RES E AR C H Open Access
In vitro antiviral activity of plant extracts from
Asteraceae medicinal plants
María F Visintini Jaime
, Flavia Redko
, Liliana V Muschietti
, Rodolfo H Campos
, Virginia S Martino
and Lucia V Cavallaro
Background: Due to the high prevalence of viral infections having no specific treatment and the constant
appearance of resistant viral strains, the development of novel antiviral agents is essential. The aim of this study was
to evaluate the antiviral activity against bovine viral diarrhea virus, herpes simplex virus type 1 (HSV-1), poliovirus
type 2 (PV-2) and vesicular stomat itis virus of organic (OE) and aqueous extracts (AE) from: Baccharis
gaudichaudiana, B . spicata, Bidens subalternans, Pluchea sagittalis, Tagetes minuta and Tessaria absinthioides.A
characterization of the antiviral activity of B. gaudichaudiana OE and AE and the bioassay-guided fractionation of
the former and isolation of one active compound is also reported.
Methods: The antiviral activity of the OE and AE of the selected plants was evaluated by reduction of the viral
cytopathic effect. Active extracts were then assessed by plaque reduction assays. The antiviral activity of the most
active extracts was characterized by evaluating their effect on the pretreatment, the virucidal activity and the effect
on the adsorption or post-adsorption period of the viral cycle. The bioassay-guided fractionation of
B. gaudichaudiana OE was carried out by column chromatography followed by semipreparative high performance
liquid chromatography fractionation of the most active fraction and isolation of an active compound. The antiviral
activity of this compound was also evaluated by plaque assay.
Results: B. gaudichaudiana and B. spicata OE were active against PV-2 and VSV. T. absinthioides OE was only active
against PV-2. The corresponding three AE were active against HSV-1. B. gaudichaudiana extracts (OE and AE) were
the most selective ones with selectivity index (SI) values of 10.9 (PV-2) and >117 (HSV-1). For this reason, both
extracts of B. gaudichaudiana were selected to characterize their antiviral effects. Further bioassay-guided
fractionation of B. gaudichaudiana OE led to an active fraction, F
=3.1 μg/ml; SI= 37.9), which showed antiviral
activity during the first 4 h of the viral replication cycle of PV-2 and from which the flavonoid apigenin (EC
3.3 μM) was isolated as a major compound.
Conclusions: The results showed that, among the species studied, B. gaudichaudiana seemed to be the most
promising species as a source of antiviral agents.
Keywords: Asteraceae, Antiviral activity, Baccharis gaudichaudiana, Poliovirus, Herpes simplex virus, Apigenin
* Correspondence: firstname.lastname@example.org
Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Junín 956, 4ºP, Ciudad de Buenos Aires, 1113, Argentina
Full list of author information is available at the end of the article
© 2013 Visintini Jaime et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Visintini Jaime et al. Virology Journal 2013, 10:245
Antiviral drugs for diseases caused by herpesviruses, ret-
roviruses, orthomyxoviruses, hepatitis B virus and hepa-
titis C virus (HCV) are currently commercially available
. However, due to the high prevalence of viral infec-
tions for which there are no specific treatment and the
constant appearance of new resistant viral strains, the
development of novel antiviral agents is essential.
Natural products have proved to be an important
source of lead molecules and many extracts and com-
pounds of plant origin with antiviral activity have been
The great diversity of plants growing in Argentina offers
interesting possibilities of finding novel antiviral com-
pounds from a natural origin. Asteraceae is the most nu-
merous and diverse plant family in our country and is
highly promising from a pharmacological perspective .
The aim of this study was to evaluate the antiviral activ-
ity against bovine viral diarrhea virus (BVDV), herpes
simplex virus type 1 (HSV-1), poliovirus type 2 (PV-2) and
vesicular stomatitis virus (VSV) of organic (OE) and aque-
ous extracts (AE) from: Baccharis gaudichaudiana, Bac
charis spicata, Bidens subalternans, Pluchea sagittalis,
Tagetes minuta and Tessaria absinthioides, all medicinal
plants belonging to the Asteraceae family (Table 1) in
which different compounds from diverse chemical groups
have been found.
The selection of the viruses was based on the clinical
importance of their infections, the type of the genome
and the strategies of viral replication. HSV-1, a DNA
virus, is responsible of viral infections that have in-
creased over the past decades  and the development
of the rapeutic agents has become necessary due to its
growing incidence and the appearance of drug-resistant
strains, especially in immunocompromised patients .
Poliovirus is an RNA virus that causes poliomyelitis for
which there are two commercially available vaccines.
Nevertheless, no complete eradication of this viral infe c-
tion has been achieved . There is a need to find ef-
fective drugs to complete the eradication plan and to
control future outbreaks .
BVDV and VSV cause serious disease in livestock and
are responsible for major losses in cattle. Both are RNA
viruses but BVDV has a positive sense genome while
VSV has a negative one . Moreover, BVDV is also ac-
cepted as a surrogate virus model for identifying and
characterizing antiviral agents to be used against HCV
 and VSV has been extensively studied as a prototype
of non-segmented, negative-strand RNA viruses .
Besides the results of the antiviral screening, the prelim-
inary characterization of the antiviral effect of the most
active extracts is reported. In addition, the bioassay-
guided fractionation of B. gaudichaudiana organic extract,
altogether with the isolation of its major antiviral com-
pound is also described.
Antiviral activity against BVDV, HSV-1, PV-2 and VSV
Six Argentinean Asteraceaes were selected for this study.
Chemical, ethnopharmacological data and previously
reported antiviral studies are shown in Table 1. The anti-
viral activ ity of plant extracts against BVDV, HSV-1,
PV-2 and VSV was assessed in vitro by the viral CPE reduc-
tion assay. Results obtained from this screening (Table 2)
showed that B. gaudichaudiana and B. spicata OE were
active against PV-2 and VSV, the AE of both species and
Table 1 Ethnopharmacological and chemical data of the medicinal plants selected
Plant species Vernacular
Popular use Chemical composition
Digestive, hepatic, antidiabetic, antidiarrheal, antiseptic in
urinary and respiratory tract infections 
hydroxycinnamic acids 
Medicinal  Diterpenoids 
“amor seco” Ciudad de
Ocular antiseptic, to treat aphthae and sore throat [7,8] Triterpenoids, steroids 
Stomachic, hepatic, choleretic, antispasmodic, digestive,
cholagogue, antipyretic, antitussive, antiseptic, for
stomachache, febrifuge, antiseptic, for venereal diseases [4,9]
essential oils, polyphenols,
tannins, triterpenes 
“chinchilla” Ibicuy, Entre
Digestive, antispasmodic, diuretic, antifungal, anthelminthic,
antiseptic, antitussive, pectoral, disinfectant, in urinary tract
Terpenoids, flavonoids, essential
(Hook. & Arn.)
Hypocholesterolemic, balsamic, expectorant, for hepatitis and
renal insufficiency 
compounds, flavonoids, essential
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 2 of 10
the AE of T. absinthioides were active against HSV-1
and T. absinthioides OE was active only against PV-2.
None of the twelve extract s was active a gainst BVDV.
To confirm the inhibitory effect detected in the screen-
ing, we evaluated the antiviral activity of the positive ex-
tracts by the plaque reduction assay and the SIs were
determined (Table 3). Regarding the active extracts, the
thin layer chromatographic (TLC) profiles of the OE of
the two Baccharis species were very similar. Bands corre-
sponding to flavonoid aglycones and terpenoids were ob-
served after spraying with NPR and anisaldehyde/H
On the other hand, Tessaria absinthioides OE showed
strong bands corresponding to flavonoid glycosides and
only weak bands corresponding to terpenoids in the OE
and AE (Additional file 1).
B. gaudichaudiana OE and AE exhibited the highest
SI values against PV-2 (10.9) and HSV-1 (>117), respect-
ively. Based on these results, both extracts were selected
to charact erize the antiviral activity.
Characterization of antiviral activity
In or der to characterize the antiviral activity against PV-
2 and HSV-1, different experimental approaches were
considered for the OE and AE of B. gaudichaudiana.
In the pretreatment assay, none of the extracts protected
Vero cells against PV-2 or HSV-1 infection after 7 h of in-
cubation at the evaluated concentrations (Figure 1).
When the virucid al activity was assessed, the OE did
not prove to have this effect against PV-2 since 10 and
20% of reduction of viral infectivity was obtained at r.t.
and 37°C, respectively. In contrast, higher values were
obtained for the AE against HSV-1 with values of 85%
and 97%, at r.t. and 37°C, respectively (Figure 1).
With the aim to determine whether the inhibitory ef-
fect of the extracts occurs during the adsorption or post-
adsorption steps of the viral cycle, different experimental
conditions were evaluated with 1xEC
of OE and AE
(Figure 2A). The results obtained demonstrated that B.
gaudichaudiana OE (30 μg/ml) reduced the formation
of PV-2 plaques when it was added after the adsorption
period. This reduction in the number of plaques was
similar to that obtained when the OE was present during
all the experimental time (Throughout) (Figure 2B).
In contrast, B. gaudichaudiana AE (35 μg/ml) inter-
fered in the adsorption step of HSV-1 to Vero cells and
caused an inhibition degree similar to that obtained
Table 2 Screening of antiviral activity of plant extracts
Plant name Extracts Yield
BVDV HSV-1 PV-2 VSV
Baccharis gaudichaudiana OE 29 - - + +
AE 10 - + - -
Baccharis spicata OE 15.5 - - + +
AE 9 - + - -
Bidens subalternans OE 8.4 - - - -
AE 6.3 - - - -
Pluchea sagittalis OE 11 - +/− -+/−
AE 11 - - - -
Tagetes minuta OE 7.5 - - +/− -
AE 7.8 - - - -
Tessaria absinthioides OE 13.5 - - + -
AE 15.3 - + - -
The antiviral activity was tested by the reduction of viral cytopathic effect
(+) positive: reduction of viral CPE higher than 50% at both
(+/−) positive/negative: reduction of viral CPE only achieved at 100 μg/ml.
(−) negative: without protection at 25 and at 100 μg/ml.
OE organic extract, AE, aqueous extract.
Yield (% w/w = g of extracts/100 g of dried and ground plant material).
Table 3 Antiviral activity of selected active extracts
Plant name Extract Virus CC
Baccharis gaudichaudiana OE PV-2 161.0 ± 2.5 30.1 ± 0.8 14.8 ± 1.5 10.9
VSV 114.0 ± 0.5 33.9 ± 3.8 4.8
AE HSV-1 > 2000 35.4 ± 1.2 17.1 ± 0.1 > 117
Baccharis spicata OE PV-2 114.3 ± 4.7 74 ± 4.7 19.3 ± 3.9 5.9
VSV 110.1 ± 1.6 23.8 ± 0.1 4.8
AE HSV-1 > 2000 61.3 ± 3.2 34.7 ± 3.2 > 57.6
Tessaria absinthioides OE PV-2 390.1 ± 3.2 61.1 ± 2.8 40.3 ± 5.6 9.7
AE HSV-1 > 2000 26.5 ± 1.5 19.1 ± 3.2 > 104
Acyclovir* HSV-1 > 9
1.9 ± 0.2
Guanidine* PV-2 > 84
0.2 ± 0.6
Ribavirin* VSV > 2
0.3 ± 1.2
cytotoxic concentration 50,
effective concentration 90,
effective concentration 50,
SI (Selectivity index) = CC
, OE organic extract, AE
aqueous extract. Results are shown as means ± SD, each time in triplicate.
*Acyclovir, Guanidine and Ribavirin were included as positive controls for the antiviral activity of HSV-1 and PV-2 and VSV, respectively. The CC
were expressed in mM (
), except for EC
of acyclovir that is expressed in μM(
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 3 of 10
when it was present throughout the experimental time.
The reduction of 25% observed in the post-adsorption
condition could be due to the inhibitory effect of this ex-
tract on the adsorption steps on the subsequent HSV-1
replication cycles occurring during the 48 h incubation
period carried out at 37°C (Figure 2B).
Bioassay-guided fractionation of B. gaudichaudiana OE
B. gaudichaudiana OE was fractionated by a silicagel
column chromatography. Eight final fractions were
obtained according with their TLC profiles. The result s
obtained in the evaluation of the anti-PV-2 activity dem-
onstrated that F
was the most active fraction with a
SI = 56.4 and EC
= 2.1 ± 0.1 μg/ml followed by F
SI = 44.1 and EC
= 2.5 ± 0.3 μg/ml (Table 4). The
HPLC profile obtained for F
was similar to that of F
but based on the SI value and the yield, F
for further characterization (Add itional file 2).
To define the post-adsorption steps of the viral cycle
that could be targeted by F
, the effect of the addition of
=22μg/ml) at different times of infection
on PV-2 production at 10 h p.i. was evaluated (Figure 3).
The results obtained demonstrated that the maximum
inhibition level was exerted when F
was present before
4 h of infection.
A semipreparative HPLC of F
was then performed and
four subfractions (F
) were collected. The antiviral
activity against PV-2 was detected in F
μg/ml) and F
= 1.8 ± 0.1 μg/ml) (Figure 4). The
value of F
was higher than 100 μg/ml and the SI
was > 55.6. A major pure compound was isolated from
by semipreparative HPLC (Figure 5) and identified as
apigenin (Figure 6) by comparison of its spectral data
(Additional file 3 and Additional file 4) with literature
values  and by HPLC comparison with a reference
standard (Additional file 5). The antiviral activity of apige-
nin was determined by plaque assay with EC
3.3 μM. Its CC
value was 230.7 ± 4.4 μM; in conse-
quence the SI was 18.9. The apigenin standard exhibited
similar values of EC
(data not shown).
Several Baccharis species have been reported to have anti-
viral activity: B. genistelloides , B. teindalensis , B.
trinervis , B. coridifolia  and B. articulata  but
this is the first report on the antiviral activity of B.
gaudichaudiana and B. spicata. Although the virucidal
activity of T. absinthioides essential oil was reported previ-
ously against HSV-1 and Junín virus , this is the first
report of the antiviral activity of the OE and AE obtained
from this plant.
In the characterization of the antiviral activity of B.
gaudichaudiana AE , this extract did not a ffe ct HSV-1
replication when it wa s adde d t o t he cell culture before
infection, thus , it is unlikely that its antiviral activity
could be due to direct effe ct s on the host ’s cell. On the
Figure 2 Effect of B. gaudichaudiana OE and AE in the adsorption and post-adsorption steps of PV-2. A. Scheme of addition of OE or AE.
Open and black arrows indicate the absence and presence of extract, respectively. B.- Percentage of viral inhibition under different experimental
was used for the experiments. Data represented % of virus inhibition compared to untreated control as mean ± SD (n = 3),
each time by quadruplicate.
Figure 1 Virucidal activity and the effect of pretreatment with
B. gaudichaudiana OE and AE. The virucidal activity and the
pretreatment of 10xEC
(OE= 300 μg/ml AE = 350 μg/ml) and
(OE= 30 μg/ml, AE = 35 μg/ml) were evaluated against PV-2
and HSV-1, respectively. Data represent % of virus inhibition
compared to untreated controls as mean ± SD (n = 3), each time
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 4 of 10
other hand, the results of the virucidal assays suggest
that this extract could interact with viral particles a nd
inactivate them. Data also indicated that HSV-1 infe c-
tion was significant impaired only if the AE wa s
present at the time of adsorption. Therefore, these
result s su ggest that AE may exe rt its antiviral activity
by inactivation of viral particles at high concentra-
tions and possibly by interference of the adsorp-
tion step of the virus to the cells at non-virucidal
Upon characterizing the antiviral activity of B. gaudi
chaudiana OE against PV-2, it could be considered that
this extract had a true antiviral activity against this virus
because of its ability to inhibit the viral cycle, parti-
cularly during the post-adsorption period. In the present
study, B. gaudichaudiana OE was selected for fur ther
purification and isolation of antiviral principles by
bioassay-guided fractionation. The most active fraction
, exerted the maximum inhibition of PV-2
replication when it was present before 4 h p.i. At this
time of the poliovirus replication cycle, the synthesis of
viral RNA is maximum [26,27]. Taking into account the
results obtained, it can be deduced that F
its antiviral activity at an intermediate stage of virus life
cycle and could interfere with viral RNA synthesis and
From this active fraction the flavonoid apigenin (5, 7-
dihydroxy-2-(4-hydroxylphenyl)-4H –chromen-4-one) was
isolated. This compound has previously been reported
from B. gaudichaudiana . It has been demonstrated
that apigenin is active against different viruses, including
avian influenza H5N1 virus strain, hepatitis C virus, HSV
and human immunodeficiency virus [29-32].
Although apigenin exhibited antiviral activity against
= 12.2 ± 3.3 μM), HPLC profile of F
showed the presence of other minor compounds which
could be responsible, altogether with apigenin, of the
antiviral activity observed.
Further studies are under way to characterize the
mechanism of action of apigenin against PV-2.
To our knowledge, this is the first time that the anti-
viral activity of B. gaudichaudiana is reported and the
anti-poliovirus activity of apigenin is determined.
In this study we have shown that the organic extract of
B. gaudichaudiana shows high antiviral effect against
PV-2 and the isolated compound, apigenin could be, at
least in part, responsible for the antiviral activity
Table 4 Antiviral activity of fractions of B. gaudichaudiana
Fraction Yield (%) CC
1.43 396.7 ± 14.4 15.4 ± 1.0 25.8
1.35 196.1 ± 6.1 7.6 ± 0.4 25.8
2.56 118.5 ± 6.5 2.1 ± 0.1 56.4
0.66 110.2 ± 7.9 2.5 ± 0.3 44.1
2.14 390.9 ± 10.9 38.4 ± 3.8 10.2
1.68 390.4 ± 10.4 33.1 ± 4.8 11.8
0.75 412.3 ± 7.1 27.4 ± 3.9 15.0
0.31 729 ± 3.1 20.8 ± 3.6 35.1
cytotoxic concentration 50,
effective concentration 50,
(Selectivity index) = CC
Yield (% w/w = g of fraction/100 g of OE).
Results are shown as mean ± SD (n=3), each time in quadruplicate.
Figure 3 Effect of F
on PV-2 replication cycle. A. Kinetics of PV-2 extracellular production during one replication cycle Vero cell monolayers
were infected with PV-2 ( m.o.i. = 10). Viral titers were determined at different hours by plaque assay. B. Effect of addition of F
on the PV-2
production during a one step replication cycle At different h p.i. after the adsorption period, F
(22 μg/ml) was added and the extracellular viral
production was determined at 10 h p.i. of incubation at 37°C, by the plaque assay. Data represent % of virus production respect to untreated
control. The viral production at 10 h p.i. in the kinetic curve of control virus was considered 100%. * p < 0.05 vs 0 and vs 2 h (one-way ANOVA
with Bonferroni a posteriori test).
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 5 of 10
observed. Further studies are necessary for a better un-
derstanding of the mechanism of action of apigenin.
Moreover, since the aqueous extract of B. gaudichau
tionation of this extract will be carried out.
Plant samples (aerial parts with flowers) were collected
between 2008 and 2010 in their places of origin in
Argentina. Voucher specimens are deposited as follows:
B. gaudichaudiana (1655): Botany Herbarium at Facultad
de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario, Argentina; B. spicata (BAF 711),
Bidens subalternans (BAF 704), Pluchea sagittalis (BAF
709) and Tagetes min uta (BAF 714): Herbarium at Museo
de Farmacobotánica, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires; Tessaria absinthioides
(Slanis-Juarez 1041): Herbarium of Fundación Miguel A.
Lillo, Universidad de Tucumán. Botanical and vernacular
names, popular uses and reported chemical composition
are shown in Table 1.
Extraction of plant material
Dried aerial parts of each plant (10 g) were reduced to pow-
der and extracted by soaking in 100 ml of dichloromethane:
methanol (1:1) at room temperature (r.t.) for 24 h and then
vacuum-filtered. The process was repeated twice and the
filtrates were combined and dried under vacuum to obtain
the organic extract (OE). The marc of the plant material
was further extracted with distilled water under the same
conditions. T he aqueous extracts (AE) were lyophilized. For
the antiviral assays, OE and AE were dissolved in dimethyl-
sulfoxide and sterile distilled water, respectively.
Cells and virus strains
Vero cells (ATCC CCL 81) were obtained from Asociación
Banco Argentino de Células and cultured in growth
medium consisting of Eagle’s Minimal Essential Medium
(E-MEM) supplemented with 10% fetal bovine serum
(FBS) (PAA), 100 μg/ml streptomycin, 100 IU/ml penicil-
lin, 2 mM L-glutamine, 2.25 g/L sodium bicarbonate and
non-essential amino acids (100 μM) (Gibco), at 37°C in a
incubator. The infection medium (IM), used for
the antiviral assays, was the same as the growth medium
but 2% FBS was added instead. The plaque medium (PM)
was IM supplemented with 1% methylcellulose (Sigma).
Madin-Darby Bovine Kidney cells (MDBK) (ATCC CLL
22) were grown in growth medium supplemented with
Figure 4 Antipoliovirus (PV-2) activity of subfractions derived
. The antiviral activity of each subfraction was determined
by the reduction of plaque assay. Results are shown as mean ± SD
(n = 3), each concentration in quadruplicate.
Figure 5 HPLC profile of F
. HPLC: RP-18 column, using a water (A)-methanol (B) gradient: 0–2 50% A; 2–15 min: 50 → 98% A, 15–25 min:
isocratic 98% A, 26–30 min: 98 → 50% A, flow rate=1 ml/min, monitored at 336 nm. The insert shows the UV adsorption spectra of the major
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 6 of 10
10% of γ-irradiated FBS. IM for the MDBK cell line was
supplemented with 2.5% horse serum (Gibco).
The herpes simplex type 1 (HSV-1) F strain, the polio-
virus type 2 (PV-2) Sabin strain and the bovine viral
diarrhea virus (BVDV:NADL strain cytopathic biotype
were kindly provided by Dr. Albert Epstein, Dr. María
Cecilia Freire (ANLIS-Instituto Dr. Carlos G. Malbrán,
Argentina) and Dr. Laura Weber (INTA, Ca stelar,
Argentina), respectively. VSV, Indiana strain (ATCC VR-
1421), was purchased from ATCC. Virus stocks of HSV-
1, PV-2 and VSV were propagated and quantified in
Vero cells. BVDV was propagated and quantified in
MDBK cells. Virus quantification was performed by
plaque assay method as number of plaque forming units
per ml (p.f.u./ml). All virus stocks were stored at −70°C
Screening of antiviral activity
The antiviral activity of each plant extract was screened in
96-well culture plates by measuring the reduction of the
viral cytopathic effect (CPE). Confluent Vero and subcon-
fluent MDBK cell monolayers were infected with HSV-1,
PV-2 or VSV or with BVDV, respectively, at a multiplicity
of infection (m.o.i.) of 0.01 p.f.u./cell in the presence of 25
and 100 μg/ml of each OE/AE. Infected cells in the ab-
sence of extract as control virus and mock-infected cells
with and without extract as control cells and cytotoxicity
control were included. Plates were incubated at 37°C in a
humidified atmosphere containing 5% CO
until 90% of
viral CPE in the CV was reached. The reduction of viral
CPE was determined by measuring cell viability by the
tetrazolium salt/phenazine methosulfate (MTS/PMS) col-
orimetric assay (CellTiter 96™ Promega, Madison, WI,
USA). The absorbance at 490 nm was measured in a
Multi-Mode microplate reader (Synergy™ HT, BioTek). Re-
sults of the screening were expressed as positive (+) (re-
duction in the CPE at both concentrations tested),
negative (−) (absence of reduction in the CPE) and (+/−)
(reduction in CPE only at 100 μg/ml).
Cytotoxicity assay: determination of cytotoxic
concentration 50 (CC
The cytotoxic eff ect of B. gaudichaudiana, B. spicat a
and T. absinthioides OE and AE on Vero cells wa s de-
termined by the MTS/PMS method, a s pre viously de-
scribed [ 33]. Briefly, sub confluent monolayers of Vero
cells/well; 24 h culture) were incubated in
quadruplicate in 96-multiwe ll plates in the presence of
two-fold dilutions of the extracts for 72 h at 37°C. Cell
viability (%) was calculated for each concentration a s
are the absorbance readings for t he wells with and
without extract, respectively. The CC
is defined as
the concentration that reduced cell viability by 50%
with respect to controls without drug. The CC
was derived from the corresponding dose–response
cur ves. The maximum non-cytotoxic concentration
(MNCC) is defined as the maximum concentration of
the extract that leaves 100% of viable cells.
Antiviral assay: determination of effective concentration
The effective concentration 50 (EC
of extract that reduces the number of viral plaques by 50%
with respect to control virus (without extract). This par-
ameter was determined by the plaque reduction assay.
Briefly, monolayers of Vero cells grown in a 24-well plate
(24 h; 5% CO
; 37°C) were infected with 100 p.f.u./well of
PV-2, VSV or HSV-1 in either the absence or presence of
serial two-fold dilutions from the MNCC of B. gaudi-
chaudiana , B. spicata and T. absinthioides extracts
(treated). After 45 min incubation at 37°C, the viral inocu-
lum was removed, and the cell monolayers were washed
with phosphate buffer saline (PBS) and overlaid with PM
supplemented with the corresponding concentrations of
each extract. PM without extract was added in CC and
CV wells. After 24 h at 37°C for PV-2 and VSV or 48 h for
HSV-1, cell monolayers were fixed and stained with 0.75%
crystal violet in methanol:water (40:60) and viral plaques
were counted. Reduction of plaques (%) was calculated as:
)] × 100. The EC
values were calculated by regression analysis of the dose–
response curves generated with the data.
The selectivity index (SI) was calculated as the CC
Acyclovir (Filaxis); Guanidine.HCl (Sigma-Aldrich, St.
Louis, MO) and Ribavirin (MP Biomedicals, LLC) were
tested simultaneously a s positive controls for HSV-1,
PV-2 and VSV, respectively.
Chromatographic profile- thin layer chromatography
Chromatographic analysis of positive OE were performed
by thin layer chromatography (TLC) on silica gel layers
Figure 6 Chemical structure of apigenin: 5, 7-dihydroxy-2-
(4-hydroxylphenyl)-4H –chromen-4-one, C
, MW: 270.24.
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 7 of 10
(Silica gel 60 F
EMD Chemicals Inc.) using a- ethyl acet-
ate:toluene:formic acid:methanol (2:2:1:1) and Natural
Product Reagent (NPR - 2-aminoethildiphenilboric acid -
Sigma) as visualization reagents; and b- toluene:ethylacetate
(5:5) and sulphuric/anisaldehyde (SAni) as reagent. The
positive AE were tested on: a) silica gel layers using
ethylacetate:methanol:water (50:6:5) and SAni reagent; and
b) Cellulose plate (Polygram® CEL 300 UV
Nagel) using acetic acid 15% and NPR as reagent. In all
cases, the TLC plates were visualized under UV light (254
and 366 nm) and visible light.
Characterization of the antiviral activity
The virucidal activity was measured by in vitro incuba-
tion of viruses with the extract s. Briefly, 10
p.f.u. of PV-
2 or HSV-1 were incubated for 30 min at r.t. or at 37°C
of B. gaudichaudiana OE (300 μg/ml ) or
AE (350 μg/ml), respectively. Simu ltaneously, the same
amount of virus was incubated with IM without extract
as control. The residual infectious viruses were quanti-
fied by viral plaque assays.
To assess the effect of the pretreatment with B.
gaudichaudiana extracts, Vero cell monolayers seeded
in 24-well plates were treated for 7 h at 37°C with two
concentrations of the extract 10xEC
300 and 30 μg/ml and AE: 350 and 35 μg/ml, respect-
ively). Then, the medium was removed and washed with
PBS, and the cell monolayers were infected with 100 p.f.
u. of PV-2 or HSV-1/well in the absence of the extracts.
Mock-infected cells (CC ) and cells pretreated with IM
(CV) were included in each assay. After 45 min at 37°C,
the viral inoculum was removed and PM without extract
was added and further incubated at 37°C for 24 or 48 h.
Finally, the number of viral plaques was determined.
To study the effect of the extracts in the adsorption and
post-adsorption events, three different treatments with
B. gaudichaudiana OE (1xEC
=30μ g/ml) against PV-
2 or AE (1xEC
=35μg/ml) against HSV-1 were carried
out. B. gaud ichaudiana OE and AE were present: (i)
only during the adsorption period (Adsorption); (ii) after
adsorption and until the end of the experiment (Post-
Adsorption), and (iii) during and after the adsorption
(Throughout). Briefly, Vero cell monolayers cultured in
24-well plates were precooled for 1 h at 4°C. Cells were
then infected with 100 p.f.u. of virus/well in the presence
or absence of OE/AE and further incubated at 4°C for 1
h allowing only the adsorption step of the viral particles
to the cells (Adsorption). Cell monolayers were washed
with PBS, and then PM with or without extract was
added. The number of viral plaques was determined
after 24 h and 48 h for PV-2 and HSV-1, respectively.
Bioassay-guided fractionation of Baccharis
B. gaudichaudiana aerial parts (500 g) were air-dried,
ground to powder and extracted with dichloromethane:
methanol (1:1) and the extract was taken to dryness. Thirty
grams of this OE was fractionated by silica gel 60 (500 g)
column chromatography eluted with a step gradient of
hexane:ethylacetate (100:0 to 0:100) and ethylacetate:
methanol (100:0 to 0:100) to afford 21 fractions of 500 ml
each. Eluates were monitored by thin-layer chromatog-
raphy (TLC) on silica gel 60 F
using toluene-ethyl acet-
ate (1:1) and cellulose layers using acetic acid 40% and
combined into eight final fractions (F
) according to
their TLC profiles.
was further fractionated by a semipreparative
reverse-phase HPLC (Waters 2996 – Photodiode Array De-
100, 5 μm, LiChroCART 125×4 – Merck). The injection vol-
ume was 50 μl. Elution was performed at a flow rate of 1 ml/
methanol (B): 0–15 min: isocratic 50% A, 15–25 min: 50 →
98% A, 25–30 min: isocratic 98% A, 30–31 min: 98 → 50%
A. Eluates were monitored at 254 nm. Eluates were collected
into four subfractions: F
(0–13 min), F
(13–20 min), F
(20–25 min) and F
subfraction was subjected to reverse-phase
HPLC on RP-18 column (LiChrospher® 100, 5 μm,
LiChroCAR T 125×4 – Merck), using a water (A)-methanol
(B) gradient: 0–2 50% A; 2–15 min: 50 → 98% A, 15–25
min: isocratic 98% A, 26–30 min: 98 → 50% A and a flow
rate=1 ml/min and a pure compound was isolated. Eluates
were monitored at 336 nm.
The anti-PV-2 activity of fractions F
and the pure compound was determined
by viral plaque reduction assay at concentrations ranging
from 100 to 0.1 μg/ml in Vero cells. The cytotoxicity and
SI were also evaluated as previously described.
Identification of apigenin
The pure compound obtained from F
was identified by
ultraviolet spectroscopy (UV) (Jasco V-630), infrared spec-
troscopy (IR) (Nicolet 380 FT-IR-Smart Multi Bruce
HATR, Zn Se 45°) and HPLC/DAD by comparison with
authentic sample (Sigma-Aldrich, St. Louis, MO) and
comparison with literature data.
One-step replication curve: effect of fraction F
Confluent Vero cell monolayersculturedina96-wellplate
were infected with PV-2 (m.o.i. = 10) for 1 h at 4°C. Follow-
ing the adsorption period, cells were washed three times, and
Visintini Jaime et al. Virology Journal 2013, 10:245 Page 8 of 10
infection (p.i): 0, 2, 4, 6 and 8 h. Cells were further incubated
up to 10 h. At this time, supernatan ts were collected and
clarified by centrifugation (3,500 × g at 4°C) and the virus
production was deter mined by viral plaque assays.
Data are presented as means ± standard deviation (SD). A
one-way ANOVA with Bonferroni a posteriori test was
used to compare differences between groups. A p < 0.05
was considered significant. The EC
were calculated using GraphPad Prism software v. 5.01.
Additional file 1: TLC profile of OE and AE of B. gaudichaudiana,
B. spicata and T. absinthioides. Right Panels (A and B) showed the OE
profiles in silica gel in (A-) ethylacetate:toluene:formic acid:methanol
(2:2:1:1) revealed with NPR at 366 nm; and (B-) toluene:ethylacetate (5:5)
revealed with AniS, at visible light. Left panels (C and D) correspond to
AEs: (C-.) silica gel and ethylacetate:methan ol:water (100:10:13) and SAni,
at visible light; and (D-) AE profile in cellulose with AcH 15% and NPR at
366 nm. BG (B. gaudichaudiana); BS (B. spicata) and TA (T. absibthioides).
Additional file 2: HPLC profile of F
from the OE of
B. gaudichaudiana. A gradient of mobile phase system consisting of
water (A) and MeOH (B) used was: 0–15 min: 2 → 98% A; 15–20 min:
isocratic 98% A; 20–21 min: 98 → 2% A.
Additional file 3: UV spectra of purified apigenin. A.- UV spectra with
methanol (MeOH) and MeOH with sodium methoxide (MeONa); B.- UV
spectra with MeOH, MeOH with aluminium chloride (AlCl3), MeOH+AlCl3+
chloridric acid (HCl) and MeOH+AlCl3+HCl 5 minutes later; C.- UV spectra
with MeOH, MeOH+ sodium acetate (AcONa) and MeOH+AcONa+ boric
Additional file 4: IR spectra of purified apigenin.
Additional file 5: HPLC of standard apigenin (Sigma). The inserts
show the UV adsorption spectra of the major peak detected. HPLC with a
RP-18 column, using a water (A)-methanol (B) gradient: 0–2 50% A; 2–15
min: 50 → 98% A, 15–25 min: isocratic 98% A, 26–30 min: 98 → 50% A,
flow rate=1 ml/min monitored at 336 nm.
The authors declare that they have no competing interests.
MFVJ designed and carried out the antiviral and cytotoxicity studies, the
extract preparation, TLC profiles and drafted the manuscript. FR carried out
the fractionation of Bg OE and the HPLC analyses. LM and RHC participated
in the design of the study. VM and LVC conceived the whole study and
edited the manuscript. All authors read and approved the final manuscript.
This work was supported by grants B045; B037 from the Universidad de
Buenos Aires (UBACyT 2008–2011); and grant PIP 112-200801-01169 from the
Consejo Nacional de Investigaciones Científicas y Tecnológicas, Argentina.
We acknowledge Martha Gattuso and Susana Gattuso from Universidad
Nacional de Rosario, Gustavo Giberti from Universidad de Buenos Aires and
Alberto Slanis from Instituto Miguel Lillo from Universidad Nacional de
Tucumán for collection and identification of plant material. We also thank
Pharm. Daiana Retta for providing B. gaudichaudiana plant material;
Dr. Eliana F. Castro for encouraging discussions Ms. María Teresa Argerich
(CONICET) for her technical assistance.
Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Junín 956, 4ºP, Ciudad de Buenos Aires, 1113, Argentina.
Cátedra de Farmacognosia, Instituto de Química y Metabolismo del
Fármaco (IQUIMEFA), Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Junín 956, 2ºP, Ciudad de Buenos Aires, Argentina.
Received: 20 November 2012 Accepted: 24 July 2013
Published: 27 July 2013
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Cite this article as: Visintini Jaime et al.: In vitro antiviral activity of plant
extracts from Asteraceae medicinal plants. Virology Journal 2013 10:245.
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