Notoginsenoside ST-4 inhibits virus penetration of herpes simplex virus in vitro

Article abstract of DOI:10.1080/10286020.2011.571645

Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in raw and treated forms for a long time, led to the isolation of a new dammarane-type saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3β,12β,20(S)-tri-hydroxydammar-24-ene-3-O-β-d- xylopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)-β-d-glu- copyranoside, based on the detailed analyses of the 1D and 2D NMR spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50% effective concentration (EC₅₀) values, determined by plaque reduction assay, were 16.47±0.67 and 19.44±1.16μM for HSV-1 and HSV-2, respectively, whereas the 50% cytotoxic concentration (CC₅₀) determined by the XTT test on Vero cells was 510.64±4.56μM. As analyzed by attachment assay and penetration assay based on plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due to the penetration inhibition effects, which was confirmed by fluorescence microscopy observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore, notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.

Full text of DOI:10.1080/10286020.2011.571645

This article was downloaded by: [McMaster University]
On: 28 October 2014, At: 07:33
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Notoginsenoside ST-4 inhibits virus
penetration of herpes simplex virus in
vitro
Ying Pei a b , Qian Du a b , Peng-Ying Liao c d , Zhen-Ping Chen a b
,
Dong Wang c , Chong-Ren Yang c e , Kaio Kitazato f , Yi-Fei Wang a b
& Ying-Jun Zhang c
a Biomedicine Research and Development Center , Jinan
University , Guangzhou, 510632, China
b National Engineering Research Center of Genetic Medicine ,
Guangzhou, 510630, China
c State Key Laboratory of Phytochemistry and Plant Resources
of West China, Kunming Institute of Botany, Chinese Academy of
Sciences , Kunming, 650204, China
d Guangxi Traditional Chinese Medical University , Nanning,
530001, China
e Weihe Biotech Research and Development Center , Yuxi, 653101,
China
f Department of Molecular Microbiology and Immunology ,
Graduate School of Biomedical Sciences, Nagasaki University ,
Nagasaki, 8528521, Japan
Published online: 25 May 2011.
To cite this article: Ying Pei , Qian Du , Peng-Ying Liao , Zhen-Ping Chen , Dong Wang , Chong-
Ren Yang , Kaio Kitazato , Yi-Fei Wang & Ying-Jun Zhang (2011) Notoginsenoside ST-4 inhibits virus
penetration of herpes simplex virus in vitro , Journal of Asian Natural Products Research, 13:06,
498-504, DOI: 10.1080/10286020.2011.571645
To link to this article: http://dx.doi.org/10.1080/10286020.2011.571645
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions

and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research
Vol. 13, No. 6, June 2011, 498–504
Notoginsenoside ST-4 inhibits virus penetration of herpes simplex
virus in vitro
Ying Peiab†, Qian Duab†, Peng-Ying Liaocd†, Zhen-Ping Chenab, Dong Wangc,
Chong-Ren Yangce, Kaio Kitazatof, Yi-Fei Wangab* and Ying-Jun Zhangc*
aBiomedicine Research and Development Center, Jinan University, Guangzhou 510632, China;
bNational Engineering Research Center of Genetic Medicine, Guangzhou 510630, China; cState Key
Laboratory of Phytochemistry and Plant Resources of West China, Kunming Institute of Botany,
d
Chinese Academy of Sciences, Kunming 650204, China; Guangxi Traditional Chinese Medical
University, Nanning 530001, China; Weihe Biotech Research and Development Center, Yuxi
e
653101, China; fDepartment of Molecular Microbiology and Immunology, Graduate School of
Biomedical Sciences, Nagasaki University, Nagasaki 8528521, Japan
(Received 13 December 2010; final version received 10 March 2011)
Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H.
Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in
raw and treated forms for a long time, led to the isolation of a new dammarane-type
saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3b,12b,20(S)-
tri-hydroxydammar-24-ene-3-O-b-D-xylopyranosyl-(1 ! 2)-b-D-glucopyranosyl-
(1 ! 2)-b-D-glu-copyranoside, based on the detailed analyses of the 1D and 2D NMR
spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its
antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50%
effective concentration (EC50) values, determined by plaque reduction assay, were
16.47 ^ 0.67 and 19.44 ^ 1.16 mM for HSV-1 and HSV-2, respectively, whereas the
50% cytotoxic concentration (CC50) determined by the XTT test on Vero cells was
510.64 ^ 4.56 mM. As analyzed by attachment assay and penetration assay based on
plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due
to the penetration inhibition effects, which was confirmed by fluorescence microscopy
observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore,
notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.
Keywords: notoginsenoside ST-4; herpes simplex virus; penetration; antiviral activity
1. Introduction
on steam-treated notoginseng led to the
isolation of four new dammarane-type
triterpenoid saponins, namely, notoginse-
nosides ST-1–ST-3 and ST-5, together
with 23 known ones [2]. In this study, a
new dammarane-type saponin, named
notoginsenoside ST-4, was further isolated
from steam-treated notoginseng. Its struc-
ture was determined on the basis of
detailed analyses of 1D and 2D NMR
spectral data and acidic hydrolysis.
Notoginseng, the root of Panax notogin-
seng (Burk.) F.H. Chen (Araliaceae)
mainly cultivated in Yunnan Province of
China, is a famous traditional Chinese
medicinal herb used both in raw and
processed forms. The raw form is mainly
used for injuries from falls and for
removing blood stasis, whereas the pro-
cessed one is used as a tonic to promote
blood circulation [1]. Our previous study
*Corresponding authors. Email: twang-yf@163.com; zhangyj@mail.kib.ac.cn
†These authors contributed equally to this work.
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.571645
http://www.informaworld.com

Journal of Asian Natural Products Research
499
The anti-herpes simplex (HSV) activity
and the underlying mechanisms of action
for notoginsenoside ST-4 against HSV-1
were also investigated.
The fragment ion peaks at m/z 784 [M-
132(pentosyl)]2 and 622 [M-132-
162(hexosyl)]2 in the FAB-MS, together
with the anomeric proton and carbon
signals at dH 4.92 (d, J ¼ 8.0 Hz), 5.51
(d, J ¼ 7.5 Hz), and 5.39 (d, J ¼ 6.5 Hz),
and dC 104.8, 103.2, and 106.5, respect-
ively, in the 1H and 13C NMR spectra, the
existence of b-pentosyl and b-hexosyl
units. Acidic hydrolysis of this compound
gave D-glucose and D-xylose as the sugar
residue. The 1H and 13C NMR spectral
data of this compound were closely related
to those of notoginsenoside Ft1 [8], except
for the chemical shifts of C-17 (dC 54.8),
C-21 (dC 27.1), and C-22 (dC 35.9). These
observations suggested that this compound
was the C-20(S) epimer of notoginseno-
side Ft1, a protopanaxadiol monodesmo-
side [9]. Furthermore, the HMBC
correlations of H-10 at dH 4.92 with C-3
at dC 88.9, H-100 at dH 5.51 with C-20 at dC
83.0, and H-1000 at dH 5.39 with C-200 at dC
84.6 confirmed the sugar sequence and
location in molecule, which were the same
as those of nogoginsenoside Ft1. Accord-
ingly, the new compound was determined
to be 3b,12b,20(S)-tri-hydroxydammar-
24-ene-3-O-b-D-xylopyranosyl-(1 ! 2)-
b-D-glucopyranosyl-(1 ! 2)-b-D-gluco-
pyranoside, and named notoginsenoside
ST-4 (Figure 1).
Herpesviridae are classified into three
subfamilies: alpha-herpesviruses (including
HSV-1, HSV-2, and varicella-zoster virus),
beta-herpesviruses and gamma-herpes-
viruses [3]. HSV-1 is normally associated
with orofacial infections and encephalitis,
whereas HSV-2 usually causes genital
infections. An infectious herpesvirus con-
sists of four structures: a linear DNA-
genome core, a capsid, a tegument, and an
outer envelope [3]. During its life cycle,
HSV-1 is first absorbed into the cell
membrane. After penetration of absorbed
virus, the incoming nucleocapsids are
transported to the nuclear pore [4,5].
Acyclovir (ACV) has been used for the
treatment ofHSV infectionand isassociated
diseases [6]. However, the efficacy of ACV
is limited by the increase in the resistance
of virus [7].
2. Results and discussion
2.1 Structural elucidation of the new
compound
The new compound was obtained as a pale
amorphous powder. Its molecular formula
C47H80O17 was elucidated by the negative
HR-ESI-MS (m/z 915.5323 [M 2 H]2).
21
24
22
26
HO
HO
20
25
27
13
11
17
18
10
19
1
15
14
30
6'
GlcI
O
3
HO
HO
5
O
6
HO
1'
O
29
28
6"
HO
O
GlcII
HO
HO
1"
O
5'"
O
HO
HO
1'"
1
OH
Xyl
Figure 1. Structure of notoginsenoside ST-4 isolated from the steam-treated notoginseng.

500
Y. Pei et al.
2.2 Assessment of cytotoxicity and anti-
HSV activity of notoginsenoside ST-4
senoside ST-4 was investigated and was
found to be ineffective on absorption, but
effective on penetration. To verify the
results of penetration assay, we performed
the immunofluorescence assay and found
that at 4 h post-infection, most viruses
(exemplified by HSV-1 capsid protein
VP5, red fluorescence) have already
penetrated into the cell, so that no viruses
(red fluorescence) were detected on the cell
membrane of infection control (Figure
2(A)). On the contrary, in the presence of
notoginsenoside ST-4, punctate red fluor-
escence signals of viruses (arrow) were
distributed over the cell membrane, which
could not penetrate into the cell (Figure
2(B)). At 12 h post-infection, massive vp5
proteins (red fluorescene) were synthesized
in the nucleus of infection control (Figure
2(C)). However, there was no vp5 protein
synthesizing in the nucleus of the treatment
group (Figure 2(D)). All the results
The CC50 of notoginsenoside ST-4 was
510.64 ^ 4.56 mM and the CC50 of ACV
was 2926.11 ^ 26.38 mM. Notoginseno-
side ST-4 inhibited HSV-1 with an EC50 of
16.47 ^ 0.67 mM and inhibited HSV-2
with an EC50 of 19.44 ^ 1.16 mM. The
EC50 for ACV was comparable to previous
studies for HSV-1, with EC50 of
0.98 ^ 0.24 mM.
2.3 Effect of notoginsenoside
ST-4-treatment on HSV-1 penetration
The cell and viruses were pre-treated with
notoginsenoside ST-4 in two ways as
described below. Notoginsenoside ST-4
inhibited HSV-1 absorption with an EC50
of 100,000.00 ^ 0.00 mM and inhibited
HSV-1 penetration with an EC50 of
16.46 ^ 0.43 mM. The effect of notogin-
Viral
Group
Notoginsenoside ST-4-treatment
4hp.i.
A
B
12hp.i.
C
D
Figure 2. Notoginsenoside ST-4 inhibited HSV-1 penetration and viral protein (vp5) synthesis.
Infected cells were incubated with notoginsenoside ST-4 (10 mM) treatment at 2 h post-infection and
detected at 4 h post-infection and 12 h post-infection (B,D), or without notoginsenoside ST-4
treatment as viral control and detected at 4 h post-infection and 12 h post-infection (A,C). Cells were
stained with antibody to vp5 (red fluorescene) and chromatin was stained with DAPI (blue
fluorescene). At 4 h post-infection, most viruses have already penetrated into the cell (A). At 12 h
post-infection, massive vp5 proteins (red fluorescene) were synthesized in the nucleus of infection
control (C). In the presence of notoginsenoside ST-4, HSV-1 attached to the membrane, but the
penetration of HSV-1 was blocked on the cell membrane (B, arrow). And at 12 h post-infection, there
was no vp5 protein synthesizing in the nucleus (D). Scale bars are 9 mm.

Journal of Asian Natural Products Research
indicated that penetration of HSV-1 was 3.2 Plant material
501
inhibited by notoginsenoside ST-4 treat-
ment. Further studies will be required to
explore the detailed antiviral mechanism of
notoginsenoside ST-4. In conclusion, this
study described that notoginsenoside ST-4
possessed an anti-HSV activity, which
inhibited viral penetration to cells.
Air-dried notoginsengs were obtained
from Wenshan County, Yunnan Province
of China in May 2006. The raw materials
were crushed into small grains, and then
steamed at 1208C for 12 h, to give the
steamed notoginseng, which were used for
extraction and isolation in this study.
3. Experimental
3.3 Extraction and isolation
3.1 General experimental procedures
As described in our previous work [2], the
EtOH extract (778 g) of air-dried steamed
notoginseng was dissolved in H2O and
passed through a macroporous adsorption
resin (D101) column, eluting with H2O and
MeOH, successively. The MeOH eluates
were combined and concentrated under
reduced pressure to give a residue (440 g),
which was further subjected to silica gel
column eluted with CHCl3:MeOH:H2O
(85:15:1) to afford nine fractions (frs 1–9).
Further purification of Fr. 7 (38 g) by
repeated CC over silica gel (CHCl3–
MeOH–H2O, 80:20:2 to 70:30:5), Rp-8
(MeOH–H2O, 1:1 to 4:1), Rp-18 (MeOH–
H2O, 1:1 to 4:1), and MCI-gel CHP20P
(MeOH–H2O, 3:7 to 0:1) afforded a new
compound (31 mg, yield: 0.00066%),
together with five known ones, which
were determined as ginsenosides Rg1
(151 mg), Rg2 (23 mg), Rf (71 mg), noto-
ginsenoside R2 (20 mg), and 20(R/S)-
ginsenoside Rg3 (238 mg), respectively,
as described in the previous report [2].
Melting points (mp) were determind using
XRC-I melting point apparatus, produced
by Sichuan University, China (uncor-
rected). Optical rotation was performed
on a P-1020 Polarimeter (JASCO, Tokyo,
Japan). IR spectrum was measured on a
Bruker Tensor 27 spectrometer with KBr
pellets. 1H and 13C NMR, HMQC, and
HMBC spectra were recorded in C5D5N
with Bruker AM-400 and DRX-500
spectrometers. Coupling constants were
expressed in Hertz, and chemical shifts
were given on a d (ppm) scale with TMS as
an internal standard. FAB-MS was
recorded on an AutoSpe-3000 spec-
trometer (VG, Manchester, UK) with
glycerol as the matrix, in m/z. HR-ESI-
MS was recorded on API Qstar Pulsa
LC/TOF spectrometer.
Column chromatography (CC) was
performed on silica gel (200–300 mesh;
Qingdao Marine Chemical and Industrial
Factory, Qingdao, China), MCI-gel
CHP20P (75–100 mm; Mitsubishi Chemi-
cal Co., Ltd., Tokyo, Japan), and Rp-8 or
Rp-18 gel (40–60 mm, Merck, Darmstadt,
Germany). Thin layer chromatography
(TLC) was performed on silica gel H
precoated plates (Qingdao Marine Chemi-
cal and Industrial Factory) with CHCl3–
MeOH–H2O (75:25:3 or 80:20:2 or
85:15:1 or 90:10:1 v/v) and CHCl3–
MeOH (90:10 to 25:1), and Rp-8 or Rp-
18 precoated plates (Merck) with MeOH–
H2O (60:40 to 100:0). Spots were detected
by spraying with 10% H2SO4 in EtOH
followed by heating.
3.3.1 Notoginsenoside ST-4 (1)
A pale amorphous powder: mp 187–1918C;
26
½aꢀD þ3.5 (c ¼ 0.209, MeOH); IR (KBr)
n
max (cm21) 3422, 2942, 2878, 1637, 1452,
1
1376, 1307, 1077, 1043, and 896; H and
13C NMR spectral data (Table 1). FAB-MS
(negative ion mode): m/z 916 [M]2, 784
[M-132(xylosyl)]2, 622 [M-132(xylosyl)-
162(glucosyl)]2; HR-ESI-MS (negative
ion mode): m/z 915.5323 [M 2 H]2
(calcd for C47H79O17, 915.5317).

502
Y. Pei et al.
Table 1. 13C and 1H NMR spectroscopic data of compound 1 (d in ppm, J in Hz, in C5D5N).
No.
1
dC
dH
No.
23
dC
dH
2.62 m
2.31 m
39.2
1.49 m
0.75 m
2.18 m
1.81 m
23.1
2
26.8
24
25
26
27
28
29
30
GlcI-10
20
126.4
130.8
25.9
17.8
28.2
16.7
17.1
104.8
83.0
78.4
71.1
78.7
62.9
5.30 t (7.0)
3
4
5
6
88.9
39.8
56.4
18.5
3.29 dd (4.5, 11.5)
1.66 s
1.63 s
1.29 s
1.11 s
0.96 s
4.92 d (8.0)
4.11 m
4.35 m
4.13 m
3.95 m
4.57 br d (7.5)
4.35 m
5.51 d (7.5)
4.19 m
4.28 m
4.20 m
3.87 m
4.49 br d
5.39 d (6.5)
4.11 m
0.69 m
1.51 m
1.38 m
1.47 m
1.24 m
7
35.2
8
9
10
11
40.0
50.4
37.0
32.1
30
1.39 m
40
50
2.03 m
1.53 m
3.95 m
2.02 m
60
12
13
14
15
71.2
48.6
51.8
31.4
GlcI0I0-100
103.2
84.6
77.8
71.8
78.0
63.0
106.5
76.0
77.8
70.8
67.5
200
3
1.60 m
1.06 m
1.91 m
1.35 m
2.35 m
0.78 s
400
500
16
26.9
600
Xyl-1000
2000
17
18
19
20
21
22
54.8
16.4
15.9
73.0
27.1
35.9
3000
4.31 m
4.12 m
4.33 m
3.68 m
0.96 s
4000
5000
1.43 s
2.03 m
1.71 m
3.4 Acid hydrolysis of compound 1
equipped with an H2 flame ionization
detector. The column was 30QC2/AC-5
quartz capillary column (30 m £ 0.32 mm)
with the following conditions: column
temperature: 1808C/2808C, programed
increase: 38C/min, carrier gas: N2
(1 ml/min), injector and detector tempera-
ture: 2508C, injection volume: 4 ml, and
split ratio: 1/50. The configuration of the
sugar moiety was determined by compar-
ing the retention time with the derivatives
of the authentic samples. The retention
times of D-/L-glucose and D-/L-xylose were
19.715/20.159 and 14.606/15.256 min,
respectively. The configurations of the
sugar moieties from the new compound
were D-glucose and D-xylose.
The compound (8 mg) was hydrolyzed
with 2 M HCl–dioxane (1:1, 4 ml) under
reflux for 8 h, respectively. The reaction
mixture was partitioned between H2O and
CHCl3 (2 ml £ 3). The aqueous layer was
neutralized with 2 M NaOH, and then dried
to give a monosaccharide mixture. A
solution of the sugar mixture in pyridine
(2 ml) was added to L-cysteine methyl ester
hydrochloride (about 1.5 mg) and kept at
608C for 1 h. Then, trimethylsilylimidazole
(about 1.5 ml) was added to the reaction
mixture and kept at 608C for 30 min. The
mixture was detected by gas chromatog-
raphy (GC) analysis. The GC analysis was
run on Agilent Technologies HP5890 GC

Journal of Asian Natural Products Research
503
3.5 Cell and virus
with minor modifications. Briefly, the Vero
cell monolayer was seeded in a 24-well
culture plate and then prechilled at 48C for
1 h. The cell monolayer was infected with
HSV-1 at a multiplicity of infection of 10 in
the absence or the presence of various
concentrations of notoginsenoside ST-4.
After further incubation at 48C for 3 h, the
medium was aspirated and the cell mono-
layer was overlaid with methylcellulose
medium. After 3 days of incubation, the
plaque reduction assay and statistical
analysiswereperformedasdescribedabove.
African green monkey kidney cells (Vero;
ATCC CCL81) were propagated in Dul-
becco’s Modified Eagle Medium (DMEM;
Invitrogen, Carlsbad, CA, USA) sup-
plemented with 10% fetal bovine serum
(FBS; Invitrogen, Carlsbad, CA, USA).
HSV-1 strain F (ATCC VR733) and HSV-2
strain 333 were propagated in Vero cells
and stored at 2808C until use. Virus titers
were obtained by plaque assay.
3.6 Cytotoxic assay, antiviral assay, and
statistical analysis
3.8 Penetration assay
The cytotoxicity in Vero cells was deter-
mined by the XTT (tetrazolium salt (2,3-
bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide)) assay [10].
Briefly, cells were seeded in a 96-well
plate. After 24 h of incubation, cells were
treated with or without various concen-
trations of notoginsenoside ST-4 and were
then incubated for 72 h. Later, the XTT
solution and phenazine methosulphate
were added. The optical densities were
measured with enzyme immunoassay
reader (BIO-RAD 550) at a test wavelength
of 480 nm and a reference wavelength of
630 nm. CC50 was calculated [11].
The penetration assay was performed
according to the published procedure [11]
with minor modifications. The Vero cell
monolayer was prechilled at 48C for 1 h.
The cell monolayer was infected with
HSV-1 at a multiplicity of infection of 10
and incubated at 48C for 3 h. And then the
infected cell monolayer was incubated at
378C with or without notoginsenoside ST-
4 for 10 min. After 10 min intervals, the
infected cell monolayer was treated with
acidic phosphate buffered saline (PBS, pH
3) for 1 min to inactivate nonpenetrating
virus. Immediately after the addition of
PBS (pH 11) to neutralize acidic PBS (pH
3), the neutral PBS was removed. The cell
monolayer was washed with PBS (pH 7)
and overlaid with methylcellulose med-
ium. After 3 days of incubation, the plaque
reduction assay and statistical analysis
were performed as described above.
Antiviral activity was assessed with
plaque reduction assay. Vero cells were
infected with HSV-1 or HSV-2 (30 pfu/-
well) in the presence or absence of
notoginsenoside ST-4 for 2 h and then
overlaid with medium containing 1% of
methylcellulose. The plate was incubated
for 3 days before being fixed with formalin
and then stained with crystal violet. EC50
was calculated by regression analysis of the
dose–response curves generated from
plaque reduction assay [11]. Each exper-
iment was performed at least three times.
Results were expressed as mean ^ SD.
3.9 Indirect immunofluorescence and
confocal microscopy
Cells were grown on coverslips and
infected with HSV-1 at a multiplicity of
infection of 10 for 2 h for viral adsorption
on ice. Cells were washed with PBS (pH 7)
and transferred into medium with or
without notoginsenoside ST-4 (10 mM)
and incubated for another 2 h at 378C.
Then the cells were fixed for 15 min with
3.7 Attachment assay
The attachment assay was conducted
according to the published procedure [11]

504
Y. Pei et al.
U0632010, 2008ZX09401-004, 2011CB915
503, the State Key Laboratory of Phytochem-
istry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Acad-
emy of Sciences (P2008-ZZ08) and ‘211 grant
of the Ministry of Education (MOE)’.
4% paraformaldehyde (PFA), blocked
with PBS containing 1% BSA for 1 h and
subsequently incubated with anti-vp5
antibody (Santa Cruz; CA, USA) at
dilutions of 1:500, followed by an Alexa
Flour 488 secondary antibody (Molecular
Probes, Invitrogen; Carlsbad, CA, USA)
for 1 h. After each step, the slides were
washed repeatedly with PBS, and finally
they were preserved with PBS. The
additional nuclear staining with 40,6-
diamidino-2-phenylindole (DAPI, Mol-
ecular Probes) was performed for 20 min.
Fluorescence was recorded with a confocal
laser scan microscope (LSM 510 meta;
Zeiss, Germany).
References
[1] D.J. Ye and S.C. Zhang, Chinese Medic-
inal Herbs Preparation 187 (1999).
[2] P.Y. Liao, D. Wang, Y.J. Zhang, and C.R.
Yang, J. Agric. Food Chem. 56, 1751
(2008).
[3] J.A. Levy, Lancet 349, 558 (1997).
[4] K. Radtke, K. Dohner, and B. Sodeik, Cell
Microbiol. 8, 387 (2006).
[5] R.D. Everett, Cell Microbiol. 8, 365
(2006).
[6] K.A. Cassady and R.J. Whitley,
J. Antimicrob. Chemother. 39, 119
(1997).
[7] J.A. Englund, M.E. Zimmerman, E.M.
Swierkosz, J.L. Goodman, D.R. Scholl,
and H.H. Balfour, Ann. Intern. Med. 112,
416 (1990).
[8] J.T. Chen, H.Z. Li, D. Wang, Y.J. Zhang,
and C.R. Yang, Helv. Chim. Acta. 89,
1442 (2006).
[9] R.W. Teng, H.Z. Li, J.T. Chen, D.Z.
Wang, Y.N. He, and C.R. Yang, Magn.
Reson. Chem. 40, 483 (2002).
Cells were grown on coverslips and
infected with HSV-1 at a multiplicity of
infection of 10 for 2 h on ice. After
absorption, cells were washed three times
with PBS (pH 7) and transferred into the
medium with or without notoginsenoside
ST-4 (10 mM) and incubated for another
10 h at 378C. Then, the cells were fixed for
15 min with 4% PFA and permeabilized
with 0.02% Triton X-100 for 2 h. And then
the cells were blocked and stained as
described above.
[10] T. Meshulam, S.M. Levitz, L. Christin,
and R.D. Diamond, J. Infect. Dis. 172,
1153 (1995).
Acknowledgements
This study was supported by the National
Nature Science Foundation of China (NSFC)
[11] H.Y. Cheng, C.C. Lin, and T.C. Lin,
Antiviral. Res. 55, 447 (2002).