KR101112675B1 - Composition comprising 3-o-glucose-isorhamnetin as an effective ingredient for treatment and prevention of inflammatory diseases, immune diseases or cancers - Google Patents

Abstract

The present invention relates to a novel use of the flavonol derivative compound, specifically, a pharmaceutical composition comprising 3-O-glucose-isorhamnetin or a pharmaceutically acceptable salt thereof It relates to a composition.

 3-O-glucose-isolamnetine of the present invention inhibits the gene expression of CXCL1 (Melanomagrowth-stimulatory active / growth-regulated protein α; Chemokine ligand 1), a regulator of immune control and inflammatory processes during immune mechanisms in the human body Since it can inhibit the initial pathways and processes of the biological inflammatory response system, it can be usefully used as a pharmaceutical composition that can prevent and treat various pathological diseases caused by the inflammatory response.

3-O-glucose-isolametine, CXCL1, TNF-α

Description

COMPOSITION COMPRISING 3-O-GLUCOSE-ISORHAMNETIN AS AN EFFECTIVE INGREDIENT FOR TREATMENT AND PREVENTION OF INFLAMMATORY DISEASES , IMMUNE DISEASES OR CANCERS}

The present invention relates to a novel use of the 3-O-glucose isorrametine as an agent for treating inflammatory diseases, immune diseases or cancer.

Harmful stimulation excessively stimulates the body's immune system, leading to excessive induction of inflammatory mediators such as tumor necrosis factor-a (TNF-a), interleukin (IL), and prostaglandin in immune cells such as macrophages. Inflammatory diseases such as inflammatory diseases and tissue transplant rejection, autoimmune diseases, immune diseases such as diabetes and neuronal cell death is known to cause.

Chemokine (CXC motif) ligand 1 (CXCL1, GRO-a, GRO-1) is a representative neutrophil activator that plays a role in attracting neutrophils, the first defense factor when inflammation occurs in the body, to the site of inflammation. It is a kind of cytokine. Chemokines are expressed in various types of cells such as macrophages, neutrophils, and epithelial cells. They have long half-lives and can induce the production of inflammatory substances as well as neutrophils, which are the earliest defenses at the onset of the body's inflammatory response. It plays an important role in the pathophysiology of various diseases resulting from inflammatory reactions, autoimmune diseases, angiogenesis, tumorigenesis, and inflammatory responses.

Chemokines, including CXCL1, act alone or in combination to play an important role in the pathophysiology of ulcerative colitis. It is also believed that the activation of chemokines, including CXCL1, plays an important role in the pathophysiology of various inflammatory diseases. In addition, CXCL1 is overexpressed in melanoma and breast cancer, and contributes to tumor metastasis by helping angiogenesis. It has been confirmed that tumor metastasis is inhibited by inhibiting the expression of CXCL1 in a mouse model.

Angiogenesis, characterized by neoformation of blood vessels, is essential for many physiological and pathophysiological phenomena, and chemokines affect all these aspects of angiogenesis through different mechanisms. Arthritis is known to be an essential step in the growth and metastasis, and it is reported that the formation of new blood vessels into the joint acts as an important pathological mechanism. Psoriasis is known to be important for exacerbation of disease by the formation of neovascularization and thereby increased vascular permeability. In addition, diabetic eye disease has been reported to be a disease caused by the rapid progression of angiogenesis, atherosclerosis is reported to play an important role in weakening the blood vessel wall of angiogenesis into the atherosclerosis, inflammation increases blood flow, Angiogenesis is known to be the cause and effect of all changes such as increased permeability of blood vessels and infiltration of leukocytes. Therefore, the treatment and prevention of these diseases requires the inhibition of chemokine activation.

Therefore, by using compounds that can regulate gene expression of CXCL1, selective control of the initial pathways and processes of various reactions can be developed as an indicator for the development of new leading agents of therapeutics as well as for the treatment of the various diseases listed above.

 The present inventors inhibit the gene expression of CXCL1, a regulator of immune control and inflammatory processes in the immune system in the human body, thereby preventing and treating various pathological diseases caused by inflammatory reactions by inhibiting the initial pathways and processes of the biological inflammatory response system. As a result of the intensive efforts to find a compound having the result, it is possible to prevent and treat various pathological diseases caused by the inflammatory response by inhibiting the gene expression of the CXCL1 induced by the TNF-α and inhibiting the early pathways and processes of the biological inflammatory response system. The present invention was completed by confirming that the effect was added.

It is therefore an object of the present invention to provide new uses of 3-O-glucose isoramnetine.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention is an inflammation containing 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient, and comprising a pharmaceutically acceptable carrier, diluent or excipient. It provides a pharmaceutical composition for the prevention and treatment of diseases or immune diseases.

The present invention also provides a food composition for preventing and / or inhibiting inflammatory diseases or immune diseases, including 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof and a food acceptable additive. do.

In addition, the present invention contains 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient, and a cancer prophylactic and / or therapeutic pharmaceutical comprising a pharmaceutically acceptable carrier, diluent or excipient. To provide a composition.

The present invention also provides a food composition for preventing and / or inhibiting cancer, comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof and a food acceptable additive.

The present invention also provides an angiogenesis inhibitor comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel use of inhibiting gene expression of CXCL1 of 3-O-glucose-isolamnetine represented by the following formula (1).

In addition, 3-O-glucose-isolametine of the present invention is a compound represented by the formula (1).

The compounds of formula (I) according to the invention can be prepared by extraction, separation from plants or by synthetic methods well known in the art. We synthesized 3-O-glucose-isolamnetine based on techniques known in JM Fuentes-alventosa et al., J. Agric. Food Chem., 56, 6977, 2008.

 3-O-glucose-isolamnetine of the present invention inhibits the gene expression of CXCL1 induced by TNF-α to prevent various pathological diseases caused by inflammatory reactions by inhibiting the early pathways and processes of the biological inflammatory response system. It can be used as a therapeutic agent for inflammatory diseases or immune diseases because it has a therapeutic effect.

The present invention provides a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases or immune diseases comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

As the pharmaceutically acceptable salt of the present invention, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol in water (eg glycol motomethylether) can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and the like may be used as the inorganic acid, and methane may be used as the organic acid. Methane sulfonic acid, p-toluen sulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, propionic acid, citric acid, lactic acid, glycolic acid acid, gluconic acid, galacturon acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid Ascorbic acid, carbonic acid, vanillic acid, hydro Hydroiodic acid may be used.

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

Pharmaceutically acceptable salts of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise noted. For example, pharmaceutically acceptable salts include sodium, calcium, and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, and hydrogen sulfate. sulfate, phosphate, hydrogen sulfate, dihydrogen sulfate, acetate, succinate, citrate, tartrate, lactate ), Methanesulfonate (mesylate, methane sulfonate) and p -toluenesulfonate (tosylate, p- toluen sulfonate) salts, and can be prepared through the preparation or manufacturing process of salts known in the art.

The 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof can be appropriately adjusted depending on the purpose of use, and there is no particular limitation.

In addition, the pharmaceutical compositions of the present invention may further comprise one or more pharmaceutically acceptable liquid or solid carriers. It may also be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, etc. that are commonly used, but is not limited thereto.

 The solid form preparations may be tablets, pills, powders, granules, capsules, pellets, granules or powders, and such solid preparations may be prepared by adding a carrier, excipients and / or diluents to the compound. The carrier, excipient and / or diluent may include lactose, sucrose, dextrose, mannitol, malitol, malitol, sorbitol, xylitol, and erythritol. (erithritol), starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, polyvinyl pyrroly Polyvinyl pyrrolidone, magnesium stearate and mineral oil.

The formulations in liquid form may be solutions, suspensions or emulsions, and may include various excipients, for example wetting agents, sweeteners, fragrances, preservatives, etc., in addition to the commonly used simple diluents, water and liquid paraffin. Aqueous suspensions suitable for oral use may be prepared by dispersing the finely divided active component in a viscous material such as natural or synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose and known suspending agents. Can be.

Examples of fillers that can be used in the pharmaceutical compositions and dosage forms disclosed herein include talc, calcium carbonate (eg, granules or powders), microcrystalline cellulose, powder cellulose, dexrate, kaolin, mannitol, silicic acid, sorbitol, Starch, pre-gelatin ring starch, and mixtures thereof, including but not limited to. The binder or filler of the pharmaceutical composition of the present invention is generally present in about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Disintegrants that can be used in the pharmaceutical compositions and dosage forms disclosed herein are intended to disintegrate when the tablet is exposed to an aqueous environment. Tablets containing too much disintegrant may disintegrate during storage, while tablets containing too little do not disintegrate at the desired rate under the desired conditions. A sufficient amount of disintegrant, therefore not too much or too little, should not be used to form the solid oral dosage form of the invention so as not to be bad for controlling the release of the active ingredient. The amount of disintegrants used varies depending on the type of formulation, and can be readily determined by one of ordinary skill in the art. Typical pharmaceutical compositions contain about 0.5 to about 15 weight percent of disintegrant, preferably about 1 to about 5 weight percent of disintegrant. The disintegrants that can be used in the pharmaceutical compositions and dosage forms of the present invention are agar- Agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, poracryline potassium, sodium starch glycolenmit, potato or tapioca starch, other starch, pre-gelatinized starch, other starch, clay, Other algins, other celluloses, gums, and mixtures thereof.

Glidants that can be used in the pharmaceutical compositions and dosage forms of the present invention are calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium Lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar , And mixtures thereof. Additional lubricants are, for example, siloid silica (AEROSIL200, manufactured by WR Grace Co., Baltimore, MD), coagulated aerosol of synthetic silica, manufactured by Degussa Co., Plano, TX, CAB- O-SIL (pyrogenic silicon dioxide product, Cabot Co., Boston, Mass.), And mixtures thereof. If used, glidants are generally used in amounts up to about 1 weight percent of the pharmaceutical composition or dosage form in which they are contained.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes, and all modes of administration can be expected. For example, it may be administered orally or parenterally, and if administered parenterally, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, intrauterine dural or intracerebroventricular injection, or the like. Can be. It is preferable that the route of administration of the pharmaceutical composition of the present invention is determined according to the type of the disease to be applied.

Examples of the parenteral administration include injections, drops, sap, ointments, sprays, suspensions, emulsions, suppositories, and the like. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate may be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a therapeutically effective amount upon administration for clinical purposes. The term “therapeutically effective amout” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective amount for a particular patient is age, sex, weight, health condition. , Time of administration, route of administration, rate of excretion, drug mixture and severity of disease. However, for the desired effect, the compound of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, divided once or several times a day. In the compositions of the present invention the compound of formula 1 should be present in an amount of 0.001 to 50% by weight, preferably 0.01 to 50% by weight relative to the total weight of the total composition.

Diseases to which the compounds according to the present invention may be applied include, but are not limited to, sepsis, arthritis, rheumatoid arthritis, rheumatic polymyalgia, arteriosclerosis, inflammatory visceral disease, ulcerative colitis, osteoporosis, Crohn's disease, encephalomyelitis, Meningitis, pancreatitis, peritonitis, osteomyelitis, encephalitis, meningitis, rhinitis, acute bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthritis, ankylosing spondylitis, psoriatic arthritis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, uveitis, ileitis, liver Inflammation, kidney inflammation, asthma, pain, septic shock, ischemia, ulcers, multiple sclerosis, sclerosis, hemorrhagic shock, anaphylactic shock, burns, infections, bacterial infections, viral infections, fungal infections and parasitic infections , Asthma, psoriasis, atopic dermatitis, leishmaniasis, schistosomiasis, hemodialysis, seizures, cardiovascular transplantation, ischemia, recurrent disease, hemochromatosis, blood Cattle include neuropathy, diabetes, Alzheimer's disease (Alzheimer) disease, Parkinson's (Parkinson) bottles, Allografts geobujeung or autoimmune disease.

The pharmaceutical compositions of the present invention can be formulated or used in combination with one or more other agents.

Examples of medicaments that may be administered in combination with the compositions of the present invention include bronchodilators, antihistamines, anti-inflammatory drugs, anticancer agents, neuroprotective agents or antibiotics.

However, the compound-containing preparation according to the present invention is not limited to those described above, and any agent may be included as long as it is useful for the treatment or prevention of an inflammatory disease or an immune disease.

The present invention also provides a food composition comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

 The food composition may be usefully used as a food composition for preventing and / or inhibiting or treating an inflammatory disease or an immune disease.

The food composition of the present invention includes all forms such as functional food, nutritinal supplement, health food and food additives. Food compositions of this type may be added as such or used with other foods or food ingredients, and may be used in various ways according to conventional methods known in the art.

For example, functional foods include meat, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen and other dairy products including noodles, gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages, It can be prepared by adding 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof to a vitamin complex or the like.

The beverage composition may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin. sugar alcohols such as polysaccharide, xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extracts, or synthetic sweeteners such as saccharin and aspartame may be used.

In addition, as a health food can be ingested by granulating, encapsulating and powdering the 3-O-glucose-isolametine of the present invention. It can also be prepared in the form of a composition by mixing with the 3-O-glucose-isolamnetine of the present invention and a known active ingredient known to have an anti-inflammatory effect.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage or vegetable beverage. These components can be used independently or in combination.

In consideration of easy access to food, the food composition of the present invention is very useful for the prevention and / or inhibition, treatment of inflammatory diseases or immune diseases.

The present invention also provides a pharmaceutical composition for preventing and treating cancer, comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition may be usefully used as an agent for preventing and / or inhibiting cancer metastasis.

"Cancer", a disease to be improved, prevented or treated by the compositions of the present invention, is an aggressive property in which cells divide and grow, ignoring normal growth limits, and an invasive property that penetrates surrounding tissues. And diseases caused by cells having metastatic properties that spread to other parts of the body. In the present specification, the cancer is also used in the same sense as a malignant tumor, and includes, but is not limited to, solid tumors and blood born tumors.

The term cancer refers to the bladder, bone or blood, brain, breast, cervix, chest, colon, endrometrium, esophagus, oral cavity, eyes, head (head or neck) kidney, small intestine, large intestine, kidney, thyroid gland, parathyroid gland , But not limited to, cancers of the endocrine tissue, bone marrow, liver, lymph nodes, lungs, tongue, neck, ovaries, pancreas, gallbladder, prostate, rectum, stomach, testes, throat, larynx, anus, central nervous system and uterus Refers to diseases of skin tissue, organs, blood and blood vessels. Certain cancers include advanced malignancy, amyloidosis, neuroblastoma, meningiomas, pericarcinoma, multiple brain metastase, glioblastoma multiforms, glioblastoma, brain stem glioma stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendrocyte Anaplastic oligodendroglioma, neuroendocrine tumor, adenocarcinoma, Dukes C & D colorectal cancer, unresectable rectal colon cancer, metastatic hepatocellular carcinoma, Kaposi's sarcoma, acute karotype Myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma, diffuse giant B-cell Diffuse large B-Cell lymphoma, low grade follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, Celiac carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma, subcutaneous vasculitis, langerhans cell histiocytosis, smooth muscle sarcoma, Fibrodysplasia ossificans progressive, hormonal unresponsive prostate cancer, surgically high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma that is difficult to remove surgically, and Waldenstrom macro Globulinemia, exposed myeloma, painless myeloma, cervical cancer, androgen-independent prostate cancer, androgen-dependent stage IV non-metastasis Prostate gland cancer, hormone-insensitive prostate cancer, chemotherapeutic-nonsensitized prostate cancer, papillary gland carcinoma, follicular gonocarcinoma, medullary goiter carcinoma, and leiomyoma ), Including but not limited to head and neck cancer.

Among the cancers, melanoma and breast cancer are preferable.

As used herein, the term "prevention" means to inhibit the occurrence of a disease or condition in an animal that has not been diagnosed as having a disease or condition but is prone to such disease or condition. As used herein, the term "treatment" refers to (1) inhibition of the development of a disease or condition; (2) alleviation of the disease or disorder; And (3) elimination of the disease or condition.

The compositions of the present invention may be administered before, simultaneously or after an anticancer agent for the prevention and / or treatment of cancer.

The pharmaceutical composition of the present invention can be effective in preventing or treating cancer by using in combination with another second anticancer agent. Other anticancer agents that can be administered in combination with the pharmaceutical compositions of the present invention are as follows.

The second anticancer agent is interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorubicin, etoposide, etoposide, Irinotecan hydrochloride (irinotecan hydrochloride), cisplatin (cisplatin), amsacrine (cysocrine), cytosine arabinoside (cytosine arabinoside), fluorouracil (fluoro uracil) or taxol, and the like.

The pharmaceutical composition may be usefully used as an agent for preventing and / or inhibiting cancer metastasis.

Cancer transmission is known to be a major cause of cancer death, which is to break away from the first place of cancer and move to other organs to cause secondary cancer there.

Since CXCL1 is overexpressed in cancer and is involved in tumor metastasis by assisting angiogenesis, 3-O-glucose-isolamnetine of the present invention which suppresses expression of CXCL1 can be usefully used for preventing and inhibiting metastasis of cancer. have.

The present invention also provides an angiogenesis inhibitor comprising the 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient.

The angiogenesis inhibitors may be usefully used for the treatment and suppression of diseases caused by angiogenesis, preferably anticancer agents, cancer metastasis inhibitors, arthritis agents, psoriasis agents, diabetic eye disease agents, arteriosclerosis agents, and inflammatory reactions. It may be usefully used in therapeutics, but is not limited thereto.

3-O-glucose-isolametine according to the present invention has a function of inhibiting the expression of CXCL1 gene, which is a regulator of immune control and inflammatory processes during immune mechanisms in the human body.

Accordingly, the present invention provides a therapeutic agent for treating inflammatory or immunological diseases, foods for treating inflammatory or immunological diseases, anticancer agents, cancer metastasis inhibitors, comprising 3-O-glucose-isolamnetine or a pharmaceutically acceptable salt thereof as an active ingredient. It can be effectively used for the development of anti-cancer foods and angiogenesis inhibitors.

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Experimental Example 1. Confirmation of gene expression inhibitory effect of CXCL1 by 3-O-glucose-isolamnetine

In the present invention, in order to observe the inhibitory effect of CXCL1 gene expression by 3-O-glucose-isolamnetine (Rhamnetin-3-O-glucoside), Luciferase reporter assay measuring the CXCL1 gene promoter activity (Luciferase reporter assay) Was used. Validation methods using promoter activity to measure increased expression of specific enzymes play a very important role. This is because increased promoter activity means increased gene expression.

Genometics software (www.genomatix.de) was used for gene sequencing. Multiple cloning site of pGL4-basic vector (Promega, Madison, Wisconsin) containing luciferase gene by PCR synthesis of transcriptional regulatory sites (base sequence -970 to +76 base pair) of human CXCL1 gene site) to prepare a pGL4 / CXCL1-Luc (-970 / + 76) reporter plasmid. Primers used for sequencing are as follows.

forward primer (-970F): 5'-ggtacccactccctggtgtc-3 '(SEQ ID NO: 1)

reverse primer (+ 76R): 5'-gctcagcaggcgggtctggc-3 '(SEQ ID NO: 2)

Using the analyzed results, 0.5 μg of pGL4 / CXCL1-Luc (-970 / + 76) plasmid containing the binding sequence of CXCL1 to human breast cancer cell line MDA-MB-231 (ATCC, American Type Culture Collection, USA) Plasmid was injected intracellularly with fugenE6 (Transfection reagent), and 48 hours later, the cell culture solution was treated with 10 ng / mL of TNF-a alone as a control. 3-O-glucose-isolamnetine (Rhamnetin) in the group treated with ng / mL TNF-a and 20 μg / mL 3-O-glucose-isolamnetine (Rhamnetin-3-O-glucoside) Inhibitory effect of -3-O-glucoside) on TNF-a activity was analyzed.

Cells treated with TNF-a and 3-O-glucose-isolamnetine (Rhamnetin-3-O-glucoside) were incubated for another 12 hours, harvested and lysed, and luciferin as a substrate in cell solution protein. ) Was added to 50 µl, and the amount of luciferin fluorescence emitted was measured. Cell lysates and substrate luciferin were obtained from the Dual-Glo Luciferase Assay System Kit purchased from Promega (Madison, WI, USA). Luciferin fluorescence measurements were measured using a Centro LB 960 luminometer (Berthold). Technology, German).

 As shown in FIG. 1, the samples treated with TNF-a alone increased the luciferase enzyme activity by 2.5-fold compared to untreated cells. These results indicate that the expression of CXCL1 gene is increased by TNF-a. At this time, the treatment of 3-O-glucose-isolamnetine (Rhamnetin-3-O-glucoside) of the present invention in parallel with TNF-a observed that the luciferase activity increased by TNF-a was inhibited by about 3.7-fold. It became.

In summary, the 3-O-glucose-isolamnetin (Rhamnetin-3-O-glucoside) of the present invention can be found to inhibit the expression of CXCL1 gene activated by TNF-a at the transcriptional level.

Examples of formulations for the compositions of the present invention are illustrated below, but are not limited thereto.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powder

2 g of 3-O-glucose-isolametine

1g lactose

After mixing the above components and filled in airtight cloth to prepare a powder.

<1-2> Preparation of the tablet

3-O-glucose-isolametine 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

<1-3> Preparation of the capsule

3-O-glucose-isolametine 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

&Lt; 1-4 >

1 g of 3-O-glucose-isolamnetine

Lactose 1.5g

1 g of glycerin

Xylitol 0.5g

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

<1-5> Preparation of granules

150 g of 3-O-glucose-isolamnetine

Soybean Extract 50mg

Glucose 200mg

Starch 600mg

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

 <1-6> Preparation of Injection Solution

3-O-glucose-isolametine 10mg

Mannitol 180mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ .12H ₂ O 26 mg

According to the conventional method for preparing an injection, it is prepared in the above-mentioned ingredient content per 2 ampules.

<1-7> Preparation of liquid

3-O-glucose-isolametine 20mg

10 g of isomerized sugar

Mannitol 5g

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, lemon juice is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the whole to 100 ml, and then sterilized by filling into a brown bottle. do.

 Preparation Example 2 Preparation of Food

 Powders, tablets, capsules, pills, and granules of Formulation Example 1 may be applied to foods, and foods containing 3-O-glucose-isolamnetine were prepared as follows.

<2-1> Preparation of Powder

 Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

 3-O-glucose-isolamnetine was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and 3-O-glucose-isolamnetine dry powder prepared above were blended in the following ratios to prepare a conventional method.

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

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

 3-O-glucose-isolamnetine dry powder (1 part by weight),

 (0.5 part by weight),

 Foxglove (0.5 part by weight)

 Preparation Example 3 Preparation of Beverage

 A beverage comprising 3-O-glucose-isolamnetine was prepared as follows.

 <3-1> Preparation of health drink

 Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight) and 3-O-glucose-isolametine (0.5 After weight sterilization by homogeneously blending parts by weight, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

<3-2> Preparation of Vegetable Juice

 0.5 g of 3-O-glucose-isolamnetine was added to 1,000 ml of a vegetable juice such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.

<3-3> Preparation of fruit juice

 0.1 g of 3-O-glucose-isolamnetine was added to 1,000 ml of fruit juice, such as apple or grape, to prepare fruit juice for health promotion in a conventional manner.

Although the composition ratio is a composition that is relatively suitable for food ingredients in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

1 is a reporter gene analysis table confirming the degree of CXCL1 expression according to formula (1).

<110> Konkuk University Industrial Cooperation Corp <120> COMPOSITION COMPRISING 3-O-GLUCOSE-ISORHAMNETIN AS AN EFFECTIVE          INGREDIENT FOR TREATMENT AND PREVENTION OF INFLAMMATORY DISEASES,          IMMUNE DISEASES OR CANCERS <130> P09-E178 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for identifying human CXCL1 promoter <400> 1 ggtacccact ccctggtgtc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for identifying human CXCL1 promoter <400> 2 gctcagcagg cgggtctggc 20  

Claims (17)

delete delete delete delete delete delete      3-O-glucose-isolamnetine represented by the following formula (1) having an inhibitory effect on tumorigenesis through inhibition of expression of the chemokine ligand 1 (CXCL1) gene induced by tumor necrosis factor-a (TNF-a) O-glucose-Isorhamnetin) A pharmaceutical composition for inhibiting and treating melanoma formation, comprising a compound or a pharmaceutically acceptable salt thereof as an active ingredient, and comprising a pharmaceutically acceptable carrier, diluent or excipient. [Formula 1]

delete delete delete The method of claim 7, wherein The pharmaceutical composition is interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorubicin, doxorubicin, etoposide, irinotecan hydro Further comprising at least one second anticancer agent selected from irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluoro uracil or taxol. Pharmaceutical composition for inhibiting and treating melanoma formation. delete delete 3-O-glucose-isolamnetine (T0F-glucose-isolamnetine) having the effect of inhibiting tumorigenesis through inhibition of expression of CXCL1 (Chemokine ligand 1) gene, which is represented by Chemical Formula 1 of claim 7 and is induced by tumor necrosis factor-a (TNF-a) A food composition for preventing and inhibiting melanoma formation, comprising a 3-O-glucose-Isorhamnetin) compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable food supplement. delete A 3-O-glucose-isorhamnetin compound represented by Formula 1 of claim 7 or a pharmaceutically acceptable salt thereof as an active ingredient and used as an anticancer agent or cancer metastasis inhibitor Angiogenesis inhibitors, characterized in that. delete

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