KR101649047B1 - Use of Kazinol C for treating or preventing cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cancer diseases including casinol C, and a health functional food for prevention and improvement of cancer diseases. More specifically, the present invention relates to a health functional food for preventing and treating cancers, As an ingredient. The present invention has the effect of preventing or treating cancer diseases by inhibiting cell migration and non-adherent growth of caspase-3 by increasing the activity of AMPK in cells by promoting apoptosis of cancer cells.

Description

Use of casinol C for the prevention or treatment of cancer [

The present invention relates to a pharmaceutical composition for preventing and treating cancer diseases including casinol C, and a health functional food for prevention and improvement of cancer diseases.

Cancer is one of the incurable diseases that humanity needs to solve. In the world, huge capital is invested in development to heal it globally. In Korea, it is the first disease among the causes of death, It is diagnosed and more than 60,000 people are dying.

Cancer is characterized by "uncontrolled cell growth." These abnormal cell growths form a mass of cells called tumors that penetrate into surrounding tissues and, in extreme cases, to other organs of the body. Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, in many cases can not be cured, causes pain to the patient, and ultimately leads to death.

Among the methods used to treat these malignant tumors, chemotherapeutic agents other than surgery or radiation therapy are collectively referred to as anticancer agents. There are two main ways in which anticancer drugs can target cancer cells. There is a direct way to kill cancer cells themselves and an indirect way to prevent the growth of cancer cells. Currently, most of the drugs used as anticancer drugs are cell killing agents, have considerable toxicity, and can not selectively remove only cancer cells, thus destroying normal cells and severely damaging the patient's health. In addition to the treatment of cancer after the occurrence of cancer, it is urgent to develop an effective anti-cancer drug that is less toxic to prevent the development of cancer.

In addition, cancer tissues are initially atrophied by chemotherapy, but they are mutated. Therefore, there is a problem of increasing the amount of anticancer drugs, and thus many side effects due to overuse of anticancer drugs have been reported. Therefore, anticancer therapy combined with various anticancer drugs is the mainstay of chemotherapy. Despite the use of multiple co-administration techniques, the cancer has not yet been conquered. In particular, the improvement in survival rate due to the use of anticancer drugs is considered to be minimal. In recent years, efforts have been made to find the active ingredient in natural products that have been used in the private sector in connection with the problem of overcoming such side effects.

On the other hand, Moraceae and Mulberry ( Broussonetia kazinoki ) is a Chinese word in the Chinese language (楮 木) is called. It is a deciduous broad-leaved shrub that can be seen from the sides of the mountain. It has short hairs on the small branches but disappears while growing. Its bark is grayish brown with a height of about 3m and is distributed in Korea, Japan and China.

Mackerel was once used to weave a type of fabric called foil, which is used as paper mill raw material. Leaves, branches and fruit of the plants have also been used as diuretics, tonic agents and edema inhibitors. According to existing studies, it is known that mackerel has antioxidant activity (non-patent document 1), cytotoxicity (non-patent document 2), antihyperglycemia (non-patent document 3) and tyrosine inhibitory activity (non-patent document 4). However, the relationship between the anticancer mechanism of mulberry trees is not known.

1. Korean Patent Publication No. 10-2010-0029559

1. Enhanced antioxidant effect of prenylated polyphenols as Fyn inhibitor. Lee, JH, Kim, SY, Lee, Free Radic Biol Med 53: 1198-1208, 2012. 2. Ko HH, Yen MH, Wu RR, Won SJ and Lin CN: Cytotoxic isoprenylated flavans of Broussonetia kazinoki. J Nat Prod 62: 164-166, 1999. 3. Cha JY, Kim YT, Kim HS and Cho YS: Antihyperglycemic effect of stem bark powder from paper mulberry (Broussonetia kazinoki Sieb.) In type 2 diabetic Otsuka Long-Evans Tokushima fatty rats. J Med Food 11: 499-505, 2008. 4. Baek YS, Ryu YB, Curtis-Long MJ, Ha TJ, Rengasamy R, Yang MS and Park KH: Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki. Bioorg Med Chem 17: 35-41, 2009. 5. Woods, Azzout-Marniche D, Foretz M, Stein SC, Lemarchand P, Ferre P, Foufelle F and Carling D: Characterization of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase. Mol Cell Biol 20: 6704-6711, 2000. 6. Ikuta J, Hano Y, Nomura T, Kawakami Y and Sato T: Components of Broussonetia kazinoki SIEB. I. Structures of two new isoprenylated flavans and five new isoprenylated 1,3-diphenylpropane derivatives. Chemical & pharmaceutical bulletin 34: 1968-1979, 1986. 7. New estrogenic compounds isolated from Broussonetia kazinoki. JH Lee, JH Lee, YH Lee, YH Lee, YH Ryu, Bioorg Med Chem Lett 20: 3764-3767, 2010.

The present invention is to provide a pharmaceutical composition for prevention and treatment of cancer diseases and a health functional food for prevention and improvement of cancer by using a natural material-derived substance with less adverse side effects and excellent effects.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases comprising casinol C or a pharmacologically acceptable salt thereof as an active ingredient.

According to one aspect of the present invention, the casinol C contained in the present invention is characterized by being represented by the formula (1).

According to one aspect of the present invention, the casinol C contained in the present invention is characterized in that it is extracted from mulberry.

According to one aspect of the present invention, the present invention provides a method for treating 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, , Hodgkin's disease, Esophageal cancer, Small intestine cancer, Endocrine cancer, Thyroid cancer, Parathyroid cancer, Adenocarcinoma, Soft tissue sarcoma, Urethral adenocarcinoma, Urethral adenocarcinoma (CNS) tumors, primary CNS lymphoma, spinal cord tumors, neoplasms of the central nervous system (CNS), cancer of the central nervous system (CNS) And at least one disease selected from the group consisting of glioma, glioma, and pituitary adenoma.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising cyclophosphamide, amsacrine, daunomycin, taxol, 5-fluorouracil (5-FU), doxorubicin At least one selected from the group consisting of Doxorubicin, Mitomycin, Cisplatin, Paclitaxel, Docetaxel, Irinotecan, Xeloda, and Oxalopatin. .

According to one aspect of the present invention, the present invention is characterized in that it has an anticancer effect through inhibition of cell proliferation, induction of apoptosis, inhibition of cell migration and non-adherent growth of cancer cells through an increase in AMPK activity .

According to one aspect of the present invention, there is provided a health functional food for preventing and ameliorating cancer diseases comprising casinol C as an active ingredient.

According to one aspect of the present invention, the casinol C is characterized by being represented by the general formula (1).

According to one aspect of the present invention, the casinol C is extracted from mulberry.

According to one aspect of the present invention, the present invention provides a method for treating 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, , Hodgkin's disease, Esophageal cancer, Small intestine cancer, Endocrine cancer, Thyroid cancer, Parathyroid cancer, Adenocarcinoma, Soft tissue sarcoma, Urethral adenocarcinoma, Urethral adenocarcinoma (CNS) tumors, primary CNS lymphoma, spinal cord tumors, neoplasms of the central nervous system (CNS), cancer of the central nervous system (CNS) Characterized by prevention and improvement of at least one cancer disease selected from the group consisting of brain glioma glioma and pituitary adenoma.

According to one aspect of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, .

The present invention provides a pharmaceutical composition for prevention and treatment of cancer diseases and a health functional food for prevention and improvement of cancerous disease, which have less adverse side effects and excellent effects by using casinol C derived from Mulberry.

FIG. 1A shows the effect of casinol C on the survival rate of HT-29 cells, which are colon cancer cells.
1B is a graph showing the effect of casinol C on the growth rate of colon cancer cells.
1C and D are graphs showing the effect of casinol C on the apoptosis of colon cancer cells.
Figure 2a shows the results of time-dependent confirmation of the effect of casinol C on the activity of AMPK.
FIG. 2B shows the effect of casinol C on the activity of AMPK in a concentration-dependent manner.
FIG. 3A shows the results of confirming that the AMPK activation of casinol C and the cancer cell survival rate inhibitory activity thereof are inhibited by the simultaneous treatment of Compound C. FIG.
3B and 3C are the results of confirming that the cancer cell killing effect of casinol C is inhibited by the simultaneous treatment of compound C.
FIGS. 4A and 4B are the results of identifying the cytotoxic effect of cancer cells through activation of AMPK of casinol C using molecular techniques.
FIGS. 5A, 5B and 5C show the effect of the concentration of casinol C, which has no effect on the cell survival rate of cancer cells, on the migration force of cancer cells induced by TPA.
FIG. 5D shows the effect of casinol C on the non-adherent growth of cancer cells.
FIG. 6 shows the results of identifying the effect of cancer cell C on the migration force of cancer cells through activation of AMPK using molecular techniques.

In order to achieve the above object, the present invention provides a pharmaceutical composition for prevention and treatment of cancer diseases comprising casinol C or a pharmacologically acceptable salt thereof as an active ingredient, and a health functional food for prevention and improvement of cancer diseases . BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.

The present invention provides a pharmaceutical composition for prevention and treatment of cancer diseases comprising casinol C or a pharmacologically acceptable salt thereof as an active ingredient.

The casinol C may be represented by the formula (1).

≪ Formula 1 >

The casinol C of the present invention can be obtained from non-polar solvent extract from mulberry through the following process.

For example, first, the mica tree of the present invention is dried to obtain a lower alcohol (C1 to C4) or a mixed solvent thereof, preferably about 1 to 20 times, preferably about 3 to 10 times the sample weight (g) Ethanol, at a temperature of extraction from 20 ° C to 100 ° C, preferably 50 ° C to 70 ° C for about 1 hour to 10 days, preferably about 2 hours to 5 hours, by hot water extraction, The supernatant is recovered by extraction, more preferably by a reflux cooling extraction method. The supernatant is recovered by repeating the above steps 2 to 7 times, and the supernatant is collected and concentrated under reduced pressure to obtain a polar solvent-soluble extract.

In addition, the non-polar solvent-soluble extract of the present invention may be obtained by suspending the polar solvent-soluble extract in distilled water and then adding the suspension to a suspension containing about 1 to 100 times, preferably about 1 to 5 times by volume of hexane, ethyl acetate, methylene chloride, The same nonpolar solvent may be added to extract and separate the nonpolar solvent soluble layer 1 to 10 times, preferably 2 to 5 times, to obtain a nonpolar soluble fraction. It is also possible to carry out a conventional fractionation process

More specifically, the ethyl acetate fraction of the non-polarized non-polar solvent extract obtained in the above process was taken and 11 fractions were obtained from the n-hexane / acetone gradient elution system (20: 1 → 1:10) Collect. The seventh fraction is subjected to silica gel column chromatography using chloroform: methanol (100: 1 - > 10: 1) to separate into six sub-fraction layers, and then a third sub-fraction layer is collected. The third sub-fraction can be subjected to a chromatographic gradient elution system (40% - > 100%) to yield casinol C.

The compounds of the present invention represented by the above formula (1) can be prepared as pharmaceutically acceptable salts and solvates according to methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, lactic acid, glycollic acid, gluconic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

The pharmaceutically acceptable salts of the above Formula 1 include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of Formula (I). For example, pharmaceutically acceptable salts include the sodium, calcium, and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Salicylate) salts, which are known in the art and which are known in the art, such as, for example, ≪ / RTI >

In order to investigate the anticancer effect and the anticancer adjuvant effect of the present invention obtained by the above method, cell death, AMPK activity, cell migration and nonadherent growth were evaluated. As a result, The compounds of the present invention can inhibit cell proliferation, induce apoptosis, inhibit cell migration and non-adherent growth through AMPK activation, and thus have excellent anticancer effects.

The pharmaceutical composition for prevention and treatment of cancer diseases of the present invention may contain 0.001 to 50% by weight of the above compound based on the total weight of the composition. The term "effective ", as used herein, refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, Lt; RTI ID = 0.0 > a < / RTI > disease or disorder. It will be apparent to those skilled in the art that the effective dose and the frequency of administration for the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

The cancer diseases include common cancer diseases, and preferably cancer such as 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, Hodgkin's disease, Esophageal cancer, Small intestine cancer, Endocrine adenocarcinoma, Thyroid cancer, Parathyroid cancer, Adrenocortical cancer, soft tissue, cancer, rectum cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma Renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, renal cell carcinoma, renal cell carcinoma, renal cell carcinoma, pancreatic cancer, Spinal cord tumors, spinal cord tumors, glioma glioma, and pituitary adenoma.

The pharmaceutical dosage forms of the compounds of the present invention may also be used in the form of their pharmaceutically acceptable salts and may also be used alone or in combination with other therapeutically active compounds such as anticancer drugs well known in the art, Cyclophosphamide, amsacrine, daunomycin, taxol, 5-FU, doxorubicin, mitomycin, cisplatin, Can be used in appropriate combinations as well as in combination with existing anticancer agents such as paclitaxel, docetaxel, irinotecan, Xeloda and oxalopatin.

Accordingly, the present invention relates to the use of cyclophosphamide, amsacrine, daunomycin, taxol, 5-fluorouracil (5-FU), doxorubicin, mitomycin The composition may include one or more selected anticancer agents selected from the group consisting of mitomycin, cisplatin, paclitaxel, docetaxel, irinotecan, Xeloda, and oxalopatin.

Therefore, the present invention relates to a pharmaceutical composition for treating cancer, which comprises the above-mentioned anticancer agent, preferably methotrexate, 6-mercaptopurine, 6-thioguanine, 5-fluorouracil or Cytarabine, Antimetabolites; Alkylating agents such as chlorambucil, cyclophosphamide, ethylene imine compound, thiotepa, alkyl sulfone, carmustine, and dacarbazine, ); 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 adrenocortical hormone or progesterone; At least one anticancer drug selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, and taxol, and anticancer activity against platinum-containing anticancer drugs such as cisplatin, more preferably adriamycin, cyclophosphamide, For example, a combination ratio of a conventional anticancer agent and a mixture of carcinol C of 1: 0.1 to 10 times.

The pharmaceutical composition containing the compound according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition including the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, 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 ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and 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. The base of suppositories may be witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The preferred dosage of the compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The compounds of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

According to still another aspect of the present invention, there is provided a health functional food for prevention and improvement of cancer diseases comprising casinol C as an active ingredient. The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for prevention and improvement of cancer.

The above-mentioned casinol C is as described above.

The present invention relates to a method for the treatment of 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 cancer, thyroid cancer, pituitary cancer, adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, endometrial cancer, endometrial carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, (CNS) tumors, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. The present invention also encompasses a method of treating a patient suffering from chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cell carcinoma, kidney pelvic carcinoma, central nervous system At least one cancer disease selected from the group can be prevented and improved.

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with the Act on Health Functional Foods, and the nutrients can be regulated on the structure and function of the human body, ≪ / RTI > is intended to be taken for the purpose of obtaining a beneficial effect for health use such as action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards. Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like. For example, the health functional food in the form of tablets may be prepared by granulating a mixture obtained by mixing Casinol C, an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives in a usual manner, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary. The hard capsule may be prepared by filling a normal hard capsule with a mixture of caspase C, an active ingredient of the present invention, with an additive such as an excipient. The soft capsule may be prepared by mixing casinol C with excipients such as excipients And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of casinol C, an active ingredient of the present invention, excipient, binder, disintegrant and the like, according to a conventionally known method, and if necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of casinol C, an active ingredient of the present invention, excipient, binder, disintegrant and the like, into granules by a known method, and if necessary, adding a flavoring agent, And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

< Example  1> Casinol  Extraction and Separation of C

Air dried bark of peanut (Reference Sheet No. SPH 07002) (0.6 kg) was extracted with ethanol. The extract (51 g) was fractionated into hexane, ethyl acetate (EtOAc), chloroform (CHCl ₃ ) and butanol (BuOH) fractions. The ethyl acetate fraction (31 g) was subjected to silica gel column chromatography with a n-hexane / acetone gradient elution system (20: 1 - &gt; 1:10) to collect 11 fractions (see Non-Patent Document 6).

Fraction 7 was subjected to silica gel column chromatography using chloroform: methanol (100: 1 - &gt; 10: 1) to obtain 6 sub-fractions. Subfraction 3 of this fraction 7 was chromatographed on a RP-C18 column under a methanol gradient elution system (40% - &gt; 100%) to yield carbinol C (260 mg).

The purity was measured by HPLC and hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectroscopy. The structure of casinol C was determined by spectral analysis and confirmed by comparison with the existing literature (non-patent document 6).

The &lt; RTI ID = 0.0 &gt; Casinol C & As the active ingredient of kazinoki , the chemical structure of casinol C used in this experiment is shown in Chemical Formula 1 above.

[Experimental Preparation and Method]

1. Preparation of reagents

Dulbecco's Modified Eagle's Medium (DMEM), neomycon (G418) glucose-free DMEM, and fetal bovine serum (FBS) were purchased from Gibco / Invitrogen (Carlsbad, CA). Propidium iodide (Propidium iodide), 12-O- tetra-decanoyl phorbol 13-acetate (12- O -tetradecanoylphorbol-13-acetate , TPA), and 3- [4,5-dimethylthiazol-2- thiazolyl] - 2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies recognizing 5-aminoimidazole-4-carboxamidine-1-beta-D-ribofuranoside (AICAR) and AMPKa-Thr ¹⁷² and ACC-Ser ⁷⁹ were purchased from Cell Signaling Technology (Boston, MA). Α-actinin, poly (ADP-ribose) polymerase (PARP) and AMP-activated protein kinase (AMPK) α were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Compound C was purchased from Calbiochem (San Diego, CA).

2. Cell culture

HT-29 colon cancer cells (ATCC, Manassas, Va.) Were cultured in DMEM containing 10% heat-activated fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin at 37 ° C under 5% Lt; / RTI &gt;

3. Measurement of apoptosis and viability

The degree of apoptosis was measured using a fluorescence-activated cell sorter. Cells were treated with Cazinol C for 24 hours and cells were obtained by trypsin treatment and washed with phosphate buffered saline (PBS). The cells thus obtained were fixed with 70% ethanol and resuspended in phosphate buffered saline containing 10 μg / ml propidium iodide. Fluorescence intensity was measured with a FACSCanto ^™ II flow cytometer (BD Biosciences, San Jose, Calif.).

Cell viability was measured by MTT assay. Cells were treated with 5 ug / ml MTT solution and cultured for 3 hours. After the cells were dissolved in dimethyl sulfoxide (DMSO), the absorbance was measured at 570 nm.

4. In vitro cell growth and soft agar colony formation assay

Cells were inoculated into 6-well plates and treated with 0 to 60 uM concentration of casinol C for 24 hours. After obtaining the cells, the cell number was measured by trypan blue exclusion test. To evaluate anchorage-dependent, 1 x 10 ³ HT-29 cells were suspended in 1.5 ml of medium containing 0.3% agar, 10% fetal bovine serum and 15 uM casinol C, -well plate onto pre-solidified 0.5% agar. After incubation for 2 weeks, soft agar colonies were observed under a phase-contrast microscope and image data were obtained.

5. Wound recovery analysis

The cells were inoculated into 6-well plates and cultured in starvation medium for 12 hours. The cell monolayer was scratched with a sterile 200 uM pipette tip and washed with starvation medium to remove the separated cells. The cells were treated with 0 to 15 μM concentration of casinol C for 30 minutes and then cultured for 36 hours in each of 40 ng / ml TPA oil-inactivated state. The medium was replaced with phosphate buffered saline and image data were obtained using a phase contrast microscope.

6. Measurement of ex vivo cancer cell migration power

A 24-well transwell unit was analyzed for migration and invasion in vitro using a polycarbonate filter (Corning Costar, Cambridge, Mass.) Having 6.5 mm diameter and 8.0 micron pore size.

For the invasion assay, the bottom of the transwell was filled with DMEM supplemented with 10% fetal bovine serum as a chemoattractant and then assembled to function as an intrusion barrier. 5 × 10 ⁴ of the cells were suspended in serum-free DMEM together with Kazinol C, then attached to the upper part of the transwell, and cultured for 48 hours. The cells attached to the upper part of the transwell were wiped off with a cotton swab and the filter was fixed with a solution of 0.2% crystal violet / 10% methanol w / v).

6. Plasmid Transformation

A plasmid of pcDNA3-AMPK dominant-negative (DN) was prepared by the method described in the prior art (non-patent document 5). The plasmid was injected into HT-29 cells using PolyFect transfection reagent (Qiagen, Valencia, Calif.). The transformed cells were selected using complete medium containing 1 mg / ml neomycin.

7. Western blotting

For preparation of whole-cell lysate, the treated cells were dissolved in a solution of PRO-PREP ^™ Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea) at 4 ° C for 30 minutes. 14,000 g × 20 min, centrifuged at 4 ° C to obtain supernatant, and protein concentration was measured using Bradford Protein Assay.

The sample was prepared with 2-mercaptoethanol and then decomposed at 95 ° C for 3 minutes. The proteins were separated into 8-12% acrylamide gels and transferred to nitrocellulose membranes. The membrane was blocked with 1% bovine serum albumin or 5% nonfat milk and bound to the primary antibody. Protein bands were detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and a LAS-3000 or LAS-4000 imaging system (FUJIFILM Corporation, Tokyo, Japan) after binding with HRP-conjugated secondary antibodies . Western blot band strength was assessed using Quantity One software (Bio-Rad Laboratories, Hercules, Calif.).

8. Statistical Analysis

Experimental results were expressed as mean ± standard deviation. Statistical significance was determined by analyzing the data using GraphPad Prism 5 software. After one-way ANOVA, statistical significance was analyzed using the Newman-Keuls multiple comparison test between the three groups or the unpaired t- test between the two groups. P value <0.05 was considered statistically significant.

<Experimental Results>

One. Casinol  Cells of C Self-destruction  Acceleration

Cells cultured in complete medium were treated with casinol C for 24 hours to induce apoptosis of casinol C.

As a result of MTT analysis, no significant increase in cell death was observed in case of treatment with low concentration of casinol C (<30 μM), but HT-29 apoptosis was significantly increased in case of treatment with high concentration of casinol C (60 to 120 μM) (Fig. 2a).

In addition, the growth of HT-29 cells was significantly decreased in the case of treatment with Kajinol C 30 to 60 μM for 48 hours, whereas the cell growth was not affected in the case of treatment with Kajinol C 30 μM for 24 hours (FIG. 1 b) .

In order to confirm the cell death-inducing ability of casinol C, FACS analysis was performed on the sub-G1 DNA content. In case of treatment with low concentration of casinol C (<30 μM), apoptosis was not affected at 24 hours, whereas when caspase C 60 μM was treated, apoptosis was significantly increased (FIG. In addition, it was confirmed that PARP decomposition was significantly increased when treated with 60 μM of casinol C (FIG. 1d).

Taken together, it shows that casinol C is capable of effectively promoting apoptosis of HT-29 cells.

2. Casinol  Of C AMPK  Activity increasing action

The activity of AMPK activity was increased according to treatment time and treatment concentration of casinol C. As a result, casinol C markedly increased the Thr ¹⁷² phosphorylation and the Ser ⁷⁹ phosphorylation in the acetyl-Co A carboxylase (ACC) in the catalytic subunit of AMPK (FIG. 2, By treatment concentration). This means that casinol C activates AMPK.

3. Casinol  By C AMPK  Cells with increased activity With apostasy  relation

In order to investigate the relationship between casinol C and apoptosis, MTT analysis was performed after treatment with AMPK inhibitor Compound C or AMPK promoter AICAR.

Experimental results showed that when Compound C was treated to inhibit AMPK activation, apoptosis induced by casinol C was significantly decreased in a concentration-dependent manner (FIG. 3A). In contrast, AICAR, an AMPK promoter, did not significantly affect caspase-C apoptosis, but it is important that AICAR increases AMPK activity at that concentration.

Meanwhile, FACS analysis of sub-G1 DNA content or PARP degradation showed that compound C inhibited caspase-C induced apoptosis (FIGS. 3B and 3C).

Experimental results on cells transformed with pcDNA3 or AMPK dominant negative (DN) plasmid of HT-29 cells are shown in Fig. As a result, the AMPK activity was attenuated by the continuous expression of AMPK dominant negative form, and the AMPK-DN expression induced by caspase-C was abruptly decreased by AMPK-DN expression (FIG. 4A).

As shown in the FACS analysis for the content of sub-G1 (sub-G1) DNA, casinol C effectively promoted apoptosis, and expression of AMPK-DN type significantly attenuated casinol C-induced apoptosis 4b).

These results indicate that casinol C significantly increases the apoptosis of HT-29 cells through AMPK activation, which is important for promoting apoptosis.

4. Casinol  Of C HT -29 cell migration and Adhesion-independent  Growth inhibitory action

Cancer metastasis is a series of multiple processes involving migration, invasion, adhesion, proliferation, and angiogenesis. In order to confirm the action of casinol C on cancer cell metastasis activity, in vitro migration and wound healing ability analysis were performed.

As shown in Fig. 5A, when treated with Kajinol C 7.5 to 30 uM for 24 hours, HT-29 cell growth was not significantly affected. Treatment of casinol C for 24 hours significantly reduced wound healing (FIG. 5b).

Similar results were obtained in in vitro cell migration experiments (FIG. 5c). Since TPA typically promotes the migration of colon cancer cells, we observed the effect of inhibiting caspase-3 C cell migration. Interestingly, TPA treatment promoted wound healing and migration of HT-29 cells, whereas casinol C markedly inhibited TPA-induced wound healing and cell migration (FIGS. 5b and 5c).

As a metastatic potential evaluation index, the ability of the cells to form colonies in a semi-solid state medium was evaluated. TPA-treated HT-29 cells produced a significant number of colonies and increased cell proliferation in soft agar (Fig. 5d). On the contrary, the basal cell proliferation and TPA - induced cell proliferation were significantly decreased when treated with casinol C treatment.

In addition, AMPK inhibition through stable transformation of AMPK DN type significantly attenuated casinol C mediated cancer cell migration (Fig. 6).

Taken together, it can be seen that casinol C inhibits cell migration and non-adherent growth through AMPK activation in HT-29 colon cancer cells.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby . It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (11)

1. A pharmaceutical composition for preventing or treating colorectal cancer, comprising casinol C represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

. delete The method according to claim 1,
Wherein said casinol C is isolated from mulberry. delete The method according to claim 1,
The composition may also include one or more of cyclophosphamide, amsacrine, daunomycin, taxol, 5-FU, Doxorubicin, Mitomycin, Characterized in that it further comprises at least one selected from the group consisting of cisplatin, paclitaxel, docetaxel, irinotecan, Xeloda and oxalopatin. . The method according to claim 1,
Wherein said casinol C has an anticancer effect through inhibition of cell proliferation, apoptosis induction, cell migration inhibition, and nonadherent growth inhibition through the increase of AMPK activity. A health functional food for preventing or ameliorating colorectal cancer comprising casinol C represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

. delete 8. The method of claim 7,
Wherein said casinol C is isolated from mulberry. delete 8. The method of claim 7,
Wherein said food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods.

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