KR20230006422A - Composition comprising nodakenin and nodakenetin for suppressing influenza virus - Google Patents

Abstract

The present invention relates to a health functional food composition for preventing or improving influenza virus infection, and a pharmaceutical composition for preventing or treating influenza virus infection, comprising a fermented product of Angelica gigas extract or a fraction thereof. In addition, the present invention relates to a health functional food composition for preventing or improving influenza virus infection and a pharmaceutical composition for preventing or treating influenza virus infection, containing nodakenetin. More specifically, the fermented product of the Angelica gigas extract or a fraction thereof, according to the present invention, inhibits the proliferation of influenza virus infection by inhibiting neuraminidase activity, and thus, the same can be usefully used as a health functional food composition for preventing or improving influenza virus infection, a pharmaceutical composition for preventing or treating influenza virus infection, and the like.

Description

Composition comprising nodakenin or nodakenetin for suppressing influenza virus

In the present invention, nodakenin or nodakenetin and a plant extract, fermented product or fraction thereof containing the same can prevent, improve or treat influenza virus infection by inhibiting neuraminidase (NA) activity, It relates to a composition capable of inhibiting influenza virus.

Influenza belongs to orthomic viruses and is a typical pathogenic virus that kills thousands to tens of thousands of people every season. Since the 20th century, influenza is a virus with a high mutation rate that crosses species, causes mutations, leads to epidemics, and renders previously discovered drugs meaningless. Commonly referred to as pandemic flu, this disease usually causes chills, fever, sore throat, muscle pain, cough, weakness and discomfort. It can cause fatal complications when accompanied by pneumonia or Reye's syndrome. Usually, respiratory infections occur mainly through coughing, sputum, or sneezing of infected people, but many variants occur when infections are transmitted to humans from other animals (especially birds). There are three types of influenza viruses: influenza virus A, influenza virus B, and influenza virus C. Among them, influenza A, also known as swine flu, which was prevalent in 2009, which caused a pandemic through transmission between animal species, was the influenza A H1N1 type that first broke out in Mexico and spread to several countries as several strains were created, and the World Health Organization (WHO) ) has defined it as a 'global trend'. Usually, in the case of flu, a purified inactivated strain is injected before the cold season, and protective treatment through prevention is common. I have to do it. In the case of ‘Tamiflu’, which stopped the spread of the swine flu, which was spreading helplessly, it is excellent in terms of effectiveness, but side effects such as hallucinations after ingestion are often reported through the media. In addition, there is no component that can be ingested for a long time in terms of prevention, rather than a drug for treatment.

Influenza virus has two surface antigens, such as hemagglutinin, hemagglutinin (HA) and neuraminidase (NA), which are glycoproteins, on the surface, and there are 8 segmented RNAs inside. HA binds to sialic acid residues on the surface of the host cell, attaching the virus to the host cell and allowing it to penetrate. NA breaks the α-ketosidic bond between the disaccharide moiety on the cell surface and the neuraminic acid residue, allowing the virus to multiply into another host cell. plays a guiding role. In vivo viral defense mechanisms act by recognizing HA and interfering with host cell binding, or by binding to NA to inhibit the proliferation of viruses in infected host cells into adjacent cells. However, through various combinations of 16 types of HA and 9 types of NA, viruses generate numerous mutations and have resistance to defense mechanisms. Influenza virus inhibitors have been developed with various drugs targeting various mechanisms, and currently used drugs include ribavirin, an inhibitor of viral gene transcription and replication, and amantadine and rimantidine, which are M2 channel inhibitors. rimantidine), NA activity inhibitors Zanamivir and oseltamivir. Among them, ribavirin is used to treat infections caused by various viruses as well as flu, but has many reported side effects such as flu-like symptoms, depression, and insomnia, and the M2 channel inhibitors amantadine and rimantidine are effective only for influenza virus A and have already lost resistance. A number of viruses have been reported, and there have been cases of causing serious side effects to the nervous system and stomach.

Under this background, it is still required to develop ingredients and preparations that are safe for the human body and can be continuously ingested as a preventive measure, while exhibiting inhibitory activity against the influenza virus.

In order to achieve the above object, as a result of intensive research efforts, the present invention was completed by confirming that extracts, fermented products, or fractions thereof of plants that have been used as medicinal plants have excellent inhibitory effects on influenza virus proliferation.

The present invention is to provide a composition for inhibiting influenza virus, preventing infection, treating or improving infection, including an active ingredient that has an effect on inhibiting influenza virus growth and safety even when ingested for a long time.

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

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

Another object of the present invention is to provide a composition for inhibiting influenza virus comprising a plant extract containing nodakenin or nodakenetin, a fermented product thereof, or a fraction thereof.

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

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating influenza virus infection containing nodakenin or nodakenetin.

Another object of the present invention is to provide a composition for inhibiting influenza virus containing nodakenin or nodakenetin.

In order to achieve the above object, a composition for inhibiting influenza virus and preventing or improving infection, including a plant extract containing nodakenin or nodakenetin, a fermented product thereof, or a fraction thereof, for treatment provides

In addition, a composition for inhibiting influenza virus and preventing or ameliorating infection or treating an influenza virus containing nodakenin or nodakenetin is provided.

fermented product of the present invention or a fraction thereof; Alternatively, when ingesting a composition containing nodakenetin, influenza virus infection can be inhibited.

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

Hereinafter, the present invention will be described in detail.

In one embodiment, a composition for inhibiting influenza virus, including a plant extract containing nodakenin or nodakenetin, a fermented product thereof, or a fraction thereof, and a health functional food for preventing or improving infection Compositions and preventive or therapeutic pharmaceutical compositions are provided.

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

The plants are Angelica gigas , Peucedanum japonicum , Peucedanum terebinthaceum , Aegopodium podagraria , Anthriscus sylvestris , Chervil ( Anthriscus cerefolium), and Angelica decursiva . Including, but not limited thereto, all containing notakenetin or notakenin may be included.

In one embodiment of the present invention, it was confirmed that the compound obtained by fractionating the fermented product of Angelica quail extract had an effect of inhibiting the growth of influenza virus, and it was experimentally confirmed that this compound was nodakenetin, nodakenetin or noda Extracts of plants containing kenin can be used in the present invention without limitation.

In one aspect, the present invention provides a composition for inhibiting influenza virus, a health functional food composition for preventing or improving infection, and a pharmaceutical composition for preventing or treating infection, including a fermented product of Angelica gigas extract or a fraction thereof.

The term " Angelica gigas " of the present invention is a perennial plant belonging to the Apiaceae family and is distributed in East Asia and the like. Angelica is called, but is not limited to, true angelica, Korean angelica, Korean angelica, Chinese angelica, Danggui, Dangui, Dang quai, Japanese angelica, Gaeganghwa, Japanese angelica, Japanese angelica, Western angelica, and angelica. It is a perennial plant, 1-2m tall, hairless, purplish, with large roots, strong scent, and straight stems. Flowers bloom in August-September and bear fruit in September-October, and young shoots are eaten as herbs. It is warm in nature and non-toxic, and the taste is spicy, sweet and bitter. It is used for poor blood circulation, physical weakness, arthralgia, headache, abdominal pain, dizziness, constipation, emaciation due to digestive weakness, bruises, arthritis, internal bleeding caused by vascular disease, blood flow congestion, swelling, headache, and is the main medicine for women's diseases. It regulates menstruation and has a calming effect. When using angelica, it is used by distinguishing parts. The upper part replenishes blood, the body part controls blood, the tail removes blood and releases blood, and the whole acts to activate blood circulation. Angelica quinoa contains 0.31% of essential oil in roots and 0.69% in fruits, and 1.38% of coumarin in outposts and 2-3% in roots. The main component of the root is called decursin, which is a pyranocoumarin-based substance, and other decursinol, umbelliferon, and β-sitosterol have been reported. . Others such as nodakenetin, nodakenin, and imparatorin have been isolated and reported. Angelica roots and leaves are edible as food and have been consumed as herbal medicines for a long time, so there are no problems with toxicity and side effects.

The term "influenza" of the present invention is commonly known as 'flu' and is an acute respiratory disease caused by an influenza virus. Influenza causes large and small epidemics around the world every year, and is a very contagious disease that infects 10 to 20% of the general population within 2 to 3 weeks when the epidemic begins.

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

Influenza viruses belong to Orthomyxoviridae and Influenzavirus, and are largely divided into A, B, and C types due to antigenic differences in nucleocapsid (NP) and matrix (M) proteins. Separated. Of these, type A is classified into subtypes from H1 to H15 depending on the antigenic characteristics of the HA protein and from N1 to N9 according to the antigenic characteristics of the NA protein. Influenza virus particles have an average diameter of 80 to 120 nm and are spherical with projections on the outside, but viruses immediately after isolation from fertilized eggs and susceptible cells may be in the form of filaments with a length of 400 nm. Virions have an envelope and are sensitive to ether, heat and pH.

The most obvious symptom is a sudden high fever of 38 to 40 ° C within 24 hours of infection, and systemic symptoms such as headache, muscle pain and fatigue, and respiratory symptoms such as sore throat, cough, expectoration and rhinitis appear. In addition, abdominal pain, vomiting and convulsions may occur rarely. Healthy people show symptoms for several days and then recover, but patients with chronic lung disease, heart disease, and immunosuppressed patients may die from complications such as pneumonia, and in addition, encephalopathy, myelitis (transverse myelitis), Reye syndrome, myositis, myocarditis and Complications such as pericarditis may occur. Children have similar symptoms as adults, but they have higher fevers, febrile seizures may occur, and otitis media, pseudomembranous laryngitis (croup), and myalgia are more common.

As used herein, the term "infection" refers to the propagation of a host organism by a parasitic species. It simply refers to the work of pathogens entering the body and multiplying.

The term "prevention" of the present invention refers to any activity that inhibits or delays infection or proliferation of influenza virus by ingestion or administration of nodakenetin or a composition containing nodakenin according to the present invention.

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

In addition, neuraminidase inhibitors (Tamiflu, Relenza), such as the composition of the present invention, have an influenza preventive effect of 70% to 90%. , It has been proven effective during influenza outbreaks in nursing homes. Neuraminidase inhibitors are an effective means of preventing the occurrence of influenza in unvaccinated persons, in high-risk groups in need of additional precautions, when the vaccine strain does not match the currently circulating virus strain. Neuraminidase inhibitors do not significantly affect the antibody production response after influenza vaccination, so they can be used for prevention until antibodies develop in the case of late vaccination during the influenza season (KCDC, National Health Information Portal of the Centers for Disease Control and Prevention, http://health.cdc.go.kr/health/HealthInfoArea/HealthInfo/View.do?idx=3980 ).

Therefore, the composition containing nodakenetin or nodakenin of the present invention has a preventive effect as well as an effect of improving or treating influenza virus infection.

The term "treatment" of the present invention refers to all activities that improve or benefit symptoms of influenza by ingestion or administration of nodakenetine or a composition containing nodakenin according to the present invention.

The term "improvement" of the present invention means alleviating symptoms of influenza, inhibiting the survival of influenza virus, and inhibiting or reducing growth and division by ingestion or administration of a composition containing notakenetin or notakenin according to the present invention. By doing so, it means the effect of alleviating the symptoms of influenza.

The term "extract" of the present invention refers to an extract obtained by extraction of the plant material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed using the extract itself and the extract solution. Specifically, the extract of the present invention may be prepared and used in the form of a dry powder after extraction.

The plant extract of the present invention may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) Washing and drying the plant and then pulverizing it into powder;

2) extracting by adding a solvent to the plant powder obtained in step 1);

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 the plant extract.

In the above method, the plant of step 1) can be used without limitation, such as cultivated or commercially available. In addition, in step 1), although not limited thereto, Angelica quail may be extracted including all stems, branches, leaves, or roots.

In the above method, the extraction solvent in step 2) may be one or more solvents selected from the group consisting of water, C ₁ to C ₄ lower alcohol, 1,3-butylene glycol, and ethyl acetate, preferably May be water or fermented alcohol, but is not limited thereto. As the extraction method, an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, or ultrasonic extraction may be used, preferably hot water extraction, but is not limited thereto. The extraction solvent may be extracted by adding 1 to 10 times the amount of the dried plant, and specifically, it may be extracted by adding 2 to 3 times. 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. In addition, the extraction time may be 2 hours to 48 hours, may be specifically 15 hours to 30 hours, and may be more specifically 24 hours, but is not limited thereto. In addition, the number of extractions may be 1 to 5 times, may be specifically 3 to 4 times, and may be more specifically 3 times, but is not limited thereto.

In the above method, step 4) may further include a process of concentrating the extract under reduced pressure, and the vacuum concentration may use a vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto.

In the method, step 5) may further include drying the extract subjected to the vacuum concentration process, and the drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying. Not limited.

The plant extract of the present invention may be treated by various methods such as hydrolysis, fermentation, heating, and pressurization to enhance the influenza virus proliferation inhibitory activity, preferably fermentation.

The term "fermentation" or "fermented product" of the present invention refers to a process of decomposing organic matter using the efficacy of microorganisms (eg, lactic acid bacteria) or a product thereof. Fermentation and decay proceed by similar processes, but as a result of decomposition, when a substance useful in our lives is produced, it is called fermentation, and when a substance that smells or is harmful is produced, it is called decay. Therefore, when the herbal medicine is fermented using microorganisms, the toxicity of the extract is reduced, the molecular weight is reduced and digested easily, or the bioavailability is improved by facilitating percutaneous absorption when applied to the skin. In addition, although natural components may exhibit pharmacological actions by themselves, the pharmacological effects may be further enhanced through fermentation, or pharmacological actions not found in the existing extract before fermentation may be exerted by metabolism of microorganisms. The fermented product may contain nodakenetin/nodakenin in a weight ratio of 0.5 to 20.

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

In one embodiment of the present invention, the extract fermented with Lactobacillus plantarum is 40 times or more in weight ratio of nodakenetin / nodakenin than the unfermented extract, and the content of nodakenetin after fermentation is It was confirmed that the increase was up to 18 times, and the influenza virus inhibitory activity of the fermented extract was excellent.

The term "fraction" in the present invention refers to a product obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture containing various components. The fraction of the present invention may include a solvent fraction and a polysaccharide fraction, but is not limited thereto. A fractionation method for obtaining the fraction may be performed according to a method commonly used in the art.

Examples of the fractionation method include a method of treating the extract or fermented product of the present invention with a predetermined solvent to obtain a fraction from the extract, and a method of immersing and recovering polysaccharides in the extract or fermented product using a cold precipitation method and a centrifugal separation method. etc., but is not limited thereto.

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 may 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 may be used alone or in combination of two or more. That is, it is obvious to those skilled in the art that fractionation using the material extracted from the plant of the present invention is included in the scope of the present invention without limitation to the extraction or fractionation method.

Specifically, in Preparation Example 3 of the present invention, the solvent fraction of each extract was obtained through the following process. After preparing each extract, preferably after filtering the suspension, fractionation according to polarity using about 1 to 5 times the volume of hexane, ethyl acetate, chloroform, C ₁ to C ₄ lower alcohol and water After passing through, a hexane fraction, an ethyl acetate fraction, a chloroform fraction, a C ₁ to C ₄ lower alcohol fraction and a water fraction can be obtained. The first fraction fractionated in the above process can be fractionated again using about 1 to 5 times the volume of hexane, ethyl acetate, chloroform, C ₁ to C ₄ lower alcohol and water to obtain a second fraction, and once again Through the above process, a third fraction may be obtained. In the case of an example carried out in Preparation Example 3, hexane, ethyl acetate, and butanol were fractionated in order to obtain a fraction having a high content of notakenetin and notakenin.

In one aspect, there is provided a health functional food composition for preventing or improving influenza virus infection comprising a plant extract containing nodakenin or nodakenetin, a fermented product thereof, or a fraction thereof.

The term "health functional food composition" of the present invention refers to a food prepared by adding a fermented product of a plant extract or a fraction thereof to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, suspended, etc. When ingested, it means that it brings a specific effect on health, but unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material.

When a fermented product of plant extract or a fraction thereof is used as a food additive, the fermented product of Angelica gigas extract or a fraction thereof may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present invention may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. .

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health foods in a conventional sense.

In one aspect, there is provided a pharmaceutical composition for preventing or treating influenza virus infection comprising a plant extract, a fermented product thereof, or a fraction thereof containing nodakenin or nodakenetin.

The term "pharmaceutical composition" of the present invention may be administered orally or parenterally, and may be administered in the form of general pharmaceutical preparations, for example, in various oral and parenteral dosage forms during clinical administration. It may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient in the pharmaceutical composition of the present invention, for example, starch, calcium carbonate, It may be prepared by mixing sucrose, lactose, gelatin, etc., but is not limited thereto.

In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid formulations for oral use include suspensions, solutions for oral use, emulsions, and syrups. 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, but Not limited.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. 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. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin, etc. may be used, but is not limited thereto.

In one aspect, there is provided a composition for inhibiting influenza virus comprising a plant extract containing nodakenin or nodakenetin, a fermented product thereof, or a fraction thereof.

The term "composition for inhibiting viruses" of the present invention interferes with replication of viruses or strengthens the immune system against viral infections. Vertebrate animals, preferably in the form of pharmaceutical compositions administered to mammals including humans, or may be used as food compositions, tableware, kitchen utensils, household items, and other various cleaning agents, disinfectants, and general antiviral agents, but are limited thereto It doesn't work.

In order to confirm the ability of the fermented product of the plant extract prepared by the above method or a fraction thereof to inhibit influenza virus growth, a neuraminidase inhibition assay, which is currently the most commonly used influenza virus growth inhibition assay, was used. .

Neuraminidase (NA) is an enzyme that proliferates in infected host cells by breaking the alpha-ketosidic bond between the disaccharide moiety on the surface of the host cell and neuraminic acid (sialic acid) residues. It is a protein that allows the population to proliferate by infecting with neuraminidase, and its inhibitors can inhibit the proliferation of influenza virus by binding to the action site of neuraminidase and inhibiting its function.

The experiment of Preparation Example 3 was conducted to track the active ingredient of the fermented product of Angelica Quail extract in the present invention. Compound A was obtained by fractionating and purifying the fermented product of Angelica gigas extract. As a result of comparing the NMR spectrum of compound A with known literature, it was identified as nodakenetin. confirmed by comparison.

The fermented product of Angelica Quail extract and its fractions had a high content of nodakenetin (Experimental Example 2), and showed a concentration-dependently significant inhibitory effect on influenza virus growth. showed (Experimental Example 1).

The fermented product of the plant extract or a fraction thereof of the present invention may contain notakenetin/notakenin in a weight ratio of 0.5 to 20, preferably 1 to 15, 2 to 10, or 4 to 9, but is limited thereto. it is not going to be

In the present invention, it was confirmed that the Angelica Quail extract before fermentation contained nodakenetin/nodakenin in a weight ratio of 0.1, and the fermented product of Angelica Quail extract or a fraction thereof contained nodakenetin/nodakenin in a weight ratio of 0.5 to 20. . The fermented product of angelica extract or its fractions satisfying this range showed a 40-fold higher weight ratio of nodakenetin/nodakenin compared to the unfermented angelica extract, and the amount of nodakenetin after fermentation increased more than 18-fold. Confirmed, it can be seen that it has a better virus infection inhibitory ability.

In one embodiment, a composition for inhibiting influenza virus containing nodakenin or nodakenetin, a health functional food composition for preventing or improving infection, and a pharmaceutical composition for preventing or treating infection are provided.

The description of the virus inhibitory composition, health functional food composition, and pharmaceutical composition is omitted to avoid redundancy, and may be applied to the composition containing notakenin or notakenetin as it is.

The "notakenetin" (Molecular formula: C ₁₄ H ₁₄ O ₄ , Molecluar weight: 246.262) is a coumarin-based non-glycoside component represented by the following [Formula 1], and notakenin (nodakenin, a glycoside represented by [Formula 2]) , Molecular formula: C ₂₀ H ₂₄ 0 ₉ , Molecular weight: 408.403) is converted by fermentation or hydrolysis.

[Formula 1]

[Formula 2]

Nodakenetin is not necessarily formed through a fermentation process, and although it is smaller than glycoside nodakenin, it is naturally present in various parts of the plant. Nodakenetin and nodakenin are mainly contained in plants of the genus Angelica sp. In addition, plants of the genus Peucedanum sp. .) It is known to be present in plants (jeonho, chervil, etc.) and body herbs.

The nodakenin or nodakenetin may be isolated from a fermented product of a plant extract or obtained through synthesis or semi-synthesis, but is not limited thereto.

The "plant" may be at least one selected from the group consisting of Angelica sp. plants, Peucedanum sp. plants, and Anthriscus sp. plants, preferably Angelica gigas , seaweed herbs ( Peucedanum japonicum ), oil herbs ( Peucedanum terebinthaceum ), Japanese wind ( Aegopodium podagraria ), Jeonho ( Anthriscus sylvestris ), chervil ( Anthriscus cerefolium ) and body herbs ( Angelica decursiva ), more preferably angelica It may be, but is not limited thereto.

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

Since the fermented product of Angelica Quail extract or a fraction thereof according to the present invention inhibits the proliferation of influenza virus infection by inhibiting neuraminidase activity, a health functional food composition for preventing or improving influenza virus infection, preventing or treating influenza virus infection It can be usefully used as a pharmaceutical composition for use.

1 is a flowchart of a method of separating and purifying nodakenetin by fractionating a fermented product of Angelica gigas extract.
Figure 2 is a result of influenza virus growth inhibitory activity (%) of Nodakenin and Nodakenetin.
3 is a result of influenza virus proliferation inhibitory ability (%) before and after fermentation of a plant extract containing Angelica gigas.
Figure 4 is a result of influenza virus proliferation inhibitory ability (%) of Compound A (Compound A, nodakenetin) and the EA fraction.

Hereinafter, the present invention will be described in detail by the following experimental examples and preparation examples. However, the following Experimental Examples and Preparation Examples are only to illustrate the present invention, and the contents of the present invention are not limited by the following Experimental Examples and Preparation Examples. In addition, since these experimental examples and preparation examples are only for the purpose of helping the understanding of the present invention, the scope of the present invention is not limited thereto in any sense.

Preparation Example 1: Preparation of Plant Extract Containing Nodakenin and Notakenetin

First, the original Angelica quail was washed and dried.

Then, 10 kg of primary distilled water was added to 1 kg of angelica raw material, and hot water extraction was performed 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 and used as a sample of Experimental Example 2. The yield of the hot water extract prepared by the above method is about 36%.

50wt% ethanol extract manufacturing method, 10kg of 50wt% ethanol aqueous solution was added to 1kg of raw material, and hot water extraction was performed at 65 ° C. for 3 hours. The 50wt% 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 50wt% ethanol extract prepared by the above method is about 40%.

Preparation Example 2: Preparation of fermented product of plant extract

MRS medium powder (peptone 10wt%, beef extract 10wt%, yeast extract 5wt%, dextrose 20wt%, polysorbate 80 1wt%, ammonium citrate 2wt%, sodium acetate 5wt%, magnesium sulfate 0.1wt%, manganese sulfate 0.05wt %, Dibasic potassium phosphate 2wt%) 55g was put into 1L of water to prepare an MRS liquid medium, and then Lactobacillus plantarum ( Lactobacillus plantarum ) Solid colonies were inoculated into 100ml of MRS liquid medium, and then OD600 was measured over time A starter culture was prepared by culturing until the solution value was 1.5. Thereafter, 1 ml of the starter culture medium was inoculated into 100 ml of MRS liquid medium containing 1 g, 2 g, 5 g, and 10 g of the culture medium, angelica extract (ethanol extract), respectively, and fermented in a 30 ° C shaking incubator at 150 rpm for 4 days to obtain a plant fermented product. was completed (Experimental Example 3).

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

Preparation Example 3: Separation and purification of fermented product fractions and active compounds

In order to track the active ingredient of the lactic acid bacteria culture containing Angelica Quail extract, 8L of MRS medium containing 80 g of Angelica Quail extract was fermented with Lactobacillus plantarum , and the resulting solution was sterile filtered (0.22um). The same amount of n-hexane (n-Hexane) was added and left to shake to fractionate, and sequentially ethyl acetate (EA) and n-butyl (n-Butanol, below to BuOH) were added and left to shake to obtain each fraction got The EA fraction (15 g) was subjected to silica gel column chromatography using n-hexane/EA (gradient) as an eluting solvent and divided into 11 small fractions. Small fraction Fr. 9 was subjected to Prep-LC using acetonitrile/water (Acetonitrile (ACN)/H ₂ O) (3:7) as an elution solvent to obtain 30 mg of compound A (FIG. 1). The chemical structure of Compound A was confirmed through NMR spectrum. From the characteristic peaks of a typical furocoumarin structure, it was found that compound A was nodakenetine. By synthesizing these spectral data, compound A was identified as nodakenetine by comparison with previously known literature, and confirmed by direct comparison through nodakenetin purchased from Biopurify, TLC and HPLC. did

NMR data

Compound A (Nodakenetin) - White amorphous powder, ESI-MS m/z : 247 [M + H]+ ; 1H-NMR (600 MHz, CD3OD) δ_H: 7.83 (1H, d, J = 9.6 Hz, 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 (150 MHz, CD3OD) δ_C: 165.4 (C-2), 163.9 (C-7), 157.1 (C-10), 146.4 (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 Inhibiting Ability

The following examples used the following influenza virus inhibitory ability evaluation method.

Influenza virus inhibitory ability was evaluated using NA activity assay kit (Sigma, #cat: MAK121-1KT) after treating the candidate substance in neuraminidase (NA). The positive control group was set with 10 nM of zanamivir, a component of a swine flu 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). Then, after mixing the reagents provided in the NA activity assay kit according to the instructions, 40 μl was added, reacted in an incubator at 30 ° C for 20 minutes, the absorbance was measured at 570 nm (A20), and wrapped in foil again. After reacting for 30 minutes in a 37° C. incubator, 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 inhibitory effects of nodakenetin (Chengdu biopurify phytochemical Ltd. Cat No. BP3273) and nodakenin (Chengdu biopurify phytochemical Ltd. Cat No. BP1002) were evaluated. Notakenetin formed through glycolysis of nodakenin increased the efficacy in a concentration-dependent manner (10, 50, 250 ppm), but in the case of nodakenin, the concentration-dependent effect was insufficient (FIG. 2).

Influenza virus proliferation inhibitory ability was evaluated before and after fermentation of the plant extract. It was confirmed that the fermented extract increased the influenza virus inhibitory ability compared to the unfermented extract in a concentration-dependent manner (FIG. 3).

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

Experimental Example 2: Changes in nodakenin and nodakenetin according to fermentation of plant extracts

In order to confirm the change in the contents of nodakenin and nodakenetin according to fermentation of the Angelica quail extract (hot water extract), the Angelica quail extract fermented by the method of Preparation Example 2 (hot water extract) and the unfermented Angelica quail extract (hot water extract) were prepared. As a result of measuring the content, as shown in Table 1 below, the notakenetin content of plant extracts including Angelica gigas increased by about 43ppm due to lactic acid fermentation, and the notakenetin content decreased by 43ppm due to notakenetin conversion.

It was confirmed that the fermented product had a better ability to inhibit viral infection.

Changes in nodakenetin and nodakenin contents before and after fermentation of Angelica quail extract content analysis Nodakenetin (ppm) Nodakenin (ppm) Before Fermentation of Angelica Quail Extract 8.3 117.0 After fermenting angelica extract 51.1 74.2

Experimental Example 3: Comparison of notakenin and notakenetin 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 gigas extract (ethanol extract), respectively, the contents of nodakenin and nodakenetin before and after fermentation were analyzed (Table 2). ).

Analysis of nodakenin and nodakenetin content before and after fermentation before fermentation after fermentation Angelica quail extract content (%) notakenin
(A, ppm) nodakenetin
(A', ppm) A'/A notakenin
(A, ppm) nodakenetin
(A', ppm) A'/A One 340 32 0.09 72 597 8.34 2 610 57 0.09 127 865 6.80 5 1053 108 0.10 224 1343 6.00 10 1420 146 0.10 346 1420 4.10

The Angelica Quacca extract is in the form of a completely concentrated, dried powder, and all of the analysis samples were analyzed in a completely dry state. As shown in Table 2 above, nodakenetin (A') / nodakenin (A ) ratio is around 0.1, but the fermented Angelica gigas extract increased A'/A by more than 41 times from 4.1 to 8.34, and thus, it can be confirmed that the fermented Angelica quail extract with a high content of nodakenetine has a better ability to inhibit viral infection. there was.

In addition, in order to examine the effect of the extraction solvent, the contents of notakenin and notakenetin in the hot water extract and the ethanol extract of Angelica gigas were analyzed and compared.

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

Comparison of the content ratio of nodakenin and nodakenetine according to the extraction solvent Nodakenin (A, ppm) Nodakenetin (A', ppm) A'/A Dong Quai 50wt% Ethanol Extract 2228 227 0.10 Dong Quai Hot Water Extract 1920 216 0.11

Claims (24)

An extract of a plant selected from the group consisting of Angelica sp. plants, Peucedanum sp. plants, and Anthriscus sp. plants containing nodakenin or nodakenetin. , Health functional food composition for preventing or improving influenza virus infection comprising a fermented product or a fraction thereof.
According to claim 1,
The plants are Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, Anthriscus cerefolium, and Angelica decursiva. Health functional food composition for preventing or improving at least one influenza virus infection selected from the group consisting of.
According to claim 1,
The extract is a health functional food composition for preventing or improving influenza virus infection, which is an extract of one or more solvents selected from the group consisting of water, C ₁ -C ₄ alcohol, 1,3-butylene glycol and ethyl acetate.
According to claim 1,
The fraction is a functional food composition for preventing or improving influenza virus infection, which is a fraction of one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform and dichloromethane for the fermented product.
According to claim 4,
The fraction is a functional food composition for preventing or improving influenza virus infection, which is a fraction of n-hexane and ethyl acetate solvent for the fermented product.
According to claim 1,
The fermented product is a health functional food composition for preventing or improving influenza virus infection, which is a fermented product by lactic acid bacteria.
According to claim 6,
The lactic acid bacteria are Lactobacillus sp. Health functional food composition for preventing or improving influenza virus infection, which is a lactic acid bacterium.
According to claim 1,
The fermented product of the plant extract or a fraction thereof is a health functional food composition for preventing or improving influenza virus infection, comprising nodakenetin / nodakenin in a weight ratio of 0.5 to 20.
An extract of a plant selected from the group consisting of Angelica sp. plants, Peucedanum sp. plants, and Anthriscus sp. plants containing nodakenin or nodakenetin. , A pharmaceutical composition for preventing or treating influenza virus infection comprising a fermented product thereof or a fraction thereof.
According to claim 9,
The plants are Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, Anthriscus cerefolium, and Angelica decursiva. A pharmaceutical composition for the prevention or treatment of at least one influenza virus infection selected from the group consisting of:
According to claim 9,
The extract is a pharmaceutical composition for preventing or treating influenza virus infection, which is an extract of one or more solvents selected from the group consisting of water, C ₁ -C ₄ alcohol, 1,3-butylene glycol and ethyl acetate.
According to claim 9,
The fraction is a pharmaceutical composition for preventing or treating influenza virus infection, which is a fraction of one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform and dichloromethane for fermented product.
According to claim 12,
The fraction is a pharmaceutical composition for preventing or treating influenza virus infection, which is a fraction of n-hexane and ethyl acetate solvent for the fermented product.
According to claim 9,
The fermented product is a pharmaceutical composition for preventing or treating influenza virus infection, which is a fermented product by lactic acid bacteria.
According to claim 14,
The lactic acid bacteria are Lactobacillus sp. A pharmaceutical composition for preventing or treating influenza virus infection, which is a lactic acid bacterium.
According to claim 9,
A pharmaceutical composition for preventing or treating influenza virus infection, wherein the fermented product of the plant extract or a fraction thereof contains nodakenetin / nodakenin in a weight ratio of 0.5 to 20.
An extract of a plant selected from the group consisting of Angelica sp. plants, Peucedanum sp. plants, and Anthriscus sp. plants containing nodakenin or nodakenetin. , A composition for inhibiting influenza virus comprising a fermented product thereof or a fraction thereof.
According to claim 17,
The plants are Angelica gigas, Peucedanum japonicum, Peucedanum terebinthaceum, Aegopodium podagraria, Anthriscus sylvestris, Anthriscus cerefolium, and Angelica decursiva. A composition for inhibiting at least one influenza virus selected from the group consisting of:
According to claim 17,
The extract is an extract of one or more solvents selected from the group consisting of water, C ₁ -C ₄ alcohol, 1,3-butylene glycol and ethyl acetate Composition for inhibiting influenza virus.
According to claim 17,
Wherein the fraction is a fraction of one or more solvents selected from the group consisting of water, alcohol, hexane, ethyl acetate, chloroform and dichloromethane for the fermented product, composition for inhibiting influenza virus.
According to claim 20,
The fraction is a composition for inhibiting influenza virus, which is a fraction of n-hexane and ethyl acetate solvent for the fermented product.
According to claim 17,
The fermented product is a composition for inhibiting influenza virus, which is a fermented product by lactic acid bacteria.
The method of claim 22,
The lactic acid bacteria are Lactobacillus sp . A composition for inhibiting influenza virus, which is a lactic acid bacterium.
According to claim 17,
The composition for inhibiting influenza virus, wherein the fermented product of the plant extract or a fraction thereof contains nodakenetin / nodakenin in a weight ratio of 0.5 to 20.

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