Antiviral Activity of Essential Oils and Essential Oil Compounds against Enveloped
Viruses
A subjective selection of interesting scientific works on this topic from the last
decade is summarized in Table 1S, Supporting Information. All of the listed viruses and EOs are summarized and characterized
in Tables 3S and 4S, Supporting Information, respectively. Some scientific papers are covered below in
more depth to give the readers an insight into the antiviral mode of action of EOs
and isolated oil components in cell-based assays (in vitro) and, in some cases, also in animal models (in vivo).
Influenza viruses
IFVs are enveloped RNA viruses that are classified as type A, B, or C by HA and NA
proteins. Only IFV-A and IFV-B are dangerous to the health of human beings [35]. The structure and replication cycle of IFV are described in detail by Samji [36].
Influenza viruses in liquid phase
The antiviral effect of TTO and its active components were explored in vitro on IFV A/PR/8 (H1N1) in MDCK cells [39]. It was demonstrated that the test compounds (TTO at 0.01%, terpinen-4-ol at 0.01%,
terpinolene at 0.005%, α-terpineol at 0.02%) inhibited early events in the virus replication cycle when added
to the cells within 1 h after the end of the virus adsorption period to MDCK cells.
The virus titer was reduced by 70 to 100%. However, no further reduction in the virus
replication was observed if the substances were added to the host cells later than
2 h after virus adsorption. Further experiments demonstrated that TTO and its components
did not interfere with the cellular attachment of the virus nor did it inhibit the
IFV neuraminidase activity or show any virucidal effects [39]. It has been shown in literature that the acidic condition in endosomes and lysosomes
is essential for the
uncoating process of the IFV inside infected host cells [40], [41]. The treatment of MDCK cells with TTO and terpinen-4-ol at 0.01% at 37 °C for 4 h
inhibited the acridine orange accumulation in cytoplasmic vesicles (cellular endosomes/lysosomes),
indicating an inhibitory effect of the test compounds on the acidification of these
intracellular compartments. The result obtained was concordant with the positive control
bafilomycin A1 (autophagy inhibitor) at 100.0 nM. From these experiments, the authors
concluded TTO and terpinen-4-ol could potentially inhibit IFV-A replication intracellularly
by inhibiting the uncoating of the viruses [39].
Li et al. [42] examined the anti-IFV (IFV-A/2009/H1N1) activity of a MAC mainly consisting of terpinen-4-ol
(56 – 58%), γ-terpinene (20.65%), and α-terpinene (9.80%). When cell-free IFV-A particles were incubated by 0.01% MAC for
1 h prior to MDCK cell infection, no cytopathic effects on host cells were detected.
In addition, electron microscopic studies showed that MAC in the virucidal concentration
of 0.01% did not damage the virus envelope. But in an in silico molecular docking experiment, the authors could demonstrate that terpinen-4-ol, the
main constituent of MAC, could bond very strongly to the receptor (membrane fusion)
site of HA of IFV-A (s. Table 1S, Supporting Information). The authors concluded from these experiments that MAC prevented
viruses from entering the cells by disrupting the normal fusion process between virions
and host cells.
Wu et al. [43] investigated the anti-IFV-A (H2N2) properties of PA in in vitro, in vivo, and in silico experiments. PA is a sesquiterpene alcohol, which is the major constituent of patchouli
oil derived from the aerial part of Pogostemon cablin (Blanco) Benth. Posttreatment of virus-infected cells with PA resulted in a strong
reduction of the viral replication inside MDCK cells, with an IC50 value of 4.03 µM (SI: 4.96). The antiviral activity was time and concentration dependent.
In the nontoxic concentration of 8.0 µM, the compound inhibited the virus replication
in a time-dependent manner, with a maximum inhibition of 97.68% after 72 h of incubation.
In contrast to this result, no effects were seen when PA was used for the pretreatment
of cells or virions or when the compound was added during the virion adsorption phase.
On the other hand, an in silico molecular docking experiment revealed a strong
chemical bond of PA to the active site of the viral NA protein (s. Table 1S, Supporting Information). NA plays a crucial role in the release of newly formed
progeny IFVs from the cell membrane of infected host cells into the intercellular
space (e.g., nasal epithelium) [44]. In addition, in a mouse model, PA protected the test animals from an influenza
infection at a concentration of 5.0 mg/kg/day. Mice were infected by intranasal instillation;
oral treatment daily for 5 days; 10 animals in total. Result (survivors/total): PA
7/10 animals; oseltamivir (1.0 mg/kg/day) 5/10 animals; infected control 0/10 animals.
Recently, the antiviral activity of PA on three different IFV-A types, H1N1 (A/Puerto
Rico/8/34), H1N1 (A/NWS/33), and H1N1 (A/Virginia/ATCC1/2009), was reexamined in in vitro and in vivo experiments [45]. PA exerted its antiviral activity in vitro by direct inactivation of cell-free virus particles, with IC50 values of 6.1, 3.5, and 6.3 µg/mL, respectively, as well as by inhibiting the early
steps of viral replication in MDCK cells. The virucidal effect of PA was not based
on a direct interaction with the NA and HA proteins on the virus surface. Therefore,
it can be speculated that the virucidal effect of PA can be caused by a destruction
of the viral envelope. The inhibition of the early steps in the viral life cycle could
also be explained by a reduced viral RNA and protein expression. In cells that were
treated with PA (40.0 µg/mL) after virus infection (posttreatment of virus-infected
cells), the viral NP
mRNA level and NP protein synthesis were significantly reduced by about 86 and
80%, respectively. IFV NPs are proteins that are associated with vRNA to form nucleocapsids.
In an in vivo experiment (mouse pneumonia model), mice were first inoculated intranasally with
IFV-A (A/PR/8/34) and subsequently divided into different experimental groups. Four
hours after inoculation, 10 mice per group received intranasal PA (20.0 or 40.0 µg/day)
or placebo or oral oseltamivir phosphate (10.0 mg/kg/day); these treatments were repeated
once daily for 4 or 7 days. In contrast to the control groups, the PA treatment (40.0 µg/mL)
significantly reduced the pulmonary virus titer (from about 5.0 × log10 PFU/mL to about 3.0 × log10 PFU/mL), increased the survival time of the infected mice (survival time 14 days
post-infection: placebo group: 30%; verum group 100%), and reduced the symptoms of
pneumonia. The effects were comparable to that of oseltamivir [45].
Paulpandi et al. [46] investigated the anti-IFV activity (IFV-A/HK/H3N2) and the inhibition of viral RNA
synthesis in MDCK cells caused by β-santalol. The compound was isolated from the essential sandalwood (Santalum album L.) oil. In a co-treatment assay (cells + virions), β-santalol reduced the virus-induced formation of CPE by 86% in a concentration of
100.0 µg/mL. In addition, using the RT-PCR method, the authors could also demonstrate
a complete inhibitory effect of β-santalol (at 100.0 µg/mL) on the late viral RNA synthesis of the M gene. The antiviral
effect of β-santalol was comparable to oseltamivir (CPE reduction by 83% at 100.0 µg/mL) in the
same test system. Additional note: The M gene encodes for two proteins, M1 (matrix
protein) and M2 (membrane protein). There are indications that the M gene may be involved
in determining host tropism (ability to infect a specific tissue) [47]. Furthermore, IFV-A M2 is able to block the fusion of autophagosomes with lysosomes
[48]. Dai et al. [50] screened natural compounds for their inhibitory effects on viral autophagy. IFVs
promote the multiplication of autophagosomes containing IFV particles. The viruses
block the fusion of autophagosomes and lysosomes and therefore they can escape the
immune system [51]. Eugenol, the main compound of essential clove (Syzygium aromaticum (L.) Merr. & L. M. Perry) oil, was found to be a potent anti-autophagy agent at the
maximal non-cytotoxic concentration of 5.0 µg/mL (cell culture: A549 cells). Eugenol
not only inhibited the viral autophagy, but also reduced the expression of autophagic
genes (measured at both the mRNA and protein levels) induced by viruses. The anti-IFV-A
activity (posttreatment of virus-infected cells) was measured by using a
plaque reduction assay in MDCK cells. The best result was achieved 1 – 3 h post-infection
with an EC50 value of 0.6392 µg/mL; SI- value: 117.8. In addition, eugenol (5.0 µg/mL) significantly
inhibited the release of the cytokines TNF-α, IL-1, IL-6 and IL-8 [50].
Germacrone, a sesquiterpene oxide from the EO of Curcumae xanthorrhizae rhizoma, inhibited,
in a co-treatment assay, the IFV-A/PR/8/34(H1N1) replication in MDCK cells in a dose-dependent
manner with an EC50 value of 6.03 µM [52]. In addition, germacrone also inhibited the replication of other IFA viruses such
as influenza A/human/Hubei/1/2009(H1N1), A/human/Hubei/3/2005(H3N2), A/human/WSN/33(H1N1,S31N,
amantadine resistant), and influenza B/human/Hubei/1/2007 in a dose-dependent manner
(for MDCK cells: ED50 values of 3.82 – 7.12 µM/SI values > 41.0; for A549 cells: ED50 values of 2.15 – 4.78 µM/SI values 93.9) [52]. Furthermore, germacrone blocked several early steps (up to 4 h post-viral infection)
of IFV-A/PR/8/34(H1N1) replication in MDCK cells as shown by time-of-drug-addition
assays and temperature-shift assays. It also impaired the attachment and penetration
of IFV-A into
the host cells. In addition, the compound reduced the viral RNA synthesis, protein
synthesis, and production of infectious progeny virions in MDCK cells in a dose-dependent
manner (similar to Ribavirin) [52].
BALB/c mice were intranasally infected with 5 × LD50 of IFV-A/PR/8/34 (H1N1). Germacrone (50.0 or 100.0 mg/kg) was administered intravenously
once daily for 5 days, starting 24 h before virus exposure. The survival of the infected
animals (16 in each group) was monitored daily for 18 days. Germacrone exhibited an
effective survival of mice by 50% when 100.0 mg/kg germacrone were administered. In
addition, the test compound at 100.0 mg/kg significantly reduced the virus titers
in the lungs of the mice without killing or intoxicating the animals [52].
In an animal study [54], the effect of 1,8-cineole (in 0.5% Tween 80) in concentrations of 30.0, 60.0, and
120.0 mg/kg on mice infected with influenza A virus/Font Monmouth/47 (H1N1) was evaluated
(IFV control group: IFV + saline in 0.5% Tween 80; positive control group: oseltamivir
10.0 mg/kg). Mice were inoculated intranasally with the virus particles. First, the
mice were treated orally with 1,8-cineole or oseltamivir for 2 days before viral infection
and for 5 days after viral infection. The survival time was monitored for 15 consecutive
days in all of the test groups. In contrast to the IFV control group (IFV-Co), 1,8-cineole
treatment significantly prolonged the survival time of the mice after virus infection
(survival time: IFV-Co: 5 days; 1,8-cineole (60.0/120.0 mg/kg) group: 10 days) [54]. In addition, the test compound reduced at 60.0 and 120.0 mg/kg the expression of
the nuclear factor-κB, the
intercellular adhesion molecule-1, and the vascular cell adhesion molecule-1
in mice lung tissues. Furthermore, it reduced the levels of proinflammatory cytokines
such as IL-4, IL-5, IL-10, and MCP-1 in nasal lavage fluids and IL-1β, IL-6, TNF-α, and IFN-γ in lung tissues of mice and relieved the pathological changes of viral pneumonia
in IFV-A-infected mice. The authors argued that 1,8-cineole protects mice from IFV-A
by means of attenuation of pulmonary inflammatory responses [54].
Choi [55] investigated 63 plant EOs for their anti-influenza (IFV-A/WS/33) properties using
MDCK cells and a CPE assay (co-treatment of cells and virions with test oils during
virus inoculation). The most active EOs were anise oil, marjoram oil, and clary sage
oil. At a concentration of 100.0 µg/mL, the EOs exhibited an anti-IFV-A effect by
more than 53.0% (CPE reduction) without any cytotoxic side effects.
Influenza virus in ambient air (airborne viruses)
In principle, IFVs can be transmitted from person to person through the air via aerosols
or large respiratory droplets and via direct contact with secretions [56]. In an animal model (ferrets), it has recently been shown that influenza A viruses
(H1N1/H3N2/H5N1) are transmitted specifically via the air from the nasal respiratory
epithelium, and not from the trachea, bronchus, or the lungs [57]. Against this background, EO aerosols and vapors can have positive effects on human
health in enclosed rooms and working spaces.
The main aim of the following study was to investigate the antiviral activity of TTO
and eucalyptus oil aerosols and vapors against airborne influenza A virus (NWS/G70C/H11N9).
TTO, when actively diffused with a nebulizer for 2 s, cleared nearly all airborne
IFV-A after 10 min, and showed zero virus after 15 min post-nebulizer treatment. Blue
mallee (Eucalyptus polybractea F. Muell. ex R. T. Baker) oil showed zero virus after 15 min following a 15-s period
of active diffusion with a nebulizer. Compared to the aerosols, the corresponding
vapors of both EOs were less effective [58].
Vimalanathan and Hudson [59] investigated the anti-influenza virus (IFV-A/Denver/1/57/H1N1) activities (virucidal
activities) of EOs and EOCs of Cinnamomum zeylanicum Blume, Citrus bergamia Risso, Cymbopogon flexuosus (Nees ex Steud.) W. Watson, Eucalyptus globulus Labill., Pelargonium graveolens LʼHér., Thymus vulgaris L., and some other plants in both the liquid and vapor phase using MDCK and A549
cells as host cell lines. The best results in the liquid phase were exhibited by the
EOs of C. zeylanicum, C. bergamia, C. flexuosus, and T. vulgaris and eugenol; they inactivated the cell-free virions by 100% (plaque reduction) at
3.1 µL/mL. In the liquid phase, the EOs of C. bergamia and E. globulus as well as the isolated EOCs citronellol and eugenol inhibited virus infectivity
by 90 – 100% after exposure of the virus to 250 µL test substance per test tube for
10 min. In both
cases, the test compounds were not toxic on epithelial cell monolayers. In addition,
in the vapor phase, the test compounds were able to inhibit HA activity. The authors
concluded that the anti-influenza properties of these compounds were based on the
interaction of EOs with HA.
Avian influenza virus
AIV is a variety of influenza A virus that has adapted to birds as host animals (e.g.,
chickens, turkeys, geese, ducks, wild waterfowl). It causes the so-called avian flu
or bird flu. The AIV-A subtype H9N2 is found in all of Eurasia among land birds. Although
the virus is not highly pathogenic, it causes severe economic losses and even the
death of the birds [62].
Pourghanbari et al. [64] investigated the antiviral effect of lemon balm oil on the AIV (AIVA/turkey/Wisconsin/1/66/H9N2)
at different concentrations (0.5 to 0.005 mg/mL). Time-of-addition assays showed that
lemon balm oil was able to inhibit various stages of the viral replication cycle in
MDCK cells. The best results were obtained when virions were pretreated with 0.5 and
0.1 mg/mL prior to host cell infection (reduction of the virus titer of about 85%).
The results were comparable with the oseltamivir control (0.5 mg/mL for 85% virus
titer reduction). The reduction of virus replication was based on the reduction of
RNA synthesis measured by an RT-PCR test.
In a standardized protocol for quantitative evaluation of anti-aerosolized AIV-A H9N2
activity by vapors of a well-characterized EO blend (using constructed impingers/glass
chambers), Kumosani et al. [65] showed that in contrast to the control (viral particles count: 1.2 × 106/cm3), within 1.5 min of contact time, a vaporized EO blend (equivalent volumes of eucalyptus
oil with 42.2% 1,8-cineole and peppermint oil with 48.7% menthol) reduced the virus
titer (aerosolized viruses) by about 84.6% at a concentration of 1.0 × 10−4 µL EO/µL air volume.
Shayeganmehr et al. [66] examined the EO of Zataria multiflora Boiss. for its anti-AIV-A H9N2 activity in virus-infected broilers. The virus replication
in the respiratory and gastrointestinal tracts of the animals was explored using RT-qPCR.
Broiler chicks (21 days old) received 20.0 or 40.0 µL/kg bw/day Zataria oil (dissolved
in corn oil) before or after (2 days) virus infection. Another group of chicks got
4.0 mg/kg bw/day amantadine (dissolved in corn oil), a known antiviral agent, from
2 days after virus challenge concurrent with the onset of H9N2 symptoms. The control
group received only corn oil in the same concentration as the verum groups. All test
compounds were administered per os. Broiler chickens treated with Zataria oil, before
or after infection with viruses, and the amantadine group showed a significant reduction
of the viral replication (decreased viral load) in the respiratory and gastrointestinal
tracts compared to
the control group. Overall, Zataria oil was slightly better than amantidine.
Severe acute respiratory syndrome coronavirus 2
SARS-CoV-2 is a novel enveloped RNA betacoronavirus that is closely related to the
SARS-CoV from 2003 (SARS-CoV-1) and is known to cause the viral disease COVID-19.
It first emerged in Wuhan, China, and is presently causing a global pandemic. The
virus can be transmitted from person to person by droplets or aerosols, as well as
by physical contact [67]. In recent months, increased efforts have been made to identify new antiviral and
virucidal substances that can prevent or at least mitigate a SARS-CoV-2 infection.
In this context, numerous extracts and isolated individual substances from medicinal
plants have been investigated in vitro and in silico for their potential antiviral properties, such as EOs and isolated oil components
[28], [68], [69], [70], [71], [72], [73], [74], [75].
ACE2 is an integral membrane glycoprotein that is known to be expressed in most human
tissues such as kidneys, endothelium, lungs, and heart. ACE2 is the host cell entry
receptor for SARS-CoV-2. Therefore, if the ACE2 protein can be inhibited, cells can
be protected temporarily from infection with SARS-CoV-2. Recently, several in silico studies were carried out to screen for EOCs that can block the activity of ACE2 as
a receptor for SARS-CoV-2. Abdelli et al. [77] investigated isothymol, thymol, limonene, p-cymene, and γ-terpinene, characteristic compounds of Ammoides verticillata (Desf.) Briq., for docking experiments. Isothymol demonstrated the best result of
all the compounds tested in binding to the ACE2 active site (s. Table 1S, Supporting Information). Furthermore, organosulfur compounds (allyl disufide, allyl
trisulfide) in garlic EO [78], (E,E)-α-farnesene,
(E)-β-farnesene, (E,E)-farnesol [79] as well as terpineol, guaiol, linalool, 1,8-cineole, β-selinenol, α-eudesmol, and γ-eudesmol, typical compounds in Melaleuca cajuputi Powell EO [80], also displayed a strong binding affinity to the human ACE2 cell receptor in in silico experiments (s. Table 1S, Supporting Information).
Kumar et al. [81] explored 30 EOs for their ability to downregulate the ACE2 receptor in HT-29 epithelial
cells. Among them, geranium and lemon oils exerted significant ACE2 inhibitory effects
in vitro. Geranium oil inhibited the ACE2 activity at 50 µg/mL by 89.37% and lemon oil at
25 µg/mL by 75.21%. Immunoblotting and an ELISA assay revealed a significant decrease
of ACE2 protein expression and RT-qPCR a significant downregulation of the corresponding
mRNA level in HT-29 cells. In addition, citronellol, limonene, and geraniol also downregulated
the ACE2 receptor as well as the protein expression and mRNA level in HT-29 epithelial
cells.
The main viral protease Mpro/3CLpro is part of the SARS CoV-2 replication cycle. This proteolytic enzyme processes the
coherent “polyproteins” into functional single molecules. Therefore, it has recently
been regarded as a suitable target for a drug design against SARS CoV-2 infections
[28]. Recently several in silico molecular docking experiments have shown that 1,8-cineole (eucalyptol) from Eucalyptus globulus Labill. [82], jensenone (4,6-diformyl-2-isopentanoylphloroglucinol) from Eucalyptus jenseni Maiden [83], carvacrol from Origanum vulgare L. and Thymus vulgaris
[84], organosulfur compounds (allyl disulfide, allyl trisulfide) from Allium sativum L. [78] as well as terpineol, guaiol, linalool, 1,8-cineole, β-selinenol, α-eudesmol, and
γ-eudesmol from M. cajuputi
[80] very effectively bound to the main SARS-CoV-2 proteinase (s. Table 1S, Supporting Information).
The spike protein (S glycoprotein) consists of two subunits, namely, the S1 unit,
which is also known as the RBD, and the S2 unit. The virus binds to the ACE2 receptor
of the host by means of the S1 subunit; the S2 subunit is responsible for the fusion
of the virus envelope and the host cell membrane [85]. The RBD seems to be of clinical relevance, since blocking this domain will probably
lead to the first step of a virus infection being prevented [86].
Kulkarni et al. [86] investigated 25 monoterpenes and phenylpropanoids for their binding affinities to
the virus RBD using in silico molecular docking experiments. The authors found that anethole, cinnamaldehyde, carvacrol,
geraniol, cinnamyl acetate, α-terpineol, thymol, and pulegone, all components of EOs from different plant families,
effectively bound to the S1 RBD of the spike (S) glycoprotein (s. Table 1S, Supporting Information)
In addition, the human serine protease TMPRSS2 is another important protein involved
in the binding of ACE2 and the spike protein. This protease primes the spike protein
before it binds to ACE2. It was found that trans-pinocarveol, eucalyptol, menthol, linalool, α-pinene, and methyl salicylate docked to TMPRSS2 with an average binding energy value
of − 4.2 kcal/mol. Of the tested substances, methyl salicylate was of particular interest
as it bound to TMPRSS2 on the same site as the known TMPRSS2 inhibitory drug camostat
(protease inhibitor) [87].
However, it must be critically noted that EOs and EOCs as COVID-19 antiviral agents
are still limited to in vitro and molecular docking research or to computer simulations conducted by equating the
active substance molecules in EOs with the SARS-CoV-2 virus protein molecules. Hitherto,
corresponding biochemical in vitro studies have not been carried out. There is also a lack of information concerning
the data at which concentration the tested compounds should be used extracellularly
(effective dose) in order to actually inhibit the viral main proteinase intracellularly
without being cytotoxic at the same time. This statement is also true for similar
in silico studies with plant-derived compounds that are currently being carried out worldwide.
Herpes simplex virus-1 and herpes simplex virus-2
HSV-1 and HSV-2 are highly prevalent in human beings all over the world. HSV-1 is
mainly responsible for HSV-induced lesions in the oral cavity and epidermis, while
HSV-2 causes genital herpes, a sexually transmitted disease (s. Table 3S, Supporting Information). After the primary infection, both viruses establish a lifelong
latent infection in the lumbosacral sensory ganglia [91], [92], [93], [94]. In the last decade, the inhibitory effects of different EOs and EOCs on the viral
replication cycle of acyclovir-sensitive and acyclovir-resistant HSV-1 and/or HSV-2
were described [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106], [107], [108], [109]. A selection of interesting scientific papers deailing with this subject is listed
in Table 1S, Supporting Information. The knowledge gained from the investigations carried out
so far allows the conclusion that significant anti-HSV effects could generally be
determined when cell-free herpes viruses were pretreated with the test oils for 1 h
before infection of the host cells. For example, pretreatment of cell-free virions
with EOs obtained from Illicium verum Hook. f., Melaleuca alternifolia (Maiden & Betche) Cheel, Leptospermum
scoparium J. R. Forst. & G. Forst., and Matricaria chamomilla L. was found to inhibit the infective ability of acyclovir-sensitive and -resistant
HSV strains, indicating the immense antiviral potential of EOs [93], [96]. In addition, when cell-free HSV-1 particles were pretreated for 1 h with the maximum
non-cytotoxic concentration of lemon balm oil, the virus replication in Vero cells
was inhibited by more than 98% [95]. It can be concluded that lemon balm oil and other EOs (e.g., manuka oil, star anise
oil, hyssop oil, TTO, thyme oil, ginger oil, and many others) develop their maximum
anti-HSV effects through direct interaction with the infectious virions outside the
host cells and thus prevent the viruses from adhering to the cells and/or prevent
the viruses from penetrating the cells. This mechanism of action is fundamentally
different from the effect of synthetic
products (e.g., acyclovir, pencyclovir), which attack only the intracellular
replication step of the viruses. This finding suggests that EOs mainly attack the
lipid envelope of the viruses. This hypothesis was examined in more detail in two
studies on HSV-1. If cell-free virions were incubated with the EO of oregano for 1 h
at room temperature prior to their exposure to host cells, the virions lost their
lipid envelope. As a result, these virions could no longer bind to the host cells
and thus lost their infectivity [99]. Lai et al. [100] reported a disruption of the lipid envelope of HSV-1 (as visualized via TEM) by
carvacrol and thymol at 100 µM after 1 h pretreatment. In contrast to the control
experiment, carvacrol disrupted the virus envelopes up to 79% and thymol reduced the
enveloped virus up to 93%.
In some cases, it could be demonstrated that several EOs and EOCs were also able to
inhibit early steps as well as late steps in the virus life cycle when added to the
host cells after virus infection (posttreatment of virus-infected cells) [92], [101], [102].
Human immunodeficiency virus
AIDS (acquired immune deficiency syndrome) is caused by infection with the HIV [111]. The HIV-Tat (trans-activator of transcription) is an RNA-binding protein that plays
a major role in HIV gene expression and replication [112]. It is a key activator of the virus transcription. Tat interacts with TAR (trans-activation-response
element), a specific sequence of the HIV-1 RNA molecule, to enhance transcription
of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral
replication and for the emergence of the virus from the latent state. Therefore, the
Tat/TAR-RNA complex could be a target of HIV-1 inhibitors. Feriotto et al. [113] examined the EO-treated Tat/TAR-RNA complex using gel electrophoresis and found
that EOs of T. vulgaris (complex inhibition at 3.0 – 6.0 µg/mL), Cymbopogon citratus (DC.) Stapf (complex inhibition at 6.0 –1 2.0 µg/mL), and
Rosmarinus officinalis L. (complex inhibition at 0.25 – 0.50 µg/mL) interacted directly with Tat protein
and destabilized the Tat/TAR-RNA complex.
Dengue virus
Dengue fever is a tropical and subtropical virus infection that is caused by four
antigenetically related serotypes of dengue viruses (DENV-1, DENV-2, DENV-3, DENV-4)
[114], [115]. The viruses can be transmitted to humans by infected Aedes mosquitoes such as Aedes aegypti and Aedes albopictus. There are no effective drugs to treat dengue infections. The current exploration
of anti-DENV agents is focused on the molecules targeting the host and the virus molecules
[114], [115].
Ocazionez et al. [117] found that Lippia EOs directly inactivated the cell-free virion particles of all four serotypes of
dengue viruses (DENV 1 – 4) with IC50 values of 0.4 – 32.6 µg/mL for Lippia alba and 1.9 – 33.7 µg/mL for Lippia citriodora. However, no reduction in virus replication was noted when the EO was added to cells
previously infected with viruses.
Pajaro-Castro et al. [118] evaluated the antiviral properties of several EO components on DENV-2 replication
and DENV-2 proteins. In several in silico molecular docking studies (virus protein-ligand docking), α-copaene, β-bourbonene, germacrene D, spathulenol, β-caryophyllene, caryophyllene oxide, and (+)-epi-bicyclosesquiphellandrene showed
significant interactions with different virus proteins [e.g., capsid (C) protein,
envelope (E) protein, NS2B, NS3, NS5 polymerase, and NS5 methyltransferase], which
are essential for a virus replication cycle (s. Table 1S, Supporting Information). Since β-caryophyllene showed promising in silico data, it was investigated for its inhibitory effects on DENV-2 replication in vitro. In a virus NS1 reduction assay, β-caryophyllene inhibited the virus replication in vitro (HepG2 cells; posttreatment of virus-infected cells) at an
IC50 value of 22.5 µM with a high SI value of 71.1. Thus, β-caryophyllene seems to be a promising anti-DENV-2 candidate that inhibts the virus
replication in vitro at an early stage (up to 4 h post-infection treatment) of the virus replication cycle.
In a more recent experiment, Flechas et al. [119] screened the antiviral activities of β-caryophyllene, citral, (R)-(−)-carvone, (S)-(+)-carvone, (R)-(+)-limonene, p-cymene, geranyl acetate, nerol, and α-phellandrene against DENV serotypes 1 – 4 in vitro by measuring the reduction of viral NS1 and cell-surface E proteins in HepG2 and
Vero cells. β-Caryophyllene was found to be the most active compound inhibiting the early steps
of the virus life cycle inside the host cells. The compound reduced the formation
of the NS1 protein and of the cell-surface E protein of DENV-2 in HepG-2 cells at
an IC50 value of 22.0 µM. In addition, in Vero cells, β-caryophyllene reduced the replication of all four serotypes at IC50 values of 8.0 to 15.0 µM with high SIs between 5.3 and 10.0.
Bovine viral diarrhea virus
The BVDV causes a dangerous diarrhea in cattle, especially in calves [122]. Pilau et al. [90] investigated the antiviral effects of Mexican oregano (Lippia graveolens Kunth) EO and its major compound carvacrol on the animal BVDV. The EO and carvacrol
were tested at different stages of the virus replication cycle. The most pronounced
antiviral effects were seen when the host cells were treated with both Lippia oil or carvacrol after viral inoculation (post-entry treatment) to MDBK cells with
EC50 values of 78.0 (SI: 7.2) and 50.7 µg/mL (SI: 4.2), respectively.
Yellow fever virus
Yellow fever is an acute viral hemorrhagic disease transmitted by infected Aedes and Haemogogus mosquitoes [125]. In different in vitro studies, the inhibitory effects of the EOs of Lippia origanoides Kunth [126], Lippia citriodora (Palau) Kunth [127] and L. alba (Mill.) N. E.Br. ex Britton & P.Wilson [126], [127] as well as of O. vulgare, Artemisia vulgaris L. [126], and the EOCs citral and limonene [127] on YFV replication were investigated.
Treatment of cell-free virions with the tested EOs for 24 h prior to host cell infection
led to a significant reduction of the virus yields with IC50 values of 3.7 µg/mL (for L. alba, L. origanoides, O. vulagare; SI values: 22.9, 26.4, 26.5) and 11.1 µg/mL (for A. vulgaris; SI value: 8.8) [126]. However, no virus yield reduction was observed when the host cells were pretreated
for 24 h with the tested EOs prior to the virus infection. The results allow the conclusion
that the inhibition of YFV infectivity in vitro is based on a direct virion inactivation preventing the adsorption to host cells
and a subsequent cellular infection [126].
In a further study, Gomez et al. [127] were able to show that the EO of L. alba inhibited the virus replication cycle before and after the entry of viruses into
the host cells. When cell-free yellow fever virions were inoculated with the test
oil before the host cell infection, the virus infectivity was significantly reduced
(IC50: 4.3 µg/mL; SI: 30.6). When the host cells were treated with the test oil after the
virus cell infection, the virus titer (PFU/mL) was reduced by 50% at 15.2 µg/mL (IC50: 15.2 µg/mL; SI: 10.8) with respect to the untreated control. L. citriodora oil and citral also reduced the viral infectivity when the virus was treated before
(IC50: 19.4/17.6 µg/mL; SI: 2.6/1.5) and after entry (IC50: 21.2/25.0 µg/mL; SI: 2.4/1.1) into the host cells. In contrast to the interpretation
of the authors, however, it must be stated that the antiviral activity of
L. citriodora oil and citral is most likely based on their cytotoxicity, since the SI values of
both substances are below the cutoff value of 4.0 (SI values for L. citriodora oil: 2.6 and 2.4; SI values for citral: 1.5 and 1.1).
West Nile virus
The West Nile virus is a mosquito-borne virus that is able to cause a neuroinvasive
disease in human beings and animals [129]. Zamora et al. [129] investigated in vitro the antiviral effects of a CMA (64% terpinen-4-ol, 14% p-cymene, 7% α-terpineol, 6% δ-cadinene, 9% other monoterpenes) derived from M. alternifolia [against two different strains of WNV (WNVKUNV and WNVNY99)]. CMA showed a significant
virucidal effect against both WNV strains in vitro (Vero cells) at the maximum non-cytotoxic concentration of 0.0075%. In comparison
to the vehicle control, CMA reduced the virus titer of both WNV strains to about 1.0 × log10 PFU/mL. In addition to its virucidal effect, CMA also exhibited an antiviral activity
that decreased the production and/or release of virions from the host cells. In comparison
to the vehicle control, the treatment of cells previously
infected with viruses (posttreatment of virus-infected cells) with CMA (0.0075%)
significantly reduced the virus titer of both WNV strains inside Vero cells by 2.0 × log10 PFU/mL.
The disease symptoms of WNV-infected mice closely resemble that of a human infection
[130]. When CMA was tested against WNV-infected mice (6-week-old male C57BL/6 IRF 3–/–/7–/–mice; 150 µL of 3.0% CMA per day i. p.) for a period of 6 days, it was found that
the test compound significantly reduced the morbidity of the animals and reduced the
loss of body weight as well as the virus titers in the brain of the mice [129].
Antiviral Activities of Essential Oils and Essential Oil Compounds against Non-enveloped
Viruses
The capsid in non-enveloped viruses serves to protect the integrity of the viral RNA/DNA
and to initiate an infection by adsorbing to the host cells. Up to now, only limited
research has looked at the efficacy of EOs against non-enveloped viruses. On the one
hand, this is due to the fact that in recent years, EOs have more often been investigated
against enveloped viruses and, on the other hand, to the fact that EOs showed little
or no effect on non-enveloped viruses. Garozzo et al. [135] found tea tree EO to be ineffective against PV1, echovirus 9, coxsackievirus B1,
and adenovirus 2. In addition, Kovac et al. [136] examined in vitro the antiviral activity of hyssop oil (Hyssopus officinalis L.) and marjoram oil (Thymus mastichina (L.) L.) at 0.02 and 0.2% on non-enveloped viruses such as the murine norovirus and
the human adenovirus at different incubation times (2 and 24 h) and temperatures
(+ 4 °C, room temperature, 37 °C). No significant reduction of the virus titers
in host cells (human lung carcinoma A-549; mouse macrophage RAW 264.7 cell lines)
was observed. Rouis et al. [137] investigated the antiviral activity of the EO from Hypericum triquetrifolium Turra against coxsackievirus B3 on Vero cells (pretreatment of virions/host cells
for 1 h prior to cell/virus infection). The authors found the EO to be ineffective
against coxsackievirus B3 in different non-cytotoxic concentrations.
However, various EOs have been identified in the last decade that have shown strong
antiviral effects against non-enveloped RNA and DNA viruses, such as the murine norovirus,
the coxsackievirus, the adenovirus, feline calcivirus, porcine parvovirus, rhinovirus,
rotavirus, and some phages (Table 2S, Supporting Information). For the listed viruses and EOs, see also Tables 3S and 4S, Supporting Information, respectively. EOs and their active compounds act on the
viral capsid to some extent. But it is difficult to determine whether such reductions
in the virus infectivity are due to actual damage to the virus particles or to a simple
inhibition of virus adsorption to the host cells. For instance, in many cases, the
viral RNA was unaffected although the virus was no longer infectious.
Murine norovirus type 1
MNV-1 infects mice and is virulent, especially in immunocompromised mice. MNV-1 is
used as a surrogate to study the biological and physiological behavior of noroviruses
[138], [139], [140]. In three different in vitro experiments, oregano oil, carvacrol [138], allspice oil, lemongrass oil-1, and citral [139] as well as Artemisia oil-2 (s. Table 4S, Supporting Information) and α-thujone [140] were evaluated for their anti-MNV-1 effects in RAW 264.7 cells. Of all the substances
tested, carvacrol was found to be the most active one. After exposure of cell-free
virions to 0.5% carvacrol for 1 h prior to the cell infection, their cell infectivity
was significantly inhibited (reduction of the virus titer by 3.87 × log10 TCID50/mL). RNase I protection
assays and TEM analyses showed that carvacrol acted directly on the virion capsid
and the RNA. Interestingly, after carvacrol treatment, the protein capsids enlarged
from about 35 nm in diameter to 800 nm [138].
Coxsackievirus
Coxsackieviruses, named after a town in New York [141], are RNA viruses that are transmitted via droplets or smear infections. There are
numerous subtypes, which are simply divided into Coxsackie A and Coxsackie B viruses
and which can trigger different clinical pictures such as colds, viral meningitis,
and myocarditis.
Elaisi et al. [142] and El-Baz et al. [106] explored in vitro the antiviral activity of EOs of several Eucalyptus species against coxsackievirus B3 and B4. Eucalyptus bicostata Maiden, Blakely & Simmonds EO showed the best antiviral activity. Pretreatment of
the cell-free coxsackievirus B3 for 1 h prior to the cell infection resulted in a
significant reduction of the viral infectivity, with an IC50 value of 0.7 mg/mL (SI: 22.8) [142].
Porcine parvovirus
PPV infections (SMEDI syndrome or porcine parvovirose) can lead to significant reproductive
failure in pigs. As non-enveloped viruses they are extremely resistant to environmental
influences and disinfectants [145]. Chen et al. [145] tested the antiviral effect of germacrone on PPV in swine testis host cells. Germacrone
was able to suppress in vitro the synthesis of viral RNA and proteins inside infected cells. In addition, germacrone
(0 – 200 µM) inhibited early stages (up to 9 h post-viral infection) of the PPV replication
cycle in a dose-dependent manner (reduction of viral titers: 6.0 log10 to 2.5 log10 TCID50/mL).
Adenovirus and rhinovirus
In most cases, rhinoviruses trigger a flu-like infection. However, they are also involved
in other respiratory diseases such as sinusitis and bronchitis. In addition to a flu-like
infection, adenoviruses can also cause other respiratory diseases (e.g., pneumonia)
and be responsible for infections of the gastrointestinal tract and the conjunctiva
[60].
Vimalanathan and Hudson [60] investigated the vapor of CLO for its virucidal effects against the virions of adenovirus
and rhinovirus 14. Since respiratory viruses often induce proinflammatory cytokine
responses, the authors also explored the anti-cytokine activity of CLO in virus-induced
human lung cells. Both viruses were completely inactivated after 60 – 120 min of exposure
to the oil vapor. Furthermore, rhinovirus-induced production of the cytokine IL-6
in human lung epithelial cell monolayers was reduced by more than 50% after 60 min
exposure to the oil vapor.
Feline calicivirus F9
FCV is a small, non-enveloped virus with single-stranded RNA and typical cup-shaped
indentations on the surface from which the name of the virus is derived. It causes
mouth ulcers, inflammation of the gums, and nasal discharge in cats [148].
Germacrone [148] as well as Artemisia princeps var. orientalis Hara EO and its main component α-thujone [140] were investigated for their anti-FCV-F9 properties. In time-of-drug-addition assays,
germacrone was shown to reduce the viral replication in CRFK cells at an early stage
of the viral replication cycle in a concentration-dependent manner (e.g., vRNA level
was reduced more than 50% at 60 to 100 µM). In contrast, germacrone did not affect
the virion attachment to the host cells or the virion entry into the host cells [148].
In a further study, Chung [140] found that the viral infectivity of FCV-F9 was moderately reduced when the cell-free
virions were treated for 1 h with 0.1% of A. princeps var. orientalis EO (plaque reduction of about 48%) or 25 mM α-thujone (virus titer reduction of about 1 × log10 PFU/mL) before cell infection.
Conclusions
Over the past decade, the antiviral effects of EOs and EOCs extracted from various
aromatic plants against a variety of RNA and DNA viruses have been well documented
in in vitro and in vivo model systems (s. Tables 1S–4S, Supporting Information). Most of the published in vitro experiments deal with enveloped viruses, in particular, with IFV, AIV, HSV-1, and
HSV-2 and to an increasing extent, with SARS-CoV-2. Only a few studies have already
evidenced the great antiviral potency of some EOs and EOCs on certain non-enveloped
viruses such as coxsackievirus and MNV-1. According to available data, the antiviral
and virucidal properties of EOs are usually based on phenolic, alcoholic, and other
oxygenated EOCs. In particular, cinnamon oil, clove oil, eucalyptus oil, lemongrass
oil, Lippia oil, oregano oil, TTO, thyme oil, and the oil compounds carvacrol, 1,8-cineole, eugenol,
germacrone, PA, thymol, terpinen-4-ol ([Fig. 2]), and some others proved to be particularly effective antiviral/virucidal substances.
Studies in mice (or chicks) infected with viruses causing respiratory diseases (e.g.,
IFV) showed that some EOs (e.g., Mosla oil, Zataria oil) and EOCs (e.g., 1,8-cineole,
germacrone, PA) were able to prolong the life of infected animals, reduce the virus
titers in brain and lung tissues and significantly inhibit the synthesis of proinflammatory
cytokines and chemokines in lung tissues. The most interesting antiviral and virucidal
effects of EOs and EOCs on enveloped and non-enveloped viruses are summarized in [Fig. 3].
Fig. 3 Summary of the main in vitro and in vivo antiviral and virucidal properties of EOs and EOSs.
Concerning isolated EOCs, it must be noted that the biochemical world is chiral. The
biosynthesis of terpenes in plant tissues is usually stereospecific, with the consequence
that many mono- and sesquiterpenes are synthesized in defined enantiomeric ratios
or as pure enantiomers. It is known from the literature that different enantiomers
of a bioactive terpene can act in different ways on microbes such as bacteria or fungi
[25]. It can therefore be assumed that this also applies to their antiviral activity.
But most existing studies on the antiviral effect of EOCs are based on commercially
purchased substances, which may not be fully consistent with their fractionated counterparts
from natural EOs. Therefore, the effects of stereoisomerism and polarity of isolated
EO components on their antiviral effectiveness need to be given more attention in
future studies.
The in vitro studies published so far clearly show that the major mechanisms through which EOs
and EOCs induce antiviral actions are a direct action on cell-free virions, inhibition
of steps involved in the virus attachment, penetration, intracellular replication,
and release from the host cells and the inhibition of the vital viral enzymes. In
most cases, the greatest antiviral effect was observed when virions were incubated
with the drugs for 1 h or more hours prior to their addition to host cells (pretreatment
of cell-free virions), thus indicating a direct effect (virucidal effect) on cell-free
virions outside the host cells. Concerning the enveloped virions, one can speculate
that the virucidal effect of EOs and EOCs is likely accounted for by disruption of
the virus envelope and its associated structures, which are necessary for the virion
adsorption to and entry into the host cells. In most cases, the in vitro studies only identified the stages of the
virus replication that were inhibited or influenced by EOs and EOCs, while the
specific sites of action on and in the host cell as well as the molecular mode of
interaction underlying the action were only more intensively investigated in a few
cases.
In silico analyzes are used to discover new antiviral and virucidal compounds and to study
their bioactive mechanisms. This approach is also useful for estimating the binding
affinity of EOCs to viral or host cell proteins to demonstrate and explain their biological
effects. Due to the current SARS-CoV-2 pandemic, particularly great efforts have been
made to identify suitable molecules that inhibit the entry and replication of the
virus in the host cells. With the molecular docking technique, it could be shown that
several molecules (e.g., isothymol, organosulfur compounds, 1,8-cineole, jensenone
and others) may exert relative strong binding to the viral and host cell-specific
target molecules that are indispensable for virus cell adsorption (ACE2, TMPRSS2,
RBD), penetration/internalization (RBD), and replication (Mpro). However, validation of the predictions by wet bench data is still missing.
In addition, it is known from available data that the effective time of antiviral
action of EOs is limited by their high volatility. The encapsulation of EOs into suitable
nano-carrier materials could increase their chemical stability, enhance their water
solubility, bioavailabilty, and their antiviral effectiveness. The few studies that
have been published on this subject support this assumption but require additional
efforts in the future.
