Melissa officinalis efficacy against human influenza virus (New H1N1) in comparison with oseltamivir

Abstract

Objective: To evaluate the antiviral activity of Melissa officinalis (MO) extract against the
influenza virus H1N1 in vitro.
Methods: The cytotoxicity of MO extract was identified on Madin-Darby canine kidney
(MDCK) cell culture by MTT assay. The virus was inoculated to the cells (multiplicity of
infection = 0.1) in two protocols. In protocol 1, the MO extracts at concentrations of 0.005,
0.050, 0.010, 0.100 and 0.500 mg/mL were incubated with the virus for one hour preinoculation.
In protocol 2, the mentioned concentrations of MO extracts were added to the
cells one-hour post infection. Furthermore, the antiviral effect of oseltamivir with different
concentrations was tested as the positive controls. The 50% tissue culture infective dose,
neutralizing index and hemagglutination titer were determined.
Results: The medicine oseltamivir and MO extracts were not toxic for MDCK at concentrations
less than 1 mg/mL. All utilized concentrations of MO extracts were vigorously efficient to
decrease the viral yield in both experiments. The 50% tissue culture infective dose of the
groups containing up to 0.100 mg/mL of MO extracts in the first experiment in compare with
0.050 mg/mL in the second experiment reduced to 0. Although hemagglutination tests showed
little titers, the viral quantity significantly decreased in both experiments. By the way, the
medicine oseltamivir could completely suppress viral replication in MDCK.
Conclusions: The present study suggests that MO extracts have a potent anti-influenza effect in
cell culture.

Full text

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Infectious disease research doi: 10.1016/S2222-1808(16)61115-5 ©2016 by the Asian Pacific Journal of Tropical Disease. All rights reserved.
Melissa ofcinalis efcacy against human inuenza virus (New H1N1) in comparison with oseltamivir
Parvane Jalali1, Afagh Moattari2, Ali Mohammadi1, Nima Ghazanfari3, Gholamhosein Pourghanbari4*
1Division of Virology, Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
2Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
3Department of Pharmacology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
4Department of Clinical Science, School of Veterinary Medicine, Ardakan University, Ardakan, Yazd, Iran
Asian Pac J Trop Dis 2016; 6(9): 714-717
Asian Pacific Journal of Tropical Disease
journal homepage: www.elsevier.com/locate/apjtd
*Corresponding author: Dr. Gholamhosein Pourghanbari, Department of Clinical
Science, School of Veterinary Medicine, Ardakan University, Ardakan, Yazd, Iran.
Tel: 0098-35-32240926
Fax: 0098-35-32240926
E-mail: hpourghanbari@ardakan.ac.ir, hpourghanbari@gmail.com
The journal implements double-blind peer review practiced by specially invited
international editorial board members.
1. Introduction
Influenza A viruses are considered to be one of the most important
human pathogens and can cause severe viral respiratory infections.
The influenza pandemics such as those occurred in 1918 resulted
in high morbidity and mortality rates mainly due to the lack of
sufficient protection against the new virus strains[1]. The pandemic
new H1N1 virus spread rapidly throughout the world in 2009 and
the virus was shown to be more transmissible than the seasonal
H1N1[2].
Two groups of antiviral compounds have been approved by the
Food and Drug Administration until now, but oseltamivir is regarded
as the drug of choice for influenza viruses[3,4].
Lemon balm is one of the most important member of the
Lamiaceae family and it is native to Europe, central Asia and Iran.
The main ingredients of the Melissa officinalis (MO) are
citral (neral and geranial), citronellal, linalool, geraniol and
β-caryophyllene-oxide. Tannins such as triterpenylic acid, bitter
principles, flavonoids including phenolic acids, terpenes, rosmarinic
acid and caffeic acids were belonged to the Lamiaceae[5-8].
Several properties of lemon balm such as antioxidant,
antihistamine, antispasmodic, anti-tumor/anticancer, antibacterial,
antifungal, antidepressant and antiviral activities were reported[9,10].
It has been shown that the extract of MO is able to prevent protein
synthesis in the herpes simplex virus type 1[11]. Some studies have
shown that the antiviral activity of lemon balm was due to tannins
and polyphenolic compounds[12].
There is no report on the efficacy of lemon balm on the
human influenza viruses. In this study, the antiviral activity of
MO hydroalcoholics extracts and their synergistic activity with
oseltamivir on the replication of the influenza virus subtype H1N1
ARTICLE INFO ABSTRACT
Objective: To evaluate the antiviral activity of Melissa officinalis (MO) extract against the
influenza virus H1N1 in vitro.
Methods: The cytotoxicity of MO extract was identified on Madin-Darby canine kidney
(MDCK) cell culture by MTT assay. The virus was inoculated to the cells (multiplicity of
infection = 0.1) in two protocols. In protocol 1, the MO extracts at concentrations of 0.005,
0.050, 0.010, 0.100 and 0.500 mg/mL were incubated with the virus for one hour pre-
inoculation. In protocol 2, the mentioned concentrations of MO extracts were added to the
cells one-hour post infection. Furthermore, the antiviral effect of oseltamivir with different
concentrations was tested as the positive controls. The 50% tissue culture infective dose,
neutralizing index and hemagglutination titer were determined.
Results: The medicine oseltamivir and MO extracts were not toxic for MDCK at concentrations
less than 1 mg/mL. All utilized concentrations of MO extracts were vigorously efficient to
decrease the viral yield in both experiments. The 50% tissue culture infective dose of the
groups containing up to 0.100 mg/mL of MO extracts in the first experiment in compare with
0.050 mg/mL in the second experiment reduced to 0. Although hemagglutination tests showed
little titers, the viral quantity significantly decreased in both experiments. By the way, the
medicine oseltamivir could completely suppress viral replication in MDCK.
Conclusions: The present study suggests that MO extracts have a potent anti-influenza effect in
cell culture.
Contents lists available at ScienceDirect
Article history:
Received 23 Jun 2016
Received in revised form 17 Jul, 2nd
revised form 8 Aug 2016
Accepted 12 Aug 2016
Available online 15 Aug 2016
Keywords:
Melissa officinalis
New H1N1
Antiviral
Cell culture

Parvane Jalali et al./Asian Pac J Trop Dis 2016; 6(9): 714-717 715
were evaluated in Madin-Darby canine kidney (MDCK) cell line and
the efficacy were analyzed by the 50% tissue culture infective dose
(TCID50) and hemagglutination (HA) tests.
2. Material and methods
2.1. Reagents
Lemon balm extracts were prepared in the Pharmacy Department
of Shiraz University of Medical Science. Antibiotic, trypsin–
ethylene diamine tetraacetic acid, fetal bovine serum (FBS) and
Dulbecco’s modified eagle medium (DMEM) were supplied by
Gibco BRL (Grand Island, NY, USA). Oseltamivir (F. Hofmann La
Roche Ltd, Switzerland) was purchased from a pharmacy in Iran.
Tissue culture plates and flasks were purchased from Falcon (BD
Biosciences, Franklin Lakes, NJ, USA). Stock solutions (10 mg/mL)
of the materials were solved in dimethyl sulfoxide (DMSO) and were
subsequently diluted in appropriate culture media. The maximum
DMSO concentration reached to 0.1%.
2.2. Cells and viruses
Human influenza A virus, 2009 pandemic new H1N1, which was
taken from Influenza Virus Research Centre of Shiraz University
of Medical Science, the Influenza virus (new H1N1) was replicated
and passaged in the cells and virus titers were evaluated by using
TCID50. MDCK cells were grown in DMEM with penicillin (100 IU/
mL), streptomycin (100 µg/mL) and supplemented with 7% heat-
inactivated FBS.
2.3. Cell viability assay
The extract and oseltamivir efficacy against the MDCK was
measured by MTT test. MDCK cells were propagated (1 × 104 cells/
well) in a 96-well plate for 26 h. The medium was taken place with
DMEM including different concentrations of the extracts. After
incubating the cells at 37 °C for 48 h, 100 µL of the Roswell Park
Memorial Institute medium (without phenol red) with 10 µL MTT (5
mg/mL in phosphate-buffered saline) was added to each well and the
cells were incubated for 4 h. Then, the supernatant was removed and
50 µL of DMSO was inoculated to the each well and incubated for 30
min. A micro plate reader at the wave length of 540 nm was used for
recording the absorbance[13].
2.4. Virus inoculation
MDCK cells were grown in the plates (including 96 wells) by using
DMEM (1 × 104/well), when the cells confluence was up to 90%.
The residual FBS was removed by washing the cells with phosphate-
buffered saline twice.
The inoculation of the cells with the virus at multiplicity of
infection = 0.1 was carried out in two protocols. In protocol one
(pre-infection), the virus was added with the different concentrations
of MO extracts (0.500, 0.100, 0.050, 0.010 and 0.005 mg/mL) for 1
h then the inoculation was occurred. In protocol two (post-infection),
the cells were incubated with the virus for 1 h, and then the medium
containing those concentrations of MO extracts were added to the
wells and incubated for 72 h. The medium applied in both protocols
contained trypsin at the concentration of 2 µg/mL. The incubations
were performed at 37 °C in 5% CO2 and 80% humidity.
The oseltamivir was also used as the positive control. The medium
containing the various concentrations (0.500, 0.100, 0.050, 0.010,
0.005 mg/mL) of oseltamivir were inoculated to the MDCK cells for
1 h after the virus inoculation (multiplicity of infection = 0.1) and
incubated for 72 h.
The cell culture supernatants were collected and the viral HA titres
and TCID50 were calculated.
2.5. TCID50 test and HA assay
A standard protocol by using a 2-fold dilution of each sample was
used for TCID50 test[14].
Cell culture supernatants containing virus was diluted 2-fold
serially and 0.5% chicken red blood cell was inoculated at an equal
amount. Then, the plate was incubated for 60 min at 4°C, the red
buttons were composed in negative wells, whereas positive wells did
not show any red buttons and the opaque appearance was observed.
HA results are given as hemagglutination units/50 µL (HAU/50 µL).
2.6. Neutralizing index (NI)
The NI test was used to obtain the antiviral activity of the extracts
or drugs. The NI of virus inactivation was calculated by subtracting
the log10 titer of collected virus from the infected MDCK cells with
extract/drug treated virus from the collected viral titer of the infected
negative control cells[1]. Inactivation of the virus was evaluated to
be effective when NI ≥ 2.8 and the NI in positive control group was
4.0[15].
3. Results
3.1. Cell viability
The safe concentration of MO extract and oseltamivir in MDCK
cells were calculated by using different concentrations of the
components and adding them to the cells and the cytotoxicity was
evaluated with MTT assay. The cytotoxicity of the MO extract and
oseltamivir on the MDCK cells was reduced under to 50% as the
concentration was decreased to 1 mg/mL.
3.2. MO extract efficacy in protocol 1
3.2.1. HA and TCID50 test
The mean virus titer in the negative control reached to 53 HAU/50
µL, while it decreased to 20 HAU/50 µL at the least concentration
0.005 mg/mL and to 15 HAU/50 µL at 0.010 mg/mL. The mean
virus titer was 5.5 HAU/50 µL at the rest concentrations.
The viral TCID50 based on log10 reached to 4 in the negative
control while it was 2 at the concentration of 0.005 mg/mL. It
decreased to 0 at the concentrations of 0.100 and 0.500 mg/mL of
MO.
3.2.2. NI
According to the findings, NI was shown to be equal or more than

Parvane Jalali et al./Asian Pac J Trop Dis 2016; 6(9): 714-717
716
2.8 in the groups containing 0.050, 0.100 and 0.500 mg/mL of MO.
3.3. MO extract efficacy in protocol 2
3.3.1. HA and TCID50 test
The mean virus titer in the negative control reached to 53 HAU/50
µL, whereas it decreased to 15 HAU/50 µL at the concentration of
0.005 mg/mL. MO extract could considerably decrease the virus HA
titer in all concentrations (Table 1).
Table 1
The effect of the MO extract and oseltamivir on the human influenza virus
subtype new H1N1 replication.
Protocol Concentration
(mg/mL)
Evaluation tests
HA (HAU/50 µL) TCID50
(Log10/mL)
NI
Pre-incubation 0.500 5.5 (ta) 0.0 4.0
0.100 5.5 (ta) 0.0 4.0
0.050 5.5 (ta) 1.2 2.8
0.010 15.0 (ta) 1.8 2.3
0.005 20.0 (ta) 2.0 2.0
Post-inoculation 0.500 3.5 (ta) 0.0 4.0
0.100 7.5 (ta) 0.0 4.0
0.050 11.0 (ta) 0.0 4.0
0.010 12.0 (ta) 1.2 2.8
0.005 15.0 (ta) 1.5 2.5
Oseltamivir 0.500 0.0 (ta) 0.0 4.0
0.100 0.0 (ta) 0.0 4.0
0.050 0.0 (ta) 0.0 4.0
0.010 0.0 (ta) 0.0 4.0
0.005 0.0 (ta) 0.0 4.0
Negative control 0.000 0.0 0.0 0.0
Positive control 0.000 53.0 (tpc) 4.0 -
ta: Log10 titer of collected virus from the infected MDCK cells with extract/
drug treated virus; tpc: Viral titer of the infected control cells (positive).
The TCID50 and HA results were decided based on log10/mL and
HAU/50 µL, respectively. NI was calculated by the equation:
NI = tpc – ta
where ta was log10 titer of collected virus from the infected MDCK
cells with extract/drug treated virus and tpc was viral titer of the
infected control cells (positive).
3.3.2. NI
The tpc was 4 TCID50/mL. NI of MO extract was equal or more then
2.8 at the concentrations equal or more than 0.010 mg/mL.
3.4. Oseltamivir efficacy on virus growth
3.4.1. HA and TCID50 test
The mean virus titer in MDCK in the presence of all concentrations
of oseltamivir reached to 0. Also the viral TCID50 based on log10 was
0 in all concentrations of oseltamivir.
3.4.2. NI
NI of oseltamivir was equal to 4 in all concentrations. Oseltamivir
had a definite inhibitory effect on the growth of influenza virus
H1N1 in MDCK.
4. Discussion
New H1N1 strain of influenza virus may spread to the environment
from humans, swines and birds. Due to antigenic changes between
subtypes of influenza A viruses, there is a probability of the
emergence of any subtype with dangerous antigenic properties
among human populations and also in the livestock and poultry
industry[16].
Recently, Food and Drug Administration has emphasized that the
drugs which affect M2 and NA influenza virus proteins have created
drug resistant viruses. According to recent reports, the majority of
viruses circulating during the years 2007–2008 and later are resistant
to oseltamivir in America and Australia[17-20]. Due to the resistance
of influenza viruses to synthetic antiviral drugs, it would be
necessary to develop other compounds such as traditional and herbal
medicines.
In this study, the effect of the hydroalcoholic extract of MO on the
growth of influenza virus subtype H1N1 in the MDCK cell culture
was evaluated and compared with oseltamivir.
Reviewing the efficacy of MO extract on the growth of flu virus
(H1N1) showed that the hydroalcoholic extract of MO reduces the
virus growth in both protocols as compared to the control group.
According to the results, it seems that the extract through several
mechanisms, including direct destructive effect on the virus and
effect on the internal mechanisms of the cell, can reduce the virus
titer of influenza A subtype H1N1, suggesting the suppressing effect
of MO extract on the growth of influenza virus H1N1. Pourghanbari
et al.[21] recently have found that MO essential oil was able to
suppress the propagation of the avian influenza virus (H9N2),
especially throughout the direct interaction with the virus particles.
Also, melissa extract exhibits virucidal activity and affects herpes
simplex virus-1 attachment to host cells in vitro[22,23]
According to many studies, lemon balm extract has antioxidant,
antihistamine, antispasmodic and anti-cancer effects. The extract
also stimulates the immune system and rosmarinic acid compound
inhibits several inflammatory pathways of the complement system,
especially C5-convertas[24,25]. Also the aqueous extract of lemon
balm has antiviral effects against HIV-1 and flu viruses. This herb
also has antibacterial and antifungal effects[26,27].
In recent years, the effects of medicinal herbs on the virus have
been studied. In a study, the antiviral activity of pomegranate extract,
pomegranate juice and fulvic acid were evaluated on the growth
of influenza viruses H1N1, H3N2, H5N1. All of these compounds
had an immediate inhibitory action on the growth of influenza
virus and the electron microscope test revealed that virus particles
were neutralized by these compounds and covered with unknown
substances and particles were damaged, but H5N1 virus was affected
less than other viruses[28].
According to Song and Choi[29], silymarin compound, which is
a flavonoid extracted from Silybum mariamum, has anti-influenza
virus efficacy when compared with oseltamivir and the inhibitory
effect of the compound is in the last stages of the virus synthesis.
The results of this study suggest MO extracts act as an antiviral
substance like oseltamivir that was shown in table 1.
In a study, one of the most important phenolic compounds called
punicalagin had synergistic effects with oseltamivir on the growth of
influenza A virus subtypes H1N1 and H3N2[13]. Song et al.[30] also
reported that polyphenolic compounds in green tea have synergistic
effects with oseltamivir.
In conclusion, Although oseltamivir had a perfect influence

Parvane Jalali et al./Asian Pac J Trop Dis 2016; 6(9): 714-717 717
to inhibit influenza virus growth in MDCK, MO extract also had
considerable effect especially in concentrations more than 0.050 mg/
mL. The studies on in vivo impact of the extract in prevention or
treatment of the disease would be necessary.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgments
This work was supported by a grant from School of Veterinary
Medicine at Shiraz University and Shiraz University of Medical
Science.
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