: 4 × 100 cm) eluting with chloroform–methanol
(v/v 8:1–3:1) to yield 29 (15.0 mg), 2 (20.0 mg), 12 (15.0 mg)
and 13 (26.0 mg), while Fr.5.4 (13.4 g, chloroform–methanol
3:1–0:1) was further purified on silica gel (150 g, column:
4 × 100 cm) with chloroform–methanol (v/v 4:1–1:2) to give
18 (108 mg), 20 (11.0 mg), 30 (15.0 mg) and 21 (21.0 mg).
2.2. Plant material
The dried aerial parts of A. sieversiana were collected in July,
2012, Heka town of Xinghai County in Qinghai Province, China.
It was identified by Prof. Hong-Fa Sun of the Northwest Institute
of Plateau Biology, Chinese Academy of Sciences, Xining, China,
and a voucher specimen (No. 12-07-01) was deposited at the
laboratory of Zhi-Xin Liao, Southeast University, Nanjing, China.
2.3. Extraction and isolation
The dried and powdered plant material (2.40 kg) of
A. sieversiana was percolated four times (7 days each time)
with petroleum ether–Et2O–MeOH (1:1:1) (8 L × 4) at room
temperature. The filtrates were consolidated and evaporated
in vacuum to give a concentrate (204 g).
The resultant extract (204 g) was subjected to column
chromatography (CC) on silica gel (2000 g, column: 10 ×
100 cm) with a petroleum ether–ethyl acetate gradient
(v/v 50:1–0:1). Five crude fractions (Fr.1–Fr.5) were obtain-
ed, which were combined according to TLC data. Specifically,
Fr.1 (55.4 g, petroleum ether-ethyl acetate 50:1–20:1) was
discarded without further separation because it was mainly
comprised of volatile oil components of low polarity. Fr.2
(25.4 g, petroleum ether–ethyl acetate 10:1–8:1) was
further separated by column chromatography over silica gel
(250 g, column: 5 × 100 cm) with a petroleum ether–ethyl
acetate gradient (v/v 20:1–4:1) to yield 22 (203 mg). For Fr.3
(6.70 g, petroleum ether–ethyl acetate 6:1–4:1), it was
rechromatographed over silica gel (100 g, column: 2.5 ×
100 cm) with petroleum ether–ethyl acetate (v/v 14:1–1:1)
mixtures to give two fractions (Fr.3.1–Fr.3.2, TCL data). In
detail, Fr.3.1 (2.10 g, petroleum ether–ethyl acetate, 14:1–
8:1) was further purified by column chromatography over
silica gel (40 g, column: 2 × 80 cm) with a petroleum ether–
ethyl acetate gradient (v/v 16:1–4:1) to yield 3 (3.00 mg) and 9
(5.00 mg) while Fr.3.2 (3.20 g, petroleum ether-ethyl acetate
6:1–1:1) being further separated over Sephadex LH-20 with
chloroform–methanol (2:1) to yield 31 (3.00 mg), 26 (4.00 mg)
and 32 (3.00 mg). In terms of Fr.4 (37.0 g, petroleum ether–
ethyl acetate 3:1–1:1), it was firstly purified over MCI GEL
eluted with 80% ethanol to remove the pigments. The remaining
materials were evaporated for further isolation. It (20.4 g)
was rechromatographed over silica gel (200 g, column: 5 ×
100 cm) with petroleum ether–ethyl acetate (v/v 6:1–1:1)
mixtures to give two fractions (Fr.4.1–Fr.4.2 TCL data). Fr.4.1
(5.40 g, petroleum ether–ethyl acetate 6:1–3:1) was further
separated by repeated column chromatography over silica
2.4. Compound characterization
2α,9α-dihydroxymuurol-3(4)-en-12-oic acid (1): Colorless
crystals (CHCl3–MeOH, 8:1), mp: 203–204 °C, Rf 0.60, silica gel
40 F254, CHCl3/MeOH (7:1), [α]20 = −75.3 (c = 1.00, MeOH),
D
IR (KBr):3400, 2920, 2880, 1700 cm−1
.
1H and 13C NMR
(DMSO) (see Table 1). HR-ESI-MS: m/z 267.1599 ([M − H]−,
C15H23O-4, calc. 267.1596).
13α-methyl-(5αH,6αH,7αH,8αH)-austricin 8-O-β-D-gluco-
pyranoside (2): white amorphous powder, mp: 306–307 °C, Rf
0.50, silica gel 40 F254, CHCl3/MeOH (7:1), [α]20 = +12.2
D
(c = 0.11, MeOH), IR (KBr): 3527, 1760, 1688, 1621 cm−1. 1H
and 13C NMR (DMSO) (see Table 1), HR-ESI-MS: m/z 425.1809
([M + H]+, C21H29O+9 , calc. 425.1813).
2.5. Cytotoxicity experiments
The cytotoxicity effects of compounds 1–2 against MCF-7,
NCI-H460 and Hep-G2 cells were tested using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay, and Cisplatin was used as a positive control.
The cell suspensions were distributed into 96-well cell
culture plates and cultured at 36–37 °C in a 5% CO2 incubator
for 24 h and each sample was dissolved with a limited
amount of DMSO, diluted to five different concentrations
with a culture medium and then added to the corresponding
well. The blank controls consisting of microbial culture were

S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
45
Table 1
Table 2
1H, 13C-NMR data of 1 and 2 at 500 and 125 MHz in DMSO at 30 °C (δ in
ppm; J in Hz).
The median inhibitory rate (IC50) (mean
and 2.
S.D., n = 5) of compounds 1
Position
1
2
Sample
Median inhibitory rate(IC50) (μM)
1H-NMR
13C-NMR 1H-NMR
30.9
13C-NMR
132.5
MCF-7a
NCI-H460a
Hep-G2a
1α
1β
1.53(1H, dd,
13.20,3.70)
1.46(1H, d, 12.95)
3.72(1H, t, 4.35)
1
2
26.8
31.4
27.3
0.6
0.8
0.5
48.7
43.4
30.9
1.2
1.2
0.7
34.5
40.0
15.2
0.9
1.1
0.2
Cisplatinb
2
3
4
5
6
66.8
194.8
134.7
170.4
50.4
a
Clinical strain.
Positive control.
135.6
128.2
34.9
6.16(1H, s)
b
5.73(1H, d,5.40)
2.34(1H, t, 5.55)
2.54(1H, dd,2.70,
7.00)
3.62(1H, d,10.05)
3.95(1H, t, 10.35)
39.7
80.7
C
15H24O4 based on the 1H- and 13C-NMR data and the
7α
7β
8α
8β
9α
9β
1.29(1H, m)
1.51(1H, m)
1.26(1H, m)
1.40(1H, m)
22.9
34.8
70.1
2.70(1H, dd,3.25,
10.40)
3.54(1H, t, 10.55)
55.8
74.6
45.0
quasi-molecular ion peak at m/z 267.1599 ([M − H]−, calc.
267.1596) in the HR-ESI-MS. The assignment was confirmed
with the aid of 2D-NMR (HSQC, HMBC and ROESY) spectra.
The NMR data further secured the structure. There were
three signals corresponding to methyl groups (δ 1.67, 1.05
and 0.99) in the 1H-NMR spectrum and 15 carbon signals
in the 13C-NMR spectrum. An olefinic proton at δ 5.73 (d, J =
5.40 Hz, 1H) with a pair of olefinic carbon signals at δ 128.2
and 135.6 indicated a double bond in the structure. The
proton signal at δ 11.90 (s, 1H) together with the carbon signal
at δ 176.7 revealed the presence of a carboxylic group. The
13C-NMR and HSQC of 1 also indicated that three methylene
(δC 30.9, C-1; 22.9, C-7; 34.8, C-8), four methine (δC 34.9, C-5;
39.7, C-6; 41.7, C-10; 39.9, C-11), one oxygen-bearing methine
(δC 66.8, C-2), and one sp3 oxygen-bearing quaternary C-atom
(δC 70.1, C-9) existed in structure 1. These data revealed that 1
was a muurolane-type sesquiterpene acid [3], and the proton
and carbon signals of 1 are shown in Table 1.
2.61(1H, dd,1.65,
11.30)
2.94(1H, dd, 7.35,
9.25)
10
11
12
13
14
15
1’
2’
3’
4’
5’
1.38(1H, m)
1.72(1H, m)
41.7
39.9
176.7
15.0
29.6
21.7
146.3
40.0
178.2
9.3
20.7
19.2
2.50(1H, m)
0.99(3H, d, 7.05)
1.05(3H, s)
1.67(3H, s)
1.15(3H, d, 7.60)
2.33(3H, s)
2.20(3H, s)
4.29(1H, d, 7.80) 104.9
2.95(1H, m)
3.16(1H, m)
3.05(1H, m)
3.15(1H, m)
3.45(1H, m)
3.69(1H, m)
73.6
76.8
70.1
76.6
61.1
6’α
6’β
In the HMBC spectrum (Fig. 1), the correlations of H-1
with C-3 and C-5; H-4 with C-2, C-6 and C-15 indicated that
the double bond was located between C-3 and C-4 and that
the hydroxyl group was linked to C-2. The correlation of H-14
with C-8 revealed that the methyl group (δC 29.6) and the
additional hydroxyl group were located at C-9. In addition,
the correlation of H-13 with C-12 suggested the presence of a
\CH(CH3)\COOH moiety, and the attachment position was
deduced to be C-6 because of the correlations of H-6 with
C-8, C-10, C-12 and C-13.
The relative configuration of 1 was determined by a ROESY
experiment. In the ROESY spectrum (Fig. 2), H-5 was coupled
with H-10, which suggested that H-5 and H-10 were α-axial
[3]. The ROESY correlations of H-1α and H-8α with H-10, H-1β
and H-8β with H-14, H-1β with H-2 in compound 1 indicated
that H-14 and H-2 were β-side. Consequently, the hydroxyl
group at C-2 and C-9 was α-equatorial. The structure of 1 was
also incubated with 0.02% DMSO under the same conditions,
and DMSO was not toxic at these limited amounts under the
experimental conditions. After 48 h of cultivation, MTT was
added to each well for another 4 h cultivation. Finally, the
supernatant was discarded, and 150 μl DMSO was added to
each well to completely dissolve the blue–violet crystals, then
the optical density (OD) values were then read on a microplate
reader. Origin7.5 computer programme (Data analysis and
Graphics Software, USA) was used to determine the median
inhibitory rate (IC50), and the results are presented in Table 2.
3. Results and discussion
Compound 1 was obtained as a colorless crystals (CHCl3–
MeOH, 8:1). The molecular formula of 1 was deduced to be
Fig. 1. Key HMBC (H → C) correlations of compounds 1–2.

46
S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
Fig. 2. Key ROESY correlations of compounds 1–2.
confirmed by X-ray crystallography (Fig. 3). On the basis of
the above analysis, compound 1 was identified as 2α,9α-
dihydroxymuurol-3(4)-en-12-oic acid.
Compound 2 was obtained as a white amorphous powder.
The molecular formula was determined to be C21H28O9 based
on the 1H- and 13C-NMR data and the quasi-molecular ion
peak at m/z 425.1809 ([M + H]+, calc. 425.1813) in the
HR-ESI-MS.
X-ray crystallographic analysis of 1: Colorless blocks,
C15H24O4, Mr = 268.34, tetragonal, space group P43212, a =
8.8251(10)Å, b = 8.8251(10)Å, c = 38.147(9)Å, α = 90.00°,
The 1H NMR spectrum (Table 1) of 2 displayed the signal
of an anomeric proton of D-glucose, the anomeric proton
doublet was well separated at δ 4.29 and its large coupling
constant (J = 7.80 Hz) indicated the β-D-glycosidic linkage
of the sugar moiety [4]. The 1H NMR spectrum (Table 1) of 2
displayed the signal of an anomeric proton of sugar, and acid
hydrolysis of 2 gave D-glucose identified by direct compar-
ison with the authentic sample. The J value of the anomeric
proton (J = 7.80 Hz) indicated the β-D-glycosidic linkage. In
addition, the 1H NMR spectrum showed three methyl protons
signals at δ 2.33 (3H, s), 2.20(3H, s) and 1.15 (3H, d, J =
7.60 Hz). 13C NMR data (Table 1) revealed the presence of 21
carbon signals, in which, the following functionalities of two
double bond (δC 134.7, C-3; 170.4, C-4; 132.5, C-1; 146.3,
β = 90.00°, γ = 90.00°, V = 2971.0 (10)Å3, Z = 8, Dx
=
1.200 mg/m3, F(000) = 1168, μ (Mo Kα) = 0.085 mm-1. Data
collection was performed on a Gemini SUltra using graphite-
monochromated Mo Kα radiation. λ = 0.71073 Å at 296 K.
20830 unique reflections were collected to θmax = 24.99°,
in which 2442 reflections were observed [F2 N 4σ(F2)]. The
structure was solved by direct methods using the SHELXS-97
program and refined by the program SHELXL-97 and full-matrix
least-squares calculations. The final refinement gave R1
=
0.0418, wR2 = 0.1152 and S = 1.050. CCDC 978094 contains
the supplementary crystallographic data for 1. These data can be
obtained free of charge from The Cambridge Crystallographic
Data Centre www.ccdc.cam.ac.uk/data_request/cif.
Fig. 3. Perspective drawing of the X-ray structure 1.

S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
47
14
10
3'
OH
O
OH
HO
H
O
2'
HO
H
14 OH
1
1
4'
H
1'
HO
2
2
9
5'
9
3
6'
10
8
O
8
O
5
OH
4
3
5
7
7
6
H
6
O
15
H
4
15
H
H
H
11
H
11
13
O
12
H
OH
O
HOOC12
1*
13
3
O
2*
4
O
O
R5
R7
OH
O
OH
R4
O
R3
R2
O
O
R6
H
H
O
R8
O
O
CH3
R1
O
O
9
8
5 R1=OH R2=OCH3 R3=OH R4=OCH3 R5=OCH3 R6=OCH3 R7=H
R8=H
R8=H
6 R1=OH R2=H
7 R1=OH R2=H
R3=OH R4=H
R3=OH R4=H
R5=OCH3 R6=OH
R5=OCH3 R6=H
R7=H
R7=OCH3 R8=OH
H3C
CH3
O
H
H
R3
H
O
OCH3
H3CO
R2
R1
O
O
O
O
O
CH3
14
H3CO
H
H
H
H
R4
OCH3
OCH3
O
O
R5
10
O
R6
OCH3
O
11 R1=OCH3 R2=OCH3 R3=OCH3 R4=OCH3 R5=OCH3 R6=OCH3
12 R1=OCH3 R2=OCH3 R3=H
13 R1=H
R4=OCH3 R5=OGlc R6=H
R5=OGlc R6=OCH3
15
R2=OH R3=OCH3 R4=H
O
O
H
N
H
HO
HN
(CH2)12CH3
OCH3
N
H
N
HO
OH
HO
O
CH2OH
OH
OH
O
H
O
OH
O
C
C
(CH2)7CH3
OH
H
OH
HO
17
COOH
16
18
OH
OH
O
H
OH
CH2NH2
O
H2C
C
OH
CH(CH2)5CH2
H3C(H2C)6HC
O
O
NH2
OH
O
HO
NH
OH
OH
OH
O
20
21
19
Fig. 4. Chemical structures of compounds 1–21.
C-10), two oxygenated methine carbons (δC 80.7, C-6; 74.6,
C-8), two carbonyl (δC 194.8, C-2; 178.2, C-12), three methyl
(δC 9.3, C-13; 20.7, C-14; 19.2, C-15) and D-glucose carbon
(δC 104.9, C-1′; 73.6, C-2′; 76.8, C-3′; 70.1, C-4′; 76.6, C-5′;
61.1, C-6′) were distinguishable.
In the HMBC spectrum (Fig. 1), the proton signal of H-1′
(δ 4.29) (the anomeric proton of D-glucose) correlated with
the carbon signal at δ 74.6 (C-8) revealed the presence of a
D-glucose at C-8. The planar structure of 2 was thus worked
out.
Compound 2 possessed the same planar structure as that of
austricin 8-O-β-D-glucopyranoside as reported by Xu (2000)
[5]. However, the chemical shift of C-7, C-8 and C-13 was
clearly different. The ROESY (Fig. 2) correlations of H-7α [5]
with H-8 and H-13; H-7α with H-6 and H-5; H-5 with H-13
in compound 2 indicated that they were located mutually on
the same α-side; consequently, H-11 was β-equatorial. Thus,
compound 2 was identified as 13α-methyl-(5αH,6αH,7αH,
8αH)-austricin 8-O-β-D-glucopyranoside. The structure having
a cis-fused γ-lactone ring at C-6 and C-7, which was reported in
literatures was rather rare [6].
Based on the comparison of the NMR data as well as
the physicochemical properties of the known compounds,
the following compounds were identified as follows: 4,10-
epizedoarondiol (3) [7]; leukodin (4) [8]; 5,7-dihydroxy-6,8,
3′, 4′-tetramethoxy flavone (5) [9]; chrysoeriol (6) [10];
5,7-trihydroxy-3′,5′-dimethoxylflavone (7) [11]; 1,8-dihy-
droxy-4-methylanthraquinone (8) [12]; sesamin (9) [13];
diayangambin (10) [14]; epiyangambin (11) [14]; phillyrin
(12) [15]; simplocosin (13) [16]; 12-epi-eupalmerone (14)
[17]; 3-oxo-11α-H-germacra-1(10) E, 4Z-dien-12,6α-olide
(15) [18]; 1-O-β-D-glucopyranosyl-(2S, 3S, 4R, 8E)-2-[(2′
R)-2′-hydroxypalmitoylamino]-8-octadecene-1,3,4-triol (16)
[19]; chisitine 2 (17) [20]; D-3-O-methyl chiroinositol (18)
[21]; (1S,5S,7R,8S)-8-(aminomethyl)-7-(pentadec-7-en-1-yl)-
2,6-dioxabicyclo [3.3.1] nonan-3-one (19) [22]; tryptophan
(20); sucrose(21) (Fig. 4); and additionally stigmasterol (22);
achillin (23) [8]; 3α,4α,10β-trihydroxy-8α-acetoxy-11Βh-guai-

48
S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
[5] Xu FL, Tian J, Li ML, Ding LS, Wu FE. Sesquiterpene lactones from
Notoseris Porphyrolepis. Chin Chem Lett 2000;11(10):905–8.
[6] Muckensturm B, Diyani F, Nouën DL, Souad FT, Reduron JP. Ammolactone,
a guaianolide from a medicinal plant, Ammodaucus leucotrichus. Phyto-
chemistry 1997;44(5):907–10.
[7] Saifudin A, Tanaka K, Kadota S, Tezuka Y. Sesquiterpenes from the
rhizomes of Curcuma heyneana. J Nat Prod 2013;76(2):223–9.
[8] Mariano M, Alejandra MZ, Pedro JN. Conformational analysis of achillin
and leukodin. J Nat Prod 1988;51(2):221–8.
[9] Chen J, Montanari AM. Isolation and identification of new poly-
methoxyflavonoids from Dancy Tangerine Leaves. J Agric Food Chem
1998;46(4):1235–8.
[10] Park Y, Moon BH, Yang H, Lee Y, Lee E, Lim Y. Complete assignments of
NMR data 13 hydroxymethoxyflavones. Magn Reson Chem 2007;45:
1072–5.
[11] Wang B, Li HL, Yang J, Zhang WD. Studies on the chemical constituents
of flavones of Veratrum nigrum L. Pharm Care Res 2007;7(5):347–9.
[12] Jaki B, Heilmann J, Sticher O. New antibacterial metabolites from the
cyanobacterium Nostoc commune (EAWAG 122b). J Nat Prod 2000;63(9):
1283–5.
[13] Zhang HD, Wang MT, Zhen MD, Zhang SJ, Chen YZ. Chemical
constituents of (Lancea tibetica Hook. f. et. Thoms). J Lanzhou Univ
(Nat Sci) 1987;23(4):156–8.
[14] Tulake A, Jiang Y, Tu PF. Nine lignans from Artemisia absinthium L. J Chin
Pharm Sci 2012;21(4):360–4.
[15] Rahman MMA, Dewick PM, Jackson DE, Lucas JA. Lignans of Forsythia
intermedia. Phytochemistry 1990;29(6):1971–80.
1-en-12,6α-olide (24) [2]; 3α,4α,10β-trihydroxy-11 β H-guai-
1-en-12,6α-olide (25) [2]; sieversol (26) [2]; 2α,4α,8α,-
trihydroxy-3α-acetoxy-11βH-guai-1(10)-en-12,6α-olide(27)
[2]; epiashchantin(28) [14]; 3α,4α-dihydroxy-8α-acetyloxy-
11βH-guai-1,9-dien-12,6α-olide(29) [2]; rutin(30) [23]; 5-
methoxysesamin(31) [2]; 11,14,15-trimethoxy-6-one-1(10),
3-diene-12,5-olide (32) [24]; 3,5-dihydroxy-6,7,3′,4′-tetra-
methoxy flavone (33) [25] and tricin (34) [26]. Compound 14
was first isolated from the soft coral Sarcophyton crassocaule
[27]. Such cembranes diterpene was often as an evidence in
chemotaxonomy found in some marine organisms(soft corals)
[28], but cembranes are not restricted to marine organisms.
Novel cembranes have been also obtained from frankincense
[29], tobacco [30,31], the bark of Croton oblongifolius [32] and
Anisomeles indica [33] of the terrestrial plant. The cembrane
diterpenoid was first found in the genus Artemisia.
In an anti-proliferative activities test, compound 1 was
found to show moderate cytotoxicity in MCF-7 cell lines with
an IC50 value of 26.8 μM and weak cytotoxic activity in
NCI-H460 (48.7 μM) and Hep-G2(34.5 μM). However, com-
pound 2 showed to have weak cytotoxic activity in MCF-7
(31.4 μM), NCI-H460 (43.4 μM) and Hep-G2(40.0 μM) cell
lines. Additionally, compounds 1–2 were evaluated for cyto-
toxic activity in vitro against MCF-7, NCI-H460 and Hep-G2 cell
lines, respectively. The choice of the cell lines was established
on the basis of cell line data with literature values of similar
compounds [34–39].
[16] Su BN, Zhu QX, Gao K, Yuan CS, Jia ZJ. Lignan and phenylpropanoid
glycosides from Lancea tibetica and their antitumor activity. Planta Med
1999;65(6):558–61.
[17] Rodriguez AD, Dhasmana H. Further bioactive cembranolide
diterpenes from the gorgonian Eunicea succinea.
1993;56(4):564–70.
J Nat Prod
[18] Tan RX, Lu H, Wolfender JL, Yu TT, Zheng WF, Yang L, et al. Mono-and
sesquiterpenes and antifungal constituents from Artemisia species.
Planta Med 1999;65(1):64–7.
[19] Kang SS, Kim JS, Xu YN, Kim YH. Isolation of a new cerebroside from the
root bark of Aralia elata. J Nat Prod 1999;62(7):1059–60.
[20] Tzouros M, Bigler L, Bienz S, Hesse M, Inada A, Murata H, et al. Two new
spermidine alkaloids from Chisocheton weinlandii. Helv Chim Acta
2004;87(6):1411–25.
[21] Parveen N, Khan NU, Inoue T, Sakurai M. Ethyl brevifolin carboxylate
and other constituents from Acer oblongum leaves. Phytochemistry
1988;27(12):3990–1.
Conflict of interest
There are no conflicts of interest of all authors with respect
to this work.
Acknowledgment
[22] Villasenor IM, Sanchez AC. Menthalactone,
a new analgesic from
Mentha cordifolia Opiz. leaves. J Biosci 2009;64(11–12):809–12.
[23] Yao LY, Lu Y, Chen ZN. Studies on chemical constituents of Hibiscus
mutabilis. Chin Tradit Herb Drugs 2003;34(3):201–3.
[24] Tang HQ. Chemical and biological studies of three species of medicinal
plants of Artemisia sieversiana, Gentiana scabra, Inula racemosa. (in Chinese)
Nanjing University; 1997.
[25] Horie T, Kawamura Y, Yamada T. Revised structure of a natural flavone
from Artemisia lanata. Phytochemistry 1989;28(10):2869–71.
[26] Li F, Liu YL. Studies on the isolation and structures of BaohuosideII, III,
IV and V. Acta Pharmacol Sin 1988;23(9):672–81.
[27] Xu XH, Kong CH, Lin CJ, Wang X, Lu JH. Isolation and identification of a
novel cembrane-type diterpenoid from the soft coral Sarcophyton
crassocaule. Chem J Chin Univ 2003;24(6):1023–5.
This work was supported by the National Natural Science
Foundation of China (30770233). We thank Prof. Hong-Fa Sun
of the Northwest Institute of Plateau Biology, Chinese Academy
of Sciences, Xining, China, for the identification of the plant
material. We also thank Dr. Hai-Bin Zhu (Southeast University,
Nanjing, PR China) for the analysis of the compound 1 X-ray
Crystallographic.
Appendix A. Supplementary data
[28] Hanson JR. Diterpenoids. Nat Prod Rep 2007;24:1332–41.
[29] Moussaieff A, Rimmerman N, Bregman T, Straiker A, Felder CC, Shoham
S, et al. Incensole acetate, an incense component, elicits psychoactivity
1D and 2D NMR spectra, as well as HR-ESI-MS spectra
and HPLC data for the new compounds (1–2) are available
as Supporting Information. Supplementary data to this article
can be found online at http://dx.doi.org/10.1016/j.fitote.
2014.05.007.
by activating TRPV3 channels in the brain. FASEB
J 2008;22(8):
3024–34.
[30] Nishida T, Wahlberg I, Nordfors K, Vogt C, Enzell CR. Application of 2D-
NMR spectroscopy in the structural determination of a new tobacco
cembranoid. Tetrahedron Lett 1984;25(12):1299–302.
[31] Wahlberg I, Arndt R, Wallin I, Vogt C, Nishida T, Enzell CR. Tobacoo
chemistry.59. Six new cembratrienetriols from Tobacco. Acta Chem
Scand B 1984;38(1):21–30.
References
[32] Pudhom K, Vilaivan T, Ngamrojanavanich N, Dechangvipart S, Sommit
D, Petsom A, et al. Furanocembranoids from the stem bark of Croton
oblongifolius. J Nat Prod 2007;70(4):659–61.
[1] Editorial Committee of Flora of China. Beijing: Science Press; 1991,76
(2): 1-10.
[33] Chen YL, Lan YH, Hsieh PW, Wu CC, Chen SL, Yen CT, et al. Bioactive
cembrane diterpenoids of Anisomeles indica. J Nat Prod 2008;71(7):
1207–12.
[2] Tan RX, Tang HQ, Hu J, Shuai B. Lignans and sesquiterpene lactones from
Artemisia sieversiana and Inula racemosa. Phytochemistry 1998;49(1):
157–61.
[34] Song FH, Fan X, Xu XL, Zhao JL, Yang YC, Shi JG. Cadinane
sesquiterpenes from the Brown Alga Dictyopteris divaricata. J Nat Prod
2004;67(10):1644–9.
[35] Chang HS, Lee SJ, Yang CW, Chen IS. Cytotoxic sesquiterpenes from
Magnolia kachirachirai. Chem Biodivers 2010;7(11):2737–47.
[3] Ngo KS, Wong WT, Brown GD. Muurolane sesquiterpenes from Illicium
tsangii. J Nat Prod 1999;62(4):549–53.
[4] Yang FY, Lü M, Su YF, Li CZ, Si CS, Bae YS. New 3-nitropropanoyl-D-
glucopyranoses in root of Indigofera kirilowii. Chin Tradit Herb Drugs
2007;38(10):1448–50.

S.-J. Liu et al. / Fitoterapia 97 (2014) 43–49
49
[36] Xu XY, Xie HH, Hao J, Jiang YM, Wei XY. Eudesmane sesquiterpene
glucosides from lychee seed and their cytotoxic activity. Food Chem
2010;123(4):1123–6.
[37] Kim DC, Kim JA, Min BS, Jang TS, Na MK, Lee SH. Guaiane
sesquiterpenoids isolated from the fruits of Torilis japonica and their
cytotoxic activity. Helv Chim Acta 2010;93(4):692–7.
[38] Strapasson RLB, Cervi AC, Carvalho JE, Ruiz ALTG, Salvador MJ, Stefanello
MÉA. Bioactivity-guided isolation of cytotoxic sesquiterpene lactones of
Gochnatia polymorpha ssp. Floccose. Phytother Res 2012;26(7):1053–6.
[39] Wang D, Huang W, Shi Q, Hong CT, Cheng YY, Ma ZJ, et al. Isolation and
cytotoxic activity of compounds from the root tuber of Curcuma wenyujin.
Nat Prod Commun 2008;3(6):861–4.