KR20100076614A - A composition comprising eupafolin isolated from artemisia princeps pampanini for preventing and treating cancer disease - Google Patents

Abstract

PURPOSE: A composition containing eupafolin isolated from Artemisia princeps Pampanini is provided to suppress proliferation of cancer cells by inducing cancer cell apoptosis. CONSTITUTION: A pharmaceutical composition for preventing and treating cancer contains eupafolin of structural formula 1 as an active ingredient, which is isolated from Artemisia princeps Pampanini. The composition contains 0.1-50 weight% of Artemisia princeps Pampanini extract. The Artemisia princeps Pampanini extract is isolated using water, low alcohol of C1-C4, or mixture solvent thereof. A health food for preventing and treating cancer contains eupafolin as an active ingredient. The health food is used in the form of powder, granule, tablet, capsule, or beverage.

Description

A composition comprising eupafolin isolated from Artemisia princeps Pampanini for preventing and treating cancer disease} which contains eupapoline [ｅｕ 〔ａｆｏｌｉｎ] isolated from lion cucurbits as an active ingredient

The present invention provides a composition for the prevention and treatment of cancer diseases, containing as an active ingredient opapaline isolated from Artemisia princeps Pampanini.

Kalechman et al., Synergistic anti-tumoral effect of paclitaxel (Taxol) + AS101 in a murine model of B16 melanoma: association with ras-dependent signal-trasduction pathways., Int. J. Cancer , 86 , pp. 281-288, 2000

Gamet-Payractre et al., Sulforaphane a naturally occurring isothiocyanate, induced arrest and apoptosis in HT29 human colon cancer cell. Cancer Res ., 60 , pp. 1426-1433, 2000

Piao et al., Induction of G (2) / M phase arrest and apoptosis by a new synthetic anti-cancer agent, DW2282, in promyelocytic leukemia (HL-60) cells. BiochemPharmacol , 62 , pp. 1439-1447, 2001

Nagata et al., Degradation of chromosomal DNA during apoptosis., Cell Death Differ , 1 , pp. 108-116, 2004

[Document 5] Orrenius S et al., Mitochondrial oxidative stresses: implications for cell death. A nnu. Rev. Pharmacol. Toxicol , 47 , pp.19.1-19.41, 2007

Kim R., Recent advances in understanding the cell death pathways activated by anticancer therapy. Cancer , 103 , pp.1551-1560, 2005

Jiang X et al., Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway, Science , 299 , pp.223-226, 2003

Kaufmann et al., Specific proteolytic cleavage of poly (ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis, Cancer Research , S3 , pp.3976-3985, 1993

Ikai et al., Induction of apoptosis, p53 and heme oxygenase-1 by cytotoxic prostaglandin Δ ¹² -PGJ ₂ in transformed endothelian cells, Prostaglandins, Leukotrienes, and Essential Fatty Acids , 58 (4) , pp.295- 300, 1998

Vispe et al., A cellular defense pathway regulating transcription through poly (ADP-ribosyl) ation in response to DNA damage, PNAS , 97 (18) , pp.9886-9891, 2000

11 Kelloff et al., Progress in cancer chemoprevention., Annals New York Academy of Sciences, 889 , pp.1-13, 1999

12, Tan, RX, et al., Biologically active substances from the genus Artemisia, Planta Medica, 64 , pp. 295-302, 1998

[13] Ryu SN., Et al., Quantitative analysis of eupatilin and jaceosidin in Artemisia herba, Kor. J. Crop Sci. , 49 , pp.452-456, 2004

[14] Xiaoyi W., et al., Phenolic constituents from Mikania micrantha, Biochem. System. Ecol. , 32 , pp.1091-1096, 2004

Carmichael et al., Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemisensitivity testing. Cancer Research , 47 , pp. 944-946, 1987

Oberhammer et al., Apoptotic death in epithelial cells: cleavage of DNA to 300 and / or 50 kb fragments prior to or in the absence of internucleosomal fragmentation, The EMBO Journal , 12 (9) , pp.3679-3684. , 1993

17, Kim, YH, et al., Expression of the murine homologue of the cell cycle control protein p32cdc2 in T lymphocytes. Journal of Immunology, 149 , pp.17-23, 1992

[18] Luo, Y et al., Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobio l. 66 , p. 479, 1997

Sambrook J., et al., Molecular Cloning Laboratory Manual, 2nd ed ., Pp. 18.60-18.71, 1989

Zamzami N., et al., Reduction of mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med ., 181 , pp. 1661-1672, 1995

21 Von Ahsen O., et al., The 'harmless' release of cytochrome c . Cell Death Differ ., 7 , pp. 1192-1199, 2000

Li P., et al., Cytochrome c and dATP-dependent formation of Apaf-1 / caspase-9 complex initiates an apoptotic protease cascade, Cell , 91 , pp. 479-489, 1997

Reed J., Bcl-2 and regulation of programmed cell death. J Cell Biol., 124 , pp . 1-6, 1994

24. Oltvai ZN., Et al., Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74 , pp. 609-619, 1993

Cancer is one of the incurable diseases that humanity has to solve, and huge capital has been invested in the development to cure it all over the world.In Korea, it is the number one disease cause of death among Koreans since 1983. More than 100,000 have been diagnosed, and more than 60,000 have died. Carcinogens, which are triggers of cancer, include smoking, ultraviolet rays, chemicals, food, and other environmental factors. However, due to various causes, the development of therapeutic agents is difficult, and the effects of the therapeutic agents also vary depending on the site of occurrence.

Currently used anticancer agents include biological preparations such as enzyme preparations or vaccines, pure synthetic medicines, and medicines derived from natural products. Among them, anticancer drugs using genes, enzymes, vaccines, etc. are not in a practical stage and are used by chemotherapy. The developed anticancer drugs have considerable toxicity and have side effects of destroying not only cancer cells but also normal cells because they cannot selectively remove only cancer cells, and recently, cancer cells have become resistant to this and are ineffective for treating cancer. . Therefore, there is an urgent need to develop an effective anticancer agent that is less toxic for preventing cancer and develops cancer cells.

Apoptosis is an important mechanism of action in which most anticancer agents exhibit the antiproliferative effect of cancer cells (Kalechman et al., Synergistic anti-tumoral effect of paclitaxel (Taxol) + AS101 in a murine model of B16 melanoma: association with ras) -dependent signal-trasduction pathways.Int. J. Cancer , 86 , pp.281-288, 2000; Gamet-Payractre et al ., Sulforaphane a naturally occurring isothiocyanate, induced arrest and apoptosis in HT29 human colon cancer cell . , 60 , pp.1426-1433, 2000), which refers to active cell death regulated by genes and plays an important role in maintaining a balance between cell proliferation and cell death in vivo (Piao et al . , Induction of G (2) / M phase arrest and apoptosis by a new synthetic anti-cancer agent, DW2282, in promyelocytic leukemia (HL-60) cells.BiochemPharmacol , 62 , pp. 1439-1447, 2001). During the apoptosis process, cells undergo a series of processes that lead to enrichment of chromatin in the nucleus, condensation of the cytoplasm, fragmentation of DNA, fragmentation of the nucleus, and formation of apoptotic bodies (Nagata et al. , Degradation of chromosomal DNA during apoptosis.Cell Death Differ , 1 , pp. 108-116, 2004). These apoptosis pathways can be divided into extrinsic pathways and intrinsic pathways. First, the death-inducing signaling complex (DISC) is formed in the exogenous pathway, ie, the cell death pathway via the death receptor, resulting in the activation of procaspase-8 (Orrenius). S et al., Mitochondrial oxidative stresses: implications for cell death.Annu . Rev. Pharmacol. Toxicol. 47 , 19.1-19.41, 2007). Activated caspase-8 directly activates procaspase-3 to target proteins (eg PARP-1: poly (ADP-ribose) polymerase-I, ICAD: inhibitor of caspase- activated DNase) to cut apoptosis (type I) (Kim R., Recent advances in understanding the cell death pathways activated by anticancer therapy. Cancer, 103 , pp. 1551-1560, 2005). However, in many cells, caspase-8 cleaves Bcl-2 homology domain 3 interacting domain death agonist (Bcl-2), a member of the Bcl-2 (B-cell lymphoma 2) protein family, into tBid (truncated Bid). tBid migrates to the mitochondrial outer membrane (type II). In addition, Bax (Bcl-2-associated X protein) in the cytoplasm, mitochondrial outer membrane, and Bak (Bcl-2 antagonist killer) in the mitochondrial outer membrane and endoplasmic reticulum are also inserted into the mitochondrial outer membrane. tBid and Bak or Bax and Bak are polymerized and inserted into the mitochondrial outer membrane to increase the outer membrane permeability of the mitochondria. As a result, cytochrome c between the outer membrane and the inner membrane of the mitochondria is released into the cytoplasm to form an apoptosome complex. In the presence of dATP, the apoptosome complex, consisting of cytochrome c , Apaf-1 (apoptosis activating factor-1) and procaspase-9, is a low-level procaspase- Activate 3 to induce cell death. Second, the intrinsic pathway causes the death signal to act on the mitochondria, releasing cytochrome c , and to form an apoptosome complex in the cytoplasm as described above. This pathway is regulated by the Bcl-2 family of proteins, inhibitors of apoptosis proteins (IAPs), second mitochondrial activator of caspases (Smac) / Diablo, and high-temperature requirement protein A2 (HtrA2) / Omi. Apoptosome complexes are also regulated by proteins such as oncoprotein prothymosin-α (Pro-T) and tumor suppressor putative HLA-DR-associated protein (Jiang X et al., Distinctive roles). of PHAP proteins and prothymosin-alpha in a death regulatory pathway, Science, 299 , pp.223-226, 2003).

The caspase that is initially activated by DNA damage is procaspase-2. Procaspase-2 forms a PIDDosome complex consisting of PID53 (p53-inducible protein with a death domain), RAIDD (RIP-associated Ich-1 / Ced-3-homologoes protein with a death domain), Cytochrome c is released into the cytoplasm to induce apoptosome formation. In other words, the cascade of caspase cascade and subsequent intracellular proteins leads to cell death.

PARP (116 kDa), known as the most representative substrate of caspases, is characterized by 85 kDa carboxyl fragments and 28 kDa amino terminal fragments by the activity of caspase-3 protease. Kaufmann et al ., Specific proteolytic cleavage of poly (ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis, Cancer Research, S3 , pp.3976-3985, 1993. PARP is an enzyme present in the nucleus of many organs, including the brain, which is activated when DNA is damaged, which not only repairs DNA but also participates in cell differentiation and gene expression (Ikai et al ., Induction of apoptosis, p53 and heme oxygenase-). 1 by cytotoxic prostaglandin △ ¹² -PGJ ₂ in transformed endothelian cells, Prostaglandins, Leukotrienes, and Essential Fatty Acids, 58 (4) , pp.295-300, 1998; Vispe et al ., A cellular defense pathway regulating transcription through poly ( ADP-ribosyl) ation in response to DNA damage, PNAS , 97 (18) , pp.9886-9891, 2000).

There are many methods for cancer treatment, including chemotherapy, radiation therapy, surgical therapy, and gene therapy, but chemotherapy using drugs is most commonly used. However, chemotherapy has many side effects and is not easily cured, requiring a new approach to cancer treatment. Two approaches are being attempted. The first is the synthesis and development of new derivatives that maintain the efficacy of existing anticancer drugs through various synthetic methods, and the side effects are significantly reduced, and the second is to prevent malignant cancer by inhibiting cancer development itself or delaying or reversing cancer progression. It is chemical chemoprevention that inhibits progression. In recent years, many countries have been paying attention to develop nontoxic and efficacious chemical cancer preventive and anticancer agents from natural resources such as vegetables, fruits and medicinal plants (Kelloff et al., Progress in cancer chemoprevention., Annals New York). Academy of Sciences, 889 , pp.1-13, 1999). Chemical cancer prevention is primarily intended to prevent cancer from appearing in healthy individuals and secondary prevention, malignancy, metastasis and complications to reverse the carcinogenic process in people with benign cancer. It can be applied step by step as a third prevention to prevent recurrence in people who have been treated. Therefore, it can be very useful as a therapeutic agent as well as cancer prevention.

Artemisia is a perennial herbaceous plant of the compositae, which is distributed in Korea, Asia and Europe, and is known to have about 350 species, of which 300 are native to Korea. Mugwort is divided into those that can be used for food, those that can be used for medicinal use, both edible and medicinal. Artemisia princeps Pampanini is wild throughout the country and is also called fortified wormwood. The leaves are broad and dark green, and split into lion-shaped leaves, and the nodes are known to give off their unique scent. The components of lion cucurbita are eupatilin, eupicin, apigenin, eupafolin, 6-methoxy 5, 7, 30, 40-tetra hydroxy flavone, amylase, choline, It contains aldehydes, eucalyptol, essential oils (65 kinds such as cineol, tricycline, terpinene, bornole, pinene), vitamins A, B, C, protein, calcium, magnesium, iron, potassium and phosphorus It is known to have superior medicinal effects compared to regular mugwort, and its pharmacological effects include anti-malarial, antiviral, anti-oxidant and anti-ulcer as well as anti-inflammatory, analgesic, cardiac and antitussive effects. anti-ulcerogenic effects are known (Tan, RX, et al., Biologically active substances from the genus Artemisia ., Planta Medica, 64 , pp. 295-302, 1998).

However, none of the above documents teaches or discloses that eupapoline isolated from lion cucurbita of the present invention can be used as a composition for preventing and treating cancer diseases by inhibiting the proliferation of cancer cells by inducing apoptosis of cancer cells.

Since the substances used as therapeutic agents are quite toxic and cannot selectively remove only cancer cells, there is an urgent need for the development of low-toxic and effective natural anticancer drugs to prevent the development of cancer as well as its treatment after the occurrence of cancer. Do.

Accordingly, the present inventors have completed the present invention by confirming that the yupapoline isolated from lion cucurbita exhibits an excellent anticancer effect of inhibiting the proliferation of cancer cells by inducing apoptosis of cancer cells.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases, containing as an active ingredient opapaline isolated from Artemisia princeps Pampanini .

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the eupapoline of the following structural formula (1) isolated from Artemisia princeps Pampanini as an active ingredient.

(1)

(One)

Cancer diseases as defined herein include 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, colon cancer, small intestine cancer, rectal cancer, Anal familial cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, 1 Primary CNS lymphoma, spinal cord tumor, brain tumor, brain stem glioma or pituitary adenoma, etc., preferably colon cancer, cervical cancer, ovarian cancer, uterine cancer, liver cancer, lung cancer, brain tumor, brain stem glioma, bladder cancer or leukemia. Preferably including cervical cancer, ovarian cancer, cervical cancer, lung cancer, or a brain tumor.

Hereinafter, the method for obtaining the compound of the present invention will be described in detail.

The compound of the present invention is a dried lion cucurbita, preferably sliced lion cucurbita, about 1 to 20 times the dry weight of water, preferably about 3 to 15 times the water, C ₁ to C ₄ lower alcohols or a mixture thereof Cold extraction, hot water extraction with a solvent, preferably water or methanol, at room temperature (20-100 ° C., preferably 20-30 ° C.) for about 1 hour to 10 days, preferably about 20 hours to 30 hours. In the first step to obtain a crude extract by repeated extraction 1 to 10 times, preferably 2 to 7 times obtained by using an extraction method, such as ultrasonic extraction, reflux cooling extraction, preferably reflux cooling extraction method ; Suspending the crude extract in water and then extracting with ethyl acetate and water, respectively; A third step of diluting the ethyl acetate solvent extract obtained in the second step with n -hexane: ethyl acetate (1-10: 1 (v / v)) by elution solvent to thin layer chromatography to 20 fractions; A fourth step of dividing the sixteenth fraction from the fractions of the three steps into chloroform: methanol (20-40: 1 (v / v)) using silica gel column chromatography as an eluting solvent and dividing it into 12 fractions; A fifth step of purifying the sixth fraction of the fraction of the fourth step by performing chromatography with methanol: water (1-3: 1 (v / v)) as an eluting solvent; The compound of the present invention can be obtained through the sixth step of preparing the separation step by the five step ODS column chromatography to obtain the eupapoline.

       The pharmaceutical composition for the prevention and treatment of cancer, which contains the eupapoline isolated from lion cucurbita of the present invention as an active ingredient, the compound comprises 0.1 to 50% by weight 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.

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.

The pharmaceutical composition for treating cancer containing the compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the invention may be used in the form of their pharmaceutically acceptable salts, or 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 of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be used. Carriers, excipients and diluents which 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 Silicates, 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, etc. 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 compositions of the present invention vary depending 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, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. 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 composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intra cerebroventricular injections.

The present invention provides a dietary supplement for the prevention and improvement of cancer diseases, containing as an active ingredient opapaline showing the preventive effect of cancer diseases.

Examples of the food to which the compound can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

In addition, the compounds of the present invention can be added to food or beverages for the purpose of preventing and improving 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 essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. 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 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

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 colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. 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.

As described above, eupafolin isolated from the Artemisia princeps Pampanini of the present invention exhibits an excellent anticancer effect of inhibiting the proliferation of cancer cells by inducing apoptosis of cancer cells. Can be used.

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, and the content of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example 1 Isolation of Euphapoline from Lion Wormwood

2.4kg of dried lion footwort used in this experiment was purchased from Ganghwa-gun Agricultural Technology Center and pulverized to a particle size of 30 mesh or less using a grinder, and 15 liter of 80% methanol solution was added to room temperature (24 After reflux extraction under reflux for 24 hours, the residue was further extracted twice in the same manner and then combined. The extract was filtered and freeze-dried to obtain crude extract of lion cucurbita (137 g).

Suspension extract of lion cucurbita obtained by the above method (137 g) was suspended in water (2 L) and placed in a separatory funnel, and then ethyl acetate (2 L × 2 times) was added thereto, followed by shaking. Concentration yielded ethyl acetate soluble extract (47 g), and water soluble extract (90 g) of lion cucurbita.

Sodium mugwort ethyl acetate soluble extract (45 g) obtained by the above method was placed on a silica gel column and n -hexane: ethyl acetate (7: 1-> 5: 1-> 3: 1 1> 1: 1 (v / v). Chromatography using)) as the elution solvent, the same phase in thin layer chromatography was combined, concentrated and divided into 20 fractions (1-20). The sixteenth fraction, 16 (1.53 g), was separated from silica gel column chromatography using chloroform: methanol (30: 1 (v / v)) as an eluting solvent. Divided by). The sixth fraction, 16-6 (558 mg), was purified from ODS column chromatography using methanol: water (2: 1 (v / v)) as an eluent to obtain Compound 1 (57 mg). As a result of the analysis, it was confirmed that it is eupapoline (eupafolin; 6-methoxy 5, 7, 30, 40-tetra hydroxy flavone) having the following physical properties (Ryu SN., Et al., Quantitative analysis of eupatilin and jaceosidin in Artemisia herba, Kor. J. Crop Sci. , 49 , pp.452-456, 2004; Xiaoyi W., et al., Phenolic constituents from Mikania micrantha, Biochem.System.Ecol . , 32 , pp.1091-1096, 2004).

Yellow powder;

R _f = 0.52 (chloroform: methanol = 5: 1); R _f = 0.63 (acetone: water = 2: 1);

M.P. 272-274 ° C;

IR (KBr, cm- ¹ ): 3410, 1662, 1601;

EI / MS m / z (70 eV): 316 [M] ⁺ , 301, 298, 273, 167;

¹ H-NMR (400 MHz, C ₅ D ₅ N, δ _H ): 7.31 (1H, s, H-2 '), 7.30 (1H, d, J = 8.0 Hz, H-6'), 6.85 (1H , d, J = 8.0 Hz, H-5 '), 6.46 (2H, s, H-3,8), 3.86 (3H, s, 6-OCH ₃ );

¹³ C-NMR (100 MHz, C ₅ D ₅ N, δ c): 183.88 (C-4), 166.10 (C-2), 158.42 (C-9), 154.34 (C-5), 153.91 (C-7 ), 150.73 (C-4 '), 146.75 (C-3'), 132.61 (C-6), 123.46 (C-1 '), 120.16 (C-6'), 116.59 (C-5 '), 113.98 (C-2 ′), 105.59 (C-10), 103.25 (C-3), 95.13 (C-8), 60.91 (OCH ₃ ).

Reference Example 1. Experiment Preparation

1-1. Experimental material

RPMI 1640 medium, fetal bovine serum, penicillin and streptomycin were purchased from Life Technologies, Grand Island, NY, and MTT ((GrandIsland, NY). 3- (4,5-Dimethylthiazol -2-yl) -2,5-diphenyl-tertazoliumbromide, DMSO (dimethyl sulfoxide), RNase A, leupeptin, aprotinin, PMSF (phenylmethylsulfonylfluoride), DAPI (4´, 6-diamidino- 2-phenylindole-dihydrochloride), Triton X-100, and propidium iodide (PI) were purchased from Sigma Chemical, St. Louis, Mo. Anti-caspase-3, anti-caspase 7, anti-PARP, anti-Lamin A / C, anti-Bax, anti-Bcl-2, anti-Apaf-1, anti-VDAC, anti- α-tubulin, anti-β-actin antibodies are from Santa Cruz Biotechnology (Santa Cruz, CA), anti-XIAP, anti-caspase 6, anti-caspase-8, anti-cytochrome c antibodies It was purchased from Science (BD Biosciences, Pharmingen, San Diego, Calif.). anti-caspsae-9, anti-Bid antibodies were purchased from Cell Signaling Technology (Beverly, Mass.). Other z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk, z-LEHD-fmk were purchased from Calbiochem, Bad Soden, Germany, respectively.

1-2. Preparation of Cancer Cell Lines

The human cervical cancer cell HeLa cell line, human ovarian cancer cell SK - OV - 3 cell line, human lung cancer cell A549 cell line and human glioblastoma cell A172 cell line were used in this experiment. , Seoul, Korea), and cell cultures were inactivated by heat treatment with RPMI 1640 or DMEM (Dulbecco's modified Eagle's minimum essential medium) and 10% fetal bovine serum (FBS) and penicillin (100 units /). ml) and streptomycin sulfate (100 / ml) were added and cultured under 37 ° C., 5% CO 2 conditions.

Reference Example 2. Statistics Processing Method

The mean values for each group were expressed as mean ± SD, and the results indicated were three or more independent experiments, with P <0.001 and P <0.001 and P, 0.00 and P, respectively, at P <0.05 and P <0.01. The statistical significance test between the experimental groups was evaluated using Student's t-test.

Experimental Example 1 Measurement of Cell Proliferation Inhibitory Effect of Euphapoline

In order to confirm the cytotoxicity of the eupapoline obtained in Example 1, the experiment was performed using the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay as follows. Carmichael et al., Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemisensitivity testing. Cancer Research, 47 , pp. 944-946, 1987.

The MTT assay is based on the reduction of water-soluble tetrazolium salt MTT (yellow) into formazan crystals (violet) by the dehydrogenase action of mitochondria in living cells. Formazan crystal production increases and absorbance is measured high.

Each cancer cell cultured by the method of Reference Example 1 was inoculated in a 96-well plate (5 x 10 ⁴ cells / well) and incubated for 24 hours in 100 ml RPMI medium containing 3% FBS. After 48 hours of treating the papapoline obtained in Example 1 at various concentrations (200, 100, 50, 25, 12.5 / ml), 50 MTT solution (5.0 mg / ml) was added to each well and further After further incubation for 4 hours, the plate supernatant was obtained, and intracellular formazan crystals produced by MTT were dissolved in precipitate by adding 100 DMSO, and the OD value was determined using a microplate reader (Molecular Devices). Measured at 540 nm. In addition, cisplatin, which is widely used as an effective anticancer agent for treating various solid cancer chemotherapy agents and various solid cancers, was set as a positive control and compared.

As a result, HeLa, SK - OV - 3, A549, and A172 cells (1.0 × 10 ⁵ cells / well) treated with papapoline for 48 hours were 26.75, 423.13, 53.34, and 145.7 μΜ, respectively, as shown in Table 1. The IC ₅₀ value was shown. Especially, the HeLa and A549 cells showed much better inhibition of cell proliferation than the cisplatin used as the positive control group. Accordingly, in the following experimental examples, the effect of opapoline was investigated using cancer cells, mainly HeLa cells (see Table 1).

Three pimp  Three forgi circle Cell proliferation inhibitory concentration (IC ₅₀ ; μΜ) Euphalin Cisplatin HeLa Cervical cancer 26.75 52.26 SK - OV - 3 Ovarian Cancer 423.13 153.80 A549 Lung cancer 53.34 82.92 A172 Glioblastoma 145.7 137.77

Experimental Example 2 Measurement of Apoptosis-inducing Effect of Euphapoline

Apoptosis is an active cell death process regulated by genes. Once a cell is signaled to die, chromatin in the nucleus is concentrated and lined up along the nuclear membrane, resulting in condensation, DNA fragmentation, and nuclear fragmentation. The surface of the cell changes prominently. This protruding part is separated into the apoptotic body surrounded by the cell membrane and undergoes a series of processes where it is separated and taken up by neighboring macrophages to disintegrate and remove. Therefore, the following experiments were carried out to determine whether the proliferation inhibitory effect of the cancer cells by the euapolin shown in Experimental Example 1 was caused by induction of apoptosis.

2-1. DNA fragmentation analysis

In order to examine whether eupapoline induces apoptosis of cancer cells, the experiment was performed using agarose gel electrophoresis to determine whether DNA fragmentation, which is one of the characteristics of apoptosis, was performed (Oberhammer et al. ., Apoptotic death in epithelial cells: cleavage of DNA to 300 and / or 50 kb fragments prior to or in the absence of internucleosomal fragmentation, The EMBO Journal , 12 (9) , pp.3679-3684, 1993).

DNA fragmentation is caused by cleavage of the linkage between nucleosomes (DNA + histone proteins), the cleavage size being a multiple of a constant size of 180-200 base pairs in length, which is an agarose gel. It appears as a band of ladder patterns in the phase.

HeLa cells (1.0 × 10 ⁵ cell / well) treated with 48 mg of papapoline (10, 20, 30, 40, and 60 μM) were washed once with PBS, followed by cold lysis buffer (10 mM Tris-HCl ( pH 7.4), 1 mM EDTA, 0.2% Triton X-100), incubated at 48 ° C. for 15 minutes, and the lysed cells were centrifuged at 14,000 rpm for 15 minutes to separate low molecular weight DNA from pure chromatin. do. After the supernatant was recovered, 0.2 mg / ml proteinase K containing 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 40 mM EDTA and 1% SDS buffer was added. The reaction is carried out at 37 ° C. for 4 hours. The DNA reactant was treated with phenol / chloroform extraction method twice to remove protein, and 140 mM NaCl and 2 times ethanol were added to the recovered supernatant overnight at -20 ° C. After allowing to stand to precipitate the DNA and centrifuged for 15 minutes at 4 ℃ at 14,000rpm and washed twice with 70% ethanol (ethanol) and dried. DNA was dissolved in 15 ml of TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) containing RNase-DNase (Boehringer Mannheim, Mannheim, Germany), followed by reaction at 37 ° C for 1 hour, followed by 40 mM Tris -After 1.5 hours agarose gel containing acetate buffer (Tris-acetate buffer (pH 7.4)) at 50V for 1 hour and photographed with ethidium bromide (ethidium bromide).

     As a result, as shown in FIG. 1, DNA fragmentation was observed in the group treated with HeLa cells for 48 hours with Eupapoline, and it was found that Eupapoline induced apoptosis of HeLa cells (FIG. 1).

2-2. Apoptosis analysis using Annexin V-PI staing

In order to quantitatively determine the degree of apoptosis of cancer cells caused by eupapoline, cell cycle analysis was carried out as described in the literature using flow cytometry (Kim, YH, et al. ., Expression of the murine homologue of the cell cycle control protein p32cdc2 in T lymphocytes.Journal of Immunology, 149 , pp. 17-23, 1992).

Annexin V is an early apoptosis indicator, propidium iodide (propidium iodide (50 ㎍ / ml)) is a substance that binds specifically to the DNA fragments in the nucleus of the cell undergoing apoptosis process and exhibits fluorescence.

100 ml of HeLa cells (1.0 × 10 ⁵ cell / well) treated with 0, 10, 20, 30, 40, and 60 μM of 0,12, 24, 36, and 48 hours of the eupapoline obtained in Example 1 were used. After floating with binding buffer (10 mM HEPES / NaOH, 140 mM NaCl, 2.5 mM CaCl ₂ , pH7.4), 5 μl of Annesine V (FITC) -FITC and 5 μl of propidium Iodide was mixed and stained. The mixture was incubated for 15 minutes at room temperature and shaded, and then analyzed within 1 hour using a flow cytometer (FACScan flow cytometer; Becton Dickinson Co, Heidelberg, Germany).

As a result of the experiment, as shown in Figure 2-A, HeLa cells treated with euphapoline at a concentration of 30 μM was found that apoptosis increases with increasing treatment time. In other words, the annexin V positive percentage of untreated control group was 6.91%, whereas in the group treated with UFAPOLIN at a concentration of 30 μM for 12, 24, 36, 48 hours, 7.36%, 11.32%, 37.5%, Gradually increased to 80.38% confirmed an increase in cell death. In addition, as shown in Figure 2-B, after treatment for 48 hours according to the concentration of eupapoline, annexin V-positive% of the control group not treated with eufapoline is 7.06%, while eupapoline 10, 20, 30 , 40, 60 μM treated group was gradually increased to 52.08%, 86.02%, 90.08%, 92.22% confirmed apoptosis (see Figure 2). Through this, it was possible to quantitatively determine the extent to which eupapoline induces apoptosis of HeLa.

2-3. DAPI staining

To determine whether eupapoline induces apoptosis of HeLa cells, DAPI (4'-6-diamidino-2-phenylindole, which binds specifically to DNA, is one of the features of apoptosis, the morphological change of the nucleus. (Luo, Y et al., Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol . 66 , p. 479, 1997).

Eupapoline was treated with a concentration of 30 μM, incubated for 48 hours, washed with PBS, and fixed in 3.7% formaldehyde for 10 minutes. After staining with DAPI, a fluorescent material specifically binding to DNA for 10 minutes, the stained cells were examined using an fluorescence microscope (META 510, Zeiss).

As a result, as shown in Figure 3, in the eupapoline treatment group showed apoptosis process while forming the condensation of HeLa cell nucleus, vesicle formation and apoptotic bodies compared to the control group (see Figure 3).

Experimental Example 3 Caspase Dependent Pathway Determination by Euphapoline

3-1. Caspase Gene Expression Patterns

In order to examine the mechanisms involved in inducing apoptosis of HeLa cells by the eupapoline identified through the results of Experimental Examples 2-1 to 2-3, specific protein detection tests ( Western blot) using the method (Sambrook J., et al., Molecular Cloning Laboratory Manual, 2nd ed ., pp18.60-18.71, 1989).

HeLa cells (1.0 × 10 ⁵ cell / well) cells were treated with eupapoline (10, 20, 30, 40 and 60 μM), respectively, and incubated for 48 hours, followed by centrifugation at 4 ° C. for 5 minutes at 600 g. . This was washed twice with cold PBS and centrifuged at 4 ° C. for 5 minutes at 600 g to collect each cell, and the cells were lysed with protein lysate (Intron, Seoul, Korea). Each protein thus prepared was quantified using the Bradford method, and the same amount of protein was separated using 10-12% sodium dodecyl sulphate (SDS) -PAGE (Polyacrylamide Gel Electrophoresis) gel, followed by PVDF membrane (BIO-). RAD, USA; 162-0177) (200 mA, 2 hours). After blocking in 5% non-fat dry milk, the primary antibodies caspase-3, -6, -7, -8, and in TTBS (0.1% Tween 20 in TBS) solution -9, PARP, Lamin A / C and β-actin were diluted 1: 1000 and reacted overnight at 4 ° C. This was washed three times with TTBS, and the secondary antibody was HRP (Horse Radish Peroxidase) -bound secondary antibody (antirabbit Ig G (Cell Signaling, USA; # 7074)) and anti-mouse IgG (American Pharmacia Biotech, USA; # 94010) was diluted 1: 2000 and reacted for 1 hour. Then, washed three times with TTBS again, reacted with ECL substrate (American Pharmacia Biotech, USA; 16021) for 1 to 3 minutes, and then photosensed on X-ray film (Amersham, Piscataway, NJ) to change the protein expression of each cell. Was analyzed.

Caspase-8 and caspase-9, the direct effectors of caspase, were the early effects of apoptosis in the HeLa cells treated with euphalin (after 48 hours). The expression of caspase-6 and caspase-7 proteins was divided and examined. As shown in Fig. 4-A, procaspase-8 exhibiting inactivity and While procaspase-9 protein gradually decreased in concentration-dependent expression, increased expression of active caspase-8 and cleaved caspase-9 protein was observed. Confirmed. In addition, as shown in Fig. 4-B, the expression of caspase-3, caspase-6 and caspase-7 proteins, which are direct effects factors of apoptosis, was confirmed. As a result, procaspase-3 and procase exhibiting inactivation. While procaspase-6 and procaspase-7 proteins gradually decreased in concentration-dependent expression, active caspase-3, active caspase- 6 and the expression of the active caspase (cleaved caspase-7) protein was confirmed to be increased (see Figure 4). In addition, the active caspase-3 and the active caspase-6 decompose the PARP protein that repairs the DNA in the nucleus and the Lamin A / C protein that constitutes the nuclear membrane. The mechanism by which the nucleus was fragmented was confirmed. Through this, the apoptosis of HeLa cells by the eupapoline was caspase-8, caspase-9, caspase-3, caspase-6 and caspase. It can be seen that it is induced by the caspase-7 protein (see FIG. 4).

3-2. Measuring caspase activity

In order to directly determine the activity of this enzyme after confirming that the mechanism involved in the apoptosis of HeLa cells by the eupapoline identified through the results of Experimental Example 3-1 was due to caspase protein expression, The same experiment was carried out.

HeLa cells (1.0 × 10 ⁵ cell / well) were incubated for 48 hours with treatment of eupapoline (10, 20, 30, 40 and 60 μM), respectively, and centrifuged at 4 ° C. for 5 minutes at 600 g. The cells were washed twice with cold PBS, centrifuged at 600 ° C. for 5 minutes at 4 ° C. to collect each cell, and then the cells were lysed with protein lysis solution (Calbiochem, Bad Soden, Germany) for measuring enzyme activity. The reaction was carried out in a bath for 5 minutes. After centrifugation at 10,000 rpm for 10 minutes, protein was obtained and quantified, 10 μg of protein was added to 80 μl of assay buffer (Calbiochem, Bad Soden, Germany) and 10 μl of substrate (substrate). Mixed and incubated for 2 hours at 37 ℃. The culture mixture was measured at a wavelength of 405 nm using a spectrophotometer to show caspase enzyme activity.

As a result, as shown in Fig. 5, caspase-3, caspase-8, and caspase-9 in HeLa cells treated with papapoline (after 48 hours) It was confirmed that the activity of the concentration-dependent increase, in particular, it can be seen that the activity of caspase (caspase-9) is excellent (see Fig. 5).

3-3. Inhibition of Apoptosis by Caspase Inhibitors

After confirming that the mechanism involved in the apoptosis of HeLa cells by the eupapoline is due to caspase protein expression, it was again verified using caspase inhibitors.

Each 50 μM z-VAD-fmk (a broad caspase inhibitor), 100 μM z-DEVD-fmk (a specific caspase-3 inhibitor; caspase) in HeLa cells (1.0 × 10 ⁵ cell / well) -3 inhibitor), 100 μM z-IETD-fmk (a specific caspase-8 inhibitor; caspase-8 inhibitor), and 100 μM z-LEHD-fmk (a specific caspase-9 inhibitor; caspase-9 inhibitor) After pretreatment for 1 hour, cells treated with 30 μM of papapoline were incubated for 48 hours, and the same procedure as in Experimental Example 2-2 was performed.

As a result, as shown in Figure 6-A, the% Annexin V positive of the control group not treated with papapoline showed 6.95%, and the Annexin V positive% of the group treated with papapoline at a concentration of 30 μM was 62.54. % Was confirmed. However, after treatment with caspase inhibitors (caspase) inhibitors, the group treated with a concentration of 30 μM of eupapoline showed a similar% of annexin V, similar to the control group not treated with eupapoline. In other words, the percentage of Annexin V positive in the group treated with the concentration of 30 μM was 62.54%, but 50 μM z-VAD-fmk, 100 μM z-DEVD-fmk, 100 μM z-IETD-fmk, and 100 μM z-LEHD After pre-treatment with -fmk, the percentage of Annexin V positive in the group treated with euphapoline at a concentration of 30 μM was 6.81%, 12.41%, 14.92%, and 11.92%, respectively. Each inhibitor had no effect on annexin V positive%. Therefore, the apoptosis effect of HeLa cells by ufapoline was again demonstrated through the mechanism by caspase protein (see FIG. 6).

3-4. Inhibitory Effect of Caspase Inhibitors on Cell Damage

Caspase inhibitors are involved in the morphological changes of HeLa cell nuclei induced by apoptotic apoptosis in order to verify that the apoptosis mechanism of HeLa cells is caused by caspase. I looked at the degree.

HeLa cells (1.0 × 10 ⁵ cell / well) were treated with 50 μM z-VAD-fmk for 1 hour, treated with opapoline at a concentration of 30 μM and incubated for 48 hours, the same as in Experimental Example 2-3. Experimental method was performed.

As shown in FIG. 7, nuclei fragmentation and condensation were observed in the group treated with the concentration of 30 μM of papapoline, but after treatment with 50 μM z-VAD-fmk, the concentration of 30 μM of papapoline was treated. As in the control group, no damage occurred to the nucleus of the HeLa cells, and the group treated with 50 μM z-VAD-fmk alone showed no effect on the nuclei of the HeLa cells.

In other words, it was confirmed that fragmentation of the nuclei of HeLa cells by eupapoline was completely inhibited by caspase inhibitors (see FIG. 7).

3-5. Inhibitory Effect of Caspase Inhibitors on Gene Expression

In order to more closely verify that the apoptosis mechanism of HeLa cells by the eupapoline identified through the results of Experimental Examples 3-1 to 3-4 is due to caspase protein expression, caspase The effects of inhibitors on the expression of genes important for the regulation of apoptosis were identified.

HeLa cells (1.0 × 10 ⁵ cell / well) were pretreated with 50 μM z-VAD-fmk, 100 μM z-IETD-fmk, and 100 μM z-LEHD-fmk for 1 hour and treated with opapoline at a concentration of 30 μM. After culturing for 48 hours, it was carried out by the same experimental method as Experimental Example 3-1. Antibodies were caspase-3, -6, -7, PARP and Lamin A / C. Β-actin was used as a control.

As shown in FIG. 8, in the group treated with HeLa cells at a concentration of 30 μM of eupapoline, active caspase-3, active caspase-6, and active form It was confirmed that the cleaved caspase-7 protein, the PARP protein for repairing the DNA in the nucleus, and the Lamin A / C protein constituting the nuclear membrane were degraded. These apoptosis-related proteins were found to be inhibited in the 50 μM z-VAD-fmk, 100 μM z-IETD-fmk, 100 μMz-LEHD-fmk treatment groups. In other words, the expression of apoptosis-induced apoptosis genes was completely inhibited by caspase inhibitors, so that apoptosis of HeLa cells induced by eupapoline was induced in the caspase apoptosis pathway. It can be confirmed that it is dependent (see FIG. 8).

Experimental Example 4 Mitochondrial Membrane Potential Difference by Euphapoline ΔΨ _m ) Change and cytochrome c Measure

The effect on membrane potential, which is one of intracellular organelles in the process-induced cell death in school morpholin mitochondria (ΔΨ _m) Change In order to find out, the experiment was performed as follows.

When mitochondria receive a cell death signal, caspase activators are rapidly released into the cytoplasm due to the decrease and permeability of the mitochondrial membrane potential difference ( ΔΨ _m ) (Zamzami N., et al., Reduction of mitochondrial potential origins an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med ., 181 , pp. 1661-1672, 1995), cytochrome c , one of the caspase activators, diffuses into the cytoplasm (Von Ahsen O., et al., The 'harmless' release of cytochrome c . Cell Death Differ ., 7 , pp. 1192-1199, 2000). In addition, cytochrome c diffused into the cytoplasm combines Apaf-1 and procaspase-9 present in the cytoplasm to form an apoptosome complex, which leads to procaspase-9. Induced with active cleaved procaspase-9. Active cleaved procaspase-9 induces apoptosis by inducing the activity of caspase-3, caspase-6 and caspase-7, which are direct effectors. (Li P., et al., Cytochrome c and dATP-dependent formation of Apaf-1 / caspase-9 complex initiates an apoptotic protease cascade, Cell , 91 , pp. 479-489, 1997).

In order to confirm the reduction of mitochondrial membrane potential ( ΔΨ _m ) and mitochondrial membrane permeability during the induction of apoptosis of HeLa cells, hepapoline was applied to HeLa cells (1.0 × 10 ⁵ cell / well), CsA (5 μM cyclosporin A, an inhibitor of mitochondrial permeability). ) Was pretreated for 30 minutes, and eupapoline was treated at 30 μM and incubated for 48 hours. Thereafter, the mitochondrial voltage difference-dependent staining agent DiOC ₆ was treated at 50 nM concentration for 30 minutes, washed twice in PBS, analyzed by flow cytometry, and the 100 μM CCCP treated group was set as a positive control.

In addition, the following experiments were carried out to investigate the effect of papapoline on the expression of the caspase activator cytochrome c and Apaf-1 and the caspase inhibitor XIAP (X-linked inhibition of apoptosis protein). It was.

HeLa cells (1.0 × 10 ⁵ cell / well) cells were treated with papapoline (10, 20, 30, 40 and 60 μM), respectively, and incubated for 48 hours, and centrifuged at 4 ° C. for 5 minutes at 600 g. Each cell was collected, washed twice with cold PBS, and then centrifuged at 4 ° C. for 5 minutes at 600 g. After 500 μl of cytosolic buffer was added, the mixture was reacted on ice for 15 minutes, homogenized using B-type flask (80 times), and then centrifuged at 10,000 g for 20 minutes at 4 ° C. After taking the fraction group (C)), the precipitate of the mitochondrial fraction group (M) was tested by homogenizing in the mitochondrial dissolution solution. The cytoplasmic fraction group (C) and mitochondrial fraction group (M) thus obtained were adjusted to the same amount of protein by protein quantification and separated by 12% SDS-PAGE, followed by the same experimental method as Experimental Example 3-1. At this time, cytochrome c , Apaf-1 and XIAP were used as primary antibodies, and VDAC, α-tubulin and β-actin as internal controls of mitochondrial fraction group (M), cytoplasmic fraction group (C), and general protein, respectively. Was used.

As a result, as shown in FIG. 9-A, the group treated with HeLa cells with eupapoline decreased mitochondrial membrane potential difference ( ΔΨ _m ), similar to CCCP (positive control), and treated with eupapoline after pretreatment of CsA. Inhibited the membrane potential difference ( ΔΨ _m ) induced by papapoline . In addition, as shown in FIG. 9-B, the amount of cytochrome c protein expression in the mitochondrial fraction group (M) in HeLa cells decreased in concentration-dependent manner in heLa cell, and the amount of cytochrome c protein expression in the cytoplasmic fraction group (C). This increase was confirmed. In addition, the expression of Apaf-1 in HeLa cells was increased by the papapoline, but the expression of XIAP, a caspase inhibitor, was decreased. Thus, when papapoline induces apoptosis of HeLa cells, the mitochondrial apoptosis pathway occurs. It can be seen (see Fig. 9).

Experimental Example 5. Measurement of Bcl-2 Protein Expression by Euphapoline

Bcl-2 family proteins are largely divided into genes that inhibit apoptosis (typically Bcl-2) and genes that induce apoptosis (typically Bax, Bak, Bid, etc.) (Reed J. Bcl-2 and regulation of programmed cell death.J Cell Biol., 124 , pp.1-6, 1994.), Intracellular balance of Bcl-2 proteins during apoptosis is an important indicator of the maintenance of mitochondrial membranes (Oltvai ZN., et al. ., Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74, pp.609-619, 1993).

In order to determine the expression changes of Bcl-2 protein, which is an important regulator related to the apoptosis mechanism of HeLa cells by eupapoline, it was performed by the same experimental method as Experimental Examples 3-1 and 4. At this time, Bid, Bcl-2 and Bax were used as primary antibodies, and α-tubulin and β-actin were used as internal controls.

As a result, as shown in Figure 10, in the mitochondrial fraction group (M) of the HeLa cell group, the expression of Bak, Bid, which are apoptosis-inducing proteins among the Bcl-2 family proteins, was increased depending on the concentration of eupapoline treatment and the cytoplasmic fraction group ( It was confirmed that the decrease in C), but Bax did not change expression. In addition, it was confirmed that the expression of Bcl-2, which is an apoptosis inhibitory protein, was reduced by euphapoline.

Through the above experiments, expression of apoptosis-inducing proteins Bak and Bid and apoptosis-inhibiting protein Bcl-2 in Bcl-2 family proteins was altered by ufapoline, and the balance of mitochondrial membranes in HeLa cells was disrupted, resulting in apoptosis-inducing factors. It was confirmed that caspase protein is activated. In addition, PARP protein repairing DNA in the nucleus and Lamin A / C protein constituting the nuclear membrane are decomposed by the caspase protein activated by papapoline, resulting in HeLa cell apoptosis. Pauline was confirmed that it can be used as a cancer prevention and treatment composition based on the excellent anti-cancer effect of inhibiting the proliferation of cancer cells.

Hereinafter, an example of the preparation of a pharmaceutical composition comprising the composition of the present invention will be described, but the present invention is not intended to limit the present invention but is only specifically intended to be described.

Formulation Example 1 Preparation of Powder

Euparoline 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 Tablet

Euparoline 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 Preparation of Capsule

Euparoline 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 Injection

Euparoline 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 Preparation of Liquid

Euparoline 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Drink

100 mg of papapoline

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 B1

0.3 g of vitamin B2

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 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 compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Figure 1 is a diagram showing the DNA fragmentation of HeLa cells by euphapoline using electrophoresis,

Figure 2 is a diagram showing the effect of the increase in the number of cells by papapoline in HeLa cells using a flow cytometer,

Figure 3 is a diagram showing apoptosis by eupapoline in HeLa cells using DAPI staining,

Figure 4 is a diagram showing the gene expression by papapoline in HeLa cells,

Figure 5 is a diagram showing caspase (caspase -3, -8, -9) activity by papapoline in HeLa cells,

Figure 6 is a diagram showing the cell cycle by the eupapoline in HeLa cells using a flow cytometer,

Figure 7 is a diagram showing the apoptosis of genes by eupapoline in HeLa cells using DAPI staining,

Figure 8 is a diagram showing caspase (caspase -3, -8, -9) activity by the papapoline and genes in HeLa cells by papapoline,

9 is a diagram showing the membrane potential and cytochrome ( c ) effect by the gene in HeLa cells caused by papapoline,

Figure 10 is a diagram showing the expression level of Bcl-2, Bid and Bax in HeLa cells by euapolin.

Claims (5)

Pharmaceutical composition for the prevention and treatment of cancer diseases containing eupapoline of the following structural formula (1) isolated from Artemisia princeps Pampanini as an active ingredient;

(One) 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, colon cancer, small intestine 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, lymph gland cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) A pharmaceutical composition, which is one or more diseases selected from the group consisting of tumors, primary CNS lymphomas, spinal cord tumors, brain tumors, brainstem glioma or pituitary adenoma. The pharmaceutical composition according to claim 1, wherein the extract comprises 0.1 to 50% by weight of the total weight of the composition. A health functional food for the prevention and improvement of cancer diseases, which contains eupapoline , isolated from Artemisia princeps Pampanini , as an active ingredient. The dietary supplement of claim 4 which is a powder, granule, tablet, capsule or beverage.

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