KR101936249B1 - Composition for preventing, improving or treating cancer comprising kumatakenin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing, ameliorating or treating cancer comprising kumatakenin or a salt thereof as an active ingredient. The Kumatakenin of the present invention is excellent in the ability to inhibit the survival ability of cancer cells and inhibit cancer malignancy, and the pharmaceutical compositions and health functional foods using the same can effectively prevent, ameliorate, and treat cancer. Specifically, Kumatakenin of the present invention induces apoptosis in cancer cells to exhibit strong cytotoxicity to cancer cells, and inhibits the expression of MCP-1 and RANTES to inhibit the migration of macrophages to the tumor site Do. In addition, it suppresses the expression of M2 phenotype markers CD206 and Trem-2, and suppresses the expression of IL-10, VEGF, MMP-2 and MMP-9, which are cancer-promoting factors, Further, the Kumatakenin of the present invention is a naturally derived compound and has no side effects, so that it can be used variously as a pharmaceutical composition, a health functional food, and the like.

Description

[0001] The present invention relates to a composition for preventing, ameliorating or treating cancer,

The present invention relates to a composition for preventing, ameliorating or treating cancer comprising kumatakenin.

Specifically, the present invention relates to a pharmaceutical composition for preventing, ameliorating or treating cancer comprising Kumatakenin or a salt thereof as an active ingredient, and a health functional food.

It is generally known that cancer is caused by an uncontrolled growth of malignant cells due to an imbalance between cell growth and apoptosis. Apoptosis is one of the best known methods of programmed cell death. Inducing apoptosis in cancer cells is widely used as a therapeutic strategy to control the growth of cancer cells. In fact, many conventional anticancer agents are known to induce the death of tumor cells through apoptotic pathways.

Apoptosis is characterized by chromatin condensation, nucleosome DNA fragmentation, apoptotic body formation and activation of caspases. The tumor microenvironment, which consists of a variety of non-cancerous cells including immune cells, fibroblasts and endothelial cells, is recognized as a major factor influencing cancer development and progression. For example, most tumors are known to contain a large number of macrophages as a major component of host leukocyte infiltration. It has also been reported that macrophages change into the tumor-associated macrophage (TAM) of the M2 phenotype, which can promote tumor growth and metastasis. In this regard, TAM is a potential therapeutic target for cancer therapy (Tsuboki J, et al.) Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages. Sci. Rep. 2016; 6: 29588).

Gynecology The major cause of death in cancer is known to be ovarian cancer, and most ovarian cancer patients are diagnosed with advanced disease (stage Ⅲ or Ⅳ) and the survival rate is only 10-30%. Conventional chemotherapeutic agents such as platinum and taxane have generally been used for the treatment of ovarian cancer. However, they have shown a wide range of side effects such as nausea and vomiting (Ferrandina G, et al. Increased cyclooxygenase-2 (COX-2) expression 2002; 13: 1205-1211), and it has been reported that resistance to chemotherapy repeatedly appears in most patients (Kigawa J. New strategy for overcoming resistance to chemotherapy of ovarian cancer. Yonago Acta Med. 2013; 56: 43-50). Thus, there is a need for the development of new therapeutic agents for ovarian cancer, especially late stage and recurrent ovarian cancer.

On the other hand, buds of Syzygium aromaticum , commonly known as cloves, are one of the most useful spices that have been used for food preservatives and medicinal purposes for centuries. Cloves have been reported to have biological activities such as antibacterial, antifungal, antioxidant and anti-inflammatory activities (Kislev ME, et al., Early domesticated fig. However, little is known about the biological activity of kumatakenin, a component isolated from cloves, and there is little known about the anticancer efficacy of Kumatakenin in particular.

It is an object of the present invention to provide a composition for preventing, improving or treating cancer including kumatakenin.

Specifically, the present invention aims to provide a pharmaceutical composition for preventing, ameliorating or treating cancer comprising Kumatakenin or a salt thereof as an active ingredient and a health functional food.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, kumatakenin or a pharmaceutically acceptable salt thereof.

The Kumatakenin of the present invention is a naturally derived compound, and its specific structure is shown in the following formula (1).

[Chemical Formula 1]

The Kumatakenin of the present invention can be chemically synthesized or isolated from natural products, and those commercially available from domestic or overseas can be purchased and used.

In an embodiment of the present invention, Syzygium aromaticum L .; Clove) was separated and purified and used.

Specifically, the dried and powdered seeds of Aromaticum (2.1 kg) purchased from the tiller extract market were extracted twice with 20 L of 70% EtOH at 60 ° C for 2 hours in a water bath, and the solvent was extracted at 40 ° C And evaporated under vacuum. The above 70% EtOH extract (625.0 g) was successively fractionated with n-hexane (2 L × 3), EtOAc (2 L × 3) and BuOH (2 L × 3), and each n-hexane extract (303.1 g), EtOAc- Soluble extracts (132.0 g), BuOH extracts (93.0 g) and aqueous extracts (95.7 g) were obtained.

The EtOAc- soluble extract _{(132.0 g) CH 2 Cl 2} - soluble fraction was fractionated by-soluble and non. The CH ₂ Cl ₂ -soluble fraction (12.2 g) was added to the mobile phase as a CH ₂ Cl ₂ -MeOH mixture (gradient from 1: 0 to 9: 1 v / v), silica gel (70-230 mesh, 44 cm) was performed to obtain 7 fractions (EM1-EM7). Elution with EM3 fractions [CH ₂ Cl ₂ -MeOH (19: 1 v / v); 7.2 g] was subjected to silica gel column chromatography (CC) (230-400 mesh,? 5.0 x 33 cm, n-hexane-EtOAc = 4: 1 to 1: 1 v / EM3-1-EM3-8). Among the lower fractions, reversed-phase CC (YMC gel 150 μm, φ3.5 × 24 cm) using MeOH-H ₂ O mixture (7: 3 v / v) against EM3-4 was performed to obtain 9.0 mg ).

The term "extract" used in the present invention refers to an extract obtained by extracting a substance, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a preparation or a purified product of the extract, Extracts themselves and extracts of all formulations which can be formed using extracts.

The method for producing the extract of the present invention is not particularly limited and may be carried out according to a method commonly used in the art. Examples of the extraction method include hydrothermal extraction method, ultrasonic extraction method, filtration method, reflux extraction method, immersion extraction method, high temperature and high pressure steam extraction method, etc. These methods can be performed alone or in combination of two or more methods have. The kind of the extraction solvent used in the present invention is not particularly limited, and any solvent known in the art can be used.

The term "fraction " as used herein means a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited and may be carried out according to a method commonly used in the art. As a non-limiting example of the above-mentioned fractionation method, there can be mentioned a method in which a fraction is obtained from the extract by treating the extract with a predetermined solvent.

The kind of the fraction solvent used for obtaining the fraction in the present invention is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fraction solvent include polar solvents such as water and alcohol; And non-polar solvents such as hexane, ethyl acetate, chloroform and dichloromethane. These may be used alone or in combination of two or more.

The inventors of the present invention have for the first time determined that Kumatakenin of the present invention has an excellent effect of inhibiting the survival ability of cancer cells and suppressing cancer malignancy, and thus, the first use of Kumatakenin for the prevention, improvement or treatment of cancer.

Specifically, in the examples of the present invention, Kumatakenin inhibitory effect on the cancer cell viability of human ovarian cancer cells (A2780 and SKOV3) was confirmed, and Kumatakenin showed strong cytotoxicity to both A2780 and SKOV3 (Fig. 2A), confirming that Kumatakenin is involved in inducing apoptosis in cancer cells (Fig. 4).

In addition, in another embodiment of the present invention, the inhibitory effect of Kumatakenin on the expression of genes involved in malignancy of tumors showed that Kumatakenin inhibited the expression of MCP-1 and RANTES, (FIG. 6) inhibited the expression of the M2 phenotype markers CD206 and Trem-2 (FIG. 7) and inhibited the expression of IL-10, VEGF, MMP-2 and MMP-9 Expression was inhibited (Fig. 8), and it was confirmed that cancer malignancy was effectively inhibited.

The term " monocyte chemoattractant protein-1 (MCP-1) " and "RANTES (Regulated on Activation, normal T cell expressed and secreted ") used in the present invention are CC- And is known to induce macrophage to migrate into the tumor site.

The term " CD206 " and "Trem-2 (Triggering Receptor expressed on myeloid cells 2) " used in the present invention refer to M2 phenotype markers. Macrophages transferred to tumor sites have M2 phenotype and tumor promoting activity by cancer cells It is known that TAM can be converted to tumor-associated macrophage (TAM), and it is known that cancer is malignant through M2 polarization of TAM.

The terms "IL-10 (interleukin-10)", "Vascular endothelial growth factor (VEGF)", "Matrix metalloproteinase-2" and "Matrix metalloproteinase- "Is known as a tumor promoting factor, and cancer is known to be malignant through the expression of these tumor promoting factors in tumor-associated macrophages.

As used herein, the term "cancer" refers to a disease caused by a disorder in the function of regulating normal division, differentiation or death of cells, resulting in abnormally excessive proliferation and invasion into surrounding tissues or organs to form lumps, It means any state that destroys or transforms the structure. Cancer is largely divided into primary carcinoma in the affected area and metastatic cancer that has spread to other parts of the body from the site of development.

The diseases to be prevented, ameliorated or treated according to the present invention are interpreted to include all of them as long as they fall within the category of cancer. Specific disease names are not particularly limited, but preferably ovarian cancer, colon cancer, pancreatic cancer, gastric cancer, liver cancer, Breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, brain tumor, prostate cancer, gallbladder cancer, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, uterine cancer, rectum cancer, fallopian tube carcinoma, endometrial carcinoma, Cancer, endocrine adenocarcinoma, adrenal cancer, and urethral cancer, and more preferably it may be an ovarian cancer.

As used herein, the term " pharmaceutically acceptable salt " refers to a salt of the form that can be used pharmaceutically, among the salts in which the cation and anion are bound by electrostatic attraction, Salts with bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Examples of the metal salt include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, barium salt, etc.), aluminum salts and the like; Examples of salts with organic bases include salts with organic bases such as triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, Salts with isoparaffin; Salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like; Salts with basic amino acids include salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid and the like.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to comprising coumadacin or a pharmaceutically acceptable salt thereof as an active ingredient.

The kind of carrier which can be used in the present invention is not particularly limited and any carrier commonly used in the technical field can be used. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, . These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be supplemented with other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, and may be used as fillers, extenders, wetting agents, disintegrants, , A binder, a lubricant, and the like may be additionally used.

In the pharmaceutical composition of the present invention, Kumatakenin or a pharmaceutically acceptable salt thereof may be contained in an amount of 0.00001% by weight to 99.99% by weight based on the total weight of the pharmaceutical composition, preferably 0.1% But it is not limited thereto, and it may vary depending on the condition of the subject to be administered, the type of the disease to be treated, the degree of progress of the disease, etc. And can be variously changed. If desired, it may also be included in the total content of the pharmaceutical composition.

The pharmaceutical composition of the present invention may be formulated into various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of oral dosage forms using the pharmaceutical compositions of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft Capsules, syrups, and elixirs.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and the like. Sweetening agents, fragrances, syrups, and the like may also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be further used.

Non-limiting examples of the parenteral preparation using the pharmaceutical composition of the present invention include injections, suppositories, respiratory inhalation powders, aerosol preparations for spraying, ointments, powder for application, oils and creams.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and external preparations may be used. Examples of the non-aqueous solutions and suspensions include propylene glycol, polyethylene Glycerol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

When the pharmaceutical composition of the present invention is formulated into an injection, the pharmaceutical composition of the present invention is mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which is used for unit administration of an ampoule or a vial Can be formulated.

When the pharmaceutical composition of the present invention is formulated into an aerosol formulation, a propellant or the like may be added together with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

When the pharmaceutical composition of the present invention is formulated into an ointment, a cream, a powder for application, an oil, an external preparation for skin or the like, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, a polyethylene glycol, , Silica, talc, zinc oxide or the like as a carrier.

The pharmaceutically effective amount and the effective dose of the pharmaceutical composition of the present invention can be varied depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and the kind of reaction to be achieved by administration of the pharmaceutical composition Including the age, weight, general health condition, degree and severity of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, and other compositional components, etc. May be varied according to factors well known in the art and in the pharmaceutical arts, and those of ordinary skill in the art can readily determine and prescribe dosages that are effective for the desired treatment.

The administration of the pharmaceutical composition of the present invention may be administered once a day or divided into several doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking into consideration all of the above factors, in such an amount as to obtain the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The Kumatakenin of the present invention is a compound derived from a natural product and can be safely used for prolonged administration for the purpose of prevention, improvement and treatment of diseases without toxicity and side effects.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and may be arbitrarily selected depending on the route of administration and the mode of administration as long as the pharmaceutical composition can reach the desired site. The pharmaceutical composition may be administered orally or parenterally.

Examples of the parenteral administration method of the pharmaceutical composition of the present invention include intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and the method of applying, spraying or inhalation the above composition But are not limited thereto.

The pharmaceutical composition of the present invention may be used alone, but may be used in combination with various cancer treatment methods such as radiotherapy and chemotherapy in order to increase the therapeutic efficiency.

The present invention provides a health functional food for preventing or ameliorating cancer comprising Kumatakenin or a pharmaceutically acceptable salt thereof as an active ingredient.

The term " pharmaceutically acceptable salt " used in the present invention means a salt which can be used in foodstuffs among salts in which cations and anions are bound by electrostatic attraction, Specific examples include examples of the above-mentioned "pharmaceutically acceptable salts ".

The term " health food "used in the present invention refers to a food having an active health promotion effect or a health promotion effect as compared to a general food, and a health supplement food means a health food. In some cases, the terms functional food, health food, and health supplement are used. The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids,

The term " functional food " as used in the present invention refers to a food for special health use (FoSHU). In addition to nutrition, the term " functional food " It means foods with high effectiveness.

The health functional food of the present invention may contain additional components that are commonly used in food compositions and can improve odor, taste, visual appearance and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like. It is also possible to use preservatives such as potassium sorbate, sodium benzoate, salicylic acid and dihydroxyacetate, disinfectants such as bleaching powder and highly bleached sodium hypochlorite, antioxidants such as butylhydroxyanilide (BHA), butylhydroxytoluene (BHT ), Coloring agent (tar color, etc.), coloring agent (such as sodium nitrite, sodium acetylate), bleaching agent (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (hypodermic, cyclamate, saccharin, (Food additive) such as flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), reinforcing agents, emulsifiers, thickeners, encapsulating agents, gum bases, foam inhibitors, Can be added. The additives may be selected and used in appropriate amounts depending on the type of food.

When the health functional food of the present invention is used as a food additive, it may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method.

In the health functional food of the present invention, the content of Kumatakenin or a pharmaceutically acceptable salt thereof is not particularly limited and may be variously changed depending on the state of the subject to be administered, the type of the specific disease, the progress of the disease, and the like. If necessary, it may also be included in the total content of the food.

The kumatakenin of the present invention is excellent in the ability to inhibit cancer cell viability and inhibit malignant cancer, and the pharmaceutical composition and health functional food using the same can effectively prevent, improve and treat cancer.

Specifically, Kumatakenin of the present invention induces apoptosis in cancer cells to exhibit strong cytotoxicity to cancer cells, and inhibits the expression of MCP-1 and RANTES to inhibit the migration of macrophages to the tumor site Do. In addition, it suppresses the expression of M2 phenotype markers CD206 and Trem-2, and suppresses the expression of IL-10, VEGF, MMP-2 and MMP-9, which are cancer-promoting factors,

Further, the Kumatakenin of the present invention is a naturally derived compound and has no side effects, so that it can be used variously as a pharmaceutical composition, a health functional food, and the like.

Figure 1 is a chemical structural formula of Kumatakenin.
Fig. 2 is a graph showing the results of the reaction of coumaracin (a), quercetin (b), pachidol (c), luteolin (d), rhamnazin-3-0- ) And laminazin-3-O-? -D-glucoside (f) on human ovarian cancer cells A270 and SKOV3.
FIG. 3 is a graph showing the results of analysis of cell cycle by performing PI staining for SKOV3 treated with coumadacin.
FIG. 4 is a graph showing the results of analysis of apoptosis by performing double-staining with annexin V and PI in SKOV3 treated with coumadacin.
FIG. 5 is a graph and a graph showing the results of confirming whether apoptosis of SKOV3 induced by Kumatakenin contained a caspase-dependent pathway (#: p <0.05 compared with the control group; *: p <0.05 compared to Kumatakenin) .
FIG. 6 is a graph showing the results of confirming the expression levels of chemokine MCP-1 (a) and RANTES (b) in SKOV3 treated with coumatakene (*: p <0.05).
FIG. 7 is a graph showing the results of confirming the expression levels of the M2 phenotype markers CD206 (a) and Trem-2 (b) in SKOV3 treated with Kumatakenin (*: p <0.05).
FIG. 8 is a graph showing the results of confirming expression levels of tumor promoting factors IL-10 (a), VEGF (b), MMP-2 (C) and MMP-9 (D) in SKOV3 treated with coumadacin (*: p < 0.05).

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

Statistical analysis

Data are presented as means ± SD (SD). Student's t-test and one-way analysis of variance were used to identify statistically significant differences. P value <0.05 was considered statistically significant.

Example  One: Of the kumatakenin  Separation purification

Example  1-1: cloves  The extract and Fraction  Produce

The preparation of the extracts and compounds used in the examples of the present invention followed conventional methods (Ryu B, et al., New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove). J Agric Food Chem. : 3048-3053).

Briefly, Syzygium aromaticum L .; Clover) buds were purchased in the Kyungdong Medicines Market in June 2013. The origin of herbal medicines was confirmed by professor Zhang Dae-shik and a sample of evidence (SYAR1-2013) was deposited with the College of Pharmacy, Kyung Hee University. The dried and powdered plant material (2.1 kg) was extracted twice with 20 L of 70% EtOH at 60 ° C for 2 hours in a water bath, and the solvent was evaporated in vacuo at 40 ° C. The above 70% EtOH extract (625.0 g) was successively fractionated with n-hexane (2 L × 3), EtOAc (2 L × 3) and BuOH (2 L × 3), and each n-hexane extract (303.1 g), EtOAc- Soluble extracts (132.0 g), BuOH extracts (93.0 g) and aqueous extracts (95.7 g) were obtained.

The EtOAc- soluble extract _{(132.0 g) CH 2 Cl 2} - soluble fraction was fractionated by-soluble and non. The CH ₂ Cl ₂ -soluble fraction (12.2 g) was added to the mobile phase as a CH ₂ Cl ₂ -MeOH mixture (gradient from 1: 0 to 9: 1 v / v), silica gel (70-230 mesh, 44 cm) was performed to obtain 7 fractions (EM1-EM7). Elution with EM3 fractions [CH ₂ Cl ₂ -MeOH (19: 1 v / v); 7.2 g] was subjected to silica gel column chromatography (CC) (230-400 mesh,? 5.0 x 33 cm, n-hexane-EtOAc = 4: 1 to 1: 1 v / EM3-1-EM3-8).

Example  1-2: Kumatakenin  And other compound separation purification

Reversed phase CC (YMC gel 150 μm, φ3.5 × 24 cm) using a MeOH-H ₂ O mixture (7: 3 v / v) was performed to obtain 9.0 mg of kumatakenin from the subfraction EM3-4 ) And pachypodol (5.5 mg). Quercetin (35.1 mg) was obtained from sub-fraction E3-5 via recrystallization (MeOH). The lower fraction E-6 was further fractionated using Sephadex LH-20 (? 2.4x40 cm) with a CH ₂ Cl ₂ -MeOH mixture (1: 1 v / v) to give luteolin (14.0 mg). Redi Sep-C18 (26g, MeOH -H 2 O, 3.5: 6.5 to 7: 3 v / v) cartridge using a sub-fraction person Rajin -3-O-β-D- glucoside from E4-5 (rhamnazin- 3-O-? -D-glucoside (20.3 mg) and rhamnazin-3-O-? -D-glucuronide-6 ""-methylester) (8.0 mg).

Example  2: Cell culture

Human ovarian cancer cell lines (A2780 and SKOV3) and human monocytic cell line (THP-1) were obtained from the American Type Culture Collection (ATCC). Ovarian cancer cells were cultured in RPMI 1640 supplemented with 5% fetal bovine serum (FBS), penicillin (100 U / mL) and streptomycin sulfate (100 ug / mL). THP-1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin (100 U / mL), streptomycin sulfate (100 μg / mL) and 0.05 mM 2-mercaptoethanol. The THP-1 cells were differentiated into macrophages for 24 hours with 100 nM PMA. Tumor-associated macrophages (TAM) were prepared from SKOV3 ovarian cancer cells by stimulation of THP-1 macrophages with conditioned media for 24 hours.

Example  3: Kumatakenin  Confirming Cancer Cell Viability Suppression Efficacy

Example  3-1: cloves Fraction  Confirming Cancer Cell Viability Suppression Efficacy

MTT assay was performed to investigate the effect of cloaking on cell viability of human ovarian cancer cells (A2780 and SKOV3).

Briefly, cells (5 x 10 ⁴ ) were dispensed into each well of a 96-well plate containing 50 μL of RPMI medium per well. After 24 hours, six compounds isolated from various concentrations of 70% EtOH extract, n-hexane fraction, EtOAc fraction, BuOH fraction, water fraction and n-hexane-soluble extract were added. After 48 hours, 25 μL of MTT (5 mg / mL stock solution) was added and the plates were incubated for an additional 4 hours. Then, after the medium was removed, the formazan blue formed in the cells was dissolved in 50 μL of DMSO. Optical density was measured at 540 nm using a microplate spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale, Calif., USA).

[Table 1]

a) IC ₅₀ means the concentration when the number of cells is reduced by 50% compared with the control. The data values represent the average of the results from three independent experiments conducted in a similar manner.

As shown in Table 1 above, only the EtOAc fraction among the four fractions showed significant cytotoxic activity with an IC50 value of less than 100 μM in both A2780 and SKOV3 cells. Thus, the cytotoxic activity of the compounds in the EtOAc fractions on ovarian cancer cells was examined as described below.

Example  3-2: Clove EtOAc From the fraction  Identification of the ability of the isolated compounds to inhibit cancer cell viability

In a previous study of the present inventors (Ryu B, et al., New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove) .J Agric Food Chem. 2016; 64: 3048-3053), among the 19 flavonoids isolated from the EtOAc fraction , 6 flavonoids (Kumatakenin, Pachidol, Quercetin, Luteolin, Rhamnazin-3-O-? -D-glucuronide-6 "-methyl ester and Rhamnazin- Glucoside) was slightly cytotoxic. Thus, among the total of 19 flavonoids for further confirming the cytotoxic activity against two ovarian cancer cells (A2780 and SKOV3), the six flavonoids were selected .

As a result of the above experiments, it was found that rhamnazine-3-O- beta -D-glucuronide-6 "-methyl ester and rhamnazine-3-O- beta -D- glucoside having a sugar residue were found to be mild in both A2780 and SKOV3 cells But not in SKOV3 cells In contrast, luteolin and pachydol significantly inhibited cell viability in A2780 cells but did not significantly inhibit cell viability In both A2780 and SKOV3 cells, Lt; / RTI &gt; (FIG. 2).

Quercetin, commonly found in many fruits, vegetables, leaves and grains, has been studied for its anticancer activity. On the other hand, Kumatakenin has little known about its biological activity. Accordingly, the present inventors have elucidated the molecular mechanism of the inhibitory effect of Kumatakenin on the cell viability of ovarian cancer cells.

Example  4: Kumatakenin  For cancer cells Apoptosis ( apoptosis ) Induction efficacy Sho sign

SKOV3 To investigate whether the inhibitory effect of Kumatakenin on the cell viability of human ovarian cancer cells is related to the cell cycle arrest or apoptosis, it is known that after the administration of propidium iodide (PI) Cell cycle distribution was determined using metric analysis, and Annexin V-FITC analysis and PI double staining were performed to analyze apoptosis.

PI staining for cell cycle analysis was performed as follows. In Example 3-2, cells with suppressed cell viability were treated with Kumatakenin and washed twice with ice-cold phosphate-buffered saline (PBS). The cells were fixed and ice-cold 70% ethanol was infiltrated at 4 ° C for 1 hour. The cells were washed once with PBS and resuspended in a staining solution containing PI (50 [mu] g / mL) and RNase (250 [mu] g / mL). The cell suspension was incubated in a dark room at room temperature for 30 minutes and then analyzed by fluorescence-activated cell sorting (FACS) Carter-plus flow cytometry (Bedton Dickinson Co., Germany) Respectively.

Annexin V and PI double staining for apoptosis analysis was performed in the following manner. For Annexin V and PI double staining, the cells were suspended in 100 μL of binding buffer (10 mM HEPES / NaOH, 140 mM NaCl, 2.5 mM CaCl ₂ , pH 7.4) and 1.25 μL of FITC-conjugated Annexin V And PI (50 mg / ml). The mixture was incubated in the dark for 15 minutes at room temperature and analyzed with a FACS Carter-Plus flow cytometer.

As a result, Kumatakenin induced an increase in sub-G1 cell group in SKOV3 cells, and no significant cell cycle arrest was observed after treatment with Kumatakenin (Fig. 3).

On the other hand, the treatment with Kumatakenin increased the population of annexin V-FITC-positive cells (apoptosis cells) to 37.25% in the right quadrant of the flow cytometry graph in a time-dependent manner (Fig. These results suggest that the inhibitory effect of Kumatakenin on the viability of human ovarian cancer cells is mediated by apoptosis rather than cell cycle arrest.

Example  5: Kumatakenine  Induced by Apoptotic Caspase ( caspase ) Dependency Check

In many cases, apoptosis is known to require caspase activation (Villaflores OB, et al. Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity against Mycobacterium tuberculosis. Pharmacogn Mag. 2010; 6: 339-344). Western blot analysis was performed as follows to evaluate the effect of kuramatake nin on the activation of caspase-3, caspase-8 and caspase-9.

SKOV3 cells in which the cell viability was suppressed by treatment with Kumatakenin in Example 3-2 were washed with ice-cold PBS and extracted from the protein lysis buffer. Protein concentrations were determined using the Bradford assay. Total protein (30 μg) was dissolved using a 10% SDS-PAGE gel, electrotransferred onto polyvinylidene fluoride membrane, and then treated with Tris buffered saline (Bust Biological Technology, Ltd., Wuhan, China) with 5% skim milk powder for 1 hour. The membranes were incubated overnight at 4 [deg.] C with the primary antibody and the actin antibody. The membrane was washed with PBS containing 5% nonfat milk and 0.1% Tween-20 and then incubated with a goat anti-rabbit immunoglobulin G antibody conjugated with horseradish peroxidase. Finally, the membrane was incubated with an enhancement chemiluminescent reagent (EMD Millipore, Billerica, MA) and exposed to Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).

As a result of the experiments, Kumatakenin markedly activated caspase-3, caspase-8 and caspase-9 in SKOV3 cells in a dose-dependent manner as their pro-form density decreased (FIG.

Z-DEVD-fmk (a specific caspase-3 inhibitor), z &lt; RTI ID = 0.0 &gt; -IEVD-fmk caspase-8 inhibitor) and z-LEHD-fmk (specific caspase-9 inhibitor) were used. As a result of the experiments, the caspase inhibitor remarkably reversed the apoptosis induced by coumathenine (Fig. 5B), indicating that apoptosis induced by coumathenine in human ovarian cancer cells contains a caspase dependent pathway .

Example  6: Kumatakenin MCP -1 and RANTES  Confirming expression suppression efficacy

MCP-1 and RANTES are highly expressed in several cancer cells and are known to play an important role in allowing macrophages to migrate to tumor sites. Thus, the present inventors investigated the effect of kumatakehenin on the expression of MCP-1 and RANTES in human ovarian cancer cells. RNA isolation and real-time RT-PCR analysis performed in the examples of the present invention were carried out in the following manner.

RNA of SKOV3 cells treated with Kumatakenin in Example 3-2 was extracted using an Easy Blue ^® kit (Intron Biotechnology, Seoul, Korea) according to the manufacturer's instructions. Total RNA was reverse transcribed according to the manufacturer's protocol with first strand cDNA (Amersham Pharmacia Biotech, Oakville, ON, Canada). The synthesized cDNA was used as a template for polymerase chain reaction (PCR) amplification. Real-time PCR was performed using a Thermal Cycler Dice Real Time PCR System (Takara, Tokyo, Japan). The primers used in the SYBR Green real-time RT-PCR were as follows: 5'-GCT CAT AGC AGC CAC CTT CA 3 '(SEQ ID NO: 1) as an MCP-1 sense primer and 5'- GGA CAC TTG CTG CTG GTG AT-3 '(SEQ ID NO: 2); 5'-CCT CAT TGC TAG GCC CTC T-3 '(SEQ ID NO: 3) as the RANTES sense primer and 5'-GGT GTG GTG TCC CGA GGA AT-3' (SEQ ID NO: 4) as the antisense primer; 5'-GAG TCA ACG GAT TTG GTC GT-3 '(SEQ ID NO: 5) as a GAPDH sense primer and 5'-TTG ATT TTG GAG GGA TCT CG-3 (SEQ ID NO: 6) as an antisense primer.

The dissociation curve analysis showed a single peak, and PCR was performed 50 times using the following conditions: denaturation at 95 ° C for 5 seconds, annealing at 57 ° C for 10 seconds, and extension at 72 ° C for 20 minutes. The mean cycle threshold (Ct) of the gene of interest was calculated from three measurements and normalized to the mean Ct of the control gene, GAPDH.

As a result, Kumatakenin significantly reduced mRNA levels of both MCP-1 and RANTES in SKOV3 cells (Fig. 6). These results indicate that Kumatakenin inhibits the secretion of MCP-1 and RANTES from cancer cells by controlling the expression of chemokine MCP-1 and RANTES in ovarian cancer cells, thereby inhibiting the migration of macrophages to the tumor.

Example  7: Kumatakenin  M2 phenotype Marker  And inhibiting the expression of cancer-promoting factors

It has been reported that macrophages that migrate to the tumor site can be converted to TAM with M2 phenotype and tumor-promoting activity by cancer cells (De Palma M, Lewis CE. Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell. 2013; 23: 277-286). We therefore investigated the effects of Kumatakenin on M2 polarization and pro-tumor activation of macrophages. The primers used for real-time RT-PCR analysis were as follows: 5'-CTT TCA CAA GTA TCC ACA CCA TC-3 '(SEQ ID NO: 7) as a CD206 sense primer and 5'- CTT TCA TCA CCA CAC AAT CCT C-3 '(Ward No. 8); 5'-TTG CCC CTA TGA CTC CAT GA-3 '(SEQ ID NO: 9) as a Trem-2 sense primer and 5'-CGC AGC GTA ATG GTG AGA GT-3' (SEQ ID NO: 10) as an antisense primer; 5'-CCA CTT GCG GTC ATC ATC GT-3 '(SEQ ID NO: 12) as an MTC-2 sense primer, 5'-ACC GCG ACA AGA AGT ATG GC-3' 5'-CGA TGA CGA GTT GTG GTC CC-3 '(SEQ ID NO: 13) as an MMP-9 sense primer and 5'-TCG TAG TTG GCC GTG GTA CT-3' (SEQ ID NO: 14) as an antisense primer; VEGF, 5'-ATG GCA GAA GGA GGA GGG CA-3 '(SEQ ID NO: 15) as a sense primer and 5'-ATC GCA TCA GGG GCA CAC AG-3' (SEQ ID NO: 16) as an antisense primer; (SEQ ID NO: 17) as 5'-GAC AAG CTG GAC AAC ATA CTG CTA A-3 '(SEQ ID NO: 17) as an IL-10 sense primer and 5'- GAT AAG GCT TGG CAA CCC AAG TAA- ).

As a result, the expression of the M2 phenotypic markers CD206 and Trem-2 was increased in TAM, a macrophage stimulated by conditioned medium of SKOV3 cells, compared with control macrophages, and Kumatakenin was increased in TAM CD206 and Trem-2 (Fig. 7). In addition, Kumatakenin significantly inhibited the expression of IL-10, VEGF, MMP-2 and MMP-9, which are known as tumor promoting factors in TAM (Fig. 8). These results suggest that Kumatakenin inhibits the selective activation of macrophages that contribute to ovarian cancer progression.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other forms without departing from the spirit or scope of the inventive concept as defined by the appended claims and their equivalents.

<110> UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY <120> Composition for prevention, improving or treating cancer          comprising kumatakenin <130> P17-042-KHU <160> 18 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MCP-1 sense primer <400> 1 gctcatagca gccaccttca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MCP-1 antisense primer <400> 2 ggacacttgc tgctggtgat 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RANTES sense primer <400> 3 cctcattgct aggccctc 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RANTES antisense primer <400> 4 ggtgtggtgt cccgaggaat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 5 gagtcaacgg atttggtcgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 6 ttgattttgg agggatctcg 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD206 sense primer <400> 7 cctcacaagt atccacacca tc 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD206 antisense primer <400> 8 ctttcatcac cacacaatcc tc 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Trem-2 sense primer <400> 9 ttgcccctat gactccatga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Trem-2 antisense primer <400> 10 cgcagcgtaa tggtgagagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 sense primer <400> 11 accgcgacaa gaagtatggc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-2 antisense primer <400> 12 ccacttgcgg tcatcatcgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 sense primer <400> 13 cgatgacgag ttgtggtccc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MMP-9 antisense primer <400> 14 tcgtagttgg ccgtggtact 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGF sense primer <400> 15 atggcagaag gaggagggca 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VEGF antisense primer <400> 16 atcgcatcag gggcacacag 20 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> IL-10 sense primer <400> 17 gaccagctgg acaacatact gctaa 25 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-10 antisense primer <400> 18 gataaggctt ggcaacccaa gtaa 24

Claims (12)

A pharmaceutical composition for preventing or treating ovarian cancer comprising as an active ingredient Kumatakenin or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has cytotoxicity against cancer cells. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition has an effect of inducing apoptosis in cancer cells. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has an effect of inhibiting the expression of a gene selected from the group consisting of MCP-1, RANTES, CD206, Trem-2, IL-10, VEGF, MMP-2 and MMP- &Lt; / RTI &gt; delete delete A health functional food for preventing or ameliorating ovarian cancer comprising Kumatakenin or a pharmaceutically acceptable salt thereof as an active ingredient. The health functional food according to claim 7, wherein the health functional food has cytotoxicity against cancer cells. The health functional food according to claim 8, wherein the health functional food has an effect of inducing apoptosis in cancer cells. The method of claim 7, wherein the health functional food has an effect of suppressing the expression of a gene selected from the group consisting of MCP-1, RANTES, CD206, Trem-2, IL-10, VEGF, MMP-2 and MMP- Having a health functional food. delete delete

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