全 文 ：药学学报 Acta Pharmaceutica Sinica 2014, 49 (10): 1433−1437 · 1433·
Two new sulfated sesquiterpenoids from Petasites tricholobus
ZHANG Yong1, GAO Yuan-yuan2, JIA Qi2, GUO Fu-jiang2, LI Bo1, XU Zhi-jian1,
LI Yi-ming2*, ZHU Wei-liang1*, CHEN Kai-xian1, 2
(1. Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China; 2. Department of Phytochemistry, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai
201203, China)
Abstract: Two new sulfated sesquiterpenoids, megastigman-7-ene-3, 5, 6, 9-tetrol-3-O-β-D-6-sulfonated-
glucopyranoside (1) and 3-O-β-D-6-sulfonated-glucopyranosyl-6-(3-oxo-2-butenylidenyl)-1, 1,
5-trimethylcy clohexan-5-ol (2), along with one known sesquitepenoid compound icariside B1 (3) were isolated
from the whole herb of Petasites tricholobus Franch. Their structures were identified by their chemical and
spectroscopic characters. All obtained compounds were tested for their cytotoxicity against four cancer cell
lines.
Key words: Petasites tricholobus; Asteraceae; sulfated sesquiterpenoids
CLC number: R284 Document code: A Article ID: 0513-4870 (2014) 10-1433-05
毛裂蜂斗菜中两个新的倍半萜硫酸酯
张 勇 1, 高媛媛 2, 贾 琦 2, 郭夫江 2, 李 波 1, 徐志建 1,
李医明 2*, 朱维良 1*, 陈凯先 1, 2
(1. 中国科学院上海药物研究所受体结构与功能重点实验室 , 上海 201203;
2. 上海中医药大学中药学院中药化学教研室 , 上海 201203)
摘要: 从毛裂蜂斗菜全草中分离得到两个新的倍半萜硫酸酯类化合物 megastigman-7-ene-3, 5, 6, 9-tetrol-3-
O-β-D-6-sulfonated-glucopyranoside (1) 、 3-O-β-D-6-sulfonated-glucopyranosyl-6-(3-oxo-2-butenylidenyl)-1, 1, 5-
trimethylcyclohexan-5-ol (2) 和一个已知化合物 icariside B1 (3)。其中化合物 icariside B1 (3) 为首次从蜂斗菜属植
物中分离得到。通过化学和波谱等方法对上述结构进行了鉴定。此外 , 还测定了 3个化合物对 4种肿瘤细胞
(HepG2、HCT 116、H460和 MCF7) 的细胞毒活性。
关键词: 毛裂蜂斗菜; 菊科; 倍半萜硫酸酯
 Petasites tricholobus Franch is a perennial plant of
Compositae as a member of Senecioneae Cass. (Tussilaginae
Dum), which is widely distributed in the southwest and
Received 2014-03-28; Accepted 2014-04-21.
Project supported by Ministry of Science and Technology (2012AA01A305),
National Natural Science Foundation (81302699,
21373258 and 81261120567).
*Corresponding author Tel: 86-21-51322191, Fax: 86-21-51322193,
E-mail: ymlius@163.com;
Tel: 86-21-50806600-1205, Fax: 86-21-50807188,
E-mail: wlzhu@mail.shcnc.ac.cn
northwest China[1]. As a traditional Chinese medicine, the
rhizomes of Petasites tricholobus have been used for the
treatment of blood stasis, fracture, and snake-bite.
Phytochemical investigation of this herb previously focused
on its fat-soluble chemical constituents, and lots of
sesquiterpenoids, especially bakkenane had been isolated
from this plant[2]. In addition, these compounds showed
multiply pharma cological activities, such as
anti-inflammatory[2], anti-tumor[3], antibacterial[4], and
neuroprotective[5, 6]. Although there are many natural
DOI:10.16438/j.0513-4870.2014.10.006
· 1434· 药学学报 Acta Pharmaceutica Sinica 2014, 49 (10): 1433−1437
sulfur sesquiterpenoids had been isolated from the genus
Petasites in the past few decades, for example S-petasin,
neo-S-petasin and S-isopetasin et al, the discovering
of sulfated sesquiterpenoids from the genus Petasites
was not reported.
Herein, we present the isolation and structure
elucidation of two new sulfated sesquiterpenoids which
are megastigman-7-ene-3, 5, 6, 9-tetrol-3-O-β-D-6-
sulfonated-glucopyranoside (1) and 3-O-β-D-6-
sulfonated-glucopyranosyl-6-(3-oxo-2-butenylidenyl)-
1, 1, 5-trimethylcyclohexan-5-ol (2), and one known
sesquitepenoid compound icariside B1 (3) which was
isolated from the genus Petasites for the first time
(Figure 1). And all obtained compounds were tested
for their cytotoxicity against four cancer cell lines.
Results and discussion
Compound 1 was isolated as a white powder, [α] 22D−28.1 (c 0.16, MeOH). Its molecular formula was
established as C19H34O12S by HR-ESI-MS. Compound
1 displayed a quasi-molecular ion peak at m/z 485.164 9
[M−H]− (calcd. 485.169 3). The IR spectrum showed
1 had absorption bands at 3 429, 1 633 and 997 cm−1,
which were assignable to hydroxyl and double bond
groups, respectively. The 1H NMR (400 MHz, CD3OD)
spectrum (Table 1) of 1 showed signals corresponding
to four methyl protons at δH 0.84 (3H, s, CH3-11), 1.14
(3H, s, CH3-13) , 1.21 (3H, s, CH3-12) and 1.26 (3H, d,
J = 6.4 Hz, CH3-10); two olefinic protons at δH 6.05
(1H, dd, J = 16.0, 1.2 Hz, H-7) and 5.77 (1H, dd, J =
16.0, 6.4 Hz, H-8) for a disubstituted trans double bond;
and an anomeric proton signal at δH 4.40 (1H, d, J = 8.0
Hz, H-1) corresponding to a β-glucopyranosyl moiety.
1H- 1H COSY experiment revealed a spin system
including signals of three methines [δH 6.05 (1H, dd,
J = 16.0, 1.2 Hz, H-7); 5.77 (1H, dd, J = 16.0, 6.4 Hz,
H-8); 4.31 (1H, quint. d, J = 6.4, 1.2 Hz, H-9)] and
a methyl [δH 1.26 (3H, d, J = 6.4 Hz, CH3-10)]. The
13C NMR (100 MHz, CD3OD) spectral data displayed
19 carbon resonances (Table 1), six of which were
attributed to a β-glucopyranosyl moiety. The remaining
thirteen signals consist of four methyls, two methylenes,
four methines, and three quaternary carbons. The
signals at δC 136.5 and 131.7 were assigned to a
disubstituted double bond. These signal patterns
indicated the presence of a C13-norisoprenoid[7]. In
addition, the downfield-shift displayed by the 6-position
of the β-glucopyranosyl moiety suggested the attachment
of sulfonyl moiety at that position (δC 68.7), which
was substantiated by the mass data. In the HMBC
experiment (Figure 2), the long-range correlations from
CH2-2 to C-3 and C-12, from H-3 to C-1, from CH2-4
to C-2 and C-3, from H-7 to C-6 and C-9, from H-9
to C-7 and C-8, from CH3-10 to C-8 and C-9, from
CH3-11 to C-2 and C-6, from CH3-12 to C-2 and C-6, as
well as from CH3-13 to C-4 and C-6 clearly supported
the structure of 1 . Based on the above data and
comprehensive 2D NMR experiments (1H-1H COSY,
HMQC, HMBC), the structure of 1 was established as
shown in Figure 1. The relative configurations of 1
were deduced from analyses of the 1H-1H coupling
constants and ROESY spectrum (Figure 2). The
key ROESY correlations of H-3/H-1, H-3/CH3-12, and
H-9/CH3-12 suggested that H-3, H-9, and CH3-12 were
cofacial and α-orientation. Moreover, the 13C NMR
spectrum displayed three important carbon signals at
δC 27.9 (C-11), 26.7 (C-12) and 27.7 (C-13) which
could improve the relative configuration of CH3-13 (α-
orientation) according to literatures[7, 8]. The 1H NMR
spectrum signal at δH 4.31 (1H, quint. d, J = 6.3, 1.9 Hz,
H-9) which also could suggest the relative configuration
of H-9 (α-orientation) according to literature [8 ] .
In addition, the relative configuration of 6-OH (β-
orientation) has been determined by comparing the
specific rotations of 1 ([α] 22D −28.1) with a known
compound (3S, 5R, 6R, 7E, 9S)-megastigman-7-ene-3,
5, 6, 9-tetrol 3-O-D-glucopyranoside ([α] 24D −38.0) at
last[8]. The sugar configuration was deduced as a β-
glucopyranosyl moiety by its anomeric proton signal at
Figure 1 Structures of compounds 1−3 from P. Tricholobus
ZHANG Yong, et al: Two new sulfated sesquiterpenoids from Petasites tricholobus · 1435·
Table 1 NMR data of compounds 1−3 (400 MHz, in CD3OD, J in Hz)
No.
1 2 3
δH δc δH δc δH δc
1 41.2 37.6 35.6
2ax 1.76 (1H, dd, 12.1, 12.1) 44.9 1.43 (1H, dd, 12.0, 11.8) 48.7 1.48 (1H, dd, 12.5, 12.1) 46.7
2eq 1.71 (1H, dd, 12.1,4.3) 2.14 (1H, ddd, 12.0, 3.3, 1.9) 2.09 (1H, ddd, 12.5, 4.2, 2.0)
3 4.17 (1H, overlapped) 74.2 4.30 (1H, overlapped) 73.8 4.35 (1H, dddd, 12.1, 11.4, 4.2, 3.8) 71.1
4ax 1.61 (1H, dd, 13.0, 12.5) 43.0 1.48 (1H, ddd, 12.6, 4.0, 1.9) 47.3 1.46 (1H, ddd, 12.6, 3.8, 2.0) 45.2
4eq 1.93 (1H, ddd, 13.0, 4.2, 2.0) 2.34 (1H, dd, 12.6, 11.8) 2.37 (1H, dd, 12.6, 11.4)
5 78.2 72.8 71.0
6 79.4 120.6 118.6
7 6.05 (1H, dd, 16.0, 1.2) 131.7 212.1 210.1
8 5.77 (1H, dd, 16.0, 6.4) 136.5 5.81 (1H, s) 101.6 5.83 (1H, s) 99.8
9 4.31 (1H, quint. d, 6.4, 1.2) 70.1 201.5 199.5
10 1.26 (3H, d, 6.4) 24.7 2.19 (3H, s) 27.0 2.19 (3H, s) 25.1
11 0.84 (3H, s) 27.9 1.15 (3H, s) 32.7 1.16 (3H, s) 30.8
12 1.21 (3H, s) 26.7 1.38 (3H, s) 29.9 1.38 (3H, s) 28.0
13 1.14 (3H, s) 27.7 1.39 (3H, s) 31.3 1.39 (3H, s) 29.4
Glc 1 4.40 (1H, d, 8.0) 103.0 4.43 (1H, d, 7.9) 103.7 4.44 (1H, d, 7.8) 101.2
2 3.15 (1H, dd, 9.2, 7.8) 75.5 3.15 (1H, dd, 9.0, 7.9) 75.5 3.15 (1H, dd, 9.0, 7.8) 73.7
3 3.36 (1H, dd, 9.2, 9.2) 78.2 3.36 (1H, dd, 9.0, 9.0) 78.2 3.37 (1H, dd, 9.0, 9.0) 76.7
4 3.36 (1H, overlapped) 71.8 3.36 (1H, overlapped) 71.9 3.34 (1H, overlapped) 70.2
5 3.45 (1H, m) 76.3 3.48 (1H, m) 76.4 3.37 (1H, overlapped) 76.5
6a 4.31 (1H, dd, 12.0, 1.8) 68.7 4.31 (1H, overlapped) 68.7 3.88 (1H, d, 11.8) 61.3
6b 4.17 (1H, overlapped) 4.15 (1H, dd, 12.0, 5.1) 3.69 (1H, d, 11.8, 4.8)
Figure 2 Key 1H-1H COSY, HMBC, ROESY and NOESY correlations of 1 and 2
δH 4.40 (1H, d, J = 8.0 Hz, H-1).
Compound 2 was obtained as a white powder,
[α] 22D −36.2 (c 0.34, MeOH), which was assigned a
molecular formula of C19H30O11S by HR-ESI-MS.
Compound 2 displayed a quasi-molecular ion peak at
m/z 489.137 3 [M+Na]+ (calcd. 489.140 7). The IR
spectrum of 2 indicated the presence of a hydroxyl
(3 433 cm−1), an allenic (1 940 cm−1), and a conjugated
carbonyl (1 655 cm−1) group. The 1H NMR (400 MHz,
CD3OD) and 13C NMR (100 MHz, CD3OD) spectrum
of 2 were very similar to 3 (Table 1), which were
assigned a C13-norisoprenoid glucoside type, compared
with that of the icariside B1 (3) isolated from peucedanum
japonicum Thunb[9]. And the only difference between
2 and 3 was the chemical shift of 6-position. The
downfield-shift displayed by the 6-position of the β-
· 1436· 药学学报 Acta Pharmaceutica Sinica 2014, 49 (10): 1433−1437
glucopyranosyl moiety also suggested the attachment of
sulfonyl moiety at that position, which was substantiated
by the measurement of the HR-ESI-MS (C19H30O11SNa
[M+Na]+). The HMBC experiment (Figure 2) further
accounted the presence of a C13-norisoprenoid glucoside.
Based on the above data and comprehensive 2D NMR
experiments (HMQC, HMBC), the structure of 2
was established as shown in Figure 1. The relative
configurations of 2 were deduced from the analyses of
the 1H-1H coupling constants and ROESY spectrum
(Figure 2). The ROESY correlations of H-3/H-1,
H-3/CH3-12, and H-8/CH3-12 indicated that H-3, H-1,
H-8 and CH3-12 were cofacial and α-orientation, while
the glycosidic bond was on the opposite side of the
molecular plane and β-orientation. In addition, from
the analysis of 1D NOESY spectrum of 2 (Figure 2),
the signal at δH 4.30 (H-3) exhibited a NOE correlation
of the signals at δH 4.43 (H-1) and δH 1.38 (CH3-12),
which also suggested the configuration of the CH3-12
to be α-orientation, and those of the CH3-11 and CH3-13
to be β-orientation.
Compound 3 was identified as icariside B1[9],
which was isolated from the genus Petasites for the first
time.
Experimental
General experimental procedures 1H NMR and
13C NMR spectra were recorded on a Bruker AM-400
spectrometer with TMS as the internal standard. 2D
NMR spectra were recorded on a Bruker DRX-500
spectrometer. ESI-MS and HR-ESI-MS were carried
out on a Waters Q-TOF Premier instrument. Optical
rotations were measured on a Perkin Elmer Model 341
polarimeter. The FT-IR spectra were recorded on an
IR Perkin-Elmer 577 spectrophotometer with KBr
pellets. An Agilent 1200 Series machine equipped with
Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm,
5 μm) was used for HPLC analysis, and semi-preparative
Agilent ZORBAX SB-C18 column (9.4 mm × 250 mm,
5 μm) was used in sample preparation.
Plant material The whole plants of P. tricholobus
Franch were collected from Chongqing, China in October
2009. The plants were identified by Prof. Li-Hong Wu,
Shanghai R&D Center for Standardization of Chinese
Medicines. A voucher specimen (No. FDC- 20091029)
was deposited in Department of Phytochemistry, School
of Pharmacy, Shanghai University of Traditional
Chinese Medicine.
Extraction and isolation Air-dried whole plants
of P. tricholobus (6.5 kg) were reflux-extracted with
95% ethanol for three times (1.5 hours each time) at
80 ℃ , and then the solvent was evaporated under
reduced pressure to give extracts (320 g). The extracts
were suspended in hot water (60 ℃ , 1.5 L) and
extracted successively with petroleum ether, EtOAc,
and n-BuOH.
The n-BuOH soluble fraction was concentrated
under reduced pressure to afford a residue (85 g). This
residue was subjected to chromatographic separation on
D101 macroporous adsorption resin using H2O, 20%
EtOH, 40% EtOH, 60% EtOH and 95% EtOH as the
eluent. The 20% EtOH fraction (11.2 g) was separated
by ODS-A column chromatography (10%−100% MeOH-
H2O) to give fractions A-F. Fraction C (54.4 mg) was
subjected to Sephadex LH-20 column using a 20%
MeOH system to give compound 1 (2.5 mg). Fraction
D (210 mg) was subjected to Sephadex LH-20 column
using a 20% MeOH system to give fractions D1-D6.
Fraction D1 (21.6 mg) was purified by semi-preparative
HPLC using MeCN−H2O (15∶85, 3 mL·min−1) as the
mobile phase to obtain compound 2 (t = 2.326 min, 7.2
mg). Fraction E (1.7 g) was subjected to silica gel
column chromatography (300−400 mesh) eluted with
CH2Cl2−MeOH (20∶1−0∶1) to give fractions E1-E11.
Fraction E8 (68.9 mg) was separated by ODS-A column
chromatography (20%−100% MeOH−H2O) to give
fractions (E8 Fr.1−Fr.4). E8 Fr.2 (21.3 mg) was
purified by semi-preparative HPLC using MeCN−H2O
(15∶ 85, 3 mL·min−1) as the mobile phase to afford
compound 3 (t = 9.226 min, 10.2 mg).
Structure elucidation
Compound 1 White powder (MeOH); [α] 22D −28.1
(c 0.16, MeOH); HR-ESI-MS m/z 485.164 9 [M−H]−
(calcd. for C19H33O12S, 485.169 3); ESI-MS m/z: 485
[M−H]−; IR (KBr) νmax 3 429, 2 922, 1 657, 1 633, 1 377,
1 259, 1 068, 997, 580 cm−1; 1H NMR (400 MHz,
CD3OD) and 13C NMR (100 MHz, CD3OD) data see
Table 1.
Compound 2 White powder (MeOH); [α] 22D −36.2
(c 0.34, MeOH); HR-ESI-MS m/z 489.137 3 [M+Na]+
(calcd. for C19H30O11NaS, 489.140 7); ESI-MS m/z: 465
[M−H]−, 931 [2M−H]−; IR (KBr) νmax: 3 433, 2 922,
1 940, 1 655, 1 637, 1 381, 1 248, 1 074, 1 001, 820, 580
cm−1; 1H NMR (400 MHz, CD3OD) and 13C NMR (100
MHz, CD3OD) data see Table 1.
Compound 3 White powder (MeOH); ESI-MS m/z:
ZHANG Yong, et al: Two new sulfated sesquiterpenoids from Petasites tricholobus · 1437·
409 [M+Na]+, 793 [2M+Na]+; 1H NMR and 13C NMR
(CD3OD, 100 MHz) data see Table 1. The above data
were identical with icariside B1[9].
Acid hydrolysis of compound 1 and 2 The acid
hydrolysis and detection of sugars were conducted
according to the method described in references[10, 11].
Compounds 1 and 2 (each 2.0 mg) were separately
hydrolyzed in 1.5 mL 2 mol·L−1 HCl at 80 ℃ for 3 h.
After cooling to room temperature, each reaction
mixture was washed with CHCl3 (1.5 mL×3) and the
aqueous layer was neutralized with Ba(OH)2 caused
white precipitates to form. Then each resulting mixture
was centrifuged. The precipitates were separated and
proved as BaSO4. It was indicated that 1 and 2 were
sulfated compounds. The supernatant was concentrated
under reduced pressure. D-glucose was observed in
the supernatant by TLC analysis method (chloroform−
MeOH−water, 7∶3∶0.5, v/v/v) with standard D-glucose
sample.
Cytotoxic activity All compounds were evaluated
for cytotoxic activity against four human cancer cell
lines, including HepG2 (human liver cancer), HCT 116
(human colon cancer), H460 (human lung cancer) and
MCF7 (human breast cancer) by using the methyl
thiazol tetrazolium (MTT) method. However, the three
compounds did not exhibit significant cytotoxicity (IC50
values > 100 μmol·L−1) against the tested tumor cells.
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