Letters in Applied Microbiology ISSN 0266-8254 In vitro antiviral activity of Melaleuca alternifolia essentialoilA. Garozzo1, R. Timpanaro1, B. Bisignano1, P.M. Furneri1, G. Bisignano2 and A. Castro1 1 Department of Microbiological and Gynaecological Sciences, University of Catania, Catania, Italy2 Department of Pharmacobiology, University of Messina, Messina, Italy antiviral activity, essential oil, influenza virus,Melaleuca alternifolia, terpinen-4-ol.
Aims: To investigate the in vitro antiviral activity of Melaleuca alternifoliaessential oil (TTO) and its main components, terpinen-4-ol, a-terpinene, c-ter- pinene, p-cymene, terpinolene and a-terpineol.
Methods and Results: The antiviral activity of tested compounds was evaluated Microbiological and Gynaecological Sciences, against polio type 1, ECHO 9, Coxsackie B1, adeno type 2, herpes simplex (HSV) type 1 and 2 viruses by 50% plaque reduction assay. The anti-influenza 95124 Catania, Italy. E-mail: [email protected] virus assay was based on the inhibition of the virus-induced cytopathogenicity.
2009 ⁄ 0924: received 26 May 2009, revised Results obtained from our screening demonstrated that the TTO and some of its components (the terpinen-4-ol, the terpinolene, the a-terpineol) have an inhibitory effect on influenza A ⁄ PR ⁄ 8 virus subtype H1N1 replication at dosesbelow the cytotoxic dose. The ID50 value of the TTO was found to be 0Æ0006% (v ⁄ v) and was much lower than its CD50 (0Æ025% v ⁄ v). All the compoundswere ineffective against polio 1, adeno 2, ECHO 9, Coxsackie B1, HSV-1 andHSV-2. None of the tested compounds showed virucidal activity. Only a slightvirucidal effect was observed for TTO (0Æ125% v ⁄ v) against HSV-1 and HSV-2.
Conclusions: These data show that TTO has an antiviral activity against influ-enza A ⁄ PR ⁄ 8 virus subtype H1N1 and that antiviral activity has been princi-pally attributed to terpinen-4-ol, the main active component.
Significance and Impact of the Study: TTO should be a promising drug in thetreatment of influenza virus infection.
Quantitative results (g% w ⁄ w) obtained by GC (FID, Flame Ionization Detector) analysis of oil samples, dem- The essential oil of Melaleuca alternifolia, also known as onstrated that TTO is characterized by a high proportion tea tree oil (TTO), is a complex mixture of terpene of terpinen-4-ol (36Æ71%) and c-terpinene (22Æ20%), and hydrocarbons and tertiary alcohols mainly distilled from plantation stands of the Australian native plant M. alt- (2Æ52%), a-terpinene (10Æ10%), terpinolene (3Æ53%) and ernifolia, a member of the Myrtaceae family. A series of a-terpineol (2Æ74%) (Shellie et al. 2003).
standards has attempted to define and limit the variation Many authors have demonstrated that TTO has a seen in this heterogeneous mixture, because it is subject broad spectrum of antimicrobial activity against Gram- to considerable batch-to-batch variation depending on positive and Gram-negative bacteria, both aerobic and growth conditions at the plantations (Kawakami et al.
anaerobic, against yeasts and fungi. It is also active 1990). The exact constituency of TTO varies, as well as against clinically isolated fluconazole-resistant Candida the antibacterial, antifungal, anti-inflammatory and anal- strains (Hammer et al. 2004; Wilkinson and Cavanagh gesic properties (Carson and Riley 1993; Hart et al. 2000; Hammer et al. 2003; Caldefie-Che´zet et al. 2006).
Recently, it was also demonstrated an in vitro antimicro- Shellie et al. identified about 72 out of 97 possible bial activity against Mycoplasma pneumonia, Mycoplasma components in M. alternifolia using GC and GC ⁄ MS.
fermentans and Mycoplasma hominis (Furneri et al. 2006).
ª 2009 The AuthorsJournal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology The aims of the present study was to compare the in vitro HSV-1 and HSV-2 viruses by 50% plaque reduction antiviral activity of TTO and its constituents, terpinen-4-ol, assay, as previously described (Cutrı` et al. 1998). The a-terpinene, c-terpinene, p-cymene, terpinolene and a-ter- anti-influenza virus assay was based on the inhibition of pineol, against some DNA and RNA viruses, including the virus-induced cytopathogenicity on MDCK cells, as influenza A ⁄ PR8 virus subtype H1N1 in Madin-Darby previously described (Garozzo et al. 2000).
Canine Kidney (MDCK) cells, Herpes simplex virus type 1 The compound concentration required to inhibit virus (HSV-1) and 2 (HSV-2) in VERO cells, Echovirus 9 plaque formation and virus-induced cytopathogenicity by (Hill strain) in LLC-MK2 cells, Poliovirus 1 (Sabin strain), 50% was expressed as the 50% inhibitory dose (ID50) and Coxsackievirus B1 and Adenovirus 2 in HEp2 cells.
calculated by dose–response curves and linear regression.
TTO employed in our experiments was provided by Noninfected and infected cells in the absence of com- Australian Botanical Products (Hallam, Australia). Terpi- pounds served as cell and virus control, respectively.
nen-4-ol, a-terpinene, c-terpinene, p-cymene, terpinolene To test possible virucidal activity, equal volumes and a-terpineol were obtained from Sigma Chemical (0Æ5 ml) of virus suspension (containing 106 PFU ml)1) Company. All the compounds were dissolved in dimethyl and medium containing various concentrations of the sulfoxide (DMSO; Sigma) to give a concentration of 10% compounds were mixed and incubated for 1 h at 37°C.
(v ⁄ v) and diluted in maintenance medium at final con- Infectivity was determined by plaque assay after dilution centrations ranging from 0Æ1% (v ⁄ v) to 0Æ0001% (v ⁄ v).
of the virus below the inhibitory concentration.
Dilution of test compounds contained a maximum con- Results obtained from our screening demonstrated that centration of 0Æ01% DMSO (v ⁄ v), which was not toxic to the TTO and some of its components have an inhibitory effect on influenza virus A ⁄ PR8 replication at doses below Viruses tested working stock solutions were prepared as cellular lysates using DMEM (or RPMI 1640 for MDCK In fact, the ID50 value was found to be 0Æ0006% (v ⁄ v) cells) supplemented with 2% heat-inactivated foetal calf and was much lower than its CD50 (0Æ025% v ⁄ v). Three of serum (FCS), 0Æ2 g l)1 of streptomycin and 200 U ml)1 the TTO components tested were effective. In particular, the ID50 values were found to be 0Æ0025% (v ⁄ v), 0Æ0012% The cytotoxicity of the test compounds was evaluated (v ⁄ v) and 0Æ025% (v ⁄ v) for terpinen-4-ol, terpinolene and by both measuring their effect on cell morphology (e.g.
a-terpineol, respectively. Finally, compounds a-terpinene, rounding up, shrinking, detachment) by light microscopy p-cymene and c-terpinene were completely ineffective.
and on cell growth by the MTT method in 96-well tissue All the compounds were ineffective against polio 1, culture plates, when compared with the control cultures, adeno 2, ECHO 9, Coxsackie B1, HSV-1 and HSV-2 after 24, 48 and 72 h. The 50% cytotoxic dose (CD50) was expressed as the concentration of the compounds The study of the effect of these compounds on neutral- that inhibited cell growth by 50% when compared with ization of virus infectivity demonstrated that none of the the control cultures (Cutrı` et al. 1998).
tested compounds showed virucidal activity against polio The antiviral activity of tested compounds was evalu- 1, adeno 2, ECHO 9, Coxsackie B1 and influenza A ⁄ PR8.
ated against polio 1, ECHO 9, Coxsackie B1, adeno 2, Only a slight reduction for the TTO (0Æ125% v ⁄ v) against Table 1 Antiviral activity of tea tree oil (TTO) HSV, Herpes simplex virus.
*Values are mean ± 0Æ5 SD (maximal SD estimated) for three separate assays.
 CD50, concentration which inhibited cells growth by 50% when compared with controlculture.
àID50, concentration which inhibited virus plaque formation and virus-induced cytopathogenicityby 50%.
Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology HSV-1 and HSV-2 viruses was demonstrated (data not Furneri, P.M., Paolino, D., Sajia, A., Marino, A. and Bisignano, G. (2006) In vitro antimycoplasmal activity of Melaleuca In contrast with the results reported by other authors alternifolia essential oil. J Antimicrob Chemother 58, (Carson et al. 2001, 2008; Schnitzler et al. 2001), TTO did not show any antiviral activity against the replicative Garozzo, A., Cutrı`, C.C.C., Castro, A., Tempera, G., Guerrera, F., Sarva`, M.C. and Geremia, E. (2000) Anti-rhinovirus In this case, the different mechanism of action could activity of 3-methylthio-5-aryl-4-isothazolecarbonitriles depend on many factors involved. In fact, the origin of derivatives. Antiviral Res 45, 199–210.
TTO, the diversity of antimicrobial components and the Hammer, K.A., Carson, C.F. and Riley, T.V. (2003) Antifungal activity of the components of Melaleuca alternifolia (tea relative concentrations or the viral serotype could influ- tree) oil. J Appl Microbiol 95, 853–860.
Hammer, K.A., Carson, C.F. and Riley, T.V. (2004) Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata andSaccharomyces cerevisiae. J Antimicrob Chemother 53, Caldefie-Che´zet, F., Fusillier, C., Jarde, T., Laroye, H., Damez, M., Vasson, M.P. and Guillot, J. (2006) Potential anti- Hart, P.H., Brand, C., Carson, C.F., Riley, T.V., Prager, R.H.
inflammatory effects of Melaleuca alternifolia essential oil and Finlay-Jones, J.J. (2000) Terpinen-4-ol, the main on human peripheral blood leukocytes. Phytother Res 20, component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator produc- Carson, C.F. and Riley, T.V. (1993) Antimicrobial activity of tion by activated human monocytes. Inflamm Res 49, the essential oil of Melaleuca alternifolia. Lett Appl Micro- Kawakami, M., Sachs, R.M. and Shibamoto, T. (1990) Volatile Carson, C.F., Ashton, L., Dry, L., Smith, D.W. and Riley, T.V.
constituents of essential oils obtained from newly devel- (2001) Melaleuca alternifolia (tea tree) oil gel (6%) for the oped tea tree (Melaleuca alternifolia) clones. J Agric Food treatment of recurrent herpes labialis. J Antimicrob Chemo- Schnitzler, P., Schon, K. and Reichling, J. (2001) Antiviral Carson, C.F., Hammer, K.A. and Riley, T.V. (2006) Melaleuca activity of Australian tea tree oil and eucalyptus oil alternifolia (tea tree) oil: a review of antimicrobial and against herpes simplex virus in cell culture. Pharmazie 56, other medicinal properties. Clin Microbiol Rev 19, 50–62.
Carson, C.F., Smith, D.W., Lampacher, G.J. and Riley, T.V.
Shellie, R., Marriott, P., Zappia, G., Mondello, L. and Dugo, (2008) Use of deception to achieve double-blinding in a G. (2003) Interactive use of linear retention indices on clinical trial of Melaleuca alternifolia (tea tree) oil for the polar and apolar columns with an MS-library for reliable treatment of recurrent herpes labialis. Contemp Clin Trials characterisation of Australian tea tree and other Melaleuca sp. oils. J Essent Oil Res 15, 305–312.
Cutrı`, C.C.C., Garozzo, A., Siracusa, M.A., Sarva´, M.C., Tem- Wilkinson, J.M. and Cavanagh, H.M. (2005) Antibacterial pera, G., Geremia, E., Pinizzotto, M.R. and Guerrera, F.
activity of essential oils from Australian native plants.
(1998) Synthesis and antiviral activity of a new series of 4-isothiazolecarbonitriles. Bioorg Med Chem 6, 2271–2280.
ª 2009 The AuthorsJournal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology

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