Antiviral Research 89(2011)83-88 Contents lists available at ScienceDirect Antiviral Research ELSEVIER journal homepage:www.elsevier.com/locate/antiviral Activity of Melaleuca alternifolia(tea tree)oil on Influenza virus A/PR/8: Study on the mechanism of action A.Garozzoa.*,R.Timpanaroa,A.Stivalaa,G.Bisignanob,A.Castroa Department of Microbiological and Gynaecological Sciences.University of Catania.Via Androne 81.95124 Catania,Italy Department of Pharmacobiology.University of Messina,Messina,Italy ARTICLE INFO ABSTRACT Article history: Our previous study demonstrated that Melaleuca alternifolia(tea tree)oil(TTO)had an interesting antiviral Received 6 August 2010 activity against Influenza A in MDCK cells.In fact,when we tested TTO and some of its components,we Received in revised form 18 October 2010 found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose: Accepted 13 November 2010 terpinen-4-ol,terpinolene,and alfa-terpineol were the main active components. The aim of this study was to investigate the mechanism of action of TTO and its active components Keywords: against Influenza A/PR/8 virus subtype H1N1 in MDCK cells. Melaleuca alternifolia Essential oil None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Antiviral activity Thus,the effect of TTO and its active components on different steps of the replicative cycle of influenza Influenza virus virus was studied by adding the test compounds at various times after infection.These experiments revealed that viral replication was significantly inhibited if TTO was added within 2h of infection,indi- cating an interference with an early step of the viral replicative cycle of influenza virus. The influence of the compound on the virus adsorption step,studied by the infective center assay. indicated that TTO did not interfere with cellular attachment of the virus. TTO did not inhibit influenza virus neuraminidase activity,as shown by the experiment measuring the amount of 4-methylumbelliferone,cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid. The effect ofTTO on acidification ofcellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control.The treatment of cells with 0.01%(v/v)of TTO at 37C for 4h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles,indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment. 2010 Elsevier B.V.All rights reserved. 1.Introduction Our interest in natural compounds with antiviral activity led us to analyze TTO against DNA and RNA viruses(Influenza A/PR/8 Over the last few years there has been an increasing interest in subtype H1N1,polio type 1,ECHO 9,Coxsackie B1,adeno type 2. the anti-microbial properties of tea tree oil (TTO),known for a long herpes simplex(HSV)type 1 and 2 viruses).Our results demon- time as a general antiseptic.TTO is extracted from the leaves and strated that TTO and some of its components (terpinen-4-ol, terminal branches of Melaleuca alternifolia,also called the tea tree. terpinolene,and o-terpineol)have an inhibitory effect on Influenza M.alternifolia grows in a restricted marshy area of northern New A/PR/8 virus subtype HIN1 replication at doses below the cyto- South Wales,in Australia. toxic dose.The ECso value of TTO was found to be 0.0006% TTO is a heterogeneous mixture,subject to considerable batch- (v/v)and was much lower than its CC5o (0.025%,v/v).All the to-batch variation depending on growth conditions at the planta- compounds were ineffective against polio 1,adeno 2,ECHO 9. tions (Kawakami et al.,1990:Shellie et al.,2003).The exact con- Coxsackie B1,HSV-1,and HSV-2 (Garozzo et al.,2009).This stituency of tea tree oil varies,as well as the antibacterial,antifun- was in contrast with the results reported by other authors who gal,anti-inflammatory and analgesic properties(Caldefie-Chezet demonstrated antiviral activity against the replicative cycle of et al.,2006;Carson and Riley,1993;Carson et al.,2006;Hammer HSV-1 and HSV-2 (Carson et al.,2001,2008:Schnitzler et al., et al.,2004;Hart et al.,2000;Wilkinson and Cavanagh,2005). 2001). The aim of the present study was to determine the mechanism of action of TTO and its active components,terpinen-4-ol,terpinolene Corresponding author.Tel.:+39095 2504720:fax:+39 095 2504721. and o-terpineol,(test compounds)against Influenza A/PR/8 virus E-mail addresses:agar@unict.it,adrianag56@yahoo.com(A.Garozzo). subtype H1N1. 0166-3542/S-see front matter 2010 Elsevier B.V.All rights reserved. doi:10.1016j.antiviraL.2010.11.010Antiviral Research 89 (2011) 83–88 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Activity of Melaleuca alternifolia (tea tree) oil on Influenza virus A/PR/8: Study on the mechanism of action A. Garozzoa,∗, R. Timpanaroa, A. Stivalaa, G. Bisignano b, A. Castroa a Department of Microbiological and Gynaecological Sciences, University of Catania, Via Androne 81, 95124 Catania, Italy b Department of Pharmacobiology, University of Messina, Messina, Italy article info Article history: Received 6 August 2010 Received in revised form 18 October 2010 Accepted 13 November 2010 Keywords: Melaleuca alternifolia Essential oil Antiviral activity Influenza virus abstract Our previous study demonstrated that Melaleuca alternifolia (tea tree) oil (TTO) had an interesting antiviral activity against Influenza A in MDCK cells. In fact, when we tested TTO and some of its components, we found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and alfa-terpineol were the main active components. The aim of this study was to investigate the mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1 in MDCK cells. None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Thus, the effect of TTO and its active components on different steps of the replicative cycle of influenza virus was studied by adding the test compounds at various times after infection. These experiments revealed that viral replication was significantly inhibited if TTO was added within 2 h of infection, indi￾cating an interference with an early step of the viral replicative cycle of influenza virus. The influence of the compound on the virus adsorption step, studied by the infective center assay, indicated that TTO did not interfere with cellular attachment of the virus. TTO did not inhibit influenza virus neuraminidase activity, as shown by the experiment measuring the amount of 4-methylumbelliferone, cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2 -O-(4-methylumbelliferyl)-N-acetylneuraminic acid. The effect of TTO on acidification of cellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control. The treatment of cells with 0.01% (v/v) of TTO at 37 ◦C for 4 h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles, indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Over the last few years there has been an increasing interest in the anti-microbial properties of tea tree oil (TTO), known for a long time as a general antiseptic. TTO is extracted from the leaves and terminal branches of Melaleuca alternifolia, also called the tea tree. M. alternifolia grows in a restricted marshy area of northern New South Wales, in Australia. TTO is a heterogeneous mixture, subject to considerable batch￾to-batch variation depending on growth conditions at the planta￾tions (Kawakami et al., 1990; Shellie et al., 2003). The exact con￾stituency of tea tree oil varies, as well as the antibacterial, antifun￾gal, anti-inflammatory and analgesic properties (Caldefie-Chézet et al., 2006; Carson and Riley, 1993; Carson et al., 2006; Hammer et al., 2004; Hart et al., 2000; Wilkinson and Cavanagh, 2005). ∗ Corresponding author. Tel.: +39 095 2504720; fax: +39 095 2504721. E-mail addresses: agar@unict.it, adrianag56@yahoo.com (A. Garozzo). Our interest in natural compounds with antiviral activity led us to analyze TTO against DNA and RNA viruses (Influenza A/PR/8 subtype H1N1, polio type 1, ECHO 9, Coxsackie B1, adeno type 2, herpes simplex (HSV) type 1 and 2 viruses). Our results demon￾strated that TTO and some of its components (terpinen-4-ol, terpinolene, and -terpineol) have an inhibitory effect on Influenza A/PR/8 virus subtype H1N1 replication at doses below the cyto￾toxic dose. The EC50 value of TTO was found to be 0.0006% (v/v) and was much lower than its CC50 (0.025%, v/v). All the compounds were ineffective against polio 1, adeno 2, ECHO 9, Coxsackie B1, HSV-1, and HSV-2 (Garozzo et al., 2009). This was in contrast with the results reported by other authors who demonstrated antiviral activity against the replicative cycle of HSV-1 and HSV-2 (Carson et al., 2001, 2008; Schnitzler et al., 2001). The aim of the present study was to determine the mechanism of action of TTO and its active components, terpinen-4-ol, terpinolene and -terpineol, (test compounds) against Influenza A/PR/8 virus subtype H1N1. 0166-3542/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2010.11.010