KR20160094332A - Pharmaceutical composition for the prevention or treatment of a cancer comprising exopolysaccharide produced by ceriporia lacerata as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer. More particularly, the pharmaceutical composition comprises an exo-biopolymer produced by Ceriporia lacerata, a Ceriporia lacerata mycelium culture medium comprising the exo-biopolymer, a dried powder of the mycelium culture medium, or an extract of the mycelium culture medium as effective components. The composition is useful for preventing or treating various cancers.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises an extracellular polysaccharide produced by Sellapora lacrosera as an active ingredient. The present invention relates to a pharmaceutical composition for preventing or treating cancer,

The present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or a pharmaceutical composition for preventing or treating cancer comprising an extract of the mycelial body as an active ingredient.

Cancer is a group of diseases in which a mass or tumor composed of undifferentiated cells that multiply unlimitedly by various causes, can destroy the life of an individual by destroying surrounding normal tissues or organs. Chemotherapy for cancer relies on the administration of anticancer agents that show strong cytotoxicity, which causes many side effects. Therefore, it is required to develop new anticancer drugs that can reduce the side effects and improve the treatment rate. Therefore, in recent years, research has been conducted to develop anticancer agents from natural products, especially native plants and plant herbal medicines. Taxol (paclitaxel), which is isolated from a tree of interest, is known to be a natural cancer-derived anticancer agent, but it has a problem that it can cause other diseases because it acts on other normal cells in the body as well as cancer cells.

It is known that serpolyla lucerata is a kind of white rot fungus and performs co-metabolism of lignin decomposition in order to utilize carbon sources such as cellulose, hemicellulose, other polysaccharides and glycerol in the ecosystem.

Regarding the medical treatment application using the serpia lacrosera, Korean Patent Registration No. 10-1031605 filed by the present inventors has only known heretofore for the diabetes treatment of the extract of the culture liquid of Serrachia lacera rata , And the effect of preventing or treating cancer by using Seripolyla lucerata has not been reported yet.

Accordingly, the present inventors have found that an extracellular polysaccharide produced by a cellulolytic enzyme; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or the extract of the mycelial culture liquid shows the effect of preventing or treating cancer.

Korean Patent No. 10-1031605

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which contains an active ingredient produced by a cerporolytic enzyme.

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

In order to achieve the above object, the present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelial body culture liquid as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

To achieve these and other objects, the present invention provides Ceriporia an extracellular polysaccharide produced by lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelium culture liquid as an active ingredient.

An extracellular polysaccharide produced by a cellulolytic enzyme according to the present invention; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or a pharmaceutical composition containing the extract of the mycelial body as an active ingredient may be useful for the prevention or treatment of various cancers.

FIG. 1 is a graph showing the cell growth inhibitory effect on the hepatocarcinoma cell line of an extracellular polysaccharide produced by seripositoria lactata.
FIG. 2 is a graph showing the cell growth inhibitory effect of an extracellular polysaccharide produced by a cerporolytic enzyme on the colon cancer cell line. FIG.
FIG. 3 is a graph showing the cell growth inhibitory effect of an extracellular polysaccharide produced by a serogroup L. serrata on a gastric cancer cell line. FIG.
FIG. 4 is a graph showing the cell growth inhibitory effect of extracellular polysaccharide produced by seripositive Lercerata on lung cancer cell lines. FIG.
FIG. 5 is a graph showing the cell growth inhibitory effect of an extracellular polysaccharide produced by a serogroup Lacarrata on a cervical cancer cell line. FIG.
FIG. 6 is a graph showing cell growth inhibitory effect of extracellular polysaccharide produced by Sellapora lacerata on a skin cancer cell line (** P <0.01).
FIG. 7 is a graph showing the cell growth inhibitory effect on the oral cancer cell line of the extracellular polysaccharide produced by the cerporolia lactase (** P < 0.01).
FIG. 8 is a graph showing the cell growth inhibitory effect of extracellular polysaccharide produced by the serogroup Lactacera on the prostate cancer cell line (** P <0.01).
FIG. 9 is a graph showing the cell growth inhibitory effect on extracellular polysaccharide produced by three lipolylacerase in the pancreatic cancer cell line (* P <0.05, ** P <0.01).
FIG. 10 is a graph showing the cell growth inhibitory effect of extracellular polysaccharide produced by the serogroup Lacarrata on the thyroid cancer cell line (** P <0.01).
FIG. 11 is a graph showing cell growth inhibitory effect on cultured skin cancer cell lines of the culture medium of Selegiolia lactaceras mycelium (** P < 0.01).
FIG. 12 is a graph showing the cell growth inhibitory effect on the oral cancer cell line of the culture medium of the S. lipolacrata mycelium (** P < 0.01).
FIG. 13 is a graph showing the cell growth inhibitory effect on the prostate cancer cell line of the culture medium of the seripositive Lactacera mycelium (** P < 0.01).
14 is a graph showing the cell growth inhibitory effect on the pancreatic cancer cell line of the culture medium of the seripositive Lactacera mycelium (* P <0.05, ** P <0.01).

Hereinafter, the present invention will be described in detail.

The present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelial body culture liquid as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

As used herein, the term "extracellular polysaccharide (EPS)" refers to a part of the cell wall of a microorganism such as fungi. The polysaccharide is secreted extracellularly to form a cavernous membrane, . The extracellular polysaccharide is secreted by the microorganism to protect itself from the external environment such as antibodies, toxins, protozoa, and bacteriophages.

Wherein the extracellular polysaccharide comprises 40 to 60 wt% sugar, 30 to 40 wt% protein, 40 to 50 wt% sugar, 32 to 38 wt% protein, 43 to 47 wt% sugar, 33 to 36 wt% Of the protein, or about 45 wt% sugar and about 34 wt% 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 about 120 kDa.

As an embodiment of the present invention, the extracellular polysaccharide may be prepared by: (a) liquid culturing mycelia lacticera mycelium to prepare a culture medium of a seriposita lactamera mycelium; (b) drying and cultivating the culture medium of the above-mentioned sera lipolactacera mycelium; And (c) a step of extracting the culture medium of the cultivated mycelium lacticera mycelium with a solvent, followed by filtration and concentration under reduced pressure.

The medium for liquid culture of the mycelium of seripositive Lactacera in the step (a) is selected from the group consisting of sugar, glucose, starch, water, barley, soybean powder, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ), and water, and may have a hydrogen ion concentration (pH) of 4.5 to 6.0.

Specifically, the culture medium contains 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% 0.05 to 0.15% by weight of magnesium sulfate (MgSO ₄ ), 0.05 to 0.25% by weight of potassium monophosphate (KH ₂ PO ₄ ), 0.05 to 0.25% by weight of potassium diphosphate (K ₂ HPO ₄ ) .

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

The liquid culture is, for example, carried out at 20 to 28 ° C with a hydrogen ion concentration of 4.5 to 6.0, a light source of a blue LED, an illuminance of 0.1 to 0.8 LUX, air is injected at 0.5 to 2.0 kgf / cm 2, The concentration can be carried out for 8 to 13 days while maintaining the concentration at 1,000 to 2,000 ppm. Specifically, it can be carried out at a temperature of 20 to 25 占 폚, a pH of 4.5 to 6.0, an air injection of 0.5 to 2.0 kgf / cm2 and a carbon dioxide concentration of 1,000 to 2,000 ppm for 5 to 15 days. When the liquid culture is carried out under the above-described conditions, the content of the extracellular polysaccharide produced is preferably high.

In step (a), the parent strain in the PDA (potato dextrose agar) medium is maintained at 25 ° C. in a shaking incubator using PDB (potato dextrose broth) medium in an Erlenmeyer flask And then cultured for 7 to 9 days. In addition, the culture broth or obtained mycelium can be used as an inoculum after the parent strain is cultured as described above. At this time, the amount of the mycelium to be added to the inoculation source is preferably about 0.5% (w / v) based on the amount of the solution to be cultured. Since the amount of extracellular polysaccharide is not so high as much as the amount of mycelium (% / 100 ml, w / v), the composition of the medium is not the best nutritional ratio and environmental condition for growth of the mycelium, It is preferable to apply selective culture conditions in which the cells are formed.

The culture solution can be separated and purified into mycelium and an aqueous solution. Specifically, the separated tablets may be purified by repeatedly removing the mycelium from the mycelium with a centrifugal separator using a multi-sheet filter press and a vibrating centrifugal separator (PALLSEP), and then irradiating ultraviolet rays (UV) for 1 minute have. In addition, the culture solution can be kept sealed after removing oxygen. If the mycelium is present in the culture solution, the content of the active ingredient may be changed by growing mycelium.

In the step (b), the mycelial culture liquid prepared in the step (a) may be dried and pulverized. The drying may be carried out at a temperature of 40 DEG C or less, more specifically, at a temperature of 30 DEG C or less for 48 to 96 hours to prevent the disappearance of the active material. Also, in the step (b), it is preferable to use a vacuum freeze dryer rather than a vacuum dryer in which the evaporation temperature is set to be relatively high, since the change in effective substance content is minimized.

In step (c), the dry powder of the mycelial liquid obtained in step (b) is extracted with a solvent, and the extracellular polysaccharide, which is an active ingredient of the composition according to the present invention, is isolated.

Specifically, 100 ml of distilled water is added to 3 to 10 g of the dried powder of the mycelial culture liquid, and the suspension is well suspended. After centrifuging at 5,000 to 10,000 rpm for 10 to 30 minutes, a supernatant is obtained. Three times as much extraction solvent may be added, and the mixture is allowed to stand in a refrigerator at 1 to 5 DEG C for 10 to 15 hours. After centrifuging the supernatant alone at 5,000 to 10,000 rpm for 10 to 30 minutes in the supernatant, the precipitate may be recovered to produce a crude extracellular polysaccharide. The extracellular polysaccharide may be lyophilized and dried at 30 DEG C or lower to obtain an extracellular polysaccharide.

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

The present invention relates to an extracellular polysaccharide produced by a cellulolytic enzyme; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or the composition for preventing or treating cancer, which comprises the extract of the mycelial body culture liquid, may further comprise a suitable carrier, excipient and diluent conventionally used.

The extracellular polysaccharide may be contained in an amount of 0.1 to 80% by weight, or 0.1 to 50% by weight, based on the total weight of the pharmaceutical composition for preventing or treating cancer. The extracellular polysaccharide may be a mycelial culture solution, a dry powder thereof, May suitably be included in an amount corresponding to the content of the extracellular polysaccharide. However, the extracellular polysaccharide; A culture fluid containing the same; A dry powder of the culture solution; Or the effective amount of the extract of the culture solution may be suitably adjusted according to the method and purpose of use of the pharmaceutical composition.

The pharmaceutical composition according to the present invention can be formulated according to a conventional method. 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 composition according to the present invention can be prepared into a suitable formulation using a pharmaceutically inert organic or inorganic carrier. That is, when the formulations are tablets, coated tablets, dragees and hard capsules, they may include lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or a salt thereof. Also, when the formulation is a soft capsule, it may include vegetable oils, waxes, fats, semi-solid and liquid polyols. Further, when the formulation is in the form of a solution or a syrup, it may contain water, polyol, glycerol, and / or vegetable oil.

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

The method of administering the pharmaceutical composition according to the present invention can be easily selected according to the formulation, and can be administered orally or parenterally. The dosage may vary depending on the patient's age, sex, weight, degree of pathology, and route of administration, but is generally in the range of 5 to 1,000 mg / kg body weight based on the extracellular polysaccharide, mg / kg body weight may be administered once or three times a day. However, the dose does not limit the scope of the present invention.

The pharmaceutical composition according to the present invention not only provides an excellent cancer prevention or therapeutic effect but also has little toxicity and side effects caused by drugs, so that it can be safely used for long-term use as an anticancer drug.

Accordingly, the pharmaceutical composition of the present invention can be used for the treatment of various cancers such as 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, Cancer, cancer, epithelial cancer, blood cancer, oral cancer, prostate cancer, and combinations thereof.

Further, the present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelium culture liquid as an active ingredient.

The health functional food according to the present invention may be in the form of a powder, a granule, a tablet, a capsule or a drink, and may be a candy, a chocolate, a gum, a tea, a vitamin complex,

Herein, the extracellular polysaccharide according to the present invention contained in the health functional food; A mycelial culture fluid containing the same; A dry powder of the mycelial culture liquid; Or the content of the extract of the mycelial culture liquid may be usually 0.01 to 50% by weight, or 0.1 to 20% by weight of the total food weight. Also, in the case of a health drink composition, it may be contained in an amount of 0.02 to 10 g, or 0.3 to 1 g, based on 100 ml of the health drink composition.

The food may be an extracellular polysaccharide of the present invention; A culture solution of mycelium of Sellapora lacera rata containing the same; A dry powder of the mycelial culture liquid; Or a food-acceptable food-aid additive together with the extract of the mycelial culture broth.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

Manufacturing example  One. Three Li Pori Lacerata  Production of Mycelial Culture Medium

The bacterium cultivated through subculture was stored frozen at -80 ℃ and the strain was stored in a potato dextrose agar (PDA) medium (Hereinafter referred to as "PDA culture broth") was stored in a refrigerator at 4 ° C for 2 to 3 times in a plastic culture dish (Difco, Becton Dickinson and Company). Then, 600 ml of a PDB (potato dextrose broth) medium (Difco, Becton Dickinson and Company) was added to the Erlenmeyer flask. One PDB culture was added to the flask, followed by shake culture at 25 ° C for 8 days to obtain a PDB culture.

On the other hand, for the culture of the strain, sugar 1.5%, glucose 0.5% potato starch 0.5% by weight, it can water 0.25 weight%, barley minutes, 0.25% by weight, soy flour 0.75% by weight, magnesium sulfate (MgSO ₄₎ 0.05 wt. Liquid culture medium containing 0.05% by weight of potassium phosphate monobasic (KH ₂ PO ₄ ), 0.05% by weight of potassium diphosphate (K ₂ HPO ₄ ) and water in the remaining amount was fed to an 800 l fermenter at a rate of 1.5 kgf / Cm 2, sterilized for 20 minutes, 600 ml of the PDB culture strain was inoculated with a starter in a state of being cooled to 23 ° C, air was passed through at 0.5 to 1.5 kgf / cm 2, The culture medium of Sellapora lactacerata was prepared by liquid culturing the mycelium of C. liparcerata at a constant temperature of 23 ° C. for 10 days while maintaining the light source of blue LED and the illuminance of 0.5 LUX and the concentration of carbon dioxide at 2,000 ppm.

Manufacturing example  2 Three Li Pori Lacerata  Preparation of dried powder of culture medium

The culture of the mycelia of the ceriplora lactaceras prepared in Preparation Example 1 was vacuum-freeze-dried at 25 ° C for 72 hours using a vacuum freeze dryer to obtain a dry powder of the culture medium of the seriposita lactamera mycelium .

Manufacturing example  3. Three Li Pori Lacerata  Preparation of culture extract

100 ml of distilled water was added to 5 g of the dry powder of the cultured mycelium lacticcerus cultured product prepared in Preparation Example 2, and the mixture was well suspended, centrifuged at 8,000 rpm for 20 minutes, Three times the amount of ethanol was added and allowed to stand at 4 占 폚 for 12 hours. Thereafter, the supernatant was taken from the column, and an extract of the cultured Mycelium lacticera mycelium was prepared.

Manufacturing example  4. Three Li Pori Lacerata  The extracellular polysaccharide ( extracellular  polysaccharide; Hereinafter referred to as "EPS")

The extract of the culture medium of the cellulolyticase obtained in Preparation Example 3 was further centrifuged at 8,000 rpm for 20 minutes, and the precipitate was recovered to obtain crude EPS. The crude EPS was vacuum-lyophilized in a vacuum freeze dryer at 25 ° C for 72 hours to obtain an EPS produced by Sera lipolactacrata.

Example  1. Characterization of EPS

1.1 Gel Permeation Chromatography, GPC Molecular weight measurement of EPS using

The EPS prepared in Preparation Example 4 was dissolved in a solution of 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ (pH adjusted to 4 with glacial acetic acid) to a concentration of 1% (w / v) . After centrifugation for 0.5 hour, only the supernatant was filtered with a 0.45 탆 syringe filter and analyzed by GPC.

GPC analysis was performed using refractive index as a detector, OHPak SB 805 HQ (Shodex, Japan) as a GPC column, 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ [pH adjusted to 4 with glacial acetic acid] And the flow rate of the mobile phase was allowed to flow at a rate of 1.0 ml / min. 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 refractive index (RI) Knauer K-2310 Germany) was used to measure 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 about 120 kDa.

1.2 Measurement of sugar and protein content of EPS

The EPS prepared in Preparation Example 4 was subjected to second purification and treated with a protein hydrolyzing enzyme to measure sugar and protein content.

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

The 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 sample diluted by concentration, 2.5 ml of sulfuric acid was added, and the solution was cooled to room temperature and absorbance was measured at 465 nm to calculate the sugar content.

Protein content was also 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 45 to 51% by weight and the protein content was 33 to 34% by weight.

yield(%) Total sugar content (%) Total protein content (%) EPS 1.22 + 0.03 45.32 + - 1.41 34.17 ± 0.73 Second refined EPS 0.78 ± 0.01 50.49 + - 0.52 33.50 ± 2.79 Enzymatically processed EPS ^* 0.24 0.06 51.39 ± 1.32 34.61 + 1.51

^* Enzyme treatment: Alkaline 0.5%, 50 ℃, 30 minutes.

Each value is mean ± SE (n ≥ 3).

Also, as a result of analyzing sugar components of EPS, it was found that EPS mainly contains mannose, galactose and glucose.

Example  2. Anticancer effect of EPS Ⅰ

2.1 MTT Assay

In order to confirm the anti-cancer effect of the EPS prepared in Preparation Example 4, the cell growth inhibitory effect on various cancer cells was measured by MTT assay according to treatment concentration of EPS.

The above-mentioned MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) is a pale yellow substrate, which is cleaved by respiratory chain enzymes in the mitochondria of living cells and produces dark blue formazan Since the reaction does not occur in dead cells, the amount of this formazan is used to measure viable cell count (see Van de Loosdrecht, AA, et al., J. Immunol . Methods , 141 (1): 15-22, 1991 ).

First, Hep3B (liver cancer cell line), DLD-1 (colorectal cancer cell line), AGS (gastric cancer cell line), A549 (lung cancer cell line) and HeLa (cervical cancer cell line) ⁴ cells / well, and cultured in a 5% CO ₂ incubator at 37 ° C for one day.

The composition of culture medium for each cancer cell is as follows.

Hep3B (Dulbecco Modified Eagle Medium), 10% (v / v) FBS (Fetal Bovine Serum), 1% (v / v) P / S (Penicillin / Streptomycin Solution)

1% (v / v) P / S (v / v), 10% (v / v) FBS,

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 changed to a new culture medium. EPS was dissolved in distilled water to give a concentration of 5, 10 or 20 mg / ml, and the cells were cultured in a 5% CO ₂ incubator at 37 ° C for 3 days. The control group was cultured under the same conditions with distilled water.

Here, the MTT solution in an amount of 1/10 (v / v) of the total medium was each treated and reacted for 4 hours in an incubator, and the formazan formation was confirmed, and the supernatant was completely removed so that the formazan was not dispersed . Then, 100 쨉 l of DMSO (dimethyl sulfoxide) was added to each well to dissolve the formazan formed in the cells. Absorbance was measured at a wavelength of 570 nm with an ELISA reader, and the cell viability of each cancer cell was measured.

2.2 Analysis of liver cancer treatment effect

As shown in Table 3 and FIG. 1, the cell survival rate of Hep3B (liver cancer cell line) increased as the treatment concentration of EPS of the present invention was increased from 5 mg / ml to 20 mg / ml as a result of the MTT assay of Example 2.1 Respectively. Especially, when treated with EPS at a concentration of 5 mg / ㎖, the cell growth inhibitory effect was about 97%. The results show that the EPS according to the present invention has excellent therapeutic effect on liver cancer.

Kinds density
(mg / ml) OD (570 nm) Cell viability (%) 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 Analysis of treatment effect of colorectal cancer

As shown in the following Table 4 and FIG. 2, when the treatment concentration of EPS according to the present invention was increased from 5 mg / ml to 20 mg / ml as a result of the MTT assay of Example 2.1, DLD-1 (colon cancer cell line) Cell survival rate was decreased. Especially, when EPS was treated at a concentration of 5 mg / ㎖, the cell growth inhibition effect was about 98%. The above results show that EPS of the present invention has excellent colorectal cancer treatment effect.

Kinds density
(mg / ml) OD (570 nm) Cell viability (%) 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 Analysis of gastric cancer treatment effect

As shown in the following Table 5 and FIG. 3, as a result of the MTT assay of Example 2.1, as the treatment concentration of EPS according to the present invention increased from 5 mg / ml to 20 mg / ml, the cell survival rate of AGS (gastric cancer cell line) Respectively. Especially, when EPS was treated at a concentration of 5 mg / ㎖, the cell growth inhibition effect was about 85%. The above results show the excellent effect of the EPS according to the present invention on gastric cancer treatment.

Kinds density
(mg / ml) OD (570 nm) cell
Survival rate (%) Cell survival rate
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 Analysis of lung cancer treatment effect

As shown in the following Table 6 and FIG. 4, as a result of the MTT assay of Example 2.1, the cell survival rate of A549 (lung cancer cell line) increased from 5 mg / ml to 20 mg / Respectively. Especially, when EPS was treated at the concentration of 5 mg / ㎖, the cell growth inhibition effect was about 43%. The results show that the EPS according to the present invention has excellent lung cancer therapeutic effect.

Kinds density
(mg / ml) OD (570 nm) cell
Survival rate (%) Cell survival rate
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 the following Table 7 and FIG. 5, as a result of the MTT assay of Example 2.1, the treatment concentration of EPS according to the present invention increased from 5 mg / ml to 20 mg / ml, the cells of HeLa (cervical cancer cell line) Survival rate decreased gradually. Especially, when treated with EPS at a concentration of 5 mg / ㎖, the cell growth inhibition effect was about 96%. The above results show that the EPS of the present invention has excellent cervical cancer treatment effect.

Kinds density
(mg / ml) OD (570 nm) cell
Survival rate (%) Cell survival rate
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. Anticancer effect of EPS Ⅱ

3.1 MTT Assay

(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 the like were used as the cancer cells and the treatment concentration of the EPS was 0.25, 0.5, or 1 mg / The anti-cancer effect of EPS was verified in the same manner as in Example 2 except that SNU-790 (thyroid cancer cell line) was used.

The composition of culture medium for each cancer cell is as follows.

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

10% (v / v) FBS, 1% (v / v) P / S (v / v), CRL-1628, PC-3, SNU-410 and SNU-

3.2 Analysis of skin cancer treatment effect

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

3.3 Analysis of oral cancer treatment effect

As shown in FIG. 7, when the EPS of the present invention was treated at a concentration of 1 mg / ml, the MTT assay of Example 3.1 showed a cell growth inhibitory effect of about 15% (EPS concentration 0.25 and 0.5 mg / ml MTT assay results).

3.4 Analysis of prostate cancer treatment effect

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

3.5 Analysis of treatment effect of pancreatic cancer

As shown in FIG. 9, the cell survival rate of the pancreatic cancer cell line gradually decreased as the treatment concentration of EPS of the present invention increased from 0.5 mg / ml to 1 mg / ml as a result of the MTT assay of Example 3.1 above. Especially, when treated with EPS at a concentration of 1 mg / ㎖, it showed a cell growth inhibition effect of about 14% (MTT assay of EPS treated concentration of 0.25 mg / ㎖).

3.6 Analysis of treatment effect of thyroid cancer

As shown in FIG. 10, the cell survival rate of the thyroid cancer cell line gradually decreased as the treatment concentration of EPS of the present invention increased from 0.5 mg / ml to 1 mg / ml as a result of the MTT assay of Example 3.1. Especially, when treated with EPS at a concentration of 1 mg / ㎖, it showed a cell growth inhibition effect of about 14% (MTT assay of EPS treated concentration of 0.25 mg / ㎖).

Example  4. Three Li Pori Lacerata  Anticancer effect of mycelial culture solution

4.1 MTT Assay

In order to confirm the anticancer effect of the culture medium of the cellulolytic enzyme Serrate produced in Preparation Example 1 (hereinafter referred to as CL01 in the tables and drawings), the treatment concentration of CL01 was set to 1, 2.5 or 5 mg / ml MTT assays were carried out as in Example 2.1 to determine cell growth inhibitory effects on various 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 as the cancer cells. The composition of the culture medium for each cancer cell line was as described in Example 3 It was the same.

4.2 Analysis of skin cancer treatment effect

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

4.3 Analysis of oral cancer treatment effect

As shown in FIG. 12, the MTT assay of Example 4.1 showed that the cell survival rate of the oral cancer cell line gradually decreased as the treatment concentration of CL01 of the present invention was 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 Analysis of prostate cancer treatment effect

As shown in FIG. 13, the MTT assay of Example 4.1 showed that the cell viability of prostate cancer cells gradually decreased as the treatment concentration of CL01 of the present invention was 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 treatment effect of pancreatic cancer

As shown in FIG. 14, 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 as a result of the MTT assay of Example 4.1. 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 culture medium of the mycelium of the cellulolytic enzyme, the dried powder of the mycelial culture solution and the extract of the culture solution of the mycelium are also effective as anticancer drugs.

Claims (11)

An extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of said mycelial liquid as an active ingredient.
The method according to claim 1,
Wherein said extracellular polysaccharide comprises from 40 to 60% by weight of sugar and from 30 to 40% by weight of protein and has a molecular weight of from 100 to 150 kDa.
3. The method of claim 2,
Wherein 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.
3. The method of claim 2,
Wherein the saccharide contains mannose, galactose and glucose.
2. The composition of claim 1, wherein the extracellular polysaccharide is selected from the group consisting of:
(a) liquid culturing the mycelium of ceriplora lactaclora to prepare a culture medium of a seriposita lactamera mycelium;
(b) drying and cultivating the culture medium of the mycelium of ceriplora lactaclora; And
(c) extracting the culture medium of the mycelium of Cerporaria lacera with a solvent, filtering it, and concentrating it under reduced pressure.
6. The method of claim 5,
The medium for the liquid culture of sugar, glucose, starch, be water, barley minutes, soy flour, magnesium sulfate (MgSO _4), 1 potassium phosphate (KH ₂ PO _{4), 2} potassium phosphate (K ₂ HPO ₄₎ and water Wherein the hydrogen ion concentration (pH) is 4.5 to 6.0.
6. The method of claim 5,
Wherein said liquid culture is performed under a blue LED light source.
8. The method of claim 7,
Wherein the liquid culture is performed while maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm.
The method according to claim 1,
Wherein the extracellular polysaccharide is contained in an amount of 0.1 to 80% by weight based on the total weight of the composition.
The method according to claim 1,
Wherein the cancer is selected from the group consisting of liver cancer, colon cancer, stomach cancer, lung cancer, cervical cancer, bladder cancer, breast cancer, ovarian cancer, thyroid cancer, central nervous system cancer, brain cancer, skin cancer, pancreatic cancer, rectal cancer, &Lt; / RTI &gt; or a combination thereof.
An extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelium culture liquid as an active ingredient.

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