WO2011068386A2 - Anticancer composition containing a kampheride compound as an active ingredient - Google Patents

Anticancer composition containing a kampheride compound as an active ingredient Download PDF

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WO2011068386A2
WO2011068386A2 PCT/KR2010/008648 KR2010008648W WO2011068386A2 WO 2011068386 A2 WO2011068386 A2 WO 2011068386A2 KR 2010008648 W KR2010008648 W KR 2010008648W WO 2011068386 A2 WO2011068386 A2 WO 2011068386A2
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cancer
compound
camphoride
anticancer
present
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French (fr)
Korean (ko)
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WO2011068386A3 (en
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이민재
박대훈
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강원대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a composition for anticancer containing a kaempferide compound as an active ingredient. More specifically, the present invention relates to an anticancer composition containing a camphoride compound as an active ingredient, a medicament for preventing or treating cancer, and a functional health food.
  • Cancer is one of the incurable diseases to be solved by humankind, and huge capital is being invested in the development to cure it all over the world.In Korea, it is the number one disease cause of death among Koreans since the 1990s. More than 100,000 people have been diagnosed with cancer, and more than 60,000 have died.
  • the cancer causing factors include smoking, ultraviolet rays, chemicals, food, stress, and other environmental factors, but the causes of the various causes are difficult to develop the treatment, and the effects of the treatment vary depending on the site of occurrence.
  • Surgical treatment is effective for eliminating the initial state of cancer, but in some cases, it is necessary to remove organs.
  • metastasis cannot be prevented, and radiation treatment has a high therapeutic effect in treating cancer of a specific site, while radiation therapy cannot prevent new carcinogenesis and metastasis. The problem is that it involves pain.
  • anticancer agent which is a chemotherapeutic agent used for the treatment of malignant tumors
  • anticancer agents are agents that exhibit anticancer activity by inhibiting various metabolic pathways of cancer cells, especially synthesis of nucleic acids.
  • anticancer drugs currently used in cancer treatment are classified into six categories of alkylating agents, metabolic antagonists, antibiotics, mitosis inhibitors, hormonal agents, and other agents according to biochemical mechanisms of action. It does not work but damages normal cells, especially tissue cells with active cell division, which has been accompanied by various side effects such as bone marrow dysfunction, gastrointestinal disorders, and alopecia. Therefore, it is very important to develop an anticancer agent that can enhance the anticancer effect without any side effects of the human body.
  • the research is to extract or process anticancer ingredients from natural materials such as plants to develop them into cancer treatments, preventive medicines or health supplements. Is actively being done.
  • the anticancer activity of the anticancer agent for the treatment of cancer can be divided in several ways from the cancer development mechanism, firstly the mechanisms that induce direct action on cancer cells, ie cytotoxicity, cell cycle regulation and apoptosis (apoptosis) The second is due to the mechanism of inhibiting the angiogenesis of cancer cells, the third is due to the mechanism of inhibiting the invasion of cancer cells, and the fourth is to increase the immunity to inhibit the metastasis of cancer cells. It can be divided into mechanism and the like.
  • each anticancer agent may be active at each of these stages, but it is common to show anticancer activity by showing a major activity at a specific stage.
  • the present inventors completed the present invention by confirming that the camphoride compound has not only an excellent anticancer activity but also no side effects during the study for developing a new anticancer agent.
  • an object of the present invention is to provide an anticancer composition containing a camphoride compound or a salt thereof as an active ingredient which is excellent in anticancer activity and has no side effects.
  • Another object of the present invention to provide a medicament for the prevention or treatment of cancer comprising the anticancer composition.
  • Another object of the present invention to provide a functional health food having an anticancer effect including the anticancer composition.
  • the present invention is effective to a camphoride (kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) compound represented by the formula (1) or a pharmaceutically acceptable salt thereof It provides the anticancer composition containing as a component.
  • camphoride kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone
  • the camphoride compound may have an activity of inhibiting cell proliferation or inducing apoptosis of cancer cells.
  • the camphoride compound may be included in a concentration of 0.1 ⁇ 0.5mM relative to the total volume of the composition.
  • the cancer is breast cancer, laryngeal cancer, lung cancer, epithelial cancer, prostate cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, cervical cancer, ovarian cancer, kidney cancer , Gallbladder cancer, oral cancer, colon cancer, liver cancer and bladder cancer.
  • the present invention also provides a medicament for the prevention or treatment of cancer comprising the anticancer composition according to the present invention.
  • the present invention provides a functional health food having an anticancer effect including the anticancer composition according to the present invention.
  • the camphoride compound according to the present invention has excellent activity of inhibiting cell proliferation of cancer cells, and excellent activity of inducing apoptosis by apoptosis, and has no side effects on the body, thereby preventing or treating cancer. And it is very useful as a material of the functional health food having an anticancer effect.
  • FIG. 1 shows a chromatogram obtained by separating and purifying a camphoride compound of the present invention by performing C18 HPLC chromatography.
  • Figure 2 shows the peak confirming whether the compound purchased on the market through the 1 H NMR analysis of the camphoride compound.
  • Figure 3 is a graph showing the degree of apoptosis by FACs method through the treatment of 0.1% DMSO and 0.33 mM of the camphoride compound of the present invention as a control for the breast cancer cell line MDA-MB-231 as a control .
  • FIG. 4 is a photograph showing the degree of migration of NF- ⁇ B to the nucleus through a fluorescence microscope after treatment with 0.1% DMSO and 7ug / ml of the camphoride compound against MDA-MB-231, a breast cancer cell line.
  • Figure 5a shows a photograph of the expression level of COX-2 in breast cancer cell line through a fluorescence microscope after treatment with 0.1% DMSO and 7ug / ml camperide compound for MDA-MB-231 breast cancer cell line.
  • FIG. 5B shows COX in breast cancer cells via Western blot with anti-COX-2 antibody after treatment with 0.1% DMSO, 7ug / ml and 70ug / ml camphoride compounds, respectively, for MDA-MB-231, a breast cancer cell line. It shows that the expression level of -2 was confirmed.
  • the present invention is characterized in that it provides an anticancer composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the camphoride compound according to the present invention may be 3,5,7-trihydroxy-4'-methoxyflavone represented by the following formula (3,5,7-trihydroxy-4'-methoxyflavone).
  • the camphoride compound having the above structural formula according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt.
  • the salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid.
  • the organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid.
  • the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
  • camphoride compound according to the present invention may be isolated from nature or manufactured and used by chemical synthesis methods known in the art, or may be used as long as it is a commercially available camphoride compound.
  • the camphoride compound of the present invention when isolated and purified from nature, it can be obtained from natural materials by using a method of extracting and separating conventional substances, and the extraction for separating and purifying camphoride compound from nature
  • the method may use a solvent extraction method through water or an organic solvent.
  • the organic solvent is not limited thereto, but methanol, ethanol, propanol, isopropanol, isopropanol, butanol, acetone, ether, ether, and benzene
  • Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.
  • the extract obtained from the above method can be used to obtain the active ingredient from the extract using a separation and purification method known in the art, generally, various synthetic resins such as silica gel or activated alumina Column chromatography and high performance liquid chromatography (HPLC) packed with may be used alone or in combination. Extraction and separation and purification of the active ingredient is not necessarily limited to the above-described method.
  • camphoride compounds have been found to have melanin synthesis inhibitory effect, antioxidant effect and blood pressure control effect (Matsuda, H. et. Al., Bioorg Med Chem ., 17, 6048 , 2009; Simoes, LMC, et.al. , J. Ethnopharm , 94, 59, 2004; Maruyama, H. et.al., Biol Pharm Bull , 32, 7, 1244, 2009).
  • these camphoride components have anticancer activity by inhibiting cell proliferation of cancer cells and inducing apoptosis has not been known until now.
  • the present invention first identified that the camphoride compound has excellent anticancer activity.
  • the camphoride compound according to the present invention is characterized by having anticancer activity through cell death by apoptosis (apoptosis) while inhibiting cell proliferation of cancer cells.
  • cancer is characterized by "uncontrolled growth and proliferation of cells", and the growth and proliferation of these abnormal cells form cell masses called tumors, which form into the surrounding tissues. In severe cases, the disease may spread to other organs in the body and cause abnormal activity of normal cells. Therefore, inhibition of abnormal cell growth and proliferation is one of the methods for preventing and treating cancer.
  • Apoptosis also refers to active death caused by the regulation and expression of several genes and proteins in response to a programmed signal within the cell. It is eliminated by phagocytosis such as macrophage and does not cause inflammation.
  • phagocytosis such as macrophage and does not cause inflammation.
  • morphological and physiological characteristics of apoptosis include cytoplasm shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and phosphatidylserine, lipids that form the cell membrane. (phosphatidylserine) exposure to the outside of the cell and the formation of atoptosome (apoptome) has been reported.
  • apoptosis can be confirmed by various methods, such as a method for confirming DNA fragmentation characteristic of apoptosis through electrophoresis, TUNEL assay (terminal dUTP nick-end labeling assay) or Flow cytometry method using Annexin V is generally used.
  • Annexin V has a property of binding to phosphatidylserine (PS), which is present in a large amount in the cell membrane, at the initial stage of apoptosis.
  • PS phosphatidylserine
  • necrosis is a passive death that occurs rapidly due to changes in the external environment, which leads to the process of irregular clumping and swelling of cytoplasm, and finally the degradation of cells.
  • Cell debris are produced and are known to cause inflammation (Earnshaw, WC, Curr. Opin. Cell Biol ., 7, pp 337-343, 1995).
  • apoptosis or cell death through apoptosis is easily observed in many normal physiological phenomena of life.
  • apoptosis is a function of various morphogenesis and functional functions of the immune or nervous system observed in the early stages of life. It plays an important role in the functional self-organization process. In addition, after adulthood it also plays an essential role in tissue homeostasis, regulation of cell numbers, removal of damaged cells and defense mechanisms against infection.
  • apoptosis is deeply involved in the development of various diseases, that is, the occurrence of abnormal cell death may be the cause of neurodegenerative disorders, immune disorders and cardiovascular diseases. Abnormal inhibition of apoptosis can cause cancer.
  • apoptosis plays an important role in maintaining the normal physiological function of life, and is closely related to the pathogenesis of various diseases, and apoptosis of each cell constituting an individual is genetically damaged cells or differentiation stimulants. It is a mechanism that can remove abnormal cells in order to prevent tumors developed by inappropriate induction of differentiation.
  • a substance having an activity that inhibits the proliferation and growth of abnormal cells and induces apoptosis can be used as an anticancer agent (Fesus, L., J. Cell Biochem. , 22, 151-161, 1995; Reddy, BS, Cancer Res ., 57, 420-425, 1997).
  • the camphoride compound is a breast cancer cell line MDA-MB-231 cells and MCF-7 cells and prostate cancer cell line PC-3
  • the extent of inhibition of proliferation of cancer cells by the camphoride compound was confirmed by the FACs method.
  • cell proliferation of MDA-MB-231 and MCF-7 cells, which are breast cancer cell lines, and PC-3 cells, which are prostate cancer cell lines were inhibited.
  • about 80% of MDA-MB-231, a breast cancer cell line was inhibited. Showed high cell proliferation inhibition (see Table 1).
  • the camphoride of the present invention is very excellent in anticancer activity.
  • the cancer cell line is treated with a camphoride compound, and stained with annexin V, a label of apoptosis, to apoptosis.
  • apoptosis-induced apoptosis was significantly increased in the breast cancer cells treated with the camphoride compound compared to the control treated with DMSO instead of the camphoride compound. It was shown to be proportionally dependent on concentration (see FIG. 3).
  • the camphoride compound of the present invention has anticancer activity by inducing apoptosis through apoptosis in cancer cells.
  • camphoride compound according to the present invention is characterized by having anticancer activity by a mechanism through inhibition of NF- ⁇ B and COX-2.
  • NF- ⁇ B the development and progression of tumors are known to be closely related to the signal transduction process by NF- ⁇ B.
  • activation of NF- ⁇ B inhibits apoptosis of cancer cells. It has been shown to promote cell proliferation by promoting progression (Monks NR et al ., J. Cell Biochem , 92, 646-650, 2004), and phosphorylated active NF- ⁇ B is a cyclooxygenase-2. Has been shown to promote the expression of genes.
  • recent studies have reported that NF- ⁇ B and I ⁇ B exist in phosphorylated and activated form in cancer cells, and activated NF- ⁇ B migrates from the cytoplasm to the nucleus (Jand BC., J. BioL . Chem ., 275, 39 507-39515, 2000; Monks NR et al ., J. Cell Biochem , 92, 646-650, 2004).
  • COX-2 cyclooxygenase-2
  • COX-2 cyclooxygenase-2
  • COX-2 cyclooxygenase-2
  • COX-2 cyclooxygenase-2
  • COX-2 is a main enzyme involved in the biosynthesis of prostaglandin, and there are two kinds of isomers in vivo.
  • COX-1 is expressed in the normal state and is involved in gastrointestinal protection and renal function control.
  • COX-2 is transient and rapid in the cell by mitogen or cytokines during inflammation or other immune reactions. Expressed.
  • substances that inhibit COX-1 or COX-2 may play an important role not only in the treatment of inflammation but also in inhibiting cancer production.
  • inhibiting the activity or expression of NF- ⁇ B and COX-2 can inhibit the development or progression of cancer.
  • the present inventors examined whether the camphoride compound according to the present invention has an activity of inhibiting NF- ⁇ B and COX-2, and when the camphoride compound was treated in breast cancer cell lines, NF- ⁇ B shifted to the nucleus and COX. The expression of -2 was shown to be more inhibited than the control group not treated with the camphoride compound (see Example 4).
  • the present inventors measured the degree of apoptosis due to necrosis when measuring the degree of apoptosis after treatment with camphoride in cancer cells to confirm whether the camphoride compound of the present invention is stable as a clinical anticancer agent. As a result of comparison with the control group, the degree of necrosis by camperide treatment was found to be almost similar to the control group (see FIG. 3).
  • camphoride compound according to the present invention can be used as an anticancer agent having relatively good stability without causing other side effects in vivo.
  • the present invention provides an anticancer composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient, and the camphoride compound may be included at a concentration of 0.1 to 0.5 mM with respect to the total volume of the composition.
  • composition of the present invention can be used as a pharmaceutical composition for preventing and treating the symptoms of cancer caused by abnormal growth and proliferation of cells.
  • the type of cancer that can be prevented or treated using the camphoride compound is not limited thereto, but is not limited thereto, breast cancer, laryngeal cancer, lung cancer, epithelial cancer, prostate cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, Skin cancer, brain tumor, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer and bladder cancer.
  • the anticancer composition according to the present invention may include a pharmaceutically effective amount of a camphoride compound alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat cancer.
  • the pharmaceutically effective amount of the camphoride compound according to the invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight.
  • the pharmaceutically effective amount may be appropriately changed according to the degree of disease symptoms, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.
  • the pharmaceutically acceptable means a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness or similar reaction when administered to a human.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • the anticancer composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on several factors.
  • the anticancer composition of the present invention can be administered in parallel with known compounds having the effect of preventing, improving or treating the symptoms of cancer.
  • the present invention may provide a medicament for preventing or treating cancer, including a composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the composition of the present invention comprising the same has a very stable feature for the body.
  • the anticancer composition of the present invention can be added to a food for the purpose of preventing or improving cancer, and thus the composition of the present invention can be used as a functional health food composition having an anticancer effect.
  • composition for functional health foods having an anticancer effect of the present invention can be easily utilized as foods, such as the main ingredients, side ingredients, food additives, functional foods or beverages that are effective in preventing and improving cancer symptoms.
  • the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, It includes all foods, food additives, functional foods and drinks.
  • Foods to which the composition for functional health foods having an anticancer effect according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and functional foods.
  • food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings.
  • Foods e.g. soy sauce, miso, red pepper paste, mixed soy sauce
  • sauces confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g.
  • fermented milk, cheese, etc. other processed foods
  • kimchi, Pickled foods various kimchi, pickles, etc.
  • beverages e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.
  • natural seasonings e.g. ramen soup, etc.
  • the food, beverage or food additives may be prepared by a conventional manufacturing method.
  • the term "functional food” refers to the control of biological defense rhythms and disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body's regulatory function regarding recovery and the like, and specifically, it may be a health functional food.
  • the functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.
  • the "beverage” refers to a generic term for drinking to quench thirst or to enjoy a taste and includes a functional drink.
  • the beverage contains, as essential ingredients, a composition for the prevention and amelioration of the cancer symptoms as an essential ingredient, and there are no particular restrictions on the other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be.
  • the food containing the functional health food composition having an anticancer enhancing effect of the present invention in addition to the above-described flavors, colorants and fillers such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors (Cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • the above components may be used independently or in combination.
  • the amount of the composition according to the present invention may be included in 0.001% to 90% by weight of the total food weight, preferably 0.1% by weight To 40% by weight, and in the case of a beverage, it may be included in a ratio of 0.01g to 20g, preferably 0.3g to 10g based on 100ml, but for health and hygiene purposes or for health control purposes.
  • the active ingredient is not limited to the above range because it can be used in an amount above the above range because there is no problem in terms of safety.
  • the present inventors have performed a C18 HPLC chromatography to determine the correct this campesterol fluoride compound a compound purchased for campesterol fluoride compound a commercially available separating campesterol fluoride compound, known in the art with a discrete compound 1 H-NMR analysis was performed.
  • camphoride compound was a camphoride (kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) having a molecular weight of 300.26 represented by the following Chemical Formula 1, and was separated by C18 HPLC chromatography.
  • the peaks for the camphoride compound were shown in FIG. 1, and the peaks of the camphoride compound by 1 H-NMR are shown in FIG. 2.
  • the activity of inhibiting cell proliferation of cancer cells with respect to the camphoride compound identified in Example 1 was investigated through FACs known in the art.
  • MDA-MB-231 cell line and MCF-7 cell line and PC-3 cell line, prostate cancer cell line which were purchased from Korea Cell Line Bank as cancer cells, were treated with 10% fetal bovine serum, 2mg / ml sodium carbonate, 100 Cultured in RPMI-1640 (Gibco / BRL, Grand Island, NY) medium containing U / mL penicillin and 100 / mL of streptomycin, each cancer cell line incubated thereafter was 1.5 ⁇ 10 4 cells / After dispensing into wells, the camphoride compound was treated at various concentrations of 0.01 to 0.33 mM and then incubated for 48 hours.
  • A is the OD value of the control cell
  • B is the OD value of the cells treated with the camphoride compound.
  • * indicates that P ⁇ 0.001 by Tukey's studentized range (HSD) test, calculated by calculating the cell activity of the control to 100%.
  • the camphoride compound isolated and purified in the present invention inhibits cell proliferation against MDA-MB-231 and MCF-7 cells, which are breast cancer cell lines, and PC-3 cells, which are prostate cancer cell lines. It can be seen that there is an effect, and in particular, the breast cancer cell line MDA-MB-231 was found to have the activity of inhibiting the proliferation of cells with very high efficiency.
  • the present inventors confirmed that the camphoride compound according to the present invention has anticancer activity through cytotoxicity and cytotoxicity of various cancer cells.
  • the present inventors investigated whether the camphoride compound has an activity of inducing apoptosis of cancer cells in order to directly confirm the anticancer activity of the camphoride compound.
  • 0.33mM camphoride compound was treated with MDA-MB-231 cell line, which was cultured under the conditions of Example 2, and cultured for 48 hours.
  • the camphoride compound of the present invention has excellent activity of killing cells by inducing apoptosis in cancer cells.
  • the present inventors when using the camphoride compound as an anticancer agent, in order to confirm the stability to the body, the degree of apoptosis due to necrosis when treated with camphoride compound compared to the control group, the control group was about 3.07% Necrosis rate of was shown, and even when treated with camphoride compound showed a necrosis rate of about 4.0% similar to the control.
  • camphoride compound according to the present invention is biologically stable through the fact that the degree of cell death by necrosis does not show a significant difference from the control group.
  • the present inventors found that the camphoride compound not only has the activity of inhibiting the cell proliferation of cancer cells and induces apoptosis by apoptosis, but also has no side effects and is excellent in stability so that it can ultimately be used as a therapeutic agent for effective cancer treatment.
  • the camphoride compound not only has the activity of inhibiting the cell proliferation of cancer cells and induces apoptosis by apoptosis, but also has no side effects and is excellent in stability so that it can ultimately be used as a therapeutic agent for effective cancer treatment.
  • NF- ⁇ B which acts as a survival factor of cancer cells, is known to regulate COX-2 gene expression while migrating to the nucleus when activated by phosphorylation. Therefore, the present inventors examined whether the camphoride compound was treated with breast cancer cells to inhibit NF- ⁇ B activity, that is, to inhibit migration to the nucleus.
  • MDA-MB-231 cells which are breast cancer cell lines, were dispensed to 100 ⁇ plate at 1.0 ⁇ 10 6 cells / well and cultured for 24 hours. Thereafter, 7ug / ml of the camphoride compound was treated, and after 90 minutes of incubation, migration of NF- ⁇ B to the nucleus was observed by immunofluorescence analysis. At this time, the immunofluorescence assay method was performed by washing the cells treated with camphoride with cold PBS solution, fixing with cold acetone solution, and using animal-free-blocker (Vector, Burlingame, CA, USA, SP-5030). Blocking for hours was followed by overnight reaction at 4 ° C.
  • animal-free-blocker Vector, Burlingame, CA, USA, SP-5030
  • NF- ⁇ B migration to the nucleus was suppressed in the breast cancer cells treated with the camphoride compound as compared with the control group.
  • the camphoride compound of the present invention has anticancer activity through the mechanism of inhibiting migration of NF- ⁇ B, which is a survival factor of cancer cells, into cancer cells in cancer cell lines.
  • the inventors performed western blot and immunofluorescence assays to investigate whether the camphoride compound has an activity of inhibiting the expression of COX-2, which is known to mediate inflammatory reactions and grow cancer cells in cancer cells.
  • the immunofluorescence assay was performed by incubating the camphoride-treated MDA-MB-231 cells for 48 hours in the same manner as in Example ⁇ 4-1>, and then centrifuging to obtain the cells, followed by 3 steps with cold PBS solution. Washed once, cells were fixed with 4% formaldehyde (Sigma Aldrich, SO57504) and then washed three times with cold PBS solution.
  • MDA-MB-231 cells were aliquoted to 100 ⁇ plate at 1.0 ⁇ 10 6 cells / well, followed by incubation for 24 hours, and 7, 70 ug each of the camphoride compound for the cells. / ml, then incubated for 48 hours, cells were removed from the plate with trypsin and washed with cold PBS solution. Thereafter, to confirm the expression of COX-2 in the cells, the cells' lysates were electrophoresed with 10% SDS-PAGE, followed by anti-COX-2 polyclonal antibody (Cayman, Ann Arbor, MI, USA). Blotting was performed by Western blotting method known in the art using Cat # CAY-160106.
  • a DMSO-treated group was used instead of the camphoride compound, and a beta-actin monoclonal antibody (Sigma Aldrich, USA, Cat # A-5316) was used to confirm the amount of the same protein in Western blot.
  • the present inventors have found that the camphoride compound of the present invention has an effect of inhibiting the growth of NF- ⁇ B and simultaneously inhibiting cancer growth by inhibiting the expression of COX-2. It was found that the camphoride compound can be used as a therapeutic agent for the prevention or treatment of cancer.

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Abstract

The present invention relates to an anticancer composition containing a kampheride compound as an active ingredient. More specifically, the present invention relates to an anticancer composition containing a kampheride compound having the structure of Chemical formula 1 as an active ingredient. The anticancer composition containing a kampheride compound according to the present invention presents the advantage that it can be used to very good effect as a material for medicines for the prevention and treatment of cancer and in functional health foods having an anticancer effect since not only does it have an outstanding action in suppressing the proliferation of cancer cells and an outstanding action in inducing cell death due to apoptosis but it also has no side effects in the body.

Description

캄페라이드 화합물을 유효성분으로 함유하는 항암용 조성물Anticancer composition containing camphoride compound as an active ingredient
본 발명은 캄페라이드(kaempferide) 화합물을 화합물을 유효성분으로 함유하는 항암용 조성물에 관한 것이다. 보다 구체적으로, 캄페라이드 화합물을 유효성분으로 함유하는 항암용 조성물, 암의 예방 또는 치료용 약제 및 기능성 건강식품에 관한 것이다. The present invention relates to a composition for anticancer containing a kaempferide compound as an active ingredient. More specifically, the present invention relates to an anticancer composition containing a camphoride compound as an active ingredient, a medicament for preventing or treating cancer, and a functional health food.
암은 인류가 해결해야 할 난치병 중의 하나로, 전 세계적으로 이를 치유하기 위한 개발에 막대한 자본이 투자되고 있는 실정이며, 우리나라의 경우, 1990년대 이후로 한국인의 사망원인 중 제 1위의 질병으로서 연간 약 10만 명 이상이 암 환자로 진단되고 있고, 약 6만 명 이상이 사망하고 있다. 이러한 암의 유발 인자로는 흡연, 자외선, 화학물질, 음식물, 스트레스 및 기타 환경인자들이 있으나, 그 유발 원인이 다양하여 치료제의 개발이 어려울 뿐만 아니라 발생하는 부위에 따라 치료제의 효과 또한 각기 다르다.  Cancer is one of the incurable diseases to be solved by humankind, and huge capital is being invested in the development to cure it all over the world.In Korea, it is the number one disease cause of death among Koreans since the 1990s. More than 100,000 people have been diagnosed with cancer, and more than 60,000 have died. The cancer causing factors include smoking, ultraviolet rays, chemicals, food, stress, and other environmental factors, but the causes of the various causes are difficult to develop the treatment, and the effects of the treatment vary depending on the site of occurrence.
한편, 암을 치료하는 방법으로는 현재 외과적 치료, 방사선 치료 및 화학적 치료가 사용되고 있는데, 외과적 치료는 암의 초기상태의 제거에는 효과적이나, 경우에 따라서는 장기를 제거해야 하는 등의 부작용과 전이를 방지할 수 없다는 문제가 있으며, 방사선 치료의 경우도 특정부위의 암 치료에는 치료효과가 높은 장점이 있는 반면 방사선의 조사로 인해 새로운 발암위험성과 전이를 방지할 수 없다는 점과 함께 치료시 환자의 고통을 수반한다는 문제가 있다. On the other hand, as a method of treating cancer, surgical treatment, radiation treatment, and chemical treatment are currently used. Surgical treatment is effective for eliminating the initial state of cancer, but in some cases, it is necessary to remove organs. There is a problem that metastasis cannot be prevented, and radiation treatment has a high therapeutic effect in treating cancer of a specific site, while radiation therapy cannot prevent new carcinogenesis and metastasis. The problem is that it involves pain.
또한, 화학적 치료는 악성종양의 치료를 위해 사용되는 화학요법제인 항암제를 사용하는 방법으로서, 대부분의 항암제는 암 세포의 각종 대사경로, 특히 핵산의 합성을 억제하여 항암활성을 나타내는 약제들이다. 또한, 현재 암 치료에 사용되고 있는 항암제는 생화학적인 작용 기전에 따라 크게 알킬화제, 대사길항제, 항생물질, 유사분열억제제, 호르몬제 및 기타 제제의 6개의 범주로 분류되며, 이들 항암제는 암 세포에만 선택적으로 작용하는 것이 아니라 정상세포, 특히 세포분열이 활발한 조직세포에도 손상을 입히기 때문에 골수기능저하, 위장장애, 탈모증 등의 여러 가지 부작용을 수반하는 문제점이 있어 왔다. 따라서 인체의 부작용이 없으면서도 항암효과를 높일 수 있는 항암제를 개발하는 것이 매우 중요하다. In addition, chemotherapy is a method using an anticancer agent, which is a chemotherapeutic agent used for the treatment of malignant tumors, and most anticancer agents are agents that exhibit anticancer activity by inhibiting various metabolic pathways of cancer cells, especially synthesis of nucleic acids. In addition, anticancer drugs currently used in cancer treatment are classified into six categories of alkylating agents, metabolic antagonists, antibiotics, mitosis inhibitors, hormonal agents, and other agents according to biochemical mechanisms of action. It does not work but damages normal cells, especially tissue cells with active cell division, which has been accompanied by various side effects such as bone marrow dysfunction, gastrointestinal disorders, and alopecia. Therefore, it is very important to develop an anticancer agent that can enhance the anticancer effect without any side effects of the human body.
따라서 최근에는 인체에 부작용을 유발하지 않으면서도 항암 효과가 우수한 항암제를 천연재료로부터 수득하기 위해 식물 등 천연재료에서 항암성분을 추출하거나 또는 가공하여 암치료제나 예방용 약제 또는 건강보조식품으로 개발하려는 연구가 활발히 이루어지고 있다.  Therefore, in recent years, in order to obtain an anticancer agent having excellent anticancer effect from natural materials without inducing side effects on the human body, the research is to extract or process anticancer ingredients from natural materials such as plants to develop them into cancer treatments, preventive medicines or health supplements. Is actively being done.
이러한 노력들로 인해 천연재료로부터 항암 활성을 가지는 물질들이 발굴되었는데, 이러한 예로 인삼의 사포닌 성분이 항암 활성이 있다고 밝혀졌으며, 동충하초 또는 상황버섯을 비롯한 버섯류 및 버섯류로부터 분리된 베타-글루칸 성분도 항암 활성이 있음이 밝혀졌고, 부추의 추출물도 항암 활성이 있음이 밝혀진 바 있다. 이 외에도 암 치료에 효과가 있다고 알려진 각종 천연재료로부터 유효성분을 추출하여 약학조성물 또는 가공식품 등의 형태로 사용하려는 연구가 계속되고 있다. Due to these efforts, substances with anticancer activity were discovered from natural materials. For example, saponin of ginseng was found to have anticancer activity, and beta-glucan component isolated from mushrooms and mushrooms including Cordyceps and S. mushrooms also had anticancer activity. It has been found that the extract of leek has also been found to have anti-cancer activity. In addition, research is being conducted to extract active ingredients from various natural ingredients known to be effective in treating cancer and to use them in the form of pharmaceutical compositions or processed foods.
한편, 암 치료를 위한 항암제의 항암 활성 작용은 암의 발전 메카니즘으로부터 몇 가지로 나눌 수 있는데, 첫째는 암 세포에 대한 직접적인 작용, 즉 세포독성, 세포주기조절 및 아폽토시스(apoptosis)를 유도하는 기작에 의한 것이고, 두 번째는 암 세포의 혈관신생(angiogenesis)을 저지하는 기작에 의한 것이며, 세 번째는 암 세포의 침윤을 저지하는 기작에 의한 것이고, 네 번째는 면역력을 높여 암 세포의 전이를 저지하는 기작 등으로 크게 나눌 수 있다. 또한, 각 항암제는 이러한 각 단계에서 모두 활성을 나타낼 수도 있지만 특정 단계에서 주요한 활성을 보여 항암 작용을 하는 것이 일반적이다.  On the other hand, the anticancer activity of the anticancer agent for the treatment of cancer can be divided in several ways from the cancer development mechanism, firstly the mechanisms that induce direct action on cancer cells, ie cytotoxicity, cell cycle regulation and apoptosis (apoptosis) The second is due to the mechanism of inhibiting the angiogenesis of cancer cells, the third is due to the mechanism of inhibiting the invasion of cancer cells, and the fourth is to increase the immunity to inhibit the metastasis of cancer cells. It can be divided into mechanism and the like. In addition, each anticancer agent may be active at each of these stages, but it is common to show anticancer activity by showing a major activity at a specific stage.
따라서 지금까지도 많은 천연재료로부터 암의 발전 메카니즘을 억제함을 통해 항암 활성을 가지는 약제들이 개발되고 있으나, 종래 발굴된 항암제의 경우 인체에 부작용을 유발시키고 항암 효과도 미비하다는 문제점이 있다. Therefore, until now, drugs having anticancer activity have been developed by suppressing cancer development mechanisms from many natural materials, but conventionally discovered anticancer drugs cause side effects in the human body and have a problem of insufficient anticancer effects.
그러므로 부작용 및 독성이 없고 우수한 항암 활성을 가지는 효과적인 암 치료를 위한 새로운 항암제의 개발이 시급한 실정이다.Therefore, there is an urgent need to develop a new anticancer agent for effective cancer treatment with no side effects and toxicity and excellent anticancer activity.
이에 본 발명자들은 새로운 항암제를 개발하기 위한 연구를 진행하던 중, 캄페라이드 화합물이 우수한 항암 활성을 가질 뿐만 아니라 부작용이 없다는 사실을 확인함으로써 본 발명을 완성하였다.  Accordingly, the present inventors completed the present invention by confirming that the camphoride compound has not only an excellent anticancer activity but also no side effects during the study for developing a new anticancer agent.
따라서 본 발명의 목적은 항암 활성이 우수하고 부작용이 없는 캄페라이드 화합물 또는 이의 염을 유효성분으로 함유하는 항암용 조성물을 제공하는 것이다. Therefore, an object of the present invention is to provide an anticancer composition containing a camphoride compound or a salt thereof as an active ingredient which is excellent in anticancer activity and has no side effects.
본 발명의 다른 목적은 상기 항암용 조성물을 포함하는 암의 예방 또는 치료용 약제를 제공하는 것이다. Another object of the present invention to provide a medicament for the prevention or treatment of cancer comprising the anticancer composition.
본 발명의 다른 목적은 상기 항암용 조성물을 포함하는 항암 효과를 갖는 기능성 건강식품을 제공하는 것이다.Another object of the present invention to provide a functional health food having an anticancer effect including the anticancer composition.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 하기 화학식 1로 표시되는 캄페라이드(kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항암용 조성물을 제공한다. In order to achieve the object of the present invention as described above, the present invention is effective to a camphoride (kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) compound represented by the formula (1) or a pharmaceutically acceptable salt thereof It provides the anticancer composition containing as a component.
화학식 1
Figure PCTKR2010008648-appb-C000001
 Formula 1
Figure PCTKR2010008648-appb-C000001
본 발명의 일실시예에 있어서, 상기 캄페라이드 화합물은 암 세포의 세포증식을 억제하거나 세포사멸을 유도하는 활성을 가질 수 있다.  In one embodiment of the present invention, the camphoride compound may have an activity of inhibiting cell proliferation or inducing apoptosis of cancer cells.
본 발명의 일실시예에 있어서, 상기 캄페라이드 화합물은 조성물 총 부피에 대하여 0.1~0.5mM의 농도로 포함될 수 있다.  In one embodiment of the present invention, the camphoride compound may be included in a concentration of 0.1 ~ 0.5mM relative to the total volume of the composition.
본 발명의 일실시예에 있어서, 상기 암은 유방암, 후두암, 폐암, 상피암, 전립선암, 식도암, 췌장암, 대장암, 간암, 위암, 설암, 피부암, 뇌종양, 자궁암, 자궁경부암, 난소암, 신장암, 담낭암, 구강암, 결장암, 간암 및 방광암으로 이루어진 군 중에서 선택될 수 있다.  In one embodiment of the present invention, the cancer is breast cancer, laryngeal cancer, lung cancer, epithelial cancer, prostate cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, cervical cancer, ovarian cancer, kidney cancer , Gallbladder cancer, oral cancer, colon cancer, liver cancer and bladder cancer.
또한, 본 발명은 본 발명에 따른 상기 항암용 조성물 포함하는 암의 예방 또는 치료용 약제를 제공한다. The present invention also provides a medicament for the prevention or treatment of cancer comprising the anticancer composition according to the present invention.
또한, 본 발명은 본 발명에 따른 상기 항암용 조성물 포함하는 항암 효과를 갖는 기능성 건강식품을 제공한다.In addition, the present invention provides a functional health food having an anticancer effect including the anticancer composition according to the present invention.
본 발명에 따른 캄페라이드 화합물은 암 세포의 세포증식을 억제하는 활성이 우수하며, 아폽토시스에 의한 세포사멸을 유도하는 활성이 우수할 뿐만 아니라 체내에 대한 부작용이 없기 때문에 암의 예방 또는 치료를 위한 약제 및 항암 효과를 가지는 기능성 건강식품의 소재로 매우 유용하게 사용할 수 있는 효과가 있다.The camphoride compound according to the present invention has excellent activity of inhibiting cell proliferation of cancer cells, and excellent activity of inducing apoptosis by apoptosis, and has no side effects on the body, thereby preventing or treating cancer. And it is very useful as a material of the functional health food having an anticancer effect.
도 1은 C18 HPLC 크로마토그래피를 수행하여 본 발명의 캄페라이드 화합물을 분리 및 정제한 크로마토그램을 나타낸 것이다.1 shows a chromatogram obtained by separating and purifying a camphoride compound of the present invention by performing C18 HPLC chromatography.
도 2는 1H NMR 분석을 통해 시중에서 구입한 화합물이 캄페라이드 화합물인지를 확인한 피크를 나타낸 것이다.Figure 2 shows the peak confirming whether the compound purchased on the market through the 1 H NMR analysis of the camphoride compound.
도 3은 유방암 세포주인 MDA-MB-231에 대하여 대조군으로 0.1% DMSO와 0.33 mM의 본 발명의 캄페라이드 화합물을 처리한 후 아넥신 염색을 통한 FACs 방법을 통해 세포사멸 정도를 비교하여 나타낸 그래프이다.  Figure 3 is a graph showing the degree of apoptosis by FACs method through the treatment of 0.1% DMSO and 0.33 mM of the camphoride compound of the present invention as a control for the breast cancer cell line MDA-MB-231 as a control .
도 4는 유방암 세포주인 MDA-MB-231에 대하여 0.1% DMSO와 7ug/ml의 캄페라이드 화합물을 처리한 후 형광현미경을 통해 NF-κB의 핵으로의 이동 정도를 관찰한 사진을 나타낸 것이다. 4 is a photograph showing the degree of migration of NF-κB to the nucleus through a fluorescence microscope after treatment with 0.1% DMSO and 7ug / ml of the camphoride compound against MDA-MB-231, a breast cancer cell line.
도 5a는 유방암 세포주인 MDA-MB-231에 대하여 0.1% DMSO와 7ug/ml의 캄페라이드 화합물을 처리한 후 형광현미경을 통해 유방암 세포주에서 COX-2의 발현 정도를 관찰한 사진을 나타낸 것이다. Figure 5a shows a photograph of the expression level of COX-2 in breast cancer cell line through a fluorescence microscope after treatment with 0.1% DMSO and 7ug / ml camperide compound for MDA-MB-231 breast cancer cell line.
도 5b는 유방암 세포주인 MDA-MB-231에 대하여 0.1% DMSO, 7ug/ml 및 70ug/ml의 캄페라이드 화합물을 각각 처리한 후, 항-COX-2 항체를 이용한 웨스턴 블럿을 통해 유방암 세포에서 COX-2의 발현 정도를 확인한 것을 나타낸 것이다.FIG. 5B shows COX in breast cancer cells via Western blot with anti-COX-2 antibody after treatment with 0.1% DMSO, 7ug / ml and 70ug / ml camphoride compounds, respectively, for MDA-MB-231, a breast cancer cell line. It shows that the expression level of -2 was confirmed.
본 발명은 캄페라이드(kaempferide) 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항암용 조성물을 제공함에 그 특징이 있다. The present invention is characterized in that it provides an anticancer composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에 따른 상기 캄페라이드 화합물은 하기 화학식으로 표시되는 3,5,7-트리히드록시-4'-메톡시플라본(3,5,7-trihydroxy-4'-methoxyflavone)일 수 있다.The camphoride compound according to the present invention may be 3,5,7-trihydroxy-4'-methoxyflavone represented by the following formula (3,5,7-trihydroxy-4'-methoxyflavone).
<화학식 1><Formula 1>
Figure PCTKR2010008648-appb-I000001
Figure PCTKR2010008648-appb-I000001
또한, 본 발명에 따른 상기 구조식을 가지는 캄페라이드 화합물은 염, 바람직하게는 약학적으로 허용 가능한 염의 형태로 사용될 수 있다. 상기 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의하여 형성된 산 부가염이 바람직하며, 상기 유리산으로는 유기산과 무기산을 사용할 수 있다. 상기 유기산은 이에 제한되는 것은 아니나, 구연산, 초산, 젖산, 주석산, 말레인산, 푸마르산, 포름산, 프로피온산, 옥살산, 트리플로오로아세트산, 벤조산, 글루콘산, 메타술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 글루탐산 및 아스파르트산을 포함한다. 또한 상기 무기산은 이에 제한되는 것은 아니나, 염산, 브롬산, 황산 및 인산을 포함한다.  In addition, the camphoride compound having the above structural formula according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and an organic acid and an inorganic acid may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.
본 발명에 따른 상기 캄페라이드 화합물은 천연으로부터 분리되거나 당업계에 공지된 화학적 합성법으로 제조하여 사용할 수 있으며, 또는 시중에서 판매되고 있는 캄페라이드 화합물이라면 모두 사용할 수 있다.  The camphoride compound according to the present invention may be isolated from nature or manufactured and used by chemical synthesis methods known in the art, or may be used as long as it is a commercially available camphoride compound.
또한, 본 발명의 캄페라이드 화합물을 천연으로부터 분리 및 정제하여 사용할 경우에는, 종래 물질을 추출하고 분리하는 방법을 이용하여 천연재료로부터 수득할 수 있으며, 캄페라이드 화합물을 천연으로부터 분리 및 정제하기 위한 추출방법은 물 또는 유기용매를 통한 용매추출법을 사용할 수 있다. 이때, 상기 유기용매로는 이에 제한되지는 않으나, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol), 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다. In addition, when the camphoride compound of the present invention is isolated and purified from nature, it can be obtained from natural materials by using a method of extracting and separating conventional substances, and the extraction for separating and purifying camphoride compound from nature The method may use a solvent extraction method through water or an organic solvent. In this case, the organic solvent is not limited thereto, but methanol, ethanol, propanol, isopropanol, isopropanol, butanol, acetone, ether, ether, and benzene Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.
또한, 상기 방법으로부터 수득한 추출물은 이후 당업계에 공지된 분리 및 정제 방법을 사용하여 상기 추출물로부터 활성 성분을 수득할 수 있는데, 일반적으로 실리카겔(silica gel)이나 활성 알루미나(alumina)등의 각종 합성수지를 충진한 컬럼 크로마토그래피(column chromatography) 및 고속액체크로마토그라피(HPLC) 등을 단독으로 혹은 병행하여 사용할 수 있다. 활성성분의 추출 및 분리정제 방법은 반드시 상기한 방법에 한정되는 것은 아니다.  In addition, the extract obtained from the above method can be used to obtain the active ingredient from the extract using a separation and purification method known in the art, generally, various synthetic resins such as silica gel or activated alumina Column chromatography and high performance liquid chromatography (HPLC) packed with may be used alone or in combination. Extraction and separation and purification of the active ingredient is not necessarily limited to the above-described method.
한편, 캄페라이드 화합물에 대하여 지금까지 알려진 약리 효과로는 멜라닌 합성 억제효과, 항산화 효과 및 혈압 조절의 효과가 있음이 밝혀진 바 있다(Matsuda, H. et. al., Bioorg Med Chem., 17, 6048, 2009; Simoes, LMC, et. al., J. Ethnopharm, 94, 59, 2004; Maruyama, H. et. al., Biol Pharm Bull, 32, 7, 1244, 2009). 그러나 이러한 캄페라이드 성분이 암 세포의 세포증식을 억제하고 세포사멸을 유도함으로써 항암 활성을 가진다는 사실은 지금까지 알려진 바 없다. On the other hand, the pharmacological effects known to the camphoride compounds have been found to have melanin synthesis inhibitory effect, antioxidant effect and blood pressure control effect (Matsuda, H. et. Al., Bioorg Med Chem ., 17, 6048 , 2009; Simoes, LMC, et.al. , J. Ethnopharm , 94, 59, 2004; Maruyama, H. et.al., Biol Pharm Bull , 32, 7, 1244, 2009). However, the fact that these camphoride components have anticancer activity by inhibiting cell proliferation of cancer cells and inducing apoptosis has not been known until now.
이에 본 발명에서는 캄페라이드 화합물이 우수한 항암활성을 가진다는 사실을 최초로 규명하였다.  Accordingly, the present invention first identified that the camphoride compound has excellent anticancer activity.
즉, 본 발명에 따른 캄페라이드 화합물은 암 세포의 세포증식을 억제함과 동시에 아폽토시스(apoptosis)에 의한 세포사멸을 통해 항암 활성을 가지는 특징이 있다.  In other words, the camphoride compound according to the present invention is characterized by having anticancer activity through cell death by apoptosis (apoptosis) while inhibiting cell proliferation of cancer cells.
일반적으로 “암”이란 "세포의 제어되지 않는 성장 및 증식"으로 특징지어지며, 이러한 비정상적인 세포의 성장 및 증식은 종양(tumor)이라고 불리는 세포덩어리를 형성하게 되고 형성된 세포덩어리는 주위의 조직으로 침투하고 심한 경우에는 신체의 다른 기관으로 전이되어 정상적인 세포들의 활성기작에 이상을 초래한다. 따라서 비정상적인 세포의 성장 및 증식의 억제는 암을 예방 및 치료할 수 있는 방법 중 하나이다. In general, "cancer" is characterized by "uncontrolled growth and proliferation of cells", and the growth and proliferation of these abnormal cells form cell masses called tumors, which form into the surrounding tissues. In severe cases, the disease may spread to other organs in the body and cause abnormal activity of normal cells. Therefore, inhibition of abnormal cell growth and proliferation is one of the methods for preventing and treating cancer.
또한, 아폽토시스(apoptosis)는 세포 내부에서 프로그램화된 신호를 따라 여러 유전자 및 단백질들의 발현과 활성이 조절되어 일어나는 능동적인 죽음을 말하며, 그 과정을 통해 생성된 아폽토좀(apoptosome)들은 주변의 세포들이나 대식세포(macrophage)등의 식세포 작용에 의해 제거됨으로서, 염증을 유발하지 않는다. 또한, 세포사멸의 형태적, 생리적 특징으로는 세포질 축소(cytoplasm shrinkage), 세포막 기포형성(membrane blebbing), 염색체의 응축(chromatin condensation), DNA의 분절(DNA fragmentation), 세포막을 이루는 지질인 포스파티딜세린(phosphatidylserine)의 세포 외부로의 노출 및 아톱토좀(apoptosome)의 형성 등이 보고되고 있다. Apoptosis also refers to active death caused by the regulation and expression of several genes and proteins in response to a programmed signal within the cell. It is eliminated by phagocytosis such as macrophage and does not cause inflammation. In addition, morphological and physiological characteristics of apoptosis include cytoplasm shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and phosphatidylserine, lipids that form the cell membrane. (phosphatidylserine) exposure to the outside of the cell and the formation of atoptosome (apoptome) has been reported.
또한, 이러한 아폽토시스의 발생은 다양한 방법을 통해 확인할 수 있는데, 이러한 방법으로는 아폽토시스의 특징인 DNA 단편화(DNA fragmentation)를 전기영동을 통하여 확인하는 방법, TUNEL assay(terminal dUTP nick-end labeling assay)나 Annexin V를 이용한 유세포 분석방법 등이 일반적으로 사용되고 있으며, 특히 Annexin V는 아폽토시스가 발생하는 초기시점에서 세포막에 다량 존재하는 포스파티딜세린(Phosphatidylserine, PS)과 결합하는 성질이 있으며, Annexin V에 형광을 표지한 후 유세포 분석기를 이용하면 빠르고 손쉽게 정량적인 분석까지 가능하므로 매우 유용하게 사용되는 방법이다.  In addition, the occurrence of such apoptosis can be confirmed by various methods, such as a method for confirming DNA fragmentation characteristic of apoptosis through electrophoresis, TUNEL assay (terminal dUTP nick-end labeling assay) or Flow cytometry method using Annexin V is generally used. Especially, Annexin V has a property of binding to phosphatidylserine (PS), which is present in a large amount in the cell membrane, at the initial stage of apoptosis. The flow cytometer is then very useful because it enables quick and easy quantitative analysis.
반면, 세포괴사(necrosis)는 외부적 환경의 변화에 의해 급격히 일어나는 수동적 죽음으로 염색사의 다형태적 덩어리(irregular clumping) 및 세포질의 팽창(swelling of cytoplasm) 과정을 거치게 되고, 최종적으로 세포들의 분해를 통해 세포파편(cell debris)이 생성되며 이들은 염증을 유발하는 것으로 알려져 있다 (Earnshaw, W.C., Curr. Opin. Cell Biol., 7, pp 337-343, 1995). On the other hand, necrosis is a passive death that occurs rapidly due to changes in the external environment, which leads to the process of irregular clumping and swelling of cytoplasm, and finally the degradation of cells. Cell debris are produced and are known to cause inflammation (Earnshaw, WC, Curr. Opin. Cell Biol ., 7, pp 337-343, 1995).
이와 같이 아폽토시스 또는 세포괴사를 통한 세포사멸은 생명체의 여러 정상적인 생리적 현상에서 쉽게 관찰되는데, 예를 들면 세포사멸은 생명체의 초기 발생단계에서 관찰되는 여러 형태적 변화과정(morphogenesis)과 면역계 또는 신경계의 기능적 자가조직화(functional self-organization)과정에서 중요한 역할을 담당한다. 또한, 성인이 된 이후에도 조직 항상성(tissue homeostasis), 세포 수의 조절, 손상된 세포의 제거 및 감염에 대한 방어 기작으로서 필수적으로 작용한다.  Thus, apoptosis or cell death through apoptosis is easily observed in many normal physiological phenomena of life. For example, apoptosis is a function of various morphogenesis and functional functions of the immune or nervous system observed in the early stages of life. It plays an important role in the functional self-organization process. In addition, after adulthood it also plays an essential role in tissue homeostasis, regulation of cell numbers, removal of damaged cells and defense mechanisms against infection.
한편, 세포사멸은 여러 질환들의 발병과정에도 깊이 관여하는데, 즉, 비정상적인 세포사멸의 발생은 퇴행성뇌신경질환(neurogegenerative disorder), 면역계 이상(immune disorder) 및 심장 혈관계질환(cardiovascular disease) 등의 원인이 될 수 있으며, 세포사멸의 비정상적인 억제는 암의 원인이 될 수 있다.  On the other hand, apoptosis is deeply involved in the development of various diseases, that is, the occurrence of abnormal cell death may be the cause of neurodegenerative disorders, immune disorders and cardiovascular diseases. Abnormal inhibition of apoptosis can cause cancer.
따라서 세포사멸은 생명체의 다양한 생리작용을 정상적으로 유지하는데 중요한 역할을 할 뿐만 아니라, 여러 질병들의 발병과정에도 밀접한 관련이 있으며, 개체를 구성하는 각 세포의 세포사멸은 유전적으로 손상을 입은 세포나 분화 자극제에 의한 부적절한 분화 유도에 의해 발달된 종양을 막기 위해 비정상적인 세포를 제거할 수 있는 기작이다.  Therefore, apoptosis plays an important role in maintaining the normal physiological function of life, and is closely related to the pathogenesis of various diseases, and apoptosis of each cell constituting an individual is genetically damaged cells or differentiation stimulants. It is a mechanism that can remove abnormal cells in order to prevent tumors developed by inappropriate induction of differentiation.
그러므로 비정상적인 세포의 증식 및 성장을 억제하고 세포사멸을 유도하는 활성이 있는 물질은 항암제로서 사용할 수 있다(Fesus, L., J. Cell Biochem., 22, 151-161, 1995; Reddy, B.S., Cancer Res., 57, 420-425, 1997).Therefore, a substance having an activity that inhibits the proliferation and growth of abnormal cells and induces apoptosis can be used as an anticancer agent (Fesus, L., J. Cell Biochem. , 22, 151-161, 1995; Reddy, BS, Cancer Res ., 57, 420-425, 1997).
이에 본 발명자들은 캄페라이드 화합물의 항암 활성을 확인하기 위해, 본 발명의 일실시예에 있어서, 캄페라이드 화합물을 유방암 세포주인 MDA-MB-231 세포 및 MCF-7 세포와 전립선암 세포주인 PC-3 세포에 처리한 후, 캄페라이드 화합물에 의한 암 세포들의 증식 억제 정도를 FACs 방법을 통해 확인하였다. 그 결과, 유방암 세포주인 MDA-MB-231 및 MCF-7 세포와 전립선암 세포주인 PC-3 세포의 세포증식이 억제되는 것으로 나타났으며, 특히 유방암 세포주인 MDA-MB-231에 대해서는 약 80%의 높은 세포증식 억제율을 보였다(표 1 참조).  In order to confirm the anticancer activity of the camphoride compound, the present inventors, in one embodiment of the present invention, the camphoride compound is a breast cancer cell line MDA-MB-231 cells and MCF-7 cells and prostate cancer cell line PC-3 After treatment with the cells, the extent of inhibition of proliferation of cancer cells by the camphoride compound was confirmed by the FACs method. As a result, cell proliferation of MDA-MB-231 and MCF-7 cells, which are breast cancer cell lines, and PC-3 cells, which are prostate cancer cell lines, were inhibited. In particular, about 80% of MDA-MB-231, a breast cancer cell line, was inhibited. Showed high cell proliferation inhibition (see Table 1).
또한, 화합물의 세포독성을 나타내는 지표인 IC50의 값을 측정한 결과, MDA-MB-231 및 PC-3 세포의 경우 캄페라이드의 IC50 값은 23uM인 것으로 나타났다. 따라서 매우 적은 농도의 캄페라이드의 처리에도 불구하고 암 세포에 대하여 높은 독성을 나타낸다는 사실을 통해 본 발명의 캄페라이드는 항암 활성이 매우 우수하다는 것을 알 수 있었다. In addition, when the IC 50 value, which is an indicator of cytotoxicity of the compound, was measured, the IC 50 value of camphoride was 23 uM for MDA-MB-231 and PC-3 cells. Therefore, the fact that despite the treatment of very small concentrations of camphoride shows high toxicity against cancer cells, it can be seen that the camphoride of the present invention is very excellent in anticancer activity.
또한, 본 발명의 다른 일실시예에 따르면, 캄페라이드의 직접적인 항암 활성을 확인하기 위해 유방암 세포주에 캄페라이드 화합물을 처리하고, 아폽토시스(apoptosis)의 표지 물질인 아넥신 V로 세포를 염색하여 아폽토시스에 의한 세포사멸을 측정한 결과, 캄페라이드 화합물을 처리한 유방암 세포들은 캄페라이드 화합물 대신 DMSO를 처리한 대조군에 비해 아폽토시스에 의한 세포사멸이 현저하게 증가하는 것으로 나타났으며, 캄페라이드에 의한 세포사멸은 농도 의존적으로 비례하는 것으로 나타났다(도 3 참조).  In addition, according to another embodiment of the present invention, in order to confirm the direct anti-cancer activity of camphoride, the cancer cell line is treated with a camphoride compound, and stained with annexin V, a label of apoptosis, to apoptosis. As a result of measuring apoptosis, apoptosis-induced apoptosis was significantly increased in the breast cancer cells treated with the camphoride compound compared to the control treated with DMSO instead of the camphoride compound. It was shown to be proportionally dependent on concentration (see FIG. 3).
따라서 본 발명자들은 상기 결과를 통해 본 발명의 캄페라이드 화합물을 암 세포에서 아폽토시스를 통한 세포사멸을 유도하여 항암 활성을 갖는다는 사실을 알 수 있었다. Therefore, the present inventors have found that the camphoride compound of the present invention has anticancer activity by inducing apoptosis through apoptosis in cancer cells.
나아가 본 발명에 따른 캄페라이드 화합물은 NF-κB 및 COX-2의 억제를 통한 기작에 의해 항암활성을 갖는 특징이 있다. Furthermore, the camphoride compound according to the present invention is characterized by having anticancer activity by a mechanism through inhibition of NF-κB and COX-2.
특히, 종양의 발생 및 진행과정은 NF-κB에 의한 신호 전달 과정과 매우 밀접한 관련이 있는 것으로 알려져 있는데, 예컨대, NF-κB의 활성화는 암 세포의 세포사멸을 억제하는 작용을 하며, 세포주기의 진행을 촉진시켜 세포의 증식을 활성화하는 것으로 밝혀졌으며(Monks NR et al., J. Cell Biochem, 92, 646~650, 2004), 인산화된 활성형의 NF-κB는 COX-2(cyclooxygenase-2) 유전자의 발현을 촉진시키는 것으로 밝혀진 바 있다. 또한, 최근 연구에 의하면 암 세포에서 NF-κB 및 IκB는 인산화되어 활성화된 형태로 존재하고, 활성화된 NF-κB는 세포질에서 핵으로 이동하는 것으로 보고된 바 있다(Jand BC., J. BioL. Chem., 275, 39 507~39515, 2000; Monks NR et al., J. Cell Biochem, 92, 646~650, 2004). In particular, the development and progression of tumors are known to be closely related to the signal transduction process by NF-κB. For example, activation of NF-κB inhibits apoptosis of cancer cells. It has been shown to promote cell proliferation by promoting progression (Monks NR et al ., J. Cell Biochem , 92, 646-650, 2004), and phosphorylated active NF-κB is a cyclooxygenase-2. Has been shown to promote the expression of genes. In addition, recent studies have reported that NF-κB and IκB exist in phosphorylated and activated form in cancer cells, and activated NF-κB migrates from the cytoplasm to the nucleus (Jand BC., J. BioL . Chem ., 275, 39 507-39515, 2000; Monks NR et al ., J. Cell Biochem , 92, 646-650, 2004).
또한, 염증반응에 관여하는 효소로 알려진 COX-2(cyclooxygenase-2: 시클로옥시게나제-2)는 종양발생 과정의 각 단계가 진행할수록 발현양이 증가하는 것으로 보고되어 있다(Kitayama W., et al., Carcinogenesis, 20:2305-2310, 1999; Takahashi M., et al., Cancer Res., 57:1233-1237, 1997). 이러한 COX는 프로스타그란딘(prostaglandin)의 생합성에 관련되는 주효소로서 생체 내에 두 종류의 이성효소가 존재한다. COX-1은 정상상태에서 발현하여 위장관 보호나 신장기능 조절 등에 관여하며, COX-2는 염증이나 기타 면역 반응시 세포분열인자(mitogen)나 사이토카인(cytokines)들에 의해 세포내에서 일시적이고 빠르게 발현된다. 따라서 COX-1또는 COX-2를 억제하는 물질은 염증의 치료뿐만 아니라 암 생성 억제의 측면에서도 중요한 역할을 할 수 있다.In addition, COX-2 (cyclooxygenase-2), known as an enzyme involved in the inflammatory response, has been reported to increase in expression level as each stage of the tumor development process (Kitayama W., et. al., Carcinogenesis , 20: 2305-2310, 1999; Takahashi M., et al., Cancer Res. , 57: 1233-1237, 1997). COX is a main enzyme involved in the biosynthesis of prostaglandin, and there are two kinds of isomers in vivo. COX-1 is expressed in the normal state and is involved in gastrointestinal protection and renal function control. COX-2 is transient and rapid in the cell by mitogen or cytokines during inflammation or other immune reactions. Expressed. Thus, substances that inhibit COX-1 or COX-2 may play an important role not only in the treatment of inflammation but also in inhibiting cancer production.
따라서 NF-κB 및 COX-2의 활성 또는 발현을 억제한다면 암의 발생 또는 진행을 억제할 수 있다. Therefore, inhibiting the activity or expression of NF-κB and COX-2 can inhibit the development or progression of cancer.
이에 본 발명자들은 본 발명에 따른 캄페라이드 화합물이 NF-κB 및 COX-2를 억제하는 활성이 있는지를 조사한 결과, 유방암 세포주에 캄페라이드 화합물을 처리하였을 경우, NF-κB의 핵으로의 이동 및 COX-2의 발현이 캄페라이드 화합물을 처리하지 않은 대조군에 비해 더 많이 억제되는 것으로 나타났다(실시예 4 참조). Therefore, the present inventors examined whether the camphoride compound according to the present invention has an activity of inhibiting NF-κB and COX-2, and when the camphoride compound was treated in breast cancer cell lines, NF-κB shifted to the nucleus and COX. The expression of -2 was shown to be more inhibited than the control group not treated with the camphoride compound (see Example 4).
한편, 종래 사용되고 있는 대부분의 항암제들은 인체에 부작용을 초래하거나 또는 독성이 강하여 암 세포 이외에도 정상세포에 이상을 초래하는 문제점들이 있었다. 이에 본 발명자들은 본 발명의 캄페라이드 화합물이 임상용의 항암제로서 안정한지를 확인하기 위해 암 세포를 대상으로 캄페라이드를 처리한 후 세포사멸 정도를 측정할 때, 괴사(necrosis)에 의한 세포사멸 정도를 대조군과 비교하여 측정한 결과, 캄페라이드 처리에 의한 괴사(necrosis)의 정도는 대조군과 거의 유사한 것으로 나타났다(도 3 참조). On the other hand, most of the anti-cancer agents used conventionally have side effects or strong toxicity to the human body, there are problems that cause abnormalities in normal cells in addition to cancer cells. Therefore, the present inventors measured the degree of apoptosis due to necrosis when measuring the degree of apoptosis after treatment with camphoride in cancer cells to confirm whether the camphoride compound of the present invention is stable as a clinical anticancer agent. As a result of comparison with the control group, the degree of necrosis by camperide treatment was found to be almost similar to the control group (see FIG. 3).
따라서 상기 결과를 통해 본 발명에 따른 캄페라이드 화합물은 생체 내에서 다른 부작용을 유발하지 않는 비교적 우수한 안정성을 갖는 항암제로 사용할 수 있음을 알 수 있었다. Therefore, the above results showed that the camphoride compound according to the present invention can be used as an anticancer agent having relatively good stability without causing other side effects in vivo.
그러므로 본 발명은 캄페라이드 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항암용 조성물을 제공하며, 상기 캄페라이드 화합물은 조성물 총 부피에 대하여 0.1~0.5mM의 농도로 포함될 수 있다.  Therefore, the present invention provides an anticancer composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient, and the camphoride compound may be included at a concentration of 0.1 to 0.5 mM with respect to the total volume of the composition.
또한, 상기 본 발명의 조성물은 비정상적인 세포의 성장 및 증식으로 발생하는 암의 증상을 예방 및 치료하기 위한 약학적 조성물로 사용될 수 있다. In addition, the composition of the present invention can be used as a pharmaceutical composition for preventing and treating the symptoms of cancer caused by abnormal growth and proliferation of cells.
본 발명에서 캄페라이드 화합물을 이용하여 예방 또는 치료할 수 있는 상기 암의 종류로는 이에 제한되지는 않으나, 유방암, 후두암, 폐암, 상피암, 전립선암, 식도암, 췌장암, 대장암, 간암, 위암, 설암, 피부암, 뇌종양, 자궁암, 자궁경부암, 난소암, 신장암, 담낭암, 구강암, 결장암, 간암 및 방광암을 포함할 수 있다.  In the present invention, the type of cancer that can be prevented or treated using the camphoride compound is not limited thereto, but is not limited thereto, breast cancer, laryngeal cancer, lung cancer, epithelial cancer, prostate cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, Skin cancer, brain tumor, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer and bladder cancer.
또한, 본 발명에 따른 항암용 조성물은 약학적으로 유효한 양의 캄페라이드 화합물을 단독으로 포함하거나 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함할 수 있다. 상기에서 약학적으로 유효한 양이란 암을 예방, 개선 및 치료하기에 충분한 양을 말한다. In addition, the anticancer composition according to the present invention may include a pharmaceutically effective amount of a camphoride compound alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat cancer.
본 발명에 따른 캄페라이드 화합물의 약학적으로 유효한 양은 0.5 ~ 100 mg/day/체중kg, 바람직하게는 0.5 ~ 5 mg/day/체중kg이다. 그러나 상기 약학적으로 유효한 양은 질환 증상의 정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료기간 등에 따라 적절히 변화될 수 있다. The pharmaceutically effective amount of the camphoride compound according to the invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed according to the degree of disease symptoms, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.
* 또한, 상기에서 약학적으로 허용되는이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. * In addition, the pharmaceutically acceptable means a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness or similar reaction when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.  In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
본 발명에 따른 항암용 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있으며, 활성 성분의 투여량은 투여 경로, 환자의 연령, 성별, 체중 및 환자의 중증도 등의 여러 인자에 따라 적절히 선택될 수 있다. 또한, 본 발명의 항암용 조성물은 암의 증상을 예방, 개선 또는 치료하는 효과를 가지는 공지의 화합물과 병행하여 투여할 수 있다. The anticancer composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, the age, sex, weight and severity of the patient. It may be appropriately selected depending on several factors. In addition, the anticancer composition of the present invention can be administered in parallel with known compounds having the effect of preventing, improving or treating the symptoms of cancer.
따라서 본 발명은 캄페라이드 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 조성물을 포함하는 암의 예방 또는 치료용 약제를 제공할 수 있다. Accordingly, the present invention may provide a medicament for preventing or treating cancer, including a composition containing a camphoride compound or a pharmaceutically acceptable salt thereof as an active ingredient.
나아가 본 발명에 따른 캄페라이드 화합물은 생체 내에서 다른 부작용을 유발시키지 않고 장기간 복용시에도 안심하고 사용할 수 있으므로 이를 포함하는 본 발명의 조성물은 체내에 대해 매우 안정한 특징이 있다. Furthermore, since the camphoride compound according to the present invention can be used safely in a long time without causing other side effects in vivo, the composition of the present invention comprising the same has a very stable feature for the body.
따라서 본 발명의 항암용 조성물은 암의 예방 또는 개선 효과를 목적으로 하는 식품에 첨가할 수 있으므로, 상기 본 발명의 조성물을 항암 효과를 갖는 기능성 건강식품용 조성물로 사용할 수 있다.  Therefore, the anticancer composition of the present invention can be added to a food for the purpose of preventing or improving cancer, and thus the composition of the present invention can be used as a functional health food composition having an anticancer effect.
그러므로 본 발명의 항암 효과를 갖는 기능성 건강식품용 조성물은 암 증상의 예방 및 개선에 효과가 있는 식품, 예컨대, 식품의 주원료, 부원료, 식품 첨가제, 기능성 식품 또는 음료로 용이하게 활용할 수 있다. Therefore, the composition for functional health foods having an anticancer effect of the present invention can be easily utilized as foods, such as the main ingredients, side ingredients, food additives, functional foods or beverages that are effective in preventing and improving cancer symptoms.
본원에서 상기 “식품”이란, 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 기능성 식품 및 음료를 모두 포함하는 것을 말한다.  As used herein, the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step, It includes all foods, food additives, functional foods and drinks.
본원발명에 따른 항암 효과를 갖는 기능성 건강식품용 조성물을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 기능성 식품 등이 있다. 추가로, 본원발명에서 식품에는 특수영양식품(예, 조제유류, 영,유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예, 라면류, 국수류 등), 빵류, 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 캔디류, 쵸코렛류, 껌류, 아이스크림류, 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예, 과실 음료, 채소류 음료, 두유류, 발효음료류 등), 천연조미료(예, 라면 스프 등)을 포함하나 이에 한정되지 않는다. 상기 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.  Foods to which the composition for functional health foods having an anticancer effect according to the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, in the present invention, food includes special nutritional products (e.g., formulated milk, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), bread, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additives may be prepared by a conventional manufacturing method.
또한, 상기 “기능성 식품”이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미하며, 구체적으로는 건강 기능성 식품일 수 있다. 상기 기능성 식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.  In addition, the term "functional food" refers to the control of biological defense rhythms and disease prevention of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body's regulatory function regarding recovery and the like, and specifically, it may be a health functional food. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.
또한, 본원발명에서 상기“음료”란 갈증을 해소하거나 맛을 즐기기 위하여 마시는 것의 총칭을 의미하며 기능성 음료를 포함한다. 상기 음료는 지시된 비율로 필수 성분으로서 상기 암 증상의 예방 및 개선용 조성물을 포함하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.  In addition, in the present invention, the "beverage" refers to a generic term for drinking to quench thirst or to enjoy a taste and includes a functional drink. The beverage contains, as essential ingredients, a composition for the prevention and amelioration of the cancer symptoms as an essential ingredient, and there are no particular restrictions on the other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be.
나아가 상기 기술한 것 이외에 본원발명의 항암 증진 효과를 갖는 기능성 건강식품용 조성물을 함유하는 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있으며, 상기 성분은 독립적으로 또는 조합하여 사용할 수 있다.  Furthermore, the food containing the functional health food composition having an anticancer enhancing effect of the present invention in addition to the above-described flavors, colorants and fillers such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors (Cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The above components may be used independently or in combination.
본원발명의 항암 효과를 갖는 기능성 건강식품용 조성물을 함유하는 식품에 있어서, 상기 본 발명에 따른 조성물의 양은 전체 식품 중량의 0.001중량% 내지 90중량%로 포함할 수 있으며, 바람직하게는 0.1중량% 내지 40중량%로 포함할 수 있고, 음료의 경우, 100ml를 기준으로 0.01g 내지 20g, 바람직하게는 0.3g 내지 10g의 비율로 포함할 수 있으나, 건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로 사용될 수 있으므로 상기 범위에 한정되는 것은 아니다. In a food containing a functional health food composition having an anticancer effect of the present invention, the amount of the composition according to the present invention may be included in 0.001% to 90% by weight of the total food weight, preferably 0.1% by weight To 40% by weight, and in the case of a beverage, it may be included in a ratio of 0.01g to 20g, preferably 0.3g to 10g based on 100ml, but for health and hygiene purposes or for health control purposes. In the case of long-term ingestion may be below the above range, the active ingredient is not limited to the above range because it can be used in an amount above the above range because there is no problem in terms of safety.
이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.  Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
캄페라이드 화합물의 확인Identification of Camperide Compounds
본 발명자들은 시중에서 구입한 캄페라이드 화합물에 대해 구입한 화합물이 캄페라이드 화합물이 맞는지를 확인하기 위해 C18 HPLC 크로마토그래피를 수행하여 캄페라이드 화합물을 분리하였으며, 분리된 화합물을 가지고 당업계에 공지된 1H-NMR 분석을 수행하였다.The present inventors have performed a C18 HPLC chromatography to determine the correct this campesterol fluoride compound a compound purchased for campesterol fluoride compound a commercially available separating campesterol fluoride compound, known in the art with a discrete compound 1 H-NMR analysis was performed.
그 결과, 시중에서 구입한 캄페라이드 화합물은 하기 화학식 1로 표시되는 분자량이 300.26인 캄페라이드(kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone)임을 확인하였으며, C18 HPLC 크로마토그래프에 의해 분리된 캄페라이드 화합물에 대한 피크들은 도 1에 나타내었으며, 1H-NMR 에 의한 캄페라이드 화합물의 피크들은 도 2에 나타내었다.As a result, it was confirmed that a commercially available camphoride compound was a camphoride (kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) having a molecular weight of 300.26 represented by the following Chemical Formula 1, and was separated by C18 HPLC chromatography. The peaks for the camphoride compound were shown in FIG. 1, and the peaks of the camphoride compound by 1 H-NMR are shown in FIG. 2.
<화학식 1><Formula 1>
Figure PCTKR2010008648-appb-I000002
Figure PCTKR2010008648-appb-I000002
<실시예 2><Example 2>
캄페라이드 화합물의 암 세포 증식 억제 활성 분석Analysis of Cancer Cell Proliferation Inhibitory Activity of Camperide Compounds
상기 실시예 1에서 확인된 캄페라이드 화합물에 대하여 암 세포의 세포증식을 억제하는 활성이 있는지를 당업계에 공지된 FACs 방법을 통해 조사하였다. 먼저 이를 위해 암 세포로서 한국세포주 은행으로부터 구입한 유방암 세포주인 MDA-MB-231 세포주 및 MCF-7 세포주와 전립선암 세포주인 PC-3 세포주들을 10% 소태아 혈청, 2mg/ml의 소듐 카보네이트, 100 U/mL 페니실린 및 100 /mL의 스트렙토마이신이 함유된 RPMI-1640(Gibco/BRL, Grand Island, NY) 배지에서 배양하였고, 이후 배양한 각 암 세포주를 96웰 플레이트에 1.5× 104 세포수/웰이 되도록 분주한 후, 캄페라이드 화합물을 0.01~0.33mM의 다양한 농도로 처리한 다음 48시간 동안 배양하였다. 이때 대조군으로는 캄페라이드 화합물 대신 0.1%의 DMSO를 처리한 것을 사용하였다. 그런 뒤, 캄페라이드 화합물에 의한 세포의 증식 억제 정도를 세포 카운팅 키트인 CCK-8(Dojindo Laboratories, Japan)을 사용하여 측정하였고, 이때 흡광도는 멀티-측정 마이크로플레이트-리더(Bio-TEK, Winooski, VT)를 이용하여 450nm의 파장에서 측정하였다. 또한, 억제율은 다음과 같은 식으로 계산하였으며, 그 결과를 하기 표 1에 나타내었다.The activity of inhibiting cell proliferation of cancer cells with respect to the camphoride compound identified in Example 1 was investigated through FACs known in the art. For this purpose, MDA-MB-231 cell line and MCF-7 cell line and PC-3 cell line, prostate cancer cell line, which were purchased from Korea Cell Line Bank as cancer cells, were treated with 10% fetal bovine serum, 2mg / ml sodium carbonate, 100 Cultured in RPMI-1640 (Gibco / BRL, Grand Island, NY) medium containing U / mL penicillin and 100 / mL of streptomycin, each cancer cell line incubated thereafter was 1.5 × 10 4 cells / After dispensing into wells, the camphoride compound was treated at various concentrations of 0.01 to 0.33 mM and then incubated for 48 hours. In this case, 0.1% DMSO treatment was used instead of the camphoride compound. Then, the degree of inhibition of cell proliferation by the camphoride compound was measured using a cell counting kit, CCK-8 (Dojindo Laboratories, Japan), where the absorbance was measured by a multi-measurement microplate-reader (Bio-TEK, Winooski, Measured at a wavelength of 450 nm using VT). In addition, the inhibition rate was calculated by the following formula, the results are shown in Table 1 below.
세포증식 억제율=(A-B)/A× 100 Cell proliferation inhibition rate = (A-B) / A × 100
여기서, 상기 A는 대조군 세포의 OD값이고, B는 캄페라이드 화합물이 처리된 세포의 OD값이다. Here, A is the OD value of the control cell, B is the OD value of the cells treated with the camphoride compound.
표 1 캄페라이드에 의한 암 세포주의 증식 억제율
암 세포주화합물 처리 MDA-MB-231 MCF-7 PC-3
캄페라이드 화합물(0.33mM) 80.98±1.11*(%) 41.73±2.76* 70.82±0.83*
0.1% DMSO 0 (%) 0 (%) 0 (%)
Table 1 Inhibition of Cancer Cell Line Proliferation by Camperide
Cancer Cell Line Compound Treatment MDA-MB-231 MCF-7 PC-3
Camperide Compound (0.33 mM) 80.98 ± 1.11 * (%) 41.73 ± 2.76 * 70.82 ± 0.83 *
0.1% DMSO 0 (%) 0 (%) 0 (%)
여기서, 상기 *는 Tukey's studentized range(HSD)테스트에 의해 P<0.001인 것을 나타낸 것이며, 대조군의 세포활성을 100%로 산정하여 계산한 것임.Here, * indicates that P <0.001 by Tukey's studentized range (HSD) test, calculated by calculating the cell activity of the control to 100%.
그 결과, 상기 표 1에 나타낸 바와 같이, 본 발명에서 분리 및 정제된 캄페라이드 화합물은 유방암 세포주인 MDA-MB-231 및 MCF-7 세포와 전립선암 세포주인 PC-3 세포에 대하여 세포증식을 억제하는 효과가 있다는 것을 알 수 있었으며, 특히 유방암 세포주인 MDA-MB-231에 대해서는 매우 높은 효율로 세포의 증식을 억제하는 활성이 있음을 알 수 있었다.  As a result, as shown in Table 1, the camphoride compound isolated and purified in the present invention inhibits cell proliferation against MDA-MB-231 and MCF-7 cells, which are breast cancer cell lines, and PC-3 cells, which are prostate cancer cell lines. It can be seen that there is an effect, and in particular, the breast cancer cell line MDA-MB-231 was found to have the activity of inhibiting the proliferation of cells with very high efficiency.
또한, 화합물의 세포독성을 나타내는 지표인 IC50의 값을 측정한 결과, MDA-MB-231 및 PC-3 세포에 대한 캄페라이드의 IC50 값은 23uM의 농도로 나타났으며, 따라서 매우 적은 농도에서도 암 세포에 대하여 독성을 나타낸다는 사실을 알 수 있었다(결과 미도시). In addition, the measurement of IC 50 , an indicator of the cytotoxicity of the compound, showed that the IC 50 value of camphoride for MDA-MB-231 and PC-3 cells was 23 μM and therefore very low. In addition, it was found that toxic to cancer cells (results not shown).
따라서 상기 결과를 통해 본 발명자들은 본 발명에 따른 캄페라이드 화합물이 각종 암세포의 세포증식 억제 및 세포독성을 통해 항암 활성을 가지고 있다는 사실을 확인하였다.  Therefore, the present inventors confirmed that the camphoride compound according to the present invention has anticancer activity through cytotoxicity and cytotoxicity of various cancer cells.
<실시예 3><Example 3>
캄페라이드 화합물의 암 세포 사멸 활성 분석Analysis of Cancer Cell Killing Activity of Camperide Compounds
나아가 본 발명자들은 캄페라이드 화합물의 항암 활성을 직접적으로 확인하기 위하여 캄페라이드 화합물이 암 세포의 세포사멸을 유도하는 활성이 있는지를 조사하였다. 상기 조사를 위해, 상기 실시예 2의 조건으로 배양된 유방암 세포주인 MDA-MB-231 세포주에 대하여 0.33mM의 캄페라이드 화합물을 처리하고 48시간을 배양하였다. 이후 트립신 처리에 의해 세포들을 각각 수득하였고, 차가운 PBS 용액으로 세포들을 세척한 다음, 0.14 M NaCl 및 2.5 mM CaCl2을 함유한 0.01 M Hepes/NaOH, pH7.4 용액으로 세포를 용해시킨 다음, 5㎕ 아넥신(Annexin) V-FITC 및 5㎕ propidium iodide(PI)를 첨가하고 천천히 혼합하여 상온의 어두운 상태에서 15분간 반응시켰다. 이후 0.01 M Hepes/NaOH, pH7.4 용액을 더 첨가한 다음, 세포의 사멸을 측정하는 지표인 Annexin V로 염색된 세포들을 BD Model FACScan(Becton Dickinson Inc., New Jersey, USA)으로 분석하여 각 세포들의 아폽토시스(apoptosis) 정도를 분석하였다. 이때, 대조군으로는 캄페라이드 화합물 대신 DMSO를 처리한 것을 사용하였다.Furthermore, the present inventors investigated whether the camphoride compound has an activity of inducing apoptosis of cancer cells in order to directly confirm the anticancer activity of the camphoride compound. For the investigation, 0.33mM camphoride compound was treated with MDA-MB-231 cell line, which was cultured under the conditions of Example 2, and cultured for 48 hours. Cells were then obtained by trypsin treatment, washed with cold PBS solution, and then lysed with 0.01 M Hepes / NaOH, pH7.4 solution containing 0.14 M NaCl and 2.5 mM CaCl 2 , and then 5 Μl Annexin V-FITC and 5 μl propidium iodide (PI) were added and slowly mixed to react for 15 minutes in the dark at room temperature. Then, 0.01 M Hepes / NaOH, pH7.4 solution was further added, and cells stained with Annexin V, an indicator of cell death, were analyzed by BD Model FACScan (Becton Dickinson Inc., New Jersey, USA). The degree of apoptosis of the cells was analyzed. At this time, the control was used to treat DMSO instead of camphoride compound.
그 결과, 도 3에 나타낸 바와 같이, 대조군인 DMSO를 처리한 경우, 약 14%의 아폽토시스가 유발된 것으로 나타난 반면, 캄페라이드 화합물을 처리한 경우에는 약 30%의 아폽토시스가 유발된 것으로 나타났다. 또한, 캄페라이드 화합물의 처리 농도가 높을수록 아폽토시스가 유발된 세포의 수도 더 증가하는 것으로 나타났다.  As a result, as shown in FIG. 3, treatment with DMSO, which was a control group, showed about 14% of apoptosis, whereas treatment with camphoride compound induced about 30% of apoptosis. In addition, the higher the treatment concentration of the camphoride compound, the higher the number of apoptosis-induced cells.
따라서 상기 결과를 통해 본 발명의 캄페라이드 화합물은 암세포에서 아폽토시스를 유도하여 세포를 사멸하는 활성이 우수함을 알 수 있었다. Therefore, the above results show that the camphoride compound of the present invention has excellent activity of killing cells by inducing apoptosis in cancer cells.
한편, 본 발명자들은 상기 캄페라이드 화합물을 항암제로 사용하였을 경우, 체내에 대한 안정성을 확인하기 위해, 캄페라이드 화합물 처리시 괴사에 의한 세포사멸 정도를 대조군과 비교하여 확인한 결과, 대조군의 경우 약 3.07%의 괴사율을 나타내었으며, 캄페라이드 화합물을 처리한 경우에도 대조군과 유사하게 약 4.0%의 괴사율을 나타내었다.  On the other hand, the present inventors, when using the camphoride compound as an anticancer agent, in order to confirm the stability to the body, the degree of apoptosis due to necrosis when treated with camphoride compound compared to the control group, the control group was about 3.07% Necrosis rate of was shown, and even when treated with camphoride compound showed a necrosis rate of about 4.0% similar to the control.
따라서 본 발명에 따른 캄페라이드 화합물은 괴사에 의한 세포사멸 정도가 대조군과 유의한 차이를 나타내지 않는다는 사실을 통해 생물학적으로 안정하다는 사실을 알 수 있었다.  Therefore, it can be seen that the camphoride compound according to the present invention is biologically stable through the fact that the degree of cell death by necrosis does not show a significant difference from the control group.
그러므로 본 발명자들은 캄페라이드 화합물이 암세포의 세포증식을 억제하고, 아폽토시스에 의한 세포사멸을 유도하는 활성을 가지고 있을 뿐만 아니라 부작용이 없어 안정성이 우수하므로 궁극적으로 효율적인 암 치료를 위한 치료제로 사용할 수 있음을 알 수 있었다.       Therefore, the present inventors found that the camphoride compound not only has the activity of inhibiting the cell proliferation of cancer cells and induces apoptosis by apoptosis, but also has no side effects and is excellent in stability so that it can ultimately be used as a therapeutic agent for effective cancer treatment. Could know.
<실시예 4><Example 4>
캄페라이드 화합물의 NF-κB 및 COX-2에 미치는 영향 분석Analysis of Effects of Camperide Compounds on NF-κB and COX-2
<4-1> 캄페라이드 화합물에 의한 NF-κB 의 핵으로의 이동 억제 활성 <4-1> Inhibitory Activity of NF-κB to the Nucleus by Camperide Compounds
암 세포의 생존 인자로 작용하는 NF-κB의 경우, 인산화에 의해 활성화가 되면 핵으로 이동함과 동시에 COX-2의 유전자 발현을 조절하는 것으로 알려져 있다. 이에 본 발명자들은 캄페라이드 화합물을 유방암 세포에 처리하였을 경우, NF-κB의 활성 억제, 즉 핵으로의 이동 억제 활성이 있는지를 조사하였다. NF-κB, which acts as a survival factor of cancer cells, is known to regulate COX-2 gene expression while migrating to the nucleus when activated by phosphorylation. Therefore, the present inventors examined whether the camphoride compound was treated with breast cancer cells to inhibit NF-κB activity, that is, to inhibit migration to the nucleus.
이를 위해, 유방암 세포주인 MDA-MB-231 세포를 100ψ 플레이트에 1.0× 106 세포수/웰이 되도록 분주한 후, 24시간 동안 배양하였다. 이후, 7ug/ml의 캄페라이드 화합물을 처리하였고, 90분간 배양한 후, 면역형광 분석방법을 통해 NF-κB의 핵으로의 이동을 관찰하였다. 이때 상기 면역형광 분석방법은 캄페라이드가 처리된 세포를 차가운 PBS 용액으로 세척한 다음, 차가운 아세톤 용액으로 고정시키고, 동물-프리-블록커(벡터, Burlingame, CA, USA, SP-5030)로 한 시간 동안 블록킹 시킨 다음, 4℃에서 항-NF-κB 래빗 항체(세포 시그널, 덴버, 미국 Cat#4764)로 밤새 반응시켰다. 이후 세포들을 항-래빗 igG FITC 항체(Cayman, Cat#CAY-10006588)로 1시간 반응시키고, 4‘6-디아미도-2-페닐인돌(DAPI)로 염색한 다음, IX51 현미경(올림포스, 일본)을 사용하여 세포내에서 NF-κB의 핵으로의 이동을 관찰하였다. 이때 대조군으로는 캄페라이드 대신 DMSO만을 처리한 것을 사용하였다.To this end, MDA-MB-231 cells, which are breast cancer cell lines, were dispensed to 100 × plate at 1.0 × 10 6 cells / well and cultured for 24 hours. Thereafter, 7ug / ml of the camphoride compound was treated, and after 90 minutes of incubation, migration of NF-κB to the nucleus was observed by immunofluorescence analysis. At this time, the immunofluorescence assay method was performed by washing the cells treated with camphoride with cold PBS solution, fixing with cold acetone solution, and using animal-free-blocker (Vector, Burlingame, CA, USA, SP-5030). Blocking for hours was followed by overnight reaction at 4 ° C. with anti-NF-κB rabbit antibody (Cell Signal, Denver, USA Cat # 4764). Cells were then reacted with anti-rabbit igG FITC antibody (Cayman, Cat # CAY-10006588) for 1 hour, stained with 4'6-dimami-2-phenylindole (DAPI), and then IX51 microscope (Olympos, Japan). Was used to observe the migration of NF-κB into the nucleus intracellularly. At this time, the control was used to treat only DMSO instead of camphoride.
그 결과, 도 4에 나타낸 바와 같이, 대조군에 비해 캄페라이드 화합물을 처리한 유방암 세포의 경우, NF-κB의 핵으로의 이동이 억제되는 것으로 나타났다. As a result, as shown in FIG. 4, NF-κB migration to the nucleus was suppressed in the breast cancer cells treated with the camphoride compound as compared with the control group.
따라서 상기 결과를 통해 본 발명자들은 본 발명의 캄페라이드 화합물은 암 세포주에서 암 세포의 생존인자인 NF-κB의 핵으로의 이동을 억제하는 기작을 통해 항암 활성을 가진다는 사실을 알 수 있었다. Therefore, the present inventors have found that the camphoride compound of the present invention has anticancer activity through the mechanism of inhibiting migration of NF-κB, which is a survival factor of cancer cells, into cancer cells in cancer cell lines.
<4-2> 캄페라이드 화합물에 의한 COX-2의 발현 억제 활성<4-2> Expression Inhibition Activity of COX-2 by Camperide Compound
나아가 본 발명자들은 캄페라이드 화합물이 암 세포에서 염증반응을 매개하고 암세포를 성장시키는 작용을 하는 것으로 알려진 COX-2의 발현을 억제하는 활성이 있는지를 조사하기 위해 웨스턴 블럿 및 면역형광 분석법을 수행하였다. 상기 면역형광 분석법은 상기 실시예 <4-1>의 동일한 방법으로 캄페라이드를 처리한 MDA-MB-231 세포를 48시간 동안 배양한 후, 원심분리하여 세포들을 수득한 다음, 차가운 PBS 용액으로 3번 세척하고, 4%의 포름알데히드(시그마 알드리치, SO57504)로 세포를 고정시킨 다음, 다시 차가운 PBS 용액으로 3번 세척하였다. 이후 밤새도록 항-COX-2항체(바이오케어:CRM 306C)를 반응시킨 다음, EnVISION+TM(다코, 덴마크)를 처리하고 액체의 DAB 기질을 사용하여 현상시킨 후, BX51 현미경(올림포스, 일본)을 사용하여 세포들을 관찰하였다. Furthermore, the inventors performed western blot and immunofluorescence assays to investigate whether the camphoride compound has an activity of inhibiting the expression of COX-2, which is known to mediate inflammatory reactions and grow cancer cells in cancer cells. The immunofluorescence assay was performed by incubating the camphoride-treated MDA-MB-231 cells for 48 hours in the same manner as in Example <4-1>, and then centrifuging to obtain the cells, followed by 3 steps with cold PBS solution. Washed once, cells were fixed with 4% formaldehyde (Sigma Aldrich, SO57504) and then washed three times with cold PBS solution. After overnight reaction with anti-COX-2 antibody (Biocare: CRM 306C), followed by treatment with EnVISION + TM (Dako, Denmark) and development using a liquid DAB substrate, followed by BX51 microscopy (Olympos, Japan). The cells were observed using.
또한, 상기 웨스턴 블럿을 위해 MDA-MB-231 세포를 100ψ 플레이트에 1.0× 106 세포수/웰이 되도록 분주한 다음, 24시간 동안 배양시키고, 상기 세포들에 대하여 캄페라이드 화합물을 각각 7, 70ug/ml씩 처리한 다음, 48시간 배양한 후, 트립신으로 세포들을 플레이트에서 떼어낸 후 차가운 PBS 용액으로 세척하였다. 이후, 세포내에서의 COX-2의 발현 확인을 위해 세포들의 용해물(laysate)를 10% SDS-PAGE로 전기영동 시킨 후, 항-COX-2 다클론 항체(Cayman, Ann Arbor, MI, USA, Cat#CAY-160106)를 사용하여 당업계에 공지된 웨스턴 블럿 방법으로 블럿팅을 수행하였다. 이때 대조군으로는 캄페라이드 화합물 대신 DMSO를 처리한 군을 사용하였고, 웨스턴 블럿 시 동일한 단백질의 양을 확인하기 위해 베타-엑틴 단클론 항체(시그마 알드리치, 미국, Cat#A-5316)를 사용하였다.In addition, for Western blot, MDA-MB-231 cells were aliquoted to 100 × plate at 1.0 × 10 6 cells / well, followed by incubation for 24 hours, and 7, 70 ug each of the camphoride compound for the cells. / ml, then incubated for 48 hours, cells were removed from the plate with trypsin and washed with cold PBS solution. Thereafter, to confirm the expression of COX-2 in the cells, the cells' lysates were electrophoresed with 10% SDS-PAGE, followed by anti-COX-2 polyclonal antibody (Cayman, Ann Arbor, MI, USA). Blotting was performed by Western blotting method known in the art using Cat # CAY-160106. In this case, a DMSO-treated group was used instead of the camphoride compound, and a beta-actin monoclonal antibody (Sigma Aldrich, USA, Cat # A-5316) was used to confirm the amount of the same protein in Western blot.
그 결과, 도 5a 및 5b에 나타낸 바와 같이, 캄페라이드를 처리하지 않은 군에 비해 캄페라이드를 처리한 군의 경우, COX-2의 발현 양이 감소한 것으로 나타났으며, 특히 캄페라이드의 처리 농도가 증가할수록 COX-2의 발현은 더욱 감소되는 것으로 나타났다. As a result, as shown in Figures 5a and 5b, the amount of expression of COX-2 was reduced in the group treated with camphoride compared to the group without camphoride, especially the concentration of camphoride Increasing expression of COX-2 was found to decrease.
따라서 상기 결과를 통해 본 발명자들은 본 발명의 캄페라이드 화합물이 NF-κB의 활성을 억제함과 동시에 COX-2의 발현 억제를 통해 암의 성장을 억제할 수 있는 효과가 있다는 것을 알 수 있었으며, 궁극적으로 캄페라이드 화합물을 암의 예방 또는 치료를 위한 치료제로 사용할 수 있음을 알 수 있었다. Therefore, the present inventors have found that the camphoride compound of the present invention has an effect of inhibiting the growth of NF-κB and simultaneously inhibiting cancer growth by inhibiting the expression of COX-2. It was found that the camphoride compound can be used as a therapeutic agent for the prevention or treatment of cancer.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.
본 연구는 농촌진흥청 바이오그린21사업(과제번호: 20070301-034-044-007-01)의 지원에 의해 이루어진 것임. This study was supported by the Rural Development Agency's Bio Green 21 project (project number: 20070301-034-044-007-01) .

Claims (6)

  1. 하기 화학식 1로 표시되는 캄페라이드(kaempferide: 3,5,7-trihydroxy-4'-methoxyflavone) 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항암용 조성물:An anticancer composition comprising a kaempferide compound represented by the following Chemical Formula 1 (3,5,7-trihydroxy-4'-methoxyflavone) or a pharmaceutically acceptable salt thereof as an active ingredient:
    <화학식 1><Formula 1>
    Figure PCTKR2010008648-appb-I000003
    Figure PCTKR2010008648-appb-I000003
  2. 제1항에 있어서,  The method of claim 1,
    상기 캄페라이드 화합물은 암 세포의 세포증식을 억제하거나 세포사멸을 유도하는 활성을 갖는 것을 특징으로 하는 항암용 조성물.The camphoride compound is an anticancer composition, characterized in that it has an activity of inhibiting cell proliferation of cancer cells or inducing apoptosis.
  3. 제1항에 있어서, The method of claim 1,
    상기 캄페라이드 화합물은 조성물 총 부피에 대하여 0.1~0.5mM의 농도로 포함되어 있는 것을 특징으로 하는 항암용 조성물. The camphoride compound is an anticancer composition, characterized in that contained in a concentration of 0.1 ~ 0.5mM relative to the total volume of the composition.
  4. 제1항에 있어서,  The method of claim 1,
    상기 암은 유방암, 후두암, 폐암, 상피암, 전립선암, 식도암, 췌장암, 대장암, 간암, 위암, 설암, 피부암, 뇌종양, 자궁암, 자궁경부암, 난소암, 신장암, 담낭암, 구강암, 결장암, 간암 및 방광암으로 이루어진 군 중에서 선택되는 것을 특징으로 하는 항암용 조성물.The cancer includes breast cancer, laryngeal cancer, lung cancer, epithelial cancer, prostate cancer, esophageal cancer, pancreatic cancer, colon cancer, liver cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, gallbladder cancer, oral cancer, colon cancer, liver cancer and Anticancer composition, characterized in that selected from the group consisting of bladder cancer.
  5. 제1항 내지 제4항 중 어느 한 항의 조성물을 포함하는 암의 예방 또는 치료용 약제.A pharmaceutical for the prophylaxis or treatment of cancer comprising the composition of any one of claims 1 to 4.
  6. 제1항 내지 제4항 중 어느 한 항의 조성물을 포함하는 항암 효과를 갖는 기능성 건강식품.Functional health food having an anticancer effect comprising the composition of any one of claims 1 to 4.
PCT/KR2010/008648 2009-12-04 2010-12-03 Anticancer composition containing a kampheride compound as an active ingredient WO2011068386A2 (en)

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KR20210021827A (en) * 2019-08-19 2021-03-02 연세대학교 산학협력단 A Composition for Preventing or Treating Metabolic Disorders Comprising Kaempferide as an Active Ingredient

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Title
E. SZLISZKA ET AL.: 'Ethanolic extract of propolis (EEP) enhances the apoptosis-inducing potential of TRAIL in cancer cells' MOLECULES vol. 14, no. 2, 13 February 2009, ISSN 1420-3049 pages 738 - 754 *
J. B. DASKIEWICZ ET AL.: 'Effects of flavonoids on cell proliferation and caspase activation in a human colonic cell line HT29: An SAR study' JOURNAL OF MEDICINAL CHEMISTRY vol. 48, no. 8, 2005, ISSN 0022-2623 pages 2790 - 2804 *
R. VACLAVIKOVA ET AL.: 'Modulation of paclitaxel transport by flavonoid derivatives in human breast cancer cells. Is there a correlation between binding affinity to NBD of P-gp and modulation of transport?' BIOORGANIC & MEDICINAL CHEMISTRY vol. 14, no. 13, 2006, ISSN 0968-0896 pages 4519 - 4525 *

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