Inhibition activities of polysaccharide (RG4-1) from Gentiana rigescens against RSV

Article abstract of DOI:10.1080/10286020.2011.573628

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infection in infants and young children. With the emergence of drug-resistant strains of RSV, new antiviral agents are needed urgently. Gentiana rigescens is a kind of Chinese herb, belonging to Gentianaceae, which has long been used as a folk medicine for curing inflammation, bacterial infection, viral infection, and so on. In this research, polysaccharide designated RG4-1 was isolated from G. rigescens by hot water extraction, ethanol precipitation, and macroreticular adsorbing resin column chromatography, and its antiviral activity, cytotoxicity, and possible antiviral mechanisms were assayed by cytopathogenic effect inhibition assay, 3-(4,5-dimethythiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, and plaque reduction assay. RG4-1 was a fructose-binding lectin. In host cell cultures, RG4-1 was found to be an effective antiviral component against RSV. It showed good inhibitory effect against RSV when it was added 2h after virus infection with 50% effective concentration of 12.86μg/ml. RG4-1 also displayed its direct inactivation, attachment inhibition effect, and penetration inhibition effect against RSV. A time-dependent experiment was set up to confirm that RG4-1 blocked RSV infection at early stages of the infection. But RG4-1 seemed to be ineffective against intracellular virus and viral biosynthesis.

Full text of DOI:10.1080/10286020.2011.573628

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Inhibition activities of polysaccharide
(RG4-1) from Gentiana rigescens against
RSV
Xin Li a , Jing-Yu Zhou a , Qing-Wei Yang a , Yang Chen a , Ying-Ai
Piao b & Hong-Yuan Li a
a Department of Epidemiology, College of Public Health, Harbin
Medical University, Harbin, 150086, China
b Department of Pharmacochemistry, Heilongjiang Provincial
Institute for Drug Control, Harbin, 150086, China
Published online: 25 May 2011.
To cite this article: Xin Li , Jing-Yu Zhou , Qing-Wei Yang , Yang Chen , Ying-Ai Piao & Hong-Yuan Li
(2011) Inhibition activities of polysaccharide (RG4-1) from Gentiana rigescens against RSV, Journal
of Asian Natural Products Research, 13:06, 512-522, DOI: 10.1080/10286020.2011.573628
To link to this article: http://dx.doi.org/10.1080/10286020.2011.573628
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Journal of Asian Natural Products Research
Vol. 13, No. 6, June 2011, 512–522
Inhibition activities of polysaccharide (RG4-1) from Gentiana rigescens
against RSV
Xin Lia†, Jing-Yu Zhoua†, Qing-Wei Yanga, Yang Chena, Ying-Ai Piaob and
Hong-Yuan Lia*
aDepartment of Epidemiology, College of Public Health, Harbin Medical University, Harbin
150086, China; bDepartment of Pharmacochemistry, Heilongjiang Provincial Institute for Drug
Control, Harbin 150086, China
(Received 21 February 2010; final version received 17 March 2011)
Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract
infection in infants and young children. With the emergence of drug-resistant strains of
RSV, new antiviral agents are needed urgently. Gentiana rigescens is a kind of Chinese
herb, belonging to Gentianaceae, which has long been used as a folk medicine for curing
inflammation, bacterial infection, viral infection, and so on. In this research,
polysaccharide designated RG4-1 was isolated from G. rigescens by hot water
extraction, ethanol precipitation, and macroreticular adsorbing resin column chroma-
tography, and its antiviral activity, cytotoxicity, and possible antiviral mechanisms were
assayed by cytopathogenic effect inhibition assay, 3-(4,5-dimethythiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay, and plaque reduction assay. RG4-1 was a
fructose-binding lectin. In host cell cultures, RG4-1 was found to be an effective antiviral
component against RSV. It showed good inhibitory effect against RSV when it was added
2 h after virus infection with 50% effective concentration of 12.86 mg/ml. RG4-1 also
displayed its direct inactivation, attachment inhibition effect, and penetration inhibition
effect against RSV. A time-dependent experiment was set up to confirm that RG4-1
blocked RSV infection at early stages of the infection. But RG4-1 seemed to be
ineffective against intracellular virus and viral biosynthesis.
Keywords: respiratory syncytial virus (RSV); Gentiana rigescens; antiviral agent
1. Introduction
recover from RSV-induced bronchiolitis are
at increased risk for the development of
recurrent wheeze and asthma in later
childhood [2]. In older patients, RSV may
exacerbate an underlying condition or
pulmonary and cardiac manifestations. It is
also reported that RSV produces significant
morbidity in those with underlying con-
ditions, especially cardiopulmonary and
immunosuppressive diseases. In transplant
units, infection of RSV may be occult and is
associated with high mortality rates [3].
Recently, only two approved products have
been found to be effective for the treatment
Human respiratory syncytial virus (RSV)
belongs to Family Paramyxoviridae, Pneu-
movirinae subfamily, and is the leading
cause of severelowerrespiratory tract illness
in infants and young children. It is reported
that RSV was detected in 77% of children
with bronchiolitis [1]. Virtually all children
experience RSV infection at least once
duringthefirst2yearsoflifeandpartofthem
develop bronchiolitis and more severe
disease requiring hospitalization, usually in
the first 6 months of life. Children who
*Corresponding author. Email: lihy00@yahoo.com.cn; lihongyuan@ems.hrbmu.edu.cn
†These authors contributed to this paper equally.
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.573628
http://www.informaworld.com

Journal of Asian Natural Products Research
513
or prevention of RSV infection: the nucleo-
side analog ribavirin for the treatment and
themonoclonalantibodypalivizumabforthe
prophylaxis [4]. However, for the significant
limitations such as poor safety and high cost
of these two agents, new therapeutics with
improved efficacy and safety are needed.
A small molecule inhibitor A-60444,
which is a highly potent inhibitor of RSV
replication, is on its phase II clinical trial. It
shows potent sub-micromolar activity in
plaque assay against RSV with the 50%
inhibitory concentration (IC50) values of
0.7–0.9 mM, approximately 30 times more
active than ribavirin. Moreover, natural
products from plants have been found to be
another potent source for the discovery of
antiviral agents now [5]. Gentianarigescens,
a Chinese medicinal herb, which is com-
monlyfoundinChina, consistedofGentiana
manshurica Kitag, Gentiana scabra Bge,
Gentiana triflora Pall, and G. rigescens
Franch.SinceitwasfirstreportedbyHayashi
in 1976 [6], many researchers have under-
taken numerous studies on G. rigescens. It
inhibited the release of several intrahepatic
enzymes, which was parenchymal injury
parameter [7], and caused a 50% increase
in benzo(a)pyrene hydroxylase activity in
rats [8]. Meanwhile, the aqueous extracts
from G. rigescens have anti-inflammatory,
antimicrobial, and antiviral effects [9].
Heilongjiang confirmed that the G. riges-
cens used in this research was genuine by
high-pressure liquid chromatography
(HPLC)-fingerprint analysis. During the
initial screening of herbal extracts for
antiviral activity, the hot water extract from
the root of G. rigescens displayed good
activity in inhibiting RSV. As little is
known about the anti-RSV activity of G.
rigescens, experiments for isolating the
active anti-RSV principle from this plant
were initiated. After ethanol precipitation,
the dark precipitate of G. rigescens aqueous
extract showed anti-RSV activity in cyto-
pathogenic effect (CPE) inhibition assay.
The anti-RSV compound present in the
precipitate was further fractionated by
filtration chromatography on a macroreti-
cular adsorbing resin column. Upon assay-
ing, the active compound was detected in
pooled fractions 1, 2, 3, 4, and 5, in which
fraction 1 (RG4-1) had the highest activity.
The purity of RG4-1 was analyzed by
HPLC and spectrophotometry. As shown
in Figures 1 and 2, RG4-1 showed a
characteristic absorption peak within 260–
280 nm in the ultraviolet spectra indicating
that RG4-1 contained no protein or nucleic
acid. Meanwhile, samples from RG4-1
contained essentially one peak on the
ODS2 column indicating that the anti-
RSV compound was relatively pure.
In previous studies, the hot water extract
from the roots of G. rigescens was found to
have good antiviral activity against RSV. It
might be a potential anti-RSV substance for
treating RSV infection. In this research,
the bioassay-directed fractionation was used
to isolate a polysaccharide from the roots
of G. rigescens which displays anti-RSV
activity. The isolation, purification, chemi-
calnature, andantiviralactivityofthewater-
soluble polysaccharide and its possible
antiviral mechanism are described.
2.2 Chemical nature of RG4-1
RG4-1 was soluble in water but insoluble
in methanol or ethanol. The Molisch test
showed the presence of a purple ring at
the junction of two layers, and this
indicated the presence of carbohydrates.
In the Iodine test, the sample turned violet
after the addition of Iodine. The result
indicated the presence of polysaccharide.
After RG4-1 was hydrolyzed in 2 N
trifluoroacetic acid at 1208C for 1 h, the
hydrolysate was analyzed by thin-layer
chromatography (TLC). One spot that had
the same Rf value of fructose standard was
identified. The results indicated that
2. Results and discussion
2.1 Extraction and isolation of RG4-1
The Traditional Chinese Drug Laboratory,
Provincial Institute for Drug Control of

514
X. Li et al.
20.00
0.00
–20.00
–40.00
–60.00
–80.00
–100.00
–120.00
–140.00
–160.00
–180.00
–200.00
–220.00
–240.00
–260.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
Minutes
Figure 1. HPLC of RG4-1. RG4-1 was dissolved in deionized water (0.2 mg/ml) and 10 ml was
injected into a Spherisorb ODS2 column. Compound was eluted with isotonic sodium chloride at a
flow rate of 1.0 ml/min and detected with Differential Refractive Index Detector.
fructose was the constituent monosacchar-
ide of RG4-1.
trations (CC50s) of RG4-1 against HeLa
cell, Hep-2 cell, and Vero cell were 4.88,
5.01, and 5.22mg/ml, respectively, and
CC50s of ribavirin against HeLa cell, Hep-
2 cell, and Vero cell were 1.09, 1.09, and
1.11mg/ml, respectively.
2.3 Biological characteristics
2.3.1 Cytotoxicity
To delineate whether the suppressive effect
of RG4-1 on RSV replication was related to
cytotoxicity, the viability of host cells was
examined after they were treated with RG4-
1 for 3 days by 3-(4,5-dimethythiazol- 2-yl)-
2,5-diphenyl tetrazolium bromide (MTT)
assay. Fifty percent cytotoxic concen-
2.3.2 Anti-RSV activity of RG4-1
RG4-1 showed good antiviral activity
against RSV with 50% effective concen-
tration (EC50) of about 15 mg/ml in MTT
assay. The activity of RG4-1 against RSV
1.0000
0.7625
0.5250
0.2875
0.0500
190.00
300.00
Detective wavelength
400.00
Figure 2. Ultraviolet spectrum of RG4-1. RG4-1 at the concentration of 0.5 mg/ml was scanned by
TU-1901 ultraviolet spectrophotometer at the range of 190–400 nm.

Journal of Asian Natural Products Research
Table 1. Anti-RSV effect of RG4-1 and ribavirin in vitro.
515
RG4-1 (mg/ml)
Ribavirin (mg/ml)
a
a
Culture cell
HeLa cell
Tested method
EC50
TIb
EC50
TIb
CPE
MTT
CPE
MTT
CPE
MTT
62.45 ^ 8.78
12.64 ^ 1.25
50.26 ^ 3.28
13.67 ^ 1.50
48.75 ^ 5.40
13.48 ^ 3.15
40.38 ^ 1.49
78.14
386.08
103.86
381.86
102.97
372.40
124.32
51.40 ^ 5.29
12.86 ^ 0.79
32.93 ^ 3.24
18.66 ^ 1.47
30.73 ^ 4.11
9.20 ^ 3.51
22.31 ^ 1.40
21.23
87.76
33.40
58.95
35.47
118.48
48.85
Vero cell
Hep-2 cell
Plaque reduction assay
Notes: Antiviral drugs were added into cells 2 h after virus inoculation. The antiviral activity of drugs was
analyzed by CPE inhibition assay, MTT assay, and plaque reduction assay, respectively, as compared with that of
control. Each value of EC50 was the mean ^ SD of three independent experiments.
a EC50 was 50% effective concentration in host cells.
b TI was the ratio of CC50 to EC50
.
was further confirmed by plaque reduction
assay. RG4-1 showed a dose-dependent
inhibition against RSV when RG4-1 was
added 2 h after infection. The therapeutic
index (TI) was calculated based on their
CC50s and EC50s. The narrowest TI of
RG4-1 was 78-fold, but the range was as
great as 386-fold, which was much higher
than ribavirin (Table 1).
volume of medium. Pre-incubation of
RSV with RG4-1 produced a significant
reduction in remaining infectivity of RSV.
The data strongly confirmed that RG4-1
acted as a viricidal agent in the research
(Table 2).
2.3.4 Attachment inhibition effect of
RG4-1
To investigate the attachment inhibition
effect of RG4-1 against RSV, serial
dilution of RG4-1 was added with RSV
simultaneously. In attachment inhibition
assays, RG4-1 displayed anti-RSV
effect remarkably. The results are given
in Table 3. In the experiment, RG4-1
2.3.3 Direct killing effects of RG4-1
To investigate the direct inactivating effect
of RG4-1 against RSV, serial dilution of
RG4-1 was incubated with RSV at 378C
for 2 h before infection. In the virus control
group, diluted virus was mixed with equal
Table 2. Viricidal effect of RG4-1 against RSV in vitro.
RG4-1 (mg/ml)
a
Culture cell
HeLa cell
Tested method
EC50
TIb
CPE
MTT
CPE
MTT
CPE
MTT
61.15 ^ 7.38
13.59 ^ 0.77
47.91 ^ 1.95
13.62 ^ 1.00
49.80 ^ 1.33
10.45 ^ 0.94
26.23 ^ 1.08
79.80
359.09
108.95
383.26
100.80
480.38
191.38
Vero cell
Hep-2 cell
Plaque reduction assay
Notes: Viral suspension was pre-incubated with different concentrations of RG4-1 at 378C for 2 h. Then the
mixture was used to infect host cells. The direct killing effect of RG4-1 was analyzed by CPE inhibition assay,
MTT assay, and plaque reduction assay, respectively, as compared with that of control. Each value of EC50 was
the mean ^ SD of three independent experiments.
a EC50 was 50% effective concentration in host cells.
b TI was the ratio of CC50 to EC50
.

516
X. Li et al.
Table 3. Inhibition effect of RG4-1 on adsorption in vitro.
RG4-1 (mg/ml)
a
Culture cell
HeLa cell
Tested method
EC50
TIb
CPE
MTT
CPE
MTT
CPE
MTT
127.25 ^ 18
34.01 ^ 2.23
82.42 ^ 3.02
47.11 ^ 4.90
93.71 ^ 5.29
27.13 ^ 1.50
46.23 ^ 2.35
38.35
143.49
63.33
110.80
53.57
Vero cell
Hep-2 cell
185.03
108.59
Plaque reduction assay
Notes: Viral suspension together with different concentrations of RG4-1 was added into host cell at 378C. After
adsorbing for 2 h, the mixture was replaced with maintenance medium. The attachment inhibiting effect of RG4-1
was analyzed by CPE inhibition assay, MTT assay, and plaque reduction assay, respectively, as compared with
that of control. Each value of EC50 was the mean ^ SD of three independent experiments.
a EC50 was 50% effective concentration in host cells.
b TI was the ratio of CC50 to EC50
.
displayed its strong attachment inhibition
effect at the smallest EC50 of 27.13 mg/ml.
2.3.6 Effect of RG4-1 on viral growth
The effect of RG4-1 was further investi-
gated in a one-step growth research in which
RG4-1 was added at 0, 2, 4, 6, and 8 h post-
infection. In the experiment, culture med-
ium instead of RG4-1 was added in virus
control group. The extracellular and intra-
cellular viral yields were measured. As
expected, the polysaccharide reduced more
than 80% of the extracellular viral yield
for each of the time points as compared
to controls, but the intracellular viral yield
was not reduced dramatically for the
respective time points. A parallel exper-
iment carried out using ribavirin showed
2.3.5 Penetration inhibition effect of
RG4-1
To investigate the penetration inhibition
effect of RG4-1, RG4-1 diluted in series
was added to cells after viral attachment at
48C. For RSV strain tested, there was
significant difference in the RG4-1 treated
cells compared with the viral control (Table
4). In the experiment, RG4-1 displayed its
strong penetration inhibition effect at the
smallest EC50 of 26.96 mg/ml.
Table 4. Inhibition effect of RG4-1 on penetration in vitro.
RG4-1 (mg/ml)
a
Culture cell
HeLa cell
Tested method
EC50
TIb
CPE
MTT
CPE
MTT
CPE
MTT
94.40 ^ 10.81
36.70 ^ 6.30
70.46 ^ 7.35
32.94 ^ 1.39
68.29 ^ 5.84
26.96 ^ 3.17
41.98 ^ 2.66
51.69
132.97
73.80
158.47
73.51
Vero cell
Hep-2 cell
186.20
119.58
Plaque reduction assay
Notes: Viral suspension was infected into host cells followed by 2 h of adsorption at 48C. After two washes with
PBS, different concentrations of RG4-1 were added at 378C. The penetration inhibiting effect of RG4-1 was
analyzed by CPE inhibition assay, MTT assay, and plaque reduction assay, respectively, as compared with that of
control. Each value of EC50 was the mean ^ SD of three independent experiments.
a EC50 was 50% effective concentration in host cells.
b TI was the ratio of CC50 to EC50
.

Journal of Asian Natural Products Research
517
Table 5. The virus titer of extracellular virus and intracellular virus at different times after viral
infection.
Extracellular virus
Intracellular virus
Time course
(h)a
Drug
Pfu/ml £ 106 Inhibition (%)b Pfu/ml £ 106 Inhibition (%)b
0
2
4
6
8
Ribavirin
RG4-1
Virus control
Ribavirin
RG4-1
Virus control
Ribavirin
RG4-1
Virus control
Ribavirin
RG4-1
1.33
1.67
24.30
2.00
2.33
24.30
2.33
3.00
24.70
3.00
3.67
95
93
–
92
90
–
91
88
–
88
85
–
1.67
22.64
33.30
2.70
25.61
33.70
2.98
24.31
33.30
7.08
26.62
33.70
12.24
24.14
34.00
95
32
–
92
24
–
91
27
–
79
21
–
**
**
**
**
**
**
*
**
**
**
**
**
**
Virus control
Ribavirin
RG4-1
24.30
3.33
4.33
87
83
–
64
29
–
**
**
Virus control
25.30
Notes: Hep-2 cells were pre-chilled at 4 8C and infected with RSV for 2 h at 4 8C to synchronize the initial stage of
infection and adsorption. Virus inoculum was then removed and cultures were washed three times with pre-
chilled PBS. At 0, 2, 4, 6, and 8 h post-infection, RG4-1 at a final concentration of 200 mg/ml was added. In the
virus control group, culture medium instead of RG4-1 was added. In the ribavirin control group, ribavirin at a final
concentration of 200 mg/ml was added. The culture was further incubated to 24 h post-infection, and then the titer
of extracellular and intracellular virus was determined by plaque assay.
aThe time when compound was added.
bPercentage of plaque inhibition is indicated in parentheses ¼ [(Plaque number in the virus control group–Plaque
number in the treatment group)/Plaque number in the virus control group] £ 100.
*Compared with virus control group, P , 0.05; compared with virus control group, P , 0.01.
**
that the extracellular viral yield was also
reduced by more than 85%. Moreover, the
intracellular viral yield was reduced by 95,
95, 91, 79 and 64% of the controls for the
respective time points. These results indi-
cated a clear difference between the effect
of RG4-1 and ribavirin on RSV growth.
RG4-1 displayed excellent inhibitory effect
on extracellular virus (Table 5).
competitor of receptor to viral glyco-
proteins, are known to affect the growth of
animal viruses [10]. Polysaccharide such as
fucoidan was found to have antiviral
activity, which was associated with its effect
on adsorption inhibition and preventing
viral penetration into the macrophages [11].
The results of our research demon-
strated that RG4-1 inhibited RSV in vitro.
The inhibition was concentration depen-
dent and specific. RG4-1 showed good
inhibitory effect against RSV with the
minimal EC50 of 12.64 mg/ml when it was
added 2 h after viral infection. Considering
its CC50 that was about 5 mg/ml, the
therapy index of RG4-1 was up to 386.08,
which was much higher than ribavirin. The
results showed that RG4-1 might be a
potent substance for treating RSV
infection.
2.4 Discussion
In this research, RG4-1 was isolated from
the roots of G. rigescens by hot water
extraction, ethanolprecipitation,andmacro-
porous adsorptive resin chromatography.
The active principle RG4-1 was a kind of
polysaccharide, asindicated bythe results of
the Molisch test and Iodine test. Polysac-
charides, which sometimes play the part of a

518
X. Li et al.
In the direct killing effects assay, RG4-1
virus-cell binding and/or virus-cell fusion
process. RG4-1 might act with viral G
glycoprotein or F glycoprotein directly. One
of the mechanisms of the anti-RSV action of
RG4-1 has been attributed to an inhibition
of virus binding to the cell membrane. This
is further supported by other studies, which
indicated that polysaccharide inhibited the
virus by inhibiting viral adsorption and
penetration [12]. Moreover, in the research,
RG4-1 also displayed its direct killing
effect. Results from this research indicated
that one mode of action of RG4-1 against
RSV appeared to be dependent on its
virucidal activity.
displayed excellent inhibitory effect on
extracellular virus with the minimal EC50 of
10.45 mg/ml and a therapy index of 480.
Besides, when RG4-1 was present during
adsorption, significant antiviral effect was
observed even at an EC50 of 27.13 mg/ml
when comparisons were made to virus
control. In penetration inhibition assay,
RSV binds to cells but does not penetrate
through the cell membrane at 48C. The virus
starts to penetrate the cell membrane at
378C. In the experiment, obvious antiviral
effect was observed in the presence of RG4-
1 at an EC50 of 26.96 mg/ml.
In the biomacromolecule synthesis
inhibition assay, RG4-1 did not show any
antiviral effect. As the experiment showed,
RG4-1 was added to the infected cells after
2 h of virus infection. Until 8 h later, it was
replaced by maintenance media. Finally,
RG4-1 did not have any inhibitory effect
on the virus biological macromolecule
synthesis. An explanation was that the
biomacromolecule synthesis processes
started at about 4 h after virus infection.
Approximately 6–8 hours later, the repro-
duction processes were done and the
filial virus was released. In the first
8 hours, RG4-1 killed parts of extracellular
virus. But 8 hours later the filial virus was
released and RG4-1 was removed, so that
the filial virus continued to infect the other
cells. That is to say, the biological
macromolecule synthesis processes as well
as following assembly release process are
not influenced.
For confidence, three different host cells
and three methods were used in the research
and the results were highly coincident. In
this research, the anti-RSV effect of RG4-1
was proved by these three methods. The
result confirmed that RG4-1 might be a
potential anti-RSV drug candidate.
A disadvantage of this research was
that the EC50 of the positive drug ribavirin
was much higher than that previously
reported, especially in CPE inhibition
assay [13]. A possible reason was that
the virus infectivity of RSV in the two
experiments was not the same, or the
therapeutic effect of ribavirin used in the
two experiments is not the same.
In conclusion, a polysaccharide from
the roots of G. rigescens had been isolated
and its strong anti-RSV activity was
observed. This polysaccharide had strong
direct killing activity, adsorption inhibition
activity, and penetration inhibition activity
against RSV. Infurther research, manyother
viruses such as influenza virus, parain-
fluenza virus, and adenovirus, will be used
to test the antiviral activity of RG4-1.
Over the time course, the dramatic
antiviral effect against RSV was observed
at no more than 6 h post-infection. That is
to say, RG4-1 impacted the early stage of
virus replication. Besides, RG4-1 only had
poor inhibitory effect against intracellular
virus but had strong efficient inhibitory
effect against extracellular virus.
3. Materials and methods
3.1 Isolation and purification of
polysaccharide from G. rigescens
In conclusion, RG4-1 inhibited RSV
in vitro when present during pre-incubation,
adsorption, and penetration. It means
that RG4-1 might have interfered with the
Dried roots of G. rigescens (500g) from
Shiyitang Pharmaceutical Factory (Harbin,
China) were authenticated by the Tra-

Journal of Asian Natural Products Research
519
ditional Chinese Drug Laboratory, Provin-
cial Institute for Drug Control of Heilong-
jiang. The washed roots were disintegrated
with a blender and extracted with 5 L of
distilled water at 1008C for 1 h. The aqueous
extract was clarified through a cotton cloth
and the residue was extracted again as
before. Then, the mixed aqueous extract was
condensed to 500 ml. The condensate was
precipitated by 90% ethanol sequentially at
48C overnight and the precipitate was
collected by centrifugation. The concen-
trated crude polysaccharide was further
fractionated on a macroreticular adsorbing
resin column and eluted with deionized
water, followed by 30, 60, and 90% ethanol.
The collected water-eluted part was further
fractionated on a macroporous resin column
(50 £ 2.5 cm, macroreticular adsorbing
resin X-5, purchased from the Chemical
Plant of Nankai University, Tianjin, China)
and eluted with deionized water. Fractions
of5 mlwerecollected and the fractions were
freeze-dried. The first part of the fractions
was an ivory powder named RG4-1.
CH3OH and water (70:30) at a flow rate
of 1.0 ml/min. Twenty microliters of the
sample were injected into the system and
detected with a UV absorbance at the
wavelength of 270 nm [14]. The operating
temperature was maintained at 25.08C.
3.3 Purity detection of RG4-1
3.3.1 High-pressure liquid
chromatography (HPLC)
For the analysis of the purity of RG4-1,
RG4-1 was dissolved in deionized water
(0.2 mg/ml) and 10 ml was injected into a
Spherisorb ODS2 column (125 £ 4.0 mm,
5 mm, Waters, MA, USA). The result is
shown in Figure 1. The compound was
eluted with isotonic NaCl at a flow rate of
1.0 ml/min and detected with 2414 Differ-
ential Refractive Index Detector (Waters,
MA, USA).
3.3.2 Ultraviolet spectra scanning
RG4-1 at the concentration of 0.5 mg/ml
was scanned by TU-1901 ultraviolet
spectrophotometer at the range of 190–
400 nm. The result is shown in Figure 2.
3.2 HPLC analysis of the extract of
G. rigescens
To identify the G. rigescens, gentiopicrin, a
chemical ingredient of G. manshurica
Kitag, was used as a marker substance for
the HPLC-fingerprint analysis. Gentiopi-
crin (110770–200510) was provided by
China Institute for Drug and Biologic
Product Standardization. The HPLC sys-
tem consisted of a Shimadzu LC-10AT
Liquid Chromatograph (Tokyo, Japan)
equipped with a Shimadzu SPD-10Avp
UV–VIS detector, a Shimadzu SCL-
10Avp system controller, a Shimadzu
CTO-10ACvp column oven, and a Shi-
madzu DUG-12A Degasser. A Diamonsil
C18 (4.6 £ 250 mm, 5 mm; DIKMA, St.
Louis, MO, USA) and security guard
cartridges Gemini C18 (4 £ 3.0 mm; Phe-
nomenex, Los Angeles, CA, USA) were
used. The sample was eluted with the
mobile phase that was composed of
3.4 Chemical tests
3.4.1 Test for polysaccharide
3.4.1.1 The Molisch test. For the analysis
of the chemical character of RG4-1, two
drops of 5% L-napthol solution (in ethanol)
was added and mixed to 2 ml of 0.1%
RG4-1. Then 2 ml of concentrated sulfuric
acid was poured to the side of the tube.
3.4.1.2 The Iodine test. One milliliter of
the sample was taken in a test tube and one
drop of iodine was added.
3.4.2 Acid hydrolysis of RG4-1
A total of 5 mg of RG4-1 was hydrolyzed in
2 N trifluoroacetic acid at 1208C for 1 h.
The hydrolysate was dried using a rotary
evaporator under reduced pressure at

520
X. Li et al.
408C and the residues obtained were
dissolved in water and analyzed by
TLC using the solvent system: n-
butanol:acetoacetate:water ¼ 5:4.5:0.5.
Glucose, galactose, xylose, mannose,
rhamnose, levulose, arabinose, and mal-
tose were used as the standards [14]. Sugars
were revealed by staining the thin-layer
plate with aniline-o-phthalic acid at 1108C
for 5 min.
concentration of 5 mg/ml was added.
Then, all wells were incubated again for
4 h. The supernatants were removed and
150 ml of dimethyl sulfoxide (DMSO) was
added to each well. Plates were incubated
in the dark for 10 min at room temperature
to solubilize the dye and then gently mixed
on a 96-well plate-adapted vortexer for
30 s. The absorption was measured at the
wavelength of 550 nm. The cytotoxicity
was expressed as CC50.
The cytotoxic effect of ribavirin was
also observed in the same way.
3.5 Materials
3.5.1 Virus and cells
RSV (Long strains) and host cells (HeLa
cell, Hep-2 cell, and Vero cell) were kindly
provided by the Institute of Virology,
Chinese Academy of Preventive Medicine.
All cells were routinely propagated in
Modified Eagle’s medium (MEM) and
supplemented with 10% heat-inactivated
fetal bovine serum (FBS), and 100 IU/ml
of penicillin G and 100 mg/ml streptomy-
cin were incubated under 5% CO2 at 378C.
The concentration of serum was reduced to
2% in antiviral assays.
3.6.2 Inhibition of virus-induced
cytopathogenic effect (CPE)
The antiviral activity of RG4-1 against
RSV was evaluated by cytopathogenic
effect (CPE) inhibition assay [15]. Con-
fluent monolayers of cultured cells were
overlaid in 96-well plate with 0.1 ml of
virus suspension (63 TCID50/ml). Serial
two-fold dilutions of RG4-1 were added
after the virus was adsorbed for 2 h.
Ribavirin was used as positive control
group. Virus-induced CPE was scored at
the time when complete cell death was
observed in the virus control group
(usually 3 days). The concentration that
reduced CPE by 50% was defined as EC50.
The inhibitory activity of that assay was
confirmed by MTT [16] assay done on the
same plate, and the EC50 was determined.
The 50% tissue culture infective dose
(TCID50) of RSV was calculated by the
Reed–Muench method according to the
scores of cytopathogenic effect (CPE) of
virus infection.
3.5.2 Positive control drug
Ribavirin used as a positive drug was
provided by Hefeng Pharmaceuticals Ltd.
(Shanghai, China).
3.6.3 Inhibition effect of RG4-1 against
RSV by plaque reduction assay
Plaque reduction assay was carried out to
confirm the antiviral activity of RG4-1,
after its anti-RSV activity was evaluated
by CPE inhibition assay and MTT
assay. Monolayers of Hep-2 cells grown
on 24-well culture plates were infected
with 0.2 ml of virus (63 TCID50/ml).
After incubation for 2 h with gentle
agitation every 15 min, the inoculum was
aspirated and the cultures were overlaid
with maintenance medium with 1%
3.6 Anti-RSV activity assays
3.6.1 Cytotoxicity test by MTT
Host cells were seeded in 96-well plates at
an initial density of 5 £ 103 cells/well.
After the cells had been incubated for 20 h
at 378C, 0.1 ml of doubled dilutions of
RG4-1 at the concentration of 25–
1.6 mg/ml were added and the incubation
was continued for 72 h. Discarding the
maintenance medium, 20 ml of MTT at a

Journal of Asian Natural Products Research
521
methylcellulose containing dilutions of
RG4-1. Ribavirin was used as positive
control group. After 96 h of incubation, the
plates were fixed with 10% formalin,
stained with 0.2% crystal violet, and air-
dried, and the number of plaques was
counted. The concentration of RG4-1 that
reduced the number of plaques to 50%
of the virus control was determined as
EC50 [17].
assay, and plaque reduction assay, the
attachment inhibition effect of RG4-1 was
investigated.
3.6.6 Penetration inhibition assay of
RG4-1
The penetration inhibition effect of RG4-1
was quoded vide the paper of Zhang [20].
Host cells were pre-chilled at 48C and were
infected with 0.1 ml of virus suspension
(63 TCID50/ml) for 2 h at 48C. After
washing with PBS twice, different con-
centrations of RG4-1 were added at 378C.
Using CPE inhibition assay, MTT assay,
and plaque reduction assay, the pen-
etration inhibition effect of RG4-1 was
investigated.
3.6.4 Direct killing effect of RG4-1
The direct killing effect of RG4-1 was
determined by the method of Oxford [18].
RSV was diluted to 2 £ 63TCID50/ml in a
medium and mixed with an equal volume of
medium containing RG4-1 to obtain final
concentrations of 200, 100, 50, 25, and
12.5mg/ml. In virus control group, diluted
virus was mixed with equal volume of
medium. After the mixtures were incubated
at 378C for 2 h, 0.1 ml of viral sample was
removed from each tube to infect confluent
host cells at 378C for 2h. After incubation,
viral samples were removed and cells were
washed with PBS and then the maintenance
medium was added. Using CPE inhibition
assay, MTT assay, and plaque reduction
assay, the direct killing effect of RG4-1 was
investigated.
3.6.7 Effect of RG4-1 on viral growth
A single-cycle growth of RSV in host cells
was performed to determine the inhibitory
effect of RG4-1 [21]. Hep-2 cells were pre-
chilled at 48C and infected with RSV for
2 h at 48C to synchronize the initial stage
of infection and adsorption. Virus inocu-
lum was then removed and culture was
washed three times with pre-chilled PBS.
At 0, 2, 4, 6, and 8 h post-infection, RG4-1
at a final concentration of 200 mg/ml was
added. In the virus control group, culture
medium instead of RG4-1 was added. The
culture was further incubated 24 h post-
infection, at which time, the titer of
extracellular and intracellular virus was
determined. In this research, the culture
media were removed for the assay of
extracellular virus, and intracellular virus
was measured following three frozen–
thawed cycles and centrifugation after the
culture was gently washed three times with
PBS. The virus titer was measured by the
plaque assay.
3.6.5 Attachment inhibition assay of
RG4-1
The attachment inhibition effect of RG4-1
was determined by the method described by
Zhang [19]. RSV was diluted to 2 £
63TCID50/ml in a medium and mixed
with an equal volume of medium containing
RG4-1 to obtain final concentrations of 200,
100, 50, 25, and 12.5mg/ml. The mixtures
in 0.1 ml volume was immediately inocu-
lated onto host cell monolayers. After 2 h of
adsorption, inocula were removed from the
cultures followed by washing with PBS
twice and the addition of maintenance
medium, and then the plate was incubated
again. Using CPE inhibition assay, MTT
For confidence, in all the experiments
described above, monolayers that were
neither infected with RSV nor received
treatment were used as the normal control,
and monolayers infected with RSV but did

522
X. Li et al.
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not receive treatment were used as the
virus control.
3.7 Statistical analysis
[12] J.F. Liao, Y.M. Jan, S.Y. Huang, H.H.
Wang, L.L. Yu, and C.F. Chen, J. Proc.
Natl Sci. Counc. Repub. Chin. 19, 151
(1995).
TCID50 of RSV in HeLa Cells, Hep-2
cells, and Vero cells was calculated by the
Reed–Muench method [22]. CC50 and
EC50 were calculated by probit regression
analysis [23]. TI was calculated using the
formula: TI ¼ CC50/EC50.
[13] J.F. Hruska, J.M. Bernstein, R.G. Dou-
glas, and C.B. Hall, J. Antimicrob. Agents
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X.K. Wang, Y. Huang, Y. Zhao, and W.F.
Gong, China J. Chin. Mater. Med. 31,
1176 (2006).
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