KR102268932B1 - Composition for preventing or treating cancer comprising compound represented by formula 1 - Google Patents

Abstract

The present invention relates to a novel use of the compound represented by the formula (1), which can be usefully used for the prevention or treatment of cancer. More specifically, the compound of the present invention can inhibit the migration and invasion of cancer cells by inhibiting the levels of MMP-9 and IL-8 or reducing the activities of PKCα and JNK.

Description

A composition for preventing or treating cancer comprising a compound represented by Formula 1 {COMPOSITION FOR PREVENTING OR TREATING CANCER COMPRISING COMPOUND REPRESENTED BY FORMULA 1}

The present invention relates to a novel use of the compound represented by the formula (1), which can be usefully used for the prevention or treatment of cancer.

Cancer is one of the leading causes of death and accounts for approximately 25.6% of deaths worldwide. In particular, breast cancer is the leading cause of cancer mortality in women worldwide. About 20,000 women suffer from breast cancer each year, and the number is expected to rise. If breast cancer is diagnosed early, it can be treated through resection after surgery. However, treatment can be difficult when breast cancer has metastasized to organs such as lymph glands, bones, lungs, brain, and skin. This refers to the process by which tumor cells from the primary site metastasize to other secondary sites in the host. In this process, invasion of cells into surrounding tissues is essential and is a key characteristic of malignant cancer cells, and degradation of the extracellular matrix (ECM) and basement membrane can promote tumor cell migration and invasion.

On the other hand, arthraxon hispidus is an annual grass of the monocotyledonous plant family, and it is a weed that grows in ditches or roadside. It is mainly distributed in warm regions of Asia such as China, Korea, Manchuria, Japan, Mongolia, Siberia, India and Malaysia, and 15 species are distributed around the world, and one species is native to Korea. The height is about 20-50 cm. The lower part of the flower stalk crawls on the ground, the root grows from the stem node and spreads, and the upper part rises obliquely. The leaves are egg-shaped, and the lower part of the leaf wraps the stem, and there are setae at the edge. Sep.~Oct. At the end of the stem, 3 to 20 spikelets of flowers hang in the shape of a palm. The spikelets form a pair and hang on the flower node, but the spikelets with stalks degenerate and become only short stalks. The dorsal surface of the outer hull is round and hairy. The leaves and stems are often used for yellow paint. In addition, the Chinese book <Bonjing> states that it is effective in stopping coughing and phlegm and has an insecticidal detoxification effect. The components of clams include aconitic acid, arthraxin, luteolin, and luteolin-7-glucoside, and the main compound is flavone. belong to the class This plant has been used to dye fabrics as a natural yellow dye.

The anticancer and anti-inflammatory effects of luteolin glycosides have already been elucidated. However, mLU8C-PU (7-methoxy-luteolin-8-C-β-6-deoxy-xylo-pyranos-3-uloside) extracted from Arthraxon hispous and orientin ( orientin) has not yet been studied on the inhibition of breast cancer metastasis. When diagnosed early, breast cancer can be treated relatively easily through simple surgical resection, but the mortality rate increases rapidly due to breast cancer metastasis. In particular, metastatic breast cancer is difficult to treat because it contains anticancer drug resistance factors. Therefore, it is essential to verify new candidates for metastasis inhibition of breast cancer targeting ER-positive breast cancer (Curr Med Chem. 2013), which accounts for more than 70% of all breast cancers. Accordingly, the present inventors completed the present invention by confirming the ER(+) breast cancer metastasis inhibitory mechanism and cancer metastasis inhibitory efficacy of mLU8C-PU and orientin extracted from clam grass.

Republic of Korea Patent Publication No. 10-2011-0015846

The present invention provides a method for preventing cancer selected from the group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient Or it provides a pharmaceutical composition for treatment.

In addition, the present invention is from the group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 2, which is a c-Jun N terminal kinase (JNK) phosphorylation inhibitor as an active ingredient. It provides a pharmaceutical composition for the prevention or treatment of selected cancer.

In addition, the present invention is a STAT3 (signal transducer and activator of transcription 3) activity inhibitor comprising a compound represented by Formula 3 as an active ingredient, a group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma It provides a pharmaceutical composition for preventing or treating cancer selected from.

In addition, the present invention relates to a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a food composition for prevention or improvement.

The present invention relates to a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma, and hepatocellular carcinoma comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for prevention or treatment is provided.

[Formula 1]

는 결합이 존재하거나 존재하지 않음을 나타내고,

는 단일결합 또는 이중결합을 나타내며, 상기 단일결합 또는 이중결합은 연속적으로 나타나지 않는다.In Formula 1, R ₁ is C ₁ -C ₃ alkyl or hydroxy C ₁ -C ₃ alkyl, R ₂ is hydrogen or C ₁ -C ₃ alkyl,

indicates the presence or absence of a bond,

represents a single bond or a double bond, and the single bond or double bond does not appear consecutively.

Preferably, in Formula 1, R ₁ may be methyl or hydroxymethyl, and R ₂ may be methyl or hydrogen.

More preferably, the compound may be a compound represented by the following Chemical Formula 2 or Chemical Formula 3.

[Formula 2]

[Formula 3]

The cancer may be breast cancer.

The active ingredient may inhibit metastasis of breast cancer.

In addition, the present invention is composed of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 2, which is a c-Jun N terminal kinase (JNK) phosphorylation inhibitor as an active ingredient. It provides a pharmaceutical composition for preventing or treating cancer selected from the group.

The present invention also provides breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 3, which is a STAT3 (signal transducer and activator of transcription 3) activity inhibitor, as an active ingredient. It provides a pharmaceutical composition for preventing or treating cancer selected from the group consisting of.

The present invention can provide compounds that can be usefully used for the prevention or treatment of cancer. In addition, since the compound of the present invention has an effect of inhibiting the migration and invasion of cancer cells, it can be usefully used as an anticancer agent, a therapeutic agent for inhibiting cancer metastasis, or a health functional food material for preventing/improving breast cancer metastasis. More specifically, the compound orientin of the present invention inhibits the membrane potential of PCKα and phosphorylation of ERK and blocks the nuclear translocation of AP-1 and STAT3, thereby inhibiting the levels of MMP-9 and IL-8, thereby inhibiting cancer cell migration and invasion. can be suppressed. In addition, the compound mLU8C-PU of the present invention can inhibit the migration and invasion of cancer cells by reducing the activity of PKCα and JNK, thereby inhibiting the translocation of AP-1 and NF-κB transcription factors to the cell nucleus.

1 schematically shows the mechanism of action of mLU8C-PU and orientin of the present invention.
Figure 2a schematically shows the manufacturing process of the clam chowder fraction.
Figure 2b schematically shows the process of obtaining an active fraction from clam grass.
Figure 3 shows the results of liquid chromatography minutes for the clam chowder extract and solvent fraction.
4 is a result of analyzing the effect of each single compound on cell viability and mRNA level through MTS analysis and RT-PCR in MCF-7 breast cancer cells treated with TPA (12-O-tetradecanoylphorbol-13- acetate). ER(+) MCF-7 cells were pretreated with various concentrations (0, 5, 10, 20 and 40 μM) of a single compound and TPA (50 nM) for 24 h, followed by each compound (mLU8C-PU(7-methoxy-) luteolin-8-C-β-(6-deoxyxylopyranos-3-uloside), AG8C-GS (apigenin-8-C-β-glucoside), AP8C-FP (apigenin-8-C-β-fucopyranoside), Orientin, The effect of KF7O-GS and LU7β-GS) on the survival rate of MCF-7 breast cancer cells was confirmed by MTS analysis, and matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) by qRT-PCR analysis. ) were quantified: * p <0.05 (TPA alone vs. TPA + mLU8C-PU), ** p <0.01 (TPA alone vs. TPA + mLU8C-PU).
5 is an analysis of the effect of mLU8C-PU and orientin on cell invasion and migration. It was evaluated by Matrigel invasion assays and wound-healing migration assays in TPA-treated MCF-7 breast cancer cells. MCF-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU or orientin followed by TPA (50 nM) for 24 h. Cell migration was confirmed by wound-healin assays ( FIGS. 5A and 5C ). Cell infiltration was performed by Matrigel invasion assay ( FIGS. 5b and 5d ). *p <0.05 (TPA alone vs. TPA + mLU8C-PU), **p <0.01 (TPA alone vs. TPA + mLU8C-PU).
6 is an analysis of the effect of mLU8C-PU and orientin on MMP-9 activity. It was evaluated by gelatin zymography and qRT-PCR analysis in TPA-treated MCF-7 breast cancer cells. MCF-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU or orientin and then treated with TPA (50 nM) for 24 h. The activity of MMP-9 was quantified by gelatin zymography. The mRNA levels of MMP-9 were quantified by qRT-PCR analysis. * p <0.05 (TPA alone vs. TPA + mLU8C-PU), ** p <0.01 (TPA alone vs. TPA + mLU8C-PU).
7 is an analysis of the effect of mLU8C-PU and orientin on IL-8 expression. Assessed by qRT-PCR analysis and ELISA in TPA-treated MCF-7 breast cancer cells. MCF-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU or orientin and then treated with TPA (50 nM) for 24 h. 7A and 7D are results of analyzing IL-8 protein levels by ELISA. 7B and 7C are results of analyzing the mRNA level of IL-8 by qRT-PCR analysis. *p <0.05 (TPA alone vs. TPA + mLU8C-PU), **p <0.01 (TPA alone vs. TPA + mLU8C-PU).
8 is an analysis of the effects of mLU8C-PU and orientin on the membrane translocation of PKCα and translocation of ERK. Western blotting was evaluated in TPA-treated MCF-7 breast cancer cells. MCF-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU or orientin followed by TPA (50 nM) for 30 min. 8A and 8C are results of analysis of protein kinase Ca (PKCα) and PKCδ protein levels by Western blotting. 8B and 8D are results of analysis of p-ERK, p-JNK and p-p38 protein levels by Western blotting. *p <0.05 (TPA alone vs. TPA + mLU8C-PU), **p <0.01 (TPA alone vs. TPA + mLU8C-PU).
9 is an analysis of the effect of mLU8C-PU and orientin on the expression of transcription factors. It was evaluated by fractionation and immunofluorescence assay in TPA-treated MCF-7 breast cancer cells. MCF-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU or orientin for 1 h and then treated with TPA (50 nM) for 30 min. 9a is a result of fractionation evaluation of the levels of activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB) and STAT3 transcription factors. 9B is a result of evaluating the levels of AP-1 and NF-κB transcription factors by immunofluorescence analysis. Figure 9c shows that MCF-7 cells were pre-treated with mLU8C-PU and JNK inhibitor (SP600125) or individually for 1 h followed by TPA (50 nM) for 30 min followed by the reduction of AP-1 and NF-κB transcription factors. It is the result of evaluating the level by fractionation method. 9d is a result of evaluating the levels of AP-1, NF-κB and STAT3 transcription factors by fractionation. 9E is a result of evaluating the levels of AP-1 and STAT3 transcription factors by immunofluorescence analysis. Figure 9f shows that MCF-7 cells were pre-treated with orientin and the ERK inhibitor PD98059 or individually for 1 h followed by TPA (50 nM) for 30 min followed by fractionation of the levels of AP-1 and STAT3 transcription factors. It is an evaluation result. *p <0.05 (TPA alone vs. TPA + mLU8C-PU), **p <0.01 (TPA alone vs. TPA + mLU8C-PU).
10 is an analysis of the effect of mLU8C-PU and orientin on cell invasion and MMP-9 and IL-8 expression. TPA-treated MCF-7 breast cancer cells were analyzed by matrigel invasion assay, qRT-PCR, ELISA and gelatin zymography using JNK inhibitor SP600125 (or ERK inhibitor PD98059). MCU-7 cells were pre-treated with various concentrations (0, 5, 10, 20 and 40 μM) of mLU8C-PU and the JNK inhibitor SP600125 (or orientin and the ERK inhibitor PD98059) together or separately followed by TPA (50 nM) ) was treated for 24 hours. 10a and 10e are results of cell invasion analysis by Matrigel invasion assay. 10B and 10F are results of analyzing MMP-9 and IL-8 mRNA levels by qRT-PCR. 10c and 10g are results of analyzing the IL-8 protein level by ELISA. 10d and 10h are results of analysis of MMP-9 activity by gelatin zymography. *p <0.05 (TPA alone vs. TPA + mLU8C-PU), **p <0.01 (TPA alone vs. TPA + mLU8C-PU).
11 is a comparative analysis of the cancer metastasis inhibitory effects of orientin, luteolin, and LU8C-FP through wound-healing analysis and invasion analysis.
12 is a zymography, qPCR, and ELISA analysis results for orientin, luteolin, and LU8C-FP.
13 is a comparative analysis of the cancer metastasis inhibitory effect of mLU8C-PU, luteolin, and LU8C-FP through wound-healing analysis and invasion analysis.
14 is a zymography, qPCR, and ELISA analysis results for mLU8C-PU, luteolin, and LU8C-FP.

Hereinafter, the present invention will be described in detail.

The present invention relates to a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma, and hepatocellular carcinoma comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for prevention or treatment is provided. In addition, the active ingredient may inhibit cancer metastasis.

[Formula 1]

는 결합이 존재하거나 존재하지 않음을 나타내고,

는 단일결합 또는 이중결합을 나타내며, 상기 단일결합 또는 이중결합은 연속적으로 나타나지 않는다.In Formula 1, R ₁ is C ₁ -C ₃ alkyl or hydroxy C ₁ -C ₃ alkyl, R ₂ is hydrogen or C ₁ -C ₃ alkyl,

indicates the presence or absence of a bond,

represents a single bond or a double bond, and the single bond or double bond does not appear consecutively.

Preferably, in Formula 1, R ₁ may be methyl or hydroxymethyl, and R ₂ may be methyl or hydrogen.

More preferably, the compound may be a compound represented by the following Chemical Formula 2 or Chemical Formula 3.

[Formula 2]

[Formula 3]

The compounds can inhibit MMP-9 and IL-8, which are metastatic factors of various cancers, and through inhibition of MMP-9 and IL-8, breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma Cancer selected from the group consisting of cancer can be prevented or treated.

Preferably, the cancer may be breast cancer, more preferably ER(+) breast cancer. In addition, the active ingredient may inhibit metastasis of cancer, preferably breast cancer, more preferably ER(+) breast cancer.

Looking at the chemical formulas included in the composition, since it is a flavone in the form of C-glycoside of a CC bond, not O-glycoside, sugar is decomposed by microbial secreting enzymes or strong acids in the intestine (Bioorg Med Chem. 2007;15:7138; J Sci Food Agric. 2015; 95:1925; Atherosclerosis. 2015; 242:418) It can be provided as an alternative medicine (health functional food).

The compounds represented by Chemical Formulas 1, 2 and 3 may be luteolin glycosides derived from clam chowder.

The compound represented by Chemical Formulas 1, 2 and 3 may be a component extracted from clam grass using a solvent including at least one selected from the group consisting of water, distilled water, and a C1 to C6 alcohol.

The composition may further include one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents.

In the present invention, "cancer metastasis" refers to a state in which a malignant tumor has spread to other tissues away from the diseased organ. As a malignant tumor that starts in one organ progresses, it spreads from the primary site, the organ, to other tissues. Spreading from the primary site to other tissues is called metastasis. Metastasis can be said to be a phenomenon accompanying the progression of malignant tumors, and metastases can occur while malignant tumor cells proliferate and acquire new genetic traits as the cancer progresses. When tumor cells that have acquired new genetic traits invade into blood vessels and lymph glands, circulate through blood and lymph, and settle and proliferate in other tissues, metastasis can occur.

The composition of the present invention can inhibit metastasis to prevent and treat the spread of cancer, as well as improve, prevent, and treat cancer-related diseases derived from metastasis.

As used herein, the term “suppression” refers to any action of inhibiting the cancer metastasis or the onset of cancer-related diseases derived from metastasis by administering the composition according to the present invention.

As used herein, the term “prevention” refers to any action of inhibiting or delaying the onset of cancer metastasis or cancer-related diseases derived from metastasis by administering the composition according to the present invention.

In the present invention, the term "treatment" refers to any action that improves or advantageously changes the symptoms of cancer metastasis or cancer-related disease derived from metastasis by administration of the composition according to the present invention.

The pharmaceutical composition of the present invention may be used as a single agent, and may be prepared and used as a combination formulation by additionally including a pharmaceutical composition known to have a recognized anti-metastatic effect.

The pharmaceutical composition of the present invention may be formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

The term "pharmaceutically acceptable" means that it does not significantly stimulate the organism and does not impair the biological activity and properties of the administered active substance.

In the present invention, the composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin, glycerogelatin, etc. may be used.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories may have the form of

In addition, in the composition of the present invention, at least one component selected from the group consisting of the compound represented by Formula 1, the compound represented by Formula 2, the compound represented by Formula 3, and pharmaceutically acceptable salts thereof is a pharmaceutical agent. It may be included in a scientifically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type and severity of the subject, age, sex, and activity of the drug. , sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The 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. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art. Preferably, it may be included in an amount of 0.001 to 50% by weight, more preferably 0.001 to 20% by weight, based on the total weight of the pharmaceutical composition in the present invention.

In another aspect, the present invention provides a method for preventing or treating cancer by administering the composition to an individual in need of preventing or treating cancer. Also provided is a method of inhibiting cancer metastasis or preventing or treating cancer-related disease derived from metastasis or inhibiting cancer metastasis by administering the composition to an individual in need of preventing or treating cancer-related disease derived from cancer metastasis inhibition or metastasis.

The subject is an individual in need of prevention or treatment of cancer, or an individual in need of prevention or treatment of cancer-related diseases derived from cancer metastasis inhibition or metastasis, as well as humans, cancer-related diseases derived from cancer metastasis or metastasis and similar symptoms It may be a mammal such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs and cats in need of treatment, but is not limited thereto.

In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through

The treatment method includes administering the composition in a pharmaceutically effective amount. It will be apparent to those skilled in the art that a suitable total daily amount can be determined by the treating physician within the scope of sound medical judgment. In addition, it can be administered once or divided into several doses. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.

In another aspect, the present invention relates to breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocytes comprising the compound represented by Formula 2, which is a c-Jun N terminal kinase (JNK) phosphorylation inhibitor as an active ingredient. It provides a pharmaceutical composition for preventing or treating cancer selected from the group consisting of cancer.

In another aspect, the present invention provides breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocytes comprising the compound represented by Formula 3, which is a STAT3 (signal transducer and activator of transcription 3) activity inhibitor, as an active ingredient. It provides a pharmaceutical composition for preventing or treating cancer selected from the group consisting of cancer.

In another aspect, the present invention provides a group consisting of breast cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, osteosarcoma and hepatocellular carcinoma comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient It provides a food composition for the prevention or improvement of cancer selected from.

The food composition may help prevent or improve cancer, or may have a function of helping to inhibit metastasis or the occurrence of metastasis-related diseases described above.

The food composition of the present invention includes the form of pills, powders, granules, needles, tablets, capsules or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example, There are beverages, gum, tea, vitamin complexes, and health supplements.

Other ingredients that do not interfere with the activity of the active ingredient may be added to the food composition, and the type is not particularly limited. For example, it may contain various herbal extracts, food-logically acceptable food supplements or natural carbohydrates as additional ingredients, such as conventional food.

In the present invention, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc., but the above examples are not limited to the types of food supplement additives of the present invention.

Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol and the like. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

The food composition of the present invention may include a health functional food. In the present invention, the term "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, food is used as a raw material, so there are no side effects that may occur when taking the drug for a long time, and it can be excellent in portability.

The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment).

In general, in the production of food, the component of the present invention (a compound represented by the following formula 1, at least one of two compounds represented by the following formula 2) is 1 to 10% by weight of the raw material composition, preferably 5 to 10% by weight % is added. However, in the case of long-term ingestion for health and hygiene purposes or for health control, the above amount may be used below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In another aspect, the present invention may provide a JNK (c-Jun N terminal kinase) phosphorylation inhibitor comprising the compound represented by Formula 2 above.

In addition, the present invention may provide a method for inhibiting JNK (c-Jun N terminal kinase) phosphorylation by treating the inhibitor in vitro.

In addition, the present invention may provide a STAT3 (signal transducer and activator of transcription 3) activity inhibitor comprising the compound represented by Formula 3 above.

In addition, the present invention may provide a method for inhibiting the activity of STAT3 by treating the inhibitor in vitro.

Hereinafter, the present invention will be described in more detail through examples. Objects, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein, and may be embodied in other forms. The embodiments introduced herein are provided so that the spirit of the present invention can be sufficiently conveyed to those of ordinary skill in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

<Example> Separation of mLU8C-PU and orientin compounds derived from clam

clam grass ( Arthraxon hispous ) preparation of extract

Arthraxon hispidus (Thunb.)) was harvested in Janghowon-eup, Icheon-si in December 2017 (A voucher specimen (KRIB 0079082), and stored in KRIBB herbarium (Daejeon, Korea). Then, clam grass (3.99 kg) was dried in a dryer (50- 55°C) to remove moisture, then pulverized to a size of about 1 cm, added 32 L of methanol based on the dry weight of the pulverized powder sample, and extracted 3 times at room temperature. Then, after filtration and concentration under reduced pressure , and dried at 40° C. to obtain 404.5 g (extraction yield of 10.37%) of clamshell extract (methanol extract).

Preparation of clam chowder fraction

Fractions were prepared from the methanol extract of clams obtained above in the following manner.

Specifically, distilled water (0.5 L) was added to the methanol extract of clam chowder (100 g) and suspended, followed by adding the same amount of hexane to separate the hexane layer and the water layer, which was filtered and concentrated under reduced pressure to obtain a hexane fraction (20.8 g). Then, the same amount of ethyl acetate was added to the water layer remaining after removing the hexane fraction to obtain an ethyl acetate fraction (30.2 g) in the same manner as above. Then, an equal amount of butanol was added to the water layer remaining after removing the ethyl acetate fraction to obtain a butanol fraction (20.1 g) in the same manner as above, and the remaining water layer was concentrated to obtain a water fraction (8.8 g) (Fig. 2a).

Preparation of active fraction

In order to separate and prepare the active fraction from the fraction, flash column chromatography was performed.

Specifically, open column [open column, 10 cm × 50 cm; resin (Resin); Silica gel, 400 g] was loaded, and the ethyl acetate layer in the fraction obtained above was loaded in an amount of 30.2 g. As a solvent, hexane/ethyl acetate (5:1→3:1→1:1), ethyl acetate/methanol (10:1→5:1→3:1→1:1), chloroform/ethyl acetate (30: 1→10:1→5:1→1:1) and chloroform/methanol/water (75:25:1) were used to obtain 16 active fractions (AE Frs.1-16) (FIG. 2b).

Analysis of clam chowder extract and column fraction

In order to analyze the clam chowder extract and column fraction obtained above, it was analyzed by ultra performance liquid chromatography (UPLC).

Specifically, after filtering the clam chowder extract and solvent fraction once with a 0.25 mm membrane filter for UPLC, a column (Waters BEH C18 column, 2.1 × 100 mm, 1.7 μm) is mounted on a UPLC device (Waters UPLC-QTOF-MS). After that, each fraction filtered was loaded in an amount of 5 μl. At this time, the solvent is water + 0.1% formic acid (A) / acetonitrile + 0.1% formic acid (B) [0-1 min, 5% B; 1-11 min, 5-100% B; 11-13.2 min, 100%; 13.2-13.33 min, 100-5%; 13.3-15 min, 5%], and the elution rate was 0.4 ml/min. As a detector, the separation degree of solvent fractions was confirmed by chromatography of the substances separated from the UPLC using UV and Mass Spectrometry (MS) (FIG. 3).

Isolation of compounds in small fractions (AE13B-2-i∼m)

Small fractions were separated from the active fraction obtained above in the following manner.

Specifically, HSCCC (TBE-1000A, hexane:EtOAc:MeOH:H2O = 1:5:3:5:, v/v/v/v, upper phase for stationary phase, lower phase for mobile phase, flow rate: 5 mL/min, 501 rpm, forward), and then repeatedly loading the active fraction AE13B-2-i∼m to the compound represented by the following formula 1 (7-methoxy-luteolin-8-C-β-6-deoxy - xylo-pyranos-3-uloside, mLU8C-PU) (Compound 1, 16.9 mg), a compound represented by Formula 2 below (luteolin 8-C-β-glucopyranoside) (Compound 2, 10.0 mg), apigenin 8-C -β fucopyranoside (apigenin 8-C-β-fucopyranoside (233 mg), apigenin 8-C-glucoside (apigenin 8-C-glucoside, 13.6 mg), luteolin 8-C-β-8 -C-β-fucopyranoside (410.0 mg) was obtained (Fig. 2b).

^{1D ( 1} H, ¹³ C, andDEPT) and 2D (COSY, HMQC, and HMBC) NMR spectra for the obtained compound were performed using TMS (tetramethylsilane) as an internal standard, Bruker AM 400/ Bruker Obtained with DRX 500 spectrometers (Bruker). HRESIMS was measured using UPLC-QTOF-MS (ultraperformance liquid chromatography quadrupole time of flight mass spectrometer) (Waters, Milford, MA, USA) in negative-ion mode.

mLU8C-PU

(7-methoxy-luteolin-8-C-β-6-deoxy-xylopyranos-3-uloside)

[Formula 2]

mLU8C-PU data

Yellow powder; mp 160-162 ° C; [α] ²⁵ _D +58.5° (MeOH,c 1.00); HRESIMS, m/z 443.0979 [MH]-(calculated for C ₂₂ H ₂₀ O ₁₀ 443.0978).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ _H 1.40 (3H, d, J = 6.0 Hz, H-6''), 3.50 (1H, dq, H-5''), 3.87 (3H, s , 7''-OCH ₃ ), 4.08(1H,dd,J= 10.0,1.0Hz,H-4''), 4.78 (1H,dd,J=10.0,1.0Hz,H-2''),4.89 (1H,d,J=10.0Hz,H-1''),6.55(1H,s,H-6),6.77(1H,s,H-3),6.92(1H,d,J=8.0Hz, H-5'), 7.50 (2H, m, H-2', 6').

¹³ C NMR (DMSO- d ₆ , 100 MHz) δ _C 19.3 (C-6''), 56.7 (7''-OCH ₃ ), 74.0 (C-2''), 75.1 (C-1'') ,78.8(C-4''),78.7(C-5''),95.1(C-6),102.9(C-3),104.4(C-8,10),114.0(C-2'), 115.8(C-5'),119.2(C-6'),121.9(C-1'),146.0(C-3'),149.9(C-4'),155.0(C-9),161.8(C -5), 163.2 (C-7), 164.4 (C-2), 182.1 (C-4).

Orientin

(luteolin 8-C-β-glucopyranoside)

[Formula 3]

NMR data: ¹ H NMR (400 MHz, DMSO- d ₆ ) δ _H 3.25 (1H, t, J = 10.95 Hz), 3.26 (1H, t, J = 7.10 Hz), 3.37 (1H, t), 3.52 ( 1H, d, J = 14.50 Hz, a), 3.77 (1H, d, J = 14.15 Hz, b), 3.82 (1H, t)m 4.68 (1H, d, J = 6.35 Hz), 6.27 (1H, s) ) 6.65 (1H, s), 6.87 (1H, d, J = 10.45 Hz), 7.48 (1H, d, J = 2.85 Hz), 7.53 (1H, dd, J = 2.80, 10.40 Hz).

¹³ C NMR (DMSO- d ₆ , 100 MHz) δC 61.6 (C-6''), 70.7 (C-4''), 70.8 (C-2''), 73.4 (C-1''), 78.8 (C-3''), 82.0 (C-5''), 98.1 (C-3), 102.4 (C-6), 104.1 (C-10), 104.6 (C-8), 114.1 (C-2) '), 115.7 (C-5'), 119.4 (C-6'), 122.0 (C-1'), 145.8 (C-3'), 149.6 (C-4'), 156.0 (C-9), 160.4 (C-5), 162.6 (C-7), 164.1 (C-2), 182.1 (C-4).

<Experimental example>

Experimental method

cell culture

MCF-7 cell line, a human-derived breast cancer, was used, and TPA (phorbol-12-myristate-13-acetate) was used as an inducer to induce metastasis to confirm the metastasis inhibitory effect. The MCF-7 cell line was purchased from Korea Cell Line Bank (KCLB, No. 40071) (Seoul, Korea), and for cell culture, 10% heat-inactivated FBS and 1% penicillin and streptomycin ( streptomycin) containing DMEM. Cells were cultured at 37° C., _{under 5% CO 2} conditions, the medium was changed every 2 days, and subcultured in order to solve the over-density phenomenon caused by the proliferation of cells.

MTS assay

As a result of HPLC analysis, orientin with a purity of 98.0% or more and mLU8C-PU were dissolved in dimethyl sulfoxide (DMSO, final concentration 0.01%) to measure the activity. This is an experiment to set the concentration of the test substance by measuring the cytotoxicity of the substance. ^{1.5 × 10 4} MCF-7 cells per well were dispensed in a 96-well plate for cell culture, stabilized at 37° C. for 24 hours, exchanged with fresh DMEM medium, and the substance was treated with the cells After incubation for 1 hour, the stimulant TPA was treated and cultured for 24 hours. 20 μL of AQueous One Solution reagent was added to each well (WELL), reacted for 1 hour, and absorbance at 492 nm was confirmed. Through this experiment, 5, 10, 20, and 40 μM without cytotoxicity were determined as experimental concentrations.

Wound-healing assay

This is an experiment to confirm the migration (migration) of breast cancer cells. Cells were placed in 1.5 × 10 ⁵ cells per well, wounds were made, and the degree of healing according to material treatment was confirmed through a microscope.

Matrigel invasion assay

This is an experiment to confirm the invasion (invasion) of breast cancer cells. The degree of invasion of MCF-7 cells was measured using Transwell chamber plates coated with an insert chamber with Matrigel. 1.5 × 10 ⁵ cells per well were treated with TPA and substances. After culturing for 24 hours, the cells that passed through Matrigel and moved to the lower chamber were confirmed through a microscope.

Gelatin zymography

This is an experiment to confirm the enzymatic activity of MMP-9, a representative enzyme involved in breast cancer metastasis. After suspending in DMEM containing MCF-7 cells, the cells were aliquoted to a cell number ^{of 6 × 10 5} _{cells/ml in a 6 well plate, and cultured at 37° C. 5% CO 2 in an} incubator for 24 hours. After exchange with new DMEM medium, the cells were treated with the material and cultured for 1 hour, then treated with the stimulant TPA and cultured for 24 hours. After 24 hours, only the supernatant was collected and electrophoresed on an SDS-polyacrylamide gel containing 0.1% gelatin to separate proteins by size. After that, it was washed 3 times for 30 minutes in 2.5% Triton X-100, and then washed with distilled water to remove SDS. _{Gelatin incubation buffer containing 5 mM CaCl 2} , 0.2 M NaCl, 50 mM Tris (gelatin incubation buffer) ( pH 7.5) and reacted by shaking at 70 rpm in an incubator at 37° C. for 20 to 24 hours. After the reaction, the gel was stained with a coomassie blue solution.

RT-PCR (Reverse transcription-polymerase chain reaction) analysis

This is an experiment to qualitatively confirm the mRNA levels of MMP-9 and IL-8, which are representative factors involved in breast cancer metastasis. After suspension in DMEM containing MCF-7 cells, the cells were aliquoted to a cell number ^{of 6×10 5} _{cell/ml in a 6 well plate, and cultured at 37° C. 5% CO 2 in an} incubator for 24 hours. After exchange with new DMEM medium, the cells were treated with the material and cultured for 1 hour, then treated with the stimulant TPA and cultured for 24 hours. After removing the supernatant, 1 ml of Easy-Blue was added, left for 2 minutes, chloroform was added, vortexed for 10 seconds, and centrifuged at 12,000 rpm for 15 minutes, The supernatant was taken, mixed with the same amount of isopropanol and shaken. The supernatant was removed by centrifugation at 12,000 rpm for 10 minutes, and the pellet was dissolved in 20 ml of DEPC (diethyl pyrocarbonate)-DW and used for RT-PCR. After reacting at 45 ° C. for 30 minutes and 94 ° C. for 5 minutes using an RT-PCR kit, denaturation at 94 ° C. for 30 seconds, and annealing at 55 to 62 ° C. for 30 seconds Next, after repeating the cycle of extension 30 to 35 times at 72° C. for 1 minute, the last extension was performed in a PCR machine at 72° C. for 5 minutes. Each PCR product (products) was analyzed through electrophoresis for 30 minutes at 100 V conditions by loading (loading) on a 2% agarose gel (agarose gel).

RNA isolation and qRT-PCR (quantitative real time reverse transcription-polymerase chain reaction qRT-PCR) analysis

This is an experiment to quantitatively confirm the mRNA levels of MMP-9 and IL-8, which are representative factors involved in breast cancer metastasis. After the MCF-7 cells were suspended in DMEM, ^{3 ml each was dispensed in a 6-well plate (Corning, USA) to a cell number of 6×10 5} cell/ml, and cultured in a 37° C. 5% CO ₂ incubator for 24 hours. After exchanging with fresh DMEM medium, the cells were treated with the material and cultured for 1 hour, then treated with the stimulant TPA and cultured for 24 hours. After removing the supernatant, 1 ml of Easy-Blue was added, left for 2 minutes, chloroform was added, vortexed for 10 seconds, and centrifuged at 12,000 rpm for 15 minutes, The supernatant was taken, mixed with the same amount of isopropanol and shaken. The supernatant was removed by centrifugation at 12,000 rpm for 10 minutes, and the pellet was dissolved in 20 ml of DEPC (diethyl pyrocarbonate)-DW and used for RT-PCR. The produced mRNA was quantitatively confirmed.

Enzyme-linked immunosorbent assay (ELISA)

This is an experiment to confirm the secretion level of IL-8, a representative cytokine involved in breast cancer metastasis. After the material was treated, the supernatant was collected and the secretion level of IL-8 was quantitatively confirmed through sandwich ELISA. The secretion amount of IL-8, a metastasis-related cytokine produced from MCF-7 cells induced by TPA invasion, was measured as follows. Inoculated on a plate adjusted to 6×10 ⁵ _{cell/mL using DMEM medium and incubated for 24 hours in a 5% CO 2} incubator incubator, treated with TPA and material, and re-cultured for 24 hours. The secretion amount of IL-8 cytokines in the cell culture was measured using an ELISA kit. To this end, anti-mouse IL-8 was dispensed on an ELISA microplate as a capture antibody and coated overnight at 4°C. This was washed with PBST containing 0.05% Tween 20 and blocked with 10% FBS solution. After washing with PBST, the culture supernatant was dispensed on each microplate and reacted at room temperature for 2 hours. After the reaction, biotinylated anti-mouse IL-8 detection antibody and streptavidin-horseradish peroxidase conjugate washed and diluted with PBST were dispensed. and reacted at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. _{After dispensing 2 NH 2} SO ₄ to terminate the reaction, the absorbance was measured at 490 nm using a microplate reader.

Western blotting

This is an experiment to check the expression level of proteins such as MAPK and PKC Families. After the MCF-7 cells were suspended in DMEM, the cells were aliquoted to a cell number ^{of 6×10 5} _{cell/ml in a cell culture dish, and cultured at 37° C. 5% CO 2 in an} incubator for 24 hours. After exchange with new DMEM medium, the cells were treated with the material and cultured for 1 hour, followed by treatment with the stimulant TPA and cultured. In order to measure MAPK activity, it was exposed to TPA for one hour. After completion of the reaction, the medium was removed, washed with cold PBS, and the cell lysates were prepared using a lysis buffer (10 mM pH 7.4 Tris-HCl, 5 mM NaF, 1 mM Na ₃ VO). _4, by the addition of 1 mM EDTA abd 1 mM EGTA) was extracted protein (protein). Protein content was quantified by Bradford method, 20-50 mg of protein was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and Hybond-PVDF membrane (Amersham, Little Chalfont) , UK). The transferred membrane was prepared in 5% skim milk dissolved in TBST (Tris-buffered saline Tween)-20 (20 mM Tris, pH 7.6, 136 mM NaCl, 0.1% Tween 20). After blocking at room temperature for a period of time, anti-phospho-ERK (anti-phospho-ERK) and anti-ERK (anti-ERK), anti-phospho-JNK (anti-phospho-JNK) and anti-JNK (anti-JNK), anti-phospho-p38 and anti-p38 MAP kinase and β-actin primary antibody (1 : 1000) dilution) at 4°C overnight, followed by washing 3 times with TBST, and HRP-conjugated secondary antibody (1:1000 dilution) for 1 hour at room temperature. reacted in After washing 3 times with TBST, immunoreactive protein bands were detected on X-ray films using enhanced chemiuminescence regents (ECL) (Amersham, Little Chalfont, UK).

Nuclear and Cytoplasmic Fractionation / Plasma Membrane and Cytoplasmic Fractionation

This is an experiment to identify the location of the protein to be identified by separating the nucleus and cytoplasm. This is an experiment to check the expression level of proteins such as MAPK and PKC Families. After the MCF-7 cells were suspended in DMEM, the cells were aliquoted to a cell number ^{of 6×10 5} _{cells/ml in a cell culture dish, and cultured at 37° C. 5% CO 2 in an} incubator for 24 hours. After exchange with new DMEM medium, the cells were treated with the material and cultured for 1 hour, followed by treatment with the stimulant TPA and cultured. In order to measure MAPK activity, it was exposed to TPA for 30 minutes. After completion of the reaction, the medium was removed, washed with cold PBS, and nuclear and cytoplasmic extraction reagents kit and plasma membrane and cytoplasmic extraction reagents kit were used to remove the nucleus, cytosol, and plasma membrane. ) was isolated.

Statistical processing

For the results of this experiment, the average value of the three experiments was used. For statistical processing, one way-ANOVA was used, and it was determined that there was significance when p <0.05.

Abbreviations

TPA, 12-O-tetradecanoylphorbol-13-acetate; IL-8, interleukin-8; MMP-9, matrix metalloproteinase-9; AP-1, activator protein-1; STAT3, signal transducer and activator of transcription 3; ERK, extracellular signal-regulated kinase; MAPKs, mitogen-activated protein kinases.

Experiment result

Analysis of cytotoxicity and ability to inhibit migration/infiltration of MCF-7 cells

Although research to develop new anticancer substances is ongoing, there is a problem that clinical access as a therapeutic agent becomes difficult if it is accompanied by strong cytotoxicity. Since Luteolin has already been known for its anticancer and anti-inflammatory effects, it was investigated whether Luteolin glycosides Orientin and mLU8C-PU inhibit cell invasion in metastatic breast cancer. In order to elucidate the mechanism by which and mLU8C-PU inhibit cancer cell invasion, the expression of MMP-9 and IL-8 important for early invasion and metastasis of cancer cells and changes in cell signaling proteins involved were investigated. First, ER(+) MCF-7 breast cancer cells were treated with Orientin and mLU8C-PU by concentration in DMEM medium, and then cell viability was measured by MTS assay. As a result, cytotoxicity was not observed at concentrations below 40 μM. After confirming that it does not, 5, 10, 20, and 40 μM were determined as experimental concentrations.

After examining the cytotoxicity of Orientin and mLU8C-PU on MCF-7 cells, it was investigated whether Orientin and mLU8C-PU inhibit the migration and invasion of MCF-7 cells increased by TPA. , the migration and invasion of MCF-7 cells was inhibited in a concentration-dependent manner at concentrations that did not show cytotoxicity ( FIGS. 4 and 5 ).

MMP-9 and IL-8 activity assay

In addition, as a result of examining the activities of MMP-9 and IL-8 through zymography, RT-qPRC, and ELISA experiments, the degree of MMP-9 and IL-8 significantly increased by TPA was found to be orientin (Orientin). ) or mLU8C-PU was confirmed to be reduced in a concentration-dependent manner ( FIGS. 6 and 7 ). From these results, orientin and mLU8C-PU decreased the activity of MMP-9 and IL-8 in MCF-7 breast cancer cells. It can be seen that it inhibits TPA-induced cell migration and invasion.

Confirmation of detailed mechanisms of anti-metastatic effect of orientin and mLU8C-PU

It has been reported that the expression and invasion of MMP-9 and IL-8 in breast cancer cells is induced by the activation of TPA-induced protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and various transcription factors. have. Therefore, experiments were conducted to confirm the detailed mechanism of the anti-metastatic effect of orientin and mLU8C-PU, and the results are shown in FIGS. 8 to 10 .

Orientin (orientin) decreases the activity of PKCα (protein kinase Cα) and ERK (extracellular signal-regulated kinase), AP-1 (activator protein-1). Translocation of the STAT3 (signal transducer and activator of transcription 3) transcription factor to the cell nucleus was suppressed. In addition, it was confirmed that ERK inhibited AP-1, STAT3, MMP-9 and IL-8 as an upstream regulator in the mechanism of orientin's anticonvulsant effect.

mLU8C-PU was confirmed to decrease the activity of PKCα (protein kinase Cα) and JNK (c-Jun N-terminal kinase). Furthermore, mLU8C-PU inhibited the translocation of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) transcription factors to the cell nucleus. In addition, it was confirmed that JNK inhibited AP-1, NF-κB, MMP-9 and IL-8 as upstream regulators in the mechanism of the anti-anxiety effect of mLU8C-PU. In summary, through this experiment, orientin and mLU8C-PU regulate the PKCα/MAPK/AP-1/NF-κb/STAT3 signaling pathway, and the migration and invasion of breast cancer cells by regulating the expression of MMP-9 and IL-8. was confirmed to have an inhibitory effect.

Comparison of breast cancer metastasis inhibition effect with orientin-like luteolin and LU8C-FP (Luteolin 8-C-β-fucopyranoside)

The effect of inhibiting breast cancer metastasis with orientin-like luteolin, LU8C-FP (Luteolin 8-C-β-fucopyranoside) was analyzed, and the results are shown in FIGS. 11, 12, Table 1 and Table 2. As a result of the analysis, it was confirmed that orientin had a superior metastasis inhibitory effect compared to luteolin (FIG. 11). In addition, it was confirmed that orientin had an excellent inhibitory effect on MMP-9 compared to LU8C-FP, and orientin had an IL-8 inhibitory effect, but luteolin did not inhibit IL-8 ( FIG. 12 ).

[Table 1]

[Table 2]

Comparison of breast cancer metastasis inhibition effect with mLU8C-PU and similar luteolin and LU8C-FP (Luteolin 8-C-β-fucopyranoside)

Luteolin similar to mLU8C-PU and LU8C-FP (Luteolin 8-C-β-fucopyranoside) were analyzed for the inhibitory effect of breast cancer metastasis, and the results are shown in FIGS. 13, 14, 3 and 4. As a result of the analysis, it was confirmed that mLU8C-PU had a superior inhibitory effect on cancer cell migration, invasion, and metastasis compared to luteolin (FIG. 13). In addition, it was confirmed that LU8C-PU had an excellent inhibitory effect on IL-8, a representative chemokine that plays an important role in cancer cell migration and metastasis, compared to LU8C-FP, and luteolin did not inhibit IL-8 (Fig. 14). ).

[Table 3]

[Table 4]

In the present invention, it was investigated whether glycosides orientin and mLU8C-PU with luteolin, which are widely used as therapeutic agents for inhibiting metastasis of breast cancer, inhibit the invasion of metastatic breast cancer cells. Orientin and mLU8C-PU inhibit the invasion of cancer cells. In order to elucidate the mechanism of suppression, changes in cell signaling proteins involved in the activity of MMP-9, IL-8, MAPK, PKC, and transcription factors were analyzed.

Orientin and mLU8C-PU significantly inhibited TPA-stimulated MCF-7 breast cancer cell invasion at non-cytotoxic concentrations, and dose-dependently inhibited the levels of MMP-9 and IL-8 increased by TPA. did. The increase in MMP-9 and IL-8 induced by treatment with TPA was confirmed to be due to the increase in transcriptional activity by AP-1, STAT3, and NF-κB through MAPK and PKCα activation. Orientin and mLU8C-PU inhibit the activities of MMP-9 and IL-8 by inhibiting PKCα, MAPK, AP-1, STAT3, and NF-κB pathways in TPA-stimulated MCF-7 cells, and consequently Invasion of breast cancer cells was inhibited. Therefore, it was confirmed that orientin and mLU8C-PU inhibit metastasis of breast cancer cells, suggesting that they can be used as novel therapeutic drugs for metastasis inhibition.

<Production Example>

1. Preparation of pharmaceutical preparations

<1-1> Preparation of powder

0.2 g mLU8C-PU or Orientin

1 g lactose

The above ingredients are mixed and filled in an airtight bag to prepare a powder.

<1-2> Preparation of tablets

mLU8C-PU or Orientin 10mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional manufacturing method of tablets.

<1-3> Preparation of capsules

mLU8C-PU or Orientin 10mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, the capsules are prepared by filling in gelatin capsules according to a conventional manufacturing method of capsules.

2. Manufacture of food

Foods containing mLU8C-PU or orientin of the present invention are prepared as follows.

<2-1> Manufacture of wheat flour food

0.05 to 5.0 parts by weight of mLU8C-PU or orientin of the present invention is added to wheat flour, and the mixture is used to prepare breads, cakes, cookies, crackers and noodles.

<2-2> Production of dairy products

0.05 to 5.0 parts by weight of mLU8C-PU or orientin of the present invention is added to milk, and various dairy products such as butter and ice cream are prepared using the milk.

Claims (8)

A pharmaceutical composition for inhibiting metastasis of breast cancer comprising a compound represented by the following [Formula 2], which is a c-Jun N terminal kinase (JNK) phosphorylation inhibitor, or a pharmaceutically acceptable salt thereof, as an active ingredient:
[Formula 2]

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