a-Bulnesene, a novel PAF receptor antagonist isolated

from Pogostemon cablin

Hui-Chun Hsu
a,b
, Wen-Chia Yang
a
, Wei-Jern Tsai
c
, Chien-Chih Chen
c
,
Hui-Yu Huang
d,
*
, Ying-Chieh Tsai
a,
*
a
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
b
Yangsen Biotechnology Co. Ltd, Taipei, Taiwan
c
National Research Institute of Chinese Medicines, Taipei, Taiwan
d
Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih-Chien University, Taipei, Taiwan
Received 13 March 2006
Available online 8 May 2006
Abstract
a-Bulnesene is a sesquiterpenoid isolated from the water extract of Pogostemon cablin. It showed a potent and concentration-depen-
dent inhibitory eect on platelet-activating factor (PAF) and arachidonic acid (AA) induced rabbit platelet aggregation. In a radioligand
binding assay for the PAF receptor, a-bulnesene competitively inhibited [
3
H]PAF binding to the PAF receptor with an IC
50
value of
17.62 5.68 lM. a-Bulnesene also dose-dependently inhibited PAF-induced intracellular Ca
2+
increase in uo-3/AM-loaded platelets
(IC
50
values of 19.62 1.32 lM). Furthermore, a-bulnesene inhibited AA-induced thromboxane B
2
(TXB
2
) formation and prostaglan-
din E
2
(PGE
2
) formation. These results indicate that the inhibitory eect of a-bulnesene on platelet aggregation was due to a dual activ-
ity; specically the chemical blocked PAF-induced intracellular signal transduction and interfered with cyclooxygenase activity, which
resulted in a decrease in thromboxane formation. This study is the rst to demonstrate that a-bulnesene is a PAF receptor antagonist
as well as an anti-platelet aggregation agent.
2006 Elsevier Inc. All rights reserved.
Keywords: a-Bulnesene; Pogostemon cablin; Sesquiterpenoid; Platelet aggregation; Platelet-activating factor (PAF); Arachidonic acid (AA)
It is well recognized that platelet aggregation plays an
important role in thrombosis and atherosclerosis. Mural
thrombus formation can restrict the ow of blood to vital
tissues or organs leading to peripheral cerebral or coronary
ischemia. Evidence has indicated that platelets contribute
signicantly to the etiology and pathogenesis of acute cor-
onary syndrome, myocardial infraction, and embolismic
stroke, which are major causes of death in developed coun-
tries [1,2]. The rst step in the response of platelets to vas-
cular injury is irreversible attachment to the altered surface
followed by platelet activation [3]. This mainly takes place
though the action of many agonists such as platelet-activat-
ing factor (PAF), arachidonic acid (AA), and others. The
activation of platelets can be counter-regulated by factors
that attenuate or prevent these agonist-induced responses
[2]. Anti-platelet agents can inhibit platelet activation by
many mechanisms. One example is aspirin, which is a
non-specic cyclooxygenase inhibitor that dampens the
production of thromboxane A
2
(TXA
2
). Aspirin has been
shown to be clinically benecial in the treatment of throm-
boembotic diseases.
Previous studies have reported that the constituents of
Melicope semecarpifolia root, aqueous extracts of plants
(alfalfa, fresh nettle, and camonile) and the constituents
of Panax notoginseng root show signicant eects on plate-
let aggregation [46]. However, current anti-platelet drugs
still have considerable limitations in their mode of action
and ecacy. A greater understanding of platelet function
at the molecular level will probably lead to the develop-
ment of novel anti-platelet drugs [3].
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.05.006
*
Corresponding authors. Fax: +886 2 28209226.
E-mail addresses: maggieh@mail.usc.edu.tw (H.-Y. Huang), tsaiyc@
ym.edu.tw (Y.-C. Tsai).
www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 345 (2006) 10331038
BBRC
The Chinese herb, Pogostemon cablin (Labiatae), is the
aerial part of P. cablin Bentham and has been principally
used to treat the common cold, vomiting, and diarrhea in
China and the surrounding region. This plant is cultivated
extensively in Indonesia, Malaysia, China, and Brazil for
its essential oil (patchouli oil), which is important to the per-
fumery industry [79]. Anumber of investigations have been
carried out on the composition of the essential oil of P. cablin
[10,11], and the presence of some sesquiterpenoids has been
reported [12]. Sesquiterpenoids frequently occur as compo-
nents of plant essential oils and have been shown to have
various biological activities including anti-fungal, antioxi-
dant, and anti-inammatory eects [1315]. Asmall oily ses-
quiterpene, a-bulnesene, has been isolated fromthe essential
oil of P. cablin (Fig. 1), and has shown a most potent inhib-
itory eect on platelet aggregation. In the present study, we
evaluated the anti-platelet mechanism of a-bulnesene and
showed it to be a PAF receptor antagonist.
Materials and methods
Materials. a-Bulnesene was isolated from the essential oil of P. cablin
and the structure was identied using GC/MS and
1
H NMR spectroscopy
(Fig. 1). PAF, AA, uo-3/AM, indomethacin, and ethylenediaminetetra-
acetic acid (EDTA, disodium salt) were purchased from Sigma Chemical
(St. Louis, MO). The thromboxane B
2
(TXB
2
) and prostaglandin E
2
(PGE
2
) enzyme immunoassay kits were obtained from Cayman Chemical
Co. [
3
H]PAF (1-O-[
3
H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine),
with a specic radioactivity of 120 Ci mmol
1
, a product of Perkin-Elmer
Science, was dissolved in absolute ethanol and diluted with saline solution
containing 2.5 mg mL
1
bovine serum albumin (BSA) immediately prior
to use. All other chemicals were of the highest purity grade available.
Preparation of the platelet suspension. The platelet suspension was
prepared according to a procedure previously described with some mod-
ications [16,17]. Blood was collected from the marginal ear vein of New
Zealand White rabbits. The blood was then anticoagulated with acid cit-
rate dextrose (ACD, 6:1, v/v) and centrifuged for 8 min at 1000g at room
temperature. The upper portion was kept as platelet-rich plasma (PRP)
after mixing with EDTA to a nal concentration of 5 mM. Platelets were
obtained from the EDTA-mixed PRP by centrifugation for 12 min at
2000g. The platelet pellets were resuspended in Ca
2+
-free Tyrodes solu-
tion, and then incubated with apyrase (1 U mL
1
) for 15 min at 37 C.
After centrifugation for 6 min at 2000g, the platelet pellets were nally
suspended in Tyrodes solution with the following composition: NaCl
(137 mM), KCl (2.8 mM), MgCl
2
(2 mM), NaH
2
PO
4
(0.33 mM), CaCl
2
(1 mM), glucose (5 mM), Hepes (10 mM), and BSA (0.35%) at pH 7.3.
The concentration of platelets was adjusted to 2.5 10
8
platelets mL
1
.
Measurement of platelet aggregation. Platelet aggregation was mea-
sured turbidimetrically with a light-transmission Platelet Aggregation
Chromogenic Kinetic System PACK4 (Helena Laboratories, Beaumont,
TX, USA) with some modications [2]. The platelet suspension was stirred
at 900 rev min
1
and incubated with an appropriate amount of isopro-
panol (vehicle) or various concentrations of a-bulnesene in isopropanol at
37 C for 2 min. Aggregation was induced with PAF (5 nM) or AA
(100 lM). The absorbance of the Tyrodes solution was taken as 0%
aggregation and that of the platelet suspension as 100% aggregation. The
extent of platelet aggregation was measured as the maximal increase in
light transmission within 4 min after the addition of an inducer. When
isopropanol was used as the solvent, the nal concentration was xed at
0.5% (v/v) to eliminate the eect of the solvent. All glassware was
siliconized.
[
3
H]PAF receptor binding assay. This experiment was performed as
detailed previously [18,19] with some modications. The reaction mixture
consisted of 400 lL of platelet suspension, 50 lL of [
3
H]PAF (0.2 nM,
100,000 dpm) with or without unlabeled PAF (1.5 lM), and 50 lL of
sample or control solution. The reaction mixture was incubated at 4 C for
2 h and then 5 mL of BSA-containing saline solution was added to ter-
minate the reaction. The free and bound ligands were separated by l-
tration using a Whatman GF/C glass ber lter that had been presoaked
with ice-cold water. The lters were rapidly washed with the same solution
and then dried and placed into vials containing 10 mL of scintillation uid.
Radioactivity was measured in a liquid scintillation counter (Beckman
LS3801). The dierence between the total radioactivity of the bound
[
3
H]PAF in the absence and the presence of excess unlabeled PAF was
dened as specic binding of the radio-labeled ligand. In this set of
experiments, [
3
H]PAF was incubated with dierent concentrations of PAF
receptor antagonists, and the eect of the antagonist on the specic
binding was expressed as a percentage inhibition of the control. The IC
50
value was dened as the nal concentration of the inhibitor required to
block 50% of specic [
3
H]PAF binding to rabbit platelet receptors.
Measurement of intracellular calcium mobilization. This used a slight
modication of a method published previously [2]. The platelets were
pelleted from PRP and resuspended in Ca
2+
-free Tyrodes solution, then
incubated with uo-3/AM (2 lM) at 37 C for 30 min. In order to prevent
leakage of the dye, probenecid (2.5 mM) was added to the buers
throughout the experiments [20]. After washing, the uo-3-loaded platelets
were nally suspended in Ca
2+
-free Tyrodes solution without heparin at a
concentration of 1.3 10
8
platelets mL
1
. The uo-3-loaded platelets were
preincubated with an appropriate amount of isopropanol (control) or with
various concentrations of a-bulnesene in isopropanol in the presence of
extracellular calcium (1 mM) at 37 C for 2 min prior to the addition of
PAF. Fluorescence (Ex 505 nm, Em 530 nm) was measured with a uo-
rescence spectrophotometer (Model F4500; Hitachi, Tokyo, Japan). At
the end of the experiment, the cells were treated with digitonin (20%)
followed by the addition of 50 mM ethyleneglycol-bis(aminoethylether)-
N,N-tetraacetic acid (EGTA) to obtain the maximal and minimal uo-
rescence, respectively. [Ca
2+
]
i
was calculated as described for uo-3/AM
using the Ca
2+
-dye dissociation constant of 864 nM.
Thromboxane B
2
and prostaglandin E
2
assay. Because TXA
2
is very
unstable and rapidly converted to the more stable metabolite TXB
2
, we
measured the amount of TXB
2
present. After challenge of the platelets
with AA (100 lM) for 6 min, 1 mM ethylenediaminetetraacetic acid
(EDTA) and 20 lM indomethacin were added to terminate the reaction.
After centrifugation for 5 min at 4 C and 10,000g, the TXB
2
and PGE
2
in
the supernatants were assayed using the appropriate EIA kit according to
the procedure described by the manufacturer (Cayman).
Statistics. All results were expressed as means standard error (SE).
The statistical signicance was analyzed by Students t-test and the sig-
nicance level set at P = 0.05.
Results
Eect of a-bulnesene on platelet aggregation
The inhibitory eect of a-bulnesene on PAF- and AA-
induced platelet aggregation was determined using 5 nM
Fig. 1. The chemical structure of a-bulnesene.
1034 H.-C. Hsu et al. / Biochemical and Biophysical Research Communications 345 (2006) 10331038
PAF and 100 lM AA, which causes about 7080%
aggregation of rabbit washed platelets and the IC
50
val-
ues were calculated to be 24.47 2.45 lM and
42.47 5.97 lM, respectively. This result was concentra-
tion-dependent and paralleled the inhibitory eect on
platelet aggregation. Fifty micromolar a-bulnesene had
the highest inhibition rate when assayed against PAF-
and AA-induced platelet aggregation (Fig. 2). In the
cytotoxicity assay, a-bulnesene (12.550 lM) did not
show any toxicity against rabbit platelets even after
30 min of treatment (data not shown), and this indicated
that the inhibitory eect of a-bulnesene on platelet aggre-
gation did not occur due to cytotoxicity.
Eect of a-bulnesene on [
3
H]PAF binding of platelets
The total binding of 0.2 nM [
3
H]PAF to intact rabbit
platelets (1.3 10
8
platelets mL
1
) in the presence of BSA
(2.5 mg mL
1
) was 109131.17 4534.02 dpm (n = 6),
whereas the non-specic binding in the presence of
1.5 lM unlabeled PAF was 2339.03 292.18 dpm. As
shown in Fig. 3, the a-bulnesene concentration was
positively correlated with PAF receptor binding to rabbit
washed platelets and also antagonized [
3
H]PAF binding
to intact rabbit platelets. Without adding a-bulnesene to
the rabbit washed platelets (control group), [
3
H]PAF
showed 100% binding ability to the PAF receptor. After
treatment with dierent concentrations of a-bulnesene
(0.5, 5, and 50 lM), [
3
H]PAF showed a decreased binding
ability to the PAF receptor. a-Bulnesene could compete
with [
3
H]PAF for PAF receptor binding. The IC
50
value
of a-bulnesene for [
3
H]PAF binding was 17.62
5.68 lM, and the K
i
value was 55.81 6.07 nM. In con-
trast, 10 lM CV-3988, a specic PAF receptor antagonist,
almost completely inhibited [
3
H]PAF binding ability. The
IC
50
value and K
i
value of CV-3988 were 3.69 0.86 lM
and 34.00 7.92 nM, respectively.
Eect of a-bulnesene on the intracellular calcium increase of
platelets
In uo-3-loaded platelets, the resting level for the
intracellular Ca
2+
concentration was 61.80 1.04 nM,
and PAF (5 nM) caused an increase to 167.01
5.17 nM (Fig. 4a). a-Bulnesene (50 lM) inhibited the
PAF-induced intracellular Ca
2+
increase with an IC
50
value of 19.62 1.32 lM (Fig. 4b). Fig. 4c shows that
the increase in Ca
2+
release was negatively correlated
with the increase in a-bulnesene concentration
(12.550 lM).
The results shown in Figs. 3 and 4 reveal a similar trend
with the a-bulnesene IC
50
values for inhibition of PAF-in-
duced platelet aggregation and decreased Ca
2+
release
being 17.62 5.68 lM and 19.62 1.32 lM, respectively.
This implies that a-bulnesene is able to compete with
PAF to bind the PAF receptor and this causes the inhibi-
tion of the PAF-induced intracellular calcium increase.
Eect of a-bulnesene on thromboxane B
2
and prostaglandin
E
2
formation
Thromboxane B
2
and prostaglandin E
2
formation in
rabbit washed platelets were measured at 6 min after AA
was added. As shown in Table 1, the resting level of
TXB
2
in rabbit platelets was 7.64 0.40 ng mL
1
. AA
(100 lM) markedly increased TXB
2
formation to
568.51 42.63 ng mL
1
. TXB
2
formation stimulated by
AA was inhibited by increasing amounts of a-bulnesene
(25100 lM). Indomethacin (1 lM) and furegrelate
(300 lM) were chosen as positive controls and gave
98.05% and 96.49% inhibition, respectively. On the other
hand, PGE
2
was also formed in the presence of AA.
PGE
2
formation in the rabbit platelet suspension was
3.45 0.12 ng mL
1
in the unstimulated condition, and
AA raised the PGE
2
level to 20.31 0.57 ng mL
1
. PGE
2
formation was inhibited by both a-bulnesene
(25100 lM) and indomethacin, but this was reversed with
furegrelate (300 lM) (Table 1).
Fig. 2. Inhibition trend for dierent concentration of a-bulnesene on the
platelet aggregation induced by PAF (5 nM) and AA (100 lM). After
washing, the platelets were incubated with isopropanol (0.5%, vehicle) or
various concentrations of a-bulnesene at 37 C for 2 min and then the
inducer was added to trigger aggregation. Values are expressed as percent
inhibition of the aggregation and are presented as means SE (n = 5).
Fig. 3. Inhibitory eect of a-bulnesene on [
3
H]PAF binding to the PAF
receptor on rabbit washed platelets. The platelet suspension was incubated
with various concentrations of a-bulnesene and [
3
H]PAF for 2 h. CV-3988
used as positive control. After ltration, the radioactivity was measured.
Data are shown as means SE (n = 6). *P < 0.05, **P < 0.01.
H.-C. Hsu et al. / Biochemical and Biophysical Research Communications 345 (2006) 10331038 1035
Discussion
In this study, we demonstrated that a-bulnesene inhibit-
ed platelet aggregation induced by PAF and AA (Fig. 2).
Therefore, we further elucidated the detail of inhibitory
mechanisms whereby a-bulnesene aects platelet activation
caused by these two agonists.
PAF is a potent phospholipid mediator that is involved
in platelet aggregation, thrombosis, and inammation
[18,21,22]. PAF binds to target cells with specic and orga-
nized binding kinetics in order to exert its physiological
and pathophysiological eects [23]. Inhibition of this spe-
cic binding eect reduces the above-mentioned patho-
physiological responses. In the platelet aggregation assay
(Fig. 2), we observed that a-bulnesene inhibited PAF
-induced platelet aggregation. This implied that a-bulne-
sene might aect PAF-mediated signal transduction in
rabbit platelets. Indeed, a-bulnesene inhibited the specic
binding of [
3
H]PAF to rabbit washed platelets in a
dose-dependent fashion (Fig. 3), and the potency was sim-
ilar to its inhibiting eect on PAF-induced platelet aggrega-
tion. These results suggested that a-bulnesene inhibited
platelet aggregation stimulated by PAF through competi-
tively inhibiting the binding of PAF to its specic receptor.
Using the intracellular Ca
2+
increase assay (Fig. 4), the
extent of PAF-elicited intracellular Ca
2+
increase was low-
ered progressively by an increase in a-bulnesene, and this
data indicated that a-bulnesene blocked the PAF-mediated
Ca
2+
increase from the dense-tubular system (DTS) by
interfering with the binding between PAF and its receptor.
Calcium is an essential second messenger that activates
myosin light chain kinase for the induction of the shape
change, phospholipase A
2
activation of arachidonic acid
release, and platelet aggregation [24]. Rosado et al. [25]
recently reported that ethanol reduces thrombin-induced
aggregation, which is likely to be due to a signicant inhi-
bition of Ca
2+
entry, as well as a reduction in the activity of
protein tyrosine kinases. However, in the present study, we
deduced that this inhibitory eect of a-bulnesene on plate-
let aggregation stimulated by PAF was due to competitive
inhibition of the specic binding of PAF to its receptor and
this causes a suppression of intracellular Ca
2+
increase.
Release of TXA
2
from the activated platelets is an
important factor in amplication of the original stimulus
Fig. 4. The eect of a-bulnesene on PAF-induced Ca
2+
mobilization. Fluo-3-loaded platelets were pretreated with (a) isopropanol (0.5%) or (b)
a-bulnesene (50 lM) at 37 C for 3 min and then activated by 5 nM PAF to trigger [Ca
2+
]
i
increase. (c) After PAF induction, the percentage of Ca
2+
released by adding dierent concentrations of a-bulnesene. Data are shown as means SE (n = 5).

P < 0.01.
Table 1
Eects of a-bulnesene on TXB
2
and PGE
2
formation induced by AA on
rabbit washed platelets
Compound TXB
2
formation
(ng/mL)
PGE
2
formation
(ng/mL)
Basal 7.64 0.40 3.45 0.12
Control 568.51 42.63 20.31 0.57
Indomethacin (1 lM) 19.93 4.41
**
7.60 0.32
**
Furegrelate (300 lM) 11.06 0.89
**
31.65 0.65
**
a-Bulnesene
25 lM 422.96 25.90
**
18.74 1.42
37.5 lM 366.09 13.72
**
15.47 0.81
**
50 lM 292.89 5.53
**
12.36 0.54
**
100 lM 160.93 3.76
**
9.57 0.14
**
Platelet suspension was treated with 0.5% isopropanol (vehicle), 1 lM
indomethacin, 300 lM furegrelate or various concentration of a-bulnesene
for 2 min. The TXB
2
and PGE
2
formation were induced by 100 lM AA
for 6 min and terminated by 1 mM EDTA and 20 lM indomethacin.
Following centrifugation, the TXB
2
and PGE
2
levels of the supernatant
were measured and expressed as means SE from two independent
experiments.
**
P < 0.01.
1036 H.-C. Hsu et al. / Biochemical and Biophysical Research Communications 345 (2006) 10331038
due to recruitment of additional platelets from the circula-
tion to the site of aggregation [26]. The half-life of TXA
2
is
very short, and so the amount of TXB
2
, a stable metabolite
of TXA
2
, was measured as an index of TXA
2
formation.
According to our data, AA-stimulated PGE
2
and TXB
2
formation were inhibited by a-bulnesene in a concentra-
tion-dependent manner (Table 1). This result implied that
a-bulnesene decreases TXB
2
and PGE
2
formation primari-
ly by inhibiting cyclooxygenase (COX) activity. Thus,
a-bulnesene inhibited AA-induced platelet aggregation by
blocking of COX activity.
When we compared the inhibitory eect of a-bulnesene
with CV-3988 on platelet aggregation, we found three phe-
nomena. First, both a-bulnesene and CV-3988 have a sim-
ilar K
i
value (55.81 6.07 nM and 34.00 7.92 nM) in the
PAF receptor binding assay (Fig. 3). Second, if we compare
the results for the PAF binding assay for a-bulnesene and
CV-3988, the IC
50
value of a-bulnesene was only ve times
lower than that for CV-3988. In addition, a-bulnesene, as
the results in Fig. 2 and Table 1 show, is able to inhibit
both PAF- and AA metabolite-induced platelet aggrega-
tion. However, CV-3988 is only able to inhibit PAF-in-
duced platelet aggregation and does not aect COX
activity. This implied that a-bulnesene might have poten-
tial to be developed as a novel anti-platelet aggregation
agent in the future.
In conclusion, a-bulnesene is a novel anti-platelet ses-
quiterpenoid that acts as a PAF receptor competitive inhib-
itor and this would seem to be the predominant method by
which there is inhibition of PAF-induced platelet aggrega-
tion. Specically, a-bulnesene competitively inhibits the
specic binding of PAF to its receptor, thus inhibiting
intracellular Ca
2+
increase. However, in addition, a-bulne-
sene also possessed an additional inhibitory eect on
AA-induced secondary platelet aggregation and TXA
2
formation because the chemical is able to inhibit COX
activity. These results indicated that the natural sesquiterp-
enoid a-bulnesene has a dual mechanism whereby it acts on
platelet aggregation through PAF competitive inhibition
and also through inhibition of COX activity. Taken
together, our study is highly indicative that a-bulnesene
has potential as an anti-platelet agent and deserves further
detailed study.
Acknowledgment
We are grateful to Dr. Kirby for critical reading and
comments on the manuscript.
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