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Comparative study on the antiviral activity of selected monoterpenes derived from essential oils

Abstract Essential oils are complex natural mixtures, their main constituents, e.g. terpenes and phenylpropanoids, being responsible for their biological properties. Essential oils from eucalyptus, tea tree and thyme and their major monoterpene compounds α‐terpinene, γ‐terpinene, α‐pinene, p‐cymene, terpinen‐4‐ol, α‐terpineol, thymol, citral and 1,8‐cineole were examined for their antiviral activity against herpes simplex virus type 1 (HSV‐1) in vitro. These essential oils were able to reduce viral infectivity by >96%, the monoterpenes inhibited HSV by about >80%. The mode of antiviral action has been determined, only moderate antiviral effects were revealed by essential oils and monoterpenes when these drugs were added to host cells prior to infection or after entry of HSV into cells. However, both essential oils and monoterpenes exhibited high anti‐HSV‐1 activity by direct inactivation of free virus particles. All tested drugs interacted in a dose‐dependent manner with herpesvirus particles thereby inactivating viral infection. Among the analysed compounds, monoterpene hydrocarbons were slightly superior to monoterpene alcohols in their antiviral activity, α‐pinene and α‐terpineol revealed the highest selectivity index. However, mixtures of different monoterpenes present in natural tea tree essential oil revealed a ten‐fold higher selectivity index and a lower toxicity than its isolated single monoterpenes. Copyright © 2009 John Wiley & Sons, Ltd.


INTRODUCTION
Essential oils, odours and volatile products of plant secondary metabolism have a wide application in folk medicine as well as in fragrance industries. Essential oils are complex natural mixtures of volatile secondary metabolites, isolated from plants by distillation. The main constituents of essential oils, e.g. monoterpenes and sesquiterpenes and phenylpropanoids including carbohydrates, alcohols, ethers, aldehydes and ketones, are responsible for the fragrant and biological properties of aromatic and medicinal plants.
Herpes simplex virus (HSV) is differentiated into two antigenic types, type 1 (HSV-1) and type 2 (HSV-2), and is an important pathogen for humans, therefore the discovery of novel anti-HSV drugs deserves great effort. HSV infects and replicates in cells at the site of entry, the mucocutaneous surface. After the acute ganglionic infection subsides, HSV establishes latency and persists in the neurons for the life-time. The latent virus is reactivated spontaneously causing frequent recurrent infections in some patients, while most people experience few recurrences. HSV-1 infections are very common and reactivations mostly affect adult people (Whitley and Roizman, 2001). The primary symptoms of herpes infection include a prodromal fl u-like syndrome with fever, headache, malaise, diffuse myalgias, followed by local symptoms consisting of itching and painful papules. Gingivostomatitis and pharyngitis are the most frequent clinical manifestations of the fi rst episodes of HSV-1 infection. The clinical manifestation of the disease exhibits different severity in immunocompetent patients and in addition some patients always encounter recurrent attacks (Whitley and Roizman, 2001).
The antiviral agents acyclovir, famciclovir and valacyclovir can be used to shorten the course and decrease the severity of these clinical symptoms and may suppress the virus itself (Whitley and Roizman, 2001). Antiviral agents licensed currently for the treatment of herpesvirus infections include acyclovir and derivatives, foscarnet and cidofovir, all of which inhibit herpesvirus DNA polymerases (De Clercq, 2004). In addition, the emergence of virus strains resistant to commonly used anti-herpesvirus drugs is of importance, particularly in immunocompromised patients (Reusser, 1996;Cassady and Whitley, 1997). The development of viral resistance towards antiviral agents enhances the need for new effective compounds against viral infections. Medicinal plants produce a variety of chemical constituents with the ability to inhibit viral replication, and compounds from natural sources are of interest as possible sources to control viral infection.
Antiherpes screening experiments on medicinal plant extracts and plant derived secondary metabolites have been reported (Reichling et al., 1999;De Logu et al., 2000). The antiherpes activity of several essential oils of different plant sources as well as of some constituents of essential oils had been demonstrated previously (Farag et al., 2004). The application of tea tree oil, the essential oil of Melaleuca alternifolia, for the treatment of recurrent herpes labialis has been reported recently (Carson et al., 2001). The antiherpes activity of eucalyptus oil, Australian tea tree oil (Schnitzler et al., 2001), thyme oil (Schnitzler et al., 2007) and manuka oil (Reichling et al. 2005) have been published previously. Some phenylpropanes (Benencia and Courrèges, 2000;Tragoolpua and Jatisatienr, 2007), triterpenes (Niedermeyer et al., 2005) and sesquiterpenes (Hayashi et al., 1996;Pusztai et al., 2008;Rollinger et al., 2008) had been tested for their antiviral activity against different herpesviruses and rhinovirus. However, monoterpenes as major constituents of essential oils, have not been analysed systematically for their antiviral potential. Only few reports describe the inhibition of viral replication by monoterpenes, e.g. isoborneol as a potent inhibitor of HSV (Armaka et al., 1999), consequently there is little information about terpenes concerning the inhibition of the viral replication cycle and their mode of antiviral action.
The goal of the present study is the evaluation of the antiviral activity of monoterpenes, major constituents of essential oils, against HSV-1 and the mode of antiviral action of these monoterpenes during the viral multiplication cycle.
Acyclovir. Acyclovir was purchased from GlaxoSmith-Kline (Bad Oldesloe, Germany) and dissolved in sterile water.
Cell culture and herpes simplex virus type 1 (HSV-1). RC-37 cells (African green monkey kidney cells) were grown in monolayer culture with Dulbecco's modifi ed Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confl uent. The cells were plated out onto 96-well and 6-well culture plates for cytotoxicity and antiviral assays, respectively, and propagated at 37 ºC in an atmosphere of 5% CO 2 . Herpes simplex virus type 1 (HSV-1) strain KOS was used for all experiments. Viruses were routinely grown on RC-37 cells and virus stock cultures were prepared from supernatants of infected cells and stored at −80 ºC. Infectivity titers were determined by a standard plaque assay on confl uent RC-37 cells .
Cytotoxicity assay. For cytotoxicity assays, the cells were seeded into 96-well plates and incubated for 24 h at 37 ºC. The medium was removed and fresh DMEM containing the appropriate dilution of the essential oils or monoterpenes was added onto subconfl uent cells in eight replicates for each concentration of the drugs. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls. After 3 days of incubation, the growth medium was removed and the viability of the drug treated cells was determined in a standard neutral red assay (Söderberg et al., 1996). Neutral red dye uptake was determined by measuring the optical density (OD) of the eluted neutral red at 540 nm in a spectrophotometer. The mean OD of the cell-control wells was assigned a value of 100%. The cytotoxic concentration of the drug which reduced the viable cell number by 50% (TC 50 ) was determined from dose-response curves. Additionally the maximum noncytotoxic concentration of each drug was determined.
Dose-response assays. Inhibition of HSV replication was measured by a plaque reduction assay. Usually 2 × 10 3 plaque forming units (pfu) were incubated with different concentrations of essential oils or monoterpenes for 1 h at room temperature, then the virus was allowed to adsorb to the cells for 1 h at 37 ºC. The residual inoculum was discarded and the infected cells were overlaid with medium containing 0.5% methylcellulose. Each concentration was performed in three replicates, virus-infected cells in wells containing medium with 1% ethanol but no drug were also included on each plate as controls. After incubation for 3 days at 37 ºC, the monolayers were fi xed with 10% formalin. The cultures were stained with 1% crystal violet and subsequently plaques were counted. By reference to the number of plaques observed in virus control monolayers (untreated cultures), the concentration of test compound which inhibited plaque numbers by 50% (IC 50 ) was determined from dose-response curves.
Time of addition assay. In order to determine the mode of antiviral action for oils and monoterpenes, the cells were pretreated with essential oils or components before viral infection, viruses were incubated with drugs before infection or infected cells were incubated immediately after penetration of the virus into cells. Essential oils and components were always used at the maximum noncytotoxic concentration. Cell monolayers were pretreated with drugs prior to inoculation with virus by adding the essential oils or components to the culture medium followed by incubation for 1 h at 37 ºC. The drugs were aspirated and the cells were washed before the HSV inoculum was added. For pretreatment of herpes simplex virus with drugs, about 2 × 10 3 pfu of HSV was incubated in medium containing the maximum noncytotoxic concentration of the drugs for 1 h at room temperature prior to infection of RC-37 cells. The effect of essential oils and components against HSV was also tested during the replication period by addition of drugs after cell infection to the overlay medium, as typically performed in antiviral susceptibility studies. Each assay was run in three replicates. Plaque reduction assays were carried out as described above and the number of plaques of drugtreated cells and viruses were compared with untreated controls. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls.

RESULTS
Eucalyptus oil, tea tree oil, thyme oil and some selected monoterpenic compounds (Fig. 1) were serially diluted in ethanol and added to the cell culture medium to examine the effect on the growth and viability of tissue culture cells, always resulting in an ethanol concentration below 1% which had no effect on cells and viruses. Cell monolayers were grown in medium containing different concentrations of these drugs. After 3 days of incubation, the cell viability of RC-37 cells was determined with the neutral red assay (Table 1). The maximum noncytotoxic concentrations of these drugs were determined between 20 μg/mL for citral and 1250 μg/mL for 1,8-cineole, the least toxic compound tested. Essential oils and monoterpenes revealed low cytotoxicity. The potential antiviral effect of different essential oils and some selected components was determined against herpes simplex virus type 1 (HSV-1) in vitro. HSV-1 was incubated for 1 h at room temperature with various concentrations of eucalyptus oil, tea tree oil, thyme oil, α-terpinene, γ-terpinene, α-pinene, p-cymene, terpinen-4-ol, α-terpineol, thymol, citral and 1.8-cineole. In all assays untreated virus-infected cells were used as a control. Subsequently, aliquots of each dilution were added on the cells for 1 h, afterwards the cells were washed and overlaid with drug-free medium and incubated for 3 days at 37 ºC. The 50% inhibitory concentrations (IC 50 ) for HSV-1 were determined in a wide range between 2.0 μg/mL for tea tree oil and 1200 μg/mL for 1,8-cineole (Table 2). The results are presented as virus reduction and represent the average of three independent experiments. In plaque reduction assays, all essential oils and monoterpenes exhibited a concentration-dependent antiviral effect, tea tree oil was the most effective drug. All essential oils were able to suppress viral multiplication by >96% (Table 2), all monoterpenes inhibited HSV by >80%, with the exception of the less active 1,8-cineole. In Table 2, the results for up to 100 μg/mL are shown, for some monoterpenes with maximum noncytotoxic concentrations beyond 100 μg/mL the data are not shown. Selectivity indices for oils and different monoterpenes were calculated as the TC 50 /IC 50 ratio (Table 3), the highest selectivity index of 60 was determined for tea tree oil. Low selectivity indices were found for eucalyptus oil, thyme oil and most monoterpenes. Herpesvirus replication is characterized by a complex sequence of different steps at which antiviral agents might interfere. In order to investigate the inhibitory effects on herpes simplex virus in detail, all drugs were added at different stages during viral infection. For comparison, all untreated controls contained the same concentration of ethanol as the drug-treated viruses, in order to exclude any infl uence of ethanol. When the host cells were pretreated with drugs prior to infection, some of the tested drugs showed minor effects on viral infection, e.g. eucalyptus oil, tea tree oil and α-pinene (Fig. 2). On the other hand, pretreatment of HSV-1 with the oils or monoterpenic compounds for 1 h prior to infection caused a signifi cant reduction in plaque formation. At maximum non-cytotoxic concentrations of the tested drugs, infectivity was reduced by >96% for all essential oils as well as for three of the monoterpenes, αterpinene, γ-terpinene and α-pinene (Fig. 3). With the exception of 1,8-cineole, all other constituents revealed a plaque reduction of herpes simplex virus between about 80% and 90%. Acyclovir showed the highest antiviral activity when added during the replication period with inhibition of the viral replication of 98.6%. This drug inhibits specifi cally the viral DNA polymerase during the replication cycle when new viral DNA is synthesized. However, only minor effects on viral infection were detected when cells or viruses were pretreated with acyclovir (data not shown). In contrast, when the oils or compounds were added to the overlay medium  after penetration of the viruses into the host cells, plaque formation was not signifi cantly reduced, only α-pinene and 1,8-cineole being moderately effective (Fig. 4).

DISCUSSION
The pharmaceutical industry is increasingly targeting medicinal plants with the aim of identifying lead compounds, focusing particularly on suitable alternative antiviral agents. Several drugs are currently available for the management of HSV infections such as acyclovir. Topical treatment of herpes labialis infection is standard, for the most part carried out with acyclovir creams, but also with phytotherapeutic preparations containing sage oil and lemon balm oil (Saller et al., 2001;Wölbling and Leonhardt, 1994;Koytchev et al., 1999). Both plant essential oils were shown to be significantly superior to placebo and equivalent to acyclovir (Wölbling and Leonhardt, 1994). Our previous in vitro    experiments revealed similar results for essential oils from eucalyptus, tea tree and thyme (Schnitzler et al., 2001;Schuhmacher et al., 2003;Koch et al., 2008).
In the present study, the inhibitory effects of several essential oils against herpes simplex virus infection were compared with the antiviral potential of their major monoterpenic compounds. All tested essential oils and most monoterpenes exhibited high levels of antiviral activity against HSV-1 in viral suspension tests. At maximum noncytotoxic concentrations plaque formation was signifi cantly reduced by >96% by eucalyptus oil, tea tree oil and thyme oil, most monoterpenic constituents were able to suppress viral infection by >80%. High antiviral activity was observed for essential oils and isolated monoterpenes when herpesvirus was incubated with these drugs prior to host cell infection. These results suggest that the investigated drugs directly inactivate herpes virus and might interfere with virion envelope structures or mask viral structures which are necessary for adsorption or entry into host cells. A virucidal activity of Melaleuca armillaris essential oil has been reported recently (Farag et al., 2004) and dissolution of the HSV envelope by treatment with oregano essential oil has been described (Siddiqui et al., 1996). Thus different mechanisms of antiviral activity of different essential oils seem to be present. The inhibition of HSV by the tested essential oils and monoterpenes appears to occur before adsorption but not after penetration of the virus into the cell. It remains to be determined whether the inhibitory effect of essential oils is due to binding of the essential oil to viral proteins involved in host cell adsorption and penetration. De Logu et al., (2000) reported an inactivation of herpesviruses and prevention of cell to cell spread by Santolina insularis essential oil. However, no antiviral effect was observed during the intracellular replication phase, which is in accordance with our results for essential oils and monoterpenes. Adenovirus, a virus without envelope, was not affected by eucalyptus essential oil due to the lack of a viral envelope (Cermelli et al., 2008). Sesquiterpenes, e.g. triptofordin C-2 and sesquiterpene coumarins inhibit cytomegalovirus, (Hayashi et al., 1996), severe acute respiratory syndrome coronavirus (Wen et al., 2007) and rhinovirus (Rollinger et al., 2008). Pusztai et al. (2008) reported a specifi c inhibition of the CMV immediate early gene expression, whereas other sesquiterpenes are moderately virucidal against different enveloped viruses, e.g. herpes simplex virus, cytomegalovirus, measles virus and infl uenza virus (Hayashi et al., 1996). Isoborneol, a monoterpene and a component of several plant essential oils, showed virucidal activity against HSV-1 and specifi cally inhibited glycosylation of viral proteins (Armaka et al., 1999). The application of cineole protects mice against infection with HSV-2 (Bourne et al., 1999). Since essential oils are able to inhibit acyclovir-resistant HSV-1 isolates (Schnitzler et al., 2007), the mechanism of interaction between these compounds and acyclovir with HSV must be different. Acyclovir inhibits virus replication by interference with the DNA polymerase inside the cell, whereas essential oils and monoterpenes probably inactivate HSV before it enters the cell. Viral resistance to acyclovir represents a particular problem, the prevalence of resistance in acyclovir-treated immunocompromised individuals is approximately 4% to 7% (Christoph et al., 1998;Stranska et al., 2005). Therefore other antiherpetic agents which are effective for viral mutants resistant to current antiviral agents are of great interest for topical treatment. The application of tea tree oil, the essential oil of Melaleuca alternifolia, for the treatment of recurrent herpes labialis has been reported recently (Carson et al., 2001(Carson et al., , 2006. Terpinen-4-ol, the principal active component of tea tree oil, was more active in antibacterial time-kill assays as an isolated compound than when present in tea tree oil (Cox et al., 2001). These fi ndings are in contrast to results of antiviral activity, where the complex mixture of the essential oil revealed a higher antiviral activity and selectivity index of 60, whereas single major monoterpenes α-terpinene, γ-terpinene and terpinen-4-ol revealed low selectivity indices of 6.5, 5.4 and 10.8, respectively. These rather low selectivity indices of monoterpenes will not permit topical application on mucous membranes. The tested monoterpenes dominate the toxicity of essential oils, e.g. eucalyptus oil consists of about 80% 1,8-cineole, both revealed very low toxicity but also low selectivity indices. For thyme oil, the TC 50 of 70 μg/mL corresponds well to its major compounds p-cymene and thymol, with TC 50 values in the same range of 65 μg/mL and 85 μg/mL. The same is true for tea tree oil, the main constituents α-terpinene, γ-terpinene and terpinen-4-ol contribute equally to a fi nal TC 50 of 120 μg/mL for the mixture in this essential oil. Taken together, the composition of monoterpenes in these essential oils dominates their toxic activity. However, the antiviral activity of single monoterpenes does not contribute equally to the antiviral activity of the essential oil. Tea tree oil is composed of about 10% α-terpinene, 20% γ-terpinene and 40% terpinen-4-ol. Although terpinen-4-ol is the major compound in tea tree oil and revealed a much lower antiviral activity than α-terpinene and γ-terpinene, the complex oil demonstrated a high antiviral activity. Cos et al. (2006Cos et al. ( , 2008 recommended IC 50 values for promising natural products against infectious diseases, e.g. for extracts below 100 μg/mL and below 25 μm for pure compounds. The essential oils in our study revealed IC 50 values of 55 μg/mL, 2 μg/mL and 11 μg/mL for eucalyptus, tea tree and thyme, respectively, and are far below the recommended cut off. However, only the monoterpene citral revealed an IC 50 value below the suggested cut off with 23 μm, all other monoterpenes displayed higher IC 50 values and do not present promising antiinfective agents according to this suggestion. However the most important predictive value for future application of these drugs is their selectivity index, Amoros et al. (1992) recommended a selectivity index of at least 4 as appropriate. According to this suggestion, all essential oils and monoterpenes, with the exception of thymol and 1,8-cineole might be suitable agents. However, drugs with a higher selectivity are preferable (Vanden Berghe et al., 1986), e.g. tea tree oil which had already been successfully applied in labial herpes infections. Other compounds of essential oils need to be screened but a mixture of different compounds in essential oils seems to be superior and preferable to single compounds. Considering the lipophilic nature of tea tree oil which enables penetration of the skin, it is a promising topical therapeutic agent in the treatment of recurrent herpes infection.