KR20150099203A - Composition containing polyphenols isolated from the fruiting bodies of Inonotus obliquus for preventing or treating of influenza virus infection - Google Patents

Abstract

Provided is a composition for inhibiting an activity of neuraminidases containing fractionated materials of ethyl acetate from fruiting bodies of Inonotus obliquus as an active ingredient and, more specifically, to a composition for inhibiting an activity of neuraminidases containing solvent extracts of Inonotus obliquus, fractionated materials and compounds having specific structures isolated therefrom as active ingredients. The composition according to the present invention can be used for preventing and treating influenza virus infection by inhibiting the activity of neuraminidases which are essential for viral replication and exist on the surface of influenza viruses, and preventing proliferation of viruses to other cells in respiratory organs.

Description

[0001] The present invention relates to a composition for preventing and treating influenza virus infection comprising a polyphenol component isolated from a mushroom fruiting body,

The present invention relates to a composition for preventing and treating influenza virus infection, which comprises a polyphenol component isolated from a mushroom fruiting body. More particularly, the present invention relates to a composition for preventing and treating infection of an influenza virus, A composition for preventing and treating influenza virus infection comprising a polyphenol component isolated from a mushroom fruiting body, which can be used for prevention and treatment of an influenza virus infection because it inhibits the influenza virus from spreading to other cells in the respiratory tract .

Influenza virus is a respiratory disease that invades the airways and causes viral infections. It is known to cause respiratory symptoms such as cold, headache, high fever, arthralgia, muscular pain and coloring, nasal discharge (Hien, TT et al. , ≪ / RTI > 350, 1179, 2004). These influenza viruses are largely classified into A, B, and C types. In particular, the A virus is infected mainly in humans and has been confirmed to be infected in pigs, other mammals and various wild birds compared to B and C.

Recently, Avian influenza, Swine influenza and Novel flu, all of which are a problem in the world, all belong to type A virus. There are surface proteins called hemagglutinin (HA) and neuraminidase (NA) on the surface of influenza virus, and various kinds of viruses exist depending on their types. Hemagglutinin binds to the terminal sialic acid residues on the surface of the host cell, attaching the virus, sequentially allowing the virus to penetrate into host cells, and the other protein, neuraminidase, Viruses replicated and propagated in infected cells are cleaved from the alpha-ketosidic bond that connects the oligosaccharide moiety of the cell surface to the terminal neuraminic acid moiety and the virus is released from the host cell (Chandrasekaran, A. et al., Nature Biotechnology 26, 107, 2008; Mark, VI Nature review 6, 967, 2007; Huberman, K. et al., Virology 214, 294, 1995). Therefore, in order to treat viral diseases, besides transcriptional and replication inhibition of genes and inhibition of protein synthesis, it is also possible to inhibit adsorption / invasion (hemagglutinin inhibitory activity) with epithelial cells and inhibition of release from cells (neuraminidase activity inhibition) , Each of which is the target of an antiviral.

Currently, the only treatment for avian influenza and swine flu is oseltamivir phosphate, an oral therapeutic agent developed as a selective inhibitor of viral originated neurominidase, and the inhaled zanamivir. However, Tamiflu, which is used as an oral therapeutic agent, has side effects such as nausea, vomiting, nervous system or mental disorder and should be prepared for the case of Tamiflu resistant virus used for oral treatment. Recently, the emergence of resistant viruses against Tamiflu, which is already lethal to the spread of the H1N1 virus, has been reported, and even human-to-human transmission of the Tamiflu-resistant virus has been found. Thus, the antiviral agents developed so far show severe side effects, and a great deal of attention must be paid to their applications. In addition, the development of a vaccine has a problem of low efficacy when the virus type of the prevalent virus is not matched with the virus of the vaccine. Therefore, there is an increasing need to develop a new influenza virus agent having excellent infection control effect and excellent stability.

Accordingly, a problem to be solved by the present invention is to provide a composition for inhibiting the spread of influenza virus to other cells in the respiratory tract by inhibiting the activity of neuraminidase, and a composition for inhibiting influenza virus infection, And to provide a therapeutic composition.

In order to solve the above-mentioned problems, the present invention relates to a method for producing a mushroom ( Inonotus obliquus ) A composition for inhibiting neuraminidase activity comprising an ethyl acetate fraction fractioned from a fruity body extract as an active ingredient.

The present invention provides a composition for inhibiting neuraminidase activity comprising, as an active ingredient, a compound represented by the following formula (1) or a compound containing the structure of the formula (1).

[Chemical Formula 1]

The present invention provides a composition for inhibiting neuraminidase activity comprising, as an active ingredient, a compound represented by the following formula (2) or a compound containing the structure of the formula (1).

(2)

The present invention provides a composition for inhibiting neuraminidase activity comprising, as an active ingredient, a compound represented by the following formula (3) or a compound containing the structure of the formula (1).

(3)

In one embodiment of the present invention, the neuraminidase is derived from H1N1 influenza virus influenza virus, and the composition for inhibiting neuraminidase activity described above is selected from the group consisting of Inonotus obliquus , It is fractionated from fruit body extract.

The present invention provides a composition for preventing and treating an influenza virus infection comprising the composition for inhibiting neuraminidase activity described above as an active ingredient, wherein the disease caused by the influenza virus infection is influenza, cold, sore throat, bronchitis, or pneumonia . In addition, the flu is avian flu, swine flu or swine flu.

The present invention provides a composition for the prevention and treatment of influenza virus infection, wherein the composition for preventing and treating influenza virus infection is a pharmaceutical composition, a food composition or a quasi-drug composition.

The present invention provides a composition for inhibiting neuraminidase activity, which contains, as an active ingredient, a mushroom solvent extract, a fraction and a compound of a specific structural formula separated therefrom. The composition according to the present invention inhibits the activity of neuraminidase, which is present on the surface of influenza virus and functions as a vital for viral replication, thereby preventing the influenza virus from diffusing into other cells in the respiratory tract. Therefore, the composition for preventing and treating influenza virus infection .

1 is a graph showing the effect of inhibiting neuraminidase enzyme activity of the mushroom solvent fraction.
FIG. 2 is a graph showing the inhibitory effect of phelligridin D, phelligridin E and phelligridin G on neuraminidase enzyme activity, which was isolated and purified from the mash ethyl acetate fraction .
Figure 3 shows the effect of pheligridin D, phelligridin E and phelligridin G, isolated and purified from chaga mushroom ethyl acetate fraction, on influenza virus A inhibitory activity in MDCK cell lines FIG.
Figure 4 shows the effect of pheligridin D, phelligridin E and phelligridin G isolated from chaga mushroom ethyl acetate fraction on influenza virus A inhibitory activity in MDCK cell lines Lt; RTI ID = 0.0 > cytoxin < / RTI > performance.

In order to fully understand the present invention, operational advantages of the present invention, and objects achieved by the practice of the present invention, reference should be made to the accompanying drawings and the accompanying drawings which illustrate preferred embodiments of the present invention.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments of the present invention with reference to the accompanying drawings. However, the present invention can be implemented in various different forms, and is not limited to the embodiments described. In order to clearly describe the present invention, parts that are not related to the description are omitted, and the same reference numerals in the drawings denote the same members.

BRIEF DESCRIPTION OF THE DRAWINGS In the entire specification, when a section includes a constituent element, it does not exclude other constituent elements, but may include other constituent elements, unless specifically stated otherwise.

In order to solve the above-mentioned technical need, the present invention provides a fractionated substance after being extracted from the fruiting body of a mushroom, and more particularly, a polyphenol compound represented by the following formulas (1) to (3) We have found that polyphenol materials are effective in inhibiting neuraminidase activity.

[Chemical Formula 1]

(2)

(3)

In the present invention, the base material of the mushroom ( Inonotus obliquus ) used for extracting the polyphenol component of Formulas 1 to 3 is black birch. If you look at the tea mushrooms native to the Kamchatka peninsula, they are attached to a living birch like a black hump, and this black hump grows gradually, and the birch is gradually dying. Fruiting bodies are formed in the bark of birch, and the epidermis is black and sclerotia is formed with yellow tissue. In particular, chaga mushrooms produce yellow pigments, and these pigments are known to have antioxidant activity (Lee IK et al, J. Bioorganic & Medicinal Chemistry Letters 2007, 17, 6678-6681).

Thus, the present inventors have found that yellow pigment compounds separated from Inonotus obiliqus fruiting body, namely phelligridin D, phelligridin E and phelligridin G, The present inventors confirmed that the inhibitory activity of neuraminidase and the antiviral activity of MDCK cells showed excellent inhibitory activity. In particular, it was confirmed that the medicines for the prevention and treatment of diseases related to the inhibition of the activity of neuraminidase, , Or a food additive can be used for the purpose of the present invention.

In one embodiment of the present invention, the compounds of Formulas 1-3 may be used in the form of pharmaceutically acceptable salts, including all salts, hydrates and solvates prepared by conventional methods. Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The acid addition salt is prepared in a conventional manner, for example, by dissolving the compound in an excess amount of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid and hydroiodic acid may be used. It is not limited.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutically acceptable salts of the compounds represented by the above general formulas (1-3) include salts of acidic or basic groups which may exist in the compounds of general formulas (1-3), unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate) and p -toluenesulfonate (tosylate) salt, and the like, and a method for producing a salt known in the art ≪ / RTI >

The compound represented by any one of Chemical Formulas 1-3 according to the present invention may be isolated and extracted from the mushroom fruiting body.

When the compound represented by any one of Chemical Formulas 1-3 according to the present invention is separated from chaga mushrooms, the method for isolating the compound may be carried out as follows. In the present invention, the extract from chaga mushroom Here's how to get it:

Chaga mushrooms can be used without restrictions, such as cultivated or marketed, cleaned and dried. The alcohol may be a lower alcohol such as methanol, ethanol, propanol, and butanol. Methanol or ethanol may be preferably used.

The step of obtaining the mushroom extract can be carried out by putting the dried mushroom in a shredded mushroom, adding an appropriate amount of alcohol, allowing it to stand at room temperature for 7 days, and filtering it with filter paper or the like. This extraction process may be repeated several times, and then a method such as concentration or lyophilization may be additionally performed.

  The chaga mushroom extract obtained in the above step is suspended in water, and fractionated successively using hexane, chloroform and ethyl acetate to obtain a mushroom fraction. At this time, a usual fractionation extraction method can be used, and a fractionation funnel can be preferably used. Chaga mushroom fraction can be obtained from hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction.

In order to separate the active substance from chaga mushroom characterized by the present invention, the methanol extract of fruiting body is extracted by an organic solvent such as hexane, chloroform, ethyl acetate, etc., a known method used for separation and extraction of components such as water distribution and column chromatography Can be obtained either singly or in a suitable combination. The details of the method are as follows.

1) extracting the crushed chaga mushroom with a solvent selected from among aliphatic alcohols having 1 to 6 carbon atoms and aliphatic hydrocarbons having 1 to 10 carbon atoms to obtain a solvent extract;

2) fractionating the solvent extract with hexane, chloroform and ethyl acetate in order to obtain an ethyl acetate fraction; And

 3) The ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography with 100% methanol and subjected to octadecylsilylated silica gel (ODS) column chromatography to obtain peligyridine D 1) and separating peligliadin D (Formula 1) and peligyridine E (Formula 2) from the mushroom.

In the present invention, the above-mentioned chaga mushroom ethyl acetate fraction is subjected to Sephadex LH-20 column chromatography using 100% methanol, and then the active eluate is purified by preparative high-performance liquid chromatography using an aqueous 55% methanol solution containing 0.04% trifluoroacetic acid (Preparative HPLC) and separation of peligyridine G (Formula 3).

The composition for the prevention and treatment of influenza virus infection according to the present invention prevents and treats influenza virus infection by inhibiting the activity of neuraminidase enzyme, and thus the composition is suitably used as an appropriate carrier, excipient And a diluent.

The composition according to the present invention may further comprise at least one selected from the group consisting of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like Formulation, external preparation, suppository, and sterile injection solution. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension agents, solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

EXAMPLES Example 1: Separation of Formula 1-3 from Chrysanthemum fruiting body extract

 1. Chaga mushroom extract manufacturing

In the step of obtaining chaga mushroom extract, 3 kg of dried chaga mushroom is shredded and placed in an extraction container, and 15 liters of methanol or ethanol is added. The resultant is left at room temperature for 7 days, filtered with a filter paper, .

The chaga mushroom extract obtained in the above step is suspended in water, and fractionated successively using hexane, chloroform and ethyl acetate to obtain a mushroom fraction.

2. Isolation, purification and identification of active compounds

The crude extract of the mushroom obtained in 1 above was subjected to Sephadex LH-20 column chromatography using methanol to obtain the first active fractions 1 and 2.

The primary active fractions 1 and 2 were subjected to Sephadex LH-20 column chromatography with a 70% methanol aqueous solution to obtain a second active fraction, and 0.04% trifluoroacetic acid ( lt; RTI ID = 0.0 > 55% < / RTI > HPLC was carried out to obtain a pure compound of the formula 1-3 described below.

Experimental Example 1: Determination of the inhibitory activity of H1N1 neuraminidase from the extracts of Chaga mushroom, fractions thereof and the compounds 1-3 isolated therefrom

To determine the inhibitory activity against neuraminidase, the recombinant neuraminidase of the virus (A / Bervig_Mission / 1/18) isolated from the Spanish influenza virus in 1918, ruH1N1 Influenza A virus neurominidys (R & D system, 4858) 4-Methylumbelliferyl-α-DN-acetylneuraminic acid (SIGMA M8639) was used as the substrate.

The mushroom extract and its fractions were dissolved in MeOH and added in a volume of 10 μL each. 50 μL of the substrate was added to a final concentration of 200 μM, mixed with 90 μL of Tris buffer (pH 7.5) containing 5 mM CaCl ₂ and 200 mM NaCl And 50 μL of the enzyme, Neuraminidase (final concentration of enzyme 50 ng / mL) was added to the reaction mixture, and the activity of the neuraminidase was measured by measuring the light absorbance at 365 nm and the luminescence at 445 nm using a fluorescence spectrometer.

The inhibitory activity (%) was expressed by the following equation, and the concentration showing the inhibition rate of 50% was described as IC ₅₀ .

Inhibition activity (%) = ((RFU of control - RFU of sample) / RFU of control) x 100

      control: an experimental sample

The chitosan ethyl acetate fraction of the present invention showed 67.9% inhibition of neuraminidase enzyme activity at a concentration of 100 μg / mL (FIG. 1).

In addition, phelligridin D, phelligridin E and phelligridin G compounds of Formula 1-3 of the present invention decreased neuraminidase enzyme activity in a concentration-dependent manner , IC ₅₀ values were measured as 8.8 (1), 6.2 (2) and 92.4 μM (3), respectively (FIG. 2).

Experimental Example 2: Measurement of the inhibitory effect on the influenza virus of the compound of the formula (1-3) isolated from the chrysanthemum extract

1. Culture of virus

The influenza viruses used in this experiment were Influenza A virus (A / WS / 33), and the influenza virus was infected with MDCK cells. MDCK cells were cultured in MEM medium supplemented with 10% FBS at 37 ° C. The medium used for the virus growth culture and the antiviral activity assay was cultured in serum-free medium with trypsin concentration adjusted to 1 μg / mL Medium was used.

2. Antiviral Suppression and Cytotoxic Performance Analysis

To measure the inhibitory effect of virus against influenza virus, MDCK cells were cultured for 24 hours in 2 wells and 10 ⁴ cells in each well of 96-well plates. After 24 hours, the culture supernatant of each well was removed, and influenza virus infectious solution was added to each well. Then, Compound 1-3 isolated from each mushroom fruiting body was dissolved in a concentration of 50 g / mL or 0.1, 1, 10, 100 Lt; RTI ID = 0.0 > L < / RTI > After 48-72 hours, the virus infection reaction was stopped, and the antiviral inhibitory activity of each of the extracts was measured and the cytotoxicity was measured according to Korean Patent Application No. 10-2004-0111298. The culture supernatant of each well was removed and the cells remaining in the wells were fixed with 100 L of 70% acetone solution. 100 L of SRB (sulforhodamine B) solution was added to each well to stain the cell protein. After removing the SRB stain that was not bound to the cell protein, 100 μL of a 10 mM Tris solution (pH 10.5) was added to each well to dissolve the stain bound to the cells, and the absorbance of each well was measured with an ELISA reader at 565 nm . Each treatment group was labeled with virus (A), extract only (B), virus only (C), virus and extract (D) (%) And the inhibitory effect of the compound 1-3 on the proliferation of the virus was calculated as the cell survival rate (%) after infection. The results are shown as the mean value and the standard deviation of the results of three experiments performed under the same conditions.

Experimental Results The phelligridin D, phelligridin E and phelligridin G compounds of Formulas 1-3 of the present invention showed antiviral activity using MDCK cells infected with influenza virus The IC ₅₀ values for the influenza A virus (A / WS / 33) were 22.6 μM (1), 32.4 μM (2) and 52.1 μM (3), respectively, and the control drug In Tamiflu (IC ₅₀ = 64.7 [mu] M) (Fig. 3 and 4).

Therefore, the composition according to the present invention is highly effective for diseases caused by influenza virus infection such as flu, cold, sore throat, bronchitis, or pneumonia, and the flu may be avian flu, swine flu or swine flu.

In addition, the composition for the prevention and treatment of influenza virus infection may be in the form of a pharmaceutical composition, a food composition or a quasi-drug composition, which will be described below.

Formulation Example 1: Preparation of Syrup

The syrup containing 2% (weight / volume) of the mushroom fruylic acid solvent extract, the fraction and the antiviral compound of the formula 1-3 or the pharmaceutically acceptable salt thereof isolated therefrom as an active ingredient was prepared as follows.

The mushroom fruiting body solvent extract, the fractions and the acid addition salts, saccharin, and saccharides of the antiviral compounds of Formulas 1-3 isolated therefrom were dissolved in 80 g of hot water. After the solution was cooled, a solution composed of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to the mixture to make 100 mL. The addition salt may be replaced with another salt according to the embodiment.

The components of the syrup are as follows.

Active ingredient: 2 g

Saccharin: 0.8 g

Per unit: 25.4 g

Glycerin: 8.0 g

Spices: 0.04 g

Ethanol: 4.0 g

Sorbic acid: 0.4 g

Distilled Water: Quantity

Formulation Example 2: Preparation of tablets

The tablet containing the mushroom fruiting body solvent extract, the fraction and the antiviral compound of the formula 1-3 or the pharmaceutically acceptable salt thereof as an active ingredient is prepared by the following method.

The mushroom fruity body extract, fractions or compounds 1-3 isolated therefrom were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 3: Preparation of injection solution

An injectable solution containing as an active ingredient an extract from chaga mushroom flesh, a fraction thereof, or a compound represented by the general formula (1-3) or a pharmaceutically acceptable salt thereof isolated therefrom was prepared by the following method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (10)

Mushroom ( Inonotus obliquus ) A composition for inhibiting neuraminidase activity comprising an ethyl acetate fraction fractioned from a fruity body extract as an active ingredient. A composition for inhibiting neuraminidase activity comprising a compound represented by the following formula (1) or a compound containing the structure represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

A composition for inhibiting neuraminidase activity comprising a compound represented by the following formula (2) or a compound containing the structure represented by the following formula (2) as an active ingredient.
(2)

A composition for inhibiting neuraminidase activity comprising a compound represented by the following formula (3) or a compound containing the structure represented by the following formula (3) as an active ingredient.
(3)

The composition according to any one of claims 1 to 4, wherein the neuraminidase is derived from H1N1 influenza virus influenza virus. A composition for inhibiting neuraminidase activity is characterized in that the composition for inhibiting neurominidase activity according to any one of claims 2 to 4 is fractionated from an extract of Inonotus obliquus fruit body. A composition for preventing and treating an influenza virus infection comprising the composition for inhibiting neurominidase activity according to claim 6 as an active ingredient. 8. The method of claim 7,
Wherein the disease caused by the influenza virus infection is influenza, cold, sore throat, bronchitis, or pneumonia. 9. The method of claim 8,
Wherein said influenza virus is avian influenza, swine flu or swine flu. The method according to claim 6,
A composition for the prevention and treatment of influenza virus infection, which comprises a pharmaceutical composition, a food composition or a quasi-drug composition.

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