Activity of Melaleuca alternifolia（tea tree）oil on Influenza virus A/PR/8：Study on the mechanism of action

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.2010.11.010

Antiviral 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

84 A Garozzo et aL Antiviral Research 89(2011)83-88 2.Materials and methods 120 110 口TT0 terpinen-4-o 2.1.Compounds 100 terpinolene 90 口ala-4 erpineol The essential oil of M.alternifolia was provided by Australian 80 Botanical Products(Hallam,Australia).Terpinen-4-ol,terpinolene 70 and o-terpineol were obtained from Sigma Chemical Company.All 60 the compounds were initially dissolved in DMSO(dimethyl sulfox- ide,Sigma)to a concentration of 5%(v/v). Oseltamivir and ribavirin were used as reference compounds: oseltamivir phosphate was used directly from the blister pack- aging of Tamiflu 75mg(Roche Product)and prepared in sterile glass-distilled water supplemented with 12.5%(v/v)DMSO to Ads 0 15 30 4560120180240300360 a concentration of 1 mM;ribavirin (1-p-D-ribofuranosyl-1,2,4- minutes triazole-3-carboxamide)was purchased from Sigma Chemical Fig.1.Effect of the addition of TTO(0.01%,v/v)and its active components,terpinen- Company.Ribavirin was prepared in sterile glass-distilled water 4-ol (0.01%.v/v).terpinolene (0.005%.v/v)and a-terpineol (0.02%.v/v)at various to a concentration of 2 mM. times during the replicative cycle of influenza virus.Time 0=post 2 h adsorption The macrolide antibiotic bafilomycin A1(Sigma)was used as ref- period at 4C.Each value represents the mean+S.E.M.of three separate assays. erence compound blocking the vacuolar H*-ATPase proton pump (Yoshimori et al.,1991).The drug was prepared in DMSO at 10 uM 2.4.Cell culture pretreatment Acridine orange (AO)(Sigma)was prepared in sterile glass- distilled water at 50 mg/ml. Pretreatment of cultures was performed by exposing the cell All the compounds were diluted in maintenance medium before monolayers to different concentrations of the test compounds in use to achieve the final concentration needed.The dilution of the maintenance medium for 1 and 2 days at 37C.After treatment the test compounds contained a maximum concentration of 0.01% cell monolayers were washed thoroughly with PBS and infected DMSO.which was not toxic to the cells. with influenza virus at an MOI(multiplicity of infection)of 0.1 to allow viral cytopathic activity.The cell monolayers grown in maintenance medium without the test compounds were used as 2.2.Cells and virus controls.Virus titration was performed as described above. Madin-Darby canine kidney (MDCK)cells were kept in a 2.5.Addition at different time intervals humidified 5%carbon dioxide atmosphere at 37C and grown in RPMI 1640 medium supplemented with 10%heat inactivated Monolayers of MDCK cells were grown to confluence in 24- fetal calf serum(FCS).2 mM L-glutamine,0.1%sodium bicarbonate, well plates and inoculated with influenza virus at an MOl of 0.1 200 wg/ml of streptomycin and 200 units/ml of penicillin G. The plates were incubated for 2h at 4C to ensure synchronous Influenza virus A/Puerto Rico/8/34 H1N1 (PR8)kindly supplied replication of the viruses,with or without the test compounds for by Prof.L Nencioni (Universita di Roma La Sapienza,Rome,Italy). the adsorption period.The inoculum was then removed and the was grown in MDCK cells at 37C. medium,with or without the test compounds,was added at various The working stock solution was prepared as cellular lysates times after the adsorption period,as indicated in Fig.1.The plates using RPMI 1640 with 2%FCS (maintenance medium)and cen- were incubated at 37C for 12 h,the cultures were then frozen and trifuged (5000 x g for 30 min at 4C). the virus yield was determined as previously described. Infectivity of virus stock and virus yield in the experiments on the mechanism of action were determined by the MTT method: 2.6.Inhibition of virus attachment assay the reciprocals of viral dilution which resulted in 50%reduction of absorbance of formazan in the infected cells at 48-72 h was deter- The infective center assay was used to study the effect of the test mined as infectivity of the virus by MTT IDs0(50%infective dose) compounds on the virus adsorption step.Briefly,a MDCK cell sus- (Garozzo et al.,2000). pension (104 cells/ml)was cooled to 4C for at least 1 h.Influenza Virus stock solution was also used as a source of neuraminidase virus(MOI=0.1)was incubated for 60min at 37C with different (NA)in the NA inhibition assay. concentrations of the test compounds,cooled to 4C,and subse- Virus yield was also evaluated by measuring haemagglutinin quently added to the cell suspension.Cells were incubated with the units (HAU).Briefly,twofold serial dilution of each test was mixed virus-compound mixtures for 120 min at 4C to prevent the virus with an equal volume (50 ul)of 0.5%suspension of human red from entering the cells.After the adsorption period,unadsorbed blood cells of the O Rh+group and incubated for 30 min at room virus and free compound were removed by washing three times temperature. with cold RPMI.The cells were then diluted serially and assayed for The viral titre of the sample was the reciprocal of the dilution cell-associated viral cytopathic activity as previously described. giving rise to complete haemoagglutination. 2.7.Haemoagglutination inhibition assay 2.3.Virucidal activity Haemoagglutination inhibition assay was used to test the effect of the test compounds on virus adsorption to target cells. To test possible virucidal activity.equal volumes(0.5 ml)of Twenty-five ul two-fold dilution in PBS of the test compounds influenza virus suspension,containing 10 CCIDso(50%cell culture with equal volumes of influenza virus solution (200 HAU/25 wl) infective dose).and RPMI containing the various concentrations were mixed and incubated for 30 min at room temperature.Fifty of the test compounds were mixed and incubated for 2h at 37C. ul of 0.5%suspension of human red blood cells of the O Rh+ Infectivity was determined as described above after dilution of the group was then added and incubated for 30min at room tem- virus below the inhibitory concentration. perature.The minimum concentration of the compounds that

84 A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 2. Materials and methods 2.1. Compounds The essential oil of M. alternifolia was provided by Australian Botanical Products (Hallam, Australia). Terpinen-4-ol, terpinolene and -terpineol were obtained from Sigma Chemical Company. All the compounds were initially dissolved in DMSO (dimethyl sulfox￾ide, Sigma) to a concentration of 5% (v/v). Oseltamivir and ribavirin were used as reference compounds: oseltamivir phosphate was used directly from the blister pack￾aging of Tamiflu 75 mg (Roche Product) and prepared in sterile glass-distilled water supplemented with 12.5% (v/v) DMSO to a concentration of 1 mM; ribavirin (1-p-d-ribofuranosyl-1,2,4- triazole-3-carboxamide) was purchased from Sigma Chemical Company. Ribavirin was prepared in sterile glass-distilled water to a concentration of 2 mM. Themacrolide antibiotic bafilomycin A1 (Sigma) was used as ref￾erence compound blocking the vacuolar H+-ATPase proton pump (Yoshimori et al., 1991). The drug was prepared in DMSO at 10 M. Acridine orange (AO) (Sigma) was prepared in sterile glass￾distilled water at 50 mg/ml. All the compounds were diluted in maintenance medium before use to achieve the final concentration needed. The dilution of the test compounds contained a maximum concentration of 0.01% DMSO, which was not toxic to the cells. 2.2. Cells and virus Madin–Darby canine kidney (MDCK) cells were kept in a humidified 5% carbon dioxide atmosphere at 37 ◦C and grown in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 2 mM l-glutamine, 0.1% sodium bicarbonate, 200 g/ml of streptomycin and 200 units/ml of penicillin G. Influenza virus A/Puerto Rico/8/34 H1N1 (PR8) kindly supplied by Prof. L. Nencioni (Università di Roma La Sapienza, Rome, Italy), was grown in MDCK cells at 37 ◦C. The working stock solution was prepared as cellular lysates using RPMI 1640 with 2% FCS (maintenance medium) and cen￾trifuged (5000 × g for 30 min at 4 ◦C). Infectivity of virus stock and virus yield in the experiments on the mechanism of action were determined by the MTT method: the reciprocals of viral dilution which resulted in 50% reduction of absorbance of formazan in the infected cells at 48–72 h was deter￾mined as infectivity of the virus by MTT ID50 (50% infective dose) (Garozzo et al., 2000). Virus stock solution was also used as a source of neuraminidase (NA) in the NA inhibition assay. Virus yield was also evaluated by measuring haemagglutinin units (HAU). Briefly, twofold serial dilution of each test was mixed with an equal volume (50 l) of 0.5% suspension of human red blood cells of the O Rh+ group and incubated for 30 min at room temperature. The viral titre of the sample was the reciprocal of the dilution giving rise to complete haemoagglutination. 2.3. Virucidal activity To test possible virucidal activity, equal volumes (0.5 ml) of influenza virus suspension, containing 10 CCID50 (50% cell culture infective dose), and RPMI containing the various concentrations of the test compounds were mixed and incubated for 2 h at 37 ◦C. Infectivity was determined as described above after dilution of the virus below the inhibitory concentration. Fig. 1. Effect of the addition of TTO (0.01%, v/v) and its active components, terpinen- 4-ol (0.01%, v/v), terpinolene (0.005%, v/v) and -terpineol (0.02%, v/v) at various times during the replicative cycle of influenza virus. Time 0 = post 2 h adsorption period at 4 ◦C. Each value represents the mean ± S.E.M. of three separate assays. 2.4. Cell culture pretreatment Pretreatment of cultures was performed by exposing the cell monolayers to different concentrations of the test compounds in maintenance medium for 1 and 2 days at 37 ◦C. After treatment the cell monolayers were washed thoroughly with PBS and infected with influenza virus at an MOI (multiplicity of infection) of 0.1 to allow viral cytopathic activity. The cell monolayers grown in maintenance medium without the test compounds were used as controls. Virus titration was performed as described above. 2.5. Addition at different time intervals Monolayers of MDCK cells were grown to confluence in 24- well plates and inoculated with influenza virus at an MOI of 0.1. The plates were incubated for 2 h at 4 ◦C to ensure synchronous replication of the viruses, with or without the test compounds for the adsorption period. The inoculum was then removed and the medium, with or without the test compounds, was added at various times after the adsorption period, as indicated in Fig. 1. The plates were incubated at 37 ◦C for 12 h, the cultures were then frozen and the virus yield was determined as previously described. 2.6. Inhibition of virus attachment assay The infective center assay was used to study the effect of the test compounds on the virus adsorption step. Briefly, a MDCK cell sus￾pension (104 cells/ml) was cooled to 4 ◦C for at least 1 h. Influenza virus (MOI = 0.1) was incubated for 60 min at 37 ◦C with different concentrations of the test compounds, cooled to 4 ◦C, and subse￾quently added to the cell suspension. Cells were incubated with the virus-compound mixtures for 120 min at 4 ◦C to prevent the virus from entering the cells. After the adsorption period, unadsorbed virus and free compound were removed by washing three times with cold RPMI. The cells were then diluted serially and assayed for cell-associated viral cytopathic activity as previously described. 2.7. Haemoagglutination inhibition assay Haemoagglutination inhibition assay was used to test the effect of the test compounds on virus adsorption to target cells. Twenty-five l two-fold dilution in PBS of the test compounds with equal volumes of influenza virus solution (200 HAU/25 l) were mixed and incubated for 30 min at room temperature. Fifty l of 0.5% suspension of human red blood cells of the O Rh+ group was then added and incubated for 30 min at room tem￾perature. The minimum concentration of the compounds that

A.Garozzo et al./Antiviral Research 89 (2011)83-88 8 completely inhibited the haemoagglutination of the virus was medium without the addition of fetal calf serum.At the end of the determined. incubation period the cells were treated with AO(diluted in ster- ile glass-distilled water to a concentration of 25 wg/ml)at 37C for 2.8.Neuraminidase inhibition assay 30 min under 5%CO2,then washed four times with warm PBS and dried.The coverslips were mounted with cells facing down onto a Influenza virus neuraminidase activity was measured by a mod- slide containing a drop of PBS and immediately examined using a ification of the method described by Potier et al.(1979).The assay Leica DM LB fluorescence light microscope.All steps were carried measures the amount of 4-methylumbelliferone(FL-4-MU)that is out in the dark.Photographs of the microscopic observations were cleaved by the influenza virus neuraminidase(NA)from the fluoro- taken with a Leica DC180 digital camera system genic substrate 2-0-(4-methylumbelliferyl)-N-acetylneuraminic Fluorometry:The measurement of fluorescence intensity was acid(FL-4-MU-NANA:Sigma,St.Louis,MO). used to identify the accumulation of protonated acridine orange Influenza virus NA activity was determined before it was used in the acid compartment by fluorescence emission (Natale and in NA inhibition tests.Briefly,75 uM FL-4-MU-NANA were used McCullough,1998;Zoccarato et al.,1999). as substrate in an enzyme buffer consisting of 33mM 2-(N- Subconfluent monolayers of MDCK cells,grown in 96-well tis- Morpholino)ethanesulfonic acid(MES;Sigma,St.Louis,MO),4mM sue culture plates,were incubated at 37C for 1,2,4,and 24h CaCl2 and pH adjusted to 6.5.Fifty ul of the substrate with 50 wl with or without 100 ul ofTTO(0.01,0.005,and 0.0025%,v/v).Plates two-fold dilutions of virus stock were mixed and incubated for 1 h were gently shaken on a mechanical vibrator after the addition of at 37C with shaking in the dark.The reaction was stopped by the 10 ul per well of freshly prepared 10x concentrate solution of AO addition of 150 ul of a stop solution(0.014 M NaOH in 83%ethanol, (100 ug/ml)in RPMI medium and were then incubated for 30 min pH10.7). at 37C in the dark.The supernatant was carefully removed and FL-4-MU was immediately quantified in RFUs(relative fluores- discarded,the cells were washed four times with warm PBS and cence units)using a Wallac Victor 2 multilabel counter with an immediately examined.The fluorescence was quantified in RFUs excitation wavelength of 365 nm and an emission wavelength of using a 96-well plate-based spectrofluorometer (Wallac Victor2 450 nm.Wells were read at a rate of 1.0s per well. 1420-Multilabel Counter,Perkin-Elmer)at Aex 490nm and Aem Various volumes(5,10,20,and 40 ul)of a 40 uM solution of 530 nm.Wells were read at a rate of 1.0s per well. free FL-4-MU(Sigma,St.Louis,MO)in 0.9%(w/v)NaCl were used In all the experiments,cells incubated at 37C for 1h with in parallel to determine the linear range of detection. bafilomycin A1 at doses(100,10 and 1 nM)that did not affect MDCK The concentration of virus versus NA activity was plotted graph- cell viability after a 24h incubation period were used as positive ically to determine the virus/enzyme concentration to be used in subsequent inhibition assays. NA inhibition assay was used to determine the test compounds A110 concentration required to reduce NA activity by 50%(ICso)(Buxton 100 et al.,2000;Gubareva et al.,2002).Equal volumes(25 wl)of twofold dilutions of test compounds and virus(10,000 RFUs)were mixed 90 在一TT0 and preincubated at 37C for 60min to allow interaction of drug 80 ●一Terpinen-4-ol and virus. alfa-terpineol 50 ul of substrate FL-4-MU-NANA(75 uM)in enzyme buffer pH % Terpinolene 6.5 was added,incubated at 37C for 60min under shaking and stopped as previously described. % RFUs of FL-4-MU were immediately quantified,and the percent- age of fluorescence inhibition of each compound was calculated by 30 the following formula:RFUvirus-RFUtest/RFUvirus x 100 whereby 20 RFUvirus and RFUtest indicated the RFU of the virus-infected-control 10 (no compound)and the virus treated with the compound,respec- tively. 02 0.00390.00780.01560.031250.06250.1250.25 0.5 Experimental RFU values were corrected by subtracting the mean RFU value for the background fluorescence (which was a %(vw) combination of the plate,buffer fluorescence,and mock-infected B110 control). 3100 The data were plotted as compound concentrations against flu- 90 orescence inhibition,and the ICsos were read from the graph. 80 Ribavirin In all the experiments,oseltamivir and ribavirin were used as 70 positive and negative compounds,respectively. 2.9.Effect of TTO on endosomal/lysosomal pH measured by acridine orange accumulation 40 30 The effect ofTTO on acidification of cellular lysosomes was stud- ied by vital staining with acridine orange(AO)using microscopy and fluorometry. Vital fluorescence microscopy:The living cultured cells were 0 十十十 stained with Ao following the method previously described 0 0.01250.050.190.783.125125 50 (Geisow et al.,1981:Yoshimori et al.,1991).Briefly,MDCK cells Conc. grown to confluence on sterilized glass coverslips placed in 24- Fig.2.Effects of TTO and its active components (A).and effects of positive con- well plates,were incubated at 37C for 1,2,4 and 24h with or trol oseltamivir(nM)and negative control ribavirin (uM)(B)on the influenza virus without the TTO compound(0.01,0.005,and 0.0025%,v/v)in RPMI neuraminidase activity.Values <50%were not considered positive

A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 85 completely inhibited the haemoagglutination of the virus was determined. 2.8. Neuraminidase inhibition assay Influenza virus neuraminidase activity was measured by a mod￾ification of the method described by Potier et al. (1979). The assay measures the amount of 4-methylumbelliferone (FL-4-MU) that is cleaved by the influenza virus neuraminidase (NA) from the fluoro￾genic substrate 2 -O-(4-methylumbelliferyl)-N-acetylneuraminic acid (FL-4-MU-NANA; Sigma, St. Louis, MO). Influenza virus NA activity was determined before it was used in NA inhibition tests. Briefly, 75 M FL-4-MU-NANA were used as substrate in an enzyme buffer consisting of 33 mM 2-(N￾Morpholino)ethanesulfonic acid (MES; Sigma, St. Louis, MO), 4 mM CaCl2 and pH adjusted to 6.5. Fifty l of the substrate with 50 l two-fold dilutions of virus stock were mixed and incubated for 1 h at 37 ◦C with shaking in the dark. The reaction was stopped by the addition of 150 l of a stop solution (0.014 M NaOH in 83% ethanol, pH 10.7). FL-4-MU was immediately quantified in RFUs (relative fluores￾cence units) using a Wallac Victor 2 multilabel counter with an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Wells were read at a rate of 1.0 s per well. Various volumes (5, 10, 20, and 40 l) of a 40 M solution of free FL-4-MU (Sigma, St. Louis, MO) in 0.9% (w/v) NaCl were used in parallel to determine the linear range of detection. The concentration of virus versus NA activity was plotted graph￾ically to determine the virus/enzyme concentration to be used in subsequent inhibition assays. NA inhibition assay was used to determine the test compounds concentration required to reduce NA activity by 50% (IC50) (Buxton et al., 2000; Gubareva et al., 2002). Equal volumes (25 l) of twofold dilutions of test compounds and virus (10,000 RFUs) were mixed and preincubated at 37 ◦C for 60 min to allow interaction of drug and virus. 50 l of substrate FL-4-MU-NANA (75 M) in enzyme buffer pH 6.5 was added, incubated at 37 ◦C for 60 min under shaking and stopped as previously described. RFUs of FL-4-MU were immediately quantified, and the percent￾age of fluorescence inhibition of each compound was calculated by the following formula: RFUvirus − RFUtest/RFUvirus × 100 whereby RFUvirus and RFUtest indicated the RFU of the virus-infected-control (no compound) and the virus treated with the compound, respec￾tively. Experimental RFU values were corrected by subtracting the mean RFU value for the background fluorescence (which was a combination of the plate, buffer fluorescence, and mock-infected control). The data were plotted as compound concentrations against flu￾orescence inhibition, and the IC50s were read from the graph. In all the experiments, oseltamivir and ribavirin were used as positive and negative compounds, respectively. 2.9. Effect of TTO on endosomal/lysosomal pH measured by acridine orange accumulation The effect of TTO on acidification of cellular lysosomes was stud￾ied by vital staining with acridine orange (AO) using microscopy and fluorometry. Vital fluorescence microscopy: The living cultured cells were stained with AO following the method previously described (Geisow et al., 1981; Yoshimori et al., 1991). Briefly, MDCK cells, grown to confluence on sterilized glass coverslips placed in 24- well plates, were incubated at 37 ◦C for 1, 2, 4 and 24 h with or without the TTO compound (0.01, 0.005, and 0.0025%, v/v) in RPMI medium without the addition of fetal calf serum. At the end of the incubation period the cells were treated with AO (diluted in ster￾ile glass-distilled water to a concentration of 25 g/ml) at 37 ◦C for 30 min under 5% CO2, then washed four times with warm PBS and dried. The coverslips were mounted with cells facing down onto a slide containing a drop of PBS and immediately examined using a Leica DM LB fluorescence light microscope. All steps were carried out in the dark. Photographs of the microscopic observations were taken with a Leica DC180 digital camera system. Fluorometry: The measurement of fluorescence intensity was used to identify the accumulation of protonated acridine orange in the acid compartment by fluorescence emission (Natale and McCullough, 1998; Zoccarato et al., 1999). Subconfluent monolayers of MDCK cells, grown in 96-well tis￾sue culture plates, were incubated at 37 ◦C for 1, 2, 4, and 24 h with or without 100 l of TTO (0.01, 0.005, and 0.0025%, v/v). Plates were gently shaken on a mechanical vibrator after the addition of 10 l per well of freshly prepared 10× concentrate solution of AO (100 g/ml) in RPMI medium and were then incubated for 30 min at 37 ◦C in the dark. The supernatant was carefully removed and discarded, the cells were washed four times with warm PBS and immediately examined. The fluorescence was quantified in RFUs using a 96-well plate-based spectrofluorometer (Wallac Victor2 1420-Multilabel Counter, Perkin-Elmer) at ex 490 nm and em 530 nm. Wells were read at a rate of 1.0 s per well. In all the experiments, cells incubated at 37 ◦C for 1 h with bafilomycin A1 at doses (100, 10 and 1 nM) that did not affect MDCK cell viability after a 24 h incubation period were used as positive 0 10 20 30 40 50 60 70 80 90 100 110 0 0.0039 0.0078 0.0156 0.03125 0.0625 0.125 0.25 0.5 % (v/v) Neuraminidase inhibition (%) TTO Terpinen-4-ol alfa-terpineol Terpinolene 0 10 20 30 40 50 60 70 80 90 100 110 0 0.0125 0.05 0.19 0.78 3.125 12.5 50 Conc. Neuraminidase inhibition (%) Oseltamivir Ribavirin B A Fig. 2. Effects of TTO and its active components (A), and effects of positive con￾trol oseltamivir (nM) and negative control ribavirin (M) (B) on the influenza virus neuraminidase activity. Values <50% were not considered positive.

86 A Garozzo et aL Antiviral Research 89(2011)83-88 Fig.3.Acridine orange staining of MDCK cells:untreated(DMSO)(A).treated with 100 and 10nM bafilomycinA1 for 1h(B and C):with .01TTO for 4h(D)and for 1h(E): with 0.01%terpinen-4-ol for 4h(F):treated with 0.01 TTO for 4 h followed by washing and incubation without TTO (G). controls.For control experiments,only DMSO was added to the cence was indicative of a diminished concentration of ao in the medium instead of the compounds. cells. The compounds terpinen-4-ol,terpinolene,and o-terpineol were used at the concentration of 0.01%(v/v),0.005%(v/v),and 2.10.Reversibility of drug effect 0.02%(v/v),respectively. The fluorescence signal depends on the AO accumulation in To study the possible reversibility of the TTO effect,MDCK cells acidified cellular compartments,therefore an increased fluores- were incubated at 37C for 4h with TTO,and terpinen-4-ol (0.01

86 A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 Fig. 3. Acridine orange staining of MDCK cells: untreated (DMSO) (A), treated with 100 and 10 nM bafilomycin A1 for 1 h (B and C); with 0.01% TTO for 4 h (D) and for 1 h (E); with 0.01% terpinen-4-ol for 4 h (F); treated with 0.01% TTO for 4 h followed by washing and incubation without TTO (G). controls. For control experiments, only DMSO was added to the medium instead of the compounds. The compounds terpinen-4-ol, terpinolene, and -terpineol were used at the concentration of 0.01% (v/v), 0.005% (v/v), and 0.02% (v/v), respectively. The fluorescence signal depends on the AO accumulation in acidified cellular compartments, therefore an increased fluores￾cence was indicative of a diminished concentration of AO in the cells. 2.10. Reversibility of drug effect To study the possible reversibility of the TTO effect, MDCK cells were incubated at 37 ◦C for 4 h with TTO, and terpinen-4-ol (0.01,

A.Garozzo et al./Antiviral Research 89 (2011)83-88 87 0.005 and 0.0025%,v/v),with bafilomycin A1 (100,10 and 1 nM) 400000 for 1 h or without compounds.After three washings with RPMI 350000 OTTO cells were incubated with fresh medium at 37C for 2 h and were stained with AO(10 ug/ml)following the method described above 300000 ☐hfm3nA and immediately examined and quantified in RFUs. 250000 200000 3.Results and discussion 150000 Recent studies performed in our laboratory revealed that TTO 100000 terpinen-4-ol,terpinolene,and o-terpineol had an inhibitory effect on Influenza A/PR8 virus replication.In fact,TTO had no adverse effect on MDCK cell cultures up to a concentration of 0.025%(v/v). not-treated Ist dose 2nd dose 3rd dose which is much higher than the 50%inhibitory concentration of influenza virus(ECso:0.0006%,v/v).Three of the TTO components Fig.4.Fluorescence signal (RFUs:relative fluorescence units)of MDCK cells incu- tested were effective.In particular,the ECso values were found to be bated without compounds.with various concentrations of TTO.and terpinen-4-ol for 4h at 37C.or bafilomycin A1 for 1 h at 37C.before treatment with acri- 0.002%(v/v),0.025%(v/v)and 0.00125%(v/v)for terpinen-4-ol,a- dine orange.TTO and terpinen-4-ol:1st dose=0.0025%(v/v):2nd dose=0.005% terpineol,and terpinolene,respectively.Compounds o-terpinene, (v/v):3rd dose=0.01%(v/v).Bafilomycin:1st dose=1 nM:2nd dose=10nM:3rd p-cymene and y-terpinene were completely ineffective (Garozzo dose=100nM.Each value represents the mean+S.E.M.of three separate assays. etal,2009). The asterisk indicates a significant difference between test samples,P<0.05. In the present study,we investigated the antiviral mechanism of action of TTO and its active components against Influenza A/PR/8 positive control oseltamivir (Fig.2B).Only a slight effect (~35%) virus subtype H1N1. was observed at a concentration of 0.5%(v/v)TTO,that was not In all the experiments,influenza virus replication was evalu- ated in MDCK cells 48-72h after infection by HAU/ml and CPEso considered inhibitory(<50%). Ribavirin was also evaluated in parallel,it is an influenza assays.The results of the two methods were consistently concor- virus inhibitor with different modes of action unrelated to inhi- dant,indicating that all observed changes pertained to both vira bition of viral neuraminidase(Eriksson et al.,1977:Wray et al., HA production and infectivity. 1985) In order to determine whether TTO and its components inhib- It has been demonstrated that the acidic condition in endosomes ited viral replication at a specific step in the virus cycle,the effect and lysosomes is essential for the uncoating process of influenza of compound addition at different time intervals using MDCK cells virus infection since it causes the viral envelope fusion activity was studied.The results demonstrated that TTO,terpinen-4-ol,ter- (Guinea and Carrasco,1995).Thus,we set up some experiments pinolene,and o-terpineol interfere with an early step of the viral in order to verify if TTO and its components exerted an inhibitory replicative cycle of influenza virus.In fact,the test compounds were effect on the acidification of intracellular compartments such as effective when added within 1 h after the end of the adsorption endosomes and lysosome inhibiting the influenza virus growth in period while no reduction was observed if they were added more MDCK cells. than 2 h after virus adsorption.Only a slight reduction of virus The effect of the compounds on acidification of lysosomes was yield was observed if the test compounds were added during the tested by vital staining with acridine orange(AO).This dye is a adsorption period and at 120 min(Fig.1). weak base,which is taken up by living cells and protonated in endo- Since the period of the most sensitivity of the compounds was somes/lysosomes,resulting in an accumulation of the AO therein. an early event in influenza virus replication,it was important to As a consequence,when untreated MDCK cells were stained establish whether our compounds produced a virucidal effect or with AO,nuclei,nucleoli and the cytoplasm,showed green fluo- a protective action for the MDCK cells.The results demonstrated rescence,whereas the granular pattern in the cytoplasm showed that TTO and its active components were not virucidal for influenza orange fluorescence,due to acidified lysosomes(Fig.3A). virus and did not exert any protective action for the cells(data not shown). The effect of TTO and its active components under different 400000 experimental conditions was studied in order to understand the mechanism of inhibition. 兰 350000 口TT0 The influence of the test compounds on the virus adsorption 30000 step,studied by infective center assays and by haemoagglutination inhibition assay,demonstrated that TTO.o-terpineol,terpinen-4- 250000 ol and terpinolene did not interfere with cellular attachment of the 200000 virus or with the viral adsorption to RBC resulting in haemagglu- tination(data not shown).Therefore,the slight reduction of virus 15000 yield observed during the adsorption period in the time of addition 100000 experiments was,probably,due to residual compounds that could interfere with the following steps. 50000 The ability of TTO and its active components to inhibit the enzy- matic activity of viral neuraminidase was tested and compared to not-treated Ist dose 2nd dose 3rd dose that of the known neuraminidase inhibitor oseltamivir(ICso value: Fig.5.Reversibility of drug effect:MDCK cells incubated without compounds,with 1.34 nM,in our experimental condition)(Hayden et al.,1999). various concentrations of TTO,and terpinen-4-ol for 4h at 37C or bafilomycin A1 As shown in Fig.2A TTO and its active components did not have for 1h at 37C,washed and incubated without compounds for 2h at 37C.before any inhibitory effect on the influenza virus neuraminidase activity treatment with acridine orange.TTO and terpinen-4-ol:1st dose=0.0025%(v/v): 2nd dose=0.005%(v/v):3rd dose=0.01(v/v).Bafilomycin:1st dose=1 nM:2nd In fact,the results indicated that there was a substantial differ- dose=10 nM:3rd dose=100 nM.Each value represents the mean+S.E.M.of three ence in the percentage of enzymatic inhibition compared with the separate assays

A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 87 0.005 and 0.0025%, v/v), with bafilomycin A1 (100, 10 and 1 nM) for 1 h or without compounds. After three washings with RPMI, cells were incubated with fresh medium at 37 ◦C for 2 h and were stained with AO (10 g/ml) following the method described above and immediately examined and quantified in RFUs. 3. Results and discussion Recent studies performed in our laboratory revealed that TTO, terpinen-4-ol, terpinolene, and -terpineol had an inhibitory effect on Influenza A/PR8 virus replication. In fact, TTO had no adverse effect on MDCK cell cultures up to a concentration of 0.025% (v/v), which is much higher than the 50% inhibitory concentration of influenza virus (EC50: 0.0006%, v/v). Three of the TTO components tested were effective. In particular, the EC50 values were found to be 0.002% (v/v), 0.025% (v/v) and 0.00125% (v/v) for terpinen-4-ol, - terpineol, and terpinolene, respectively. Compounds -terpinene, p-cymene and -terpinene were completely ineffective (Garozzo et al., 2009). In the present study, we investigated the antiviral mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1. In all the experiments, influenza virus replication was evalu￾ated in MDCK cells 48–72 h after infection by HAU/ml and CPE50 assays. The results of the two methods were consistently concor￾dant, indicating that all observed changes pertained to both viral HA production and infectivity. In order to determine whether TTO and its components inhib￾ited viral replication at a specific step in the virus cycle, the effect of compound addition at different time intervals using MDCK cells was studied. The results demonstrated that TTO, terpinen-4-ol, ter￾pinolene, and -terpineol interfere with an early step of the viral replicative cycle of influenza virus. In fact, the test compounds were effective when added within 1 h after the end of the adsorption period while no reduction was observed if they were added more than 2 h after virus adsorption. Only a slight reduction of virus yield was observed if the test compounds were added during the adsorption period and at 120 min (Fig. 1). Since the period of the most sensitivity of the compounds was an early event in influenza virus replication, it was important to establish whether our compounds produced a virucidal effect or a protective action for the MDCK cells. The results demonstrated that TTO and its active components were not virucidal for influenza virus and did not exert any protective action for the cells (data not shown). The effect of TTO and its active components under different experimental conditions was studied in order to understand the mechanism of inhibition. The influence of the test compounds on the virus adsorption step, studied by infective center assays and by haemoagglutination inhibition assay, demonstrated that TTO, -terpineol, terpinen-4- ol and terpinolene did not interfere with cellular attachment of the virus or with the viral adsorption to RBC resulting in haemagglu￾tination (data not shown). Therefore, the slight reduction of virus yield observed during the adsorption period in the time of addition experiments was, probably, due to residual compounds that could interfere with the following steps. The ability of TTO and its active components to inhibit the enzy￾matic activity of viral neuraminidase was tested and compared to that of the known neuraminidase inhibitor oseltamivir (IC50 value: 1.34 nM, in our experimental condition) (Hayden et al., 1999). As shown in Fig. 2A TTO and its active components did not have any inhibitory effect on the influenza virus neuraminidase activity. In fact, the results indicated that there was a substantial differ￾ence in the percentage of enzymatic inhibition compared with the Fig. 4. Fluorescence signal (RFUs: relative fluorescence units) of MDCK cells incu￾bated without compounds, with various concentrations of TTO, and terpinen-4-ol for 4 h at 37 ◦C, or bafilomycin A1 for 1 h at 37 ◦C, before treatment with acri￾dine orange. TTO and terpinen-4-ol: 1st dose = 0.0025% (v/v); 2nd dose = 0.005% (v/v); 3rd dose = 0.01% (v/v). Bafilomycin: 1st dose = 1 nM; 2nd dose = 10 nM; 3rd dose = 100 nM. Each value represents the mean ± S.E.M. of three separate assays. The asterisk indicates a significant difference between test samples, *P < 0.05. positive control oseltamivir (Fig. 2B). Only a slight effect (∼35%) was observed at a concentration of 0.5% (v/v) TTO, that was not considered inhibitory (<50%). Ribavirin was also evaluated in parallel, it is an influenza virus inhibitor with different modes of action unrelated to inhi￾bition of viral neuraminidase (Eriksson et al., 1977; Wray et al., 1985). It has been demonstrated that the acidic condition in endosomes and lysosomes is essential for the uncoating process of influenza virus infection since it causes the viral envelope fusion activity (Guinea and Carrasco, 1995). Thus, we set up some experiments in order to verify if TTO and its components exerted an inhibitory effect on the acidification of intracellular compartments such as endosomes and lysosome inhibiting the influenza virus growth in MDCK cells. The effect of the compounds on acidification of lysosomes was tested by vital staining with acridine orange (AO). This dye is a weak base, which is taken up by living cells and protonated in endo￾somes/lysosomes, resulting in an accumulation of the AO therein. As a consequence, when untreated MDCK cells were stained with AO, nuclei, nucleoli and the cytoplasm, showed green fluo￾rescence, whereas the granular pattern in the cytoplasm showed orange fluorescence, due to acidified lysosomes (Fig. 3A). Fig. 5. Reversibility of drug effect: MDCK cells incubated without compounds, with various concentrations of TTO, and terpinen-4-ol for 4 h at 37 ◦C or bafilomycin A1 for 1 h at 37 ◦C, washed and incubated without compounds for 2 h at 37 ◦C, before treatment with acridine orange. TTO and terpinen-4-ol: 1st dose = 0.0025% (v/v); 2nd dose = 0.005% (v/v); 3rd dose = 0.01% (v/v). Bafilomycin: 1st dose = 1 nM; 2nd dose = 10 nM; 3rd dose = 100 nM. Each value represents the mean ± S.E.M. of three separate assays.

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. 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