Effects of Oils and Essential Oils from Seeds of Zanthoxylum schinifolium against Foodborne Viral Surrogates

Human noroviruses are the most frequent cause of foodborne viral disease and are responsible for the vast majority of nonbacterial gastroenteritis. However, no specific therapies are available for the efficient control or prevention of foodborne viral disease. Here, we determined the antiviral activities of oils from seeds of Zanthoxylum schinifolium (ZSO) against foodborne viral surrogates, feline calicivirus-F9 (FCV-F9), and murine norovirus-1 (MNV-1), using plaque assay. Time-of-addition experiments were designed to determine the antiviral mechanism of action of ZSO against the surrogates. Maximal antiviral effect was observed upon pretreatment of FCV-F9 or MNV-1 with ZSO, which comprised oleic acid, linoleic acid, palmitic acid, and linolenic acid as the major fatty acids. FCV-F9 was more sensitive to ZSO than MNV-1, and the 50% effective concentration of ZSO against pretreatment of FCV-F9 was 0.0007%. However, essential oils from Z. schinifolium (ZSE), which comprised 42% estragole, showed no inhibitory effects against FCV-F9 and MNV-1. These results suggest that the inhibitory activities of ZSO were exerted by direct interaction of FCV-F9 or MNV-1 virion with ZSO, which may be a food material candidate for control of foodborne viral disease.


Introduction
Human norovirus, a member of the Caliciviridae family, is responsible for approximately 90% of epidemic nonbacterial outbreaks of gastroenteritis globally in people of all ages [1,2]. It is highly contagious, often leading to large outbreaks in institutional food services such as schools, hospitals, childcare centers, nursing homes, and military camps, in which young children, the elderly, soldiers, and immunosuppressed patients are high-risk populations [3]. Norovirus genus is classified into five genogroups, which can be further divided into different genotypes. Genogroup II is the most prevalent, and genogroup II genotype 4 (G II-4) is responsible for most infections worldwide [4]. However, human norovirus has been relatively understudied due to lack of a suitable in vitro culture system. Feline calicivirus (FCV) and murine norovirus-1 (MNV-1) from STAT1-deficient (STAT1 −/− ) mice have been used as surrogates to elucidate norovirus biology and replication [5][6][7]. However, no specific therapies are yet available to efficiently control or prevent foodborne noroviral disease.
Seeds and pericarp of Zanthoxylum schinifolium, which belongs to the Rutaceae family, are widely consumed in Korea, China, and Japan as a spice. Z. schinifolium has been used in folk medicine for treatment of vomiting, diarrhea, and abdominal pain [8]. The pericarp of Z. schinifolium is also used as an antimicrobial and antioxidant. These biological functions are due to pericarp essential oils [9]. Essential oils, which are secondary metabolites of aromatic plants, have a distinct odor and are extracted from various parts of plants by hydrodistillation. Their main constituents, for example, terpenes, alcohols, aldehydes, and esters, are responsible for their biological properties such as antimicrobial activity.
Essential oils have been applied as flavoring agents to foods such as meat and have shown a wide spectrum of antimicrobial activity against several foodborne pathogens and spoilage bacteria, both in vitro and in food matrices [10]. Several research groups have demonstrated antiviral activities of essential oils against FCV [11], MNV-1 [12], herpes simplex virus type 1 (HSV-1) [13], HSV-2 [14], dengue virus type 2, and Junin virus [15]. Even though essential oils from Z. schinifolium have been demonstrated as having antibacterial 2 Evidence-Based Complementary and Alternative Medicine  [16], no information is available on the antiviral effects of oils and essential oils from seeds of Z. schinifolium against FCV-F9 and MNV-1. In the present study, antiviral activities of oils (ZSO) and essential oils (ZSE) from seeds of Z. schinifolium against FCV-F9 and MNV-1 were analyzed using cytopathic effect assay and plaque assay. Maximal antiviral effect was observed upon pretreatment of FCV-F9 or MNV-1 with ZSO.

Cytopathic Effect (CPE) Assay.
For CPE assay of ZEO or ZSO, cell viability was quantitatively determined using MTT assay [20]. RAW 264.7 and CRFK cells were seeded in 96well plates (2 × 10 5 cells and 0.5 × 10 4 cells per well, resp.) in DMEM containing 10% FBS and 1% PS. After incubation for 12 h, media were removed and 10 L of ZSO (0.0001%, 0.001%, and 0.01%) or ZSE (0.00001%, 0.0001%, and 0.001%) and 10 L of FCV-F9 or MNV-1 were added to cells containing 80 L of fresh media for 48-72 h. Then, 10 L of MTT solution was added to each well and incubated at 37 ∘ C for 2 h. After removal of supernatant, DMSO was added and incubated for 30 min. The absorbance at 570 nm was determined using a microplate reader. All determinations were performed in triplicate.

Plaque Assay.
To evaluate the antiviral activity of ZSO against FCV-F9 and MNV-1, the effect of ZSO was evaluated at different time points during virus infection using plaque assay [21]. Pretreatment of cells and viruses with ZSO was carried out separately. Pretreatment of RAW 264.7 cells was Evidence-Based Complementary and Alternative Medicine 3 conducted as follows: ZSO in DMEM containing 10% FBS and 1% PS was added to confluent monolayers of RAW 264.7 cells and incubated at 37 ∘ C in a CO 2 incubator for 1 h with gentle shaking. After complete aspiration of cell media containing the ZSO, a 10-fold serial dilution of virus stock (2∼3 log 10 PFU/mL) prepared in DMEM containing 10% FBS and 1% PS was inoculated into each well. After viruses were adsorbed for 1 h at 37 ∘ C in a CO 2 incubator and the inocula were removed, 1 mL of DMEM containing 1.5% agarose, 5% FBS, and 0.5% PS was added to each well. The plates were then incubated for 42 h at 37 ∘ C in a CO 2 incubator, after which cells were stained with 0.5% crystal violet and the number of plaques counted. DMSO and 2-thiouridine (2TU) were used as negative and positive controls, respectively. Pretreatment of MNV-1 with ZSO was performed by mixing equal volumes of ZSO and MNV-1, followed by incubation at room temperature for 1 h. Ten-fold serial dilutions of virus stock (2∼ 3 log 10 PFU/mL) were then inoculated onto confluent RAW 264.7 cell monolayers for 1 h at 37 ∘ C in a CO 2 incubator. After virus adsorption, the same procedure as that described for pretreatment of RAW 264.7 cells was carried out. For cotreatment, the same procedure as that described for pretreatment of 264.7 cells was carried out, except that confluent RAW 264.7 cell monolayers were infected with 100 L of virus stock (2∼3 log 10 PFU/mL), which was simultaneously incubated with ZSO for 1 h at 37 ∘ C in a CO 2 incubator. For posttreatment, inocula were completely removed after virus adsorption (2∼3 log 10 PFU/mL) to cells, which were incubated with ZSO for 1 h. The same procedure as that described for pretreatment of cells was then carried out. FCV-F9 plaque assay using CRFK cells was also performed following the method described for MNV-1 and RAW 264.7 cells. An effective concentration to reduce the 50% plaque number (EC 50 ) was calculated by regression analysis of the dose-response curves generated from these data [22].

Preparation and Identification of Fatty Acid Methyl Esters.
Fatty acid methyl esters (FAMEs) were prepared according to AOAC Official Method 969.33 [23]. Briefly, 500 mg of ZSO was placed in a boiling flask to which 4 mL of 0.5 M sodium hydroxide in methanol was added. The flask was refluxed for 10 min, after which 5 mL of 12.5% boron trifluoride (Sigma-Aldrich Co. LLC, St Louis, MO, USA) in methanol was added. After refluxing for 2 min, 5 mL of n-hexane was added, and the flask was again refluxed for 1 min. Saturated sodium chloride solutions were then added to the flask. The upper layer containing FAMEs was pipetted into a vial containing anhydrous sodium sulfate to remove water. The solution was finally filtered and used for subsequent gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS (Agilent 6890 GC/5973 MSD) was used to analyze fatty acid composition with an Omegawax column (30 m length × 0.25 mm i.d. × 0.25 m film thickness; Sigma-Aldrich Co.). Injection volume was 1 L, and the flow rate of helium as a carrier gas was 5.0 mL/min. The oven temperature was held constant at 140 ∘ C for 5 min, increased to 240 ∘ C at a rate of 5 ∘ C/min, and then held constant at 240 ∘ C for an additional 20 min. The temperatures of the injector and detector were 250 ∘ C. The MS was operated in electron ionization mode at 70 eV, scanning masses from 33 to 330 m/z. Identification of peaks on the chromatogram was performed the same way as that described for essential oils.

Statistical Analysis.
Experimental results are expressed as mean ± SD. Data were analyzed using ANOVA with SAS software (version 9.2, SAS Institute, Cary, NC, USA), and the means were separated with Duncan's multiple range test. Means with a value of < 0.05 were considered statistically significant.

ZSE Do Not Inhibit FCV-F9 and MNV-1 Infectivity.
We first evaluated the effect of ZSE on RAW 264.7 or CRFK cell viability. Cells were exposed to ZSE at concentrations of 0.00001%, 0.0001%, and 0.001% for 72 h. RAW 264.7 or CRFK cells exhibited >85% cell viability upon treatment with 0.001% ZSE (data not shown). Next, antiviral activity of ZSE was examined using CPE assay. We found that when FCV-F9 or MNV-1 was incubated with up to 0.001% ZSE for 48-72 h, inhibition of cytopathic effects on CRFK or RAW 264.7 cells could not be detected (data not shown). These results suggest that ZSE do not inactivate the foodborne viral surrogates FCV-F9 and MNV-1. In accordance with our results, previous reports have also shown that MNV-1 is unaffected by hyssop and marjoram essential oils at 0.02% [12]. Further, FCV has been shown to survive on inoculated baby-leaf salad during refrigerated storage for 9 days in the presence of clove or zataria essential oils at 10% concentration [11]. However, the titer of FCV was found to be significantly reduced with essential oils from oregano, clove, and zataria at 37 ∘ C [24]. Oregano essential oil and its primary active component, carvacrol, were shown to be effective against MNV [25]. In addition, several research groups have demonstrated antiviral activities of essential oils from tea tree, chamomile, and Lantana grisebachii against HSV-1, HSV-2, and dengue virus type 2, respectively [13][14][15]. The major constituents of essential oils from pericarp of Z. schinifolium are linalool, d-limonene, and sabinene [9]. In this study, the main compound of essential oils from seeds of Z. schinifolium was found to be estragole (42%), which has a sweet-herbaceous anise-fennel type odor (Table 2).

Protective Effect of ZSO against MNV-1 and FCV-F9
Infectivity. RAW 264.7 or CRFK cells showed >87% cell viability upon exposure to 0.01% ZSO. ZSO at a concentration of 0.01% also resulted in 33% and 52% inhibition against FCV-F9 and MNV-1, respectively, via CPE assay. Next, to assess the antiviral mechanism of action of ZSO against FCV-F9 and MNV-1, the antiviral effect of ZSO was examined at different time points during virus infection using plaque assay. Plaque assay can be used to target the multiplication cycle of the calicivirus attachment of the viral protein to the cellular receptor, internalization of virion into the cell, replication of the virus, and release of the mature virion from the cell [26]. Pretreatment can assess the ability to 4 Evidence-Based Complementary and Alternative Medicine inhibit attachment of the virus to cells, and the co-and posttreatments can test inhibition of virus internalization and replication, respectively. For FCV-F9, strong inhibition of FCV-F9 was achieved upon pretreatment with 0.01% ZSO in a dose-dependent manner, resulting in 70% inhibition (Figure 1), whereas 2TU as a positive control showed 25% inhibition at 200 M. Norovirus RNA-dependent RNA polymerase (RdRp) is involved in the synthesis of viral genomic RNA. RdRp, encoded by open reading frame 1 of the norovirus genome, is one of the key targets for the development of novel antiviral agents [27]. Ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3carboxamide) and 2TU, which are nucleoside analogs, inhibit viral replication by blocking the active site of RdRp [28]. In addition, 2TU has a stronger inhibitory effect on MNV-1 replication than ribavirin in RAW 264.7 cells [29]. In the present study, 2TU was used as a positive control. Pretreatment of CRFK cells with 0.01% ZSO resulted in 60% inhibition. The EC 50 values of ZSO were 0.0007% and 0.001% upon pretreatment of FCV-F9 and CRFK cells, respectively. In comparison, co-and posttreatments with 0.01% ZSO resulted in 44 and 43% inhibition, respectively. These results suggest that the inhibitory activity of ZSO was exerted by direct interaction with FCV-F9 virion.
The effect of ZSO on another foodborne viral surrogate, MNV-1, was also examined using plaque assay. Upon pretreatment of MNV-1, ZSO showed a moderate inhibitory effect, reaching 47% at 0.01% concentration, whereas 2TU at 50 M resulted in 24% inhibition (Figure 2). Pretreatment of RAW 264.7 cells with ZSO exhibited 32% inhibition at 0.01%. When ZSO was added simultaneously to the cells with virus or the virus was posttreated with ZSO, plaque formation was inhibited by 36% and 20%, respectively, at a concentration of 0.01%. Likewise, these data suggest that the inhibitory activity of ZSO was exerted by direct interaction with MNV-1 virion.
The antiviral activities of ZSO against FCV-F9 and MNV-1 increased in a dose-dependent manner up to maximal antiviral activity upon pretreatment. Furthermore, FCV-F9 was more sensitive to ZSO than MNV-1, and EC 50 of ZSO upon pretreatment with FCV-F9 was 0.0007%. Similar results have reported that FCV-F9 is more significantly inhibited by black raspberry juice or grape seed extract as compared with MNV-1 [21,30]. In addition, MNV-1 is known to be more resistant to pH, heat [31], and environmental conditions [32] than FCV-F9. Taken together, our results suggest that ZSO can affect FCV-F9 and MNV-1 possibly by blocking virus attachment to host cells. It is therefore possible that ZSO can be used to control foodborne viral infection. Further studies are in progress to characterize active compounds of ZSO and their specific antiviral mechanisms against FCV-F9 and MNV-1.

Fatty Acid Composition of ZSO.
As ZSO showed inhibitory activities against FCV-F9 and MNV-1, we analyzed the fatty acid composition of ZSO. The fatty acid methyl esters (FAMEs) of ZSO were prepared and analyzed by GC/MS. A total of eight fatty acids were identified, representing 98.31% of the total amount (Table 3). Oleic acid (35.36%), linoleic acid (22.6%), palmitic acid (18.5%), and linolenic acid (15.6%) were found to be the major fatty acids in ZSO, followed by palmitoleic acid (3.0%) and stearic acid (2.7%). In addition, arachidic acid and myristic acid were minor fatty acids. Among the identified fatty acids, unsaturated fatty acids constituted 76.6% of the oils.

Conclusions
In the present study, we determined the antiviral effects of oils (ZSO) and essential oils (ZSE) from seeds of Z. schinifolium against foodborne viral surrogates. ZSE, which comprised 42% estragole, showed no inhibitory effect against FCV-F9 or MNV-1. However, maximal antiviral effect was observed upon pretreatment of FCV-F9 or MNV-1 with ZSO, which contained oleic acid, linoleic acid, palmitic acid, and linolenic acid as the major fatty acids. These results suggest that the inhibitory activity of ZSO was exerted by direct interaction with FCV-F9 or MNV-1 virion. Therefore, ZSO may be a food material candidate for control of foodborne viral disease.