KR20150138842A - Antiviral composition against norovirus and feline calicivirus comprising resveratrol as an active ingredient - Google Patents

Abstract

The present invention relates to an antiviral composition comprising red wine against norovirus or feline calicivirus. In addition, the present invention relates to a pharmaceutical composition and a food composition for preventing, alleviating or treating norovirus or feline calicivirus infectious diseases.

Description

Antiviral composition against norovirus and feline calicivirus comprising resveratrol as an active ingredient}

The present invention relates to an antiviral composition against norovirus and feline calicivirus comprising resveratrol as an active ingredient. In addition, the present invention relates to a pharmaceutical composition and a food composition for the prevention, amelioration or treatment of norovirus and feline calicivirus infection diseases.

Food hazard pathogens can be broadly divided into bacteria and viruses. Norovirus is known to be the cause of more than 90% of non-bacterial enteritis in developed countries, and is a major cause of food poisoning that occurs in all age groups around the world. According to a report by the U.S. Centers for Disease Control and Prevention in January 2011, norovirus occupies the first place with 46% of the causes of food poisoning, and long-term nursing homes account for the highest number of food poisoning causes, followed by restaurants, ships, and schools. It can be seen that mainly occurs in group catering facilities. Each year in the United States, about 1 in 6 Americans or 48 million people get food poisoning, 128,000 are hospitalized, and 3,000 die. 90% of people with food poisoning that lead to death are caused by norovirus pathogens. In Korea, the number of patients with norovirus food risk continues to increase, accounting for about 30% of all food poisoning patients except 2009, which was affected by the swine flu since 2006. It is counted as a causative agent.

Norovirus is a virus that can survive in dry and cold winter. It has a physically and chemically stable structure, so it can survive for a long time under conditions of 10 days at room temperature, 30 to 40 days in 10℃ seawater, and below -20℃. have. It can be transmitted to humans through food and water, or it can be transmitted through direct contact with the patient or through the air. Even if only 10 particles of norovirus are ingested, disease can be caused, and even after symptoms disappear, it has a strong infectious power that can be transmitted for two weeks.

Norovirus is a single-stranded positive RNA virus and has a 7.5 kb RNA genome, and among the three primary ORFs, ORF2 and ORF3 have capsid protein genetic information. ORF2 and ORF3 express VP1 and VP2, respectively, and VP1 is a major capsid protein and is involved in viral capsid assembly, and the function of VP2 is still uncertain. Norovirus has a variety of genotypes, but studies on several strains are still limited because viral cell culture is difficult.

In particular, human norovirus proliferates only in the human intestine, and until now, since cell culture is not possible, related studies are insufficient, so there are no vaccines or treatments. However, as the murine norovirus as a surrogate can be cultured in RAW 264.7 cells, related virus research is becoming active. RAW 264.7 cells are ^{mouse leukemic monocyte macrophage cell lines obtained from STAT1 deficient (STAT1 -/-} ) mice with weakened immunity, and are the only cells in which murine norovirus-1 (MNV-1) is cultured. System. STAT1 is one of the gene proteins highly expressed by antiviral interferon signaling.

Regarding the treatment of food poisoning, antibiotics (antibiotic drugs) are excellent in treating food poisoning caused by bacteria, but they have no curative effect on food poisoning caused by viruses. In particular, they can prevent infection of norovirus, which is the main cause of food poisoning caused by viruses. A vaccine or therapeutic agent that can be used has not yet been developed. Therefore, at the time when norovirus infections due to changes in lifestyle patterns in advanced countries are increasing rapidly, discovering natural products that can control noroviruses and ingesting them in a group feeding center can reduce norovirus food poisoning, so development is very meaningful in terms of socioeconomics. It is expected to be.

Meanwhile, feline calicivirus, known as a food poisoning replacement virus, is a virus that causes infection in animals belonging to the feline family, and is characterized by respiratory infections rather than digestive organs. The virus is a very important virus that accounts for about 50% of the causes of respiratory infections in cats. Recently, a vaccine for cats applied to domestic pets has been developed. Therefore, when an effective therapeutic agent for the feline calicivirus is developed, it is expected that it can be widely applied not only to domestic cats, but also to various feline animals such as cheetahs reared in zoos.

It is an object of the present invention to provide an antiviral composition against norovirus or feline calicivirus, including red wine.

Another object of the present invention is to provide a food composition for preventing or improving norovirus or feline calicivirus infection disease, including red wine.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of norovirus or feline calicivirus infection disease, including red wine.

In order to achieve the above object, the present inventors have made intensive research efforts to develop natural biomaterials based on murine norovirus cell culture research. As a result, red wine is superior to red wine through research on inhibitors using cell-based antiviral activity studies. It was confirmed that it has norovirus activity, and it was also confirmed that it has excellent antiviral activity against feline calicivirus, which is an alternative model of the norovirus, to complete the present invention.

Accordingly, an embodiment of the present invention relates to an antiviral composition against norovirus or feline calicivirus, including red wine.

The antiviral composition is in the form of a pharmaceutical composition administered to vertebrates, preferably mammals including humans, or food compositions, tableware, kitchen utensils, daily necessities, cleaning agents for various items, disinfectants, conventional antiviral agents, etc. It can be applied in all known forms.

Another embodiment relates to a pharmaceutical composition for the prevention or treatment of norovirus or feline calicivirus infection disease, including red wine.

Another embodiment relates to a food composition for preventing or improving norovirus or feline calicivirus infection disease, including red wine.

Hereinafter, the present invention will be described in more detail.

In the present invention, the term'red wine' is made from red grapes such as Cabernet Sauvignon, Merlot, Pinot Noir, Syrah, Shiraz, etc. It can be a reddish wine made with some or all of them. Red wine is a mixed extract of various components, specifically catechin, gallic acid, 3.4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid), resveratrol (resveratrol), quercetin-3 -Glucuronide (quercetin-3-glucuronide), caffeic acid, quercetin, myricetin, p-coumaric acid, chlorogenic acid, cyanide It may contain polyphenolic compounds of nidin-3-glucoside and cyanidin-3-rutinoside (FIG. 2), in addition to fermentation or in particular for grapes. Through the maturation process, tannins, minerals such as potassium and zinc, and vitamins such as vitamins B1, B2, and E may be included.

Specifically, the red wine may be red wine containing alcohol or red wine from which alcohol has been removed.

More specifically, red wine from which alcohol has been removed may be prepared by concentrating conventional red wine and then adding tertiary distilled water.

Conventionally, red wine has been reported to have a good effect on cardiac circulatory diseases, cancer, diabetes and neurological diseases (Rastija and Medic-Saric. 2009; Gonzalez-Burgos et al. 2013). In the present invention, it was further revealed for the first time that red wine has excellent antiviral activity against caliciviridae, particularly norovirus and feline calicivirus.

Specifically, the present inventors measured red wine and red wine from which alcohol was removed by pre-treatment, simultaneous treatment, or post-treatment, respectively, to measure the inhibitory activity of viral lysis spot formation, and the red wine and red wine from which alcohol was removed. It was confirmed that the viral lysed plaque formation inhibitory activity of the sample was very excellent in the case of pretreatment with the virus and simultaneously treatment (FIGS. 7 and 8). In addition, red wine and alcohol-removed red wine strongly reduced RdRp mRNA, which is involved in the replication of MNV-1 and FCV-F9, in a concentration-dependent manner, thereby confirming an important inhibitory activity in the initial mechanism of virus replication (Fig. 13 and Fig. 14).

Accordingly, a specific embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of norovirus or feline calicivirus infection disease comprising red wine. The infectious disease may preferably be food poisoning or enteritis caused by norovirus, and preferably may be respiratory disease of feline animals caused by feline calicivirus.

In addition, specifically, one embodiment of the composition of the present invention may exhibit the activity of inhibiting cell adhesion, intracellular invasion, or replication of virus of norovirus or feline calicivirus.

In the present specification,'treatment' refers to relief or improvement of symptoms, reduction of the range of the disease, delay or alleviation of disease progression, improvement, alleviation or stabilization, partial or complete recovery, prolongation of survival, and other beneficial treatment results, etc. It is used in a sense that includes all of.

The composition for the prevention or treatment of norovirus or feline calicivirus infection disease according to an embodiment of the present invention may contain red wine alone, but in addition to the formulation, method of use and purpose of use, A pharmaceutically acceptable carrier, excipient, or diluent may be further included according to a conventional method used. The composition may be administered orally or parenterally according to a conventional method. For example, when administered orally, it may be provided in a dosage form such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, or the like.

When the composition for preventing or treating norovirus or feline calicivirus infectious diseases is provided as a mixture in which other ingredients other than the red wine are added, the composition for preventing or treating calicivirus infectious diseases is based on the total weight of the composition. The red wine may be included in an amount of 0.001% to 99.9% by weight, preferably 0.1% to 99% by weight, and more preferably 1% to 50% by weight.

A preferred dosage may be an amount suitable for the treatment or prevention of a subject and/or a disease, which is the age, sex, general health condition and weight of the subject to be administered, the type and severity of the disease, the type of formulation, and the composition contained. It can be adjusted according to various factors, including the type and content of other ingredients, the secretion rate of the composition, the route and duration of administration, etc., and may be appropriately selected by those skilled in the art, but preferably 50 to 100 mg per day for an adult Can be administered.

In the present invention, the'norovirus' is a virus belonging to the family Caliciviridae (Caliciviridae) taxonomically and having a single-stranded RNA, and is also called a small round structured virus (SRSV) due to its appearance, but recently became a norovirus. It is an officially named food poisoning virus. Norovirus food poisoning is caused by vomit, feces, and body contact of an infected patient. If foods that can be eaten immediately such as drinking water, fish and shellfish, fruits, vegetables, etc. are contaminated, even 10-100 small particles can cause disease. Because it is small and floats in the air in the form of an aerosol, group infection occurs rapidly, which poses a great threat to public health. Long-term nursing homes had the highest number of norovirus food poisoning outbreaks, followed by restaurants, ships, and schools, indicating that norovirus food poisoning mainly occurs in group feeding facilities.

'Feline calicivirus' belongs to the family calicivirus along with Norovirus. It has a single-stranded RNA and its genetic, biochemical, and physicochemical properties are similar to norovirus, so it is a replacement for norovirus. It is being used as a model.

Another embodiment of the present invention relates to a food composition for preventing or improving norovirus or feline calicivirus infection disease comprising red wine. The food composition may further include a suitable carrier, excipient, and diluent commonly used in its preparation.

In the present specification, the term food refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain amount of processing process, and food as a general meaning , Food additives, health functional foods, beverages and beverage additives are all intended to be included.

In the present invention, a beverage refers to a generic term for drinking to quench thirst or to enjoy the taste, and is intended to include a functional beverage. In addition to including the red wine as an essential component in the indicated ratio, the beverage is not particularly limited to other components, and may contain various flavoring agents or natural carbohydrates as an additional component, as in ordinary beverages. Examples of the above natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides, such as dextrin, cyclodextrin, and the like, and conventional sugars, and These are sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. It may contain additional flesh.

In the food composition according to an embodiment of the present invention for the purpose of preventing or improving norovirus or feline calicivirus infection disease, the amount of red wine is 0.00001% to 100% by weight of the total food weight, preferably It may be included in 0.00001% to 50% by weight, and in the case of a beverage composition, preferably 0.01 to 100% by weight, more preferably 1 to 10% by weight, specifically, the volume of the whole food 100 ml It may be included in a ratio of 0.01 g to 100 g, preferably 1 g to 10 g, but is not limited thereto.

The composition containing red wine of the present invention is very excellent in inhibiting the adhesion, proliferation and replication of calicivirus, so it can be used as an effective therapeutic agent for norovirus and feline calicivirus for which no vaccine or therapeutic agent has been developed yet. In addition to being able to, there is an advantage that stability is recognized.

1 shows a schematic diagram for measuring antiviral activity of red wine, red wine from which alcohol has been removed, and resveratrol samples.
2 shows the results of analysis of a polyphenol compound in red wine.
3 shows the cytotoxicity results of red wine and alcohol-removed red wine on the CRFK cell line according to the concentration.
4 shows the cytotoxicity results of red wine and alcohol-free red wine on RAW 264.7 cell lines according to concentration.
5 shows the cytotoxicity results of resveratrol on the CRFK cell line according to the concentration.
6 shows the cytotoxicity results of resveratrol on the RAW 264.7 cell line depending on the concentration.
FIG. 7 shows the inhibitory activity of viral lysis plaque formation when a sample of red wine or alcohol-removed red wine is pretreated with FCV-F9 and a sample of red wine or alcohol-removed red wine is treated with FCV-F9. It was measured and shown.
FIG. 8 shows the inhibitory activity of viral lysis spot formation in the case of pretreatment of a red wine sample from which red wine or alcohol is removed from MNV-1 and treatment of a red wine sample from which red wine or alcohol is removed with MNV-1. It was measured and shown.
Figure 9 shows by measuring the lysed plaque formation inhibitory activity of FCV-F9 when a CRFK cell line is pretreated with red wine or red wine samples from which alcohol has been removed, and when red wine or red wine samples from which alcohol has been removed are post-treated. will be.
FIG. 10 is a measurement of the inhibition activity of MNV-1 in the case of pretreatment of red wine or red wine sample from which alcohol is removed in RAW264.7 cell line, and post-treatment of red wine or red wine sample from which alcohol is removed. Is shown.
11 is a case in which a resveratrol sample was pretreated in a CRFK cell line, a resveratrol sample was pretreated with FCV-F9, a case where FCV-F9 and a resveratrol sample were simultaneously treated, and a case where a resveratrol sample was treated after infection with FCV-F9, respectively. It is shown by measuring the inhibitory activity of lysis plaque formation
12 is a case where a resveratrol sample was pretreated in RAW264.7 cell line, a resveratrol sample was pretreated with MNV-1, a case where MNV-1 and a resveratrol sample were simultaneously treated, and a resveratrol sample was treated after infection with MNV-1. In each case, it is shown by measuring the activity of inhibiting lysis plaque formation.
FIG. 13 shows the results of RT-PCR analysis after adding red wine, alcohol-removed red wine, and resveratrol by concentration while infecting RAW 264.7 cells with MNV-1.
14 shows the results of performing RT-PCR analysis after infecting CRFK cells with FCV-F9 and simultaneously adding red wine, alcohol-removed red wine, and resveratrol by concentration.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of red wine and resveratrol samples

Red wine (12.5% alcohol, v/v) was used in the experiment using Chateau Bel Air, Bordeaux superieur 2007, (France). Alcohol-removed red wine is Boban, N et.al., 2010 (Antimicrobial effects of wine: separating the role of polyphenols, pH, ethanol, and other wine components.Journal of Food Science 75 (5), 322-326. According to the method described in ), the volume of the red wine used in the experiment was concentrated to about 50% using a rotary evaporator at 30° C., and then tertiary distilled water equal to the lost volume was added to prepare it. Resveratrol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate buffered saline (PBS) and used in the experiment.

As a sample for evaluating the activity against murine norovirus and feline calicivirus in Examples to be described later, the red wine, alcohol-removed red wine and resveratrol were filtered through a 0.2 μm syringe filter, It was dispensed, stored in a cryogenic freezer, and used.

Example 2. Analysis of Polyphenol Compounds in Red Wine

2-1. Experiment method

Polyphenols of red wine using the method of Cantos et al. (2000) (Effect of 　postharvest ultraviolet irradiation on resveratrol and other phenolics of cv.Napoleon table grapes.Journal of Agricultural Food Chemistry 48 (10), 4606-4612.) Compound analysis was performed. LCMS-8040 Liquid Chromatograph Mass Spectrometer (Shimazu, Japan) and Shim-pack XR-ODSII column (2.0 x 75 mm, 2.2 μm, Shimazu) equipped with Nexera UHPLC system (Shimazu) For the mobile phase, solvent A (0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid) were used, and solvents A and B were 95:5 for 1.5 minutes, and then 65 for 8 minutes. :35, and the ratio of 0:100 was adjusted for 10 minutes, and the flow rate of the mobile phase was 0.4 mL/min, and the detection wavelength was 280 nm.

2-2. result

A total of 90.39 μg/mL of polyphenol compound was identified in red wine, and the identified polyphenol compound was 46.04 μg/mL of catechin, 19.35 μg/mL of gallic acid, 6.06 μg/mL of 3.4. -Dihydroxybenzoic acid (3,4-dihydroxybenzoic acid), 5.27 μg/mL resveratrol, 5.20 μg/mL quercetin-3-glucuronide, 4.49 μg/mL Caffeic acid, 2.28 μg/mL quercetin, 0.92 μg/mL myricetin, 0.72 μg/mL p-coumaric acid, 0.04 μg/mL Chlorogenic acid, 0.01 μg/mL of cyanidin-3-glucoside, and 0.01 μg/mL of cyanidin-3-rutinoside (FIG. 2) ).

Example 3. Antiviral activity evaluation

3-1. Virus culture

MNV-1, a murine norovirus as a substitute for human norovirus ((sold from Dr. Herbert Virgin (Washington University School of Medicine, St Louis, USA)), FCV-F9 (VR-782, ATCC, Manassas, VA, USA) were used as host cells for each virus, in the case of murine norovirus, a RAW 264.7 cell line (ATCC TIB-71), and in the case of FCV, a CRFK cell line (ATCC CCL-94) was used. Antibiotic-Antimycotic (Gibco, Grand Island, NY), 10% (v/v) fetal bovine serum (FBS), Hyclone Lab, Logan, Utah) added Dulbecco's modified eagle medium (DMEM) (Gibco, Grand Island, NY) as a basic medium was subcultured at intervals of 24-72 hours _{in a 37°C, 5% CO 2 incubator.}

RAW 264.7 and CRFK cells were aliquoted into a tissue culture plate at 2.0x10 ⁶ and 2.0x10 ⁵ cells/ml, respectively, and cultured in an incubator at 37°C and 5% CO ₂ ^{for 24 hours, and MNV-1 5.0x10 5} PFU/ml, FCV-F9 2x10 ⁶ PFU/ml was diluted according to the multiplicity of infection (MOI) under the experimental conditions and inoculated, and then _{incubated in an incubator at 37°C and 5% CO 2} for 1 hour to incubate each virus. Infected.

3-2. Cytotoxicity measurement

Red wine to be used in antiviral experiments, red wine from which alcohol is removed by measuring cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The cytotoxicity of wine and resveratrol samples was measured.

Specifically, RAW 264.7 and CRFK cells were seeded into 96 wells at 1.5x10 ⁶ cells/ml and 2.0x10 ⁵ cells/ml, respectively, and after 24 hours, each sample was added by concentration for 1-72 hours. Until 37 ℃, 5% CO ₂ was incubated in an incubator. Thereafter, MTT was added at a final concentration of 0.5 mg/mL, and after 2 hours, absorbance was measured at 570 nm with a UV-Vis spectrophotometer. The cell viability was calculated and expressed as a relative% to the non-added group.

As a result of performing the above analysis, red wine and red wine from which alcohol was removed were not cytotoxic to CRFK cells and RAW 264.7 cells at 10% concentrations, respectively, and resveratrol was at 100 μM and 50 μM concentrations for CRFK cells and RAW 264.7 cells. It was found that each was not toxic, and it was confirmed that it can be used very safely at each corresponding concentration (FIGS. 3 to 6).

3-3. Determination of antiviral activity by yongbacilli test

Red wine and red wine samples from which alcohol has been removed were added at different time points in the concentration range confirmed to be non-toxic to cells according to 3-2 above, and then the antiviral activity according to the time point of addition of the sample was determined as a candidate substance. The difference between the added and the non-added group was compared and analyzed (calculated as 100% of the degree of lysis of the non-added group).

(1) Measurement of antiviral activity during sample pre-treatment on cells

Red wine, red wine from which alcohol has been removed, or resveratrol sample and cells or viruses are mixed and incubated for 1 hour, and then the degree of virus inhibition is checked. The effect on the virus was confirmed.

First, the effect of pre-treatment of red wine, alcohol-removed red wine, or resveratrol samples on cells was observed.

In the pre-treatment of cells, RAW 264.7 cells and CRFK cells were dispensed into 24 wells, respectively, and after incubating the sample and the cells for 1 hour, the sample was removed, and the virus was not mixed with the sample. Infected.

(2) Measurement of antiviral activity during sample pre-treatment on virus

Each of RAW 264.7 and CRFK cells was dispensed into 24 wells (DMEM medium containing 10% FBS, 1.5×10 ⁶ viable cells were dispensed per well), mixed with the sample as described above, and cultured for 1 hour MNV- 1 and FCV-F9 virus were infected with the dispensed cells, followed by incubation at 37° C. for 1 hour. Thereafter, 1.5% (w/v) agarose, 5% (v/v) FBS and 1% (v/v) PS (penicillin-streptomycin) were The included DMEM medium was added and cultured in an incubator at 37° C. for 42 hours, followed by staining with 0.5% (v/v) crystal violet to measure the lysed plaque.

(3) Antiviral activity measurement when viral infection and sample are treated simultaneously (co-treatment)

Each of the RAW 264.7 and CRFK cells was distributed and cultured in 24 wells as in (1) above, and virus and samples were simultaneously added at the concentrations described above, and cultured at 37°C for 1 hour. Afterwards, DMEM medium containing 1.5% (w/v) agarose, 5% (v/v) FBS and 1% (v/v) PS (penicillin-streptomycin) was added and incubated in an incubator at 37° C. for 42 hours. By staining with 0.5% (v/v) crystal violet, the lysing spot was measured.

(4) Measurement of antiviral activity during post-treatment of samples after virus infection

Each of the RAW 264.7 and CRFK cells was distributed and cultured in 24 wells as in (1) above, and after infection with the virus, the cells were cultured at 37° C. for 1 hour. After completely removing the virus, the sample was cultured at 37° C. for 1 hour by adding DMEM medium added at the concentrations described above, and then the medium was completely removed. After adding DMEM medium containing 1.5% (w/v) agarose, 5% (v/v) FBS and 1% (v/v) PS (penicillin-streptomycin), cultured in a 37°C incubator for about 42 hours. Then, the lysing spot was measured by staining with 0.5% (v/v) crystal violet.

(5) Experiment result

The above (1) to (4) were repeated three times in consideration of accuracy, and the plaque counting result was analyzed by SAS software and ANOVA, and the result was derived by Duncan's multiple range test. The resulting data have a reliability of error p<0.05.

The results of measuring virus inhibitory activity using red wine and red wine samples from which alcohol has been removed by the above-described method are shown in FIGS. 7 to 10. After dispensing the cells, as a result of measuring the activity of inhibiting lysis plaque formation of the viruses in the cases (2) and (3) for each of the viruses of FCV-F9 and MNV-1, the red wine, red wine from which alcohol was removed It was found that the viral lysed plaque formation inhibitory activity of the wine sample exhibited high inhibitory activity in both the cases (2) and (3) (FIGS. 7 and 8 ). However, as a result of measuring the activity of inhibiting lysis plaque formation of the virus in the cases (1) and (4) for each of the viruses of FCV-F9 and MNV-1 (FIGS. 9 and 10), the red wine and alcohol were removed. The red wine samples showed low inhibitory activity in both cases (1) and (4) above. From this, the present inventors confirmed that red wine and alcohol-removed red wine samples exhibit important inhibitory activity during the life cycle of norovirus, particularly for viral cell adhesion and internalization mechanisms.

Using the resveratrol sample, the results of measuring the viral lysed plaque formation inhibitory activity by the above-described method are shown in FIGS. 11 and 12. As a result of measuring the viral lysis plaque formation inhibitory activity in the cases (1) to (4) for each of the viruses of FCV-F9 and MNV-1, the viral lysis plaque formation inhibitory activity of the resveratrol sample was shown in (2). ) And (3) showed high inhibitory activity. From this, the present inventors confirmed that the resveratrol sample exhibits an important inhibitory activity during the life cycle of norovirus, particularly for the mechanism of viral cell adhesion and internalization. 2-TU (2-thiouridine) (Berry & Associates, Inc., USA) in FIGS. 7 to 12 was used as a positive control.

3-4. Measurement of antiviral activity by RT-PCR

RAW 264.7 cells or CRFK cells were spread on 12-well plates containing DMEM, 5% FBS, and 1% PS (penicillin-streptomycin) at a ^{density of 1×10 6} /well or 1×10 ⁵ /well, and MNV-1 (MOI 0.01) or FCV-F9 (MOI 0.001), respectively, while simultaneously infecting cells with red wine (0.1, 1 and 10%), alcohol-removed red wine (0.1, 1 and 10%) and resveratrol (1, 10). And 100 μM) were each added and reacted for 2 hours. After the reaction, a medium was added and cultured for 24 hours, and viral RNA was extracted from the cells using an RNeasy kit (QIAGEN, Valencia, CA, USA).

Oligo dT, random hexamer, RdRp primers (Bioneer, Daejon, Korea, primer sequences) for each of MNV-1 and FCV-F9, MultiScribe ^TM After cDNA was synthesized using reverse transcriptase (RT-Master Mix kit, ABI, USA), RT-PCR analysis was performed. The primers used are as follows.

RdRp forward primer for MNV-1 (SEQ ID NO: 1); 5'-GGAATTCCATATGCTTCCCCGCCCCTCAGGCACCTAT-3'

RdRp reverse primer for MNV-1 (SEQ ID NO: 2); 5'-ATAAGAATGCGGCCGCATCCTCATTCACAAAGACTGCTGA-3'.

RdRp forward primer for FCV-F9 (SEQ ID NO: 3); 5'-CGGTGTTTGATTTGGCC-3'

RdRp reverse primer for FCV-F9 (SEQ ID NO: 4); 5'-GACACCATCATCCCCGTA-3'

GAPDH (MNV-1) and beta-actin (FCV-F9) genes were used as endogenous controls. The forward and reverse primers of GAPDH (MNV-1) and beta-actin (FCV-F9) are as follows.

Forward primer for GAPDH (MNV-1) (SEQ ID NO: 5); 5'-AACGACCCCTTCATTGAC-3'

Reverse primer for GAPDH (MNV-1) (SEQ ID NO: 6); 5'-TCCACGACATACTCAGCAC-3'

forward primer for beta-actin (FCV-F9) (SEQ ID NO: 7); 5'-ACGAGCGGTTCCGC-3'

reverse primer for beta-actin (FCV-F9) (SEQ ID NO: 8); 5'-GATCTTGATCTTCATCGTGC-3'

For the amplification of cDNA, the first reaction was performed at 95℃ for 10 minutes, 95℃ for 15 seconds (denaturation), 60℃ for 1 minute (annealing), 72℃ for 1 minute (extension), 35 cycles, and then at 72℃. After reacting for 5 minutes (final extension), 1.5% agarose gel electrophoresis was performed.

13 and 14 show the results of analyzing the inhibitory activity of red wine, alcohol-free red wine, and resveratrol samples on RdRp mRNA involved in the replication of MNV-1 and FCV-F9 by RT-PCR. 13 and 14, it was found that red wine, red wine from which alcohol was removed, and resveratrol strongly reduced RdRp mRNA involved in the replication of MNV-1 and FCV-F9 in a concentration-dependent manner. From this, it was confirmed that red wine, red wine from which alcohol was removed, and resveratrol samples exhibited an important inhibitory activity in the early mechanism of replication of the virus, especially during the life cycle of norovirus.

<110> CHUNG, MI-SOOK <120> Antiviral composition comprising red wine <130> DPP20140243KR <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> MNV-1 RdRp forward primer <400> 1 ggaattccat atgcttcccc gcccctcagg cacctat 37 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> MNV-1 RdRp reverse primer <400> 2 ataagaatgc ggccgcatcc tcattcacaa agactgctga 40 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> FCV-F9 RdRp forward primer <400> 3 cggtgtttga tttggcc 17 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FCV-F9 RdRp reverse primer <400> 4 gacaccatca tccccgta 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH(MNV-1) forward primer <400> 5 aacgacccct tcattgac 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH(MNV-1) reverse primer <400> 6 tccacgacat actcagcac 19 <210> 7 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> beta-actin(FCV-F9) forward primer <400> 7 acgagcggtt ccgc 14 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin(FCV-F9) reverse primer <400> 8 gatcttgatc ttcatcgtgc 20

Claims (6)

An antiviral composition for norovirus and feline calicivirus comprising resveratrol as an active ingredient. The antiviral composition according to claim 1, wherein the composition is for preventing or treating food poisoning or enteritis caused by norovirus. The antiviral composition according to claim 1, wherein the composition is for preventing or treating respiratory diseases of feline animals by calicivirus. The antiviral composition according to claim 1, wherein the composition inhibits cell adhesion, intracellular entry or viral replication of norovirus or feline calicivirus. A food composition for prevention or improvement of norovirus and feline calicivirus infectious disease comprising resveratrol as an active ingredient. A pharmaceutical composition for preventing or treating a norovirus or feline calicivirus infection disease comprising resveratrol as an active ingredient.

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