KR20050081212A - Pharmaceutical composition comprising the extract of african phellinus mushroom for the treatment and protection of cold - Google Patents

Abstract

The present invention relates to a composition containing an extract of African Phellinus genus having an influenza virus inhibitory activity. Since it suppresses, it can be usefully used for medicines or functional foods for the treatment and prevention of colds.

Description

 Pharmaceutical composition comprising the extract of African Phellinus mushroom for the treatment and protection of cold}

The present invention relates to a composition for the treatment and prevention of colds containing African situation mushroom extract having the ability to suppress influenza virus.

Common cold is a generic term for acute inflammatory or allergic diseases of the respiratory mucosa, such as the nose, throat, and bronchus. Acute rhinitis, acute pharyngitis, acute laryngitis, acute Tonsillitis, bronchitis, influenza, other viral upper respiratory tract infections, and the like.

The causes of colds are various viruses, bacteria, stimulation of cold and dust, imbalance of body temperature distribution and allergens, but only one of them causes colds. Stimulation of the virus, imbalance of body temperature distribution, and the like have been the main cause of infection by the virus.

There are about 50 kinds of viruses that cause colds, but the main ones are influenza virus, adenovirus, RS virus, and rhinovirus. Typhoid bacteria usually respond to pathogens and symptoms, such as getting typhoid, but it is not always possible to cope with the symptoms of colds and hospital viruses, and because many hospital viruses cause similar cold symptoms, sometimes symptoms cannot be distinguished alone. many. Bacteria, such as Streptococcus, Staphylococcus aureus, Pneumococcal pneumoniae, and influenza bacteria, are often caused by colds, such as in the case of viruses. Bacteria, however, are usually involved in viral infections and are involved in cold complications.

Physical and chemical stimuli, such as cold and dust, can also cause mild cold symptoms, but stimuli caused by them only return to normal without significant symptoms. A common cold is caused by a temporary anemia of the respiratory mucosa caused by cold or dust stimulation, and the resistance of the part decreases, and the virus begins to infect and become active.

These diseases are common cold syndromes such as runny nose, sneezing, coughing, fever or sore throat. Called as In addition, the symptoms are a little unusual, such as acute rhinitis, acute pharyngitis, acute laryngitis, acute tonsillitis, pharyngeal conjunctivitis, bronchitis, etc., tend to be called a disease caused by a painful area. Other viral cold) means.

Colds and flu have a lot in common, but there are many differences in complications, so it's good to distinguish them. (15%) and other viruses are the main cause. Influenza viruses that cause pandemic influenza are classified into three types, A, B, and C, depending on the antigen type, of which A is the most frequent mutation, and is found to account for 90% of the worldwide pandemic viruses. Antigen alterations are proteins that are on the surface of a virus or RNA that contains the genetic information of a virus, causing them to have different traits. They are a survival strategy for survival or evolution.

Sichuan mushrooms are different from China since ancient times, and they grow on the heartwood of hardwoods such as mulberry, elm, aspen and alder, and are mud mushrooms of Hymenochaetaceae A white fungus belonging to the genus Phellinus Ouel.Em.lmaz . 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 B ₂ , B ₃ , 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 a mushroom extract as an anticancer agent is disclosed, and Korean Patent Registration No. 10-0348115 discloses a hair regrowth 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.

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.

Phellinus genus ( Phellinus genus ) of the present invention belongs to the pine scales ( Hymenochaetaceae ) mud mushrooms ( Phellinus ), mushrooms that grow on the hardwoods, etc. belonging to the mulberry family that grows wild in the high alpine area of Kenya, Africa to be. The ecological feature of this area is the alpine region of 5000m above sea level, although it is tropical near the equator, it has the best 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, 11 cm high, 20 cm wide, and 8 cm 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.

Influenza viruses occur worldwide every year, causing respiratory illnesses, which are associated with high mortality in children and the elderly with weak immunity and complications of bronchial diseases such as pneumonia and cardiopulmonary disease. In this case, the mortality rate is higher, which has a big impact on public health.

The pandemic flu virus, which appears worldwide every 10 to 20 years, is particularly deadly, especially the Spanish flu virus, which killed about 20 million people in 1918. It has not occurred since the Hong Kong flu virus in the 60s. In the near future, the emergence of deadly flu virus is expected in the near future.

Influenza viruses can infect the respiratory tract, cause systemic symptoms, change their appearance periodically, and move to other hosts without killing them before they die. Although it is the virus that causes the greatest economic loss to human beings and preventive vaccines have been developed, they have not caught up with the mutation of the virus, and the basic virus treatment is not yet performed.

Therefore, the present inventors took the boiled water of the situation mushroom in a specific region and showed the flu treatment effect, and on the basis of many cases that the intake group was not infected with the flu, the effect of inhibiting the influenza virus of the African situation mushroom extract and fractions As a result of the experiment, the excellent efficacy was confirmed to complete the present invention.

The present invention is to provide a pharmaceutical and dietary supplement as a composition for the prevention and treatment of colds containing African situation mushroom extract.

In order to achieve the above object, the present invention is an African situation mushroom ( Phellinus It provides a composition for the prevention and treatment of colds containing genus) extract.

The situation mushroom includes a situation mushroom native to Southeast Africa, preferably Kenya, such as Africa Kenya, Uganda and Tanzania.

The situation mushroom extract is a crude extract or a non-polar solvent soluble extract, the crude extract means a lower alcohol such as water, ethanol, methanol, butanol or a mixed solvent thereof, preferably an extract available in water or methanol, non-polar solvent Soluble extract means an extract soluble in a nonpolar solvent such as hexane, dichloromethane or ethyl acetate, preferably hexane or ethyl acetate.

In addition, the African situation mushroom extract includes an African situation mushroom hydrothermal extract having a polymer polysaccharide (molecular weight 12,000 or more) fraction and an African situation mushroom cold extract having a polymer protein (molecular weight 12,000 or more) fraction.

Hereinafter, the present invention will be described in detail.

The crude mushroom extract of the present invention, the nonpolar solvent soluble extract, the hydrothermal extract having the polymer polysaccharide fraction and the cold needle extract having the polymer protein fraction can be obtained as follows.

After collecting the situation mushroom of the present invention, washed with water and dried, pulverized by using a homogenizer and the like, and then powdered, the volume of the mushroom mushroom is about 2 to 30 times the dry weight, preferably about 15 to 30 times Lower alcohols such as water, methanol, ethanol and butanol or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably with an extraction temperature of 20 to 100 캜, preferably 70 to 85 캜 with methanol Extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction for about 0.5 hours to 2 days, preferably 1 hour to 1 day, preferably 1 to 5 times, preferably 3 Continuous extraction and filtration under reduced pressure, and the filtrate was concentrated under reduced pressure at 20 to 100 ℃, preferably 20 to 70 ℃ with a vacuum rotary concentrator to water, lower alcohol or a mixed solvent thereof, preferably It can be obtained as a crude extract of Phellinus extracts available to all.

The non-polar solvent soluble extract of the present invention is obtained by suspending the crude mushroom crude extract in water and then extracting it using a non-polar solvent such as hexane, ethyl acetate, dichloromethane and chloroform to obtain the non-polar solvent soluble extract of the present invention. Can be. More specifically, the crude mushroom extract, preferably the mushroom mushroom methanol extract in a volume of water of about 2 to 30 times the dry weight of the mushroom, preferably about 15 to 30 times, and then stir well to dissolve After mixing a certain amount of hexane or ethyl acetate, and repeated three to four times, fractionation can be obtained non-polar solvent soluble extracts such as each hexane soluble extract and ethyl acetate soluble extract.

In addition, the African mushroom mushroom hot water extract is washed with water and dried African mushroom mushrooms with water, and finely pulverized the dried mushroom mushrooms using a homogenizer, and then about 2 to 60 times the dry weight of the mushroom mushrooms, preferably Is about 30 to 50 times the volume of water and hydrothermal extraction for about 1 to 24 hours, preferably 6 hours using a heating mantle, and then filtrate 1 to 5 times, preferably in the hydrothermal extract obtained by filtration Is precipitated at about 0 to 10 ° C., preferably 4 ° C. for about 12 to 48 hours, preferably 24 hours by adding 3 times lower alcohol, preferably cold ethanol, and then removing the supernatant. And centrifugation to collect the precipitate to evaporate the solvent, and then re-dissolve the pellet with distilled water at about 50 to 100 ℃, preferably 60 ℃ centrifugation The supernatant obtained by repeating five times was dialyzed with distilled water at 0 to 10 ℃, preferably 4 ℃ for about 12 to 72 hours, preferably 48 hours, and then lyophilized to obtain a polymer polysaccharide (molecular weight of 12,000 or more). An African situation mushroom hydrothermal extract of the present invention having a fraction can be obtained.

In addition, the African mushroom mushroom extract of the present invention, the African mushroom mushroom extract, washed with water and dried thoroughly with water, and after grinding the dried mushrooms in a fine state using a homogenizer, adding a buffer solution about 0 to 10 After cold extraction with standing at 12 DEG C., preferably at 4 DEG C for about 12 to 24 hours, the extract was filtered and added with a protein precipitant to a salt such as ethanol, acetone or ammonium sulfate, preferably ammonium sulfate, and saturated. After centrifugation, the supernatant was removed and the precipitate obtained was redissolved with a buffer solution, and the redissolved extract was then buffered at 0 to 10 ° C., preferably at 4 ° C. for 50 to 100 hours, Preferably, after dialysis for 74 hours, the amount of protein quantitatively determines the amount of protein (molecular weight 12,000 or more) African situation mushroom having a fraction It is possible to obtain a saliva extract.

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

In addition, the present invention provides an effective composition for the prevention and treatment of colds containing the African situation mushroom extract of the present invention as an active ingredient.

The composition for the prevention and treatment of colds 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. Oral liquid preparations include suspensions, solvents, emulsions, and syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. 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 in general, the amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 to 200 mg / kg Can be administered in several divided doses. 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 little toxicity and side effects, so can be used with confidence even for long-term administration for the purpose of prevention.

The present invention provides a health functional food comprising a food supplement acceptable food additives in addition to the African situation mushroom extract exhibiting the prevention and improvement effect of the cold described above.

Compositions comprising the extracts of the present invention can be used in a variety of foods and drinks for the prevention and improvement of colds. Foods to which the African situation mushroom extract of the present invention can be added include various foods, for example, candy, chocolate, beverages, gums, teas, vitamin complexes, health functional foods, and the like, powders, granules, tablets, It can be used in the form of a capsule or a beverage.

At this time, the amount of the extract in the food or beverage, in the case of the health functional food composition of the present invention can generally be added to 0.01 to 50% by weight, preferably 0.1 to 20% by weight of the total food weight, In the case of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional beverage composition of the present invention is not particularly limited in the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and sugar alcohols such as xylitol, sorbitol, erythritol and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

Other flavors may be advantageously used natural flavors (tautin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.). The composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. Other compositions of the present invention may include natural fruit juices and fruit juice beverages and vegetable beverages. The components may be used independently or in combination The ratio of these additives is not critical but is zero per 100 parts by weight of the composition of the present invention. It is generally selected from the range of about 20 parts by weight.

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

Example  1. African situation mushroom Hydrothermal  Preparation of Extract

10 kg of African mushrooms (collected from Kenya, Africa) were collected, washed with water, dried (dried in shaded shades), and then 200 g of dried mushrooms were finely ground using a homogenizer. After that, 1 liter of water per 20 g of mushrooms were put into a heating mantle, and hot water was extracted at 100 ° C. for 6 hours, followed by three times with 0.8 L of hot water extract obtained by filtering with filter paper. Cold 95% ethanol was added and precipitated at 4 ° C. for 24 hours, then the supernatant was removed and centrifuged at 2800 rpm, 4 ° C. for 20 minutes to collect only the precipitate. After evaporation of ethanol, the supernatant obtained by re-dissolving pellets with distilled water at 60 ° C. and centrifuged three times was dialyzed using distilled water at 4 ° C. for 48 hours (dialysis membrane Mw). More than 12,000) After the end of freeze-dried to give 1 g African Phellinus hot-water extract with the polysaccharide polymer fraction (molecular weight above 12,000).

Example  2. African situation mushroom Cold  Preparation of Extract

Ten kilograms of African situation mushrooms (collected from Kenya, Africa) were collected, washed with water, dried (dried in shaded shade), and then 50 g of dried mushrooms were finely ground using a homogenizer. Then, 0.2 liters of 50 mM Tris-HCl (pH 8.0) buffer solution per 20 g of the situation mushroom was added and left overnight at 4 ° C. for extraction. The extract was filtered, and ammonium sulfate (Ammonium Sulfate) was added thereto. After saturation, the supernatant was removed by centrifugation (8000 g, 20 minutes), and the obtained precipitate was redissolved with 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer, and then the redissolved extract. Dialysis (at dialysis membrane Mw 12,000 or more) at 4 ° C. using 50 mM tris-hydrochloric acid (pH 8.0) and 50 mM sodium chloride buffer solution for 72 hoursHaving a stroke to give the African linteus extract naengchim 15 ㎖ (concentration 250 ㎍ / ㎖).

Example  3. African situation mushroom Crude extract  Produce

Take African situation mushrooms (gathered from Kenya, Africa), wash them with water, dry them (dried in shaded shades), and crush 200 g of dried mushrooms using a homogenizer and heat them. Into a mantle (heating mantle) and 4 liters of methanol, the extract obtained by extracting three times at regular intervals (12 h) at 79 ℃ repeatedly filtered and then filtered using a rotary vacuum evaporator (rotary vacuum evaporator) Concentration under reduced pressure yielded 7 g of crude mushroom extract.

Example  4. African Mushrooms Hexane  Availability Fraction  Produce

The crude mushroom extract obtained in Example 3, that is, 6.5 g of methanol extract of mushroom mushrooms was dissolved in 300 ml of tertiary distilled water, and stirred for about 1 hour. Then, 1.5 L of hexane was added, mixed and repeated three times to obtain a hexane-soluble fraction. Thereafter, the hexane soluble fraction was concentrated under reduced pressure after filtration to obtain 0.5 g of a mushroom hexane soluble extract, which was used as a sample while being stored in a 4 ° C. refrigerator.

Example  5. Ethyl acetate solubility in African situation mushrooms Fraction  Produce

The crude mushroom extract obtained in Example 3, that is, 6.5 g of methanol extract of mushroom mushrooms was dissolved in 300 ml of tertiary distilled water, and stirred for about 1 hour. Then, 1.5 L of ethyl acetate was added to the mixture, and the mixture was repeatedly mixed and fractionated three times. After obtaining the soluble fraction, the ethyl acetate soluble fraction was concentrated under reduced pressure after filtration to obtain 2.5 g of the ethyl acetate soluble extract of the situation mushroom, which was used as a sample, and used as a sample while being stored in a 4 ° C. refrigerator.

Experimental Example  1. Determination of Antiviral Activity of Extracts from African Mushroom

In order to search for antiviral efficacy of influenza virus type A / Bayern / 05/97 (H1N1), the degree of CPE inhibition of virus-infected MDCK cells (Marbin & Darby Canine Kidney) was measured by MTT assay. Inoculate each well with influenza virus type A / Bavarian virus diluted in DME / 2% FBS culture and inoculate 100 CCID ₅₀ (50%) into aliquots of MDCK cells (6 × 10 ³ / ml) in 96-well plates. 50 μl was inoculated to make a cell culture inhibiotory dose), and the culture medium was removed after adsorption at 37 ° C. for 1 hour. 100 μl of the drug diluted to each concentration (0.00001-100 μg / ml) was added to each well as a duplicate and incubated in CO ₂ incubator at 37 ° C. for 3 days, and then 0.1 mg of MTT solution was added to all wells. After addition to the incubation at 37 ℃ for 2 hours. Centrifuge the plate at 450 × g for 5 minutes, remove all but only 30 μl of medium, add 100 μl of DMSO to each well, shake until formazan crystals melt, and then use a microplate reader. The absorbance was measured at 540 nm using a reader).

In order to determine the effect of drug toxicity in the results of the drug efficacy evaluation, the culture was added to the cells without virus addition at the time of virus inoculation (mock-infected) and then treated in the same manner as the cells inoculated with the virus. That is, after one hour of incubation, the medium was removed, the drug diluted with the culture medium was added as a duplicate, and then cultured for three days, and the number of surviving cells in each well to which the drug was added was compared with the cell control well without the drug. Compared.

Experimental results showed that the inhibitory capacity against influenza virus type A was evenly expressed in all the detailed fractions. Cytotoxic concentration (CC), effective concenration (VC) and selectivity index (SI) were determined by Equation 1 below.

CC = O.D / cell control O.D with only sample added

VC = {(sample and virus added O.D-virus control O.D) / (cell control O.D-virus control O.D)} × 100 (%)

SI = CC / VC

Hydrothermal extraction number Proper concentration Treatment (ug / ml) CC VC SI One 10 ( ^-9 ) 0.00001 77 75 1.031 2 10 ( ^-8 ) 0.0001 82 75 1.091 3 10 ( ^-7 ) 0.001 88 76 1.156 4 10 ( ^-6 ) 0.01 86 76 1.130 5 10 ( ^-5 ) 0.1 86 91 0.945 6 10 ( ^-4 ) One 86 83 1.028 7 10 ( ^-3 ) 10 83 80 1.035 8 10 ( ^-2 ) 100 94 36 2.575

Cold extraction number Proper concentration Treatment (ug / ml) CC VC SI One 10 ( ^-9 ) 0.00001 76 37 2.069 2 10 ( ^-8 ) 0.0001 86 70 1.222 3 10 ( ^-7 ) 0.001 89 85 1.044 4 10 ( ^-6 ) 0.01 89 79 1.129 5 10 ( ^-5 ) 0.1 83 65 1.282 6 10 ( ^-4 ) One 77 49 1.568 7 10 ( ^-3 ) 10 94 38 2.449 8 10 ( ^-2 ) 100 70 36 1.933

Crude extract number Proper concentration Treatment (ug / ml) CC VC SI One 10 ( ^-9 ) 0.00001 78.34 74.04 1.06 2 10 ( ^-8 ) 0.0001 75.45 62.54 1.21 3 10 ( ^-7 ) 0.001 78.23 76.66 1.02 4 10 ( ^-6 ) 0.01 74.60 86.12 0.87 5 10 ( ^-5 ) 0.1 82.41 64.44 1.28 6 10 ( ^-4 ) One 80.59 67.93 1.19 7 10 ( ^-3 ) 10 79.30 73.89 1.07 8 10 ( ^-2 ) 100 84.98 34.16 2.49

Hexane extract number Proper concentration Treatment (ug / ml) CC VC SI One 10 ( ^-9 ) 0.00001 85.51 69.24 1.24 2 10 ( ^-8 ) 0.0001 82.30 73.60 1.12 3 10 ( ^-7 ) 0.001 72.35 79.13 0.91 4 10 ( ^-6 ) 0.01 73.95 75.20 0.98 5 10 ( ^-5 ) 0.1 77.48 80.88 0.96 6 10 ( ^-4 ) One 82.30 75.93 1.08 7 10 ( ^-3 ) 10 72.67 77.24 0.94 8 10 ( ^-2 ) 100 85.83 46.83 1.83

Ethyl acetate extract number Proper concentration Treatment (ug / ml) CC VC SI One 10 ( ^-9 ) 0.00001 75.56 76.51 0.99 2 10 ( ^-8 ) 0.0001 80.48 86.85 0.93 3 10 ( ^-7 ) 0.001 83.37 84.81 0.98 4 10 ( ^-6 ) 0.01 73.63 85.10 0.87 5 10 ( ^-5 ) 0.1 67.10 90.19 0.74 6 10 ( ^-4 ) One 68.71 87.87 0.78 7 10 ( ^-3 ) 10 75.02 94.12 0.80 8 10 ( ^-2 ) 100 73.10 72.15 1.01

Experimental Example  2. Determination of Toxicity of African Situation Mushroom Extract

2-1. Cytotoxicity of African Situation Mushroom Extracts

In order to measure the cytotoxicity of African linteus extract, the sensitive mammalian origin in enterovirus cell line ^{RD (Rabdomyosaarcoma, 6 × 10 4} / cell) using the in the laboratory African Phellinus each extract, that is, hot water Cytotoxicity of the extract, crude extract, hexane extract, ethyl acetate extract was measured by MTT assay (MTT assay), wherein the concentration of each extract was 1 mg / ㎖.

First, the cell monolayers (6 × 10 ⁴ / cells) were incubated overnight at 37 ° C., and then the samples of African mushrooms were diluted 10-fold, 100 μl of the diluted samples were incubated at 37 ° C. for 3 days. Then, 50 μl of MTT staining (MTT staining) was added, incubated at 37 ° C. for 2 hours, and then the medium was discarded and 150 μl of DMSO was added, stirred for 1 hour, and the absorbance was measured at 540 nm. And the OD value compared to the average of the cell control (PBS) of each extract was compared, and the average of the cell control is represented by a constant line, based on which the higher than this line is not cytotoxic, the lower than this line is determined to be cytotoxic It was.

As a result, as shown in Table 6 below showing the cytotoxicity of the hydrothermal extract of African situation mushroom, and Table 7 below showing the cytotoxicity of the crude extract, hexane extract, ethyl acetate extract, African situation mushroom No significant cytotoxicity of each extract was found (see FIGS. 6 and 7).

number Titer process Average O.D One Cell control 0 mg / ml 0.409 2 Dilution 10 mg / ml 0.119 3 Sample 10 ^(-1) 1 mg / ml 0.738 4 Sample 10 ^(-2) 100 μg / ml 0.774 5 Sample 10 ^(-3) 10 μg / ml 0.580 6 Sample 10 ^(-4) 1 μg / ml 0.553 7 Sample 10 ^(-5) 100 ng / ml 0.468 8 Sample 10 ^(-6) 10 ng / ml 0.430 9 Sample 10 ^(-7) 1 ng / ml 0.441 10 Sample 10 ^(-8) 100 pg / ml 0.436 11 Sample 10 ^(-9) 10 pg / ml 0.560 12 Sample 10 ^(-10) 1 pg / ml 0.448

number Titer process Average O.D Crude extract Hexane extract Ethyl acetate extract One Cell control 0 mg / ml 0.630 0.630 0.630 2 Sample 10 ^(-1) 1 mg / ml 0.267 0.320 0.074 3 Sample 10 ^(-2) 100 μg / ml 0.509 0.444 0.365 4 Sample 10 ^(-3) 10 μg / ml 0.650 0.494 0.365 5 Sample 10 ^(-4) 1 μg / ml 0.638 0.440 0.520 6 Sample 10 ^(-5) 100 ng / ml 0.669 0.426 0.659 7 Sample 10 ^(-6) 10 ng / ml 0.720 0.420 0.682 8 Sample 10 ^(-7) 1 ng / ml 0.771 0.442 0.720 9 Sample 10 ^(-8) 100 pg / ml 0.772 0.428 0.689 10 Sample 10 ^(-9) 10 pg / ml 0.652 0.563 0.696 11 Sample 10 ^(-10) 1 pg / ml 0.716 0.491 0.681

2-2. Histotoxicity of Situation Mushroom Extract

In order to measure the histotoxicity of the extracts of African situational mushrooms, crude extracts of African situational mushrooms were made by using a mouse (Nude mouse: normal colon), which is a tissue derived from mammals that was determined to be normal in the tissues extracted during surgery. In order to measure the tissue toxicity experiments of the hexane extract and the ethyl acetate extract, MTT assay was performed, and it was determined that the extract was toxic only when the inhibition rate was 30% or more in consideration of various errors. Inhibition rate was calculated by the following equation (2).

       Inhibition rate (%) = 100-((O.D of the extract / O.D of the control) × 100]

As a result, the histotoxicity of the crude extract of the African situation mushroom was shown in Table 8a, the histotoxicity of the hexane extract in 8b, the histotoxicity of the ethyl acetate extract in 8c. At this time, the time when the inhibition rate is 30% is indicated by a constant line, it was determined that the extract with the rod above this line is toxic. As can be seen in Tables 8a to 8c (see FIGS. 8a to 8c), the extracts of the African situation mushrooms were not toxic in normal tissues because they showed no inhibition of more than 30% except for 10 ㎍ of ethyl acetate. It was judged that there was no.

number process IR Survival rate One PBS 0 % 100% 2   1 mg One % 99% 3 100 μg 25% 75% 4  10 μg 14% 86% 5   1 μg -11% 111%

number process IR Survival rate One PBS 0 % 100% 2   1 mg 0 % 100% 3 100 μg One % 99% 4  10 μg 14% 86% 5   1 μg 14% 86%

number process IR Survival rate One PBS   0 % 100% 2   1 mg  14% 86% 3 100 μg 19% 81% 4  10 μg  34% 66% 5   1 μg  One % 99%

Reference Example  1. Identification of African Mushrooms

In order to identify the kinds of African mushrooms of the present invention, DNA sequences were analyzed by Micro ID Co., Ltd. and identified by ITS rDNA sequencing. In this case, first, the% similarity value is obtained, and the evolutionary distance is based on the Kimuras two-parameter model (Kimura, MA, J. Mol . Evol., 16 , pp111-120, 1980). (evolutionary distance) and then phylogenetic with the neighbor-joining method (Saitou, N. and Nei, M., Mol . Biol . Evol ., 4 , pp406-425, 1987). tree). On the other hand, the scale bar of the tree represents a substitution per site of 0.1 or 0.01, and the number next to each branch in the tree represents the bootstrap percentage. do.

As a result of NCBI blast search of the strain by the nucleotide sequence of the ITS portion, the determined number of nucleotide sequences was 701 bp, and the nucleotide sequences thereof are shown in FIG. 9 and SEQ ID NO: 1. This strain is Felinus It was found to be a fungus belonging to the genus ( phellinus genus ).

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

100 mg of African situation mushroom extract of Example 3

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 Tablets

African situation mushroom extract 200 mg of Example 3

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 African situation mushroom extract of Example 3

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. Liquid  Produce

1000 mg of crude situation mushroom extract of Example 3

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 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.

Formulation example  5. Manufacture of Healthy Foods

1000 mg of crude situation mushroom extract of Example 3

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

   Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  6. Manufacture of health drinks

1000 mg of crude situation mushroom extract of Example 3

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

The situation mushroom extract of the present invention inhibits the proliferation of influenza virus by enhancing the antiviral effect and immune activity function, it can be usefully used in pharmaceutical compositions for the treatment and prevention of colds.

1 is a diagram observing the antiviral activity of the hydrothermal extract of African situation mushroom,

2 is a diagram observing the antiviral activity of the African mushroom extracts,

3 is a diagram observing the antiviral activity of the crude extract of African situation mushroom,

4 is a diagram observing the antiviral activity of the African situation mushroom hexane extract,

5 is a diagram illustrating the African situation mushroom ethyl acetate antiviral activity,

Figure 6 is a diagram measuring the cytotoxicity of the hydrothermal extract of African situation mushroom,

7 is a diagram measuring the cytotoxicity of each solvent extract of African situation mushroom,

8a to 8c is a measure of the histotoxicity of each solvent extract of the African situation mushroom, Figure 8a is a measure of the histotoxicity of the crude extract, Figure 8b is a diagram measuring the histotoxicity of the hexane extract, 8c is a diagram measuring the histotoxicity of ethyl acetate extract,

9 is a diagram showing the nucleotide sequence of the African situation mushroom.

<110> KIM, JIN-DONG LEE, JONG-SUNG <120> Pharmaceutical composition comprising the extract of African Phellinus mushroom for the treatment and protection of cold <130> DIF / 2003-07-0010-1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Phellinus genus <400> 1 gaacctgcgg aaggatcatt atcgagtttt tgaaagcgag gcttgctgct ggcgcagaaa 60 cgcgcatgtg cacggccttc gcgctcaaat ccactcaacc ccctgtgcac ctttatcctg 120 tcggagcgag tagcttgaga cctttttggg acgtagtagc cggtaatagt agaaaggagg 180 gtaacagctc cattcgaaag gcgaaaaggc cctaactcga gcgaaaactt tggcttgtat 240 tttataaacc acatttgttg tcctgtgaat gttaatgctc cttgtgggcg agaataaata 300 caactttcaa caacggatct cttggctctc gcatcgatga agaacgcagc gaaatgcgat 360 aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac cttgcgcccc 420 ttggtattcc gaggggcatg cctgtttgag tgtcatgtta acctcaaatc acacttgtct 480 tttggggctt tgtggtttgg acctggaggt ttctgctggc cctgcggtcg gctcctctta 540 aatgcattag ctgggtttcg gctcgcgttt gtggtgtaat agttaattct ttcgcctgag 600 cgcttgcctg atgggcccgc ttctaattgt ctgcttggtc ggacaaggtc ctttggcctt 660 cttgactctt ttgacctcaa atcaggtagg actacccgct g 701

Claims (7)

A pharmaceutical composition for the prevention and treatment of colds containing Phellinus genus extract, native to Kenya, Africa. The pharmaceutical composition according to claim 1, wherein the situation mushroom is a situation mushroom having a nucleotide sequence of SEQ ID NO: 1. The pharmaceutical composition according to claim 1, wherein the situation mushroom extract is a crude extract or a nonpolar solvent soluble extract. The pharmaceutical composition of claim 3, wherein the crude extract is extracted with water or methanol. The pharmaceutical composition according to claim 3, wherein the nonpolar solvent soluble extract is extracted with hexane or ethyl acetate. The pharmaceutical composition according to claim 1, wherein the extract is a situation mushroom hydrothermal extract having a polymer polysaccharide (molecular weight of 12,000 or more) fraction. The pharmaceutical composition according to claim 1, wherein the extract is a situation mushroom cold extract having a fraction of high molecular protein (molecular weight 12,000 or more).