KR20180039337A - Method of Manufacturing Sparassis crispa Extract using Organic Solvent - Google Patents

Abstract

The present invention relates to a method of preparing an extract of Sparassis crispa by using an organic solvent. More specifically, the present invention relates to a pharmaceutical composition for treating cancer and a food for alleviating cancer, comprising an extract of Sparassis crispa having an anticancer activity, an anti-angiogenic effect, and a neuronal cell protective effect, wherein the extract of Sparassis crispa is prepared using an organic solvent containing ethanol, distilled water, and chloroform. The extract of Sparassis crispa according to the present invention inhibits metastatic and propagation abilities of cancer cells such as stomach cancer, skin cancer, cervical cancer, breast cancer, liver cancer, and brain cancer; inhibits the ability of vascular endothelial cells to differentiate such as invasiveness and tube formation of vascular endothelial cells; and shows a protective effect against death of neuroglial cells induced by a neurotoxin, glutamate, and thus may be beneficially used as a pharmaceutical composition for treating cancer and a food for alleviating cancer, comprising an extract of Sparassis crispa as an effective component.

Description

Technical Field [0001] The present invention relates to a method for producing an extract of Sparassis crispa using an organic solvent,

The present invention relates to a method for producing a sparassis mushroom More particularly, the present invention relates to a method for producing an extract of Chrysanthemum morbus by using an organic solvent including ethanol, distilled water and chloroform, and a method of producing an extract of Zygomycorrhizae mushroom having anticancer activity, And to a food for cancer improvement.

Mushrooms contain essential nutrients such as essential amino acids, fatty acids, and vitamins, and have a unique taste and aroma. They have been used mainly for edible purposes, and only a few have been used for medicinal purposes. However, with the development of science and technology, the systematic research has clarified the pharmacological components of mushrooms and attempts to use them for various disease prevention and treatment have led to a growing interest in mushrooms as medicinal as well as edible.

Sparassis crispa is a mushroom belonging to Aphyllophorales, Sparassidaceae and Sparassis. It is distributed in Korea, Japan, China, Europe and North America. It is a stubble of conifer or It is known to occur around it and parasitize its roots.

Recently, we found that the content of β-1,3-D-glucan, which is effective for improving the immune system, is about 3 to 4 times higher than other mushrooms by about 45% This is because there was a research report of Japan. In particular, the results of various pharmacological activities of Beta Glucan of Zygomycetes mushroom showed that it inhibited tumor growth (Ohno et al., 2000), allergic symptom improving action (Yiu and Ushio, 1989), human NK cell activating action (Harada T. et al , 2002), inhibition of blood glucose level elevation, inhibition of cholesterol elevation, and inhibition of hypertension (Morigiwa et al., 1986). In addition, it has been confirmed that the blossom mushroom contains a large amount of functional vitamins such as natural tocopherol, and it is evaluated as having high value as an edible mushroom because it is rich in minerals and has a higher amino acid content than other mushrooms.

At present, products processed by formulations such as granules and capsules have been developed in Japan. Recently, antioxidant activity, collagen production promoting action, and whitening action function of P. falciparum have been confirmed and commercialized as a health food for cosmetics.

In 1985, the artificial cultivation of chrysanthemum mushroom was carried out in China, and in 1997, the success of sawdust cultivation in Japan began to become widespread. In Korea, the degree of culture characteristics of P. falciparum began to be studied in the 1990s, and studies on cultivation and physiological activity have been actively conducted since the 2000s. In Korea, several patents related to the optimal cultivation method of the mushroom were registered, but the cultivation methods have not been widely spread yet.

Recently, a nutritional additive for cultivating mushroom cultivated with vegetable lactic acid bacteria and yeast, a method of producing the same, and a method of cultivating mushroom cultivated with the same have been developed, and a high quality blossom having 45% to 50% of a core functional ingredient beta glucan The technology for mass production of mushrooms has been secured, which has laid the foundations for the development of medicinal mushroom products with high quality and low prices (Korean Patent No. 1480396).

Methods for extracting active ingredients and taking them for a long period of time have been researched since the absorption of the active ingredient by the human body is small when the mushroom is directly consumed with fresh juice. However, when the hot water extraction method is used, it is difficult to extract the nonpolar low-molecular compounds showing useful activity and denaturing the effective components of the mushroom. In order to overcome the drawbacks of hydrothermal extraction, another extraction method using an organic solvent such as alcohol is used for the production of the mushroom extract. However, for the process technology obtained by concentrating only the fractions containing the components having specific effects, There is no known.

The present inventors have made intensive efforts to obtain the fractions in which the components exhibiting specific effects have been supplemented to overcome the disadvantages of the hot water extraction method. As a result, it has been found that the extract of Zanthoxylum mushroom prepared using an organic solvent can be used for gastric cancer, skin cancer, cervical cancer, , Can inhibit the metastatic ability and proliferative capacity of cancer cells such as brain cancer, inhibit the differentiation ability of vascular endothelial cells such as invasiveness and tubular formation of vascular endothelial cells, and inhibit glutamate-induced neuroblast cell death The present inventors confirmed that they have a protective effect and completed the present invention.

It is an object of the present invention to provide a method for producing a mushroom extract of Rosemary mushroom using an organic solvent.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of cancer, which comprises an extract of Zanthoxylum mushroom, produced by the above method, as an active ingredient.

Another object of the present invention is to provide a cancer-improving food comprising an effective amount of a mushroom extract prepared by the above method.

In order to accomplish the above object, the present invention provides a method for producing a mushroom of the present invention, comprising the steps of: (a) extracting the mushroom powder with ethanol and then separating the ethanol extract; (b) extracting the ethanol extract with a mixed solvent of ethanol and hexane, and then removing the hexane layer; (c) removing the hexane layer, extracting the remaining residue with a mixed solvent of distilled water and chloroform, and then removing the distilled water layer; And (d) removing the distilled water layer, adding 1% sodium chloride to the remaining residue, and then extracting the chloroform layer to obtain a mushroom extract of P. japonica.

The present invention also provides a pharmaceutical composition for the treatment of cancer, comprising an extract of P. falciparum prepared by the above method as an active ingredient.

The present invention also provides a food for cancer improvement comprising an extract of Zanthoxylum mushroom produced by the above method as an active ingredient.

The mushroom extract according to the present invention inhibits the metastatic and proliferative capacity of cancer cells such as stomach cancer, skin cancer, cervical cancer, breast cancer, liver cancer and brain cancer and inhibits the differentiation ability of vascular endothelial cells such as vascular endothelial cell invasion and tube formation And can be effectively used as a pharmaceutical composition for treating cancer and a composition for improving cancer, which contains a Zygomycorr mushroom extract as an active ingredient, since it has a protective effect against glutamate-induced neurotrophic factor death, which is a neurotoxin.

FIG. 1 is a schematic view illustrating a method of manufacturing a mushroom extract according to an embodiment of the present invention.
FIG. 2 shows HPLC analysis results of the mushroom extract prepared according to an embodiment of the present invention.
FIG. 3 shows the result of the preparative-HPLC analysis and the amount of each fraction obtained from the mushroom extract prepared according to an embodiment of the present invention.
FIG. 4 is a graph illustrating the results of measuring the growth inhibitory activity of cancer cells after treating the extract of Zanthoxylum mushroom, prepared according to one embodiment of the present invention, at different concentrations.
FIG. 5 shows the results of measuring the activity of inhibiting the migration of cancer cells in the gastric cancer cell line (AGS) after treatment of the mushroom extract prepared according to one embodiment of the present invention at a concentration of 100 μg / ml.
FIG. 6 is a graph showing the results of measuring the inhibitory activity of the vascular endothelial cells (HUVEC) after treating the mushroom extract prepared according to an embodiment of the present invention at a concentration of 25 μg / ml.
FIG. 7 shows the results of measuring the inhibitory activity of vascular endothelial cells (HUVEC) after treatment of P. japonicus extract prepared according to one embodiment of the present invention at a concentration of 25 μg / ml.
FIG. 8 shows the results of measuring the protective activity against the extinction of C6 glial cells induced by excessive glutamate treatment after treating the extract of Zanthoxylum mushroom, prepared according to one embodiment of the present invention, .

The present invention can be all accomplished by the following description. It is to be understood that the following description is only illustrative of preferred embodiments of the invention, but the invention is not necessarily limited thereto. It is to be understood that the accompanying drawings are included to provide a further understanding of the invention and are not to be construed as limiting the present invention. The details of the individual components may be properly understood by reference to the following detailed description of the related description.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, the extract of Zanthoxylum mushroom was prepared using an organic solvent. The Zanthoxylum mushroom extract inhibited the metastatic and proliferative capacity of cancer cells such as gastric cancer, skin cancer, cervical cancer, breast cancer, liver cancer and brain cancer, And tubular formation, and has a protective effect against neuroblastoma induced by glutamate which is a neurotoxin. Therefore, it is possible to provide a pharmaceutical composition for treating cancer comprising an extract of Zygomycorr mushroom as an active ingredient, It can be usefully used as a cancer-improving food.

In one aspect, the present invention provides a method for producing a mushroom, comprising: (a) extracting a mushroom powder with ethanol and then separating the ethanol extract; (b) extracting the ethanol extract with a mixed solvent of ethanol and hexane, and then removing the hexane layer; (c) removing the hexane layer, extracting the remaining residue with a mixed solvent of distilled water and chloroform, and then removing the distilled water layer; And (d) removing the distilled water layer, adding 1% sodium chloride to the remainder, extracting the chloroform layer to obtain an extract of P. falciparum mushroom.

In the present invention, it is preferable that (e) the obtained Rosemary mushroom extract further comprises fractionating the fraction containing the active ingredient by preparative HPLC.

In the present invention, the mixed solvent of step (b) preferably has a volume ratio of ethanol and hexane of 1: 0.1 to 1: 5, and most preferably ethanol: hexane of 2: 3.

The ethanol used for the mixed solvent is more preferably 90% ethanol, but not limited thereto.

In the present invention, the mixed solvent of step (c) preferably has a volume ratio of distilled water and chloroform of 1: 0.1 to 1: 5, and most preferably distilled water: chloroform = 2: 3.

In another aspect, the present invention relates to a pharmaceutical composition for treating cancer, which comprises an extract of Zanthoxylum mushroom, produced by the above method, as an active ingredient.

In the present invention, it is preferable that the cancer is gastric cancer, skin cancer, cervical cancer, breast cancer, liver cancer or brain cancer.

The pharmaceutical composition for treating cancer, which contains the above-described mushroom extract as an active ingredient, may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the pharmaceutical composition for treating cancer comprising the extract of Mushroom Mushroom of the present invention as an active ingredient can be administered in the form of powders, pills, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, , A suspension, a freeze-drying agent, and a suppository.

In the case of formulation, a diluent or excipient such as a commonly used filler, an extender, a binder, a wetting agent, a disintegrant or a surfactant may be used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 60, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be determined depending on the health condition of the patient, The dosage, the excretion rate, the degree of disease, the type of drug, the time of administration, the route of administration, and the period of administration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the mushroom extract of the present invention contains 0.1 mg / kg (body weight) to 500 mg / kg (body weight), more preferably 0.5 mg / kg mg / kg (body weight), and the administration may be carried out once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of cancer diseases.

In another aspect, the present invention relates to a food for cancer improvement comprising an extract of Zanthoxylum mushroom, produced by the above method, as an active ingredient.

The cancers whose symptoms can be improved by the food according to the present invention are as described above.

In the present invention, the food includes all forms such as functional food, nutritional supplement, health food, and food additives. These types of health functional foods can be prepared in various forms according to conventional methods known in the art. For example, as a health food, the mushroom extract of the present invention may be prepared in the form of tea, juice, and drink and then consumed for drinking, granulated, encapsulated and powdered. Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (such as canned fruits, bottled, jam, marmalade, etc.), fish, meat and processed foods such as ham, sausage, (Such as butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, fruit juice, fruit juice, Frozen foods, various kinds of seasonings (for example, soybean paste, soy sauce, sauces, etc.) by adding the mushroom extract of the present invention.

The health functional food also includes various forms such as a functional food, a nutritional supplement, a health food, a food additive and the like as a food composition. The health functional food may be prepared in various forms according to conventional methods known in the art, such as the above- The present invention relates to a method for producing the above-mentioned compound or extract in the form of tea, juice or drink, granulating, encapsulating or pulverizing the same, Or by adding them.

As used herein, the term " active ingredient "alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which is itself inactive.

[ Example ]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example  1: Manufacture of Extract of Mushroom

In this Example, the extract of Zygomycorrhizae mushroom was prepared using Zygomorph mushroom powder and organic solvent (see Fig. 1).

10 g of the dried mushroom powder was added to 1 L of ethanol (CH ₃ CH ₂ OH), and the mixture was stirred at room temperature for 24 hours. The mixture was then filtered through a glass filter to remove the powder of mushroom. The sample obtained by evaporating the ethanol extract from which the mushroom powder was removed by evaporation was dissolved in 200 mL of 90% ethanol, 300 mL of hexane (C ₆ H ₁₄ ) was added to the dissolved sample, and the mixture was stirred for 3 hours. . 200 mL of distilled water and 300 mL of chloroform (CHCl ₃ ) were added to the sample from which the hexane layer was removed, and the mixture was stirred for 3 hours, and the distilled water layer was separated. 200 mL of 1% sodium chloride (NaCl) was added to the sample from which the distilled water layer had been removed, and the mixture was stirred for 0.5 hour. Then, the chloroform layer was separated, and the chestnut mushroom extract contained in the separated chloroform layer was obtained.

Example  2: Analysis and isolation of Mushroom extract

In order to analyze the physiologically active substance contained in the chloroform layer obtained in Example 1, the extract of Zygomorph mushroom was analyzed and separated by using a partition type high performance liquid chromatography.

Example  2-1: Analysis of Mushroom Extract

Analysis was performed using high performance liquid chromatography (HPLC) in order to analyze the physiologically active substance contained in the mushroom extract obtained from Example 1. Shimadzu LC-2030C 3D was used for the sample analysis, and 10 μl was injected by filtration through a 0.45 μm membrane filter. The column was subjected to Shim-peck GIS (5 μm ODS, 250 × 4.6 mm id 10-70101-05) (1 ml / min). The solvent was analyzed for 60 minutes using a gradient (A) solution (water containing 0.05% trifluoroacetic acid) and B solution (acetonitrile). Samples were analyzed using a UV detector, and the samples extracted at 200, 230, 250, 270, 290 and 300 nm of the analytical wavelength were respectively identified (see FIG. 2).

Example  2-2: Extract of Mushroom Extract Fraction  detach

In order to analyze the physiologically active substances contained in the mushroom extract obtained from Example 1, seven sections were separated using preparative HPLC (Thermo Ultimate 3000 prep). The column was packed with YMC-Pack ODS-AQ-HG (250 x 20 mm lD S-10 μm, 12 nm, AQG12S11-2520WT, No.2025002045) at a flow rate of 10 ml / (Water containing 0.05% trifluoroacetic acid) and B solution (acetonitrile) for a total of 120 minutes using a gradient program. The samples were analyzed using a UV detector and the analysis wavelengths were 200, 230, 250, 270, 290, and 300 nm.

(Fraction 1), 2 mg (fraction 2), 2 mg (fraction 2), 1.3 mg (fraction 3), and 4 mg 4), 9.6 mg (fraction 5) in the 5th zone, 17.4 mg (fraction 6) in the 6th zone and 3.5 mg (fraction 7) in the 7th zone.

Example  3: Anticancer Activity of Extract of Mushroom Extract

In this Example, the anticancer activity of the mushroom extract (fractions 1 to 7) obtained in Example 2-2 was confirmed. The cancer cell cultures were purchased from Korean Cell Line Bank, B16F10 malignant melanoma cell line, AGS cancer cell line, HepG2 liver cancer cell line, HeLa cervical cancer cell line, MCF-7 breast cancer cell line, and U87MG brain tumor cell line. DMEM medium containing 10% (v / v) FBS and 1X Pen / Strep / Amph B was used for B16F10, HepG2, HeLa and MCF-7. U87MG was used for MEM medium and AGS was used for RPMI medium And cultured in a carbon dioxide cell incubator at 37 ° C in a 5% CO ₂ atmosphere.

Example  3-1: Cancer cell proliferation inhibitory activity

The cancer cell proliferation inhibitory activity of P. falciparum mushroom extract was measured by MTT assay. (2 × 10 3 cells / well) were seeded in 96-well plates and cultured for 24 hours. Then, each of the cells was subjected to serial dilution (100 μg / ml) Lt; / RTI > After incubation for 3 days, 50 μl of MTT solution (2 mg / ml) was added and reacted for 3 hours. After carefully removing the medium, 100 μl of dimethylsulfoxide (DMSO) was added to dissolve the formazan precipitate produced by MTT reduction, and then the absorbance was measured at 540 nm using a microplate reader. The concentration of the extract (100 μg / ml , 50 / / ㎖, 25 / / ㎖, 12.5 ㎍ / ㎖, 6.25 ㎍ / ㎖, 3.125 ㎍ / ㎖).

FIG. 4 shows the results of measuring the inhibitory activity of the growth of cancer cells after treatment of each of the cancer cells with the concentration of the extract of Zygomycorruscinus japonica (fractions 1 to 7). In all of the six carcinomas, fraction 4 showed the highest cancer cell proliferation inhibitory activity .

Example  3-2: Cancer cell transfer inhibitory activity of extract

The inhibitory effect on the cancer cell metastasis ability of the mushroom extract was investigated using a wound healing assay. The cells were inoculated (3x105 / well) in a 24-well plate to confluent more than 90% of AGS gastric cancer cells. After attachment of the cells, the bottom of the plate was scratched using a pipette tip to make a wound. Cells rinsed off with PBS were removed and the medium treated with the extract was added. After incubation for 24 hours, the wound area was photographed with a microscope to observe the inhibitory effect on the migration ability of cancer cells.

FIG. 5 shows the results of measuring the inhibitory activity against the movement of cancer cells after treatment with a concentration of 100 μg / ml of Zygomycota mushroom extract (fractions 1 to 7). In fractions 4 and 5, the highest inhibitory activity against cancer cell transformation I could see what was visible.

Example  4: Extract of Mushroom Extract Angiogenesis  activation

In this Example, the vascular endothelial cell (HUVEC) invasion and the ability to inhibit the tube formation ability were confirmed in order to confirm the anti-angiogenic activity of the mushroom extract (fractions 1 to 7) obtained in Example 2-2.

Example  4-1: Suppression of vascular endothelial cell invasion by the extract of Mushroom mushroom

Inhibition of vascular endothelial cell penetration by Zygomyksis mushroom extract was observed by invasion assay. The EBM-2 medium of the cultured HUVECs was removed, treated with serum-free EBM-2 medium, and cultured for 24 hours. Invasion transwell membrane (8 μm pore) was coated with 10 μl of gelatin (1 ㎎ / ㎖), dried and coated with 10 μl of Matrigel (3 ㎎ / ㎖) and dried. Wells were treated with 600 μl of serum-free EBM-2 medium and 25 μg / ml of Mushroom mushroom extract, respectively, and treated with VEGF (30 ng / ml), an angiogenic factor. HUVECs (7 × 10 4 cells) were added to each transwell and cultured for 18 hours. 70% methanol was used to immobilize the invasion cells. Cells were fixed with transwell membrane using hematoxylin and eosin to stain nuclei and cytoplasm and dehydrated with ethanol. The inside of each well was carefully wiped off with a cotton swab and the degree of cell permeation inhibiting activity was observed using a microscope.

FIG. 6 shows the results of measuring the inhibitory activity against the infiltration of vascular endothelial cells (HUVEC) after treatment with 25 μg / ml of Zygomycorr mushroom extract (fractions 1 to 7). In the fraction 4, the highest inhibition of vascular endothelial cell invasion Activity.

Example  4-2: Vascular endothelial cell line of Brassica juncea Extract Formability  control

The inhibitory activity of vascular endothelial cell line formation inhibited by the extract of Mushroom Mushroom Extract was observed using tube formation assay. The EBM-2 medium of the cultured HUVECs was removed, treated with serum-free EBM-2 medium, and cultured for 24 hours. Each well of a 48-well plate was coated with Matrigel (10 μg / μl), and then 400 μl of serum-free EBM-2 medium containing 25 μg / ml of Mushroom Extract and VEGF (30 ng / , And HUVECs (7 x 10 < 4 > cells) were subcultured for 3 hours. The degree of inhibition of tube formation was observed using a microscope.

FIG. 7 shows the results of measuring the inhibitory activity of vascular endothelial cells (HUVEC) against vascular endothelial cells after treating 25 mu g / ml of the extract of Brassica juncea (fractions 1 to 7). In the fraction 4, Formation inhibitory activity.

Example  5: Extract of Mushroom Extract Cranial nerve cell protection  activation

In this example, the protective effect of glutamate-induced glial cell glial cells, C6 glial cells, was evaluated using the MTT assay in order to measure the neuroprotective effect of Brassica juncea extract. C6 cells were cultured in DMEM supplemented with 10% FBS and 1% Pen / Strep / Amph B. The incubator maintained a temperature of 37 ° C, and 5% CO ₂ was continuously supplied to obtain appropriate conditions for cell culture. The C6 cells were cultured for 1 day at a density of 5 × 10 3 cells / well in a 96-well plate. Then, the extracts of Zygomycorrusca japonica were cultured at concentrations of 50 μg / ml, 25 μg / ml, 12.5 μg / ㎍ / ㎖, 3.125 ㎍ / ㎖) for 1 hour, and 23 mM L-glutamate was added to induce cytotoxicity, followed by further culture for 24 hours. The MTT solution was added and reacted at 37 ° C. for 3 hours. After the formazan formed by DMSO was completely dissolved, the absorbance at 540 nm was measured. Cell viability is expressed as a percentage of the survival rate of the control.

FIG. 8 shows the results of measuring the protective activity against the extinction of C6 glial cells induced by excessive glutamate treatment after treating the extracts of Foliar mushroom (fractions 1 to 7) by concentration. Fractions 4, 5, and 7, respectively, showing protective activity against neuronal cell death.

Claims (8)

A method for preparing a mushroom extract comprising the following steps:
(a) extracting the mushroom powder with ethanol and separating the ethanol extract;
(b) extracting the separated ethanol extract with a mixed solvent of ethanol and hexane, and then removing the hexane layer;
(c) removing the hexane layer, extracting the remaining residue with a mixed solvent of distilled water and chloroform, and then removing the distilled water layer; And
(d) removing the distilled water layer, adding 1% sodium chloride to the remaining residue, and then extracting the chloroform layer to obtain a mushroom extract.
The method according to claim 1, further comprising the step of (e) separating the fractions containing the active ingredient using the fractionated HPLC of the obtained mushroom extract.
[6] The method according to claim 1, wherein the mixed solvent of step (b) comprises ethanol and hexane in a volume ratio of 1: 0.1 to 1: 5.
The method according to claim 1, wherein the mixed solvent of step (c) comprises distilled water and chloroform in a volume ratio of 1: 0.1 to 1: 5.
A pharmaceutical composition for the treatment of cancer, comprising an effective amount of a mushroom extract prepared by the method of any one of claims 1 to 4.
[Claim 7] The pharmaceutical composition according to claim 5, wherein the cancer is gastric cancer, skin cancer, cervical cancer, breast cancer, liver cancer or brain cancer.
6. A cancer-improving food comprising an effective amount of a mushroom extract prepared by the method of any one of claims 1 to 4.
The cancer-improving food according to claim 7, wherein the cancer disease is gastric cancer, skin cancer, cervical cancer, breast cancer, liver cancer or brain cancer.

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