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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
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Antithrombotic eects and related
mechanisms of Salvia deserta
Schang root EtOAc extracts
Rena Kasimu1, Xinling Wang1, Xiaomei Wang1, Junping Hu1, Xiaoqing Wang1 & Yuming Mu2
Salvia deserta Schang (SDS) belongs to the same family as Salvia miltiorrhiza bunge, one of the
antithrombotic Chinese herbal medicines. In our study, EtOAc root extracts were analyzed for their
eects on adenosine diphosphate (ADP)-induced platelet aggregation in rabbits and FeCl3-induced
rat common carotid artery thrombosis as well as on rat blood plasma concentrations of thromboxane
B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1α), antithrombin-III (AT-III), protein C (PC),
plasminogen (PLG), plasminogen activator inhibitor (PAI-1), von Willebrand factor (vWF) and tissue-
type plasminogen activator (t-PA). EtOAc extracts from SDS roots had signicant inhibitory eects on
ADP-induced maximum platelet aggregation rate (10.2 ± 2.6 vs control 35.7 ± 5.2; P < 0.05), reduced
the FeCl3-induced rat common carotid artery thrombus weight and thrombus area ratio (P < 0.05),
signicantly decreased plasma TXB2, vWF and PAI-1 levels and increased 6-keto-PGF1α and t-PA levels
in a dose dependent manner (all P < 0.05). Thus, the ratio of TXB2/6-keto-PGF1α was signicantly
decreased (P < 0.05), while the ratio of t-PA/PAI-1 was signicantly increased (P < 0.05). In addition,
enhanced AT-III and PC activities indicated coagulation inactivation eects of EtOAc SDS root extracts.
EtOAc extraction from SDS showed antithrombotic eects, which are likely due to platelet adhesion
and aggregation inhibition as well as anticoagulant activities.
Numerous genetic, acquired and environmental factors can tip the homeostatic balance in favor of coagulation
and thus lead to the formation of thrombi, which is a common pathology underlying ischemic heart disease,
stroke and venous thromboembolism. It has been reported that ischemic heart disease and stroke collectively are
responsible for one in four deaths worldwide1,2. us, despite the existing available antithrombotic agents, new
eective drugs are still urgently required.
Salvia deserta Schang (SDS) is a perennial plant belonging to the Lamiaceae family and is widely distributed
in the Gobi wilderness of Xinjiang province. SDS is a species of the Salvia genus like Salvia miltiorrhiza bunge,
whose root extracts are an important Chinese herbal medicine called Danshen, which can aect hemostasis by
several mechanisms including inhibition of platelet aggregation, interference with extrinsic blood coagulation,
antithrombin III-like activity and promotion of brinolytic activity. erefore, it is commonly used in Chinese
clinics as antithrombotic therapy3–6. However, whether SDS extracts also have anti-thrombotic eects has rarely
been investigated.
e chemical composition of whole plant SDS was previously systematically studied. About 30 dierent com-
pounds including phenolic acids, diterpenoid quinones, avonoids, triterpenoids and others were isolated of
which the hydrosoluble phenolic acids and liposoluble terpenoids were also found in Salvia miltiorrhiza bunge7–9.
e diterpenoid quinones 6,7-dehydroxyleanone and 6,7-dehydroroyleanone have eects in preventing myocar-
dial ischemia, inhibiting platelet aggregation and inducing nitric oxide synthase in vitro10,11, and the triterpenoid
oleanolic acid can signicantly inhibit collagen and ADP-induced platelet aggregation to protect the heart12,13. In
the present study, we hypothesized that SDS might have similar antithrombotic eects to Salvia miltiorrhiza bunge
and we compared the SDS extract and Danshen application outcomes on thrombosis and related factor patterns
in rabbit and mouse models.
1College of Pharmacy, Xinjiang Medical University, No. 393 Xinyi Road, Urumqi, 830011, China. 2The First Aliated
Hospital Of Xinjiang Medical University, No. 137 South Liyushan Road, Urumqi, 830054, China. Rena Kasimu
is deceased. Correspondence and requests for materials should be addressed to Xinling W. (email: 365021216@
qq.com)
Received: 1 December 2016
Accepted: 1 August 2018
Published: xx xx xxxx
OPEN
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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
Methods and Materials
Experimental animal ethics. The study was approved by the ethical committee of Xinjiang Medical
University and all procedures involving animals were performed in accordance with the ethical standards of the
Guidelines for the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People’s
Republic of China, Policy No. 2006 398).
Rabbits. Two hundred SPF grade healthy New Zealand white rabbits were provided by the animal research
center of the Xinjiang Medical University (certicate number: SCXK (Xin) 2011-0004). e animals were male
and female, weighing 2.0 ± 0.2 kg, and were kept at 21 ± 2 °C, with a light cycle of 12 hours/day at 40–45% humid-
ity and free access to water and food.
Rats. One hundred and thirty healthy male Sprague-Dawley (SD) rats (SPF grade), weighing 250–300 g were
provided by the experimental animal center of Xinjiang Medical University (license number: SCXK (Xin) 2011-
0004) and kept under the same conditions as the rabbits but in separate holding rooms.
Preparation of dierent SDS components for dierent solvent extractions. SDS plants were sep-
arated into roots, stems, leaves and owers, and then dried in the shade and nally pulverized.
H2O extraction. Roots, stems, leaves and owers were respectively extracted 3 times for 1 hour by a reux
extraction method in water at 80 °C. e extracts were combined, concentrated and freeze-dried to obtain the
water extracts of roots, stems, leaves and owers.
Ethanol extraction. Roots, stems, leaves and owers were extracted 3 times using a method involving 95%
ethanol, 1 hour per extract; the 3 extracts were combined, concentrated in a low-temperature vacuum under
reduced pressure and dried to obtain ethanol extracts of roots, stems, leaves and owers.
EtOAc soluble fraction (ESF). Roots, stems, leaves and owers were reux extracted 3 times for 1 hour
with acetate (EtOAc); the 3 extracts were combined, concentrated under atmospheric pressure and dried to obtain
ESFs of roots, stems, leaves and owers.
Adenosine diphosphate (ADP)-induced platelet aggregation test. For each SDS plant component
(root, stem, leaves and ower) 50 New Zealand white rabbits were randomly divided into aspirin (10 mg/kg), high
(40–50 mg/kg), middle (20–25 mg/kg) and low (10–12.5 mg/kg) SDS extract doses as well as control groups; each
group was comprised of 10 rabbits. Intragastric administration was carried out 3 times a day for the controls and
SDS low/middle/high dose groups, and once a day for the aspirin group for 3 consecutive days. An additional
dose was administered 1 hour before the operation.
For the horminone, 7-O-acetylhorminone and 6,7-dehydeoroyleanone experiments, 1,000 µg/mL, 100 µg/mL
and 10 µg/mL of each chemical dissolved in 5% methyl alcohol-saline water was administered to 7 rabbits in each
dosage group, with one group for each chemical acting as the control (n = 7) (5% methyl alcohol-saline water
only).
Blood was collected by cardiac puncture and coagulation prevented by 3.8% sodium citrate (the volume ratio
of blood with anticoagulant was 9:1), centrifuged at 1,000 rpm at room temperature for 10 min, aer which the
upper plasma layer was aspirated as platelet-rich plasma (PRP). e remaining sample was centrifuged again at
4,000 rpm at room temperature for 15 min and the upper plasma layer was aspirated as the platelet-poor plasma
(PPP) fraction.
e PRP was adjusted to a platelet concentration of 4 ~ 5 × 108/mL with PPP. e ADP-induced platelet max-
imum aggregation rate (MAR) was determined using Born’s turbidimetric method14. ADP was purchased from
Chrono-Log Corp. (Havertown, PA, US: lot number 3427) and data are presented as the maximum aggregation
inhibition rate (MAIR) according to the following formula: MAIR (%) = platelet aggregation rate of the control
group%- platelet aggregation rate of test drug treated group%/platelet aggregation rate of the control group%.
FeCl3-induced rat common carotid artery thrombosis experiment. Dierent root extraction method
measurements. Seventy male rats were randomly divided into 7 groups with 10 rats in each group, including
a group that did not receive FeCl3 (0.5% saline) (Sham), a control group (0.5% saline) (Model), a composite
Danshen droplet pills group (Tianjin Tasly Pharmaceutical Group Co., Ltd., batch number: 20130522) dissolved
as 85 mg/kg in 0.5% saline as a positive control (CDDP), a SDS water extraction group (SDS-W), 80 mg/kg in
0.5% saline, a SDS 95% ethanol extraction group (SDS-E), 80 mg/kg in 0.5% saline, a SDS n-butanol soluble frac-
tion group (SDS-BuSF), 80 mg/kg in 0.5% saline) and a SDS EtOAc soluble fraction group (SDS-ESF), 80 mg/kg
in 0.5% saline. All the animals underwent intragastric administration for 20 days, once daily (Fig.1).
Dose-dependent SDS ESF measurements. For dose-dependent measurements, rats were treated with high
(160 mg/kg, SDS-HD), middle (80 mg/kg, SDS-MD) and low (40 mg/kg, SDS-LD) doses of SDS root ESF dis-
solved in 0.5% saline as well as a no FeCL3 group (0.5% saline) (Sham), a control group (0.5% saline) (Model) and
a 85 mg/kg composite Danshen droplet pills group dissolved in 0.5% saline as a positive control (CDDP). Again,
all the animals underwent intragastric administration for 20 days, once daily (Fig.1).
Common carotid artery thrombosis rat model after 20 days of treatment. A common carotid
artery thrombosis model was established by reference to previous studies15–17. In order to determine the time
to blood vessel occlusion (TTO), in preliminary experiments the necessary FeCl3 concentration and exposure
duration of the common carotid artery have been measured with Doppler ultrasound (Supplementary Table1,
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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
Supplementary Figure1). Isolated 2 cm segments of the le common carotid artery, removed under deep gen-
eral anesthesia induced by intraperitoneal injection of urethane (1.5 g/kg), had a small piece of plastic lm
(3 cm × 1.5 cm) placed under them to protect tissues surrounding the blood vessels. en a small piece of lter
paper (1 cm × 1 cm) soaked in 2.16 mol/L FeCl3 solution was applied onto the exposed surface of the artery, which
was replaced with saline soaked (lter paper in the sham group. e lter paper was positioned close to the blood
vessel wall. e lter paper was removed aer 20 min (the start time was when the lter paper was positioned on
the artery). e le blood vessel on the site of thrombosis was removed and the residual blood blotted on lter
paper and weighted. In addition, the thrombosis arteries were xed in 10% formalin solution for 24 hours and
washed in running water for 4 hours, followed by paran sections, HE staining and thrombosis analysis using
light microscopy. e thrombus area determination has been performed with image pro plus 6.0 soware (Silver
Spring, MD, USA)
Detecting levels of thrombus related factors. Blood was collected via the abdominal aorta of
FeCl3-induced common carotid artery thrombosis rats and anticoagulated with sodium citrate (1:9) aer removal
of the thrombosis arteries. e blood was centrifuged at 3,000 rpm at 4 °C for 15 min and the supernatant plasma
collected for thrombus factor determinations. TXB2 and 6-keto-PGF1α radioimmunoassay kits were purchased
from North Biotechnology Institute (Beijing, China). PC-C, AT-III, vWF ELISA kits as well as PAI-1 and t-PA
test kits, and a PLG immunoassay quantitative test kit were purchased from West Tang Biological Technology Co.,
Ltd. (Shanghai, China). All measurements were carried out according to the manufacturer’s instructions.
High performance liquid chromatography (HPLC)/gas chromatography mass spectrometry
(CG-MS) SDS extract analysis. Chromatographic separation of SDS root extracts was performed using a
Surveyor HPLC system (ermoFisher Scientic, San Jose, CA, USA) composed of an autosampler and an HPLC
pump. e column used was an Atlantis® HILIC Silica, 4.6 mm × 250 mm, 5 µm (Waters Corporation, Milford,
MA). e analytes were separated with isocratic elution: mobile phase water (A) methanol solution (B) gradient
(0~19 min, 25% A, ow rate 0.8 mL/min; 19~22 min; 25%~13% A, ow rate 0.8~1.0 mL/min; 22~30 min, 13% A,
ow rate 1.0 mL/min. Measurement wavelength was 272 nm (0~22.00 min for hominone, 7-O-acetylhorminone),
330 nm (22.01~30.00 min for 6,7-dehydeoroyleanone) and the column temperature was 40 °C with a sample
injection volume of 10 μL. For the MS/MS analysis, a TSQ Quantum Ultra triple quadrupole mass spectrometer
(ermoFisher Scientic, San Jose, CA, USA) was used.
Statistical analysis and data processing. Stataistical analyses were pereformed with SPSS for Windows
(Ver. 16.0. Chicago, SPSS Inc.). Continuous variables are presented as mean ± standard deviation (
X
± SD),
Dierences between dierent doses were analysed using one-way ANOVA and comparisons between two groups
were performed using bonferroni post hoc-test. e ratio variables were analysed with chi-square test. P < 0.05
was considered to be statistically signicant.
Results
SDS root EtOAc extract significantly inhibited platelet aggregation. First, we investigated
whether the dierent plant parts and extraction methods yielded eective treatments to inhibit platelet aggre-
gation in rabbits (data not shown), when we found that only root ESF inhibited platelet aggregation. Further
investigations revealed that MAR/% in high doses of root extracts was 10.2 ± 2.6, compare to MAR/% in control
Figure 1. Flow chart of the rat experiments.
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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
(35.7 ± 5.2), which showed signicant inhibition of platelet aggregation (P < 0.05) to a similar extent as aspirin
(P > 0.05) (Table1).
Eects of H2O, ethanol, n-butanol and EtOAc SDS root extracts on rat carotid artery throm-
bosis. Rats in the sham group showed no common carotid endovascular thrombosis, while FeCl3 successfully
induced rat endovascular thrombosis in the other groups, with weights up to 9 times that of the sham group.
(Fig.2). Compared with the model group, the thrombi in the CDDP and SDS groups were signicantly smaller
and the thrombus bodies were looser, especially in the SDS root ESF group. Consistent with the morphological
results, thrombus weights in each test and CDDP group were signicantly lower than that in the model group and
thrombosis was the least in the SDS root ESF group (P < 0.05, Fig.2B). In addition, the ratio of thrombus area
in each test and CDDP were all signicantly lower than that in the model group, expecially SDS root ESF group
showed lowest arear ratio (*P < 0.05, Fig.2C).
Eects of dierent SDS root ESF doses on rat carotid artery thrombosis. We further compared
the inhibitory eects on thrombosis of dierent doses of SDS root ESF. As shown in Fig.3A, the low, middle and
high dose groups (40 mg/kg, 80 mg/kg and 160 mg/kg, respectively) of SDS root ESFs all signicantly inhibited
FeCl3-induced thrombosis in a dose-dependent manner, losing and/or reducing the weight and area ratio of
thrombus bodies (P < 0.05, Fig.3B,C). It is noteworthy that thrombus weight and area ratio in the high dose SDS
group was signicantly lower than that in the CDDP group, suggesting that its inhibitory eect on thrombosis
was stronger than that in the CDDP group (Fig.3B,C).
Inuences of SDS root ESF on rat plasma ET-1, TXB2, 6-keto-PGF1α and vWF levels. TXB2
and 6-keto-PGF1α are stable metabolites of TXA2 and prostaglandin I2 (PGI2), respectively. TXA2 is mainly pro-
duced by platelets and is a vasoconstrictor that also stimulates platelet aggregation in vivo; vascular endothelial
cells mainly produce PGI2. e function of PGI2 is opposite to that of TXA2 with a strong eect on expanding
capillaries and inhibiting platelet aggregation. TXA2/PGI2 imbalance is one cause of platelet aggregation, vascular
Faction Groups Dose/mg·kg
ADP
MAR/% MAIR/%
Root
Aspirin 10 15.2 ± 6.5 57.4 ± 12.9
Control 35.7 ± 5.2Δ
STD Low Dose 10 40.8 ± 11.2Δ0.0 ± 0.0Δ
STD Middle Dose 20 29.8 ± 8.7Δ16.5 ± 7.9Δ
STD High Dose 40 10.2 ± 2.6*71.4 ± 4.1Δ
p-value (STD dose) <0.0001
p-value (Aspirin + STD dose) <0.0001 <0.0001
Stem
Aspirin 10 13.3 ± 8.1 67.2 ± 11.7
Control 40.6 ± 11.53Δ
STD Low Dose 10 42.9 ± 17.3Δ0.0 ± 0.0Δ
STD Middle Dose 20 43.1 ± 9.5Δ0.0 ± 0.0Δ
STD High Dose 40 35.5 ± 7.4Δ12.6 ± 5.8Δ
p-value (STD dose) 0.4667
p-value (Aspirin + STD dose) <0.0001 <0.0001
Leaf
Aspirin 10 8.2 ± 3.6 70.8 ± 5.1
Control 28.1 ± 8.1Δ
STD Low Dose 10 30.4 ± 10.7Δ0.0 ± 0.0Δ
STD Middle Dose 20 24.5 ± 7.3Δ12.8 ± 1.8Δ
STD High Dose 40 22.8 ± 6.3Δ18.8 ± 2.4Δ
p-value (STD dose) 0.1780
p-value (Aspirin + STD dose) <0.0001 <0.0001
Flower
Aspirin 10 8.2 ± 3.6 70.8 ± 5.1
Control 28.1 ± 8.1Δ
STD Low Dose 12.5 35.6 ± 6.6Δ0.0 ± 0.0Δ
STD Middle Dose 25 27.1 ± 7.6Δ3.5 ± 1.6Δ
STD High Dose 50 20.8 ± 2.7Δ25.9 ± 12.3Δ
p-value (STD dose) 0.0002
p-value (Aspirin + STD dose) <0.0001 <0.0001
Table 1. Eect of EtOAc extracts of indicated SDS parts on New Zealand white rabbit platelet aggregation (
X
mean ± SD, n = 10). *P < 0.05 signicant dierence compared to control group; ΔP < 0.05 signicant dierence
compared to aspirin group. MAIR(%) = platelet aggregation rate of the control group% - platelet aggregation
rate of test drug treated group% / platelet aggregation rate of the control group%.
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spasm or thrombosis. As shown in Fig.4A, compared with the sham group, plasma TXB2 levels were signicantly
higher (P < 0.05) and 6-keto-PGF1α levels signicantly lower (P < 0.05) in the model group, which resulted in
a signicantly increased TXB2/6-keto-PGF1α ratio in the model group (P < 0.05, Fig.4B). CDDP signicantly
reduced plasma TXB2 levels (P < 0.05) and the TXB2/6-Keto-PGF1α ratio (P < 0.05) compared to the model
group. Similarly, the high dose group of SDS signicantly reduced plasma levels of TXB2 (P < 0.05) and its eects
were somewhat stronger than CDDP.
von Willebrand factor (vWF) is a multimeric plasma protein that mediates platelet adhesion as well as
platelet aggregation at sites of vascular injury and acts as a carrier of factor VIII18. Compared with the sham
group, the vWF level in the model group was signicantly increased (P < 0.05). CDDP treatment appeared to
reduce the increase in vWF concentration compared to the model group, but statistical signicance was not
achieved, whereas SDS treatment signicantly suppressed the increase of vWF concentrations (P < 0.05) in a
dose-dependent manner (Fig.4C).
Inuence of SDS root ESF on rat plasma PC and AT-III levels. Protein C (PC) system and AT-III are
important physiological anticoagulants in vivo. Compared with the sham group, PC (P < 0.05, Fig.5A) and AT-III
(P < 0.05, Fig.5B) were signicantly reduced in the model group. Compared with the model group, the AT-III
concentration decreased signicantly in the CDDP group (P < 0.05), but the PC concentration did not change. In
contrast, compared to the model group the PC (P < 0.05, Fig.5A) and AT-III (P < 0.05, Fig.5B) serum concentra-
tions were signicantly increase particularly in the high dose group.
Inuences of SDS root ESF on rat plasma PLG, t-PA and PAI-1. Plasminogen (PLG) is the precursor
of plasmin, which is a brin hydrolase and mainly produced by the actions of the serum tissue-type plasminogen
activator (t-PA). e resulting plasmin dissolves brin in blood clots. In contrast, the plasminogen activator
inhibitor (PAI-1) can inhibit the activation of t-PA. Compared with the sham group, the plasma t-PA/PAI-1 ratio
showed signicant decrease (P < 0.05, Fig.6B), while the PLG concentration did not show a signicant decrease
in the model group (Fig.6C) and the t-PA concentration was signicantly reduced (P < 0.05, Fig.6A). Compared
with the model group, PLG (P < 0.05) and t-PA (P < 0.05) concentrations in the CDDP group were increased and
the PAI-1 concentration was decreased, resulting in a signicantly increased t-PA/PAI-1 ratio (P < 0.05, Fig.6B).
Figure 2. Extracts of SDS roots signicantly inhibited FeCl3-induced rat carotid artery thrombosis. (A) HE
staining of rat carotid artery thrombosis in each group; (B) comparison of the thrombus weight in each group;
(C) comparison of the ratio of thrombosis area in each group. Before the establishment of FeCl3-induced rat
carotid artery thrombosis models, the rats in each group were treated with the indicated solvent extract of
SDS roots or CDDP for 20 days, once a day. n = 10, data are presented as the mean ± SD; Signicant dierence
compare to model group *P < 0.05, and compre to sham group ΔP < 0.05.
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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
Although in contrast to the CDDP group, PLG concentrations showed no enhancement but rather signicant
decreases (P < 0.05), the PAI-1 concentrations in each SDS dose group were signicantly decreased and the con-
centration of t-PA signicantly increased in a dose-dependent manner, resulting in signicantly increased t-PA/
PAI-1 ratios in all dose groups (Fig.6C).
Horminone, 7-O-acetylhormione and 6,7-dehydeoroyleanone were active ingredients in
SDS root extracts. In order to further analyze active ingredients in SDS, we subjected SDS extracts to a
HPL-C/CG-MS analysis and found that horminone, 7-O-acetylhormione and 6,7-dehydroroyleanone were com-
pounds (Supplementary Figure2). As shown in Table2, apart from low doses of 6,7-dehydroroyleanone and
7-O-acetylhorminone all other doses of the 3 tested chemicals led to signicant reductions in MAIR% compared
to the controls.
Discussion
In the present study, we extracted dierent plant parts of SDS using a number of polar solvents, including water,
ethanol n-Butanol and EtOAc, and then determined the antithrombotic eects of SDS extracts in New Zealand
white rabbit anti-platelet aggregation and FeCl3-induced rat carotid artery thrombosis models.
e anti-platelet aggregation eective substances were rich in root SDS and EtOAc was the eective extraction
solvent for it. In the New Zealand white rabbit anti-platelet aggregation model, SDS root ESF (40 mg/kg) signi-
cantly inhibited ADP-induced platelet aggregation, which was equivalent to the anti-platelet aggregation eects of
aspirin (10 mg/kg). ese ndings suggested that SDS root extracts have similar anti-platelet aggregation eects
as Salvia miltiorrhiza bunge19. Also in the FeCl3-induced rat common carotid artery thrombosis model group,
particularly high doses of SDS root extracts inhibited thrombus development, which is in agreement with a pre-
vious study in which anti-platelet drugs could extend the time until occlusion in a FeCl3 common carotid artery
thrombosis model20. It is noteworthy that this eect was more pronounced with SDS root ESF than with CDDP
(Figs2 and 3). Consistent with a previous study, we observed that the serum concentration of TXB2 increased
Figure 3. Dierent doses of SDS root ESF suppressed FeCl3-induced rat carotid artery thrombosis. (A) HE
staining of rat carotid artery thrombosis in each group; (B) comparison of the rat thrombus weight in each
group; (C) comparison of the ratio of thrombosis area in each group. Before the establishment of FeCl3-induced
rat carotid artery thrombosis models, rats in each group were treated with dierent doses of SDS root ESF or
CDDP for 20 days, once a day, n = 10; data are presented as the mean ± SD; *P < 0.05, signicant dierence
compared to the model group; ΔP < 0.05 compared to the sham group.
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in the FeCl3 common carotid artery thrombosis model group, while the activities of 6-keto-PGF1α, t-PA, AT-III
and PC were reduced21. Moreover, treatment particularly with high doses SDS root ESF reduced plasma TXB2
and vWF, and increased 6-keto-PGF1α, leading to a signicantly reduced TXB2/6-keto-PGF1α ratio, which was
similar to the eect of CDDP and underlined the anti-platelet aggregation eects of SDS extract shown in the
rabbit and rat models. However, in contrast to the CDDP formulation, vWF expression was signicantly and
dose-dependently reduced in SDS treated rats, indicating that SDS might have a stronger antithrombotic eect
than CDDP (Fig.4). As an indicator of coagulation inactivation, PC and AT-III serum levels were increased in
SDS treated rats with the highest levels found in the SDS-HD group, indicationg a trigger of anti-coagulation by
SDS root extracts.
ese data are contrary to the CDDP-induced changes, since AT-III serum concentrations were signicant
lower and PC serum levels remained the same as the model group in CDDP treated rats (Fig.5). Taken together,
the antithrombotic eect of SDS root ESF can be attributed to anti-platelet activity and anti-coagulation actions,
similar to but not the same as CDDP. However, these ndings supported our hypothesis that SDS can induce
similar eects on thrombus development as Salvia miltiorrhiza bunge.
In order to conrm the platelet aggregation inhibitory eect of 6,7-dehydroroyleanone11 and the two other
diperpenoid quinones, horminone and 7-O-acetylhormione, which we isolated from SDS roots and which have
been described as constituents in a previous study9, we carried out a dose increasing experiment in rabbits and
Figure 4. Eects of SDS root ESF on FeCl3 induced rat plasma levels of TXB2 and 6-keto-PGF1α. (A) TXB2/6-
keto-PGF1α ratio; (B) and vWF (C). Before the establishment of FeCl3-induced rat carotid artery thrombosis
models, rats in each group were lavaged with dierent doses of SDS root EtOAc extract or CDDP for 20 days,
once a day. n = 10; data are presented as the mean ± SD; *P < 0.05, signicant dierence compared to the model
group; ΔP < 0.05 compared to the sham group.
Figure 5. Eects of SDS root EtOAc extracts on FeCl3-induced rat plasma levels of PC. (A) and AT-III (B).
Before the establishment of the FeCl3-induced rat carotid artery thrombosis models, rats in each group were
treated with dierent doses of SDS root EtOAc extract or CDDP for 20 days, once a day, n = 10; data are
presented as the mean ± SD; *P < 0.05, signicant dierence compared to the model group; ΔP < 0.05 compared
to the sham group.
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found that all 3 chemicals signicantly inhibited platelet aggregation in a dose-dependent manner (Table2).
Tanshinone IIA is a diperpenoid quinone in Salvia miltiorrhiza bunge and has been attributed to be a major
active compound of Danshen with antiplatelet and anticoagulant effects via tubulin acetylation and Erk-2
Figure 6. Eect of SDS root ESF on the activity of the brinolytic system. Inuences of each dose of EtOAc
extract on FeCl3-induced rat plasma levels of (A) t-PA and PAI-1, (B) t-PA/PAI-1 ratios and (C) PLG. Before the
establishment of FeCl3-induced rat carotid artery thrombosis models, rats in each group were treated with dierent
doses of SDS root EtOAc extract or CDDP for 20 days, once a day, n = 10; data are presented as the mean ± SD;
*P < 0.05, signicant dierence compared to the model group; ΔP < 0.05 compared to the sham group.
Group Concentration/µg/mL MAR/% MAIR/%
6,7-dehydroroyleanone
Aspirin 100 16.7 ± 4.31 62.3 ± 11.5
Control — 97.1 ± 1.86Δ—
High 1000 50.6 ± 10.20*Δ47.8 ± 10.5Δ
Medium 100 73.0 ± 5.90*Δ24.7 ± 6.07Δ
Low 10 94.4 ± 6.08Δ1.20 ± 5.31Δ
p-value (Dose) <0.0001
p-value
(Aspirin + Dose) <0.0001 <0.0001
Horminone
Aspirin 100 17.35 ± 4.63 61.70 ± 5.82
Control — 91.00 ± 2.89Δ—
High 1000 66.10 ± 4.80*Δ27.50 ± 5.28Δ
Medium 100 79.10 ± 1.21*Δ13.00 ± 1.36Δ
Low 10 85.80 ± 1.68*Δ6.10 ± 2.13Δ
p-value (Dose) <0.0001
p-value
(Aspirin + Dose) <0.0001 <0.0001
7-O-acetylhorminone
Aspirin 100 18.03 ± 5.06 58.63 ± 8.91
Control — 65.1 ± 7.15Δ—
High 1000 42.60 ± 7.02*Δ34.50 ± 10.80Δ
Medium 100 51.80 ± 6.25*Δ17.80 ± 4.70Δ
Low 10 63.40 ± 4.93Δ2.40 ± 7.60Δ
p-value (Dose) <0.0001
p-value
(Aspirin + Dose) <0.0001 <0.0001
Table 2. Eects of 6,7-dehydroroyleanone, horminone and 7-O-acetylhorminone on platelet aggregation
(n = 7,
x
± s). *P < 0.05 signicant dierence compared to control group; ΔP < 0.05 signicant dierence
compared to aspirin group. MAIR (%) = platelet aggregation rate of the control group% - platelet aggregation
rate of test drug treated group% / platelet aggregation rate of the control group%.
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Scientific RepoRts | (2018) 8:17753 | DOI:10.1038/s41598-018-36026-7
phosphorylation inhibition22. However, whether the same mechanisms are valid for 6,7-dehydroroyleanone,
horminone and 7-O-acetylhormione actions requires further investigation, while other compounds may also
have an eect on blood coagulation.
One drawback of our study was that the time between injury and data collection was short, which may have
had an inuence, particularly on brinolytic enzyme data.
Conclusion
In conclusion, the present study demonstrated that SDS root ESF had signicant antithrombotic eects, with
EtOAc being the most eective extraction solvent. e antithrombotic eect could be attributed to enhanced
anti-platelet aggregation and coagulation inactivation. us, further studies on SDS ESF as an antithrombotic
traditional Chinese medicine are warranted.
Data Availability
e datasets supporting the conclusions of this article is included within the article.
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Acknowledgements
is study was supported by the Scientic Research Program of the Higher Education Institution of XinJiang
(Grant No. XJEDU2012I23).
Author Contributions
R.K. and X.L.W. designed the experiments; R.K., X.L.W., X.M.W. and Y.M. performed the experiments; All
authors analyzed the data; R.K. and X.L.W. obtained funding and wrote the manuscript; all authors have read and
approved the nal version of the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-36026-7.
Competing Interests: e authors declare no competing interests.
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