KR20040042119A - Composition having immune-enhancing and anticancer activity comprising the extracts of CHAGA mushroom - Google Patents

Abstract

PURPOSE: Provided is a pharmaceutical composition comprising the extract of Inonotus obliquus which has immune-enhancing and anticancer activities. The extract of Inonotus obliquus is effectively used for health foods and medicaments for prevention and treatment of adult diseases, such as immunodeficiency diseases and cancers. CONSTITUTION: A pharmaceutical composition is characterized by containing the extract of Inonotus obliquus or Fuscoporia obliquus, Fuscoporia punctata. It is effective for prevention and treatment of both solid tumor, such as lung cancer, stomach cancer, lymphoma, liver cancer, and nonsolid tumors, such as leukaemia. It also has immune-enhancing activity.

Description

Composition having immune-enhancing and anticancer activity comprising the extracts of CHAGA mushroom}

The present invention relates to a composition having an immune enhancing and anticancer activity using natural chaga.

Since all aerobic organisms inevitably carry harmful free radical species during metabolism, all organisms must have a mechanism to remove these free radical species in order to survive. Active oxygen species can be broadly classified into two types: compounds that form oxygen radical species, such as hydrogen peroxide, and oxygen radical species. As already known, the hydrogen peroxide is catalase (catalase) or the case of peroxidase (peroxidase) oxygen, but the conversion of water, oxygen radical species are removed by the mechanism, O ₂ ^- other oxygen radicals other than the mechanism of removing (superoxide anion) Species removal mechanisms are not yet clear.

Hydroxy radicals, a type of oxygen radical species, are formed by the reaction of H ₂ O ₂ and O ₂ in living things and are known to be present in almost all biomolecules [Fridovich et al., Ann. Rev. Biochem ., 44 , pp 147-159, 1975; Tauber et al., Blood , 53, pp 666-676, 1979]. Therefore, it is obvious that defense against highly reactive hydroxy radicals is required in order to maintain homeostasis in vivo. In this respect, organic materials such as ascorbic acid and uric acid are known to remove hydroxy radicals by chemical reaction, but this is not fundamentally to remove hydroxy radicals but to convert them into less reactive radicals, as well as oxidizing agents in vivo. It has been reported that it can react with a transition metal such as iron to promote the production of reactive oxygen radical species [Borg, DC and Schaich, KM, Handbook of Free Radicals and Antioxidant in Biomedicine, Vol. I, pp 63-80, CRC press, 1989].

Physiological processes radicals generated during ^{_{(O 2 -, H 2 O}} 2, OH and O ₂₎ is a free radical reaction in which a very strong reactivity caused by these represents the destruction effect of major macromolecules, including lipids [Davies , KJA and Goldberg, AL, J. Biol. Chem., 262 , p8220-8226, 1987]. Their production is increased by a number of factors leading to abnormal metabolic processes, photosensitization reactions, drug metabolism processes and other cellular metabolisms, and thus the living body is always exposed to the harmful effects of free radical reactions caused by them. Morehouse, LA and Thomas, CE, J. Free Radicals Biol. Med., 1, p 8, 1985]. In particular, as the cells age, the harmful effects of their harmful radicals continue to accumulate, leading to overall aging of the cells as well as diseases related to various adult diseases such as carcinogenesis, arteriosclerosis, heart disease and skin aging. It has been suggested as a cause of human disease occurrence and aging [Harman, D., Free Radicals in Biology, Academic Press, New York, pp 255-275, 1982]. Therefore, the study aims to find candidates that are expected to be useful in the treatment of diseases such as lipid metabolism, immune diseases and cancer by inhibiting free radical reactions in vivo by reactive oxygen metabolites or removing harmful free radicals accumulated in the human body. Have been going on.

Chaga has already been widely used as a medicinal cure in the Kamchatka Peninsula in the Far East, the Northern Hemisphere of Japan, Mongolia, and the volcanic region of the altitude of 45 degrees north latitude. It is written that this is a widely known background. Therefore, this chaga is a parasitic mushroom that grows on the birch and is called 'chaga' in Russia and 'Kabanoanatake' in Japan, and it has begun artificial culture of chaga by cultivating strains. Is said to be in progress. Already, natural chaga is recognized as a raw material for food as the Food and Drug Administration has proved its stability in food and medicine. As this fact became known, many researchers in Japan, Europe and the United States became interested in the preparation of chaga.

The present inventors focused on finding anti-inflammatory and anti-cancer effects from chaga, and as a result, the present invention was completed by confirming the anti-inflammatory and anti-cancer activity of chaga of chaga.

An object of the present invention to provide a pharmaceutical composition and health supplements effective for treating various inflammation.

It is also an object of the present invention to provide a pharmaceutical composition and health supplements effective for the prevention and treatment of various cancers.

1 is a graph showing the proliferation test of splenocytes after intraperitoneal injection of chaga mushroom extract according to the present invention,

Figure 2 is a graph showing the activity of IL-2 of splenocytes after intraperitoneal injection of chaga mushroom extract according to the present invention,

Figure 3 is a graph showing the NO production capacity of celiac cells after intraperitoneal injection mouse chaga extract according to the present invention,

Figure 4 is a graph showing the efficacy of the chaga mushroom extract according to the present invention on the inhibition of cancer cell proliferation in KATO3 human gastric cancer cells,

5 is a graph showing the efficacy of the chaga mushroom extract according to the present invention on cancer cell proliferation inhibition in Raw 264.7, a mouse mononuclear macrophage,

FIG. 6 is a graph showing the efficacy of chaga extract according to the present invention on cancer cell proliferation inhibition in JurKAT which is human acute T leukemia cells.

FIG. 7 is a graph showing the efficacy of chaga extract according to the present invention on cancer cell proliferation inhibition in EL4, a mouse lymphoma cell.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for immuno-enhancing containing chaga extract.

The present invention also provides a pharmaceutical composition effective for the treatment of cancer containing chaga extract.

The cancer includes solid cancers such as lung cancer, stomach cancer, lymph gland cancer, liver cancer, and non-solid cancers such as leukemia.

The chaga extract is extracted from mushrooms selected from artificially cultivated or wild mushrooms of the genus Puscoporia oblquus, Fuscoporia punctata and Inonotus obliquus. Containing extracts.

In addition, the chaga extract includes the surface portion and flesh portion of the mushroom.

In addition, the present invention provides a method for preparing the chaga extract.

More specifically,

(a) about 1 to 1000 times the volume of chaga mushroom weight, preferably about 10 to 100 times the volume of water, and then 20 to 300 ℃, preferably 60 to 200 ℃ 1 minute to 10 days, preferably Preferably, the first step of performing extraction by autoclaving, reflux cooling extraction, preferably extraction by autoclaving with stirring for 12 hours to 3 days,

(b) a second step of centrifuging the extract to recover only the precipitate and re-extracting according to the first step process;

(c) Suspension is added to the precipitate by adding about 1 to 1000 times the volume by weight, preferably about 10 to 100 times the volume of methanol, ethanol, butanol and the like, and a lower alcohol, preferably ethanol, and centrifuging the suspension. A third step of concentrating the obtained supernatant under reduced pressure,

(d) provides a method for extracting chaga extract comprising the fourth step of purifying by adding the same amount of an organic solvent, preferably ethanol to the concentrate.

In another aspect, the present invention provides a pharmaceutical composition for treating immunodeficiency and cancer containing chaga mushroom extract prepared by the above method as an active ingredient.

The composition comprising the mushroom extract of the present invention of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

As a means for verifying the immunity enhancement and anticancer efficacy of the present invention, the proliferation test and IL-2 activity test of the splenocytes after intraperitoneal injection also compared to the normal culture and scavengerae mushroom used as control in the NO production capacity test of the peritoneal cells When extracts of natural chaga show excellent activity. In addition, in the experiment of inhibiting cancer cells, Chaga mushroom extract was weaker than TAXOL and mistlettoe extracts used as anticancer drugs in a mouse exposed to cancer cells after administration of various cancer cell lines to mice. It can be seen that there is better cancer cell suppression than mushrooms.

The composition comprising the extract of the present invention may further comprise a suitable carrier, excipient and diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the composition comprising the extract of the present invention 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 formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used.

The amount of the extract of the present invention may vary depending on the age, sex, and weight of the patient, but may be administered once to several times in an amount of 0.1 to 100 mg / kg. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

In addition, chaga extract of the present invention can be used as a main, secondary ingredients and food additives of other foods.

In another aspect, the present invention provides a dietary supplement containing chaga extract and food-acceptable food supplement additives for immune enhancement and cancer prevention.

The composition comprising the extract of the present invention can be used in various ways, such as drugs, foods and beverages for immune enhancement or cancer prevention and treatment. Examples of the food to which the extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

Extract itself of the present invention is a drug that can be used with confidence even for long-term administration for the purpose of prevention because there is little toxicity and side effects.

The extract of the present invention may be added to food or beverages for the purpose of immune enhancement and cancer prevention. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 1 g.

The health 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; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and 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, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention is described in more detail based on the following examples, although the present invention is not limited thereto.

Example 1. Preparation of chaga hot water extract (1)

1 kg of dried chaga mushrooms from Siberia and China were selected to remove the birch bark, and the coal-like surface (black) was separated and chopped, and then 10 liters of purified water was added and 4 hours at 100 ° C. Extracted by autoclaving with stirring for a while, the extracted extract was centrifuged to recover the precipitate and the obtained supernatant, and the supernatant was used as a sample.

Example 2. Preparation of Chaga Hot Water Extract (2)

1 kg of dried chaga mushrooms from Siberia and China were selected and the birch bark was removed, and the contents of chaga, ie, flesh parts (yellow), were separated and chopped, and then 10 liters of purified water was added at 100 ° C. The mixture was extracted by autoclaving with stirring for 4 hours, the extracted extract was centrifuged to recover the precipitate and the obtained supernatant, and the supernatant was used as a sample.

Comparative Example 1. Preparation of chaga cold water extract (1)

Select 1 kg of dried chaga from Siberia and China, remove the birch bark, remove the coal-like surface (black), and then chop each, and add 10 liters of purified water for 4 hours at room temperature. The mixture was extracted by autoclaving with stirring, the extracted extract was centrifuged to recover the precipitate and the obtained supernatant, and the supernatant was used as a sample.

Comparative Example 2. Preparation of Chaga Mushroom Cold Water Extract (2)

Select 1 kg of dried chaga mushroom from Siberia and China, remove the birch bark, separate the contents of chaga, ie, meat parts (yellow), and then cut them separately. Add 10 liter of purified water and 4 hours at room temperature. Extracted by autoclaving with stirring for a while, the extracted extract was centrifuged to recover the precipitate and the obtained supernatant, and the supernatant was used as a sample.

Experimental Example 1. Immune Enhancement Efficacy Experiment

In order to confirm the efficacy of the immunity enhancement of the mushroom extracts of the present invention, the following experiment was performed.

The experimental animals used in the animal experiments were collected four weeks old BALB / C rats (30 ± 10g ♂, Daehan Biolink), the mice's abdominal cells three days after the intraperitoneal injection of the functional chaga supernatant of the present invention to mice Cell counts were plated at 2 × 10 ⁴ cells / well and plated at 2 × 10 ⁴ cells / well, incubated at 37 ° C., 5% carbon dioxide for 44 hours, and shaken at room temperature for 15 minutes after 4 hours of incubation with MTT. The absorbance at 550 nm was then measured with an ELISA reader for a 96 well plate. This absorbance represents the amount of MTT reduced by the cells and is proportional to the number of viable cells present in each well [Mosmann, T., J. Immunol. Methods, 65, pp 55-58, 1983; Boekhorst, PAW, Cancer. Chemother. Pharmacol., 30, p238, 1992].

Intraperitoneal and splenic cells of the same mouse were incubated for 16 to 20 hours at 37 ° C. and 5% carbon dioxide by adding LPS or a sample to a 96 well plate at 2 × 10 ⁴ cells / well, and then gently shaken with grease reagent. , The absorbance was measured at 550nm to determine the content of NO [Miesel, R, et al., Free Radical Biology & medicine, 20 (1), pp75-81, 1996].

In addition, Ab was diluted 1: 250 using a coating buffer (coating buffer), and the wells were dispensed into the wells, and the lid was left overnight. The wells were washed with the wash buffer the next day. After the assay diluent was added and allowed to stand at room temperature for 1 hour, 100 μl of the sample was added to each well, which was left at room temperature for 2 hours, washed five times, and the TMB substrate solution was added and the lid was not closed. After leaving in the dark for 30 minutes, the reaction stop solution was added, and the absorbance at 450 nm was measured to measure IL-2 production capacity [Oyama, Y, et al., Jpn. J. Pharmacol, 72, pp 381-385, 1996].

Proliferation of Spleen Cells after Intraperitoneal Injection

After 3 days of 100 μL intraperitoneal injection at a concentration of 5 mg / mL of the sample, spleen cells were stimulated with LPS or ConA to measure lymphocyte activity in the splenocytes by MTT method.

For reference, LPS (Lipopolysaccharide) is a glycolipid extracted from Gram negative bacteria cell wall, a mitogen that activates B cells, and ConA (Concanavalin A) is a glycoprotein derived from soybean, which activates T cells. The state is mitogen.

Comparing the results of Figure 1, chaga 1 is chaga extract extract (Example 1), chaga 2 is chaga extract extract (Example 2), roe deer 1 and roe deer 2 is a fruiting body Mycelium is the medium and normal culture is used as a control. Chaga extract induced the activity of splenocytes compared to the control, and was similar or slightly higher than the roe deer 1 and roe deer 2 used as the control.

IL-2 Activity Test of Splenocytes after Intraperitoneal Injection

After 3 days of intraperitoneal injection at 100 μL sample concentration of 5 mg / ml, splenocytes were stimulated with ConA to measure the activity of IL-2 (interleukin-2) in splenocytes. The function of IL-2 is generally known to activate T cells and induce differentiation of B cells. The medium used as a control and the extracts of the roe deer mushroom show higher activity.

NO Production of Peritoneal Cells after Intraperitoneal Injection

After 3 days of intraperitoneal injection at 200 μl sample concentration of 5 mg / ml, the peritoneal cells were stimulated with LPS, and the results of measuring NO (nitric oxide) production in the peritoneal cells were shown in FIG. 3. NO is known to act as a catalyst for antimicrobial activity and macrophage activation due to its antimicrobial activity. When stimulated with LPS, NO production in peritoneal cells was higher than that in the control group.

Experimental Example 2. Anticancer Test

In order to confirm the therapeutic efficacy of the mushroom extracts of the present invention against cancer, the following experiment was performed.

The experimental animals used in this animal experiment was a human gastric carcinoma cell line (human gastric carcinoma, KATO3-ATCC HTB103, USA, Figure 4) using a 4 week old BALB / C rat (30 ± 10g ♂, Korea Biolink) Mouse macrophage cell line, Raw264.7 (B, C) -ATCC TIB 103, USA, see FIG. 5), human acute T leukemia cell line, Jurkat cell line (E) )-ATCC TIB 152 (US, see FIG. 6), mouse lymphoma cell line (EL4 (D)-ATCC TIB 39, USA, see FIG. 7), 22 hours (B) Or after 44 hours (all others) incubation, three days after the intraperitoneal injection of the functional chaga supernatant of the present invention, mouse peritoneal cells were collected and cell counted (plating) at 2 × 10 ⁴ cells / well After 44 hours of incubation at 37 ° C. and 5% carbon dioxide, MTT was added for 4 hours, followed by shaking for 15 minutes at room temperature. Absorbance at 550 nm was measured with an ELISA reader for a 96 well plate. This absorbance represents the amount of MTT reduced by the cells and is proportional to the number of viable cells present in each well [Mosmann, T., J. Immunol. Methods, 65, pp 55-58, 1983; Boekhorst, PAW, Cancer. Chemother. Pharmacol., 30, p238, 1992]. As a control, a sample of the indicated concentrations of Taxol (ng / mL), mistletoe (Mistletoe extract) (μg / mL) and Agaricus mushroom (mg / mL) used as an anticancer agent was measured.

As a result of the experiment, it was confirmed that chaga mushroom extract using the same concentration (mg / ㎖) showed an excellent cell growth inhibition than the extract of Agaricus mushroom.

Experimental Example 3. Acute Toxicity Test

1. Oral administration

ICR mice and Sprague Dawley (Central Experimental Animals) were divided into four groups of ten animals each, orally administered with the extracts of Example 1 of the present invention at doses of 500, 725, 1000 and 5000 mg / kg, respectively. After two weeks of toxicity, none of the four groups died and no symptoms were found.

2. Intraperitoneal administration

ICR mice (25 ± 5 g) and Sprague Dawley (Central Experimental Animal) were divided into four groups of 10 animals each, and the extracts of Example 1 of the present invention were 25, 250, 500 and 725 mg, respectively. Toxicity was observed for 24 hours after intraperitoneal administration at / kg. There were no deaths in all four groups and no symptoms were apparent in the control group.

From the above results, it was confirmed that the extract of the present invention has little acute toxicity.

Hereinafter, an example of the preparation of the pharmaceutical composition will be described, but it is not intended to limit the present invention but merely to explain in detail.

Formulation Example 1 Preparation of Injection

Example 1 100 mg of dry extract

Sodium Metabisulfite3.0mg

Methylparaben 0.8mg

Propylparaben 0.1mg

Sterile distillation volume for injection

The above ingredients were mixed and adjusted to 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Example 1 200 mg of dry extract

Lactose 100mg

Starch 100mg

Magnesium stearate

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

Formulation Example 3 Preparation of Capsule

Example 1 100 mg of dry extract

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate

The capsules were prepared by mixing the above components and filling the gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Example 1 1000 mg of dry extract

20g sugar

Isomerized sugar 20g

Lemon flavor

Purified water was added to adjust the total volume to 100 ml.

The above-mentioned components were mixed according to a conventional method for preparing a liquid solution, filled into a 100 ml brown bottle, and sterilized to prepare a liquid solution.

Formulation Example 5 Preparation of Health Beverage

Example 1 Dry Powder 0.1-80%

Sugar 5 ~ 10%

Citric acid0.05 ~ 0.3%

Caramel 0.005 ~ 0.02%

Vitamin C0.1 ~ 1%

79 to 94% of purified water is mixed here to make a syrup, and the syrup is sterilized at 85 to 98 ° C. for 20 to 180 seconds, mixed with cooling water at a ratio of 1: 4, and then prepared by injecting carbon dioxide to 0.5 to 0.82%. A carbonated beverage containing mushroom dry extract was prepared.

Instant sterilization by homogeneously mixing the dry powder of Example 1 with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) This was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

Chaga mushroom according to the present invention is a material that exists in nature, and thus has an excellent effect as an antioxidant, lipid metabolism, immunity enhancement, and anti-cancer material, and thus is a very useful invention in the biopharmaceutical industry.

Claims (9)

Pharmaceutical composition for immuno-enhancement containing chaga extract. A pharmaceutical composition for treating cancer containing chaga extract. The composition of claim 2, wherein the cancer is solid cancer such as lung cancer, gastric cancer, lymph gland cancer, liver cancer, or non-solid cancer such as leukemia. The composition of claim 1 or 2, wherein the chaga is artificially cultivated or is naturally produced. The method of claim 1 or 2, wherein the chaga is a mushroom of the genus Fuscoporia, Fuscoporia oblquus, Fuscoporia punctata and Inonotus obliquus The composition is the selected mushroom. The composition of claim 1 or 2, wherein the chaga extract is extracted from the surface and flesh portions of the mushroom. Dietary supplement comprising chaga extract and immunologically acceptable food supplement additives for boosting immunity. Dietary supplement comprising chaga extract and cancer food supplement additives for cancer prevention. The health supplement food according to claim 7 or 8, which is a health drink.