全 文 ： 药学学报 Acta Pharmaceutica Sinica 2016, 51 (8): 1285−1289 · 1285 ·




A new anti-hypoxia sesquiterpene from the rhizome of Petasites japonicus
XIE Yao-yu1†, LI Yu-xian2†, SUN Yi-ming1, WANG Yan3*, GUO Mei-li1*
(1. School of Pharmacy, Second Military Medical University, Shanghai 200433, China;
2. Department of Plant Science, Jilin Agricultural Science and Technology College, Jilin 132101, China;
3. Department of Pharmacy, Shanghai Putuo District Liqun Hospital, Shanghai 200333, China)
Abstract: A new sesquiterpene, bakkenolide-Ⅵa (1), was isolated from the rhizome of Petasites japonicas
(Sieb. et Zucc.) Maxim. The structure was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY,
HMQC, HMBC and NOESY) and mass spectrometry data. Bakkenolide-Ⅵa showed potent cerebral hypoxia-
ischemia protective activity in mice subjected to decapitation through prolonging the survival time and gasping
time. It also exhibited a protective activity against hypoxia injury in PC12 cells in anaerobic culture by
inhibition of lactate dehydrogenase (LDH) release.
Key words: anti-hypoxia; bakkenolide-Ⅵa; Petasites japonicus; sesquiterpene
CLC number: R284 Document code: A Article ID: 0513-4870 (2016) 08-1285-05
蜂斗菜根茎中的一个新倍半萜成分及其抗缺氧活性
谢曜宇 1†, 李余先 2†, 孙一鸣 1, 王 燕 3*, 郭美丽 1*
(1. 第二军医大学药学院, 上海 200433; 2. 吉林农业科技学院植物科学学院, 吉林 吉林 132101;
3. 上海普陀区利群医院药学部, 上海 200333)

摘要: 从蜂斗菜 [Petasites japonicas (Sieb. et Zucc.) Maxim.] 根茎中分离得到一个新倍半萜成分蜂斗菜内酯
Ⅵa (bakkenolide-Ⅵa), 其结构经核磁共振谱 (1H NMR、13C NMR、1H-1H COSY、HMQC、HMBC、NOESY) 和
质谱数据确证。蜂斗菜内酯Ⅵa 对断头小鼠脑缺氧缺血有明显保护作用, 能够明显延长断头小鼠的生存时间和
喘息时间; 并有保护缺氧细胞的作用, 能显著提高缺氧处理后 PC12 细胞株的存活率, 抑制细胞中乳酸脱氢酶的
释放。
关键词: 抗缺氧; 蜂斗菜内酯Ⅵa; 蜂斗菜; 倍半萜内酯

Petasites species (Asteraceae) are distributed
mainly in the northern Eurasian and North American
continents. The reported characteristic chemical
components of Petasites species include sesquiterpenes
(bakkenolides) and triterpenoids[1−12]. P. japonicus
(Sieb. et Zucc.) Maxim. is distributed widely in central,

Received 2016-04-15; Accepted 2016-05-16.
Project supported by the National Major Projects for New Drugs Innova-
tion and Development 2014ZX09J14102-07C.
†These two authors equally contributed to this work.
*Corresponding author Tel / Fax: 86-21-81871302,
E-mail: mlguo@126.com; wangyansjtu@163.com
DOI: 10.16438/j.0513-4870.2016-0346
southern and southwest China. Russian Far-East,
Japan, Korea are also distributed. Its rhizomes have
been applied as a folk medicine for the treatment of
traumatic injury, parotitis and snakebite[13]. In our
previous study, we reported the structural elucidation of
three known bakkenolides and five novel bakkenolides
(bakkenolide- a, a, a, a and aⅠ Ⅰ Ⅰ Ⅰ Ⅰ ) from P.
tricholobus Franch.[9, 14−16]. We also reported three
known bakkenolides from P. japonicus[17]. In the
present study, we isolated and identified a novel
sesquiterpene from the rhizome of P. japonicus. Its
anti-hypoxic activity was tested in vivo and in vitro.
· 1286 · 药学学报 Acta Pharmaceutica Sinica 2016, 51 (8): 1285−1289

Results and discussion
1 Bakkenolide- aⅥ
HPLC data suggested that the purity of compound
1 was 99.3%. 1 was obtained as colorless needles (in
MeOH), mp 120−123 ℃; UV (MeOH) λmax (204, 230,
289 nm). The IR spectrum indicated the presence of
ketone (1 763 cm−1). Its HR-ESI-MS exhibited a quasi
molecular ion peak at m/z 455.203 0 [M+Na]+ (Calcd.
455.204 6), which was compatible with the molecular
formula C24H32O7. The 13C and DEPT NMR spectra of
1 indicated 24 carbon signals, belonging to six methyl
(δ 8.4, 8.5, 15.7, 19.1, 19.8 and 20.6), three methylene
(δ 25.7, 25.9 and 38.8), seven methine (δ 35.8, 36.3,
68.2, 72.2, 113.5, 138.3 and 151.1), and eight quaternary
carbons (δ 41.6, 115.3, 119.7, 127.7, 150.4, 165.6, 167.4
and 174.3). Referring to our previous study, these
findings above suggested an eremophilenolide skeleton
of the molecule. Careful analysis of the NMR spectra
of 1, including 2D-NMR, allowed the 1H and 13C NMR
signals (Table 1) being assigned. In the HMBC
spectrum, the methyl proton CH3-14 at δ 0.98 correlated
with the carbon signals at δ 35.8 (C-4), 41.6 (C-5), 72.2
(C-3); the methyl proton CH3-15 at δ 1.05 correlated
with the carbon signals at δ 36.3 (C-10), 41.6 (C-5).
Thus, the two methyl groups were located at C-4 and
C-5, respectively. The position of the side chain was
determined by HMBC spectrum. The H-3 at δ 5.38
had a HMBC correlation with the carbon signal at
δ 167.4 (C-1″) and the H-6 at δ 6.45 had a HMBC
correlation with the carbon signal at δ 165.6 (C-1′).
Some other key HMBC correlations were observed and
listed in Table 1.
The relative stereochemistry of 1 was determined
by NOESY experiment, in which the correlations
between H-14 and H-6, H-10, H-15; H-6 and H-3
were observed. Thus H-3, H-6, H-10, H-14 and H-15
adopted the same orientation and were arbitrarily
designated as the β-orientation (Figure 1). Therefore, 1
was a novel sesquiterpene, named as bakkenolide-Ⅵa.
2 Pharmacologic actions
Compound 1 significantly increased the survival
time and gasping time of the mice after decapitation.
1 also increased creatine kinase (CK) content and
decreased lactic acid content in brain tissues of mice at
the same time (Table 2 and 3). These results indicated
that 1 possess potent cerebral hypoxia-ischemia activity
in mice.
Compound 1 significantly increased the cell
viability of the PC12 cells after anaerobic cultivation
(Table 4). 1 also increased lactate dehydrogenase
(LDH) content in PC12 cells (Table 5). These results
indicated that 1 can effectively protect PC12 cells
from hypoxia injury.

Table 1 The 1H NMR (500 MHz), 13C NMR (125 MHz) and
HMBC spectral data of compound 1 in CDCl3 (J in Hz)
Position δC δH (J/Hz) HMBC (H→C)
1 25.9 2.36 (m), 2.84 (m)
2 25.7 1.68 (m)
3 72.2 5.38 (m) 1
4 35.8 1.96 (m)
5 41.6
6 68.2 6.45 (s) 10, 11
7 150.4
8 115.3
9 38.8 1.93 (m) 8
10 36.3 2.10 (m) 4
11 119.7
12 174.3
13 8.5 1.87 (s) 11
14 8.4 0.98 (d, 5.6) 3, 4, 5
15 19.8 1.05 (s) 5, 6, 10
1′ 165.6
2′ 127.7
3′ 138.3 6.08 (q) 4′, 5′
4′ 15.7 2.01 (t) 2′, 3′
5′ 20.6 1.92 (s) 2′, 3′
1″ 167.4
2″ 151.1 7.03 (d, 16.0)
3″ 113.5 5.81 (d, 16.0) 2″
4″ 19.1 2.37 (s) 2″, 3″


Figure 1 The main NOESY correlations of compound 1

Table 2 Effects of compound 1 on survival time and gasping
time by decapitation in mice. n = 10, x ± s. *P < 0.05 vs
vehicle group
Group Survival time/s Gasping time/s
Vehicle group 10.6 ± 4.7 7.3 ± 2.9
Nimodipine group (18 mg·kg−1) 15.0 ± 3.8* 10.5 ± 2.5*
High dose group (30 mg·kg−1) 14.5 ± 2.7* 10.4 ± 2.9*
Middle dose group (15 mg·kg−1) 14.1 ± 2.8* 9.8 ± 1.8*
Low dose group (7.5 mg·kg−1) 12.4 ± 6.7 8.5 ± 4.9

XIE Yao-Yu, et al: A new anti-hypoxia sesquiterpene from the rhizome of Petasites japonicus · 1287 ·

Table 3 Effects of compound 1 on creatine kinase (CK) activity
and lactic acid content in brain tissues of mice. n = 10, x ± s.
*P < 0.05, **P < 0.01 vs vehicle group
Group CK/U·mg−1 Lactic acid /mmol·mg−1
Vehicle group 4.3 ± 1.7 0.87 ± 0.28
Nimodipine group (18 mg·kg−1) 6.8 ± 1.3** 0.44 ± 0.13**
High dose group (30 mg·kg−1) 6.0 ± 1.4* 0.56 ± 0.15**
Middle dose group (15 mg·kg−1) 6.0 ± 1.4* 0.60 ± 0.22*
Low dose group (7.5 mg·kg−1) 4.8 ± 1.2 0.82 ± 0.21

Table 4 Effects of compound 1 on cell viability in PC12 cells.
n = 5, x ± s. **P < 0.01 vs hypoxia group. Data are expressed
as percentages of normals
Group Cell viability (% Normals)
Normal group 100.0 ± 2.2**
Hypoxia group 35.1 ± 4.5
2 000 ng·mL−1 69.3 ± 7.1**
1 000 ng·mL−1 57.4 ± 3.7**
500 ng·mL−1 55.2 ± 1.6**
250 ng·mL−1 49.8 ± 1.6**
125 ng·mL−1 46.6 ± 2.3**
63 ng·mL−1 44.1 ± 1.3**
31 ng·mL−1 37.9 ± 2.1
16 ng·mL−1 36.1 ± 0.8

Table 5 Effects of compound 1 on lactate dehydrogenase (LDH)
activity in PC12 cells. n = 5, x ± s. **P < 0.01 vs normal
group; ΔP < 0.05, ΔΔP < 0.01 vs hypoxia group
Group LDH/U·L−1
Normal group 391 ± 14
Hypoxia group 1 324 ± 48**
High dose group (2 000 ng·mL−1) 777 ± 20ΔΔ
Middle dose group (250 ng·mL−1) 941 ± 26ΔΔ
Low dose group (31 ng·mL−1) 1 204 ± 10Δ

The above results confirmed compound 1 possessed
the anti-hypoxic activity.

Experimental
UV spectra were recorded on a WFH-203 UV
spectrophotometer (Shanghai Jingke Industrial Co.
Ltd). EI-MS and HR-ESI-MS spectra were obtained
on a MAT-212 mass spectrometer (Finnigan) and a
micromass Q-TOF spectrometer (Waters), respectively.
Melting points were measured with a Yanaco MS-S3
(Yanaco Co. Ltd) micro-melting point apparatus
(uncorrected). The 1H NMR, 13C NMR, and 2D-NMR
spectra were recorded on a Bruker AVANCE-500 FT-
NMR spectrometer with superconducting, ultra shielded
magnet (Bruker) using TMS as an internal standard.
Silica gel H (200−300 mesh) and TLC plates (HSGF254)
were made by Qingdao Haiyang Chemical Corp.
Model 1029 anaerobic system (Thermo Forma), CO2
cell incubator (Thermo Forma), IX50 Inverted phase-
contrast microscope (Olympus Company) and ELx800
enzyme sign meter (Bio-Rad) were used during PC12
cells culture experiment.
Ethanol (AR) and petroleum ether (60−90 ℃, AR)
were made by Sinopharm Chemical Reagent Co. Ltd.
DMEM medium, sugar-free DMEM medium, fetal
bovine serum and horse serum were purchased from
Gibco Company. Nimodipine was provided by Bayer
Healthcare. Methyl thiazolyl tetrazolium salt (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,
MTT assay) and dimethyl sulfoxide (DMSO) were
purchased from Pik-Day. Lactate dehydrogenase (LDH)
test kit was provided by Nanjing Jiancheng Bioengi-
neering Institute.
The rhizome of P. japonicas was collected from
Huzhou, Zhejiang Province of China in September
2006. It was identified by professor Mei-li Guo from
Department of Pharmacognosy, Second Military Medi-
cal University. A voucher specimen (PJ. 20060905)
was stored in our lab.
6-week-old ICR mice (SPF) with half male and half
female weighing (21 ± 1) g were provided by Shanghai
SLACOM Experimental Animal Company. Animal
permit number: SCXK (Hu) 2007-0005. PC12 cells
were provided by Shanghai Institute of Cell Biology,
Chinese Academy of Sciences.
1 Extraction and isolation
Dry rhizomes of P. japonicus (10.0 kg) were ground
and extracted with 95% ethanol (30 L×6) by percolation.
The solvent was evaporated under reduced pressure to
give an extract (625.4 g), equivalent to 6.3% of the
weight of the dry sample. This extract were suspended
in H2O (6 L) and extracted with petroleum ether (6 L×3).
The petroleum ether extract (315.2 g) was subjected to
column chromatography packed with 3.6 kg of silica
gel H and gradiently eluted with mixed petroleum ether
and EtOAc (10∶1, 8∶1, 5∶1, 2∶1 and 1∶1; 15 L of
eluent for each step) to give six fractions according to
TLC analysis. The fifth fraction (eluted with petroleum
ether and EtOAc 2∶1, 3.8 g) was loaded on a silica gel
H column (250 g, 2.5 cm × 80 cm), eluted with petroleum
ether-EtOAc 5∶1, filtered and crystallized repeatedly
to give compound 1 (350 mg).
2 Animal experiment
Fifty healthy ICR mice were partitioned into five
groups (n = 10 in each group, 5 female and 5 male) at
· 1288 · 药学学报 Acta Pharmaceutica Sinica 2016, 51 (8): 1285−1289

random, three test groups for compound 1 were low
dose group, middle dose group, high dose group with
dose of 7.5, 15 and 30 mg·kg−1, bw, respectively, a
nimodipine positive group (18 mg·kg−1, bw), and a
vehicle group. All the test groups were treated as above
by intragastric administration. The vehicle group
was treated with an equal volume of vehicle. After
administration for 5 days, fifty mice were subjected to
decapitation. The effect of 1 on the survival time and
gasping time of the mice were observed. The content
of creatine kinase (CK) and lactic acid in brain tissues
homogenate were measured.
3 PC12 cells culture experiment
PC12 cells were cultured in medium of 10% fetal
bovine serum, 10% horse serum and 80% sugar-free
DMEM, and grown in a 5% CO2 incubator at 37 ℃.
They were digested by 0.25% trypsin to passage once
per 2-3 days. Medication was during logarithmic phase.
Hypoxia-reoxygenation model was established as
follow. PC12 cells were washed in sugar-free DMEM
twice and randomly grouped. The hypoxia group
was added sugar-free DMEM medium 200 μL, and
subjected to anaerobic cultivation at 37 ℃ in an
incubator of 5% CO2 and 95% N2. Test groups were
added sugar-free DMEM medium supplemented with
different concentrations of compound 1 200 μL at
37 ℃ in a 5% CO2 incubator 30 min before anaerobic
cultivation. The normal group was out of anaerobic
cultivation, while other treatments were the same as the
hypoxia group. 1 h after anaerobic cultivation, all
groups were turned into a 5% CO2 incubator at 37 ℃.
24 h later, cell viability and LDH content of each group
were determined using methods below.
Quantification of cell viability was made using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. 96-well microassay culture
plates were inoculated with PC12 cells in logarithmic
phase (1×105 cells per well). These cells were grown
at 37 ℃ in a 5% CO2 incubator overnight and partitioned
into ten groups. They got different treatments ut supra
as compound 1 was added to the wells to get eight
test groups (16, 31, 63, 125, 250, 500, 1 000 and 2 000
ng·mL−1). Wells containing culture medium without
cells were used as blanks. After treatments, stock
MTT dye solution (20 μL, 5 mg·mL−1) was added
to each well. After 4 h incubation at 37 ℃ in a 5%
CO2 incubator, 100 μL DMSO was added to solubilize
the MTT formazan. The absorbance intensity of
each well was measured on the enzyme sign meter at
a wavelength of 570 nm. Reference wavelength was
630 nm. Each experiment was repeated five times
to get the mean values.
LDH content was measured with the test kit.
96-well microassay culture plates were inoculated
with PC12 cells in logarithmic phase (5×104 cells per
well). They were grouped and treated with different
conditions ut supra as compound 1 was added to the
wells to get three test groups (31, 250 and 2 000
ng·mL−1). After treatments, supernatant 100 μL was
taken from each well to operate following the instruct-
tions of the test kit strictly. The absorbance intensity
was measured on the UV spectrophotometer at a wave-
length of 440 nm and LDH content was calculated by
formula. Each experiment was repeated five times to
get the mean values.
4 Statistical analysis
The results were presented as Mean ± SD. The
differences among the means were analyzed using one-
way analysis of variance (ANOVA) followed by post
hoc Dunnett’s multiple comparison test at the 95%
(P < 0.05) and 99% (P < 0.01) confidence levels.
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