ArticlePDF Available

Effect on platelet aggregation activity: extracts from 31 Traditional Chinese Medicines with the property of activating blood and resolving stasis

Authors:

Abstract and Figures

Objective: To evaluate the anti-platelet aggregation effects of extracts from 31 Traditional Chinese Medicines (TCM) with the property of activating blood and resolving stasis in terms of TCM theory. Methods: The 31 TCMs extracts were prepared using water, 90% ethanol and ethyl acetate., and the effects on anti-platelet aggregation were tested on a platelet aggregation analyzer in vitro with adenosine 5'-diphosphate, bovine thrombin and arachidonic acid (AA) as aggregation inducers, respectively. Aspirin was the positive control. Results: Lots of the tested TCMs had inhibitory effects with concentration-dependent manner on platelet aggregations induced by various agonists. Especially, some of the TCMs such as Chuanxiong (Rhizoma Chuanxiong), Yanhusuo (Rhizoma Corydalis Yanhusuo) and Danshen (Radix Salviae Miltiorrhizae) showed good anti-platelet aggregation effect similar or higher than that in positive control group. Conclusion: The study provided scientific references that several TCMs such as Chuanxiong (Rhizoma Chuanxiong), Yanhusuo (Rhizoma Corydalis Yanhusuo) and Danshen (Radix Salviae Miltiorrhizae), possess the property of anti-platelet aggregation.
Content may be subject to copyright.
TOPIC
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Online Submissions: http://www.journaltcm.com J Tradit Chin Med 2017 February 15; 37(1): 64-75
info@journaltcm.com ISSN 0255-2922
EXPERIMENTAL STUDY
Effect on platelet aggregation activity: extracts from 31 Traditional
Chinese Medicines with the property of activating blood and resolv-
ing stasis
Chen Cen, Wang Fengqin, Xiao Wen, Xia Zhining, Hu Guang, Wan Jianbo,Yang Fengqing
aa
Chen Cen, Wang Fengqin, Xiao Wen, Xia Zhining, School
of Chemistry and Chemical Engineering, Chongqing Univer-
sity, Chongqing 400030, China
Hu Guang, School of Pharmacy and Bioengineering,
Chongqing University of Technology, Chongqing 400054,
China
Wan Jianbo, State Key Laboratory of Quality Research in
Chinese Medicine, Institute of Chinese Medical Sciences,
University of Macau, Macao, China
Supported by the National Natural Science Foundation of
China (Magnetic Nanoparticles-based Cell Affinity Capillary
Electrochromatography and Its Applications, No. 21275169
and Screening of Bioactive Compounds From Typical Huox-
ue Huayu Medicine by Platelet Based Capillary Electrochro-
matography, No. 81202886); the Fundamental Research
Funds for the Central Universities (Study on the Quality Con-
trol Method for the Animal Glue Medicine, No. CQDX-
WL-2012-028); the Chongqing Postdoctoral Funds (No.
RC20120027)
Correspondence to: Prof. Yang Fengqing, School of Chem-
istry and Chemical Engineering, Chongqing University,
Chongqing 400030, China. fengqingyang@cqu.edu.cn
Telephone: +86-23-6510-6615
Accepted: November 7, 2015
Abstract
OBJECTIVE: To evaluate the anti-platelet aggrega-
tion effects of extracts from 31 Traditional Chinese
Medicines (TCM) with the property of activating
blood and resolving stasis in terms of TCM theory.
METHODS: The 31 TCMs extracts were prepared us-
ing water, 90% ethanol and ethyl acetate., and the
effects on anti-platelet aggregation were tested on
a platelet aggregation analyzer in vitro with adenos-
ine 5'-diphosphate, bovine thrombin and arachi-
donic acid (AA) as aggregation inducers, respective-
ly. Aspirin was the positive control.
RESULTS: Lots of the tested TCMs had inhibitory ef-
fects with concentration-dependent manner on
platelet aggregations induced by various agonists.
Especially, some of the TCMs such as Chuanxiong
(Rhizoma Chuanxiong), Yanhusuo (Rhizoma Coryda-
lis Yanhusuo) and Danshen (Radix Salviae Miltiorrhi-
zae) showed good anti-platelet aggregation effect
similar or higher than that in positive control group.
CONCLUSION: The study provided scientific refer-
ences that several TCMs such as Chuanxiong (Rhi-
zoma Chuanxiong), Yanhusuo (Rhizoma Corydalis
Yanhusuo) and Danshen (Radix Salviae Miltiorrhizae),
possess the property of anti-platelet aggregation.
Key words: Platelet aggregation inhibitors; Ade-
nosine diphosphate; Thrombin; Arachidonic acid;
Blood activating stasis removing ; Medicine, Chi-
nese traditional
INTRODUCTION
Blood stasis patter (BSP) or thrombosis in biomedical
terms, which refers to the block in the circulation of
blood or meridians,1is attributed to a very complicated
pathogenesis which mainly involves hemorheology dis-
order, microcirculatory disturbance, vascular endotheli-
al cell injury and platelet aggregation.2Actually, BSP is
considered to be closely related to senile diseases such
as atherosclerosis, ischemic heart disease, and stroke,3
as well as rheumatoid arthritis, hyperuricemia, and vari-
64
© 2016 JTCM. This is an Open Access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/4.0/).
© 2016 JTCM. This is an Open Access article under the CC BY-NC-ND
License (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
ous inflammatory conditions.4Platelet aggregation in
blood vessels plays a crucial role in the formation of
thrombosis. Inhibiting platelet activation is considered
to be an effective way to stop or slow down thrombo-
sis.5,6 Therefore, anti-platelet aggregation tests were
widely applied to evaluate and investigate cardiovascu-
lar disease both in experimental7-9 and clinical observa-
tions.10-12 The adhesiveness and aggregation of platelets
will happen due to the impairment of blood vessels or
emergency of platelet activation factor, such as adrena-
line, adenosine 5-diphosphate (ADP), 5-hydroxytrypta-
mine (5-HT), thromboxane A2(TXA2) and thrombin
(THR) besides collagen.13 Therefore, there are four
main action mechanisms of anti-platelet medicines
have been reported such as inhibiting the metabolism
of platelet arachidonic acid (AA), increasing the level
of platelet cyclic adenosine monophosphate (cAMP),
inhibiting the activation of ADP, and blocking the
platelet membrane glycoprotein b/a.14
Traditional Chinese Medicines (TCM) have a long his-
tory. According to records in Huang Di Nei Jing in Chi-
nese),15 Shang Han Za Bing Lun16 in Chinese and Jin
Kui Yao Lue17 in Chinese in the Eastern Han Dynasty,
there were several TCMs such as Chuanxiong (Rhi-
zoma Chuanxiong) and Yanhusuo (Rhizoma Corydalis
Yanhusuo) had been used for the treatment of BSS. In
Tang and Song Dynasty, more TCMs such as Moyao
(Myrrha) and Jianghuang (Rhizoma Curcumae Longae)
were applied to treat BSS. Actually, in Shen Nong Ben
Cao Jing in Chinese,18 83 of 365 TCMs were recorded
with the function of activating blood and resolving sta-
sis. However, to date, the systematic investigation and
comparison on the anti-platelet aggregation activities
of activating blood and resolving stasis TCMs within
one study has not been reported.
In the present study, 31 TCMs with the property of ac-
tivating blood and resolving stasis were selected based
on their traditional medical literature. Their extracts
were prepared with 90% ethanol (further liquid-liquid
extracted by ethyl acetate) and water. The inhibitory ef-
fects on rabbit platelet aggregation were induced by
AA, ADP and THR of those extracts were investigated.
MATERIALS AND METHODS
Chemicals and reagents
AA, THR and pentobarbital sodium were obtained
from Sigma (St Louis, MO, USA). ADP was the prod-
uct of Wuhu Huaren Technology Company (Wuhu,
Anhui, China). Aspirin and ethanol was purchased
from Chengdu Kelong Chemical Reagent Factory
(Chengdu, China) and sodium citrate was obtained
from Chengdu Aikeda Chemical Reagent Company
(Chengdu, China). All other chemicals and reagents
were of analytical grade.
Thirty-one raw materials of TCMs were obtained from
local drugstores (Chongqing Xhoo Medicin Co., Ltd.,
and Chongqing Hongshengqiao drugstore, Chongq-
ing, China) in Chongqing during autumn of 2012.
The voucher specimens of those TCMs were deposited
at the School of Chemistry and Chemical Engineering
(Chongqing University, Chongqing, China).
Preparation of the extracts
The dried materials of 31 TCMs were ground into fine
powder in a pulverizer, respectively. 20 g of the powder
was extracted with 60 mL 90% ethanol in an ultrason-
ic cleanser tank for 20 min, along with reflux extrac-
tion for 1 h at 80 , filtered and another fresh 60 mL
90% ethanol was added into the residue, repeated the
reflux extraction and filtered. The filtrate was com-
bined and dried at 45 using a rotary evaporator, and
then the residue was re-suspended in 10 mL water and
liquid-liquid extraction with 10 mL ethyl acetate for
two times. The ethyl acetate extract and the rest solu-
tion were separately dried at 45 using a rotary evapo-
rator and further dried in an oven at 50 . After 90%
ethanol extraction, the material residue continued to
be reflux extracted by 60 mL × 2 distilled water for
twice at 100 , filtered and combined the filtrate. The
filtrate solution was evaporated by a rotary evaporator
to produce aqueous extract. Finally, the aqueous ex-
tract, the rest part after ethyl acetate extraction and eth-
yl acetate extract of 90% ethanol extract were labeled
as A1, A2 and A3, respectively. The extraction yields of
each extract for 31 TCMs were between 0.03% to
29.10% . The yields of some extracts such as the A2
and A3 extracts of Xuejie (Sanguis Draconis) were very
low, so the anti-platelet aggregation tests for those ex-
tracts were done only with one concentration each. A1
extract was dissolved in phosphate buffer saline, A2 in
60% ethanol and A3 in diluted dimethyl sulfoxide
(DMSO) before use, respectively.
Preparation of blood plasma sample
Rabbits, males, weighing [(2.2 ± 0.4) kg] were pur-
chased from Animal farm in Chongqing. All experi-
mental procedures were approved by the Institutional
Animal Ethical Committee of Chongqing University
and were conducted according to the Guide for the
Care and Use of Laboratory Animal of the National In-
stitute of Health (Publication No. 80-23, revised 1996).
Rabbit blood samples were collected in 3.8% sodium
citrate with the ratio of 91 (bloodanticoagulant)
from carotid artery after anesthetizing by 1% pentobar-
bital sodium. Platelet-rich plasma (PRP) was obtained
by centrifugation at 93 ×g for 15 min at room tempera-
ture, and platelet-poor plasma (PPP) was obtained by
further centrifugation from the remaining blood at
2325 ×g for 15 min. The concentration of PRP was ad-
justed to 3 × 1011/L by PPP.19
In vitro platelet aggregation assay
Platelet aggregation test was performed using the meth-
od described in previous report with some modifica-
tions.20 In brief, the level of light transmission was cali-
65
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Chen C et al. / Experimental Study
February 15, 2017
|
Volume 37
|
Issue 1
|
brated as 0% by PPP (300 μL) using SC-2000 platelet
aggregometer (Beijing Success Technology Develop-
ment Co., Ltd., Beijing, China). PRP (300 μL) and dif-
ferent concentrations of extracts (10 μL) were incubat-
ed for 3 min at 37 . After incubation, platelet aggre-
gation was induced by adding 10 μL of ADP, THR
and AA, respectively, and monitored for 5 min with
stirring. DMSO (final concentration: 1.7%), 60% eth-
anol and PBS were used as blank control for A3, A2
and A1 extracts, respectively. Aspirin was used as the
positive control.
The percentage of aggregation inhibition is calculated
by the following formula:
I% = A-B
A× 100
where I% is the inhibitory percentage, A is maximal ag-
gregation of the blank control test and B is maximal ag-
gregation of drug-treated PRP. Data is expressed as
mean ± standard deciation ( x
ˉ±s).
RESULTS
Effects of A1 extracts of 31 TCMs on the platelet
aggregation
The results (Table 1) indicated that positive drug aspi-
rin showed a strong inhibitory effect on all the platelet
aggregation induced by AA, ADP and THR with a con-
centration-dependent manner. The aqueous extracts
(A1) of 31 TCMs exhibited different effects on the
platelet aggregation with different inducers (AA, ADP
and THR). For ADP as aggregation inducer, Huaihua
(Flos Sophorae) had the strongest anti-aggregation effect
(even stronger than that of aspirin), while other TCMs
with strong anti-aggregation effect (inhibition ratio
higher than 40% and with dose-dependent manner)
were in the order: Chuanxiong (Rhizoma Chuanx-
iong) ≈ Xuanshen (Radix Scrophulariae) ≈ Danggui (Ra-
dix Angelicae Sinensis) ≈ Jianghuang (Rhizoma Curcum-
ae Longae) ≈ Zelan (Herba Lycopi Hirti) > Honghua
(Flos Carthami) ≈ Sanleng (Rhizoma Sparganii) ≈Mu-
danpi (Cortex Moutan Radicis) ≈ Jixueteng (Caulis
Spatholobi) > Sanqi (Radix Notoginseng). However,
some TCMs such as Chongweizi (Fructus Leonuri Ja-
ponici) and Xuejie (Sanguis Draconis) showed promot-
ing instead of inhibiting effect on the platelet aggrega-
tion. For THR as aggregation inducer, most of the test-
ed TCMs had inhibitory effect on the aggregation with
the order: Chuanxiong (Rhizoma Chuanxiong) > Huai-
huami (Flos Sophorae) ≈ Sanqi (Radix Notoginseng) ≈
Danggui (Radix Angelicae Sinensis) ≈ Jianghuang (Rhi-
zoma Curcumae Longae) ≈ Wulingzhi (Faeces Trogoptero-
ri) ≈ Zelan (Herba Lycopi Hirti) ≈ Yimucao (Herba
Leonuri Japonici) > Honghua (Flos Carthami) ≈ Xuan-
shen (Radix Scrophulariae) ≈ Puhuang (Pollen Typhae) ≈
Zicao (Radix Lithospermi) > Danshen (Radix Salviae
Miltiorrhizae) ≈ Chishao (Radix Paeoniae Rubra) >
Chongweizi (Fructus Leonuri Japonici) ≈ Sanleng (Rhi-
zoma Sparganii) ≈ Dahuang (Radix Et Rhizoma Rhei
Palmati) > Ezhu (Rhizoma Curcumae Phaeocaulis) >
Mudanpi (Cortex Moutan Radicis) > Yanhusuo (Rhi-
zoma Corydalis Yanhusuo). For AA as aggregation induc-
er, few of the tested TCMs had inhibitory effect on ag-
gregation with the order: Chuanxiong (Rhizoma Ch-
uanxiong) > Danshen (Radix Salviae Miltiorrhizae) ≈
Zicao (Radix lithospermi) > Yimucao (Herba Leonuri Ja-
ponici) > Xuejie (Sanguis Draconis) > Jixueteng (Caulis
Spatholobi). It is worthy to note that some TCMs such
as Niuxi (Radix Achyranthis Bidentatae) (with THR
and AA as aggregation inducer) and Chishao (Radix
Paeoniae Rubra) (with AA as aggregation inducer) had
strong anti-aggregation effect at lower concentrations,
but the effect were decreased with the increase of drug
concentrations. In addition, the A1 extracts of Huai-
hua (Flos Sophorae), Honghua (Flos Carthami), Xuansh-
en (Radix Scrophulariae), Danggui (Radix Angelicae Si-
nensis), Jianghuang (Rhizoma Curcumae Longae), San-
leng (Rhizoma Sparganii), Sanqi (Radix Notoginseng)
and Zelan (Herba Lycopi Hirti) selectively inhibited the
platelet aggregation induced by ADP and THR, while
Yimucao (Herba Leonuri Japonici), Danshen (Radix Sal-
viae Miltiorrhizae) and Zicao (Radix lithospermi) had
the inhibitory effects on THR and AA induced aggrega-
tion and Jixueteng (Caulis Spatholobi) had the inhibito-
ry effects on ADP and AA induced aggregation. Ch-
uanxiong (Rhizoma Chuanxiong) had inhibitory effects
on all the three agonists induced aggregation.
Effects of A2 extracts of 31 TCMs on platelet
aggregation
As shown in Table 2, the rest part of 90% ethanol ex-
tract after liquid-liquid extracting by ethyl acetate (A2)
had much weaker anti-aggregation effects. For ADP as
aggregation inducer, ten of the tested TCMs had
strong inhibitory effect on the aggregation with the or-
der: Yanhusuo (Rhizoma Corydalis Yanhusuo) > Shuizhi
(Hirudo) > Danshen (Radix Salviae Miltiorrhizae) >
Danggui (Radix Angelicae Sinensis) > Sanqi (Radix No-
toginseng) > Chuanxiong (Rhizoma Chuanxiong) ≈ Xu-
anshen (Radix Scrophulariae) ≈ Puhuang (Pollen
Typhae) ≈ Yimucao (Herba Leonuri Japonici) ≈ Ezhu
(Rhizoma Curcumae Phaeocaulis). For THR as aggrega-
tion inducer, seven of the tested TCMs had strong in-
hibitory effect on the aggregation with the order: Ch-
uanxiong (Rhizoma Chuanxiong) ≈ Yanhusuo (Rhizoma
Corydalis Yanhusuo) > Yimucao (Herba Leonuri Japoni-
ci) > Ezhu (Rhizoma Curcumae Phaeocaulis) > Danshen
(Radix Salviae Miltiorrhizae) > Puhuang (Pollen
Typhae) ≈ Sanqi (Radix Notoginseng). For AA as aggre-
gation inducer, five of the tested TCMs had strong in-
hibitory effect on the aggregation with the order: Yan-
husuo (Rhizoma Corydalis Yanhusuo) > Chuanxiong
(Rhizoma Chuanxiong) ≈ Yujin (Radix Curcumae We-
nyujin)> Shuizhi (Hirudo) > Ezhu (Rhizoma Curcumae
Phaeocaulis). In addition, Danshen (Radix Salviae Milt-
iorrhizae), Puhuang (Pollen Typhae), Yimucao (Herba
66
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
TCMsa
Aspirin
Cebaiye
(Cacumen Platycladi)
Jixueteng
(Caulis Spatholobi)
Mudanpi
(Cortex Moutan Radicis)
Wulingzhi
(Faeces Trogopterori)
Honghua
(Flos Carthami)
Huaihua
(Flos Sophorae)
Chongweizi
(Fructus Leonuri Japonici)
Yimucao
(Herba Leonuri Japonici)
Zelan
(Herba Lycopi Hirti)
Shuizhi
(Hirudo)
Moyao
(Myrrha)
Final concentration
(mg/mL)
0.3125
0.7813
1.5625
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
Inhibition of platelet aggregation (%, n= 3)
ADP
5 μmol/L
7.5±4.7
41.8±13.3
78.4±7.2
5.5±11.2
11.6±3.6
5.9±3.8
23.0±11.8
20.3±9.1
51.8±0.1
7.3±8.5
27.4±10.0
47.8±6.7
37.1±1.9
2.3±1.2
6.6±7.5
16.5±5.8
33.7±10.6
50.7±1.0
31.2±3.0
98.2±1.6
97.4±4.5
9.7±2.6
25.4±6.2
73.1±27.5
7.6±2.2
10.8±4.7
24.5±4.3
41.4±6.0
30.4±5.6
58.1 ±0.9
8.8±4.0
4.4±5.8
15.4±8.2
2.2±8.7
1.6±7.9
22.5±4.2
THR
0.25u/mL
12.3±1.3
62.8±6.8
97.7±2.3
3.1±3.1
7.1±6.0
4.3±4.6
36.9±4.9
22.9±1.3
33.4±8.8
7.1±1.4
18.1±2.3
58.6±4.6
10.8±3.8
49.1±5.4
95.2±1.7
19.5±7.5
71.2±9.3
85.1±2.4
37.9±6.2
80.6±3.7
92.3±4.3
26.8±1.9
32.9±15.3
69.1±10.5
1.6±4.0
28.8±4.8
94.3±5.4
26.2±0.5
91.2±1.3
94.2±5.1
9.5±3.4
15.8±8.8
17.1±8.8
6.8±1.4
24.5±12.7
5.2±1.8
AA
0.205 mmol/L
42.3±2.5
82.9±3.9
96.9±3.9
1.8±4.2
8.9±5.2
22.5±11.8
14.9±4.0
12.1±5.2
51.0±1.2
14.0±9.2
14.9±3.4
29.1±0.3
1.6±8.8
2.7±2.7
16.7±3.0
23.3±8.9
2.6±19.1
10.0±19.8
29.7±6.6
19.5±3.8
33.3±13.8
3.0±5.3
12.3±8.7
14.6±8.3
22.5±7.7
55.4±9.0
77.8±6.0
11.7±4.9
19.1±7.5
18.0±5.4
19.4±5.3
0.3±2.3
13.8±3.0
15.2±11.4
10.7±3.5
11.9±5.2
TCMs
Zicao
(Radix lithospermi)
Yujin
(Radix Curcumae)
Chuanniuxi
(Radix Cyathulae)
Sanqi
(Radix Notoginseng)
Dahuang
(Radix Et Rhizoma Rhei
Palmati)
Chishao
(Radix Paeoniae Rubra)
Danshen
(Radix Salviae
Miltiorrhizae)
Xuanshen
(Radix Scrophulariae)
Yanhusuo
(Rhizoma Corydalis
Yanhusuo)
Ezhu
(Rhizoma Curcumae
Phaeocaulis)
Jianghuang
(Rhizoma Curcumae
Longae)
Huzhanggen
(Radix Polygoni Cuspidati)
Final
concentration
(mg/mL)
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
Inhibition of platelet aggregation (%, n= 3)
ADP
5 μmol/L
10.3±3.4
2.4±7.3
34.1±6.5
10.8±2.6
25.2±3.3
32.3±4.8
22.1±5.4
21.9±2.4
19.2±8.6
9.5±8.9
30.8±5.0
41.6±9.3
32.3±0.6
44.0±2.2
33.6±1.4
3.4±8.7
16.7±11.6
11.4±6.8
14.5±8.1
0.7±3.0
8.9±6.5
26.0±0.7
29.6±6.7
61.0±1.9
19.6±3.5
17.1±9.5
11.0±3.8
3.0±8.1
1.2±6.0
6.0±5.1
3.4±3.5
23.5±4.9
64.6±6.7
5.0±7.5
12.6±2.7
13.0±6.8
THR
0.25u/mL
3.1±9.4
25.5±12.6
88.7±11.3
2.8±11.6
22.3±17.3
21.3±2.4
9.5±9.4
21.9±2.0
18.1±2.9
7.8±2.6
42.3±7.6
92.3±7.3
10.0±2.1
10.8±4.4
67.3±13.1
14.1±3.8
43.0±20.8
77.9±11.1
9.6±8.9
34.1±9.3
74.7±7.9
10.0±15.2
69.6±5.4
84.3±8.2
1.8±13.0
35.5±15.8
52.2±10.4
1.6±1.8
25.0±0.3
63.6±6.2
10.9±4.6
42.6±3.2
99.6±0.6
0.3±5.8
3.9±2.6
3.9±1.7
AA
0.205 mmol/L
15.6±3.4
67.1±12.4
97.2±3.9
15.7±5.0
15.8±12.9
17.3±10.7
18.7±13.2
7.1±2.0
12.8±7.0
10.2±1.3
5.1±3.7
8.9±3.4
8.2±0.8
13.5±3.3
11.4±10.4
40.5±7.8
13.4±2.3
4.9±7.3
5.6±8.0
92.2±10.6
98.0±3.5
4.8±1.9
0.7±2.7
15.5±12.0
7.0±4.7
17.1±12.6
3.8±5.1
0.2±1.9
5.3±1.6
12.4±1.7
5.8±11.9
6.1±6.8
14.6±3.1
9.9±7.6
21.4±0.6
10.6±2.2
Table 1 Inhibition effects on the platelet aggregation of aqueous extracts (A1) of 31 TCMs ( x
ˉ±s)
67
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Chen C et al. / Experimental Study
Leonuri Japonici) and Sanqi (Radix Notoginseng) have
inhibitory effects on two inducers (ADP and THR),
while Chuanxiong (Rhizoma Chuanxiong), Yanhusuo
(Rhizoma Corydalis Yanhusuo) and Ezhu (Rhizoma Cur-
cumae Phaeocaulis) showed strong anti-platelet aggrega-
tion effects induced by all the three agonists.
Effects of A3 extracts of 31 TCMs on platelet
aggregation
As shown in Table 3, the ethyl acetate extracts from
90% ethanol extracts (A3) had stronger anti-aggrega-
tion effects than that of A1 and A2 when using AA as
inducer. Fifteen of the tested TCMs had strong inhibi-
tory effect on the aggregation induced by AA with the
order: Chuanxiong (Rhizoma Chuanxiong) ≈ Puhuang
(Pollen Typhae) ≈ Yanhusuo (Rhizoma Corydalis Yanhu-
suo) ≈ Huzhanggen (Radix Polygoni Cuspidati) > Hong-
hua (Flos Carthami) ≈ Mudanpi (Cortex Moutan Radi-
cis) > Xuanshen (Radix Scrophulariae) > Dahuang (Ra-
dix Et Rhizoma Rhei Palmati) > Chishao (Radix Paeoni-
ae Rubra) > Danggui (Radix Angelicae Sinensis) ≈ Wul-
ingzhi (Faeces Trogopterori) > Danshen (Radix Salviae
Miltiorrhizae) ≈ Huaihua (Flos Sophorae) ≈ Yujin (Ra-
dix Curcumae Wenyujin) ≈ Cebaiye (Cacumen Platy-
cladi). For ADP as aggregation inducer, fifteen of the
tested TCMs had strong inhibitory effect on the aggre-
gation with the order: Yanhusuo (Rhizoma Corydalis
Yanhusuo) > Chuanxiong (Rhizoma Chuanxiong) >
Honghua (Flos Carthami) ≈ Xuanshen (Radix Scrophu-
lariae) > Danshen (Radix Salviae Miltiorrhizae) ≈
Puhuang (Pollen Typhae) ≈ Huzhanggen (Radix Polygo-
ni Cuspidati) > Cebaiye (Cacumen Platycladi) ≈ Mudan-
pi (Cortex Moutan Radicis) > Sanqi (Radix Notogin-
seng) ≈ Dahuang (Radix Et Rhizoma Rhei Palmati) >
Danggui (Radix Angelicae Sinensis) ≈ Huaihua (Flos So-
phorae) > Chongweizi (Fructus Leonuri Japonici) ≈
Moyao (Myrrha). For THR as aggregation inducer,
twelve of the tested TCMs had strong inhibitory effect
on the aggregation with the order: Yanhusuo (Rhizoma
Corydalis Yanhusuo) > Sanqi (Radix Notoginseng) > Xu-
anshen (Radix Scrophulariae) ≈ Danshen (Radix Salviae
Miltiorrhizae) > Huzhanggen (Radix Polygoni Cuspida-
ti) > Puhuang (Pollen Typhae) ≈ Chishao (Radix Paeoni-
ae Rubra) > Honghua (Flos Carthami) > Huaimi (Flos
Sophorae) ≈ Mudanpi (Cortex Moutan Radicis) ≈ Dang-
gui (Radix Angelicae Sinensis) ≈ Wulingzhi (Faeces Trog-
opterori). Furthermore, Sanqi (Radix Notoginseng) had
inhibitory effects on both ADP and THR as aggrega-
tion inducers. Chuanxiong (Rhizoma Chuanxiong) and
Dahuang (Radix Et Rhizoma Rhei Palmati) inhibited
ADP and AA induced aggregation. Wulingzhi (Faeces
Trogopterori) and Chishao (Radix Paeoniae Rubra) can
inhibit the THR and AA induced aggregation. In addi-
tion, Honghua (Flos Carthami), Xuanshen (Radix
Scrophularia), Danshen (Radix Salviae Miltiorrhizae),
Huaihua (Flos Sophorae), Danggui (Radix Angelicae Si-
nensis), Puhuang (Pollen Typhae), Yanhusuo (Rhizoma
Corydalis Yanhusuo), Mudanpi (Cortex Moutan Radicis)
Table 1 Inhibition effects on the platelet aggregation of aqueous extracts (A1) of 31 TCMs ( x
ˉ±s)continuted)
TCMs
Chuanxiong
(Rhizoma Chuanxiong)
Sanleng
(Rhizoma Sparganii)
Xuejie
(Sanguis Draconis)
Taoren
(Semen Persicae)
Final
concentration
(mg/mL)
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
Inhibition of platelet aggregation (%, n= 3)
ADP
5 μmol/L
21.5±2.2
56.7±11.6
61.3±4.0
13.0±9.8
18.3±4.4
47.7±4.0
2.9±7.9
9.5±5.6
42.3±8.2
7.5±8.2
7.8±15.4
29.0±7.5
THR
0.25u/mL
13.7±3.2
97.9±3.8
97.4±2.7
15.6±11.4
43.0±19.1
71.2±4.4
7.3±0.6
5.1±1.4
5.5±6.3
17.9±5.5
34.8±12.4
35.9±7.8
AA
0.205 mmol/L
22.8±5.3
35.6±2.1
100.0±0
30.6±2.4
14.5±5.4
27.9±5.0
10.8±8.9
36.8±7.9
67.9±3.8
4.9±11.7
9.0±4.7
20.5±5.1
TCMsa
Ruxiang
(Olibanum)
Puhuang
(Pollen Typhae)
Niuxi
(Radix Achyranthis
Bidentatae)
Danggui
(Radix Angelicae Sinensis)
Final concentration
(mg/mL)
0.3125
1.5625
3.1250
0.3125
0.7813
1.5625
0.3125
1.5625
3.1250
0.3125
1.5625
3.1250
Inhibition of platelet aggregation (%, n= 3)
ADP
5 μmol/L
49.4±3.5
27.8±2.7
24.2±7.2
31.9±3.4
20.0±4.1
11.5±4.4
19.7±7.3
27.6±12.5
22.1±6.7
16.0±4.4
21.5±4.2
61.5±4.5
THR
0.25u/mL
10.4±8.1
7.0±1.1
55.1±7.8
34.2±5.1
52.0±11.1
84.2±16.6
78.5±4.7
33.1±4.4
3.5±12.8
5.6±4.3
70.3±8.1
92.7±3.2
AA
0.205 mmol/L
12.3 ±6. 9
16.7±6.8
27.6±5.2
7.7±5.8
5.4±1.9
2.8±5.4
63.2±15.7
29.8±5.7
22.0±22.9
7.0±8.5
25.2±18.2
20.1±8.6
Notes: aTCMs were listed in alphabetical order.TCMs: Traditional Chinese Medicines; ADP: adenosine 5-diphosphate; THR: thrombin; AA: arachidonic acid.
68
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
TCMs
Zicao
(Radix lithospermi)b
Yujin
(Radix Curcumae)
Chuanniuxi
(Radix Cyathulae)
Sanqi
(Radix Notoginseng)
Dahuang
(Radix Et Rhizoma Rhei
Palmati)
Chishao (Radix Paeoniae
Rubra)
Danshen
(Radix Salviae Miltiorrhizae)
Xuanshen
(Radix Scrophulariae)
Yanhusuo (Rhizoma Corydalis
Yanhusuo)
Ezhu
(Rhizoma Curcumae
Phaeocaulis)
Jianghuang
(Rhizoma Curcumae Longae)
Huzhanggen
(Radix Polygoni Cuspidati)b
Final
concentration
(mg/mL)
0.4063
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
Inhibition of platelet aggregation (%, n= 3)
ADP
10 μmol/L
35.7±8.5
21.8±4.1
19.9±3.8
38.9±12.7
21.0±7.2
19.2±9.4
28.2±8.4
7.0±1.7
38.8±1.8
59.7±15.5
6.6±2.4
14.3±6.6
22.3±7.0
6.2±10.5
13.6±16.3
17.6±7.0
27.1±2.9
45.5±2.5
70.3±5.4
29.9±8.7
40.0±8.7
53.8±5.4
57.1±1.7
97.2±3.4
100.0±0.0
12.9±4.3
27.0±12.6
50.5±3.3
22.6±4.5
25.8±2.2
18.6±5.1
9.1±9.8
THR
0.25u/mL
7.2±1.7
6.7±8.5
19.7±8.4
26.8±5.4
12.5±2.8
12.2±1.8
19.6±2.0
26.8±5.4
42.8±4.2
58.0±7.4
8.4±5.6
9.6±3.8
-
10.9±4.6
9.6±3.8
19.0±3.5
24.2±3.8
40.0±1.9
62.3±3.1
13.2±3.5
13.8±4.7
20.6±3.6
45.4±3.8
63.6±1.4
93.4±5.8
20.5±2.4
45.6±2.0
73.6±11.0
10.9±5.9
29.1±8.1
33.6±3.5
23.7±7.2
AA
1.205 mmol/L
1.6±5.5
6.3±1.6
9.1±5.8
57.7±0.8
0.8±1.5
0.0±2.1
2.6±3.6
6.1±4.6
27.0±6.5
35.0±2.3
8.6±4.3
0.6±3.1
-
10.4±4.2
1.0±8.8
2.1±4.4
10.9±3.8
14.1±10.0
35.2±1.2
8.9±6.4
10.1±7.6
15.3±3.8
29.3±8.7
75.5±1.4
100.0±0.0
10.3±0.6
24.3±3.0
46.9±0.7
4.0±4.6
8.4±1.9
27.8±4.9
4.0±4.9
TCMsa
Aspirin
Cebaiye
(Cacumen Platycladi)
Jixueteng
(Caulis Spatholobi)
Mudanpi
(Cortex Moutan Radicis)
Wulingzhi
(Faeces Trogopterori)
Honghua
(Flos Carthami)
Huaihua
(Flos Sophorae)
Chongweizi
(Fructus Leonuri Japonici)
Yimucao
(Herba Leonuri Japonici)
Zelan
(Herba Lycopi Hirti)
Shuizhi
(Hirudo)
Moyao
(Myrrha)
Final
concentration
(mg/mL)
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
Inhibition of platelet aggregation (%, n= 3)
ADP
10 μmol/L
7.5±4.7
41.8±13.3
78.4±7.2
7.0±7.8
18.5±11.5
17.8±15.3
15.2±6.0
7.4±11.6
16.9±5.6
29.8±4.9
5.1±1.9
4.0±7.4
18.3±7.0
13.5±9.4
21.5±5.5
9.3±14.2
15.4±1.8
14.5±3.3
26.1±2.7
22.0±1.8
23.3±4.8
22.3±4.0
13.4±10.7
28.6±5.5
48.2±7.3
22.4±1.3
19.5±5.0
31.9±5.0
16.1±2.4
62.3±1.0
95.0±3.9
2.9±3.0
15.5±1.7
25.6±0.6
THR
0.25u/mL
12.3±1.3
62.8±6.8
97.7±2.3
10.6±3.8
10.9±2.8
14.4±0.6
4.5±2.3
12.0±1.7
18.9±5.7
11.9±6.3
5.2±5.8
12.0±1.6
13.2±3.4
6.2±1.1
11.4±4.6
35.1±8.1
20.9±4.3
19.9±1.8
28.3±2.7
19.7±3.8
18.9±3.7
37.2±1.8
17.3±6.8
54.2±10.7
89.3±1.9
8.5±4.2
13.0±7.9
10.4±3.2
19.9±5.3
19.9±8.7
37.8±3.1
0.9±2.9
4.6±3.5
11.5±4.8
AA
1.205 mmol/L
42.3±2.5
82.9±3.9
96.9±3.9
2.1±10.5
0.6±8.1
3.3±1.6
15.0±18.6
4.2±2.0
7.7±0.9
5.9±0.5
3.0±4.3
8.9±11.4
12.2±1.6
1.4±3.8
4.3±1.7
5.1±3.7
4.1±9.0
11.6±8.6
7.1±7.9
9.3±2.8
10.7±2.6
22.5±2.8
2.2±0.5
7.2±5.8
31.2±3.8
6.1±3.5
3.1±2.7
6.0±8.5
21.0±11.1
22.1±11.2
43.8±2.9
1.7±2.8
19.3±3.8
10.2±2.6
Table 2 Inhibition effects on the platelet aggregation of 90% ethanol extracts (A2) of 31 TCMs ( x
ˉ±s)
69
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Chen C et al. / Experimental Study
and Huzhanggen (Radix Polygoni Cuspidati) inhibited
platelet aggregation induced by all the three inducers.
DISCUSSION
In this study, we evaluated the anti-platelet aggregation
effects of extracts from 31 TCMs with the property of
activating blood and resolving stasis in terms of TCM
theory.
Our results show that 20 aqueous extracts inhibited
platelet aggregation with more than 40% inhibitory ra-
tio induced by THR, which may due to the polypep-
tides in those aqueous extracts. Furthermore, the meta-
bolic pathway of AA depends on the bioactivity of
COX-1 as mentioned above. So it is an effective way to
inhibit platelet aggregation by blocking the activity of
COX-1. It was reported that natural COX-1 inhibitors
usually were flavonoids or polyphenols, pentacyclic trit-
erpenoids, alkaloids, organic acids and so on,30 which
can be extracted by ethyl acetate. Therefore, ethyl ace-
tate extracts (A3) showed higher inhibitory ratio on
AA-induced platelet aggregation than A1 and A2 ex-
tracts.
The results (Table 4) indicate that most of the tested
TCMs have anti-platelet aggregation activities, especial-
ly for Danshen (Radix Salviae Miltiorrhizae),31 Dang-
gui (Radix Angelicae Sinensis),32 Xuanshen (Radix
Scrophulariae)33 and Sanqi (Radix Notoginseng)34 have
strong inhibitory activities on all the aggregation in-
duced by the three inducers (ADP, THR and AA).
Therefore, it can be concluded that the anti-platelet ag-
gregation may be one of the most important approach-
es for the activating blood and resolving stasis activity.
Furthermore, the activating blood and resolving stasis
TCMs can be classified into four group based on their
action mechanisms,18 and our present results indicated
that the anti-platelet aggregation activities of those four
groups' TCMs had significant difference. The first
group (nourishing and promoting blood circulation)
TCMs including Danshen (Radix Salviae Miltiorrhi-
zae), Danggui (Radix Angelicae Sinensis), Chishao (Ra-
dix Paeoniae Rubra), Jixueteng (Caulis Spatholobi), Zi-
cao (Radix lithospermi), Xuanshen (Radix Scrophulari-
ae), Huaimi (Flos Sophorae) and Cebaiye (Cacumen
Platycladi) had the highest level on the inhibition of
platelet aggregation. The second group (promoting
blood circulation and removing blood stasis) TCMs in-
cluding Chuanxiong (Rhizoma Chuanxiong), Honghua
(Flos Carthami), Puhuang (Pollen Typhae), Sanqi (Radix
Notoginseng), Yujin (Radix Curcumae), Zelan (Herba Ly-
copi Hirti), Yimucao (Herba Leonuri Japonici) and Mu-
danpi (Cortex Moutan Radicis) also showed high inhibi-
tory rate on platelet aggregation. Among them, Ch-
uanxiong (Rhizoma Chuanxiong) displayed distinct an-
ti-platelet aggregation effect induced by various ago-
nists. The bioactive components were demonstrated as
ligustrazine and ferulic acid in Chuanxiong (Rhizoma
Table 2 Inhibition effects on the platelet aggregation of 90% ethanol extracts (A2) of 31 TCMs ( x
ˉ±s) (continuted)
TCMsa
Ruxiang
(Olibanum)
Puhuang
(Pollen Typhae)
Niuxi
(Radix Achyranthis
Bidentatae)
Danggui
(Radix Angelicae Sinensis)
Final
concentration
(mg/mL)
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
Inhibition of platelet aggregation (%, n= 3)
ADP
10 μmol/L
9.2±2.2
10.9±3.2
9.6±5.5
7.3±3.9
26.9±2.5
48.5±5.8
0.9±4.3
4.5±2.4
3.3±1.2
17.2±1.9
31.5±5.8
64.2±14.8
THR
0.25u/mL
11.3±4.8
6.6±6.5
1.8±4.0
8.2±10.0
19.3±2.0
55.5±1.5
3.0±4.7
11.0±4.5
9.4±6.4
14.2±4.2
24.4±3.7
38.3±3.6
AA
1.205 mmol/L
3.7±5.3
5.6±2.0
16.9±2.8
0.6±1.2
10.1±11.2
38.1±9.6
0.9±3.6
4.5±2.0
10.2±3.5
1.7±1.9
18.2±3.4
34.8±3.1
TCMs
Chuanxiong
(Rhizoma Chuanxiong)
Sanleng (Rhizoma Sparganii)
Xuejie
(Sanguis Draconis)b
Taoren
(Semen Persicae)
Final
concentration
(mg/mL)
0.3125
0.7813
1.5625
0.3125
0.7813
1.5625
0.3875
0.3125
0.7813
1.5625
Inhibition of platelet aggregation (%, n= 3)
ADP
10 μmol/L
27.4±11.0
33.9±4.9
53.2±5.1
20.6±6.4
24.9±12.0
25.3±3.6
9.7±2.7
9.9±9.6
13.5±13.6
20.2±3.0
THR
0.25u/mL
44.7±3.4
88.4±4.5
93.9±6.9
11.5±3.6
9.9±4.4
30.0±2.3
0.8±3.5
8.1±4.8
22.5±5.9
29.7±3.8
AA
1.205 mmol/L
13.8±4.2
24.7±5.6
56.7±5.8
2.9±1.1
9.8±4.8
25.4±1.5
5.5±13.0
4.8±3.6
10.2±3.1
8.8±1.0
Notes: aTCMs were listed in alphabetical order; bonly one concentration was tested for those extracts with poor solubility or very low yielded amounts. TCMs: Traditional Chinese Medicines; ADP: adenosine
5-diphosphate; THR: thrombin; AA: arachidonic acid.
70
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Table 3 Inhibition effects on the platelet aggregation of acetate ethyl extracts (A3) of 31 TCMs ( x
ˉ±s)
TCMs
Zicao
(Radix lithospermi)
Yujin
(Radix Curcumae)
Chuanniuxi
(Radix Cyathulae)
Sanqi
(Radix Notoginseng)
Dahuang
(Radix Et Rhizoma Rhei Palmati)
Chishao
(Radix Paeoniae Rubra)
Danshen
(Radix Salviae Miltiorrhizae)
Xuanshen
(Radix Scrophulariae)
Yanhusuo
(Rhizoma Corydalis Yanhusuo)
Ezhu
(Rhizoma Curcumae Phaeocaulis)
Jianghuang
(Rhizoma Curcumae Longae)
Huzhanggen
(Radix Polygoni Cuspidati)
Final
concentration
(mg/mL)
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.2656
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
Inhibition of platelet aggregation (%, n=3)
ADP
10 μmol/L
1.8±2.1
1.9±0.5
2.5±5.4
4.2±3.3
6.5±2.6
5.7±2.4
3.4±9.4
7.4±4.1
19.7±4.5
65.5±7.2
25.2±2.4
38.6±2.1
63.7±7.0
25.2±3.2
39.7±1.6
30.8±2.2
59.2±3.7
67.1±4.8
79.3±9.1
56.7±8.0
62.4±12.4
84.3±10.2
93.9±5.4
86.5±9.3
97.6±2.9
10.1±3.1
7.4±6.6
8.1±7.7
2.3±1.1
0.8±12.9
0.6±5.9
53.7±5.1
66.2±4.1
79.8±2.6
THR
0.25μ/mL
3.1±5.0
3.4±0.9
6.4±0.1
3.6±1.2
2.4±11.7
7.3±7.7
5.0±8.2
4.4±4.3
23.7±0.6
92.6±6.6
8.5±3.1
15.6±2.3
27.6±0.8
40.5±11.6
51.6±7.8
70.0±7.3
44.0±10.1
71.4±0.4
93.0±9.6
58.2±5.1
73.4±1.7
91.7±0.9
77.0±1.0
83.7±1.1
97.2±4.9
6.5±3.2
4.8±0.8
15.6±6.0
0.8±2.6
5.2±5.3
3.1±8.3
42.7±3.7
63.2±1.3
81.6±5.4
AA
1.205 mmol/L
7.8±1.7
8.3±2.5
12.0±0.4
10.0±2.4
20.5±2.7
40.0±1.6
19.2±2.8
28.6±2.3
36.6±1.9
31.2±9.6
27.4±1.9
41.5±9.0
61.7±7.7
19.0±2.9
32.8±6.3
57.7±4.5
15.7±1.5
29.6±2.2
44.4±3.3
7.4±1.6
23.1±0.3
64.8±0.8
88.1±4.0
88.4±4.1
94.1±7.5
4.1±1.9
7.8±2.0
9.1±2.8
0.6±2.2
8.2±1.8
14.4±2.1
95.4±8.0
84.3±8.4
98.9±1.9
TCMsa
Aspirin
Cebaiye
(Cacumen Platycladi)
Jixueteng
(Caulis Spatholobi)
Mudanpi
(Cortex Moutan Radicis)
Wulingzhi
(Faeces Trogopterori)
Honghua
(Flos Carthami)
Huaihua
(Flos Sophorae)
Chongweizi
(Fructus Leonuri Japonici)
Yimucao
(Herba Leonuri Japonici)
Zelan
(Herba Lycopi Hirti)
Shuizhi
(Hirudo)
Moyao
(Myrrha)
Final
concentration
(mg/mL)
0.3125
0.7813
1.5625
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.3375
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
Inhibition of platelet aggregation (%, n=3)
ADP
10 μmol/L
7.5±4.7
41.8±13.3
78.4±7.2
20.6±1.6
34.4±3.6
72.4±4.2
4.6±5.3
2.4±6.1
16.6±7.7
17.1±1.6
45.6±4.9
71.2±9.3
16.6±3.1
18.7±3.2
30.3±2.7
34.5±3.0
47.3±1.2
83.2±3.6
57.9±15.0
15.0±5.6
22.9±5.4
40.3±0.3
0.3±3.8
6.9±13.1
14.2±0.5
2.0±5.3
2.7±3.4
20.3±1.3
5.6±2.2
0.2±7.0
6.1±3.1
25.2±2.4
26.6±3.1
41.1±4.8
THR
0.25μ/mL
12.3±1.3
62.8±6.8
97.7±2.3
14.0±1.7
29.0±2.7
24.4±5.5
6.2±2.4
6.0±1.6
2.3±3.8
23.1±2.4
39.5±8.5
54.4±4.0
41.9±2.8
42.6±3.4
56.8±6.4
28.8±2.1
53.4±6.3
60.4±4.5
54.4±2.5
5.5±2.6
23.9±6.0
32.7±4.6
6.4±7.6
11.7±6.2
16.5±8.7
4.3±4.3
1.5±6.7
3.4±5.2
4.0±2.8
6.7±2.2
13.7±3.5
10.6±0.4
36.4±1.5
4.6±0.8
AA
1.205 mmol/L
42.3±2.5
82.9±3.9
96.9±3.9
14.3±1.8
14.7±3.4
43.5±1.0
3.7±2.2
10.2±2.3
8.0±1.2
63.3±4.8
78.5±2.4
90.6±0.6
28.2±3.2
31.5±2.6
55.3±4.7
42.3±11.8
63.6±9.7
89.7±7.4
43.2±2.9
12.1±1.4
18.7±2.1
21.3±2.7
2.1±0.7
16.2±2.2
27.4±0.9
10.0±5.9
11.3±4.6
17.0±15.0
5.3±3.2
5.7±1.8
28.7±1.0
10.8±4.9
15.6±4.1
33.4±1.1
71
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Chen C et al. / Experimental Study
Chuanxiong).35,36 The way for Ligustrazine to inhibit
platelet aggregation is to extend platelet aggregation
time induced by ADP and liberate the aggregative
platelet, while ferulic acid can inhibit platelet release of
TXA2and increase the contents of cAMP. The third
group (removing blood stasis and relieving pain) TC-
Ms including Yanhusuo (Rhizoma Corydalis Yanhusuo),
Ruxiang (Olibanum), Moyao (Myrrha), Jianghuang
(Rhizoma Curcumae Longae), Huzhanggen (Radix
Polygoni Cuspidati), Niuxi (Radix Achyranthis Bidenta-
tae), Chuanniuxi (Radix Cyathulae), Chongweizi (Fruc-
tus Leonuri Japonici) and Wulingzhi (Faeces Trogoptero-
ri) had relative weak inhibitory effect on the platelet ag-
gregation. The last group (breaking and scattering
blood thrombosis) TCMs such as Sanleng (Rhizoma
Sparganii), Ezhu (Rhizoma Curcumae Phaeocaulis), Tao-
ren (Semen Persicae), Shuizhi (Hirudo), Longxuexie
(Sanguis Draconis) and Dahuang (Radix Et Rhizoma
Rhei Palmati) also showed relatively weak inhibitory ef-
fect on the platelet aggregation.
In addition, there are some tested TCMs such as Ch-
uanniuxi (Radix Cyathulae), Niuxi (Radix Achyranthis
Bidentatae), Chishao (Radix Paeoniae Rubra), Ruxiang
(Olibanum), and Cebaiye (Cacumen Platycladi) didn't
show (or with very weak) anti-platelet aggregation ac-
tivities. As mentioned, the therapeutic mechanisms of
activating blood and resolving stasis TCMs on BSP re-
lied on various approaches besides anti-platelet aggrega-
tion. For example, Chishao (Radix Paeoniae Rubra)
cures BSPmainly through reducing erythrocyte aggrega-
tion and blood viscosity, and improving abnormal
blood rheology.37 Furthermore, platelet adherence is
one of the major reasons causing BSP and Ruxiang
(Olibanum) takes effect on this pathway instead of an-
ti-platelet aggregation.38 In addition, Chuanniuxi (Ra-
dix Cyathulae) and Niuxi (Radix Achyranthis Bidenta-
tae) display curative effects on BSP mainly through im-
proving blood hemorheology.39
In conclusion, several TCMs such as Chuanxiong (Rhi-
zoma Chuanxiong), Yanhusuo (Rhizoma Corydalis Yan-
husuo) and Danshen (Radix Salviae Miltiorrhizae), pos-
sess the property of anti-platelet aggregation.
REFERENCES
1Chen KJ. Blood stasis syndrome and its treatment with ac-
tivating blood circulation to remove blood stasis therapy.
Zhong Guo Zhong Xi Yi Jie He Za Zhi 2012; 18(12):
891-896.
2Guo QZ, Li YY. Research progress of blood stasis syn-
drome. Liaoning Zhong Yi Yao Da Xue Xue Bao 2012; 14
(8): 45-49.
3Ma J, Chen JQ. Review of clinical and experimental stud-
ies on treatment of atherosclerosis with expelling phlegm
and relieving blood stasis principle. Zhong Guo Zhong Xi
Yi Jie He Za Zhi 2006; 26(12): 1135-1138.
4Matsumoto C, Kojima T, Ogawa K, et al. A proteomic ap-
proach for the diagnosis of 'Oketsu' (blood stasis), a patho-
Table 3 Inhibition effects on the platelet aggregation of acetate ethyl extracts (A3) of 31 TCMs ( x
ˉ±s) (continuted)
TCMsa
Ruxiang
(Olibanum)
Puhuang
(Pollen Typhae)
Niuxi
(Radix Achyranthis Bidentatae)
Danggui
(Radix Angelicae Sinensis)
Final
concentration
(mg/mL)
0.6250
0.7813
1.0406
0.6250
0.7813
1.0406
0.6250
0.6250
0.7813
1.0406
Inhibition of platelet aggregation (%, n=3)
ADP
10 μmol/L
2.2±3.9
5.3±1.0
8.1±7.4
20.7±0.4
53.0±5.1
79.2±5.4
3.0±3.0
40.0±4.9
43.5±0.1
56.4±5.9
THR
0.25u/mL
3.9±3.4
5.1±6.1
3.7±0.9
50.6±3.2
38.4±6.9
70.3±7.0
19.5±5.0
28.3±1.8
28.6±5.7
56.9±0.3
AA
1.205 mmol/L
3.9±0.4
10.5±2.7
4.3±0.7
85.5±3.3
93.8±5.9
97.4±4.5
26.6±2.8
7.1±1.5
42.2±1.7
55.3±9.5
TCMs
Chuanxiong
(Rhizoma Chuanxiong)
Sanleng
(Rhizoma Sparganii)
Xuejie
(Sanguis Draconis)
Taoren
(Semen Persicae)
Final
concentration
(mg/mL)
0.6250
0.7813
1.0406
0.1078
0.3469
0.6250
0.7813
1.0406
Inhibition of platelet aggregation (%, n=3)
ADP
10 μmol/L
56.3±11.8
72.2±1.7
93.5±11.3
13.5±3.7
29.1±3.8
36.0±2.0
46.2±3.6
35.8±8.4
THR
0.25u/mL
9.4±5.3
25.2±4.7
26.9±3.3
37.8±1.6
23.9±2.1
16.3±1.8
11.1±3.2
29.5±3.6
AA
1.205 mmol/L
92.6±3.2
91.7±7.4
94.9±2.8
36.0±7.3
19.9±1.4
11.2±4.3
16.1±2.0
37.3±3.0
Notes: aTCMs were listed in alphabetical order; bonly one concentration was tested for those extracts with poor solubility or very low yielded amounts. TCMs: Traditional Chinese Medicines; ADP: adenosine
5-diphosphate; THR: thrombin; AA: arachidonic acid.
72
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Table 4 Inhibitory strength of 31 TCMs on the platelet aggregation
TCMsa
Aspirin
Cebaiye (Cacumen Platycladi)
Jixueteng (Caulis Spatholobi)
Mudanpi
(Cortex Moutan Radicis)
Wulingzhi
(Faeces Trogopterori)
Honghua (Flos Carthami)
Huaihua (Flos Sophorae)
Chongweizi
(Fructus Leonuri Japonici)
Yimucao
(Herba Leonuri Japonici)
Zelan (Herba Lycopi Hirti)
Shuizhi (Hirudo)
Moyao (Myrrha)
Ruxiang (Olibanum)
Extracts
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
Inhibitory strength
ADP
++
-
+
++
++
+
-
++
+
++
-
+
+
++
-
+++
+++
+
++
-
+
+
+
++
+
-
+
+
-
+++
-
-
+
++
-
-
-
THR
+++
-
+
+
-
-
-
-
-
++
+++
+
++
+++
+
++
+++
+
++
++
+
+
+++
+++
+
+++
-
-
+
+
+
-
+
-
-
-
-
AA
+++
+
-
++
++
+
-
+
-
+++
+
+
++
-
-
+++
-
-
++
+
+
+
++
+
+
-
-
+
-
++
+
-
-
+
+
-
-
TCMs
Zicao (Radix lithospermi)
Yujin (Radix Curcumae)
Chuanniuxi
(Radix Cyathulae)
Sanqi (Radix Notoginseng)
Dahuang (Radix Et
Rhizoma Rhei Palmati)
Chishao
(Radix Paeoniae Rubra)
Danshen (Radix Salviae
Miltiorrhizae)
Xuanshen
(Radix Scrophulariae)
Yanhusuo (Rhizoma
Corydalis Yanhusuo)
Ezhu (Rhizoma Curcumae
Phaeocaulis)
Jianghuang (Rhizoma
curcumae Longae)
Huzhanggen (Radix
Polygoni Cuspidati)
Chuanxiong
(Rhizoma Chuanxiong)
Extracts
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
Inhibitory strength
ADP
-
+
-
+
+
+
-
+
+
++
++
++
-
+
++
-
+
+
-
++
++
++
++
+++
-
+++
+++
-
++
-
++
-
-
+
-
++
++
++
+++
THR
+++
+
-
+
+
-
-
+
+
+++
++
+++
++
+
+
++
+
++
++
++
+++
+++
+
+++
++
+++
+++
++
++
-
+++
+
-
-
+
+++
+++
+++
+
AA
++
-
+
+
++
+
-
-
+
-
+
+
+
-
++
-
-
++
+++
+
++
-
+
++
-
+++
+++
+
++
+
-
+
+
-
-
+++
+++
++
+++
73
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
Chen C et al. / Experimental Study
physiologic concept of Japanese traditional (Kampo) medi-
cine. eCAM 2008; 5(4): 463-474.
5Jennings LK. Mechanisms of platelet activation: need for
new strategies to protect against platelet-mediated athero-
thrombosis. Thromb Haemost 2009; 102(2): 248-257.
6Cheng PF, Wei ZQ. Research progress of anti-platelet ag-
gregation medicines. Shi Jie Zhong Xi Yi Jie He Za Zhi
2012; 7(7): 639-642.
7Xia Q, Wang X, Xu DJ, Chen XH, Chen FH. Inhibition
of platelet aggregation by curdione from Curcuma wenyu-
jin essential oil. Thromb Res 2012; 130(3): 409-414.
8Schemmer P, Zhang Z, Galli U, et al. Glycine reduces
platelet aggregation. Amino Acids 2013; 44(3): 925-931.
9Yi T, Chen HB, Zhao ZZ, Yu ZL, Jiang ZH. Comparison
of the chemical profiles and anti-platelet aggregation ef-
fects of two 'Dragon's blood' drugs used in Traditional
Chinese Medicine. J Ethnopharmacol 2011; 133(2):
796-802.
10 Wurtz M, Hvas AM, Kristensen SD, Grove EL. Platelet
aggregation is dependent on platelet count in patients
with coronary artery disease. Thromb Res 2012; 129(1):
56-61.
11 Wurtz M, Hvas AM, Wulff LN, Kristensen SD, Grove
EL. Shear-induced platelet aggregation in aspirin-treated
patients: initial experience with the novel Placor PRT de-
vice. Thromb Res 2012; 130(5): 753-758.
12 Nagata Y, Inomata JI, Kinoshita M, et al. Impact of pro-
ton pump inhibitors or famotidine on the platelet actions
during dual-antiplatelet therapy in Japanese patients. Car-
diovasc Interv Ther 2013; 28(1): 22-29.
13 Jackson SP, Nesbitt WS, Kulkarni S. Signaling events un-
derlying thrombus formation. J Thromb Haemost 2013; 1
(7): 1602-1612.
14 Chen X, Chen WZ, Zeng GY. Cardiovascular Pharmacol-
ogy. 3rd ed. Beijing: People's Health Publishing House,
2002: 551-562.
15 Li CY. Shi Zai-xiang's academic thoughts and the theoreti-
cal and clinical studies on filling up Qi collapse and remov-
ing blood stasis for the treatment of cardiovascular diseas-
es. Beijing: China Academy of Chinese Medical Sciences,
2013: 14-18.
16 Dai SP. Establishing and mining database for activating
blood and resolving stasis Traditional Chinese Medicines.
Beijing: Beijing University of Chinese Medicine, 2007:
12-13.
17 Zhao JJ. The research of activating blood circulation to
dissipate stasis therapy in Jin Kui Yao Lue. Nanjing: Nan-
jing University of Chinese Medicine, 2009: 4-6.
18 Liu Y. Study on the knowledge discovery from the "Huox-
ue huayu" TCM database. Beijing: Beijing University of
Chinese Medicine, 2006: 3-4.
19 Iwashita M, Oka N, Ohkubo S, Saito M, Nakahata N.
Piperlongumine, a constituent of Piper longum L., inhib-
its rabbit platelet aggregation as a thromboxane A2 recep-
tor antagonist. Eur J Pharmacol 2007; 570(1-3): 38-42.
20 Kim YS, Pyo MK, Park KM, et al. Antiplatelet and anti-
thrombotic effects of a combination of ticlopidineand Gink-
go biloba Ext (EGb 761). Thromb Res 1998; 91(1): 33-38.
21 Ju HK, Lee JG, Park MK, et al. Metabolomics investiga-
tion of the anti-Platelet aggregation activity of ginsenoside
Rk1 reveals attenuated 12-HETE production. J Proteome
Res 2012; 11(10): 4939-4946.
22 Andrews RK, Berndt MC. Platelet physiology and throm-
bosis. Thromb Res 2004; 114(5-6): 447-453.
23 Gachet C, Leon C, Hechler B. The platelet P2 receptors
in arterial thrombosis. Blood Cells Mol Dis 2006; 36(2):
223-227.
24 Li JZ, Hou M, Bao CX. Platelet disorders. Beijing: Sci-
ence and Technology Literature Publishing House, 2009:
26-27.
Table 4 Inhibitory strength of 31 TCMs on the platelet aggregation
TCMsa
Ruxiang (Olibanum)
Puhuang
(Pollen Typhae)
Niuxi (Radix Achyranthis
Bidentatae)
Danggui (Radix Angelicae
Sinensis)
Extracts
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
Inhibitory strength
ADP
-
-
-
-
++
++
-
-
-
++
++
++
THR
-
-
-
+++
++
++
-
-
+
+++
+
++
AA
+
-
-
-
+
+++
-
+
+
-
+
++
TCMs
Chuanxiong
(Rhizoma Chuanxiong)
Sanleng
(Rhizoma Sparganii)
Xuejie (Sanguis Draconis)
Taoren (Semen Persicae)
Extracts
A1
A2
A3
A1
A2
A3
A1
A2
A3
A1
A2
A3
Inhibitory strength
ADP
++
++
+++
++
+
+
-
+
+
-
+
+
THR
+++
+++
+
++
+
+
-
-
+
+
+
+
AA
+++
++
+++
-
+
+
++
-
+
+
-
+
Notes: aTCMs were listed in alphabetical order; "-": promoting platelet aggregation or with irregular inhibitory effect; "+": the maximum
inhibitory rate lower than 40%; "++": the maximum inhibitory rate between 40% and 80%; "+++": the maximum inhibitory rate higher
than 80%; TCMs:Traditional Chinese Medicines; ADP: adenosine 5-diphosphate; THR: thrombin; AA: arachidonic acid; A1: aqueous ex-
tract; A2: the rest part after ethyl acetate extraction; A3: ethyl acetate extract of 90% ethanol extract.
74
Chen C et al. / Experimental Study
JTCM
|
www. journaltcm. com February 15, 2017
|
Volume 37
|
Issue 1
|
25 Jurk K, Kehrel BE. Platelets: physiology and biochemis-
try. SeminThromb Hemost 2005; 31(4): 381-392.
26 Wu Y, Guo HB, Wang TJ, et al. Comparative study on ef-
fects of active ingredients of several Traditional Chinese
Medicines on rabbit platelet aggregation in vitro. Zhong
Guo Lin Chuang Yao Li Xue Yu Zhi Liao Xue 2007; 12
(9): 1047-1051.
27 Mazziero AM, Lorenzetti R, Donato JL, Lilla S, Nucci
GD. Inhibition of human platelet aggregation by eosino-
phils. Life Sci 2013; 93(9-11): 416-422.
28 Han GM, Yao Q, Li HL, et al. Inhibitory effect of pani-
coin on platelet activation and its mechanism. Zhong Guo
Zhong Xi Yi Jie He Za Zhi 2000; 20(7): 527-529.
29 Schror K. Study on the progress of antiplatelet drugs.
Guo Wai Yi Xue Yao Xue Fen Ce 1996; 23(3): 156-159.
30 Chen KL, Chen G. Distribution and action of natural cy-
cloxygenase and lypoxygenase inhibitor in traditional Chi-
nese materia medica. Zhong Nan Min Zu Da Xue Xue
Bao 2009; 28(1): 42-47.
31 Park JW, Lee SH, Yang MK, et al. 15, 16-Dihydrotanshi-
none I, a major component from Salvia miltiorrhiza
Bunge (Dansham), inhibits rabbit platelet aggregation by
suppressing intracellular calcium mobilization. Arch
Pharm Res 2008; 31(1): 47-53.
32 Zhan JYX, Zheng KYZ, Zhu KY, et al. Chemical and bio-
logical assessment of Angelicae Sinensis Radix after pro-
cessing with wine: an orthogonal array design to reveal the
optimized conditions. J Agric Food Che 2011; 59(11):
6091-6098.
33 Hu YY, Huang Z. Advances in research of chemical con-
stituents and pharmacological effects of Scrophularia ning-
poensis Hemsl. Zhejiang Zhong Yi Yao Da Xue Xue Bao
2008; 32(2): 268-270.
34 Dong TTX, Zhao KJ, Huang WZ, Leung KW, Tsim
KWK. Orthogonal array design in optimizing the extrac-
tion efficiency of active constituents from roots of Panax
notoginseng. Phytother Res 2005; 19(8): 684-688.
35 Li JM, Zhao YH, Ma FS, et al. Synthesis of ligustrazine-ar-
omatic acid derivatives and their inhibitory effect on plate-
let aggregation. You Ji Hua Xue 2008; 28(9): 1578-1583.
36 Li QY, Gan GP, Liu YW. Research progress of the pharma-
cology and chemical components of Ligusticum wallichii.
Shi Zhen Guo Yi Guo Yao 2006; 17(7): 1298-1299.
37 Wang LL, Ding AW. Research on effects of total paeony
glycoside on rat blood stasis model. Nanjing Zhong Yi Yao
Da Xue Xue Bao 2011; 27(6): 552-554.
38 Guan, HZ, Peng ZC, Zhang SW. Comparison between ef-
fect of the un-processed Ruxiang on rabbit's platelet adher-
ence and that of the vinegar-processed one. Zhong Guo Yi
Yuan Yao Xue Za Zhi 2000; 20(9): 524-525.
39 Gao CK, Gao J, Ma RL, Xu XX, Huang P, Ni SD. Re-
search on analgesic and anti-inflammatory and invigorate
circulation effects of total saponins of Achyranthes. Anhui
Yi Yao 2003; 7(4): 248-249.
75
... Therefore, it has become a trend to search for THR and FXa inhibitors in natural sources to provide a reference for the development of anticoagulant drugs [9]. Danshen (Salviae Miltiorrhizae Radix et Rhizoma, DS) and Chuanxiong (Chuanxiong Rhizoma, CX) are both effective for invigorating blood circulation and eliminating blood stasis [10]. DS and CX have attracted widespread attention as primary materials for the treatment of cardiovascular diseases (CVDs) [11], tumor disorders [12] and inflammatory diseases [13]. ...
Article
Full-text available
In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid; Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC50 of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC50 of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol−1; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.
... The formation of QSBS is accompanied by the following pathological changes: increased platelet aggregation or decreased release function, microcirculation disturbance, and abnormal hemorheology (Huang et al., 2020). Several TCMs with the property of activating blood and resolving stasis in terms of TCM theory, such as Chuanxiong (Rhizoma Ligusticum chuanxiong), Yanhusuo (Rhizoma Corydalis Yanhusuo) and Danshen (Radix Salviae Miltiorrhiza), possess the property of anti-platelet aggregation (Chen et al., 2017). It has been indicated that QSBS primary dysmenorrhea is related to platelet aggregation. ...
Article
Background Curcumae Rhizoma (CR) has a clinical efficacy in activating blood circulation to dissipate blood stasis and has been used for the clinical treatment of qi stagnation and blood stasis (QSBS) primary dysmenorrhea for many years. However, its molecular mechanism is unknown. Objective The present study aimed to demonstrate the multicomponent, multitarget and multipathway regulatory molecular mechanisms of CR in the treatment of QSBS primary dysmenorrhea. Methods Observations of pathological changes in uterine tissues and biochemical assays were used to confirm that a rat model was successfully established and that CR was effective in the treatment of QSBS primary dysmenorrhea. The main active components of CR in rat plasma were identified and screened by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS). The component-target-disease network and protein-protein interaction (PPI) network of CR were constructed by a network pharmacology approach. Then, we performed Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was adopted to verify the interactions between the core components and targets of CR to confirm the accuracy of the network pharmacology prediction results. Furthermore, we evaluated the bioactive constituents of CR and molecular mechanism of by which CR promote blood circulation and remove blood stasis via platelet tests in vivo and in vitro and Western blot analysis. Results The results of HE staining and biochemical assays of PGF2α, TXB2 and Ca²⁺ showed that CR was effective in the treatment of QSBS primary dysmenorrhea. A total of 36 active components were identified in CR, and 329 common targets were obtained and used to construct the networks. Of these, 14 core components and 10 core targets of CR in the treatment of primary dysmenorrhea were identified. The GO and KEGG enrichment analyses revealed that the common targets were involved in multiple signaling pathways, including the calcium, cAMP, MAPK, and PI3K-Akt signaling pathways, as well as platelet activation, which is closely related to platelet aggregation. The molecular docking results showed that the 14 core components and 10 core targets could bind spontaneously. Two core targets (MAPK1 and CCR5) and 7 core components (Isoprocurcumenol, Curcumadione, Epiprocurcumenol, (+)-Curdione, Neocurdione, Procurcumenol, and 13-Hydroxygermacrone) were closely related to CR in the treatment of primary dysmenorrhea. Furthermore, the in vivo platelet test showed that CR clearly inhibited platelet aggregation. Five core components ((+)-Curdione, Neocurdione, Isoprocurcumenol, Curcumadione and Procurcumenol) obviously inhibited platelet aggregation in vitro. In addition, based on the relationships among the signaling pathways, we confirmed that CR can effectively inhibit the expression of MAPK and PI3K-Akt signaling pathway-related proteins and decrease the protein expression levels of ERK, JNK, MAPK, PI3K, AKT and CCR5, thereby inhibiting platelet aggregation. Conclusion This study demonstrated the bioactive constituents and mechanisms of CR in promoting blood circulation and removing blood stasis and its multicomponent, multitarget and multipathway treatment characteristics in primary dysmenorrhea. The results provide theoretical evidence for the development and utilization of CR.
... A large body of studies has shown that Chuanxiong possesses multifarious pharmacological effects, including protective effects on neuron [16], heart [17], liver [18] and kidney [19], as well as antioxidation [20,21], anti-inflammation [22][23][24], etc. In our previous studies, Chuanxiong extracts had inhibitory effects on platelet aggregation [25] and THR activity [12], while there are few studies on its effects on THR and FXa so far. It is of reasonable to screen THR/FXa inhibitors from Chuanxiong. ...
Article
Full-text available
Background: The dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong. Methods: Four extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC-MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis. Results: Chuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibiting FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a-h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than - 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was - 9.39 kcal/mol. The IC50 was determined as 0.56 mM. Conclusions: THR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.
... This research takes DS−CX as an example to investigate the herb−drug interactions, in terms of supposed antithrombotic effect and the real wide popularity. Existing literature shows that both DS and CX are effective on invigorating blood circulation and eliminating stasis [16][17][18]. Meanwhile, a pairing (a basic unit of complex herbal formulae) of the two herbs was one of the most frequently used herbal pairs in the best-selling herbal formulae released by the China Association of Chinese Medicine in 2017 [19]. ...
... Therefore, the above important factors were verified and can be used as the laboratory diagnostic indexes for the invigorating blood and dissolving stasis effect of hawthorn leaves. In addition, anti-platelet aggregation tests were widely applied in the evaluation and research of cardiovascular disease [37]. The strong correlation indexes PC, MPV, and PCT found in this work coincided with it. ...
Article
Full-text available
Hawthorn leaves, officially listed in the 2015 Chinese Pharmacopoeia, comes from Crataegus pinnatifida Bge. var. major N. E. Br. and Crataegus pinnatifida Bge. It has the effect of invigorating blood and dissolving stasis, regulating qi and dredging veins, turbid, and lipid-lowering. This work was designed to explore the spectrum–effect relationship between high-performance liquid chromatography (HPLC) fingerprint and the invigorating blood and dissolving stasis effect of hawthorn leaves. Four extracts from hawthorn leaves, 50% ethanol extract (EE), macroporous resin extract (MRE), ethyl acetate extract (EAE), and n-butanol extract (BAE) were administered to rats at three doses of 0.4 g·kg⁻¹, 0.6 g·kg⁻¹, and 0.8 g·kg⁻¹ (crude medicine weight/rat weight), respectively. Twenty-seven indexes were obtained and analyzed by principal component analysis (PCA) and one-way ANOVA. The spectrum–effect relationship was constructed by multiple linear regression (MLR) analysis. With the above separation method, HPLC fingerprints of 12 batches (three concentrations of four extracts) of hawthorn leaves extracts were obtained, and 32 common peaks were marked by similarity analysis. Additionally, 9 indexes of fibrinogen (FIB), erythrocyte total (ET), platelet count (PC), mean platelet volume (MPV), plateletcrit (PCT), whole blood viscosity-WBV(LSR-20S⁻¹), WBV(LSR-60S⁻¹), WBV(LSR-150S⁻¹), and plasma viscosity (PV) were selected as the relevant indexes with pertinence and statistical significance (p < 0.05) on invigorating blood and dissolving stasis, and taken for the subsequent spectrum–effect experiments. Finally, the results of spectrum–effect relationships between 32 common peaks and 9 indexes showed the main peaks responsible for invigorating blood and dissolving stasis were 1–6, 8–11, 13–15, 17, 19, 21, 23, and 27–32. Compared with the standards and other references, peaks 3, 14, 17, and 19 were, respectively, identified as chlorogenic acid, vitexin glycoside, vitexin, and rutin. This work was helpful to the discovery of the active substance and quality control of hawthorn leaves. Graphic Abstract
... Charcoal treatment is a major category of Chinese medicine, with 2000 years of historical application, especially in the treatment of haemorrhagic diseases that show a convergent haemostatic effect [1][2][3]. The haemostatic effect of charcoal has been included in the Chinese Pharmacopoeia [4]. Modern pharmacological studies [5] have proven that Schizonepetae herba carbonisata can increase the FIB and platelet content in rats to achieve haemostatic purposes. ...
Article
Full-text available
High-temperature carbonisation is used to prepare many traditional Chinese medicine charcoal drugs, but the bioactive haemostatic substances of these medicines and their mechanisms are still unknown. This study developed and evaluated nanoparticles (NPs) derived from Selaginella pulvinate Carbonisata (STC) for the first time. The haemostatic effect of STC-NPs prepared at 300, 350, and 400 °C were investigated in mouse tail amputation and liver scratch experiments. STC-NPs obtained at 400 °C had the strongest haemostatic effect, and were accordingly characterised by ultraviolet-visible spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, high resolution transmission electron microscopy, X-ray diffractometry, and X-ray photoelectron spectroscopy. STC-NPs averaged 1.4-2.8 nm and exhibited a quantum yield of 6.06% at a maximum excitation wavelength of 332 nm and emission at 432 nm. STC-NPs displayed low toxicity against mouse monocyte macrophage RAW 264.7 cells by CCK-8 assay, and STC-NP treatment significantly shortened bleeding time in rat and mouse models. Coagulation assays showed that the haemostatic effects of STC-NPs were related to improving the fibrinogen and platelet contents, as well as decreasing the prothrombin time that resulted from stimulating extrinsic blood coagulation and activating the fibrinogen system. The STC-NPs had remarkable haemostatic effects in the tail amputation and liver scratch models; these effects may be associated with the exogenous coagulation pathway and activation of the brinogen system, according to the evaluation of the mouse coagulation parameters. This novel evaluation supports the material basis of STC use in traditional Chinese medicine, and this article is worthy of study by authors of clinical pharmacy.
Article
Background : Coronary thrombosis and its correlated disorders are main healthcare problems globally. The therapeutic effects of current treatments involving antiplatelet drugs are not fully satisfactory. Danshensu (DSS) is an important monomer obtained from Salvia miltiorrhiza roots that have been widely employed for vascular diseases in medicinal practices. Nonetheless, the underlying mechanisms of DSS are not fully unraveled. Purpose : The objective of this study was to penetrate the antithrombotic and antiplatelet mechanisms of DSS. Methods : Network pharmacology assay was used to forecast the cellular mechanisms of DSS for treating thrombosis. The work focused the impacts of DSS on platelet activation by analyzing aggregation and adhesion in vitro. Flow cytometry, western blotting, CM-H2DCFDA staining and mitochondrial function assays were performed to reveal the molecular mechanisms. The model of common carotid artery thrombus induced by ferric chloride was established. The wet weight of thrombus was measured, and the thrombosis was observed by hematoxylin and eosin (H&E) staining, in order to support the inhibitory effect of DSS on thrombosis. Results : Data mining found the antithrombotic effect of DSS is related to platelet activation and the core target is silent information regulator 1 (SIRT1). We confirmed that DSS dose-dependently inhibited platelet activation in vitro. DSS was further demonstrated to induce the expression of SIRT1 and decreased reactive oxygen species (ROS) burden and thereby prevented mitochondrial dysfunction. Mitochondrial function tests further indicated that DSS prevented mitochondrial DNA (mtDNA) release, which induced activation of platelet in a dendritic cell specific intercellular-adhesion-molecule-3 grabbing non-integrin (DC-SIGN)-dependent manner. In carotid artery injury model induced by ferric chloride, DSS inhibited the development of carotid arterial thrombosis. More encouragingly, in tail bleeding time assay, DSS did not augment bleeding risk. Conclusion : These findings indicated that DSS effectively inhibited platelet activation by depressing the collection of ROS and the release of platelet mtDNA without arousing hemorrhage risk. DSS might represent a promising candidate drug for thrombosis and cardiovascular disease therapeutics.
Article
This study evaluated for the first time the inhibitory effect on human platelet aggregation, blood coagulation, and the antioxidant activity of Canna x generalis fractions and its phytoconstituents. The antiplatelet effect was evaluated using the turbidimetric method to determine the percentage inhibition (%I), the area under aggregation curve (AUC) and the aggregation velocity (slope). Prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were measured to assess the inhibitory effect on blood coagulation. ABTS and DPPH assays were performed for evaluation of the antioxidant effect. Compounds structures were determined by NMR and MS spectroscopic methods. The ethyl acetate fraction exhibited the most potent antiplatelet (%I, 98.2 ± 1.7% at 4 mg/mL), anticoagulation (mean APTT, 42.97 s) and antioxidant activity (IC50, 58 ± 0.1 and 12.5 ± 2.0 μg/mL for DPPH and ABTS, respectively). Thirteen compounds were found for the first time from aerial CG: 16α-hydro‑ent-kauran-3β-ol-7,19-dioic acid, named cannagelic acid (a new ent‑kaurane diterpenoid) (1), 16α-hydro‑ent-kauran-17,19-dioic acid (2), ent‑kauran-15-en-17,19-dioic acid (3), 3-methoxybenzaldehyde (4), 2-methoxynaphtalene (5), 4-ketopinoresinol (6), p-coumaric acid (7), indole-3-carboxylic acid (8), procatechuic aldehyde (9), astragalin (10), isoquercitrin (11), corchoionol C (12) and rutin (13). Compounds 7, 9–11, 13 possess good antiplatelet, anticoagulant and antioxidant activity. This study demonstrated that compound 1 did not show any tested effects. Compounds 4 and 5 had a very weak antiplatelet effect. Compound 8 expressed the highest capacity to inhibit platelet aggregation for both ADP and collagen (%I = 70.2 ± 1.8 and 79.2 ± 1.4 at 0.4 mg/mL, respectively), followed by compound 6. Moreover, these two compounds at 0.4 mg/mL could prolong APTT compared to the negative control (p < 0.05). Compound 12 had weak antiplatelet activity, but its antioxidant effect was better than other tested compounds. In conclusion, CG would be a good natural resource of bioactive molecules or dietary supplements to prevent and/or treat cardiovascular and free radicals-associated diseases.
Article
Full-text available
Cardio-cerebrovascular diseases (CVDs) are a serious threat to human health and account for 31% of global mortality. Ligusticum chuanxiong Hort. (CX) is derived from umbellifer plants. Its rhizome, leaves, and fibrous roots are similar in composition but have different contents. It has been used in Japanese, Korean, and other traditional medicine for over 2000 years. Currently, it is mostly cultivated and has high safety and low side effects. Due to the lack of a systematic summary of the efficacy of CX in the treatment of CVDs, this article describes the material basis, molecular mechanism, and clinical efficacy of CX, as well as its combined application in the treatment of CVDs, and has been summarized from the perspective of safety. In particular, the pharmacological effect of CX in the treatment of CVDs is highlighted from the point of view of its mechanism, and the complex mechanism network has been determined to improve the understanding of CX’s multi-link and multi-target therapeutic effects, including anti-inflammatory, antioxidant, and endothelial cells. This article offers a new and modern perspective on the impact of CX on CVDs.
Article
Full-text available
Atherosclerotic thrombotic disease continues to maintain a high morbidity and mortality rate worldwide at present. Aspirin, which is reckoned as the cornerstone of primary and secondary prevention of atherosclerotic cardiovascular diseases (ASCVDs), has been applied in clinics extensively. However, cardiovascular events continue to occur even though people utilize aspirin appropriately. Therefore, the concept of aspirin resistance (AR) was put forward by scholars, which is of great significance for the prediction of the clinical outcome of diseases. The pathogenesis of AR may be incorporated with low patient compliance, insufficient dose, genetic polymorphism, increased platelet transformation, inflammation, and the degenerative changes and calcification of platelets. The improvement of AR in the treatment of ASCVDs has gradually become a research hot spot in recent years. Traditional Chinese medicine (TCM) regards individuals as a whole and treats them from a holistic view, which has been found to have advantages in clinical studies on the treatment of AR. Many kinds of blood-activating TCM have the effect of improving AR. The potential mechanism for the improvement of AR by blood-activating herbs combined with aspirin was explored. The combination of blood-activating herbs and aspirin to improve AR is likely to turn into a hot topic of research in the future.
Article
Full-text available
Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross talk to other blood and vascular cells. Stimulated “sticky” platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.
Article
Ligustrazine and ferulic acid, which are useful compounds of Chinese traditional medicine, were used as leading compounds. Six ligustrazine-aromatic acid derivatives were designed and synthesized based on the twin drug principle. Their structures were characterized by IR, 1H NMR, 13C NMR and mass spectra. In the in vivo experiment, some compounds have significant inhibitory effect on adenosine diphosphate (ADP) induced platelet aggregation. Among them, the inhibitory effect of compound 1a was 5.7 times than ozagrel.
Article
Single spiral tube heat exchanger has been developed for improving heat exchange efficiency of shell-and-tube heat exchanger and reducing complexity of structure of heat exchanger. Flow field and temperature field of the heat exchanger have been calculated by using finite element software, and it has been investigated of the influence on heat exchange efficiency of inlet flow of tube side, temperature and the cycle number of spiral pipe. The results showed that the heat exchange efficiency of spiral-tube heat exchanger has been much better than that of straighttube heat exchanger, but the cycle number of spiral pipe has no significant impact for heat exchange efficiency, and has remarkably increased the pressure drop along the way of the tube side. The heat exchange between shell and tube has mostly happened near the entrance, thus it could make axial dimension of the heat exchanger reduced further. The greater the temperature difference of the entrance between shell side and tube side has been, the better the heat exchange efficiency of spiral-tube heat exchanger has been. However, for avoiding excessive thermal stress concentration near entrance of spiral tube, it has been necessary of adjusting entrance orientation of shell side properly for fear that cold air at entrance of shell side has directly scoured the entrance of tube side.
Article
The relationship between the activity of eosinophils and platelets has been observed in recent decades by many investigators. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with L-Nitro-Arginine-Methyl-Ester (L-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition.