WO2017222252A1 - Pharmaceutical composition for preventing or treating colon cancer, containing extract of telectadium dongnaiense as active ingredient - Google Patents

Abstract

The present application relates to a pharmaceutical composition and a health functional food for preventing and treating colon cancer, containing an extract of Telectadium dongnaiense as an active ingredient. The extract targets the Wnt/β-catenin signaling pathway, thereby being usable as a treatment for cancer caused by an abnormality of the Wnt signaling pathway or as an anti-cancer adjuvant.

Description

A pharmaceutical composition for preventing or treating colorectal cancer using the extract of Teleectadidium dongnaesu as an active ingredient

The present invention relates to a composition for the prevention and treatment of colorectal cancer comprising the extract of telenecdium dongnae.

As the incidence and mortality rate of cancer continues to increase worldwide, the importance of developing effective anticancer drugs is also increasing. Globally, about 10 million people were diagnosed with cancer in 2000 and are estimated to reach about 15 million by 2020. According to the National Statistical Office's "Cause Cause of Death Statistics 2013", the total number of deaths was 26,257, 964 (-0.4%) decreased from the previous year, and 526.6 deaths per 100,000 population were reported. The mortality rate from cancer was 149.0, an increase of 6.2 per 100,000 population compared to the previous year. In terms of the three major causes of death among Koreans, the highest cancer mortality rate (per 100,000 population) was 142.8, followed by cerebrovascular disease and heart disease, and the incidence and mortality rate of cancer patients continued to increase. It is reported that medical and social expenses amount to more than 2 trillion a year, and the 5-year survival rate is very low.

In particular, the incidence of colorectal cancer is on the rise, and the consumption of animal fats, sugars and alcohols is increasing due to Western diets, and colorectal cancer is rapidly increasing due to reduced intake of selenium, calcium, vegetables, and fiber. About 5-15% of all colorectal cancers are predicted to be due to genetic factors.

Statistics Korea estimates that colorectal cancer is the fastest growing rate in the last decade compared to other cancers, and that colorectal cancer incidence and mortality will continue to increase, more than double in the next 20-30 years. . Treatment for colorectal cancer can be classified into surgery, radiation therapy, chemotherapy, etc. In most cases, additional chemotherapy drugs are used to obtain higher therapeutic effects.In advanced colorectal cancer, chemotherapy or radiation therapy is mainly used. It is a cure.

Recently, molecular targeted therapy (Molecular targeted therapy), which aims to reduce specific biological mechanisms to increase the therapeutic effect and reduce side effects, is being developed. Targets associated with colorectal cancer treatment include EGFR and VEGFR. Irressa and Avastin have been applied in clinical trials, respectively.

Another target is also the Wnt / β-catenin signaling pathway, which is a cause of cancer because abnormal activation of the Wnt / β-catenin signaling pathway is often caused by mutations in genes involved in the pathway. The key is to find an anti-inhibitor as an anticancer agent for colorectal cancer. Distrelled, GSK3β and β-catenin are involved in the Wnt signaling pathway, Wnt reduces GSK3 activity and GSK3 downregulates Wnt signaling. Currently, APC and β-catenin are well known substrates of GSK3β (Liu C et al (2002) Cell 108: 837-847; Amit S et al (2002) Genes Dev 16: 1066-1076; Hart MJ et al (1998) Curr Biol 8: 573-581: Behrens J et al (1998) Science 280: 596-599). The dysregulation of the Wnt / β-catenin signaling pathway plays a very important role in early-stage colorectal cancer, and when APC binds to β-catenin, it promotes the breakdown of β-catenin, which keeps the β-catenin concentration low. GSK3β phosphorylates APC to increase binding to β-catenin and consequently promotes degradation of β-catenin (Latres E et al (1999) Oncogene 18: 849-854; Kitagawa M et al. (1999) EMBO J 18: 2401-2410. Therefore, Wnt is thought to increase the intracellular amount of β-catenin by inhibiting the activity of GSK3β, thereby reducing the phosphorylation of APC. Β-catenin binds to several proteins in addition to APC, and TCF / There is LEF, and the increase of β-catenin by Wnt is expected to move the β-catenin-TCF / LEF complex to the nucleus and promote the oncogene transcription.

In other words, Wnt is a pathway through which cancer stem cells and proteins bind, and this pathway must be blocked to prevent protein from being supplied to cancer stem cells, resulting in the death of cancer stem cells. More than 80% of colon cancer patients have mutations in Wnt signaling. Has been shown to cause Wnt is a target for colorectal cancer treatment. Therefore, the development of drugs that block the Wnt signaling pathway could lead to a breakthrough in cancer treatment (Clevers H et al (2006) Cell 127: 469-480; Mlodzik M (2002) Trends Genet 18: 564-). 571; Huelsken J et al (2001) 11: 547-553; MacDonald BT et al (2009) Dev Cell 17: 9-26; Fearnhead NS (2001) Mol Genet 10: 721-733; Klaus A et al (2008) Nat Rev Cancer 8: 387-398; Barker N et al (2006) Nat Rev Drug Discov 5: 997-1014).

Korean Unexamined Patent Publication No. 2015-0037024 relates to a composition for the treatment or prevention of cancer diseases using weighted tree extract as an active ingredient, and treatment of colorectal cancer that inhibits the Wnt / β-catenin signaling system comprising the weighted wood extract or Disclosed is a prophylactic composition.

Korean Unexamined Patent Publication No. 2015-0037024 (published on April 8, 2015) discloses a composition for treating or preventing cancer diseases that inhibits the Wnt signaling pathway using the weighted tree extract as an active ingredient.

In order to prevent or treat colorectal cancer, it is necessary to suppress the Wnt / β-catenin signaling system, and thus, it is necessary to develop a therapeutic agent based on various naturally occurring substances that target the Wnt / β-catenin pathway other than the agar tree extract. .

The present application is to provide a composition for the prevention and treatment of colorectal cancer using the extract of telectadium Dong Nai.

In one embodiment the present application provides a telectadium Dongnaiense ) provides a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising the extract as an active ingredient.

In another embodiment, the present disclosure provides a telectadium dongnaiense ) extract for the prevention or treatment of colorectal cancer.

The extract according to the invention is particularly effectively extracted in organic solvents. Such organic solvents include, but are not limited to, for example, one or more mixed solvents selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, and ethers. .

In one embodiment, in particular, as the organic solvent, a mixed solvent in which chloroform, acetone, and a lower alcohol having 1 to 4 carbon atoms, in particular methanol, is mixed in a ratio of 10 to 1: 1 to 10: 1 to 10 (v / v) is used.

Extract according to the present invention can effectively inhibit the proliferation of colorectal cancer cells by inhibiting the Wnt / β-catenin signaling system or pathway, and verified the effect on various transcription factors involved in the Wnt / β-catenin signaling system . Colorectal cancer according to the present application is particularly colorectal cancer in which the Wnt / β-catenin signaling system is overactivated. Whether the Wnt / β-catenin signaling system is overactivated is referred to, for example, the constituents constituting the Wnt / β-catenin signaling system with reference to the contents described herein and the documents described herein. Can be determined by measuring the expression of genes and proteins, or by measuring the mutations of genes and proteins, and comparing them with corresponding measurements in appropriate controls such as normal colon cells.

In another aspect, the present application also provides a Wnt target anticancer agent or anticancer agent that inhibits the Wnt / β-catenin signaling system of a cell comprising the above-described teletadium dongnae extract as an active ingredient. Although not limited to this, Wnt-targeted anticancer agents modulate the activity of one or more molecules involved in the Wnt / β-catenin signaling system, such as the proliferation of cancer cells, ultimately affected by the Wnt / β-catenin signaling system. To regulate biological effects.

Wnt target anticancer agent or anticancer agent according to the present invention can be particularly useful for inhibiting the proliferation of colorectal cancer cells that are highly related to the abnormality of the Wnt / β-catenin signaling system.

In another aspect, the present invention also provides a dietary supplement for preventing or ameliorating colon cancer, comprising the teletadium dongnae extract according to the present application as described above.

In another aspect the present disclosure provides a method for treating colorectal cancer, comprising administering an extract according to the present application to a subject in need thereof.

Pharmaceutical composition for the prevention or treatment of colorectal cancer, including the extract of Telectadium dongnaiense of the present invention and dietary supplements have a mechanism of action that inhibits the Wnt / β-catenin signaling pathway, especially cancer Since it has an anticancer effect against cancer, it can be usefully used as an adjuvant to prevent and treat colorectal cancer or an existing anticancer agent.

Figure 1 shows the analysis of TCF / LEF inhibitory activity in the human kidney cell line of the Teletadium Dong Naien extract of the present invention. These results indicate that Wnt signal overexpression increases β-catenin, which promotes the transcription of oncogenes after β-catenin-TCF / LEF complex formation in the nucleus. Signals may be useful in the treatment of overexpressed cancer.

Figure 2 shows the cytotoxicity to colorectal cancer cells upon 24 hours treatment of the teletadium Dong Naien extract of the present invention. These results indicate that the IC ₅₀ value of 1.5 μg / ml for the 50% survival rate of the colorectal dongnae extract extract of the present application for the colorectal cancer cell line, indicating that it is effective for the death of colorectal cancer cells.

Figure 3 shows the inhibitory activity in the TCF / LEF transcriptional analysis for colorectal cancer cells upon 24 hours treatment of the teletadium Dong Naien extract of the present invention. This indicates that the extract according to the present application can be useful for the treatment of colorectal cancer in which the Wnt signal is overexpressed.

Figure 4 shows the effect on c-Myc, Cyclin D1 and Survivin gene expression, which is a sub-regulatory of Wnt / β-catenin signaling by the lectrectium dongnae extract in colorectal cancer cell line. This indicates that the lectrectium dongnae extract according to the present application inhibits the proliferation of colorectal cancer through the inhibition of the Wnt / β-catenin signaling system.

Figure 5 shows the concentration-dependent effect on the expression of c-Myc, Cyclin D1 and Survivin proteins, which are sub-regulators of Wnt / β-catenin signaling by the Telectium dongnae extract in colorectal cancer cell line (HCT116). This indicates that the lectrectium dongnae extract according to the present application inhibits the proliferation of colorectal cancer through the inhibition of the Wnt / β-catenin signaling system.

The present invention is based on the discovery that the extract of Telectadium dongnaiense has a mechanism of action that inhibits the Wnt / β-catenin signaling pathway and has anticancer efficacy against cancer, particularly colorectal cancer.

The dysregulation of Wnt / β-catenin signaling pathways plays a very important role in early-stage colorectal cancer. Wnt inhibits GSK3β activity and reduces APC phosphorylation, resulting in intracellular amounts of β-catenin. Increasing and the increase of β-catenin by Wnt, β-catenin is believed to be a transcription factor TCF / LEF complex, β-catenin-TCF / LEF complex to the nucleus to promote the oncogenic transcription. The present inventors screened over 11,000 species of foreign plants obtained from four major regions of the world, and as a result, the above-mentioned teletadium dongnae extract inhibits the activation of the Wnt / β-catenin signaling system, in particular TCF / LEF transcription inhibition Activity, and c-Myc, Cyclin D1 and Survivin gene expression inhibitory activity, and inhibited the proliferation of colorectal cancer through the inhibition of the Wnt / β-catenin signaling system.

Therefore, in one embodiment, the present application relates to a pharmaceutical composition for preventing and treating colorectal cancer, which contains as an active ingredient telerectium dongnae extract.

The plant telelectadidium dongien used in the preparation of the extract according to the present invention is a plant native to the province of Dongnai of Vietnam belonging to the Park Juryaceae. The teletadium dongensu is a ground, root stem, or root, more preferably, above ground, roots, or roots, including flowers, branches, stems, leaves, immature fruit, or fruit of the teletadium dongenance Even more preferably, the ground portion can be used. In one embodiment of the present application, but the ground portion of the teletadium Dong Nience is used, but is not limited thereto.

Cancer disease studies targeting the Wnt / β-catenin signaling pathway of Teletadium Dong Naien have not been reported in the present invention.

Extracts herein can be prepared using known general methods of preparing conventional plant extracts, and are not particularly limited. For example, the method may be prepared by a known method using an ultrasonic extraction method, reflux cooling extraction method, hot water extraction method, or an extraction solvent described later (for example, Nomura T. Phenolic compounds of the mulberry tree and related plants. Progress in the Chemistry of Organic Natural Products (Herz W, Grisebach H, Kirby GW, Tamm Ch, eds.), Vol. 53. Springe: Vienna, 1988; 87-201).

For example, the teleectadidium dongnae extract of the present invention can be prepared as follows. After washing and sintering the dried teleectadidium copper ions, lower alcohols having 1 to 4 carbon atoms, chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, including methanol, ethanol, butanol, propanol and isopropanol, including purified water And one or more mixed solvents selected from the group consisting of ethers, preferably chloroform, acetone, methanol or mixed solvents thereof, more preferably 10-1: 1-10: 1 of chloroform, acetone, and methanol A mixed solvent of a -10 (v / v) mixing ratio, even more preferably a 5-1: 1-5: 1-5 (v / v) mixing ratio of chloroform, acetone, and methanol can be mixed several times.

Extract preparation temperature may be 4 to 150 ℃, but is not limited to this may be extracted at a temperature of 30 ℃ to 150 ℃, preferably 50 ℃ to 100 ℃.

Extraction time is not particularly limited, but may be 10 minutes to 30 days, preferably 30 minutes to 48 hours, more preferably 1 hour to 12 hours, conventional ultrasonic extraction, hot water extraction, room temperature extraction or The reflux extraction method, preferably the ultrasonic extraction method may be repeated about 1 to 20 times, preferably 2 to 10 times. The telelectadidium dongnae extract of the present invention prepared by the above method may then be removed under reduced pressure filtration or / and lyophilization to remove the solvent.

In one embodiment, the teletadium dong extract extract according to the present application is an organic solvent extract. The organic solvent is one or more selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, and ether, including methanol, ethanol, butanol, propanol, isopropanol, and the like. Mixed solvents, preferably 10-1: 1-10: 1-10 (v / v) mixing ratio of chloroform, acetone, methanol or a mixed solvent thereof, more preferably chloroform, acetone, and methanol, even more preferred Preferably, it is a mixed solvent of 5-1: 1-5: 1-5 (v / v) mixing ratio of chloroform, acetone, and methanol. In one embodiment of the present invention, the mixed solvent is chloroform, acetone and methane is used in a mixing ratio of 1: 1: 1 (v / v).

Telectium dongnae extract according to the present application can inhibit colon cancer proliferation by inhibiting the Wnt / β-catenin signaling system.

As used herein, the term “treatment” is a concept that includes inhibiting, eliminating, alleviating, alleviating, ameliorating, and / or preventing a disease or condition or condition resulting from the disease.

The compositions herein may be used alone or in combination with other conventional chemotherapy or radiation therapy for the treatment or prevention of colorectal cancer. Anticancer agents commonly used in chemotherapy include: antimetabolites such as folate derivatives (methotrexate), purine derivatives (6-mercaptopurine, 6-thioguanine), and pyrimidine derivatives (5-fluorouracil, cytarabine); Alkylating agents such as nitrogen mustard compounds (chlorambucil, cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), triazene compounds (dacarbazine) ); Mitosis inhibitors such as actinomycin D, anti-cancer anticancer agents such as doxorubicin, bleomycin, mitomycin, plant alkaloids such as vincristine and vinblastine, and mitosis inhibitors including taxane rings antimitotic drugs); Or hormones such as corticosteroids or progesterone; Platinum-containing compounds such as cisplatin and the like.

The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as necessary. Other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The method of administration of the composition of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method. Administration by intravenous injection is preferred, in order to obtain a rapid therapeutic effect. The dosage may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the subject, and is generally 0.01 to 1,000 mg / day based on an adult patient weighing 70 kg. Preferably it is 0.01-500 mg / day, and can be divided into once or several times a day. In one embodiment, the daily dosage of the extract or anticancer and anticancer agents, or 1/2, 1/3 or 1/4 thereof, per unit dosage form is contained, and at a predetermined time interval according to the judgment of a doctor or pharmacist. It may be administered in 1 to 6 divided doses.

The telelectium dongnae organic solvent extract of the present application has the effect of inhibiting the Wnt signaling system. Specifically, Wnt signaling is inhibited by inhibiting TCF / LEF luciferase activity in β-catenin-induced HEK293 cell lines and human colorectal cancer cell lines, and by decreasing the expression of c-Myc, Cyclin D1 and Survivin genes, which are β-catenin target genes. Inhibit the system.

In another aspect, the present application is a Wnt target anticancer agent, in particular Wnt containing an organic solvent extract of teletadium Dong Naien, the extract of the organic solvent of the Teletadium Dong Naien inhibits the Wnt / β-catenin signaling system It relates to a target colorectal cancer anticancer agent.

The organic solvent extract of teleectadium dongnae of the present invention is as described above.

The target anticancer agent refers to a therapeutic agent that selectively attacks only a specific target of the carcinogenic process, and the target anticancer agent of the present application is a Wnt target anticancer agent that targets the Wnt / β-catenin signaling system involved in the colorectal carcinogenesis process.

Since the extract of the present invention has a Wnt signaling inhibitory effect, it can be used as an anticancer adjuvant for cancer cells.

In yet another aspect, the present application is to the Wnt target anticancer adjuvant containing an organic solvent extract of Telectidinium dong eunsu, the organic solvent extract of telectadidium dong eunsu inhibits the Wnt / β-catenin signaling system It is about.

The organic solvent extract of teleectadium dongnae of the present invention is as described above.

The anticancer adjuvant of the present invention targets the Wnt / β-catenin signaling system, and can be used to increase the anticancer effect of the anticancer agent and to suppress or improve the side effects of the anticancer agent.

In the present invention, the "inhibition" means all the actions of reducing the side effects of the anticancer agent by the administration of the anticancer adjuvant, the term "improvement" in the present invention, the reduction of the anticancer agent side effects or the reduction of the anticancer agent side effects by the administration of the anticancer adjuvant Means any behavior that improves or beneficially changes the symptoms caused by cancer.

In addition, in the present invention, the "individual" means all animals including humans who may develop or may develop cancer, and by administering the anticancer adjuvant of the present invention to an individual, the cancer may be effectively treated while minimizing side effects of the anticancer agent. For example, the anticancer adjuvant of the present invention can be administered to a human suffering from colorectal cancer who is receiving an anticancer agent to effectively treat cancer while reducing side effects of anticancer.

Acceptable carriers, formulations, administration methods and dosages of the target anticancer agents or anticancer adjuvants of the present invention are as described in the pharmaceutical composition.

The compositions of the present invention may also be prepared as food, in particular functional food compositions. Therefore, the present invention relates to a dietary supplement for preventing and improving colorectal cancer, which contains an organic solvent extract of telenecdium dongnae.

The organic solvent extract of teleectadium dongnae of the present invention is as described above.

Health functional food containing the extract of the present invention can be used in a variety of drugs, foods and beverages for the prevention and improvement of colorectal cancer. Examples of the food to which the compound of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

The functional food composition of the present invention includes ingredients that are commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the extract as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As flavoring agents, natural flavoring agents (e.g., taumartin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used. Considering easy access to food, the nutraceutical of the present invention is very useful for the treatment or prevention of cancer.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The composition of the present invention comprises 0.01 to 99% by weight of the compound relative to the total weight of the composition. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

In another aspect the invention relates to a method of treating colorectal cancer comprising administering an extract according to the present invention to a subject in need thereof in a therapeutically effective amount.

For the extracts used in the methods according to the invention, the characteristics and dosages of colorectal cancer, reference may be made to the foregoing.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Example 1.Preparation of Telectadium dongnaiense Pierre ex Costantin Extract

Telectium Dong Naien (origin of Vietnam) is a ground part including flowers, branches, stems, leaves, immature fruit, fruit; Rhizome; And roots were harvested and washed after selection of one or more assayed test materials selected.

The ground portion of the dried teleectadidium Dongens, which is a sample; Rhizome; And 10 L of chloroform: acetone: methanol (1: 1: 1, v / v) in each 1 kg of the roots, using a ultrasonic extractor (SDN-900H, SD-ULTRASONIC CO. LTD.) Three times in a water bath for 3 hours. Extracted. The extract was filtered, concentrated under reduced pressure, and freeze-dried to give a brown Telectadium dongnaiense Pierre ex Costantin ground extract; Rhizome extracts; And root extracts were obtained, respectively, and stored at −20 ° C., and ground extracts were used as samples for analysis of the following experimental examples.

Example 2 Confirmation of the TCF / LEF Luciferase Activity Inhibitory Effect in the HEK293 Cell Line Incorporated with β-Catenin of Terectadidium dongens extract

Since it is known that β-catenin expression is overactivated by mutations in Wnt signaling proteins in some cancer cell lines, especially colorectal cancer, it is meaningful to find a substance that can inhibit the action of β-catenin. In order to determine whether the extract of the Teletadium dongnae extract has such an effect, β-catenin was introduced into HEK293 cells and overactivated to evaluate whether the extract was suppressed by the telectadidium dongnae extract. The method of the prior art (Kang YJ et al (2012) Mol Pharm 82: 168-177) was tested with some modifications.

In order to confirm whether the teletadium dong extract extract of Example 1 is related to the regulation of transcriptional activity of TCF / LEF (T-cell factor / lymphoid enhancer factor), a transcription factor to which β-catenin binds, HEK293 cells TCF4 and pcDNA β-catenin and plasmid (TOPFlash) containing a TCF binding site were transiently transfected into a Human Embryonic Kidney cell (ATCC, USA), and then the Teletadium Dongnae extract was used for the transcriptional activity of TCF. The effect on the luciferase activity with increasing was tested as follows.

HEK293 cells were adjusted to 1.5 × 10 ⁴ per well in 48 well plates with DMEM medium (# 12800-017, Gibco) containing 10% FBS, incubated for 24 hours at 37 ° C., 5% CO ₂ , and lucifer 100ng of plasmid with TRE binding site attached to an aze, 8ng of TCF4 plasmid, 20ng of β-catenin and 1/20 of the total amount of each DNA, pRL-SV40 vector (E2231, Promega) and lipofectamine 2000 (# 11668-019 , Invitrogen), mixed with Opti-MEM (31985-070, Gibco), transfected for 24 hours, and then diluted with telelectadidium dongnae extract (5, 10, 20 μg / ml), incubate for 24 hours, remove all supernatant, suspend in 1X Passive Lysis Buffer (E194A, Promega), mix for 15 minutes, and then use Luminometer (Cetro LB) using Dual Luciferase Kit (E1910, Promega). 960, Berthold, Germany) to measure luciferase activity, transfection efficiency was pRL-SV40 lucifer It was corrected in cyclase activity.

As shown in Table 1 and FIG. 1, when TCF4, pcDNA β-catenin, and TOPFlash were introduced together in HEK293 cells, luciferase activity was increased as compared to the control in which TOPFlash alone or TOPFlash and TCF4 plasmid were introduced. In the presence of TCF4 and pcDNA β-catenin in the HEK293 cells, the increased TCF / LEF luciferase activity was increased by treatment of the lectredium dongena extract, as shown in FIG. , 5 μg / ml was added, and TCF / LEF transcriptional activity was statistically significant (** P <0.01) and showed transcription-inhibitory activity in a concentration-dependent manner. It showed 35.2% when adding / ml, 55.6% when adding 2.5μg / ml, 73.4% when adding 5μg / ml, and TCF / LEF transcription inhibitory activity. From the above results, the transcription inhibitory activity IC ₅₀ of the T. pectium dongnae extract showed excellent inhibitory activity at 2.1 μg / ml. In addition, when transfected plasmid (FOPFlash) mutated TCF binding site by using Tele-Titanium Dong Naien extract, TCF4 and pcDNA β-catenin were also found to have no effect on luciferase activity. These results indicate that Wnt signal overexpression increases β-catenin, which promotes the transcription of oncogenes after β-catenin-TCF / LEF complex formation in the nucleus. Signals may be useful in the treatment of overexpressed cancer.

TABLE 1

Example 3 Confirmation of Growth Inhibitory Effect of Tecterdium Dongnae Enthusiast Extract on Human Colon Cancer Cells in Vitro

In order to confirm the growth inhibitory effect on human colon cancer cells, etc. in vitro of the sample obtained in Example 1, the experiment was performed as follows by applying the method described in the literature (Lee SK et al (2008) Chem Biol Interact 115 : 215-28).

Human colorectal cancer cell line HCT116 was sold by the Bank of Korea Cell Line (CCL-247, ATCC, USA).

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

[Equation 1]

% Survival = (OD (sample)-OD (0-day)) / (OD (10% DMSO)-OD (0-day)} X 100

When the control group was not treated with the sample at 100%, the value of the sample treatment group was expressed as a percentage of the control group, and each test substance treatment was calculated as the average value ± SEM of the double or triple test, and the results are shown in Table 2 and FIG. 2. Shown in

In detail, as shown in FIG. 2, 1, 2, 4, and 8 μg / ml of telectadinium dong extract extract were evaluated and cytotoxicity against human colorectal cancer cell lines was statistically significant (** P <0.01) showed cytotoxicity in a concentration-dependent manner, specifically 12.4% with 1 μg / ml of Telactium dongnae extract, 83.5% with 2μg / ml, 92.4% with 4μg / ml, 8μg / ml The addition showed 97.2% and cytotoxicity, respectively. From the above results, the concentration of test substance for 50% viability of H. 116 cell line of the Tecterdium dongnae extract extract showed an excellent cytotoxicity of 1.5 μg / ml.

TABLE 2

Example 4 Identification of TCF / LEF luciferase Activity Inhibitory Effect of Terectadidium dongnae extracts in Human Colon Cancer Cell Lines

Based on the results of Example 2 that inhibit the action of overactivated β-catenin, HCT116 colorectal cancer cell line known to be overexpressed of β-catenin due to genetic variation in GSK3β and β-catenin, which are Wnt signaling related proteins TCF / LEF luciferase activity was assessed at.

Human colon cancer cell line HCT 116 (5 X 10 ³ cells / mL) cells were placed in RPMI 1640 medium (# 31800-022, Gibco) containing 10% FBS in 300 μL of each well in a 48-well plate at 37 ° C, 5% Incubated in CO ₂ conditions. 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 diluted in Opti-MEM (# 31985-070, Gibco). After transfection using lipofectamine 2000 (# 11668-019, invitrogen). After 24 hours, a sample of pre-diluted telenecdium dong extract extract was treated, and then incubated for 24 hours in an incubator. The medium in the wells was removed and cells were lysed using 1X passive lysis buffer (E194A, Promega). 10 μl of the cell lysate was transferred to a white 96 well plate, and then confirmed luciferase activity using a dual luciferase reporter assay system (E1910, Promega), and corrected by Renilla luciferase activity, and the results are shown in Table 3 and FIG. 3. It was.

As shown in FIG. 3, 1, 2.5, and 5 μg / ml of telectadinium dongens extracts were added and TCF / LEF luciferase activity inhibitory effect was evaluated in human colon cancer cell lines. 0.05) TCF / LEF transcription inhibitory activity was dependent on the concentration, and in detail, 27.5% when 1.0 μg / mL was added to the Tectadidium dong extract, 37.7% when 2.5 μg / mL was added, and 49.7% when 5 μg / mL was added. Transcriptional activity was inhibited. From the above results, it can be seen that the extract of T. T. dianthus has excellent TCF / LEF transcription inhibitory activity in colorectal cancer cell lines. This indicates that the extract according to the present application can be useful for the treatment of colorectal cancer in which the Wnt signal is overexpressed.

TABLE 3

Example 5 Confirmation of Reduction of Expression of β-Catenin Target Genes c-Myc, Cyclin D1, and Survivin Genes in Human Colorectal Cancer Cell Lines by the Terectadidium dongnae extract

The c-Myc, Cyclin D1, and Survivin genes are known as 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 (1999). 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 on β-catenin target gene expression in the colorectal cancer cell line of the sample obtained in the above example, the experiment was performed by applying the method described in the literature (Kang YJ et al (2012) Mol Pharm 82: 168-177).

Incubate HCT 116 (1 X 10 ⁵ cells / ml) cells in RPMI medium containing 10% FBS for 24 hours at 37 ° C and 5% CO ₂ in a 100 mm dish. It was. The attached cells were washed twice with PBS, and the cells were collected by TRI reagent (TRIZol (# 15596-026, Invitrogen)), collected, and then extracted with RNA by adding CHCl ₃ and precipitated using isopropyl alcohol. The RNA precipitate was washed with 70% ethanol and dried in air, and then dissolved in nuclease-free water (AM9937, Ambion). Heated at 55 ℃ for 10 minutes, and heated at 70 ℃ for 5 minutes to allow RNA to exist in a single strand state. Total RNA was quantified using NanoDrop (ND-1000, Dae Myung Sceince Co., Ltd), diluted to 1 μg / μl, and then avian myeloblastosis virus (AMV) reverse transcriptase (M5108, Promega) and oligo (dT) ₁₅ primer ( CDNA was prepared using C110B, Promega). Amplify target genes in 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] I was. Real-time PCR conditions were denatured at 95 ° C for 20 seconds, followed by 40 cycles of 20 seconds of denaturation at 95 ° C, 20 seconds at 56 ° C, annealing at 30 ° C and 30 seconds at 72 ° C. The final stage was then followed by 1 minute at 95 ° C and 1 minute at 55 ° C. The Ct value was determined by the method of Libak et al. (Livak KJ et al (1996) Method 25: 402-408) using MJ Opticon Monitor software (Opticon Monitor 3.1.32, Bio-rad), and β-actin of the sample. It is shown in Figure 4 that the c-Myc, Cyclin D1 and Survivin gene expression is reduced by the Telectadidium Dong Naise extract by correcting the Ct value of.

Table 4 Real-time PCR primer

As shown in Figure 4, the addition of 1, 2.5, 5 μg / ml of the extract of telectadium Dong Nai and the expression of β-catenin target genes C-Myc, Cyclin D1 and Survivin gene expression in human colorectal cancer cell lines , Concentration-dependently exhibited expression inhibitory activity, and in detail, c-Myc inhibited the expression of 39.8% of the Tectadidium dongens extract, 80.2% of the addition of 5μg / ml, and 80.2% of the gene expression; In case of Cyclin D1, 12.4 g / ml of telenecdium dong extract was added 22.4%, 40.2% of 2.5 μg / ml, 54.0% of 5 μg / ml, and gene expression was inhibited; Survivin gene expression was inhibited by 26.3% when 1 μg / mL was added, 49.7% when 2.5 μg / mL was added, and 88.7% when 5 μg / mL was added. In particular, c-Myc and survivin showed statistical significance at 99% confidence interval when 2.5 and 5 μg / ml were added. Therefore, the cytotoxicity of colorectal cancer cell lines of the present invention of the teletadium Dong Naien extract is related to c-Myc, Cyclin D1 and Survivin, which are sub-regulators of Wnt / β-catenin signaling pathway related to cancer cell proliferation I could confirm it.

Example 6 Confirmation of Concentration-dependent Inhibition of the Expression of β-Catenin Target Proteins c-Myc, Cyclin D1, and Survivin Proteins in Human Colon Cancer Cell Lines by Terectadinium Dongniens Terrestrial Extract

In order to examine the effect on the protein factor of the sub-regulatory factor involved in cell proliferation in the Wnt / β-catenin signaling pathway, the experiment was performed as follows (Hong JY et al (2011) J Nat Prod). 74: 2102-8).

Colon cancer cell line (HCT116) was placed in DMEM medium containing 10% FBS at a concentration of 1 × 10 ⁵ in a 100 mm culture dish, and cultured for 24 hours at 37 ° C. and 5% CO ₂ , followed by phosphate buffered saline (PBS). ) Twice. 10 ml of DMEM medium containing 10% FBS in which the samples were previously diluted by concentration was added thereto, and then cultured for a predetermined time. Collect unattached cells and adhered cells in cell medium, wash twice with PBS, and then boil 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) were added to disrupt the cells and placed at 100 ° C. for 5 minutes. After cooling, the product was stored at -20 ° C and dissolved at 37 ° C immediately before use to use for protein quantification and electrophoresis. 80 μg of protein was electrophoresed at 138 V for 2 hours using 5-12% SDS-polyacrylamide gel (# 456-1036, Bio-rad, Hercules, CA). The isolated protein was transferred to a PVDF membrane (membrane, ISEQ15150, Millipore, Bedford, Mass.) For 1 hour at 100 V, washed twice with TBST and washed with blocking buffer (5% Albumin from Bovine Serum in PBS containing 0.1% Tween-20 ( TBST)) and incubated in shaker for 1 hour at room temperature. After washing 3 times with TBST for 5 minutes, the single antibody was diluted with 3% Albumin from Bovine Serum (BSA (A9647, Sigma)) with TBST and reacted with a membrane for 18 hours at 4 ° C. After washing the membrane (Membrane, ISEQ 1S150, Millipore, Bedford, MA) three times for 5 minutes with TBST, the HRP (horseradish peroxidase) -binding secondary antibody was diluted with TBST in a ratio of 1: 1,000 to 1: 2,000 with 3% BSA. The reaction was performed at room temperature with a membrane for 3 to 4 hours. After washing 3 times with TBST for 5 minutes and treated with Western blotting substrate (WEST-ZOL PLUS (16021, Intron)) to determine the degree of protein expression in LAS-3000 (Fuji Film Corp., Japan).

In detail, the colon cancer cell line (HCT116) was treated with the lectaddium dong extract extract 1, 2.5, 5 μg / ml and cultured for 24 hours, followed by the expression analysis of the colon cancer cell line (HCT116). As a result of confirming the inhibition of the signaling system in, it was confirmed that the expression of c-Myc, Cyclin D1 and Survivin protein was inhibited in a concentration-dependent manner, and in particular, the expression of the survivin gene was remarkably suppressed. Taken together, it is believed that the lectrectium dongnae extract according to the present application inhibits the proliferation of colorectal cancer by inhibiting the Wnt / β-catenin signaling system.

Hereinafter, the preparation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to limit the present invention but only to explain in detail.

Formulation Example 1 Preparation of Powder

Telectium Dong Naien Extract 200 mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Telectium Dong Naien Extract 200 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3 Preparation of Capsule

Telectium Dong Naien Extract 200 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Telectium Dong Naien Extract 200 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Telectium Dong Naien Extract 200 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, lemon flavor is added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by adding purified water, and then filled into a brown bottle and sterilized. To prepare a liquid solution.

Formulation Example 6 Preparation of Healthy Food

Telectium Dong Naien Extract 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic 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 mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Telectium Dong Naien Extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and then stored in the present invention For the preparation of healthy beverage compositions.

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

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Claims (15)

A pharmaceutical composition for preventing or treating colorectal cancer comprising an extract of Telectadium dongnaiense as an active ingredient. The pharmaceutical composition for preventing or treating colorectal cancer according to claim 1, wherein the extract is an organic solvent extract. The method of claim 1, wherein the organic solvent is one or more mixed solvents selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, chloroform, acetone, propylene glycol, ethyl acetate, butylene glycol, and ethers. Or therapeutic pharmaceutical compositions. The method of claim 3, wherein the organic solvent is chloroform, acetone, and a lower alcohol having 1 to 4 carbon atoms in a mixed solvent in a ratio of 10 to 1: 1 to 10: 1 to 10 (v / v). Or therapeutic pharmaceutical compositions. The pharmaceutical composition for preventing or treating colorectal cancer according to claim 1, wherein the colorectal cancer is colorectal cancer in which the Wnt / β-catenin signaling system is overactivated. Wnt target anticancer agent that inhibits the Wnt / β-catenin signaling system of the cell containing the teletadium Dong Naien extract as an active ingredient. The Wnt target anticancer agent of claim 6, wherein the cell is a colorectal cancer cell. Wnt target anticancer adjuvant that inhibits the Wnt / β-catenin signaling system of cells containing an organic solvent extract of Teletadium Dong Naien as an active ingredient. The Wnt target anticancer agent according to claim 8, wherein the cells are colorectal cancer cells. A dietary supplement for preventing or improving colorectal cancer, which comprises teleectadidium dongnae extract. The health functional food according to claim 10, wherein the extract is an organic solvent extract. Telectadium Use of the extract of dongnaiense ) for the prevention or treatment of colorectal cancer. Use of claim 12, wherein the colorectal cancer is colorectal cancer with a Wnt / β-catenin signaling system overactivated. Telectadium A method for treating colorectal cancer comprising administering an extract of dongnaiense ) to a subject in need of treatment for colorectal cancer. The method of claim 14, wherein the colorectal cancer is colorectal cancer with a Wnt / β-catenin signaling system overactivated.

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