KR100550822B1 - Composition comprising the extract of African Phellinus mushroom for the treatment and protection of hepatitis B - Google Patents

Abstract

The present invention relates to a composition containing an extract of African Phellinus genus having the ability to inhibit hepatitis B virus (HBV), wherein the African situation mushroom extract of the present invention is proliferated by hepatitis B virus by an antiviral effect. Since it inhibits, it can be usefully used in medicines or functional foods for the treatment and prevention of hepatitis B.

Hepatitis B, virus, African, mushrooms, extracts, pharmaceutical compositions.

Description

Composition comprising the extract of African Phellinus mushroom for the treatment and protection of hepatitis B}             

1 is a diagram observing the HBV inhibitory ability of the African hot spring mushroom hot water extract,

Figure 2a to 2b is to measure the HBV DNA inhibitory ability of each solvent extract of African situation mushroom,

Figure 2a is a result of the reaction of hepatitis B patients serum and each extract for 2 hours,

Figure 2b is a result of the reaction of hepatitis B patients serum and each extract for 8 hours,

Figure 3 is a diagram measuring the cytotoxicity of the hydrothermal extract of African situation mushroom,

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

5a to 5c are measured the histotoxicity of each solvent extract of African situation mushroom,

Figure 5a is a diagram measuring the histotoxicity of crude extracts,

Figure 5b is a diagram measuring the histotoxicity of the hexane extract,

Figure 5c is a diagram measuring the histotoxicity of the ethyl acetate extract,

Figure 6 shows the nucleotide sequence of the African situation mushroom.

The present invention relates to a composition for the treatment and prevention of hepatitis B containing an African situation mushroom extract having a hepatitis B virus inhibitory ability.

Hepatitis B is a major problem in Korea, China, India and Southeast Asia. Hepatitis B virus (hereinafter referred to as HBV virus) belongs to the family hepadnaviruses and is a non-cytopathic DNA virus that causes not only acute hepatitis but also chronic liver disease including cirrhosis and liver cancer. (genome) is an incomplete double helix structure of about 3,200 bp, maintaining a relaxed circle (Peutherer JF., Medical Microbiology , 14th Ed ., pp 527-540, 1994).

Self-replicating of HBV virus begins with the attachment of virions to hepatocytes. Once the synthesis of the plus strand of HBV viral DNA is completed in the nucleus of the hepatocytes, the viral gene is covalently closed. circular DNA, cccDNA). This cccDNA becomes a template of pregenomic RNA, which is then reverse transcribed into the minus strand of HBV viral DNA. This cccDNA has two origins: partly double-stranded DNA, either a new virus particle entering the hepatocyte or in an immature nucleocapsid in the cytoplasm. )to be. Most antiviral agents are known to have little effect on this cccDNA (Locarnini S., et al., J. Hepatol. , 30 , pp536-550, 1999), which would stop treatment with antiviral agents. It is considered to be the reason for the emergence of serum HBV virus DNA quickly.

The pathogenesis of chronic hepatitis B has not been elucidated, but the decisive factor is presumably due to the impairment of the immune response, which continues the proliferation of the virus and the immune system's response to incomplete control. Under these theoretical backgrounds, many studies and attempts have been made on drugs that enhance immunity and antiviral agents that can directly inhibit the proliferation of viruses for the treatment of chronic hepatitis B. The purpose of chronic hepatitis B treatment is to inhibit self-replicated HBV virus and maintain remission of hepatitis.

Currently, interferon (IFNs) and lamivudine are mainly used to treat chronic hepatitis B. Interferon has antiviral effects, proliferation suppression, and immunomodulatory functions. In particular, IFN-α is known to be very effective in inhibiting HBV virus self-replicating and inducing remission of liver disease. However, there is a limit that the efficacy of IFN-α is limited to only a few patients who are well selected. It is also known to be less effective in Asian patients, who are expensive and have many side effects, and whose infections that begin shortly after birth are problematic (Wong DK. Et al., Ann.Intern.Med . , 119 , pp312-323, 1993). ).

Lamivudine (brand name Zeffix, glaxo) is the negative enantiomer of 2'-3'-idedeoxy-3'-thiacytidine. Active triphosphate (3TC-TP), which binds to the DNA chain and leads to premature chain termination, inhibiting the synthesis of HBV viral DNA and inhibiting the synthesis of HBV virus DNA in the RNA pregenome during HBV virus propagation. Interfering with the reverse transcription process to produce minus strand DNA (HBV virus) has the effect of blocking the growth of HBV virus. However, it is known to have no effect on the production of HBsAg or HBeAg (Raffanti S. et al., Goodman &Gilman's Pharmacological Basis of Therapeutics , 10th ed ., Pp1349-1380, 2001). In addition, mutants (YMDD variant (M204V / I)) are resistant to long-term dosing (Allen MI., Et al., Hepatology , 27 , pp1670-1677, 1998; Stuyver LJ., Hepatology , 33 , pp751). -757, 2001). Lamivudine is bound to the YMDD variant (motif) and clings to reduce the action of the DNA polymerase to inhibit the growth of the virus. However, patients with YMDD strains are slightly deformed, which is less effective when lamivudine adheres to them , resulting in a slight gap ( Hyewon , a record of the symposium held in Seoul , Korea on 8th Feb , 2002; GlaxoSmithKelein).

In addition, hepatitis B is important for treatment, but more important than that, it is also very important to prevent the hepatitis B virus involved in the conversion to liver cancer. Most of the growth of HBV virus occurs in the liver, but some occur in the spleen, kidney, pancreas and bone marrow. However, proliferation in these organs other than liver does not cause tissue damage, only HBV virus.

As described above, chronic hepatitis B is characterized by systemic diseases, has a very complex and complex pathogenesis, and ultimately, disorders of the immune system are important causes. However, laboratory methods for the immunological pathophysiology of HBV virus have not been fully developed compared to other research fields. Thus, the pathophysiological and immunological mechanisms of hepatitis B are still far from established. There are also few treatments that can help those who are infected.

Current treatments have been reported to temporarily inhibit the virus, but withdrawal of the virus causes the virus to multiply and develop resistance.

Sichuan mushrooms, which are known in China since ancient times, are native to the heart of hardwoods such as mulberry, elm, aspen and alder. ( Phellinus Ouel. Em.lmaz ) is a white fungus belonging to. 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 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 that 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 ), a mushroom that grows 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 will be 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 is 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 therapeutic effect of hepatitis from the African situation mushroom has not been studied until now.

The present inventors focused on the excellent therapeutic effect of the local residents suffering from various viral diseases while taking boiled water of the situation mushroom, and tested the HBV inhibitory ability of the African situation mushroom extracts and subfractions, and its excellent efficacy It was confirmed to complete the present invention.

The present invention provides a pharmaceutical and dietary supplement as a composition for the prevention and treatment of hepatitis B containing African situation mushroom extract.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of hepatitis B containing an African Phellinus 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 crude mushroom extract, is dissolved 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. After mixing a certain amount of hexane or ethyl acetate, and repeated three to four times, fractionation can be obtained to obtain a non-polar solvent soluble extract 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 the hot water extraction using a heating mantle at about 60 to 100, preferably 100 ℃, about 1 to 24 hours, preferably 6 hours, and then filtered through About 1 to 5 times, preferably 3 times lower alcohol, preferably cold ethanol is added to the obtained hot water extract, about 12 to 48 hours at about 0 to 10 ℃, preferably 4 ℃, preferably Precipitate for 24 hours, and then remove the supernatant and centrifuged to collect the precipitate to evaporate the solvent, and then pellet the pellet with distilled water at about 50 to 100 ℃, preferably 60 ℃ The supernatant obtained by dissolution and centrifugation 2 to 5 times was dialyzed with distilled water at about 0 to 10 ° C, preferably at 4 ° C for about 12 to 72 hours, preferably 48 hours. Lyophilization can yield the hydrothermal extract of African situation mushroom of the present invention having a polymer polysaccharide (molecular weight of 12,000 or more) fraction.

In addition, the African mushroom mushroom extracts of the present invention, after collecting the African mushroom mushrooms washed with water and dried with water, and then crushed the dried mushrooms in a dry state using a homogenizer, about 2 to about the weight of the dried mushrooms 30 times, preferably 5 to 10 times the buffer solution was added and left for about 12 to 24 hours at about 0 to 10 ℃, preferably 4 ℃ to extract cold extract, the extract was filtered and ethanol, acetone or sulfuric acid with a protein precipitant After adding a salt such as ammonium sulfate, preferably ammonium sulfate, and saturating, centrifuging to remove the supernatant, and redissolved the obtained precipitate with a buffer solution, and then the redissolved extract was added to the buffer solution. Dialyzed at about 0 to 10 ° C., preferably at 4 ° C. for about 50 to 100 hours, preferably 72 hours, and then It is possible to obtain a high molecular protein African Phellinus having a (molecular weight of 12,000 or more) fractions naengchim extract.

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

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

The composition for the prevention and treatment of hepatitis B of the present invention, the total weight of the composition comprises 0.1 to 80% by weight, preferably 1.0 to 50% by weight of the extract.

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 showing the prevention and improvement effect of hepatitis B described above.

Compositions comprising the extracts of the present invention can be used in a variety of food and beverages for the prevention and improvement of hepatitis B. 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, and tablets. , Capsules or beverages.

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 be used in natural fruit juices and fruit juice beverages and vegetable beverages. The components may be used independently or in combination The ratio of such 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. Preparation of hydrothermal extract of African situation mushroom

Take 10 kg of African mushrooms (collected from Kenya, Africa), wash them with water, dry them (dried in shaded shade), and then crush 200 g of dried mushrooms using a homogenizer. 1 liter of water per 20 g of mushrooms in a heating mantle, followed by hot water extraction at 100 ° C. for 6 hours, followed by three times cold in 0.8 L of hot water extract obtained by filtering with filter paper. (cold) 95% ethanol was added to precipitate for 24 hours at 4 ℃, the supernatant was removed, centrifuged at 2800 rpm, 4 ℃, 20 minutes conditions to collect only the precipitate (harvesting), the ethanol of the precipitate After evaporation of the pellet, the pellet was re-dissolved with distilled water at a high temperature of 60 ° C., and centrifuged three times. The supernatant obtained was dialyzed using distilled water at 4 ° C. for 48 hours (dialysis membrane Mw 12,000 Over) and dialysis After lyophilization, 1 g of African situation mushroom hot water extract having a polymer polysaccharide (molecular weight of 12,000 or more) fraction was obtained.

Example 2 Preparation of Extract from African Mushrooms

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 at 4 ° C. overnight for cooling extraction. The extract was filtered and added with ammonium sulfate (Ammonium Sulfate). 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 hours, and after the dialysis, the protein amount was quantified to determine the high molecular protein (molecular weight 12,000 or more). minute 15 ml (concentration 250 µg / ml) of African situation mushroom extracts having a stroke were obtained.

Example 3. Preparation of African Mushroom Extract

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) Concentrated under reduced pressure to give 7 g of crude mushroom extract.

Example 4 Preparation of Hepatic Mushroom Hexane Fractions from Africa

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 Preparation of Ethyl Acetate Soluble Fraction from African Mushrooms

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. Hepatitis B virus inhibitory effect of African situation mushroom extract

1-1. DNA Quantitative Methods

In order to measure the inhibitory ability of the extract against the DNA of HBV virus, the DNA of hepatitis B virus present in the serum was quantitated by chemiluminescence on a microplate. This experiment was carried out using a RNA probe (ad & av stralms) capable of binding to the DNA of HBV virus contained in the serum to make a DNA: RNA hybrid, and then to a microplate containing specific antibodies. Capture and bind the captured hybrid to an antibody conjugated with alkaline phosphatase, and then measure the amount of HBV virus DNA in the serum using a chemiluminescent material as a light intensity and use a calibration curve. As a method of determining the concentration through), a hybrid capture technology (hybrid capture technology, Digene) was used for this experiment.

In more detail, the test method is described in detail. First, the microplate heater is removed from the microplate heater, and then the sealer is immediately removed and the calibrator (Calibrators; Cal 1, Cal 2, Cal 3, Cal 4). And Cal 5), controls (P3 and P2) and experimental groups (Experimental Group S and Experimental Group A) were each transferred to 75 μl by Capture microplate (microplate coated with anti-RNA: DNA hybrid antibody). The capture microplate was covered using a microplate sealer and incubated for 60 minutes at 1100 ± 100 rpm, 18-25 ° C. using a rotary stirrer. The filtrate of the capture microplate was removed using a pipette and then tapped on clean blotter paper 2-3 times to completely remove the filtrate.

In a buffer containing sodium azide, 75 μl of the detection reagent combining RNA: DNA hybrid with an antibody conjugated with alkaline phosphatase was dispensed into each capture microplate well, and micro Cover the capture microplate wells using a plate sealer and incubate for 30 minutes at 20-25 ° C. After the filtrate of the capture microplate well was completely removed, the capture microplate well was flooded with dilution washing solution containing sodium azide, and then washed six times by turning it upside down. The washed capture plate was left inverted for 10-15 minutes on a clean blotter paper to absorb the remaining filtrate, and 75 μl of chemiluminescent material (CDP-Star ^™ , Chemilumlnescent substrate) was dispensed into each capture microplate well.

The microplate sealer was used to cover the capture microplate wells and incubated for 15 minutes at room temperature under direct sunlight. Within 15 minutes after the reaction was completed, the amount of DNA of the HBV virus present in the serum was measured by light intensity using a DML 2000 Luminometer.

In the experiment, P3 and P2 were positive controls and included the infectious HBV virus and sodium azide. Calibrator 1 (Cal 1) is the carrier DNA and sodium azide in HBV negative human serum, Calibrator 2 (Cal 2) is 1.42 × 10 ⁵ copies / mL (0.5 pg / ml) in HBV negative human serum. ) HBV virus plasmid DNA and carrier DNA and sodium azide, Calibrator 3 (Cal 3) is 2.83 × 10 ⁷ copies / ml (100 pg / ml) HBV virus plasmid DNA and carrier DNA in HBV negative human serum. Sodium azide, Calibrate 4 (Cal 4), contains 5.66 × 10 ⁸ copies / ml (2000 pg / ml) HBV virus plasmid DNA and carrier DNA and sodium azide in HBV negative human serum. (Ccal 5) comprises 1.70 × 10 ⁹ copies / ml (100 pg / ml) HBV virus plasmid DNA in HBV negative human serum and carrier DNA and sodium azide.

Gepix, the control drug, was used in one experiment, of which 10 μl was adjusted to a concentration of 100 μg / ml as fractions.

1-2. HBV Inhibitory Activity of Hot Water Extracts from African Situation Mushroom

As a preliminary experiment for measuring HBV inhibitory capacity of African mushrooms, HBV DNA quantitative tests were used to randomly select serum containing HBV DNA, and then the sera of 11 selected patients (patients with chronic active hepatitis B) were subjected to HBV. From the serum containing less DNA to the serum containing more DNA, the serum HBV DNA inhibitory effect of the hydrothermal extract of African situation mushroom was observed for each serum by the following method.

First, 20 [mu] l of each selected serum was treated with 10 [mu] l of African hot mushroom extracts (concentration: 50 g / 1.5 l) for 2 hours at 37 [deg.] C., and the amount of HBV DNA in the serum was immediately determined. Measured by the method of 1-1, and compared with the control group (10 μl of PBS in 20 μl serum) to observe the HBV DNA inhibitory effect of the extract, the results are shown in FIG.

As a result, as can be seen in Figure 1, the African hot mushroom extract was not only inhibits HBV DNA, but also showed a continuous virus inhibitory effect over time.

1-3. HBV inhibitory activity of each extract of African situation mushroom

As a result of Experimental Example 1-2, HBV DNA inhibitory ability of African situation mushroom was confirmed, HBV of each extract, that is, hot water extract, cold extract, crude extract, hexane extract, ethyl acetate extract, in order to select the effective extract In order to observe the inhibitory ability against DNA, one kind of serum was treated with 10 μl of each extract (concentration: 50 g / 1.5 L), followed by reaction at 37 ° C. for 2 hours and 8 hours, respectively, followed by HBV DNA The amount of was measured (using the method of Experimental Example 1-1 below). At this time, a positive control was used (zeffix, glaxo company, denoted by Gepic in the figure), the negative control (indicated as a control in the figure) includes infectious HBV virus and sodium azide. Each inhibition rate was calculated by the following equation.

       % Inhibition = {(value of control-value of extract) / value of control} × 100

As a result, as shown in Table 1 below, after 2 hours, Gepix inhibited 1.33% of HBV DNA, while 22% of African mushroom extracts, 15.5% of hexane extracts, and 23.8% of ethyl acetate extracts. (See FIG. 2A).

In addition, as can be seen in Table 2 below, after 8 hours, Gepix inhibited 2.20% of HBV DNA, while 37.2% of crude extract, 25.5% of hexane extract, and 31.4% of ethyl acetate extract (FIG. 2 b). Reference).

In other words, the extracts of each of the African situation mushrooms generally showed better effects than Gepix.

sample Replication number (1000 / ml) pg / ml  % Inhibition Control group (2 hours) 66,825.8 266.134     0.0 Hydrothermal extract 66,432.2 264.595     0.58 Cold extract 75,338.5 266.214     0.0 Crude extract 52,124.2 207.575    22.0 Hexane extract 56,467.9 224.978    15.5 Ethyl acetate extract 50,921.3 202.671    23.8 Gepix 65,937.0 262,607     1.33

 sample Replication number (1000 / ml) pg / ml  % Inhibition Control group (8 hours) 73,675.3 278.337 0.0 Hydrothermal extract 78,854.0 278.636 0.0 Cold extract 72,010.3 272.041  2.26 Crude extract 46,268.1 174.580 37.2 Hexane extract 54,888.1 207.197 25.5 Ethyl acetate extract 50,541.3 190.783 31.4 Gepix 72,054.5 272,199  2.20

Experimental Example 2. Determination of Toxicity of African Situation Mushroom

2-1. Cytotoxicity of African Situation Mushroom Extracts

In order to measure the cytotoxicity of African extracts, the extracts of each of the African mushrooms, ie, hot water, were used in the laboratory using RD (Rabdomyosarcoma, 6 × 10 ⁴ / cell), a mammalian-derived cell line sensitive to enteric viruses. 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 monolayers of cells (6 × 10 ⁴ / cell) 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 3 showing the cytotoxicity of the hydrothermal extract of African situation mushroom, and Table 4 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 Figures 3 and 4).

number Titer process Average. OD 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.OD Methanol 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 African situation mushroom extract, the crude extract of African situation mushroom was extracted using a colon mouse (Nude mouse: normal colon), which was found to be normal tissue by staining. In order to measure the tissue toxicity experiments of hexane extracts and ethyl acetate extracts, MTT assays were performed, and it was determined that the extracts were toxic only when the inhibition rate (IR) 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 is shown in Tables 5a and 5a, the histotoxicity of the hexane extract is 5b and 5b, the histotoxicity of the ethyl acetate extract is 5c and 5c. In this case, the time when the inhibition rate (IR) is 30% is indicated by a constant line, and it was determined that the extract having the rod above this line was toxic. As can be seen in Tables 5a to 5c (see FIGS. 5a to 5c), the extracts of the African situation mushrooms were not toxic in normal tissues because they showed no inhibition of more than 30% except 10 ㎍ of ethyl acetate. It was judged that there was no.

number process OD Crude extract IR Survival rate One PBS 0.358   0 % 100% 2   1 mg 0.354  One %  99% 3 100 μg 0.267  25% 75% 4  10 μg 0.309  14%  86% 5   1 μg 0.399 -11% 111%

number process OD Hexane extract IR Survival rate One PBS 0.358  0 % 100% 2   1 mg 0.358  0 % 100% 3 100 μg 0.355  One % 99% 4  10 μg 0.309  14% 86% 5   1 μg 0.307  14% 86%

number process OD Ethyl acetate extract IR Survival rate One PBS 0.358   0 % 100% 2   1 mg 0.309  14%  86% 3 100 μg 0.289  19%  81% 4  10 μg 0.236  34%  66% 5   1 μg 0.355   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 evolution is obtained by Kimuras two-parameter model (Kimura, MA, J. Mol. Evol., 16 , pp111-120, 1980). After calculating the evolutionary distance, the phylogenetic tree is determined by the neighbor-joining method (Saitou, N. and Nei, M., Mol. Biol. Evol., 4 , pp406-425, 1987). tree). On the other hand, the tree's scale bar represents 0.1 or 0.01 substitution per site, 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. 6 and SEQ ID NO: 1. This strain 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

Proper 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

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 Preparation of Liquid

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 Preparation of Healthy Food

1000 mg of crude situation mushroom extract of Example 3

Vitamin Mixture

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 Preparation of Healthy Drink

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 in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and stored in the refrigerator and then Used to prepare the healthy beverage composition of the invention.

The situation mushroom extract of the present invention has the effect of inhibiting the proliferation of hepatitis B virus. In addition, since it can be safely administered for a long time because there is no side effect in vivo and does not cause drug resistance, it can be usefully used as a pharmaceutical composition for the treatment and prevention of hepatitis B.

<110> KIM, JINDONG          LEE, JONGSUNG <120> Composition comprising the extract of African Phellinus mushroom          for treatment and protection of hepatitis B <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 (10)

delete delete delete delete delete delete delete delete A health functional food for the prevention and improvement of hepatitis B, which is native to Kenya, Africa, and which contains food supplements which are food-acceptable food additives in addition to the African situation mushroom extract having the nucleotide sequence of SEQ ID NO: 1. delete

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