Full text of DOI:10.1078/09447110260571634

Phytomedicine 9: 405–409, 2002
Urban & Fischer Verlag
http://www.urbanfischer.de/journals/phytomed
©
Phytomedicine
Effect of salvianolic acids from Radix Salviae miltiorrhizae
on regional cerebral blood flow and platelet aggregation
in rats
Min-Ke Tang, De-Cheng Ren, Jun-Tian Zhang, and Guan-Hua Du
Pharmacology Department 2, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing, P.R. China
Summary
This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood
flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was pro-
duced in rats by occluding of the right middle cerebral artery, together with the right common
carotid artery. rCBF was monitored by H clearance method with a tissue blood-flow meter.
2
Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by
platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. ( i.v.) improved rCBF in rats after
ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the
platelet aggregation induced by collagen, ADP, and AA with IC values of 0.197, 2.22 and 3.29 ×
5
0
3
1
0 mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the
platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably
platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the is-
chemic hemisphere and inhibit platelet aggregation in rats.
Key words: Salvianolic acids (SA), Radix Salviae miltiorrhizae, cerebral ischemia, regional cerebral
blood flow (rCBF), and platelet aggregation.
ꢀ
Introduction
Cerebral ischemic damage is initiated by a remark-
able decrease in regional cerebral blood flow in ischemic cerebral damage. They are activated
rCBF). The decrease of rCBF in the affected hemi- following cerebral ischemia, and the activated
Platelets are considered to play an important role
(
sphere in stroke patients may result in an energy fail- platelet may result in thrombosis formation in the
ure and over-release of excitatory neurotransmitters, ischemic hemisphere. The thrombosis then further
and lead to an activation of the toxic intracellular aggravates the decrease of rCBF in the ischemic
pathway (Jorgensen et al., 1982). Cells in the affect-
hemisphere, and finally exacerbates the develop-
ed hemisphere may die via multiple processes, and ment of ischemic brain damage (Turcani et al.,
neurological deficits in the patients (such as motor 1988; Obrenovitch et al., 1985).
and cognitive dysfunction) may follow, according to
clinical research (Mori et al., 1994; Rodriguez et al., nents extracted from a Chinese traditional drug,
993). To investigate the rCBF change after ischemia Radix Salviae miltiorrhizae. SA composed mainly of
Salvinolic acids (SA) are water-soluble compo-
1
is a reliable way to understand the protective effect
of drugs against cerebral ischemia.
two compounds, salvinolic acid A (SalA, 7%) and
salvinolic acid B (SalB, 50%) (Li, 1997). The other
0
944-7113/02/09/05-405 $ 15.00/0

4
06
M.-K. Tang et al.
components of SA are also various polyphenolic Cerebral Ischemia Preparation and rCBF Monitoring
acids. Most of them are derivatives of 3,4-hydroxy- Rats were anesthetized using Ethyl Carbamate (25%,
3
benzyl-lactic acid. In a previous study, it was found 1 × 10 mg/kg body wt.). The right common carotid
that SA could decrease the infarct area and inhibit artery (CCA) was dissociated. A thread was set under
the CCA in order to occlude it later. The right middle
cerebral artery (MCA) was occluded by a modifica-
tion of the method described by Bederson et al.
cerebral edema of the ischemic rat (Wang et al.,
999a, b). In order to find the mechanisms by which
SA protected rats from cerebral ischemic damage,
we observed the effect of SA on rCBF and platelet
aggregation in this study.
1
(
1986). Briefly, the animal was placed in the right
lateral position, and a skin incision (1.5 cm length)
was made between the ear and the eye. The right tem-
poral muscle was exposed and partially excised. The
right MCA was exposed through a 2-mm hole drilled
ꢀ
Materials and Methods
2
-mm rostral and 5-mm ventral to the fusion point of
the zygomatic arch with the squamosal skull bone.
During drilling, care was taken to preserve a thin
layer to avoid physical injury to the cortex. The bone
layer was then carefully removed with forceps, with-
out disrupting the dura. The dura overlaying the
MCA was pierced. The head of the animal was then
placed in a stereotaxic unit, and an additional hole (Φ
Drugs and Reagents
Salvianolic acids (SA) were supplied by the Depart-
ment of Phytochemistry of our institute (Fig. 1) and
were prepared using a standardized protocol (Li,
1
984). Briefly, slices of the dried roots of Salvia mil-
tiorrhiza were percolated with 95% ethanol at room
temperature. After evaporation of the solvent, the
residue was extracted thoroughly with hot water. The
aqueous extract was left overnight, filtered, and con-
centrated to yield SA. SA was dissolved in normal
saline (NS). Adenosine diphosphate (ADP) and
arachidonis acid (AA) were purchased from Sigma
and Fluke, respectively. Collagen was prepared in
our laboratory.
2
mm, 2-mm lateral and 3-mm posterior to the breg-
ma) for rCBF monitoring was drilled. The rCBF sen-
sor probe was placed into cortex through the up hole
with a length of 0.8 mm, and the reference probe was
placed under the skin behind the skull. Cerebral is-
chemia was produced by ligating the thread under the
right CCA and occluding the right MCA with elec-
trocautery. Sham operation without artery occlusion
was processed in the group without ischemia. SA or
NS was given by i.v. 30 min after cerebral ischemia
or sham operation. rCBF was monitored using an LS-
3
tissue blood-flow meter (LiKe Tech., Co, Beijing)
as follows: pre-artery occlusion, 15 min, and 30 min
after SA or NS administration. The experiment was
performed in parallel, but not blinded.
Platelet Aggregation in vitro
Rats were anesthetized using trichloroaldehyde (10%,
i.p., 350 mg/kg body wt.). Platelet-rich plasma (PRP)
and platelet-poor plasma (PPP) were prepared as fol-
lows: rat blood from CCA was anti-coagulated with
3
1
.8% sodium citrated (1:1, v/v), and centrifuged at
80 × g for 5 minutes to get PRP. The precipitate was
further centrifuged at 1600 × g for 10 minute to yield
11
PPP. PRP platelets were adjustd to 3 × 10 /l using PPP
before the test. PRP was incubated with NS or various
concentrations of SA at 37 °C for 5 minutes. Then the
aggregation-inducing agent, collagen, ADP, or AA,
was added. Aggregation was measured using a platelet
aggregometer (NKK, HEMA TRACER1, Japan).
Animals
Platelet Aggregation in vivo
Wistar rats, weighing 250 ± 20 g each, were provided Rats were scarified 30 minutes after i.v. SA administra-
by the Experimental Animal Center, Chinese Acade- tion at 3, 6, or 10 mg/kg body wt. PRP and PPP were
my of Medical Sciences. The certificate number is prepared with blood from CCA. Aggregation was mea-
0
1-3008.
sured as above.

Effect of salvianolic acids
407
Statistical Analysis
14.7%, 63.7 ± 10.0%, 15 min after drug; and 66.8 ±
To diminish the individual deviation of rCBF from rat 16.9%, 61.5 ± 14.4%, 30 min after drug, respectively).
to rat, the relative value of rCBF (RVrCBF) in each Identical doses of SA had no effect on the rCBF of
rat was calculated in this study using the following sham-operated rats (Tables 1 and 2).
formula:
RVrCBF = (rCBF value after-occusion/rCBF value Effect of SA on Platelet Aggregation in vitro
pre-occlusion) × 100%.
The RVrCBF in the same group were averaged. The
mean values were compared between groups.
All data in this study were expressed as mean ± SD;
In vitro, SA inhibited platelet aggregation induced by col-
lagen, ADP, and AA, in a concentration-dependent man-
3
ner. Their IC values were 0.197, 2.22 and 3.29 × 10
50
mg/l, respectively. SA was more potent in inhibiting
platelet aggregation induced by collagen than byADP and
AA (Table 3).
t-test was used for comparison between groups.
ꢀ
Results
Effect of SA on Platelet Aggregation in vivo
Effect of SA on rCBF of Ischemic Rats
5 minutes after MCA occlusion, together with CCA
occlusion, rCBF of the rats decreased to a very low
level, about 48 percent of the pre-occlusion level. The
rCBF persisted at about 44 percent of the pre-occlusion
In vivo, SA at 6 and 10 mg/kg body wt. significantly in-
hibited platelet aggregation induced by collagen, and
SA at 10 mg/kg body wt. inhibited platelet aggregation
induced by ADP remarkably (47.4 ± 5.9% vs. 63.2 ±
4
5
.1%, p < 0.01). SA at 3 mg/kg body wt. had no obvi-
level during the monitoring period. SA at 6 and 10 mg ous effect on platelet aggregation induced by either
body wt. (i.v., 30 min after arteries’ occlusion) im- collagen or ADP (56.1 ± 3.1% vs. 57.8 ± 5.0%, 60.6 ±
proved the rCBF of ischemic rats significantly (67.1 ± 5.7% vs. 63.2 ± 5.1%, respectively) (Table 4).
Table 1. Effect of SA administration on regional cerebral blood flow (rCBF) in rats. Acute focal cerebral ischemia was pro-
duced by right CCA occlusion and right MCA electrocautery occlusion. SA or NS was given (i.v.) 30 min after cerebral is-
chemia or sham operation. rCBF was monitored using a LS-3 tissue blood-flow meter at the following time points: pre-artery
occlusion, 15-min, and 30-min post-SA or NS administration. Values were averaged in the same group, and presented as X ±
SD.
group
animal
number
n
doses
(mg/kg body wt.)
rCBF (ml/100 g/min)
15 min after drug
pre-occlusion
30 min after drug
sham
ischemia
SA + ischemia
SA + ischemia
SA + sham
SA + sham
8
8
8
8
8
7
NS
NS
6
10
6
142.1 ± 83.7
136.6 ± 68.7
176.4 ± 88.7
141.3 ± 46.7
151.7 ± 60.9
127.9 ± 60.7
140.4 ± 77.2
62.8 ± 30.0
120.7 ± 78.6
88.2 ± 25.6
132.8 ± 49.7
121.9 ± 51.5
134.7 ± 84.4
58.9 ± 28.9
117.4 ± 70.5
83.3 ± 18.5
128.8 ± 28.0
112.2 ± 62.1
10
Table 2. Effect of SA administration on the relative value of regional cerebral blood flow (RVrCBF) in rats. The RVrCBF
were calculated using the following formula; RVrCBF in the same group were averaged; Mean values were compared between
groups. *P < 0.05, **P < 0.01 vs ischemia.
Relative rCBF value = (rCBF value after-occlusion)/(rCBF value pre-occlusion) × 100%.
group
animal number
n)
doses
(mg/kg body wt.)
RVrCBF (%)
(
15 min after drug
30 min after drug
sham
ischemia
SA + ischemia
SA + ischemia
SA + sham
SA + sham
8
8
8
8
8
7
NS
NS
6
10
6
100.8 ± 10.1**
47.9 ± 12.1
67.1 ± 14.7*
63.7 ± 10.0*
91.4 ± 25.4**
99.2 ± 29.6**
94.0 ± 12.3**
44.2 ± 10.6
66.8 ± 16.9*
61.5 ± 14.4*
91.9 ± 23.1**
88.2 ± 22.9**
10

4
08
M.-K. Tang et al.
Table 3. Effect of SA administration on rat platelet aggrega-
tion induced by collagen, ADP, or AA in vitro. PRP was incu-
bated with NS or various concentrations of SA at 37 °C for
ꢀ
Discussion
The principal pathophysiological processes in cerebrl
ischemia are energy failure, loss of cell-ion home-
ostasis, acidosis, increase of intracellular calcium, ex-
citotoxicity, and free-radical overgeneration. The rel-
ative contribution of each process to ischemic cell
5
min, then collagen, ADP or AA was added. Aggregation
was measured using a platelet aggregometer (NKK, HEMA
TRACER1, Japan), n = 4. Values were presented as X ± SD.
*P < 0.05, **P < 0.01, vs vehicle.
group doses
Platelet aggregation damage is believed to be associated with the level of
3
(10 mg/l)
(%)
cerebral blood flow. In the core of infarct, blood flow
is very low, and cells will die through of necrosis. In
A. Effect of SA on rat platelet aggregation induced by the ischemia penumbra, however, where the blood
collagen in vitro
flow is not very low, cells can be salvaged by reperfu-
vehicle
SA
SA
SA
SA
NS
61.0 ± 2.2
56.7 ± 5.5
51.3 ± 6.0*
47.3 ± 6.7**
36.7 ± 7.0**
28.3 ± 9.3**
0 ± 0**
sion or by pharmacological therapies. Our previous
study indicated that Salvianolic acids have a very
strong antioxidant effect (Li, 1997), and could protect
rats from cerebral ischemic damage caused by MCAO
0.092
0.123
0.164
0.205
0.256
0.320
(
Wang et al., 1999a, b). It was demonstrated in this
SA
SA
study that SA improved rCBF in the ischemic hemi-
sphere of MCAO rats after artery occlusion. The
B. Effect of SA on rat platelet aggregation induced by rCBF maintained at a relatively higher level during
ADP in vitro
the monitoring period as compared to untreated
MCAO rats. This might promise an opportunity for
the neurons in the ischemic area to be rescued by ther-
apeutic methods. The pathophysiological processes
caused by ischemia may then be prevented. This
might also support the common use of Saliva miltior-
rhiza as an effective drug against cerebrovascular dis-
eases in Traditional Chinese Medicine.
It is reported that the rCBF decreased immediately
after bilateral carotid artery occlusion and that it de-
creased to 48 percent of pre-occlusion levels 20 min
after arteries’ occlusion. The rCBF decreased further
after a duration of ischemia and did not restore even if
reperfusion was conducted (Fukuchi et al., 1998).
This finding was also supported by our present re-
sults: the rCBF decreased to 47% of pre-occlusion
levels, and did not apparently restore during the mon-
itoring period. Our research also demonstrated that
cerebral ischemia could cause deposition of platelets
in the brain (Littleton-Kearney et al., 1998), and en-
hance platelet-aggregation response (Turcani et al.,
vehicle
SA
SA
SA
SA
NS
66.0 ± 1.6
62.0 ± 2.8
0.54
0.78
1.11
1.59
2.27
3.24
56.3 ± 4.7**
55.5 ± 2.9**
50.3 ± 1.9**
38.3 ± 9.0**
13.5 ± 5.0**
SA
SA
C. Effect of SA on rat platelet aggregation induced by
AA in vitro
vehicle
SA
SA
SA
SA
NS
80.0 ± 4.0
77.0 ± 5.1
0.84
1.20
1.72
2.45
3.50
5.00
70.0 ± 6.2*
67.0 ± 4.1**
53.3 ± 3.8**
37.8 ± 4.9**
21.3 ± 3.9**
SA
SA
1
986; Li et al., 1998). In clinical research it was found
Table 4. Effect of SA on rat platelet aggregation induced by that platelet activation is also present in patients after
collagen and ADP in vivo. Rats were scarified 30 min after stroke, and participates in the further decrease of
SA administration at 3, 6, or 10 mg/kg body wt. (i.v.). PRP rCBF after ischemia (Van Kooten et al., 1999). All
was prepared with blood from CCA, n = 8. Values were pre-
these researches suggest that platelet activation re-
sented as X ± SD. **P < 0.01, vs. vehicle.
sults in the further decrease of rCBF and prevents its
restoration after ischemia. Our previous studies also
group
doses
mg/kg
body wt.)
platelet aggregation (%)
(
found that SA could inhibit thrombosis formation
(Wang et al., 1999c). Together with the present results
of SA inhibiting platelet aggregation and improving
rCBF of MCA rats, we suggest that SA improve the
rCBF of ischemic rats partially by inhibiting the
platelet aggregation and thrombosis formation after
ischemia. Also, it should be studied in the future
collagen
ADP
Vehicle
SA
SA
SA
NS
3
6
57.8 ± 5.0
56.1 ± 3.1
63.2 ± 5.1
60.6 ± 5.7
13.0 ± 3.5**
5.40 ± 2.2**
59.3 ± 4.8
47.4 ± 5.9**
10

Effect of salvianolic acids
409
whether SA can dilate the microvessel that might
contribute to the restoration of rCBF after ischemia.
Why does SA inhibit the platelet aggregation? Our
previous studies indicated that SA could inhibit the in-
crease of TXB2 in MCAO rats, but has no apparent ef-
fect on 6-keto-PGF1α (Wang et al., 1999c). In other
words, TXA2 was inhibited and PGI2 was not affected.
This is quite different from Aspirin, which can inhibit
both TXA2 and PGI2. The results suggest that the in-
hibitory effect of SA on platelet aggregation may be re-
lated to its inhibitory effect on TXA2. More interesting
biology in neonatal and adult sheep. Am J Physiol 274 (4
pt 2): H1293–H1300
Mori S, Sadoshima S, Ibayashi S, Lino K, Fujishima M
(
1994) Relation of cerebral blood flow to motor and cog-
nitive functions in chronic stroke patients. Stroke 25:
09–317
3
Obrenovitch TP, Hallenbeck JM (1985) Platelet accumula-
tion in regions of low blood flow during the postischemic
period. Stroke 16: 224–234
Rodriguez G, Nobili F, De Carli F, Francione S, Marenco S, Ce-
lestino MA, Hassan K, Rosadini G (1993) Regional cerebral
blood flow in chronic stroke patients. Stroke 24: 94–99
is that although SA has anti-thrombosis effects, it has Turcani P, Gotoh F, Ishihara N, Tanaka K, Gomi S,
Takashima S, Mihara B (1986) Dual effect of naloxone on
no influence on the coagulates system of normal ani-
blood platelet aggregation and cerebral blood flow in ger-
mals. This property of the drug may avoid hemorrhagic
bils. Thromb Res 44: 817–828
risk in future clinical trials.
Turcani P, Gotoh F, Ishihara N, Tanaka K, Gomi S,
Takashima S, Mihara B (1988) Are blood platelets in-
volved in the pathogenesis of ischemic brain edema in ger-
ꢀ
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ꢀ
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