Anti-influenza virus effect of aqueous extracts from dandelion

Abstract

Human influenza is a seasonal disease associated with significant morbidity and mortality. Anti-flu Traditional Chinese Medicine (TCM) has played a significant role in fighting the virus pandemic. In TCM, dandelion is a commonly used ingredient in many therapeutic remedies, either alone or in conjunction with other natural substances. Evidence suggests that dandelion is associated with a variety of pharmacological activities. In this study, we evaluated anti-influenza virus activity of an aqueous extract from dandelion, which was tested for in vitro antiviral activity against influenza virus type A, human A/PR/8/34 and WSN (H1N1).
Results obstained using antiviral assays, minigenome assay and real-time reverse transcription-PCR analysis showed that 0.625-5 mg/ml of dandelion extracts inhibited infections in Madin-Darby canine kidney (MDCK) cells or Human lung adenocarcinoma cell line (A549) of PR8 or WSN viruses, as well as inhibited polymerase activity and reduced virus nucleoprotein (NP) RNA level. The plant extract did not exhibit any apparent negative effects on cell viability, metabolism or proliferation at the effective dose. This result is consistent with the added advantage of lacking any reported complications of the plant's utility in traditional medicine over several centuries.
The antiviral activity of dandelion extracts indicates that a component or components of these extracts possess anti-influenza virus properties. Mechanisms of reduction of viral growth in MDCK or A549 cells by dandelion involve inhibition on virus replication.

Full text

RESEARC H Open Access
Anti-influenza virus effect of aqueous extracts
from dandelion
Wen He
1,2,3
, Huamin Han
1,2
, Wei Wang
1,2
and Bin Gao
1,4*
Abstract
Background: Human influenza is a seasonal disease associated with significant morbidity and mortality. Anti-flu
Traditional Chinese Medicine (TCM) has played a significant role in fighting the virus pandemic. In TCM, dandelion
is a commonly used ingredient in many therapeutic remedies, either alone or in conjunction with other natural
substances. Evidence suggests that dandelion is associated with a variety of pharmacological activities. In this
study, we evaluated anti-influenza virus activity of an aqueous extract from dandelion, which was tested for in vitro
antiviral activity against influenza virus type A, human A/PR/8/34 and WSN (H1N1).
Results: Results obstained using antiviral assays, minigenome assay and real-time reverse transcription-PCR analysis
showed that 0.625-5 mg/ml of dandelion extracts in hibited infections in Madin-Darby canine kidney (MDCK) cells
or Human lung adenocarcinoma cell line (A54 9) of PR8 or WSN viruses, as well as inhibited polymerase activity and
reduced virus nucleoprotein (NP) RNA level. The plant extract did not exhibit any apparent negative effects on cell
viability, me tabolism or proliferation at the effective dose. This result is consistent with the added advantage of
lacking any reported complications of the plant’s utility in traditional medicine over several centuries.
Conclusion: The antiviral activity of dandelion extracts indicates that a component or components of these
extracts posse ss anti-influenza virus properties. Mechanisms of reduction of viral growth in MDCK or A549 cells by
dandelion involve inhibition on virus replication.
Keywords: Dandelion, Anti-influenza virus, Traditional Chinese Medicine
Background
Influenza A viruses are negative strand RNA viruses with a
segmented genome that belong to the family of orthomyx-
oviridae. Both influenza A and B viruses can infect
humans and cause annual influenza epidemics which
result in significant mobidity and mortality worldwide.
There are 16 hemagglutinin (HA) and 9 neuraminidase
(NA) subtypes of the influenza A virus that i nfect a wide
variety o f species [1]. The introduction of avian virus
genes into the human population can happen at any time
and may give rise to a new pand emic. Ther e is even the
possibility of a direct infection of humans by avian viruses,
as evidenced by the emergence of the highly pathogenic
avian influenza viruses of the H5N1 subtype that were
capable of infecting and killing humans [2].
Vaccines are the best option for the prophylaxis and
control of a pandemic; however, the lag time between
virus identification and vaccine distribution exceeds 6
months and concerns regarding vaccine safety are a grow-
ing issue leading to vaccination refusal. In the short-term,
antiviral therapy is vital to control the spread of influenza.
To date, on ly two classes of anti-influenza drugs have
been approved: inhibitors of the M2 ion channel, such as
amantadine and rimantadine, or neuraminidase inhibitors,
such as oseltamivir or zanamivir [3]. Treatment with
amantadine, and its derivatives, rapidly results in the
emergence of resistant variants and is not recommended
for general or uncontrolled use [4]. Among H5N1 isolates
from Thailand and Vietnam, 95% of the strains have been
shown to harbor ge netic mutations associated with resis -
tance to the M2 ion channel-blocking amantadine and its
derivative, rimantadine [5]. Furthermore, influenza B
viruses are not s ensitive to amantadine derivatives [6].
Recent studies have reported that the development o f
resistance can also occur against neuraminidase inhibitors
* Correspondence: bgao2004@gmail.com
1
CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI),
Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen West
Road, Beijing 100101, PR China
Full list of author information is available at the end of the article
He et al. Virology Journal 2011, 8:538
http://www.virologyj.com/content/8/1/538
© 2011 He et al; lic ensee BioMed Central Ltd. Th is is an Open Access article d istribute d under the terms of the Creative Commons
Attribution License (http://creativecommons.or g/licenses/by/2.0), which permits unrestricted use, di stribution, and reproduction in
any medium, provided the or iginal work is properly cited.

[7]. According to a recent study, oseltamivir-resistant
mutants in children being treated for influenza with osel-
tamivir appear to arise m ore fr equently than previously
reported [8]. In addition, there are several reports suggest-
ing that resistance in H5N1 viruses can emerge during the
currently recommended regimen of oseltamivir therapy
and that such resistance may be associated with clinical
deterioration [9]. Thus, it has been stated that the treat-
ment strategy for influenza A (H5N1) viral infections
should include additional antiviral agents. All these high-
light the urgent need for new and a bundantly available
anti-influenza agents.
A number of anti-flu agents have been discovered from
Traditional Chinese Medicine (TCM) he rbs. Ko et al.
found that TCM herbal extracts derived from Forsythia
suspensa (’Lianqiao’), Andrographis pa niculata (’Chuan-
xinlian’ ), and Glycyrrhiza uralensis (’Gancao’)suppressed
influenza A virus-induced RANTES secretion by human
bronchial epithelial cells [10]. Mantani et al. reported
that the growth of influenza A/PR/8/34 (H1N1) ( PR8)
virus was inhibited when the cells were treated with an
extract of Ephedraspp (‘ Mahuang ’ )[11].Hayashietal.
found that trans-cinnamaldehyde of Chinese cinnamon
(’ Rougui’) could inhibit the growth of influenza A/PR/8
virus in vitro and in vivo [12]. Park et al. found that Alpi-
nia Katsumadai extracts and fractions had strong a nti-
influenza virus activity in vitro [13]. Many TCM herbs
have been found to be anti-flu agents, but their mechan-
isms of action have not yet been elucidated [14,15].
Plants have a long evolutionary history of developing
resistance against viruses and have increasingly drawn
attention as potential sources of antiviral drugs [16,17].
Dandelion belongs to the Compositae family, which
includes ma ny types of tr aditional Chine se herbs [18].
Dandelion is a rich source of vitamins A, B complex, C,
andD,aswellasmineralssuchasiron,potassium,and
zinc. Its leaves are often used to add flavor to salads,
sandwiches, and teas. The roots can be found in some
coffee substitutes, and the flowers are used to make cer-
tain wines. Therapeutically, dandelion has the ability to
eliminate heat and toxins, as well as to reduce swelling,
choleresis, diuresis, a nd inflammation [ 19]. Dandelion
has been used in Chinese folklore for the treatment of
acute mastitis, lymphadenitis, hepatitis, struma, urinary
infections, cold, and f ever. Choi et al. found that dande-
lion flower ethanol extracts inhibit cell proliferation and
induce apoptosis in human ovarian cancer SK-OV-3 cells
[20]. Hu et al. detected antioxidant, pro-oxidant, and
cytotoxic a ctivities in solvent-fractionated dandelion
flower extracts in vitro [21]. Ki m et al. dem onstrated
antioxidative, a nti-inflammatory and antiatherogenic
effects of dandelion (Taraxacum officinale)extractsin
C57BL/6 mice, fed on an athe rogenic diet [22]. Ovadje
et al. suggested that aqueous da ndelion root extracts
contain components that induce apoptosis selectiv ely in
cultured leukemia cells, emphasizing the importance of
this traditional medicine [23]. Furthermore, there are no
side effects a ssociated with the prolonged use of dande-
lion for therapeutic purposes.
In this report, we attempted to analyze whether dande-
lion have anti-influenza virus activity in cell culture. We
found dandelion could inhibit the influenza virus infection.
We further identified the inhibition of viral polymerase
activity and the reduction of the virus nucleoprotein (NP)
RNA level contributed to the antiviral effect. Thus, dande-
lion may be a promising approach to protect against influ-
enza virus infections.
Methods
Evaluation and extraction of plant materials
Extracts made by boiling the herb in water. The voucher
speci men of the plant material was deposited i n the CAS
Key Laboratory of Pathogenic Microbiology and Immunol-
ogy (CASPMI), Institut e of Microbiology, Chinese Acad-
emy of Sciences. Dandelion, purchased from a medicine
store, was dissolved in sterile H
2
O (100 mg/ml) at room
temperature for 2 h and then extracted twice with water at
100°C for 1 h. The aqueous extracts were filtered through
a0.45μm membrane. This aqueous dandelion extract lyo-
philized, and the resulting light yellow powder ( 17% w/w
yield) was dissolved with cell culture medium when
needed.
Viruses, cells and viral infections
Human influenza virus A/Puerto Rico/8/34 (H1N1) (PR8)
and A/WSN33 (WSN) were grown in 10-day old fertilized
chicken eggs. After incubation at 37°C for 2 days, the
allantoic fluid was harvested and used for infection.
All cell lines were purchased f rom ATCC (Rockville,
MD, USA). Madin-Darby canine kidney (MDCK) cells or
Human lung adenocarcinoma cel l line (A549) were cul-
tured in Dulbecco’s modified eagle medium (DMEM ) or
RPMI-1640 medium, respectively, with 10% fetal bovine
serum (FBS, Gibco, USA), penicillin 100 U/ml, and strep-
tomycin 10 μg/ml. Prior to infection, the cells were
washed with phosphate-buffered saline (PBS) and were
cultured in infection medium (DMEM without FBS, 1.4%
BSA) supplemented wit h antibiotic s and 2 μg/ml of tryp-
sin (Gibco; Invitrogen, Carlsbad, CA).
Hemagglutination inhibition test
Influenza viruses are characterized by their ability to
agglutinate e rythrocytes. This hemagglutination activity
can be visualized upon mixing virus dilutions with
chicken erythrocytes in microtiter plates. The chicken
erythrocytes were supplemented with 1.6% sodium citrate
(Sigma, USA) in sterile water, separated by centrifugation
(800 × g, 10 min, room temperature) and washed three
He et al. Virology Journal 2011, 8:538
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times with sterile PBS. Serial two-fold dilutions of dande-
lion extracts were made in 25 μl of PBS in 96-well V-bot-
tom plates. Influenza viruses in 25 μlofPBS(4HAU)
were added to each dilution, and the plates were incu-
bated for 1 h at room temperature. 25 μl of 1% (v/v)
chicken erythrocytes in PBS was added to each well. The
hemagglutination pattern was read following the incuba-
tion o f the plates for 0.5 h at room temperature. The
highest dilution t hat completely inhibited hemaggl utina-
tion was defined as the hemagglutination inhibition (HI)
titer.
Cell viability assay
A549 or MDCK cells were left untreated or treated with
the indicated amounts of dandelion extracts ranging
from 20 to 0.1563 mg/ml, and oseltamivir ranging from
12.5 to 0.098 mg/ml for 48 h; MDCK cells were left
untreated or treated with 0.1 mg/ml oseltamivir, 2.5 mg/
ml and 15 mg/ml dandelion extracts for 72 h. All drugs
were multiproportion diluted in serum-free medium.
Cell-proliferation and metabolism were measured using
the CCK8-assay. Briefly, the cells were treated with CCK-
8 solution (dojindo, 10 μl/well) and incubat ed for 4 h at
37°C. The absorbance was measured using a microplate
reader (DG5032, Huadong, Nanjing, China) at 450 nm.
The untreated control was set at 100%, and the treated
samples were normalized to this value according to the
following equation: Survival rate (%) = optical density
(OD) of the tr eated cells - OD of blank control/OD o f
negative control - OD of blank control × 100.
Plaque titrations and antiviral assays
Plaque titrations: MDCK cells grown to 90% confluency in
96-well dishes were washed with PBS and infected with
serial dilutions of the supernatants in PBS for 1 h at 37°C.
The inoculum was aspirated and cells were incubated with
200 μl DMEM (medium containing 1.4% BSA, 2 μg/ml of
trypsin and antibiotics) at 37°C, 5% CO
2
for 2-3 days.
Virus plaques were visualized by staining with trypan blue.
Antiviral assay: MDCK cells were infected with the influ-
enza A virus strain PR8 or WSN (1 × 10
6
PFU) and were
left untreated or treated with dandelion extracts (0.0782-5
mg/ml), oseltamivir (0.0047-0.3 mg/ml) (Sigma), or sux-
iaoganmaojiaonang (0.069-4.375 mg/ml). At 16 h post
infection supernatants were taken. This procedure was
repeated two times in triplicate. Supernatants were assayed
for progeny virus yields by standard plaque titrations.
Virus yields of mock-treated cells were arbitrarily set as
100%.
Simultaneous treatment assay: dandelion extracts (2.5
mg/ml), oseltamivir (0.1 mg/ml) or suxiaoganmaojiaonang
(4.375 mg/ml) was mixed with virus individually and incu-
bated at 4°C for 1 h. The mixture was inoculated onto
near confluent MDCK cell monolayers (1 × 10
5
cells/well)
for 1 h with occasional rocking. The solution was
removed, the cells were washed twice with PBS and the
inoculum was aspirated, and then the cells were incubated
with 2 ml of DMEM supplemented with 1.4% BSA, anti-
biotics, 2 μg/mL trypsin at 37°C under 5% CO
2
atm.
Post treatment assay: Influenza viruses (1 × 10
6
PFU)
were inoculated onto near co nfluent MDCK cell mono-
layers (1 × 10
5
cells/well) for 1 h with occasional rock-
ing. The media was removed and replaced by DMEM
containing 1.4% BSA, antibiotics, 2 μg/mL trypsin and
dandelion extracts (2.5 mg/ml), or oseltamivir (0.1 mg/
ml), or suxiaoganmaojiaonang(4.375 mg/ml). Th e cul-
tures were incubated at 37°C under 5% CO
2
atm.
After 6, 12, 24 , 36 and 4 8 h incuba tion in all antiviral
assays, t he supernatant was analyzed for the production
of progeny virus using the hemagglutinin test and was
compared with th e untreated control cells. Cell prolifera-
tion and metabolism were analyzed by the CCK8-assay at
48 h post-treatment. Virus yields from the mock-treated
cells were normalized to 100%.
Real-time reverse transcription-PCR analysis
MDCK cells were grown to about 90% confluence infected
with influenza virus (1 × 10
6
PFU). Medium was removed
after 1 h, and cultured in the presence of dandelion
extracts (2.5 mg/ml) 13 h. The inoculum was aspirated
after 13 h. Cells were scraped off, washed twice with PBS,
and collected by centrifugation (500 g for 5 min). Total
RNA was prepared using the RNApure total RNA fast iso-
lation kit (Shanghai Generay Biotech Co., Ltd). The primer
sequence used for quantitative real-time PCR of viral RNA
were 5’ -TGTGTATGGACCTGCCGTAGC - 3’ (sense)
and 5’ - CCATCCACACCAGTTG ACTCTTG - 3’ (ant i-
sense). The Canis familiaris beta-actin was used as internal
control of cellular RNAs, with primer sequences of 5’
-CGTGCGTGACATCAAGGAAGAAG - 3’ (sens e) and
reverse: 5’ -GGAACCGCTCGTTGCCAATG - 3 ’ (anti-
sense). The primer sequences used in real-time PCR were
designed using Beacon Designer 7 software.
Real-time reverse transcription-PCR was performed
using 100 ng of RNA and the One-step qPCR kit (RNA-
direct SYBR Green Real-time PCR Master Mix,
TOYOBO). Cycling conditions fo r real-time PCR were as
follows: 90°C for 30 s, 61°C for 20 min, and 95°C for 1
min, followed by 45 cycl es of 95°C for 15 s, 55°C for 15 s
and 74°C f or 45 s. As the loading control, we measured
the level of Canis familiaris beta-actin mRNA. Real-time
PCR was co ndu cted using the AB I Prism 7300 seque nce
detection system, and the data were analyzed using ABI
Prism 7300 SDS software (Applied Biosystems).
Minigenome assay and transient transfection
To test the transcription efficiency of the influenza virus
polymerases after drug treatment, a minigeno me assay
He et al. Virology Journal 2011, 8:538
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was performed in Human embryonic kidney (2 93T)
cells. Briefly, ambisense plasmids encoding PB2, PB1,
PA and NP were cotransfected together with the i nflu-
enza virus replicon reporter plasmid pPOLI-luciferase.
The reporter p lasmid pPOLI-luciferase was constructed
by inserting the luciferase protein open reading frame
(ORF) flanked by the noncoding regions of t he M gene
of influenza A virus between th e BamHIand NotI site of
the pPOLI vector (a generous present from Dr. Edward
Wright). Calcuim phosphate transfection was used.
Briefly, the cell culture was replaced by Opti-medium;
0.5 μg of each plasmid was mixed, incubated at room
temperature for 15 min, and added over 80% confluent
293T ce lls seeded the day before in six-well plates. Six
hours later, the DNA-transfection mixture was replaced
by DMEM containing 10% FBS. At 48 h posttransfec-
tion, the cells were treated with cell lysis buffer, centri-
fuged, and supernatant was collected. Add 5 μlaliquots
of cell lysate to individual luminometer tubes containing
180 μl of luciferase a ssay buffer at room temperature.
To start the assay, inject 100 μl of luciferin solution into
the luminometer tube and measure the light output in
the luminometer.
Statistical analysis
Data were presented as mean ± SD. The data were sta-
tistically evaluated using a one-way ANOVA to compare
difference s between the groups. A p-value of < 0.05 was
considered to be significant. The IC50 and CC50 values
were calculated using GraphPad Prism programme.
Results
Treatment with aqueous dandelion extracts results in a
reduction of progeny virus titers
Treatment with aqueous dandelio n extracts results in an
efficient and concentration-dependent reduction of pro-
genyvirustitersininfectedlung epithelial cells (A549)
or Madine-Darby canine kidney (MDCK) cells; both of
which are standard host cell lines for influenza virus
propagation. These cells were treated with dandelion
extracts at various concentrations (0.0 782-5 mg/ml) 1 h
post-infection with different influenza A virus strains,
including human prototype isolate A/Puerto-Rico/8/34
(PR8) and A/WSN33 (WSN) (H1N1). The concentra-
tions of the plant extract dilutions were kept constant in
each sample throughout the experiment and showed a
dose-dependent change in virus titer. Oseltamivir
(0.0047-0.3 mg/ml) was used as a positive control and
suxi aoganmaojiaonang (0.069-4.375 mg/ml) was used as
anegativecontrolfortheinhibition of virus replication
(Figure 1). The maximum inhibitory effect (100%) was
obtained with 5 mg/ml, and the IC
50
of dandelion
extracts was 0.99 mg/ml.
Dandelion treatment does not affect cell morphology,
viability, or negatively interfere with proliferation and
metabolism
A major prerequisite for an antiviral agent is safety.
Thus, we tested whether therapeutic concentra tions of
dandelion extracts would have any harmful effects on
healthy cells. Initially, cells treated with dandelion
extracts at the indi cated concentrations were examined
for changes in morphology. No differences in cell shape
or cell number could be observed compared with
untreated control cells. The same cells were treated with
the CCK-8 solution to detect cell proliferation and
metabolism in each sample (Figure 2). The CC
50
of dan-
delion extracts was 8.47 mg/ml. SI = CC50/IC50 = 8.47/
0.99 = 8.4.
Inhibitory activity of dandelion extracts on influenza virus
replication
The post treatment assay was performed to evaluat e
whether dandelion extracts are able to inhibit replication
of influenza virus A/PR/8/34 a nd WSN (H1N1) in
MDCK cells. Dandelion showed a strong antiviral activ-
ity against A/PR/8/34 and WSN (H1N1) at concentra-
tion 2.5 mg/mL (Figure 3B).
Dandelion extracts does not block the hemagglutination
activity of pre-treated virus particles
To determine whether dandelion extracts would prevent
the ability of virus particles to bind to cell surface recep-
tors, we used simultaneous treatment assay and hemagglu-
tination inhibition (HI) assays. The simultaneous
treatment assay results indicated that treatment with dan-
delion extracts on virus entry couldn’t inhibit virus infec-
tivity (Figure 3A). Influenza A viruses a re able to
agglutinate red blood cells (RBCs) by means of hemagglu-
tinin, a viral glycoprotein that binds to N-acetylneuraminic
acid at the cell surface. The RBCs become cross-linked by
the virus and will form a type of lattice. This cross-linking
results in a diffuse distribut ion of the RBCs in a round-
bottom vial, as compared with the spot-like appearance of
RBCs in the absence of any virus. Pretreatment with dan-
delion extracts could not preven t the binding of different
virusestoRBCsinthisassay(Figure4).Thesefindings
suggest that aqueous dandelion extracts do not block
bindi ng of viruses to cel l receptors by directly interfering
with viral HA.
Viral RNA synthesis is affected in the treatment of
dandelion extracts
The levels of influenza viral RNA were compared between
dandelion extracts -treated and untrea ted infected cell s.
RNAextractionwasperformedat16hafterinfluenza
virus infecti on and the levels o f intracell ular influenza
He et al. Virology Journal 2011, 8:538
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RNA were measured. Quantitative real-time PCR showed
a reduction of influenza RNA from dandelion extracts (2.5
mg/mL) treated cells comparison with the non-treated
cells in both A/PR/8/34 (H1N1) and WSN. There were
marked difference s in NP RNA level be tween dandelion
extracts-treated virus-infected cells and untreated virus-
infected cells (Figure 5). These results indicate that block-
age of virus replication is one of mechanisms, by which
dandelion exerts antiviral effects.
Treatment with dandelion extracts inhibit viral
polymerase activity
To evaluate if dandelion extracts influenced the poly-
merase activity, we performed a flu minigenome repor-
ter assay (Figure 6A). The flu m inigenome plasmid
containing the luciferase reporter gene was cotransfected
into 293T cells together with the four plasmids neces-
sary for viral polymerase activity (PB2, PB1, PA and
NP). The luciferase expression was quantified as
0
1
2
3
4
5
0.069 0.137 0.274 0.547 1.094 2.188 4.375
Suxiaoganmaojiaonang
0
1
2
3
4
5
6
0.0782 0.1563 0.3125 0.625 1.25 2.5 5
dandelion
PR8 infectivity
WSN infectivity
0
1
2
3
4
5
0.0047 0.0094 0.0188 0.0375 0.075 0.15 0.3
Oseltamivir
PR8 infectivity
WSN infectivity
Concentration(mg/ml)
Virus titers (Pfu/ml, lg)
A
B
Virus titers (Pfu/ml, lg)
0
1
2
3
4
5
6
7
8
6 12243648
mock drug
1.25mg/ml
0.625mg/ml
Infection time(h)
Figure 1 Dandelion extracts inhibit influenza virus propagation. Influenza virus (A/PR/8/34 [H1N1]) (1 × 10
6
PFU) were inoculated in MDCK
cells. After 1 h, viruses were removed. (A) MDCK cells were treated with suxiaoganmaojiaonang (0.069-4.375 mg/ml), dandelion (0.0782-5 mg/
ml), ostalmivir (0.0047-0.3 mg/ml) individually. The cultures were incubated for 24 h at 37°C under 5% CO
2
atm. (B) MDCK cells were treated with
dandelion (1.25 mg/ml, 0.625 mg/ml) individually. The cultures were incubated for 6, 12, 24, 36 and 48 h at 37°C under 5% CO
2
atm. The yield of
progeny viruses in MDCK supernatants was determined by plaque titrations assay. Each concentration of drugs was assayed two times in
triplicate.
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described in Materials and Methods. There were marked
differences between dandelion extracts treated virus-
infected cells and non-treated or ostalmivir treated
virus-infected cells (Figure 6B). These results indicate
that dandelion inhibited the viral polymerase activity,
then to exert antiviral effects.
Discussion
Outbreaks of avian H5N1 pose a public health risk of
potentially pandemic proportions. Infections with
influenza A viruses are still a major health burden, and the
options for the control and treatment of the disease are
limited. Natural products and their derivatives have, his-
torically, been invaluable sources of therapeutic agents.
Recent technological advances, coupled with unrealized
expectations from current lead-generation strategies, have
led to renewed interest in natural products in drug discov-
ery. This is also true in the field of anti-influenza research
[24]. Here, we show that aqueous dandelion extracts exert
potent antiviral activity in cell culture.
0
20
40
60
80
100
120
0.1563 0.3125 0.625 1.25 2.5 5 10 20
Dandelion
Viability rate (%)
Concentration (mg/ml)
0
20
40
60
80
100
120
0.098 0.195 0.391 0.781 1.563 3.125 6.25 12.5
oseltamivir
0
10
20
30
40
50
60
70
80
90
100
untreated dandelion(2.5mg/ml) oseltamivir(0.1mg/ml) dandelion(15mg/ml)
*
Viability rate (%)
Figure 2 Cytotoxicity assay of dandelion extracts. (A) MDCK cells were left untreated (negative control) or trea ted with the indicated
amounts of dandelion extracts or oseltamivir (2-fold dilutions) for 48 h. (B) MDCK cells were left untreated (negative control) or treated with 0.1
mg/ml oseltamivir, 2.5 mg/ml dandelion extracts and 15 mg/ml dandelion extracts (positive control) for 72 h. The cells were treated with CCK-8
solution (10 μl/well) and incubated for 4 h at 37°C. The absorbance was measured using a microplate reader at 450 nm. The untreated control
was set at 100%. (* p > 0.05)
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Dandelion is a natural diuretic that increases urine
production by promoting the excretion of salts and
water from the kidney. Dandelion extracts may b e used
for a wide range of conditions requiring mild diuretic
treatment, such as poor digestion, liver disorders, and
high blood pressure. Dandelion is also a source of potas-
sium, a nutrient often lost through the use of other nat-
ural and synthetic diuretics. Additionally, fresh or dried
dandelion herb is used as a mild appetite stimulant and
to improve stomach symptoms, including feelings of
fullness, flatulence, and constipation. The root of the
dandelion plant is believed to have mild laxative effects
and is often used to improve digestion.
Dandelion has a very high polyphenol content [18]. It
is well known that polyphenols have protein-binding
capabilities, which suggests that components of dande-
lion extracts may interact with pathogens through physi-
cal, non-specific interactions. Two potential advantages
of this non-specific mechanism of action may be that
resistant variants only emerge rarel y and that dandelion







S D O untreated







S D O untreated
Virus titers (Pfu/ml, lg)
A
B
treatment
*
Figure 3 Antiviral as say strategies with drugs on different stages of virus infection. (A) Simultaneous treatment assay: MDCK cells were
inoculated with PR8 treated with suxiaoganmaojiaonang (S, 4.375 mg/ml), dandelion (D, 2.5 mg/ml), ostalmivir (O, 0.3 mg/ml), or untreated with
drugs (negative control) for 1 h, the media was removed and replaced by DMEM without any drugs; (B) Post treatment assay: Influenza viruses
(1 × 10
6
PFU) were inoculated in MDCK cells. After 1 h, viruses were removed and MDCK cells were treated with suxiaoganmaojiaonang (S, 4.375
mg/ml), dandelion (D, 2.5 mg/ml), ostalmivir (O, 0.1 mg/ml) or untreated with drugs (negative control). The cultures were incubated for 16 h at
37°C under 5% CO
2
atm. The yield of progeny viruses in MDCK supernatant was determined by plaque assay. Each concentration of drugs was
assayed two times in triplicate.
He et al. Virology Journal 2011, 8:538
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extracts may also act against bacterial co-infections that
represent a major complication in severe influenza virus
infections. A non-specific interaction with viral HA has
been report ed for the polyphenolic compound epigallo-
catechin-gallate [17]. Simultaneous treatment was used
to identify whether dandelion extracts block the viral
adsorption to cells. The simultaneous treatment assay
did not show significant antiviral activity (Figure 3A).
These data indicate that dandelion extracts can not
directly interfere with viral envelope protein at the cell
surface. Therefore, we used HI assays to determine
whether dandelion extracts interacted with HA of influ-
enza virus (Figure 4). Dandelion extracts did not exhibit
inhibition of viral H A in both A/PR/8/34 and WSN

Dandelion, concentration (mg/ml)
Virus control
Serum( mice immunized by PR8), two-fold dilution

Ostalmivir, concentration (mg/ml)
Figure 4 Effect of dandelion extracts on agglutination with viral hemagglutinin and chicken RBC (cRBC). Four HAU of influenza virus (A/
PR/8/34 [H1N1]) were mixed with an equal volume of 2-fold diluted dandelion extracts, ostalmivir (negative control), serum (mice immunized by
PR8, positive control) or PBS (virus control) and incubated for 1 h at room temperature. The hemagglutination activity was tested by incubation
with 1% (v/v) cRBC in PBS for 1 h at room temperature. We found dandelion extracts couldn’t inhibit the viral hemagglutination.
He et al. Virology Journal 2011, 8:538
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(H1N1), which agrees with the simultaneo us treatment
assay results.
To evaluate the anti-influenza activity after virus infec-
tion, we employed the post treatment assay (Figure 3B),
quantitative real-time PCR (Figure 4) and minigenome
assay (Figure 6) to test the in vitro effect of dandelion
extracts on viral replication. Our studies do not show
the prevention of receptor binding of the virus after
dandelions treatment, but reduction of the nucleopro-
tein (NP) RNA level and the viral polymerase activity
are obvious. Currently, anti-influenza targets include
viral factors (such as hemagglutinin (HA), M2 ion chan-
nel protein, RNA-depende nt RNA polymerase (RdRp),
nucleoprotein (NP), non-structural protein (NS) and
neuraminidase (NA) and host factors (such as v-ATPase,
protease, i nosine monophosphate dehydrogenase
(IMPDH) and intracellular signalling cascades), and
their relevant inhibitors [25]. In virus particles, t he
genomic RNAs (vRNAs) are associated with the RNA-
dependent RNA polymerase proteins and the NP, which
together form the ribonucleoprotein (RNP) com plexes.
The NP viral RNA level reflected the RNP complexes’s
action. Our resu lts indicate that dandelion extracts inhi-
bit influenza virus infection probably by decreasing the
NP viral RNA level and viral polymerase activity, and
thus affecting the RNP complexes’ activities, further to
inhibit viral RNA replication.
Vaccines play an important role in combating in flu-
enza. However, vaccination has only been able to pro-
vide a limited control of the infection, because the virus
has a tendency to mutate and thus, escape the immune
system . Plants have a long evolutionary history of devel-
oping resistance against viruses and have increasingly
drawn a ttention as potential sources of a ntiviral drugs
[24,26]. Many p lant extracts and compounds of plant
origin have been shown to possess activity against influ-
enza viruses. Our results indicate that aqueous dande-
lion extracts can inhibit influenza virus infections.
Dandelion is composed of multiple compounds that are
able to regulate multiple targets for a range of medical
indications and that are able to be titrated to the speci-
fic symptoms of an individual.
Conclusion
This study has shown that dandelion extracts can inhibit
both A/PR/8/34 and WSN (H1N1) influenza viruses by
inhibiting viral nucleoprotein synthesis a nd polymerase
activity. These results lead to further investigati on about
characterization of active compounds and their specif ic
mechanism against influenza virus. Given the urgent
need for new and abundantly available anti-influenza
drugs, dandelion extracts appear to be a promising
option as a replacement or supplemental strategy to cur-
rently available anti-influenza therapies.






PR8 infected Dandelion(2.5mg/ml) mock infected
RQ max
*
Figure 5 Real-time reverse transcription-PCR of influenza viral Nucleoprotein (NP) RNA levels normalized to beta-actin. MDCK cells were
infected with influenza viruses A/PR/8/34 (H1N1) (1 × 10
6
PFU). After 1 h, viruses were removed. MDCK cells were treated with dandelion
extracts (2.5 mg/ml) or untreated with drugs. Total RNA extraction was performed at 16 h after influenza virus infection and the levels of
intracellular influenza viral RNA were measured. Influenza viral RNA levels normalized to beta-actin. (* p < 0.01). Mock-infected cells were also
analyzed.
He et al. Virology Journal 2011, 8:538
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Acknowledgements
This work was supported by grants 2008ZX10003-012 and 2009ZX10004-305.
Author details
1
CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI),
Institute of Microbiology, Chinese Academy of Sciences, 1 Beichen West
Road, Beijing 100101, PR China.
2
Graduate University of Chinese Academy of
Sciences, 1 Beichen West Road, Beijing 100101, PR China.
3
Biochemistry
Teaching and Research office of Hebei Medical University, Zhongshan East
Road, Shijiazhuang 050017, PR China.
4
China-Japan Joint Laboratory of
Molecular Immunology and Microbiology, Institute of Microbiology, Chinese
Academy of Sciences, Beijing, PR China.
Authors’ contributions
Conceived and designed the experiments: WH, BG. Performed the
experiments: WH, HMH, WW. Contributed reagents/material/analysis tools:
BG, WH, HMH, WW. Wrote the paper: WH, BG, HMH. All authors have read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 6 August 2011 Accepted: 14 December 2011
Published: 14 December 2011
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doi:10.1186/1743-422X-8-538
Cite this article as: He et al.: Anti-influenza virus effect of aqueous
extracts from dandelion. Virology Journal 2011 8:538.
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