ABSTRACT

Foodborne viruses, particularly human norovirus, are a concern for public health, especially in fresh vegetables and other minimally processed foods that may not undergo sufficient decontamination. It is necessary to explore novel nonthermal techniques for preventing foodborne viral contamination. In this study, aqueous extracts of six raw food materials (flower buds of clove, fenugreek seeds, garlic and onion bulbs, ginger rhizomes, and jalapeño peppers) were tested for antiviral activity against feline calicivirus (FCV) as a surrogate for human norovirus. The antiviral assay was performed using dilutions of the extracts below the maximum nontoxic concentrations of the extracts to the host cells of FCV, Crandell-Reese feline kidney (CRFK) cells. No antiviral effect was seen when the host cells were pretreated with any of the extracts. However, pretreatment of FCV with nondiluted clove and ginger extracts inactivated 6.0 and 2.7 log of the initial titer of the virus, respectively. Also, significant dose-dependent inactivation of FCV was seen when host cells were treated with clove and ginger extracts at the time of infection or postinfection at concentrations equal to or lower than the maximum nontoxic concentrations. By comprehensive two-dimensional gas chromatography–mass spectrometry analysis, eugenol (29.5%) and R-(-)-1,2-propanediol (10.7%) were identified as the major components of clove and ginger extracts, respectively. The antiviral effect of the pure eugenol itself was tested; it showed antiviral activity similar to that of clove extract, albeit at a lower level, which indicates that some other clove extract constituents, along with eugenol, are responsible for inactivation of FCV. These results showed that the aqueous extracts of clove and ginger hold promise for prevention of foodborne viral contamination.

Enteric viruses, particularly human norovirus (NoV), are the leading causes of foodborne illnesses (16). Human NoV, one of the highest-ranking pathogens with respect to the total cost of foodborne illness in the United States, belongs to the family Caliciviridae and is a well-known cause of “winter-vomiting disease” or “stomach-flu” (15). It has been reported that NoV is responsible for 19 to 21 million cases of acute gastroenteritis annually in the United States and that it leads to 1.7 to 1.9 million outpatient visits, 56,000 to 71,000 hospitalizations, and 570 to 800 deaths, mostly among young children (7). More than 50% of all foodborne disease outbreaks due to a known cause that were reported to the Centers for Disease Control and Prevention from 2006 to 2010 were attributed to NoV. Foodborne viruses are transmitted through the fecal-oral route, and food processing workers have been identified as a major source for foodborne NoV outbreaks (26). Several types of fresh produce (e.g., green salad vegetables, lettuce, raspberries, strawberries, cabbage, kimchi, pomegranate seeds, and raw frozen fruit mixes), contaminated by polluted water or virus-infected food handlers, have been implicated in NoV outbreaks (8). Between 1998 and 2005, NoV-contaminated green salads, lettuce, fruits, and vegetables were implicated in 24, 5.1, 3.2, and 2.3% of all produce-based outbreaks, respectively (12).

Recent experiments with NoV in a variety of fresh and minimally processed foods revealed that minimal food processing is not significantly effective for inactivation of NoV or its surrogates (29). Therefore, the development of novel nonthermal technologies for viral decontamination of foods is of great interest to food scientists and food producers. A few studies are available on the virucidal effects of such technologies to combat foodborne viral contamination, e.g., ozone treatment (20), gamma irradiation (17), lactic acid bacteria used as an antiviral biopreservative (1), pulsed-light technology (38), and, most recently, cold atmospheric pressure gaseous plasma (2). Another growing trend of investigation is the exploration of natural compounds for their antiviral properties. Plant extracts have been the main focus of these studies because they are the most abundant source of potential antimicrobial natural substances, they can usually be produced at low cost, and their natural origin provides a high level of acceptance by most consumers (26).

Most of the studies on plant antimicrobials have involved bacteria, yeasts, and molds. Many studies have reported significant antiviral activity of some natural extracts, such as the antiviral activity of garlic extract and the fructan from Welsh onion against influenza virus type A (24, 28). In addition, an aqueous extract of fresh ginger was found to have antiviral activity against human respiratory syncytial virus (10). Grape seed extract, mulberry juice, green tea catechins, and peptide derivatives of soybean have shown significant antiviral activity against feline calicivirus (FCV) and murine norovirus (MNV), surrogates for human NoV (27, 30, 36). Because research on the antiviral effects of natural bioactive substances is still in its infancy, Li et al. (26) have advocated the exploration and use of new natural antiviral compounds with low toxicity and low cost. This study was designed to investigate in vitro antiviral effects of six common plant materials against FCV. Aqueous extracts were obtained from onion and jalapeño peppers, plant materials that are consumed regularly as constituents of fresh vegetable salads. An additional four extracts are used commercially as components of salad dressings (flower buds of clove, fenugreek seeds, garlic, and ginger). Additional aims were to study the mechanism of virus inactivation by treating the virus at different stages of its interaction with the host cells and to identify the antiviral bioactive compound in the most promising extract.

MATERIALS AND METHODS

Virus propagation.

Strain 255 of FCV was used as a surrogate of NoV. The virus was propagated and titrated in Crandell-Reese feline kidney (CRFK) cells. The cells were grown in minimum essential medium (MEM) with Earle's salts and L-glutamine (Mediatech, Inc., Herndon, VA) supplemented with 8% fetal bovine serum, neomycin (90 U/ml), gentamicin (50 μg/ml), penicillin (455 IU/ml), streptomycin (455 μg/ml), and fungizone (1.5 μg/ml). For virus propagation, confluent CRFK cell monolayers were infected with FCV, followed by incubation at 37°C under a 5% CO2 atmosphere. The incubated cells were examined daily under an inverted microscope for the appearance of cytopathic effects, which usually appeared 1 to 2 days after infection. Infected cells were frozen and thawed three times, followed by centrifugation at 3,000 × g for 15 min at 4°C. The virus-containing supernatant was aliquoted and stored at −80°C until it was used. For virus titration, serial 10-fold dilutions of all samples were prepared in MEM containing 4% fetal bovine serum. The dilutions were inoculated in CRFK cell monolayers prepared in 96-well microtiter plates, using three wells per dilution. The plates were incubated at 37°C under 5% CO2 and were examined daily for the development of cytopathic effects for up to 5 days. Viral titers were calculated by the Kärber method and were expressed as 50% tissue culture infective dose (TCID50) per 0.1 ml.

Preparation of natural plant extracts.

Several raw products (flower buds of clove, Syzygium aromaticum; fenugreek seeds, Trigonella foenum-graecum; garlic, Allium sativum; ginger, rhizomes of Zingiber officinale; jalapeño pepper, Capsicum annuum; and onion, Allium cepa) were obtained from local supermarkets in Saint Paul, MN. Two methods were used for extraction, based on the characteristics of each raw material. For supple and tender materials, the raw products (garlic, jalapeño pepper, and onion) were dehusked (except jalapeño pepper), washed with distilled water, and chopped using sterile scalpels and razor blades. From each product, 25 g was homogenized in 25 ml of sterile distilled water, using a sterile ceramic mortar and pestle and then a glass homogenizer. Homogenized mixtures were squeezed through four overlapping layers of sterile gauze to remove larger residues, followed by centrifugation at 2,200× g for 15 min. The supernatants were filter sterilized, distributed in 5-ml aliquots, and stored at −20°C until they were used. This method was developed to extract bioactive compounds from supple and tender raw materials because the bioactive compounds are located mainly in the cytoplasm of the tissue's cells. For hard and fibrous raw products (clove buds, fenugreek seeds, and ginger), 10 g of each was boiled in 40 ml of distilled water for 20 min under reflux to avoid water loss due to evaporation. Ginger was chopped, whereas cloves and fenugreek seeds were added whole. The crude aqueous extracts were centrifuged at 2,200 × g for 15 min. The supernatants were filter sterilized, distributed in 5-ml aliquots, and stored at −20°C until they were used.

Cytotoxicity assay.

The maximum nontoxic concentrations (MNTCs) of all extracts were determined based on cellular morphological alterations. Several dilutions of the aqueous extracts were prepared in MEM. The initial extract was taken as a reference of “100%,” and the percentages of subsequent dilutions were calculated based on that reference. Then monolayers of CRFK cells prepared in 96-well microtiter plates were inoculated with 100 μl per well of each of the tested concentrations. The plates were incubated in 5% CO2 at 37°C for 48 h. Concentrations that were not toxic to cells were labeled as “nontoxic” and were also compared with nontreated cells (negative control) for confirmation. The MNTCs were used for further antiviral tests. A similar assay was used to evaluate the cytotoxicity of pure eugenol [2-methoxy-4-(2-propenyl) phenol, 4-allyl-2-methoxyphenol, 4-allylguaiacol] (Sigma-Aldrich, St. Louis, MO) on CRFK cells.

Antiviral assays.

The anti-FCV activity of aqueous extracts was assayed by four different methods. (i) In the first, CRFK cells were pretreated with extracts. After discarding the growth medium, the CRFK monolayers were inoculated with 100 μl of three nontoxic dilutions (one equal to and two less than MNTC) of each natural extract, separately. After incubation at 37°C in 5% CO2 for various times (30, 60, and 90 min), the monolayers were washed with sterile phosphate buffer saline (PBS). Immediately, the washed monolayers were infected with 10-fold serial dilutions of FCV 255 prepared in MEM. The plates were then incubated at 37°C in 5% CO2. The FCV titers in pretreated and nontreated (control) monolayers were compared after 5 days of incubation. (ii) In the second method, FCV was pretreated with extracts. Aliquots (250 μl) of FCV suspension were mixed separately with equal volumes of crude extracts (undiluted) in 1.5-ml sterile Eppendorf tubes. After incubation at 4°C for 24 h, 10-fold serial dilutions were prepared from each mixture, followed by inoculation of CRFK monolayers. The titers of treated and nontreated (control) FCV were compared after 5 days of incubation. (iii) The third method used cotreatment with FCV and extracts. Three different nontoxic concentrations of each extract were prepared in MEM. Serial 10-fold dilutions of FCV were prepared in each extract concentration separately. From each virus dilution, 100 μl was used to infect the CRFK monolayers. The titers of FCV prepared in each natural extract and those prepared in MEM (control) were compared after 5 days of incubation. (iv) In the fourth method, CRFK cells were treated with extracts postinfection. Three different nontoxic concentrations of each extract were prepared in MEM. CRFK monolayers were inoculated with serial 10-fold dilutions of FCV, followed by incubation at 37°C for 2 h. The cells were then washed with sterile PBS and were covered with 100 μl of nontoxic concentrations of natural extracts. The titers of FCV were then calculated after 5 days of incubation at 37°C in 5% CO2 and were compared with the nontreated control.

GC-MS analysis of clove and ginger ethanolic extracts.

One volumetric unit of clove or ginger extract was diluted in 10 volumetric units of HPLC-spectrophotometric grade ethanol (Sigma-Aldrich). After vortexing for 5 min, the mixture was centrifuged at 12,000 × g for 10 min. Of the top (ethanolic) layer, 50 μl was collected in a dry screw-cap vial (12 by 33 mm). For gas chromatography–mass spectrometry (GC-MS) analysis, samples were diluted 1 to 5 (vol/vol) in hexane. GC-MS was performed using the LECO Pegasus 4D comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer platform (LECO Corporation, St. Joseph, MI). An Agilent 6890N gas chromatograph (Agilent, Santa Clara, CA) was fitted with a Gerstel thermal desorption unit in-line with a cold injection system (CIS-4, Gerstel Inc., Baltimore, MD). After injection of 1 μl of the thermal desorption unit ramped from 40 to 300°C at 60°C/min, the cold injection system was ramped from −125 to 300°C at 12°C/s. Sample was injected from the cold injection system in splitless mode at a constant 1 ml/min (helium carrier gas) onto the first of two columns (0.25-mm inside diameter (i.d.), 0.25-μm film, 30 m, Agilent DB-5MS; and 0.1-mm i.d., 0.1-μm film, 1.2 m; BPX50 column, SGE Analytical Science, Austin, TX) separated by cold-hot modulator. For the heating ramp in the first column, the initial temperature was 40°C for 0.2 min, followed by 5°C/min to 200°C and 10°C/min to 300°C (~33 min); in the second column, 4 s at 320°C was followed by 4 s (1 s per hot-cold pulse). Mass spectrometry detection began after a delay of 280 s, with mass range of 35 to 600 m/z; electron energy, 70 eV; ion source temperature, 200°C; acquisition rate, 50 per s; and signal-to-noise ratio of 50. A total ion chromatogram was used for area quantification, and i.d. values were taken from the National Institute of Standards and Technology mass spectral library.

Validation of antiviral activity of eugenol.

A cytotoxicity test of pure eugenol (Sigma-Aldrich) was carried out as described in “Cytotoxicity assay.” The MNTC of eugenol was used to perform antiviral assays, following the same methods used with extracts.

Statistical analysis.

Each titration was carried out in triplicate, and each experiment was done in triplicate. The results shown are the arithmetic means of the surviving FCV titers ± standard deviation. The difference between the mean of each considered treatment and the control was assessed for significance using a two-sample t test. The statistical analysis was carried out using Statistica software version 10 (Statsoft, Inc., Tulsa, OK).

RESULTS

Cytotoxicity of the tested aqueous extracts against CRFK cells.

The level of cytotoxicity of tested aqueous extracts on CRFK cells varied (Table 1). Ginger and jalapeño pepper extracts showed the least toxicity because they were only toxic in the nondiluted form. Onion, clove, and fenugreek extracts showed toxicity at 10, 5, and 2.5% concentrations, respectively. The highest toxicity was seen with garlic extract; it was toxic at a concentration of 2%. Based on the abovementioned results, the MNTCs were 3.3, 2, 1, 20, 20, and 5% for clove, fenugreek, garlic, ginger, jalapeño pepper, and onion extracts, respectively. Three nontoxic concentrations (one equal to and two less than MNTC) were used to determine the effect of concentration on the antiviral activities of the extracts: 3.3, 1.7, and 1% for clove; 2, 1, and 0.5% for fenugreek; 1, 0.7, and 0.5% for garlic; 20, 10, and 5% for ginger; 20, 10, and 5% for jalapeño pepper; and 5, 2.5, and 1.7% for onion.

TABLE 1.

Cytotoxicity of natural extracts and eugenol to CRFK cellsa

Cytotoxicity of natural extracts and eugenol to CRFK cellsa
Cytotoxicity of natural extracts and eugenol to CRFK cellsa
TABLE 2.

Anti-FCV activity following pretreatment of CRFK cells with natural extractsa

Anti-FCV activity following pretreatment of CRFK cells with natural extractsa
Anti-FCV activity following pretreatment of CRFK cells with natural extractsa

Pretreatment of FCV with nondiluted extracts.

Variation was observed in virucidal activities after pretreatment of FCV with undiluted extracts (Fig. 1). The virus titer was not significantly (P ≥ 0.05) reduced after pretreatment with undiluted fenugreek, garlic, ginger, and jalapeño pepper extracts. However, significant (P < 0.01) decreases in FCV titer were obtained with clove and ginger extracts (reduction of ~6 and 2.7 log TCID50, respectively).

FIGURE 1.

Effect of pretreatment of feline calicivirus (FCV) with natural extracts on virus titer. FCV was pretreated with nondiluted extracts for 24 h at 4°C. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are the significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus.

FIGURE 1.

Effect of pretreatment of feline calicivirus (FCV) with natural extracts on virus titer. FCV was pretreated with nondiluted extracts for 24 h at 4°C. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are the significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus.

Coinfection treatment of CRFK with extracts.

When CRFK was treated with fenugreek, garlic, and jalapeño pepper extracts at the time of infection with FCV, no significant (P ≥ 0.05) decrease in FCV titer was seen at any of the nontoxic concentrations used (Fig. 2). Cotreatment with FCV and clove or ginger extract showed significant (P < 0.01) concentration-dependent decreases in FCV titer. The highest virucidal activity (~4.8, 4.2, and 4 log TCID50) was seen with clove extract at concentrations of 3.3, 1.7, and 1%, respectively. Ginger extract showed a significant reduction (P < 0.01; ~3.16 log TCID50) only at MNTC (20%), whereas lower concentrations did not show significant decreases (P ≥ 0.05).

FIGURE 2.

Effect of cotreatment of Crandell-Reese feline kidney (CRFK) cells with feline calicivirus (FCV) and natural extracts. Three concentrations (MNTC and two lower concentrations) of each extract were used for treating CRFK simultaneously with viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

FIGURE 2.

Effect of cotreatment of Crandell-Reese feline kidney (CRFK) cells with feline calicivirus (FCV) and natural extracts. Three concentrations (MNTC and two lower concentrations) of each extract were used for treating CRFK simultaneously with viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

Postinfection treatment of CRFK with extracts.

Postinfection treatment with fenugreek, garlic, jalapeño pepper, and onion extracts did not show significant (P ≥ 0.05) effects on FCV titers at any of the tested concentrations (Fig. 3). A significant (P < 0.01) decrease in FCV titer was seen when CRFK cells were treated postinfection with clove and ginger extract at their MNTCs. The highest decreases seen (~3.8 and 2.7 log TCID50) were for clove extract at 3.3 and 1.7%, respectively; there was no significant decrease at 1%. Ginger extract showed a significant reduction (P < 0.01; ~2.5 log TCID50) only at its MNTC (20%) but not at lower concentrations.

FIGURE 3.

Effect of postinfection treatment of Crandell-Reese feline kidney (CRFK) with feline calicivirus (FCV) and natural extracts. Three concentrations (MNTC and two lower concentrations) of each extract were used for treating CRFK 2 h after viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

FIGURE 3.

Effect of postinfection treatment of Crandell-Reese feline kidney (CRFK) with feline calicivirus (FCV) and natural extracts. Three concentrations (MNTC and two lower concentrations) of each extract were used for treating CRFK 2 h after viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

GC×GC-MS analysis of the aqueous extract of clove and ginger.

A number of compounds were separated from clove and ginger extracts using two-dimensional GC-MS (GC×GC-MS) analysis. Figures 4 and 5 show the GC×GC-MS chromatograms (contour [A] and surface [B] views) of clove and ginger extracts, respectively. Of all separated compounds, only the top 200 were recognized. For compounds to be identified by name, data processing parameters required a similarity score of greater than 800 for clove and 750 for ginger (scores ranged from 0 to 999) between the detected compounds and the library spectra. If the similarity score was lower than the prescribed level, the compounds were reported as “unknown.” The identified compounds of the top 200 detected were tabulated (Tables 3 and 4) after elimination of all solvent-contaminated compounds, which were identified by running the solvents as background. For clove extract, the peak area of identified compounds was 67.6% of the total area of whole separated compounds (Table 3). The major components in clove extract were eugenol (29.5%), (S)-(+)-1,2-propanediol (6.9%), and eugenol acetate (3.3%). The peak area of identified compounds of ginger extract was 35.5% of the total area of whole separated compounds (Table 4). The major identified constituents of ginger were R-(-)-1,2-propanediol (10.7%), 2,3-butanediol, [S-(R*, R*)]- (6.3%), and 1,2-benzenedicarboxylic acid (2.9%).

FIGURE 4.

GC×GC-MS chromatogram of the aqueous extract of clove buds (Syzygium aromaticum) showing eugenol as the major constituent. (A) Contour view. (B) Surface view.

FIGURE 4.

GC×GC-MS chromatogram of the aqueous extract of clove buds (Syzygium aromaticum) showing eugenol as the major constituent. (A) Contour view. (B) Surface view.

FIGURE 5.

GC×GC-MS chromatogram of the aqueous extract of fresh ginger (Zingiber officinale). (A) Contour view. (B) Surface view. White arrows refer to the major peaks representing 1-chloroundecane and nonanal. These two peaks belong to the solvent background and so are not included in the component list of ginger extract (Table 3).

FIGURE 5.

GC×GC-MS chromatogram of the aqueous extract of fresh ginger (Zingiber officinale). (A) Contour view. (B) Surface view. White arrows refer to the major peaks representing 1-chloroundecane and nonanal. These two peaks belong to the solvent background and so are not included in the component list of ginger extract (Table 3).

TABLE 3.

Chemical composition of the aqueous extract of clove buds (Syzygium aromaticum)

Chemical composition of the aqueous extract of clove buds (Syzygium aromaticum)
Chemical composition of the aqueous extract of clove buds (Syzygium aromaticum)
TABLE 4.

Chemical composition of the aqueous extract of fresh ginger (Zingiber officinale)

Chemical composition of the aqueous extract of fresh ginger (Zingiber officinale)
Chemical composition of the aqueous extract of fresh ginger (Zingiber officinale)

Cytotoxicity and antiviral activity of eugenol.

Eugenol was cytotoxic at a concentration of as low as 0.2%. The concentration of 0.1% was nontoxic and was, therefore, recognized as MNTC and selected for antiviral assay (Table 1). As shown in Figure 6, eugenol did not show any significant decrease in FCV infectivity when it was used for pretreating CRFK cells (P ≥ 0.05). The pretreatment of FCV for 24 h at 4°C with concentrated eugenol resulted in a reduction of 3.78 log TCID50/0.1ml in FCV titer. When CRFK cells were cotreated with FCV and eugenol (at MNTC), a reduction of 3.34 log TCID50 was seen in FCV titer. When eugenol was used in a postinfection treatment test, there was a decrease of only 2.56 log TCID50/0.1ml of FCV.

FIGURE 6.

Effect of pretreatment of Crandell-Reese feline kidney (CRFK), pretreatment of feline calicivirus (FCV), and coinfection and postinfection treatment of CRFK with eugenol. The maximum nontoxic concentration (MNTC) of eugenol was used to pretreat CRFK for 30, 60, and 90 min before viral infection. The undiluted eugenol was used to pretreat the virus at 4°C for 24 h. For coinfection treatment, the MNTC was used to treat cells simultaneously with viral infection. For postinfection treatment, MNTC was used to treat cells after 2 h of viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

FIGURE 6.

Effect of pretreatment of Crandell-Reese feline kidney (CRFK), pretreatment of feline calicivirus (FCV), and coinfection and postinfection treatment of CRFK with eugenol. The maximum nontoxic concentration (MNTC) of eugenol was used to pretreat CRFK for 30, 60, and 90 min before viral infection. The undiluted eugenol was used to pretreat the virus at 4°C for 24 h. For coinfection treatment, the MNTC was used to treat cells simultaneously with viral infection. For postinfection treatment, MNTC was used to treat cells after 2 h of viral infection. Data are the average of triplicate experiments. Error bars represent standard deviations. Column labels are significance levels of the difference between treatment and control (ns, nonsignificant at P ≥ 0.05; *, significant at P < 0.05; **, significant at P < 0.01). TCID50, 50% tissue culture infectious dose of the virus. MNTC, maximum nontoxic concentration.

DISCUSSION

In this study, FCV was chosen as a surrogate for NoV because the former does not grow in vitro. In addition, FCV belongs to the same Caliciviridae family as does human NoV (13). The expected mode of action of the antiviral activity of the natural extracts is the chemical interaction between the bioactive chemical compounds of the extracts and the virus or its hosting cells. Therefore, as a worst-case scenario, we selected FCV in our study because it is known to have higher resistance to chemical disinfectants as compared to the other NoV surrogates, such as murine norovirus (2, 5, 6, 33, 34). The plant materials were chosen from among those that are consumed commonly as constituents of food, such as fresh vegetable salads. In addition, extracts that are used commercially as components of salad dressings were also tested. Some of the selected extracts are known to have a high content of some bioactive molecules that antagonize bacteria, fungi, and viruses. We decided to use the aqueous extracts instead of using essential oils or organic-nonpolar solvent extracts (i) to meet the needs of food consumers who want a very simple method to prepare extracts for home use, (ii) to minimize extraction costs for industrial-scale production, and (iii) to mimic the actual form in which these plant materials are commonly used.

In a preliminary experiment, we found that the use of PBS as a solvent for preparation of extracts was not appropriate because it inhibited the antiviral activity of all tested plant material extracts, including clove and ginger extracts (data not shown). Therefore, distilled water was selected as an extraction solvent to perform the subsequent experiments.

To unambiguously assess the effect of the studied extracts on FCV, cytotoxicity was determined prior to performing the antiviral assays. The results showed that four of the six aqueous extracts (fenugreek, garlic, jalapeño pepper, and onion) did not show any significant antiviral activity against FCV, although an antibacterial effect of fenugreek extract has been reported against Pseudomonas spp., Escherichia coli, and Salmonella Typhi (11). There are no published reports on the antiviral activity of fenugreek extracts. Seeds of fenugreek are rich in saponins, a group of bioactive glycosidic compounds, which are known to have antibacterial and antiviral effects against Escherichia coli, Staphylococcus aureus, Candida albicans (35), rotavirus (37), and human hepatitis C (23) virus. However, our results showed no antiviral effect of fenugreek extract on FCV.

Although many reports have confirmed the antiviral effects of garlic extract on herpes simplex virus types 1 and 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2 (39), our results show that garlic extract has no effect on FCV. This is in agreement with the results reported by Oh et al. (30). In our study, jalapeño pepper extract did not show anti-FCV activity, although it is known that hot peppers are rich in capsaicinoids, which have antibacterial effects against many foodborne bacterial pathogens (31). To date, there are no available data on the antiviral activity of jalapeño pepper extract or capsaicinoids, except one investigation that reported an antiviral effect of capsaicin against herpes simplex type 1 and parainfluenza type-3 (32). In one study, no significant anti-FCV effect of the ethanolic extract of red pepper was seen (30). We did not observe anti-FCV activity of onion extract, although fructan from Welsh onion has been reported to have an anti-influenza A virus effect (24).

Both clove and ginger extracts significantly decreased the infectivity of FCV in pretreatment of virus (Fig. 1) and in coinfection and postinfection treatments (Figs. 2 and 3) but not in pretreatment of cells (Table 2). In a similar study, Elizaquível et al. (14) found that pretreatment of FCV with 1% (wt/vol) clove bud essential oil for 2 h at 37°C resulted in 3.75-log reduction in FCV titer, whereas no significant reduction was seen when the virus was pretreated at 4°C. In our study, there was a decrease in FCV titer of ~6 log when the virus was pretreated with the concentrated aqueous extract of clove at 4°C for 24 h. Thus, the use of aqueous extract of clove buds seems to be more advantageous than the use of essential oils for FCV inactivation. Its higher solubility in water and its higher antiviral activity at low temperature are advantageous for food processing purposes because it can be used as a natural disinfectant in washing water of fresh vegetables. The antiviral effect of ginger extract has been reported against human respiratory syncytial virus (10). On the other hand, a methanolic extract of ginger did not show significant anti-FCV activity (30), which is in contrast to our results. These contradictory results may be attributable to the different treatment methods and the different solvent (methanol) used.

To understand the possible mode of action by which the natural extracts may affect the virus, we performed antiviral assays at four different stages of virus infection, e.g., pretreatment of CRFK cells, pretreatment of virus, coinfection treatment, and postinfection treatment. In all tests, clove extract was more efficient than ginger extract (P < 0.05), regardless of their concentration. Absence of antiviral effects following pretreatment of the host cells with the two extracts indicates that the extracts do not affect the cell receptors for FCV. The antiviral effect seen following pretreatment of virus with the extracts is probably due to alterations in the viral capsid, thereby inhibiting virus attachment to cells. Inhibition of FCV attachment by clove essential oil and of human respiratory syncytial virus attachment by fresh ginger extract has been reported (10, 14). The dose-dependent reduction in FCV titer when cells were infected with FCV and simultaneously were treated with extracts (coinfection treatment) may be due to suppression of virus attachment and/or suppression of viral entry to the cell. The antiviral effect seen with postinfection treatment indicates that it suppresses virus replication even if the virus has successfully attached to the host cells. A similar mechanism was hypothesized to explain the antiviral effect of clove extract against hepatitis C virus (21) and of fresh ginger extract against human respiratory syncytial virus (10).

Several studies have reported that the antiviral activity of natural extracts is due to the presence of bioactive molecules, such as polyphenols (25), proanthocyanins (36), polysaccharides (27), and some other constituents (26). We analyzed the chemical composition of clove and ginger extracts using the GC×GC-MS technique to identify the bioactive molecules that may be responsible for the observed antiviral activity. Eugenol, (S)-(+)-1, 2-propanediol, and eugenol acetate were the major components of the aqueous extract of clove buds, in addition to lesser amounts of some other components (Table 3). Although there are no published data on the composition of the aqueous extract of clove for comparison, the abundance of eugenol and eugenol acetate is consistent with the published data on the essential oil of clove buds (9). The antiviral activity of pure eugenol has been reported against human herpes simplex virus (3). However, the amount of eugenol in the used aqueous extract (29.5%) was much lower than in the essential oil of clove (47.64 to 78%) (9). This may be due to the hydrophobic nature of eugenol, which may make it water insoluble (18), although it could be partially extracted in boiled water.

The GC×GC-MS analysis of ginger aqueous extract identified R-(-)-1,2-propanediol (10.7%), 2,3-butanediol[S-(R*R*)] (6.3%), and 1, 2-benzenedicarboxylic acid (2.9%). Some phytochemical reports have shown a large variability in the identified compounds of ginger extract. The reported major components of ginger essential oil are gingerols, shogaols, zingerone, and paradol (22) and camphene (6.39%), p-cineole (5.96%), and zingiberene (4.19%) (19). In comparison with these reports of ginger oil composition, the aqueous extract of ginger has a different composition. This may be due to the aqueous extraction method, which restricts the isolation of hydrophobic constituents.

Because of the superior antiviral activity of clove extract, we focused mainly on this extract to determine whether its anti-FCV activity was indeed due to eugenol. Eugenol was selected because it is the most abundant compound in clove extract (Table 3) and because of its reported antiviral activity against human herpesvirus (4). Antiviral assays were done with pure eugenol, following the same approach as used with the extracts. The results were similar to those obtained with clove extract (Fig. 6). The maximum decrease in FCV titer (3.78 log TCID50/0.1ml) was obtained upon pretreatment of FCV with eugenol, which is lower than that obtained in the corresponding experiment performed with the clove extract. This indicates that eugenol may not be the only antiviral compound in the clove extract; and, hence, the crude aqueous extract of clove buds should be used for virus inactivation rather than pure eugenol.

In conclusion, we report for the first time, to our knowledge, the antiviral activity of clove buds and ginger aqueous extracts against FCV used as a human NoV surrogate. The aqueous extracts of clove and ginger hold promise for preventing foodborne viruses. Based on our findings, many potential home and industrial applications of the aqueous extracts of clove and ginger are possible, but they need further verification studies. For instance, these extracts can be used as antiviral vegetable salad dressing solutions industrially and at home. These extracts can be added to the washing water of vegetables and fruits that are eaten fresh, to provide viral decontamination. Also, wipes impregnated with these antiviral natural extracts can be produced for decontamination of food contact surfaces and the environment.

ACKNOWLEDGMENTS

Partial funding provided by the Cultural Affairs and Mission Sector, Ministry of Higher Education and Scientific Research, Egypt, is gratefully acknowledged. The work was done at the Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul. We thank Stephen Harvey, Center for Mass Spectrometry and Proteomics, University of Minnesota, for technical help.

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