PHYTOTHERAPY RESEARCH
Phytother. Res. (in press)
Published online in Wiley InterScience
ANTIVIRAL ACTIVITY OF SOUTH AMERICAN PLANTS
(www.interscience.wiley.com) DOI: 10.1002/ptr.2198
1
Evaluation of Antiviral Activity of South
American Plant Extracts Against Herpes
Simplex Virus Type 1 and Rabies Virus
Vanessa Müller1, Juliana H. Chávez2, Flávio H. Reginatto1,3, Silvana M. Zucolotto1,
Rivaldo Niero4, Dionezine Navarro5, Rosendo A. Yunes5, Eloir P. Schenkel1,
Célia R. M. Barardi2, Carlos R. Zanetti2 and Cláudia M. O. Simões1*
1
Departamento de Ciências Farmacêuticas, CCS, Universidade Federal de Santa Catarina (UFSC), Florianópolis 88040-900,
SC, Brasil
2
Departamento de Microbiologia e Parasitologia, CCB, Universidade Federal de Santa Catarina (UFSC), Florianópolis
88040-900, SC, Brasil
3
Curso de Farmácia, ICB, Universidade de Passo Fundo (UPF), Passo Fundo, 99010-080, RS, Brasil
4
Centro de Ciências da Saúde, CCB, Universidade do Vale do Itajaí (UNIVALI), Itajaí 88302-202, SC, Brasil
5
Departamento de Química, CFM, Universidade Federal de Santa Catarina (UFSC), Florianópolis 88040-900, SC, Brasil
This paper describes the screening of different South American plant extracts and fractions. Aqueous and
organic extracts were prepared and tested for antiherpetic (HSV-1, KOS and 29R strains) and antirabies (PV
strain) activities. The evaluation of the potential antiviral activity of these extracts was performed by using an
MTT assay for HSV-1, and by a viral cytopathic effect (CPE) inhibitory method for rabies virus (RV). The
results were expressed as 50% cytotoxicity (CC50) for MTT assay and 50% effective (EC50) concentrations
for CPE, and with them it was possible to calculate the selectivity indices (SI = CC50/EC50) of each tested
material. From the 18 extracts/fractions tested, six extracts and four fractions showed antiviral action. Ilex
paraguariensis, Lafoensia pacari, Passiflora edulis, Rubus imperialis and Slonea guianensis showed values of
SI > 7 against HSV-1 KOS and 29-R strains and Alamanda schottii showed a SI of 5.6 against RV, PV strain.
Copyright © 2007 John Wiley & Sons, Ltd.
Keywords: plant extracts; antiviral activity; MTT assay; viral cytopathic effect; HSV-1; rabies virus.
INTRODUCTION
Infection by viral diseases remains as an important
worldwide health problem and the control of viral diseases is the subject of constant scientific endeavor.
Additionally, the appearance of viral strains resistant
to antiviral agents is an emerging problem. As a consequence, there are only few antiviral drugs available
for the treatment of virus diseases. Therefore, the search
for more effective antiviral agents is a necessary and
highly desirable task (De Clercq, 2004).
Herpes simplex viruses (HSV) are DNA viruses
belonging to the family Herpesviridae and are responsible for a variety of mild to severe diseases, which
are sometimes life threatening, especially in immunocompromised patients (Snoeck, 2000).
On the other hand, rabies is a neurotropic RNA
virus of the Rhabdoviridae family, in an acute, progressive and, in most cases, incurable encephalitis
(Rupprecht et al., 2002; Willoughby et al., 2005).
* Correspondence to: Dr Cláudia M. O. Simões, Laboratório de Virologia
Aplicada/UFSC, Departamento de Ciências Farmacêuticas, CCS,
Universidade Federal de Santa Catarina (UFSC), Campus Universitário
da Trindade, Florianópolis 88040-900, SC, Brazil.
E-mail: claudias@reitoria.ufsc.br
Contract/grant sponsor: CNPq/MCT/Brazil.
Copyright © 2007 John Wiley & Sons, Ltd.
Copyright © 2007 John Wiley & Sons, Ltd.
Although the incubation period varies from 1 to 3
months, it has been reported that the disease can occur
days or years after exposure. Additionally, pathogenetic
mechanisms remain barely understood and current care
entails only palliative methods (Rupprecht et al., 2002;
Hendekli, 2005).
According to the literature many traditional medicinal plants have been reported to have strong antiviral
activity and some of them have already been used
to treat animals and people who suffer from viral
infections inhibiting the replication cycle of various types
of DNA or RNA viruses. Additionally, different secondary metabolites, including lignans, tannins, saponins,
flavonoids and phenolic acids exhibit promising antiviral activity (Almeida et al., 1998; Charlton, 1998; Abad
et al., 2000; Chiang et al., 2003; Jassim and Naji, 2003;
Palomino et al., 2005).
In the search for new antiviral agents, the antiviral
activity of natural products, including Brazilian medicinal plants, was evaluated (Simões et al., 1999a, 1999b;
Andrighetti-Frohner et al., 2003, 2005; Bettega et al.,
2004).
This paper describes the inhibitory activity of several
South American plant extracts against herpes simplex
virus type 1 and rabies virus. As far as we are aware,
this is the first report of the detection of antiviral activity of these plants, and the first screening of medicinal
plants for antirabies activity.
Received Res.
20 April
2006
Phytother.
(in press)
Accepted
30 March
2007
DOI:
10.1002/ptr
2
V. MULLER ET AL.
Table 1. Plants selected for antiviral screening
Family
Botanical name
Local name
Plant part used
Traditional use
Apocynaceae
Alamanda
Leaves and flowers
Ornamental (Pio Correa, 1978)
Aquifoliaceae
Lythraceae
Allamanda blanchettii A. DC.
Allamanda schottii Pohl
Ilex paraguariensis A. St.Hil.
Lafoensia pacari St.Hil.
Erva-mate, mate
Mangava-brava
Leaves
Leaves
Passifloraceae
Rosaceae
Elaeocarpaceae
Passiflora edulis Deg.
Rubus imperialis Chum. Schl.
Sloanea guianensis Aubl. Benth
Leaves
Aerial parts
Leaves and stems
Leguminosae
Glycine max L.
Maracujá-azedo
Amora-branca,
Amora-do-mato
Sapopema
Soja
Stimulant (Gosmann et al., 1995)
Tonic and febrifuge; for gastric
ulcers and inflammations
(Sólon et al., 2000)
Sedative (Pio Correa, 1978)
Diabetes (Niero et al., 1999)
For wood extraction
(Pio Correa, 1978)
Food; to treat menopause
symptoms (Murphy et al., 1999)
MATERIAL AND METHODS
Plant material. Allamanda blanchettii A. DC., Allamanda
schottii Pohl, Ilex paraguariensis St.Hil., Lafoensia pacari
St.Hil., Passiflora edulis Deg., Rubus imperialis Chum.
Schl., Sloanea guianensis L. and Glycine max L. were
collected in Brazil. The plant materials were identified
and voucher specimens have been deposited at the
Herbariums of the Universidade de Passo Fundo (RSPF/
UPF/RS), Universidade Federal de Santa Catarina
(FLOR/UFSC/SC) and Herbário Barbosa Rodriguez
(HBR/ITAJAÍ/SC). General information about these
plants is listed in Table 1.
Extract preparation. Aqueous and organic extracts of
the tested medicinal plants were prepared according
to the procedures previously described by De Oliveira
et al. (2005) with modifications. Briefly, different parts
of the plants were extracted with 1000 mL of distilled
water, hydroethanol (40%), ethanol (EtOH) or methanol (MeOH) for 1 h. The methanol extracts of leaves
were successively partitioned (3 × 50 mL) with hexane,
chloroform, ethyl acetate and n-BuOH yielding HX,
CH, EA and BuOH fractions, respectively. These
extracts and fractions were filtered and concentrated
under reduced pressure (Büchi®-R200) – organic extracts – or lyophilized (Edwards®) – aqueous extracts.
The extracts were suspended in DMSO 1%, dissolved
in culture medium, filtered (Millipore® 0.22 µm) and
stored (4 °C) until used.
Cell culture and viruses. The used cell lines were VERO
(ATCC: CCL81) and McCoy (ATCC: CRL1696) grown,
respectively, in MEM Medium (Sigma®) and DMEM
(Gibco® BRL) both supplemented with 10% fetal
bovine serum (FBS – Gibco® BRL), penicillin G (100 U/
mL), streptomycin (100 µg/mL) and amphotericin B
(0.025 µg/mL) (Gibco® BRL). Cell cultures were maintained at 37 °C under a humidified 5% CO2 atmosphere.
The following viruses were used: herpes simplex virus
type 1 (HSV-1), strains KOS and 29-R/acyclovir resistant (Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Rennes, France), and rabies virus,
PV strain (Pasteur Institute, Sao Paulo, Brazil). HSV-1
and rabies virus were propagated in VERO and
McCoy cells, respectively. Stock viruses were prepared
as previously described (Simões et al., 1999a) and the
Copyright © 2007 John Wiley & Sons, Ltd.
Seeds, soybeans
supernatant fluids were collected, titrated and stored
at −80 °C until used. Virus titers were obtained by
the limit-dilution method and expressed as 50% tissue
culture infections dose per mL (TCID50/mL) (Reed and
Müench, 1938).
Cytotoxicity evaluation – Cell viability test. The
cytotoxicity evaluation was performed by MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
method, according to Takeuchi et al. (1991) and
Sieuwerts et al. (1995) with minor modifications. Briefly,
VERO and McCoy cell cultures (2 × 105 cells/mL) were
prepared in 96-well tissue culture plates (Corning®).
After a 24 h period of incubation at 37 °C under a
humidified 5% CO2 atmosphere, the cell monolayers
were confluent, the medium was removed from the
wells, and 200 µL of each extract/fraction dilutions (1:2
– ranging from 2000 to 15.6 µg/mL prepared in cell
culture medium) was added to each well. As a cell control only 200 µL of medium was added to the cells. The
plates were incubated under the same conditions cited
above. After 4 days, the medium was removed by
suction from all wells and 50 µL of MTT (Sigma®, 1 mg/
mL) solution prepared in cell culture medium were
added to each well and the plates were incubated once
more for 4 h. After the MTT solution was removed
without disturbing the cells and 100 µL of DMSO was
added to each well to dissolve the formazan crystals.
After gently shaking the plates, the crystals were completely dissolved, and the absorbances were read on
a multiwell spectrophotometer (Bio-Tek®, Elx 800)
at 540 nm. The CC50 was defined as the cytotoxic concentration of each extract/fraction that reduced the
absorbance of treated cells to 50% when compared with
that of the cell control.
Antiherpes assay. VERO cell cultures (2 × 105 cells/
mL) were prepared in the same way as described above
and, when the cell monolayers were confluent, the
medium was removed from the wells and 100 µL/well
of non-cytotoxic concentrations (≤CC50 values) of the
extracts/fractions and 100 µL/well of HSV-1 (KOS and
29-R strains) at a MOI of 0.5 were added simultaneously to the cells. Cell and viral controls were performed
by adding only 200 µL of MEM medium or 200 µL of
viral suspension, respectively. The plates were incubated
for 96 h. The same MTT method used to evaluate cell
viability was followed. The percentages of protection
Phytother. Res. (in press)
DOI: 10.1002/ptr
3
ANTIVIRAL ACTIVITY OF SOUTH AMERICAN PLANTS
were calculated as [(A − B) × 100/(C − B)], where A, B
and C indicate the absorbances of the extracts/fractions,
virus and cell controls, respectively. Each obtained EC50
value was defined as the effective concentration that
reduced the absorbance of infected cells to 50% when
compared with cell and virus controls. Acyclovir [9-(2hydroxyethoxymethyl) guanosine, Sigma®, 10 µg/mL]
was used as a positive control for HSV-1 (KOS strain)
inhibition.
Antirabies virus assay. The viral cytopathic effect (CPE)
inhibitory assay as previously described by Simões
et al. (1999b) was used with minor modifications. McCoy
cell culture was prepared similarly to VERO cell culture. 100 µL/well of non-cytotoxic concentrations (≤CC50
values) of the extracts/fractions and 100 µL/well of
rabies virus (PV strain) at a MOI of 1.0 were added
simultaneously to the cells. Cell and viral controls were
performed by adding, respectively, 200 µL of DMEM
medium or 200 µL of viral suspension. Isoprinosine
(UNIBIOS®, 1800 µM) and ketamine (DOPALEN®,
3000 µM) were used as positive controls for rabies virus
inhibition, according to Hernandez-Jaurégui et al. (1980)
and Lockhart et al. (1992). After 96 h, the cells were
visually scored for the inhibition of CPE and EC50
values were estimated in relation to the controls. The
results were expressed by using the selectivity index
(SI = CC50/EC50) of each tested extract.
Data analysis. The 50% cytotoxic (CC50) and 50%
effective (EC50) concentrations were calculated from
concentration-effect curves after linear regression analysis. The results represent the mean ± standard error
of the mean values of three different experiments.
RESULTS AND DISCUSSION
Eighteen plant extracts and fractions were investigated
for their antiviral activity against herpes simplex
virus type 1 (HSV-1, KOS and 29-R/acyclovir resistant
strains). Additionally, four of these extracts were tested
against rabies virus (PV strain). Although there is no
evidence that these plants have been used as antiviral
agents, several medicinal plants belonging to this genus
have long been used in folk medicine (Pio Correa, 1978;
DerMarderosian and Beutler, 2002).
Before the evaluation of the antiviral activity, the
cytotoxic effects of the selected extracts/fractions on
VERO and McCoy cells were investigated. For this
purpose, the MTT colorimetric assay was used, and for
each tested material a CC50 value after 96 h of incubation was calculated. This assay has several advantages:
it is easy to perform, the evaluations are objective, it
can be automated using a personal computer and the
cytotoxicity evaluation can be made in parallel with
antiviral activity evaluation (Takeuchi et al., 1991;
Andrighetti-Frohner et al., 2005; Palomino et al., 2005).
The results of the cytotoxicity evaluation of the tested
extracts and fractions are shown in Tables 2 and 3,
respectively.
The antiherpes activity was also evaluated by MTT
assay in VERO cells inoculated with both virus strains
at a MOI of 0.5. Nevertheless, considering that for
antirabies activity the MTT assay did not show significant differences between the absorbances of cell control and viral infected cells (data not shown), for PV
rabies strain, the studies were based on the viral
cytopathic effect inhibitory method by using McCoy
cells and the PV strain at a MOI of 1.0 (Consales et al.,
1990; Nogueira, 1992).
From the crude tested extracts, Ilex paraguariensis
(aqueous extract – leaves), Lafoensia pacari (MeOH
extract – leaves), Passiflora edulis (aqueous extract –
roots), Rubus imperialis (MeOH extract-leaves) and
Sloanea guianensis (MeOH extract – leaves) were the
most active against both strains of HSV-1. Their EC50
values ranged from 60 to 170 µg/mL, and their SI were
higher than 7. Nevertheless, the antirabies virus activity
was detected only for Allamanda schottii (MeOH extract
– leaves) with a SI = 5.6. The results of the antiviral
evaluation of the tested extracts are shown in Table 2.
For aqueous extract from the leaves of Ilex
paraguariensis, it was possible to verify strong antiviral activity against HSV-1 KOS (IS = 15.8) and 29-R
(IS = 12.6) strains. It has been reported that caffeoyl
acids and triterpenoid saponins possess strong antiviral
Table 2. Antiviral activity of crude extracts of South American plants against Herpes and Rabies Virus
Plant
Alamanda blanchetti
Alamanda schottii
Ilex paraguariensis
Glycine max
Lafoensia pacari
Passiflora edulis
Rubus imperialis
Sloanea guianensis
Plant part
used
Extract
Roots
Leaves
Flowers
Leaves
Seeds
Leaves
Roots
Roots
Leaves
Leaves
Stems
EtOH
MeOH
MeOH
Aqueous
EtOH 40%
MeOH
EtOH 40%
Aqueous
MeOH
MeOH
MeOH
CC50 (µg/mL)
VERO cells
1900
1190
1220
1260
3000
1140
1230
1600
1390
1400
610.0
±
±
±
±
±
±
±
±
±
±
±
0.2
0.2
0.4
0.6
0.8
0.6
0.6
0.3
0.8
0.5
0.5
HSV-1 (KOS)
Acyclovirsensitive
HSV-1 (29R)
Acyclovirresistant
EC50
(µg/mL)
EC50
(µg/mL)
I
719.7 ±
487.7 ±
80.0 ±
I
60.0 ±
I
290.9 ±
70.0 ±
318.2 ±
381.2 ±
0.6
0.4
0.2
0.5
0.4
0.2
0.6
0.8
SI
–
2.6
2.4
15.8
–
19.0
–
5.5
19.8
4.4
1.6
I
I
I
100.0 ±
I
170.1 ±
I
89.9 ±
90.0 ±
140.0 ±
160.5 ±
0.4
0.7
0.5
0.8
0.6
0.7
SI
–
–
–
12.6
–
6.7
–
17.8
15.4
10.0
3.8
Rabies
virus (PV)
CC50 (µg/mL)
McCoy cells
NT
1290 ±
1542 ±
NT
NT
NT
NT
1713 ±
NT
NT
1543 ±
0.3
0.5
0.7
0.5
EC50
(µg/mL)
SI
NT
230 ± 0.06
500 ± 0.11
NT
NT
NT
NT
800 ± 0.2
NT
NT
430 ± 0.14
NT
5.6
3.1
NT
NT
NT
NT
2.1
NT
NT
3.6
I, inactive; NT, not tested; SI = CC50/EC50; MeOH, methanol; EtOH, ethanol.
Copyright © 2007 John Wiley & Sons, Ltd.
Phytother. Res. (in press)
DOI: 10.1002/ptr
4
V. MULLER ET AL.
Table 3. Antiviral activity of fractions of selected South American plants against herpes virus (HSV-1)
Plant
Fraction
HSV-1 (KOS)
Acyclovir-sensitive
HSV-1 (29R)
Acyclovir-resistant
CC50 (µg/mL)
VERO cells
EC50 (µg/mL)
SI
EC50 (µg/mL)
SI
Lafoensia pacari
HX
CH
EA
1050 ± 0.2
600.0 ± 0.2
1030 ± 0.4
I
500.0 ± 0.8
490.5 ± 0.5
–
1.2
2.1
–
500 ± 0.7
100 ± 0.6
–
1.2
10.3
Sloanea guianensis
HX
CH
EA
380.0 ± 0.2
640.0 ± 0.2
500.0 ± 0.2
290.1 ± 0.7
I
63.9 ± 0.6
1.3
–
7.8
I
I
I
–
–
–
Rubus imperialis
BuOH
1390 ± 0.6
300 ± 0.2
4.6
53 ± 0.3
26.2
I, inactive; SI = CC50/EC50; HX; CH; EA; BuOH (see experimental section).
activity, respectively, against HSV-1 and adenovirus
(Chiang et al., 2002) and HSV-1 (Hostettmann and
Marston, 1995; Simões et al., 1999b). In view of that
caffeoylquinic acids and derivatives (Filip et al., 2001)
and triterpenoid saponins (Gosmann et al., 1995) are
considered the major compounds present in this plant,
the antiviral effect observed for Ilex paraguariensis could
be associated with the presence of these compounds.
Otherwise, the detected antiherpes activity of MeOH
extract obtained from the leaves of Lafoensia pacari
(SI / KOS = 19 and SI / 29R = 6.7) could be related to
the presence of tannins, as other authors have also
reported the antiviral activity of tannins (Fukuchi et al.,
1989; Solon et al., 2000; Fortin et al., 2002; Cheng
et al., 2004).
For Passifora edulis, the presence of saponins was
described (Zucolotto et al., 2006; Yoshikawa et al., 2000)
and flavonoids (Petry et al., 2001; De Paris et al., 2002).
So, the observed antiviral effect (SI / KOS = 5 and
SI / 29R = 17.8) could be associated with these compounds, since for these groups of metabolites similar
antiherpes activity has been already reported (Almeida
et al., 1998; Simões et al., 1999b; Gonçalves et al., 2001;
Gosse et al., 2002).
Rubus imperialis is the crude extract that showed
higher activity against HSV-1 (SI / KOS = 19.8 and
SI / 29-R = 15.4). For this genus, the occurrence of tannins and flavonoids was reported (Gudej and Tomczyk,
2004) leading us to propose that these compounds
could be related to the detected antiviral activity for
this extract (Hudson, 1990).
The methanol extracts from leaves that showed
highest activity against HSV-1 were partitioned with
solvents of increasing polarity: hexane (HX), chloroform (CH), ethyl acetate (EA) and n-butanol (BuOH).
From the seven tested fractions, EA fractions of
Lafoensia pacari (anti-HSV-1/29R strain, SI = 10.3) and
Sloanea guianensis (anti-HSV-1/KOS strain, SI = 7.8)
showed promising antiherpes activity, and the n-BuOH
fraction of Rubus imperialis, with polar substances as
the main components (Niero et al., 1999), was the most
active against HSV-1 (anti- HSV-1/29R, SI = 26.2). The
results of the antiviral evaluation of the tested fractions
are shown in Table 3.
Considering the results obtained, it can be stated that
these extracts protect against viral infection, but the
mechanism of their antiviral action and the active substances are not yet identified. Further studies are needed
in order to verify which compounds could be responsible for this activity and how they exert their antiviral
effects. Studies with these goals are under development
in our laboratory.
Acknowledgements
This work was supported by CNPq/MCT/Brazil. The authors E. P.
Schenkel, R. A. Yunes, C. R. M. Bararadi, C. R. Zanetti and C. M.
O. Simões are also grateful to CNPq for their research fellowships.
F. H. Reginatto also thanks to CNPQ and FUPF for his Postdoctoral
fellowship. We are also grateful to Dr Daniel de Barcellos Falkenberg
(UFSC), Oscar Benigno Isa (ITAJAÍ) and Branca Severo (UPF) for
identifying plant materials.
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