KR20230001589A - Nature extract of anti influenza virus and composition containing the same - Google Patents

Abstract

The present invention relates to a use of an antiviral agent against influenza viruses, which comprises a composite extract of Epimedium koreanum and Hovenia dulcis Thunb. and, more specifically, to an antiviral pharmaceutical composition against influenza viruses, which comprises the composite extract of Epimedium koreanum and Hovenia dulcis Thunb., a pharmaceutical composition for preventing or treating an influenza virus-caused infectious disease, a method for preventing or treating an influenza virus-caused infectious disease by using the same, a functional health food composition comprising the composite extract of Epimedium koreanum and Hovenia dulcis Thunb. for preventing or alleviating an influenza virus-caused infectious disease, and a feed composition comprising the composite extract of Epimedium koreanum and Hovenia dulcis Thunb. for preventing or alleviating an influenza virus-caused infectious disease. The composite extract of natural materials according to the present invention has the effect of inhibiting infection caused by influenza viruses, so the composite extract can be used in developing an antiviral agent for preventing or treating an influenza virus-caused infectious disease.

Description

Natural extract having anti influenza virus effect and composition thereof {Nature extract of anti influenza virus and composition containing the same}

The present invention relates to an antiviral use against influenza virus comprising a composite extract of Eumyanggwak and Hovenia japonica.

Virus means toxic substance in Latin, and is a group of infectious pathogenic particles that pass through a bacterial filter (0.22 μm). Viruses are also classified into bacteriophages, plant viruses, and animal viruses according to the type of host cell, and can be classified into DNA viruses and RNA viruses according to the type of nucleic acid. Recently, various viral diseases such as swine flu, AI, and foot-and-mouth disease have caused great social problems, and accordingly, concerns about effective countermeasures against viral diseases are arousing great social interest.

Vaccination is currently the best way to prevent viral diseases, but in the case of viral diseases, the effectiveness of vaccines due to the generation of many virus serotypes (subtypes) has been raised as an important issue. It is important to develop and distribute inhibitors for preventing viruses that can compensate for the problems of these vaccines, and for this purpose, preventive agents that enhance the immunity of individual animals by stimulating the innate immune system in vivo, which is the initial defense system against viruses The discovery and development of drugs can be an important pharmaceutical development method.

On the other hand, due to the concentration of population due to the development of transportation and urbanization, airborne respiratory infectious diseases caused by influenza, pneumococcal bacteria, etc. are becoming a serious threat to the public.

Influenza virus is an RNA virus belonging to the Orthomyxoviridae family, and its serotypes are classified into three types: A, B, and C. Among them, types B and C have been confirmed to be infected only in humans, while type A has been confirmed to be infected in humans, horses, pigs, other mammals, and various types of poultry and wild birds (Selmons et al., Avian Dis. , 18(1), p.119-124, 1974; Webster RG et al., Microbiol Rev., 56(1), p.152-179, 1992).

Serotypes of type A influenza virus are classified according to the types of two proteins on the surface of the virus, Hemagglutinin (HA) and Neuraminidase (NA), and there are 144 types (HA type) according to the serotype. 16 types and 9 NA types). HA protein serves to attach viruses to somatic cells, and NA proteins allow viruses to penetrate cells (Alexander DJ, Vet. Microbiol., 74(1-2), p.3-13, 2000).

Influenza viruses are classified into several subtypes according to the antigenicity of HA and NA. For example, in the case of H1N1, which was first detected in San Diego, California, USA in March 2009, 14,000 people were infected in 48 countries, including Korea, within just two months. It has spread worldwide, infecting more than 130,000 people and killing more than 300 ( http://www.who.int/influenza ). In addition, in the case of avian influenza (Avian influenza A; H5N1), which was first reported in 1997, infection in livestock, including birds, continues to be reported, and mortality rates of more than 60% in cases of human infection have been reported. are subject to serious scrutiny.

Representative antiviral agents that inhibit the proliferation of influenza virus include amantadine and rimantadine, but these two antiviral agents are effective only for serotype A influenza virus and serotype B without M2 protein. It has been confirmed that there is no effect on influenza virus. In addition, it has been confirmed that amantadine and rimantadine have a disadvantage in that the emergence of mutant viruses that do not affect the ion channel function of the influenza virus M2 protein occurs very easily when used. In order to compensate for these disadvantages, zanamivir and oseltamivir have been developed as antiviral agents effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, and oseltamivir can be administered orally, but has recently been pointed out as a disadvantage due to reports of the appearance of resistant viruses and side effects such as vomiting and dizziness during oral administration.

In this situation, many efforts have recently been made to overcome the disadvantages of existing antiviral agents overseas and domestically, and as one of them, research on the antiviral efficacy of herbal extracts and plant extracts is ongoing in Korea. , Since it is still incomplete, it is necessary to develop a composition containing herbal extracts as active ingredients capable of exhibiting excellent innate immunity enhancing effect and antiviral activity with little toxicity and side effects by overcoming the disadvantages of existing antiviral preparations. .

On the other hand, “Yinyanggwak” refers to the above-ground parts (stems and leaves) of Epimedium koreanum Nakai, a perennial plant of the dicotyledonous plant Buttercup Orchidaceae, or other plants close to the same genus. Warm. Yin-Yang Kwak is used for erectile dysfunction, oil wells, cold cervix, cold limbs, skin paralysis, Guanwasa, forgetfulness, hemiplegia, back and knee weakness, high blood pressure, and polio. Pharmacological actions include semen secretion promotion, blood pressure enhancement, coronary blood flow increase, blood sugar lowering, cholesterol lowering, immune function enhancement, antitussive, expectorant, flatulence, sedation, antibacterial, anti-inflammatory action, femur growth of chickens, activation of protein polysaccharide synthesis, etc. This has been reported (Jeong Bo-seop Jeong, Min-gyo Shin, Hyang-yak Daesajeon, Younglimsa, pp461-462, 1998).

In addition, "guja" refers to a fruit or seed with a fruit of a barberry tree of the buckthorn family, and is also called wood wheat, meaning honey from a tree because it tastes sweet. In China, it is called Hyeonpo-ri, which means a pear tree in the garden of freshmen, and it is also called Baekseokmok because it is white and hard like a stone.

It is used for fever, thirst, hiccups, vomiting, etc. caused by fever, and is known to help diuresis, treat liver damaged by alcoholism, and is known to help blood circulation and relax muscles. In addition, Korean Patent No. 0588759 discloses a food composition for cognitive improvement containing earthworms and health functional food using the same, and Korean Patent No. 1314749 discloses a method for producing a composition for tea bags containing raisins and earthworms. and Korean Patent Registration No. 1306878 discloses a composition for preventing and treating bone diseases containing an extract of Hovenia japonica as an active ingredient.

However, there has been no report on the antiviral activity against the influenza virus using the composite extract of Eumyanggwak and Hovenia japonica.

Under this background, the present inventors, as a result of continuing efforts to develop a novel influenza virus therapeutic agent, confirmed that the composite extract of Eumyanggwa and Hovenia japonica with guaranteed stability has an excellent effect of inhibiting the proliferation of influenza virus. The invention was completed.

One object of the present invention is to provide an antiviral pharmaceutical composition for influenza virus containing a complex extract of Eumyanggwa and Hovenia japonica as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of influenza virus infectious diseases comprising a complex extract of Eumyanggwa and Hovenia japonica as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating influenza virus infectious diseases using the pharmaceutical composition.

Another object of the present invention is to provide a health functional food composition for preventing or improving influenza virus infectious diseases comprising a complex extract of Yinyanggwak and Hovenia japonica as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving influenza virus infectious diseases comprising a complex extract of Yinyanggwak and Hovenia japonica as an active ingredient.

One embodiment of the present invention for achieving the above object provides an antiviral pharmaceutical composition for influenza virus comprising a composite extract of Eumyanggwa and Hokkienja as an active ingredient.

Another embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating influenza virus infectious diseases comprising a complex extract of Eumyanggwak and Hovenia japonica as an active ingredient.

Specifically, since the pharmaceutical composition of the present invention exhibits an effect of inhibiting influenza virus, the present invention can be usefully used for preventing or treating infectious diseases caused by influenza virus.

The term of the present invention, "Eumyanggwak" refers to the above-ground parts (stems and leaves) of Epimedium koreanum Nakai, a perennial plant of the Dicotyledonous plant Buttercup Barberry family, or other plants close to the same genus, and has no odor and has a spicy taste. It is sweet and warm in nature. Yin-Yang Kwak is used for erectile dysfunction, oil wells, cold cervix, cold limbs, skin paralysis, Guanwasa, forgetfulness, hemiplegia, back and knee weakness, high blood pressure, and polio. Pharmacological actions include semen secretion promotion, blood pressure enhancement, coronary blood flow increase, blood sugar lowering, cholesterol lowering, immune function enhancement, antitussive, expectorant, flatulence, sedation, antibacterial, anti-inflammatory action, femur growth of chickens, activation of protein polysaccharide synthesis, etc. This has been reported (Jeong Bo-seop Jeong, Min-gyo Shin, Hyang-yak Daesajeon, Younglimsa, pp461-462, 1998).

The term of the present invention, "district japonica" refers to a fruit or seed with a fruit of a barberry tree of the buckthorn family, and is also called wood wheat in the sense of honey from a tree because it tastes sweet. In China, it is called Hyeonpo-ri, which means a pear tree in the garden of freshmen, and it is also called Baekseokmok because it is white and hard like a stone.

However, there has been no report on the antiviral activity against the influenza virus using the composite extract of Eumyanggwak and Hovenia japonica.

The term "composite extract" of the present invention refers to a combination of the extracts of Yinyanggwak and Hovenia japonica provided by the present invention.

In the present invention, the composite extract may be obtained by combining the extracts of Yinyanggwak and Hovenia japonica, respectively, or may be obtained by extracting a composite of Yinyanggwak and Hokkienji.

The composite ratio of the natural substances of Yin Yang Kwak and Ho Chi Minh Plant included in the complex extract is not particularly limited thereto, but as an example, the composite ratio of Yin Yang Kwak and Ho Chi Minh Ji is 10:0 to 0:10, more specifically 9:1 to 1:9 , 8:2 to 2:8, 7:3 to 3:7, 6:4 to 4:6 or 5:5 ratio by weight, specifically 8:2, 7:3, 5:5 or 4: It may be preferable to extract after preparing the mixed natural product complex at a ratio of 6 parts by weight, but is not limited thereto.

As described above, the term "extract" of the present invention has a meaning commonly used as a crude extract in the art, but in a broad sense also includes fractions obtained by further fractionating the crude extract. That is, the natural product composite extract of the present invention includes not only those obtained using the above-described extraction solvent, but also fractions obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (designed for separation according to size, charge, hydrophobicity or affinity), the extraction solvent described above Fractions obtained through various additional purification methods, such as secondary or tertiary additional extraction fractions by , are also included in the extract of the present invention.

In the natural product complex extract of the present invention, a method for extracting the natural product complex is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.

In the present invention, the type of solvent used for the extraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, alcohol, or a combination solvent thereof, which may be used alone or in combination with one or more, and specifically, water may be used. When alcohol is used as a solvent, alcohol having 1 to 4 carbon atoms may be specifically used.

For example, the natural product complex extract is a method of extracting for 2 to 4 hours at a temperature of 70 ° C. to 125 ° C. after adding 2 to 30 times the amount of water of the natural product composite consisting of Yin-Yang Kwak and Earth Ja in the above-described ratio. It may be extracted, but is not limited thereto.

In addition, the natural product composite extract shall include any one of an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product or a purified product.

The term of the present invention, "influenza virus infectious disease" refers to a disease caused by an infection in which an influenza virus invades the body of a host organism. Specifically, the influenza virus is a group consisting of PR8, H1N1 and H3N2. It may be one or more selected from.

The term of the present invention, “influenza virus” is an RNA virus belonging to the family Orthomyxoviridae, and is classified into three serotypes, such as type A, type B, and type C. Among them, types B and C have been confirmed to be infected only in humans, while type A has been confirmed to be infected in humans, horses, pigs, other mammals, and various types of poultry and wild birds (Selmons et al., Avian Dis. , 18(1), p.119-124, 1974; Webster RG et al., Microbiol Rev., 56(1), p.152-179, 1992).

Serotypes of type A influenza virus are classified according to the types of two proteins on the surface of the virus, Hemagglutinin (HA) and Neuraminidase (NA), and there are 144 types (HA type) according to the serotype. 16 types and 9 NA types). HA protein serves to attach viruses to somatic cells, and NA proteins allow viruses to penetrate cells (Alexander DJ, Vet. Microbiol., 74(1-2), p.3-13, 2000).

Influenza viruses are classified into several subtypes according to the antigenicity of HA and NA. For example, in the case of H1N1, which was first detected in San Diego, California, USA in March 2009, 14,000 people were infected in 48 countries, including Korea, within just two months. It has spread worldwide, infecting more than 130,000 people and killing more than 300 ( http://www.who.int/influenza ). In addition, in the case of avian influenza (Avian influenza A; H5N1), the first human infection was reported in 1997, infection in livestock, including birds, is continuously reported, and mortality rates of more than 60% when infected to humans have been reported. are subject to serious scrutiny.

In the present invention, the pharmaceutical composition for preventing or treating influenza virus infectious diseases comprising the composite extract of Yinyanggwak and Hokkienji as an active ingredient is an influenza virus infectious disease, such as any one or more viruses selected from the group consisting of PR8, H1N1 and H3N2. It is characterized by suppressing. Specifically, in one embodiment of the present invention, it was confirmed that the combined extract of Eumyangkwak and Hovenia japonica has an ability to inhibit influenza virus infection, and this was compared with single extracts of Eumyanggwak and Hovenia japonica, respectively, compared to the single extract of the complex of the present invention. It was confirmed that the extract was excellent in inhibiting influenza virus infection (Example 3).

As used herein, the term "infectious disease" refers to a disease caused by infection.

The term "infection" refers to a state in which a pathogenic microorganism invades the body of a host organism and develops and proliferates.

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of infectious diseases caused by influenza virus by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of suspected or affected subjects of influenza virus infection by the administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid ( Poly Lactic Acid), poly-L-lactic acid, and mineral oil.

The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively, and the carrier In the form of may include various irregular carriers, microspheres, nanofibers, and the like.

When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used.

Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The content of the complex extract of Eumyanggwak and Hocus japonica included in the pharmaceutical composition of the present invention is not particularly limited, and may include a pharmaceutically effective amount of the complex extract of natural substances.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, generally in an amount of 0.001 to 1000 mg/kg, specifically 0.05 mg/kg. to 200 mg/kg, more specifically, an amount of 0.1 to 100 mg/kg may be divided and administered once a day to several times. However, for purposes of the present invention, a specific therapeutically effective amount for a particular patient is determined by the type and extent of the response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general state of health, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without causing side effects in consideration of all of the above factors, and can be easily determined by those skilled in the art.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through various routes.

The administration method of the pharmaceutical composition according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various formulations depending on the desired administration method.

The dosage of the pharmaceutical composition of the present invention is, for example, 1 to 20 mg / kg, more specifically, 1 to 10 mg / kg of the pharmaceutical composition of the present invention can be administered to mammals, including humans, during the day , The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose.

Another embodiment of the present invention for achieving the above object is prevention or treatment of influenza virus infectious disease comprising the step of administering the pharmaceutical composition to a subject other than a human who has symptoms of influenza virus infectious disease or is likely to appear provides a way

At this time, the description of the "pharmaceutical composition", "influenza virus", "infection", "prevention" and "treatment" is as described above.

As used herein, the term "subject" may refer to all animals, including humans, who show symptoms of influenza virus infection or are likely to appear. The animal may be not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but are not limited thereto.

Specifically, the preventive or therapeutic method of the present invention may include the step of administering the composition in a pharmaceutically effective amount to a subject who has or is likely to have symptoms of an influenza virus infection.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject who has or is likely to exhibit symptoms of an influenza virus infection by any appropriate method, and the administration route of the composition of the present invention can be administered through various routes, oral or parenteral, as long as it can reach the target tissue.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for preventing or improving influenza virus infectious diseases comprising a complex extract of Yinyanggwak and Hocus japonica as an active ingredient.

At this time, the description of the "composite extract of Yinyanggwak and Hovenia japonica", "a fraction thereof", "influenza virus", "infection" and "prevention" is as described above.

Since the composite extract of Yinyanggwak and Hovenia japonica according to the present invention exhibits excellent influenza virus inhibitory effect, it can be included in a food composition for the purpose of preventing or improving infectious diseases caused by influenza virus, and the food composition can be consumed on a daily basis. Therefore, a high effect can be expected for the prevention or improvement of infection by influenza virus.

As used herein, the term "improvement" refers to all actions that at least reduce the degree of a parameter related to a condition to be treated by administering the pharmaceutical composition according to the present invention, for example, the severity of a symptom.

The term of the present invention, "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Act No. 6727 on Health Functional Food, and 'functional' refers to the structure of the human body. and to obtain useful effects for health uses such as regulating nutrients with respect to functions or physiological actions, and the like. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement food refers to food for the purpose of supplementing health. In some cases, the terms of health functional food, health food, and health supplement food can be mixed.

The complex extract of Yinyanggwak and Hokkien japonica of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, bactericides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, emulsifiers, thickeners, coating agents, gum base agents, foam inhibitors, solvents, and improvers. can include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material, and has excellent portability, so the present invention Of the foods can be consumed as supplements to enhance the effect of preventing or improving infectious diseases caused by influenza virus.

The composite extract of Yinyanggwak and Hovenia japonica of the present invention can be included in various weight percents in food compositions if it can exhibit an effect of preventing or improving infectious diseases caused by influenza virus. Specifically, it may be included in 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto. In the case of long-term intake for health and hygiene purposes, the content below the above range may be included, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object provides a feed composition for preventing or improving influenza virus infectious diseases comprising a composite extract or a fraction thereof of Eumyanggwak and Hovenia japonica as an active ingredient.

At this time, the description of the "composite extract of Yinyanggwak and Hokkien japonica", "a fraction thereof", "influenza virus", "infectious disease", "prevention" and "improvement" is as described above.

Since the composite extract of Yinyanggwak and Hovenia japonica according to the present invention exhibits excellent influenza virus inhibitory effect, it can be included in a feed composition for the purpose of preventing or improving infectious diseases caused by influenza virus, and the feed composition is a food composition that animals routinely consume. Since it is possible, a high effect can be expected for the prevention or improvement of infectious diseases caused by influenza virus.

As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals.

The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

Since the composite extract of Eumyanggwak and Hovenia japonica exhibits an effect of inhibiting influenza virus infection, it can be used in the development of antiviral agents for the prevention or treatment of infections caused by influenza virus.

Figure 1 is a graph showing the results of confirming the toxicity to mouse macrophage RAW 264.7 cells according to the concentration of the composite extract of the present invention.
Figure 2 is a fluorescence micrograph showing the result of confirming the antiviral efficacy against influenza virus (PR8-GFP virus) of the composite extract of the present invention.
3 is a graph showing the results of confirming the antiviral efficacy of the composite extract of the present invention against influenza virus (PR8-GFP virus).
4 is a graph showing the results of confirming the antiviral efficacy of the composite extract of the present invention against influenza virus (PR8-GFP virus).
Figure 5 is a graph showing the results of confirming the infection inhibitory efficacy against influenza viruses (H1N1 and H3N2) of the composite extract of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Example 1: Preparation of Natural Composite Extracts Consisting of Yin-Yang Kwak and Geukja

All herbal medicines were purchased domestically and used as samples. After adding 1 liter of 20 times the volume of bottled water to 50 g of a mixture of Yin Yang Kwak and Geulja having the mixing ratio shown in Table 1, the mixture was left at room temperature for one day. Thereafter, hot water extraction was performed at 115 ° C. for 3 hours (using Cosmos-600 Extractor, Gyeonseo Co., Korea) to obtain a composite extract, and the composite extract was filtered with a standard testing sieve (150 μm, Retsch, Haan, Germany) Freeze-dried to obtain a lyophilizate. The lyophilizate was stored at -20°C, and the composite extract or lyophilizate thereof was used in the present invention. Hereinafter, it was described as the composite extract of the present invention, and the extract of Eumyangkwak alone was named Eumyangkwak, the extract of Hovenia japonica alone, and the complex extracts were named EH 1 to EH 4 according to the mixing ratio.

complex extract Mixing ratio (weight ratio) Yin-Yang Kwak earth ruler Yin-Yang Kwak 10 0 EH 1 8 2 EH 2 7 3 EH 3 5 5 EH 4 4 6 earth ruler 0 10

Example 2: Confirmation of Toxicity of Complex Extracts to Mouse Macrophage RAW 264.7 Cells

Eumyangkwak alone extract prepared in Example 1, Hovenia japonica extract alone; And treatment with CCK reagent to confirm the toxicity of EH 1 to EH 4 complex extracts EH 1 to EH 4 on mouse macrophage RAW 264.7 cells by concentration (0, 10, 100, 200, 500, 1000 (μg/ml)) and analyzed.

Specific results are shown in Figure 1. The right vertical axis (Cell viability, assay) represents the cytotoxicity (%) of the sample.

As a result, as can be seen in Figure 1, in the case of the composite extract of Eumyanggwa and Hokkeumjae prepared in Example 1, the cytotoxic concentration (CC50) that reduces the viability of 50% cells is 1000 μg / ml or more, the highest experimental concentration. It was confirmed that

Example 3: Confirmation of influenza antiviral efficacy of composite extracts

Example 3-1: Analysis of antiviral activity against influenza virus (PR8-GFP virus)

RAW 264.7 cells were used to confirm the inhibitory effect on influenza virus, and the specific experimental method is as follows.

After culturing Raw 264.7 cells (2X10 ⁵ cell/well) in a 96-well plate, Eumyangkwak alone extract and Hovenia dulcis extract prepared in Example 1; And the extracts EH 1 to EH 4 of Eumyanggwa and Hokkeum japonica were co-infected with influenza virus (PR8-GFP), and after 24 hours, the inhibitory effect on influenza virus was observed under a fluorescence microscope (FIG. 2).

Next, the GFP expression of each cell observed under the fluorescence microscope was confirmed using a flow cytometer, and was shown as a histogram (FIG. 3).

Subsequently, the fluorescence value of the GFP protein expressed through the flow cytometer was quantified, and the fluorescence value by the PR8-GFP virus was relatively compared with 1 as a standard, and shown in a graph (FIG. 4).

Specific results are shown in Figures 2 to 4. Mock is a negative control group, PR8-GFP is an influenza virus infection group, PR8-GFP/Eumyangkwak is a group treated with influenza virus and Eumyangkwak alone extract 20 μg/ml, PR8-GFP/Dukja is influenza virus and a globe extract alone 20 μg/ml The ml simultaneous treatment group, PR8-GFP/EH 1 to PR8-GFP/EH 4, represents a simultaneous treatment group of influenza virus and 20 μg/ml of complex extracts of Eumyanggwa and Hokkeum japonica.

As a result, as can be seen in FIGS. 2 to 4, as a result of simultaneous infection with the extract of Eumyangkwak and Hovenia dulcis and the PR8 influenza virus, the GFP fluorescence value by the influenza virus in the complex extract treatment group was higher than that of the single extract of Eumyanggwak and Hokkot. It was confirmed that it significantly decreased.

Through this, it was confirmed that the natural compound extract composed of Yinyanggwak and Hovenia japonica of the present invention has an effect of effectively inhibiting the infection rate of influenza virus.

Example 3-2: Infection control efficacy analysis against influenza viruses (H1N1 and H3N2)

Raw 264.7 cell line was used to confirm the efficacy of inhibiting the cytopathic effect caused by the proliferation of H1N1 and H3N2 influenza viruses, and the specific experimental method is as follows.

Raw 264.7 cell line extract prepared in Example 1 alone, extract alone extract of Hovenia japonica; And 24 hours after the simultaneous treatment of EH 1 to EH 4 and the virus, the extracts EH 1 to EH 4 of Eumyanggwa and Hokkotja, and 1 hour after treatment with CCK-8 reagent to measure cytotoxicity, the absorbance was measured at 450 nm. The more living cells, the higher the absorbance, and when the cytopathic effect of cell death by the virus appears, the absorbance decreases. A negative control (mock) without virus was graphed by comparing the relative absorbance based on 1.

Specific results are shown in FIG. 5 . Mock is a negative control group, H1N1 and H3N2 are an influenza virus infection group, Yinyanggwak is a group treated with 20 μg/ml of influenza virus and extract alone from Yinyangkwak, and Guggul is a group treated with influenza virus and 20 μg/ml of extract alone, EH 1 to 10 EH 4 represents a group simultaneously treated with 20 μg/ml of influenza virus and 20 μg/ml complex extracts of Eumyanggwa and Hokkotja.

As a result, as can be seen in FIG. 5, as a result of analyzing the cell viability by virus infection by simultaneously infecting the extract of Eumyanggwa and Hokkeum japonica and H1N1 and H3N2 influenza viruses, the cell viability was about 10 by infection with H1N1 and H3N2 viruses. %, and in the case of the group treated with the complex extract of the present invention and 1 MOI virus at the same time, the cell viability was about 70% or more, confirming that the decrease in cell viability due to virus infection was prevented. It was confirmed that the cell viability increased to the level of the negative control group in the complex extract treatment group compared to the single extract and Hovenia dulcis single extract treatment groups.

Through this, it was confirmed that the natural compound extract composed of Yinyanggwak and Hovenia japonica of the present invention has an effect of effectively inhibiting influenza virus infection.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (8)

An antiviral pharmaceutical composition for influenza virus comprising a complex extract of Eumyanggwa and Hovenia japonica as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the influenza virus is any one or more selected from the group consisting of PR8, H1N1 and H3N2.
The pharmaceutical composition according to claim 1, wherein the composite extract is prepared by extraction with water, C1-4 alcohol or a combination solvent thereof.
The pharmaceutical composition according to claim 3, wherein the extraction is hot water extraction, ultrasonic extraction, room temperature extraction, cold extraction, reflux cooling extraction or steam extraction.
A pharmaceutical composition for the prevention or treatment of influenza virus infectious diseases comprising a complex extract of Yinyanggwak and Hovenia japonica as an active ingredient.
A method for preventing or treating an influenza virus infectious disease, comprising administering the pharmaceutical composition according to any one of claims 1 to 5 to a subject other than a human having symptoms of an influenza virus infectious disease or having a risk of appearing.
A functional health food composition for preventing or improving influenza virus infectious diseases, comprising a complex extract of Yinyanggwak and Hocus japonica as an active ingredient.
A feed composition for preventing or improving influenza virus infectious diseases comprising a complex extract of Yinyanggwak and Hovenia japonica as an active ingredient.

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