INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 25, no.

I, 259-266 (2012)

PROTECTIVE ACTIVITY OF THE ETHANOL EXTRACT OF CYNANCHUM

PANICULATUM (BUNGE) KITAGAWA ON TREATING HERPES SIMPLEX
ENCEPHALITIS

x.-r. LI', Y.-J, GV02, D.-M. ZHANG!, Z. CHEN!, X. WEI',Y.-H. LI',

S.-L. ZHANG3, J.-Y. TA04, J.-H. DONGS, Y.-W. MEF, L.-L. LI' and L. ZHA03

'Department ofNeurology, the People 50 Hospital ofGuangxi Zhuang Autonomous Region, Nanning,
P.R. China; 'Department ofNeurology, Union Hospital, Tongji Medical College, Huazhong
University ofScience and Technology, Wuhan, P.R. China; 3Department ofInfectious Disease
and Hepatology, Union Hospital, Tongji Medical College, Huazhong University ofScience and
Technology, Wuhan, P.R. China; 'Faculty ofPharmacy, Hubei University ofChinese Medicine,
Wuhan, PR China; 'Central Lab, Union Hospital, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan, P.R. China

Received June 6, 2011 - Accepted December 15, 2011

The first two authors contributed equally to this study

To date there has been no valid treatment for herpes simplex encephalitis (HSV). This study explores
the protective activity of ethanol extract of Cynanchum paniculatum (bunge) kitagawa for treatment of
HSV. Cell models and animal models were established and divided into 4 groups: normal group, virus
group, cynanchum paniculatum group and Dexamethasone group. Flow cytometry was employed to
detect apoptosis of cell model and TUNEL assay was chosen to detect apoptosis of animal tissues. The
survival time of the animal models was observed. ELISA was used to measure TNF -a expression and
the Greiss method to measure Nitric Oxide (NO) expression in the mouse brain. As a result, it was
found that extract of Cynanchum paniculatum can improve the survival rate of HSV-infected mice. The
extract could prevent apoptosis in the neuron cell model and reduce apoptosis rate in brain tissue after
HSV infection. With the extract intervention, TNF-a and NO levels in brain tissue were significantly
decreased in the animal model. In conclusion, the extract of Cynanchum paniculatum can prevent HSV-
inducing impairment in the cell and animal model of HSE.

Herpes simplex encephalitis (HSE) is a kind of older children and adults (l) and is a significant
viral encephalitis that attacks neonates as well as cause of morbidity and mortality (2). Nowadays, the
Key words: Cynanchum paniculatum (bunge) kitagawa, herpes simplex virus, apoptosis, TNF-a., NO
Mailing addresses: Dr. Lei Zhao,
Department of Infectious Disease and Hepatology,
Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology,
Wuhan, 430022, P.R. China
Tel: +86 27 85726783 Fax: +86 2787716917
e-mail: chinesemd@hotmail.com
Prof. Lv-Li Li, 0394-6320 (2012)
Department of Neurology, the People's Hospital Copyright © by BlOLlFE, s.a.s.
This publication and/or article is for individual use only and may not be further
of Guangxi Zhuang Autonomous Region,
reproduced without written permission from the copyright holder.
Nanning, 530021, P.R. China Unauthorized reproduction may result in financial and other penalties
Tel: +8613617811502. Fax: +86 0771 2186018. 259 DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF
e-mail: 450211034@qq.com INTEREST RELEVANT TO THIS ARTICLE.
260 X.-F. LI ET AL.
mechanism of HSE is not fully understood although
research for this disorder proceeds fast. However,
diffuse inflammation of the brain parenchyma has
been confirmed for cerebral dysfunction (3).
Cynanchumpaniculatum (bunge) kitagawa, aplant
grown across all of China and other Asian countries,
is one of the Chinese traditional medicinal herbs. In
traditional medicine, Cynanchum paniculatum has
been used in China for gastritis and relieving pain,
including rheumatic arthralgia, lumbago, pain due to
traumatic injuries, abdominal pain, toothache, and
other kinds of pain, as well as skin diseases such as
eczema, rubella, neurodermatitis and snake bite (4).
Studies have shown that the ethyl acetate fraction
of the root of Cynanchum paniculatum has anti-
inflammatory and anti-nociceptive effects (4). Other
investigations showed that an alkaloid antofine from
Cynanchum paniculatum can inhibit cell growth and
potentiate tumor necrosis factor-a (TNF-a)-induced
apoptosis in human colon cancer cells (5). However,
there have been no reports related to the protective
effect of the extract of Cynanchum paniculatum on
treating herpes simplex encephalitis. In this study,
we initiate the exploration.
MATERIALS AND METHODS
Chemicals and reagents
DMEM was purchased from Gibico (Grand Island,
NY, USA). Methyl thiazolyl tetrazolium (MTT) was
obtained from Sigma (St. Louis, MO, USA). Cell TNF-a
ELISA kit was purchased from Quantikine, R&D Systems
(Minneapolis, MN, USA). Annexin V-FITC kit was
obtained from Bender Medsystem (Vienna, Austria). The
Griess reagent nitric oxide assay kit was obtained from
Nanjing Jiancheng Biotech (Jiangsu P R, China). TUNEL
kit was taken from Roche China (Shanghai).
Preparation of ethanol extract from Cynanchum
paniculatum (bunge) kitagawa
The preparation of ethanol extract from Cynanchum
paniculatum (bunge) kitagawa abided by our previous
study (6, 7). Cynanchum paniculatum (bunge) kitagawa
was identified by Prof. Ke-Li Chen (School of Pharmacy,
Hubei University of Chinese Medicine) according to the
Dictionary of Traditional Chinese Medicine (Jiangsu
College of New Medicine, 1985) and was extracted by
Dr. Jun-Yan Tao. The plant materials were stored in the
plant specimen department, School of Pharmacy, Hubei
University of Chinese Medicine. 50 g air-dried whole
grass of Cynanchum paniculatum (bunge) kitagawa was
powdered and extracted with 70% ethanol at 85°C (2 x
500 mL, 1.5 h each). Then the extracted liquid was filtered,
combined and concentrated in vacuum. Subsequently the
concentrated liquid was diluted by deionizer water to a
concentration of I g herb weight per I mL water.
Cell culture
PC-12 cells were bought from Shanghai Institutes
for Biological Sciences, CAS. HSV-1 virus was cultured
with VERa cells which were from the laboratory of the
virus facility of Wuhan Union Hospital. DMEM medium
was mixed with 10% newborn calf serum for cell culture.
When the cells attained a density of 80-90% on the bottle,
the cells were split and continued to be cultured. The cells
were then cultured in the incubator at 37°C, 5% COz and
saturated humidity. Medium was changed every 2-3 days
or the cells were subcultured.
MrT assay on cytotoxicity
The procedure has been reported in our previous
studies (8). Cells were inoculated into 96-well plate with
the density of 1x 104 • Each group had 8 wells, and the
volume ofeach well was 200 ul, The cells were incubated
for 24 h under the condition of 37°C, 5% COz' Then
the extracts of cynanchum paniculatum with different
concentrations were added. After 12 h, 24 hand 48 h, the
plates were taken out respectively and 20 ~l 5mg/ml MTT
solution were added to each well, followed by 4-hour
incubation at 37°C. Then culture supernatant in the wells
was carefully removed and 200 ul DMSO were added
followed by 10 min vibration to completely dissolve the
crystals. The Ol) at 490 nm was measured by ELISA
reader. The experiment was repeated three times.
Cell grouping
After the cells grew to 70% density after subculturing
in a 24-well plate for 24 h, they were divided into a
normal group, a virus group, a Cynanchum paniculatum
group (l Oug/ml)and a Dexamethasone group (500~g/ml),
and herpes simplex virus-1 (HSV-I) at 10·z/0.1 ml was
added to the latter three groups. Then the medium of
cells was replaced at I h after adding the virus, and the
different interventions in each group were carried out. The
solution of intervention included DMEM without serum.
Normal cells without HSV-I stimulation were prepared as
the normal group.
Flow cytometry assay on cell apoptosis
PC-12 cells were vaccinated in 6-well plates. After 24
h, they were activated with virus and added with different
drugs according to the experimental design; the cells
were then continuously incubated for 12 h, 24 hand 48
 lot J. Immuoopathol. Pharmacol.
h. After thorough washing with PBS, cells at the bottom
and in suspension at 1x 106/ml were obtained for testing
and suspended again with 250 I!I mixture buffer. Then 195
I!I of the cell suspension were mixed with 5 I!I Annexin
V-FITC and washed once with 190 ul mixture buffer. The
cells were again suspended with 190 ul mixture buffer and
10 I!I (l20I!g/ml) Propidium Iodide solution was added
(final concentration was lug/nil). All mixtures were then
incubated away from light for 30 min at room temperature.
FACS was used to analyze the apoptosis rate.
Animals
Sixty 3-week old Balblc male mice weighing 11-13g
were purchased from Hubei provincial laboratory animal
center. They were divided into 4 groups with 5 mice in
each group. The groups were normal group, virus group,
cynanchum paniculatum group (0.4 mg leach mouse) and
Dexamethasone group (2 ug leach mouse). Twenty mice
were used to perform animal experiments and the other 40
were used for observing survival time.
Animal model
The mice were peritoneally injected and anesthetized
with 10% chloral hydrate (3.5mllkg). Then HSV-l was
directly injected into the intracalvarium at the midpoint of
the line from the right canthus to external auditory canal.
The virus group and treatment group were injected with
20 I!I 100LDso (1010.1 ml) viral suspension. The normal
control group was treated with 20 I!I cell supernatant. The
treatment group were given intragastric administration
after model establishment. On the 4th day after model
establishment, brain tissues of 20 mice were taken for
testing. The others were fed for observing survival time.
TUNEL test for measuring apoptosis ofbrain tissues
The test was carried out according to the experimental
procedure afforded by TUNEL in situ apoptosis kit. The
brain tissues were paraffin-embedded, fixed and sliced,
and then added with TdT enzyme buffer, TaT enzyme
reactive solution and peroxidase-labeled anti-digoxin-Ab.
After DAB coloration, hematine staining, the cell was
observed under microscopy. The cells with brown stains
in the nucleus were regarded as positive cells.
ELISA assay on TNF-a
The procedures were carried out according to our
previous study (9). TNF-a was determined by sandwich
ELISA. On the 4th day after model establishment, 0.2
g brain tissues were taken and homogenated with cold
buffer solution. After ultrasonic disposal for 20 seconds
and centrifugation with 12000 rpm at 4°C for 15 min,
the supernatant was collected and assayed for TNF-a by
ELISA kits. The procedure was carried out according to
261
the manufacturer's instructions.
Greiss method assay on NO
The procedure was carried out according to our previous
study (10). On the 4th day after model establishment, 0.2g
brain tissues were taken and homogenated with cold
buffer solution. After ultrasonic disposal for 20 seconds
and centrifugation with 12000 rpm at 4°C for 15 min, the
supernatant was collected and assayed for nitric oxide by
Griess reagent NO assay kit. The supernatant was taken
out and Greiss reagent I and II were added to supernatant.
Concentrations of 0, 1,2,5, 10,20,40,60 and 100 umol/L
of standard samples were placed in a 96-well plate with
50 I!I in each well and Griess Reagent 1(50 ul) and Griess
Reagent II (50 ul) added to each well at room temperature.
The absorbance was read at 570 nm.
Statistical analysis
Each test cell experiment was carried out three times.
Data were presented as mean ± SD. Comparisons of
measurement data between multiple groups were analyzed
with one-way ANOVA test. Rates were compared with
Chi-square test. Statistical significance was considered
significant when P < 0.05. The statistical process was
performed with SPSS 12.0 software.
RESULTS
Apoptosis rate ofpc- J2 cells
After PC-12 cells were stimulated by HSV-I
virus, the apoptosis rate was significantly increased
(P<0.05), which implied that the neurocytes were
significantly damaged by the virus. However,
compared to the virus group, the apoptotic rate in
the Cynanchum paniculatum group was significantly
decreased (P<0.05), which indicated that the extract
had a protective effect on neurocytes (Fig. I).
Survival time ofinfected mice
It was found that the mice in the virus group died
over time. On the 4th day one mouse died and on the
7th day all mice in the virus group were dead. In the
Cynanchum paniculatum group, on the 6th day the
mortality was zero and from the 9th day the survival
rate remained 70%. In the Dexamethasone group, on
the 5th day some mice died and from the 9th day the
remaining 50% survived to the end of observation
(Fig. 2).
Apoptosis rate ofbrain tissue
Brown-stained cells were TUNEL positive and
262 X.-F. LI ET AL•

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~:. : : . :
I 11 11111 I I 111111 I I 111 1111 I I 1111111 I I 1 111111 I 1 11111 I I 1111 111 I I 111 1111 I
2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10
Annexi n- FITC-A Ann exin- FITC-A

A B

11 111111 11 111111 1 1 11 11111 11111 111 1 I 1 1 111111 1 1 111111 1 11 111111 1 1 1 11111 1 I

2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10
Annexin- FITC-A Annexin- FITC-A

c D

Fig. 1. PC-12 cell apoptosis stimulated by virus at 24 h. A) The apoptosis rate of the normal group was 6.5%; B) The
apoptosis rate ofthe virus group was 16.8%; C) The apoptosis rate ofthe Cynanchum paniculatum group was 9.6%; D)
The apoptosis rate ofthe Dexamethasone group was 13.5%.

uncolored cells were normal brain tissue. Neuron (Fig. 3).

structure appeared in the normal group where
obvious positive cells could not be found. In the Level ofTNF-a in brain tissue
virus group, there were a large number of brown- Comparison between the normal group and
stained apoptotic cells which were not intact in the virus group for TNF-a expression showed a
morphous, reduced in volume, cytoplasm condensed significant difference (P<O.05), which demonstrated
and nuclear chromatin maldistributed, and typical the inflammation reaction during virus invasion.
apoptotic bodies could be found. It was significant TNF-a expression in the Dexamethasone group was
that the level of positive cells was decreased in reduced notably when compared to that of the virus
the Cynanchum paniculatum and Dexamethasone group (P<O.05), which showed an anti-inflammatory
groups when compared to that in the virus group effect. Comparison between the Cynanchum
 Int. J. Immunopalhol. Pharmacol. 263

SJrvi val ti rre of infected nice

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Fig. 2. The survival time ofinfected mice. On the 4th day one mouse died and on the 7th day all mice in the virus group
were dead. In the cynanchum p aniculatum group, on the 6th day the mortality was zero andfrom the 9th day the survival
rate was 70%. In the Dexamethasone group , on the 5th day som e mice died andfrom the 9th day the remaining 50% mice
survived to the end ofthe observation.

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Fig. 3. TUNEL assay for measuring the effect ofcynanchum paniculatum on brain tissue apoptosis infected by HS V- I. A)
The normal group showed no obvious po sitive cells could befound; B) the virus group showed there were a large number
of brown-stained apoptotic cells with unintact morphous, decreased volume, condensed cytoplasm and maldistributed
nuclear chromatin , and typical apoptotic bodies could be found; C) the cynanchum paniculatum group showed the level
ofpositive cells decreased significantly when compared to that in the virns group; D) the Dexamethasone group showed
the stain was similar to that in the Cynanchum paniculatum group.
264 X.-F. LI ET AL.

1l'f.a in brain tissue

ssn
I
3000 f

2~ u §

200l "
1:
c
c
1~ '

H)(Xlf

so
0
Nxmll \l rus cynarchLrn IXIni cui at urn D3xarret hasme

Fig. 4. TNF-a secretion in brain tissue. The data were expressed as mean ± SD. 0 P<O.05, cy nanchum paniculatum group
vs virus group; UP<0.05, normal group vs virus group ; § P<0.05, Dexamethasone group vs virus group.

1. 8

1. 6

1.4

~ 1.2 r ** §§

~ 1
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=
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0.2 1
0
Nxmll \l rus cynarchurn IXlni cui at urn D3xarret hasone

Fig. 5. NO secretion in brain tissue. The data were exp ressed as mean ± SD. 0 0 P<O.OJ, cynanchum paniculatum group
vs virus group; ** P<O.OJ, normal group vs virus group ; §§ P<O.OJ, Dexamethasone group vs virus group .
 Int. J. Immunopathol. PharmacoI.
paniculatum group and the virus group also showed
a significant decrease in TNF-a expression (P<0.05).
This indicated that the extract of Cynanchum
paniculatum could down-regulate TNF-a expression
markedly (Fig. 4).
Level ofNO in brain tissue
The level of NO in brain tissue was significantly
increased when the virus group was compared to
the normal group (P<O.O 1). There was significant
decrease of NO in the Dexamethasone group
compared to that in the virus group (P<O.Ol).
Similarly, NO secretion in the Cynanchum
paniculatum group was also decreased as compared
to that in the virus group (P<O.Ol), which showed
that the effect of the extract on suppressing NO was
similar to that of Dexamethasone (Fig. 5).
DISCUSSION
Herpes simplex encephalitis (HSE) leads to
one of the most devastating intracranial infections
despite available antiviral therapy (11). Increasing
evidence supports that the existence of apoptosis
of neurons caused by virus-evoking inflammation
is the main pathophysiological process in HSE (12).
Therefore, we examined the effect of ethanol extract
of Cynanchum paniculatum on anti-apoptosis and
anti-inflammation in this experiment.
PC-12, a cell line derived from a
pheochromocytoma of the rat adrenal medulla, is
regarded as a neuron cell model in experiments
(13). In our research, the data showed that the
apoptosis rate in the Cynanchum paniculatum group
was markedly lower than that in the virus group,
demonstrating that the extract had a protective effect
on neurons when infected by HSV.
In an animal model, we used the ethanol extract
of Cynanchum paniculatum on mice infected by
HSV and found the extract could prolong survival
time and prevent HSV-infected mice from death. The
TUNEL assay further showed the apoptosis in the
Cynanchum paniculatum group was prevented by the
extract, which confirmed the extract had a protective
activity on HSV-invading neurons.
In addition to the effect in apoptosis, we further
investigated the effect of the extract of Cynanchum
paniculatum on inflammation-related cytokines and
265
mediators. It has been found that the expression of
TNF-a and nitric oxide (NO) is elevated in HSV
infection in the brain (14, 15). Both cytokines and
mediators are markers of inflammatory stress, and
could be used to evaluate the extent of inflammatory
reaction that HSV induces in the brain. It has been
reported that after HSV-induced encephalitis occurs,
the expression ofTNF-a strongly rises in the brain of
an experimental model (14, 16), which demonstrates
that TNF-a plays a critical role in brain damage.
Similarly, NO also plays a similar role in HSE.
The overproduction of NO in the brain may lead to
deleterious consequences although reactive nitrogen
species is a component ofinnate defense against viral
infection (15). The iNOS-mediated NO production
may possibly be involved in secondary mechanisms
of brain injury following virus infection, which
may account for treatment failures in human herpes
simplex virus encephalitis (17-19). In our research,
the results showed that the expression of TNF-a
and NO was elevated significantly after HSV-1
infection. However, with the extract of Cynanchum
paniculatum intervention the pro-inflammatory
cytokine and mediator were inhibited, which showed
that the extract had the potential to control the HSV-
induced inflammatory damage in the brain to some
degree.
In our experiment, the data showed that the
extract of Cynanchum paniculatum could alleviate
HSV-induced brain injury, which was based on
inhibiting pro-inflammatory cytokines. Our study
shows the extract of Cynanchum paniculatum might
be an effective agent to protect neurons in viral
encephalitis. Based on our previous studies on virus
and inflammation (20-22), the next step is to find out
the exact ingredient in the extract of Cynanchum
paniculatum and the further mechanism of the
extract which protect brain damage in HSE.
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