KR20100067305A - Pharmaceutical composition with antiviral activity extracted from phellinus pini - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing extract having antiviral activity, which is obtained from Phellinus pini is provided to suppress enzyme activity relating to viral infection mechanism and to prevent viral diseases. CONSTITUTION: A pharmaceutical composition for preventing or treating diseases by infection of coxsackievirus, influenza virus, or herpes simplex virus contains Phellinus pini-derived antiviral extract as an active ingredient. The extract is obtained from a fruit body of the Phellinus pini by adding polar solvent and centrifuging. The polar sovent is alcohol of 1-4 carbon atoms. The extract is contained in a concentration of 0.001-5.0 mg/ml. The extract contains water soluble polysaccharide of beta-(1,3)(1,6)-glucan.

Description

Pharmaceutical Composition with Antiviral Activity Extracted from Phellinus pini}

The present invention relates to a pharmaceutical composition, and more particularly to a pharmaceutical composition for the treatment or prevention of viral diseases containing as an active ingredient an extract having antiviral activity obtained from deciduous fungi.

Although various types of preventive vaccines and antibiotics have been developed, diseases caused by bacteria (bacteria) have been prevented to some extent, but treatment and prevention of various diseases caused by viruses have not yet been effective, but rather recently Due to environmental pollution, viral diseases are increasing.

In particular, in addition to the existing diseases caused by influenza virus causing flu, herpes virus causing hepatitis, and hepatitis virus, recent avian influenza (AI) and Severe Acute Respiratory Syndrome (SARS) The emergence of new viruses that cause cancer is a serious threat to human and livestock life. Accordingly, there is a worldwide interest in the prevention and treatment of various viral diseases. Most antiviral agents currently commercialized include nucleoside derivatives such as iododeoxyuridine (IDU), acyclovir (ACV), azidothymidine (AZT), and proteins such as interferon (IFN) Oseltamivir phosphate (so-called Tamiflu), which has been developed as a treatment for human influenza virus A and B and avian influenza virus, also has vomiting, bronchitis, Side effects such as headaches, coughs and dizziness have been reported. In addition, the emergence of resistance strains for existing drugs has been reported to increase (Coen, EM 1986. General aspects of virus resistance with special reference to herpes simplex virus. J. Antimicrob. Chemother. 18 (Suppl. B) : 1 -10; Hardman, JG, Limbird, LE, Molinoff, PB, Ruddon, RW and Gilman, AG 1991.The pharmacological basis of therapeutics (9th eds.). 1191-1223.McGraw-Hill.New York, Because of the limited efficacy, there are many limitations in treating viral diseases efficiently. Therefore, the necessity of developing a new antiviral substance with low side effects and low cost is continuously required. In order to circumvent the side effects of existing chemical synthetic drugs, a research for searching for substances having antiviral activity from natural substances with low side effects and safety Is actively underway.

Among natural products, since mushrooms have been ingested by food or folk medicine since ancient times, the safety is high, and there is a high possibility of developing new medicines derived from natural products. In particular, mushrooms are known to be effective in anti-cancer, lowering blood pressure, lowering cholesterol, etc. with excellent physiological activity, and extracts isolated from various mushrooms have been reported to have immune enhancement and anti-cancer activity (Bohn, JA, and JN BeMiller). 1995. (1-> 3) -β-D-Glucans as biological response modifiers: a review of structure-functional activity relationships.Carbohyd . Pol. 28: 3-14.).

However, studies on the antiviral activity of mushrooms have been carried out since Cochran et al. Isolates from Lentinus edodes to increase immune responses (KW Cochran, T. Nishikawa and ES Beneke, 1966, Botanical sources of influenza inhibitors, Antimicrob. Agents Chemother. (Bethesda) 6: 515 ?? 520), Lentinus edodes (Tochikura, TS, H. Nakashima, Y, Kaneko, N. Kobayashi, and N. Yamamoto. 1987a, Suppression of human immunodeficiency virus replication by 3'-azido-3 'deoxythymidine in various human haematopoietic cell lines; Augmentation of the effect by lentinan, Japanese J. of Cancer Research (Gann) 8: 583-589), Coriolus versicolor (Tochikura, TS, H. Nakashima, K. Hirose, and N. .... Yamamoto 1987b A biological response modifier, PSK, inhibits human immunodeficiency virus infection in vitro, Biochem Biophys Res Commun 148:.. 726-733), Elfvingia applanata (Eo, SK, YS Kim, KW Oh, C. K Lee, YN Lee, and SS Han. 2001.Mode of Antiviral Activity of Water soluble components isolated from Elf vingia applanata on vesicular stomatitis virus.Arch . Pharm.Res . 24 (1): 74-78), Ganoderma spp. (Kandefer, SM, Kawecki, Z. and Guz, M. 1979. Fungal nucleic acids as interferon inducers.Acta Microbiol. Pol. 28 (4) : 277-291), Cordyceps militaris (Muller, WEG, BE Weiler, R. Charubala, W. Piferiderer, L. Lserman, RW Sobol, J. Suhadolnik, and HC Schroder. 1990. Cordycepin analogues of 2'5'-oligoadenylate inhibitor human immunodeficiency virus infection via inhibition of reverse transcriptase, Biochem. 30: 2027-2033 The antiviral activity of extracts obtained from mushrooms such as) has been reported.

However, until now, antiviral active substances obtained from mushrooms have been reported mainly as mixed extracts or modifiers. Purified purified pure substances have a clear structural characterization, physical and chemical characterization, and mass production techniques. Few examples have been developed as antiviral drugs that have not been achieved.

On the other hand, leaves mud mushroom (Phellinus pini) is a mushroom belonging to the Democratic name mushroom neck (Aphyllophorales) pine scales mushrooms and (Hymenochaetacae) mud mushrooms in (Phellinus sp.) Of the Basidiomycetes, anti-cancer action of the fruit body extracts from (Ikegawa, T , Nakanishi, M., Uehara, N., Chihara, G. and Fukuoka, F. 1968.Antumor action of some Basidiomucetes, especially Phellinus linteus.Gann 59 (2): 155-157), immunopotentiation (Jeong, SC, Cho, SP, Yang, BK, Jeong, YT, Ra, KS and Song, CH 2004. Immunomodulating Activity of the Exopolymer from submerged Mycelial Culture of Phellinus pini , Journal of Microbiology and Biotechnology 14 (1): 15-21. , Blood lipid reduction effect (Yang, BK, Park, JB and Song, CH 2002. Hypolipidemic effect of exopolymer produced in Submerged Mycelial Culture of five different mushroom.Journal of Microbiology and Biotechnology 12 (6): 957-961) The study was reported.

On the other hand, Japanese Patent Laid-Open No. 2006-340277 describes that a fat-soluble extract derived from a situation mushroom of the genus Phellinus functions as a stress inhibitor on the vesicles and is useful for the prevention or treatment of neurodegenerative diseases such as senile dementia. 10-0504272 proposes a pharmaceutical composition in which the extract of P. vulgaris and Ganoderma lucidum extract inhibits the activity of viruses related to swine or canine diseases such as PMWS virus and PRRS virus.

In addition, the Republic of Korea Patent Publication No. 10-2004-0069422 describes that the extracellular polysaccharide (exopolymer) obtained from the mycelium culture of larch larvae mushroom has an immunomodulatory activity, the Republic of Korea Patent No. 10-0695996 It is described that a cosmetic composition containing niosome is excellent in skin moisturizing, elasticity and whitening effects.

However, to date, deciduous mud mushrooms have been inhibited by other fungi in the timber industry, and most of them have been studied for their strains, and the evaluation of physiological activity of fruiting bodies is insignificant compared to other mushrooms.

The present invention has been proposed to solve the above problems, an object of the present invention can treat or prevent diseases caused by the infection of a virus involved in various diseases of human, such as influenza virus, herpes virus, coxsackie virus, The purpose of the present invention is to provide a pharmaceutical composition containing as an active ingredient an extract having antiviral activity from Phellinus pini .

Another object of the present invention is to identify the chemical composition or structure of the antiviral active ingredient that inhibits the infection of various viruses in the hydrothermal extract of Phellinus pini , as well as the biological and chemical properties, as well as the active ingredient It is to provide a pharmaceutical composition having an antiviral activity that can be industrially mass-produced by purifying only.

Other advantages and objects of the present invention will become more apparent from the following detailed description of the invention and the accompanying drawings.

According to the present invention having the above object, to treat the disease caused by the infection of influenza virus, herpes simplex virus or coxsackie virus containing an extract having antiviral activity derived from Phellinus pini as an active ingredient Or provide a pharmaceutical composition to prevent.

At this time, the extract with antiviral activity is obtained from the fruiting body of the deciduous fungi ( Phellinus pini ), the extract with antiviral activity can be obtained by adding a polar solvent to the hydrothermal extract of the deciduous fungi fruiting body powder after centrifugation. .

In this case, the extract having antiviral activity is obtained from the precipitate component among the components separated by centrifugation, and the polar solvent may be selected from alcohols having 1 to 4 carbon atoms.

On the other hand, the extract from the deciduous mud mushrooms may have antiviral activity by inhibiting the activity of neuraminidase to prevent the infectious activity of the influenza virus, or by killing the virus.

In particular, the antiviral extract derived from the deciduous mud mushrooms is preferably contained at a concentration of 0.001 ~ 5.0 mg / ㎖.

At this time, according to the embodiment of the present invention, the extract having the antiviral activity is a water-soluble polysaccharide, characterized in that the β- (1,3) (1,6) -glucan of the heterotype.

In the present invention, the extract obtained through a simple method of hydrothermally extracting the fruiting body powder of the deciduous fungi ( Phellinus pini ) inhibits the infection of the virus closely related to various diseases caused by livestock, birds, etc., which is very beneficial to humans as well as humans It was confirmed that it contained the active ingredient which has an effect.

In particular, according to the embodiment of the present invention, the hot water extract of the deciduous mud mushroom fruiting body is cardiomyopathy, such as myocarditis / dilated cardiomyopathy, coxsackie virus involved in diabetes, as well as influenza virus, oralitis causing the flu By inhibiting the mechanism of infection of the herpes virus causing the virus can be treated and / or prevent various diseases caused by these viruses.

Particularly, in the present invention, by partially purifying a component having antiviral activity in the hot water extract of Phellinus pini , the active ingredient is produced in large quantities and mixed with other carriers, diluents and the like used in the pharmaceutical field for human or It is expected that the method may be applied industrially through administration to animals.

The present inventors intensively studied the industrial applicability of the deciduous fungi ( Phellinus pini ), and found that they have antiviral activity as a pharmacological effect of the extracts from deciduous fungi. That is, according to the present invention, it was found that the hot water extract of the deciduous mud mushroom fruiting body can effectively suppress the infection caused by viruses causing various diseases of humans such as influenza virus, herpes virus, coxsackie virus, As part of the pharmacological applicability of the extract, the structure of the active substance was investigated through the isolation and purification of the antiviral active substance. EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

1. Preparation of antiviral active ingredients derived from deciduous mud mushrooms

The method for obtaining the antiviral active ingredient from Phellinus pini according to the present invention is schematically illustrated in FIG. 1. First, distilled water is added to the crude powder obtained by cutting and grinding the fruiting body of the deciduous mud mushroom to obtain a water-soluble extract of the deciduous mud mushroom. At this time, distilled water may be added at a ratio of 1 to 5 L based on 100 g of fruiting body coarse ground. The hot water extract thus obtained is dried or lyophilized to be used in powder form.

In order to separate macromolecules and micromolecules from the hot water extracts of the deciduous mud mushroom fruiting bodies thus obtained, a precipitate or supernatant (separated), which is separated by centrifugation after addition of a polar solvent, is obtained in a dry powder form. In this case, the polar solvent used to separate the active ingredient in the hot water extract may be a polar organic solvent and / or water, such as alcohol having 1 to 4 carbon atoms (for example, 60 to 100% ethanol). At this time, according to a preferred embodiment, distilled water is first added to the powdered hot water extract, and a polar organic solvent of 2-5 times may be used with respect to distilled water.

In order to separate the active ingredient, it is separated into supernatant (ES) and precipitate (pellet, EP) by polar solvent addition / centrifugation, each of which is lyophilized to be used in powder form or in liquid state. Can be used.

2. Antiviral Activity

end. Suppression of Coxsackie Virus Infection

Coxsackievirus is a non-enveloped RNA virus that has no envelope lipids and has a single-stranded single-stranded positive sense RNA genome (about 7.4 kb). Coxsackie virus belongs to the Picornaviridae family and is a human enterovirus belonging to the genus Enterovirus genus, such as poliovirus and echovirus. Coxsackievirus is classified into A-type and B-type according to the presence or absence of pathogenicity causing flaccid paralysis when infected with young mice.

Coxsackievirus A (CVA) belonging to A-type is mainly infected through the skin, causing herpangina, acute hemorrhagic conjunctivitis (AHC), etc. Coxsackievirus B (CVB) belongs to acute aseptic meningtis, meningoencephalitis, acute pancreatitis, type-1 diabetes (insulin-dependent diabetes mellitus, IDDM). Related to In particular, CVB is known to be a major cause of cardiomyopathy, such as myocarditis, dilated cardiomyopathy and the like. CVB has been reported six serotypes (CVB1 ~ CVB6), three types of CVB1, CVB3, CVB5 is closely related to heart disease, as confirmed by the present invention, deciduous mudslide fruiting body derived Among the hot water extracts, ethanol precipitate (EP), which is a polar solvent, was found to inhibit the activity of coxsackievirus (FIG. 3B, FIG. 3C).

I. Herpes Simplex Virus

Herpes simplex virus (HSV) is a virus belonging to the herpesviridae, which has a relatively large double-stranded linear DNA genome inside a capsid wrapped in envelope lipids. In HSV, the glycoprotein 'glycoprotein C' in the envelope lipids binds to a particle called 'heparan sulfate' on the surface of the host cell, and the second glycoprotein 'glycoprotein D' is called a herpesvirus entry mediator receptor (HVEM). It is known to specifically induce HSV to adhere strongly to host cells, thereby invading into the host cells.

There are two known types of HSV, HSV-1 and HSV-2. Infection with HSV causes various diseases. For example, infection of the mucous membranes or skin can cause cold sore, fever blisters, whitlow, or keratitis, or genital herpes, leading to genital herpes. In addition, it penetrates the central nervous system and is known to cause herpes encephalitis, schizophrenia, and Alzheimer's syndrome. To date, vaccines against HSV have not been developed, and only treatment to inhibit the proliferation of the virus is possible.

However, in the case of the ethanol precipitate obtained from the hot water extract of the deciduous mushroom fruiting body according to the present invention, both plaque formation of HSV-1 is greatly increased when pre-cultured with HSV-1 or pre-cultured with host cells. It can be seen that the antiviral activity by HSV-1 is excellent (Figs. 4B to 4E).

All. Influenza virus

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae. Influenza virus A, influenza virus B, and influenza virus C cause influenza as well as mammals, including humans. Known. Influenza viruses have a linear, negative-sense, single-stranded RNA genome, and hemagglutinin (HA) and neuramini, the spike proteins of the viral envelope, among many glycoproteins that exist outside the outer envelope. Neuraminidase (NA) plays an important role in the mechanism of infection of influenza viruses.

Hemagglutinin is a lectin that binds the virus to the host cell and mediates the entry of the viral genome into the host cell.The surface of epithelial cells where hemagglutinin is present in the lungs of hosts such as humans, mammals, and birds When bound to the sialic acid sugar receptor formed at the end of the carbohydrate residues present in the virus, virus enters the host cell by endocytosis, and viral RNA and protein pass through the cytoplasm of the host cell. It is transferred to the nucleus of the host cell to initiate transcription of the viral genome, thereby replicating and propagating by synthesizing the viral protein, thereby infecting the host cell (Ko, HR, H. Kakeya, A. Yoshida, R. Onese, M Ueki). , M. Muroi, A. Takatsuki, H. Matsuzaki, and H. Osada, 2002, PC-766B 'and PC-766B, 16 membered macrolide angiogenesis inhibitors produced by Nocardia sp.RK97-56, J. Micobiol.Biotechnol. 12 : 829-833; Willey, DC and JJ Skehel. 1987.The structure and function of the haemagglutinin membrane glycoprotein of influenza virus.Annu. Rev. Biochem. 56: 365-394.).

On the other hand, neuraminidase, also called sialidase, is the α (2-6) or α (2-3) between the terminal sialic acid residues of the host cell and neighboring sugars that bind to mature viral particles. By cleaving the binding site, the virus propagated from the infected cells is allowed to escape to the infected cell surface in the form of virions so that they can emerge through the cell membrane.

According to the present invention, the deciduous mud mushroom-derived hot water extract was found to specifically inhibit the activity of neuraminidase, which is involved in the emergence of influenza viruses from outside the infected host cells in the form of mature virions after infecting the host cells. (FIGS. 5-7)

In order to suppress the toxicity against the above-mentioned viruses while suppressing the toxicity to the host cell according to the present invention, the antiviral extract derived from the deciduous mud mushroom has a concentration of 0.001 to 5.0 mg / ml, preferably 0.01 to 2.0 mg / ml. It can be used at a concentration of.

3. Purification of the Active Ingredient

Analysis of the constituents of ethanol precipitates (EP) having the highest antiviral activity among the hot water extracts from the deciduous mudslide fruiting bodies was carried out using various methods. Basically, the substances having antiviral activity were water-soluble glycoproteins (FIG. 8). In particular, in the polysaccharides, the content of glucose is 50% or more, and heteropolysaccharides containing a certain amount of galactose, xylose, mannose, etc. (FIG. 9), and two active ingredients have molecular weights of about 1,006 KDa and about 100 KDa. (FIG. 10), in particular β- (1,3) (1,6) with at least partially based on β-1,3-glucose bond structure and glucose-branched structure with β-1,6- bond It is inferred to be-(1,3) (1,6) -glucan of heterotype containing many other monosaccharides as -glucan (FIG. 11).

4. Application as a pharmaceutical composition

According to the present invention, when pharmaceutically administering a composition containing an extract having antiviral activity derived from Phellinus pini as an active ingredient, it can be used in the form of a pharmaceutically acceptable salt of these extracts. These extracts may be used alone or in combination with pharmaceutically active fractions.

The composition comprising the extract having the antiviral activity of the deciduous fungi ( Phellinus pini ) of the present invention can be prepared in a pharmaceutically acceptable formulation according to methods commonly used in connection with pharmaceutical compositions. In other words, the extract of the present invention may be used in admixture with a suitable carrier, excipient or diluent conventionally used in the manufacture of a pharmaceutical composition, diluted, or enclosed in a carrier in the form of a container. In particular, when the carrier is used as a diluent, it may be a solid, semisolid, or liquid substance which acts as a vehicle, excipient or medium for the active ingredient extract.

Such carriers, excipients and diluents that may be included in the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol erythritol, maltitol, starch, acacia rubber. , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like, but the present invention It is by no means limited to this.

On the other hand, the pharmaceutical composition comprising an extract having antiviral activity derived from deciduous fungi ( Phellinus pini ) according to the present invention, can be administered orally or parenterally in accordance with conventional pharmaceutical administration methods. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or topical injection.

It can be used in various pharmaceutically acceptable formulations. For example, powders, powders, granules, tablets, soft or hard gelatin capsules, elixirs, suspensions, emulsions (emulsions), syrups, solutions, aerosols ( oral formulations such as aerosol), ointments, suppositories or sterile injectable solutions. When formulated in this way it can be prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations include at least one excipient such as starch, calcium carbonate, extracts of Phellinus pini extract of the present invention. It may be prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, Preservatives and the like.

Formulations for parenteral administration include sterile aqueous solutions, infusion solutions such as non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. During parenteral administration, pharmaceutical compositions containing an antiviral extract derived from the deciduous fungus of the present invention for topical administration are, for example, in the form of ointments, creams, emulsions, powders, pads, solutions, gels, sols, lotions, suspensions. It may be provided in the form of microspheres or nanospheres or lipid or polymeric vesicles, or hydrogels, which may be supplied or have controlled release.

As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. When used as a suppository, the pharmaceutical composition containing the antiviral extract derived from the deciduous fungus of the present invention may be, for example, a tablet, a capsule in gelatin form, a syrup, a suspension, a liquid, a granule, an emulsion, a microsphere with controlled release. Or in nanosphere form.

Hereinafter, although the preferable Example of this invention is described, this invention is not limited to these Examples at all.

Example 1 Separation of Hot Water Extract of Mushroom and Active Material

3L distilled water was added to 200 g of the coarsely ground crushed by a grinder after cutting the fruiting body of Phellinus pini (Brot. Ex Fr.) Ames from Alaska. The filtrate filtered with filter paper (Whatman # 2) was lyophilized at -80 ° C to obtain a dry powdered hot water extract (41 g, yield 20.5%).

Then, macromolecules and micromolecules were separated by precipitating by adding ethanol as a polar solvent to the hot water extract of the mushroom to separate the active ingredient. First, 600 mg of the dried hot water extract of the mushroom obtained above was dissolved in 50 ml of distilled water, and then 3 times (150 ml) of 75% ethanol was added thereto, and the mixture was left at 4 ° C for 24 hours, followed by centrifugation (8,000 rpm, 30 min, 4 ° C.). The precipitate was dissolved in distilled water, dialyzed (MWCO 14,000), lyophilized and stored in dry powder form (EP) for use in experiments (0.26g, yield 43.3%). The supernatant was concentrated under reduced pressure (Rotary evaporator, Eyela, USA) After removing it, it was dissolved in distilled water and lyophilized and used in experiments in dry powder form (ES) (0.17 g, yield 14.2%).

Example 2 Cytotoxicity Test

A cytotoxicity test was performed on HeLa cells, which are host cells of coxsackievirus B3 (CVB3), using the precipitated dry powder (EP) obtained in Example 1. RPMI 1640 (Roswell Park Memorial Institute medium) with 10% (v / v) heat inactivated (56 ° C, 30 min) fetal bovine serum (FBS), penicillin and streptomycin (Gaithersburg, MD, USA) 1640) (Gibco, USA) medium was incubated in 37 ℃, 5% CO ₂ incubator. To determine the cytotoxicity of the extract, HeLa cells were dispensed in a 96-well plate at a concentration of 1.0 × 10 ⁵ cells / well, and cultured at 5% CO ₂ , 37 ° C. for 24 hours to obtain a cell monolayer. Removed and prepared a sample solution of various concentrations using a maintenance medium (maintenance medium, MM) containing 2% FBS, 100 μl per well and incubated at 5% CO ₂ , 37 ℃ 24 hours. After the incubation, the culture medium was removed and 20 μl of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] (Sigma) (Scubiero et al., 1988) was added to each well. Added and incubated for 4 hours. After incubation, excess MTT was removed and dissolved fromazan crystal formed intracellularly with DMSO. After measuring the absorbance at 570 nm with an ELISA palte reader, the cell viability was measured by converting the percentage of cell proliferation into percentage compared to the absorbance of the control group.

In order to measure cytotoxicity, the concentration of the sample required to inhibit the growth of HeLa cells by 50% is shown as 50% cytotoxic concentration (CC ₅₀ ). As a result of the MTT assay, the cytotoxic CC ₅₀ of HeLa cells of EP, the ethanol precipitate of Example 1, was 2.25 mg / ml (FIG. 2).

Example 3 Antiviral Activity of Coxsackievirus of Hot Water Extract

In this example, the antiviral activity of coxsackie virus known to be involved in heart disease such as myocarditis and insulin-dependent diabetes mellitus was measured using the hot water extract of the deciduous mud mushroom.

Coxsackievirus B3 (CVB3) H3 strain (AAB02228) was supplied from the Catholic University Molecular Virus Laboratory and stored stock at -70 ° C. Antiviral activity against CVB3 was measured by plaque reduction assay (Shigeta et al., 1992). HeLa cell monolayers (1.1 × 10 ³ cells / well) were formed on 6-well culture plates, and then samples (EP, ES) obtained from hot water extraction at various concentrations (0, 0.1, 0.5, 1.0 mg / ml) were obtained. Added and inoculated with 150 pfu / well of CVB3. The virus was adsorbed to the cells in a 5% CO ₂ , 37 ° C incubator for 90 minutes, and then agar overlay medium (RPI 1640 containing 2% FBS, 0.5% agarose) was added to the cell monolayer at 3 ml / well, followed by 37 ° C, 5% Incubated in CO ₂ . When viral plaque was formed (formed after 48 hours of incubation), the agar overlay medium was removed, the cells were fixed with 5% formalin-PBS, and stained with crystal violet to count the number of plaques. The antiviral activity indicated that the sample concentration required to reduce the number of viral plaques by 50% was 50% effective concentration (EC ₅₀ ).

Antiviral activity against CVB3 in HeLa cells of supernatant (ES) and sediment (EP) samples obtained from ethanol precipitation from hydrothermal extracts was investigated by plaque reduction assay, and in case of ES, inhibition of plaque formation by CVB3 in HeLa cells Ability was found to be inadequate (FIG. 3A). However, EP, an ethanol precipitate, showed a significant inhibitory effect in concentration dependent on plaque formation by CVB3 (FIG. 3B). In particular, when the ethanol precipitates (EP) obtained from the hot water extract derived from the deciduous mudslide fruiting body of the present invention at concentrations of 0.1, 0.5, and 1.0 mg / ml, up to 7%, 56%, and 66.4% of the control group, respectively, were not treated. inhibited plaque formation. On the other hand, hot water extract, as can be seen in the plaque formation results (ethanol precipitate (Fig. 3c; control (a), 0.1 mg / ml (b), 0.5 mg / ml (c), 1.0 mg / ml (d))) by the ethanol precipitate As the concentration of was increased, it was confirmed that the formation of plaque was reduced. The EC ₅₀ (50% effective concentration), the concentration of EP that inhibits the growth of the virus by 50% compared to the Plaque number, was 0.45 mg / ml.

Example 4 Antiviral Activity of Herpes Virus from Hot Water Extract

In this example, the antiviral activity against herpes simplex virus 1 (HSV-1) was measured using the ethanol precipitate (EP) of the hot water extract prepared in Example 1.

(1) degree of plaque formation according to the degree of dilution of HSV

The cell monolayer of the host cell Vero cell was cultured in a 6-well culture plate to be 1 × 10 ⁶ cells / well. Subsequently, HSV-1 stock (5/13/08, vial No. 4/23) was taken out of the deep freezer, melted rapidly, and serially dilution from 10 ^-1 to 10 ^-6 and prepared on ice. 0.4 ml of serum free DMEM and 0.1 ml of HSV-1 diluent (10 ^-4 , 10 ^-5 , 10 ^-6 ) were inoculated into each well (6 well plates) of the formed Vero cell monolayer and 5% CO at 37 ° C. _The virus was adsorbed while incubated for 1 hour in a ₂ incubator. Virus dilutions were removed and agarose overlay medium (DMEM medium containing 2% FBS and 0.25% agarose) was provided 2.5 ml per well and incubated in a 37% 5% CO ₂ incubator. When the viral plaque was formed (incubated for about 50 hours), the cells were fixed with 10% formalin, stained with 0.02% crystal violet, the agarose overlay medium was removed, and the formed plaque was counted. The measurement results are shown in FIG. 4A and Table 1 below.

Table 1. Number of plaques according to the dilution concentration of HSV-1

HSV-1 dilution factor 10 ^-4 10 ^-5 10 ^-6 Mock Counted Plaque Number About 280, about 250 32, 40 3 0 Average plaques
(PFU / well) 265 36 3 0

From this result, it was confirmed that the concentration of HSV-1 stock was about 3.6 × 10 ⁷ PFU / mL.

(2) antiviral activity (HSV activity) against HSV-1 of ethanol precipitates

In this example, in order to measure the virucidal activity of the deciduous mud mushroom-derived hot water extract, ethanol precipitate (EP) obtained from the hot water extract was mixed with the virus, and then the degree of plaque reduction was measured. Of Vero cell monolayer ^{(1 × 10 6 cells / well} ) to each EP 0.05, 0.01, and 0.005 ㎎ / ㎖ was added to a concentration of and, 265 PFU / well (10 ^-4 dilution of 0.1 ㎖ HSV-1) the HSV -1 was inoculated at the same time and infected for 1 hour in a 5% CO ₂ incubator at 37 ℃, the degree of plaque reduction was measured. The measurement results are shown in FIG. 4B and Table 2 below.

Table 2. Plaque Reduction for Hot Water Extract HSV-1

Concentration of EP (µg / mL) 50 10 5 0 Counted Plaque Number 0, 0 7, 6 11, 16 About 280, about 250 Average plaques (PFU / well) 0 6.5 13.5 265 Plaque Formation Inhibitory Activity (%) 100 97.5 94.9 0

From this result, the ethanol precipitate EP obtained by 75% ethanol precipitation from the deciduous mud mushroom hot water extract began to inhibit the growth of the HSV-1 virus by more than 95% at a concentration of 5 µg / ml, and completely prevented the virus growth when the concentration was 50 µg / ml. Inhibition confirmed that the virucidal effect was excellent.

(3) Antiviral Activity (Pre-culture) of HSV-1 of Ethanol Precipitate (EP)

In this example, in order to determine the components showing antiviral activity in the deciduous mud mushroom-derived hot water extracts, ethanol precipitates and host cells were first cultured, and then the degree of plaque reduction of HSV-1 was measured. EP was added to the Vero cell monolayers (1 × 10 ⁶ cells / well) at concentrations of 50, 10, and 5 ㎍ / ml, respectively, and incubated in a 5% CO ₂ incubator at 37 ° C for 1 hour. gently washed and infected for one hour at _{265 PFU / well 5% CO 2} incubator at 37 ℃ inoculated with the HSV-1 (HSV-1 of the ^10-4 dilution 0.1 ㎖). The measurement results are shown in FIG. 4C and Table 3 below.

Table 3. HSV-1 plaque reduction after pre-culture treatment

Concentration of EP (µg / mL) 50 10 5 Counted Plaque Number 9, 15 240, 228 280, 248 Average plaque number (PFU / well) 13 234 269 Plaque Formation Inhibitory Activity (%) 95.2 13.0 0

Plaque reduction was not good in the case of virus infection after EP reaction to host cells compared to the case of simultaneous EP and virus infection, but when the concentration of EP was incubated at a concentration of 50 ㎍ / ml It was confirmed that the degree of plaque formation was reduced by more than 95%.

(4) Antiviral Activity (Pre-Reaction) of HSV-1 of Ethanol Precipitate

Antiviral activity was measured by reacting ethanol precipitate (EP) with the virus and then infecting it. 50 and 10 μg / ml of EP were added to HSV-1 dilutions (2 × 10 ^-3 and 2 × 10 ^-4 ), respectively, and reacted in the tube for 1 hour at room temperature, followed by Vero cell monolayer (1 ×). 10 ⁶ cells / well) and infected for 1 hour in a 5% CO ₂ incubator at 37 ℃. The measurement results are shown in FIG. 4D (concentration 2 × 10 ⁻³ of HSV-1), FIG. 4E (concentration 2 × 10 ⁻⁴ of HSV-1), and Table 4 below. When the virus was reacted with the EP, the virus proliferation was completely inhibited at the concentration of 50 µg / ml.

Table 4. HSV-1 Plaque Reduction Following EP and Virus Pre-Reaction

HSV-1 dilution factor 2 × 10 ^-3 2 × 10 ^-4 Concentration of EP (µg / mL) 50 10 - 50 10 - Counted Plaque Number 0, 0 10, 10 uncountable 0, 0 0, 1 uncountable Average plaques
(PFU / well) 0 10 uncountable 0 0.5 uncountable

(5) Antiviral Activity of Ethanol Precipitate (EP) Against HSV-1 Virus

In conclusion, when EP was treated with HSV-1 cells simultaneously, EP and cell pre-culture, and EP and HSV-1 after HSV-1 infection, EP was induced by HSV-1 in Vero cells. plaque formation showed inhibitory activity. In particular, when the cells were treated with the cells simultaneously with HSV-1 at the concentrations of 50, 10, and 5 ㎍ / ml, EP, 100, 97.5 and 94.9%, respectively, exhibited plaque formation inhibitory activity. Inoculation of HSV-1 after pretreatment showed 95.2 and 13.0% plaque formation inhibitory activity, respectively. In addition, when 50 μg / ml EP and HSV-1 were infected with the cells after preculture, the plaque formation inhibitory activity was 100%, indicating that the growth of HSV-1 was completely inhibited.

As described above, the number of plaques of viruses decreased with the concentration of hot water extract derived from deciduous mud mushrooms.The antiviral active material of the hot water extract is a polymer material, which prevents the virus from penetrating into the host cell from outside the host cell. Inferred to exhibit antiviral activity.

Example 5 Inhibition of Neuraminidase Activity of Hot Water Extract

In this example, influenza virus (Flu), neuraminidase whether the antiviral activity against viruses using the hemagglutinin (HA) and neuraminidase (NA) protein system on the surface of the virus for invasion and proliferation of the virus neuraminidase (NA) inhibition assay (Myers et al ., 1990).

Neuraminidase (from Clostridium perfringens ) and the standard substrate 2- (4-methylumbelliferyl) -α-DN-acetylneuraminic acid (MUNANA) (Potier et al ., 1979) were purchased from Sigma (USA). 10 μl of NA enzyme solution (2.5 × 10 ⁻³ U), 40 μl of 0.2 mM substrate (MUNANA), and 10 μl of sample solution (mushroom extract, purified) by concentration to 40 mM sodium acetate buffer (pH 5.0) After reaction at 15 ° C., 2.94 ml of 0.1 M glycine-NaOH buffer (pH 10.4) was added to terminate the reaction, followed by spectrofluorophotometer (Bio-Rad, USA). 360 nm / Em. Inhibition of enzyme activity was investigated by measuring fluorescence at 440 nm. The fluorescence value measured by the control without the sample was 100%, and sialyllactose (Sigma) was used as a standard inhibitor for NA.

(1) Neuraminidase Inhibitory Activity against Various Mushroom Hot Water Extracts

Initial experiments to compare neuraminidase inhibitory activity from various mushrooms were carried out including H. erinacium , C. vesicolor , P. linteus , G. applanatum , and Chaga ( I. obliquus ) and Alaska deciduous fungi ( P. pini ) fruiting body lysate were extracted from 100 L each of 1.5 L of distilled water, 100 ° C. for 4 hours, followed by hot water extraction for each hot water extract obtained by neuraminidase inhibitory activity. Investigate. Treatment with 1.7 mg / ml of hot water extract obtained from each mushroom resulted in inhibition of deer worm mushroom 0%, fingerling mushroom 0%, situation mushroom 0%, Jannabibulocho 12.0%, chaga mushroom 18.2%, deciduous mud mushroom 58.8% It was shown that the activity of inhibiting the enzyme activity of the deciduous mud mushroom hot water extract showed the best (Fig. 5)

(2) Neuraminidase Inhibitory Activity of Ethanol Precipitate (EP)

The neuraminidase inhibitory activity was measured using a simple hot water extract obtained in Example 1, a precipitate obtained after ethanol precipitation from the hot water extract, and a supernatant sample (ES) at a concentration of 1.7 mg / ml, respectively. ES showed no inhibitory activity even at 1.7 mg / ml, but EP showed an inhibitory effect of about 80% (FIG. 6).

On the other hand, the enzyme activity inhibition was measured by treating the concentrations of hot water extract and EP at 0.4, 0.8, 1.7, 3.4 mg / ml concentrations, respectively. As a result, the hot water extract showed inhibitory activity up to 33.3, 42.1, 56.4, and 68.8%. In the case of EP, up to 48.3, 60.2, 74.9, and 86.7%, respectively, both hot water extract and EP significantly inhibited NA enzyme activity in a concentration-dependent manner (FIG. 7). Therefore, 75% ethanol precipitation after hydrothermal extraction was shown to be effective for the purification of antiviral actives from P. pini fruiting body, and P, pini fruiting body components showed widespread antiviral activity not only in CVB3 but also influenza virus. It was confirmed that it can have.

Example 6: Isolation and Purification of the Active Ingredient

The active substance was purified by gel permeation chromatography from ethanol precipitates (EP) having antiviral activity. Sepharose CL-4B column to (3 × 120 ㎝) was equilibrated with _{0.05% NaN 3/50 mM NaCl} , 1% (w / v) injecting the sample solution 5 ㎖ the column and eluted with 1.43 ㎖ / min speed in the same solution 7 mL each. For each fraction, carbohydrates were analyzed by the phenol-sulfuric acid method (Dubois et al., 1956) and proteins were analyzed by Bradford assay (Bradford, 1976). Fractions of the major carbohydrate peaks were collected together, dialyzed in distilled water (MWCO 3,500), lyophilized, and stored at -20 ° C. 50 mg of ethanol precipitates (EP) having antiviral activity were fractionated by Sepharose CL-4B column, and each fraction was detected for carbohydrates by phenol-sulfuric acid method. As a result, two major carbohydrate fractions were eluted (FIG. 8). Each fraction was combined, dialyzed in distilled water (MWCO 3,000), lyophilized and purified to obtain 20 mg of Peak 1 and 10 mg of Peak 2. The first polymer, which is relatively polymer, is named (peak 1) as EP-AV1 and the second, relatively small molecule (peak 2) is named as EP-AV2. On the other hand, as the amount of elution of these two carbohydrate fractions increased, the amount of protein eluted also increased. In addition, as a result of examining the amount of protein in the lyophilized purified product, it was found that about 3.4% of EP-AV1 and about 2.5% of protein were contained in EP-AV2 (Table 5). It is believed that these two fractions (EP-AV1, EP-AV2) may be a kind of glycoprotein containing very small amount of protein or small peptide.

As expected, the purified EP-AV1 and EP-AV2 are polysaccharides purified from extracts of hot water extracted from mushroom fruiting bodies, and precipitates are formed even when the purified dry matter is dissolved in distilled water or HeLa cell culture medium for antiviral experiments at room temperature. It melted well without being able to see that these two polysaccharides were water soluble.

In order to confirm the antiviral activity of purified EP-AV1 and EP-AV2, the inhibition of plaque formation on CVB3 was investigated in HeLa. When treated with 0.5 and 1.0 mg / ml, EP-AV1 was 1.8% and 31.6, respectively. %, EP-AV2 showed 43.9%, 84.2% of inhibitory activity (Table 5), and both polysaccharides showed antiviral activity, but EP- was present in a relatively small amount in mushroom fruiting bodies compared to EP-AV1. AV2 has been shown to have significantly higher antiviral activity.

In addition, as a result of investigating NA enzyme inhibitory activity on these purified polysaccharides, both polysaccharides showed a concentration-dependent inhibitory activity. Showed 54.1% enzyme inhibition activity of 44.1% of EP-AV1 and 73% of EP-AV2.

Example 7 Composition Analysis of Components of Purified Polysaccharides

1% (w / v) sample solution was mixed 1: 1 with 4 M trifluoroacetic acid TFA) for analysis per component of antiviral active substance, and hydrolyzed at 100 ° C for 4 hours and then After drying in Vac, it was dissolved in 200 μl of distilled water to make acid hydrolysate, labeled with PMP (1-phenyl-3-methyl-5-pyrazolone) according to the method of Honda et al. (1989). HPLC analysis on a pack CLC-ODS column (6.0 mm × 15 cm, Shimadzu, Japan). Elution with 0.1 M sodium phosphate buffer / 10% acetonitrile (pH 7.0) was detected at 245 nm with a UV detector (Dionex, USA). Standard samples were labeled with PMP using fucose, mannose, glucose, galactose, and xylose at 20 mM. Standard monosaccharides (fucose, mannose, glucose, galactose, xylose) and gentiobiose (Glcβ1-6Glc) were all purchased from Sigma (St. Louis, MO, USA).

In the case of EP-AV1, the highest molar percentage of glucose was 53.4%, and galactose (19.2%), xylose (17%), mannose (5.8%), and fucose (4.6%). Heteropolysaccharide consisting of a) in order, EP-AV2 also has the highest glucose content of 56.1%, the main component sugar is the same heteropolysaccharide as EP-AV1, but the content of each component is somewhat different, in particular, It was shown that the content ratio of galactose was significantly lower than that of EP-AV1 (FIG. 9, Table 5; in Table 5, the molar ratio of monosaccharides was calculated from each peak region on the HPLC chromatogram of acid hydrolyzate of purified polysaccharide, and the total main peak region was calculated. Antiviral activity against different concentrations of EP-AV1 and EP-AV2 was determined by plaque reduction assay in HeLa cells for CVB3, Fuc was fructose, Gal was galactose, Xyl Silver xiloo Glc stands for glucose and Man stands for mannose.)

Table 5. Chemical Composition Analysis and Antiviral Activity of Purified Polysaccharides

Polysaccharides protein
(mass%) Monosaccharide ratio ((mole%) Antiviral activity (%) Fuc Gal Xyl Glc Man 0.5 (mg / ml) 1.0 (mg / ml) EP-AV1 3.4 4.6 19.2 17.0 53.4 5.8 2 32 EP-AV2 2.5 5.1 10.3 20.1 56.1 8.4 44 84

Example 8 Determination of Molecular Weight of Purified Polysaccharide

The molecular weights of the purified polysaccharides EP-AV1 and EP-AV2 were determined using HPLC (Dionex, USA) on a PolySep-GFC-P3000 size-exclusion column (300 × 7.8 mm, Phenomenex, USA). 10 μl of 1% solution of each sample (H ₂ O) was injected, eluted with 0.8 mL / min of water, and detected by ELSD (Evaporative light scattering detector, Altech) .The standard molecular weight markers dextran were 464,336, 188,000, 71,327, 43,000 Da. (Sigma, USA) was used. The analysis showed that EP-AV1 was about 1,006 kDa and EP-AV2, which showed better antiviral activity against CVB3, was about 100 kDa (FIG. 10).

Example 9 Partial Binding Structure Analysis of Purified Polysaccharides

Most known anti-cancer polysaccharides derived from fruiting bodies of mushrooms are known to be β-glucans having a β- (1,3) (1,6) -glucan structure (Bohn and BeMiller, 1995), and in this example, Alaska Antivirally active EP-AV1 and EP-AV2 polysaccharides isolated from acid P. pini fruiting bodies also contain the most glucose among their constituent sugars (Fig. 9, Table 1). As a result, after digestion with laminarinase (Sigma), an enzyme that specifically cleaves β-1,3-binding, the presence of β-1,3-binding structure was examined by TLC analysis (FIG. 11). Laminarinase and laminarin, a standard substrate, were used by Sigma (St. Louis, Mo., USA).

After dissolving 10 mg of sample in 1 ml of 50 mM Sodium acetate buffer (pH 5.0) and adding 2.5 units of laminarinase (Sigma, USA), an enzyme that specifically degrades β-1,3-binding, 48 hours at 37 ℃ During hydrolysis. The degradation product was developed on a Kiesel Gel 60 (Merk, Germany) TLC plate with a mixed solution of 1-butanol: 1-propanol: acetic acid: H ₂ O (3: 1: 1: 1, v / v), followed by 100 Dried for 10 minutes at ℃ and sprayed with diphenylamine / aniline / phosphoric acid mixed solution was developed for 10 minutes at 100 ℃. Glucose, gentiobiose, and laminarin were used as standards, and the control group was hydrolyzed with the same enzyme, laminarin (Sigma, USA), a representative β-1,3-glucan. The TLC analysis of the product of two purified polysaccharides, EP-AV1 and EP-AV2, as a control, laminarin with the same enzyme is shown in Figure 11 (Lane 1: glucose; Lane 2: gentiobiose; Lane 3). : laminarin; Lane 4: laminarin treated with laminarinase; Lane 5: EP-AV1 treated with laminarinase; Lane 6: EP-AV2 treated with laminarinase.G1 is glucose; G2 is gentiobiose)

As in laminarin (Lane 4), EP-AV1 (Lane 5) and EP-AV2 (Lane 6) both produced glucose as a major degradation product. This shows that both EP-AV1 and EP-AV2 are β-glucans with at least partially β-1,3-bond structure. In addition, in view of the presence of β-1,6-linked disaccharide gentiobiose as an enzyme decomposition product, these polysaccharides are based on glucoseβ-1,3-glucose binding structure and glucose is β-1, It was shown to be a kind of β- (1,3) (1,6) -glucan with a 6-bond branched structure.

However, as shown in the constituent sugar composition of these polysaccharides (Fig. 9, Table 1), these polysaccharides are present in a significant proportion of sugars such as galactose, xylose, mannose, fucose in addition to glucose, Lentinus edodes , Scleorotium glucanicum , Heterotype β- (with a slightly different structure than the typical β- (1,3) (1,6) -glucan (Bohn & BeMiller, 1995) reported in many conventional mushrooms, such as Schizophyllum commune . 1,3) (1,6) -glucan

In the above, the present invention has been described in detail based on the preferred embodiments of the present invention, but the present invention is not limited thereto. Rather, those skilled in the art will be able to easily make various modifications and changes based on the embodiments described above. However, it will be more apparent through the appended claims that such variations and modifications fall within the scope of the present invention.

1 is a diagram schematically showing the process up to the preparation of the extract with antiviral activity from the fruiting body of Alaska deciduous mud mushrooms according to the present invention.

Figure 2 is a graph measuring the cytotoxicity test for HeLa cells of the ethanol precipitate obtained from the deciduous mud mushroom hot water extract obtained according to an embodiment of the present invention.

Figure 3a to 3c is a measure of the antiviral activity of the coxsackie virus of the hot water extract of the deciduous mud mushroom of the present invention, Figure 3a shows the results of PFU measurement using the ethanol precipitate (EP) obtained from the hot water extract One graph, Figure 3b is a graph showing the results of PFU measurement using the ethanol supernatant (ES) obtained from the hot water extract, Figure 3c is a photograph of the degree of plaque formation in a state of varying the concentration of ethanol precipitates.

Figures 4a to 4e are photographed the degree of plaque generation under predetermined conditions to measure the degree of antiviral activity against herpes simplex virus (HSV-1) of ethanol sediment obtained from the hot water extract of the deciduous mud mushroom of the present invention, respectively. One picture.

5 is a graph measuring the degree of activity inhibition against neuraminidase of hot water extracts obtained from various mushrooms.

Figure 6 is a graph measuring the activity inhibitory effect on the neuraminidase of the supernatant and sediment fraction obtained by ethanol precipitation from the hot water extract of the deciduous mud mushrooms according to the present invention.

7 is a graph measuring the degree of inhibition of neuraminidase activity according to the concentration of ethanol precipitates obtained from the hot water extract and hot water extract of the deciduous mud mushroom.

8 is a graph showing the analysis results of the fraction component of the antiviral active polysaccharides fractionated by column chromatography of the ethanol precipitate obtained from the hot water extraction of deciduous mud mushrooms.

9 is a graph showing the results of HPLC analysis of the composition of purified polysaccharides (EP-AV1, EP-AV2) in the ethanol precipitate obtained from the hot water extraction of deciduous mud mushrooms.

10 is a graph showing the molecular weight measurement results using HPLC of polysaccharides (EP-AV1, EP-AV2) purified from ethanol precipitates obtained from hot water extraction of deciduous mud mushrooms.

Figure 11 is a photograph showing the results of TLC analysis for the polysaccharides (EP-AV1, EP-AV2) purified from the ethanol precipitate obtained from the hot water extraction of deciduous mud mushrooms.

Claims (10)

A pharmaceutical composition for treating or preventing diseases caused by infection with influenza virus, herpes simplex virus, or coxsackie virus, which contains as an active ingredient an extract having antiviral activity from Phellinus pini . The method of claim 1, Extract having the antiviral activity is a pharmaceutical composition obtained from the fruiting body of the deciduous fungi ( Phellinus pini ). The method of claim 1, The extract having the antiviral activity is obtained by centrifugation after adding a polar solvent to the hydrothermal extract of the deciduous mushroom fruiting body powder. The method of claim 3, wherein The extract having the antiviral activity is obtained from the precipitate component among the components separated by the centrifugation. The method of claim 3, wherein The polar solvent is selected from alcohols having 1 to 4 carbon atoms. The method of claim 1, The extract derived from the deciduous mud mushrooms inhibits the activity of neuraminidase and prevents the infection activity of influenza virus. The method of claim 1, The extract from the deciduous mud mushrooms show antiviral activity by killing the virus. The pharmaceutical composition according to claim 1, wherein the extract from the deciduous mud mushroom is contained at a concentration of 0.001 to 5.0 mg / ml. The method of claim 1, The extract having the antiviral activity comprises a water-soluble polysaccharide pharmaceutical composition. The method of claim 9, The water-soluble polysaccharide is a pharmaceutical composition, characterized in that the hetero type β- (1,3) (1,6) -glucan.

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