Journal of Ethnopharmacology 155 (2014) 1053–1060

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Huangqi
Honghua combination and its main components ameliorate

cerebral infarction with Qi deficiency and blood stasis syndrome
by antioxidant action in rats
Jinyi Cao a,1, Zhengyu Chen b,1, Yanrong Zhu a,1, Yuwen Li a, Chao Guo a, Kai Gao a, Lei Chen a,
Xiaopeng Shi a, Xiaofang Zhang a, Zhifu Yang a,n, Aidong Wen a,nn
a
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Shaanxi, Xi'an 710032, PR China
b
Health Department of General Logistics Department, CPLA, Beijing 010842, PR China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Combination of Radix Astragali (Huangqi) and Carthamus tinctorius L.
Received 16 December 2013 (Honghua) has been extensively used as traditional herb medicine in China for the treatment of stroke
Received in revised form and myocardial ischemia diseases with Qi deficiency and blood stasis (QDBS) syndrome.
22 May 2014
Aim: To investigate the effect of Huangqi
Honghua combination (HH) and its main components
Accepted 31 May 2014
Available online 21 June 2014
astragaloside IV (AS-IV) and Hydroxysafflor yellow A (HSYA) on cerebral ischemia-reperfusion (IR) with
QDBS in rat model.
Chemical compounds studied in this article: Materials and methods: Male rats were randomly divided into the following six groups: sham group,
Astragaloside IV (PubChem CID: 13943297) QDBS þI/R model group and treatment group including AS-IV, HSYA, AS-IV þHSYA and HH. The whole
Hydroxysafflor yellow A (PubChem CID:
blood viscosity (WBV), plasma viscosity (PV), neurological examination, infarct volume, histopathology
6443665)
changes and some oxidative stress markers were assessed after 24 h of reperfusion.
Keywords: Results: HH and its main components AS-IV þ HSYA could significantly decrease WBV, PV, and also
Radix Astragali significantly ameliorate neurological examination and infarct volume after 24 h of reperfusion. They also
Carthamus tinctorius L. significantly increased expression of Nuclear factor erythroid 2-related factor 2 (Nrf2), activities of
Astragaloside IV antioxidants, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px), led to
Hydroxysafflor yellow A decrease levels of malondialdehyde (MDA) and reactive oxygen species (ROS).
Cerebral infarction
Conclusion: AS-IV and HSYA are responsible for the main curative effects of HH. The study may provide
Qi deficiency and blood stasis syndrome
scientific information to further understanding the mechanism(s) of HH and its main components in
removing blood stasis and ameliorating cerebral infarction. Additionally, AS-IV and HSYA appear to have
synergistic effects on neuroprotection.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction theory, Qi deficiency and blood stasis (QDBS) syndrome is at the

Stroke is one of the leading cause of morbidity and mortality in
the both developing and developed countries (Truelsen et al.,
2005; Liu et al., 2007a, 2007b) and is a major challenge to public
health due to its high incidence and life-threatening nature (Rossi
et al., 2007). Nearly 80% of stroke is ischemic (Donnan et al., 2008),
a consequence of a transient or permanent reduction in cerebral
blood flow that is restricted to the territory of a major brain artery
(Mahajan et al., 2004). In Traditional Chinese Medicine (TCM)
n
Corresponding author. Tel./fax: þ 86 29 84775471.
nn
Corresponding author. Tel./fax: þ86 29 84773636.
E-mail addresses: Yangtian_1973@163.com (Z. Yang),
adwen-2012@hotmail.com (A. Wen).
1
These authors contributed equally to this work.
core of ischemic stroke. QDBS is one of the common syndromes,
characterized by short breath, pale complexion, tiredness, dark
tongue and deep and thin pulse, etc., that often appear in the
course of diseases such as cerebrovascular and cardiovascular
diseases (Liu et al., 2007a, 2007b; Miao et al., 2008). Radix Astragali
(Huangqi), the dry root of Astragalus membranceus (Fisch) Bge, has
been routinely used in China for patients with stroke or chronic
debilitating diseases, because it can reinforce Qi, strengthen
superficial resistance and promote the discharge of pus and
the growth of new tissue (Lin et al., 2000; Guo et al., 2012).
Carthamus tinctorius L. (Honghua), a dried flower, is popularly used
to promote blood circulation to remove blood stasis and alleviate
pain (Li et al., 2009; Han et al., 2013). Huangqi
Honghua
combination (HH) widely used in clinical practice for treating
cerebrovascular and cardiovascular diseases with QDBS, such as

http://dx.doi.org/10.1016/j.jep.2014.05.061
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
1054 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060
Buyang Huanwu decoction, a well-known TCM formula (Cai et al.,
2007; Zhang et al., 2010).
In clinical, Huangqi Injection (HQI) and Honghua Injection
(HHI), which are the water extract of Huangqi and Honghua,
respectively, both are the most popular TCM injections in China.
Moreover, the combination of HQI and HHI is widely used and the
protection for cerebral infarction with QDBS model is remarkable
(Liang et al., 2007;Hu et al., 2008a, 2008b; Lai et al., 2008). Recent
research works show that, astragaloside IV(AS-IV), one of the
major and active components of Huangqi, has the anti-infarction
effect by its antioxidant properties and attenuates permeability of
the blood-brain barrier (Luo et al., 2004; Qu et al., 2009). It has
been reported that Hydroxysafflor yellow A (HSYA) could protect
rat brains against ischemia-reperfusion injury by antioxidant
action (Wei, et al., 2005; Yang, et al., 2010; Chen, et al., 2013).
But the further biochemical mechanism(s) of AS-IV and HYSA in
treating cerebral infarction with QDBS is still unreported. In
addition, as the main components of Huangqi and Honghua, their
synergism on cerebral infarction with QDBS is little known, either.
The widely used modeling method of QDBS is exhaustive
swimming exercising (Li, 1991), and the routine cerebral infarction
modeling method is middle cerebral artery occlusion (MCAO) (Guo
et al., 2012; Raza et al., 2013). In this study, the animals are used
in the QDBS model firstly, then operated the MCAO; finally, the
animals would become the cerebral infarction with QDBS model.
This model is more similar to the stroke patients syndrome in
clinical (Liu et al., 2007a; Zhang et al., 2008) and reflects the
cerebral protection of the subject drugs more accurately.
Oxidative stress is a major contributor in the pathogenesis of I/
R injury (Janardhan and Qureshi, 2004) and one of the main causes
of tissue damage following ischemic injury in the brain (Chen et
al., 2011). Antioxidant enzymes are the primary means by which
neuronal cells protect themselves from toxic reactive oxygen
species (ROS).The induction of such enzymes is governed by the
transcription factor nuclear factor erythroid-2-related factor 2
(Nrf2). Nrf2 can promote the transcription of cytoprotective
genes in response to oxidative and electrophilic stresses, and is
becoming a promising therapeutic target for neuroprotection (Jing
et al., 2013.)
In this paper, the effects of HH and their main components
AS-IV, HSYA on cerebral infarction with QDBS were studied and
their synergistic effect was assessed. The aim of this study is to
provide scientific information to further understand the mechan-
isms of HH and their main components in neuroprotection,
exploring the effective basic material of HH, and promoting the
modernization of Traditional Chinese Medicine.
2. Materials and methods
2.1. Materials
Adult male Sprague-Dawley rats weighing 280 720 g were
supplied by the Experimental Animal Center of the Fourth Military
Medical University. All experimental procedures were carried out
according to protocols approved by the Ethics Committee for
Animal Experimentation of the Fourth Military Medical University
(Xi'an, Shaanxi, China) and in accordance with the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals.
Huangqi Injection (HQI) from Chiatai Qingchunbao Pharma-
ceutical Co., Ltd. (China), Honghua Injection (HHI) was purchased
from Ya'an Sanjiu Pharmaceutical Co., Ltd. (China). Standard
substances of astragaloside IV (Lot number: 110781-200613)
and Hydroxysafflor yellow A(Lot number: 111637-201106) were
obtained from the Chinese National Institute for the Control of
Pharmaceutical and Biological Products. The chemical structure is
shown in Fig. 1.
2.2. Quantitative analysis of marker components
2.2.1. Content of astragaloside IV in Huangqi Injection (HQI)
Analyses were performed on a Shimadzu Instruments (Kyoto,
Japan) liquid chromatographic system which was composed of a
LC-10Avp binary pump, an evaporative light-scattering detector
and a computer system for data acquisition (LC-Solution). The
analytical column employed was an Agilent Zorbax Extend C18
column (250 mm  4.6 mm, 5 mm, Agilent Corporation) and
acetonitrile
water (36:64) as mobile phase, at a flow rate of
1.0 mL/min. The injection volume was 10 mL. The detector tem-
perature was 40 1C, and the pressure was 0.35 MPa.
2.2.2. Content of Hydroxysafflor yellow A in Honghua Injection (HHI)
Analyses were performed on an Agilent 1100 series system
(Agilent Corporation, USA) with a diode-array detector. The
analytical column employed was an Agilent Zorbax Extend C18
column (250 mm  4.6 mm, 5 mm, Agilent Corporation). The
mobile phase was composed of acetonitrile (A) and 0.02 M
NaH2PO4 (adjusted to pH 3.5 with ortho-phosphoric acid (B)).
Gradient elution was employed and the gradient program was set
as follows: initial 0 min at 10% solvent A; 0–16 min, linear
increased from 10% to 22% solvent A. The system was balanced
for 5 min by the initial mobile phase (A:B ¼10:90) after detecting
each sample. The flow rate was set at 0.8 mL/min and the injection
volume was 10 mL. A detection wavelength was set at 403 nm.
2.3. Animal model
The rats were divided into six groups randomly: (1) sham
group, (2) QDBS þ I/R model group, (3) AS-IV group, (4) HSYA
group, (5) AS-IV þ HSYA group, and (6) HH group. The injection
volume was 3 mL/kg, throughout. The dosage of HQI and HHI in
clinical is 0.3 ml/kg (20 ml/60 kg) for both (Liang et al., 2007; Hu
et al., 2008a, 2008b). The most effective dosage in QDBS þI/R rats,
confirmed through preliminary experiments, was 3 ml/kg.
Rats in AS-IV group, HSYA group, AS-IVþ HSYA group and HH
group were treated with AS-IV (equal to the contents of HQI),
HSYA ( equal to the contents of HHI), AS-IV þHSYA (equal to the
contents of HQI and HHI) and HQI þHHI via tail intravenous
injection, respectively, other groups were treated with normal
saline once a day. 40 min after administration, except the sham
group, rats in other groups were treated with exhaustive swim-
ming exercising once a day so that they were in a chronic state
with QDBS (Li, 1991). On the 22nd day, the middle cerebral artery
occlusion (MCAO) was performed using a traluminal filament
model (Longa et al., 1989) as described previously (Khan et al.,
2009). The animals were anesthetized by intraperitoneal injection
of chloral hydrate (400 mg/kg) and placed in dorsal recumbency. A
3-0 monofilament nylon suture (Ethicon, Inc., Osaka, Japan) was
introduced from the external carotid artery (ECA) to the right
internal carotid artery (ICA) to occlude the origin of the right
middle cerebral artery. 2 h after the induction of ischemia, the
suture was slowly withdrawn. At the same time, the rats were
administrated with AS-IV, HSYA, AS-IV þHSYA and HQI þHHI via
tail intravenous injection and other groups were treated with
normal saline. The neck incision was closed. Sham group rats went
the same surgical procedures except the monofilament insertion.
Body temperature was maintained at 37 1C with a heating light
during surgery. Thereafter the rats were returned to their cages
and given free access to food and water.
 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060
Fig. 1. Chemical structure of astragaloside IV (A, AS-IV, molecular weight¼ 784) and Hydroxysafflor yellow A (B, HSYA, molecular weight¼ 612).
2.4. Neurological examination
Neurological tests were analyzed in a blinded fashion with
regard to treatments before the rats were killed. Neurological
function was evaluated using a 0–5-point scale neurological score:
0 ¼no neurological dysfunction; 1 ¼failure to extend left forelimb
fully when lifted by tail; 2 ¼circling to the contralateral side;
3 ¼falling to the left; 4¼ no spontaneous walk or in a comatose
state; 5 ¼death. The scoring was based on the method of Masuo
et al. (1997).
2.5. Blood collection
Rats were anesthetized with chloral hydrate (400 mg/kg) by
intraperitoneal injection after the neurological tests, and blood
was drawn from the abdominal aorta to determine hemorheolo-
gical variables. Blood was collected into dry vacuum tubes with
heparin lithium for whole blood viscosity (WBV). Then plasma was
separated from blood by centrifugation at 3000 rpm for 10 min
and detected for plasma viscosity (PV). All experiments were
completed within 3 h after blood collection.
2.6. Viscosity determination
A total of 400 mL blood or plasma was used to determine
the viscosity with a cone-plate viscometer (Model ZL9000, Zonci,
Co., China) at different shear rates maintained at 37 1C. WBV was
measured with shear rates' varying from 1 to 200/s.
2.7. Infarct volume measurement
The brains were dissected out after the blood drawn and kept
at
20 1C for 10 min. Coronal sections (2 mm) of the frozen brains
were cut with the sharp blades and stained with 1% 2,3,5-
triphenyltetrazolium chloride (TTC) at 37 1C for 10 min. Viable
tissues stained deep red while the infarcts remain unstained.
The TTC-stained sections were photographed with a digital camera
and the infarct volume of each section was calculated using an
image analysis system (Adobe Photoshop 9.0, Adobe Systems
Incorporated, San Jose, CA). To compensate for the effect of brain
edema, corrected infarct volumes were calculated as previously
described using the following equation (Belayev et al., 2003).
Infarct volumes were expressed as percentages of contralateral
hemispheric volumes. Corrected infarct area ¼measured infarct
1055
area  {1
[ (ipsilateral hemisphere area contralateral hemisphere
area)/contralateral hemisphere ] }.
2.8. Histological studies
After 24 h of reperfusion the rats were killed and then perfused
with physiological saline solution at 4 1C, followed by freshly
prepared 4% (v/v) paraformaldehyde in 0.1 M phosphate buffered
saline (PBS) buffer (pH 7.4). The brain was removed and fixed in 4%
(w/v) paraformaldehyde for 24 h. Then the brain block was
embedded in paraffin and cut into 5 mm coronal sections. Then
the sections were stained with hematoxylin–eosin (H–E) using
standard methods.
2.9. Tissue preparation
After the neurological tests, the rats were killed and their
brains were taken out to dissect the ipsilateral hemisphere to give
5% (w/v) homogenate (10 mM Tris–HCl, pH 7.4 having 10 ml/ml
protease inhibitors: 5 mM leupeptin, 1.5 mM aprotinin, 2 mM
phenylethylsulfonylfluoride (PMSF), 3 mM peptastatin A, 0.1 mM
EGTA, 1 mM benzamidine and 0.04% butylated hydroxytoluene)
and centrifuged at 1000 rpm for 5 min at 4 1C to separate the
debris. This supernatant 1 (S1) was used for the measurement of
mitochondria-generated reactive oxygen species (ROS) and the
remaining was recentrifuged at 10,500g for 15 min at 4 1C. The
post mitochondrial supernatant (PMS) was used for the estimation
of antioxidant enzyme SOD and GSH.
2.10. Measurement of mitochondria-generated reactive oxygen
species
Mitochondria are major generators of reactive oxygen species
(ROS) in cells and tissues (Moro et al., 2005). Mitochondria in brain
tissues were isolated according to a previously described method
(Sims and Anderson, 2008). ROS were measured by using the
indicator MitoSOX Red (Molecular Probes), according to manufac-
turer's instructions. Briefly, cells were washed with warm Hank's
Buffered Salt Solution (HBSS) containing calcium and magnesium,
and then incubated with 5 μM MitoSOX reagent working solution
for 10 min at 37 1C. After removing MitoSOX Red, cells were
washed with warm HBSS, and fluorescence was read at 510 nm
for excitation and 580 nm for emission with a fluorescence plate
reader. ROS level was expressed as percentage in fluorescence
relative to model group.
1056 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060

2.11. Measurement of antioxidant enzyme activity blots were visualized with ECL-Plus reagent (Santa Cruz, USA) and
and malondialdehyde content analyzed with Quantity One System image analysis software (Bio-
Rad, USA).
Both the measurements of antioxidant enzyme activities and
malondialdehyde (MDA) content in tissue homogenate were
performed according to the technical manual of the detection kit
2.13. Statistical analysis
(Jianchen Biological Institutes, China) (Dong et al., 2002). MDA
content was measured by using the thiobarbituric acid reactive
The results, except for neurologic scores, were expressed as
substances assay and expressed as nmol/mg protein. Super-oxide
mean 7standard deviation (S.D.) and analyzed with one-way
dismutase (SOD) activity was measured following the reduction of
analysis of variance (ANOVA) based on Student's two-tailed
nitrite by a xanthine–xanthine oxidase system. One unit of SOD is
unpaired t-test or Dunnett's multiple comparisons test. Neurologic
defined as the amount that shows 50% inhibition. The SOD activity
scores were expressed as median (range) and were compared
was expressed as U/mg protein. Catalase (CAT) activity was
using a nonparametric method (Kruskal–Wallistest) followed by
assayed by measuring absorbance at 240 nm using an ultraviolet
the Mann–Whitney U statistic with Bonferronicorrection. P-value
light spectrophotometer (Beckman, USA) and was expressed as
less than 0.05 presented statistical significance.
U/g protein. The definition of its activity was based on the
hydrogen peroxide decomposition rate at 240 nm in the reactive
mixture, of which the absorbance was between 0.5 and 0.55.
Glutathioneperoxidase (GSH-Px) activity was measured by mea- Table 1
suring the absorbance at 412 nm and was expressed as U/mg Effects on WBV (mPa s) at various shear rats (n¼ 8 in each group).
protein. One unit of GSH-Px activity was defined as the GSH-Px
Group 1/s 5/s 30/s 200/s
in1 g protein that led to the decrease of 1 mmol/L GSH in the
reactive system per minute. Sham 26.317 2.56 11.517 0.95 6.11 7 0.36 4.34 7 0.31
QDBSþ I/R 34.41## 72.53 13.62# 7 0.96 7.50## 70.57 5.41## 7 0.43
2.12. Western Blotting analysis AS-IV 30.53 7 1.14 12.167 0.54 6.86 7 0.27 4.75 7 0.21
HSYA 29.007 1.67 11.667 0.82 6.36* 7 0.22 4.54* 70.29
AS-IV þHSYA 27.80* 7 2.12 11.93* 7 0.46 5.83** 7 0.42 4.68* 70.12
Proteins were extracted from cerebral cells and fractionated by HH 20.46** 7 2.34 8.64** 7 0.68 4.76** 7 0.21 3.47** 7 0.08
10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis
(SDS-PAGE) and then transferred onto PVDF membranes Sham¼ sham group; QDBSþ I/R ¼QDBSþ I/R model group; AS-IV ¼AS-IV group;
(Millipore, USA). The membranes were blocked with 5% nonfat HSYA ¼ HSYA group; AS-IVþ HSYA ¼AS-IV þHSYA group; HH¼ HQI þ HHI group.
Data were represented as mean7 S.D. n¼ 8.
dried milk and incubated overnight with primary antibodies (Nrf2, ##
Po 0.01 vs. sham group.
Cell Signaling, USA) at 4 1C. The primary antibody was used at a #
Po 0.05 vs. sham group.
dilution of 1:1000. Subsequently, membranes were incubated with nn
P o 0.01 vs. QDBSþI/R model group.
secondary antibody at a 1:5000 dilution at 37 1C for 30 min. The n
P o0.05 vs. QDBSþ I/R model group.

Fig. 2. HPLC chromatogram of HQI (A), HHI (C), standard (B, D): AS-IV (1), HSYA (2).
 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060
Fig. 3. Effects on PV. ♯♯P o0.01 vs. sham group, nPo 0.05 vs. QDBSþ I/R model
group, nnPo 0.01 vs. QDBSþ I/R model group.
Fig. 4. Neurological scores after MCAO in each group (n¼ 8). Neurological scores
were expressed as median (range); nPo 0.05, nnP o 0.01 compared with QDBS þ I/R
model group,♯♯Po 0.01 compared with sham group.
Fig. 5. Infarct size in each group. (A) TTC staining of the cerebral infarct in the rat brain at 24 h after reperfusion. (B) Statistical analysis of the percentage of infarct volume
was assessed for each group. All data were expressed as mean 7 S.D. ( n¼ 8 ); nP o 0.05, nnPo 0.01 compared with QDBS þI/R model group, ♯♯P o0.01 compared with
sham group.
1057
3. Results
3.1. HPLC analysis
The main components contents of HQI and HHI were measured
by an HPLC method as described in materials and methods.
The value of AS-IV and HSYA given was the equivalent amount
of the components presented in HQI and HHI used for animal
treatment. The content of AS-IV in HQI was 0.15 mg/ml, and the
content of HSYA in HHI was 0.24 mg/ml. The quantitatively results
are show in Fig. 2.
3.2. Effects on WBV
The effects on WBV and PV are shown in Table 1. In the QDBS þ
I/R model group, WBV significantly increased at all shear rates in
the blood stasis. After administration, WBV at high shear rate
significantly decreased except for AS-IV group and significantly
decreased at low shear rate in HH as well as AS-IVþ HSYA groups.
3.3. Effects on PV
The Effects on PV are shown in Fig. 3. In the QDBS þI/R model
group, PV significantly increased, while in HH and AS-IVþ HSYA
groups, PV significantly decreased compared with the QDBS þI/R
model group.
3.4. Neurological deficit evaluation
Neurological scores assessed at 24 h after reperfusion are
shown in Fig. 4. The scores in HH and AS-IVþ HSYA groups were
significantly better than those in the QDBS þI/R group (P o0.05).
3.5. Infarction volume assessment
The representative photographs of coronal sections from each
group are shown in Fig. 5A. All treatment groups significantly
decreased the infarct volume compared with the QDBS þ I/R model
group (Fig. 5B). The group with the best protection effect was the
HH group, as well as the AS-IV þHSYA group.
1058 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060

Fig. 6. Hematoxylin–eosin stains of coronal sections of brain after 24 h reperfusion. (A) Sham group; (B) QDBS þ I/R model group; (C) AS-IV group; (D) HSYA group;
(E) AS-IV þHSYA group; (F) HH group.

3.6. Histopathology analysis

Fig. 6 shows the histopathological results from each group.

In the sham group, no histopathological abnormalities were
observed, and the neurons of those were arranged orderly and
had abundant cytoplasm and clear nucleolus (Fig. 6A). On the
contrary, in the QDBS þI/R model group, most neurons in the
damage area appeared shrunken with eosinophilic cytoplasm and
triangulated pyknotic nuclei at 24 h after reperfusion. Edema of
the neuropile was observed in the ischemic zone, furthermore
there were no neurons with normal morphology in the deep-
ischemic zone. The infarct core was surrounded with ischemic
injured neurons (penumbra) (Fig. 6B). After treatment, the central
regions of infarct core in the ischemic brain tissue were signifi-
cantly shrunken. The pathological changes involving cell swelling,
eosinophilic cytoplasm and pyknotic nuclei significantly decreased
(Fig. 6C
F). Fig. 7. Mitochondria-generated reactive oxygen species level determined after 24 h
reperfusion. All data were expressed as mean 7 S.D. ( n¼ 8 ); ♯♯Po 0.01 compared
with sham group, nnP o0.01 compared with QDBS þI/R model group.
3.7. Effect on mitochondria-generated reactive oxygen species

ROS production in the mitochondria of the QDBS þI/R model

group significantly increased compared with the sham group
as shown in Fig. 7. In the HH group and AS-IV þHSYA group, ROS
were markedly decreased (P o0.01) compared with the QDBS þI/R
model group.
3.8. Effect on antioxidant enzyme activities and MDA content
After 2 h of ischemia followed by 24 h reperfusion, the activ-
ities of SOD, catalase and GSH-Px were significantly decreased in
the QDBS þI/R model group compared with the sham group
as shown in Table 2. Nevertheless, after treatment with HH, it
showed a markedly increase compared with the QDBS þI/R model
group (P o0.01). MDA was used as a maker of lipid peroxidation.
After 24 h reperfusion, the MDA contents of the AS-IV þHSYA
and HH groups were obviously decreased compared with the
QDBS þI/R model group (P o0.01).
3.9. Western blotting analysis
We examined the expression level of Nrf2 to explore the
mechanisms responsible for the beneficial effects of HH. The level
of Nrf2 plays an important role in oxidative damage. As shown
in Fig. 8, administration of AS-IV þHSYA or HH, individually,
significantly increased the expression of Nrf2 compared with the
QDBS þI/R model group.
4. Discussion and conclusion
In the TCM theory, Qi and blood dysfunction is one major
pathogenesis of cerebral diseases. The treatment of cerebral
ischemia with QDBS by combination of Qi-tonifying drug
(e.g. Huangqi) and drug for invigorating blood circulation and
eliminating stasis (e.g. Honghua) has shown satisfactory efficacy in
 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060
Table 2
Effects on the antioxidant activities and MDA content of brain tissue after ischemia/reperfusion injury in rats (n¼ 8 in each group).
Group SOD(m/mg.prot)
Sham 204.60 728.03
QDBS þI/R 80.46## 7 28.53
AS-IV 128.53 712.14
HSYA 122.00 710.67
AS-IV þ HSYA 155.80* 7 20.25
HH 200.46** 7 31.34
Data were shown as mean7 S.D. n¼ 8.
##
P o0.01 vs. sham group.
n
Po 0.05 vs. QDBS þI/R model group.
nn
Po 0.01 vs. QDBSþ I/R model group.
Fig. 8. Effects on the levels of Nrf2. nPo 0.05 vs. QDBSþ I/R model group, nnPo 0.01
vs. QDBS þI/R model group.
clinical practice in China (Liang et al., 2007; Ma et al., 2013). In the
present study, our results indicated that HH treatment could
significantly ameliorate cerebral ischemia and up-regulate Nrf2
to coordinately increase expression of several anti-oxidative
enzymes such as SOD and CAT, which play important role in
combating oxidative stress. Furthermore, AS-IV and HSYA are
mainly responsible for curative effects of HH and play significant
roles. Sometimes two main components show synergistic effects.
WBV is one of the measurement indexes of hemorheology, the
higher the viscosity, the smaller the liquidity, the more easily to
form tissue ischemia (Liu et al., 2012). PV plays an important role
in WBV. In the present study, our results showed that the level of
WBV and PV increasing significantly in the QDBS þ I/R model
compared with the control group and HH treatment could
significantly decrease WBV at all shear rates and PV. HSYA and
AS-IV þHSYA treatments could decrease WBV at high shear rate
and PV, while AS-IV had no effect on PV and WBV, which
suggested that HSYA may contribute to the effect of HH in
decreasing WBV and PV. The above results showed that AS-IV
and HSYA had no synergistic effect in decreasing WBV and PV.
In the current study, the results of infarct size and histological
damage demonstrated that HH or AS-IV þHSYA treatment could
significantly decreased the infarct volume and shrink the central
region of infarct focus in the ischemic brain tissue. However,
treatment with AS-IV or HSYA alone had worse cerebral protective.
The results showed that AS-IV and HSYA had a synergetic effect on
cerebral protection.
1059
CAT(m/g.prot) GSH-px(vital unit) MDA(nmol/mg.prot)
42.357 8.05 66.787 9.30 11.52 73.21
13.59## 7 1.96 17.10## 7 3.57 45.33## 7 9.43
20.93 7 8.48 26.36 7 9.25 34.75 76.31
19.167 6.54 24.977 8.22 33.7875.12
29.36 7 6.72 38.83* 74.42 20.54** 73.39
38.64** 7 6.08 54.76** 7 5.71 14.47** 75.08
Mitochondria are the main sites of ROS generation, and they
function in several defense mechanisms under normal conditions
(Li et al., 2012). ROS can lead to the free radical attack of
membrane phospholipids and loss of mitochondria membrane
potential (Li et al., 2006). During exhaustive swimming exercising
and I/R injury, high concentrations of ROS cannot be efficiently
removed by the endogenous antioxidant systems, thus the accu-
mulation of ROS resulted in oxidative damage to cellular mem-
brane, DNA and protein. In our results, treatment with HH and
AS-IVþ HSYA markedly decreased ROS level compared with the
QDBS þI/R model group. What is more, treatment with AS-IV or
HSYA alone could not decrease ROS. The results demonstrated that
AS-IV and HSYA had a synergetic effect on decreasing ROS.
Nrf2 is a transcription factor that regulates an expansive set of
antioxidant-related genes, which acts in synergy to remove ROS
through sequential enzymatic reactions (Qin et al., 2009). Among
the spectrum of anti-oxidant genes, expression of those encoding
catalase, SOD, glutathione reductase, GSH-Px is all controlled by Nrf2
(Dai et al., 2012). In our findings, Nrf2 expression was significantly
increased by treatment with AS-IVþHSYA and HH, followed by
increasing the activities of SOD, catalase and GSH-Px, decomposing
O2 and H2O2 before their interaction to form the more harmful
hydroxyl (OH þ ) radical (El-Missiry et al., 2001). MDA, a crucial product
and one of the most sensitive indicators of lipid peroxidation, also
indirectly reflects the production of intracellular ROS (Gutteridge,
1995; Qin et al., 2009). The content of MDA increased remarkably in
rats after I/R injury, and AS-IVþHSYA and HH treatments significantly
reduced the level of MDA. These data suggested that AS-IVþHSYA
could play an important role in the antioxidant property of HH,
possibly increasing the endogenous defensive capacity of the brain to
combat oxidative stress induced by cerebral I/R (Yang et al., 2012).
The synergistic protective effect of AS-IV and HSYA is related to
ameliorate cerebral infarction followed by increasing the activity
of SOD, catalase and GSH-Px, decreasing MDA and ROS, up-
regulating the expression of Nrf2. While, there is no synergistic
protective effect on activating blood to dissipate blood stasis. Thus
AS-IV and HSYA are mainly responsible for curative effects of HH
and play important roles in neuroprotection, the effect of HH in
promoting blood circulation may be associated with other com-
ponents from Huangqi or Honghua besides HSYA.
Acknowledgment
This work was supported by National Natural Science Founda-
tion of China (Nos. 81173514 and 81373947), Xijing Research
Booting Program (No. XJZT10D01) and Excellent Civil Service Train-
ing Fund of Forth Military Medical University (No. 4138C4IDK6).
We thank our colleagues for their constructive advices on our
experiments.
1060 J. Cao et al. / Journal of Ethnopharmacology 155 (2014) 1053–1060
References
Belayev, L., Khoutorova, L., Deisher, T.A., Belayev, A., Busto, R., Zhang, Y., Zhao, W.,
Ginsberg, M.D., 2003. Neuroprotective effect of SolCD39, a novel platelet
aggregation inhibitor, on transient middle cerebral artery occlusion in rats.
Stroke 34, 758–763.
Cai, G., Liu, B., Liu, W., Tan, X., Rong, J., Chen, X., Tong, L., Shen, J., 2007. Buyang
Huanwu Decoction can improve recovery of neurological function, reduce
infarction volume, stimulate neural proliferation and modulate VEGF and Flk1
expressions in transient focal cerebral ischaemic rat brains. Journal of Ethno-
pharmacology 113, 292–299.
Chen, H., Yoshioka, H., Kim, G.S., Jung, J.E., Okami, N., Sakata, H., Maier, C.M., 2011.
Oxidative stress in ischemic brain damage: mechanisms of cell death and
potential molecular targets for neuroprotection. Antioxidants & Redox Signal-
ing 14, 1505–1517.
Chen, L., Xiang, L., Kong, L., Zhang, X., Sun, B., Wei, X., Liu, H., 2013. Hydroxysafflor
yellow A protects against cerebral ischemia-reperfusion injury by anti-
apoptotic effect through P13K/Akt/GSK3β pathway in rat. Neurochemical
Research 38, 2268–2275.
Dai, D.F., Rabinovitch, P.S., Ungvari, Z., 2012. Mitochondria and cardiovascular aging.
Circulation Research 110, 1109–1124.
Dong, H., Xiong, L., Zhu, Z., Chen, S., Hou, L., Sakabe, T., 2002. Preconditioning with
hyperbaric oxygen and hyperoxia induces tolerance against spinal cord ische-
mia in rabbits. Anesthesiology 96, 907–912.
Donnan, C.A., Fisher, M., Macleod, M., Davis, S.M., 2008. Stroke. Lancet 371,
1612–1623.
El-Missiry, M.A., El-Sayed, I.H., Othman, A.I., 2001. Protection by metal complexes
with SOD-mimetic activity against oxidative gastric injury induced by indo-
methacin and ethanol in rats. Annals of Clinical Biochemistry 38, 694–700.
Guo, C., Tong, L., Xi, M.M., Yang, H.F., Dong, H.L., Wen, A.D., 2012. Neuroprotective
effect of calycosin on cerebral ischemia and reperfusion injury in rats. Journal of
Ethnopharmacology 144, 768–774.
Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue
damage. Clinical Chemistry 41, 1819–1828.
Han, S.Y., Li, H.X., Ma, X., Zhang, K., Ma, Z.Z., Jiang, Y., Tu, P.F., 2013. Evaluation of the
anti-myocardial ischemia effect of individual and combined extracts of Panax
notoginseng and Carthamus tinctorius in rats. Journal of Ethnopharmacology
145, 722–727.
Hu, Q., Zhao, J.H., Zou, L.J., 2008a. 32 cases of acute cerebral infarction treated with
Huangqi and Honghua Injection. China Modern Doctor 46 (2), 70.
Hu, Q., Zhao, J.H., Zou, L.J., 2008b. Huangqi Injection combined Honghua Injection
treatment of 32 cases of acute cerebral infarction. China Modern Doctor 46,
70–71.
Janardhan, V., Qureshi, A.I., 2004. Mechanisms of ischemic brain injury. Current
Cardiology Reports 6, 117–123.
Jing, X., Ren, D.M., Wei, X.B., Shi, H.Y., Zhang, X.M., Perez, R.G., Lou, H.Y., Lou, H.X,
2013. Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral
ischemic injury. Toxicology and Applied Pharmacology 273, 672–679.
Khan, M.M., Ahmad, A., Ishrat, T., Khuwaja, G., Srivastava, P., et al., 2009. Rutin
protects the neural damage induced by transient focal ischemia in rats. Brain
Research 1292, 123–135.
Lai, Z., Li, L.S., Cheng, S.B., 2008. The effect of Radix Astragali and Safflower Injection
on neuron apoptosis and caspase-8 after local cerebral ischemia/reperfusion in
rats. Chinese Journal of Arteriosclerosis 16, 885–888.
Li, H.X., Han, S.Y., Wang, X.W., Ma, X., Zhang, K., Wang, L., Ma, Z.Z., Tu, P.F., 2009.
Effect of the carthamins yellow from Carthamus tinctorius L. on hemorheolo-
gical disorders of blood stasis in rats. Food and Chemical Toxicology 47,
1797–1802.
Li, J., Ma, X.S., Yu, W., Lou, Z.Q., Mu, D.L., Wang, Y., Shen, B.,Z., Qi, S.H., 2012.
Reperfusion promotes mitochondrial dysfunction following focal cerebral
ischemia in rats. Plos One 7, 1–10.
Li, J.J., Tang, Q., Li, Y., Hu, B.R., Ming, Z.Y., Fu, Q., Qian, J.Q., Xiang, J.Z., 2006. Role of
oxidative stress in the apoptosis of hepatocellular carcinoma induced by
combination of arsenic trioxide and ascorbic acid. Acta Pharmacologica Sinica
27, 1078–1084.
Li, Y.K., 1991. Experimental Methodology of TCM Pharmacology. Shanghai Science
and Technology Publishing House, Shanghai.
Liang, Y.J., Li, J., Liang, J.C., 2007. The clinical observation of Huangqi Injection and
Honghua Injection combination for treatment the cerebral ischemia. Modern
Journal of Integrated Traditional Chinese and Western Medicine 16, 196–197.
Lin, L.Z., He, X.G., Lindenmaier, M., Nolan, G., Yang, J., Cleary, M., Qiu, S.X., Cordell, G.
A., 2000. Liquid chromatography–electrospray ionization mass spectrometry
study of the flavonoids of the roots of Astragalus mongholicus and A.
membranaceus. Journal of Chromatography A 876, 87–95.
Liu, L., Duan, J.A., Tang, Y.P., Guo, J.M., Yang, N.Y., Ma, H.Y., Shi, X.Q., 2012.
Taoren
Honghua herb pair and its main components promoting blood
circulation through influencing on hemorheology, plasma coagulation and
platelet aggregation. Journal of Ethnopharmacology 139, 381–387.
Liu, S.Z., Bao, Z.X., Zhang, R.L., 2007a. Theory discuss of the Pathogenesis theory of
deficiency of qi and blood stasis in ischemic stroke. Chinese Archives of
Traditional Chinese Medicine 25, 97–98.
Liu, M., Wu, B., Wang, W.Z., Lee, L.M., Zhang, S.H., Kong, L.Z., 2007b. Stroke in China:
epidemiology, prevention, and management strategies. The Lancet Neurology 6,
456–464.
Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle
cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–89.
Luo, Y.M., Qin, Z., Hong, Z., Zhang, X.M., Ding, D., Fu, J.H., Zhang, W.D., Chen, J., 2004.
Astragaloside IV protects against ischemic brain injury in a murine model of
transient focal ischemia. Neuroscience Letters 363, 218–223.
Ma, Q.R., Sun, J.P., Ma, J.B., Liu, G.H., Liang, L., Zhao, J., Ma, X.W., Qin, Y., 2013. The
effect observation of Astragahs mongholicus and red flower on apoptosis of
nerve cells around the cerebral hemorrhage hemorrhage in rat. Chinese Journal
of Clinical Rational Drug Use 6 (12A), 1–3.
Mahajan, S.K., Kashyap, R., Sood, B.R., Jaret, P., Mokta, J., Kaushik, N.K., Prashar, B.S.,
2004. Stroke at moderate altitude. Journal of the Association of Physicians of
India 52, 699–702.
Masuo, Y., Matsumoto, Y., Morita, S., Noguchi, J., 1997. A novel method for counting
spontaneous motor activity in the rat. Brain Research 1, 321–326.
Miao, Y., Zhao, W.J., Jing, L., 2008. Retrospective analysis on integrative medicinal
treatment of chronic heart failure. Zhongguo Zhong Xi Yi Jie He Za Zhi 28,
406–409.
Moro, M.A., Almeida, A., Bolanos, J.P., Lizasoain, I., 2005. Mitochondrial respiratory
chain and free radical generation in stroke. Free Radical Biology and Medicine
39, 1291–1304.
Qin, F., Liu, Y.X., Zhao, H.W., Huang, X., Ren, P., Zhu, Z.Y., 2009. Chinese medicinal
formula Guan-Xin-Er-Hao protects the heart against oxidative stress induced
by acute ischemic myocardial injury in rats. Phytomedicine 16, 215–221.
Qu, Y.Z., Li, M., Zhao, Y.,L., Zhao, Z.W., Wei, X.Y., Liu, J.P., 2009. Astragaloside IV
attenuates cerebral ischemia-reperfusion-induced increase in permeability of
the blood-brain barrier in rats. European Journal of Pharmacology 606,
137–141.
Raza, S.S., Khan, M.M., Ahmad, A., Ashafaq, M., Islam, F., Wagner, A.P., Safhi, M.M.,
Islam, F., 2013. Neuroprotective effect of naringenin is mediated through
suppression of NF-kB signaling pathway in experimental stroke. Neuroscience
230, 157–171.
Rossi, D.J., Brady, J.D., Mohr, C., 2007. Astrocyte metabolism and signaling during
brain ischemia. Nature Neuroscience 10, 1377–1386.
Sims, N.R., Anderson, M.F., 2008. Isolation of mitochondria from rat brain using
percoll density gradient centrifugation. Nature Protocols 3, 1228–1239.
Truelsen, T., Ekman, M., Boysen, G., 2005. Cost of stroke in Europe. European Journal
of Neurology 12, 78–84.
Wei, X.B., Liu, H.Q., Sun, X., Fu, F.H., Zhang, X.M., Wang, J., An, J., Ding, H., 2005.
Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury
by antioxidant action. Neuroscience Letters 386, 58–62.
Yang, J.H., Li, J.H., Lu, J., Zhang, Y.Y., Zhu, Z.H., Wan, H.T., 2012. Synergistic protective
effect of astragaloside IV–tetramethylpyrazine against cerebral ischemic-
reperfusion injury induced by transient focal ischemia. Journal of Ethnophar-
macology 140, 64–72.
Yang, Q., Yang, Z.F., Liu, S.B., Zhang, X.N., Hou, Y., Li, X.Q., Wu, Y.M., Wen, A.D., Zhao,
M.G., 2010. Neuroprotective effect of hydroxysafflor yellow A against excito-
toxic neuronal death partially through down-regulation of NR2B-containing
NMDA receptors. Neurochemical Research 35, 1353–1360.
Zhang, H., Wang, W.,R., Lin, R., Zhang, J.,Y., Ji, Q.,L., Lin, Q.,Q., Yang, L.,N., 2010.
Buyang Huanwu decoction ameliorates coronary heart disease with Qi defi-
ciency and blood stasis syndrome by reducing CRP and CD40 in rat. Journal of
Ethnopharmacology 130, 98–102.
Zhang, J., Zhang, Y.L., Xiu, L., Jin, X.L., Zheng, H., Hu, X.G., 2008. Study on a rat model
of Qi deficiency and blood stasis syndrome with cerebral ischemic stroke 31,
590
593.