Neuroscience & Medicine, 2013, 4, 166-171
http://dx.doi.org/10.4236/nm.2013.43027 Published Online September 2013 (http://www.scirp.org/journal/nm)
To Optimize the Therapeutic Dose and Time Window of
Picroside II in Cerebral Ischemic Injury in Rats by
Orthogonal Test
Hui Huang1, Li Sun1*, Ling Wang1, Lei Fang1, Li Zhao2, Yan Li2
1Department of Neurology, The Second Affiliated Hospital, Qingdao University Medical College, Qingdao, China; 2Institute of Inte-
grative Medicine, The Second Affiliated Hospital, Qingdao University Medical College, Qingdao, China.
Received July 4th, 2013; revised August 5th, 2013; accepted August 22nd, 2013
Copyright © 2013 Hui Huang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The paper aims to optimize the therapeutic dose and time window of picroside II by orthogonal test in cerebral ischemic
injury in rats. The forebrain ischemia models were established by bilateral common carotid artery occlusion (BCCAO)
methods. The successful models were randomly divided into sixteen groups according to orthogonal experimental de-
sign and treated by injecting picroside II intraperitonenally at different ischemic time with different dose. The concen-
trations of neuron-specific enolase (NSE), neuroglial marker protein S100B and myelin basic protein (MBP) in serum
were determined by enzyme linked immunosorbent assay to evaluate the therapeutic effect of picroside II in cerebral
ischemic injury. The results indicated that best therapeutic time window and dose of picroside II in cerebral ischemic
injury were ischemia 1.5 h with 20 mg/kg body weight according to the concentrations of NSE, S100B and MBP in se-
rum. It is concluded that according to the principle of lowest therapeutic dose with longest time window, the optimized
therapeutic dose and time window are injecting picroside II intraperitonenally with 20 mg/kg body weight at ischemia
1.5 h in cerebral ischemic injury in rats.
Keywords: Picroside II; Cerebral Ischemia; Therapeutic Dose; Time Window; Rats
1. Introduction
S100B, a neuroglial marker protein, participates in cell
multiplication, cytoskeleton regulation and other bio-
logical activities. When there are plentiful S100B be-
tween cells, it may promote the expression of inflamma-
tory reaction factors, and induce neuron apoptosis [1].
Clinical study [2] has found that S100B, a neuroglial
marker protein, elevates significantly in the serum of
patients with ischemic stroke, and S100B concentration
is closely related to ischemic stroke type, severity, infarc-
tion volume and mortality [3]. Neuron-specific enolase
(NSE), which exists specifically in the neurons and neu-
roendocrine cells [4], involves in the formation of mem-
brane structures and the repair of brain cells [5]. The
level of NSE in CSF reaches its peak on the third day after
cerebral ischemia in rats, and is positively correlated with
the final infarction volume [6]. The up-regulation of NSE
level in serum lags behind that in brain tissue for 2 hours
in permanent cerebral ischemia model [7,8]. Myelin ba-
sic protein (MBP) is an important myelin structural pro-
tein. It is beneficial to stabilize the structure and function
of the central nerve system (CNS) [9], and its level
change can reflect the severity of the injury in CNS and
myelin damage [10]. The mRNA expression of MBP is a
small amount in normal adult rat brain, and the expres-
sion decreases after cerebral ischemic injury, however, as
ischemic time goes on, the expression of MBP mRNA
increases slowly [11]. Our previous studies have shown
that in rats with cerebral ischemia for 1.5 h, intraperito-
neal injection of picroside II (20 mg/kg) can suppress the
expression of inflammatory cytokines and neuronal apop-
tosis [12-15]. This study attempts to detect the changes
of NSE, S100B and MBP levels in serum to further
explore the optimal therapeutic dose and the time win-
dow of picroside II for treatment of cerebral ischemic
injury.
*Corresponding author.

To Optimize the Therapeutic Dose and Time Window of Picroside
II in Cerebral Ischemic Injury in Rats by Orthogonal Test
167
2. Material and Methods
2.1. Animal Model Establishment
Thirty healthy, male, specific-pathogen free, Wistar rats,
weighting 230 - 250 g, were provided by the Laboratory
Animal Center of Qingdao Drug Inspection Institute
(certificate No. SCXK (Lu) 20100010). Five rats were
selected for the sham control group randomly, and the
remaining 25 were used to establish the model of cere-
bral ischemia. The rats were deprived of food for 12
hours before surgery, and anesthetized by intraperitoneal
injection of 10% chloral hydrate (0.3 ml/kg), and the
bilateral carotid arteries were isolated and occluded to
establish forebrain ischemia model [16]. Body tempera-
ture was monitored using a rectal probe, and was main-
tained at 36˚C - 37˚C using a homeothermic blanket con-
trol unit during surgery. 4 rats were excluded for uncon-
sciousness in two hours after the surgery or death, and
the remaining 21 successful models were included in the
study. The sham control group also had the operation, but
no occlusion of the bilateral carotid arteries were per-
formed.
2.2. Group Design
The 21 successful animal models were randomly divided
into 5 for model group and 16 for treatment group. Ani-
mal models of the treatment group were grouped by four
levels and two factors [L16(45)] orthogonal experimental
design. Therapeutic time window was factor A, and four
levels were set including cerebral ischemia for 1.0 h, 1.5
h, 2.0 h, 2.5 h. Therapeutic dose was factor B, and 5, 10,
20, 40 mg/kg body weight were set as four levels.
2.3. Interventions
Picroside (CAS ⅡNo.: 39012-20-9, purity> 98%) was
provided by the Tianjin Kuiqing pharmaceutical com-
pany, and was diluted to 1% solution with saline solution.
According to [L16(45)] orthogonal experimental design,
appropriate dose of picroside was injected intraⅡperi-
toneally on corresponding ischemic time. Equal amount
of saline were injected at cerebral ischemia 2 h for sham
control group and model group (Table 1).
2.4. ELISA
The rats were anesthetized by intraperitoneal injection of
10% chloral hydrate (0.3 ml/kg) 24 hours after drug ad-
ministration. 4 ml blood was taken through the heart and
centrifuged at 4000 r/min for 10 minutes, and the serum
were separated. And then the levels of NSE (E02N0025),
S100B (E02S0042), MBP (E02M0034) were measured
in serum by the enzyme-linked immunosorbent assay kit
(Blue Gene biotech company). The ELISA plate coated
Table 1. [L16(45)] orthogonal experimental design.
Therapeutic
dose
Ischemia
1.0 h (A1)
Ischemia
1.5 h (A2)
Ischemia
2.0 h (A3)
Ischemia
2.5 h (A4)
5 mg/kg (B1)1.0 × 5 1.5 × 5 2.0 × 5 2.5 × 5
10 mg/kg (B2)1.0 × 10 1.5 × 10 2.0 × 10 2.5 × 10
20 mg/kg (B3)1.0 × 20 1.5 × 20 2.0 × 20 2.5 × 20
40 mg/kg (B4)1.0 × 40 1.5 × 40 2.0 × 40 2.5 × 40
with anti NSE, S100 and MBP specific antibody was
used, 100 μl standard solution was added in the blank
micropores according to the order of the samples, 100 μl
samples were added in the blank micropores, and 100 μl
distilled water was added in the blank control, and 50 μl
enzyme marker solution was added in each hole (the
blank control holes were not included); the enzyme label
plate was sealed with sealing compound, and incubated
for 1 hour at 37˚C. The microtiter plate was washed five
times using distilled or de-ionized water, and dried thor-
oughly with absorbent paper; 50 μl substrate A & B was
added to each well (the blank control holes were not in-
cluded) and incubated for 10 minutes in darkness at 20˚C
- 25˚C, followed by addition of 50 μl stop solution to
each well; The OD value was calculated by an enzyme
monitor (Bio-Rad 680, USA) at 450 nm for each set of
reference standards and samples; the concentration can
be found in the standard curve according to the OD value
of the samples, expressed in ng/ml.
2.5. Statistical Analysis
SPSS 17.0 statistical software was applied to process
statistical analysis, to analyze the contribution of differ-
ent levels of ischemia (drug administration) time and
dose to the test indicators, as well as the interaction of
ischemia time and dose to the test indicators, and comes
to the best dose and the combination of the time window
according to the results.
3. Results
3.1. Test Results
The model animal group had significantly elevated serum
NSE (6.773 ± 0.812), S100B (0.762 ± 0.110) and MBP
(0.675 ± 0.083) levels compared with the sham control
group, in which the serum NSE, S100B and MBP levels
were 2.368 ± 0.532, 0.234 ± 0.051 and 0.227 ± 0.042 (t =
2.79 - 5.97，P < 0.05). And the model animal group af-
ter treatment had more decreased serum NSE (5.613 ±
1.362), S100B
（0.634 ± 0.153）
and MBP (0.305 ± 0.099)
levels than before (t = 2.33 - 3.91, P < 0.05).
Table 2 is an orthogonal experiment of [L16(45)].

To Optimize the Therapeutic Dose and Time Window of Picroside
II in Cerebral Ischemic Injury in Rats by Orthogonal Test
168
Table 2. [L16(45)] orthogonal design and test results.
Rank No. Serum Serum Serum
Test
No. A B C D E NSE S100 MBP
1 1 1 1 1 1 4.784 0.631 0.565
2 1 2 2 2 2 4.976 0.651 0.655
3 1 3 3 3 3 4.494 0.640 0.517
4 1 4 4 4 4 5.074 0.710 0.573
5 2 1 2 3 4 5.268 0.696 0.598
6 2 2 1 4 3 4.122 0.537 0.387
7 2 3 4 1 2 4.076 0.415 0.341
8 2 4 3 2 1 5.385 0.461 0.404
9 3 1 3 4 2 7.086 0.753 0.756
10 3 2 4 3 1 6.396 0.409 0.623
11 3 3 1 2 4 3.290 0.375 0.526
12 3 4 2 1 3 7.478 0.664 0.603
13 4 1 4 2 3 7.319 0.780 0.759
14 4 2 3 1 4 7.523 0.783 0.722
15 4 3 2 4 1 5.433 0.740 0.726
16 4 4 1 3 2 7.098 0.891 0.823
Ⅰ 19.328 24.457 19.294 23.861 21.998 89.802 10.136 9.578
Ⅱ 18.851 23.017 23.155 20.970 23.236
Ⅲ 24.250 17.293 24.488 23.256 23.413
Ⅳ 27.373 25.035 22.865 21.715 21.155
SS 12.544 9.407 3.696 1.343 0.857
Test No. are rat models (No. 1 - 16) and Rank No. are
impact factor’s levels. Data listed in the table of serum
NSE, S100 and MBP are the experimental results of cor-
responding rat models. Data listed in line I, II, III, IV and
SS are the ANOVA result of serum NSE, the ANOVA
results of Serum S100 and MBP are omitted.
3.2. NSE Analysis
Significant difference was showed in the content of NSE
in serum (Table 3) on different levels of administration
time (factor A) and dosage (factor B) (P < 0.05), but the
interaction of administration time and dosage (factor C)
showed no significant difference (P > 0.05). With the
method of least significant difference (LSD) in each two
groups, only in the levels of administration time of 1.0 h
(A1) and 1.5 h (A2), 2.0 h (A3) and 2.5 h (A4), the con-
tent of NSE were not different significantly (P > 0.05),
and other levels of administration time all showed sig-
Table 3. Variance analysis of the content of NSE in serum.
source of variation SS df MS F P
ischemia time 12.5443 4.181 11.410.01
drug dose 9.4073 3.136 8.55 0.01
time × dose 3.6963 1.232 3.36 0.10
deviation 2.1996 0.367
nificant difference (P < 0.05); no significance was found
in administration dose of 5 mg/kg (B1) and 10 mg/kg
(B2), 10 mg/kg (B2) and 40 mg/kg (B4), 5 mg/kg (B1)
and 40 mg/kg (B4) (P > 0.05), and other levels of ad-
ministration dose all showed significant difference (P <
0.05). From the point of view of the maximum therapeu-
tic time window and minimum administration dose,
A2B3 is the optimal combination, in other words, the
best treatment time window and dose were ischemia for

To Optimize the Therapeutic Dose and Time Window of Picroside
II in Cerebral Ischemic Injury in Rats by Orthogonal Test
169
1.5 h, 20 mg/kg, respectively.
3.3. S100B Analysis
Significant difference was showed in the content of
S100B (Table 4) on different levels of factor A (P =
0.05), but the factor B, as well as time-dose interaction
(C), had not taken effect on serum S100B levels after
ischemia significantly (P > 0.05). With the method of
least significant difference (LSD) in each two groups,
only in the levels of administration time of 1.5 h (A2)
and 2.5 h (A4), 2.0 h (A3) and 2.5 h (A4), the content of
NSE were different significant (P < 0.05) , and none of
the other levels of administration time showed any dif-
ference (P > 0.05); significance (P < 0.05) was found in
administration dose of 5 mg/kg (B1) and 20 mg/kg (B3),
and other levels of administration dose showed no dif-
ference (P > 0.05). From the point of view of the maxi-
mum therapeutic time window and minimum administra-
tion dose, A2B3 is the optimal combination, namely, the
best treatment time window and dose were ischemia for
1.5 h, 20 mg/kg, respectively.
3.4. BMP Analysis
Significant difference was showed in the content of BMP
(Table 5) on different levels of factor A (P < 0.05), but
no significance was found between BMP levels after
ischemia, and there were no significant difference in
time-dose interaction (C) (P > 0.05). With the method of
least significant difference (LSD) in each two groups,
only in the levels of administration time of 1.0 h (A1)
and 2.0 h (A3), the content of NSE were not different
significantly (p > 0.05), and other levels of administra-
tion time all showed significant difference (p < 0.05);
Table 4. Variance analysis of the content of S100B in serum.
source of variation SS df MS F P
ischemia time 0.184 3 0.061 5.87 0.03
drug dose 0.075 3 0.025 2.38 0.17
time × dose 0.029 3 0.010 0.93 0.48
deviation 0.063 6 0.010
Table 5. Variance analysis of the content of BMP in serum.
source of
variation SS df MS F P
ischemia time 0.216 3 0.072 15.080.01
drug dose 0.040 3 0.013 2.81 0.13
time × dose 0.013 3 0.004 0.93 0.48
deviation 0.029 6 0.005
significance was found in administration dose of 5 mg/kg
(B1) and 20 mg/kg (B3) (p < 0.05), and other levels of
administration dose showed no difference (p > 0.05).
From the point of view of the maximum therapeutic time
window and minimum administration dose, A2B3 is the
optimal combination, that is, the best treatment time
window and dose were ischemia for 1.5 h, 20 mg/kg,
respectively.
4. Discussion
S100B mainly exists in the astrocytes and Schwann cells
of nervous system. Tracing S100B may advance the ex-
tension of neuron axons and enhance the survival rate of
neurons, while a great deal of it may generate toxic and
side-effect [1]. A large number of activated S100B pro-
tein which is produced by glial cells is released into the
extracellular after brain tissue injury [17], and then leaks
into CSF and blood through damaged blood brain barrier,
and S100B level in serum is positively correlated with
the severity of the injury of cerebral ischemia [18].
Brouns and his group have also found that the concentra-
tion of S100B, which is in the cerebral spinal fluid (CSF)
of the patients with acute ischemic stroke, is highly cor-
related to the severity of the injury and the prognosis of
cerebral ischemia [19]. Under normal circumstances,
NSE content is not high in blood, however, along with
the necrosis of neurons and the disintegration of nerve
myelin, NSE is released into CSF from the cells after
cerebral ischemia injury, and then leaks into blood
through the blood brain barrier. The more severe the
brain tissue is injured, the more nerve cells are damaged,
and the more NSE is released into the blood, therefore,
the detection of changes of NSE levels in CSF or in se-
rum becomes diagnosis markers of neuronal damage [20].
Normal MBP content in CSF is less than 6.95 mg/L,
however, when the ischemic cerebral injury happens,
cerebral ischemia and hypoxia may be due to oligoden-
drocyte death and demyelination. At the same time, MBP
will flow into the CSF and further into blood with the
damage of the blood brain barrier, which is caused by
brain injuries. Otherwise, the increase of MBP synthesis
may be stimulated by stress conditions themselves, in-
cluding ischemia, hypoxia and others [21]. So to a certain
degree, the detection of the serum MBP can show
whether there are brain injuries, and MBP becomes a
specific protein marker of demyelination [22]. Our study
confirmed that the content of MBP in serum of the model
group was significantly higher than that in the sham con-
trol group, and reduced in different degrees after treat-
ment. Our results showed that the contents of NSE,
S100B and MBP in serum of the model group were sig-
nificantly higher compared with that of the sham control
group, and decreased significantly after treatment, sug-

To Optimize the Therapeutic Dose and Time Window of Picroside
II in Cerebral Ischemic Injury in Rats by Orthogonal Test
170
gesting a protective role of picroside II on both ische-
mic brain tissue and blood brain barrier.
This experiment was a [L16(45)] orthogonal experi-
ment design, in which four time points of 1 h, 1.5 h, 2 h
and 2.5 h after brain ischemia were set and four levels of
picroside II dose were given (5 mg/kg, 10 mg/kg, 20 mg/kg
and 40 mg/kg). The expressions of NSE, S100B and
MBP in serum were measured, and the optimal therapeu-
tic dosage and time window of picroside II were analysed.
These results showed that there was a significant differ-
ence for the therapeutic effects of picroside II in admini-
stration time and the dose. From the principle of the low-
est therapeutic dose with the longest time window, A2B3
was the optimal combination and the best treatment time
window and dose were ischemia for 1.5 h and intraperi-
toneal injection of 20 mg/kg. For the mechanism of cere-
bral ischemic injury was very complicated, only four
indexes, as mentioned here, were observed in this study,
which must bring some variances. Therefore, the exact
mechanism and optimal therapeutic time window and
dosage of picroside II should be evaluated combined
with other detection indexes.
5. Acknowledgements
This study was supported by grant-in-aids for The Na-
tional Natural Science Foundation of China (No. 8104
1092, 81274116).
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