KR100701607B1 - Composition comprising cudraflavanone A isolated from Cudrania tricuspidata CARR. showing topoisomerase ? inhibiting activity for the prevention and treatment of cancer disease - Google Patents

Abstract

A cudraflavanone A isolated from Cudrania tricuspidata CARR. is provided to show topoisomerase I inhibiting activity and be able to induce apoptosis of tumor cell, thereby being used for medicines and health functional foods for preventing and treating cancer. The pharmaceutical composition for preventing and treating cancer comprises cudraflavanone A showing topoisomerase 1 inhibiting activity and inducing apoptosis of tumor cell, and a pharmaceutically acceptable salt thereof. The cudraflavanone A is represented by the formula(1) and isolated from Cudrania tricuspidata CARR.

Description

Composition comprising cudraflavanone A isolated from Cudrania tricuspidata CARR. showing topoisomerase Ⅰ inhibiting activity for the prevention and treatment of cancer disease}

1 is a diagram showing the effect of inhibiting topoisomerase I activity by Coudraflavanone A,

Figure 2 is a diagram showing the effect of killing the leukemia cell line U937 cell by Kudraflavanone A,

Fig. 3 shows the apoptosis result of leukemia cell line U937 cells by cudraflavanone A.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing cudraflavanone A, which is a compound isolated from the locust tree that inhibits topoisomerase I and induces apoptosis of cancer cells. It is about.

Cancer is one of the incurable diseases that humanity has to solve, and huge capital is invested in the development to cure it all over the world.In Korea, it is the number one disease death cause and more than 100,000 people a year It is diagnosed and about 60,000 or more die. Carcinogens, which cause cancer, are smoking, ultraviolet rays, chemicals, foods, and other environmental factors. However, various causes are not only difficult to develop a therapeutic agent, but also effective effects vary depending on the site of occurrence. Currently, the substances used as therapeutic agents are extremely toxic and cannot selectively remove only cancer cells. Therefore, there is an urgent need for the development of low-toxic and effective anti-cancer agents to prevent cancer after treatment. .

DNA replication must replenish many substances during S phase of the cell cycle, a process that is tightly regulated (Brush GS, et al., Proc . Natl . Acad . Sci . USA., 91 , pp12520-12524, 1994). During the S phase of the cell cycle, the DNA of the chromosome is accurately replicated and ready to be separated into daughter cells during mitosis. If DNA replication is interrupted by inhibitors or the template is damaged by radiation or other factors, signals are produced that can induce cell cycle inhibition or apoptosis (Pizer ES, et al. ., Cancer Res ., 58 , pp 4611-4615, 1998). SV40 in In vitro DNA replication systems are widely used as models for understanding nucleated DNA (eukaryotic DNA) replication and are also used in the analysis of anticancer agents (Snapka RM, et al . , Academic) . Press , London , pp 127-148, 1996; Ishimi Y, et al., Mol . Cell . Biol ., 12 , pp 4007-4014, 1992). The system uses host metabolic machinery for DNA replication with the viral protein T40 (virally encoded SV40-antigen, SV40 T-Ag) of SV40 (Stillman B, et al., Mol . Cell . Biol ., 5 , pp2051-2060, 1985). In replication, replication protein A is a single stranded DNA binding protein that mediates the release of SV-40 origin-containing DNA in the presence of SV40 T-Ag and topoisomerase. T-Ag interacts with DNA polymerase α-primease complexes, which are necessary to initiate SV40 DNA replication (Collins KL, et al., Mol . Cell . Biol ., 11 , pp 2108-2115, 1991 Melendy T, et al., J. Biol . Chem ., 268, pp 3389-3395, 1993). Once replication is initiated, extension of replication is performed by DNA polymerases I α and δ. Three S-specific anticancer agents currently widely used, 1-β-D-arafinofuranosylcytosine (1-β-D-arabinofuranosylcytosine, ara-C), camptothecin (CPT), and doxorubicin , DOX) is. the three different enzymes (DNA polymerase α, topoisomerase I and II) and the targeted (Abdel-Aziz W, et al ., Biochem. Pharmacol, 68, pp11-21 ,, 2004).

In some studies, topoisomerase I has been isolated as a target molecule of camptothecins such as topotecan and irinotecan among anticancer agents (Hsiang YH, et al., Cancer Res ., 48 , pp 1722-1726, 1988).

Topoisomerase is classified into two types according to the DNA strand cleavage method. Type I topoisomerase I changes the phase of DNA after instantaneous single-strand nick on the DNA substrate. Type II topoisomerase II changes the phase of DNA after cutting all the DNA double strands. During cleavage of DNA by topoisomerase I, tyrosine residues in the topoisomerase I active site act as nucleophiles to break the phosphodiester bond of the DNA backbone and Phosphotyrosyl linkages and free 5'-hydroxyl groups recombine to tyrosine residues, which are nucleophiles, to return DNA to its original state. In this way, intracellular topoisomerase I functions to remove the torsional pressure that accumulates in the DNA molecule during replication and transcription.

The study of DNA topoisomerase has influenced pharmacology and clinical medicine by identifying bacterial gyrases that target antibiotics and eukaryotic topoisomerases I and II that target anticancer agents. . The mechanism by which the topoisomerase I inhibitor can be used as an anticancer agent is because the DNA cut by topoisomerase I prevents recombination and eventually induces apoptosis of cancer cells. Drugs such as camptothecin (CPT), topotecan (TPT), and SN-38 (an active metabolite of irinotecan) are well known topoisomerase I inhibitors, including thymocytes and leukemia. Apoptosis in cells such as), cortical neurons, etc., and is known to show various anticancer effects in clinical or preclinical stages.

Meanwhile Cudrania tree used in the present invention ( Cudrania tricuspidata CARR.) is a deciduous broad-leaved arborescent or shrub, and the foliage has morin, kaferferol-7-glucoside, that is, populnin, statachydrine and proline. Contains glutamic acid, arginine, and aspartic acid.Anti-inflammatory, analgesic and buccal and inflammatory, eczema, mumps, pulmonary tuberculosis, chronic back pain, bruises, and joint acute sprains. it is known that the effects (positive boseop, new mingyo, illustrations hyangyak (herbal) Dictionary, Younglim four, pp.544-545, 1998).

However, in all of the above documents, the Kudraflavanone A (2S-2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) -6-prenyl flavanone) compound isolated from the Koji mulberry tree There is no teaching or disclosure that it can be used as a composition for the prevention and treatment of cancer diseases by inhibiting topoisomerase I in mammals and inducing apoptosis.

Therefore, the present inventors completed the present invention by confirming that kudraflavanone A inhibits the activity of topoisomerase I derived from calf thymus and induces apoptosis of leukemia cell line U937.

The present invention relates to Cudrania which induces apoptosis of cancer cells by inhibiting topoisomerase I. A pharmaceutical composition and a dietary supplement for the prevention and treatment of cancer diseases containing Cudraflavanone A having proliferation inhibitory activity isolated from tricuspidata CARR.) are provided.

In order to achieve the above object, the present invention comprises a kudraflavanone A of formula (1) or a pharmaceutically acceptable salt thereof that induces apoptosis of cancer cells by inhibiting topoisomerase I It provides a pharmaceutical composition for the prevention and treatment of cancer diseases.

(1)

(One)

The cancer disease includes a general cancer disease, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer , Anal leiomyoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, One or more diseases selected from brain stem glioma, pituitary adenoma, and the like.

Specifically, the Cudraflavanone A of the present invention contains Cudraflavanone A as an active ingredient that induces apoptosis of cancer cells by inhibiting topoisomerase I, and includes a pharmaceutically acceptable carrier, Provided are pharmaceutical compositions for the prevention and treatment of cancer diseases, including diluents or excipients.

Kudraflavanone A of the present invention is a deciduous tree of small mulberry family old tree mulberry (Maclura tricuspidata CARR.).

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

For example, after drying and cutting the bark of Koji mulberry root, a volume of water of about 2 to 20 times the sample weight and a polar solvent of C ₁ to C ₄ lower alcohols such as methanol, ethanol and butanol or the like A mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably extracted with chloroform for 1 hour to 15 days, preferably 3 to 10 days, and the extract is loaded on a column filled with silica gel. Chloroform: eluted by increasing the polarity sequentially in the developing solvent of methanol (30: 1 → 1: 1), and then eluted fraction was identified by TLC, divided into 11 fractions (F1-F11), concentrated under reduced pressure, and fraction 6 Was subjected to column chromatography by gradually increasing the polarity (20: 1 → 1: 1) under hexane: ethyl acetate (EtOAc) condition, dividing it into 80 subfractions, Fractions 31 to 36 Concentration under reduced pressure can yield the compound Coudraflavanone A of the present invention.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the kudraflavanone A isolated from the Koji mulberry tree obtained by the above production method as an active ingredient.

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, and citric acid may be used as the organic acid. , Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, 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).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

The pharmaceutical composition for preventing and treating cancer of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may 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 at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the compound and a food acceptable food supplement additive exhibiting a prophylactic effect of cancer disease. Examples of the food to which the compound of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing cancer diseases. At this time, the amount of the compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the compound of the present invention.

In addition to the above, the compounds of the present invention include 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. In addition, the compounds of the present invention may contain flesh 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 usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example  One. From old mulberry Kudraflabanon  Separation of A

After drying the bark (3kg) of Koji mulberry roots and cutting it, extracted with chloroform for 1 week, the solvent was concentrated under reduced pressure. Chloroform concentrate (86 g) was loaded on a column (760 cm) filled with silica gel (silica gel, 230-400 mesh, Merk) to sequentially increase the polarity in the developing solvent of CHCl ₃ : CH ₃ OH (30: 1 → 1: 1) eluted. The eluted fractions were identified by TLC, eleven fractions (F1-F11) and concentrated under reduced pressure. Fraction 6 (F6) was subjected to column chromatography by gradually increasing the polarity (20: 1 → 1: 1) under hexane: EtOAc conditions, and divided into a total of 80 subfractions. Cudraflavanone A (2S-2 ', 5,7-trihydroxy-4', 5 '-(2,2-dimethylchromeno) having the following physical properties represented by the formula (1) by concentrating -36 under reduced pressure -6-prenyl flavanone) 32 mg was obtained.

(1)

(One)

1) Molecular Formula: C ₂₅ H ₂₆ 0 ₆

2) Molecular Weight: 422

3) Melting Point (m.p.): 197 ℃

4) Natural Light Rotational Speed: [α] _D ²⁰ = -102.8 ° (c = 1.00, CHCl ₃ )

5) Infrared (IR) spectrum absorption maxima (cm ^-1 ): 3430, 2973, 2931, 1635, 1499, 1450

6) UV spectrum absorption maximum (nm): UV (λ _max CHCl ₃ ): 291, 338

7) ¹ H-NMR (500 MHz, CDCl ₃ , ppm): 1.41 (6H, s), 1.76 (3H, s), 1.81 (3H, s), 2.87 (1H, dd, J = 17.3, 3.0 Hz), 3.13 (1H, dd, J = 17.3, 12.7 Hz), 3.35 (2H, d, J = 7.1 Hz), 5.25 (1H, t, J = 7.1 Hz), 5.49 (1H, d, J = 9.8 Hz), 5.56 (1H, dd, J = 12.6, 2.9 Hz), 6.01 (1H, s), 6.25 (1H, d, J = 9.8 Hz), 6.33 (1H, s), 6.88 (1H, s)

8) ¹³ C-NMR (125 MHz, CDCl ₃ , ppm): 17.9, 21.2, 25.8, 28.1, 28.1, 41.9, 76.7, 77.1, 95.7, 103.0, 104.9, 107.5, 114.8, 116.4, 121.3, 121.5, 124.7, 128.5 , 135.8, 154.6, 154.6, 160.5, 161.4, 163.7, 196.4

9) ¹ H- ¹ H COSY spectrum signal group (ppm): To the ¹ H- ¹ H COSY spectrum major peak cross (cross peak in the nucleus on) are shown in Table 1 below.

¹ H- ¹ H COSY main peak cross (cross peak) of the spectrum of the nuclear Reference Core (ppm) Cross peak (ppm) 1.76 (C ₄ -H) 3.35 (C ₁ -H), 5.25 (C ₂ -H) 1.81 (C ₅ -H) 3.35 (C ₁ -H), 5.25 (C ₂ -H) 2.87 (C ₃ -H _a ) 3.13 (C ₃ -H _b ), 5.56 (C ₂ -H) 3.13 (C ₃ -H _b ) 2.87 (C ₃ -H _a ), 5.56 (C ₂ -H) 3.35 (C ₁ -H) 1.76 (C ₄ -H), 1.81 (C ₅ -H), 5.25 (C ₂ -H) 5.25 (C ₂ -H) 1.76 (C ₄ -H), 1.81 (C ₅ -H), 3.35 (C ₁ -H) 5.56 (C ₂ -H) 2.87 (C ₃ -H _a ), 3.13 (C ₃ -H _b ) 5.49 (C ₂ -H) 6.25 (C ₁ -H) 6.25 (C ₁ -H) 5.49 (C ₂ -H)

10) ¹ H- ¹³ C COSY Spectrum Signal Group (ppm): The major nuclear cross peaks on the ¹ H- ¹³ C COSY spectrum are shown in Table 2 below.

Major nuclear cross peak on ¹ H- ¹³ C COSY spectrum Carbon core (ppm) Cross peak observed proton nucleus (ppm) 17.9 (C ₅ ) 1.41 (C ₅ -H) 21.2 (C ₁ ) 5.25 (C ₁ -H) 25.8 (C ₄ ) 1.41 (C ₄ -H) 28.1 (C ₄ , ₅ ) 1.76 (C ₄ -H), 1.81 (C ₅ -H) 41.9 (C ₃ ) 2.87 (C ₃ -H _a ), 3.13 (C ₃ -H _b ) 76.7 (C ₂ ) 5.56 (C ₂ -H) 95.7 (C ₈ ) 6.01 (C ₈ -H) 104.9 (C ₃ ) 6.33 (C ₃ -H) 121.3 (C ₂ ) 5.25 (C ₂ -H) 121.5 (C ₁ ) 6.25 (C ₁ -H) 124.7 (C ₆ ) 6.88 (C ₆ -H) 128.5 (C ₂ ) 5.49 (C ₂ -H)

11) HMBC Spectrum Signal Group (ppm): The cross peaks of the major nuclei on the HMBC spectrum are shown in Chemical Formulas 2 and 3.

 Cross peak of major nucleus on HMBC spectrum 4th carbon core (ppm) Cross peak observed proton nucleus (ppm) 77.1 (C ₃ ) 6.25 (C ₁ -H), 1.41 (C ₄ -H) 103.0 (C _4a ) 2.87 (C ₃ -H _a ), 3.13 (C ₃ -H _b ), 6.01 (C ₈ -H) 107.5 (C ₆ ) 3.35 (C ₁ -H), 5.25 (C ₂ -H), 6.01 (C ₈ -H) 114.8 (C ₅ ) 6.25 (C ₁ -H), 6.33 (C ₃ -H) 116.4 (C ₁ ) 5.56 (C ₂ -H), 6.88 (C ₆ -H) 135.8 (C ₃ ) 3.35 (C ₁ -H), 1.76 (C ₄ -H) 154.6 (C ₄ ) 6.25 (C ₁ -H), 6.33 (C ₃ -H) 154.6 (C ₂ ) 5.56 (C ₂ -H), 6.33 (C ₃ -H), 6.88 (C ₆ -H) 160.5 (C _8a ) 5.56 (C ₂ -H) 161.4 (C ₅ ) 3.35 (C ₁ -H) 163.7 (C ₇ ) 3.35 (C ₁ -H), 6.01 (C ₈ -H) 196.4 (C ₄ ) 5.56 (C ₂ -H), 2.87 (C ₃ -H _a ), 3.13 (C ₃ -H _b )

Experimental Example  One. Kudraflabanon  A ( Cudraflavanone  Determination of Activity Inhibitory Effect on Topoisomerase I from Calf Thymus of A)

10 mM Tris-HCl (pH 7.5), 10 mM KCl, 4 mM MgCl ₂ , 1 µg of ultra-stranded pUC118 DNA and topoisomerase I were mixed in a buffer containing 0.1 mM EDTA, 10 µg / ml bovine serum albumin, and incubated at 30 ° C. for 1 hour. It was. In some cases, an appropriate amount of the compound was added. After the reaction, 1/4 volume of stop buffer (50 mM EDTA), 1% SDS, 50% glycerol, 0.05% bromophenol blue was added, followed by 0.7% agarose. Electrophoresis was performed on the gel. After electrophoresis gels were stained with ethidium bromide (0.5 μg / ml), pUC118 DNA was observed under ultraviolet light of 300 nm.

As a result of measuring the activity of topoisomerase I using supercoil pUC118 DNA as a substrate, as shown in FIG. 1, topoisomerase I was compared with the case where no topoisomerase I was added (lane 1). When the reaction was performed (lane 2), a relaxed form of DNA appeared. When 0.1-1.2 mM Cudraflavanone A was added (lanes 3-7), the inhibition of topoisomerase I activity was increased in proportion to the concentration of 50% at a concentration of about 0.4 mM. Topoisomerase I inhibitory effect.

Experimental Example  2. Human leukemia leukemia ) Cell line U937 For Kudraflabanon  Apoptosis effect experiment of A (Cudraflavanone A)

100 µl of the medium was added to a 96-well plate and 1 × 10 ⁴ U937 cells, which are human leukemia cells, were incubated for 16 hours, and the kudraflavanone A obtained in Example 1 was obtained. 5-30 μM was added and cultured in a 37 ° C., 5% CO ₂ incubator. After 48 hours, 20 μl of MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium] solution was added per well and 37 ° C. After standing for 2 hours in a 5% CO ₂ incubator, the absorbance was measured by an ELISA reader.

As a result of examining cancer cell killing ability by Cudraflavanone A, about 50% of U937 cells were killed at the drug concentration of 15 μM as shown in FIG. 2.

Experimental Example  3. Human leukemia leukemia ) Cell line U937 For Kudraflabanon  Of A (Cudraflavanone A) Apoptosis ( apoptosis Effect experiment

Topoisomerase I inhibitors, commonly used as anticancer agents, have been reported to kill cancer cells by apoptosis. Therefore, experiments were conducted to investigate whether apoptosis by cudraflavanone A was caused by apoptosis. The degree of apoptosis was measured because poly (ADP-ribose) polymerase (PARP) cleavage characteristically occurs when cells die from apoptosis. U937 cells treated with Cudraflavanone A obtained in Example 1 and U937 cells treated for 48 hours were collected and lysed, respectively, and cell extracts were prepared and electrophoresed on an SDS-polyacrylamide gel. The separated protein bands were transferred to a nitrocellulose membrane, and then Western blotting was performed with an antibody against poly (ADP-ribose) polymerase.

As shown in FIG. 3, 89 kDa fragments from which poly (ADP-ribose) polymerase (PARP) was cleaved were detected in cells treated with kudraflavanone A as compared to the control. This result indicates that kudraflavanone A kills U937 cells by apoptosis.

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

Cudraflavanone A 20 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 Tablets

Cudraflavanone A 10 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. Manufacture of capsule

Cudraflavanone A 10 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 Injectables

Cudraflavanone A 10 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. Liquid  Produce

Cudraflavanone A 20 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 and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of health drinks

Cudraflavanone A 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

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 by sterilization in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

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

As mentioned above, the compound of the present invention, kudraflavanone A, isolated from gujippongna or radish, exhibits topoisomerase I inhibitory effect and thus induces apoptosis of cancer cells. It can be used as a pharmaceutical composition and health functional food for prevention and treatment.

Claims (5)

Cancer disease comprising Cudraflavanone A of Formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient that exhibits topoisomerase I inhibitory effect and induces apoptosis of cancer cells Pharmaceutical composition for the prevention and treatment of:

(One) The method of claim 1, wherein the compound is Maclura tricuspidata CARR.). The method of claim 1, wherein the cancer disease is gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer , Fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , Prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem glioma or Pharmaceutical composition comprising pituitary adenoma disease. delete delete

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