KR20120053415A - Composition for prevention and treatment of cancer comprising isoegomaketone as an active ingredient - Google Patents

Abstract

PURPOSE: A composition containing isoegomaketone is provided to induce cancer cell apoptosis and to be used as an active ingredient of a health food. CONSTITUTION: A pharmaceutical composition for preventing and treating cancer contains isoegomaketone(IK) or pharmaceutically acceptable salt thereof as an active ingredient. The isoegomaketone is isolated from Perilla frutescens (L.) Britt. cv. Chookyoupjaso. The cancer includes colon cancer, prostate cancer, uterine cancer, or osteosarcoma. A health food for preventing and treating cancer contains isoegomaketone or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Composition for prevention and treatment of cancer comprising isoegomaketone as an active ingredient}

The present invention relates to a composition for preventing and treating cancer, or a health food containing the isoegomaketone (isoegomaketone) isolated from the main leaf.

Colorectal cancer is not only a major cause of cancer-related deaths in the United States, but it is also estimated to have approximately 102,900 colon cancers diagnosed in the United States in 2010 and an estimated 51,370 deaths (Jemal et al., 2010). The incidence is on the rise due to dietary habits (Park et al., 2008). Cancer is currently treated using surgical methods, chemotherapy and radiation therapy. However, these treatments have the problem of limited effect in advanced or metastatic cancer (Lee et al., 2008; Tan et al., 2009). Recently, alternative therapies, such as traditional Chinese medicine, are increasing rapidly in Asia as well as in western countries. To date, many natural compounds derived from plants have been shown to exhibit anti-cancer effects by inducing apoptosis, and these natural compounds can be used as new and effective natural compounds based on the discovery of new drugs for the treatment and prevention of cancer (Athar et. al., 2007; Nobili et al., 2009).

Apoptosis is known as programmed cell death, which is a genetic regulatory mechanism that plays an important role in maintaining cell homeostasis. Apoptosis can generally be induced through the exogenous (killing receptor) pathway or the endogenous (within the mitochondria) pathway. In the exogenous pathway, ligation of the TNF / Fas-receptor by its ligand causes cleavage of the caspase-8 initiator and directly activates effector caspase-3, or Bcl-2 After inducing cleavage of the family member Bid, the translocation of Bax is induced into the mitochondrial membrane (Mellier et al., 2010; Tan et al., 2009; Wu, 2009). In contrast, the endogenous pathway is mediated by mitochondria, and in response to atopotic stimulation, cytochrome c and apoptosis-inducing factor (AIF) are released from mitochondria, which factors are caspase-dependent and independent. Is involved in apoptosis. Cytochrome c binds with Apaf-1 to form a structure called apoptosome, activates caspase-9, then activates caspase-3, and finally causes apoptotic cell death . AIF is a sign of caspase-independent apoptosis, which is translocated into the nucleus following apoptotic stimulation, leading to DNA fragmentation (Huerta et al, 2006; Millan and Huerta, 2009).

Meanwhile, Perilla frutescens (L.) is an annual herb with a distinctive aroma and taste, its leaves are widely used as edible vegetables, and its seeds are used in edible oils in Korea, and are traditional Chinese medicine. It is also used in (Seo and Baek, 2009). In Korea, it began to cultivate from the Silla Dynasty. The height is 60 ~ 90 centimeters, the stem is square and straight, with long hairs, the leaves are facing each other, pointed in ovate circles, and the bottom is round. Leaves are 7-12 centimeters long, 5-8 centimeters long, with serrates and green on the front, but purple on the back. The flowers are in inflorescences, white, and small lip-shaped flowers bloom. Corolla 4-5 mm long, slightly lower lip petals. Two of the four stamens are long and the branch is inside the calyx and is round and has a net pattern. In Korea, most brown cultivars are grown, and they are cultivated as paid crops, have a peculiar smell on leaves, and are edible. They are often used as cooking ingredients in Korea, Japan, China, Vietnam, etc. Seeds are ground and used in food, or with perilla oil to be used for herbs. It is also used as a fat paper for feeding oil on white paper.

Many compounds such as rosmarinic acid, luteolin, apigenin, ferulic acid, catechin, and caffeic acid are found in perilla frutescens (L.)) (Peng et al., 2005) and among these various components, rosmarinic acid, luteolin, apigenin and caffeic acid It has been reported that apoptosis can be induced in various cancer cell lines (Hsu, 2010; Hur, 2004; Lim, 2007; Moon, 2010; Selvendiran, 2006).

Isoegomarketing (IK) is a perilla leaf As an essential oil component of frutescens (L.), it has been reported from Park et al (2009) in the literature, which has been reported to have anti-inflammatory effects in RAW 264.7 macrophages (Jin et al., 2010). ). However, it has not been reported that isoegomarketone is associated with apoptosis of cancer cells.

Therefore, the present inventors are studying the relationship between iso-automarket and cancer, and iso-e-marketing (IK) to DLD1 and HT29 colorectal cancer cell lines, PC-3 and DU145 prostate cancer cell lines, Hela uterine cancer cell lines and U20S osteosarcoma cell lines. The treatment resulted in a killing effect by inhibiting the survival and growth of cancer cells in a concentration-dependent manner, it was confirmed that this cancer cell killing effect is due to the induction of apoptosis (apoptosis) of cancer cells. In addition, poly (ADP-ribose) polymerase (PARP) is cleaved upon isoegomarketing (IK) treatment of DLD1 cells, and caspase-8, -9, and- 3 activates, causes Bid cleavage and Bax translocation, promotes the release of cytochrome C from the mitochondria into the cytoplasm, as well as an apoptosis induction factor, a caspase-independent mitochondrial apoptosis factor. inducing factor (AIF) was transduced from the mitochondria into the nucleus. Through this, the present inventors have confirmed that isoeegomarketing induces apoptosis of cancer cells through a caspase dependent or non-dependent pathway, thereby exhibiting a killing effect of the cancer cells, thereby making the isoegomarketing a composition for preventing and treating cancer, or The present invention was completed by revealing that it can be usefully used as an active ingredient of health food.

It is an object of the present invention to provide a composition for preventing and treating cancer, or health foods containing isoegomaketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating cancer using isoegomaketone (IK) or a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer containing isoegomaketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health food for cancer prevention and improvement containing isoego maketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a method of treating cancer comprising administering to a subject a pharmaceutically effective amount of isoegomaketone (IK) or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of isoegomaketone (IK) or a pharmaceutically acceptable salt thereof.

Isoegomaketone (IK) of the present invention is effective in inducing apoptosis of cancer cells through a caspase-dependent or non-dependent pathway in various cancer cell lines, thereby killing cancer cells. May be usefully used as an active ingredient of the composition for preventing and treating the same in various carcinomas.

1 shows the effect of IK on DLD1 colorectal cancer cell line:
(A) DLD1 cells were treated with various concentrations of IK or 0.1% DMSO for 24 hours, and the cell viability was measured using an EZ-Cytox cell survival assay kit. ) Is the mean ± standard deviation from three separate experiments;
(B) DLD1 cells were treated with various concentrations of IK or 0.1% DMSO for 24 hours, and cell growth inhibition and morphological changes were confirmed by reversed phase microscopy (× 100);
C: control; And
D: DMSO treatment group.
2 is a graph showing the results obtained by measuring various concentrations of IK or 0.1% DMSO in HT-29 cells for 24 hours, and measuring the cell viability using the EZ-Cytox cell survival assay kit. (bar) is the mean ± standard deviation from three separate experiments;
C: control; And
D: DMSO treatment group.
3 is a graph showing the results obtained by measuring various concentrations of IK or 0.1% DMSO in PC-3 cells for 24 hours, and measuring the cell viability using the EZ-Cytox cell survival assay kit. (bar) is the mean ± standard deviation from three separate experiments;
C: control; And
D: DMSO treatment group.
FIG. 4 is a graph showing treatment results of DU145 cells treated with various concentrations of IK or 0.1% DMSO for 24 hours, and their cell viability using the EZ-Cytox cell survival assay kit. ) Is the mean ± standard deviation from three separate experiments;
C: control; And
D: DMSO treatment group.
FIG. 5 is a graph showing the results obtained by treating Hela cells with various concentrations of IK or 0.1% DMSO for 24 hours, and measuring the cell viability using the EZ-Cytox cell survival assay kit. ) Is the mean ± standard deviation from three separate experiments;
C: control; And
D: DMSO treatment group.
FIG. 6 shows U20S cells treated with various concentrations of IK or 0.1% DMSO for 24 hours, and the cell viability was measured using an EZ-Cytox cell survival assay kit. ) Is the mean ± standard deviation from three separate experiments;
C: control; And
D: DMSO treatment group.
7 shows the results confirming that IK induces apoptosis in DLD1 cells;
After 24 hours of treatment with various concentrations of IK or 0.1% DMSO on DLD1 cells, apoptotic cells via annexin V FITC staining with PI were identified by flow cytometry and error bars Mean ± standard deviation from three separate experiments;
C: control, and
D: DMSO.
8 shows that IK-induced apoptosis in DLD1 cells is mediated by the activity of caspase-3;
(A) IK-treated DLD1 cells were harvested and the samples obtained were cleaved by Western blot analysis using PARP-1 antibody, and β-tubulin was used as a loading control;
(B) Caspase-3 activity was measured spectrophotometrically using Caspase-3 assay kit after 25 uM and 50 uM IK or 0.1% DMSO for 24 h in DLD1 cells;
(C) Caspase-3 activity was measured spectrophotometrically using Caspase-3 assay kit after treatment of DLD1 cells with 50 uM of IK for 3, 6, 12, 16 and 24 hours; And
* P <0.01 significant compared to control.
9 shows the results confirming that IK activates caspase in DLD1 cells;
(A) Western blot was performed by treating DLD1 cells with 25 uM and 50 uM IK or 0.1% DMSO for 24 hours; And
(B) Western blot was performed by treating DLD1 cells with 50 uM of IK for 3, 6, 12, 16 and 24 hours, and β-tubulin was used as a loading control.
10 shows that IK induces apoptosis via mitochondrial pathway in DLD1 cells;
(A) After 24 hours of 25uM and 50 uM of IK or 0.1% DMSO in DLD1 cells, cytochrome c released from the mitochondria into the cytoplasm was measured by Western blot using a cytochrome c release assay kit. to be;
COX4 and β-tubulin were used as loading controls for mitochondrial fraction and cytoplasmic fraction, respectively;
(B) DLD1 cells were treated with 25 uM and 50 uM of IK or 0.1% DMSO for 24 hours, resulting in cleavage of Bid and translocation of BAX from the cytoplasm to the mitochondria, resulting in β-tubulin loading Used as a control.
FIG. 11 shows that IK induces apoptosis via a caspase-independent pathway in DLD1 cells. After treatment with 25 uM and 50 uM of IK or 0.1% DMSO for 24 hours, nuclear from mitochondria. The translocation of AIF was measured by Western blot, and Lamin B1 and β-tubulin were used as loading controls.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing and treating cancer containing isoegomaketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.

It is preferred that the isoegomarket is a compound represented by the following [Formula 1]:

The iso-automarket is a compound produced by the synthesis through the synthesis and fractionation method of natural products or conventional substituents that are separated from the plant can be used, the perilla leaf ( Perilla frutescens (L.) Britt. cv. Chookyoupjaso) is more preferably, but not limited to.

The isoegomarkettone of the present invention represented by the above [Formula 1] may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. . Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is dissolved in a conventional method, for example, the isoegomarketton of [Formula 1] in an excess of an aqueous acid solution, and the salt is a water miscible organic solvent such as methanol, ethanol, acetone. Or by precipitation with acetonitrile.

The same amount of isoegomarketone of [Formula 1] and acid or alcohol in water may be heated and then the mixture may be evaporated to dryness or dried, or the precipitated salt may be produced by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

In addition, the isoego marketer represented by the above [Formula 1] includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, the isoegomarket of [Formula 1] is dissolved in a water miscible organic solvent, such as acetone, methanol, ethanol or acetonitrile and the excess It can be prepared by adding an organic acid or adding an aqueous acid solution of an inorganic acid and then precipitating or crystallizing it. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.

The cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer It is preferably one selected from the group consisting of species, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, osteosarcoma and central nervous system tumor. More preferably, but not limited to any one selected from the group consisting of cancer, prostate cancer, uterine cancer and osteosarcoma.

In a specific embodiment of the present invention, DLD1 and HT29 colorectal cancer cell line, PC-3 and DU145 prostate cancer cell line, Hela uterine cancer cell line and U20S osteosarcoma cell line in the concentration-dependent survival and growth of cancer cells during the treatment Inhibition showed a killing effect, it was confirmed that this cancer cell killing effect is due to the induction of apoptosis (apoptosis) of cancer cells. In addition, poly (ADP-ribose) polymerase (PARP) is cleaved upon isoegomarketing (IK) treatment of DLD1 cells, and caspase-8, -9, and- 3 activates, causes Bid cleavage and Bax translocation, promotes the release of cytochrome C from the mitochondria into the cytoplasm, as well as an apoptosis induction factor, a caspase-independent mitochondrial apoptosis factor. inducing factor (AIF) was transduced from the mitochondria into the nucleus.

Thus, by confirming that the isoegomarketone of the present invention induces apoptosis of cancer cells in various cancer cell lines through caspase dependent or non-dependent pathways, the isoegomarketone causes cancer. It can be usefully used as an active ingredient of prophylactic and therapeutic compositions.

In the case of using the composition of the present invention as a medicine, the pharmaceutical composition containing the isoego market to be represented by the above [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient is oral or various at the time of clinical administration It may be formulated and administered in a parenteral dosage form, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition comprising the isoegomarket represented by the above [Formula 1] as an active ingredient can be administered parenterally, and the parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. All.

At this time, in order to formulate into a formulation for parenteral administration, the isoegomarketton of the formula [1] or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, which is ampoule or vial It may be prepared in unit dosage form. The composition may be sterile or contain preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating or It can be formulated according to the coating method.

In addition, the dosage of the compound of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally based on an adult patient having a weight of 60 kg. 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once a day to several times at regular intervals according to the judgment of a doctor or pharmacist.

In addition, the present invention provides a health food for cancer prevention and improvement containing isoego maketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.

It is preferred that the isoegomarket is a compound represented by the following [Formula 1]:

[Formula 1]

.

.

The iso-automarket is a natural compound that is separated from a plant or a compound produced by the synthesis through the synthesis and fractionation method of conventional substituents can be used both, perilla leaf ( Perilla frutescens (L.) Britt. cv. Chookyoupjaso) is more preferably, but not limited to.

The cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer It is preferably one selected from the group consisting of species, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, osteosarcoma and central nervous system tumor. More preferably, but not limited to any one selected from the group consisting of cancer, prostate cancer, uterine cancer and osteosarcoma.

By confirming that the isoegomarketone of the present invention induces apoptosis of cancer cells in various cancer cell lines through caspase dependent or non-dependent pathways, thereby causing the death of cancer cells, thereby preventing and It can be usefully used as an active ingredient for improving health food.

The health food of the present invention may contain various flavors or natural carbohydrates as additional ingredients. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is preferably selected from 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention, but is not limited thereto.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the health food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is preferable to select from 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention, but is not limited thereto.

The present invention also provides a method of treating cancer comprising administering a pharmaceutically effective amount of isoegomaketone (IK) or a pharmaceutically acceptable salt thereof to a subject having the cancer.

In addition, the present invention provides a method for preventing cancer, comprising administering to a subject a pharmaceutically effective amount of isoegomaketone (IK) or a pharmaceutically acceptable salt thereof.

The iso-automarket is a natural compound that is separated from a plant or a compound produced by the synthesis through the synthesis and fractionation method of conventional substituents can be used both, perilla leaf ( Perilla frutescens (L.) Britt. cv. Chookyoupjaso) is more preferably, but not limited to.

The subject is applicable to all mammals, such as mice, rats, livestock or humans.

The cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer It is preferably one selected from the group consisting of species, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, osteosarcoma and central nervous system tumor. More preferably, but not limited to any one selected from the group consisting of cancer, prostate cancer, uterine cancer and osteosarcoma.

The isoegomarketone of the present invention is confirmed to induce apoptosis of cancer cells in various cancer cell lines through caspase-dependent or non-dependent pathways, thereby causing the death of cancer cells. And methods for treatment.

Hereinafter, Examples, Experimental Examples, and Production Examples are given to explain the present invention in more detail. However, the following Examples, Experimental Examples and Preparation Examples are provided only to explain the present invention more easily, and the content of the present invention is not limited by Examples, Experimental Examples, and Preparation Examples.

< Example  1> Of isoegomaketone (IK)  detach

Goma in isopropyl ketone (isoegomaketone, IK) is character leaflets (Perilla frutescens (L.) Britt. cv. Chookyoupjaso).

Specifically, the ground portion (3 kg) of dried Perilla frutescens (L.) Britt. Cv.Chookyoupjaso was left in 10 L of 95% methanol for 3 days and filtered through a filter paper. This solution was concentrated under reduced pressure to give a concentrate (45.6 g). Thereafter, the obtained extract was suspended in 400 ml of water and fractionated with 400 ml of ethyl acetate to obtain 17 g of an ethyl acetate fraction. 17 g of the obtained powder was subjected to silica gel column chromatography (silica gel: Merk, Art 9385, column size: 7 × 40 cm) using chloroform as a mobile phase to obtain fractions 1-6. The obtained fraction 1 (3 g) was subjected to silica gel column chromatography (silica gel: Merk, Art 9385, column size: φ7 × 40 cm) using a mixed solvent of ethyl acetate nucleic acid as a mobile phase. At this time, the mobile phase solvent was obtained with 10 mg of IK compounds, the most effective active substance in ethyl acetate: nucleic acid = 98: 2 (v / v), and separated iso-Egaketone by spectroscopic analysis, dissolved in sterile DMSO Was prepared.

< Example  2> Cancer cell line  culture

Human colorectal cancer DLD1 and HT-29 cell lines, PC-3 and DU145 prostate cancer cell lines, Hela uterine cancer cell lines, and U20S osteosarcoma cell lines (Chosun University) were heat-inactivated 10% fetal bovine serum (FBS) (Hyclone, Utah, USA) , 100 units / ml penicillin, and 100 ug / ml streptomycin (Invitrogen, Calif., USA) were incubated in RPMI1640 medium, incubator at 5% CO ₂ -95% oxygen, 37 ° C. Maintained at.

< Experimental Example  1> Of isoegomaketone (IK)  Confirmation of cancer cell viability and growth inhibition effect

In order to confirm the effect of the isoegomarketing (IK) of the present invention on cancer cell survival and growth in various cancer cell lines, each cell line was treated with IK, and then the viability and growth inhibition of the cancer cell line were confirmed. .

Specifically, each of the cancer cells (human colon cancer DLD1 and HT-29 cell lines, PC-3 and DU145 prostate cancer cell lines, Hela uterine cancer cell lines, and U20S osteosarcoma cell lines) cultured by the method of <Example 2> were 5 × 10 ^4. After 24 hours of incubation in 96 well plates at a concentration of cells / ml, 10, 20, 40, and 80 uM of isoegoma in each cell except DLD1 cell lines (10, 25, 50, and 100 uM isoeegomarketing treatment) Ketones (IK) were treated and then further incubated for 24 or 48 hours. Then, 10 ul of the EZ-Cytox Cell Viability Assay Kit (DAEILLAB, Korea) solution was added to each well, and further incubated at 37 ° C. for 4 hours, followed by a microplate reader (Benchmark plus, Absorbance at 480 nm was measured using Bio-Rad). At this time, the reference absorbance was 650 nm, and the result was expressed as a percentage of the value relative to that of the untreated control cells, and all the results were expressed by integrating the results obtained in at least three individual experiments.

As a result, it was confirmed that cell survival decreased concentration-dependently by treatment with isoegomarketing in all cancer cell lines (FIGS. 1A and 2 to 6).

The DLD1 cell lines were also treated with 10, 25, 50, and 100 uM of isoeegomarket, and the morphological changes of the cells were confirmed by phase contrast microscopy, resulting in blebbing of the cell surface during IK treatment and In addition to morphological changes such as shrinkage (shrinkage), it was confirmed that cell growth also inhibited concentration (Fig. 1B).

Through this, it was confirmed that the isoegomarketing (IK) of the present invention inhibits cell survival and growth in various cancer cell lines.

< Experimental Example  2> Of isoegomaketone (IK) Apoptosis ( apoptosis ) Induced effect

In order to confirm whether inhibition of cell viability induced by isoeegomarketing (IK) of the present invention is related to apoptosis, DLD1 cells were treated with IK, and then the degree of apoptosis was checked.

Specifically, DLD1 cells were cultured in 6-well plates at a concentration of 2 × 10 ⁵ cells / ml, and incubated with 10, 25, 50 and 100 uM concentrations of isoegomarket (IK) for 24 hours. The cells were washed and resuspended with 85 ul of binding buffer. 10 ul of Annexin V-FITC and 5 ul of propidium iodide were then added and incubated for 15 minutes at room temperature in the dark. After incubation, 400 ul of MEBCYTO Apoptosis Kit (MBL International, Nagoya, Japan) binding buffer was added and apoptotic cells were analyzed using a flow cytometer (Cytomics FC500, Beckman).

As a result, necrotic cells do not increase in proportion to the concentration of IK, while apoptotic cells increase depending on the concentration of treated IK upon isoegomarketing (IK) in DLD1 cells. It was confirmed (Fig. 7).

In addition, Western blot analysis using anti-PARP antibodies (Pharmingen, San Diego, Calif., USA) by treating IK to DLD1 cells revealed that PARP was cleaved, another sign of apoptosis when treated with 50 uM of IK. It was confirmed (Fig. 8A).

Through this, it was confirmed that the isoeegomarketing of the present invention has a cancer cell killing effect of inducing apoptosis and inhibiting the viability of cancer cells.

< Experimental Example  3> Of isoegomaketone (IK) Caspase ( caspase ) Check the activity inducing effect

Because caspases play an essential role in apoptosis, to determine whether apoptosis of cancer cells induced by isoegomarketing (IK) is related to caspase activity, IK may be applied to IK in DLD1 cells. Was treated and the activity of caspase was measured.

Caspase-3 colorimetric assay kit (Sigma-Aldrich, St. Louise, Mo.) is a peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) hydrolyzed by caspase-3 Based on, the pNA moiety of the peptide substrate is released, meaning that caspase-3 is activated.

Specifically, DLD1 cells are treated with 25 and 50 uM of isoegomarketing (IK) for 24 hours, or 50 uM of isoegomarketing (IK) is 0, 3, 6, 12, 18, and 24 hours After treatment for a while, cells were harvested and washed with PBS. After adding the cell crushing buffer solution to the obtained pellet (pellets) and maintained for 20 minutes in ice, centrifuged for 15 minutes at 4 ℃ at 16,000 × g to obtain a cell crushing solution, the protein contained in the cell crushing solution The concentration was measured. The cell lysate was then mixed with 2 mM substrate Ac-DEVD-pNA assay buffer solution and incubated at 37 ° C. for 2 hours, after which the value of pNA was measured at 405 nM absorbance of the microplate reader (Benchmark plus, Bio-Rad). Each pNA value was calculated from the calibration curve prepared by monitoring and standard pNA solution.

As a result, it was confirmed that the caspase-3 activity was increased in DLD1 cells treated with IK, and the caspase-3 activity was significantly increased in a concentration and time dependent manner, especially when treated with 50 uM of IK (FIG. 8 B and C).

In addition, anti-caspase-3, anti-caspase-8, anti-caspase-9, and anti-cleaved caspase-3 antibodies to cell extracts obtained by treating ILD to DLD1 cells (Cell Signaling Technology, Western blot analysis was performed using Danvers, MA, USA) as the primary antibody.

To obtain cell extracts, DLD1 cancer cells were incubated in a 100 mm dish at a concentration of 2 × 10 ⁶ cells / ml, washed with PBS, and harvested. Whole cell extract was obtained by adding NP40 cell lysis buffer containing 1 mM PMSF and protease inhibitor cocktail (Sigma-Aldrich, St. Louise, MO) to the cells, lysing for 30 minutes in ice conditions, and then centrifuging. Supernatants were harvested and prepared.

In addition, for Western blot analysis of the obtained extracts and fractions, first prepare 50 ug of protein per lane and boil in Laemmli sample buffer for 5 minutes, followed by electrophoresis on 10-15% SDS-PAGE gel It was. The protein was then blotted onto a nitrocellulose membrane (Hybond ECL Nitrocellulose, Amersham, USA). Thereafter, the membrane was blocked with TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk, according to the manufacturer's method. The reaction was diluted overnight at 4 ° C. with diluted primary antibody. After washing the membrane, the HRP conjugated anti-mouse IgG antibody (Zymed, San Francisco, CA, USA) or anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, CA, USA) was diluted 1: 4,000 at room temperature. The reaction was carried out for 2 hours. The membrane was washed and the blotted protein was purified using an enhanced chemiluminescence detection system (iNtRON Biotech). Detection was carried out.

As a result, it was confirmed that the initiators of caspase-8 and -9 and the effectors of caspase-3 were cleaved in a concentration- and time-dependent manner treated with isoegomarketone (FIG. 9A). Cleavage of caspase-8 was clearly identified in DLD1 cells treated with 50 uM of IK, and time kinetics of this cleavage were initiated 6 hours after treatment with 50 uM of IK, caspase-9 and Cleavage of -3 also showed a pattern similar to cleavage of caspase-8 (FIG. 9B).

In addition, after IK treatment to DLD1 cells, the downstream activity of effector caspase-3 was quantified, and it was confirmed that the caspase-3 activity was significantly increased in DLD1 cells treated with IK. .

This confirmed that apoptosis induced by treatment of IK in DLD1 cells activates caspases associated with exogenous and endogenous apoptosis pathways.

< Experimental Example  4> Of isoegomaketone (IK) Caspase ( caspase Of cancer cells through dependent pathways Apoptosis  Check the induction effect

<4-1> Confirmation of correlation with mitochondrial pathway (exogenous pathway)

Mitochondria play an essential role in apoptosis triggered by chemicals, and cytochrome c is released from mitochondria during apoptosis in the cell. In the cytoplasm, cytochrome c is released from the mitochondrial construct apoptosome complex with Apaf-1 and caspase-9 and activates caspase-3, leading to cell death (Mellier et. al., 2010; Tan et al., 2009; Wu, 2009).

Since apoptosis induced by IK activates caspase-9 (FIG. 5), in order to determine whether release of cytochrome c from the mitochondria into the cytoplasm occurs in the apoptosis induced by IK, anti-mitochondrial and cytoplasmic fractions are tested against the mitochondrial and cytoplasmic fractions. Western blot was performed in the same manner as in <Experimental Example 3> using cytochrome c antibody (clontech, Mountain View, CA, USA) as the primary antibody.

As a result, IK-treated DLD1 cells decreased in proportion to the concentration of IK in the mitochondria, whereas in the cytoplasm, the concentration was reversed, thereby confirming that cytochrome c was released from the mitochondria into the cytoplasm during IK treatment (FIG. 10A).

Thus, it was confirmed that apoptosis of cancer cells induced by the isoegomarketone of the present invention is related to the mitochondrial pathway.

<4-2> mitochondria Upstream  Correlation with Signal Path

Caspase-8 is a protein associated with mitochondrial upstream signals that mediate Bid cleavage and then transpose Bax to mitochondria via exogenous pathways (Mellier et al., 2010; Tan et al., 2009; Wu, 2009).

To measure the translocation of bax into the mitochondria and the release of cytochrome c from the mitochondria, 25 and 50 uM of IK were incubated with DLD1 cells. Thereafter, mitochondria and cytoplasmic fractions were prepared from the treated cells using a mitochondrial separation kit (Pierce, Rockford, IL), and anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Bid antibodies were prepared. Western blot was performed in the same manner as in <Experimental Example 3> using (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the primary antibody.

As a result, when IK was treated with DLD1 cells, Bax protein was increased in the mitochondria, decreased in the cytoplasm, and Bid protein was decreased in ILD-treated DLD1 cells (FIG. 10B). Through this, it can be seen that apoptosis of cancer cells induced by isoegomarketing of the present invention has cancer cell killing effect through cleavage of Bid and translocation of Bax.

< Experimental Example  5> Of isoegomaketone (IK) Caspase ( caspase ) Independent  Of cancer cells through the pathway Apoptosis  Check the induction effect

AIF is a sign of apoptosis through a caspase-independent pathway, which is known to release a response to a killing stimulus from the mitochondria after the apoptotic stimulus into the cytoplasm and then translocated into the nucleus, causing nuclear condensation (Huerta et al, 2006; Millan and Huerta, 2009).

To determine whether this AIF is associated with apoptosis induced by isoegomarketone (Ik), the amount of AIF translocated into the nucleus was measured.

Specifically, after treating the DLD1 cells with 25 and 50 uM of isoegomarket, DLD1 cell extract was obtained by the method of <Example 3>, and then NuCLEAR extraction kit (Sigma-Aldrich, St. Louise, MO). Nucleus and cytoplasmic fractions were isolated using), and the concentration of protein was quantified using the Bio-Rad protein assay (Bio-Rad). Then, Western blot was performed using the anti-AIF antibody (Pharmingen, San Diego, CA, USA) as the primary antibody, in the same manner as in <Experimental Example 3>.

As a result, when treated with isoegomarketing (IK), AIF decreased in the mitochondria and increased in the nucleus depending on the concentration of IK treatment (FIG. 11).

These results indicate that intranuclear translocation of AIF occurs in the induction of apoptosis by IK in DLD1 cells, and thus, the isoegomarketone of the present invention induces apoptosis of cancer cells via a caspase-independent pathway. Can be.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Powder  Produce

200 mg of the compound of formula 1 of the present invention

Lactose 100 mg

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of the compound of formula 1 of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

&Lt; 1-3 > Preparation of capsules

100 mg of the compound of formula 1 of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

100 mg of the compound of formula 1 of the present invention

Lactose 150 mg

Glycerin 100 mg

Xylitol 50 mg

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the compound of formula 1 of the present invention

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  2> Manufacture of food

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the compound of [Formula 1] of the present invention was added to the flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1 to 5.0 parts by weight of the compound of [Formula 1] of the present invention was added to soups and broth to prepare meat products for health promotion, soup of noodles and broth.

<2-3> ground Beef  Produce

10 parts by weight of the compound of Formula 1 of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> dairy products ( dairy products Manufacturing

5 to 10 parts by weight of the compound of [Formula 1] of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The compound of [Formula 1] of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh using a grinder to obtain a dry powder.

The grains, seeds, and the compound of [Formula 1] prepared above were formulated in the following ratio.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Compound (3 parts by weight) of Formula 1,

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

1 g of a compound of the formula 1 of the present invention is homogeneously mixed with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). After instant sterilization it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of Vegetable Juice

1 g of the compound of [Formula 1] of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

1 g of the compound of [Formula 1] of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

As seen above, the isoegomaketone (IK) of the present invention can be usefully used for the development of cancer prevention and treatment, or health food for cancer prevention and improvement.

Claims (11)

A pharmaceutical composition for preventing and treating cancer, comprising isoegomaketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Claim 2] The pharmaceutical composition for preventing and treating cancer according to claim 1, wherein the isoegomarket is a compound represented by the following [Formula 1]:
[Formula 1]

.
The method of claim 1, wherein the isoegomarket is Perilla leaf ( Perilla frutescens (L.) Britt. cv. Chookyoupjaso) is a pharmaceutical composition for the prevention and treatment of cancer, characterized in that it is separated from.
According to claim 1, wherein the cancer is any one selected from the group consisting of colorectal cancer, prostate cancer, uterine cancer and osteosarcoma, cancer prevention and treatment pharmaceutical composition.
Health food for the prevention and improvement of cancer containing isoegomaketone (IK) or a pharmaceutically acceptable salt thereof as an active ingredient.
The method for preventing and improving cancer of claim 5, wherein the isoegomarket is a compound represented by the following [Formula 1]:
[Formula 1]

.
The method of claim 5, wherein the isoegomarket is Perilla leaf ( Perilla frutescens (L.) Britt. cv. Chookyoupjaso) cancer prevention and improvement health food, characterized in that separated from.
According to claim 5, The cancer is cancer cancer prevention and improvement health food, characterized in that any one selected from the group consisting of colon cancer, prostate cancer, uterine cancer and osteosarcoma.
A method of treating cancer comprising administering a pharmaceutically effective amount of isoeegomaketone (IK) or a pharmaceutically acceptable salt thereof to a mammal, except for a human suffering from cancer.
A method of preventing cancer, comprising administering a pharmaceutically effective amount of isoeegomaketone (IK) or a pharmaceutically acceptable salt thereof to a mammal, except for a human.
The method of claim 9 or 10, wherein the cancer is any one selected from the group consisting of colorectal cancer, prostate cancer, uterine cancer and osteosarcoma.

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