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Article
Inhibitory Effect of Triterpenoids from Panax ginseng
on Coagulation Factor X
Lingxin Xiong 1,2,3 , Zeng Qi 1,2 , Bingzhen Zheng 1,2 , Zhuo Li 1,2 , Fang Wang 3 , Jinping Liu 1,2, *
and Pingya Li 1,2, *
1
2
3
*
School of Pharmaceutical Sciences, Jilin University, Fujin Road 1266, Changchun 130021, China;
[email protected] (L.X.); [email protected] (Z.Q.); [email protected] (B.Z.);
[email protected] (Z.L.)
National and Local Joint Engineering Research Center for Ginseng Innovative Drugs Development,
Western Chaoyang Road 45, Changchun 130021, China
Department of Pathogen Biology, Basic Medical College, Jilin University, Changchun 130021, China;
[email protected]
Correspondence: [email protected] (J.L.); [email protected] (P.L.); Tel.: +86-431-8561-9803 (J.L. & P.L.)
Academic Editors: Anusha Chaparala, Lorne Hofseth and Woo-Sik Jeong
Received: 23 February 2017; Accepted: 11 April 2017; Published: 24 April 2017
Abstract: Enzymes involved in the coagulation process have received great attention as potential
targets for the development of oral anti-coagulants. Among these enzymes, coagulation factor Xa
(FXa) has remained the center of attention in the last decade. In this study, 16 ginsenosides and two
sapogenins were isolated, identified and quantified. To determine the inhibitory potential on FXa,
the chromogenic substrates method was used. The assay suggested that compounds 5, 13 and 18 were
mainly responsible for the anti-coagulant effect. Furthermore, these three compounds also possessed
high thrombin selectivity in the thrombin inhibition assay. Furthermore, Glide XP from Schrödinger
was employed for molecular docking to clarify the interaction between the bioactive compounds and
FXa. Therefore, the chemical and biological results indicate that compounds 5 (ginsenoside Rg2),
13 (ginsenoside Rg3) and 18 (protopanaxtriol, PPT) are potential natural inhibitors against FXa.
Keywords: ginseng; triterpenoids; coagulation factor Xa; inhibitors; thrombin; molecular docking
1. Introduction
Thrombosis is a common pathology characterized by the formation of a blood clot inside a blood
vessel and the obstruction of blood flow through the circulatory system that underlies three major
cardiovascular diseases including acute coronary syndrome, stroke and venous thromboembolism [1].
Venous thromboembolism affects millions of people every year and is responsible for hundreds of
thousands of deaths in both the United States and Europe annually [2]. Warfarin, vitamin K antagonists
and low-molecular-weight heparins are the most commonly used drugs to reduce thromboembolic
occurrence. Despite the prevalence of these anti-coagulants, their employment actually increases the
risk of bleeding, brings about the inconvenience of frequent monitoring, interacts with many drugs
and foods, as well as has slow onset of action [3], which hinders further clinical application in the
treatment of thrombosis. To address the difficulties, more selective anti-coagulants are needed to meet
the requirement of efficacy and safety.
Coagulation factor X (FXa) is a vitamin K-dependent serine protease and is one of various enzymes
involved in the process of blood coagulation cascade as a catalyst in the conversion of prothrombin to
thrombin, which enables FXa to be a potent attractive target for novel anti-coagulant. Compared with
the previously prevalent targeting drug thrombin (factor IIa) inhibitors, much evidence has shown that
FXa inhibitors were more effective than direct thrombin inhibitors due to FXa’s upstream position from
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thrombin in the coagulation cascade. Selective inhibition of FXa was unable to affect the pre-existing
thrombin level, and activation and aggregation of the platelets reduced the risk of bleeding when
compared with traditional anti-coagulants [2]. In addition, FXa plays a vital role in amplifying the
process, and is endued with the ability to produce more than one thousand thrombin molecules [2,4].
Furthermore, currently available FXa inhibitors approved by Food and Drug Administration such
as rivaroxaban, apixaban and epibaxaban, have been reported to still demonstrate flaws including
bleeding risks, narrow clinical applications and drug-drug interactions [2]. Thus, there is a need to
develop novel FXa inhibitors with better efficacy and less side-effects.
Panax ginseng C. A. Mey, the Araliaceae plant, has been considered as a health food and traditional
herbal medicine in Eastern Asia including China, Korea and Bhutan over the past centuries [5].
Nowadays P. ginseng is also accessible in small doses in commercial energy beverages or herbal
teas. In a previous study, water extracts of P. ginseng were reported to possess anti-coagulation
effect in vitro [6]. In addition, some Chinese herbal formulas containing ginsenosides also displayed
a modulatory effect on the blood coagulation system. For example, a traditional Chinese medicine
remedy, Fufang Xueshuantong (including various kinds of ginsenosides) was reported to ameliorate the
disorders of the blood coagulation system in a lipopolysaccharide-induced disseminated intravascular
coagulation rat model via modulating the activation of the coagulation system [7]. In our previous
study, we demonstrated that another traditional Chinese herbal formula Xueshuan Xinmaining Tablets
(containing a total ginsenoside of ginseng stems and leaves) enhanced protective activities and
anti-oxidative effect of vascular endothelial cells in vitro and removed blood stasis syndrome in
murine models, indicating a potential anti-coagulation role in clinical application [8,9]. Furthermore,
ginsenosides Rg1 and Rg2 have been reported to possess anti-coagulation properties in vitro [6].
Although P. ginseng and part of its major functional components (ginsenosides) have shown
anti-coagulation activity in previous studies, whether ginsenosides possess anti-FXa activity and, if so,
how ginsenosides interact with surrounding residues have not been reported. Therefore, we designed
and conducted a series of anti-coagulation experiments to provide theoretical support for the further
development of novel oral-administrated ginsenosides-based FXa inhibitors.
In this study, we aimed to structurally elucidate the components of ginseng, and to determine
the content of these ginsenosides through high performance liquid chromatography (HPLC) analysis.
Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) assays
were performed to determine the plasma anti-coagulation activity of ginsenosides in vitro, and among
ginsenosides demonstrating significant in vitro anti-coagulation effects, the in vitro bioactivities
against anti-coagulation factor Xa (FXa) were assessed. Subsequently, ginsenosides that possessed the
best bioactivity were molecularly docked with the receptor protein FXa via Schrödinger software to
observe the ligand-protein interactions.
2. Results
2.1. Isolation and Characterization
The purified products above-mentioned were each characterized by NMR analyses. The structures
of compounds 1–18 were identified as follows: ginsenoside Rg1 (1, yield 0.328%), Re (2, yield 0.088%),
Rf (3, yield 0.071%), Rh1 (4, yield 0.008%), Rg2 (5, yield 0.011%), Rb1 (6, yield 0.524%), Rc (7, yield 0.121%),
Ro (8, yield 0.006%), F1 (9, yield 0.114%), Rb2 (10, yield 0.114%), Rb3 (11, yield 0.013%), Rd (12, yield 0.123%),
Rg3 (13, yield 0.021%), 20(R)-Rg3 (14, yield 0.001%), Rh2 (15, yield 0.001%), F2 (16, yield 0.001%),
protopanaxdiol (PPD, 17, yield 0.071%), protopanaxtriol (PPT, 18, yield 0.001%) [10–18].
2.2. Determination of Content for the Compounds from Ginseng
The dried ginseng root powder (1.0 g, 60 mesh sieve) was accurately weighed and was added to
two volumes of water. The mixture was heated under 100 ◦ C for 2 h. Next, the filtration was added
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to a D101 macroporous adsorption resin column eluted with water and 80% ethanol consecutively.
to80%
D101
macroporous
adsorption
resin column
column
eluted with
with
water
and
80% ethanol
ethanol
consecutively.
to
aa D101
macroporous
adsorption
resin
eluted
water
80%
consecutively.
The
ethanol
elution was
concentrated
to the residue,
which
wasand
dissolved
in 10 mL
of methanol.
The
80%
ethanol
elution
was
concentrated
to
the
residue,
which
was
dissolved
in
10
mL
of
methanol.
The
80%
ethanol
elution
was
concentrated
to
the
residue,
which
was
dissolved
in
10
mL
of
methanol.
Some standard compounds were also used in this study. Ginsenoside Rg1 (110703-200726),
Some
standard
compounds
were
also
used
in
this
study.
Ginsenoside
Rg1
(110703-200726),
Some Re
standard
compoundsginsenoside
were also used
in this study. Ginsenoside
Rg1
ginsenoside
(110754-200822),
Rb2 (111715-201203),
ginsenoside
Rb3(110703-200726),
(111686-201203),
ginsenoside Re
Re (110754-200822),
(110754-200822), ginsenoside
ginsenoside Rb2 (111715-201203),
(111715-201203), ginsenoside
ginsenoside Rb3
Rb3 (111686-201203),
ginsenoside
ginsenoside
Rd (111818-201302),
ginsenosideRb2
Rg3 (110804-201504),
ginsenoside Rf(111686-201203),
(111719-201505),
ginsenoside Rd
Rd (111818-201302),
(111818-201302), ginsenoside
ginsenoside Rg3
Rg3 (110804-201504),
(110804-201504), ginsenoside
ginsenoside Rf
Rf (111719-201505),
(111719-201505),
ginsenoside
ginsenoside Rh2 (111748-200501), ginsenoside Rg2 (111779-200801), ginsenoside Ro (111903-201604),
ginsenoside Rh2
Rh2 (111748-200501),
(111748-200501), ginsenoside Rg2
Rg2 (111779-200801), ginsenoside
ginsenoside Ro
Ro (111903-201604),
(111903-201604),
ginsenoside
protopanaxdiol
(111747-200501) andginsenoside
protopanaxtriol (111779-200801),
(111755-200601) were purchased
from the National
protopanaxdiol
(111747-200501)
and
protopanaxtriol
(111755-200601)
were
purchased
from the
the
protopanaxdiol
(111747-200501)
and
protopanaxtriol
(111755-200601)
were
purchased
from
Institutes for Food and Drug Control (Beijing, China). Ginsenosides Rh1, Rb1, Rc, F1, 20(R)-Rg3
and F2
National
Institutes
for
Food
and
Drug
Control
(Beijing,
China).
Ginsenosides
Rh1,
Rb1,
Rc,
F1,
20(R)-Rg3
National Institutes for Food and Drug Control (Beijing, China). Ginsenosides Rh1, Rb1, Rc, F1, 20(R)-Rg3
were
provided by the New Drug Research and Development Laboratory of Jilin University.
and F2
F2 were
were provided
provided by
by the
the New
New Drug
Drug Research
Research and
and Development
Development Laboratory
Laboratory of
of Jilin
Jilin University.
University.
and
The determination of the saponins was performed using a HPLC system. The detection
The determination
determination of
of the
the saponins
saponins was
was performed
performed using
using a HPLC
HPLC system.
system. The
The detection
detection
The
◦ C. The amobile
wavelength
was 40
40 °C.
phasewas
wascomprised
comprisedofof
wavelengthwas
was203
203nm.
nm. Column
Column temperature
temperature was
was
The mobile
mobile phase
phase
wavelength
was
203
nm.
Column
temperature
40 °C.
The
was comprised
of
acetonitrile
(A)
and
1%
acetic
acid
ininwater
solvent
(B).
The
gradient mode
modewas
wasas
asfollows:
follows:initial
initial20%
20%
acetonitrile
(A)
and
1%
acetic
acid
water
solvent
(B).
The
gradient
acetonitrile (A) and 1% acetic acid in water solvent (B). The gradient mode was as follows: initial 20%
A linear
gradient
to
22%
A
in
25
min;
linear
gradient
to
28%
A
in
55
min;
linear
gradient
to
35%
A
A linear
linear gradient
gradient to
to 22%
22% A
A in
in 25
25 min;
min; linear
linear gradient
gradient to
to 28%
28% A
A in
in 55
55 min;
min; linear
linear gradient
gradient to
to 35%
35% A
A in
inin
A
95 95
min;
linear
gradient
gradient
to 100%
100% A
Ain
in135
135min.
min.The
Theflow
flowrate
rate
min;
linear
gradienttoto
to60%
60%AA
Ain
in112
112min;
min;and
and linear
linear gradient
gradient to
to
95 min;
linear
gradient
60%
in
112
min;
and
linear
100% A
in 135
min. The
flow rate
was
1.3
mL/min.
The
components
were
identified
through
comparison
of
the
retention
time
from
the
was 1.3
1.3 mL/min.
mL/min. The
The components
components were
were identified
identified through
through comparison
comparison of
of the
the retention
retention time
time from
from the
the
was
chromatograms
with
of Rg1,
Rg1,Re,
Re,Rf,
Rf,Rh1
Rh1+++Rg2,
Rg2,Rb1,
Rb1,
Rc,
Ro,
F1,
Rb2,
chromatograms
withknown
knownstandards.
standards.The
Thecontents
contents of
Re,
Rf,
Rh1
Rg2,
Rb1,
Rc,
Ro,
F1,
Rb2,
chromatograms
with
known
standards.
The
contents
Rc,
Ro,
F1,
Rb2,
Rb3,
Rd,
Rg3,
20(R)-Rg3,
Rh2,
F2,
PPD
and
PPT
were
0.4659%,
0.1061%,
0.0908%,
0.0230%,
0.6145%,
Rb3,
Rd,
Rg3,
20(R)-Rg3,
Rh2,
F2,
PPD
and
PPT
were
0.4659%,
0.1061%,
0.0908%,
0.0230%,
0.6145%,
Rb3, Rd, Rg3, 20(R)-Rg3, Rh2, F2, PPD and PPT were 0.4659%, 0.1061%, 0.0908%, 0.0230%, 0.6145%,
0.1412%,
0.0017%,
0.1581%,
0.1497%,
0.0152%,
0.0342%,
0.0001%,0.0001%,
0.0001%,
0.0001%,
0.1412%,
0.0017%,
0.1581%,
0.1497%,
0.0152%,0.1546%,
0.1546%,0.0021%,
0.0021%, 0.0342%,
0.0342%, 0.0001%,
0.0001%,
0.0001%,
0.0001%,
0.1412%,
0.0017%,
0.1581%,
0.1497%,
0.0152%,
0.1546%,
0.0021%,
0.0001%,
0.0377%
and
0.0958%,respectively.
respectively.The
Thefingerprints
fingerprints
ofofthe
the
mixed
standard
compounds
andand
theextract
extract
of
0.0377%
and
0.0958%,
The
fingerprintsof
themixed
mixed
standard
compounds
the
extract
0.0377%
and
0.0958%,
respectively.
standard
compounds
and
the
of
ginseng
are
shown
in
Figures
1
and
2.
ofginseng
ginsengare
areshown
shown
in
Figures
1
and
2.
in Figures 1 and 2.
RR-R-Rgg33
S -R g 3
PPPPTT S -R g 3
RRdd
RRbb22
RRbb33
RRcc
RRoo
FF11
RRhh11/R/Rgg22
72.00 74.00 76.00 78.00 80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00 96.00 98.00 100.00 102.00 104.00 106.00 108.00 110.00 112.00
72.00 74.00 76.00 78.00 80.00 82.00 84.00 86.00 88.00 90.00 92.00 94.00 96.00 98.00 100.00 102.00 104.00 106.00 108.00 110.00 112.00
MIN
20.00
20.00
30.00
30.00
40.00
40.00
50.00
50.00
60.00
60.00
70.00
70.00
MIN
80.00
80.00
90.00
90.00
MIN (min)
Time
Time
(min)
Figure2.
2.HPLC
HPLC record
record of
of ginseng.
ginseng.
Figure
record
of
ginseng.
Figure
2.
100.00
100.00
110.00
110.00
120.00
120.00
Oleanic
Oleanicacid
acid
PPD
PPD
F2
F2
Rh2
Rh2
R-Rg3
R-Rg3
PPT
S-Rg3
PPTS-Rg3
Rd
Rd
Rh1/Rg2
Rh1/Rg2
Rf
Rf
Rg1
Rg1
Re
Re
10.00
10.00
Rb1
Rb1
Rc
Rc
Ro
Ro F1
F1
Rb2
Rb2
Rb3
Rb3
MIN (min)
Time
Time
(min)
AU
AU
0.30
0.30
0.28
0.28
0.26
0.26
0.24
0.24
0.22
0.22
0.20
0.20
0.18
0.18
0.16
0.16
0.14
0.14
0.12
0.12
0.10
0.10
0.08
0.08
0.06
0.06
0.04
0.04
0.02
0.02
0.00
0.00
-0.02
-0.02
0.00
0.00
RRbb11
Figure
1.High
Highperformance
performanceliquid
liquidchromatography
chromatography (HPLC)
(HPLC) record
record
of
mixture
of
standards.
Figure
1.1.
(HPLC)
recordof
ofmixture
mixtureof
ofstandards.
standards.
Figure
High
performance
liquid
chromatography
130.00
130.00
Molecules 2017, 22, 649
4 of 17
2.3. In VitroMolecules
Effects
of 22,
Ginsenosides
on Human Blood Clotting Time
2017,
649
4 of 17
At final
concentrations
of 0.05onmg/mL,
nine
outTime
of 18 ginseonsides showed significant
2.3. In
Vitro Effects of Ginsenosides
Human Blood
Clotting
anti-coagulant effects in vitro compared to the normal control (p < 0.05) (Figure 3) and were selected to
At final concentrations of 0.05 mg/mL, nine out of 18 ginseonsides showed significant antibe further coagulant
detectedeffects
for their
anti-FXa
in vitro.
Among
nine3)ginsenosides
with
in vitro
comparedactivity
to the normal
control
(p < 0.05)the
(Figure
and were selected
to beexcellent
anti-coagulation
activityfor
in their
vitro,
Rg2, Rg3
and
the ginsenosides
best bioactivity
(p < 0.01)
further detected
anti-FXa
activity
in PPT
vitro. possessed
Among the nine
with excellent
anti-and other
coagulation
activity
in Rh1,
vitro, F1,
Rg2,Rh2,
Rg3 and
PPT possessed
the best
bioactivity (p
< 0.01) and other activity
ginsenosides
including
Rg1,
F2 and
PPD showed
significant
anti-coagulation
including Rg1, Rh1, F1, Rh2, F2 and PPD showed significant anti-coagulation activity
compared ginsenosides
to the normal
control (p < 0.05) in APTT, PT and TT tests. In contrast, the solvent group,
compared to the normal control (p < 0.05) in APTT, PT and TT tests. In contrast, the solvent group, Rf
Rf and 20(R)-Rg3
displayed
insignificant
anti-coagulation
effects
when
compared
to the normal
and 20(R)-Rg3 displayed
insignificant
anti-coagulation effects
when
compared
to the normal
control control
in all threeincoagulation
parameters
(p
>
0.05).
all three coagulation parameters (p > 0.05).
80
**
A
Clotting time(s) in APTT test
70
60
**
50
40
*
*
30
**
**
**
*
*
*
*
*
*
20
10
0
Clotting time (s) in PT test
Groups
20
18
16
14
12
10
8
6
**
B
**
**
*
*
*
4
2
0
Molecules 2017, 22, 649
35
5 of 17
Groups
C
**
30
Clotting time (s) in TT test
**
*
*
**
**
*
25
*
*
**
*
20
15
10
5
0
Groups
3. Inanti-coagulation
vitro anti-coagulation
activities of
ginsenosides.
(A) APTT
(B) PTtest;
test; (B)
(C) TT
Figure 3. Figure
In vitro
activities
of1111
ginsenosides.
(A)test;
APTT
PTtest.
test; (C) TT
* p < 0.05, ** p < 0.01 versus the normal control. APTT: activated partial thromboplastin time; PT:
test. * p < 0.05, ** p < 0.01 versus the normal control. APTT: activated partial thromboplastin time; PT:
prothrombin time; TT: thrombin time.
prothrombin time; TT: thrombin time.
2.4. Effects of Ginsenosides on FXa Activities In Vitro
Among the nine ginsenosides showing excellent anti-coagulant activities and the two ineffective
ginsenosides in vitro, ginsenosides Rg2, Rg3 and PPT exhibited the best anti-FXa activities with fifty
percent of inhibitory concentration (IC50) of 135.9, 126.7 and 140.7 nM, respectively (Table 1). The
positive control drug showed higher activity with IC50 of 1.9 nM compared to all test ginsenosides
(Figure 4).
Clotting time (s) in
00 0
20
15
Groups
Groups
Groups
10
5 vitro
Figure
3. In
In
vitro
anti-coagulation
activities
of 11
11
ginsenosides.
(A)(A)
APTT
test;test;
(B) (B)
PT PT
test;test;
(C)(C)
TT TT
test.test.
Figure
3.vitro
In
anti-coagulation
activities
of ginsenosides.
11 ginsenosides.
APTT
Figure
3.
anti-coagulation
activities
of
(A)
APTT
test;
(B)
PT
test;
(C)
TT
test.
0.05,
**0pp**<<p0.01
0.01
versus
thethe
normal
control.
APTT:
activated
partial
thromboplastin
time;
PT:PT:
Molecules 2017, 22, 649
< 0.05,
< 0.01
versus
normal
control.
APTT:
activated
partial
thromboplastin
time;
** pp *<<p0.05,
**
versus
the
normal
control.
APTT:
activated
partial
thromboplastin
time;
PT:
5 of 17
prothrombin
time;
TT:TT:
thrombin
time.
prothrombin
time;
thrombin
time.
prothrombin
time;
TT:
thrombin
time.
Groups
2.4.2.4.
Effects
of Ginsenosides
Ginsenosides
on FXa
FXa
Activities
In Vitro
Vitro
Effects
of Ginsenosides
on FXa
Activities
In Vitro
2.4.
Effects
of
on
Activities
In
2.4. Effects of Ginsenosides
on3. InFXa
Activitiesactivities
In Vitro
Figure
vitro anti-coagulation
of 11 ginsenosides. (A) APTT test; (B) PT test; (C) TT test.
Among
the
nine
ginsenosides
showing
excellent
anti-coagulant
activities
and
the
twotwo
ineffective
* p the
<nine
0.05,
**
p ginsenosides
< 0.01 versus showing
the
normal control.
APTT:
activated
partial thromboplastin
time;
PT:
Among
nine
showing
excellent
anti-coagulant
activities
and
the
ineffective
Among
the
ginsenosides
excellent
anti-coagulant
activities
and
the
two
ineffective
prothrombin
time; TT: thrombin
time.Rg3 and PPT exhibited the best anti-FXa activities with fifty
ginsenosides
in vitro,
vitro,
ginsenosides
Rg2,
in vitro,
ginsenosides
Rg2,
Rg3
PPT
exhibited
anti-FXa
activities
with
fifty ineffective
in
ginsenosides
Rg2,
Rg3
andand
PPT
exhibited
thethe
bestbest
anti-FXa
activities
fifty
Among ginsenosides
theginsenosides
nine ginsenosides
showing
excellent
anti-coagulant
activities
andwith
the
two
percent
of inhibitory
inhibitory
concentration
(IC(IC
50)) 50
of) 135.9,
135.9,
126.7
andand
140.7
nM,
respectively
(Table
1). 1).
TheThe
percent
of
inhibitory
concentration
of 135.9,
126.7
140.7
nM,
respectively
(Table
percent
of
concentration
(IC
50
of
126.7
and
140.7
nM,
respectively
(Table
1).
The
2.4.ginsenosides
Effects of Ginsenosides on
FXa Activities
In Vitro
ginsenosidespositive
in vitro,
Rg2,
Rg3
and
PPT
exhibited
the
best
anti-FXa
activities
with fifty
control
drug
showed
higher
activity
with
IC50
50
of
1.9
nMnM
compared
to all
all
testtest
ginsenosides
positive
control
drug
showed
higher
activity
with
ICof
50 1.9
of 1.9
compared
to all
ginsenosides
positive
control
drug
showed
higher
activity
with
IC
nM
compared
to
test
ginsenosides
Among the nine ginsenosides showing excellent anti-coagulant activities and the two ineffective
percent of inhibitory
concentration
(IC50 ) ofRg2,
135.9,
126.7 and 140.7 nM, respectively (Table 1). The positive
(Figure
4).ginsenosides
(Figure
4).
(Figure
4).
in vitro, ginsenosides
Rg3 and PPT exhibited the best anti-FXa activities with fifty
of inhibitory
concentration
(IC ) ofof
140.7 nM, respectively
The
control drug showedpercent
higher
activity
with IC
1.9126.7
nMandcompared
to all (Table
test 1).
ginsenosides
(Figure 4).
50 135.9,
50
Table
1. The
The
structures
of 11
11
ginsenosides
and
their
anticoagulation
activities
against
coagulation
positive
control
drug of
showed
activity
with
ICtheir
50 anticoagulation
of 1.9
nM compared
to
all test
ginsenosides
Table
1. The
structures
of ginsenosides
11higher
ginsenosides
and
anticoagulation
activities
against
coagulation
Table
1.
structures
and
their
activities
against
coagulation
(Figure
4).
factor
Xa (FXa).
(FXa).
factor
Xa
(FXa).
factor
Xa
Table 1. The structures of 11 ginsenosides and their anticoagulation activities against coagulation factor
Table 1. The structures of 11 ginsenosides and their
R3O
Oanticoagulation activities against coagulation
R
3 R3O
OH OH
Xa (FXa).
factor Xa (FXa).
OH
OH
R3O
R1O
O
R
1 R 1O
R 1O
R2 R
R
2
2
R2
Compound
Compound
Compound
Compound Compound
R1
R11 RR11
R
R222 R2
R
R
R
2
HO HO
HO
HO
Rg1
Rg1
Rg1
Rg1 22,
Molecules2017,
2017,
649
Molecules
22, 649
Rg1
HO
O OO
O O O
OH O O
OH OH
OHOH
OH
OH
OH OH
OH OH
OH
H
H H
H H
HO
Molecules
2017,
22,22,
649
Molecules
2017,
649
Molecules
Molecules 2017,
2017, 22,
22, 649
649
Rh1
H
Rh122, 649
MoleculesRh1
2017,
Molecules 2017, 22, 649
H H
H H
Rh1 Rh1
Rg2
Rg2
H
H
Rg2
Rg2
Rg2
Rg2
HH
H
H
Rg2
Rg2
Rg2
H HH
F1
F1
F1F1
F1
Rg3
Rg3
Rg3
Rg3
Rg3
Rg3 Rg3
Rh2
Rh2
Rh2
Rh2
Rh2
Rh2
Rh2
O
O
OH
OH
OH
OH
HO
HO
O
HO O
O
HOOHOHO
OO
OH
OH
OHO
OH
O
O
OH
OH
OH
OH
OH
OH
HO
HO OH
OH OH OH
HO HO
OO
OH
OHOH
OH
HO
HO
OO O
O
O
O O
OH
OHO
O
OH
HO OH
OH
OH
OH
OH
HO
OH OHOH
OH
OH
OH
OH OH
OH O
OHO
HH
H
H H
H
Rh2
Rh2
PPD
PPD
H
PPT
PPD
PPD
PPT
PPD
PPD
PPT
PPT
H
H
H
H
H
H H
PPT
PPT
PPT
PPT
PPT
20(R)-Rg3
20(R)-Rg3
20(R)-Rg3
20(R)-Rg3
HO
HO
20(R)-Rg3
20(R)-Rg3
20(R)-Rg3
20(R)-Rg3
20(R)-Rg3
Rf
Rf
O
H
H
HH
H
H
HO O
HO
OH O
O
OH
O
OH
OH
HO OH
HO HO
HO
HO OH OO OH
O
O
OH
HOO
HO
O
HO
O
OH
OO
OH OH
O
OH
OHO
OH
O
OH OH
OH OH
OHOH
OH
HO
HO
OH
OH
OH
OH
OH
O OH
HO
OHO O O O
HO
O
OH
OHO
OH O
OH
OH
OH
OH
OH
OH
OH
OH
OH
HH H H
H
RfRf
Rf
Rf
Rivaroxaban
Rivaroxaban
Rivaroxaban
Rivaroxaban
Rivaroxaban
HH
H
H
O O
O
OH OH
OH
OH
OH
OH OH
OH OH
OH
H
334.7
334.7
334.7
334.7
334.7
H H
H
HO
HO
235.8
235.8
235.8
235.8
6 of 17
6 of 17
H
H
135.9
135.9
HH
H
H
135.9
135.9
135.9
135.9
H
H H
135.9
135.9
135.9
OH
OH
HO
HO
HO
OH
HO
OH
OO
O
O
OH
OH
O
OH
OH O O
OH
O
OH
OH
OH
OH
OH OH
OH
OH
OH
OH OH OH
OH
OH
HO HO
H
H
HH
H
HH H
HH
HH
HH
H
H
H
H
H
HH
HH
H
H
HO
HO
HHH
H
HH
OO
H
H
H H
HHH
H
H
H
H
HO HOOH
OH
HO
HO
HO
OH O O
HO
OH
O
O
OH
OH OHO
OH
OH
OH O
OH OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H H
OH
HH
OH
H
H
OH
OH
OH
OH
OH
OH
H
H
H
H
HOH
OH
H
OH
OH
H H
H
H
H
H
H
HHH
227.0
227.0
227.0
227.0
227.0
227.0
227.0
227.0
227.0
126.7
126.7
348.7
348.7
348.7
348.7
348.7
348.7
348.7
348.7
348.7
425.2
425.2
425.2
425.2
425.2
425.2
425.2
425.2
425.2
339.4
339.4
339.4
339.4
339.4
140.7
339.4
339.4
140.7
339.4
339.4
140.7
140.7
140.7
HH
H
H
H
HHH
140.7
140.7
140.7
140.7
815.3
815.3
815.3815.3
HH
H
H
815.3
815.3
815.3
815.3
HH
H
H
HO
HO
HO
HO O
O
OH O
OH
O
OH
OH
HO OH
OH
HO
O
OH
HO
HO O OH O
O
O
O
HO
OHHO
O
O
HO
OH
OH
OH O O
HO OH
OH
OH
OH
OH
OHO
OH
OH
OH
OH O
OH
126.7
126.7
126.7
126.7
126.7
126.7
126.7
H
H
HH
HH
H
HOHO OHOH
OHOH O O
HO
OHO O O
HO
O
OHOHO
OH O
OHOH
OH
OH
OHOH
OH
OH
OH
HH
H
H
815.3
1034.0
1034.0
1034.0
1034.0
1034.0
1034.0
1034.0
1034.0
1.91034.0
1.9
1.9
1.9
1.9
IC50 : fifty
of inhibitory
concentration;
PPD:protopanaxdiol;
protopanaxdiol;
protopanaxtriol.
ICpercent
50: fifty percent
of inhibitory
concentration; PPD:
PPT: PPT:
protopanaxtriol.
IC50
: fifty percent
of inhibitory
concentration;
PPD:
protopanaxdiol;PPT:
PPT: protopanaxtriol.
protopanaxtriol.
IC50: fifty
percent
of inhibitory
concentration;
PPD:
protopanaxdiol;
IC50: fifty percent of inhibitory concentration; PPD: protopanaxdiol; PPT: protopanaxtriol.
Rivaroxaban
1.9
Rivaroxaban
1.9
Rivaroxaban
1.9
Rivaroxaban
1.9
ICIC
50:50fifty
percent
PPT:
: fiftypercent
percentof
ofinhibitory
inhibitoryconcentration;
concentration;PPD:
PPD:protopanaxdiol;
protopanaxdiol;
PPT:protopanaxtriol.
protopanaxtriol.
B
IC
: fifty
A of
IC50
50: fifty percent
of inhibitory
inhibitory concentration;
concentration; PPD:
PPD: protopanaxdiol;
protopanaxdiol;
PPT: protopanaxtriol.
protopanaxtriol.
B PPT:
AA
AA
A
A
A
BB
BB
B
B
of17
17
66of
of
1717
6 of
666 of
of 17
17
235.8
O
HOHO
OH
OH
HO
HO
HO
OH
O O
HO
OH
O
O
OH
OH
OH
OH
O
O
OH
OH
OH
OH
OH
OHOH
OH
OH
OH
OH
HOH
OH H
OH
OH
OH
H
F2F2
O
OH
OH
IC50
50
(nM)
IC(nM)
50 (nM)
IC50 (nM)IC
IC50 (nM)
HO HO
HO
OH
H
H
OH
OH
HO
HO HO HO
OH
OH
HO
OH
HO
OH
O
OO O
O
O OH
OH
OHOH
OHOH OH
OH
OH
OH
HO
HO
OH
OH
OHOH OH
OH
OH
OH
OO
F2
PPD
PPD
Rf
Rf
Rf
HOH
H
OH OH
OH
HH
H
HOH
HO H H H
F2
F2
PPD
OH
OH OH
OH
HO
HO
Rg3
Rg3
O
OH OH O O
OH
OHOH
OH
H
H
F1F1
F1
F1
F2F2
F2
F2
HO O O
HO
HO
HO
HO
OH
OOOO
OOOO
OH
O
O
OHOH
OH
OH
OH
OH
HO
HO
OH
HO
OH
OH
OH
OH
HO
OO
OH
OOOO
OH
OH
O OH
O
OH
OHO
OH
OHO
OOH
HO
OH
OH
OH
O
OH
OH
OOO OO
O
OH
OHOH OOH
O
OH OH
O
OH
OH
OH
OH
OH
O
OH
O
O
R33 R3
R
R3
R3
HO
O
Rf
H
HO
OH
OH
O
H
O
1034.0
OH
OH
OH
Rivaroxaban
1.9
Molecules 2017, 22, 649
IC50: fifty percent of inhibitory concentration; PPD: protopanaxdiol; PPT: protopanaxtriol.
6 of 17
B
A
Molecules 2017, 22, 649
7 of 17
D
C
F
E
H
G
J
I
L
K
Figure 4. Cont.
Molecules 2017, 22, 649
7 of 17
Molecules 2017, 22, 649
8 of 17
M
N
P
O
Q
R
S
T
U
V
Figure 4. Cont.
Molecules
Molecules 2017,
2017, 22,
22, 649
649
98 of
of 17
17
Molecules 2017, 22, 649
9 of 17
W
W
X
X
Figure 4. The inhibition profile figures against coagulation factor X. (A,B) Rg1; (C,D) Rh1; (E,F) Rg2;
(G,H)
F1;
(I,J)
Rg3;
(K,L)profile
Rh2; figures
(M,N)
F2;
(O,P)coagulation
PPD; (Q,R)factor
PPT;X.
(S,T)
20(R)-Rg3;
(U,V)
Rf;
(W,X)
Figure
The
inhibition
profile
figures
against
coagulation
factor
X.(A,B)
(A,B)
Rg1;(C,D)
(C,D)
Rh1;
(E,F)
Rg2;
Figure
4. 4.
The
inhibition
against
Rg1;
Rh1;
(E,F)
Rg2;
10, OD: optical density.
Rivaroxaban.
lg:
log
(G,H)F1;F1;(I,J)
(I,J)Rg3;
Rg3;(K,L)
(K,L)Rh2;
Rh2;(M,N)
(M,N)F2;
F2;(O,P)
(O,P)PPD;
PPD;(Q,R)
(Q,R)PPT;
PPT;(S,T)
(S,T)20(R)-Rg3;
20(R)-Rg3;(U,V)
(U,V)Rf;
Rf;(W,X)
(W,X)
(G,H)
1010
, OD:optical
opticaldensity.
density.
Rivaroxaban.
log
Rivaroxaban.
lg:lg:log
, OD:
2.5. Selectivity Versus Thrombin
2.5.Selectivity
VersusThrombin
Thrombin
2.5.
Versus
InSelectivity
the previous
anti-coagulant
assay in vitro, Rg2, Rg3 and PPT demonstrated excellent FXa
inhibitory
activity
andanti-coagulant
were
further selected
assess
their
selectivity
versus
thrombin. excellent
Rg2,
Rg3 and
the
previous
anti-coagulant
assayinto
invitro,
vitro,Rg2,
Rg2,Rg3
Rg3and
andPPT
PPT
demonstrated
excellent
FXa
InInthe
previous
assay
demonstrated
FXa
PPT
displayed
a
high
level
of
selectivity
against
thrombin
with
IC
50 values
of
81.3,
92.6,
82.0
μM,
inhibitory
activity
and
were
further
selected
to
assess
their
selectivity
versus
thrombin.
Rg2,
Rg3
and
inhibitory activity and were further selected to assess their selectivity versus thrombin. Rg2, Rg3
respectively,
whicha were
higher
than
that of ximelagatran
with ICwith
50 values
27.1 μM
2, Figure
PPT
displayed
high
level
against
with
IC50of
values
of (Table
81.3, 92.6,
82.0 5).
μM,
and
PPT
displayed
a high
levelof
ofselectivity
selectivity
against thrombin
thrombin
IC
50 values of 81.3, 92.6, 82.0 µM,
The
evidence
showed
that
Rg3
showed
the
best
anti-coagulant
activity
in
vitro
and
also
demonstrated
respectively,
which
were
higher
than
that
of
ximelagatran
with
IC
50 values
of
27.1
μM
(Table
2,
Figure
respectively, which were higher than that of ximelagatran with IC50 values of 27.1 µM (Table 2, Figure 5).5).
the
highest
selectivity
against
thrombin.
The
evidence
showed
that
Rg3
showed
thebest
bestanti-coagulant
anti-coagulant
activity
vitroand
andalso
alsodemonstrated
demonstrated
The
evidence
showed
that
Rg3
showed
the
activity
ininvitro
the
highest
selectivity
against
thrombin.
the highest selectivity against thrombin.
Table 2. Selectivity versus thrombin.
Table 2. Selectivity versus thrombin.
Table 2. SelectivityThrombin
versus thrombin.
Compound
IC50 (μM)
Rg2
81.3 IC50 (μM)
Compound
Thrombin
Compound
Thrombin IC50 (µM)
Rg3
92.6
Rg2
81.3
Rg2
81.3
PPT
82.0
Rg3
92.6
Rg3
92.6
Ximelagatran
27.1
PPT
82.0
PPT
82.0
Ximelagatran
27.1
Ximelagatran
27.1
B
A
A
B
C
C
D
D
Figure 5. Cont.
Molecules 2017, 22, 649
10 of 17
Molecules 2017, 22, 649
Molecules 2017, 22, 649
E
F
E
9 of 17
10 of 17
F
G
H
G
H
Figure 5. The inhibition profile figures of ginsenoside (Rg2) (A,B); ginsenoside (Rg3) (C,D); protopanaxtriol
Figure 5. The inhibition profile figures of ginsenoside (Rg2) (A,B); ginsenoside (Rg3) (C,D);
(PPT) (E,F) and Ximelagatran (G,H) against thrombin.
Figure
5. The inhibition
figures
of ginsenoside
(Rg2)
(A,B);
ginsenoside (Rg3) (C,D); protopanaxtriol
protopanaxtriol
(PPT)profile
(E,F) and
Ximelagatran
(G,H)
against
thrombin.
(PPT) (E,F) and Ximelagatran (G,H) against thrombin.
2.6. Interactions between Ginsenosides and FXa Protein
2.6. Interactions between Ginsenosides and FXa Protein
2.6. Molecular
Interactions docking
between Ginsenosides andwas
FXa Protein
toclarify
clarifythe
theinteraction
interaction
modes
of the
Molecular dockinginvestigation
investigation was performed
performed to
modes
of the
mostmost
active
ginsenosides
inin
anan
anti-coagulation
on FXa
FXa
protein
andtointeraction
to
measure
the
relative
Molecular
docking
investigation
was assay
performed
toprotein
clarifyand
the
modes
of binding
the binding
most
active
ginsenosides
anti-coagulation
assay
on
measure
the
relative
energies
and
localize
accurate
binding
sites
in
the
active
pocket.
As
shown
in
Figure
6,
Rg3
showed
energies
and localize
accurate
binding sites
in the
pocket. and
As shown
in Figure
6, Rg3 showed
active
ginsenosides
in an
anti-coagulation
assay
onactive
FXa protein
to measure
the relative
binding
the
most
hydrogen
nearby
residues
amongpocket.
thebest
best
anti-coagulation
the
most
hydrogen
bondswith
with
nearby
residues
the
anti-coagulation
ginsenosides
in in
energies
and
localizebonds
accurate
binding
sites
in the among
active
As
shown
in Figure
6,ginsenosides
Rg3 showed
assays.
Among
thebinding
binding
amino
acid among
residues
between
ligands
and
thethe
protein,
GLY-216
bioactivity
assays.
Among
amino
acid
residuesthe
between
ligands
and
protein,
GLY-216
thebioactivity
most hydrogen
bondsthe
with
nearby
residues
best anti-coagulation
ginsenosides
in
was
commonly
shared
among
the three
three
ginsenosides.
Furthermore,
the
same
GLU-97
waswas
was
commonly
shared
among
the
ginsenosides.
Furthermore,
the
same
residue
GLU-97
bioactivity
assays.
Among
the binding
amino
acid residues
between ligands
andresidue
the
protein,
GLY-216
shared
ligands
Rg2and
andRg3.
Rg3.the three ginsenosides. Furthermore, the same residue GLU-97 was
was
commonly
shared
among
shared
by by
ligands
Rg2
shared by ligands Rg2 and Rg3.
(A)
(A)
Figure 6. Cont.
Molecules 2017, 22, 649
10 of 17
Molecules 2017, 22, 649
11 of 17
(B)
(C)
Figure
6. Interaction
modes
of ginsenosides
5, 5,
1313and
pocket(A–C).
(A–C).(A)
(A)H-bonds
H-bonds
Figure
6. Interaction
modes
of ginsenosides
and18
18within
withinFXa
FXa binding
binding pocket
between
Rg2Rg2
(5) and
FXaFXa
pocket;
(B)(B)
H-bonds
(C)H-bonds
H-bondsbetween
between
between
(5) and
pocket;
H-bondsbetween
betweenRg3
Rg3(13)
(13)and
and FXa
FXa pocket;
pocket; (C)
PPTPPT
(18)(18)
andand
FXa
pocket;
light
blue,
ligands
(Rg2,
Rg3
and
PPT);
pink,
ligands;
orange,
residues
FXa pocket; light blue, ligands (Rg2, Rg3 and PPT); pink, ligands; orange, residues of theof
the binding
protein(FXa);
(FXa);green
greendashed
dashed
line,
H-bond.
binding protein
line,
H-bond.
3. Discussion
3. Discussion
TheThe
anti-coagulation
effects
ofof
1818compounds
among which
whichRg1,
Rg1,Rg2,
Rg2,Rg3,
Rg3,
anti-coagulation
effects
compoundswere
wereinvestigated,
investigated, among
Rh2,
PPD
and
PPT
possessedsignificant
significantanti-coagulation
anti-coagulation activities
toto
Rh1,Rh1,
F1, F1,
Rh2,
F2, F2,
PPD
and
PPT
possessed
activitiesin
invitro
vitrocompared
compared
normal
control
< 0.05).
Next,
the
Rg2,Rg3
Rg3and
andPPT
PPT that
that displayed
displayed the
the the
normal
control
(p <(p0.05).
Next,
the
Rg2,
the best
bestanti-FXa
anti-FXaactivity
activity
< 0.01)
were
further
molecularly
dockedwith
withhuman
human FXa
FXa protein. The result
(p <(p0.01)
were
further
molecularly
docked
resultby
byGlide
GlideXP
XPfrom
from
Schrödinger
revealed
the
interactions
and
accurate
binding
sites
between
the
bioactive
FXa
and
Schrödinger revealed the interactions and accurate binding sites between the bioactive FXa andthe
the
three
ginsenosides,
whichfurther
furthersupported
supported the
assays
andand
also also
pointed
out the
three
ginsenosides,
which
thedata
dataofofthe
thebioactivity
bioactivity
assays
pointed
out
chemical
modification.
All All
the above-mentioned
evidence
indicates
that
the potential
potentialfuture
futuredirection
directionofof
chemical
modification.
the above-mentioned
evidence
indicates
Rg2,
Rg3
and
PPT
may
be
potential
natural
FXa
inhibitors.
that Rg2, Rg3 and PPT may be potential natural FXa inhibitors.
In a previous study of the water extract of P. ginseng, together with water extracts of the
In a previous study of the water extract of P. ginseng, together with water extracts of the same
same family (Araliaceae genus Panax quinquefolius L. and Panax notoginseng (Burk.) F.H. Chen),
family (Araliaceae genus Panax quinquefolius L. and Panax notoginseng (Burk.) F.H. Chen), it was
it was observed to significantly extend blood clotting time of activated partial thromboplastin,
observed to significantly extend blood clotting time of activated partial thromboplastin, prothrombin
prothrombin and thrombin in in vitro human plasma coagulation assays [6]. Similarly, the ethyl acetate
and thrombin in in vitro human plasma coagulation assays [6]. Similarly, the ethyl acetate fraction of
fraction of Korean red ginseng methanol extracts showed potent anti-coagulant activity via markedly
Korean
red ginseng
methanol
showed by
potent
anti-coagulant
viareport,
markedly
prolonged
clotting
time thatextracts
was measured
thrombin
time [19]. activity
In another
0.05prolonged
mg/mL
clotting
time
that
was
measured
by
thrombin
time
[19].
In
another
report,
0.05
mg/mL
(final
concentration)
(final concentration) Rg1 and Rg2 exhibited significantly better anti-coagulant activities in vitro
Rg1compared
and Rg2 to
exhibited
significantly
better anti-coagulant
activities
compared
to 0.1
mg/mL
0.1 mg/mL
(final concentration)
heparin that was
choseninasvitro
the positive
control
drug
[20].
(final
concentration)
heparin
that
was
chosen
as
the
positive
control
drug
[20].
In
the
present
study,
In the present study, nine ginsenosides exhibited significant anti-coagulation activity (Figure 3), which
ninewas
ginsenosides
exhibited
significant
anti-coagulation
activity
(Figure
3), whichthe
was
consistent with
consistent with
the previous
findings.
However, not
all evidence
supported
anti-coagulation
the previous
findings. However,
not
all evidence
supported
the anti-coagulation
ginsenosides
role of ginsenosides
in the blood
coagulation
cascade.
In contrast
to our data, Wee role
et al.of
[19]
observed
in the blood coagulation cascade. In contrast to our data, Wee et al. [19] observed that ginsenosides
did not inhibit the blood coagulation process. In this investigation, a 50% MeOH subfraction
demonstrated higher potent inhibition against blood coagulation compared to the 100% MeOH
subfraction; nevertheless, ginsenosides Rf, Rh1 and Rg3 that were present in the EtOAc fraction were
weakly detected in the 50% MeOH subfraction, indicating that saponins did not contribute to the
Molecules 2017, 22, 649
11 of 17
that ginsenosides did not inhibit the blood coagulation process. In this investigation, a 50% MeOH
subfraction demonstrated higher potent inhibition against blood coagulation compared to the 100%
MeOH subfraction; nevertheless, ginsenosides Rf, Rh1 and Rg3 that were present in the EtOAc fraction
were weakly detected in the 50% MeOH subfraction, indicating that saponins did not contribute to the
anticoagulation activity of the EtOAc fraction. In the current study, nine ginsenosides that showed
significant anti-coagulation activity in vitro were subsequently studied for their anti-FXa effect in vitro.
FXa inhibitors have emerged as new anti-coagulant drugs and when compared to previous
anti-coagulants, they possess the following advantages: more rapid onset and offset of action; reduction
in need for “bridging” with a parenteral anti-coagulant; less clinical monitoring; and less interactions
with other drugs and food. However, for current FXa inhibitors there are still a few drawbacks, such
as a lack of antidotes, short half-life affecting efficacy, as well as high acquisition costs which limits
wider clinical application. Therefore, natural occurring FXa inhibitors may be an ideal substitute.
Many studies have revealed that natural polypeptides isolated from organisms such as bloodsuckers
and Ancylostoma caninum could act as direct inhibitors against factor Xa [21]. However, due to the
near impossibility of these polypeptides being developed for oral administration, attention may focus
instead on natural small molecular compounds. In a study published in 2014, a natural small molecule
(glycyrrhetinic acid) derived from the Chinese herb Glycyrrhiza glabra was described in [21]. The orally
administered compound was reported to significantly inhibit FXa activity in vitro with specificity,
and reduce thrombus weight in rat models despite the unequalable anticoagulation property with
commercially accessible drugs rivaroxaban and apixaban, suggesting that glycyrrhetinic acid could
be considered as a promising natural FXa inhibitor [21,22]. In this study, ginsenosides that mainly
originate from herbs of the Panax species and possess a molecular weight less than 1000 demonstrated
significant inhibitory activities against FXa in vitro. Among the nine ginsenosides with significant
anti-coagulation effects in vitro, Rg2, Rg3 and PPT showed the highest affinity with FXa receptors
(Table 1 and Figure 4), and also displayed excellent thrombin selectivity (Table 2). FXa is structurally
similar tothrombin (coagulation factor II), which may result in the binding of FXa inhibitors to thrombin
and subsequent risk of bleeding. Furthermore, selectivity is an important issue for the development
of FXa inhibitors [2]. Hence, there is a need to address the selectivity issue for the development of
newer FXa inhibitors by understanding structural differences between FXa and thrombin, and for the
reason we designed and conducted the experiment to test whether ginsenosides showing significant
anti-FXa activity were unable to affect thrombin level. The data showed that Rg2, Rg3 and PPT were
potent FXa inhibitors with high thrombin selectivity, indicating that these three ginsenosides may be
potent anti-coagulants with less risk of bleeding. Further molecular docking by Schrödinger software
confirmed the data obtained in the anti-coagulation activity assays and pointed out the specific binding
protein residues around the active pocket, which may form the basis for future chemical modification
in ginsenosides.
Despite our finding that micromolecule direct FXa inhibitors in ginseng was accomplished in the
anti-coagulation assay in vitro, and that anti-FXa and thrombin selectivity assays may reveal potent
anti-coagulation activity in the future treatment of coagulation-associated disorders, nevertheless,
there are some unresolved issues. First, the phenomenon that multiple ginsenosides possess
anti-coagulation properties have been reported in the current research; however, the underlying
action mechanism of the anti-coagulation effect of ginsenosides still remains unclarified and requires
further investigation. Additionally, the unstable chemical structure of ginsenosides such as Rg3 under
acidic and high-temperature conditions, especially in the stomach, may hinder its further application
in scientific research and trials [20], furthermore, ginsenosides have demonstrated incomparable
bioactivity with the positive drug heparin in an anti-coagulation assay in vitro and rivaroxaban in
an anti-FXa test in vitro. Therefore, to develop novel anti-coagulants with better efficacy and lower
side effects, future work is required.
Molecules 2017, 22, 649
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4. Materials and Methods
4.1. General
A Bruker DRX 500 spectrometer (Bruker Biospin, Rheinstetten, Germany), using tetramethylsilane
(TMS) as an internal standard, was used for NMR spectra. The qualitative, quantitative analyses and
HPLC separations were all performed on a Waters 1525 Binary HPLC Pump equipped with Waters
2998 Photodiode Array Detector (Waters Corporation, Milford, MA, USA). SunFire Prep C18 Column
(10 mm × 150 mm, silica gel particle size: 10 µm) was used for HPLC separation and Diamonsil C18
(4.6 mm × 250 mm, 5 µm) was used for HPLC analyses. Silica gel for column chromatography was
provided by the Qingdao Ocean Chemical Group Co. (Qingdao, China). Pyridine-d5 was purchased
from the Sigma-Aldrich Company (St. Louis, MO, USA). For plasma preparation, the micro-centrifuge
was obtained from Thermo Fisher Scientific (Heraeus Fresco 21, Harz, Germany). For blood clotting
time assays, a coagulometer was purchased from Beckman Coulter Inc. (Beckman ACL TOP-700, CA,
USA), and the positive control drug heparin was obtained from YM Biological Technology Company
Limited. For the anti-FXa activity assay, purified FXa and FXa substrate CS-11(22) were purchased
from New England Biolabs (Hitchin, UK) and American Diagonostica Inc. (Stamford, CT, USA),
respectively. An automated microplate reader Epoch was obtained from Bio-Tek Instruments Inc.
(Winooski, VT, USA) and the positive drug rivaroxaban was obtained from Bayer Health Care AG
(Wupertal, Germany). For the thrombin selectivity assay, FIIa and chromogenic substrate CS-01(38)
were purchased from Hyphen BioMed (Paris, France), and the positive drug ximelagatran was
purchased from AstraZeneca LP (Molndal, Sweden). For the anti-coagulation activity assay in vitro,
the solvent dimethyl sulfoxide (DMSO) and heparin sodium salt were obtained from Sigma-Aldrich
Company (St. Louis, MO, USA). For data analysis, the software SPSS 19.0 was obtained from SPSS Inc.
(Chicago, IL, USA).
4.2. Plant Material
Ginseng quinquennium, was collected in Ji’an City in Jilin Province, China, in August 2015.
The plant material was identified by Professor. Pingya Li (School of Pharmaceutical Sciences,
Jilin University). A voucher specimen (No. 20150928) was deposited in the School of Pharmaceutical
Sciences, Jilin University.
4.3. Preparation of the Total Saponins of Ginseng
The powdered air-dried root of ginseng (300 g) was extracted with 70% ethanol under reflux for
3 h and repeated for three times. The extracted solution was then concentrated under reduced pressure.
The extract (63 g) was purified in an AB-8 macroporous adsorption resin column, eluted with water,
20% ethanol and 85% ethanol, respectively. Finally, the 85% ethanol elute (25.1 g) was collected as the
total saponins from ginseng.
4.4. Extraction and Isolation
The total saponins from ginseng were subjected to column chromatography in a silica
gel column gradiently eluted with chloroform/methanol repeatedly to give eight fractions,
Fraction A (chloroform/methanol (100:4, v/v)), Fraction B (chloroform/methanol (100:8, v/v)),
Fraction C (chloroform/methanol (100:9, v/v)), Fraction D (chloroform/methanol(100:12, v/v)),
Fraction E (chloroform/methanol (100:15, v/v)), Fraction F (chloroform/methanol (100:18, v/v)),
Fraction G (chloroform/methanol (100:20, v/v)), and Fraction H (chloroform/methanol (100:30, v/v)).
Compounds 17 and 18 were recrystallized from Fraction A using ethyl acetate. Compound 15 was
purified from Fraction B by using reverse phase silica gel chromatography eluted with methanol/water
gradiently. Fraction C was purified with octadecyl-bonded silica gel (ODS) column eluted with
methanol/water (55:45, v/v) to obtain compounds 4 and 9. Compound 5 was obtained from Fraction D
using column chromatography on silica gel repeatedly using acetone and ethyl acetate. Fraction E was
Molecules 2017, 22, 649
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separated on normal phase (using a mixture of chloroform, ethyl acetate, methanol and water as elution)
and reverse phase (using gradient methanol/water as elution) silica gel column chromatography to
obtain compounds 1, 3, 13, 14 and 16. Compounds 7, 2 and 12 were purified from Fraction F by
column chromatography on silica gel repeatedly using dichloromethane and methanol, respectively.
Compounds 10 and 11 were yielded from Fraction G by column chromatography on ODS, eluted
using gradient methanol and water. Fraction H was separated in reverse phase (using gradient
methanol/water as elution) silica gel column chromatography to obtain compounds 6 and 8.
4.5. Animal Preparation
Male Sprague-Dawley rats weighing 180–220 g were purchased from the Animal Center of
Norman Bethune Medical College of Jilin University in China. The rats were maintained under
controlled environment (22 ± 2 ◦ C, relative humidity 40–60%, 24 h light-dark cycles, and ad libitum
access to food and water). The animal experiments were conducted based on the guide for the
administration of laboratory animals (Directive 86/609/EEC in the Protection of Animals Used for
Experimental and Other Scientific Purposes, 1986) and were approved by the Institutional Animal
Care and Use Committee (IACUC) of Jilin University (No. SCXK-2013-0001).
4.6. Blood Collection and Preparation of Plasma Samples
Rats were anaesthetized with 10% chloral hydrate (3 mL/kg). The blood obtained from the
abdominal aorta was collected directly into citrated tubes containing 3.8% sodium citrate (1:9 (v/v)) and
was used immediately after collection [23]. Platelet poor plasma (PRP) was prepared by centrifuging
the blood at 3000 rpm for 20 min at 20 ◦ C [24]. Plasma samples with jaundice, chylus, hemolysis
and blood clot were excluded prior to the assays. Plasma mixtures were 90 µL of PRP with 10 µL of
tested compounds (0.5 mg/mL, dissolved in DMSO). For PRP in the solvent group, the sample was
90 µL of PRP with 10 µL of DMSO. For PRP in the normal control group, the plasma was not-treated
PRP sample. For PRP in the heparin group, the sample was 90 µL of PRP with 10 µL of heparin
(0.1 mg/mL), which was used as the positive control drug.
4.7. Measurement of Blood Clotting Time
Blood coagulation assays were carried out using a coagulation analyzer at the Blood Center clinical
laboratory in the Second Hospital affiliated to Jilin University (Changchun, China). Measurements
of APTT, PT and TT were performed according to the manufacturer’s recommended protocols.
The anti-coagulant activity was expressed as clotting time ± standard deviation (S.D.). Briefly, for APTT
assay, 50 µL of the plasma mixture was incubated at 37 ◦ C for 60 s and the mixture was added to the
APTT reagent (50 µL) and incubated at 37 ◦ C for 15 min. Finally, APTT values were recorded. For the
PT assay, 50 µL of the plasma mixture was incubated at 37 ◦ C for 60 s and then added into the PT
reagent (100 µL) prior to incubation at 37 ◦ C for 15 min before the PT values were recorded. For the TT
assay, 80 µL of the plasma mixture was incubated at 37 ◦ C for 60 s before the addition of 80 µL of TT
reagent for 100 s. TT values were determined by the coagulation analyzer.
4.8. FXa Activity Assay of Compounds In Vitro
Test compounds and the positive control drug rivaroxaban were dissolved in DMSO at
a concentration of 1 mM and then serially diluted to a range of 3 nM to 10 µM, respectively. 10 µL of
FXa (final concentration of 0.5 nM), 40 µL of Tris buffer (adjusted to pH 7.4 with HCl containing 0.3 M
NaCl and 50 mM Tris) and 10 µL of test compounds were added to the well, respectively. The negative
control was performed using the same mixed solutions except that the test compound was replaced
with DMSO. The positive control was composed of the same mixed solutions except that the test
compound was replaced with rivaroxaban. After 15 min of incubation at 37 ◦ C, FXa substrate (40 µL,
final concentration of 0.25 nM) was added and then was incubated 37 ◦ C for 25 min [3]. The optical
density (OD) values at 405 nm were evaluated by an automated microplate reader. The time-absorbance
Molecules 2017, 22, 649
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curve and the slope of curve reflecting enzymatic activity were observed in test groups (Vi ), positive
control group (Vi ) and negative control group (V 0 ). Inhibition rate was calculated by the following
formula: Anti-FXa activity = (V 0 − Vi )/V 0 [21]. The IC50 value was subsequently calculated by
SPSS 19.0.
4.9. Thrombin Inhibition In Vitro of Compounds
The inhibition of thrombin was evaluated by human FIIa and chromogenic substrate CS-01(38) in
96-well microtiter plates at room temperature. Compounds 5, 13 and 18 as well as the positive reagent
Ximelagatran were dissolved in DMSO to a concentration of 1mM and then serially diluted to a range
of 10 µM to 100 µM, respectively. 8 µL of FIIa (3 NIH U/mL), 80 µL of Tris buffer (adjusted to pH
7.4 with HCl) containing 0.3 M NaCl and 50 mM Tris and 8 µL of test compounds were added to the
well, respectively. The negative control was performed using the same mixed solutions except that
tested compounds were replaced with DMSO. The positive control was performed using the same
mixed solutions except that the test compounds were replaced with ximelagatran. After incubation at
37 ◦ C for 15 min, 12 µL of FIIa substrate solution (4 mM) was added and then incubated at 37 ◦ C for
25 min [3]. The anti-FIIa activity was measured at 405 nm using a microplate reader.
4.10. Molecular Docking of Rg2, Rg3 and PPT within FXa
Molecular docking study of bioactive compounds was performed using GLIDE (Grid-based
Ligand Docking with Energetics) (GLIDE, version 6.7, Schrödinger, LLC, New York, NY, USA) software
developed by Schrödinger. Maestro (version 2015-2, Schrödinger, LLC, New York, NY, USA) was
used for all the steps involving protein and ligand preparation, receptor grid generation and docking.
The X-ray crystal structure of FXa (Protein Data Bank (PDB) code: 2w26) complexed with an oral and
direct FXa inhibitor Bay59-7939 was retrieved from the PDB database (http://www.rcsb.org/pdb)
based on a previous study [2]. The Protein Preparation Wizard in the GLIDE software was used to
prepare the receptor FXa. The structure of FXa was optimized after a series of processes including
assigning bond orders and water orientations, removing water molecules, adding hydrogens, creating
zero-order bonds to metals and disulfide bonds. The protein was then energy minimized using
a default constraint of 0.30 Å root-mean-square deviation (RMSD) using the optimized potentials for
liquid simulations 3 (OPLS3) force field. When performing receptor grid generation, a present ligand
in the retrieved protein-ligand complex was identified prior to setting the center and the size of the box.
The grid box was limited to the size of 20 Å at the active site. Crystal coordinates of compounds (ligands)
were pre-drawn in Maestro Elements (Maestro Elements, version 2.2, Schrödinger, LLC, New York, NY,
USA) prior to the molecular docking study. Three-dimensional (3D) structures of all 18 compounds
were generated using LigPrep module (2015-2) from the Schrödinger Suite (LLC) by assigning the
bond orders and angles. In addition, these compounds were subjected to minimization using the
OPLS3 force field. For GLIDE docking, the prepared structure of FXa and ligands (compounds) were
imported to the workspace using GLIDE v.6.7 from the Schrödinger Suite [25–27]. Extra precision
(XP) docking was carried out and the parameters of scaling factor and partial charge cutoff were set
at the default values 0.80 and 0.15, respectively. At least the top ten ranking conformations for each
ligand was chosen in the output tab to set the output numbers. Figures of the docking results were
subsequently prepared using PyMol (Schrödinger).
4.11. Statistical Analysis
All values were expressed as means ± standard deviation (SD), and one-way analysis of variance
(ANOVA) and student’s t-test were performed by SPSS19.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 was
considered statistically significant.
Molecules 2017, 22, 649
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5. Conclusions
In summary, triterpenoids from ginseng are potential natural coagulation factor Xa
(FXa)-inhibitors with high thrombin selectivity and prolongation of coagulation time. The bioactivity
studies and HPLC analysis also suggested that despite the low content in total saponins, the three
triterpenoids, Rg2 (5, 0.0230%), Rg3 (13, 0.0021%) and PPT (18, 0.0958%), maybe responsible for
the anti-coagulant effect. Hence, the total saponins and the effective components could be used as
a potential natural anticoagulation therapy.
Acknowledgments: We thank Hang Su, Shuxue Zou and Zhenzhou Wang in preparing the figures of molecular
docking. This work was supported by the National Science and Technology Major Project for new drug
development in China (No. 2010ZX09401-305-26) and Science and Technology Development Program of
Jilin Province (Grant No. 20150519015JH).
Author Contributions: Pingya Li and Jinping Liu conceived and designed the experiments; Lingxin Xiong and
Zeng Qi performed the experiments; Zeng Qi, Bingzhen Zheng, Zhuo Li and Fang Wang were responsible for data
analysis. Lingxin Xiong wrote the paper. Pingya Li, Jinping Liu, Zhuo Li and Fang Wang assisted paper revision.
Conflicts of Interest: The authors declare no conflict of interest.
Chemical Compounds
Rg1 (PubChem CID: 441923)
Re (PubChem CID: 441921)
Rf (PubChem CID: 441922)
Rh1 (PubChem CID: 122130363)
Rg2 (PubChem CID: 12912322)
Rb1 (PubChem CID: 122130642)
Rc (PubChem CID: 122130031)
Ro (PubChem CID: 11815492)
F1 (PubChem CID: 9809542)
Rb2 (PubChem CID: 6917976)
Rb3 (PubChem CID: 12912363)
Rd (PubChem CID: 11679800)
Rg3 (PubChem CID: 9918693)
20(R)-Rg3 (PubChem CID: 46887680)
Rh2 (PubChem CID: 119307)
F2 (PubChem CID: 9918692)
protopanaxdiol (PubChem CID: 9920281)
protopanaxtriol (PubChem CID: 22392424).
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Sample Availability: Samples of all the compounds are available from the authors.
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