Fitoterapia 82 (2011) 692695

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Two new -pinene derivatives from Angelica sinensis and their

anticoagulative activities
Nian-Yun Yang a, Gui-Sheng Zhou a, Yu-Ping Tang a, Hui Yan a, Sheng Guo a, Pei Liu a,
Jin-Ao Duan a,, Bing-Sheng Song b, Zi-Qing He b
a
Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China
b
Gansu Minggui Traditional Chinese Medicine Technology Co. Ltd, Lanzhou 430072, China

a r t i c l e i n f o a b s t r a c t

Article history: Two new -pinene derivatives (12) were isolated from the aerial parts of Angelica sinensis.
Received 28 December 2010 Their structures were determined by spectroscopic and chemical methods to be 6,9-
Accepted in revised form 15 February 2011 dihydroxy-(+)--pinene (1) and 9-hydroxy-(+)--pinene-6-O-D-glucoside (2). In the
Available online 25 February 2011
anticoagulative assay, compounds 1 and 2 exhibited weak antithrombin activity and strong
antiplatelet aggregation activity in vitro.
Keywords: 2011 Elsevier B.V. All rights reserved.
Angelica sinensis
The aerial parts
-pinene derivatives
Antithrombin
Antiplatelet aggregation

1. Introduction (+)--pinene (1) and 9-hydroxy-(+)--pinene-6-O-D-

The dried radix of Angelica sinensis (Oliv.) Diels (Umbelli-
ferae) is one of the most important crude drugs in traditional
Chinese medicine and has been used as a hematopoietic and
anti-inammatory agent for the treatment of menstrual
disorders such as amenorrhea and dysmenorrhea [1]. Mean-
while, it is also used as a health food product in Asia and as a
dietary supplement in Europe and America [2,3]. In our serial
research on active constituents of the radix of A. sinensis, we
have reported before polysaccharides, oligosaccharides,
amino acids, phthalides, organic acids and ceramides from
its different solvent extracts [48]. Chemical studies on the
aerial parts of A. sinensis were only limited to its volatile
constituents [9], so we investigated the involatile constitu-
ents. In this study, we deal with the isolation and structural
elucidation of the two new -pinene derivatives from the
aerial parts of A. sinensis, and report some of their antic-
oagulative activities. They were identied as 6,9-dihydroxy-
Corresponding author.
E-mail address: dja@njutcm.edu.cn (J.-A. Duan).
glucoside (2) (Fig. 1) by means of chemical and extensive
spectroscopic analyses.
2. Experimental
2.1. General experimental procedures
Silica gel for column chromatography (CC) (200
300 mesh) and TLC plates (1040 m) were the product of
Qingdao Haiyang Chemical (Qingdao, China). All solvents
used were of analytical grade (Nanjing Chemical Plant,
Nanjing, China). Adenosine diphosphate (ADP) was pur-
chased from Beijing Zhongqin Scientic Instrument Co. Ltd
(Beijing, China). Sodium citrate was purchased from Shang-
hai Lingfeng Chemical Reagent (Shanghai,. China). Aspirin
was purchased from Hebei Jingye Co. Ltd (Pingshan, China). A
rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimen-
tal Animal (Shanghai, China). Optical rotations were mea-
sured on a JASCO P-1020 polarimeter. IR spectra were taken
on a Nicolet IR-100 FT-IR spectrometer in KBr discs. NMR
spectra were measured on a Bruker AV-500 MHz (500 MHz

0367-326X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.tote.2011.02.007
 N.-Y. Yang et al. / Fitoterapia 82 (2011) 692695 693

Fig. 1. Structures of compounds 12.

for 1H NMR and 125 MHz for 13C NMR), using TMS as internal C NMR: Table 1; ESIMS m/z: 329 [MH], 365 [M + Cl];
13

standard; chemical shifts were recorded as values. ESIMS HRESIMS m/z: 329.1610 (calcd.: 329.1600 [MH]).
and HRESIMS spectra were obtained on a Micromass Q/TOF
mass spectrometer. Anticoagulative assay was performed on 2.3. Anticoagulative bioassay
an LG-PABER-I coagulation-analysis instrument. The blood
sample was treated on an Anke TDL-40B centrifuge. Evaluation of anticoagulative activities of compounds 12
was performed by using thrombin time (TT) and platelet
aggregation methods. All samples were dissolved in absolute
2.2. Extraction and isolation ethanol respectively. Two male New Zealand white rabbits
(22.5 kg) were used for anticoagulation assay in vitro,
The aerial parts of A. sinensis (10 kg), collected in
November 2009 from Gansu province of China, were cut off
Table 1
and extracted with 80% C2H5OH (2 100 L) for 2 h under 1
H (300 MHz) and 13C (75 MHz) NMR spectral data of compounds 1 (in
reux, and the combined extracts were concentrated under CDCl3) and 2 (in DMSO-d6) (, ppm; J, Hz).
reduced pressure. The resulting extract (1206 g) was sus-
1 2
pended in H2O and extracted successively with petroleum
1 13 1 13
ether, ethyl acetate and n-butanol to give the respective H (m, J Hz) C H (m, J Hz) C
extracts after solvent removal. The combined ethyl acetate 1 2.20 (br s) 50.3 2.30 (br s) 48.0
extracts were evaporated under reduced pressure to leave a 2 141.9 142.1
residue (75 g) which was loaded on a silica gel (300 3 5.24 (br s) 118.1 5.23 (br s) 117.9
4 2.30 (m) 31.8 2.25 (m) 31.4
400 mesh) column (4 80 cm) eluting with a CH2Cl2
5 2.20 (br s) 43.5 2.20 (br s) 40.6
CH3OH stepwise gradient (100:02:1), and 8 fractions 6 3.88 (br s) 78.1 3.78 (br s) 84.2
were collected with 150 mL each. Fraction 3 was further 7 42.3 42.3
separated using a silica gel (300400 mesh) column 8 1.09 (s) 18.3 0.88 (s) 18.3
(2 30 cm) eluted with CHCl3CH3OH (20:1, 1.5 L) to afford 9 4.11 (m) 69.8 4.00 (dd, 10.7, 5.7), 67.4
3.81 (dd, 10.7, 6.2)
compound 1 (20 mg), and fraction 7 was subjected to a silica 10 1.67 (s) 22.6 1.62 (s) 22.7
gel (300400 mesh) column (2 30 cm) eluted with CHCl3 9-OH 4.22 (t, 5.9)
CH3OH (4:1, 2.0 L) to get compound 2 (30 mg). 1 4.13 (d, 7.9) 102.3
6,9-dihydroxy-(+)--pinene (1): Colorless oil, []20D +
2 2.99 (m) 73.7
3 3.16 (m) 76.9
67.5 (c 0.16, CH3OH); IR(lm): 3430 (OH), 1645 (C=C),
4 3.06 (m) 70.5
1042, 837 (NC=CH) cm1; 1H and 13C NMR: Table 1; ESIMS 5 3.02 (m) 77.7
m/z: 167 [MH], 203 [M + Cl]; HRESIMS m/z: 167.1079 6 3.66 (m), 3.38 (m) 61.4
(calcd.: m/z 167.1072 [MH]). 2-OH 4.94 (d, 5.1)
9-hydroxy-(+)--pinene-6-O-D-glucoside (2): Color- 3-OH 4.91 (d, 4.8)
4-OH 4.87 (d, 4.9)
less blocky crystalline, []20
D -14.9 (c 0.15, CH3OH); IR(lm):
6-OH 4.50 (t, 5.7)
3435 (OH), 1646 (C=C), 1040, 838 (NC=CH) cm1; 1H and
694 N.-Y. Yang et al. / Fitoterapia 82 (2011) 692695
Fig. 2. Key HMBC and NOESY correlations of compounds 12.
obtained from the experimental animal center of Nanjing
University of Chinese Medicine and approved by the Animal
Ethics Committee of Nanjing University of Chinese Medicine
on April 29, 2010. Rabbit common carotid artery was cut off to
take sample of blood, which was mixed with anticoagulant
(3.8% sodium citrate) in the proportion of 9 to 1, then
centrifuged at 1000 rpm for 10 min at room temperature to
give platelet-rich plasma (PRP). The plasma (50 L) was put
in plastic cup for 3 min at 37 C, and 100 L thrombin solution
of 15 U mL1 diluted by 0.1 mol mL1 pH 7.4. TrisHCl buffer
was also put in the plastic cup along with 10 L sample
solution, meanwhile, the coagulation-analysis instrument
was started up so that the thrombin clotting time was
recorded. The same experiment was done for the positive
control drug heparin sodium and the blank solvent ethanol.
Each analyte was tested several times, and an average value
was applied. TT prolongation rate was calculated to assess the
antithrombin activities of the samples.
Centrifugation of the remaining blood at 3000 rpm for
10 min yielded platelet-poor plasma (PPP). Platelet count
was adjusted to 3 105 with PPP. The samples was added to
the PRP (0.200 mL), then the mixture was incubated at 37 C
with stirring for 1 min before the addition of ADP (10 M) as
inducer of platelet aggregation. Aggregation was measured by
a turbidimetric method. Changes in light transmission caused
by platelet clotting were measured using an aggregometer.
Platelet aggregation was expressed as the percentage change
with the difference of light transmittance between PRP and
PPP as 100%. The same experiment was done for the positive
control drug aspirin and the blank solvent ethanol. Each
analyte was tested several times, and an average value was
applied. Analytical data were expressed as the inhibition rate
and IC50 to evaluate the antiplatelet aggregation activities.
3. Results and discussion
Compound 1 was isolated as a colorless oil. The molecular
formula of 1 was established as C10H16O2 from HRESIMS
peak at m/z 167.1079. The negative ESIMS showed a quasi
molecular ion peak at m/z 167 [MH], 203 [M + Cl]. The IR
absorption at 3430 and 1645 cm1 suggested the presence of
hydroxyl group and olenic bond. The 1H and 13C NMR spectra
of 1 showed some characteristics of -pinene (Table 1), and
there were two additional hydroxyl groups in 1. The HMBC
spectrum (Fig. 2) showed a proton at 3.88 (br s) correlated
with an olenic carbon ( 141.9), a quaternary carbon ( 42.3)
and one methene carbon ( 31.8), which established that the
hydroxyl group was located at C-6. Detailed analysis of NMR
also determined another hydroxyl group to be situated at C-9.
()--pinen-4-one, 3-hydroxy-()-pinane, 9-hydroxy-
()--pinene, 4-hydroxy-()--pinen-6-one and ()--
pinene are all levorotatory [10], while (+)--pinen-10-al,
(+)--pinen-4-one, 10-hydroxy-(+)--pinene, 10-hydroxy-
(+)-pinane, (+)--pinene and 1 are all dextrorotatory [11],
which suggested that 1 was the derivatives of (+)--pinene.
The structure and relative conguration of 1 were analyzed by
NOESY experiment (Fig. 2). In its NOESY spectrum, H-6 (
3.88) correlated with H-4 at 2.30 (m), so H-6 is -oriented.
Assignments of all protons and carbons in 1 can be made by
NOESY, HMQC and HMBC spectra. Consequently, compound 1
was identied as 6,9-dihydroxy-(+)--pinene.
Compound 2 was isolated as a colorless blocky crystalline,.
The molecular formula of 2 was established as C16H26O7 from
HRESIMS peak at m/z 329.1610. The negative ESIMS
showed a quasi molecular ion peak at m/z 329 [MH], 365
[M + Cl]. The IR absorption at 3435 and 1646 cm1
suggested the presence of hydroxyl group and olenic bond.
Its 1H NMR and 13C NMR spectra (Table 1) revealed the
characteristic signals of 1 and glucose. The C-6 chemical shift
of 2 shifted towards downeld by 6.1 ppm, which suggested
the glucose attached to C-6. When 2 was hydrolyzed with 10%
hydrochloric acid, 1 was obtained together with glucose.
HMBC and HSQC experiments (Fig. 2) allowed us to make an
unambiguous and complete assignment of the 1H and 13C
NMR spectra of 2. So compound 2 was identied as 9-
hydroxy-(+)--pinene-6-O-D-glucoside.
-Pinene was the main component of the essential oil of A.
sinensis [9], and it was reported to possess antimalarial,
Table 2
TT rate of compounds 12 (x s, n = 68).
Sample TT prolongation rate (%)
25 g mL 1 50 g mL 1 100 g mL 1
Compound 1 1.10 0.11 3.79 0.34
Compound 2 0.50 0.06 1.73 0.10
Heparin sodium 11.01 1.14 56.61 5.04 160.05 9.11
 N.-Y. Yang et al. / Fitoterapia 82 (2011) 692695 695

Table 3
Platelet aggregation rate of compounds 12 (x s, n = 68).

Sample Platelet aggregation rate

Inhibition ratio (%) IC50 (g mL 1)

2.5 g mL 1 5.0 g mL 1 10.0 g mL 1 20.0 g mL 1 40.0 g mL 1

Compound 1 6.94 28.40 68.10 85.15 95.04 8.23

Compound 2 2.78 11.02 32.52 58.25 74.04 18.13
Aspirin 11.94 37.88 68.30 86.14 95.73 7.01

antimicrobial and antioxidative activities [12]. In vitro antic- References

oagulative activities of compounds 12 were tested using TT
and platelet aggregation methods in this study. As can be
appreciated by the data summarized in Tables 2 and 3,
compounds 1 and 2 could slightly prolong thrombin time and
strongly inhibit platelet aggregation. The results suggested
that the two pinane monoterpenes are the potential antic-
oagulative constituents of the aerial parts of A. sinensis, and the
antiplatelet aggregation activity of the compounds is mainly
related to -pinene unit and might be due to inhibition of
thromboxane A2 formation or Ca2+ activation in platelet.
Acknowledgements
This research was nancially supported by the National
Science & Technology Supporting Program for the 11th Five-
Year Plan (2006BAI09B05-1, 2007BA137B02) and 2006 Great
Basic Science Research Project of Jiangsu College and
University (06KJA36022). We also thank Mr Dong-Jun Chen
and Zhen-Hua Zhu for other helpful assistance.
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