Animal Cells and Systems

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Antiplatelet activities of hyperoside in vitro and in

vivo

Sae-Kwang Ku, Hayoung Yoo, Wei Zhou, MinKyun Na & Jong-Sup Bae

To cite this article: Sae-Kwang Ku, Hayoung Yoo, Wei Zhou, MinKyun Na & Jong-Sup Bae (2014)
Antiplatelet activities of hyperoside in�vitro and in�vivo , Animal Cells and Systems, 18:3, 204-209,
DOI: 10.1080/19768354.2014.925970

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© 2014 Korean Society for Integrative

Biology

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 Animal Cells and Systems, 2014
Vol. 18, No. 3, 204–209, http://dx.doi.org/10.1080/19768354.2014.925970

Antiplatelet activities of hyperoside in vitro and in vivo

Sae-Kwang Kua, Hayoung Yoob, Wei Zhouc, MinKyun Nac* and Jong-Sup Baeb*
a
Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea;
b
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic
of Korea; cCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
(Received 28 March 2014; received in revised form 1 May 2014; accepted 5 May 2014)

Thrombosis and thromboembolic occlusions of major and minor blood vessels are a major complication in
various peripheral vascular diseases. Antiplatelet agents, key tools in the treatment of atherothrombosis,
therefore became a mainstay medication for a wide range of vascular diseases. Hyperoside, an active
compound from the Rhododendron brachycarpum G. Don (Ericaceae), was reported to have antioxidant, anti-
hyperglycemic, anticancer, anti-inflammatory, and anti-coagulant activities. However, antiplatelet properties of
hyperoside have not been studied. In this study, the antiplatelet activities of hyperoside were measured by
thrombin- or collagen-induced platelet aggregation in vitro, adenosine diphosphate-induced platelet aggrega-
tion in vivo, and the thrombus formation in vivo. Our data showed that in vitro assays using freshly isolated
human platelets, hyperoside showed the inhibition of the thrombin- or collagen-induced platelet aggregation. In
accordance with these enhanced in vitro antiplatelet activities, hyperoside showed enhanced anti-thrombotic
effects in vivo pulmonary embolism model and arterial thrombosis model. Collectively, these results indicate
that hyperoside possesses antiplatelet activities and might offer enhanced anti-thrombotic efficacies without
increasing side effects.
Keywords: hyperoside; antiplatelet; antithrombotic; aggregation

Introduction stroke by prolonged antiplatelet therapy in various categories

NEUROBIOLOGY &

of patients. Antiplatelet Trialists’ Collaboration 1994).

PHYSIOLOGY

Platelets are blood cells that participate in the human

primary hemostatic mechanism. Platelets need to be acti-
vated to perform all of their functions, and this activation
can be initially produced after an endothelial injury that
exposes subendothelial structures to the blood flow (Albers
1995). Aggregation (platelets–platelets interaction) has the
final purpose to produce a platelet thrombus that cons-
titutes the primary hemostatic plug (Albers 1995). In
addition, platelet aggregation plays a critical role in the
pathophysiology of thrombotic diseases, and as a con-
sequence, antiplatelet agents have been used clinically in
patients at risk of brain ischemia, unstable angina, and
acute myocardial infarction (Albers 1995, George 2000).
Therefore, the interaction between platelets and blood
vessels is important in the development of thrombosis and
cardiovascular diseases (George 2000). Uncontrolled plate-
let aggregation is critical in arterial thrombosis and
may cause life-threatening disorders such as heart
attacks, unstable angina, and reocclusion after angio-
plasty (Davies & Thomas 1985). Hence, in the treatment
and prevention of these cardiovascular diseases the inhibition
of platelet aggregation is of fundamental importance (Col-
laborative overview of randomised trials of antiplatelet
therapy–I: Prevention of death, myocardial infarction, and
The search for antithrombotic agents from natural
herbal medicines is an area of considerable interest (Li
et al. 2009). Flavonoids are representative secondary
metabolites widely distributed in plants and plant-based
foods. Flavonoids are known to have a wide range of
bioactivities that include cytoprotective, antioxidant, anti-
platelet, antithrombotic, and anticarcinogenic activities
(Middleton & Drzewiecki 1984, Mukaida 2000). Flavo-
noids also display a variety of anti-inflammatory proper-
ties, some of which are suggested to affect the function of
immune system (Hirano et al. 2006). Hyperoside is a
flavonol glycoside characterized as 5,7,3′,4′-tetrahydroxy-
flavone-3-O-β-D-galactopyranoside. It has been mainly
found in the genera of Hypericum and Crataegus (Zou
et al. 2004). Our recent study resulted in a large-scale
isolation of hyperoside from the leaves of Rhododendron
brachycarpum (Zhou et al. 2013). Pharmacological invest-
igation on hyperoside has demonstrated a variety of
biological activities that include antioxidant (Zhou et al.
2013), anti-hyperglycemic (Verma et al. 2013), anticancer
(Li et al. 2012), anti-inflammatory (Kim, Um et al. 2011),
and cardioprotective activities (Li et al. 2013). However,
to our knowledge, the anticoagulant and antiplatelet
functions of hyperoside are not studied. Since the leaves

*Corresponding authors: Emails: mkna@cnu.ac.kr and baejs@knu.ac.kr

© 2014 Korean Society for Integrative Biology


of R. brachycarpum have been traditionally used to treat
cardiovascular and inflammatory diseases, we assumed
that hyperoside, a major constituent in the plant, might be
related to its traditional application. Therefore, in this
study, we test the antiplatelet activity of hyperoside by
measuring platelet aggregation and thrombus formation in
vitro and in vivo.
Materials and methods
Reagents
Thrombin, collagen, epinephrine, adenosine diphosphate
(ADP) were purchased from Sigma (St. Louis, MO, USA).
Extraction and isolation
Hyperoside (Figure 1) was available from our previous
study (Zhou et al. 2013). Briefly, dried leaves of R.
brachycarpum (25 kg) were extracted with MeOH (250 L)
at room temperature for one week and filtered to acquire
the MeOH extract (6 kg). Half of the extract (3 kg) was
suspended in H2O (10 L) and partitioned with n-hexane
(10 L × 3), CHCl3 (10 L × 3), EtOAc (10 L × 3), and
BuOH (10 L × 3) to obtain four solvent extracts (438, 140,
450, and 320 g, respectively). The EtOAc fraction was
further subjected to vacuum liquid chromatography
employing a silica gel column and eluted with CHCl3-
MeOH (90:10→0:100) to generate 10 fractions (Fr. E1–
E10). Fr. E4 (143.77 g) was subjected to silica gel column
(18 × 30 cm) chromatography and eluted with CH2Cl2-
MeOH (9:1→8:2) to yield seven subfractions (Fr. E41–
E47). Hyperoside (7.6 g) was purified from Fr. E47 (9.97 g)
by medium pressure liquid chromatography (MPLC) (KP-
C18-HS [340 g] column) eluting with a gradient of MeOH-
H2O (3:7→5:5). The chemical structure of hyperoside was
identified by analysis of nuclear magnetic resonance
(NMR) data (Zhou et al. 2013).
Figure 1. Chemical structure of hyperoside.
Animal Cells and Systems 205
Animals and husbandry
Male C57BL/6 mice (6- to 7-week-old, weighting 18–20 g)
purchased from Orient Bio Co. (Sungnam, KyungKiDo,
Republic of Korea) were used in this study after a 12-day
acclimatization period. Animals were housed five per
polycarbonate cage under controlled temperature (20–
25°C) and humidity (40–45%) and a 12:12-hr light/dark
cycle. During acclimatization, animals were supplied a
normal rodent pellet diet and water ad libitum. All animals
were treated in accordance with the Guidelines for the
Care and Use of Laboratory Animals issued by Kyung-
pook National University (IRB No. KNU2012-13).
Cell culture
Primary human umbilical vein endothelial cells
(HUVECs) were obtained from Cambrex Bio Science
(Charles City, IA) and were maintained as described
previously (Lee et al. 2013).
Ex vivo clotting time
Male ICR mice were fasted overnight and hyperoside in
0.5% dimethyl sulfoxide (DMSO) was administered by
intravenous injection. One hour after administration,
arterial blood samples (0.1 mL) were withdrawn into
3.8% sodium citrate (1/10; v/v) for ex vivo activated
partial thromboplastin time (aPTT) and prothrombin time
(PT) determination. aPTT and PT were determined using a
Thrombotimer (Behnk Elektronik, Germany), according to
the manufacturer’s instructions as described previously
(Kim et al. 2012a, 2012b, Lee et al. 2012). In brief,
citrated mouse plasma (100 µL) was incubated for 3 min
at 37°C. aPTT assay reagent (100 µL) was added and
incubated for 1 min at 37°C, and then 20 mM CaCl2 (100
µL) was added. Clotting times were recorded. For PT
assays, citrated mouse plasma (100 µL) was incubated for
3 min at 37°C. PT assay reagent (200 µL), which has been
preincubated for 10 min at 37°C, was then added and
clotting time was recorded. PT results are expressed in
seconds and as international normalized ratio (INR), and
aPTT results are expressed in seconds. INR = (PT sample/
PT control)ISI, where ISI is the international sensitivity
index. The concentrations to double clotting time were
calculated from the equation of each line. All animals
were treated in accordance with the Guidelines for the
Care and Use of Laboratory Animals issued by the
Kyungpook National University (IRB No. KNU2012-13).
Platelet aggregation assay in vitro
Blood was collected from human volunteers by venipunc-
ture and it was drawn into a plastic syringe containing
3.8% trisodium citrate solution (1:9 citrate/blood, v/v).
The healthy, male volunteers had not taken any drugs for
206 S.-K. Ku et al.
at least seven days. Platelet-rich plasma (PRP) was pre-
pared by centrifugation at room temperature for 15 min
at 150 g. PRP was adjusted to a concentration of 1 × 109
platelets/mL with use of a hemocytometer for cell
counts. PRP was washed once with HEPES buffer
(5 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 0.42 mM
NaH2PO4, 2 mM MgCl2, 5.6 mM glucose, 0.1% BSA
(w/v), pH to 7.45) in the presence of 1 mM CaCl2. The
platelet aggregation study was carried out according to
a method previously reported (Kim, Kim et al. 2011).
Washed platelets were incubated with indicated hypero-
side in Tris-buffered saline (TBS) for 3min, and then
stimulated by thrombin (3U/mL, Sigma) in 0.9% saline
solution at 37°C for 5 min or collagen (1 µg/mL) at 37°C
for 15min. Platelet aggregation was recorded using an
aggregometer (Chronolog, Havertown, PA, USA).
ADP-induced platelet aggregation assay ex vivo
Male C57BL/6 mice were fasted overnight and indicated
hyperoside in TBS was administered by intravenous
injection. After 24 hr, PRP (109 platelets/mL) in a volume
of 240 µL were incubated at 37°C for 1.5 min in the
aggregometer with continuous stirring at 1000 rate per
minute and then stimulated with 10 µL of ADP. Changes
in light transmission were recorded for 7 min after
stimulation with ADP. The extent of aggregation was
expressed as a percentage of the maximum light transmit-
tance, obtained with the washed platelet aggregation.
Determination of cytotoxicity
Platelet cytotoxicity was determined by the leakage of
lactate dehydrogenase (LDH) from platelets. After incuba-
tion with indicated hyperoside at 37°C for 3 min, aliquots
were collected and centrifuged at room temperature for
2 min at 12,000 g. A 50 µL aliquot of the resulting
supernatant was added to 1 mL of Tris-EDTA-NADH
buffer [56 mM Tris (hydroxymethyl)aminomethane, 5.6
mM EDTA, 0.17 mM β-NADH, pH 7.4] and then
incubated for 10 min at 37°C. After incubation, 100 µL
of 14 mM pyruvate solution that had been preincubated at
37°C was added. The reduction in absorbance at 340 nm
by the conversion of NADH to NAD+ was measured for
the evaluation of LDH activity in the aliquots. The extent
of LDH leakage was expressed as percentage of total
enzyme activity measured in platelets completely lysed
with 0.3% Triton X-100.
Cell viability assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) was used as an indicator of cell viability.
HUVECs were grown in 96-well plates at a density of 5 ×
103/well. After 24 hr, cells were washed with fresh
medium, followed by treatment with hyperoside. After a
48-hr incubation period, cells were washed, and 100 µL of
1 mg/mL MTT was added, followed by incubation for 4 hr.
Finally, 150-µL DMSO was added to solubilize the
formazan salt formed, the amount of which was deter-
mined by measuring the absorbance at 540 nm using a
microplate reader (Tecan Austria GmbH, Austria).
Arterial thrombosis animal model
The mouse model of ferric chloride (FeCl3)-induced
carotid artery thrombosis. The FeCl3-induced thrombosis
model was established as previously described (Izuhara
et al. 2008). Male C57BL/6 mice were fasted overnight
and indicated hyperoside in TBS was administered by
intravenous injection. Then, mice were anesthetized using
3% isoflurane (Forane®, Choongwae Pharma. Corp.,
Seoul, Korea) and injected intravenously with 0.1 mL of
0.1% rhodamine 6G (Sigma). A testicular artery (200 µM
in diameter) was carefully exposed and a cotton thread
(0.2 mm in diameter) saturated with 0.25 mol/L FeCl3 was
applied to the adventitial surface. After 5 min, the cotton
thread was removed, and the wound was flushed with
saline solution. Thrombus formation was monitored at 35°C
by 3-dimensional imaging as previously described (Goto
et al. 2004). The size and time of thrombus formation
was monitored, and the findings categorized as follows: no
thrombus is 0; small thrombus (50 × 75 µM) is 1; medium-
sized thrombus (100 × 150 µM) is 2; and large thrombus
(200 × 300 µM) is 3 as the score of thrombus. The time from
FeCl3-mediated endothelial injury to occlusion of the testicu-
lar artery by a large thrombus was measured.
Acute thrombosis induced by a combination of collagen
and epinephrine in mice
Male C57BL/6 mice were fasted overnight and divided
into groups of 10 animals. Hyperoside suspended in TBS
was administered to mice intravenously. A mixture of
collagen (500 µg/kg) plus epinephrine (50 µg/kg) was
injected to the tail vein of mice to induce acute thrombosis
1 hr later. Each mouse was carefully watched for 15 min
to determine whether the mouse was paralyzed, dead, or
recovered from the acute thrombotic challenge. For
statistical analysis, five separated experiments were
fulfilled.
Statistical analysis
Data are expressed as the means ± SEM of at least three
independent experiments. Statistical significance between
two groups was determined using the Student’s t-test.
Statistical significance was accepted for p values < 0.05.
 Animal Cells and Systems 207

Results and discussion

Recently, we reported the anti-coagulant activities of
hyperoside (Ku et al. 2012) but to our knowledge, the
antiplatelet functions of hyperoside are not studied.
Therefore, in this study, we sought to test the antiplatelet
functions of hyperoside for the first time.

Figure 3. Cytotoxicity and viability of hyperoside. (A) Effects of

Effects of hyperoside on platelet aggregation in vitro hyperoside on LDH release in platelets were monitored as
described in “Materials and methods” section. (B) Effect of
and in vivo hyperoside on cellular viability was measured by MTT assay
We monitored the effects of hyperoside on thrombin- using human endothelial cells. Data represent the means ± SEM
or collagen-mediated platelet aggregation. The results of three independent experiments performed in triplicate. **p <
(Figure 2) showed that hyperoside significantly inhib- 0.01 as compared to 0 (A).
ited mouse platelet aggregation induced by thrombin (final
concentration: 3U/mL) or collagen (1 µg/mL) in a concen- performed in HUVECs treated with hyperoside for 24 hr.
tration dependent manner. Based on the inhibition curves, At concentrations up to 50 µM, hyperoside did not affect
the IC50 (the half maximal inhibitory concentration) of cell viability (Figure 3B).
hyperoside in PRP was 45.5 µM and 40.6 µM for thrombin
and collagen, respectively. The IC50 of integrilin, as a
positive control, was 434 nM. It is well known that ADP In vivo effects of hyperoside arterial thrombosis and
could induce the platelet aggregation in vivo (Boullin et al. pulmonary thrombosis model
1972). In this study, ADP at 5 µM was used to induce
The rat model of FeCl3-induced carotid artery thrombosis
platelet aggregation ex vivo (Figure 2). Figure 2 illustrated
(Izuhara et al. 2008) has been commonly used to assess
the inhibition of ex vivo platelet aggregation triggered by
antiplatelet effects. The time to thrombus formation and the
hyperoside at 20 and 50 µM. Because the average weight
size of the resulting thrombi are summarized in Figure 4.
of a mouse is 20 g and the average blood volume is 2 mL,
the injected hyperoside (1.86, 4.64, 18.6, or 46.4 µg/
mouse) produced a concentration maximum of 2, 5, 20, or
50 µM in peripheral blood.
To examine the cytotoxicity of hyperoside, we meas-
ured the LDH release in platelet for the determination of
cell lysis. Hyperoside did not induce LDH release while
positive control, digitonin (50 µM) increased the LDH
release significantly (Figure 3A) reflecting that hyperoside
treatment does not affect cell integrity. And to test the
viability of hyperoside, cellular viability assays were

Figure 4. Effects of hyperoside on arterial thrombosis and on

Figure 2. Effects of hyperoside on platelet aggregation in vitro
and ex vivo. (A) Washed platelets were incubated with indicated
hyperoside in TBS for 3 min, and then stimulated by 3U/mL
thrombin (Th, white bar) at 37°C for 5 min or 1 µg/mL collagen
(Col, black bar) at 37°C for 15min. Then, aggregation was
recorded. (B) Indicated hyperoside in TBS was injected intrave-
nously. Effects of hyperoside on mouse platelet aggregation
induced by 5 µM ADP were monitored ex vivo. Data represent
the means ± SEM of three independent experiments performed in
triplicate. **p < 0.01 as compared to thrombin/collagen alone
(A) or ADP alone (B).
acute thrombosis. (A) Time to large thrombus formation.
Tirofiban (Tiro) was used as a positive control. (B) The size
score of the thrombus at 10 min (white bar) and 60 min (black
bar) after FeCl3 treatment as described in “Materials and
methods” section (C) After hyperoside was injected intrave-
nously, a mixture of collagen (C, 500 µg/kg) plus epinephrine
(E, 50 µg/kg) was injected to the tail vein of mice to induce acute
thrombosis 6 hr later. Then, mice (10 mice per group) were
carefully watched for 15 min to determine whether the mouse
was paralyzed, dead, or recovered from the acute thrombotic
challenge. Data represent the means ± SEM of three independent
experiments. *p < 0.05 or **p < 0.01 as compared to control
(A, B) or C + E (C).
208 S.-K. Ku et al.
Table 1. Ex vivo coagulation time of hyperosidea.
Sample Dose aPTT (s) PT (s)
Control Saline 33.8 ± 1.2 14.6 ± 0.8
Hyperoside 1.9 µg/mouse 34.2 ± 0.6 14.4 ± 1.2
4.6 µg/mouse 35.6 ± 1.6 15.0 ± 0.8
18.6 µg/mouse 54.2 ± 1.4* 23.2 ± 1.2
46.4 µg/mouse 60.6 ± 1.4* 27.8 ± 1.4
Note: aEach value represents the means ± SEM (n = 10).
*p < 0.01 as compared to control.
Data showed that endothelial injury after FeCl3 treatment
in control mice led to the growth of large thrombi in 9.5 ±
2.3 min and the antiplatelet GP IIb/IIIa inhibitor, tirofiban,
significantly slowed the growth of large thrombi to 53.2 ±
9.4 min. Hyperoside significantly slowed the growth of
thrombi (Figure 4A). We also examined the effect of
hyperoside on thrombus size at 10 min and 60 min after
FeCl3-induced endothelial injury (Figure 4B). Results
showed that hyperoside could reduce FeCl3-induced
thrombus formation.
The results of in vivo pulmonary thrombosis model are
shown in Figure 4C. An intravenous injection of a mixture
of collagen and epinephrine to mice induced massive
pulmonary thrombosis causing acute paralysis, leading
ultimately to a sudden death (87% ± 6% of mortality). The
mortalities in hyperoside-treated group were reduced
significantly compared to collagen and epinephrine-treated
group (Figure 4C). To confirm the inhibitory effects of
hyperoside on pulmonary thrombosis, we determined the
effects of hyperoside on ex vivo coagulation time. As
shown in Table 1, the anticoagulation in response to
hyperoside was also observed ex vivo in mice with dose-
dependent prolongation of the aPTT and PT, which is
consistent with the previous reports (Ku et al. 2012).
It is well recognized that polyphenols have antithrom-
botic effects that appear to be the result of reduced sus-
ceptibility to platelet activation and aggregation, reduced
synthesis of prothrombotic mediators, and decrease gene
expression of tissue factor (Vilahur & Badimon 2013). In
our study, hyperoside that bears four phenolic hydroxyl
groups at 5, 7, 3′, and 4′ positions of flavonol agly-
cone demonstrated to be potential in inhibiting platelet
aggregation. Epidemiological evidence has supported that
polyphenol-rich diet is beneficial to the development and
progression of cardiovascular disease. Although the poly-
phenols have proven effective in decreasing platelet
activation through a variety of mechanisms, most data
are based on in vitro experiments and only few are based
on animal studies (Vilahur & Badimon 2013). Our study
clearly shows that hyperoside is one of the specific
polyphenols capable of reducing platelet aggregation in
animal model, suggesting benefits in attenuating the risk
of thrombosis and cardiovascular disease.
PT
In conclusion, this study shows the antiplatelet effects
(INR)
of hyperoside using freshly isolated human platelet system
1.00 in vitro and in vivo thrombosis model. Based on the
0.97 cellular toxicity and viability assays, we believe that the
1.06 antiplatelet effects of hyperoside were not due to any
2.77 cytotoxic effect. We hope this study will prove helpful to
4.12* those involving in pharmacological strategies for the treat-
ment or prevention of vascular diseases via the regulation
of platelet functions and may be of importance for the
future design of antiplatelet therapy using hyperoside.
Acknowledgments
This study was supported by the National Research Foundation
of Korea (NRF) funded by the Korea government [MSIP] (Grant
No. 2013-067053).
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