88 A Garozzo et aL /Antiviral Research 89 (2011)83-88 The treatment of the cells with 0.01%(v/v)TTO at 37Cfor 2 and Eriksson,B..Helgstrand.E.,Johansson.N.G..Larsson,A..Misiorny.A..Noren.J.O. 4h before acridine orange-staining caused complete disappear- Philipson,L.Stenberg.K..Stening.G..Stridh.S,Oberg.B..1977.Inhibition of ance of the orange fluorescence,in contrast the green fluorescence influenza virus ribonucleic acid polymerase by ribavirin triphosphate.Antimi- crob.Agents Chemother.11.946-951. remained(Fig.3D).The same results were observed ifthe cells were Garozzo.A..Cutri,C.C.C.,Castro,A.,Tempera,G.Guerrera,F.,Sarva,M.C.,Geremia.E.. treated with 100 and 10 nM of bafilomycin A1 at 37C for 1 h(Fig.3B 2000.Anti-rhinovirus activity of 3-methylthio-5-aryl-4-isothazolecarbonitriles derivatives.Antiviral Res.45,199-210. and C). Garozzo.A.,Timpanaro,R..Bisignano,B.,Furneri.P.M.,Bisignano.G..Castro,A.. The treatment of the cells with TTO at the same concentration 2009.In vitro antiviral activity of Melaleuca alternifolia essential oil.Lett.Appl for 1 h or with a lower concentration(0.0025%,v/v),did not cause Microbiol.49.806-808. the disappearance of the cytoplasmic orange fluorescence(Fig.3E) Geisow.M.I,D'Arcy Hart,P.,Young.M.R.,1981.Temporal changes of lysosome and phagosome pH during phagolysosome formation in macrophages:studies by The data obtained by treating the cells with terpinen-4-ol,ter- fluorescence spectroscopy.I.Cell Biol.89.645-652. pinolene,and o-terpineol before AO staining demonstrated that Gubareva,LV.Webster.R.G.Hayden.F.G.,2002.Detection of influenza virus resis- terpinen-4-ol,one of the major components of TTO,had an impor- tance to neuraminidase inhibitors by an enzyme inhibition assay.Antiviral Res. 53.47-61 tant role in the anti influenza virus activity as only in the cells Guinea,R.,Carrasco,L..1994.Concanamycin A blocks influenza virus entry into cells treated with this compound did we observe a lack of the orange under acidic conditions.FEBS Lett.349,327-330. fluorescence (Fig.3F). Guinea,R..Carrasco,L.1995.Requirement for vacuolar proton-ATPase activity dur ing entry of influenza virus into cells.J.Virol.69.2306-2312. These results were confirmed by measuring the fluorescence Hammer,K.A..Carson,C.F..Riley.T.V..2004.Antifungal effects of Melaleuca alterni- intensity by fluorometry,indicating that TTO and terpinen-4-ol folia (tea tree)oil and its components on Candida albicans,Candida glabrata and clearly inhibited acridine orange accumulation in acid cytoplasmic Saccharomyces cerevisiae.J.Antimicrob.Chemother.53,1081-1085. vesicles,in fact,the data obtained were consistently concordant Hart.P.H,Brand,C..Carson,C.F..Riley.T.V..Prager.R.H.Finlay-Jones,J.J,2000 Terpinen-4-ol,the main component of the essential oil of Melaleuca alternifolia with the positive control bafilomycin A1(Fig.4). (tea treeoil),suppresses inflammatory mediator production by activated human The acidification of lysosomes in MDCK cells recovered com- monocytes.Inflamm.Res.49,619-626. pletely when the cells were treated with 0.01%(v/v)TTO for Hayden,F.G..Atmar.R.L.Schilling.M..Johnson,C..Poretz.D..Paar.D..Huson.L.. 4h,washed and then incubated for 2h without the compound Ward,P..Mills,R.G..1999.Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza.N.Engl.J.Med.341.1336-1343. (Figs.3G and 5),suggesting that the cells can re-acidify after treat- Imanishi,N..Tuji,Y..Katada,Y.,Maruhashi,M..Konosu,S..Mantani,N..Terasawa ment with TTO,and that cell morphology is not influenced by K,Ochiai.H..2002.Additional inhibitory effect of tea extract on the growth of influenza A and B viruses in MDCK cells.Microbiol.ImmunoL 46,491- treatment.A similar result was observed for terpinen-4-ol (Fig.5) 494 Our results indicate that TTO inhibited the influenza virus Kawakami,M..Sachs,R.M..Shibamoto,T,1990.Volatile constituents of essential growth in MDCK cells by an interference with acidification of the oils obtained from newly developed tea trea(Melaleuca alternifolia)clones.J. Agricult.Food Chem.38,1657-1661. intralysosomal compartment that could inhibit viral uncoating. Mantani,N.,Andoh,T.,Kawamata,H.,Terasawa.K..Ochiai,H..1999.Inhibitory effect Other studies also reported that Ephedrae herba(Mantani et al., of Ephedrae herba,an oriental traditional medicine,on the growth of influenza 1999)and green-tea (Imanishi et al.,2002)extracts inhibit the A/PR/8 virus in MDCK cells.Antiviral Res.44.193-200. growth of influenza virus with a similar mechanism of action. Natale,V.A..McCullough.K.C.1998.Macrophage cytoplasmic vesicle pH gradients and vacuolar H-ATPase activities relative to virus infection.J.Leucocyte Biol. Further experiments should be performed to clarify the exact 64.302-310. mechanism of action ofTTO that,as demonstrated for the macrolide Ochiai.H.,Sakai.S.,Hirabayashi,T..Shimizu,Y.,Terasawa,K..1995.Inhibitory effect antibiotics bafilomycin Al and concanamycin A (Guinea and of bafilomycin A1.a specific inhibitor of vacuolar-type proton pump.on the growth of influenza A and B viruses in MDCK cells.Antiviral Res.27,425- Carrasco,1994:Natale and McCullough,1998;Ochiai et al.,1995). 430. could affect the vacuolar proton-ATPase activities. Potier.M..Mameli.L.Belisle M.Dallaire,L.Melanc S.B.,1979.Fluori- metric assay of neuraminidase with a sodium (4-methylumbelliferyl-o-D-N- acetvlneuraminatel substrate.Anal Biochem.94 287-296 References Schnitzler.P,Schon,K..Reichling.J..2001.Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture.Pharmazie 56. Buxton,R.C..Edwards.B..Juo.R.R.,Voyta,J.C..Tisdale.M..Bethell,C..2000.Develop- 343-347 ment of a sensitive chemiluminescent neuraminidase assay for determination Shellie,R..Marriott,P..Zappia,G..Mondello,L.Dugo,G.,2003.Interactive use of of influenza virus susceptibility to Zanamivir.Anal.Biochem.280,291-300. linear retention indices on polar and apolar columns with an MS-library for Caldefie-Chezet.F Fusillier,C..Jarde,T.,Laroye,H..Damez,M.Vasson,M.P..Guillot reliable characterisation of Australian tea tree and other Melaleuca sp.oils.J. J..2006.Potential anti-inflammatory effects of Melaleuca alternifolia essential oil Essent.Oil Res.15,305-312. on human peripheral blood leukocytes.Phytother.Res.20,364-370. Wilkinson,J.M.Cavanagh,H.M..2005.Antibacterial activity of essential oils from Carson,C.F Riley.T.V..1993.Antimicrobial activity of the essential oil of Melaleuca Australian native plants.Phytother.Res.19,643-646. alternifolia.Lett.Appl.Microbiol.16.49-55. Wray.S.K..Gilbert,B.E.Noall,M.W.,Knight,V..1985.Mode of action of ribavirin Carson,C.F..Ashton,L.Dry,L.Smith,D.W..Riley.T.V.,2001.Melaleuca alternifolia effect of nucleotide pool alterations on influenza virus ribonucleoprotein syn- (tea tree)oil gel(6%)for the treatment of recurrent herpes labialis.J.Antimicrob. thesis.Antiviral Res.5,29-37. Chemother.48,450-451. Yoshimori,T.Yamamoto,A.,Moriyama,Y.,Futai,M.Tashiro,Y,1991.Bafilomycin Carson,C.F.Hammer,K.A,Riley.T.V..2006.Melaleuca alternifolia (tea tree)oil:a A1,a specific inhibitor of vacuolar-type H(+)-ATPase,inhibits acidification review of antimicrobial and other medicinal properties.Clin.MicrobioL.Rev.19. and protein degradation in lysosomes of cultured cells.J.Biol.Chem.266. 50_62 17707-17712. Carson,C.F.Smith,D.W..Lampacher.G.J.,Riley.T.V..2008.Use of deception to Zoccarato,F..Cavallini,L.Alexandre,A..1999.The pH-sensitive dye acridine orange achieve double-blinding in a clinical trial of Melaleuca altemifolia (tea tree)oil as a tool to monitor exocytosis/endocytosis in synaptosomes.J.Neurochem.72. for the treatment of recurrent herpes labialis.Contemp.Clin.Trials 29.9-12 625-633.88 A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 The treatment of the cells with 0.01% (v/v) TTO at 37 ◦C for 2 and 4 h before acridine orange-staining caused complete disappear￾ance of the orange fluorescence, in contrast the green fluorescence remained (Fig. 3D). The same results were observed if the cells were treated with 100 and 10 nM of bafilomycin A1 at 37 ◦C for 1 h (Fig. 3B and C). The treatment of the cells with TTO at the same concentration for 1 h or with a lower concentration (0.0025%, v/v), did not cause the disappearance of the cytoplasmic orange fluorescence (Fig. 3E). The data obtained by treating the cells with terpinen-4-ol, ter￾pinolene, and -terpineol before AO staining demonstrated that terpinen-4-ol, one of the major components of TTO, had an impor￾tant role in the anti influenza virus activity as only in the cells treated with this compound did we observe a lack of the orange fluorescence (Fig. 3F). These results were confirmed by measuring the fluorescence intensity by fluorometry, indicating that TTO and terpinen-4-ol clearly inhibited acridine orange accumulation in acid cytoplasmic vesicles, in fact, the data obtained were consistently concordant with the positive control bafilomycin A1 (Fig. 4). The acidification of lysosomes in MDCK cells recovered com￾pletely when the cells were treated with 0.01% (v/v) TTO for 4 h, washed and then incubated for 2 h without the compound (Figs. 3G and 5), suggesting that the cells can re-acidify after treat￾ment with TTO, and that cell morphology is not influenced by treatment. A similar result was observed for terpinen-4-ol (Fig. 5). Our results indicate that TTO inhibited the influenza virus growth in MDCK cells by an interference with acidification of the intralysosomal compartment that could inhibit viral uncoating. Other studies also reported that Ephedrae herba (Mantani et al., 1999) and green-tea (Imanishi et al., 2002) extracts inhibit the growth of influenza virus with a similar mechanism of action. Further experiments should be performed to clarify the exact mechanism of action of TTO that, as demonstrated for the macrolide antibiotics bafilomycin A1 and concanamycin A (Guinea and Carrasco, 1994; Natale and McCullough, 1998; Ochiai et al., 1995), could affect the vacuolar proton-ATPase activities. References Buxton, R.C., Edwards, B., Juo, R.R., Voyta, J.C., Tisdale, M., Bethell, C., 2000. Develop￾ment of a sensitive chemiluminescent neuraminidase assay for determination of influenza virus susceptibility to Zanamivir. Anal. Biochem. 280, 291–300. Caldefie-Chézet, F., Fusillier, C., Jarde, T., Laroye, H., Damez, M., Vasson, M.P., Guillot, J., 2006. Potential anti-inflammatory effects of Melaleuca alternifolia essential oil on human peripheral blood leukocytes. Phytother. Res. 20, 364–370. Carson, C.F., Riley, T.V., 1993. Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett. Appl. Microbiol. 16, 49–55. Carson, C.F., Ashton, L., Dry, L., Smith, D.W., Riley, T.V., 2001. Melaleuca alternifolia (tea tree) oil gel (6%) for the treatment of recurrent herpes labialis. J. Antimicrob. Chemother. 48, 450–451. Carson, C.F., Hammer, K.A., Riley, T.V., 2006. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev. 19, 50–62. Carson, C.F., Smith, D.W., Lampacher, G.J., Riley, T.V., 2008. Use of deception to achieve double-blinding in a clinical trial of Melaleuca alternifolia (tea tree) oil for the treatment of recurrent herpes labialis. Contemp. Clin. Trials 29, 9–12. Eriksson, B., Helgstrand, E., Johansson, N.G., Larsson, A., Misiorny, A., Noren, J.O., Philipson, L., Stenberg, K., Stening, G., Stridh, S., Oberg, B., 1977. Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate. Antimi￾crob. Agents Chemother. 11, 946–951. Garozzo, A., Cutrì, C.C.C., Castro, A., Tempera, G., Guerrera, F., Sarvà, M.C., Geremia, E., 2000. Anti-rhinovirus activity of 3-methylthio-5-aryl-4-isothazolecarbonitriles derivatives. Antiviral Res. 45, 199–210. Garozzo, A., Timpanaro, R., Bisignano, B., Furneri, P.M., Bisignano, G., Castro, A., 2009. In vitro antiviral activity of Melaleuca alternifolia essential oil. Lett. Appl. Microbiol. 49, 806–808. Geisow, M.J., D’Arcy Hart, P., Young, M.R., 1981. Temporal changes of lysosome and phagosome pH during phagolysosome formation in macrophages: studies by fluorescence spectroscopy. J. Cell Biol. 89, 645–652. Gubareva, L.V., Webster, R.G., Hayden, F.G., 2002. Detection of influenza virus resis￾tance to neuraminidase inhibitors by an enzyme inhibition assay. Antiviral Res. 53, 47–61. Guinea, R., Carrasco, L., 1994. Concanamycin A blocks influenza virus entry into cells under acidic conditions. FEBS Lett. 349, 327–330. Guinea, R., Carrasco, L., 1995. Requirement for vacuolar proton-ATPase activity dur￾ing entry of influenza virus into cells. J. Virol. 69, 2306–2312. Hammer, K.A., Carson, C.F., Riley, T.V., 2004. Antifungal effects of Melaleuca alterni￾folia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J. Antimicrob. Chemother. 53, 1081–1085. Hart, P.H., Brand, C., Carson, C.F., Riley, T.V., Prager, R.H., Finlay-Jones, J.J., 2000. Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes. Inflamm. Res. 49, 619–626. Hayden, F.G., Atmar, R.L., Schilling, M., Johnson, C., Poretz, D., Paar, D., Huson, L., Ward, P., Mills, R.G., 1999. Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza. N. Engl. J. Med. 341, 1336–1343. Imanishi, N., Tuji, Y., Katada, Y., Maruhashi, M., Konosu, S., Mantani, N., Terasawa, K., Ochiai, H., 2002. Additional inhibitory effect of tea extract on the growth of influenza A and B viruses in MDCK cells. Microbiol. Immunol. 46, 491– 494. Kawakami, M., Sachs, R.M., Shibamoto, T., 1990. Volatile constituents of essential oils obtained from newly developed tea trea (Melaleuca alternifolia) clones. J. Agricult. Food Chem. 38, 1657–1661. Mantani, N., Andoh, T., Kawamata, H., Terasawa, K., Ochiai, H., 1999. Inhibitory effect of Ephedrae herba, an oriental traditional medicine, on the growth of influenza A/PR/8 virus in MDCK cells. Antiviral Res. 44, 193–200. Natale, V.A., McCullough, K.C., 1998. Macrophage cytoplasmic vesicle pH gradients and vacuolar H+-ATPase activities relative to virus infection. J. Leucocyte Biol. 64, 302–310. Ochiai, H., Sakai, S., Hirabayashi, T., Shimizu, Y., Terasawa, K., 1995. Inhibitory effect of bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, on the growth of influenza A and B viruses in MDCK cells. Antiviral Res. 27, 425– 430. Potier, M., Mameli, L., Belisle, M., Dallaire, L., Melancon, S.B., 1979. Fluori￾metric assay of neuraminidase with a sodium (4-methylumbelliferyl--d-N￾acetylneuraminate) substrate. Anal. Biochem. 94, 287–296. Schnitzler, P., Schon, K., Reichling, J., 2001. Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture. Pharmazie 56, 343–347. Shellie, R., Marriott, P., Zappia, G., Mondello, L., Dugo, G., 2003. Interactive use of linear retention indices on polar and apolar columns with an MS-library for reliable characterisation of Australian tea tree and other Melaleuca sp. oils. J. Essent. Oil Res. 15, 305–312. Wilkinson, J.M., Cavanagh, H.M., 2005. Antibacterial activity of essential oils from Australian native plants. Phytother. Res. 19, 643–646. Wray, S.K., Gilbert, B.E., Noall, M.W., Knight, V., 1985. Mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein syn￾thesis. Antiviral Res. 5, 29–37. Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., Tashiro, Y., 1991. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. 266, 17707–17712. Zoccarato, F., Cavallini, L., Alexandre, A., 1999. The pH-sensitive dye acridine orange as a tool to monitor exocytosis/endocytosis in synaptosomes. J. Neurochem. 72, 625–633.