KR101545507B1 - A Composition comprising an extract of Ailanthus altissima for treating or preventing cancer disease - Google Patents

Abstract

The present invention relates to a method for the treatment or prophylaxis of a cancer containing a pharmaceutically acceptable carrier whose active ingredient is a weighted tree ( Ailanthus altissima (Mill.) Swingle) extract inhibiting the Wnt protein signal transduction pathway, And to a health functional food. The weighted tree extract according to the present invention can be used as an anticancer composition or anticancer adjuvant useful for the treatment of cancer, for example, colon cancer, as a substance that inhibits cancer cells by targeting the Wnt / beta -catenin signal transduction system.

Description

[0001] The present invention relates to a composition for treating or preventing a cancer disease using an extract of a weighted tree as an active ingredient,

The present invention relates to a Wnt / beta-catenin signaling target for cancer prevention and treatment. Specifically, the present invention relates to a pharmaceutical composition for treating or preventing a cancerous disease and a health functional food, which is a Wnt / β-catenin signaling inhibitor containing a weighted tree extract as an active ingredient.

[Document 1] Clevers H et al (2006) Cell 127: 469-480

[Document 2] Mlodzik M (2002) Trends Genet 18: 564-571

[Document 3] Huelsken J et al (2001) 11: 547-553

[4] MacDonald BT et al (2009) Dev Cell 17: 9-26

[Literature 5] Fearnhead NS (2001) Mol Genet 10: 721-733

[Literature 6] Klaus A et al (2008) Nat Rev Cancer 8: 387-398

[Literature 7] Barker N et al (2006) Nat Rev Drug Discov 5: 997-1014

[Literature 8] Liu C et al (2002) Cell 108: 837-847

[Literature 9] Amit S et al (2002) Genes Dev 16: 1066-1076

[Literature 10] Hart MJ et al (1998) Curr Biol 8: 573-581

[Document 11] Behrens J et al (1998) Science 280: 596-599

[Literature 12] Latres E et al (1999) Oncogene 18: 849-854

[Literature 13] Kitagawa M et al (1999) EMBO J 18: 2401-2410

[Literature 14] Shih YL et al (2007) Inter J Cancer 121: 1028-1035

[15] Miller JR et al (1999) Oncogene 18: 7860-7872

[16] Staal FJ et al (2000) Hematol J 1: 3-6

[Literature 17] Logan CY et al (2004) Cell Dev Biol 20: 781-810

[Literature 18] Kang YJ et al (2012) Mol Pharm 82: 168-177

[Literature 19] Hong JY et al (2011) J Nat Prod 74: 2102-2108

[Literature 20] Livak KJ et al (1996) Method 25: 402-408

The present invention relates to compositions that modulate the < RTI ID = 0.0 > Wnt / b-catenin < / RTI >

As cancer incidence and mortality continue to increase globally, the importance of developing effective anti-cancer drugs is also increasing. The cancer drug market is the third largest market in the world, with cardiovascular and neurological diseases. Globally, about 10 million people have been diagnosed with cancer in 2000 and estimated to reach about 15 million by 2020 . The market size is estimated to be 35 trillion won in 2003 and to reach 60 trillion won in 2010. These findings demonstrate that research for the development of cancer drugs is being actively carried out globally.

In addition, the incidence of colorectal cancer is on the rise. In particular, the consumption of animal fat, sugar, and alcohol is increasing due to the effect of westernized diets, and there is a sharp increase in colorectal cancer due to the decrease of selenium, calcium, About 5% to 15% of all colon cancers are predicted by genetic factors.

According to the National Statistical Office, it is estimated that colorectal cancer has the highest rate of increase in the last 10 years compared to other cancers, and the incidence of colorectal cancer and mortality will continue to increase and increase to more than twice the current level in 20 to 30 years . Treatment of colorectal cancer can be divided into surgery, radiation therapy, and chemotherapy. However, in most cases, chemotherapy or chemotherapy is more effective in the treatment of colorectal cancer. In advanced colorectal cancer, It is a cure.

Molecular targeted therapy, a new concept of therapy, has recently been developed that aims to inhibit certain biological mechanisms and thereby increase the therapeutic effect and reduce side effects. In addition, many drugs are in clinical trials for the treatment of colorectal cancer, among which Wnt is a pathway that combines cancer stem cells with proteins. This pathway must be blocked to prevent the supply of proteins to cancer stem cells, resulting in death of cancer stem cells . It is believed that cancer treatment can be dramatically advanced if more than 80% of colorectal cancer patients develop a mutation of the Wnt signaling pathway and block the Wnt signaling pathway (Clevers H et al (2006) Cell 127: (2001) 11: 547-553; MacDonald BT et al (2009) Dev Cell 17: 9-26; Fearnhead NS (2001) Trends Genet 18: 564-571; Huelsken J et al Mol Genet 10: 721-733; Klause et al (2008) Nat Rev Cancer 8: 387-398; Barker N et al (2006) Nat Rev Drug Discov 5: 997-1014). Other target factors related to colon cancer treatment include EGFR and VEGFR, and Iressa and Avastin have been applied to clinical practice.

The abnormal activation of the Wnt / beta -catenin signaling system by mutation of the genes involved in the Wnt / beta -catenin signaling system is often the cause of cancer, so it is important to find inhibitors for this signal transduction system. Wnt signal transduction systems involve disheveled, GSK3β and β-catenin, Wnt reduces GSK3 activity, and GSK3 down-regulates Wnt signaling. Currently, APC and β-catenin are well known GSK3β substrates (Liu C (1998) Curr Biol 8: 573-581: Behrens J et al. (1998) Science (2002) Cell 108: 837-847; Amit S et al (2002) Genes Dev 16: 1066-1076; Hart MJ et al 280: 596-599). Regulation of Wnt / β-catenin pathway plays a crucial role in colon cancer at an early stage. APC binds to β-catenin to accelerate the degradation of β-catenin. This is the mechanism by which β-catenin levels are kept low (1999) Oncogene 18: 849-854; Kitagawa M et al. (1999), which catalyzes the phosphorylation of APC to increase the binding of β-catenin to GSK3β ) EMBO J 18: 2401-2410). Therefore, Wnt inhibits the activity of GSK3β and decreases APC phosphorylation, which may increase the intracellular level of β-catenin. Β-catenin binds to various proteins besides APC, and the transcription factor TCF / If LEF is present and β-catenin is increased by Wnt, it is presumed that the β-catenin-TCF / LEF complex will go to the nucleus and promote transcription of the oncogenic gene. It would be of critical importance to assess the effects of the weighted tree extract on this series of Wnt-related signaling systems and to discover natural products targeted to Wnt / β-catenin signaling.

Weighted trees (= leather trees, Ailanthus altissima (Mill.) Swingle) is a deciduous tree belonging to the Simaroubaceae. Its roots are called submaxillary blood, the leaves are low-leafed, and the fruit is called fenugreek. Mersosin, tannin, (such as phlobaphene, ailanthone, amarolide, acetylqmarolide, and quassin), antimicrobial activity and bactericidal action are known, (1997), Ailanthus altissima (Mill) Swingle) and domesticated domestic leather breed varieties Ailanthus altissima f. There are erythrocarpa Rehder and there are no native varieties in Korea, but Ailanthus There is altissima . erythrocarpa (Carri) Rehder, Ailanthus altissima there is. leucoxyla BC Ding & TB Chao, Ailanthus There is altissima . microphylla BC Ding & TB Chao, Ailanthus altissima there is. myriocephala BC Ding & TB Chao, Ailanthus There is altissima . ramosissima BC Ding & TB Chao, Ailanthus There is altissima . sutchuenensis (Dode) Rehder & EH Wilson, Ailanthus There is altissima . Tanakae (Hayata) Kaneh. There is a variant like & Sasaki.

However, no disclosure has been suggested or taught about the efficacy of the weighted wood as a therapeutic agent for Wnt /? - catenin signaling target cancer disease.

Using the Wnt / β-catenin signaling reporter cell line, the present inventors screened 6,000 kinds of foreign plant materials obtained from the four major regions in the world. Among them, the Wnt / β-catenin signal transduction system , The most effective Wnt / β-catenin-targeted anticancer efficacious substance among the 400 candidate species inhibiting the growth of the colon cancer cells, and the weighted tree extracts showed the inhibitory activity against TCF / LEF transcription, cytotoxicity against colon cancer cells, , Cyclin D1 and Survivin gene expression inhibiting activity, and Wnt /? - catenin signaling pathway inhibitory activity, thus completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for the treatment and prevention of cancer diseases, which comprises a weighted tree extract as an active ingredient.

In addition, the present invention provides a health functional food for improving and preventing cancer diseases containing a weighted tree extract as an active ingredient.

The present invention also provides a winter-targeted anticancer agent comprising a weighted tree extract as an active ingredient.

The anticancer agent defined in the present application is characterized by being a "Wnt / β-catenin targeted anticancer agent as a winter target anticancer agent.

As used herein, a " weighted tree " refers to a tree of the species Ailanthus altissima (Mill) Swingle, Ailanthus altissima f. Erythrocarpa Rehder, Ailanthus altissima var. erythrocarpa (Carri) Rehder), Isle of Lantus Ali Tea Island ryukok Silas (Ailanthus altissima var. leucoxyla BC Ding & TB Chao), the Isle of Lantus Ali Tea Island US Chloe Pilar (Ailanthus altissima var. microphylla BC Ding & TB Chao ), Isle of Lantus Ali Tea Shima pre-Osage Palacio (Ailanthus altissima var. myriocephala BC Ding & TB Chao), the Isle of Lantus Ali Tea Island L'City Island (Ailanthus altissima var. ramosissima BC Ding & TB Chao), the Isle of Lantus Ali Tea Island suchu N-Sys (Ailanthus altissima var. sutchuenensis (Dode ) Rehder & EH Wilson), tee Island or Isle of Lantus Ali Tanaka at (. Ailanthus altissima var. tanakae ( Hayata) Kaneh & Sasaki), Preferably from, Isle of Lantus Ali Tea Island seuwinggeul (Ailanthus altissima (Mill) Swingle) , Isle of Lantus Ali Tea Island erythromycin Carpathian (Ailanthus altissima f. Erythrocarpa Rehder) , or the Isle of Lantus Ali Tea Island erythromycin Carpathian (Ailanthus altissima var. erythrocarpa (Carri) Rehder), and more preferably, Ailantus altissima (Mill) Swingle.

The weighted wood stocks as defined herein may be groundwood, rootstocks, or roots, more preferably ground, rootstocks, or roots, including flowers, branches, stems, leaves, immature fruits, ≪ / RTI >

The term " Ailanthus " The extract of " altissima (Mill) Swingle" is one or more mixed solvents selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, chloroform, acetone, propylene glycol, ethyl acetate, butyleneglycol, ether and chloroform (V / v) mixing ratio of chloroform, acetone, methanol or a mixed solvent thereof, more preferably chloroform, acetone, and methanol, more preferably 10: 1 to 10: Acetone, and methanol, in a mixed solvent of 5-1: 1-5: 1-5 (v / v).

Hereinafter, the present invention will be described in more detail.

The extract of the present invention can be obtained by the following production method.

For example, the present invention will be described in detail below.

The weighted tree extract of the present invention can be prepared as follows. The dried weighted wood is washed with water and then washed with water containing purified water, a lower alcohol having 1 to 4 carbon atoms, a mixed solvent selected from the group consisting of chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, ether and chloroform , Preferably 10-1: 1-10: 1-10 (v / v) mixture ratio of chloroform, acetone, methanol or a mixed solvent thereof, more preferably chloroform, acetone, and methanol, , A mixed solvent of chloroform, acetone, and methanol in a mixing ratio of 5-1: 1-5: 1-5 (v / v) of methanol is mixed several times and then heated at a temperature of 30 ° C to 150 ° C, preferably 50 ° C to 100 ° C For example, by repeating ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction, preferably ultrasonic extraction, for about 1 to 20 times, preferably 2 to 10 times, for 30 minutes to 48 hours, preferably 1 hour to 12 hours The extract of the present invention can be filtered, concentrated under reduced pressure, and dried to obtain the weighted tree extract of the present invention.

The present invention provides a pharmaceutical composition and a health functional food for the treatment and prevention of cancer diseases containing the weighted tree extract prepared by the above production method and the above production method as an active ingredient.

The anticancer agent comprising an effective ingredient according to the above embodiment as an active ingredient, the pharmaceutical composition for treatment and prevention and the functional food may contain the weighted tree extract in an amount of 0.01 to 90% by weight.

The cancer diseases as defined herein include, but are not limited to, colorectal cancer, stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Endometrioid adenocarcinoma, adenocarcinoma, adenocarcinoma, adenocarcinoma, adenocarcinoma, adenocarcinoma, endometrial adenocarcinoma, adenocarcinoma, vulvar carcinoma, vulval carcinoma, Hodgkin's disease, Cancer of the kidney or kidney, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, cancer of the central nervous system (CNS), cancer of the central nervous system , Primary CNS lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma, preferably colon cancer, and further characterized by a functional group of the Wnt signaling inhibitory effect.

In particular, the cancer cell line as a Wnt signal transduction inhibitor targeted by the present invention is characterized by being selected as a colon cancer cell line.

Accordingly, the present invention provides a pharmaceutical composition for the treatment and prevention of cancer diseases containing a weighted tree extract as an active ingredient and containing a pharmaceutically acceptable excipient or carrier.

The extract may be used as an anti-cancer adjuvant for cancer cells.

Accordingly, the present invention provides anticancer adjuvants for the treatment and prevention of cancer diseases containing the weighted tree extract as an active ingredient.

The present invention also provides a combination comprising an extract of weight trees and an existing anticancer agent as active ingredients.

Existing anticancer agents defined herein include antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, 5-fluorouracil, and cytarabine; An alkylating agent such as a chlorambucil, a cyclophosphamide, an ethylene imine compound, an alkyl sulfonate compound, a carnustine compound or a dacarbazine compound, ); Antitumor agents such as actinomycin D, doxorubicin, bleomycin and mitomycin, plant alkaloids such as vincristine and vinblastine, and mitotic inhibitors such as taxoid, a mitotic inhibitor containing a taxane ring antimitotic drugs); Hormones such as corticosteroids or progesterone; And platinum-containing compounds such as cisplatin.

The weighted tree extract of the present invention inhibits TCF / LEF transcriptional inhibition activity, cytotoxicity against colon cancer cells, inhibition of c-Myc, cyclin D1 and Survivin gene expression inhibition, inhibition of Wnt / HCT116) proliferation inhibitory activity, and thus it was found to be useful for the treatment and prevention of cancer diseases.

The composition of the present invention contains 0.01 to 99% by weight of the compound, based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The extract of the present invention is characterized by showing cytotoxicity in cancer cells.

The efficacious substance isolated from the extract of the present invention can be prepared as a pharmaceutically acceptable active ingredient according to a method common in the art.

The present invention also provides a method for treating cancer diseases comprising administering to an individual exhibiting drug resistance to the target anticancer agent at an effective concentration that inhibits drug resistance.

In addition, the present invention provides a method for treating cancer diseases, comprising administering a weighted tree extract to a patient suffering from cancer.

The anticancer adjuvant of the present invention may further contain one or more active ingredients showing the same or similar functions. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components.

In addition, the present invention provides a method for treating a cancerous disease comprising administering to the individual an anti-cancer adjuvant and at least one anti-cancer agent at an effective concentration of the present invention.

The method of administering the extract or the anti-cancer adjuvant and the anticancer agent of the present invention is not particularly limited, but may be parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally administered according to a desired method , Parenteral administration is preferable, and administration by intravenous injection is more preferable.

The dose of the extract or the anti-cancer adjuvant and the anti-cancer agent of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. Based on the adult patient weighing 70 kg 1, 2, 3, or 1 / day of the extract or the anticancer adjuvant and the anticancer drug per unit dosage form, usually 0.1 to 1,000 mg / day, preferably 1 to 500 mg / 4, and may be administered one to six times a day at regular intervals according to the judgment of a doctor or a pharmacist.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound is preferably administered at 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

In addition, the present invention provides a health functional food for improving and preventing cancer diseases containing a weighted tree extract as an active ingredient.

The health functional food containing the extract of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of objective diseases. Examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in powder, granule, tablet, have.

Accordingly, the present invention also provides a food or food additive containing, as an active ingredient, a weighted tree extract having an effect of preventing and improving target cancer disease.

The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.

The extract of the present invention can be added to food or beverage for the purpose of prevention and improvement of the objective disease. At this time, the amount of the compound in the food or beverage may generally be from 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia compounds (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

It was confirmed from the weighted tree extract of the present invention that the cancer cell exhibits a strong anticancer effect and is useful for the treatment and prevention of cancer diseases by determining the action mechanism as a Wnt target anticancer agent.

Brief Description of the Drawings Fig. 1 is an analysis of TCF / LEF transcriptional inhibitory activity in the human kidney cell line of the weighted tree extract of the present invention;
FIG. 2 is a graph showing inhibitory activity in the TCF / LEF transcriptional analysis of colon cancer cells treated with the weighted tree extract of the present invention for 24 hours;
FIG. 3 is a graph showing cytotoxicity against colon cancer cells in the treatment of the weighted tree extract of the present invention for 24 hours; FIG.
FIG. 4 shows the effect of the weighted tree extract on the expression of c-Myc, Cyclin D1 and Survivin genes, sub-regulators of Wnt /? -Catenin signaling in colorectal cancer cell lines;
FIG. 5 is a graph showing the effect on the expression of c-Myc, Cyclin D1 and Survivin proteins as sub-regulators of Wnt /? -Catenin signaling by weighted tree extract in colon cancer cell line (HCT116).

Hereinafter, the present invention will be described in detail with reference to the following Reference Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following reference examples and experimental examples.

Example  1. Weighted tree ( Ailanthus altissima  ( Mill ) Swingle ) Preparation of extract

The weighted tree (YAAS, Yunnan Provincial Agricultural Research Institute, China) is a ground-breaking section containing flowers, branches, stems, leaves, immature fruit, and fruit; Rootstock; And roots were harvested and one or more of the assay materials selected were washed and cut and used.

A ground portion of a dried weighted tree as a sample; Rootstock; (SDN-900H, SD-ULTRASONIC CO. LTD.) With 10 L of chloroform: acetone: methanol (1: 1: 1, v / v) . The extract was filtered, concentrated under reduced pressure, and then lyophilized to obtain a brown weighted tree ( Ailanthus altissima (Mill) Swingle) overhead extract; Rootstock extract; (Hereinafter referred to as AAA; AAS; and AAR, respectively) were stored at -20 ° C, and the above-ground extract was used as a sample for analysis in the following experimental examples .

Experimental Example 1. Inhibition of TCF / LEF luciferase activity of a weighted tree in HEK293 cell line introduced with? -Catenin

Since it is known that the expression of β-catenin is hyperactivated by the mutation of Wnt signaling-related proteins in some cancer cell lines, especially in colorectal cancer, it is meaningful to find a substance that can inhibit the action of β-catenin . In order to confirm whether the weighted tree extracts showed such an effect, β-catenin was introduced into HEK293 cells and overactivated, and evaluated for inhibition by weighted tree extract. (Kang YJ et al (2012) Mol Pharm 82: 168-177).

In order to confirm whether the weighted tree extracts are involved in the transcriptional regulation of TCF / LEF, a transcription factor that binds to β-catenin, TCF4 and pcDNA β (HEK293 cells, ATCC; Manassas, VA, USA) The effect of TCF transcriptional activity on the activity of luciferase activity after transfection of a plasmid (TOPFlash) containing a -catenin and TCF binding site was investigated in the following We tested together.

HEK293 cells with 10% FBS in DMEM medium (# 12800-017, Gibco) containing the control such that the 48 well plate in 1.5 ⅹ 10 ⁴ per well and incubated for 24 hours at 37 ℃, 5% CO ₂ conditions, and luciferase 100 ng of plasmid with TRE binding site, 8 ng of TCF4 plasmid, 20 ng of β-catenin and 1/20 of pRL-SV40 vector (E2231, Promega) and lifofectamine 2000 (# 11668-019, (Invitrogen) were mixed in Opti-MEM (31985-070, Gibco), transfected for 24 hours, and the weighted tree extract (5, 10, 20 μg / ml) previously diluted in DMEM medium containing 10% (Cetro LB 960, Berthold, Germany) using a Dual Luciferase Kit (E1910, Promega). The resulting mixture was incubated for 24 hours, Germany), and the transfection efficiency was calibrated to pRL-SV40 luciferase activity.

As shown in Table 1 and FIG. 1, when TCF4, pcDNA beta -catenin, and TOPFlash were introduced into HEK293 cells, luciferase activity was increased compared to the control group in which TOPFlash alone or TOPFlash and TCF4 plasmids were introduced together. As shown in Fig. 1, increased TCF / LEF luciferase activity was obtained by adding the 5%, 10%, and 20 μg / ml of the weighted tree extract to the HEK293 cells, / LEF transcriptional activity was statistically significant (* P <0.05; ** P <0.01). In detail, when 5 μg / ml of the weighted tree extract was added, 17.8% , 39.9% when 10 μg / ml was added, and 62.5% when 20 μg / ml was added, indicating TCF / LEF transcription inhibitory activity. From the above results, the transcription inhibition activity IC ₅₀ of the weighted tree extract was 13.8 μg / ㎖, showing excellent inhibitory activity. In addition, TCF4 and pcDNA β-catenin did not affect luciferase activity when plasmid (FOPFlash) mutated TCF binding site was transfected using the weighted tree extract.

Weighted tree extract
Treatment concentration (μg / ml) TCF / LEF transcription inhibitory activity (%) 5 17.8 10 39.9 20 62.5

Experimental Example 2. Measurement of Growth Inhibitory Effect on Human Colon Cancer Cells in vitro

In order to confirm the growth inhibitory effect on the human colorectal cancer cells and the like in the test tube of the sample obtained in the above examples, experiments were carried out as described below by applying the method described in the literature. (Lee SK et al (2008) Chem Biol Interact 115: 215-28)

Human colon cancer cell line HCT116 was distributed from Korean Cell Line Bank (CCL-247, American Type Culture Collection, ATCC; Manassas, VA, USA).

Human colon cancer cells used in the present invention were subcultured in an RPMI medium containing 10% FBS, 1% PSF and the like. To each well of a 96-well plate, 10 μl of a sample dissolved in 10% DMSO and 190 μl (5 × 10 ⁴ cells / ml) of the cell suspension were added and cultured for 3 days. 190 μl of the cell suspension was added to at least 16 wells and incubated for 30 minutes, and used as a zero-day control before the experiment. The cultured cells were fixed with 10% TCA (trichloroacetic acid), stained with SRB solution (S9012, Sigma), dissolved in 10 mM Tris base, and then absorbed at 515 nm. 10% DMSO was used as a control, and the cell survival rate according to the treatment of each test substance was measured using the following equation (1).

When the control group without the sample was taken as 100%, the value of the sample treatment group was expressed as a percentage of the control group, and the treatment of each test substance was a mean value ± SEM of the double or triple test. Respectively.

In detail, as shown in FIG. 2, the addition of 0.8, 4, and 20 μg / ml of the weighted tree extract and cytotoxicity of the human colon cancer cell line were statistically significant (* P <0.05; ** P <0.01). In detail, cytotoxicity was 12.9% at the addition of 0.8 μg / ml of the weighted tree extract, 24.9% at the addition of 4 μg / ml and 86.1% at the addition of 20 μg / ml . From the above results, the IC _{50, which} is the concentration of the test substance to the 50% survival rate of the HCT116 cell line, was 10.6 μg / ml, indicating excellent cytotoxicity.

Weighted tree extract
Treatment concentration (μg / ml) Cancer cell growth inhibition (%) 0.8 12.9 4.0 24.9 20.0 86.1

Experimental Example  3. In the human colon cancer cell line, TCF / LEF luciferase  Evaluation of activity inhibition efficacy

In the HCT116 colon cancer cell line, which is known to have overexpression of β-catenin due to gene mutations in GSK3β and β-catenin itself, Wnt signaling-related proteins, TCF / LEF luciferase activity was assessed.

Human colonic cancer cell line HCT 116 cells (5 × 10 ³ cells / mL) were added to each well of a 48-well plate with RPMI 1640 medium (# 31800-022, Gibco) containing 10% FBS and incubated at 37 ° C, And cultured at 5% CO ₂ . After 24 hours, 100 ng of TOPflash (β-catenin / TCF reporter plasmids) (# 21-170, upstate biotechnology) and 5 ng of pRL-SV40 reporter plasmids (E2231, Promega) were mixed with Opti-MEM (# 31985-070, Gibco) After dilution, transfection was performed using lipofectamine 2000 (# 11668-019, Invitrogen). After 24 hours, the medium was diluted beforehand and then cultured for 24 hours in the incubator. Cells were lysed using 1X passive lysis buffer (E194A, Promega). 10 μl of cell lysate was transferred to a 96-well white plate and luciferase activity was confirmed using a dual luciferase reporter assay system (E1910, Promega). Renilla luciferase activity, and the results are shown in Table 3 and Fig. 3 below.

As shown in FIG. 3, the addition of 1, 2.5, 5 and 10 μg / ml of the weighted tree extract resulted in statistically significant inhibition of TCF / LEF luciferase activity in human colorectal cancer cell lines (* P <0.05; ** P <0.01). In detail, 25.0%, 43.0%, and 65.7% of the addition of 5 μg / , And the addition of 10 μg / ml inhibited the transcriptional activity by 83.1%. From the above results, it can be confirmed that the weighted tree extract has excellent TCF / LEF transcription inhibiting activity.

Weighted tree extract
Treatment concentration (μg / ml) TCF / LEF transcription inhibitory activity (%) 1.0 25.2 2.5 43.0 5.0 65.7 10.0 83.1

Experimental Example  In human colon cancer cell lines, Catechin  Measurement of effect on target gene regulation

C-Myc, Cyclin D1, and Survivin genes are known to be sub-target genes for β-catenin and are associated with cell proliferation (Shih YL et al (2007) Inter J Cancer 121: 1028-1035; Miller JR et al Oncogene 18: 7860-7872; Staal FJ et al (2000) Hematol J 1: 3-6; Logan CY et al (2004) Cell Dev Biol 20: 781-810). In order to confirm the effect of β-catenin target gene expression in a colon cancer cell line of the sample obtained in the above example, the following procedure was performed by applying the method described in the literature. (Kang YJ et al (2012) Mol Pharm 82: 168-177)

HCT 116 cells (1 × 10 ⁵ cells / ml) were cultured in RPMI medium containing 10% FBS in a 100 mm dish at 37 ° C, 5% CO ₂ After 24 hours of incubation, diluted samples were added to the medium and cultured for another 24 hours. The cells were washed twice with PBS, and the cells were harvested using TRI reagent (TRIZol (# 15596-026, Invitrogen)), and then CHCl ₃ was added to extract the RNA and precipitate with isopropyl alcohol. The RNA precipitate was washed with 70% ethanol, dried in air and dissolved in nuclease-free water (AM9937, Ambion). Heated at 55 ° C for 10 minutes and heated at 70 ° C for 5 minutes to allow the RNA to remain in a single stranded state. The Total RNA NanoDrop (ND-1000, Dae Myung Sceince Co., Ltd) , and then by quantitatively diluted with 1 μg / μl using avian myeloblastosis virus (AMV) reverse transcriptase (M5108, Promega) and the oligo (dT) ₁₅ primer (C110B, Promega). Target genes were amplified by MiniOpticon ^™ Real-time PCR Detection System (CFB-3120, Bio-Rad) using iQ ^™ SYBR Green Supermix (# 170-8880, Bio-Rad) and target gene-specific primers [Table 4] . Real-time PCR conditions were denaturation at 95 ° C for 20 seconds, followed by denaturation at 95 ° C for 20 seconds, annealing at 56 ° C for 20 seconds, and elongation at 72 ° C for 30 seconds. Was repeated 40 times and then finalized at 95 ^° C for 1 minute and at 55 ^° C for 1 minute. The Ct value was determined by the method of Leak et al. (Livak KJ et al (1996) Method 25 : 402-408) using MJ Opticon Monitor software (Opticon Monitor 3.1.32, Bio-rad) Ct value of c-Myc, Cyclin D1 and Survivin gene was decreased by weighted tree extract.

Real-time PCR primer Genes Sequences c-Myc Sense 5'-CACCAGCAGCGACTCTGA-3 ' Antisense 5'-GATCCAGACTCTGACCTTTTG-3 ' Cyclin D1 Sense 5'-CAGGTTGGACAGTTCACAGG-3 ' Antisense 5'-ACAGCTGGAGTTGGATGGAC-3 ' Survivin Sense 5'-CCGACGTTGCCCCCTGC-3 ' Antisense 5'-TCGATGGCACGGCGCAC-3 ' beta -actin Sense 5'-AGCACAATGAAGATCAAGAT-3 ' Antisense 5'-TGTAACGCAACTAAGTCATA-3 '

As shown in FIG. 4, the expression of C-Myc, Cyclin D1 and Survivin genes of β-catenin target genes in human colon cancer cell lines was evaluated by adding weighted tree extracts at 1, 5 and 10 μg / In detail, in the case of c-Myc, gene expression was inhibited by adding 11.1%, 20.8%, and 25 μg / ml of 1 μg / ml of the weighted tree extract and 10 μg / ml of 5 μg / ml, respectively ; Cyclin D1 inhibited gene expression by 7.2% at 1 μg / ml, 14.2% at 5 μg / ml and 24.9% at 10 μg / ml, respectively. Survivin gene expression was inhibited by 23.9% at 1 μg / ml, 24.4% at 5 μg / ㎖ and 28.9% at 10 μg / ㎖. Especially when 10 μg / ㎖ was added, all 3 genes showed statistical significance at 95% confidence interval. Therefore, it was confirmed that the cytotoxicity of the weighted tree extract of the present invention to the colon cancer cell line is related to c-Myc, Cyclin D1 and Survivin, which are subordinate regulators of the Wnt / beta -catenin signaling pathway involved in cancer cell proliferation.

Experimental Example 4. Measurement of effect of β-catechin on target protein regulation in human colon cancer cell line

In order to investigate the effect of the sub-regulatory factors related to cell proliferation in the Wnt /? -Catenin signaling pathway on the protein factor, the following experiment was conducted by applying the method as described below (Hong JY et al (2011) J Nat Prod 74: 2102-8).

Colon cancer cells (HCT116) were plated in DMEM medium containing 10% FBS at a concentration of 1 × 10 ⁵ in a 100 mm culture dish and cultured at 37 ° C and 5% CO ₂ for 24 hours. Phosphate buffered saline (PBS). 10 ml of DMEM medium containing 10% FBS diluted by concentration in advance was added and cultured for a certain period of time. Cells that were not attached to the cell culture medium and attached cells were collected and washed twice with PBS, and then boiled 2 × sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue , 2% β-mercaptoethanol, 50 mM sodium fluoride and 5 mM sodium orthovanadate), and the cells were disrupted for 5 minutes at 100 ° C. After cooling, the solution was stored at 20 ° C and dissolved at 37 ° C just before use. The solution was used for protein determination and electrophoresis. 80 μg of the protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-Rad, Hercules, Calif.). The separated proteins were transferred to a PVDF membrane (ISEQ15150, Millipore, Bedford, MA) for 1 hour at 100 V, washed twice with TBST and blocked with 5% Albumin from Bovine Serum in PBS containing 0.1% Tween- TBST)) and cultured in a shaker at room temperature for 1 hour. After washing three times for 5 minutes with TBST, the monoclonal antibody was diluted with TBST in 3% Albumin from Bovine Serum (BSA (A9647, Sigma)) and reacted at 4 ° C for 18 hours with a membrane. After washing the membranes (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times for 5 minutes with TBST, the HRP (horseradish peroxidase) -binding secondary antibody was diluted with TBST to a ratio of 1: 1,000 to 1: 2,000 with TBST And reacted with the membrane at room temperature for 3 to 4 hours. After washing with TBST three times for 5 minutes, the western blotting substrate (WEST-ZOL PLUS (16021, Intron)) was treated to confirm protein expression level on LAS-3000 (Fuji Film Corp., Japan).

In detail, inhibition of the signal transduction system in the colon cancer cell line (HCT116) of the weighted tree extract was confirmed by protein expression analysis after treating the colon cancer cell line (HCT116) with 1, 5, and 10 μg / As a result, as shown in FIG. 5, the expression of c-Myc, cyclin D1 and Survivin protein was inhibited in a concentration-dependent manner, and it was confirmed that the expression of Survivin gene was remarkably suppressed. Taken together, the weighted tree extracts inhibited the proliferation of colon cancer cells (HCT116) by inhibiting the Wnt / β-catenin signaling pathway.

Hereinafter, formulation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Preparation Example 1. Preparation of powder

Weighted tree extract ------------------------------------------ 200 mg

Lactose ------------------------------------------------- ---- 100 mg

Talc ------------------------------------------------- ----- 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

Weighted tree extract ------------------------------------------ 200 mg

Corn starch ----------------------------------------------- 100 mg

Lactose ------------------------------------------------- ---- 100 mg

Magnesium stearate ---------------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation Example 3. Preparation of capsules

Weighted tree extract ------------------------------------------ 200 mg

Crystalline cellulose ------------------------------------------ 3 mg

Lactose ------------------------------------------------ 14.8 mg

Magnesium stearate 0.2 mg &lt; RTI ID = 0.0 &gt;

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation Example 4. Preparation of injection

Weighted tree extract ------------------------------------------ 200 mg

Mannitol ------------------------------------------------- - 180 mg

Sterile sterile distilled water for injection -------------------------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ------------------------------------------ ---- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation Example 5. Preparation of a liquid preparation

Weighted tree extract ------------------------------------------ 200 mg

Isseong Party ------------------------------------------------ --- 10 g

Mannitol ------------------------------------------------- ----- 5 g

Purified water ------------------------------------------------- ----

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation Example 6. Preparation of Healthy Foods

Weighted tree extract ---------------------------------------- 1000 mg

Vitamin mixture ---------------------------------------------

Vitamin A Acetate -------------------------------------- 70 g

Vitamin E ------------------------------------------------ 1.0 mg

Vitamin B1 ---------------------------------------------- 0.13 mg

Vitamin B2 ---------------------------------------------- 0.15 mg

Vitamin B6 ----------------------------------------------- 0.5 Mg

Vitamin B12 ---------------------------------------------- 0.2 g

Vitamin C ------------------------------------------------ - 10 mg

Biotin ------------------------------------------------- - 10 [mu] g

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------------- ---- 50 μg

Calcium pantothenate ------------------------------------------- 0.5 mg

Inorganic mixture ---------------------------------------------

Ferrous sulfate ---------------------------------------------- 1.75 mg

Zinc oxide ----------------------------------------------- 0.82 Mg

Magnesium carbonate - 25.3 mg

Potassium monophosphate ---------------------------------------------- 15 mg

Calcium Phosphate ---------------------------------------------- 55 mg

Potassium citrate ----------------------------------------------- 90 Mg

Calcium Carbonate ------------------------------------------------ 100 mg

Magnesium chloride ------------------------------------------- 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Weighted tree extract --------------------------------------- 1000 mg

Citric acid ------------------------------------------------ 1000 Mg

Oligosaccharide ------------------------------------------------ 100 g

Plum concentrate ------------------------------------------------ 2 g

Taurine ------------------------------------------------- --- 1 g

Purified water was added.

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated for about 1 hour at 85 DEG C with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (13)

After washing and shredding the dried weighted wood ground part, a mixed solvent of chloroform, acetone, and methanol in a mixing ratio of 10-1: 1-10: 1-10 (v / v) was mixed several times and then heated at 30 ° C to 150 ° C Β-D- glucan extract containing an extract of Ailanthus altissima (Mill) Swingle obtained by filtering, concentrating under reduced pressure, and drying an extract obtained by repeating ultrasonic extraction for 1 to 20 times for 30 minutes to 48 hours as an active ingredient, A method for the treatment and prevention of colorectal cancer, which comprises administering a therapeutically effective amount of a compound of formula delete delete delete delete After washing and shredding the dried weighted wood ground part, a mixed solvent of chloroform, acetone, and methanol in a mixing ratio of 10-1: 1-10: 1-10 (v / v) was mixed several times and then heated at 30 ° C to 150 ° C Β-D- glucan extract containing an extract of Ailanthus altissima (Mill) Swingle obtained by filtering, concentrating under reduced pressure, and drying an extract obtained by repeating ultrasonic extraction for 1 to 20 times for 30 minutes to 48 hours as an active ingredient, A cancer-targeting anticancer agent characterized by inhibiting a catenin signaling system. After washing and shredding the dried weighted wood ground part, a mixed solvent of chloroform, acetone, and methanol in a mixing ratio of 10-1: 1-10: 1-10 (v / v) was mixed several times and then heated at 30 ° C to 150 ° C Β-D- glucan extract containing an extract of Ailanthus altissima (Mill) Swingle obtained by filtering, concentrating under reduced pressure, and drying an extract obtained by repeating ultrasonic extraction for 1 to 20 times for 30 minutes to 48 hours as an active ingredient, Wherein the therapeutic agent for colon cancer is selected from the group consisting of: delete delete After washing and shredding the dried weighted wood ground part, a mixed solvent of chloroform, acetone, and methanol in a mixing ratio of 10-1: 1-10: 1-10 (v / v) was mixed several times and then heated at 30 ° C to 150 ° C Β-D- glucan extract containing an extract of Ailanthus altissima (Mill) Swingle obtained by filtering, concentrating under reduced pressure, and drying an extract obtained by repeating ultrasonic extraction for 1 to 20 times for 30 minutes to 48 hours as an active ingredient, A catenin signal transduction system, characterized by suppressing the system for the improvement and prevention of colorectal cancer health functional food. delete delete delete

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