Anti-influenza virus activity of essential oils and vapors

Abstract

Few satisfactory therapeutic agents are available for the control of Influenza virus, largely because of
the continual emergence of drug-resistant mutants. Some essential oils (EOs) have demonstrated
effective antimicrobial and antiviral properties in experimental conditions, but most of these studies
tested the liquid oil phases, which are generally less practical and are potentially toxic for oral
applications. In the present study, we evaluated several EOs and some of their major constituents for
their possible anti-influenza virus properties in both liquid and vapor phases. In vapor phase Citrus
bergamia, Eucalyptus globulus, and the isolated compounds citronellol and eugenol were very active
against influenza virus following exposures of only 10 minutes. Pelargonium graveolens,
Cinnamomum zeylanicum, Cymbopogon flexuosus were also very active with 30 minutes exposure. In
liquid phase, Cinnamomum zeylanicum, Citrus bergamia, Cymbopogon flexuosus and Thymus vulgaris
displayed 100% inhibitory activity at 3.1 µL/mL concentration. Under these conditions the vapors
showed no measurable adverse effect on epithelial cell monolayers. This suggests that these oils in their
vapor phases could be potentially useful in influenza therapy. The oil vapors were also evaluated for
possible direct effects on the principal external proteins of the influenza virus, namely the HA
(hemagglutinin) and NA (Neuraminidase). Several of the vapors inhibited the HA activity, but not the
NA activity, suggesting that interaction with HA is a possible mechanism for the antiviral activity.
Thus some of these oil vapors could have therapeutic benefits for people suffering from influenza, and
possibly other membrane containing respiratory viruses.

Full text

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American Journal of Essential Oils and Natural Produ cts 2014; 2 (1): 47-53
ISSN: 2321 9114
AJEONP 2014; 2 (1): 47-53
© 2014 AkiNik Publications
Received: 22-06-2014
Accepted: 25-08-2014
Selvarani Vimalanathan
Department of Pathology &
Laboratory Medicine, University
of British Columbia, Vancouver,
BC, V6T 1Z1, Canada.
James Hudson
Department of Pathology &
Laboratory Medicine, University
of British Columbia, Vancouver,
BC, V6T 1Z1, Canada.
Correspondence:
Selvarani Vimalanathan
Department of Pathology &
Laboratory Medicine, University
of British Columbia, Vancouver,
BC, V6T 1Z1, Canada.
Email: vimrani1@mail.ubc.ca
Anti-influenza virus activity of essential oils and vapors
Selvarani Vimalanathan, James Hudson.
Abstract
Few satisfactory therapeutic agents are available for the control of Influenza virus, largely because of
the continual emergence of drug-resistant mutants. Some essential oils (EOs) have demonstrated
effective antimicrobial and antiviral properties in experimental conditions, but most of these studies
tested the liquid oil phases, which are generally less practical and are potentially toxic for oral
applications. In the present study, we evaluated several EOs and some of their major constituents for
their possible anti-influenza virus properties in both liquid and vapor phases. In vapor phase Citrus
bergamia, Eucalyptus globulus, and the isolated compounds citronellol and eugenol were very active
against influenza virus following exposures of only 10 minutes. Pelargonium graveolens,
Cinnamomum zeylanicum, Cymbopogon flexuosus were also very active with 30 minutes exposure. In
liquid phase, Cinnamomum zeylanicum, Citrus bergamia, Cymbopogon flexuosus and Thymus vulgaris
displayed 100% inhibitory activity at 3.1 µL/mL concentration. Under these conditions the vapors
showed no measurable adverse effect on epithelial cell monolayers. This suggests that these oils in their
vapor phases could be potentially useful in influenza therapy. The oil vapors were also evaluated for
possible direct effects on the principal external proteins of the influenza virus, namely the HA
(hemagglutinin) and NA (Neuraminidase). Several of the vapors inhibited the HA activity, but not the
NA activity, suggesting that interaction with HA is a possible mechanism for the antiviral activity.
Thus some of these oil vapors could have therapeutic benefits for people suffering from influenza, and
possibly other membrane containing respiratory viruses.
Keywords: Essential oil, Vapor phase, antiviral activity, Hemagglutination (HA) Inhibition
1. Introduction
Influenza viruses continue to pose threats of epidemics, resulting from mutated viruses, to
which we have inadequate therapeutic remedies, largely because of the continuing emergence
of drug-resistance. Thus alternative therapies, targeting the viruses themselves rather than
their individual genes, could be useful.
Essential oils (EOs) of plants have been used traditionally for numerous applications in
health-related areas, and in foods and commercial uses [1, 2]. In most medical applications the
oils were applied directly to the skin, although the potential cytotoxicity of EOs precluded
internal consumption [3]. This problem could, at least in theory, be avoided by inhalation of
the vapors of EOs, as practiced in aromatherapy. Furthermore in many traditional remedies
for colds and respiratory disorders, formulations often included plant EOs to provide relief
through inhalation of the vapors [3].
Recently a number of studies reported the presence of antimicrobial and antiviral activities in
certain EOs and their components, such as monoterpenes. However, those studies were
carried out with the liquid phases of the oils and their components [3, 4, 5, 6, 7, 8, 9, 10, 11].
In contrast, Inouye et al. [12] Thyagi and Malik [13], Hudson et al. [14] and Vimalanathan and
Hudson [15] demonstrated that the vapor or “gaseous” phase of certain EOs showed good
antibacterial, antifungal and antiviral activity, sometimes better than the corresponding liquid
phase of the oil.
Some studies also indicated that the whole unfractionated oil was as potent as any of the
individual components, suggesting synergism [3 , 10].These observations clearly indicate that,
while there is great potential for the use of EOs as antimicrobials and antivirals, there is still
scope for further evaluation of the optimal methods for their applications.

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In order to address these issues, we compared the anti-
influenza virus activities, and relative cytotoxic potentials, of a
number of commercial EOs, and some of their pure
compounds in their liquid and vapor phases. In addition we
also examined their effects on the influenza neuraminidase and
hemagglutinin, the major viral antigens.
2. Materials and methods
2.1 Test materials
All the essential oils (EOs) were standard commercial brands
purchased from local suppliers (Table 1).
Table 1: Essential oils used and their suppliers, major constituents and references.
Scientific name and family Common name
Supplier and origin Major components
(%) References
Lavandula officinalis
Lamiaceae
Lavender
Fresh flowering heads
Julia lawless
Aqua Oleum
Bulgaria
Linalyl acetate
Linalool
[24]
Pelargonium graveolens
Geraniaceae
Geranium
Leaves and flowering branchlets
Aura Cacia
China
Citronellol
Geraniol
[25, 26]
Cinnamomum zeylanicum
Lauraceae
Cinnamon leaf oil
Leaves Aura Cacia
Sri Lanka
Eugenol
[23]
Salvia officinalis
Lamiaceae
Sage
Partially dried leaves
Aura Cacia
Austria, Croatia
1,8
–
Cineole
α-Thujone
[7, 22]
Eucalyptus globulus
Myrtaceae
Eucalyptus
Leaves and twigs
Julia lawless
Aqua Oleum
South Africa
1,8
-
Cineole
α-Pinene
[13]
Cymbopogon flexuosus
Poaceae
Lemongrass
Freshly cut grass
Aura Cacia
India
Geranial
Neral
[27, 28]
Thymus vulga
ris
Lamiaceae
Red Thyme
Partially dried above ground plant parts.
Aura Cacia
Spain
1,8
-
cineole
Terpenyl acetate
Borneol
[21]
Citrus bergamia
Rutaceae
Bergamot
Fruit Peel
Aura Cacia
Italy
(
–
)
-
linalyl acetate
(–)-linalool
(+)-limonene
γ-Terpinene,
β-Pinene
α-pinene
α-terpinene
[29, 30]
Cupressus sempervirens
Cupressaceae
Cypress oil
Needles & Twigs
Aura Cacia
Morocco
α
-
pinene
α-terpinene
[7]
2.2 Cells and virus
Madin-Darby canine kidney cells (MDCK) and A549 human
lung epithelial cells were acquired originally from ATCC
(American Type Culture Collection, Rockville, MD), and were
passaged in Dulbecco MEM (DMEM), in cell culture flasks,
supplemented with 5% fetal bovine serum, at 37 °C in a 5%
CO2 atmosphere (cell culture reagents were obtained from
Invitrogen, Ontario CA). No antibiotics or antimycotic agents
were used.
Influenza virus A1/Denver/1/57 (H1N1) was acquired from
BC Centre for Disease Control, Vancouver, and was grown in
MDCK cells with TPCK (L-1-Tosylamide-2-phenylethyl
chloromethyl ketone; Sigma Chemical co.) treated trypsin (2
µg/mL) and assayed by plaque formation. The following pure
compounds citronellol and eugenol were kindly supplied to us
by Dr. Murray Isman, University of British Columbia. The
infectious titer of stock virus varied from 105-106 PFU/mL.

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2.3 Virucidal activity (liquid phase)
The assay technique was based on our standard techniques for
the evaluation of plant extracts for antiviral activity [16, 1 7]. The
experimental procedure consisted of incubating two-fold
dilutions of the test oil or compound in phosphate buffered
saline, in 96-well trays, with 20 µL of virus containing 800
plaque forming unit of virus. The mixtures, in triplicate, were
incubated for 60 min at 22 ºC. The total volume of 120 µL
from each mixture was then transferred into confluent MDCK
cells containing 1mL PBS, and incubated at 37 ºC to allow
adsorption of remaining virus. After 60 min the inocula were
removed and replaced with 0.5% agarose in MEM and 2
µg/mL trypsin. Monolayers were fixed with 3.6% formalin
after 48 h and stained with 0.1% crystal violet. Virus plaques
were counted. Inhibitory concentration was calculated as
MIC100 by comparison with untreated virus controls.
2.4 Virucidal activity (vapor phase)
The method used was a modification of the standard plaque
reduction assay described above. Aliquots (20 µL) of virus
(10000 pfu) were individually dried on the underside of the
caps from sterile Eppendorf tubes, within the biosafety cabinet
(10 min). Test oils (250 µL) were carefully added to each tube,
the caps were replaced with caps containing dried virus film
and exposure to oil allowed for 10 or 30 min, at 37 ºC. Caps
were removed again and each dried exposed film was
reconstituted in 1 mL of PBS. All samples (in triplicate) were
then assayed for virus plaque formation in MDCK cells as
described above. Canola oil, which does not inhibit influenza
virus, was used as a negative control. The reduction of viral
titer was quantified and viral infectivity loss due to drying was
determined to be ≤ 1.0 log10. Starting from a titer of 10,000
PFUs, the virus titer was reduced to ± 1,000 PFUs after drying,
before Eos vapor exposure.
2.5 Neuraminidase Assay (liquid phase)
The Amplex Red Neuraminidase (Sialidase) Assay Kit from
Invitrogen (Ontario) was used. Briefly, equal volumes (25 µL)
of two fold dilutions of EOs and virus (1:4 dilution of stock
virus) were mixed and incubated for at 37 °C with continuous
shaking. After 60 min, 50 µL of 2X working solution of 100
μM Amplex Red reagent containing 0.2 U/mL HRP, 4 U/mL
galactose oxidase and 500 μg/mL fetuin was added and the
mixtures incubated overnight. Absorbance (A) was measured
at 550 nm in a plate reader. The percentage of Neuraminidase
inhibition was calculated by the following formula: Avirus-
Atest/Avirus×100.
2.6 Hemagglutination (HA) Inhibition Assay (Liquid and
Vapor phases)
Since all the liquid EOs were toxic to erythrocytes on direct
exposure, HA inhibition was measured only in dried films of
virus exposed to the vapor phases, as described above for
antiviral activity of EO vapors in Eppendorf tubes.
Reconstituted exposed virus (50 µL) were mixed with 50 µL
of 0.75% suspension of human type O Rh+ erythrocytes and
incubated at 22 ºC for 60 min [18]. The hemagglutination
reaction was observed after 60 min incubation.
2.7 Cytotoxicity assay (Liquid and Vapor phases)
The Cell Proliferation Assay Kit (XTT) (ATCC, Manassas,
VA) was used according to the manufacturer’s instructions.
Human lung epithelial cells (A549 cell line) were used as the
indicator cells. The cells (5× 103) were seeded in each well
containing 100 μL of the MEM medium supplemented with
5% FBS in a 96-well plate. Cells were grown for 48 h and the
test materials, prepared as a series of two-fold dilutions in
MEM, without phenol red, were added to the cells and
incubated for 15 min, followed by removal of test material
and incubation for a further 24 h in MEM, followed by
measurement of cell viability. For cytotoxicity of EOs vapor,
monolayers of human lung A549 cells grown to confluence in
6-well trays for 48 hour. For EOs exposure, the media were
removed by aspiration, and the moist cells were exposed to
EOs vapor. Following further 24 h incubation in normal
medium, cell viability was measured. The results were
measured as absorbance at 490 nm in a plate reader, in
comparison with similar cells exposed to medium only.
Cytotoxicity is expressed as the concentration of test sample
inhibiting cell growth by 50% (TC50). All tests were run in
triplicate and mean values recorded.
3. Results
3.1 Antiviral Activities of Liquid Phase EOs
Three of the oils, Cinnamomum zeylanicum, Citrus bergamia
and Thymus vulgaris (Figure 1, 2), were able to completely
inactivate (IC100) the virus at high dilutions, down to <3.1 µL
per mL or less (Figure 3). Lavandula officinalis and
Eucalyptus globulus also showed excellent activity at higher
concentrations, but were much less effective at the lower
concentrations. Salvia was only partly active at the
concentrations tested. In similar experiments liquid phase
Pelargonium graveolens (Geranium) oil also showed good
antiviral activity. Cupressus sempervirens and its main
constituent α-pinene did not show antiviral activity even at
very low dilutions. Among the pure components tested, only
eugenol showed 100% plaque reduction (Figure 3) but
citronellol displayed partial inactivation. Liquid canola oil,
used as a negative control, had no activity even at very high
concentration (100%)
Fig 1: Antiviral Activities of serial dilutions of EOs. Each sample, in
triplicate, was serially diluted 2x and incubated with a standard
amount of H1N1 virus (800 pfu per reaction). Remaining infectious
viruses were measured by plaque assay on MDCK cells.

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Fig 2: Antiviral Activities of serial dilutions of EOs. Each sample, in
triplicate, was serially diluted 2x and incubated with a standard
amount of H1N1 virus (800 pfu per reaction). Remaining infectious
viruses were measured by plaque assay on MDCK cells.
Fig 3: Antiviral Activities of EOs and selected monoterpenes against
H1N1 virus with 3.1 µL/mL. Each sample, in triplicate, was diluted
to 3.1 µL/mL and incubated with a standard amount of H1N1 virus
(800 pfu per reaction). Remaining infectious viruses were measured
by plaque assay on MDCK cells. Results are expressed as percentage
of plaque reduction.
3.2 Antiviral Activities of EO Vapor Phases
Two EOs, C. bergamia, E. globulus, and the tested pure
compounds citronellol and eugenol showed significant activity
against influenza virus following exposures of only 10
minutes.
Several of the oil vapor phases were able to completely
inactivate influenza virus following exposures of 30 minutes,
as shown in Figure 4. These were, C. zeylanicum, C.
flexuosus, L. officinalis and P. graveolens. T. vulgaris and S.
officinalis showed only partial activity, and the two negative
controls, olive oil and canola oil, showed no antiviral activity.
Thus the relative activities did not reflect the corresponding
activities of the liquid phases.
Fig 4: Antiviral Activities of EOs in vapor phase. Aliquots of
influenza virus, in triplicate dried films of H1N1 virus (1000 pfu of
H1N1 per reaction) were exposed to EO vapors for 10 and 30
minutes, reconstituted in PBS, and remaining infectious viruses
measured by plaque assays on MDCK cells.
3.3 Activities Against Viral HA (Hemagglutinin) and NA
(Neuraminidase)
HA and NA are the two most important proteins of the
influenza virus which together determine successful infection
and dissemination of the virus.
Most of the oil vapors tested showed anti-hemagglutination
activity against the indicator human erythrocytes type O Rh+,
as shown in Table 2 and Figure 5. Since most of the tested Eos
in liquid phase had hemolytic effect, that disabled further
testing of HAI assay with liquid oils.
In contrast to the anti-HA activities, most of the oils, even in
liquid phase, were not able to inhibit NA activity, as shown in
Figure 6. The exception was Cinnamomum zeylanicum, which
showed complete inhibition at least down to 1.5 µL/mL. The
positive control anti-influenza compound zanamivir was
effective as expected.
Human lung epithelial cells (A549) were exposed to EOs for
15 min, followed by incubation in normal medium for 24 h.
They were then assayed for cell viability. Results are shown in
Table 3. All the EOs showed some toxicity at the high
concentrations but were non-toxic at concentrations less than
10 µL/mL. The 24 hour exposures showed greater degrees of
cytotoxicity, except for olive oil, which remained non-
cytotoxic.
In contrast to the results with the liquid phases, when cell
monolayers were exposed to each of the oil vapors for 10 or
30 min there were no adverse effects on cell appearance or
viability, as determined by XTT measurements.

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Fig 5: Viral HAI Assay plate. Inhibitory activity of EOs’ vapor on agglutination with viral hemagglutinin and human type O Rh+ erythrocytes,
as described in Methods (and in WHO Manual, 2011). The presence of conspicuous red buttons in the well indicates absence of agglutination,
i.e. inhibition of HA activity. The other wells show normal hemagglutination.
Fig 6: Effect EOs and zanamivir on the influenza virus Neuraminidase activity. Only the Cinnamon and the positive control zanamivir were
active.
Table 2: Inhibitory activity of EO vapors on agglutination with viral
hemagglutinin and human RBC O Rh+.
EOs vapor
Hemagglutination
inhibition activity (HAI)
C. bergamia
+
C. fle
xuosus
+
C. sempervirens
-
C. zeylanicum
+
E. globulus
+
L. officinalis
-
P. graveolens
+
S. officinalis
+
T. vulgaris
+
Olive oil
-
Virus control
-
Cell control
+
Table 3: TC50 values of EOs in human lung epithelial cells.TC50 =
50% tissue culture cytotoxicity, or concentration giving 50%
reduction in cell viability
Eos liquid
TC
50
Viability (µL/mL)
Citrus bergamia
15.98±6.92
Cymbopogon flexuosus
12.21±2.64
Cupressus sempervirens
75±7.98
Cinnamomum zeylanicum
12.3± 2.9
Eucalyptus globulu
s
26.51± 4.9
Lavandula officinalis
26.46± 3.9
Pelargonium graveolens
67.46± 6.85
Salvia officinalis
26.85± 7.2
Thymus vulgaris
14.34± 3.32
Olive oil
>100±0
Cell control
100
All EO vapors
No cytotoxicity

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4. Discussion
Respiratory viruses continue to cause problems within the
general population, as a result of frequent acute and chronic
infections, including occasional epidemics. Few satisfactory
therapeutic agents are available, in part because of the
diversity of replication schemes among these viruses, and
consequently the lack of a generic molecular target; and partly
because of the continual emergence of drug-resistant mutants
in the viral populations. These problems are well illustrated by
influenza viruses.
Some essential oils have demonstrated effective antimicrobial
and antiviral properties, and in a few cases beneficial anti-
inflammatory properties [4, 8, 19, 6, 3, 20]. However, these studies
tested the liquid oil phases, which are generally less practical
and potentially toxic for nasopharyngeal or oral applications.
A few reports have indicated that the vapors of some oils
might be useful for this purpose [3 , 13, 14 ], and this type of
application would be in accord with anecdotal reports of the
usefulness of inhaled vapors [3, 14].
In the present study we evaluated several essential oils (EOs)
for their possible anti-influenza virus properties under
conditions that are relevant to potential applications. Four of
the EOs, Cinnamomum zeylanicum, Citrus bergamia and
Thymus vulgaris were very active against influenza virus at
relatively low concentrations (MIC100 3.1 µL/mL) in the liquid
phase, but only Citrus bergamia showed prominent activity
(95% ± 2) in its vapor phase (10 min exposure), the rest
showed activity only after 30 min exposure. On the other hand,
Eucalyptus globulus had less activity (MIC100 50 µL/mL) in
liquid phase but showed prominent activity in 10 min vapor
phase (94% ± 3). Interestingly, although 1,8-cineole is the
major component [ 13, 21, 7, 22] of Eucalyptus globulus, Thymus
vulgaris and Salvia officinalis, only Eucalyptus globulus and
Thymus vulgaris exhibited antiviral activity, suggesting that
1,8- cineole might not be responsible for the antiviral property
of Eucalyptus globulus and Thymus vulgaris.
Under these conditions the vapor showed no measurable
adverse effect on epithelial cell monolayers. This suggests that
these oils in their vapor phases could be potentially useful in
influenza therapy. In addition Cinnamomum zeylanicum and
Thymus vulgaris were also very active in the liquid phase,
although they were only partially effective in the vapor phase.
However eugenol, the major component [23] of Cinnamomum
zeylanicum possessed the most potent anti-influenza activity in
both liquid and vapor phases. This suggests that eugenol might
be one of the major components responsible for antiviral
property of Cinnamomum zeylanicum.
Conceivably a longer exposure to these oil vapors could result
in complete virus inactivation. In contrast Salvia officinalis
displayed only slight antiviral activity, only 20% plaque
reduction following 30 minutes exposure. Cupressus
sempervirens oil did not show any activity, this may be
correlated to the inactivity of the main component α-pinene in
both phases. Liquid canola oil, included as a negative control
[19] showed no activity.
The oils were also evaluated for possible direct effects on the
principal external proteins of the influenza virus, namely the
membrane proteins HA (hemagglutinin) and NA
(Neuraminidase). It was not possible to test the liquid oils
against HA because all of them lysed the indicator
erythrocytes. However, we were able to evaluate the vapors,
which did not affect the integrity of the test erythrocytes, and
in this situation most of the oil vapors inhibited viral HA
activity, with the exception of Lavandula officinalis.
In contrast none of the liquid oils, except Cinnamon, were
able inhibit NA activity. This suggests that a primary target
for most of the oils is the viral HA, and this activity was
demonstrated with the EO vapors. However Cinnamon may
be able to target both external proteins. Since the HA and NA
proteins of influenza virus are responsible for virus entry and
exit into and from cells respectively, then inhibition of either
of these viral functions would decrease the growth and
dissemination of the virus.
Most of the liquid phase oils showed cytotoxic effects in
human lung epithelial cells, but in contrast the vapor phases
did not appear to show adverse effects following exposures of
at least 10 minutes.
5. Conclusion
Several of the essential oil vapors evaluated possess potent
anti-influenza virus activity, under conditions that did not
adversely affect cultured epithelial cells. The hemagglutinin
protein of the virus appeared to be a major target. Thus some
of these oil vapors could have therapeutic benefits for people
suffering from influenza, and possibly other respiratory
viruses.
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