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Iranian Journal of Virology 2009;3(1): 19-23
©2009, Iranian Society for Virology
Original Article
Antiviral Activity of Garlic Extract on Influenza Virus
Mehrbod P1, Amini E1, Tavassoti-Kheiri M1*
1. Influenza Unit Pasteur Institute of Iran, Tehran, Iran.
Abstract
Background and Aims: Influenza virus is the most important cause of annual morbidities and
mortalities worldwide with numerous antigenic drifts and shifts. Inaccessibility to effective drugs and
vaccines has made world health authorities to be interested in traditional medicine in order to prevent
spread of the infectious agent. Garlic is one of the most famous of all plants in human history. It has
been shown that garlic extract has various effects on different diseases. The aim of this study was to
evaluate garlic extract antiviral activity against influenza virus in cell culture.
Methods: To study the potential antiviral activity, MDCK (Madin-Darbey Canine Kidney) cells were
treated with effective minimal cytotoxic concentration of the extract and 100 TCID50 (50% Tissue
Culture Infectious Dose) of the virus during infection at different time periods. The viral titers were
determined by hemagglutination (HA) and TCID50 assays. The antiviral effect of the extract was
studied at 1, 8 and 24 hours after treatment on the culture. To measure the amount of the viral genome
synthesized at different times after treatment, RNA extraction, Reverse Transcription-Polymerase
Chain Reaction (RT-PCR) and free band densitometry software were performed.
Results: Although the precise mechanism has not been defined yet, it was found that garlic extract
with a good selectivity index (SI) has inhibitory effect on the virus penetration and proliferation in cell
culture.
Conclusion: The biochemical and molecular methods used to evaluate the antiviral activity of Garlic
extract demonstrated that this compound could be suggested as a suitable potent antiseptic agent.
Keywords: Influenza virus; Garlic Extract; hemagglutination; Reverse Transcription-Polymerase
Chain Reaction
Introduction
nfluenza virus is capable of causing
respiratory diseases worldwide. This virus
is constantly evolving and new antigenic
variants give rise to epidemics and pandemics.
Influenza virus is unique among respiratory
tract viruses because of its considerable
antigenic variations. These mutations make it
extremely difficult to develop effective
vaccines and drugs against the virus (1).
Therefore it is an essential need to use some of
the traditional medicines and combine them
with modern medicine to inhibit the viral
activity (2).
Garlic may be one of the most famous herbal
remedies to be used by in human history-
dating back to ancient cultures. Garlic has been
an interesting plant for centuries as a medicinal
panacea. Broad range of pathogenic organisms,
including bacteria, fungi, protozoa and viruses
have been shown to be sensitive to fresh
crushed garlic (3). Fresh garlic extract with
allicin as the main active component of it has
been shown to have antiviral activity in vitro
and in vivo. Its beneficial effects may be due in
part to sulfur-containing compounds such as
allicine, diallyl disulfide, diallyl trisulfide (4)
that react with thiol groups of various enzymes
which are critical for microorganism
I
*Corresponding author: Masoumeh Tavassoti-
Kheiri, Influenza Unit Pasteur Institute of Iran,
Tehran, Iran. Tel: (+98)21-66 95 33 11
Email: mtkheiri@yahoo.com
Iranian Journal of Virology Volume 3, Number 1, Spring 2009 19
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Antiviral Activity of Garlic Extract on Influenza Virus
surveillance (5). Influenza virus surface
glycoproteins are hemagglutinin (HA) and
neuraminidase (NA). HA structure consists of
HA1 and HA2 subunits linked by disulfide
bond. In this study various biochemical,
virological, and molecular techniques were
used to evaluate the antiviral activity of the
garlic extract on Influenza virus.
Methods
Virus
Influenza A/New Caledonia/20/99 (H1N1)
standard virus was obtained from National
Institute for Biological Standards and Control
(NIBSC). The virus was grown in MDCK
cells in presence of 2 µg/ml of Trypsin TPCK
(Sigma, St.Louis, MI).
Cell culture
Continuous MDCK cells were grown in
Dulbecco's Modified Eagle’s Medium
(DMEM) (ICN) containing 10% heat-
inactivated Fetal Bovine Serum (FBS) (Gibco,
Gaithersburg, MD), 100 Units/ml Penicillin G
and 100µg/ml Streptomycin (Sigma Co.) at
37°C in a humidified 5% CO2 incubator.
Garlic Extract
Garlic extract was obtained from Shahed
University. Briefly, the fresh garlic bulbs were
stripped off the outer theca and then chopped,
mixed and sterilized by filtration. The
transparent extract was preserved at 4°C for
further use.
Cytotoxicity assay
MDCK cells were seeded at 5×104 cells/well in
96-well microplates, incubated for 24 h at
37oC.
Different concentrations of the extract were
added to the culture and incubated for 48 h at
37oC in presence of 5% CO2.
The 50% cytotoxic concentration (CC50),
effective minimal cytotoxic concentration
(EMCC) and viability of the cells were defined
by the MTT method.
MTT assay
Colorimetric MTT assay was performed
according to Levi Raphael, et al. Briefly,
yellow color of MTT (3- [4, 5-dimethylthiazol-
2-yl]-2, 5-diphenyl-tetrazolium bromide,
Sigma) is changed to purple by mitochondrial
succinate dehydrogenase of living cells. The
medium of the confluent cells was removed,
then 100µl of 1x MTT was added to each well.
Following incubation at 37°C with 5% CO2 for
2 h, 100 μl of acidic isopropanol was added
and mixed to release the color from the cells.
Optical density at 540 nm was measured using
ELISA reader (Stat Fax-200).
Inhibitory effect on the virus titer
Equal volume of Influenza virus (100 TCID50)
and EMCC of the extract were mixed and
incubated at 37°C for 1, 8 and 24 h. They were
added to the cells with different multiplicity of
infection (MOI). Following one hour
incubation at 37°C unabsorbed viruses were
removed, then the cells were washed with
phosphate buffer saline (PBS) and TPCK-
containing medium was added (100μl/well).
After 48h incubation at 37°C, viability of the
infected and non-infected cells was evaluated
by MTT method.
The virus titer was determined by HA assay
and Karber formula. The viral RNA was
extracted and amplified by RT-PCR and
evaluated by band densitometry software.
Percent protection
Percent protection of the extract was calculated
by Anova test (Tukey) using viability of mock-
infected and infected cells after 48h exposure
(6).
Hemagglutination assay
To evaluate presence of the virus in cell
culture, serial dilutions of the culture media
were added to 96-well U-shape microplates.
Chicken red blood cells (cRBCs) (0.5%) were
added to each well. Following incubation at
least for one hour at room temperature,
precipitation of the RBCs demonstrated
absence of the virus while hemagglutination
indicated presence of the virus (7).
RNA Extraction
Viral genomic RNA was extracted from the
infected cells using Viral RNA Extraction Kit
(Qiagen,Hilden,Germany). Briefly, 70µl cell
culture media was used and RNA was bound to
glass fibers fixed in a column and finally was
isolated and eluted in 30µl of elution buffer.
Since influenza A virus was assessed in this
study, influenza B virus was added to the
samples as internal control (70µl).
20 Iranian Journal of Virology, Volume 3, Number 1, Spring 2009
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Mehrbod P. et al
RT-PCR
Complementary DNA of the viral RNA was
synthesized by cDNA synthesis Kit
(Fermentas,Vilnius, Lithuania). Ten microliter
RNA sample along with random hexamer
primers and dNTPs mix were incubated at
56°C for 5 min, added to a mixture of RNase
free buffer using RT-kit. The cDNA products
were stored at -20°C.
PCR
PCR reaction was performed as described by
Shahidi, et al (8). Primers used in this study
amplified NP gene of influenza B and M gene
of influenza A.
PCR products were visualized on 1.5%
agarose gel by ethidium bromide staining after
electrophoresis.
Semi-Quantitative analysis
The end of exponential phase of the virus
amplification cycles by Real-time PCR was
determined at 24 cycles. PCR products were
measured semi-quantitavely by determining
band densitometry ratio using “Image Tool”
software.
Results
1 2 3
HA assay
0
10
20
30
40
50
60
70
treatment24
virus24
treatment8
virus8
treatment1
virus1
HA Titer
Fig. 1: HA of Garlic extract treated influenza virus A
at different time periods.
Effect of the Garlic extract on HA titer of influenza
virus at three time points: 1) 24h, 2) 8h, 3) 1h,
treatments.
Table 3: Antiviral activity of Garlic extract against
influenza virus A (H1N1) by HA test. Values are
avera
g
es of four inde
p
MEAN ± SD
TIME Virus Extract treated
Virus
1h 0.156±0.018 0.253±0.013*
8h 0.175±0.023 0.275±0.051*
24h 0.168±0.029 0.274±0.063*
MEAN ± SD
TIME Virus Extract treated
Virus
1h 0.156±0.018 0.253±0.013*
8h 0.175±0.023 0.275±0.051*
24h 0.168±0.029 0.274±0.063*
Virus CC50 EMCC
(EC50) SI
A/H1N1100
µg/ml
10 µg/ml 10
Band Density
(Mean ± SD)
time Virus Extract treated
Virus
1h 0.123±0.003 0.077±0.002*
8h 0.184±0.001 0.097±0.001*
24h 0.079±0.001 0.042±0.002*
endent examinations for HA
* Significantly different from values obtained for
extract-treated compared to untreated samples
(p<0.05).
Gel electrophoresis of RT-PCR products of the viral RNA
after exposure to the extract (Mann-Whitney U test) at time
points.
Values are averages of four independent examinations for RT-
PCR.
* Significantly different values obtained for extract-treated
compared to untreated samples (p<0.0001).
Table 1: Cytotoxicity of Garlic extract on
MDCK cells (MTT test)
Table 4: Quantitative analysis of RT-PCR products.
CC50: concentration which causes 50%
cytotoxic effect
EC50: concentration of the drug required to
inhibit 50% of virus-induced CPE
SI: Selectivity Index: CC50 EC50
Table 2: Percent protection results (MTT test).
Values are averages of four independent
e
x
pe
rim
e
n
ts
Iranian Journal of Virology, Volume 3, Number 1, Spring 2009 21
* Significantly different values obtained for
extract-treated compared to untreated samples
(p<0.0001).
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Antiviral Activity of Garlic Extract on Influenza Virus
Cytotoxicity
The viabilities were evaluated by MTT
method. The results showed that Garlic extract
had no serious effect on viabilities of MDCK
cells at concentration up to 100µg/ml. EMCC
of the extract was determined 10µg/ml through
MTT standard curve by Anova test (Tukey)
(Table1). It did not show any serious
cytopathic effects on the uninfected cells as
control while reduced CPE of the virus
infected cells.
Percent protection
The extract was exposed to the infected cells
and after 48h viability of mock-infected and
infected cells measured by MTT. Percent
protection was obtained by analysis of MTT
results using SPSS software version 11.0
(Table 2).
It was found that extract-treated virus
compared to untreated samples had
significantly different MTT results using
Anova test (Tukey) analysis (p<0.0001).
Hemagglutination assay
Antiviral effect of the extract was determined
against influenza A (H1N1) in different sets of
experiments. Inhibitory effect of the extract
was shown by reducing HA titer (Figure 1 and
Table 3).
Semi-Quantitative analysis
The PCR was ceased at this cycle to be able to
have semiquantitative results. The effect of the
extract on the viral genome was shown by
reduction in the content of the PCR products
on gel electrophoresis (data not shown). Semi-
Quantitative analysis using band densitometry
on PCR products showed statically meaningful
decrease in genome content after 1, 8 and 24 h
direct exposure of the extract to the virus by
Mann-Whitney U test. (Fig. 2 and Table 4).
Discussion
As long as influenza virus remains a serious
cause of disease, there will be a need to
identify and develop some anti-influenza
compounds with distinct mechanisms of action
(9). Current therapeutic antiviral drugs have
limited clinical efficiency and toxic side effects
(10). Antiviral agents of plant origin are easily
accessible, mostly nontoxic, and inexpensive
(2). Garlic, with a long history of traditional
medications, is shown to be effective against
different microorganisms including viruses.
Sulfur containing compounds in garlic extract
such as allicine, diallyl disulfide, diallyl
trisulfide and others (4) react with thiol groups
of various enzymes, e.g. alcohol
dehydrogenase, thioredoxin reductase and even
disulfide bonds which are critical for
microorganism surveillance (5).
We studied garlic extract's inhibitory effect
against influenza virus A/H1N1. The virus was
cultured on MDCK cells. CC50 value of this
extract was obtained nearly100µg/ml by MTT
method. The concentration of 10µg/ml
(EMCC) was found to inhibit virus growth to a
large extent as was measured by reduction in
HA titer and CPE induction.
Inhibition of influenza virus adsorption to
MDCK cells and chicken red blood cells by the
Garlic extract was estimated by MTT and HA
methods. Its effect on the viral genome
replication was determined by RT-PCR
method. Our results showed concordance
among methods used in this study; high
percent protection following exposure of
Garlic extract to the virus cell culture using
MTT method is in concordance with reducing
HA titer of the virus cell culture after
incubation with this extract. The inhibitory
effect of the extract was seen in RT-PCR
products, too. By semi-quantitative analysis of
PCR products with free band densitometry
software, decrease in the content of the
treatment bands was obvious in compared with
untreated virus band.
For all three time periods of treatments, 1, 8
and 24h exposures, viral infectivity was
reduced. Although it was effective and
meaningful even after one hour contact
compared with 8 and 24h exposures. This may
involve interference of viral membrane fusion
by inhibition of penetration phase (11).
In conclusion, the biochemical and molecular
methods used to evaluate the antiviral activity
of Garlic extract demonstrated that this
compound could be suggested as a suitable
potent antiseptic agent.
22 Iranian Journal of Virology, Volume 3, Number 1, Spring 2009
www.SID.ir
Archive of SID
Mehrbod P. et al
The molecular basis of antiviral effect of Garlic
extract on influenza virus would be an
interesting field for future studies.
5. Ankri S, Mirelman D. Antimicrobial
properties of allicin from garlic. Microbes
Infect. 1999 Feb;1(2):125-9.
6. Shigeta S, Mori S, Watanabe J, Soeda
S, Takahashi K, Yamase T. Synergistic anti-
influenza virus A (H1N1) activities of PM-523
(polyoxometalate) and ribavirin in vitro and in
vivo. Antimicrob Agents Chemother. 1997
Jul;41(7):1423-7.
Acknowledgement
This study was supported by grant no. 392
from Pasteur Institute of Iran.
7. Karber G. 50% endpoint calculation,
Arch. Exp Pharmak. 1931;162:480-3.
References
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Iranian Journal of Virology, Volume 3, Number 1, Spring 2009 23
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Mathematical models can be used to predict the emergence and transmission of antiviral resistance. Previously it has been predicted that high usage of antivirals (in immunocompetent populations) to treat Herpes Simplex Virus type 2 (HSV-2) would only lead to fairly low levels of antiviral resistance. The HSV-2 predictions were based upon the assumption that drug-resistant strains of HSV-2 would be less infectious than drug-sensitive strains but that the drug-resistant strains would not be impaired in their ability to reactivate. Recent data suggest that some drug-resistant strains of HSV-2 are likely to be impaired in their ability to reactivate. Objectives: (1) To predict the effect of a high usage of antivirals on the prevalence of drug-resistant HSV-2 under the assumption that drug-resistant strains will be less infectious than drug-sensitive strains of HSV-2 and also have an impaired ability to reactivate. (2) To compare predictions with previous published predictions. We generated theoretical drug-resistant HSV-2 strains that were attenuated (in comparison with drug-sensitive strains) in both infectivity and ability to reactivate. We then used a transmission model to predict the emergence and transmission of drug-resistant HSV-2 in the immunocompetent population assuming a high usage of antivirals. Our predictions are an order of magnitude lower than previous predictions; we predict that even after 25 years of high antiviral usage only 5 out of 10,000 immunocompetent individuals will be shedding drug-resistant virus. Furthermore, after 25 years, 52 cases of HSV-2 would have been prevented for each prevalent case of drug-resistant HSV-2. The predicted levels of drug-resistant HSV-2 for the immunocompetent population are so low that it seems unlikely that cases of drug-resistant HSV-2 will be detected.
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Polyphenolic compound catechins ((-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin (EGC)) from green tea were evaluated for their ability to inhibit influenza virus replication in cell culture and for potentially direct virucidal effect. Among the test compounds, the EGCG and ECG were found to be potent inhibitors of influenza virus replication in MDCK cell culture and this effect was observed in all influenza virus subtypes tested, including A/H1N1, A/H3N2 and B virus. The 50% effective inhibition concentration (EC50) of EGCG, ECG, and EGC for influenza A virus were 22-28, 22-40 and 309-318 microM, respectively. EGCG and ECG exhibited hemagglutination inhibition activity, EGCG being more effective. However, the sensitivity in hemagglutination inhibition was widely different among three different subtypes of influenza viruses tested. Quantitative RT-PCR analysis revealed that, at high concentration, EGCG and ECG also suppressed viral RNA synthesis in MDCK cells whereas EGC failed to show similar effect. Similarly, EGCG and ECG inhibited the neuraminidase activity more effectively than the EGC. The results show that the 3-galloyl group of catechin skeleton plays an important role on the observed antiviral activity, whereas the 5'-OH at the trihydroxy benzyl moiety at 2-position plays a minor role. The results, along with the HA type-specific effect, suggest that the antiviral effect of catechins on influenza virus is mediated not only by specific interaction with HA, but altering the physical properties of viral membrane.
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Traditional methods used to monitor influenza infection typically require 2-5 days to perform, prompting a need for more rapid and quantitative methods for monitoring viral infection in 96-well formats. Such assays would find application in high-throughput screening for novel antiviral agents. A new method, based on branched DNA (bDNA) technology, is described for the specific detection of negative strand RNA of influenza A strains using a set of oligonucleotides designed for the A/PR/8/34 nucleoprotein (NP) transcript. By detecting the genomic strand, this signal amplification assay is appropriate for monitoring the kinetics of viral replication. Assay performance was monitored following infection of MDCK cells. The assay exhibited high reproducibility, good sensitivity over a range of multiplicity of infection and has a lower limit of detection of approximately 5 x 10 (5) RNA copies. Designed to quantitate the H1N1 strain A/PR/8/34, the assay also detects other influenza A subtypes, but not the evolutionarily more distant strain B/Yamagata/16/88. Validation as an antiviral assay was demonstrated with two influenza antivirals, zanamivir and rimantadine. The EC(50) values calculated following bDNA detection for zanamivir (265 nM) and rimantadine (9.4 microg/ml) in A/PR/8/34 infection correlate closely to data previously reported from visual CPE determinations, neutral red dye uptake and plaque assays, respectively. The advantages over the more time-consuming traditional assays suggest that the influenza bDNA assay is applicable to rapid screening of compound collections for antiviral activity.
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Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation namely A/H1N1, A/H3N2, and B. The objective of this study was to determine the human influenza virus genotypes in Shiraz, the capital of the Fars province of Iran. Three hundred patients suspected with human influenza virus infection were enrolled in this survey (2004-2005). The throat samples were cultured and titrated by hemagglutination (HA) assay. Typing and subtyping were performed by an in-house developed multiplex RT-PCR. Moreover, the phylogenetic analysis was carried out for HA gene. A total of 24 samples were found to be positive for human influenza virus infection, 17 H1N1 and 7 H3N2. These results were in agreement with the HI assay. The phylogenetic analysis results revealed that the Iranian H1N1 isolated were close to the A/New Caledonia/20/99 vaccine strain genetically and the Iranian H3N2 isolates were also related closely to the Fujian/411/021 and California/7/2004 vaccine strains. However, a slight genetic drift was found in these isolates. In conclusion, it was demonstrated that both influenza A subtypes A/H1N1 and A/H3N2 were dominant among Iranian patients in Shiraz during the 2004/5 winter season.
Evaluation of potential antiviral activity of hydroalcoholic extract of Lemon Balm L. against Herpes Simplex Virus type-I Development of a novel influenza A antiviral assay
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Shahrabadi M, Monavari SH, Sajjadi SE. Evaluation of potential antiviral activity of hydroalcoholic extract of Lemon Balm L. against Herpes Simplex Virus type-I. Iranian Journal of Virology. 2007;1:28-32. 9. Wagaman PC, Leong MA, Simmen KA. Development of a novel influenza A antiviral assay. J Virol Methods. 2002
Antimicrobial properties of Allium sativum (garlic) Applied Microbiology and Biotechnology Pharmacokinetics of silybin in bile following administration of silipide and silymarin in cholecystectomy patients
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Predicting the emergence of drug-resistant HSV-2: new predictions Antiviral effect of catechins in green tea on influenza virus
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Arzneimittelforschung. 1992 Jul;42(7):964-8. 10. Gershengorn HB, Darby G, Blower SM. Predicting the emergence of drug-resistant HSV-2: new predictions. BMC Infectious Diseases. 2003;3(1):186-9. 11. Song JM, Lee KH, Seong BL. Antiviral effect of catechins in green tea on influenza virus. Antiviral Res. 2005 Nov;68(2):66-74.