JP2006528173A - Elderly Kyokyo Extract prevents angiogenesis - Google Patents

Abstract

The present invention relates to an extract from a long-life doraji having an angiogenesis inhibitory effect, and more particularly to an extract from a long-life doraji having an effect of inhibiting angiogenesis involved in the growth and metastasis of cancer cells. The long-life Doraj extract of the present invention can suppress cell migration, cell adhesion, cell infiltration and lumen formation involved in angiogenesis. Therefore, an angiogenesis inhibitor containing the long-life Doraj extract of the present invention will be useful for cancer prevention and treatment.

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Description

  The present invention relates to an extract from a long-life doraji having an angiogenesis inhibitory effect, and more particularly to an extract from a long-life doraji having an inhibitory effect on angiogenesis involved in the growth and metastasis of cancer cells.

  The roots of the asteraceae (scientific name: Platycodon grandiflorum A.DC) have a main root length of about 10 to 15 cm and a root diameter of 1 to 3 cm. It has a part, and there are a wrinkle in the longitudinal direction and a shell and a wrinkle in the transverse direction. In addition, they are hard but inherently non-fibrous, so they are easy to break, and have a slight aroma and bitterness (Luc, Chang-soo et al., “Modern Pharmacognosis”, Hakuchangsa, Seoul, p.460-461. 1993 (Ryuk, Chang-Soo, et al., Modern Pharmacognosy, Hackchangsa Co., Seoul, 460-461, 1993)).

  As the main pharmaceutical ingredient of fennel, there are triterpene saponins (platicodine A, C, D, D2 and D3), which constitute about 2% of the root. According to animal studies, these pharmaceutical ingredients have various effects such as antitussive and expectorant effects, central nervous system inhibitory effects (sedation, pain relief and antipyretic), anti-inflammatory action against acute chronic inflammation, anti-ulcer action, gastric juice It has been confirmed that it has a secretory inhibitory action, an anticholinergic action that lowers blood pressure by vasodilation, a blood glucose lowering action, and a cholesterol metabolism promoting action (“Chemical & Pharmaceutical Bulletin” No. 20, p. 1952, 1972; “Chemical & Pharmaceutical Bulletin” 23, p. 2965, 1975; “Journal of Chemical Society, Perkin Transaction I” p. 661, 1984; “Comprehensive Medicine” 3 1, 1951; "Journal of Pharmaceutical Society of Colli 19, 164, 1975 (Chem., Pharm. Bull., 20, 1952 (1972); Chem. Pharm. Bull., 23, 2965 (1975); J. Chem. Soc., Perkin trans I, 661 (1984); Sogoigaku, 3,1 (1951); J. Pharm., Soc. Kor., 19, 164 (1975)). Steroid compounds such as stigmast-7-enol and α-spinasterol glucoside, and polysaccharides such as inulin and betulin.

  In the present specification, the term “long-life doraji” is the name of the root of more than 20 years old, but despite its various pharmacological effects, it has been difficult to grow long-lived kyoto. However, a technology that enables the cultivation of long-grown doraji, which is more than 20 years old, has recently been developed (Korea Patent No. 045731, Lee, Sung-Ho), and the name of the invention “Method for Produce”. Sing Perennial Balloon Flowers (a method for producing perennial balloonflowers)), which has enabled mass production of long-lived drage. Such long-lived doraji is superior in pharmacological effect and physiological action as compared with ordinary root roots cultivated for 2 to 4 years.

  The research on the long-term Draj has so far been a study on anti-diabetic activity (Journal of the Korean Society of Food Science and Nutrition No. 25, p. 986, 1996 (J. Korean Soc. Food Sci. Nutri., 25, 986 (1996)), and studies on antihyperlipidemia (Journal of Pharmaceutical Society of Japan No. 93, 1973, p. 1188 to 1194 (J. Pharmaceutical Society of Japan 93 (1973), 1188-1194)), and research on the prevention and control of liver damage has also been conducted. Ethanol extract suppresses mold aplatoxin, inulin fraction has phagocytosis and potent anti-tumor activity against sarcoma, solid tumor, and kyoto root These 40% extracts are known to have an alcohol absorption inhibitory effect (“Mycopathologia” No. 66, p. 16, 1979, “Biopharmaceutical Journal” No. 40, p. 367, 1986) , “Biopharmaceutical Journal”, No. 40, p.375, Japanese Patent Application Laid-Open No. 60-89427, 1985 (Mycopathologia, 66, 16 (1979), Shoyakugaku Zasshi, 40, 367 (1986), Shoyakugaku Zasshi, 40, 375 (1986), Japanese Patent No. 60-89427 (1985)).

  The reason cancer is perceived as a dying disease is that in the case of solid cancer, a potentially small metastatic cancer is a threat compared to a primary mass that has a size that can be diagnosed medically. is there. Cancer that metastasizes through blood vessels may always propagate to another organ or tissue even if it is removed, so once metastasis conditions are met, new cancer is induced. For these reasons, even if the cancer treatment rate is only over 50%, it is actually good, and the cancer treatment rate for which surgery or other anti-cancer treatments can be performed depends on the type of cancer or the condition of the patient. Will be extremely limited.

  On the other hand, growth of cancer tissue requires supplementation of oxygen and nutrients, and angiogenesis occurs to create a pathway for such supplements. Regarding the angiogenesis process in cancer tissue, first, cancer cells secrete angiogenesis inducing factors, which in turn bind to receptors present in endothelial cells, and at the same time, the formation process of new blood vessels is started. Stimulated endothelial cells begin to proliferate while secreting proteinase, and basement membrane degradation by proteinase occurs, whereby the endothelial cells migrate toward cells that secrete angiogenesis-inducing factors (cell migration). The migrated endothelial cells adhere to the blood vessel (cell adhesion), move to an infiltrating position, and then invade the basement membrane (cell penetration). The infiltrated cells combine with each other to form a lumen (lumen formation), and finally the end portions of the 24 hollow shoots combine with each other to form a loop through which blood flows. By blocking any one of these processes, angiogenesis can be reduced, thereby providing an anticancer effect.

  Furthermore, since cancer cells metastasize to other tissues through new blood vessels, inhibiting angiogenesis necessary for cancer growth and metastasis is recognized as a new cancer treatment.

  An object of the present invention is to provide a therapeutic agent for suppressing angiogenesis that occurs in the process of proliferation and metastasis of cancer cells, and the therapeutic agent includes a safe, non-toxic natural substance from Nagasei Doraj. An extract is included as an active ingredient.

  In order to achieve the above object, the present invention provides an angiogenesis inhibitor comprising an extract from Chosei Doraj as an active ingredient.

  Hereinafter, the present invention will be described in detail.

  The present inventors have conducted extensive research to develop a preparation that suppresses angiogenesis involved in cancer cell proliferation and metastasis using safe and non-toxic natural substances. Was found to block the processes involved in angiogenesis such as cell migration, cell adhesion, cell invasion and lumen formation. Based on this discovery, the present invention has been completed.

  The long-grown doraj extract according to the present invention is produced by any method conventionally used in the art. The long-life Doraj extract is preferably a hot water extract or an organic solvent extract, which can be prepared alone or mixed with a pharmaceutically acceptable herbal material. When extracting the long-life drage, it is preferable to use the long-life draj powder, but the powder is dried to a moisture content of less than 5%, and the dried material is powdered to obtain a particle size. Is a powder prepared by sorting out powders of less than 0.6 mm.

  The hot water extract is preferably prepared by adding about 5 to 10 times the volume of water to the long-life Doraj powder and extracting the solution twice at a temperature of 90-100 ° C. for 4-6 hours. . The resulting filtrate is used alone or mixed with one selected from acceptable herbal materials or herbal extracts.

  The organic solvent extract is preferably added about 3 times the volume of organic solvent to the long-life Doraj powder, the solution is extracted twice at room temperature, and the resulting filtrate is concentrated under reduced pressure and then acceptable A selection of fresh herbal materials or herbal extracts is prepared by adding to this concentrate. As organic solvent, it may be preferred to use an alcohol, preferably methanol or ethanol. .

  The long-life Doraj extract of the present invention may be formulated by known methods using food hygiene acceptable or pharmaceutically acceptable excipients, thereby preparing a pharmaceutical formulation. When preparing a pharmaceutical preparation, the long-lived Doradi extract as an active ingredient is preferably mixed or diluted with a carrier, or enclosed in a container-like carrier. When a carrier is used as a diluent, the carrier may be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium. Accordingly, the preparation may be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, syrups, aerosols, soft or hard capsules, sterile injections, sterile powders and the like. Examples of suitable carriers, excipients or diluents include lactose, glucose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, hydroxybenzoic acid Examples include propyl, talc, magnesium stearate, and mineral oil. The preparation may contain fillers, anti-aggregating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. An angiogenesis inhibitor comprising the long-lived Doraj extract of the present invention can be formulated by any method well known in the art to release an active ingredient rapidly, continuously or delayed after administration to a mammal. is there.

  The angiogenesis inhibitor contained as an active ingredient in the long-life Doraj extract of the present invention can be administered via various routes, oral, transdermal, subcutaneous, intravenous, and intramuscular routes depending on the purpose. . For humans, the daily dose of the extract of the present invention may be usually 10 to 1,000 mg / kg body weight, preferably 10 to 100 mg / kg body weight, and administered once or several times. Also good. However, the actual dosage of the extract of the present invention should be determined in view of various factors including the route of administration, patient age, gender and weight, health status, disease severity and the like.

  Hereinafter, the present invention will be described in more detail by way of examples and test examples. However, it should be understood that these examples are presented for the purpose of illustrating a preferred embodiment of the invention and are not intended to limit the scope of the invention.

Example 1 Production of Hot Water Extract from Chosei Doraji 2 liters of distilled water was added to 0.2 kg of Chosei Doraji powder and the solution was extracted twice at 90-95 ° C. for 5 hours and filtered. The filtrate was lyophilized to obtain 100 g of long-life Doraj extract.

  The following tests were conducted to examine the inhibitory effect of the angiogenesis inhibitor of the present invention on tube formation, cell invasion, cell adhesion, and cell migration involved in angiogenesis in human cancer cells.

Test Example 1: Inhibitory effect on adhesion of cancer lymphocytes (HL-60) to human endothelial cells (HMEC-1) Reagents The following reagents were purchased and used.
HT-1080, HMEC-1 and HL-60 cell lines, human cancer cell lines were purchased from Korea Cell Line Bank, Matrigel, RPMI-1640 medium, Calcein-AM, trypsin, and bovine Fetal serum was purchased from Sigma Chemical Co.

2. Test Method In order to examine the effect of inhibiting the adhesion of the lymphocyte cancer cell HL-60 to the human endothelial cell HMEC-1 with the long-life Doraj extract of the present invention, HMEC-1 cells were seeded in a 48-well plate, The extract was pretreated for 24 hours. Next, HL-60 was labeled with a fluorescent substance (Calcein-AM) and co-cultured with HMEC-1 cells for 6 hours. The culture was separated, washed with phosphate buffered saline (PBS) and then observed with a fluorescence microscope, thereby counting the number of adherent HL-60 cells. The result is shown in FIG.

  FIG. 1 is a graph showing the number of cancer cells adhering to human endothelial cells according to the treatment concentration of the long-life Doraj extract. As shown in FIG. 1, the adhesion of HL-60 cells to human endothelial cells HMEC-1 was greater in the group treated with long-lived Doraj extract than in the control group and the group treated with angiogenesis promoting factor (V-EGF). It was observed that it was suppressed. Further, the suppression of cell adhesion was different depending on the treatment concentration of the long-life Doraj extract (CK100: 100 μg / ml; CK200: 200 μg / ml and CK500: 500 μg / ml).

Test Example 2: Inhibitory effect of long-life drage on invasion of cells and cell membrane of human cancer cell line (HT-1080) Test method In order to examine the inhibitory effect on the invasion of cell membranes by long-life Doraj extract, subculture the cancer cell line HT-1080 in a 48-well plate coated with matrigel, and then put each concentration sample into the well plate. It was. After 18 hours, cell invasion was observed under a microscope, thereby counting the number of infiltrated HT-1080 cells. The results are shown in FIGS.

  FIG. 2 is a photograph showing cells infiltrated into Matrigel after treatment with the control group and 800 μg / ml long-life Doraj extract. FIG. 3 is a graph showing the effect of suppressing cell membrane infiltration for each treatment of long-life Doraj extract. As shown in FIG. 3, the cell and cell membrane infiltration suppressing effect of the long-lived doraj extract was shown as compared to the control group that was not treated with the long-lived doraj extract. Such an inhibitory effect increased as the treatment concentration of the long-grown Doraj extract increased from 100 μg / ml to 200 μg / ml, 400 μg / ml and 800 μg / ml.

Test Example 3: Inhibitory effect of long-life Doraj extract on cell migration of human cancer cell line HT-1080 Test Method To examine the inhibitory effect of the sample on the cell migration of human cancer cell line HT-1080, HT-1080 was subcultured in a 48-well plate and scratched with a blue chip. After 6 hours, the number of migrated HT-1080 cells was counted using a microscope. The results are shown in FIGS.

  FIG. 4 is a photograph showing a cancer cell line that has moved to the space left after the cancer cell line has been detached with a chip. FIG. 5 is a graph showing the number of migrated cells of the human cancer cell line according to the treatment concentration of the long-life Doraj extract. As shown in FIG. 5, the cell migration of the human cancer cell line HT-1080 was greatly suppressed depending on the treatment concentration of the long-life Doraj extract.

Test Example 4: Antiangiogenic effect of long-life Doraj extract Test Method In order to examine the angiogenesis inhibitory effect of the sample against the human cancer cell line HT-1080, HT-1080 was subcultured in a 48-well plate coated with Matrigel, and each concentration of the sample was placed in a well plate. After 18 hours, angiogenesis (lumen formation) was observed with a microscope. As a positive control, VEGF which is a pro-angiogenic factor was used. The results are shown in FIGS.

  FIG. 6 is a photograph showing a cancer cell line in which a lumen is formed in Matrigel. As shown in FIG. 6, the lumen formation of the group treated with the long-life Doraj extract was suppressed as compared to the control group and the group treated with the angiogenesis promoting factor. FIG. 7 is a graph showing tube formation (%) of a cancer cell line according to the treatment concentration of the long-life Doraj extract. As shown in FIG. 7, HT-1080 lumen formation during angiogenesis was significantly suppressed depending on the treatment concentration of the long-life Doraj extract.

  As described above, the inhibitory effect of long-life Doraj extract on the lumen formation of human cancer cell line HT-1080 was examined. As a result, the lumen formation (%) of human cancer cell line HT-1080 was the long-life Doraj extract. It was shown that it was suppressed according to the treatment concentration. In addition, the inhibitory effect of long-lived doraji on the cell invasion of human cancer cell line HT-1080 was also examined. As a result, the cell invasion of human cancer cell line B16F10 was remarkably reduced depending on the treatment concentration of the long-lived doraji extract. Indicated. Furthermore, the measurement results relating to the migration of the human cancer cell line HT-1080 showed that the migration of the human cancer cell line HT-1080 decreased according to the treatment concentration of the long-life Doraj extract. On the other hand, the measurement results regarding the adhesion of HL-60 cells to human endothelial cells HMEC-1 showed that the adhesion of HL-60 cells to HMEC-1 was different depending on the treatment concentration of the long-life Doraj extract.

  That is, the long-life Doraj extract of the present invention suppresses all the following processes: cell migration in which the endothelial cells migrate toward the site where the angiogenesis-inducing factor is released; cell adhesion in which the migrated cells adhere to the blood vessel; adhesion Cell invasion into the infiltrated endothelial cells, and then infiltrate the basement membrane; and lumen formation where the infiltrated endothelial cells bind to each other thereby forming a lumen. Therefore, it will be suggested that the long-life Doraj extract of the present invention suppresses angiogenesis, and as a result, the long-life Doraj extract has an anticancer effect.

Test Example 5: Acute toxicity test Oral administration 50 ICR mice weighing 25 g were divided into 5 groups of 10 mice each. The long-life Doraj extract of the present invention was orally administered to the groups of 5 animals at 1, 5, 25, 125 and 625 mg / kg, respectively. Thereafter, toxicity was observed over 2 weeks. The results showed that none of the 5 groups died.

2. Abdominal administration 40 ICR mice weighing 25 g were divided into 4 groups of 10 mice each. The long-life Doraj extract of the present invention was administered to the abdominal cavity of the four animal groups at 0.2, 2, 20, and 200 mg / kg, respectively. Thereafter, toxicity was observed over 24 hours. The results showed that none of the four groups died.

  As a result, it was confirmed that the long-life Doraj extract of the present invention has little or no acute toxicity.

  In the following formulation examples, a formulation of an anticancer agent containing the long-life Doraj extract of the present invention as an active ingredient will be described. However, as a matter of course, the anticancer agent of the present invention is not limited by these preparation examples.

Formulation Example 1: Tablet preparation Chosei Doraji extract 50mg
Lactose 20mg
Starch 20mg
Magnesium stearate appropriate amount The above-mentioned composition containing the long-grown doradi extract of the present invention as an active ingredient was tableted by a conventional tableting method, and a tablet was prepared.

Formulation Example 2: Preparation of capsule preparation Nagai Doraji extract 50 mg
Lactose 20mg
Starch 20mg
Talc 1mg
Magnesium stearate appropriate amount Gelatin capsules were filled with the above-mentioned composition containing the long-grown Doradi extract of the present invention as an active ingredient by a conventional method to prepare a capsule preparation.

  As described above, the angiogenesis inhibitor of the present invention containing, as an active ingredient, the long-life Dorazi extract of the present invention, which is a safe and non-toxic natural substance, can be used for the growth and metastasis of cancer cells generated by foreign body or virus infection. Can inhibit angiogenesis involved. Therefore, the angiogenesis inhibitor of the present invention will be clinically useful for cancer treatment and prevention.

  As described above, the long-lived Doraj extract of the present invention inhibits angiogenesis by suppressing cell migration, cell adhesion, cell infiltration and lumen formation involved in cancer cell proliferation and metastasis. Therefore, an anticancer agent comprising the present long-grown Dorazi extract as an active ingredient will be useful for cancer prevention and treatment.

It is a graph which shows the number of the cancer cells adhere | attached on the human endothelial cell according to the process density | concentration of a long life Doraj extract. It is a photograph which shows the cell which infiltrated the matrigel after each processing with a control group and 800 micrograms / ml long life Doraj extract. It is a graph which shows a cell membrane infiltration suppression effect according to a long-life Doraj extract process. It is a photograph which shows the cancer cell line which moved to the remaining space after peeling a cancer cell line with a chip | tip. It is a graph which shows the cell number which the human cancer cell line moved according to the treatment density | concentration of a long life Doraj extract. It is a photograph which shows the cancer cell line formed in the lumen shape in Matrigel. It is a graph which shows the luminal formation (%) of a cancer cell line according to the process density | concentration of a long life Doraj extract.

Claims (2)

  An angiogenesis inhibitor comprising an extract from Chosei Doraj as an active ingredient. The angiogenesis inhibitor according to claim 1, wherein the extract from the long-life Doraj is a hot water extract or an organic solvent extract.