The antithrombotic activity of corilagin purified from korean herb-Phyllanthus ussuriensis

Abstract

Plasminogen activator inhibitor-1(PAI-1) is a main negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we revealed that corilagin which purified from Korean herb, Phyllanthus ussuriensis, inhibited plasma PAI-1 and increased the activity of t-PA in vitro and in vivo. We then observed that when injected more than 6mg/kg of corilain into the rat electrically stimulated carotid artery thrombosis rat, the rethrombosis ratio was lower than 28.6%, and it was much lower than when 20000IU/kg of urokinase was injected. At the end we found out that corilagin remarkably decreased the content of plasma fibrin in the thrombosis rat and the decrease at dose of more than 6mg/kg was about 4.4 fold than the control. Our data show that corilagin is a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential and has great prospect as thrombosis therapeutic drug.

Full text

The antithrombotic activity of corilagin purified from korean
herb-Phyllanthus ussuriensis
Kum Chol Ri1,*, Chol Ho Ju1
Abstract: Plasminogen activator inhibitor-1(PAI-1) is a main negative regulator of the
fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous
thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In
this study, we revealed that corilagin which purified from Korean herb, Phyllanthus
ussuriensis, inhibited plasma PAI-1 and increased the activity of t-PA in vitro and in vivo. We
then observed that when injected more than 6mg/kg of corilain into the rat electrically
stimulated carotid artery thrombosis rat, the rethrombosis ratio was lower than 28.6%, and it
was much lower than when 20000IU/kg of urokinase was injected. At the end we found out
that corilagin remarkably decreased the content of plasma fibrin in the thrombosis rat and the
decrease at dose of more than 6mg/kg was about 4.4 fold than the control. Our data show that
corilagin is a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential and
has great prospect as thrombosis therapeutic drug.
Key Words:Plasminogen activator, Plasminogen activator inhibitor-1 (PAI-1), corilagin, thrombosis
Introduction
Fibrinolysis is an important physiologic mechanism
that removes intravascular thrombi to maintain vascular
patency in the setting of thrombosis. Plasminogen
activator inhibitor-1(PAI-1) is an immediate inhibitor of
tissue-type plasminogen activator (t-PA) and urokinse-
type plasminogen activator (u-PA), and therefore is a
negative regulator of the fibrinolytic system.
Recent studies suggest that PAI-1 also contributes
directly to the complications of obesity, including type 2
diabetes and coronary arterial thrombi, and may even
influence the accumulation of visceral fat [1]. Other
studies substantiate PAI-1 plays an important role in
cellular motility and tumor angiogenesis, and an orally
active PAI-1 inhibitor prevents angiogenesis in a
Matrigel implant [2].
It was found out that high levels of PAI-1 reduce
fibrinolytic potential and contribute to the development
of thrombosis. In the clinic, elevated levels of PAI-1
antigen and activity have been reported in patients with a
variety of thrombotic diseases including deep vein
thrombosis, disseminated intravascular coagulation,
unstable angina, myocardial infarction and coronary
artery disease [4, 5]. In animal studies, transgenic mice
expressing high levels of PAI-1 develop spontaneous
thrombosis, whereas PAI-1-deficient mice are more
resistant to venous or arterial thrombosis induced by
either endotoxin or chemical and electric injury [6].
1 Life Science Department, Univercity of Science, Pyongyang 999093,
Democratic People’s Republic of Korea .* address for correspondence:
15124579835 @163.com..Kum Chol Ri
Inhibition of PAI-1 activity by monoclonal or polyclonal
antibodies prevents thrombus formation in animal models,
indicating that inhibition of PAI-1 is a potential strategy
to prevent thrombosis. Mechanistically, the anti-
thrombotic effects of PAI-1 inhibition are achieved by
enhancing endogenous fibrinolytic potential without
directly affecting blood coagulation or platelet function.
In PAI-1-deficient mice, for example, no significant
defects were found in coagulation assays. PAI-1-deficient
mice showed no significant abnormalities in bleeding
tests and exhibited no spontaneous bleeding [7, 17].
These data suggest that an antithrombotic agent based on
PAI-1 inhibition may have a lower risk of bleeding than
that of conventional antiplatelet and anticoagulant drugs,
and therefore have a better therapeutic index.
Therefor, some studies to find out small molecule
compounds which inhibit either PAI-1 production or
activity have been performed [3]. Recently, we have
identified a small molecule PAI-1 inhibitor, corilagin,
among different compounds purified from korean herbs
by a high-throughput screening. We now report
antithrombotic characteristic of corilagin.
Materials and methods
The corilagin was prepared by filtering the extract of
Korean herb Phyllanthus ussuriensis at the gel
chromatography, and its chemical structure was verified
by nuclear magnetic resonance spectro-scopy and mass
spectrometry. To study the effects of corilagin on the
activity of PAI-1 in vitro, we collected blood from a
healthy rat, prevented coagulation by using 3.8% citric

Fig. 1 The structure of corilagin
sodium solution, and obtained a platelet-free plasma by
centrifugating for 30 minutes in 4℃, 2500rpm. The
plasma was mixed with different concentrations of
corilagin and kept for 45 minutes. The activities of t-PA
and PAI-1 were measured in the 96-well microtiter plate
according to the chromogenic substrate analysis kits
(Chromogenix) [3]. ThermoMax plate reader(Molecular
Devices, Sunnyvale, CA) was used for the chromogenic
analysis.
We also studied the effects of corilagin on the activities
of PAI-1 and tPA of the rat plasma in vivo. We divided
rats into two groups and injected 0.9% saline solution
and corilagin respectively for 20 minutes at the rate of
0.5mg/kg/min. We gathered blood before injection and
10 minutes, 30 minutes, 60 minutes, 90 minutes,
120minutes after injection respectively, and measured the
the activities of PAI-1 and tPA in the plasma.
To examine the thrombolysis effect of corilagin in the
thrombosis animals, electrically stimulated carotid artery
thrombosis rats [9] were used. In the electrically
stimulated carotid artery thrombosis rats, we divided rats
into 5 groups and 0.9% saline solution, 20000 IU/kg of
urokinase(Sigma, St.Louis, MO), 3mg/kg of corilagin, 6
mg/kg of corilagin, 9 mg/kg of corilagin were injected
respectively. At one hour after injection, the hematocele
was measured with supersonic tachometer (DVM-4200,
Hayashi Denki). We evaluated the vessels to be opened if
the hematocele is over half of the original hematocele
(the hematocele measured before the thrombus was
formed) and to fail to be opened if the hematocele is less
than half of the original hematocele. The vessels which
was opened at first but decreased to less than 25% at 1
hour after successful reperfusion were evaluated as re-
occlusion.
In order to study the effect of corilagin on the content
of the plasma fibrin, we divided thrombosis rats into 4
groups and 0.9% saline solution, 3 mg/kg of corilagin, 6
mg/kg of corilagin and 9 mg/kg of corilagin were
injected respectively. At 1 hour after that, we gathered
blood from each rat, prevented coagulation by using
citric sodium solution, and obtained a platelet-free
plasma by centrifuging for 15 minutes in 2500rpm. 1ml
of 0.025M CaCl2solution was added to each of the 1.9ml
of plasma from test groups in order to form the fibrin.
After centrifuging the fibrin fibers, the wet and dried
weight was measured with an electronic balance.
The MDA content of the brain was measured using the
TBA method [15].
Results and Discussion
- The chemical structure of corilagin
The chemical structure of corilagin is shown in Fig. 1.
- The effects of corilagin on the activities of PAI-1
and tPA of the rat plasma in vitro
We examined the effects of corilagin on the activities
of PAI-1 and tPA of the rat plasma, and the results were
shown in Table 1.
Table 1. The effect of corilagin on rat plasma PAI-1 and
tPA activities in vitro
( * P<0.05, ** P<0.01(comparison with saline solution)
As Table 1 shows, corilagin markedly inhibits the
activity of PAI-1 of the rat plasma and raises that of tPA
in vitro. The IC50 on the PAI-1 of the rat plasma
calculated from Table 1 was 18mg /L.
- The effects of corilagin on the activities of PAI-1
and tPA of the rat plasma in vivo
Table 2 and Table 3 show the effects of corilagin on
the activities of PAI-1 and tPA of the rat plasma in vivo,
respectively .
As Table 2 and Table 3 show, corilagin decreased
remarkably the activity of PAI-1 of the plama and
increased that of tPA of the plasma. At 90 minutes after
injection, the rat plasma PAI-1 activity was decreased to
the minimum value of 16.1±3.7 IU/ml and the activity of
the tPA of the plasma was mostly increased.
corilagin
mg/L
PAI-1 activity
IU/mL
tPA activity
IU/mL
0
10
20
40
80
160
19.2±2.5
16.1±2.4＊
11.5±2.2＊＊
7.4±3.5＊＊
4.5±1.6＊＊
3.7±1.4＊＊
3.3±1.4
4.7±1.6＊
5.2±1.8＊
5.7±1.6＊＊
6.5±1.7＊＊
7.0±1.8＊＊

Table 2. The change in the activity of the rat plasma PAI-1 according to the time intervals
since injection of corilagin
The unit of PAI-1 activity is IU/ml.
*P<0.05, **P<0.01(comparison with the same time of saline solution), #P<0.01(comparison with 0 min)
Table 3. The change in the activity of the rat plasma tPA according to the time intervals
since injection of corilagin
The unit of t-PA activity is IU/ml.
**P<0.01(comparison with the same time of saline solution), #P<0.01(comparison with 0 min)
- The thrombolysis effect of the corilagin on the
thrombus in the carotid artery of electrically stimulated
carotid artery thrombosis rats
We examined the thrombolysis effect of the corilagin
on the thrombus in the carotid artery of electrically
stimulated carotid artery thrombosis rat, and the results
were shown in Table 4.
Table 4. The thrombolysis effect of the corilagin on the
carotid artery thrombus in rats
Medicine
dose,
mg/kg
reperfusion
/total
reocclusion
/reperfusion
saline
corilagin
urokinase
-
3
6
9
20000
IU/kg
0/10
2/10＊
7/10＊
7/10＊
7/10＊
0/0
2/2＊
2/7＊
2/7＊
3/7＊
* P<0.05(comparison with saline)
As Table 4 shows, when injected 6mg/kg of corilagin,
reperfusion ratio was 70%, and reocclusion ratio was
28.6%, which is better than the injection of 20000IU/kg
of urokinase (reocclusion ratio was 42.9% in this case).
- The change in the content of the plasma fibrin
according to the variation in the corilagin content in the
thrombosis rats
The change in the content of the plasma fibrin
according to the variation in the corilagin dose in the
thrombosis rats was shown in Table 5.
Table 5 shows that the plasma fibrin content was
markedly reduced in thrombosis rats injected with
corilagin in comparison with the control. When more
Table 5. The change in the content of the plasma fibrin
according to the variation in the corilagin dose in the
thrombosis rats
dose,
mg/kg
the content of the plasma fibrin
wet weight,
mg/ml
dried weight,
mg/ml
saline
3
6
9
26.9±1.9
17.8±1.2＊
6.5±0.7＊＊
6.4±0.9＊＊
9.3±0.8
5.1±0.5＊
2.1±0.3＊＊
2.0±0.2＊＊
*P<0.05, **P<0.01(comparison with saline solution)
than 6mg/kg of corilagin was injected, the fibrin content
was decreased by 4.4 times in comparison with the
control.
- The effect of corilagin on MDA content of the brain
tissue in thrombosis rats
Table 6 shows the effect of corilagin on MDA content
of the brain tissue in thrombosis rats.
Table 6. The effect of corilagin on MDA content of the
brain tissue
corilagin dose,
mg/kg
MDAcontent,
nmol/g
saline
3
6
9
555.6±14.4
513.9±13.7＊
395.7±15.8＊
392.6±14.2＊
*P<0.05(comparison with the same time of saline solution)
As Table 6 shows, the MDA content of the brain tissue
in the rats was decreased according to the increase in the
corilagin dose.
time, min
medicine
0
30
60
90
120
saline
corilagin
26.4±4.8
26.7±5.2
25.9±5.0
24.1±4.6♯
27.4±4.3
20.3±5.4＊♯
26.9±5.7
16.1±3.7＊＊♯
27.4±4.9
18.8±3.5＊♯
time, min
medicine
0
30
60
90
120
saline
corilagin
2.4±0.6
2.3±0.7
1.9±0.3
3.6±1.3＊＊♯
2.0±0.5
7.3±1.4＊＊♯
1.9±0.2
8.3±1.7＊＊♯
2.1±0.4
5.8±1.5＊＊♯

Conclusions
Through our experiments we revealed that corilagin
inhibited plasma PAI-1 and increased the activity of t-PA
in vitro and in vivo. We then observed that when injected
more than 6mg/kg of corilagin into the electrically
stimulated carotid artery thrombosis rats, the
rethrombosis ratio was lower than 28.6%, and it was
much lower than when 20000IU/kg of urokinase was
injected. At the end we found out that corilagin
remarkably decreased the content of plasma fibrin in the
thrombosis rats and the decrease at dose of more than
6mg/kg was about 4.4 fold than the control.
Thrombosis is a major cardiovascular disease,
afflicting millions people in the world. Currently used
antithrombotic drugs are inhibitors of either platelets or
blood clotting factors. More recently, several small
molecule PAI-1 inhibitors have been identified [9]. Most
of them have been reported that inhibit PAI-1 activity in
vitro [8, 10–12]. One compound series among them,
represented by XR5118, was shown to have
antithrombotic efficacy in the electrically stimulated
arterial thrombosis rats [13]. Another PAI-1 inhibitor,
WAY-140312, was shown to enhance arterial blood flow
and reduce venous thrombus weight when it was
administered orally in the thrombosis rats [14]. These
studies have demonstrated the feasibility of developing
nonpeptic small molecule PAI-1 inhibitors as a new class
of antithrombotic agents[16, 18, 19, 20]. Our data show
that corilagin is a new class of small molecule PAI-1
inhibitors with anti-thrombotic potential and has great
prospect as thrombosis therapeutic drug.
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