WO2016122261A1 - Pharmaceutical composition for preventing or treating cancer containing extracellular polysaccharide produced by ceriporia lacerata as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing, as an active ingredient: an extracellular polysaccharide produced by Ceriporia lacerate; a mycelium culture of Ceriporia lacerate comprising said extracellular polysaccharide; a dried powder of said mycelium culture; or an extract of said mycelium culture. Said composition can be useful in the prevention or treatment of various kinds of cancers.

Description

Pharmaceutical composition for the prevention or treatment of cancer containing the extracellular polysaccharide produced by Ceriporia laccerata as an active ingredient

The present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it relates to a pharmaceutical composition for preventing or treating cancer containing the extract of the mycelia culture medium as an active ingredient.

Cancer refers to a group of diseases in which a tumor or tumor composed of undifferentiated cells that proliferate indefinitely for a variety of reasons can kill an individual's life by destroying surrounding normal tissues or organs. Chemotherapy for cancer relies on the administration of anti-cancer substances that show strong cytotoxicity, which causes a number of side effects. Therefore, there is a need for the development of new anticancer drugs that can increase the treatment rate while reducing side effects. Therefore, in recent years, research has been made to develop anticancer agents from conventional natural products, especially native plants and herbal medicines. Taxol (Taxol; paclitaxel) isolated from the yew tree is known as a natural-derived anticancer agent, but it has a problem that can cause other diseases because it acts on other normal cells in the body as well as cancer cell death.

Ceriporia laccerata is a type of white fungus and is known to perform co-metabolism called lignin decomposition in order to use carbon sources such as cellulose, hemicellulose, other polysaccharides and glycerol in the ecosystem.

Regarding the medical treatment using Ceriporia laccerata, only the use of Ceriporia laccerata culture extract in Korean Patent No. 10-1031605, filed by the present inventors, is known so far. The effect of cancer prevention or treatment with Ceriporia lacserata has not been reported.

Thus, the present inventors are extracellular polysaccharide produced by the seriporia lacserata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Alternatively, the present invention has been completed by confirming that the extract of the mycelium culture has a cancer prevention or treatment effect.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cancer containing the active ingredient produced by Seriphoria laccerata.

Another object of the present invention to provide a health functional food for cancer prevention or improvement.

In order to achieve the above object, the present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a pharmaceutical composition for cancer prevention or treatment containing the extract of the mycelia culture medium as an active ingredient.

In order to achieve the above object, the present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a functional food for preventing or improving cancer containing the extract of the mycelia culture medium as an active ingredient.

In order to achieve the above object, the present invention is an extracellular polysaccharide produced by Seriporia laccerata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a method for treating cancer comprising administering the extract of the mycelium culture to a subject in need of anticancer activity.

In order to achieve the above object, the present invention is an extracellular polysaccharide produced by Ceriporia laccerata for use in the manufacture of a drug for anticancer; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a use of the extract of the mycelium culture.

Extracellular polysaccharide produced by the seriporia laccerata according to the present invention; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Alternatively, the pharmaceutical composition containing the extract of the mycelium culture medium as an active ingredient may be usefully used for the prevention or treatment of various cancers.

Figure 1 is a graph showing the cell growth inhibitory effect on the hepatocellular carcinoma cell line of the extracellular polysaccharide produced by Seriphoria lac serrata.

Figure 2 is a graph showing the cell growth inhibitory effect on the colon cancer cell line of the extracellular polysaccharide produced by Seriporia laccerata.

Figure 3 is a graph showing the cell growth inhibitory effect on the gastric cancer cell line of the extracellular polysaccharide produced by Seriphoria lac serrata.

Figure 4 is a graph showing the cell growth inhibitory effect on the lung cancer cell line of the extracellular polysaccharide produced by Seriporia laccerata.

Figure 5 is a graph showing the cell growth inhibitory effect on the cervical cancer cell line of the extracellular polysaccharide produced by Seriporia laccerata.

Figure 6 is a graph showing the cell growth inhibitory effect on the skin cancer cell line of the extracellular polysaccharide produced by Seriphoria lac serrata (** P <0.01).

Figure 7 is a graph showing the cell growth inhibitory effect on oral cancer cell lines of the extracellular polysaccharide produced by Seriporia laccerata (** P <0.01).

Figure 8 is a graph showing the cell growth inhibitory effect on the prostate cancer cell line of the extracellular polysaccharide produced by Seriphoria lac serrata (** P <0.01).

Figure 9 is a graph showing the cell growth inhibitory effect on the pancreatic cancer cell line of the extracellular polysaccharide produced by Seriporia laccerata (* P <0.05, ** P <0.01).

Figure 10 is a graph showing the cell growth inhibitory effect on the thyroid cancer cell line of the extracellular polysaccharide produced by Seriphoria lac serrata (** P <0.01).

Figure 11 is a graph showing the cell growth inhibitory effect on the skin cancer cell line of the seriporia laccerata mycelium culture medium (** P <0.01).

12 is a graph showing the cell growth inhibitory effect on oral cancer cell lines of the seriporia laccerata mycelium culture medium (** P <0.01).

Figure 13 is a graph showing the cell growth inhibitory effect on the prostate cancer cell line of the seriporia laccerata mycelium culture medium (** P <0.01).

Figure 14 is a graph showing the cell growth inhibitory effect on the pancreatic cancer cell line of Seriporia laccerata mycelium culture (* P <0.05, ** P <0.01).

Hereinafter, the present invention will be described in detail.

The present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a pharmaceutical composition for cancer prevention or treatment containing the extract of the mycelia culture medium as an active ingredient.

As used herein, the term "extracellular polysaccharide (EPS)" is a part of the cell wall of a microorganism such as a fungus, and the polysaccharide is secreted to the outside of the cell to form a capillary around it or secreted into the periphery or medium as a slime. Means. The extracellular polysaccharide is secreted by the microorganism to protect itself from the external environment such as antibodies, toxic substances, protozoa and bacteriophage.

The extracellular polysaccharide is 40 to 60% by weight sugar and 30 to 40% by weight protein, 40 to 50% by weight sugar and 32 to 38% by weight protein, 43 to 47% by weight sugar and 33 to 36% by weight Protein, or about 45% by weight sugar and about 34% by weight protein.

The sugar may contain mannose, galactose and glucose.

The extracellular polysaccharide may have a molecular weight of 100 to 150 kDa, 110 to 140 kDa or 115 to 125 kDa, and more specifically may have a molecular weight of about 120 kDa.

In one embodiment of the present invention, the extracellular polysaccharide comprises the steps of: (a) liquid culture of the Ceriporia laccerata mycelium to prepare a Ceriporia laccerata mycelium culture solution; (b) drying and powdering the Ceriporia laccerata mycelium culture solution; And (c) extracting the Ceriporia laccerata mycelium culture powder with a solvent, filtering the same, and concentrating under reduced pressure.

The medium for the liquid culture of the seriporia laccerata mycelium in the step (a) is sugar, glucose, starch, hydrate, soybean meal, soybean meal, magnesium sulfate (MgSO ₄ ), potassium phosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and water, and the hydrogen ion concentration (pH) may be 4.5 to 6.0.

Specifically, the medium is 0.2 to 3% by weight of sugar, 0.2 to 3% by weight of glucose, 0.2 to 4% by weight of starch, 0.1 to 0.5% by weight of water, 0.1 to 0.5% by weight of wheat flour, 0.2 to 3% of soy flour %, 0.05 to 0.1 wt% magnesium sulfate (MgSO ₄ ), 0.05 to 0.25 wt% potassium monophosphate (KH ₂ PO ₄ ), 0.05 to 0.25 wt% potassium phosphate (K ₂ HPO ₄ ) and the balance of water Can be.

The liquid culturing in step (a) may be performed under a blue LED light source, and specifically, the liquid culturing may be performed by maintaining a concentration of carbon dioxide at 1,000 to 2,000 ppm under a blue LED light source.

The liquid culture, for example, at 20 to 28 ℃, hydrogen ion concentration of 4.5 to 6.0, light source to maintain a blue LED, illuminance of 0.1 to 0.8 LUX and air is injected at 0.5 to 2.0 kgf / ㎠, The concentration may be performed for 8 to 13 days while maintaining at 1,000 to 2,000 ppm. Specifically, it may be performed for 5 to 15 days at the conditions of 20 to 25 ℃, pH 4.5 to 6.0, 0.5 to 2.0 kgf / cm2 of air injection and 1,000 to 2,000 ppm carbon dioxide concentration. When the liquid culture under the conditions described above, the content of the extracellular polysaccharide produced is high.

In step (a), the parent strain maintains a constant temperature of 25 ° C. in a shaker incubator using PDB (potato dextrose broth) medium in a conical flask with a good strain stored at 1 to 5 ° C. in a PDA (potato dextrose agar) medium. It can be used after the culture process for 7 to 9 days. In addition, after culturing the parent strains, the culture solution or the obtained mycelium can be used as the inoculum. At this time, the amount of mycelia to be added to the inoculum is preferably about 0.5% (w / v) based on the amount of the solution to be cultured. The amount of extracellular polysaccharides does not increase as the amount of mycelia (% / 100 ml, w / v) increases, so the medium composition has the highest content of extracellular polysaccharides, not the best nutritional ratio and environmental conditions for the growth of mycelia. It is preferable to apply the selective culture conditions to form.

The culture solution can be separated and purified into a mycelium and an aqueous solution. In detail, the separation and purification may be performed by repeatedly purifying a solution obtained by removing the mycelium by a centrifuge with a multi-sheet filter press and a vibration centrifugal separator (PALLSEP), and then irradiating ultraviolet (UV) light for 1 minute. have. In addition, the culture solution may be sealed and stored after removing the oxygen, which may bring a change in the content of the active ingredient due to the growth of the mycelia when the mycelium is present in the culture solution.

In step (b), the mycelia culture solution prepared in step (a) may be dried and powdered. The drying may be performed for 48 to 96 hours at a temperature of 40 ℃ or less, more specifically 30 ℃ or less to prevent the disappearance of the active material. In addition, the drying of step (b) is preferable to use a vacuum freeze dryer rather than a vacuum dryer that sets the evaporation temperature relatively high because the change in the effective substance content is minimized.

In the step (c), after extracting the dry powder of the mycelia culture solution obtained in step (b) with a solvent, the extracellular polysaccharide which is the active ingredient of the composition according to the present invention is separated.

Specifically, 100 ml of distilled water is added to 3 to 10 g of the dry powder of the mycelium culture medium, followed by well suspension, followed by centrifugation at 5,000 to 10,000 rpm for 10 to 30 minutes to obtain a supernatant. After adding three times the extraction solvent, it can be placed in a refrigerator at 1 to 5 ° C and allowed to stand for 10 to 15 hours. After only centrifuging the supernatant again at 5,000 to 10,000 rpm for 10 to 30 minutes in the stationary material, the precipitate may be recovered to prepare a crude extracellular polysaccharide. The crude extracellular polysaccharide can be vacuum-dried at 30 ° C. or lower to obtain extracellular polysaccharide.

The extraction solvent may be a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, acetone, ether, chloroform and ethyl acetate or a mixed solvent thereof, and more specifically, water, methanol, ethanol, butanol , Acetone and ethyl acetate may be a solvent selected from the group consisting of, or a mixed solvent thereof, more preferably, water or 50 to 99% (v / v) of ethanol aqueous solution.

The present invention is an extracellular polysaccharide produced by the seriporia lacserata; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Alternatively, the composition for preventing or treating cancer, including the extract of the mycelium culture, may further include a carrier, excipient, and diluent, which are commonly used.

The extracellular polysaccharide may be included in 0.1 to 80% by weight, or 0.1 to 50% by weight relative to the total weight of the pharmaceutical composition for preventing or treating cancer, the mycelium culture medium, the dry powder thereof or the extract of the mycelium culture medium May be appropriately included in an amount corresponding to the content of the extracellular polysaccharide. However, the extracellular polysaccharide; Culture solution comprising the same; Dry powder of the culture solution; Alternatively, the effective amount of the extract of the culture solution may be appropriately adjusted according to the method of use and purpose of the pharmaceutical composition.

The pharmaceutical compositions according to the invention can each be formulated and used according to conventional methods. Suitable formulations include, but are not limited to, tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and the like.

The pharmaceutical compositions according to the invention can be prepared in suitable formulations using pharmaceutically inert organic or inorganic carriers. That is, when the formulation is a tablet, coated tablet, dragee and hard capsule, it may include lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof. In addition, when the formulation is a soft capsule, it may include polyols of vegetable oils, waxes, fats, semisolids and liquids. Furthermore, when the formulation is in the form of a solution or syrup, it may include water, polyols, glycerol, and / or vegetable oils and the like.

The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a humectant, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic agent, an antioxidant, and the like, in addition to the carrier.

The method of administering the pharmaceutical composition according to the present invention can be easily selected according to the dosage form, and can be administered orally or parenterally. The dosage may vary depending on the age, sex, weight, severity, and route of administration of the patient, but is generally 5 to 1,000 mg / kg body weight based on the extracellular polysaccharide, which is an active ingredient, specifically 10 to 600 The amount of mg / kg body weight may be administered once to three times a day. However, the above dosage does not limit the scope of the present invention.

The pharmaceutical composition according to the present invention not only provides excellent cancer prevention or treatment effect, but also has little toxicity and side effects due to the drug, and thus can be used safely even when taken as an anticancer agent for a long time.

Therefore, the pharmaceutical composition of the present invention is a variety of cancers, for example, liver cancer, colon cancer, stomach cancer, lung cancer, cervical cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, central nervous cancer, brain cancer, skin cancer, pancreatic cancer, rectal cancer, esophageal cancer, kidney It can be used for the prevention and treatment of cancer selected from the group consisting of cancer, epithelial cancer, hematologic cancer, oral cancer, prostate cancer and combinations thereof.

Furthermore, the present invention is an extracellular polysaccharide produced by Ceriporia lacerata ( Ceriporia lacerata ); Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or it provides a functional food for preventing or improving cancer containing the extract of the mycelia culture medium as an active ingredient.

The health functional food according to the present invention may be in the form of powder, granules, tablets, capsules or beverages, and may be candy, chocolate, gum, tea, vitamin complexes, health supplements, and the like.

At this time, the extracellular polysaccharide according to the invention contained in the health functional food; Mycelium culture medium containing this; Dry powder of the mycelia culture medium; Alternatively, the content of the extract of the mycelium culture may be included as typically 0.01 to 50% by weight, or 0.1 to 20% by weight of the total food weight. In addition, in the case of a health beverage composition, it may be included in an amount of 0.02 to 10 g, or 0.3 to 1 g based on 100 ml of the health beverage composition.

The food is an extracellular polysaccharide of the present invention; Mycelium culture medium of Seriporia laccerata comprising the same; Dry powder of the mycelia culture medium; Or it may further include a food supplement acceptable food additive with the extract of the mycelia culture medium.

The present invention is an extracellular polysaccharide produced by the seriporia lacserata; Mycelium culture medium of Seriporia laccerata comprising the same; Dry powder of the mycelia culture medium; Or it provides a method for treating cancer comprising administering the extract of the mycelium culture to a subject in need of anticancer activity.

The subject in need of anticancer activity may be a mammalian animal, specifically, a human.

The present invention also provides an extracellular polysaccharide produced by Ceriporia laccerata for use in the manufacture of a medicament for the prevention or treatment of cancer; Mycelium culture medium of Seriporia laccerata comprising the same; Dry powder of the mycelia culture medium; Or it provides a use of the extract of the mycelium culture.

Extracellular polysaccharide produced by the seriporia laccerata; Mycelium culture medium of Seriporia laccerata comprising the same; Dry powder of the mycelia culture medium; Alternatively, the extract of the mycelium culture solution is as described above.

In addition, the anti-cancer activity is a variety of diseases that need to prevent or treat cancer, for example, liver cancer, colon cancer, stomach cancer, lung cancer, cervical cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, central nerve cancer, brain cancer, skin cancer, It can be used for the prevention or treatment of cancers such as pancreatic cancer, rectal cancer, esophageal cancer, kidney cancer, epithelial cancer, blood cancer, oral cancer, prostate cancer and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

[ Example ]

Production Example  One. Serifolia Lakserata  Preparation of Mycelia Culture

The seedlings grown in subcultured oaks from Gyeongsangbuk-do (Korea) were grown by subculture and stored at -80 ° C, and the stored strain was stored in PDA (potato dextrose agar) medium ( 87 plastic cultures; after 2-3 passages in Difco, Becton Dickinson and Company, strains (hereinafter referred to as “PDA culture strains”) were stored in a 4 ° C. refrigerator and used. Then, 600 mL of PDB (potato dextrose broth) medium (Difco, Becton Dickinson and Company) was prepared in an Erlenmeyer flask, and one PDA culture strain was added thereto, followed by shaking culture at 25 ° C. for 8 days to obtain a PDB culture strain.

Meanwhile, for culturing the strain, 1.5% by weight sugar, 0.5% by weight glucose, 0.5% by weight potato starch, 0.25% by weight moisture, 0.25% by weight wheat flour, 0.75% by weight soy flour, 0.05% magnesium sulfate (MgSO ₄ ) %, 0.05 wt% potassium monophosphate (KH ₂ PO ₄ ), 0.05 wt% potassium diphosphate (K ₂ HPO ₄ ) and residual water in a 800 L fermenter with 1.5 kgf / After injecting in cm 2 and sterilizing for 20 minutes, inoculated with 600 ml of the PDB culture strain with a starter in a state of cooling at 23 ° C., while the air was aerated at 0.5 to 1.5 kgf / cm 2, the light source was a blue LED and the illuminance was 0.5 LUX. The concentration of carbon dioxide was maintained at 2,000 ppm, and the culture of Ceriporia laccerata mycelia was prepared by liquid culture at 10 ° C. for 10 days in liquid culture.

Production Example  2. Serifolia Lakserata  Preparation of Culture Powder Dry Powder

The dry powder of the seriporia laccerata mycelium culture medium was prepared by pulverizing the lyophoric lactera mycelium culture medium prepared in Preparation Example 1 by vacuum freeze drying at 25 ° C. for 72 hours using a vacuum freeze dryer. .

Production Example  3. Serifolia Lakserata  Preparation of culture extract

100 ml of distilled water was added to 5 g of the dry powder of the Ceriporia laccerata mycelium culture medium prepared in Preparation Example 2, and then suspended well, followed by centrifugation at 8,000 rpm for 20 minutes, and then the amount of 2- to the supernatant thereof. Three times of ethanol was added and left at 4 ° C. for 12 hours. Subsequently, the supernatant was taken from the stationary material to prepare an extract of the culture medium of the seriporia laccerata.

Production Example  4. Serifolia Lakserata  From culture Extracellular polysaccharide ( extracellular  polysaccharide; (Hereinafter referred to as "EPS")

After centrifugation of the extract of the seriporia laccerata mycelium culture medium obtained in Preparation Example 3 again at 8,000 rpm for 20 minutes, the precipitate was recovered to obtain a crude EPS. The crude EPS was vacuum-dried at 25 ° C. for 72 hours using a vacuum freeze dryer to obtain EPS produced by Ceriporia laccerata.

Example  1. Characterization of EPS

1.1 Gel Permeation Chromatography, GPC Molecular weight of EPS using

The EPS prepared in Preparation Example 4 was dissolved in 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ [adjusting pH to 4 with glacial acetic acid] to 1% (w / v), and then 4,000 rpm. After centrifugation for 0.5 hours, only the supernatant was filtered with a 0.45 μm syringe filter and analyzed by GPC.

GPC analysis conditions using a refractive index as a detector, GPC column was used OHpak SB 805 HQ (Shodex, Japan), mobile phase 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ [adjust the pH to 4 with glacial acetic acid] Was used, and the flow rate of the mobile phase was flowed at a rate of 1.0 mL / minute. Standard curves were prepared using Dextran (American Polymer Corporation, USA) with different molecular weights (130 kDa, 400 kDa, 770 kDa, or 1,200 kDa), and Knauer K-2310 (refractive index (RI) measuring instrument). Germany) to determine the molecular weight of EPS. The measurement conditions are summarized in Table 1 below.

Measurement of molecular weight GPC system Knauer K-501 System column OHpak SB 805 HQ (Shodex, Japan) Mobile phase 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ / pH 4 Flow rate 0.1 ml / min Measuring instrument RI (Knauer K-2310)

As a result, the EPS molecular weight of the present invention was found to be about 120 kDa.

1.2 Determination of sugar and protein content in EPS

The EPS prepared in Preparation Example 4 was second purified and treated with proteolytic enzymes to determine sugar and protein content.

Specifically, the first purified EPS (EPS prepared in Preparation Example 4) is dissolved in distilled water and centrifuged at 8,000 rpm for 20 minutes to separate the supernatant, and the amount of the supernatant is 2-3 times the amount of the separated supernatant. Ethanol was added and placed in a 4 ° C. refrigerator for 12 hours. Thereafter, only the supernatant was taken from the stationary material, which was again centrifuged at 8,000 rpm for 20 minutes, and the precipitate was recovered to obtain a second purified EPS. After dissolving the second purified EPS in distilled water, the proteolytic enzyme alcalase was treated at 50 ° C. for 30 minutes at a concentration of 0.5% (w / v).

Sugar content was measured by the phenol-sulfuric acid method. Specifically, 25 μl of 80% (v / v) phenol was added to 1 mL of the diluted sample, 2.5 mL of sulfuric acid was added, cooled to room temperature, and absorbance was measured at 465 nm to calculate the sugar content.

In addition, protein content was measured by the BCA method (Smith PK et al., Analytical Biochemistry, 150 (1): 76-85, 1985) and bovine serum albumin was used as a standard.

The sugar content and the protein content measured as described above are shown in Table 2 below, and the sugar content was found to be 45 to 51 wt% and the protein content was 33 to 34 wt%.

yield(%) Total sugar content (%) Total protein content (%) EPS 1.22 ± 0.03 45.32 ± 1.41 34.17 ± 0.73 Secondary Refined EPS 0.78 ± 0.01 50.49 ± 0.52 33.50 ± 2.79 Enzyme-treated EPS ^* 0.24 ± 0.06 51.39 ± 1.32 34.61 ± 1.51

^* Enzyme treatment: 0.5% Al Calle Izu, 50 ℃, 30 minutes.

Each figure is mean ± SE (n≥3).

In addition, the analysis of sugar components of EPS revealed that EPS mainly contains mannose, galactose and glucose.

Example  2. Anti-cancer effect verification of EPS Ⅰ

2.1 MTT Assay

In order to confirm the anticancer effect of the EPS prepared in Preparation Example 4, the cell growth inhibition effect on various cancer cells was measured by performing MTT assay for each treatment concentration of the EPS.

The MTT (3- (4,5-dimetylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) is a pale yellow substrate, cleaved by respiratory chain enzymes in the mitochondria of living cells, and produces dark blue formazan (formazan). Since no reaction occurs in dead cells, the amount of formazan produced is used to determine the number of live cells (Ref. Van de Loosdrecht, AA, et al., J. Immunol.Methods , 141 (1): 15-22, 1991 ).

First, Hep3B (liver cancer cell line), DLD-1 (colon cancer cell line), AGS (gastrointestinal cancer cell line), A549 (lung cancer cell line) and HeLa (cervical cancer cell line) obtained from the Korea Cell Line Bank were 5 × 10 plates, respectively. After dispensing at a concentration of ⁴ cells / well, the cells were cultured in a 37 ° C., 5% CO ₂ incubator for one day.

The composition of each cancer cell line is as follows.

Hep3B (Liver cancer cell line): DMEM (Dulbecco Modified Eagle Medium), 10% (v / v) FBS (Fetal Bovine Serum), 1% (v / v) P / S (Penicillin / Streptomycin Solution)

-DLD-1 (colon cancer cell line), AGS (gastric cancer cell line) and A549 (lung cancer cell line): RPMI (Roswell Park Memorial Institute medium), 10% (v / v) FBS, 1% (v / v) P / S

HeLa (cervical cancer cell line): MEM (Minimum Essential Medium), 10% (v / v) FBS, 1% (v / v) P / S

After confirming cell adhesion, the cells were replaced with new culture solution, dissolved in distilled water, and treated with EPS to each well to a concentration of 5, 10, or 20 mg / ml, and incubated in a 37 ° C., 5% CO ₂ incubator for 3 days. The control was incubated under the same conditions by the addition of distilled water.

Here, MTT solution of 1/10 (v / v) of the total medium was treated, and reacted for 4 hours in an incubator to confirm formazan formation, and the supernatant was completely removed so that the formazan did not scatter. . Thereafter, 100 μl of DMSO (dimethyl sulfoxide) was added to each well to dissolve formazan formed in the cells. The absorbance was measured at a wavelength of 570 nm with an ELISA reader to determine the cell viability of each cancer cell line.

2.2 Effect of Liver Cancer Treatment

As shown in Table 3 and FIG. 1, as a result of the MTT assay of Example 2.1, as the treatment concentration of the EPS of the present invention was increased from 5 mg / ml to 20 mg / ml, the cell viability of Hep3B (liver cancer cell line) was increased. It gradually decreased. In particular, when EPS was treated at a concentration of 5 mg / ㎖, showed a cell growth inhibition of about 97%. The results show excellent liver cancer treatment effect of EPS according to the present invention.

Kinds Concentration (mg / ml) OD (570 nm) Cell survival rate (%) Standard deviation of cell viability (%) 1 time Episode 2 Average Standard Deviation control 0 0.747 0.754 0.751 0.00 100.00 0.66 EPS 5 0.024 0.025 0.025 0.00 3.26 0.09 10 0.005 0.004 0.005 0.00 0.60 0.09 20 0.005 0.004 0.005 0.00 0.60 0.09

2.3 Effect of Colorectal Cancer Treatment

As shown in Table 4 and Figure 2, the MTT assay result of Example 2.1 DLD-1 (colon cancer cell line) as the treatment concentration of EPS according to the present invention increases from 5 mg / ㎖ to 20 mg / ㎖ The cell viability of decreased gradually. In particular, when EPS was treated at a concentration of 5 mg / ㎖, showed a cell growth inhibition of about 98%. The results show an excellent colorectal cancer treatment effect of the EPS according to the present invention.

Kinds Concentration (mg / ml) OD (570 nm) Cell survival rate (%) Standard deviation of cell viability (%) 1 time Episode 2 Average Standard Deviation control 0 0.480 0.488 0.484 0.01 100.00 1.17 EPS 5 0.011 0.011 0.011 0.00 2.27 0.00 10 0.010 0.009 0.095 0.00 1.96 0.15 20 0.006 0.005 0.006 0.00 1.14 0.15

2.4 Effect of Gastric Cancer Treatment

As shown in Table 5 and Figure 3, the MTT assay results of Example 2.1 cell survival rate of AGS (gastric cancer cell line) as the treatment concentration of EPS according to the present invention increases from 5 mg / ml to 20 mg / ml This gradually decreased. In particular, when the EPS was treated at a concentration of 5 mg / ㎖, showed a cell growth inhibitory effect of about 85%. The results show excellent gastric cancer treatment effect of EPS according to the present invention.

Kinds Concentration (mg / ml) OD (570 nm) Cell survival rate (%) Cell viability standard deviation (%) 1 time Episode 2 3rd time Average Standard Deviation control 0 0.902 0.844 0.973 0.906 0.065 100.00 7.13 EPS 5 0.124 0.126 0.124 0.125 0.001 13.8 0.13 10 0.113 0.112 0.113 0.113 0.001 14.0 0.06 20 0.101 0.110 0.115 0.109 0.007 12.0 0.78

2.5 Lung Cancer Treatment Effectiveness Analysis

As shown in Table 6 and Figure 4, the MTT assay results of Example 2.1 cell survival rate of A549 (lung cancer cell line) as the treatment concentration of EPS according to the present invention increases from 5 mg / ml to 20 mg / ml This gradually decreased. In particular, when the EPS was treated at a concentration of 5 mg / ㎖, showed a cell growth inhibition of about 43%. The results show excellent lung cancer treatment effect of the EPS according to the present invention.

Kinds Concentration (mg / ml) OD (570 nm) Cell survival rate (%) Cell viability standard deviation (%) 1 time Episode 2 3rd time Average Standard Deviation control 0 0.804 0.795 0.825 0.808 0.015 100.0 1.91 EPS 5 0.425 0.503 0.459 0.462 0.039 57.2 4.84 10 0.152 0.162 0.160 0.158 0.005 19.6 0.65 20 0.100 0.109 0.114 0.108 0.007 13.3 0.88

2.6 Analysis of cervical cancer treatment effect

As shown in Table 7 and Figure 5, the results of the MTT assay of Example 2.1 cells of HeLa (cervical cancer cell line) as the treatment concentration of EPS according to the present invention increases from 5 mg / ml to 20 mg / ml Survival decreased gradually. In particular, when the EPS treatment at a concentration of 5 mg / ㎖, showed a cell growth inhibition of about 96%. The results show an excellent cervical cancer therapeutic effect of the EPS according to the present invention.

Kinds Concentration (mg / ml) OD (570 nm) Cell survival rate (%) Cell viability standard deviation (%) 1 time Episode 2 Average Standard Deviation control 0 0.429 0.437 0.433 0.01 100.00 1.31 EPS 5 0.016 0.016 0.016 0.00 3.70 0.00 10 0.013 0.013 0.013 0.00 3.00 0.00 20 0.008 0.008 0.008 0.00 1.85 0.00

Example  3. Verification of anticancer effect of EPS Ⅱ

3.1 MTT Assay

The treatment concentration of EPS was 0.25, 0.5 or 1 mg / ml, and as cancer cells, Melanoma B16 (skin cancer cell line), CRL-1628 (oral cancer cell line), PC-3 (prostate cancer cell line), SNU-410 (pancreatic cancer cell line) and Except for using SNU-790 (thyroid cancer cell line), the anticancer effect of EPS was verified in the same manner as in Example 2.

The composition of each cancer cell line is as follows.

Melanoma B16: DMEM (Dulbecco Modified Eagle Medium), 10% (v / v) FBS (Fetal Bovine Serum), 1% (v / v) P / S (Penicillin / Streptomycin Solution)

CRL-1628, PC-3, SNU-410 and SNU-790: Roswell Park Memorial Institute medium (RPMI), 10% (v / v) FBS, 1% (v / v) P / S

3.2 Effect of Skin Cancer Treatment

As shown in FIG. 6, as a result of the MTT assay of Example 3.1, as the treatment concentration of the EPS of the present invention increased from 0.25 mg / ml to 1 mg / ml, the cell survival rate of the skin cancer cell line gradually decreased. In particular, when the EPS was treated at a concentration of 1 mg / ㎖, showed a cell growth inhibition of about 14%.

3.3 Analysis of Oral Cancer Treatment Effect

As shown in FIG. 7, the MTT assay of Example 3.1 showed a cell growth inhibitory effect of about 15% when the EPS of the present invention was treated at a concentration of 1 mg / ml (EPS treatment concentrations 0.25 and 0.5). mg / ml MTT assay results not shown).

3.4 Effect of Prostate Cancer Treatment

As shown in FIG. 8, as a result of the MTT assay of Example 3.1, as the treatment concentration of the EPS of the present invention increased from 0.25 mg / ml to 1 mg / ml, the cell survival rate of the prostate cancer cell line gradually decreased. In particular, when the EPS was treated at a concentration of 1 mg / ㎖, showed a cell growth inhibition of about 49%.

3.5 Analysis of Pancreatic Cancer Treatment Effect

As shown in FIG. 9, as a result of the MTT assay of Example 3.1, as the treatment concentration of the EPS of the present invention increased from 0.5 mg / ml to 1 mg / ml, the cell viability of the pancreatic cancer cell line gradually decreased. In particular, when EPS was treated at a concentration of 1 mg / ㎖, showed a cell growth inhibitory effect of about 14% (not shown in the MTT assay results of 0.25 mg / ㎖ concentration of EPS).

3.6 Analysis of thyroid cancer treatment effect

As shown in FIG. 10, as a result of the MTT assay of Example 3.1, as the treatment concentration of the EPS of the present invention increased from 0.5 mg / ml to 1 mg / ml, the cell survival rate of the thyroid cancer cell line gradually decreased. In particular, when EPS was treated at a concentration of 1 mg / ㎖, showed a cell growth inhibitory effect of about 14% (not shown in the MTT assay results of 0.25 mg / ㎖ concentration of EPS).

Example  4. Serifolia Lakserata  Validation of anticancer effect of mycelial culture

4.1 MTT Assay

In order to confirm the anticancer effect of the seriporia laccerata mycelia culture solution prepared in Preparation Example 1 (hereinafter, denoted by CL01 in the tables and figures), the treatment concentration of CL01 was set to 1, 2.5 or 5 mg / ml. By performing the MTT assay as in Example 2.1, the effect of inhibiting cell growth on various cancer cells was measured.

As cancer cells, Melanoma B16 (skin cancer cell line), CRL-1628 (oral cancer cell line), PC-3 (prostate cancer cell line), and SNU-410 (pancreatic cancer cell line) were used, and the composition of each cancer cell line was described in Example 3. It was like

4.2 Effect of Skin Cancer Treatment

As shown in FIG. 11, as a result of the MTT assay of Example 4.1, as the treatment concentration of CL01 of the present invention increased from 1 mg / ml to 5 mg / ml, the cell survival rate of the skin cancer cell line was slightly decreased. In particular, when CL01 was treated at a concentration of 5 mg / ml, cell growth inhibition of about 14% was observed.

4.3 Analysis of Oral Cancer Treatment Effect

As shown in FIG. 12, as a result of the MTT assay of Example 4.1, the cell viability of the oral cancer cell line gradually decreased as the treatment concentration of CL01 of the present invention increased from 1 mg / ml to 5 mg / ml. In particular, when CL01 was treated at a concentration of 5 mg / ml, the cell growth inhibitory effect was about 48%.

4.4 Effect of Prostate Cancer Treatment

As shown in FIG. 13, as a result of the MTT assay of Example 4.1, the cell survival rate of the prostate cancer cell line gradually decreased as the treatment concentration of CL01 of the present invention increased from 1 mg / ml to 5 mg / ml. In particular, when CL01 was treated at a concentration of 5 mg / ml, the cell growth inhibitory effect was about 48%.

4.5 Analysis of Pancreatic Cancer Treatment Effect

As shown in FIG. 14, as a result of the MTT assay of Example 4.1, the cell survival rate of the pancreatic cancer cell line gradually decreased as the treatment concentration of CL01 of the present invention increased from 1 mg / ml to 5 mg / ml. In particular, when CL01 was treated at a concentration of 5 mg / ml, the cell growth inhibitory effect was about 54%.

From this, it can be seen that not only the EPS according to the present invention but also the mycelium culture medium of the Seriphoria laccerata containing the EPS, the dry powder of the mycelium culture medium, and the extract of the mycelium culture medium are effective as anticancer agents.

Claims (13)

Extracellular polysaccharides produced by Ceriporia lacerata ; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or a pharmaceutical composition for cancer prevention or treatment containing the extract of the mycelia culture medium as an active ingredient. The method of claim 1, The extracellular polysaccharide comprises 40 to 60% by weight of sugar and 30 to 40% by weight of protein, having a molecular weight of 100 to 150 kDa, cancer prevention or therapeutic pharmaceutical composition. The method of claim 2, The extracellular polysaccharide comprises 43 to 47% by weight of sugar and 33 to 36% by weight of protein, and has a molecular weight of 115 to 125 kDa, cancer prevention or therapeutic pharmaceutical composition. The method of claim 2, A pharmaceutical composition for preventing or treating cancer, wherein the sugar contains mannose, galactose, and glucose. The method of claim 1, wherein the extracellular polysaccharide, (a) liquid culturing the Ceriporia laccerata mycelium to prepare a Ceriporia laccerata mycelium culture solution; (b) drying and powdering the Ceriporia laccerata mycelium culture solution; And (c) extracting the seriporia laccerata mycelium culture powder with a solvent, and then filtering and concentrating the same under reduced pressure, the pharmaceutical composition for preventing or treating cancer. The method of claim 5, The medium for culturing the liquid is sugar, glucose, starch, hydrated water, soybean meal, soybean meal, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and water To include, the hydrogen ion concentration (pH) is 4.5 to 6.0, cancer prevention or treatment pharmaceutical composition. The method of claim 5, The liquid culture is performed under a blue LED light source, cancer prevention or treatment pharmaceutical composition. The method of claim 7, wherein The liquid culture is carried out by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm, cancer prevention or treatment pharmaceutical composition. The method of claim 1, The extracellular polysaccharide is contained in 0.1 to 80% by weight based on the total weight of the composition, cancer prevention or therapeutic pharmaceutical composition. The method of claim 1, The cancer may include liver cancer, colon cancer, stomach cancer, lung cancer, cervical cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, central nerve cancer, brain cancer, skin cancer, pancreatic cancer, rectal cancer, esophageal cancer, kidney cancer, epithelial cancer, blood cancer, oral cancer, prostate cancer, and A pharmaceutical composition for preventing or treating cancer, selected from the group consisting of a combination thereof. Extracellular polysaccharides produced by Ceriporia lacerata ; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or a health functional food for cancer prevention or improvement containing the extract of the mycelia culture medium as an active ingredient. Extracellular polysaccharides produced by Ceriporia lacerata ; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or administering an extract of the mycelium culture to a subject in need of anticancer activity. Extracellular polysaccharides produced by Ceriporia lacerata for use in the manufacture of anticancer drugs; Mycelium culture medium of Seriporia laccerata containing the extracellular polysaccharide; Dry powder of the mycelia culture medium; Or the use of an extract of the mycelium culture.

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