JP7618334B2 - Composition for inhibiting influenza virus containing nodakenin and/or nodakenetin - Google Patents

Description

The present invention relates to a composition that can inhibit influenza viruses by inhibiting neuraminidase (NA) activity using nodakenin and/or nodakenetin and/or a plant extract, fermentation product and/or fraction thereof that contain the same, and can prevent, ameliorate and/or treat influenza virus infection.

Influenza belongs to the orthomyxoviruses and is a typical pathogenic virus that takes the lives of thousands to tens of thousands of people every season. Since the 20th century, influenza has mutated beyond the species barrier, leading to epidemic colds and a virus with such a high mutation rate that it makes previously developed medicines useless. This disease, often called epidemic flu, generally induces chills, fever, sore throat, muscle pain, cough, fatigue and discomfort, and these non-specific symptoms are only more severe than those of a common cold and are not life-threatening, but when accompanied by pneumonia or Reye's syndrome, it can induce fatal complications. Usually, respiratory infection occurs mainly through the cough, phlegm and sneezing of infected people, and many mutations occur when humans are infected from other animals (especially birds). There are three types of influenza viruses: influenza virus A, influenza virus B and influenza virus C. Among these, the 2009 influenza epidemic, also known as swine flu, which caused a pandemic through interspecies transmission, was an influenza A/H1N1 type that first emerged in Mexico and spread to various countries as various variants were generated, and the World Health Organization (WHO) once defined it as a "global pandemic." In general, in the case of swine flu, it is common to administer defensive treatment through prevention by injecting purified inactivated strains before the cold season, but in the case of patients who have already developed symptoms during the swine flu season, prevention is not possible and symptoms must be alleviated through treatment. In the case of "Tamiflu," which stopped the spread of the swine flu that continued to spread hopelessly, it is outstanding in terms of effectiveness, but cases of side effects such as hallucinations after taking it are frequently reported through the media. In addition, there are no ingredients that can be taken for a long time as a preventative rather than a therapeutic drug.

Influenza virus has two surface antigens, hemagglutinin (HA) and neuraminidase (NA), which are glycoproteins, on its surface, and eight separate RNAs are present inside. HA binds to sialic acid residues on the surface of host cells, allowing the virus to attach to and penetrate host cells. NA plays a role in inducing the virus to further grow in other host cells by cleaving the α-ketosidic bond between the disaccharide moiety on the cell surface and neuraminic acid residues after the virus has finished growing in the infected host cell. The body's defense mechanism for viruses works by recognizing HA and preventing it from binding to host cells, or by binding to NA, inhibiting the virus in the infected host cell from growing in nearby cells. However, through various combinations of 16 types of HA and 9 types of NA, the virus generates numerous mutations and has resistance to defense mechanisms. Various drugs targeting various mechanisms have been developed as influenza virus inhibitors, and currently used drugs include ribavirin, which is an inhibitor of viral gene transcription and/or replication, amantadine and rimantadine, which are M2 channel inhibitors, and zanamivir and oseltamivir, which are NA activity inhibitors. Of these, ribavirin is used to treat not only the flu but also various other viral infections, but has many reported side effects such as flu-like symptoms, depression, and insomnia. Amantadine and rimantadine, which are M2 channel inhibitors, are only effective against influenza virus A, and many viruses have been reported to be resistant to them, and there have been cases of serious side effects on the nervous system and/or gastrointestinal tract.
Under these circumstances, there remains a need for the development of ingredients and/or preparations that exhibit inhibitory activity against influenza viruses, are safe for the human body, and can be taken continuously from a preventive standpoint.

KR10-2016-0150250A KR10-2019-0041130A KR10-2012-0133133A KR10-2016-0036863A KR 10-2018-0065458 A

As a result of intensive research and efforts to achieve the above-mentioned objectives, the present invention was completed by confirming that extracts, fermented products and/or fractions of plants that have been used as medicinal plants have excellent inhibitory effects against influenza virus proliferation.

The present invention provides a composition for inhibiting, preventing infection with, treating and/or ameliorating influenza viruses, which contains an active ingredient that is effective in inhibiting influenza virus proliferation and/or has no significant safety issues even when taken over the long term.

To this end, specifically, a functional health food composition for preventing and/or improving influenza virus infection is provided, which contains a plant extract containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

Yet another object of the present invention is to provide a pharmaceutical composition for preventing and/or treating influenza virus infection, comprising an extract of a plant containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

Yet another object of the present invention is to provide a composition for inhibiting influenza viruses, which contains an extract of a plant containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

Yet another object of the present invention is to provide a health functional food composition containing nodakenin and/or nodakenetin for preventing and/or improving influenza virus infection.

Yet another object of the present invention is to provide a pharmaceutical composition for preventing and/or treating influenza virus infection, comprising nodakenin and/or nodakenetin.

Yet another object of the present invention is to provide a composition for inhibiting influenza viruses, comprising nodakenin and/or nodakenetin.

Yet another object of the present invention is to provide a method for ameliorating, preventing and/or treating a viral infection comprising administering to an individual a therapeutically effective amount of a plant extract containing nodakenin and/or nodakenetin.

Yet another object of the present invention is to provide a method for ameliorating, preventing and/or treating a viral infection comprising administering to an individual a therapeutically effective amount of nodakenin and/or nodakenetin.

Yet another object of the present invention is to provide a use of a plant extract containing nodakenin and/or nodakenetin for producing a virus therapeutic agent.

Yet another object of the present invention is to provide a use of nodakenin and/or nodakenetin for producing a virus therapeutic agent.

Yet another object of the present invention is to provide a use of a plant extract containing nodakenin and/or nodakenetin for use in ameliorating, preventing and/or treating viral infections.

Yet another object of the present invention is to provide a use of nodakenin and/or nodakenetin for use in ameliorating, preventing and/or treating viral infections.

In the present invention, the plant may be a plant selected from the group consisting of plants of the genus Angelica (Angelica sp.), plants of the genus Peucedanum (Peucedanum sp.) and/or plants of the genus Anthrisscus (Anthriscus sp.).

In the present invention, the extract may be an extract of one or more solvents selected from the group consisting of water, C ₁ -C ₄ alcohol, 1,3-butylene glycol, and/or ethyl acetate.

In the present invention, the fraction may be a fraction of one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform and/or dichloromethane for the fermentation product.

In the present invention, the fraction may be a fraction of n-hexane and/or ethyl acetate solvent for the fermentation product.

In the present invention, the fermented product may be a fermented product produced by lactic acid bacteria.

In the present invention, the lactic acid bacteria may be lactobacillus sp. lactic acid bacteria.

In the present invention, the fermented product of the plant extract and/or its fraction may contain nodakenetin/nodakenin in a weight ratio of 0.5 to 20.

In the present invention, the nodakenin and/or nodakenetin may be isolated from a fermentation product of a plant extract, or may be obtained through synthesis and/or semi-synthesis.

In the present invention, the plant may be one or more selected from the group consisting of Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, chervil (Anthriscus cerefolium) and/or Angelica decursiva.
In the present invention, the nodakenetin/nodakenin may be contained in a weight ratio of 0.5 to 20.

To achieve the above object, the present invention provides a composition for inhibiting and/or preventing and/or improving infection with influenza virus, and treating the same, which contains a plant extract containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

The present invention also provides a composition for inhibiting and/or preventing and/or improving infection with influenza viruses, which contains nodakenin and/or nodakenetin.

When a composition containing the fermentation product of the present invention and/or its fraction and/or nodakenetin is ingested, influenza virus infection can be inhibited.

The composition of the present invention may be a pharmaceutical composition, a functional health food composition and/or a quasi-drug composition.

The present invention will be described in detail below.

In one embodiment, the present invention provides a composition for inhibiting influenza viruses, which contains a plant extract containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof, a functional health food composition for preventing and/or improving infection, and/or a pharmaceutical composition for prevention and/or treatment.

The plant may be a plant selected from the group consisting of Angelica sp., Peucedanum sp. and/or Anthrisscus sp. plants.

The plants include, but are not limited to, Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, chervil, and/or Angelica decursiva, and may include any plant that contains nodakenetin and/or nodakenin.

In one embodiment of the present invention, it was confirmed that a compound obtained by fractionating the fermentation product of Angelica sinensis extract has the effect of inhibiting the proliferation of influenza viruses, and it was experimentally confirmed that such a compound is nodakenetin. Therefore, plant extracts containing nodakenetin and/or nodakenin can be used in the present invention without any restrictions.

In one aspect, the present invention provides a composition for inhibiting influenza viruses, a functional health food composition for preventing and/or ameliorating infection, and/or a pharmaceutical composition for prevention and/or treatment, which contains a fermented product of Angelica sinensis extract and/or a fraction thereof.

The term "Angelica gigas" in the present invention is a perennial plant belonging to the family Apiaceae, distributed in East Asia and the like. Angelica gigas is also called, but not limited to, Korean angelica, Chinese angelica, Dang gui, Dan gui, Dang quai, Japanese angelica, Shirane cnikyu, Japanese angelica, Japanese angelica, Western angelica, and angelica. It is a perennial plant that is 1-2m tall, completely hairless, reddish purple in color, has large roots, a strong fragrance, and a straight trunk. It flowers in August-September and bears fruit in September-October, but the new shoots can also be eaten as a vegetable. It is warm in nature, non-toxic, and has a spicy, sweet, and bitter taste. It is used for poor circulation, physical weakness, joint pain, headaches, stomachaches, dizziness, constipation, emaciation due to weak digestive function, bruises, arthritis, internal bleeding, blood stagnation, swelling, and headaches caused by vascular disease, and is a main medicine for gynecological diseases, regulating menstruation and acting as a sedative. When using Angelica sinensis, different parts are used; the upper part replenishes blood, the trunk part regulates blood, the roots remove stasis and smooth blood flow, and the whole plant stimulates blood circulation. The components of Angelica sinensis include 0.31% essential oil in the roots and 0.69% in the fruits, 1.38% coumarin in the whole plant and 2-3% in the roots. The main component of the roots is called decursin, a pyranocoumarin-based substance, and other reported components include decursinol, umbelliferone, and beta-sitosterol. Other reported components include nodakenetin, nodakenin, and imperatorin. The roots and leaves of Angelica gigantea can be used as food, and have long been taken as a herbal medicine, so there are no problems with toxicity and/or side effects.

The term "influenza" as used herein is commonly known as the "flu" and is an acute respiratory disease caused by the influenza virus. Influenza is a highly contagious disease that causes epidemics of various sizes around the world every year, and once an epidemic begins, 10-20% of the population is usually infected within 2-3 weeks.

Influenza virus was first isolated from pigs by Shope in 1931, and from humans by Smith, Andrews, and Laidlow in 1933. Since then, through several mutations, it has become a cause of increasing morbidity and/or mortality in humans worldwide.

Influenza viruses belong to the family Orthomyxoviridae and/or the genus Influenzavirus, and are largely classified into types A, B, and/or C according to the difference in antigenicity of the nucleocapsid (NP) and matrix (M) proteins. Of these, type A is classified into subtypes H1 to H15 according to the antigenic properties of the HA protein, and N1 to N9 according to the antigenic properties of the NA protein. Influenza virus particles have an average diameter of about 80 to 120 nm and are spherical with external protrusions, but viruses immediately after separation from fertilized eggs and/or susceptible cells may be in the form of filaments 400 nm long. Virions have an envelope and are sensitive to heat and/or pH.

The most obvious symptom is a sudden high fever of 38-40°C within 24 hours of infection, followed by systemic symptoms such as headache, muscle pain and/or fatigue, and respiratory symptoms such as sore throat, cough, expectoration and/or rhinitis. Abdominal pain, vomiting and/or convulsions may also occur in rare cases. Healthy people recover after showing symptoms for a few days, but those with chronic lung disease, heart disease and/or immunosuppressed individuals may develop complications such as pneumonia and die, and may also develop other complications such as encephalopathy, transverse myelitis, Reye's syndrome, myositis, myocarditis and/or pericarditis. Children show similar symptoms to adults, but may have higher fevers, fever convulsions, and more frequent otitis media, crowd and/or muscle pain.
The term "infection" as used herein refers to the proliferation of a host organism by a parasitic species, or simply the entry of a pathogen into the body and proliferation thereof.

The term "prevention" as used herein refers to any action of inhibiting and/or delaying infection and/or proliferation of influenza virus by ingestion and/or administration of a composition containing nodakenetin and/or nodakenin according to the present invention.

In the present invention, it was experimentally confirmed that nodakenetin and nodakenin inhibit influenza virus proliferation through inhibition of neuraminidase activity, and can be used as antiviral agents.

In addition, neuraminidase inhibitors (Tamiflu, Relenza) such as the composition of the present invention have an influenza prevention effect of 70% to 90%, and the use of neuraminidase inhibitors for preventive purposes has been proven to be effective when influenza occurs within a family, when influenza epidemics occur in the community, and when influenza epidemics occur in nursing homes. Neuraminidase inhibitors are an effective means of preventing influenza outbreaks in people who have not been vaccinated, and in high-risk groups who require additional preventive measures when the vaccine strain does not match the currently circulating virus strain. Since neuraminidase inhibitors do not have much effect on antibody production response after influenza vaccination, they can be used for prevention before antibodies are produced when vaccination is administered one step late during the influenza epidemic (KCDC, National Health Information Portal of the Korea Centers for Disease Control and Prevention, http://health.cdc.go.kr/health/HealthInfoArea/HealthInfo/View.do?idx=3980 ).

Therefore, the composition of the present invention containing nodakenetin and/or nodakenin has preventive effects as well as ameliorative and/or therapeutic effects against influenza virus infection.

The term "treatment" as used herein refers to any action that improves or favors influenza symptoms by taking and/or administering a composition containing nodakenetin and/or nodakenin according to the present invention.

The term "improvement" in the present invention means the effect of alleviating influenza symptoms by ingestion and/or administration of a composition containing nodakenetin and/or nodakenin according to the present invention, inhibiting the survival of influenza viruses, and inhibiting and/or reducing their growth and/or division and proliferation, thereby alleviating influenza symptoms.

The term "extract" in the present invention includes the extract itself and/or all formulations that can be formed using the extract, such as the extract obtained by the extraction process of the plant material, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, and/or a mixture of these. Specifically, the extract of the present invention can be manufactured and used in the form of a dry powder after extraction.

The plant extract of the present invention can be produced by a production method including, but not limited to, the following steps:
1) washing and/or drying the plant, followed by grinding into powder;
2) adding a solvent to the plant powder obtained in step 1) to extract it;
3) filtering the extract of step 2); 4) concentrating the filtered extract of step 3);
5) Drying the concentrated extract of step 4) to obtain a dry powder of plant extract.

In the above method, the plant in step 1) can be a cultivated plant and/or a commercially available plant, without any limitation. In addition, in step 1), the Angelica acutiloba may be extracted including the trunk, branches, leaves and/or roots, without being limited thereto.

In the method, the extraction solvent in step 2) may be one or more solvents selected from the group consisting of water, _C1 - _C4 lower alcohol, 1,3-butylene glycol, and/or ethyl acetate, preferably water and/or fermented alcohol, but is not limited thereto. The extraction method may be hot water extraction, immersion extraction, reflux cooling extraction, and/or ultrasonic extraction, preferably hot water extraction, but is not limited thereto. The extraction solvent may be added in an amount of 1 to 10 times the amount of the dried plant, specifically 2 to 3 times the amount of the dried plant. The extraction temperature may be 20°C to 100°C, specifically 60°C to 80°C, and more specifically 70°C, but is not limited thereto. The extraction time may be 2 hours to 48 hours, specifically 15 hours to 30 hours, and more specifically 24 hours, but is not limited thereto. In addition, the number of extractions may be 1 to 5 times, specifically 3 to 4 times, and more specifically 3 times, but is not limited thereto.

In the above method, step 4) may further include concentrating the extract under reduced pressure, which may be performed using, but is not limited to, a vacuum concentrator and/or a vacuum rotary evaporator.

In the above method, step 5) may further include a step of drying the extract that has undergone the reduced pressure concentration process, and the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying and/or freeze drying, but is not limited thereto.

The plant extract of the present invention can be treated in various ways, such as hydrolysis, fermentation, heating, and pressurization, to improve the influenza virus proliferation inhibitory activity, and preferably fermentation.

The terms "fermentation" and/or "fermented product" in the present invention refer to the process of decomposing organic matter using the efficacy of microorganisms (e.g., lactic acid bacteria) and/or the product thereof. Fermentation and putrefaction proceed through similar processes, but fermentation is when a substance that is useful for human life is formed as a result of decomposition, and putrefaction is when a substance that emits a foul odor or is harmful is formed. Therefore, when herbal materials are fermented using microorganisms, the toxicity of the extract is reduced or the extract is made into a low molecular weight substance, making it easy to digest, or it is easy to absorb through the skin when applied to the skin, improving the bioavailability. In addition, natural ingredients can have pharmacological effects by themselves, but the pharmacological effects are further strengthened through fermentation, or they can exhibit pharmacological effects that the extract does not have before fermentation due to the metabolism of microorganisms. The fermented product may contain nodakenetin/nodakenin in a weight ratio of 0.5 to 20.

Lactic acid bacteria can be used for fermentation, preferably Lactobacillus lactic acid bacteria, more preferably Lactobacillus plantarum, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the extract fermented with Lactobacillus plantarum had a weight ratio of nodakenetin/nodakenin that was 40 times higher than the non-fermented extract, and the nodakenetin content increased by up to 18 times after fermentation, confirming that the fermented extract has excellent influenza virus inhibitory activity.

The term "fraction" as used herein means a result obtained by fractionating a mixture containing a variety of components to separate a specific component and/or a specific group of components. The fraction of the present invention may include, but is not limited to, a solvent fraction and a polysaccharide fraction. The fractionation method for obtaining the fraction may be a method commonly used in the art.

Examples of the fractionation method include, but are not limited to, a method in which the extract and/or fermented product of the present invention is treated with a specific solvent to obtain a fraction from the extract, and a method in which the extract and/or fermented product is soaked and recovered from the polysaccharides using a cold soaking method and a centrifugation method.

In the present invention, the type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These can be used alone or in combination of two or more. In other words, it is obvious to those skilled in the art that when fractionation is performed using a substance extracted from the plant of the present invention, the extraction and/or fractionation method is not limited and is included in the scope of the present invention.

Specifically, in Preparation Example 3 of the present invention, the solvent fractions of each extract were obtained through the following process. After preparing each extract, preferably the suspension is filtered, and then fractionated according to polarity using about 1 to 5 volumes of hexane, ethyl acetate, chloroform, _C1 to _C4 lower alcohol and/or water to obtain a hexane fraction, an ethyl acetate fraction, a chloroform fraction, a _C1 to _C4 lower alcohol fraction and/or a water fraction. The first fraction fractionated in the above process can be further fractionated using about 1 to 5 volumes of hexane, ethyl acetate, chloroform, _C1 to _C4 lower alcohol and/or water to obtain a second fraction, and the same process can be further carried out to obtain a third fraction. In one example carried out in Production Example 3, fractionation was carried out in the order of hexane, ethyl acetate, and butanol, and a fraction having a high content of nodakenetin and/or nodakenin could be obtained.

In one embodiment, a functional health food composition for preventing and/or improving influenza virus infection is provided, which contains a plant extract containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

The term "health functional food composition" in the present invention refers to a food product obtained by adding fermented plant extracts and/or fractions thereof to food ingredients such as beverages, teas, spices, gums, and confectioneries, or by encapsulating, powdering, or manufacturing them into a suspension, etc., and when ingested, it brings about a specific health effect. However, unlike general medicines, since it is made from food ingredients, it has the advantage of being free of side effects that can occur when taking medicines for a long period of time.

When the fermented product of the plant extract and/or its fraction is used as a food additive, the fermented product of the Angelica extract and/or its fraction can be added as it is or with other foods and/or food ingredients, and can be used appropriately by a conventional method. The amount of the active ingredient to be mixed can be determined to suit the purpose of use (prevention, health and/or therapeutic treatment). In general, when producing foods and/or beverages, the composition of the present invention can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials, but is not limited thereto. However, in the case of long-term intake for the purpose of health and/or hygiene and/or health regulation, the amount may be less than the above range, and since there is no problem from the viewpoint of safety, the active ingredient can be used in an amount greater than the above range.

There are no particular limitations on the type of food. Examples of foods to which the substance can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other dairy products including noodles, gums, ice cream, various soups, beverages, tea, energy drinks, alcoholic beverages and/or vitamin complexes, including all health foods in the usual sense.

In one embodiment, a pharmaceutical composition for preventing and/or treating influenza virus infection is provided, which comprises an extract of a plant containing nodakenin and/or nodakenetin, a fermented product thereof, and/or a fraction thereof.

The term "pharmaceutical composition" as used herein can be administered orally and/or parenterally, and can be administered in the form of a general pharmaceutical formulation, for example, in various oral and/or parenteral dosage forms during clinical administration, and when formulated, can be prepared using diluents and/or excipients, such as, but not limited to, commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one or more excipients, such as, but not limited to, starch, calcium carbonate, sucrose and/or lactose, gelatin, etc.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to the commonly used simple diluents water and liquid paraffin, but are not limited to these.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include, but are not limited to, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

In one embodiment, a composition for inhibiting influenza viruses is provided that contains an extract of a plant containing nodakenin and/or nodakenetin, a fermentation product thereof, and/or a fraction thereof.

The term "virus-inhibiting composition" as used herein refers to a composition that interferes with the replication of viruses or strengthens the immune system against viral infections. It may be in the form of a pharmaceutical composition administered to vertebrates, preferably mammals including humans, or may be used in food compositions, tableware, kitchen utensils, household items, and various other cleaning agents, disinfectants, and conventional antiviral agents, but is not limited thereto.

To confirm the influenza virus growth inhibitory ability of the fermented plant extract and/or its fraction produced by the above method, the neuraminidase inhibition assay, which is currently the most widely used method for evaluating influenza virus growth inhibition, was used.

Neuraminidase (hereafter referred to as NA) is a protein that allows the virus to infect host cells and grow in numbers by cleaving the alpha-ketosidic bond between the disaccharide moiety on the surface of the host cell and neuraminic acid (sialic acid) residues when the virus grows in the infected host cell. Inhibitors of neuraminidase bind to the active site of neuraminidase and inhibit its function, thereby inhibiting influenza virus growth.

In order to trace the active ingredients of the fermented product of the Angelica acutiloba extract in the present invention, the experiment of Preparation Example 3 was carried out. The fermented product of the Angelica acutiloba extract was fractionated and purified to obtain compound A, and the NMR spectrum of compound A was compared with conventional literature, and it was identified as nodakenetin, which was then directly compared with a purchased nodakenetin standard through HPLC analysis to confirm it.

The fermented product of the Touki extract and/or its fraction had a high content of nodakenetin (Experimental Example 2) and showed a significant concentration-dependent inhibitory effect on the proliferation of influenza viruses. In particular, the fermented product of the Touki extract and/or its fraction showed a high inhibitory effect on the proliferation of influenza viruses (Experimental Example 1).

The fermented plant extracts and/or fractions thereof of the present invention may contain, but are not limited to, a weight ratio of nodakenetin/nodakenin of 0.5-20, preferably 1-15, 2-10, and/or 4-9.

In the present invention, it has been confirmed that before fermentation, the Angelica extract contains nodakenetin/nodakenin in a weight ratio of 0.1, and that the fermented product of the Angelica extract and/or fractions thereof contain nodakenetin/nodakenin in a weight ratio of 0.5 to 20. It has been confirmed that the fermented product of the Angelica extract and/or fractions thereof that satisfy this range has a weight ratio of nodakenetin/nodakenin that is 40 times higher than that of the unfermented Angelica extract, and that after fermentation, the amount of nodakenetin increases by 18 times or more, demonstrating that it has even better viral infection inhibitory ability.

In one embodiment, the present invention provides a composition for inhibiting influenza viruses, a functional health food composition for preventing and/or improving infection, and a pharmaceutical composition for preventing and/or treating infection, each of which contains nodakenin and/or nodakenetin.

The descriptions of the virus-inhibiting composition, health functional food composition, and pharmaceutical composition are omitted to avoid duplication, and can be directly applied to the composition containing nodakenin and/or nodakenetin of the present invention.

The "nodakenetin" (Molecular formula: _{C14H14O4 , Molecular} weight: 246.262) is a coumarin-based non-glycosidic component represented by the following [Chemical formula 1], and is converted by fermentation or hydrolysis of nodakenin (Molecular formula: _C20H2409 , Molecular weight _: 408.403) _, a glycoside represented by [Chemical formula 2].

Nodakenetin is not necessarily formed only through the fermentation process, and is naturally present in various parts of the plant body, although in small amounts compared to the glycoside nodakenin. Nodakenetin and nodakenin are mainly contained in Angelica sp. plants, but are also known to exist in other plants such as Peucedanum sp. plants (peucedanum, peucedanum, rock quince, etc.), Anthriscus sp. plants (peucedanum, chervil, etc.) and nodake.

The nodakenin and/or nodakenetin may be, but is not limited to, those isolated from the fermentation of plant extracts or obtained through synthesis and/or semi-synthesis.

The "plant" may be one or more selected from the group consisting of Angelica sp., Peucedanum sp., and/or Anthriscus sp. plants, and is preferably Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, Anthriscus cerefolium, and/or Anthriscus schidigera. decursiva), and more preferably, it may be, but is not limited to, Angelica sinensis.

In the present invention, the nodakenetin/nodakenin may be present in a weight ratio of 0.5 to 20, preferably 1 to 15, 2 to 10, and/or 4 to 9, but is not limited thereto.

The fermented product of the Angelica acutiloba extract and/or its fraction according to the present invention inhibits neuraminidase activity and inhibits the infection and proliferation of influenza viruses, and can therefore be usefully used in functional health food compositions for preventing and/or improving influenza virus infections, pharmaceutical compositions for preventing and/or treating influenza virus infections, etc.

1 is a flow chart of a method for separating and purifying nodakenetin by fractionating a fermented product of a Dongqi extract. 1 shows the results of the influenza virus proliferation inhibition ability (%) of nodakenin and nodakenetin. This shows the results of the influenza virus proliferation inhibition ability (%) of plant extracts containing Angelica sinensis before and after fermentation. 1 shows the results of the influenza virus proliferation inhibitory activity (%) of Compound A (nodakenetin) and/or the EA fraction.

Hereinafter, the present invention will be described in detail with reference to the following experimental examples and/or manufacturing examples. However, the following experimental examples and/or manufacturing examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following experimental examples and/or manufacturing examples. Furthermore, these experimental examples and/or manufacturing examples are merely illustrative for the purpose of aiding in the understanding of the present invention, and therefore do not limit the scope of the present invention in any sense.

Preparation Example 1: Preparation of a plant extract containing nodakenin and/or nodakenetin First, Angelica sinensis was washed and/or dried.

Next, 10 kg of primary distilled water was added to 1 kg of the raw Angelica acutiloba plant and extracted with hot water at 80°C for 3 hours.

The hot water extract was filtered under reduced pressure using filter paper, and the filtered extract was completely concentrated using a rotary evaporator to be used as a sample in Experimental Example 2. The yield of the hot water extract produced by the above method was about 36%.

The 50 wt% ethanol extract was produced by adding 10 kg of 50 wt% ethanol aqueous solution to 1 kg of the raw plant and extracting it with hot water at 65°C for 3 hours. The 50 wt% ethanol extract was filtered under reduced pressure using filter paper and completely concentrated using a rotary evaporator to be used as a sample (Experimental Example 3). The yield of the 50 wt% ethanol extract produced by the above method was about 40%.

Preparation Example 2: Preparation of fermented plant extract 55 g of MRS medium powder (peptone 10 wt%, beef extract 10 wt%, yeast extract 5 wt%, dextrose 20 wt%, polysorbate 80 1 wt%, ammonium citrate 2 wt%, sodium acetate 5 wt%, magnesium sulfate 0.1 wt%, manganese sulfate 0.05 wt%, dipotassium phosphate 2 wt%) was added to 1 L of water to prepare MRS liquid medium, and then a solid colony of Lactobacillus plantarum was inoculated into 100 ml of MRS liquid medium. OD600 was measured at regular intervals and cultured until the value reached 1.5 to prepare a starter culture. Thereafter, 1 ml of the starter culture was inoculated into 100 ml of MRS liquid medium containing 1 g, 2 g, 5 g, and 10 g of the main culture solution, Angelica sinensis extract (ethanol extract), and fermented in a 30°C shaking incubator at 150 rpm for 4 days to complete the plant fermentation product (Experimental Example 3).

1 ml of the starter culture was inoculated into 100 ml of MRS liquid medium containing 5 g of Angelica extract (hot water extract), and fermented in a 30°C shaking incubator at 150 rpm for 4 days to complete the plant fermentation product (Experimental Example 2).

Preparation Example 3: Separation and purification of fermentation fractions and/or active compounds In order to trace the active ingredients of the lactic acid bacteria culture containing the Angelica acutiloba extract, 80 g of the Angelica acutiloba extract was fermented with Lactobacillus plantarum in 8 L of MRS medium, and the resulting solution was sterilized and filtered (0.22 μm). The same amount of n-hexane was added to the solution, and the solution was shaken and left to stand for fractionation, and then ethyl acetate (hereinafter referred to as EA) and n-butyl (hereinafter referred to as BuOH) were added in sequence, and the solution was shaken and left to stand to obtain each fraction. The EA fraction (15 g) was subjected to silica gel column chromatography using an elution solvent of n-hexane/EA (gradient) to separate it into 11 small fractions. The small fraction Fr. 9 was subjected to Prep-LC using an elution solvent of acetonitrile (ACN)/H2O (3:7) to obtain 30 mg of compound A (Figure 1). The chemical structure of compound A was confirmed through NMR spectrum. From the characteristic peaks of a typical furocoumarins structure, compound A was found to be nodakenetin. The spectral data was collected and compared with conventional literature to identify compound A as nodakenetin, and the compound A was confirmed by direct comparison with nodakenetin purchased from Biopurify through TLC and/or HPLC.

NMR data
Compound A (Nodakenetin)-White amorphous powder, ESI-MS m/z: 247 [M+H]+; ¹ H-NMR (600MHz, CD3OD) δ_H: 7.83 (1H, d, J = 9.6Hz, H-4), 7.38 (1H, S, H-5), 6.70 (1H, S, H-8), 6.18 (1H, d, J = 9.6 Hz, H-3), 4.75 (1H, t, J=9.0 Hz, H-2'), 3.24 (2H, m, H-3'), 1.29 (3H, s, CH3), 1.23 (3H, s, CH3); 13C -NMR (150MHz, CD3OD) δ_C: 165.4 (C-2), 163.9 (C-7), 157.1 (C-10), 14 6.4 (C-4), 127.4 (C-6), 125.1 (C-5), 114.2 (C-9), 112.3 (C-3), 98.3 ( C-8), 92.7 (C-2'), 72.4 (C-4'), 30.4 (C-3'), 25.5 (CH3), 25.5 (CH3).

Experimental Example 1: Confirmation of influenza virus inhibitory activity In the following examples, the following method for evaluating influenza virus inhibitory activity was used.

To evaluate the influenza virus inhibition ability, candidate substances were treated with neuraminidase (NA) and then an NA activity assay kit (Sigma, #cat: MAK121-1KT) was used. The positive control group was set to 10 nM of zanamivir, an ingredient in a new influenza drug, and 5 μl of sample was taken to match the concentration for each experimental group and mixed with wild-type NA (R&D systems). After that, the reagents provided by the NA activity assay kit were mixed according to the instructions, 40 μl was added, and the mixture was reacted in a 30° C. incubator for 20 minutes, after which the absorbance was measured at 570 nm (A20). The mixture was then wrapped in foil and reacted in a 37° C. incubator for 30 minutes, after which the absorbance was measured at 570 nm (A50). The wild-type NA activity of each treatment group was calculated by subtracting A20 from A50, and the experiment was repeated three times.

First, the influenza proliferation inhibitory ability of nodakenetin (Chengdu biopurify phytochemical Ltd. Cat No. BP3273) and nodakenin (Chengdu biopurify phytochemical Ltd. Cat No. BP1002) was evaluated. Nodakenetin formed through sugar separation from nodakenin showed increased efficacy in a concentration-dependent manner (10, 50, 250 ppm), but nodakenin showed insufficient concentration-dependent effect (Figure 2).

The influenza virus growth inhibitory ability of the plant extracts was evaluated before and after fermentation. It was confirmed that the fermented extracts showed a concentration-dependent increase in influenza virus inhibitory ability compared to the unfermented extracts (Figure 3).

It was confirmed that the NA activity inhibitory ability of EA fractions containing nodakenetin (250, 500, 1000 ppm) and nodakenetin obtained after fractionation (Compound A, 25, 50, 100 ppm) increased in a concentration-dependent manner (Figure 4).

Experimental Example 2: Changes in nodakenin and/or nodakenetin due to fermentation of plant extract In order to confirm the change in the nodakenin and/or nodakenetin content due to fermentation of Angelica acutiloba extract (hot water extract), the contents were measured using Angelica acutiloba extract (hot water extract) fermented by the method of Preparation Example 2 and Angelica acutiloba extract (hot water extract) that was not fermented. As a result, as shown in Table 1 below, the nodakenetin content of the plant extract containing Angelica acutiloba increased by about 43 ppm due to fermentation by lactic acid bacteria, and the nodakenin content decreased by 43 ppm due to conversion to nodakenetin.

It was confirmed that the fermented product has even greater ability to inhibit viral infection.

Experimental Example 3: Comparison of nodakenin and/or nodakenetin contents before and after fermentation After fermentation with Lactobacillus plantarum KCTC 3104 strain in 100 ml of MRS liquid medium containing 1 g, 2 g, 5 g, and 10 g of Angelica sinensis extract (ethanol extract), the nodakenin and/or nodakenetin contents before and after fermentation were analyzed (Table 2).

The Angelica extract is in the form of a fully concentrated, dried powder, and all the analytical samples were analyzed in a completely dried state. As shown in Table 2, the ratio of nodakenetin (A')/nodakenin (A) in the unfermented Angelica extract is around 0.1, while the A'/A in the fermented Angelica extract is 4.1-8.34, an increase of more than 41 times. Therefore, it was confirmed that the fermented Angelica extract with a high nodakenetin content has an even more excellent ability to inhibit viral infection. In addition, to investigate the effect of the extraction solvent, the nodakenin and/or nodakenetin contents of the Angelica hot water extract and the Angelica ethanol extract were analyzed and compared.

As shown in Table 3 below, there was no significant difference in the A'/A ratio.

Claims (6)

A functional health food composition for preventing or ameliorating influenza virus infection, comprising a fermented product of a plant extract of Angelica gigas containing nodakenin and nodakenetin, or a fraction of said fermented product, wherein the fermented product of said plant extract or the fraction of said fermented product has a weight ratio of nodakenetin to nodakenin of 0.5 to 20. The health functional food composition according to claim 1, wherein the extract is an extract of one or more solvents selected from the group consisting of water, _C1 - _C4 alcohol, 1,3-butylene glycol, or ethyl acetate, the fermented product is a fermented product by lactic acid bacteria, and the fraction is a fraction of the fermented product with one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform, or dichloromethane. A pharmaceutical composition for preventing or treating influenza virus infection, comprising a fermentation product of a plant extract of Angelica gigas containing nodakenin and nodakenetin, or a fraction of said fermentation product, wherein the fermentation product of said plant extract or the fraction of said fermentation product has a weight ratio of nodakenetin to nodakenin of 0.5 to 20. The pharmaceutical composition according to claim 3, wherein the extract is an extract of one or more solvents selected from the group consisting of water, _C1 - _C4 alcohol, 1,3-butylene glycol or ethyl acetate, the fermented product is a fermented product by lactic acid bacteria, and the fraction is a fraction of the fermented product with one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform or dichloromethane . A composition for inhibiting influenza viruses, comprising a fermentation product of a plant extract of Angelica gigas containing nodakenin and nodakenetin, or a fraction of said fermentation product, wherein the fermentation product of said plant extract or the fraction of said fermentation product has a weight ratio of nodakenetin to nodakenin of 0.5 to 20 . The composition according to claim 5, wherein the extract is an extract of one or more solvents selected from the group consisting of water, _C1 - _C4 alcohol, 1,3-butylene glycol, or ethyl acetate, the fermented product is a fermented product by lactic acid bacteria, and the fraction is a fraction of the fermented product with one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform, or dichloromethane.

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