KR20140042011A - Antiviral composition for calicivirus or orthomyxovirus comprising rubus coreanus and/or morus alba active materials - Google Patents

Abstract

The present invention relates to an antiviral composition against calicivirus and orthomyxovirus. Since the composition in the present invention has an excellent activity for inhibiting the propagation and replication of the calcivirus and orthomyxovirus, the composition can be used as an effective drug against norovirus and feline calicivirus, for which no vaccine or treatment exists at present time. Moreover, the composition is safe since natural plants are used as active ingredients for an antiviral drug against avian influenza virus and human influenza virus belonging to Orthomyxoviridae, thereby being expected to reduce an occurrence of norovirus-induced food poisoning and be a very significant development socioeconomically as an alternative material against resistant viruses and new mutant viruses.

Description

ANTIVIRAL COMPOSITION FOR CALICIVIRUS OR ORTHOMYXOVIRUS COMPRISING RUBUS COREANUS AND / OR MORUS ALBA ACTIVE MATERIALS}

FIELD OF THE INVENTION The present invention relates to antiviral compositions against calicivirus or Oscomycin virus, and more particularly to antiviral compositions against calicivirus or orthomyxia virus comprising brambles and / or audi as an active ingredient.

Influenza virus

Influenza virus kills 25-500,000 people every year, infects 10-40% of children worldwide and causes serious global illnesses that cause national economic losses. After the Spanish flu pandemic in 1918, influenza pandemic occurred in decades, including the Asian flu in 1957 and the Hong Kong flu in 1968.In 2009, the emergence of the H1N1 influenza virus (2009pdm) affected 270,000 people worldwide, and more than 3,200 were infected. Died. Despite the pandemic in the southern hemisphere due to the possibility of new outbreaks of highly contagious mutants, social waves and concerns have not diminished.

Influenza is an acute respiratory disease with chills, fever, myalgia, and cough, which is usually transmitted by human-human propagation through the aerosol, which is released into the air through an infected person's cough or sneeze. It can also be transmitted from bird droppings, etc. Compared with the common cold, cold is a respiratory disease caused by infections such as adenovirus, rhinovirus, cocksackievirus, and coronavirus.

Influenza infection is caused by the influenza virus belonging to the orthomyxoviridae, and since the antigenic mutation is relatively easy as an RNA virus, repeated reinfection in the infected population is possible. Influenza viruses that cause human infection are A and B types, and viral antigen mutations are determined by hemagglutinin (NA) and neuraminidase (NA) on the surface of the virus. Genetic combinations of viral surface proteins HA and NA produce H1-H16 and N1-N9 subtypes, of which subtypes of the human influenza A virus are H1, H2, H3, N1, and N2. Determined by other combinations. The reason why influenza B does not cause pandemic is that unlike type A, it only infects humans, so it does not obtain genes from animal viruses.

Influenza antiviral agents, oseltamivir and amantadine, act on the NA and M2 channels, respectively. Although oseltamivir and zanamivir are still effective in pig-derived 2009pdm H1N1, H5N1, H1N1, and zanamivir resistant to oseltamivir have already been reported. It is becoming.

The life cycle of influenza viruses takes approximately 2-3 days in human cells. Human HA receptors recognized by viral HA are terminal sialic acid (SA) present in respiratory epithelial cells and bind a-2,6 to galactose (Gal) exposed to glycoproteins or glycolipids on the surface of human cells. However, avian influenza virus HA generally recognizes SAa-2,3-Gal. HA must bind to human receptors for the virus to successfully invade the cell, but at the same time the virus particles must enter the cell. That is, the membrane fusion according to the structural change of the influenza virus HA is achieved, and the cell must be invaded by endocytosis.

HA, which plays such an important role, is subjected to selection pressure due to the attack of the human immune system and is very important for virus evolution compared to other proteins. However, antiviral agents targeting HA have not been developed yet. Vaccination due to high mutation rate of HA is difficult and development of new antiviral agent is urgent as resistance virus to existing antiviral agents such as Tamiflu and relenza based on NA enzyme inhibitor is appeared. .

Norovirus

Food-borne pathogens Noroviruses are known to cause more than 90% of non-bacterial enteritis in developed countries and are the leading cause of food poisoning in all age groups around the world. According to a January 2011 report by the US Centers for Disease Control and Prevention, norovirus was the number one cause of food poisoning, with 46% .Norovirus food poisoning facilities were the highest in long-term nursing homes, followed by restaurants, ships, and schools. Can be seen mainly in group feeding facilities. Each year in the United States, 48 million people, or 1 in 6 Americans, get food poisoning, 128,000 are hospitalized and 3,000 die. 90% of food poisoning deaths are due to norovirus pathogens (CDC December 15, 2010). In Korea, the number of patients with norovirus food risk has increased steadily, accounting for about 30% of all food poisoning cases except 2009, which was affected by the H1N1 flu since 2006. It is counted in cause.

Norovirus is a virus that can survive dry and low temperature in winter. It has a physical and chemically stable structure, so it can survive for 10 days at room temperature, 30-40 days in 10 ℃ seawater, and below 20 ℃ below zero temperature. have. It can be transmitted to humans through food and water, and can also be transmitted through direct contact with the patient or through the air. Ingestion of 10 particles of norovirus can cause food poisoning and has a strong infectivity enough to allow infection for 2 weeks after symptoms disappear.

Norovirus is a single strand positive RNA virus with a 7.5 kb RNA genome, and among the three major ORFs, ORF2 and ORF3 contain capsid protein genetic information. ORF2 and ORF3 express VP1, VP2, respectively, VP1 is a major capsid protein involved in viral capsid assembly, and the function of VP2 is still uncertain. Noroviruses have a variety of genotypes, but research on many strains is still limited because of the difficulty in culturing viral cells.

In particular, human norovirus grows only in the human intestine, and since cell culture is impossible to date, there is insufficient research to develop vaccines or therapeutics. However, as a surrogate, murine norovirus can be cultured in RAW 264.7 cells, related viral research is being actively conducted. RAW 264.7 cells are a mouse leukemic monocyte macrophage cell line from mice with weakened STAT1-deficient (STAT1-/-) immunity, the only cells cultured with murine norovirus-1 (MNV-1) System.

Antibiotic drugs have an excellent effect on the treatment of food poisoning caused by bacteria, but have no therapeutic effect on viral food poisoning. In particular, antibiotic drugs can prevent the infection of norovirus, a major cause of food poisoning caused by viruses. No vaccines or therapeutics have yet been developed. Therefore, when norovirus infections are rapidly increasing due to the change in living patterns of advanced countries, the discovery of natural products that can control the norovirus and ingesting them in the group feeding center can reduce norovirus food poisoning. It is expected to be. The demand for natural foods is expected to explode due to the increase in the elderly population due to rapid aging and the change in dietary habits. This is because natural foods obtained in the natural state have fewer side effects than processed foods. to be.

On the other hand, feline calicivirus known as a food poisoning replacement virus (feline calicivirus) is a virus that causes infection of animals belonging to the feline, characterized by respiratory infections rather than digestive organs. The virus is a very important virus that accounts for about 50% of the causes of cat respiratory infections. Recently, a vaccine for cats for home pet cats has been developed. Therefore, when developing an effective treatment for the cat calcivirus, it will be widely applicable to various cats such as cheetahs raised in zoos as well as domestic cats.

Accordingly, the present invention has been made to solve the problems of the prior art, there is no fear of side effects or toxicity, and the inhibitory effect on the respiratory tract infection virus caused by norovirus, cat calcivirus and influenza virus An object of the present invention is to provide an antiviral composition comprising an excellent natural product as an active ingredient.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, and searched for native native plants in influenza virus, calcivirus, and norovirus through screening inhibitors that inhibit cell-based virus activity. , Especially Bokbunja ( Rubus Coreanus ) and Audi ( Morus) alba ) was confirmed to have excellent antiviral activity to complete the present invention.

Accordingly, a preferred embodiment of the present invention provides an antiviral composition for Calicivirus or orthomyxovirus comprising at least one selected from the group consisting of bokbunja and Audi as an active ingredient.

The antiviral composition is usually in the form of a pharmaceutical composition administered to vertebrates, preferably mammals, including humans, or is commonly used in food compositions, tableware, kitchen utensils, household goods, and various other objects such as cleaning agents, disinfectants, and conventional antiviral agents. All known forms of application are possible.

Another preferred embodiment of the present invention provides a composition for the prevention or treatment of a calcivirus or orosomicovirus infection disease comprising at least one selected from the group consisting of bokbunja and audi as an active ingredient.

Another preferred embodiment of the present invention provides a food composition for preventing or ameliorating a calcivirus or orosomicovirus infection disease comprising at least one selected from the group consisting of bokbunja and audi as an active ingredient.

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

Natural products

Since Korea is a treasure trove of various native plants, and has accumulated hundreds of years of experience, mainly from oriental medicine, it is in a very advantageous position where information and resources necessary to develop natural food materials exist more than any other country. However, in Korea, many native plants have not yet completed activity-based antiviral screening. In particular, domestic native plants such as Bokbunja and Audi have been found to be very useful in recent years, as well as barren native plants have not yet been actively studied for viral disease screening. Compounds that will be isolated and isolated from bokbunja and by-products or juices with antiviral activity will be high value materials that will be of great interest to multinational pharmaceutical companies.

In particular, the demand for natural food materials is expected to explode due to the increase in the elderly population due to the rapid aging of society and the increase of chronic disease patients due to the change in diet. This is because the natural material obtained in the natural state has relatively fewer side effects than the compound material. Mortality from norovirus and influenza virus infections is known to be very high in older adults and immunocompromised patients. It is expected that the industrial utilization will be very high if the anti-norovirus or anti-influenza virus natural material using by-products or juices of bokbunja and Audi can be directly used in group feeding facilities, or if it is used as a control material for the elderly and immunocompromised patients. .

Bokbunbun and Audi

Bokbunja ( Rusbus) coreanus ) is the main production area of Jeju Island and southern part of Korea. It harvests dark red fruits in early summer and eats them. In oriental medicine, it is used as tonic (The oriental medicine dictionary, 2001). Bokbunja contains various polyphenolic compounds, which are known to have antioxidant activity, prevent cardiovascular disease, prevent and treat anxiety and depression, strengthen urinary function, diabetic complications, and anti-helicobacter activity. Bokbunja fruits contain quercetin and various phenolic acids and anthocyanin pigments cyanidin-3-glucoside (C3G) and cyanidin-3-rhamnoglucoside (cyanidin-3- rhamnoglucoside) (Korean J. Pharmacogn. 1996). Bokbunja fruits are often used as raw fruits, but most are juiced or used as a raw material for alcohol. Juices and health supplements using bokbunja seeds are being produced (J. Cleanberry), but little research has been conducted to apply them as antiviral materials.

Recent studies have reported antioxidant activity, anticancer effect, and antibacterial activity against Escherichia coli and Salmonella, etc., and herpes simplex virus type 1 and hepatitis B virus. It has been reported to have antiviral effects (Phytother Res. 2001).

Audi Morus alba ), which contains a large amount of pigments such as cyanidin-3-glucoside and rutin, and contains bioactive substances such as resveratrol, which are involved in antioxidant activity (Korean J. Crop Sci. 2011), which is used as raw material for fermented broth, vinegar, vinegar, liquor and cosmetics, and its output is increasing every year.

The antiviral action of plant extracts containing various polyphenolic compounds has been suggested so far, but the peculiarity is that the antiviral action of single polyphenolic compounds isolated does not show antiviral activity, whereas the plant extracts containing various phenolic compounds have high effect. Is the point. It is predicted that antiviral action is exhibited by synergistic effect of each phenolic compound (J. Med. Plants Res. 2009).

On the other hand, the antiviral natural product material may preferably be a juice and by-products of the vegetable material as the main raw material. In this regard, bokbunja or audi have a large amount of by-products (seeds, shells, etc.) that are discarded after making juice and liquor, and seeds make up the most part. Seeds account for about 14% in the fruit of Bokbunja, and given the continuous increase in juice and alcohol consumption of Bokbunja or Audi, it can be seen that the amount of by-products discarded after juice and sake production is also increasing. Therefore, there is an advantage that the raw material is easy to obtain and very economical.

In the present invention, the bokbunja ( Rubus coreanus ) and Audi ( Morus) alba ), particularly by-products or juices thereof, have excellent antiviral activity against influenza viruses belonging to the calciviridae, norovirus and feline calicivirus, and the orthomyxoviridae. First revealed.

Therefore, a preferred embodiment of the present invention is an antiviral composition for a calcivirus or orosomicovirus comprising at least one selected from the group consisting of bokbunja and audi as an active ingredient, the prevention of a calcivirus or orosomicovirus infection disease Or a therapeutic composition and a food composition for preventing or ameliorating a calicivirus or orosomicovirus infection disease.

As used herein, "treatment" refers to alleviating or ameliorating symptoms, reducing the extent of disease, delaying or alleviating disease progression, ameliorating, reducing or stabilizing a disease state, partial or complete recovery, prolonging survival, or any other beneficial treatment outcome. It is used to mean all.

Bokbunja and Audi may preferably be included in the form of juices or extracts obtained from one or more selected from the group consisting of fruits, shells and seeds thereof.

The juices of bokbunja and audi may preferably be juices whose fruits are squeezed by using a machine such as a juicer or by applying physical force by hand.

In addition, the extracts of Bokbunja and Audi are preferably those obtained by extracting their fruits or by-products using various methods used in the preparation of conventional natural extracts such as steam extraction, hot water extraction, ultrasonic extraction, solvent extraction, or reflux cooling extraction. It may be, preferably an ultrasonic extract, a solvent extract or a hydrothermal extract, more preferably may be a hydrothermal extract.

When the ultrasonic extraction is preferably carried out for 15 to 35 minutes at 10 ℃ to 35 ℃ 30 to 70 kHz. If it is less than the above range, there is a problem that the extraction amount of useful functional components such as polyphenol component is not sufficient, and if the above range is exceeded, the effect of increasing the amount of the effective active ingredient extraction is insignificant while the manufacturing cost increases, which is not preferable.

Solvent extraction may preferably be carried out using a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms (e.g. methanol, ethanol, propanol, butanol, etc.), acetic acid and mixtures thereof, more preferably By using an ethanol aqueous solution, preferably 65 to 75% (v / v) of ethanol, it is possible to extract a greater amount of components such as polyphenols.

In the case of extracts obtained from the by-products of bokbunja and audi, preferably by-products, that is, may be obtained through the extraction step after the production of a powder form by applying a physical force to the seed or shell.

Juice or extract of the bokbunja and / or Audi can be prepared by further performing a filtration, heat treatment, concentration and / or lyophilization process. By performing the filtration process it is possible to remove the residues and the like remaining in the extract, and to perform the heat treatment to stabilize the juice by destroying the enzyme, it is possible to remove the moisture by performing the concentration and / or lyophilization. .

An antiviral composition for Calicivirus or orosomicovirus according to an embodiment of the present invention is characterized by exhibiting antiviral activity by inhibiting the proliferation and replication of the Calicivirus or orosomicovirus.

The calciviruses are preferably noroviruses or feline caliciviruses, and the orthomyxoviruses are preferably various influenza viruses, such as avian influenza or human influenza.

Norovirus is a food poisoning virus that belongs to the class of Caliciviridae and is also called small round structured virus (SRSV) with single stranded RNA and recently officially named norovirus. In addition to the norovirus, feline calicivirus, a member of the Calicivirus family, has single-stranded RNA, and its genetic, biochemical and physicochemical properties are similar to those of the norovirus and are used as an alternative to the norovirus. . Norovirus is a pathogen that causes acute gastroenteritis after human cell infection, and thus no effective treatment or vaccine has been developed.

Influenza viruses consist of eight negative strand RNA segments, each containing one or two protein genes. Influenza viruses have a high mutation rate and are very fast to evolve. In particular, hemagglutinin, a surface glycoprotein, is highly evolved compared to other viral proteins due to selection pressure by the host cell's immune system, and serves as a major antigen recognition epitope-providing protein. This is very important.

Anti-viral composition according to an embodiment of the present invention can be seen in Example 3 and FIGS. 3A-3D to be described later, the juice or extract of the bokbunja and / or the audio before or during the pre-treatment of the virus In the case of treatment (co-treatment) appeared to have a high anti-viral activity, it was confirmed that the characteristics of inhibiting the growth and replication of the virus by inhibiting the cell attachment or internalization step of the virus.

Antiviral activity against the influenza virus of the composition of the present invention showed an increase with the concentration of juice or extract of bokbunja and / or audi when co-treatment (Figs. 6-9). It can be seen that the inhibitory activity shows a different pattern. In addition, treatment with various types of influenza viruses (H1N1, H3N2, 2009pdm H1N1, and influenza B subtypes) also showed high antiviral activity, indicating that they have a wide range of antiviral activities regardless of the type of influenza virus. have.

In the case of Audi, the total phenol content of the juice was higher than that of the extract of the by-product, and in the case of bokbunja, the total phenol content of the water and ethanol extract of the bokbunja by-product was higher than that of the bokbunja juice (Example 2). The antiviral activity of bokbunja was shown in the order of seed ethanol extract, seed methanol extract, seed water extract, and juice. In particular, anti-influenza activity was determined by the total phenol content of the sample, the type of phenol compound contained, or the synergistic effect of phenolic compounds. ) Seems to be related.

The antiviral composition may further comprise appropriate carriers, excipients and diluents, and the like, commonly used in the field to which the composition is applied, in the form of pharmaceutical compositions administered to mammals including vertebrates, preferably humans, or dishes, kitchenware. It can be prepared in all conventionally known forms such as cleaning agents, disinfectants, conventional antiviral agents, etc. of articles, household goods, and various other things, in this case, at least one member selected from the group consisting of bokbunja and audi for the total weight of the composition It may be included in the weight% to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1% to 50% by weight.

Calicivirus infection diseases are preferably food poisoning or enteritis caused by noroviruses, and also respiratory diseases of feline animals caused by feline caliciviruses. Oxomyxovirus infectious diseases may preferably be digestive or respiratory diseases caused by influenza virus, digestive and / or respiratory diseases in animals including birds, and respiratory diseases in humans. Therefore, the antiviral composition of the present invention may be preferably for preventing or treating such a disease.

The composition for the prevention or treatment of a calcivirus or oxomyxovirus infection disease according to an embodiment of the present invention may include one or more selected from the group consisting of bokbunja and audi, preferably juice or extract thereof. In addition, the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent according to conventional methods used in the art, depending on the dosage form, the method of use, and the purpose of use.

The composition may be administered orally or parenterally according to a conventional method, for example, may be provided in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups and the like during oral administration.

When the composition is provided as a mixture in which other components are added in addition to the juice or extract of the bokbunja and / or audi, which is an active ingredient of the composition for the prevention or treatment of the calcivirus or oxomyxovirus infection disease, It may include at least one selected from the group consisting of bokbunja and Audi with respect to 0.001% by weight to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1% to 50% by weight.

Preferred dosages may be amounts suitable for the treatment or prophylaxis of the subject and / or disease, including the age, sex, general health and weight of the subject, the type and severity of the disease, the type of formulation, the composition contained in the composition It can be adjusted according to various factors including the type and content of other ingredients, the ratio of the composition, the route of administration and duration, and may be appropriately selected by those skilled in the art, but preferably 50 to 100 mg per adult May be administered.

According to another preferred embodiment of the present invention, a food composition for preventing or ameliorating food poisoning or influenza infectious disease, comprising a juice or extract obtained from one or more selected from the group consisting of fruits, skins and seeds of bokbunja or a mulberry Is provided.

The calcivirus or oxomyxovirus infection disease may preferably be norovirus or oxomyxovirus infection disease, as described above.

More preferably, it may be a food poisoning caused by norovirus infection or a respiratory disease caused by influenza virus infection, and since both diseases are caused by viruses, antibiotics are not effective unless they are secondary infection problems such as bacterial diseases. The present inventors can effectively prevent the infection by providing a food composition of the present invention containing the juice or extract of the bokbunja and / or Audi as an active ingredient. The food composition may further comprise suitable carriers, excipients and diluents conventionally used in the production thereof.

As used herein, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process, and food in a conventional sense. , Food additives, health functional foods, beverages and beverage additives, and the like.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include functional beverages. The beverage includes, as an essential ingredient, the juice or extract of Bokbunja and / or Audi as an essential ingredient as an active ingredient, and there are no particular restrictions on the other ingredients, and various flavors or natural carbohydrates, such as ordinary drinks, may be used. It may contain as an additional component. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, 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, in addition to the composition of the present invention, the flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage It may further contain.

In a food composition according to one embodiment of the present invention for the purpose of preventing or ameliorating a norovirus or influenza virus infection disease, the amount of juice or extract of the bokbunja and / or audio is 0.00001% by weight of the total food weight. To 100% by weight, preferably 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, 0.01 g to 100 g, preferably 1 g to 10 g, based on 100 ml of the total food volume, but is not limited thereto.

Since the composition comprising the bokbunja and the audio component of the present invention is very effective in inhibiting the proliferation and replication of calcivirus or orosomic sovirus, it can be used as an effective therapeutic agent for influenza viruses including norovirus, cat calcivirus and avian influenza. In addition, because it uses natural plant bokbunja by-products or juice and audio by-products or juice, such as food poisoning or avian influenza caused by norovirus when providing food containing the composition of the present invention is recognized in terms of recyclability and safety It is expected to be a very meaningful development socio-economically because it can reduce influenza disease.

Figure 1 shows the antiviral activity (viral plaque formation inhibitory activity) measuring scheme (sample) of the samples prepared in Example 1 (products and juices of bokbunja and Audi). Specifically, when each sample (inhibitor) was incubated with each cell or virus before infection (pre-cell (A) / virus (B) treatment), when the sample and virus were infected with the cell at the same time ((C co-treatment) is an approximate experimental scheme of ((D) post-treatment) when samples were added after 1 hour of viral cell infection.
Figure 2a shows the results of cytotoxicity test on RAW 264.7 cells (top), CRFK cells (middle), MDCK cells (bottom) by the bokbunja by-product and Audi by-product prepared in Example 1 (0.0625-1% at a concentration of 1 Measured for hours).
Figure 2b shows the results of cytotoxicity experiments on the raw juices prepared in Example 1 on RAW 264.7 cells (top), CRFK cells (middle), MDCK cells (bottom) (0.0002-0.1%, 0.3-10%, respectively) , And 1 hour at 0.25-4% concentration).
Figure 2c shows the results of cytotoxicity experiments on bokbunja juice prepared in Example 1 on RAW 264.7 cells (top), CRFK cells (bottom) (measured for 1 hour at 0.75-6% and 1-10% concentration, respectively) ).
Figure 3a and 3b are MOI values of antiviral activity against MNV-1 (top) and FCV-F9 (bottom) of seed ethanol extract (A) and ethanol by-product seed ethanol extract prepared in Example 1, respectively It is shown as plaque reduction activity at 0.001.
Figure 3c shows the antiviral activity of the lycicidal MNV (top) and FCV-F9 (bottom) of the mulberry juice prepared in Example 1, respectively, as a lytic plaque reduction activity. (MOI value 0.001 for MNV, MOI value 0.001 and 0.01 for FCV-F9)
Figure 3d shows the antiviral activity against calicivirus MNV (top) and FCV-F9 (bottom) of Bokbunja juice prepared in Example 1, respectively, as a lytic plaque reduction activity. (MOI values 0.001 and 0.001 in MNV, MOI values 0.0001, 0.001 and 0.01 in FCV-F9)
Figure 4 is a result of quantitatively showing the antiviral activity against various influenza viruses of each sample prepared in Example 1 as a cell lesion inhibition (CPE reduction) activity ((a) methanol extract of seed as a bokbunja by-product, (b) Water extract of bokbunja byproduct, (c) mulberry juice; MDCK + IV is a sample-free group infected with influenza virus in MDCK).
Figure 5 shows the antiviral activity of two kinds of influenza virus of the seed extract which is a by-product of bokbunja prepared in Example 1 as a micrograph of the degree of cytopathic inhibitory activity ((A) of bokbunja seed against seasonal strain of influenza virus Cytopathic inhibitory activity of the water extract (top) and methanol extract (bottom), (B) cytotoxic activity of the water extract (top) and methanol extract (bottom) of Bokbunja seed against 2009pdm influenza virus.
Figure 6 shows the anti-viral activity against the various influenza viruses of the mulberry juice prepared in Example 1 (A) when the juice was pre-treated with the juice, (B) the mulberry juice and virus were simultaneously infected with the cells The results of quantitative analysis of lytic plaque formation inhibition in each mutant strain were treated with co-treatment and (C) virus after infection.
Figure 7 is to confirm the antiviral activity against the various influenza virus of the bokbunja juice prepared in Example 1 (A) when the bokbunja juice pre-treated with the virus, (B) bokbunja juice and virus were infected with the cells at the same time When co-treatment and (C) virus were infected, Bokbunja juice was treated quantitatively to show lytic plaque formation inhibitory activity in each mutant strain.
Figure 8 is to confirm the antiviral activity against influenza virus of the bokbunja by-product seed extract prepared in Example 1, when the bokbunja by-product seed extract and the virus at the same time (co-treatment) in each mutant strain The results showed quantitatively inhibiting bacteriostatic activity ((A) 70% (v / v) ethanol extract, (B) water extract).
9 shows graphs and photographs of the results of inhibition of lytic plaque formation against (A) influenza virus seasonally modified strains and (B) 2009pdm influenza virus according to antiviral activity of the seed extract, a bokbunja byproduct prepared in Example 1. FIG. The seed extract and virus were simultaneously infected with the cells (co-treatment), showing the effects of 70% (v / v) methanol extract (left) and water extract (right) in each mutant strain.
FIG. 10 is a graph showing the results of inhibition of lysogenic plaque formation against influenza virus seasonally modified strain (sH1N1) and 2009pdm influenza virus (pH1N1) according to antiviral activity of the seed extract which is the by-product of the mulberry extract prepared in Example 1. FIG. The result of pre-virus treatment of virus and seed extracts before infection (pre-virus treatment), showed the effect of 70% (v / v) ethanol extract in each mutant strain.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Preparation of Seed Extract and Fruit Juice of Bokbunja and Audi

Bokbunja and Audi were harvested in July-August and purchased from Sununsan Agricultural Cooperative (Geochang-gun Jeollabuk-do) and used in this example.

1-1. Preparation of By-products (Seed Extract) of Bokbunja and Audi

Seeds of Bokbunja and Audi were selected, sprinkled, lyophilized, and then ground by a homogenizer. The seed powder in the form of ground powder was selected using a mesh (1 mm pore size), and 1 g of the selected seed powder was selected from 10 ml of 70% (v / v) methanol, 10 ml of 70% (v / v) ethanol, and After mixing with 10ml of water and extracting for 20 minutes at 20 ℃ at about 50kHz using ultrasonic sound (ultrasonic sound) (Powersonic 420, Hwashin Instruments, Korea), centrifugation was performed at about 10,000rpm at 4 ℃ for 60 minutes. The supernatant was collected. The supernatant was removed at about 40 ° C using a rotary evaporator (Eyela, NN series, Japan), followed by lyophilization, and the by-products of bokbunja and mulberry (bokbunja seed ethanol extract, bokbunja seed methanol extract, bokbunja seed water). Extract, Audi C. ethanol extract).

1-2. Bokbunja and Audi Juice (Juice) manufacturing

After washing each raw fruit of bokbunja and Audi and wrapped in gauze, 100% juice was juiced and treated at 63 ° C. for 30 minutes to remove enzymatic activity and destroy microorganisms. After 30 minutes centrifugation at 4 ℃ at about 15,000rpm, the supernatant was collected to prepare a juice.

Subsequently, as a sample for evaluating antiviral activity against bokbunja by-products or juice and udi by-products and calichevirus (murine norovirus, feline calcivirus) and influenza virus, respectively, by-products of the bokbunja and udi prepared above And juice was filtered using a 0.2 μm syringe filter and then dispensed and stored in an cryogenic freezer.

Example  2. Determination of total polyphenol content in bokbunja by-products or juice and audio by-products and juice

The total polyphenol compound content of the by-products and juices of bokbunja and Audi prepared in Example 1 was evaluated by the Folin-Ciocalteu method.

That is, 5 μl of distilled water and 5 μl of Folin-Ciocalteu's reagent (Sigma) were added to 5 μl of each sample prepared in Example 1, and then maintained at room temperature for 1 minute, followed by 5 μl of 20% (w / v) Na ₂ CO ₃ . After the reaction mixture was allowed to stand at room temperature for 2 hours, absorbance of 765 nm was measured with a spectrophotometer (SpectraMax M2, Molecular Devices Corp. USA). As a standard, gallic acid was used to prepare a standard curve, and then the content of the polyphenolic compound was calculated (J. Korean Soc. Food Sci. Nutr. 2003, 32, 338-345; Korean J. Food Sci. Technol 2011, 43, 341-347), the calculated polyphenolic compound content was calculated as gallic acid equivalents (GAEs) Bokbunja byproduct ethanol extract 1.70 ± 0.09mg / ml, Bokbunja byproduct water extract 2.39 ± 0.02mg / ml, Audi byproduct ethanol The extract was 0.30 ± 0.00mg / ml, bokbunja juice 0.64 ± 0.01mg / ml, and the mulberry juice was 2.54 ± 0.15mg / ml.

Example  3. Evaluation of antiviral activity

3-1. Cultivation of virus

To assess antiviral activity against Calicivirus, murine noroviruses MNV-1 (Sogang University) and FCV-F9 (ATCC, Manassas, VA, USA) were used as replacements for human noroviruses, respectively. As the host cells for each virus, RAW 264.7 cell lines (ATCC, Manassas, VA, USA) were used for murine norovirus, and CRFK cell lines (ATCC, Manassas, VA, USA) were used for FCV. Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand) added with antimycotic (Gilbo, Grand Island, NY), 10% (v / v) fetal bovine serum (FBS), Htclone Lab, Logan, Utah Island, NY) was passaged at 24-72 hours intervals in a 37 ℃, 5% CO ₂ incubator as a basal medium.

In addition, to assess antiviral activity against orthomyxoviruses, especially influenza viruses, Madx-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA, USA) were used as host cells to identify 1x antibodies (Gilbo, Grand Island, NY), Dulbecco's modified Eagle's medium (DMEM) (Gibco, MD) supplemented with 10% (v / v) fetal bovine serum (FBS), Htclone Lab, Logan, Utah Incubated at 37 ° C. in ₂ incubators.

Then, RAW 264.7, CRFK, and MDCK cells were divided into 2.0 × 10 ⁶ cells / ml and 2.0 × ^{10 5} cells / ml on a tissue culture plate and incubated for 24 hours at 37 ° C. in a 5% CO ₂ incubator. , And MNV-1 5.0 × 10 ⁵ PFU / ml, FCV-F9 2 × 10 ⁶ PFU / ml and influenza virus 2 × 10 ⁶ PFU / ml were diluted according to the multiplicity of infection (inoculated virus / cell number) After inoculation, the cells were incubated in a 5% CO ₂ incubator at 37 ° C for 1 hour to infect each virus.

Influenza viruses of A / Brisbane / 59/2007 (sH1N1), A / Korea / 01/2009 (pdmH1N1), A / Brisbane / 10/2007 (H3N2), and B / Florida / 4/2006 Four strains were used in this experiment. (Su X and D'Souza DH. Grape seed extract for control of human enteric viruses.Appl Environ Microbiol 2011; 77: 3982-3987. Sangster MY, and D'Souza DH.Time-dependent effects of pomegranate juice and pomegranate polyphenols on foodborne viral reduction.Foodborne Pathog Dis 2011; see 8: 1177-1183)

3-2. Cytotoxicity measurement

A. Experimental Method

Cytotoxicity experiments of by-products (70% (v / v) ethanol extract) and juice of Bokbunja and Audi prepared in Example 1 were cell viability by MTT assay (Applied Microbiology, 2009, 49, 806-808). ) Was measured. Specifically, RAW 264.7, CRFK, and MDCK cells in 96 wells were seeded at 1.5x10 ⁶ , 2.0x10 ⁵ , and 2.0x10 ⁵ cell / ml, respectively, and the samples of Example 1 (Blood and Audi) after 24 hours. By-product and juice) were added by concentration and incubated at 37 ° C., 5% CO ₂ incubator for 1-72 hours. MTT was added at a final concentration of 0.5 mg / ml and absorbance was measured (MTT assay) at 570 nm UV-Vis spectrophotometer (SpectraMax M2, Molecular Devices Corp. USA) after 2 hours. Cell viability is calculated and expressed as% relative to no addition group.

B. Results

As shown in Figure 2a, by-products (seed extract) of Bokbunja and Audi were not cytotoxic to RAW 264.7 cells and CRFK cells at 0.0625-1% concentration, Bokbunja seed extract about 0.1- about MDCK cells It was found that there was no cytotoxicity at 100ug / ml and 1-100ug / ml of Audi seed extract.

In addition, as shown in Figure 2b, in the case of audible juice showed no toxicity to RAW 264.7 cells at about 0.0002-0.1% (v / v), about 0.3-10% (v / v) for CRFK cells There was no cytotoxicity at the concentration. In addition, the juice was found to be cytotoxic at a concentration ranging from 0,25% to 4% (v / v) for MDCK cells, it was confirmed that it can be used very safely.

In the case of Bokbunja juice, as shown in FIG. 2C, it was shown that there was no cytotoxicity up to a concentration of 6% (v / v) for RAW 264.7 cells, and also 10% (v / v) for CRFK cells. It was found that there is no cytotoxicity even at the concentration of, so that it can be used very safely.

3-3. Anti-calichevirus  Active measurement

A. Experimental Method

Bokbunja and Audi samples prepared in Example 1 were added in different time points as measured in the concentration range confirmed to be non-toxic to cells according to Example 3-2, and then the terms according to the addition time of the by-products Viral activity was compared with the addition of the candidate substance and the difference in lysate plaques of the no-added group (calculated with the degree of lytic plaque formation in the no-added group as 100%).

i) Pre-treatment ( Pre - treatment ) Antiviral activity measurement (A and B in Fig. 1)

After incubating each of the cells (A in FIG. 1) or the virus (B in FIG. 1) to the bokbunja and the Audi sample of Example 1 which is a natural plant, and then incubated for 1 hour, the degree of virus inhibition was confirmed. The effects of seed extracts and juices, which are by-products of natural plant bokbunja or audi, on the viruses and cells before the virus infection was examined.

Specifically, each cell was dispensed into 24 wells (1.5 × 10 ⁶ viable cell aliquots per well of DMEM medium containing 10% FBS), and the bokbunja and Audi samples of Example 1 were added to RAW 264.7 or CRFK cells or virus, respectively. (Added at the concentration shown in Figs. 3A-D) and incubated for 1 hour, the virus was then infected with cells and incubated at 37 ° C for 1 hour. Then add DMEM medium (Gilbo, Grand Island, NY) containing 1.5% (w / v) agarose, 5% (v / v) FBS, and 1% (v / v) PS. Incubation was performed by incubation in an incubator and stained with 0.5% (v / v) crystal violet.

ii At the same time as virus infection Co - treatment ) Antiviral activity measurement (FIG. 1C)

Each cell was cultured by dispensing in 24 wells as i) above, and the virus and Bokbunja and Audi samples of Example 1 were simultaneously added at the concentrations described above and incubated at 37 ° C for 1 hour. Then add DMEM medium containing 1.5% (w / v) agarose, 5% (v / v) FBS, and 1% (v / v) PS, incubate in a 37 ° C. incubator for 42 hours, and then 0.5 The lysates were measured by staining with% (v / v) crystal violet.

iii ) Post treatment ( Post - treatment ) Antiviral activity measurement (D in Fig. 1)

Cells were cultured by dispensing in 24 wells as in i), and incubated at 37 ° C. for 1 hour after virus infection. After complete removal of the virus, the bokbunja and Audi samples of Example 1 were added to each of the above-described concentrations of DMEM medium and incubated at 37 ° C. for 1 hour before the medium was completely removed. After adding DMEM medium containing 1.5% (w / v) agarose, 5% (v / v) FBS, and 1% (v / v) PS, incubated in a 37 ° C. incubator for about 42 hours. , Lysate was measured by staining with 0.5% (v / v) crystal violet.

The above i) ~ iii) was repeated three times in consideration of the accuracy and the plaque counting results were analyzed by SAS software (version 9.2, SAS Institute, Cary, NC, USA) and ANOVA and Duncan's multiple range test The results were derived. The resulting data has a reliability of error p <0.05.

iv ) Lysis tables  inspection( Plaque assay )

Cells were infected with virus at 37 ° C. and 5% CO ₂ incubator for 1 hour, followed by 2 days of incubation, and then stained with monolayers with 0.5% (v / v) crystal violet. ) Was analyzed.

B. Results

3A to 3D show the results of measuring the virus inhibitory activity by using the bokbunja and Audi samples.

As a result of measuring the antiviral activity by pre-treatment, co-treatment, and post-treatment using cells, the antiviral activity of the bokbunja and Audi samples prepared in Example 1 was excellent at all stages. In particular, it showed a relatively higher inhibitory activity during co-treatment. Therefore, bokbunja and audi samples were found to show the most important inhibitory activity during the life cycle of norovirus, especially for the replication and proliferation mechanism. In addition, the samples showed antiviral activity against feline calicivirus (FCV) in addition to murine norovirus, it was confirmed that it is very useful as a food poisoning inhibitor through the activity against food poisoning replacement virus (surrogate). On the other hand, unlike the anti-murine norovirus activity, it was confirmed that the inhibitory activity of the sample was higher in the pre-treatment of the pre-FCV for FCV. In other words, the sample prepared in Example 1 was shown to directly inhibit the viral particles or cell receptor (receptors) binding with respect to FCV, showing a different aspect from the inhibitory activity against RAW 264.7 cells and MNV-1 Could know.

3-4. Anti-influenza virus  Active measurement

In order to evaluate the anti-influenza virus activity of the bokbunja and Audi samples prepared in Example 1 was compared by the CPE reduction assay (plaque reduction assay) method (Fig. 4-10).

Another experimental method is the method of Example 3, the cytopathic inhibitory activity of the influenza virus was measured by using a medium added 2 ug / ml trypsin to DMEM-25 mM HEPES, 2% BSA 1% PS 2 and 4 As shown in FIG. 5, the lysis plaque was added to 2 ug / ml trypsin in 2 x L-15 media containing 1.5% (w / v) agarose, 25 mM HEPES, and 2% BSA 1% PS. After the culture medium was incubated in a 37 ° C. incubator for about 48-72 hours, and stained with 0.5% (v / v) crystal violet, lysate plaques were measured and shown in FIGS. 6 to 10, respectively.

Samples of bokbunja and mulberry juice at concentrations of 1-4% were 0.5-50 ug / mL for 70% (v / v) ethanol extract and 70% (v / v) methanol extract of bokbunja byproduct. 0.1% (v / v), water extracts at concentrations of 0.01-1% (v / v), and 70% (v / v) ethanol extracts of seed by-products of Audi, respectively, at concentrations of 1-100 ug / mL. Antiviral experiments were performed.

As a result of experiments with different influenza virus types and extracting solvents with respect to the inhibition of virus growth using MDCK cells, as shown in FIGS. Could.

As described above, it can be seen that the antiviral activity of bokbunja and mulberry that can be easily ingested as food, such as beverages, is particularly excellent. It is expected to be applicable as an effective functional food for food poisoning or influenza disease.

Claims (10)

Antiviral composition for a calcivirus or orosomicovirus comprising at least one selected from the group consisting of bokbunja and audi as an active ingredient.
The composition of claim 1, wherein the bokbunja and audi are included in the form of a juice or extract obtained from one or more selected from the group consisting of fruits, shells and seeds thereof.
The method of claim 2, wherein the extract is steam extraction, hot water extraction, ultrasonic extraction, water, solvent extraction using a solvent selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, acetic acid and mixtures thereof, or reflux cooling extraction Obtained.
The composition of claim 1, wherein the composition exhibits antiviral activity by inhibiting the proliferation and replication of calicivirus or orosomicovirus.
The composition of claim 1, wherein the calicivirus is norovirus or feline calicyvirus and the orosomicovirus is influenza virus.
The composition of claim 1, wherein the composition is for preventing or treating food poisoning or enteritis caused by norovirus, respiratory disease of a feline animal by calcivirus, or digestive or respiratory disease caused by influenza virus.
The composition of claim 1, comprising 0.001% to 99.9% by weight of one or more selected from the group consisting of bokbunja and Audi.
Food composition for the prevention or amelioration of food poisoning or influenza infectious disease, comprising a juice or extract obtained from one or more selected from the group consisting of fruit, shell and seed of bokbunja or mulberry.
The composition of claim 8, wherein the food is a food additive, beverage or beverage additive.
The composition of claim 8 comprising 0.00001% to 100% by weight of the juice or extract.

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