KR100512315B1 - Composition comprising the extract of African phellinus mushroom for protecting and treating viral diseases - Google Patents

Abstract

The present invention relates to a composition containing an extract of African Phellinus baumi. Pilat , which exhibits antiviral activity.

African situation mushroom extract according to the present invention exhibits excellent antiviral activity, it can be used as an effective and safe medicine for the prevention and treatment of viral diseases.

Description

Composition comprising the extract of African phellinus mushroom for protecting and treating viral diseases}

The present invention relates to a composition containing an extract of Phellinus baumi. Pilat , which exhibits antiviral activity, and more particularly, to an African situation mushroom that exhibits excellent antiviral activity against enterovirus and bullous stomatitis virus. It relates to a pharmaceutical composition containing an extract.

As modern medicine advances, bacterial diseases are markedly suppressed by the development of antibiotics and preventive vaccines, but viral diseases are not yet conquered, but rather are reported to increase through air pollution and environmental pollution. However, only a few viral diseases in which prophylactic vaccines are developed, and fundamental treatments for many diseases, including viral flu, hepatitis, aseptic meningitis, and the like, have not yet been developed.

Enteroviruses are single-stranded (+) RNA viruses belonging to the family Picornaviridae . It is a representative human pathogen that causes aseptic meningitis, epidemic keratoconjunctivitis (Apollo's eye disease), and hand, foot and mouth disease in children and infants. In Japan, 5000-6000 cases (5804 cases in 1998) are isolated from enteroviral diseases every year, and sporadic cases of enteroviral cases have been reported in Korea since the early 1990s.

However, isolation and identification of viruses through cell culture for diagnosis in the event of disease (Kim et al. 1994. Journal of Korea Society of Virology 24: 77-86), or genetic diagnosis using RT-PCR methods are only used. Zoll et al. 1992. Journal of Clinical Microbiology 30: 160-165. Vaccine preparations aimed at defense against enteric viral diseases have not yet been developed.

Vesicular Stomatitis Virus (hereinafter referred to as VSV virus) belongs to Rhabdoviridae , Vesiculovirus , and the virus particles exhibit a shell type of 170 × 70 nm. Single-stranded RNA virus with encapsulation and high sensitivity to lipid solvents and fungicides.

In particular, bullous disease caused by bullous stomatitis virus in cows and pigs has been of constant concern due to the difficulty in differential diagnosis between foot-and-mouth disease and clinical trials. In addition, it is a native to the heart of hardwoods such as mulberry, elm, aspen and alder, and is a white species belonging to the genus Phellinus Ouel.Em.lmaz It is a fungus. In Korea, the situation mushroom refers to the woody mud mushroom ( Phellinus Linteus ). Situation mushrooms have been used for the treatment of gynecological diseases such as uterine bleeding and menstrual irregularities. Pharmacological and medical researches on the pharmacological action of situational mushrooms have been known to activate immune function following chemotherapy following liver cancer surgery, including gastric cancer, esophageal cancer, duodenal cancer, colon cancer and rectal cancer, which are cancers of the digestive system . Since the efficacy of the situation mushroom was found to be caused by polysaccharide beta glucan, research on polysaccharides has been actively conducted. In general, mushrooms contain potassium, calcium, magnesium, vitamins B2, B3 C, and fibrous amino acids. In addition to these ingredients, mushrooms contain polysaccharide β-glucan. Beta-glucan reinforces the body's immunity, repels bacteria and foreign substances that have invaded the body, and acts as a suppressor of infection. As a conventional invention related to a situation mushroom, Korean Patent Application No. 2001-0003264 discloses a Pelinus linteus strain and an anticancer immunoactive polysaccharide isolated and purified therefrom, and Korean Patent Application No. 1998-0015617 discloses a genus of Pelinus. Disclosed is a novel separation and purification of a novel polysaccharide that exhibits an immunostimulating activity by mass culturing mycelium or fruiting body from a strain. Korean Patent Registration No. 10-0174433 discloses an anticancer active polysaccharide isolated from Mycelium of Felinus linteus strain, a preparation method thereof, and a pharmaceutical composition comprising the same, and Korean Patent Application No. 2000-0054319 describes The use of the mushroom extract as an anticancer agent is disclosed, and Korean Patent Registration No. 10-0348115 discloses a seedling composition containing a natural situation mushroom extract and a method of preparing the same. In addition, a lot of research on domestic and international medicines and health foods using the situation mushroom and its extracts are being made.

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However, not all mushrooms belonging to mud mushrooms have this effect, and to date, it has been found to be wood mud mushrooms, horse mud mushrooms and fir mud mushrooms.

African Phellinus of the present invention (Phellinus baumi. Pilat) is pine scales belonging to mushrooms and (Hymenochaetaceae) mud mushrooms in (Phellinus), growing the parasitic like hardwood belonging to Moraceae, which grows in the wild in alpine give way zone of Kenya It is a mushroom. The ecological feature of this area is the altitude of 5000m above sea level, although it is a tropical region near the equator, it has the optimal environment for mushroom growth. Perennial, the surface is grayish brown or dark gray, and has a hard shell and smooth. The underside is hollow, light brown, and porous. Spores are small and globose. Each year there is an increase in layers, and the increased layers are clearly distinguished and can be easily counted so that age can be accurately measured. Fruiting bodies are perennial hardwoods. The lampshade is generally horseshoe-shaped, 11cm high, 20cm wide and 8cm thick. When grown, the upper part is blackened, dark gray, and the surface is rough. When fully grown, the lampshade is tanned and flattened. African situation mushrooms decay live or dead wood, producing white decay in white matter and heartwood.

The medical efficacy of treating diseases has been well known in oriental medicine for a long time. It has anti-cancer effects, and the extract has also been proved by clinical trials as having an excellent therapeutic and preventive effect against liver cancer, lung cancer and many other cancers. However, the antiviral activity of the African situation mushroom has not been studied.

Kenya is known to have more than 50% of residents living with viral diseases. The inventor of the present invention focuses on the fact that the mortality rate of the inhabited area of African situation mushrooms is significantly lower than that of other regions, resulting in viral mortality, and thus, the antiviral activity of the mushrooms used as a traditional treatment of the inhabitants. As a result of the study, it was confirmed that the African situation mushroom extract exhibited excellent antiviral activity against antiviral activity, in particular, enteric virus and VSV virus, and completed the present invention.

The present invention is to provide a pharmaceutical composition for the prevention and treatment of viral diseases containing African situation mushroom extract showing excellent antiviral activity.

In order to achieve the above object, the present invention contains an African situation mushroom extract as an active ingredient, and provides a pharmaceutical composition effective for the prevention and treatment of viral diseases.

Such viruses include enterovirus or bullous stomatitis virus (VSV).

The extract refers to an extract in which African situation mushroom is soluble in water, a lower alcohol having 1 to 4 carbon atoms, or a polar solvent selected from a mixed solvent thereof.

Hereinafter, the present invention will be described in detail. The African mushroom mushroom extract of the present invention is pulverized and crushed after drying the dried African mushroom mushroom, about 5 to 25 times the weight (kg), preferably about 10 to 15 times water, lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof having a mixing ratio (kg / l) of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5 50 Water, lower alcohols or mixed solvents thereof by extraction methods such as solvent extraction and hot water extraction for about 1 hour to 1 day, preferably 2 hours to 4 hours at an extraction temperature of 100 to 100 ° C, preferably 70 to 80 ° C. Soluble extract according to the present invention can be obtained by extraction, concentrated under reduced pressure and lyophilized.

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The present invention provides an African situation mushroom extract obtained by the above method and effective for the prevention and treatment of viral diseases.

In addition, the present invention provides a pharmaceutical composition containing the mushroom extract of the present invention as an active ingredient and effective for the prevention and treatment of viral diseases.

The composition for preventing and treating a viral disease of the present invention comprises 0.1 to 80% by weight of the extract, preferably 1.0 to 50% by weight, based on the total weight of the composition.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active fractions, as well as in any suitable collection.

The composition comprising the African situation mushroom extract of the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the manufacture of a pharmaceutical composition. Carriers, excipients and diluents that may be included in the composition comprising 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, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Compositions comprising extracts according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories or sterile injectable solutions according to conventional methods. Can be used. Specifically, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used when formulated. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of African situation mushroom extract may vary depending on the age, sex and weight of the patient, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, divided once or several times daily. can do. In addition, the dosage of the extract may be increased or decreased depending on the route of administration, the type and severity of the disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

African situation mushroom extract itself of the present invention has almost no toxicity and side effects, so can be used with confidence even for prolonged administration.

The present invention is described in more detail based on the following examples and examples, but the present invention is not limited to the examples or experimental examples.

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Example 1. Preparation of African Mushroom Extract

2.1 kg of dried African mushrooms (collected from Phellinus baumi. Pilat, Kenya, Africa) were chopped and powdered, and 5 L of methanol was added thereto and then deposited at 70 ° C. for 2 hours. The methanol solution extracted from the sample was filtered through a filter paper (filter No. 1, Whatama, USA), concentrated under reduced pressure using a vacuum condenser (EYELA, Japan), and lyophilized to extract 85 g of African situation mushrooms. Got.

Example 2. Preparation of African Mushroom Extract

50 g of dried African mushrooms were hydrothermally extracted from a 1000 ml of distilled water using an electronic shaker (Heating mentle, manufactured by M-tops) for at least 3 hours. The extract was filtered, concentrated under reduced pressure using a vacuum condenser, and lyophilized to obtain an African mushroom extract having a dry weight of 20.8 g.

Experimental Example 1. Intestinal virus inhibitory activity of the situation mushroom extract

Each of the intestinal viruses of the African situation mushroom extract obtained in Example 1 was tested using a cell viability test (MTT Assay). The MTT test is a colorimetric measurement of the activity of mitochondrial dehydrogenase, which is mainly used to determine mitochondrial redox potential or cell viability (Mosmann et al. 1983).

For this experiment, RD-A cell line (Rhabdomysarcoma, National Institute of Health pediatric machine virus, October 18, 2002) was used, and enterovirus as coxsackie virus (Coxsackie B Virus 100 TCID50 (10 ^-5 / ml), Hereinafter, CB2 virus) was used (National Institutes of Pediatric Virology, October 18, 2002).

RD-A cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS (Fetal Bovine Serum). When the cells grew properly, the cells were washed three times with PBS (Phosphate Buffered Saline) and then PBS was removed. Trysin was treated to allow cells to fall out of the culture plate (culture plate) to be separated by one colony, followed by centrifugation (1000 rpm / 10 minutes) to form a pallet. A certain amount of DMEM was added, mixed well, and then transferred to a culture plate. African Dense Mushroom Extract solution of Example 1 in which RD-A cells (3 × 10 ⁵ cells / well) were serially diluted four-fold from the same concentration of CB2 virus (10 ⁻⁵ ) and a reference concentration of 30 ug / ml Was added to a microtiter plate with 96 wells and incubated at 37 ° C. in a 5% CO ₂ incubator for 3-4 days. At this time, both the cell control group (hereinafter referred to as CC) and the virus control group (hereinafter referred to as VC) were added with PBS solution instead of the extract of the present invention, and the cell control group was not infected with the virus. MTT reagent (Sigma, USA); 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) After dissolving in PBS (phosphate buffered saline) at a concentration of mg / ㎖ was used by sterile filtration with a 0.22um membrane filter (membrane filter). 50 μl of MTT (0.5 mg / ml) was added to each well, incubated at 37 ° C. for 3 hours, and then 10 μl of 0.1M HCl was added to stop MTT-formazan formation. Live cells with active mitochondria break down the tetrazolium ring to form a dark blue formazan, so to dissolve it, add 100 μl of 10% sodium dodecylsulfate (SDS) overnight. Was allowed to fully dissolve MTT-formazan and then absorbance (OD) was measured at a wavelength of 570 nm.

When the virus penetrates the cells, the cells are killed and the OD value is significantly decreased during MTT staining. However, cells containing drugs will maintain a constant OD value at a certain concentration by antiviral activity.

No. Titer Treatment Average Absorbance (Ave.OD) One Cell control CC 1.178 2 Virus control group (10 ^-5 ) VC 0.264 3 Extract of Reference Concentration 30 µg / ml 0.477 4 4 ^(-1) of the reference concentration 7.5 µg / ml 0.642 5 4 ^(-2) of the reference concentration 1.8 µg / ml 0.699 6 4 ^(-3) of the reference concentration 450ng / ml 1.315 7 4 ^(-4) of the reference concentration 110ng / ml 1.362 8 4 ^(-5) of the reference concentration 20ng / ml 1.534 9 4 ^(-6) of the reference concentration 5ng / ml 1.791 10 4 ^(-7) of the reference concentration 1.25ng / ml 1.604 11 4 ^(-8) of the reference concentration 312 pg / ml 1.645 12 4 ^(-9) of the reference concentration 78 pg / ml 0.115

CC: Cell control without added virus and extract.

VC: Virus control group with only virus but no extract.

Experimental results, as shown in Table 1 and Figure 1, in the case of cells without addition of the extract of the present invention, the OD value is significantly decreased during MTT staining, the cells added with the extract of the present invention is the term of the African situation mushroom extract OD values were kept constant at constant concentrations by viral activity.

The results of three replicates showed that the extract of the African situation mushroom (Example 1) showed antiviral activity at a concentration of 1.8 ug / ml-0.4 pg / ml against the CB2 virus.

Experimental Example 2. VSV virus inhibitory activity of African situation mushroom extract

In order to measure the inhibitory ability of the extract of the present invention against VSV virus, MDCK (Marbin & Darby Canine Kidney, National Institute of Health respiratory tract virus, October 18, 2002) cell line was used, and VSV virus (Vesicular Stomatitis Virus 100 TCID50). (10 ^-5 / mL), National Institute of Health Gastroenterology, October 18, 2002).

The experiment was conducted in the same manner as in Experimental Example 1. No. Titer Treatment Average Absorbance (Ave.OD) One Cell control CC 1.178 2 Virus control group (10 ^-5 ) VC 0.297 3 Extract of Reference Concentration 30 µg / ml 0.078 4 4 ^(-1) of the reference concentration 7.5 µg / ml 0.416 5 4 ^(-2) of the reference concentration 1.8 µg / ml 0.945 6 4 ^(-3) of the reference concentration 450ng / ml 0.988 7 4 ^(-4) of the reference concentration 110ng / ml 1.815 8 4 ^(-5) of the reference concentration 20ng / ml 1.164 9 4 ^(-6) of the reference concentration 5ng / ml 1.964 10 4 ^(-7) of the reference concentration 1.25ng / ml 1.378 11 4 ^(-8) of the reference concentration 312 pg / ml 1.000 12 4 ^(-9) of the reference concentration 78 pg / ml 0.262

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CC: Cell control without added virus and extract.

VC: Virus control group added only virus but no extract. Results of three replicates showed antiviral activity at a concentration of 0.5 μg / ml to 7.3 pg / ml for VSV virus as shown in Table 2 and FIG. Indicated.

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Experimental Example 3. Toxicity Test

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In order to test the toxicity of the African situation mushroom extract of the present invention, animal experiments were performed.

The African situation of the present invention by dividing 25 ± 5g of ICR mice (Central experimental animals) and 235 ± 10g of SPF Sprague Dawley (Biogenomics) rats into three groups of 3 mice each Mushroom extract (Example 1) Toxicity was observed for 24 hours after intraperitoneal administration at a dose of 20 mg / kg, 10 mg / kg, 1 mg / kg, respectively.

As a result, no deaths were observed in all three groups, and no significant symptoms were found in weight gain and feed intake. Therefore, it could be confirmed that the African situation mushroom extract of the present invention is a safe drug.

African situation mushroom extract of the present invention can be administered in the following formulations, the following formulation examples are merely to illustrate the invention, whereby the content of the present invention is not limited. Formulation Example 1 Preparation of Injection

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100 mg of extract of Example 1

Sodium Metabisulfite 3.0 mg

Methylparaben 0.8 mg

Propylparaben 0.1 mg

Appropriate sterile distilled water for injection

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

200 mg of extract of Example 1

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

100 mg of extract of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate appropriate amount

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Liquid

1000 mg of extract of Example 1

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

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African situation mushroom extract according to the present invention exhibits antiviral activity and can be used as a useful medicine for the prevention and treatment of various viral diseases.

1 is a diagram showing the antiviral activity of the African situation mushroom extract against Koksagi virus,

Figure 2 is a diagram showing the antiviral activity of the African situation mushroom extract against VSV virus.

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Claims (8)

A pharmaceutical composition for the prevention and treatment of viral diseases comprising the extract of African situation mushroom. The pharmaceutical composition according to claim 1, wherein the virus is enteric virus or Vesicular Stomatitis Virus (VSV). The pharmaceutical composition according to claim 1, wherein the African situation mushroom extract is contained in an amount of 0.1 to 80 wt% based on the total weight of the composition. The pharmaceutical composition according to claim 1, wherein the African situation mushroom extract is an extract obtained by using the African situation mushroom in a polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition of claim 1, further comprising a carrier, excipient or diluent. The pharmaceutical composition according to claim 1, which is formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols such as aerosols, external preparations, suppositories or sterile injectable solutions. delete delete