Food Environ Virol DOI 10.1007/s12560-015-9181-9
ORIGINAL PAPER
Evaluation of Natural Compounds of Plant Origin for Inactivation of Enteric Viruses G. Sa´nchez • R. Aznar
Received: 15 December 2014 / Accepted: 23 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Essential oils (EOs) and some of their main compounds have demonstrated extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess the antiviral activity of Zataria multiflora Boiss. (zataria) and Origanum vulgare (oregano) EOs on hepatitis A virus (HAV) and the effect of thymol, an active compound of Thymus vulgaris and oregano, on norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), and HAV. Initially, each virus at titers of ca. 6 log TCID50/ml was exposed to different concentrations of natural compounds and incubated for 2 h at 37 °C. Treatment with oregano and zataria EOs resulted in slight reductions on HAV infectivity with a maximum reduction of less than 0.5 log TCID50/ml at 0.1 % zataria EO. Thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner. Concentrations of thymol at 0.5 and 1 % reduced FCV titers to undetectable levels, while for MNV, thymol at concentrations of 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. However, for HAV, no effect was observed at any of the concentrations tested. These results improve the knowledge about the antiviral activity of EO and their compounds and their potential in food sanitation.
G. Sa´nchez R. Aznar Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain G. Sa´nchez (&) R. Aznar Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustı´n Escardino, 7, 46980 Paterna, Valencia, Spain e-mail: [email protected]
Keywords Natural compounds Norovirus Hepatitis A virus Essential oils Thymol
Introduction Enteric viruses, in particular human norovirus (NoV), are the leading causes of foodborne illnesses in industrialized countries (Anonymous 2013; EFSA 2014), whereas hepatitis A virus (HAV) has recently been considered as a reemerging foodborne public health threat in Europe (Sprenger 2014) due to the number of foodborne outbreaks associated to imported foods (Collier et al. 2014; GuzmanHerrado et al. 2014; Wenzel et al. 2014). The transmission of enteric viruses associated with the consumption of contaminated produce is regularly reported after contamination by food handlers or by the use of polluted irrigation water. Since products considered healthy food i.e., fresh cut vegetables are prone to be contaminated, it is important to find natural antiviral additives which are safe, environment friendly and preferably inexpensive for use in the food industry. The growing demand for the use of natural additives in food applications has generated a substantial increase in the number of studies based on natural extracts such as essential oils (EOs) or their main compounds. They are categorized as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration, and are therefore potential alternatives to chemical additives. EOs extracted from plants are rich sources of biologically active compounds, such as phenolic acids and terpenoids, and it has been long recognized that some of them show antimicrobial properties. Furthermore, the antibacterial, antifungal, insecticidal, antiparasitic and antitoxigenic activities of these compounds have extensively been reported (Burt
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Food Environ Virol
2004). In contrast, reports on the antiviral effects of EOs or their compounds are somewhat limited and are mainly based on NoV surrogates (Table 1). In a previous study, our group reported the antiviral activity of clove, oregano and zataria EOs against norovirus surrogates (Elizaquı´vel et al. 2013). Therefore, in this study, the effect of zataria (Zataria multiflora Boiss.) and oregano (Origanum vulgare) EOs on the infectivity of HAV has been assessed. We also studied the efficacy of thymol, one of the main components of oregano and thyme EO, on the
infectivity of HAV and two norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV).
Materials and Methods Viruses, Cells and Infections The cytopathogenic HM-175 strain of HAV (ATCC VR1402), the MNV-1 strain (kindly provided by Prof. H. W.
Table 1 Inactivation rates of essential oils and their main compounds on different enteric viruses or norovirus surrogates Target virus
Inactivation rates (log reduction)
Experimental setup (temperature, time)
References
Allspice (4 %)
MNV
3.41
24 °C, 24 h
Clove (1 %)
FCV, MNV
3.75; 0.67
37 °C, 2 h
Gilling et al. (2014b) Elizaquı´vel et al. (2013)
Hyssop (0.2 %)
AdV-2, MNV
No effect
Essential oils (EO)
4, 25 and 37 °C
Kovac et al. (2012)
2 and 24 h Lemongrass (4 %)
MNV
2.74
24 °C, 24 h
Gilling et al. 2014b
Marjoram (0.2 %)
AdV-2, MNV
No effect
4, 25 and 37 °C
Kovac et al. (2012)
Mexican oregano (Lippia graveolens)
RV
No effect
Mint (2 %)
MNV
0.87
37 °C, 1 h
Oregano (2 %) (Origanum compactum)
FCV, MNV
3.75; 1.62
37 °C, 2 h
Oregano (4 %)
MNV
1.10
24 °C, 6 h
Gilling et al. (2014a)
MNV
0.75
37 °C, 1 h
El Moussaoui et al. (2013)
Tea tree (0.025 %)
PV, Echo 9, Coxsackie B1, AdV-2
No effect
37 °C, 1 h
Garozzo et al. (2009)
Thyme (2 %)
MNV
0.5
37 °C, 1 h
Zataria (0.1 %)
FCV, MNV
4.51; 0.25
37 °C, 2 h
El Moussaoui et al. (2013) Elizaquı´vel et al. (2013)
Carvacrol (0.5 %)
MNV
[4.15
24 °C, 3 h
Carvacrol (0.5 %)
MNV, FCV
[3.57, [4.53
37 °C, 2 h
Carvacrol (1 %)
HAV
0.97
37 °C, 2 h
Carvacrol
RV
Effective
Citral (4 %)
MNV
3.00
2 and 24 h
Oregano (2 %) (Origanum elongatum)
Pilau et al. (2011) El Moussaoui et al. (2013) Elizaquı´vel et al. (2013)
Main compounds of EO Gilling et al. (2014a) Sa´nchez et al. (2015) Sa´nchez et al. (2015) Pilau et al. (2011) 24 °C, 24 h
Gilling et al. (2014b)
MNV murine norovirus, FCV feline calicivirus, AdV human adenovirus, RV human rotavirus, PV poliovirus, Echo echovirus, Coxsackie virus, HAV hepatitis A virus
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Food Environ Virol
Virgin, Washington University School of Medicine, USA) and the F9 strain of FCV (ATCC VR-782) were propagated and assayed in FRhK-4 (courtesy of Prof. Albert Bosch, University of Barcelona), RAW 264.7 (kindly provided by Prof. H. W. Virgin) and CRFK cell monolayers (ATCC CCL-94), respectively. Virus stocks were obtained from the same cells by centrifugation of infected cell lysates at 6609g for 30 min at 2–3 days postinfection for MNV and FCV and 10–12 for HAV. Infectious viruses were quantified by determining the 50 % tissue culture infectious dose (TCID50) with eight wells per dilution and 20 ll of inoculum per well (Sa´nchez et al. 2011; Pinto´ et al. 1994). Natural Compounds Origanum vulgare (oregano) essential oil provided by Pronaroˆm International (Ghislenghien, Belgium) and an inhouse produced zataria essential oil (from Z. multiflora Boiss., collected in the Shiraz Province of Iran and kindly provided by Maryam Azizkhani) were used in this study. Main compounds of oregano EO are carvacrol (46.88 %), thymol (15.26 %), p-cymene (13.10 %) and c-terpinene (11.61 %) (www.pranarom.com) and in zataria, carvacrol (71.12 %), c-terpinene (7.34 %), a-pinene (4.26 %), eucalyptol (3.37 %) and globulol (2.32) (Elizaquı´vel et al. 2013). Thymol (C99 % purity) was provided by Sigma Aldrich. Oregano and zataria EOs were diluted in 70 and 50 % ethanol, respectively, whereas thymol was diluted in 25 % ethanol and kept at 4 °C before use. Cytotoxicity Determination of Natural Compounds on Cell Monolayers Natural compounds at various concentrations were added to individual wells of confluent RAW 264.7, CRFK and FRhK-4 cell monolayers grown in 96-well microtiter plates (SPL Life Sciences) and incubated 2 h under 5 % CO2. Subsequently, 150 ll of DMEM supplemented with 2 % of fecal calf serum (FCS) was added per well. After incubation at 37 °C in a CO2 incubator for 2–15 days, the cell monolayers were observed for cytotoxicity effects by visual inspection under the optical microscope only. Antiviral Effects of Natural Compounds Natural compounds at different concentrations were added to virus suspensions (ca. 6 log TCID50/ml) in DMEM with 2 % FCS and further incubated at 37 °C in a water-bath shaker at 150 rpm for 2 h. Tenfold dilutions of treated and untreated virus suspensions were inoculated into confluent monolayers in 96-well plates. Then, infectious viruses were
quantified by cell culture assays as described above. Each treatment was done in triplicate. Positive controls were virus suspensions added with ethanol in amounts corresponding to the highest quantity present. The decay of viruses was calculated as log10 (Nt/N0), where N0 is the infectious virus titer for untreated sample and Nt is the infectious virus titer for treated sample. Statistical Analysis The significance of differences among the mean numbers of viruses determined after the various treatments was estimated by the Student’s t test with a significance level of P \ 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA).
Results Determination of Cytotoxicity of EOs and Thymol on Cell Monolayers Oregano and zataria EOs resulted cytotoxic for FRhk-4 cells at concentrations that exceeded 2 and 0.1 %, respectively. Thymol was found to be cytotoxic at concentrations that exceeded 2 %, for the three cell lines. Therefore, 2, 0.1 and 2 % were the maximum concentration of oregano EO, zataria EO and thymol tested to evaluate their effect on HAV, MNV and FCV. Antiviral Efficacy of Oregano and Zataria EOs on HAV Incubation of HAV with oregano EO at concentrations of 0.5, 1 and 2 % for 2 h at 37 °C did not decreased the HAV titers (Table 2). Zataria EO at 0.1 % reduced HAV titers by 0.42 TCID50/ml (P \ 0.05). At lower zataria EO concentrations (i.e., 0.05 and 0.01 %), no differences in titer reduction were observed between HAV suspensions treated or non-treated with the tested concentrations of zataria EO (Table 2). Effect of Thymol on the Infectivity of HAV, MNV and FCV Incubation of FCV and MNV with thymol at concentrations from 0.5 to 2 % for 2 h at 37 °C decreased the titer of the two norovirus surrogates (Table 3). Thymol at 0.5 and 1 % reduced FCV titers to undetectable levels. On the other hand, MNV titers, the hardier NoV surrogate, were reduced by 0.50 log TCID50/ml after treatment with thymol at 0.5 %, while thymol at 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. For HAV, no differences in titer reduction were observed between HAV
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Food Environ Virol Table 2 Effect of oregano (O) and zataria (Z) essential oils against Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
HAV (log10 TCID50/ml) Treatment
Recovered titer
Reduction
Treatment
Recovered titer
Reduction
Control
5.82 ± 0.17A
Control
6.20 ± 0.21A
0.5 % O
5.45 ± 0.35A
0.37
0.01 % Z
6.11 ± 0.07A
0.09
1%O
5.70 ± 0.12A
0.12
0.05 % Z
6.24 ± 0.19A
0.00
2%O
5.70 ± 0.33A
0.12
0.1 % Z
5.78 ± 0.19B
0.42
Table 3 Effect of thymol against feline calicivirus (FCV), murine norovirus (MNV-1) and Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
Treatment Control
FCV
MNV
HAV
Log10 TCID50/ml
Log10 TCID50/ml
Log10 TCID50/ml
Recovered titer 5.88 ± 0.00A
Reduction a
Recovered titer
Reduction
6.07 ± 0.01A
Recovered titer
Reduction
6.41 ± 0.19A
0.1 % T
5.86 ± 0.26A
0.02
5.99 ± 0.38A
0.08
6.41 ± 0.14A
0.00
0.5 % T
\2.13 ± 0.00B
[3.57
5.57 ± 0.45A
0.50
5.29 ± 0.07A
0.12
1%T
\2.13 ± 0.00B
[3.57
2%T
NT
4.41 ± 1.16B
1.66
6.20 ± 0.12A
0.21
3.62 ± 0.72B
2.45
6.39 ± 0.17A
0.01
NT non tested a
Each treatment was done in triplicate. Within each column for each virus, different letters denote significant differences between treatments (P \ 0.05)
suspensions treated or non-treated with thymol at 0.1, 0.5, 1 or 2 % (Table 3).
Discussion The food industry is currently exploring new ways to use effective natural antimicrobials to improve food safety, and plant EOs or their compounds are potential alternative natural ingredients (reviewed by Gyawali and Ibrahim 2014). Regarding enteric viruses, oregano and zataria EO revealed efficient to reduce infectivity of MNV and FCV (Elizaquı´vel et al. 2013). The initial aim of the present study was to evaluate them on HAV, since it has recently been considered as a re-emerging foodborne public health threat in Europe (Sprenger 2014). Oregano EO was ineffective on HAV at the maximum concentration tested, unlike our previous results where oregano EO reduced the infectivity of MNV and FCV by 1.62 and 3.75 log, respectively (Elizaquı´vel et al. 2013). For zataria EO, although statistically significant, HAV infectivity was only reduced by 0.42 log TCID50/ml, while in our previous study, 0.1 % zataria EO reduced FCV and MNV infectivity by 4.51 and 0.25 TCID50/ml, respectively. As an alternative, one of the active compounds, thymol, was tested. Thymol is present in oregano EO, and its isomer, carvacrol, is the main active component of
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zataria. It is a phenolic monoterpene that has been identified as a natural economical food preservative (Lu and Wu 2010) with potential for incorporation in food packaging (Ramos et al. 2012). Following, antiviral activity of thymol was explored on NoV surrogates and HAV. This study clearly shows that thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner, where increasing concentrations of thymol resulted in increased reduction in viral titers. Furthermore, HAV was resistant to thymol treatment at any concentration tested. Similar trends have recently been reported for carvacrol treatment; however, carvacrol was more effective than its isomer thymol against NoV surrogates and HAV. Sa´nchez et al. (2015) reported that carvacrol at 0.5 % completely inactivated FCV and MNV, whereas 1 % concentration was required to achieve ca. 1 log reduction of HAV. Moreover, Sa´nchez et al. (2015) reported the potential of carvacrol to be used as a natural disinfectant of produce. Overall, this study showed that the evaluated natural compounds resulted in slight reductions of HAV infectivity, while they were effective in NoV surrogates with higher reduction in FCV than MNV titers. These results resemble those reported for carvacrol (Sa´nchez et al. 2015). However, Su and D’Souza (2011), when testing the antiviral activity of grape seed extract (GSE), reported that MNV was more resistant than HAV.
Food Environ Virol
Taking together these results and previous studies (Elizaquı´vel et al. 2013; Sa´nchez et al. 2015; Gilling et al. 2014a, b; Pilau et al. 2011), the use of plant extracts as antivirals in food safety should focus on the purified active compounds as they exhibited stronger activity than EO. Nevertheless, complete inactivation of foodborne viral contamination with plant extracts either EOs or active compounds cannot be foreseen since they slightly affect HAV infectivity. The application of plant antimicrobials in food sanitation should combine them with other natural compounds, e.g., GSE, to achieve a synergistic effect, and be used as part of hurdle approaches to improve food safety. Acknowledgments G. Sa´nchez was supported by the ‘‘Ramo´n y Cajal’’ Young Investigator program of the Spanish Ministry of Economy and Competitiveness. This study was supported by grant AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. We thank Carlos Sa´nchez for technical support.
References Anonymous. (2013). Surveillance for foodborne disease outbreaks— United States, 2009–2010. Morbidity and Mortality Weekly Report, 62, 41–47. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology, 94, 223–253. Collier, M. G., Khudyakov, Y. E., Selvage, D., Adams-Cameron, M., Epson, E., Cronquist, A., et al. (2014). Outbreak of hepatitis A in the USA associated with frozen pomegranate arils imported from Turkey: An epidemiological case study. The Lancet Infectious Diseases, 14, 976–981. EFSA (2014). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA Journal, 12(2), 3547, 312. El Moussaoui, N., Sanchez, G., El Ouardy, K., Idaomar, M., Mansour, A. I., Abrini, J., & Aznar, R. (2013). Antibacterial and antiviral activities of essential oils of northern moroccan plants. British Biotechnology Journal, 3(3), 318–331. Elizaquı´vel, P., Azizkhani, M., Aznar, R., & Sanchez, G. (2013). The effect of essential oils on norovirus surrogates. Food Control, 32, 275–278. Garozzo, A., Timpanaro, R., Bisignano, B., Furneri, P. M., Bisignano, G., & Castro, A. (2009). In vitro antiviral activity of Melaleuca alternifolia essential oil. Letters in Applied Microbiology, 49, 806–808. Gilling, D. H., Kitajima, M., Torrey, J. R., & Bright, K. R. (2014a). Antiviral efficacy and mechanisms of action of oregano essential
oil and its primary component carvacrol against murine norovirus. Journal of Applied Microbiology, 116, 1149–1163. Gilling, D. H., Kitajima, M., Torrey, J. R., & Bright, K. R. (2014b). Mechanisms of antiviral action of plant antimicrobials against murine norovirus. Applied and Environmental Microbiology, 80, 4898–4910. Guzman-Herrado, B., Jensvoll, L., Eino¨der-Moreno, M., Lange, H., Myking, S., Nyga˚rd, K., Stene-Johansen, K., & Vold, L. (2014). Ongoing hepatitis A outbreak in Europe 2013 to 2014: Imported berry mix cake suspected to be the source of infection in Norway. Eurosurveillance, 19(15): pii = 20775. http://www. eurosurveillance.org/ViewArticle.aspx?ArticleId=20775. Gyawali, R., & Ibrahim, S. A. (2014). Natural products as antimicrobial agents. Food Control, 46, 412–429. Kovac, K., Diez-Valcarce, M., Raspor, P., Herna´ndez, M., & Rodrı´guez-La´zaro, D. (2012). Natural plant essential oils do not inactivate non-enveloped enteric viruses. Food and Environmental Virology, 4, 209–212. Lu, Y., & Wu, C. (2010). Reduction of Salmonella enterica contamination on grape tomatoes by washing with thyme oil, thymol, and carvacrol as compared with chlorine treatment. Journal of Food Protection, 73(12), 2270–2275. Pilau, M. R., Alves, S. H., Weiblen, R., Arenhart, S., Cueto, A. P., & Lovato, L. T. (2011). Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Brazilian Journal of Microbiology, 42(4), 1616–1624. Pinto´, R. M., Diez, J. M., & Bosch, A. (1994). Use of the colonic carcinoma cell line CaCo-2 for in vivo amplification and detection of enteric viruses. Journal of Medical Virology, 44, 310–315. Ramos, M., Jime´nez, A., Peltzer, M., & Garrigo´s, M. C. (2012). Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging. Journal of Food Engineering, 109, 513–519. Sa´nchez, G., Aznar, R., Martı´nez, A., & Rodrigo, D. (2011). Inactivation of human and murine norovirus by high-pressure processing. Foodborne Pathogens and Disease, 8, 249–253. Sa´nchez, C., Aznar, R., & Sa´nchez, G. (2015). The effect of carvacrol on enteric viruses. International Journal of Food Microbiology, 192, 72–76. Sprenger, M. (2014). More can be done to stop ‘silent disease’ of hepatitis. The Parliament Magazine394/395: 9. http://viewer. zmags.com/publication/39b84223#/39b84223/9 (accessed Oct 27, 2014). Su, X., & D’Souza, D. H. (2011). Grape seed extract for control of human enteric viruses. Applied and Environmental Microbiology, 77, 3982–3987. Wenzel, J. J., Schemmerer, M., Oberkofler, H., Koidl, C., & Allerberger, F. (2014). Hepatitis A Outbreak in Europe: Imported Frozen Berry Mix Suspected to be the Source of At least One Infection in Austria in 2013. Food Environmental Virology, 6(4), 297–300.
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ORIGINAL PAPER
Evaluation of Natural Compounds of Plant Origin for Inactivation of Enteric Viruses G. Sa´nchez • R. Aznar
Received: 15 December 2014 / Accepted: 23 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Essential oils (EOs) and some of their main compounds have demonstrated extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess the antiviral activity of Zataria multiflora Boiss. (zataria) and Origanum vulgare (oregano) EOs on hepatitis A virus (HAV) and the effect of thymol, an active compound of Thymus vulgaris and oregano, on norovirus surrogates, feline calicivirus (FCV) and murine norovirus (MNV), and HAV. Initially, each virus at titers of ca. 6 log TCID50/ml was exposed to different concentrations of natural compounds and incubated for 2 h at 37 °C. Treatment with oregano and zataria EOs resulted in slight reductions on HAV infectivity with a maximum reduction of less than 0.5 log TCID50/ml at 0.1 % zataria EO. Thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner. Concentrations of thymol at 0.5 and 1 % reduced FCV titers to undetectable levels, while for MNV, thymol at concentrations of 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. However, for HAV, no effect was observed at any of the concentrations tested. These results improve the knowledge about the antiviral activity of EO and their compounds and their potential in food sanitation.
G. Sa´nchez R. Aznar Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain G. Sa´nchez (&) R. Aznar Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustı´n Escardino, 7, 46980 Paterna, Valencia, Spain e-mail: [email protected]
Keywords Natural compounds Norovirus Hepatitis A virus Essential oils Thymol
Introduction Enteric viruses, in particular human norovirus (NoV), are the leading causes of foodborne illnesses in industrialized countries (Anonymous 2013; EFSA 2014), whereas hepatitis A virus (HAV) has recently been considered as a reemerging foodborne public health threat in Europe (Sprenger 2014) due to the number of foodborne outbreaks associated to imported foods (Collier et al. 2014; GuzmanHerrado et al. 2014; Wenzel et al. 2014). The transmission of enteric viruses associated with the consumption of contaminated produce is regularly reported after contamination by food handlers or by the use of polluted irrigation water. Since products considered healthy food i.e., fresh cut vegetables are prone to be contaminated, it is important to find natural antiviral additives which are safe, environment friendly and preferably inexpensive for use in the food industry. The growing demand for the use of natural additives in food applications has generated a substantial increase in the number of studies based on natural extracts such as essential oils (EOs) or their main compounds. They are categorized as Generally Recognized as Safe (GRAS) by the US Food and Drug Administration, and are therefore potential alternatives to chemical additives. EOs extracted from plants are rich sources of biologically active compounds, such as phenolic acids and terpenoids, and it has been long recognized that some of them show antimicrobial properties. Furthermore, the antibacterial, antifungal, insecticidal, antiparasitic and antitoxigenic activities of these compounds have extensively been reported (Burt
123
Food Environ Virol
2004). In contrast, reports on the antiviral effects of EOs or their compounds are somewhat limited and are mainly based on NoV surrogates (Table 1). In a previous study, our group reported the antiviral activity of clove, oregano and zataria EOs against norovirus surrogates (Elizaquı´vel et al. 2013). Therefore, in this study, the effect of zataria (Zataria multiflora Boiss.) and oregano (Origanum vulgare) EOs on the infectivity of HAV has been assessed. We also studied the efficacy of thymol, one of the main components of oregano and thyme EO, on the
infectivity of HAV and two norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV).
Materials and Methods Viruses, Cells and Infections The cytopathogenic HM-175 strain of HAV (ATCC VR1402), the MNV-1 strain (kindly provided by Prof. H. W.
Table 1 Inactivation rates of essential oils and their main compounds on different enteric viruses or norovirus surrogates Target virus
Inactivation rates (log reduction)
Experimental setup (temperature, time)
References
Allspice (4 %)
MNV
3.41
24 °C, 24 h
Clove (1 %)
FCV, MNV
3.75; 0.67
37 °C, 2 h
Gilling et al. (2014b) Elizaquı´vel et al. (2013)
Hyssop (0.2 %)
AdV-2, MNV
No effect
Essential oils (EO)
4, 25 and 37 °C
Kovac et al. (2012)
2 and 24 h Lemongrass (4 %)
MNV
2.74
24 °C, 24 h
Gilling et al. 2014b
Marjoram (0.2 %)
AdV-2, MNV
No effect
4, 25 and 37 °C
Kovac et al. (2012)
Mexican oregano (Lippia graveolens)
RV
No effect
Mint (2 %)
MNV
0.87
37 °C, 1 h
Oregano (2 %) (Origanum compactum)
FCV, MNV
3.75; 1.62
37 °C, 2 h
Oregano (4 %)
MNV
1.10
24 °C, 6 h
Gilling et al. (2014a)
MNV
0.75
37 °C, 1 h
El Moussaoui et al. (2013)
Tea tree (0.025 %)
PV, Echo 9, Coxsackie B1, AdV-2
No effect
37 °C, 1 h
Garozzo et al. (2009)
Thyme (2 %)
MNV
0.5
37 °C, 1 h
Zataria (0.1 %)
FCV, MNV
4.51; 0.25
37 °C, 2 h
El Moussaoui et al. (2013) Elizaquı´vel et al. (2013)
Carvacrol (0.5 %)
MNV
[4.15
24 °C, 3 h
Carvacrol (0.5 %)
MNV, FCV
[3.57, [4.53
37 °C, 2 h
Carvacrol (1 %)
HAV
0.97
37 °C, 2 h
Carvacrol
RV
Effective
Citral (4 %)
MNV
3.00
2 and 24 h
Oregano (2 %) (Origanum elongatum)
Pilau et al. (2011) El Moussaoui et al. (2013) Elizaquı´vel et al. (2013)
Main compounds of EO Gilling et al. (2014a) Sa´nchez et al. (2015) Sa´nchez et al. (2015) Pilau et al. (2011) 24 °C, 24 h
Gilling et al. (2014b)
MNV murine norovirus, FCV feline calicivirus, AdV human adenovirus, RV human rotavirus, PV poliovirus, Echo echovirus, Coxsackie virus, HAV hepatitis A virus
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Virgin, Washington University School of Medicine, USA) and the F9 strain of FCV (ATCC VR-782) were propagated and assayed in FRhK-4 (courtesy of Prof. Albert Bosch, University of Barcelona), RAW 264.7 (kindly provided by Prof. H. W. Virgin) and CRFK cell monolayers (ATCC CCL-94), respectively. Virus stocks were obtained from the same cells by centrifugation of infected cell lysates at 6609g for 30 min at 2–3 days postinfection for MNV and FCV and 10–12 for HAV. Infectious viruses were quantified by determining the 50 % tissue culture infectious dose (TCID50) with eight wells per dilution and 20 ll of inoculum per well (Sa´nchez et al. 2011; Pinto´ et al. 1994). Natural Compounds Origanum vulgare (oregano) essential oil provided by Pronaroˆm International (Ghislenghien, Belgium) and an inhouse produced zataria essential oil (from Z. multiflora Boiss., collected in the Shiraz Province of Iran and kindly provided by Maryam Azizkhani) were used in this study. Main compounds of oregano EO are carvacrol (46.88 %), thymol (15.26 %), p-cymene (13.10 %) and c-terpinene (11.61 %) (www.pranarom.com) and in zataria, carvacrol (71.12 %), c-terpinene (7.34 %), a-pinene (4.26 %), eucalyptol (3.37 %) and globulol (2.32) (Elizaquı´vel et al. 2013). Thymol (C99 % purity) was provided by Sigma Aldrich. Oregano and zataria EOs were diluted in 70 and 50 % ethanol, respectively, whereas thymol was diluted in 25 % ethanol and kept at 4 °C before use. Cytotoxicity Determination of Natural Compounds on Cell Monolayers Natural compounds at various concentrations were added to individual wells of confluent RAW 264.7, CRFK and FRhK-4 cell monolayers grown in 96-well microtiter plates (SPL Life Sciences) and incubated 2 h under 5 % CO2. Subsequently, 150 ll of DMEM supplemented with 2 % of fecal calf serum (FCS) was added per well. After incubation at 37 °C in a CO2 incubator for 2–15 days, the cell monolayers were observed for cytotoxicity effects by visual inspection under the optical microscope only. Antiviral Effects of Natural Compounds Natural compounds at different concentrations were added to virus suspensions (ca. 6 log TCID50/ml) in DMEM with 2 % FCS and further incubated at 37 °C in a water-bath shaker at 150 rpm for 2 h. Tenfold dilutions of treated and untreated virus suspensions were inoculated into confluent monolayers in 96-well plates. Then, infectious viruses were
quantified by cell culture assays as described above. Each treatment was done in triplicate. Positive controls were virus suspensions added with ethanol in amounts corresponding to the highest quantity present. The decay of viruses was calculated as log10 (Nt/N0), where N0 is the infectious virus titer for untreated sample and Nt is the infectious virus titer for treated sample. Statistical Analysis The significance of differences among the mean numbers of viruses determined after the various treatments was estimated by the Student’s t test with a significance level of P \ 0.05 (Microsoft Office Excel; Microsoft, Redmond, WA, USA).
Results Determination of Cytotoxicity of EOs and Thymol on Cell Monolayers Oregano and zataria EOs resulted cytotoxic for FRhk-4 cells at concentrations that exceeded 2 and 0.1 %, respectively. Thymol was found to be cytotoxic at concentrations that exceeded 2 %, for the three cell lines. Therefore, 2, 0.1 and 2 % were the maximum concentration of oregano EO, zataria EO and thymol tested to evaluate their effect on HAV, MNV and FCV. Antiviral Efficacy of Oregano and Zataria EOs on HAV Incubation of HAV with oregano EO at concentrations of 0.5, 1 and 2 % for 2 h at 37 °C did not decreased the HAV titers (Table 2). Zataria EO at 0.1 % reduced HAV titers by 0.42 TCID50/ml (P \ 0.05). At lower zataria EO concentrations (i.e., 0.05 and 0.01 %), no differences in titer reduction were observed between HAV suspensions treated or non-treated with the tested concentrations of zataria EO (Table 2). Effect of Thymol on the Infectivity of HAV, MNV and FCV Incubation of FCV and MNV with thymol at concentrations from 0.5 to 2 % for 2 h at 37 °C decreased the titer of the two norovirus surrogates (Table 3). Thymol at 0.5 and 1 % reduced FCV titers to undetectable levels. On the other hand, MNV titers, the hardier NoV surrogate, were reduced by 0.50 log TCID50/ml after treatment with thymol at 0.5 %, while thymol at 1 and 2 % resulted in reductions of 1.66 and 2.45 log TCID50/ml, respectively. For HAV, no differences in titer reduction were observed between HAV
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Food Environ Virol Table 2 Effect of oregano (O) and zataria (Z) essential oils against Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
HAV (log10 TCID50/ml) Treatment
Recovered titer
Reduction
Treatment
Recovered titer
Reduction
Control
5.82 ± 0.17A
Control
6.20 ± 0.21A
0.5 % O
5.45 ± 0.35A
0.37
0.01 % Z
6.11 ± 0.07A
0.09
1%O
5.70 ± 0.12A
0.12
0.05 % Z
6.24 ± 0.19A
0.00
2%O
5.70 ± 0.33A
0.12
0.1 % Z
5.78 ± 0.19B
0.42
Table 3 Effect of thymol against feline calicivirus (FCV), murine norovirus (MNV-1) and Hepatitis A virus (HAV) after 2 h of incubation at 37 °C
Treatment Control
FCV
MNV
HAV
Log10 TCID50/ml
Log10 TCID50/ml
Log10 TCID50/ml
Recovered titer 5.88 ± 0.00A
Reduction a
Recovered titer
Reduction
6.07 ± 0.01A
Recovered titer
Reduction
6.41 ± 0.19A
0.1 % T
5.86 ± 0.26A
0.02
5.99 ± 0.38A
0.08
6.41 ± 0.14A
0.00
0.5 % T
\2.13 ± 0.00B
[3.57
5.57 ± 0.45A
0.50
5.29 ± 0.07A
0.12
1%T
\2.13 ± 0.00B
[3.57
2%T
NT
4.41 ± 1.16B
1.66
6.20 ± 0.12A
0.21
3.62 ± 0.72B
2.45
6.39 ± 0.17A
0.01
NT non tested a
Each treatment was done in triplicate. Within each column for each virus, different letters denote significant differences between treatments (P \ 0.05)
suspensions treated or non-treated with thymol at 0.1, 0.5, 1 or 2 % (Table 3).
Discussion The food industry is currently exploring new ways to use effective natural antimicrobials to improve food safety, and plant EOs or their compounds are potential alternative natural ingredients (reviewed by Gyawali and Ibrahim 2014). Regarding enteric viruses, oregano and zataria EO revealed efficient to reduce infectivity of MNV and FCV (Elizaquı´vel et al. 2013). The initial aim of the present study was to evaluate them on HAV, since it has recently been considered as a re-emerging foodborne public health threat in Europe (Sprenger 2014). Oregano EO was ineffective on HAV at the maximum concentration tested, unlike our previous results where oregano EO reduced the infectivity of MNV and FCV by 1.62 and 3.75 log, respectively (Elizaquı´vel et al. 2013). For zataria EO, although statistically significant, HAV infectivity was only reduced by 0.42 log TCID50/ml, while in our previous study, 0.1 % zataria EO reduced FCV and MNV infectivity by 4.51 and 0.25 TCID50/ml, respectively. As an alternative, one of the active compounds, thymol, was tested. Thymol is present in oregano EO, and its isomer, carvacrol, is the main active component of
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zataria. It is a phenolic monoterpene that has been identified as a natural economical food preservative (Lu and Wu 2010) with potential for incorporation in food packaging (Ramos et al. 2012). Following, antiviral activity of thymol was explored on NoV surrogates and HAV. This study clearly shows that thymol was effective in reducing the titers of norovirus surrogates in a dose-dependent manner, where increasing concentrations of thymol resulted in increased reduction in viral titers. Furthermore, HAV was resistant to thymol treatment at any concentration tested. Similar trends have recently been reported for carvacrol treatment; however, carvacrol was more effective than its isomer thymol against NoV surrogates and HAV. Sa´nchez et al. (2015) reported that carvacrol at 0.5 % completely inactivated FCV and MNV, whereas 1 % concentration was required to achieve ca. 1 log reduction of HAV. Moreover, Sa´nchez et al. (2015) reported the potential of carvacrol to be used as a natural disinfectant of produce. Overall, this study showed that the evaluated natural compounds resulted in slight reductions of HAV infectivity, while they were effective in NoV surrogates with higher reduction in FCV than MNV titers. These results resemble those reported for carvacrol (Sa´nchez et al. 2015). However, Su and D’Souza (2011), when testing the antiviral activity of grape seed extract (GSE), reported that MNV was more resistant than HAV.
Food Environ Virol
Taking together these results and previous studies (Elizaquı´vel et al. 2013; Sa´nchez et al. 2015; Gilling et al. 2014a, b; Pilau et al. 2011), the use of plant extracts as antivirals in food safety should focus on the purified active compounds as they exhibited stronger activity than EO. Nevertheless, complete inactivation of foodborne viral contamination with plant extracts either EOs or active compounds cannot be foreseen since they slightly affect HAV infectivity. The application of plant antimicrobials in food sanitation should combine them with other natural compounds, e.g., GSE, to achieve a synergistic effect, and be used as part of hurdle approaches to improve food safety. Acknowledgments G. Sa´nchez was supported by the ‘‘Ramo´n y Cajal’’ Young Investigator program of the Spanish Ministry of Economy and Competitiveness. This study was supported by grant AGL2009-08603 from the Spanish Ministry of Science and Innovation, and ACOMP/2010/279 and ACOMP/2012/199 from the Generalitat Valenciana. We thank Carlos Sa´nchez for technical support.
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