KR20240015984A - Plant-derived compounds with anti-influenza virus activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating viral infection containing a plant extract or a solvent fraction thereof as an active ingredient. The plant extract or a fraction thereof according to the present invention can also be used as a food composition for improving or preventing viral infection. You can.

Description

Plant-derived compounds with anti-influenza virus activity}

The present invention relates to a pharmaceutical composition for preventing or treating viral infections containing plant-derived substances as active ingredients.

Influenza virus infection invades the upper respiratory tract mucosa and is accompanied by cold symptoms such as cough, runny nose, and phlegm, as well as serious symptoms such as fever, chills, muscle pain, headache, and loss of appetite, which is called the flu. Influenza viruses cause complications in people with weak immunity, and are highly contagious compared to other respiratory viruses, threatening the health of not only humans but also animals around the world.

Influenza viruses are classified into types A and B, which use RNA as a nucleic acid and encode eight segments with different functions, and type C, which encodes seven segments. Among them, types A and B are the main influenza viruses that mainly infect humans. They are classified according to the type of antigen, which is a viral surface glycoprotein called hemagglutinin (HA) and neuraminidase (NA). The name is written in the following order: type, natural host, subtype, site of isolation, isolation number, and year of isolation (HA and NA serotypes).

The type A influenza virus genome consists of approximately 13,500 RNA bases, and mutant strains with new hemagglutanin and neuraminidase, that is, new antigens, can easily appear due to mutation or genome segment exchange, causing a global pandemic. On the other hand, the type B influenza virus is less likely to undergo large antigenic mutations, and only two strains are known, so the possibility of a pandemic is much lower than that of the type A virus. Influenza A viruses are composed of 6 types of HA and 3 types of NA in humans (H1N1, H2N2, H3N2, H5N1, H7N7, H3N7), and in birds, there are 16 types of HA and 9 types of NA, 144 Different serotypes of the species exist.

Meanwhile, type 2 severe acute respiratory syndrome coronavirus (SARS-Cov2) is a single-stranded RNA virus that belongs to the coronavirus subfamily and is the cause of the outbreak in Wuhan, China in December 2019. The causative virus was first discovered in a patient with unidentified pneumonia. To avoid confusion with Severe Acute Respiratory Syndrome infection, the World Health Organization (WHO) also calls it “2019 Coronavirus Disease Virus” or “2019 Coronavirus Disease Responsibility Virus.”

From December 2019 to early July 2020, 11.57 million confirmed cases occurred in 214 countries around the world, of which 537,138 died, resulting in a fatality rate of 4.64. The clinical symptoms of SARS-CoV-2 are mainly respiratory symptoms accompanied by fever, and the main vulnerable targets are the elderly and those in their 60s or older suffering from underlying diseases and immune diseases. Unlike previous viruses, it showed strong contagiousness and rapid worsening of symptoms, and in March 2020, just over three months after the first report of the outbreak, WHO declared it a global pandemic or pandemic.

As virus-specific treatments, the 3CL protease inhibitor nirmatrelvir and ritonavir combination Paxrovid and the polymerase inhibitor remdesivir have received emergency approval and are being used. However, treatments known to be effective so far are problematic because they have minimal effectiveness in actual clinical trials for seriously ill patients or can cause various side effects.

In Japan, Professor Suzuki reported that a substance called mumefual, which is produced during the process of concentrating plums, is active against the type A influenza virus. However, in Korea, the antiviral activity of plums and plum products that did not contain any mumefural was discovered without any concentration process, and as a result of identifying antiviral substances, substances completely different from the existing mumefural were found to be effective in antiviral activity. discovered excellence.

Accordingly, the present inventors confirmed that the antiviral activity was significantly superior when plum ( Prunus mume ) extract or its fraction was treated with a virus.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating viral infection containing plum extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing viral infection containing plum extract or a fraction thereof as an active ingredient.

Another object of the present invention is (+)-epicatechin, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin and epicatechin-(2β→O→7, 4β→8)-ent-epicatechin. To provide a pharmaceutical composition for preventing or treating viral infection containing at least one member selected from the group.

Another object of the present invention is to provide a method for extracting antiviral active substances.

The present invention relates to a composition for improving, preventing, or treating viral infection containing plum extract or a fraction thereof as an active ingredient. The composition containing a plum extract or a fraction thereof according to the present invention as an active ingredient is a composition for improving, preventing, or treating viral infection. It shows excellent effects in treatment, etc.

The present inventors confirmed that plum extract or fractions thereof are significantly superior in improving, preventing, or treating various viral infections.

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

One aspect of the present invention relates to a pharmaceutical composition for preventing or treating viral infection comprising plum ( Prunus mume ) extract or a fraction thereof as an active ingredient.

In the present invention, the plum extract may be one or more selected from the group consisting of plum juice, plum syrup, plum concentrate, and plum fruit extract, but is not limited thereto.

In the present invention, the plum extract may be a crude extract obtained by extraction with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, acetic acid, butanol, or combinations thereof, but is limited thereto. That is not the case.

In the present invention, the plum extract may be a crude extract obtained by extraction with methanol, but is not limited thereto.

In the present invention, the plum extract may be fractionated using one or more solvents selected from the group consisting of ethyl ether, ethyl acetate, and butanol, for example, ethyl acetate, but is not limited thereto.

In the present invention, the fraction may be obtained by performing chromatography, but is not limited thereto.

In the present invention, the virus may be one or more types selected from the group consisting of influenza and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), but is not limited thereto. no.

In the present invention, the influenza virus may include influenza virus type A or B, but is not limited thereto.

In the present invention, influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or H10N7 serum. It may have a type, but is not limited to this.

In the present invention, type 2 severe acute respiratory syndrome coronavirus is a single-stranded RNA virus that belongs to the coronavirus subfamily, and the causative virus was first discovered in a patient with pneumonia of unknown cause in Wuhan, China in December 2019. It has been done. To avoid confusion with Severe Acute Respiratory Syndrome infection, the World Health Organization (WHO) also calls it “2019 Coronavirus Disease Virus” or “2019 Coronavirus Disease Responsibility Virus.”

As used herein, the term “prevention” refers to infection of influenza virus or type 2 severe acute respiratory syndrome coronavirus by administration of the pharmaceutical composition; It refers to any action that suppresses or delays disease caused by influenza virus or type 2 severe acute respiratory syndrome coronavirus infection. In addition, the term "treatment" refers to the treatment of symptoms caused by influenza virus or type 2 severe acute respiratory syndrome coronavirus infection or disease caused by influenza virus or type 2 severe acute respiratory syndrome coronavirus infection by administration of the pharmaceutical composition. It means any action that improves or changes something beneficial.

In the present invention, the pharmaceutical composition includes plum extract or fractions thereof; In addition, it may contain pharmaceutically acceptable carriers, excipients, or diluents.

In the present invention, the dosage form of the pharmaceutical composition may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate combination. The salt is not particularly limited as long as it is pharmaceutically acceptable, and examples include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, and methane. Sulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc. may be used.

The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include one or more compounds and at least one excipient; For example, it can be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

Liquid preparations for oral administration of the pharmaceutical composition include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, Preservatives, etc. may be included. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Non-aqueous and suspending agents for the pharmaceutical composition may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. The base of the suppository may be witepsol, macrogol, tween61, cacao, laurel, glycerogelatin, etc.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the pharmaceutical composition of the present invention may be administered in an amount of 0.001 to 100 mg/kg once or several times a day.

The dosage form of the pharmaceutical composition may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination.

The pharmaceutical composition can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, intraperitoneal, rectal or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

Another aspect of the present invention relates to a food composition for improving or preventing viral infection comprising plum ( Prunus mume ) extract or a fraction thereof as an active ingredient.

In the present invention, the plum extract may be one or more selected from the group consisting of plum juice, plum syrup, plum concentrate, and plum fruit extract, but is not limited thereto.

In the present invention, the plum extract may be a crude extract obtained by extraction with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, acetic acid, butanol, or combinations thereof, but is limited thereto. That is not the case.

In the present invention, the plum extract may be a crude extract obtained by extraction with methanol, but is not limited thereto.

In the present invention, the plum extract may be fractionated using one or more solvents selected from the group consisting of ethyl ether, ethyl acetate, and butanol, for example, ethyl acetate, but is not limited thereto.

In the present invention, the fraction may be obtained by performing chromatography, but is not limited thereto.

In the present invention, it may be one or more types selected from the group consisting of influenza and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), but is not limited thereto.

In the present invention, the influenza virus may include virus type A or B, but is not limited thereto.

In the present invention, influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or H10N7 serum. It may have a type, but is not limited to this.

In the present invention, the food composition may be a plum extract or a fraction thereof used alone, in combination with other foods, or in combination with other food ingredients, and may be used appropriately according to conventional methods.

The food composition includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. In addition, there are no special restrictions on the type of food, but it includes meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. These include alcoholic beverages and vitamin complexes, but may also include health foods in the conventional sense.

The food composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame.

The food composition refers to a food manufactured by adding plum extract or a fraction thereof to a food material, or by encapsulating, powdering, or suspending it, and when ingested, it has a specific health effect. However, unlike general drugs, the food composition It may have the advantage of not having any side effects that may occur during long-term use of the drug as it is made from .

Another aspect of the present invention consists of (+)-epicatechin, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin, and epicatechin-(2β→O→7, 4β→8)-ent-epicatechin. It relates to a pharmaceutical composition for preventing or treating viral infection comprising at least one member selected from the group.

In the present invention, (+)-epicatechin is referred to as JA1.

In the present invention, (+)-epicatechin, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin is referred to as JA2.

In the present invention, epicatechin-(2β→O→7, 4β→8)-ent-epicatechin is referred to as JA3.

In the present invention, (+)-epicatechin has the following formula (I).

[Formula I]

In the present invention, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin has the following formula II.

[Formula Ⅱ]

In the present invention, epicatechin-(2β→O→7, 4β→8)-ent-epicatechin has the following formula III.

[Formula Ⅲ]

remind The virus may be one or more types selected from the group consisting of influenza virus and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), but is not limited thereto.

In the present invention, the influenza virus may include influenza virus type A or B, but is not limited thereto.

In the present invention, influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or H10N7 serum. It may have a type, but is not limited to this.

Another aspect of the present invention is a method for extracting an antiviral active substance comprising the following steps:

An extraction step of extracting plums with plum extract; and

Fractionation step of fractionating the plum extract with solvent.

In the present invention, the extraction step includes extracting plums with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, acetic acid, butanol, or combinations thereof, for example, methanol. It may be done, but it is not limited to this.

In the present invention, the fractionation step may include fractionating the plum extract using one or more solvents selected from the group consisting of ethyl ether, ethyl acetate, and butanol, but is not limited thereto.

In the present invention, the fractionation step may further include the following steps:

A first fractionation step of obtaining a first fraction by using the plum extract with at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol;

A second fractionation step of performing column chromatography on the first fraction to obtain a second fraction;

A third fractionation step of performing column chromatography on the second fraction to obtain a third fraction;

A fourth fractionation step of performing column chromatography on the third fraction to obtain a fourth fraction; and

A fifth fractionation step of subjecting the fourth fraction to membrane chromatography to obtain the fifth fraction.

In the present invention, the second to fourth fractionation steps may be performed using column chromatography based on a physical method of stationary phase and mobile phase, but are not limited thereto.

In the present invention, the second fractionation step may be performed using C18-column chromatography, but is not limited thereto.

In the present invention, the second fractionation step may use methanol as a mobile phase in a concentration gradient (0.001 to 100%), but is not limited thereto.

In the present invention, the third fractionation step may be performed using silica gel column chromatography (Flash Silica gel column chromatography), but is not limited thereto.

In the present invention, in the third fractionation step, silica gel, Sephadex, or agarose gel may be used as the silica gel column chromatography stationary phase, and hexane, ethyl acetate, ethanol, methanol, methylene chloride, or Ether, etc. may be used, and for example, a mixture of chloroform and methanol may be used, but is not limited thereto.

In the present invention, the third fractionation step may be a mixture of chloroform and methanol in a ratio of 9:1 (v/v), but is not limited thereto.

In the present invention, silica gel column chromatography may be filled with 230 to 400 mesh silica gel, but is not limited thereto.

In the present invention, silica gel column chromatography may be performed by placing 250 g of 230 to 400 mesh silica gel in a glass column and filling the column with chloroform and methanol at a ratio of 9:1 (v/v), but is limited to this. It doesn't work.

In the present invention, the fourth fractionation step may be separation using a Sephadex column (sephadex LH 20), but is not limited thereto.

In the present invention, Sephadex column is a brand name of dextran for gel filtration with a three-dimensional network structure, and the LH type may be an alkylated derivative of dextran that has both lipophilic and hydrophilic characteristics.

In the present invention, the Sephadex column may use 100% methanol as a mobile phase, but is not limited thereto.

In the present invention, the temperature of the Sephadex column sample and mobile phase may be maintained at 5 to 15°C, 5 to 10°C, for example, 10°C, but is not limited thereto.

In the present invention, the Sephadex column is a form of action that causes adsorption, such as a force that leads between molecules, and may include electrostatic attraction, complexation, or hydrogen bonding, but is not limited thereto.

In the present invention, the fifth fractionation step may be separation using membrane chromatography, but is not limited thereto.

In the present invention, membrane chromatography may be used for simple preparative purposes rather than for qualitative confirmation. Therefore, the fraction eluted in the fifth fractionation step can be used alone, or the individual fractions can be used in combination, but the present invention is not limited thereto.

In the present invention, membrane chromatography may be performed using chloroform, methanol, and ethyl acetate as the mobile phase under solvent conditions of 8:2:1 (v/v/v), but is not limited thereto.

In the present invention, the fifth fraction obtained in the fifth fractionation step is (+)-epicatechin, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin, and epicatechin-(2β→O→7, 4β→8) )-ent-epicatechin may include one or more selected from the group consisting of, but is not limited to this.

The present invention relates to a composition for improving, preventing or treating influenza virus infection comprising a plum extract or a fraction thereof as an active ingredient. The plum extract or a fraction thereof according to the present invention is a composition for improving, preventing or treating influenza virus, especially type A or type B influenza virus. It can be used as a pharmaceutical composition or food composition useful for improving, preventing, or treating infections.

Figure 1 is a schematic diagram showing the separation process of an anti-influenza material according to an embodiment of the present invention.
Figure 2 shows pure isolated JA1 ((+)-epicatechin) (a), JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) (b) and This is a graph showing the HPLC analysis results of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) (c).
Figure 3 shows the results of electrospray ionization-TOF-MS analysis in negative ion mode and positive ion mode of pure isolated JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 4 is a ¹ H-NMR spectrum of JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 5 is a ¹³ CH-NMR spectrum of JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 6 is an HSQC spectrum of JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 7 is a ¹ H- ¹ H COZY spectrum of JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 8 is an HMBC spectrum of JA1 ((+)-epicatechin) according to an embodiment of the present invention.
Figure 9 is a structural formula showing the chemical structure of (+)-Epicatechin according to an embodiment of the present invention.
Figure 10 shows the results of electrospray ionization-TOF-MS analysis in negative ion mode and positive ion mode of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 11 is ^{a 1} H-NMR spectrum of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 12 is a ¹³ CH-NMR spectrum of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 13 is an HSQC spectrum of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 14 is ^{a 1} H - ¹ H COZY spectrum of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 15 is an HMBC spectrum of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 16 is a structural formula showing the chemical structure of JA2 (epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 17 shows electrospray ionization-TOF-MS analysis results in negative ion mode and positive ion mode of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 18 is ^{a 1} H-NMR spectrum of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 19 is a ¹³ CH-NMR spectrum of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 20 is an HSQC spectrum of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 21 is ^{a 1} H - ¹ H COZY spectrum of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 22 is an HMBC spectrum of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 23 is a structural formula showing the chemical structure of JA3 (epicatechin-(2β→O→7, 4β→8)-ent-epicatechin) according to an embodiment of the present invention.
Figure 24 shows the antiviral activity of JA1, JA2, JA3, and remdesvir isolated from plum according to an embodiment of the present invention against coronavirus.

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

Preparation Example 1. Preparation of plum extract

After removing the seeds from the plum, 5 kg was finely ground. Next, 15 L of methanol (concentration and unit) was added to the ground plums and then sonicated at room temperature for 60 minutes. Next, the filtrate was concentrated under reduced pressure using Whatman No 2 filter paper to completely remove methanol, and then distilled water was added to obtain 1 L of methanol extract water suspension.

Preparation Example 2. Preparation of plum organic solvent fraction

After fractionating 1 L of the water suspension of the plum extract extracted with methanol twice with an equal volume of 100% ethyl acetate, a plum ethyl acetate fraction (A-EtOAc) was obtained from the ethyl acetate layer. Then, the remaining recovered water layer was re-fractionated twice with 800 ml of 100% butanol (BuOH) to obtain plum butanol fraction (A-BuOH) from the butanol layer, and the remainder was used as plum water fraction (A-water). It was obtained with The obtained ethyl acetate, butanol, and water fractions were concentrated under reduced pressure to obtain 14 g, 21 g, and 32 g, respectively.

Example 1. Isolation of anti-influenza substances

The active substance was separated from 14 g of plum ethyl acetate fraction (A-EtOAc) using various chromatography methods, and the entire separation process is shown in Figure 1.

Specifically, the C18-column (C18-column, 50 g, Cosmosil 75C18-OPN; Japan, 4.5 cm Then, F3 (1120 mg) and F4 (490 mg) were combined and purified on a silica gel column (250 g, 230 to 400 mesh, 5.5 cm /v) the column was packed. F341 (20 mg), F342 (120 mg), F343 (162.4 mg), F344 (143.1 mg), F345 (121.1 mg), F346 (97.3 mg), F347 (156 mg), F348 (89.8 mg), F349 (92.6 mg) and F3410 (115.4 mg) were obtained.

Among the obtained products, F342 (120 mg), F343 (162.4 mg), and F344 (143.1 mg) were combined and separated using a Sephadex LH-20 column (3.5 cm . The column size was 3.5 cm (inner diameter) 93.6 mg), F34H1-4 (93.6 mg), and F34H1-5 (53.8 mg) were obtained, and the F34H1-3 fraction was selected and fractionated according to polarity, and the fractionated material was designated as JA1 (60 mg). .

Among the obtained products, F345 (121.1 mg), F346 (97.3 mg), and F347 (156 mg) were combined and separated using Sephadex LH-20 column, 3.5 cm The column size was 3.5 cm (inner diameter) 21.7 mg), F365 (70.6 mg), and F366 (73.8 mg) were obtained, and the F362 (80.2 mg) fraction was selected and fractionated according to polarity, and the fractionated material was designated as JA2 (35.6 mg). Additionally, the F365 (70.6 mg) fraction was selected and fractionated according to polarity, and the fractionated material was referred to as JA3 (28.8 mg).

High-performance liquid chromatography (HPLC) analysis was performed to confirm the purity of the fractionated substances JA1, JA2, and JA3. The injection volume was set to 10 ul, and specific elution conditions are shown in Table 1 below.

Time Flow %A %B 0.01 1.00 90 10 30.00 1.00 0 100

Atlantis T3 5 um (Atlantis T3, 4.6 used. It was confirmed that the material was separated through HPLC analysis in a concentration gradient solvent system (Figure 2).

Example 2. Confirmation of chemical structure of isolated anti-influenza substance

2-1. Confirmation of the structure of JA1

To determine the molecular weight of the fractionated material JA1, high-resolution electrospray in positive and negative ion modes was performed using a Synapt G2 HDMS quadrupole time-of-flight (QTOF) equipped with an electron ionization source (waters, Crop.). Mass spectrometry (HR-ESI-MS) was performed.

As a result, in negative ion mode analysis, the [M-1] ^- ion peak appeared at 289.0711, and in positive ion mode, the [M+1] ^- ion peak appeared at 291.0858, confirming that the molecular weight was 290 (Figure 3). To determine the exact chemical structure, it was dissolved in methanol-d4 (Cambridge Isotope Laboratories) and subjected to ¹ H-nuclear magnetic resonance spectroscopy (NMR) and ¹³ C using a Bruker Advance III HD 500 MHz instrument (Bruker Biospin GmbH, Rheinstetten, Germany). -NMR and 2D-NMR analysis results were recorded, and tetramethylsilane (TMS) was used as an internal standard during NMR analysis.

¹ H-NMR and ¹³ C-NMR analyzes were performed and are shown in Figures 4 and 5, respectively. In order to see the connectivity between carbon and hydrogen, HSQC was analyzed to obtain a spectrum (Figure 6), and the connection between carbons with hydrogen attached was confirmed through the ¹ H- ¹ H COZY spectrum (Figure 7). And long-range coupling between hydrogen and carbon was confirmed through the HBMC spectrum (Figure 8). Combining all analysis results, JA1 was classified as (+)-Epicatechin[(2 S ,3 S )-2-(3,4-dihydroxyphenyl)-3,4-dihydro- 2H -chromene-3,5,7-triol ] was identified (Figure 9). In addition, the NMR data of JA1, an antiviral substance isolated from plum extract, is summarized in Table 2 below.

JA1 ¹³ C-NMR ¹ H-NMR, d4, 500 MHz HMBC COZY One 2 78.29 4.82, s, 1H C4, C3, C2', C6', C1' H6', H2', H3, H4 3 66.36 4.18, br s, 1H C4a H2, H4 4α 28.07 2.74, dd, 1H, 17.0; 3.0 Hz C3, C2, C4a, C8a, C5 H3, H2 4β 28.07 2.87, dd, 1H, 17.0; 4.5 Hz C3, C2, C4a, C8a, C5 H3, H2 4a 98.65 5 156.26 6 94.92 5.95, d, 1H, 2.0 Hz C8, C5, C7 7 156.59 8 94.47 5.92, d, 1H, 2.0 Hz 8a 155.94 One' 130.93 2' 113.91 6.98, dd, 1H, 1.5 Hz C2, C6', C1', C3' 3' 144.34 4' 144.51 5' 114.44 6.77, d, 1H, 8.5 Hz C1', C3', C4' 6' 118.04 6.81, dd, 1H, 8.5; 1.5 Hz C2, C5', C1', C4'

2-2. Confirmation of the structure of JA2

In order to determine the molecular weight of the fractionated material JA2, high-resolution electrospray mass spectrometry (HR-ESI-MS) was performed in positive and negative ion modes. In negative ion mode analysis, the [M-1] ^- ion peak appeared at 559.1248, and the positive ion peak was found at 559.1248. In mode, the [M+1] ^- ion peak appeared at 561.1391, confirming that the molecular weight was 560 (FIG. 10). To identify the exact chemical structure, ¹ H-NMR and ¹³ C-NMR analysis were performed (Figures 11 and 12). In order to see the connectivity between carbon and hydrogen, a spectrum (FIG. 13) was obtained by analyzing HSQC, and the connection between carbons with hydrogen attached was confirmed with ^{a 1} H- ¹ H COZY spectrum (FIG. 14). And long-range coupling between hydrogen and carbon was confirmed through the HBMC spectrum (FIG. 15). Through all analysis results and literature search, JA2 was identified as epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin [(IUPAC name: 8,14-Methano-2H,14H-1-benzopyrano[7,8- d][1,3]benzodioxocin-3,5,11,13,15-pentol, 2-bis(3,4-dihydroxyphenyl), 8-bis(4-hydroxyphenyl)-3,4-dihydro-, (2R , 3R, 8S, 14R, 15R)- (9CI, ACI)] (Figure 16). The NMR data of JA2, an antiviral substance isolated from plum extract, is summarized in Table 3 below. This substance is JA3. Compared to other substances, it is a new substance as it is missing the -OH group at carbon number 11.

JA2 1H-NMR 13C-NMR HMBC COZY 2 99.15 3 4.16, d, 1H, 3.5 Hz 66.34 C4a H4 4 4.41, d, 1H, 3.5 Hz 27.91 C2, C3, C4a, C8' H3 4a 102.67 5 156.74 6 5.9, d, 1H, 2.0 Hz 96.58 H8 7 157.54 8 6.07, d, 1H, 2.0 Hz 95.15 C4a H6 8a 155.34 9 130.33 10 7.51, d, 1H; 8.5 Hz 128.10 H11 11 6.84, d, 1H, 8.5 Hz 114.11 C9, C12, C13 H10 12 144.80 13 6.84, d, 1H, 8.5 Hz 114.11 C9, C11, C12 H14 14 7.51, d, 1H, 8.5 Hz 128.10 H13 2' 5.03, s, 1H 79.45 C9', C10', C14' H3' 3' 4.25, br s, 1H 65.74 H4' 4' 2.89, m2H 28.07 C4a' H3', H2' 4'a 100.53 5' 155.26 6' 6.09, s, 1H 95.08 C5', C7', C8', C4a' 7' 150.68 8' 105.55 8'a 152.76 9' 130.02 10' 7.14, d, 1H; 2.0 Hz 114.78 C12', C14' 11' 149.88 12' 144.80 13' 6.85, d, 1H, 8.5 Hz 113.84 C9', C12' 14' 6.95, dd, 1H, 8.5, 2.0 Hz 118.02 C10', C12'

2-3. Confirmation of the structure of JA3

As a result of performing high-resolution electrospray mass spectrometry (HR-ESI-MS) in positive and negative ion modes to determine the molecular weight of the fractionated material JA3, the [M-1] ^- ion peak appeared at 575.1190 in the negative ion mode analysis, and the positive ion peak was found at 575.1190. In mode, the [M+1] ^- ion peak appeared at 577.1335, confirming that the molecular weight was 576 (FIG. 17). In order to identify the exact chemical structure, ¹ H-NMR and ¹³ C-NMR analysis were performed to confirm the results shown in Figures 18 and 19, respectively. And in order to see the connection between carbon and hydrogen, HSQC was analyzed to obtain a spectrum as shown in Figure 20, and the connection between carbons with hydrogen attached was confirmed through the ¹ H- ¹ H COZY spectrum (Figure 21). And long-range coupling between hydrogen and carbon was confirmed through the HBMC spectrum (Figure 22). Based on all analysis results, the JA3 substance was identified as epicatechin-(2β→O→7, 4β→8)-ent-epicatechin, which is a dimer of epicatechin. This substance has been reported in other plants, but this is the first reported substance in plum. The NMR data of JA3, an antiviral substance isolated from plum extract, is summarized in Table 4 below.

JA3 1H-NMR 13C-NMR HMBC COZY 2 99.06 3 4.16, d, 1H, 3.5 Hz 66.34 C4a H4 4 4.41, d, 1H, 3.5 Hz 27.91 C2, C3, C4a, C5, C8a, C8', C8a', C7' H3 4a 102.67 5 155.37 6 5.89, d, 1H, 2.0 Hz 96.58 C4a, C5, C7 H8 7 156.78 8 6.07, d, 1H, 2.0 Hz 95.15 C4, C7, C8a H4', H6 8a 152.74 9 130.97 10 7.16, d, 1H, 2.0 Hz 114.32 C2, C14, C11, C12 11 145.42 12 144.28 13 6.85, d, 1H, 8.5 Hz 114.83 C9, C11, C12 14 7.04, dd, 1H, 8.5, 2.0 Hz 118.50 C2, C10, C12 2' 5.03, s, 1H 79.45 C9', C10', C14' H3' 3' 4.25, br s, 1H 65.74 H4' 4' 2.88, m, 2H 28.07 C3', C2', C4a' H3', H2' 4'a 100.53 5' 155.26 6' 6.09, s, 1H 95.08 C5', C7', C8' 7' 150.72 8' 105.55 8'a 149.90 9' 130.02 10' 7.14, d, 1H, 2.0 Hz 113.84 C2', C14', C11', C12' H14' 11' 144.85 12' 144.85 13' 6.82, d, 1H, 8.5 Hz 114.24 C9', C11', C12' H14' 14' 6.95, dd, 1H, 8.5, 2.0 Hz 118.09 C2', C10', C11', C12' H13', H10'

Example 3. Comparison of isomers of JA1

^{The 1} H-NMR structures between JA1, identified as (+)-epicatechin in Example 2, and its isomer (-)-epicatechin (Sigma Aldrich) were compared and analyzed. JA1 derived from plum extract and (-)-epicatechin as a control were dissolved in methanol-d4 (Cambridge Isotope Laboratories) and ¹ H-nucleus was obtained using a Bruker Avance III HD 500 MHz instrument (Bruker Biospin GmbH, Rheinstetten, Germany). The results of magnetic resonance spectroscopy (NMR), ¹³ C-NMR, and 2D-NMR analysis were recorded, and tetramethylsilane (TMS) was used as an internal standard during NMR analysis, and the results are shown in Table 5 below.

JA1 (-)-Epi-catechin Acetone (d6) 1H-NMR (MeOH) Acetone (d6) 1H-NMR (MeOH) 2 4.88 (s) 4.82 (s) 4.88 (s) 4.81 (s) 3 4.21 (m, J _3,4α = 3.0;
J _3,4β = 4.8) 4.18 (brs) 4.21 (brd, J = 3.0) 4.18 (brd, 9.0) 4α 2.74 (dd, J _4α,4β = 16.8;J _4,3 = 3.0) 2.74 (dd, J _4α,4β = 17.0;
J _4,3 = 3.0) 2.75 (dd, J _4α,4β = 16.8;
J _4,3 = 3.0) 2.73 (dd, 17.0; 3.0) 4β 2.87 (dd, J _4β,4α = 16.8;
J _4,3 = 4.8) 2.87 (dd, J _4β,4α = 17.0;
J _4,3 = 4.5) 2.87 (dd, J _4β,4α = 16.8;
J _4,3 = 3.0) 2.86 (dd, 17.0; 4.5) 4a 5 6 6.03 (d, J _6,8 = 2.4) 5.95 (d, J _6,8 = 2.0) 6.03 (d, J _6,8 = 2.4) 5.94 (d, J = 2.5) 7 8 5.92 (d, J _8,6 = 2.4) 5.92 (d, J _8,6 = 2.0) 5.92 (d, J _8,6 = 2.4) 5.91 (d, J = 2.5) 8a One' 2' 7.05 (d, J _6',2' = 1.8) 6.98 (d, J _6',2' = 1.5) 7.06 (d, J _6',2' = 1.8) 6.98 (d, J = 2.0) 3' 4' 5' 6.79 (d, J _5',6' = 8.4) 6.77 (d, J _5',6' = 8.5) 6.79 (d, J _5',6' = 8.4) 6.76 (d, J = 8.0) 6' 6.84 (ddd, J _6',5' = 8.4;
J _6',2' = 1.8, J _6',2 = 2.4,) 6.81 (dd, J _6',5' = 8.5;
J _6',2' = 1.5) 6.84 (ddd, J _6',5' = 8.4;
J _6',2' = 1.8, J _6',2 = 2.4,) 6.80 (ddd, J =8.0; 5.5; 2.0)

In the case of (+)-Epicatechin and (-)-epicatechin, the stereochemistry of carbon 2 is different. In the case of (+)-epicatechin, it is S, whereas in (-)-epicatechin, it is R. Therefore, the coupling constant of hydrogen at positions 3 and 4 of (-)-epicatechin purchased from Sigma-Aldrich is different from that of (+)-epicatechin isolated from plums. Accordingly, the substance isolated from plum was finally confirmed to be (+)-epicatechin.

Example 4. Evaluation of anti-influenza virus activity of plum extract-derived substances

Plum extract-derived substances JA1, JA2, and JA3 were 100.0 ug/mL, 33.3 ug/mL, 11.1 ug/mL, 3.7 ug/mL, 1.2 ug/mL, 0.4 ug/mL, 0.1 ug/mL, 0.05 ug/mL. , antiviral activity against MDCK (Madin-Darby Canine Kidney; purchased from American Type Culture Collection) cells was evaluated at concentration levels of 0.02 ug/mL and 0.005 ug/mL.

Specifically, MDCK cells were distributed in _a 96-well plate at a level of ³ Then, 0.001 MOI H1N1 influenza A virus (PR8, A/Puerto Rico/8/34), H3N2 influenza A virus (HK, A/Hong Kong/8/68), and influenza B virus (B/Lee), respectively. /40) and the control group (MOCK) were mixed with plum extract-derived materials in MEM (Minium Essential Media, Invitrogen) and incubated at 37°C for 1 hour. Additionally, amantadine (AMT), ribavirin (RBV), and oseltamivir carboxylate (OSV-C) were used as reference drugs.

The pre-cultured virus was infected with MDCK cells washed with PBS (phosphate buffered saline) and cultured in an incubator at 5% CO ₂ and 35°C for 2 days. Next, MDCK cell survival rate was measured through FDA (fluorescein diacetate) assay, and the results are shown in Table 6 below.

EC ₅₀ indicates 50% effective concentration and CC ₅₀ indicates 50% cytotoxicity. SI is the selectivity index and is expressed as CC ₅₀ /EC ₅₀ . CC ₅₀ is the concentration at which about 50% shows a cytotoxic reaction, and represents the concentration at which 50% of viable cells die. EC ₅₀ is an indicator of the drug's effectiveness and means the concentration at which only half of the drug's effect appears. It represents the antiviral activity value, and the lower the value, the better the antiviral effect.

No. Code Toxicity
CC ₅₀ (ug/ml) Antiviral activity
(EC ₅₀ :ug/ml) Selectivity index Remark FluA FluA FluB FluA FluA FluB H1N1 H3N2 - H1N1 H3N2 - PR8 Hong kong Lee PR8 Hongkong Lee One JA1 232.8 0.2 6.2 30.3 1,164 37.5 7.7 2 JA2 96.2 1.1 2.5 9.2 87.5 38.5 10.5 3 JA3 67.4 0.5 0.3 5.7 134.8 224.7 11.8 4 (+)-Catechin >100.0 >100.0 >100.0 >100.0 N.D. N.D. N.D. uM 5 (-)-Catechin >100.0 >100.0 >100.0 >100.0 N.D. N.D. N.D. uM 6 (-)-epi-Catechin >100.0 >100.0 >100.0 >100.0 N.D. N.D. N.D. uM 7 AMT >100.0 >100.0 0.5 >100.0 N.D. >200.0 N.D. uM 8 RBV >100.0 34.9 18.9 23.8 >2.9 >5.3 >4.2 uM 9 OSV-C >100.0 0.01 <0.005 2.21 >10,000 >20,000 >45.2 uM

As can be seen in Table 6, the cytotoxicity of JA1 was 232.8 ug/ml, which was higher than that of JA2 and JA3, but the anti-influenza activity was 0.2 ug/ml in FluA H1N1 PR8, showing strong anti-influenza virus activity. The cytotoxicity of JA2 was 96.2 ug/ml, and the anti-influenza activity was 2.5 ug/ml for FluA H3N2 Hong Kong, showing strong anti-influenza virus activity. In addition, JA3 showed lower cytotoxicity than JA1 or JA2 at 67.4 ug/ml, and anti-influenza activity was 0.5 ug/ml for FluA H1N1 PR8, 0.3 ug/ml for FluA H3N2 Hong Kong, and 5.7 ug/ml for FluB Lee. It showed strong anti-influenza virus activity per ml. Considering the selectivity index, that is, the value of CC ₅₀ /EC ₅₀ , excellent antiviral efficacy was shown in the non-toxic range in the order of JA1, JA3, and JA2. In particular, JA1 showed a selectivity value of over 1,000 against H1N1 influenza, showing a particularly high antiviral effect.

Example 5. Evaluation of anti-coronavirus activity of plum extract-derived substances

To measure antiviral activity against coronavirus COVID-19 (SARS-CoV-2), the virus was distributed by the Korea Disease Control and Prevention Agency (BetaCov/Korea/KCDC/03/2020), and Vero cells (ATCC) were purchased. used. After ^culturing the cells in a 96-well plate for one day (2 Cells were treated for 2 days. All experiments were conducted in a biosafety level 3 facility. On the second day, the infected cells were immobilized with acetone:methanol (1:3) solution and then incubated with anti-spike protein antibody and (Genetex, Irvine, CA, USA) Alexa Fluor 488-conjugated goat anti-mouse IgG. (Invitrogen, Carlsbad, CA, USA) EC ₅₀ was determined by detection with secondary antibody. The number of nuclei was determined by staining with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) and CC ₅₀ . Each experiment measured the average value of three independent repeated experiments, and quantitative analysis of imaging was performed using the Operetta high content screening system (Perkin Elmer, Waltham, MA, USA).

As a result of the experiment, JA1, JA2, and JA3 all showed anti-corona activity in the non-toxic range, and remdesivir was used as a positive control (Figure 24).

Claims (22)

A pharmaceutical composition for preventing or treating viral infection comprising plum ( Prunus mume ) extract or a fraction thereof as an active ingredient. The pharmaceutical composition for preventing or treating viral infection according to claim 1, wherein the plum extract is one or more selected from the group consisting of plum juice, plum syrup, plum concentrate, and plum fruit extract. The virus according to claim 1, wherein the plum extract is a crude extract obtained by extraction with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, acetic acid, butanol, or combinations thereof. Pharmaceutical composition for preventing or treating infection. The pharmaceutical composition for preventing or treating viral infection according to claim 3, wherein the plum extract is a crude extract obtained by extraction with methanol. The pharmaceutical composition for preventing or treating viral infection according to claim 1, wherein the plum extract is fractionated using at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol. The pharmaceutical composition for preventing or treating viral infection according to claim 1, wherein the fraction is obtained by performing chromatography. The method of claim 1, wherein the virus is one or more types selected from the group consisting of influenza and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), viral infection. Pharmaceutical composition for prevention or treatment. The pharmaceutical composition for preventing or treating viral infection according to claim 1, wherein the influenza virus is influenza virus type A or B. The method of claim 8, wherein the influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or A pharmaceutical composition for preventing or treating viral infection, having the serotype H10N7. A food composition for improving or preventing viral infection comprising plum ( Prunus mume ) extract or a fraction thereof as an active ingredient. The food composition for improving or preventing viral infection according to claim 10, wherein the plum extract is one or more selected from the group consisting of plum juice, plum syrup, plum concentrate, and plum fruit extract. The virus according to claim 10, wherein the plum extract is a crude extract obtained by extraction with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms, acetic acid, butanol, or combinations thereof. Food composition for improving or preventing infection. The food composition for improving or preventing viral infection according to claim 12, wherein the plum extract is a crude extract obtained by extraction with methanol. The food composition for improving or preventing viral infection according to claim 10, wherein the plum extract is fractionated using at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol. The food composition for improving or preventing viral infection according to claim 10, wherein the fraction is obtained by performing chromatography. The method of claim 10, wherein the virus is one or more types selected from the group consisting of influenza and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), viral infection. Food composition for improvement or prevention. The food composition for improving or preventing viral infection according to claim 16, wherein the influenza virus is influenza virus type A or B. The method of claim 17, wherein the influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or A food composition for improving or preventing viral infection, having the serotype H10N7. (+)-epicatechin, epiafzelechin-(2β→O→7, 4β→8)-ent-epicatechin, and epicatechin-(2β→O→7, 4β→8)-ent-epicatechin. A pharmaceutical composition for preventing or treating viral infection, comprising: The method of claim 19, wherein the virus is one or more types selected from the group consisting of influenza virus and type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2). Pharmaceutical composition for preventing or treating infection. The pharmaceutical composition for preventing or treating viral infection according to claim 20, wherein the influenza virus is influenza virus type A or B. The method of claim 21, wherein the influenza virus type A is H1N1, H3N2, H1N2, H2N2, H2N3, H3N1, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or A pharmaceutical composition for preventing or treating viral infection, having the serotype H10N7.