New Biotechnology  Volume 26, Number 6  December 2009 RESEARCH PAPER

Antiviral activity of ethanol extracts of

Research Paper
Ficus binjamina and Lilium candidum
in vitro
Ludmila Yarmolinsky1, Michele Zaccai2, Shimon Ben-Shabat3,
David Mills4 and Mahmoud Huleihel1
1
Department of Virology and Developmental Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
2
Department of Life Sciences and Biotechnology Engineering, The Jacob Blaunstein Institutes of Desert Research, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
3
Department of Pharmacology, Faculty of Health Sciences, The Jacob Blaunstein Institutes of Desert Research, Ben-Gurion University of the Negev,
Beer-Sheva, Israel
4
Albert Katz Department of Dryland Biotechnologies, The Jacob Blaunstein Institutes of Desert Research, Ben-Gurion University of the Negev, Beer-Sheva, Israel

The antiviral activity of plant ethanol extracts against Herpes Simplex Virus-1 and -2 (HSV-1 and HSV-2)
and Varicella-Zoster Virus (VZV) was investigated in vitro. Ficus binjamina, resistant to plant viruses, and
Lilium candidum, which has a high susceptibility to plant viruses were used. Leaf extracts of F. binjamina
inhibited all studied viruses, while its fruit extracts inhibited only VZV. L. candidum leaf extracts had no
effect on VZV but strongly inhibited HSV-1 and slightly HSV-2. None of the extracts showed significant
cytotoxic effect on uninfected Vero cells even at a concentration of 250 mg/ml (CC50 > 400 mg/ml). The
greatest antiviral effect was obtained when extracts were added to cells at the time of infection, whereas a
partial inhibitory effect was observed when they were added post-infection. There was indirect evidence
for strong interactions between the plant extracts and the viruses and weak interactions with the cell
surface.

Introduction
It is well known that plants defend themselves, in an ecological
sense, with a rich variety of secondary metabolites which have
negative effects on insects, microbes, and others. Phytoalexins
are secondary metabolites that are absent in healthy plants and
accumulate only in response to microorganism attack or stress [1–3].
Many plants also synthesize antimicrobial compounds as a part of
their normal growth and development [4]. The presence of antiviral
secondary phytochemicals is unknown either in healthy plants or in
infected plants. We hypothesized that plants which are tolerant to
various plant viruses might produce antiviral compounds, which in
turn could also inhibit animal and human viruses.
White lily (Lilium candidum) is known in folk medicine as anti-
inflammatory and cosmetic remedy. Bulbs and flowers of this
plant have been used for the treatment of ulcers, furuncles, finger
Corresponding author: Huleihel, M. (mahmoudh@bgumail.bgu.ac.il)
ulcers, reddened skin, burns and injuries [5]. L. candidum has a
high susceptibility to plant viruses and fungi [6]. Its antiviral
properties have not been investigated to date. Although weeping
fig (Ficus binjamina) has not been mentioned as a medicinal plant,
it is not susceptible to various plant viruses unlike other decorative
species of Ficus [7].
Acyclovir (ACV) is the most prescribed anti-HSV drug [8–10].
Although ACV and other nucleoside derivatives have been
approved for therapeutic use against HSV-1 and HSV-2 worldwide
[8], the search for new effective antiherpetic drugs is important for
the following reasons: (a) development of ACV-resistant herpes
viruses mutants [11], (b) side effects, such as nausea, vomiting
and headache, rash and diarrhea, associated with the available drugs
and (c) ACV is not highly effective in recurrent HSV attacks [12].
Therefore, there is a need for novel antiherpetic agents with high
efficacy, low toxicity and a different mode of action from ACV and
other nucleoside derivatives.

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 RESEARCH PAPER
In the present study we investigated the antiviral activity of
ethanolic extracts of F. binjamina and L. candidum on various
members of the herpes family of viruses (HSV-1, HSV-2 and VZV)
in vitro.
Materials and methods
Plant material
F. binjamina plants and L. candidum bulbs were obtained from
nurseries and grown in a controlled greenhouse at the Ben-Gurion
University, Beer-Sheva, Israel.
Research Paper
Preparation of plant ethanolic extracts
Ethanolic extracts were prepared from different plant organs (stems,
leaves and fruit of F. binjamina; bulbs, petals, leaves of L. candidum).
Plant tissues were destroyed in 95% ethanol solution, incubated at
RT for 48 hours, and centrifuged at 2000 rpm for 10 min, and the
supernatant evaporated by lyophilizer. The pellet was dissolved in a
minimal amount of 95% ethanol (0.5 ml) and diluted with water to
a final concentration of 10 mg/ml. The extracts were sterilized by
filtration and diluted with medium containing 2% Newborn calf
serum (NBCS) to the appropriate concentrations.
Fractionation of ethanolic plant extract
The obtained plant ethanolic extracts were separated into different
fractions by reverse phase chromatography (RP-C18 Sepack) with a
stepwise methanol gradient: 0%, 20%, 40%, 60%, 80% and 100%.
Cells and viruses
African green monkey kidney (Vero) cells were purchased from the
American Type Culture Collection (ATCC), Rockville, MD, USA.
Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal calf serum (FCS), 1% glutamine, 50 U/ml
penicillin, 50 mg/ml streptomycin and incubated at 378C in a
humidified air containing 5% CO2. HSV-1 was obtained from the
ATCC (VR-735), HSV-2 and VZV were obtained from the virology
laboratory Soroka University Medical Center, Beer-Sheva, Israel. All
of these viruses are susceptible to ACV. The viruses were propagated
to >108 plaque forming units (PFU) per ml in Vero cells and their
concentration was estimated by a standard plaque assay [13].
Cytotoxicity examination
Vero cells were treated with various concentrations of plant
extracts and the toxicity of the extracts was tested by three
methods: 1. Direct count. The cells were counted by Neubauer
hemacytometer indicating their replication rate. 2. Morphological
changes were daily observed by optical inverted microscope. 3.
MTT assay was performed as previously described [14]. Briefly,
Vero cells were incubated with 50 mg/ml MTT solution at 378C for
five hours. This solution was converted by mitochondrial succi-
nate dehydrogenase enzyme into insoluble formazan (purple). The
MTT solution was removed and replaced with SDS solution to
dissolve the formazan. After overnight incubation at 378C, absor-
bance was measured at 570 nm, indicating the metabolic activity
of the cells.
Antiviral activity examination
The antiviral activity of the tested plant extracts was evaluated by
plaque assay and cytopathic effect (CPE) development as follows.
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New Biotechnology  Volume 26, Number 6  December 2009
Vero cells were seeded at 0.15  106 cells/well in 24-well culture
plates, in RPMI with 10% FCS and antibiotics. Following overnight
incubation, medium was removed and each well was infected at a
multiplicity of infection (m.o.i.) of 0.1 PFU/cell in the presence or
absence of the examined extract for two hours at 378C. The
unabsorbed virus was removed and cells were overlaid with either
a layer of CMC (for plaque assay) or RPMI containing 2% FCS and
antibiotics in the presence or absence of increasing concentrations
of the extract under test. Two to three days post-infection (p.i.) the
CMC overlay was removed, cell monolayers were fixed with 10%
formalin in saline, stained with crystal violet and plaques were
counted. The development of CPE was evaluated daily by micro-
scopic observation and expressed as the %age of damaged cells.
IC50s were calculated by determining the extract concentration
required to confer a 50% plaque inhibition of the herpes virus
infected cell monolayers. To elucidate the antiviral mechanism of
the plant extracts, infected cells were treated with increasing
concentrations (0.01–500 mg/ml) of the extracts at various periods
of time before, during or after infection. To examine possible
interactions between the plant extract and cells, Vero cells were
incubated in medium containing the appropriate dose of extract at
378C for two hours, washed three times with saline and infected
with virus without further treatment. To study possible direct
effect of these extracts on viral infectivity, 107 PFU of the appro-
priate virus particles were preincubated with 1 mg/ml of the
extract at 48C or 228C for different periods of time (15, 30, 60,
90 and 120 min). Then these mixtures were diluted 104 times with
fresh medium (to minimize the concentration of the extract at the
time of infection) and cell monolayers were infected with the
diluted mixture.
Acyclovir [9-(2-hydroxyethoxymethyl) guanosine, Sigma, 10 mg/
ml] was used as a positive control for HSV-1, HSV-2 and VZV.
Statistical analysis
All data were analyzed using Statistica for Windows software
(StatSoft, Inc., Tulsa, Oklahoma), and P < 0.05 was chosen as
the minimal acceptable level of significance. Simple regression
models were subsequently used to eliminate nonsignificant
effects. Values are presented as means  SD.
Results
Cytotoxicity examination
Ethanolic extracts were prepared from various parts (leaves, stems,
bulbs and fruits) of F. binjamina and L. candidum. Different con-
centrations of each extract were added to Vero cell monolayers for
three days and the cytotoxicity of these extracts was evaluated by
different assays as described above. No cytotoxicity was observed
at concentrations below 100 mg/ml in all examined extracts and
the concentrations found to cause 50% toxicity (CC50) of these
extracts are summarized in Table 1.
Antiviral activity of extracts against herpes viruses
Vero cell monolayers were treated with increasing concentrations
of the appropriate extracts at the time of infection with 0.1 m.o.i.
of HSV-1, HSV-2 or VZV. The treatment was terminated immedi-
ately post-infection and the antiviral activity was evaluated by
plaque assay. The results demonstrated significant and reprodu-
cible antiviral activity of F. binjamina leaf extracts against HSV-1,
New Biotechnology  Volume 26, Number 6  December 2009 RESEARCH PAPER

TABLE 1
CC50 of L. candidum and F. binjamina extracts from different plant
organs
Species Plant organ CC50 (mg/ml)
L. candidum Bulb >500
# Petals >500
# Leaves 700
F. binjamina Stems 120
# Leaves 490
# Fruits >1000

Research Paper
HSV-2 and VZV with the respective IC50 of 0.5 mg/ml, 1.7 mg/ml
and 35 mg/ml (Fig. 1). L. candidum leaf extracts had no effect on
VZV, while they strongly inhibited HSV-1 and HSV-2 with IC50 of 8
and 20 mg/ml, respectively.
The selectivity index (SI), (determined as CC50/IC50) of F. bin-
jamina leaf extract against these herpes viruses was significantly
higher than that of ACV, while the efficiency of L. candidum leaf
extract was much lower compared to F. binjamina and ACV (Fig. 2).
Extract of F. binjamina fruit had no effect on HSV-1 and HSV-2 FIGURE 2

infection, while significantly inhibiting VZV infection with an Selectivity index (SI) of plant extracts and acyclovir against HSV-1, HSV-2 and
VZV. Vero cell monolayers were treated with different concentrations of the
appropriate extract during and after infection. SI was determined as CC50/
IC50. Values are means  SD (n = 5).

IC50 of 10 mg/ml (P < 0.001). Stems of F. binjamina did not show

significant antiviral properties, but had a high toxicity (Table 1);
neither bulbs nor petals of L. candidum had any significant anti-
herpetic activity.
In addition, a strong synergistic antiviral activity between ACV
and F. binjamina was found (Fig. 3). When each was added at low
concentration (0.01 mg/ml) to the cells at the time of infection with
the different examined viruses, a low antiviral activity (10–20%) was
observed. However, when the cells were treated with a mixture of
both ACV and F. binjamina extract at the same concentrations,
significant inhibition (70%) of the viral infection was obtained.
Similar results were observed with L. candidum and ACV (data not
shown).

FIGURE 1
Antiviral activity of leaf extracts on herpes viruses. Vero cells were infected
with 0.1 m.o.i. of the tested viruses in the presence or absence of increasing
doses of leaf extracts from F. binjamina (A) or L. candidum (B). Extract
application was terminated immediately post-infection. Plaque numbers
(PFU) of the treated cultures are presented as a percentage of the positive
control (infected but untreated cell cultures). Values are means  SD (n = 5).
FIGURE 3
Synergistic effect of leaf extract of F. binjamina and acyclovir against HSV-1,
HSV-2 and VZV. Vero cells were infected with 0.1 m.o.i. of the tested viruses in
the presence of 0.01 mg/ml of leaf extract of F. binjamina or 0.01 mg/ml of ACV
or a mixture of both components at the same concentrations. Plaque numbers
(PFU) of the treated cultures are presented as a percentage of the positive
control (infected but untreated cell cultures). Values are means  SD (n = 5).

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 RESEARCH PAPER New Biotechnology  Volume 26, Number 6  December 2009

TABLE 2
Effect of time of leaf extract addition on herpes virus infections
Leaf extract (100 mg/ml) Virus (0.1 m.o.i.) Plaque forming units (% of control)
Before During After During and
infection only infection only infection only after infection
F. binjamina HSV-1 84.2  6.30 0.5  0.04 37.1  2.01 0  0.08
HSV-2 94.6  5.27 1.8  0.28 41.1  3.29 1.6  1.16
VZV 100.1  0.47 31.6  4.25 29.7  4.16 26.8  3.64
L. candidum HSV-1 99.3  3.11 18.7  1.75 42.5  2.11 13.4  0.91
HSV-2 100.0  0.62 23.6  2.19 56.3  3.18 20.1  3.02
VZV 100.0  0.09 97.5  1.29 100.2  1.14 96.1  2.14
Research Paper

Vero cell monolayers were treated with 100 mg/ml of the appropriate extract for different periods of time before, at or after infection with 0.1 m.o.i. of HSV-1, HSV-2 or VZV. Antiviral activity
was evaluated by plaque assay. Values are means  SD (n = 5).

Effect of time of leaf extract addition on herpes virus infection
Vero cells were infected with 0.1 m.o.i. of the herpes viruses and
treated with 100 mg/ml leaf extracts for various periods of time
before, at, or after infection. When the cells were treated with leaf
extracts only at the time of infection or both at the time of
infection and post-infection, inhibition of all viruses tested was
the highest (P < 0.01) (Table 2). It can also be seen that F. binjamina
leaf extract was significantly more effective against HSV-1 and
HSV-2 than against VZV. At a concentration of 100 mg/ml F.
binjamina completely inhibited the development of HSV-1 and
HSV-2 infections as opposed to about 70% VZV inhibition
(Table 2). By contrast, L. candidum leaf extract had no significant
effect on VZV (P > 0.1), but strongly inhibited HSV-1 and HSV-2 at
a concentration of 100 mg/ml (P < 0.01). At 500 mg/ml, L. candi-
dum leaf extract almost completely inhibited HSV-1 (0  0%) and
HSV-2 (1.08  0.07%).
Preincubation of cells with extracts had no significant effect on
the development of infection induced by all examined viruses
(P > 0.1). However, when the infected cells were treated with the
extract only p.i., there was a partial reduction of the plaques
number induced by the different examined viruses (Table 2).
with F. binjamina extract completely inhibited plaque formation
while 90 min with L. candidum extract was required for such
inhibition (Fig. 4). The results obtained with both incubation
temperatures (48C or 228C) were similar.Effect of leaf extracts on
development of viral CPE
Vero cells were infected with the different herpes viruses (HSV-
1, HSV-2 or VZV) in the presence or absence of different concen-
trations of either F. binjamina or L. candidum leaf extract. At the
end of the infection period, the cell culture media were removed
and replaced by fresh medium with or without leaf extract. CPE
development was evaluated every 24 hours by inverted micro-
scopy and expressed as the percentage of damaged cells in the
culture. Significant prevention of HSV-1 and HSV-2 CPE develop-
ment during the whole period was observed when the cells were
treated continuously with F. binjamina (50 mg/ml) or L. candidum

Effect of leaf extracts on virus adsorption

The question was raised whether leaf extracts prevented virus
infection by blocking adsorption to the host cells, and if so,
whether the extract acts through interaction with the virus par-
ticles, with the host cells or both. To test leaf extract–cell inter-
action, Vero cells were first incubated in medium containing the
extract and then infected with the virus without further extract
addition. As mentioned above (Table 2), preincubation of the cells
with the leaf extracts had no effect on the viral infection of all the
tested viruses.
To examine possible interaction between viral particles and
leaf extracts, the appropriate viruses were preincubated with the
extracts for different periods of time as detailed in ‘Materials and
Methods’ section. Then these mixtures were diluted with a fresh
medium and used to infect Vero cell monolayers. Our results
show a considerable inhibition of plaque formation by the
highly diluted virus–leaf extracts mixtures (Fig. 4). In the case FIGURE 4
of HSV-1 (Fig. 4A), incubation of 15 min was sufficient to inhibit Effect on virus infectivity of preincubation of leaf extracts with the virus.
1000 mg/ml of leaf extracts was incubated with 5 m.o.i. of HSV-1 (A) or HSV-2
about 90 or 75% of plaque formation by F. binjamina and L.
(B) at 48C for different periods of time (15, 30, 60, 90 and 120 min). This
candidum leaf extracts, respectively. Incubation for 30 min was mixture was diluted 104 times with fresh medium and the cells were infected
enough for complete inhibition of plaque formation by both with the diluted mixture. PFU were evaluated by the standard plaque assay
plant extracts. In the case of HSV-2 (Fig. 4B), 30 min incubation and presented as means  SD (n = 5).

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Research Paper
FIGURE 5
Effect of the removal of leaf extracts on development of herpes virus cytopathic effect (CPE). Vero cells were infected with 0.1 m.o.i. of herpes viruses (HSV-1, HSV-2
or VZV) in the absence (control) or presence of F. binjamina (50 mg/ml) (A, C, E) or L. candidum (100 mg/ml) (B, D) leaf extracts. At the end of the infection period, the
cell culture media were removed and replaced with fresh medium with or without leaf extract. Treatment with the extracts was terminated three or ten days post-
infection. CPE development was evaluated every 24 hours by inverted microscopy and expressed as the percentage of damaged cells in the culture. Data are
means  SD of three independent experiments.

(100 mg/ml) leaf extracts (Fig. 5). In the case of VZV infected cells,
F. binjamina leaf extract provided significant protection against the
development of CPE, while L. candidum leaf extract had almost no
effect on this parameter. When the treatment with the leaf extracts
was only applied at the time of infection, there was a significant
gradual increase in CPE development of tested viruses. When the
plant extract was removed even at ten days p.i., an increase in CPE
was also observed (Fig. 5). It seems that the continuous presence of
the leaf extracts in the cell culture medium is essential for con-
tinuous protection of Vero cells against the CPE of the virus.
Antiviral activity of F. binjamina and L. candidum ethanolic
extracts fractions
Vero cells were treated with 10 mg/ml of leaf extract fractions of F.
binjamina and L. candidum at the time of infection with 0.1 m.o.i.
of the herpes viruses. The results (Fig. 6) showed that fraction
eluted with 80%-MeOH of both F. binjamina (Fig. 6A) and L.
candidum (Fig. 6B) ethanol extracts completely blocked viral infec-
tion (by HSV-1 and -2) but only slightly inhibited VZV, whereas
fractions 0 and 20% MeOH significantly inhibited VZV.
Discussion
In the present study we have demonstrated that leaf ethanolic
extract of F. binjamina effectively inhibited infection of Vero cells
by HSV-1, HSV-2 and VZV in vitro, while fruit extract inhibited only
VZV. The leaf extracts of L. candidum had no effect on VZV while
strongly inhibited HSV-1 and slightly HSV-2. Although HSV-1,
HSV-2 and VZV belong to the subfamily Alphaherpesvirinae and
have many similar characteristics, they have different clinical
manifestations, as well as different biochemical and serological
properties [15,16]. Previous studies have reported differential anti-
viral activity of plant extracts on HSV-1, HSV-2 and VZV. For
instance, an aqueous extract of Yin Chen Hao Tang (YCHT), a
Chinese prescription containing Artemisia capillaries, Rheum offi-
cinale, and Gardenia jasminoids inhibited HSV-2 infection more
effectively than HSV-1 in vitro [17]. Flavonoids from Capparis

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Research Paper
FIGURE 6
Antiviral activity of fractionated leaf extracts on HSV-1, HSV-2 and VZV
infection. Vero cells were infected with 0.1 m.o.i. of the viruses in the presence
of 10 mg/ml of the appropriate chromatographic fractions of leaf extracts of F.
binjamina (A) or L. candidum (B). Plaque numbers (PFU) of the treated
cultures is presented as a percentage of the positive control (infected but
untreated cell cultures). Values are means  SD (n = 5).
spinosa buds were active against HSV-2, but did not show activity
against HSV-1 [18]. Many biflavonoids were isolated from Rhus
succedanea and Garcinia multiflora. Amenthoflavone and robust-
flavone demonstrated moderate activities against HSV-1 and HSV-
2 but did not affect VZV infection. Rhustflavanone inhibited only
HSV-2 whereas succedaneaflavanone inhibited only VZV [19].
Acetone, ethanol and methanol extracts of Phyllanthus urinaria
inhibited HSV-2 but not HSV-1 infection [20].
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