KR100658950B1 - A composition comprising the extract of corallina pilulifera for the prevention and treatment of cancer - Google Patents

Abstract

A composition for the prevention and treatment of cancer comprising the extract of Corallina pilulifera is provided to inhibit proliferation and induce apoptosis of Hela cell line without toxicity, thereby being able to be used for preventing and treating cancer diseases. The composition for the prevention and treatment of cancer comprises the extract of Corallina pilulifera as an effective ingredient, wherein the cancer is liver cancer, thyroid cancer, ovarian cancer, gallbladder carcinoma, large intestine cancer, leukemia, esophageal cancer, stomach cancer, breast cancer, cervical cancer, prostatic cancer, pancreas cancer or laryngeal cancer; the Corallina pilulifera extract is prepared by extracting Corallina pilulifera with water, methanol or ethanol; and the amount of Corallina pilulifera extract is 0.02-50 wt.% based on the total weight of the composition.

Description

A composition comprising the extract of Corallina pilulifera for the prevention and treatment of cancer}

Figure 1 is a diagram showing the inhibition rate of HeLa cell proliferation according to the concentration of small bead corpuscle extract,

Figure 2 is a diagram showing the analysis of the cell cycle of HeLa cells,

3 is a diagram observing the morphological changes of HeLa cells treated with 48 hours of small bead coral extract through DAPI staining,

FIG. 4 is a diagram showing the TUNEL staining for DNA fragmentation of HeLa cells treated with dimethyl sulfoxide (DMSO) (A), small bead eosin extract (B) and DNase I (C). FIG.

5 is a diagram of Western blot analysis of HeLa cells treated with small bead corpuscle extract for each concentration for 48 hours,

Figure 6 is a diagram comparing the size of the cancer tissue showing the mouse in vivo anti-cancer activity of small bead Coral extract.

Apoptosis inducing substances have recently emerged as new targets for the treatment of cancer. Apoptosis is the most important process leading to cell death and plays a very important role in the maintenance of eukaryotic homeostasis and tissue growth control (Douglas R Green et al., Science , 281 , pp 1309-1312, 1998; Michael O et al., Nature, 407 , pp 770-776, 2002). Proliferation and killing of cells is an essential process for maintaining intracellular homeostasis and for normal operation the cells need to be in a steady state. However, disruption of intracellular homeostasis leads to various diseases including cancer (Thompson CB et al., Science , 267 , pp 1456-1462, 1995). Apoptosis occurs through death receptor dependent pathways outside the cell and mitochondrial dependent pathways inside the cell. This apoptosis is also induced by treatment with chemotherapeutic agents (Kaufmann SH et al., Exp . Cell Res., 256 (1) , pp 42-49, 2000; Walker PR et al., Cancer Research, 51 , pp 1078-1085, 1991). In the cytoplasm, caspases belonging to the species of cysteine proteases play an important role in the execution of apoptosis. Procaspase-8, the first implementer, is converted to caspase-8, an active form by self-processing for stimuli leading to cell death. Procaspase-9, on the other hand, is activated by the mitochondrial pathway (Denecker G et al., Cell. Mol . Life Sci ., 58 , pp356-370, 2001). this is Mitochondria also play an important role in inducing cells into cell death by signal transduction of growth factor deficiency, ultraviolet light, cyclophosphamide, and anticancer agents such as etoposide (Wilson SE, Exp. Eye Res., 69, pp255-266, 1999). Mitochondrial pathways occur with the mediation of Bcl-2 family proteins. Bcl-2 protein is part of the jong Bcl-2 and Bax protein, and these proteins in the mitochondrial cytochrome-regulates the movement of the c (cytochrome- c) (Kelekar A et al, Trends Cell Biol, 8, pp.324.. -330, 1998). Self-apoptosis occurs in cells that receive various apoptosis induction signals, in which cytochrome- c is released from the mitochondria into the cytoplasm (Newmeyer DD et al., Cell, 112 , pp.481-490, 2003). Released Cytochrome- c interacts with deoxyadenosinetriphosphate (dATP), apoptosis protease activating factor 1 (apaf1) and procaspase-9 in the cytoplasmic apoptosome complex, and consequently procaspase- 9 is transformed into the active caspase-9 (Chandra J et al., Free Radic . Biol . Med ., 29 , pp323-333, 2000). Activated caspase-9 activates practitioners such as caspase-3, caspase-6, caspase-7 (Salvesen GS et al., Cell, 91 , pp443-446, 1997). Performer caspases cleave poly ADP-ribose polymerase (PARP), cell membranes (laminas) and inhibitor of caspase activated DNase (ICAD). The cleaved proteins are used as target proteins for apoptosis. Due to various phenomena due to apoptosis, cells have morphological characteristics such as condensation of chromosomes, fragmentation of DNA, and apoptotic body formation (Germain M et al., J. Biol . Chem ., 274 , pp 28379). -28384, 1999).

Marine life is recognized as a report of new bioactive substances due to species diversity and biochemical diversity. More than 3,000 new substances have been reported from marine organisms in recent decades by several researchers, some of which have been used for clinical applications (Cragg GM et al., Semin . Oncol . , 24 , pp156-163, 1997).

Algae among marine organisms have been traditionally used as food in Asia, including Korea, as well as folk remedies for diseases such as roundworm and gout (Michanek G, Seaweed resources for pharmaceutical uses, Marine algae in pharmaceutical science). , edited by Hoppe, HA, Levring, T., Tanaka, Y. and Welter de Gruyter, Berlin, New York, pp203-234, 1979).

Research on the extraction of useful substances from seaweed has been carried out by several researchers (Scheuer PJ, Marine natural products, chemical and biological perspectives vol. 1, Academic Press , New York, pp 44-171, 1978; Faulkner DJ, Nat) Prod.Rep . , 17 , pp1-6, 2000; Faulkner DJ, Antonie Van Leeuwenhoek , 77 (2) , pp135-145, 2000; Schwartsmann, G et al., Lancet Oncol . , 2 (4) , pp221- 225, 2001;.... da Rocha AB et al, Curr Opin Pharmacol, 1 (4), pp364-369, 2001) to cancer cells experiments on human cancer cells using the algae extract to develop an anticancer agent from the last algae there are reports of separating the growth inhibition or a substance having in vivo (in vivo) tumor growth inhibitory effect (Furusawa E et al, Oncology, 42, pp364-369, 1985;.. Nagumo T et al, Kitasato Arch Exp. Med . , 61 (1) , pp 59-67, 1988; Noda H et al., Nippon Suisan Gakkaishi , 55 , pp 1259-1264, 1989a; Bohn and Be Miller et al., Carbohydr . Polym . , 28 , pp 3-14, 1995).

Small beaded coral is a red algae widely distributed in intertidal zones off the coast of Korea. Intertidal Group Inhabits the tidal flats of the upper rock, and the height is as small as 3 ~ 4 cm. To date, there has been no disclosure or teaching about the anticancer activity of small bead coral.

In this regard, the present inventors have investigated the cytotoxic effects of small beaded horsetail extract against various cancer cell lines, inhibiting cell proliferation against HeLa cell lines, inducing apoptosis and carcinogenesis with sarcoma 180 cells. The present invention was completed by confirming anticancer activity in vivo).

       An object of the present invention is to provide a composition for the prevention and treatment of cancer diseases comprising a small bead acid horsetail extract having a proliferation inhibitory activity, cancer killing induction activity and anti-cancer activity against cancer cells.

In accordance with the above object, the present invention is a small bead coral ( Corallina It provides a composition for preventing and treating cancer diseases containing the extract of pilulifera ) as an active ingredient.

The extracts include water, methanol, lower alcohols such as ethanol or a mixture thereof, preferably extracts soluble from ethanol.

The cancer diseases include liver cancer, thyroid cancer, ovarian cancer, gallbladder cancer, colon cancer, leukemia, esophageal cancer, stomach cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer and laryngeal cancer, preferably uterine cancer.

Hereinafter, the present invention will be described in detail.

Small bead esoteric extract of the present invention can be obtained as follows.

Small bead coral extract of the present invention, the small bead coral extract of the coastal Busan, washed and dried after drying, about 1 to 10 times the weight (kg), preferably about 2 to 8 times the water, C About 10 hours to 40 hours, preferably 20 hours to 30 hours at a extraction temperature of 0 to 100 ° C., preferably 10 to 50 ° C., with a solvent selected from ₁ to C ₄ lower alcohols or a mixed solvent thereof, preferably ethanol. Extraction methods such as cold sediment extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc. during the time period, preferably repeated several times by cold extraction, additionally 0 to 100 ℃ preferably about 1 at an extraction temperature of 50 to 70 ℃ Extraction methods such as cold sedimentation, hot water extraction, ultrasonic extraction, reflux cooling extraction, for several hours to 24 hours, preferably 10 hours to 15 hours, preferably repeated extraction several times by reflux cooling extraction , Filtered through a small bead of the present invention can be obtained coralline extract.

In addition, conventional fractionation processes can also be carried out (Harborne JB, Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed ., Pp 6-7, 1998).

By confirming the proliferation inhibitory activity, self-killing induction activity and in vivo anti-cancer activity in the mouse of the small bead acid horsetail extract of the present invention obtained by the method as described above, for the prevention and treatment of cancer diseases It was confirmed that it can be usefully used as a composition.

In addition, the small bead coral is a seaweed that has been used for a long time, such as a roundworm, the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects.

It provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the small bead acid horsetail extract obtained in the extraction process of the present invention as an active ingredient.

Pharmaceutical composition for the treatment and prevention of cancer diseases of the present invention, the extract based on the total weight of the composition 0.02 to 50% It is included by weight percentage.

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

The pharmaceutical dosage forms of the extracts 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.

Pharmaceutical compositions comprising extracts according to the invention, 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 extract 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 the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the extracts and fractions. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. 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 oil such as olive oil, injectable ester 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 extracts 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 extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 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 extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or Intracerebroventricular injection.

In addition, the present invention provides a health functional food for preventing and improving cancer diseases containing the small bead acid horsetail extract obtained in the extraction process of the present invention as an active ingredient.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

Health functional foods for the prevention and improvement of cancer diseases of the present invention, the extract comprises 0.01 to 95% by weight, preferably 1 to 80% by weight relative to the total weight of the composition.

In addition, it is possible to manufacture and process as a health functional food in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of improving and preventing cancer diseases.

For example, the health functional food in the form of tablets may be prepared as it is or granularly mixed with an excipient, a binder, a disintegrating agent or other additives in a suitable manner, and then compressed into a glidant, etc. It is made by directly compressing the functional food as it is or by mixing it evenly with an excipient, binder, disintegrant or other suitable additives, or mixing the health functional food as it is or evenly adding the appropriate additive to the prepared granules, The excipient, binder or other suitable additives are added to the functional food, and the powder mixed evenly is wetted with a solvent, the wet powder is molded into a mold at low pressure, and then dried and prepared by a suitable method. In addition, the nutraceutical can be added to the health functional food in the form of tablets, if necessary, can be avoided with a suitable epidermis.

Among the health functional foods in the form of capsules, the hard capsules are usually prepared by mixing the capsules evenly with the health functional foods or excipients suitable for the health functional foods, granulated by a suitable method, or granulated with a suitable epidermal agent as it is or Soft capsules are prepared by filling them, and soft capsules are usually filled with capsules containing glycerin or sorbitol in a capsule form containing gelatin or appropriate excipients suitable for health functional foods or health functional foods. It is molded and prepared, and a coloring agent preservative etc. can be added to the said capsule base as needed.

Circular functional foods are usually mixed with excipients, binders, disintegrants, etc., and then molded into a spherical form in a suitable manner, and the coating is carried out with white sugar or other suitable coating agent, or starch, talc or You can also be greeted with a suitable substance.

The health functional food in the form of granules is usually made by mixing the health functional food as it is or by adding excipients, binders, disintegrating agents, etc., evenly and then granulating it in a proper way to make the particles as even as possible. , Copulation agent, etc. For the health functional food in the form of granules, No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) were used for the next particle size test. It should be less than 5.0% and pass through No.45 to less than 15.0% of the total amount.

The definitions of the excipients, binders, disintegrants, lubricants, copulation agents, flavoring agents, etc. of the present invention are those described in the literature known in the art and include those having the same or similar functions. , Korean College of Pharmacy, 5th Edition, p33-48, 1989).

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  One. Small Bead Coral  Ethanol extract manufacturer

10 kg of small bead corals collected from March to May from the intertidal rocks near Busan were washed with purified water, and 30 liters of ethanol were added and repeated cold extraction three times at room temperature for 24 hours. The extraction was repeated twice for 12 hours under reflux conditions, and the mixture was filtered through a filter paper (Whatman, USA), concentrated under reduced pressure with a vacuum condenser (EYELA, N-1000, Japan), and freeze-dried. 101.8 g of extract were obtained. This extract was dissolved in dimethyl sulfoxide (DMSO; Sigma, St Louis, MO) and stored at 4 ° C. to be used as a sample of the present invention (hereinafter referred to as EECP).

Reference Example  1. Cell Lines and Culture Media

1-1. HeLa  Cell culture

The cervical cancer cell line, HeLa cell line, was purchased from the American Type Culture Collection, Rockville. HeLa cells were treated with DEME (Dulbecco's modified Eagle's medium, Sigma, Germany) at 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamicin (gentamycine, Sigma, Germany). ) Was used and cultured under 37 ℃, 5% CO ₂ conditions.

1-2. HT29  Cell culture

HT29 cells, a human colorectal cancer cell line, were purchased from the American Type Culture Collection, Rockville. HT29 cells were supplemented with RPMI 1640 (Hyclone, USA) with 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamycine (gentamycine, Sigma, Germany). Medium was used and incubated under 37 ° C., 5% CO ₂ conditions.

1-3. HepG2  Cell culture

HepG2 cells, a human liver cancer cell line, were purchased from the American Type Culture Collection, Rockville. HepG2 cells were treated with DEME (Dulbecco's modified Eagle's medium, Sigma, Germany) at 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamycin (gentamycine, Sigma, Germany). ) Was used and cultured under 37 ℃, 5% CO ₂ conditions.

1-4. A549 cell culture

A549 cells, a human lung cancer cell line, were purchased from the American Type Culture Collection, Rockville. A549 cells were supplemented with RPMI 1640 (Hyclone, USA) with 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamycine (gentamycine, Sigma, Germany). Medium was used and incubated under 37 ° C., 5% CO ₂ conditions.

1-5. T24  Cell culture

T24 cells, a human bladder cancer cell line, were purchased from the American Type Culture Collection, Rockville. T24 cells were added 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamycin (gentamycine, Sigma, Germany) to RPMI 1640 (Hyclone, USA). One medium was used and incubated under 37 ° C., 5% CO ₂ conditions.

1-6. Jurket E6 -1 cell culture

Jurket E6-1 cells, a human hematologic cancer cell line, were purchased from the American Type Culture Collection, Rockville. Jurket E6-1 cells contained 10% (v / v) fetal bovine serum (FBS, Fetal bovine serum, USBio-TECHNOLOGIES, Inc) and 0.1% gentamycin (gentamycine, Sigma, Germany) in RPMI 1640 (Hyclone, USA). The added medium was used and cultured under 37 ℃, 5% CO ₂ conditions.

Experimental Example  1. Cytotoxicity ( cytotoxic  activity) and cytostatic activity

1-1. Cytotoxicity Measurement

The cytotoxicity of EECP against each cancer cell was measured. Cytotoxicity was determined by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) assay (Mosmann T., J. immunol.methods , 65 , pp55-63, 1983). 1 × 10 ⁴ initial cell numbers were cultured by aliquoting to a microtiter plate. EECP was treated at different concentrations (0-500 μg / ml), and the control group was treated with 0.1% dimethyl sulfoxide (DMSO). After treatment for 24 hours at 37 ℃ MTT solution was added and further incubated for 4 hours at 37 ℃. Optical concentration was measured at 550 nm using an Enzyme linked immunosolvent assay (ELISA) reader. Cytotoxicity of EECP for each cell line showed an IC ₅₀ of 50-110 μg / ml in most cells and each IC ₅₀ is shown in Table 1 below. Among them, HeLa cells had the highest sensitivity to EECP compared to other cells, and the growth inhibition effect of the cells was examined.

Cell line IC ₅₀ (ug / ml) HT29 61.5 HepG2 103.6 A549 60.2 T24 93.3 HeLa 58.8 Jurket E6-1 54.4

1-2. Cell proliferation inhibitory measure

To examine the effect of EECP on the inhibition of proliferation in HeLa cell lines, trypan blue exclusion assay (Jones KH et al., J. Histochem . Cytochem., 33 , pp77-79. 1985) was used. .

HeLa cells were dispensed into 35 mm culture dishes, treated with different concentrations of EECP (50, 100, 150 μg / ml) and incubated for 7 days, and trypan blue staining was performed at 2-day intervals to measure viable cells. blue exclusion) was used. The cells were trypsinized and washed with phosphate buffer (PBS, phosphate-buffered saline), and the cells were suspended by the addition of trypan blue staining solution. Viable cells were measured using a hemocytometer. Cells treated with EECP showed a significant difference from day 1 compared to control cells, and when treated with 50 μg / ml, the survival rate was 69.7% compared to 100% of the control cells. In addition, the EECP treatment concentration was gradually increased to 50, 100, 150 ㎍ / ㎖ and cultured for 7 days, 61.8 ± 3.7%, 26.2 ± 4.3%, 6.56 ± 2.0% survival rate as shown in Figure 1 It was confirmed that the lowering dependent.

Experimental Example  2. Cell Cycle Analysis cytometry  analysis)

The cell cycle of HeLa cells was analyzed using flow cytometry. HeLa cells were cultured in a 35 mm dish at a concentration of 5 × 10 ⁵ cells and treated with EECP at a concentration of 50 and 100 μg / ml, and the control group was treated with 0.1% dimethyl sulfoxide (DMSO) for 48 hours. The cells were detached with trypsin, washed once with phosphate buffer solution (PBS), and the cells were collected and resuspended in phosphate buffer solution. DNA staining was performed using CycleTEST ^™ PLUS kit (Becton Dickinson, Heidelberg). Nuclear fractions stained with propidium iodide (PI) were obtained. The intensity of fluorescence was determined using a FACScan flow cytometer (Becton Dickinson, FACS Calibur, USA) and analysis was performed using CellQuest software (Becton Dickinson, Heidelberg). As shown in FIG. 2, the sub-G1 population showing self-killing by EECP treatment showed a significant increase compared to the control group 2.5% (50 ㎍ / mL: 8.2%, 100 ㎍ / mL: 11.6%). ). In the non-self killing population, the treatment of 50 μg / ml EECP increased the G1 cell population (65.4%), but decreased to 48.5% when 100 μg / ml. On the other hand, at the 50 ㎍ / ml EECP concentration, the G2 / M cell group was 19.2%, similar to the control group (19.7%). It was. As a result of cell cycle analysis, it was confirmed that high concentration of EECP in the HeLa cell line stops the cell cycle in the G2 / M phase (see arrow in FIG. 2) and induces apoptosis.

Experimental Example  3. EECP Induction of self-killing by

4'6-Diamidino-2-phenylindole (DAPI; 4'6-diamidino-2-) was used to treat HeLa cells treated with EECP for 48 hours in order to confirm that the cytostatic effect of EECP was caused by apoptosis. phenylindole, Sigma) was used for nuclear staining. HeLa cells were washed once with phosphate buffer and the cells were fixed for 10 minutes at room temperature using 3.7% paraformaldehyde. Fixed cells were washed with phosphate buffer and treated with 4'6-diamidino-2-phenylindole (DAPI) solution and stained for 10 minutes at room temperature. Stained cells were washed twice with phosphate buffer and analyzed by fluorescence microscopy (OLYMPUS BH2, Japan). As a result, as shown in FIG. 3, HeLa cells treated with 150 μg / ml EECP (B) than the 0.1% DMSO treated control group (A) condensed chromatin in the nucleus, and apoptotic bodies (typical characteristics of typical self-killing) 4B; see also arrows), and with increasing the concentration of EECP, autologous apoptosis cells also increased.

Experimental Example  4. Detection of self-killing TUNEL  assay)

DNA fragmentation is another hallmark of self-killing (Bowen ID et al., Chapman and Hall , London, 1998), cell apoptosis was determined by confirming DNA fragmentation. DNA fragmentation was confirmed by TUNEL assay using the Deadend ™ Colometric Tunel System (Promega). Cells were dipped in a slide glass coated with poly-L-lysine and dried in a tissue culture hood for 1 hour, followed by washing the cells twice with phosphate buffer. After fixing for 25 minutes at room temperature using 4% paraformaldehyde solution and washed twice with phosphate buffer. The cells were soaked in 0.2% Triton X-100 solution for 5 minutes and washed with phosphate buffer. Negative controls and positive controls were prepared by treating dimethyl sulfoxide (DMSO) (A) and DNase I (C), respectively. After the above process, the cells were equilibrated for 5 minutes at room temperature using equilibration buffer. Next, a TdT enzyme reaction mixture and a biotinylated nicleotide mixture were added to the cells, covered with a cover glass, and treated at 37 ° C. for 1 hour. The reaction was terminated by immersing the slide in 2 x SSC solution for 15 minutes. The slides were washed with phosphate buffered solution, treated with 0.3% (v / v) hydrogen peroxide solution for 5 minutes, rinsed with phosphate buffered solution, and then dyed to light brown with diaminobenzidine (DAB). Treated and stained cells were observed by light microscopy. As shown in FIG. 4, the nucleus (B) of the cells treated with EECP was confirmed to be dark brown.

Experimental Example  5. EECP Up-down Regulatory Effects of Self-killing Associated Proteins Following

In order for apoptosis to be carried out, activation of the caspase family is required. Procaspase-8 and procaspase-9 are early implementers that are activated in their processes and produce downstream procaspase-3, an active dimer, caspase-3, an execution factor for self-killing. To verify that EECP induces the activation of these caspases, Western blot analysis was performed using Procaspase-3, Procaspase-8, Procaspase-9 and cytochrome- c antibodies after exposure of HeLa cells to EECP. It was. HeLa cells were treated with 0.1% Triton X-100, 1 mM ATP and protease inhibitors in CSK buffer (10 mM Pipes, pH 6.8, 100 mM NaCl, 1 mM MgCl ₂ , 1 mM EGTA, 1 mM dithiothreitol and 1 mM phenylmethanesulfonyl fluoride). It was suspended using a solution (Pharminogen A) added and then crushed by an ultrasonic digester. The crushed cells were centrifuged at 20,000 × g for 30 minutes, and the protein concentration of the supernatant was measured using a BCA protein assay kit (Bio-Rad). The same amount of protein (40 μg) was used for SDS-PAGE, and actin was used as a control. Caspase-3, caspase-8, caspase-9, cytochrome- c and actin 15%, poly ADP-ribose polymerase (PARP) using 8% separation gel It was. After protein electrophoresis, the protein in the gel was transferred to a PVDF membrane and blocked for 1 hour at room temperature using a blocking solution (BlockaceTM, Dai-Nippon). The primary antibody was reacted at 37 ° C. for 1 hour and the PVDF membrane was washed using a solution in which 0.1% Triton X-100 was added to TBS (50 mM Tris / HCl, pH 7.5, and 0.15 M NaCl). Peroxidase-conjugated secondary antibody (Pierce) was used to react at 37 ° C for 1 hour, and the immunoreactive protein was detected by a chemiluminescence system (SuperSignal West Femto Maximum sensitivity Substrate, Pierce). It was. The degree of reaction was quantified using Fluorchem ^™ 5500 (Alpha Innotech). Actin, poly ADP-ribose polymerase, caspase-8 and caspase-9 antibodies were purchased from Santa Cruz Biotechnology Inc., and antibodies of caspase-3 and cytochrome- c proteins were obtained from BD Biosciences Pharminogen.

As a result, as shown in FIG. 5, procaspase-8 (53 kDa) was activated with caspase-8 (20 kDa), and procaspase-9 (43 kDa) was activated with caspase-9. (37kDa) and decreased the expression of procaspase-3. The release of cytochrome- c from the mitochondria into the cytoplasm suggests that apoptosis is induced by the mitochondrial dependent autoapoptosis pathway. When EECP was treated at a concentration of 50-200 μg / ml, it was confirmed that the release of cytochrome- c into the cytoplasm was increased in a concentration-dependent manner. Cleavage of poly ADP-ribose polymerase (PARP), one of the substrates of caspase-3, is also a feature found in self-killing. Activated caspase-3 cleaves 116 kDa poly ADP-ribose polymerase into fragments of 85 kDa. After the EECP treatment as shown in Figure 5 it can be seen that the poly ADP-ribose polymerase is cut to 85 kDa. These results confirm that EECP induces apoptosis of HeLa cells, which is a mitochondria dependent signal pathway.

Experimental Example  6. DNA chip analysis

Differences in gene expression variation between HeLa cells treated with EECP and untreated controls were confirmed using Platinum Biochip cancer 3.0 K oligo chip manufactured by Genochem. Total RNA samples were synthesized with complementary DNA (cDNA) attached to specific primers through reverse transcription process, and the hybridization was carried out on the Platinum oligo chip using the first hybridization. Labeling was done during the procedure. The genotyping Platinum Biochip Human cancer 3.0K oligo chip used in this experiment is known gene (2959) and unknown EST gene (81) and housekeeping gene of human cancer oligo set of Array_Ready Oligo set provided by Qiagen Operon. A total of 3096 spots containing Arabidopsis DNA, housekeeping genes and control genes, are composed of 23 columns and 23 rows in one block. . Each oligonucleotide was dissolved in 15 μl of spotting solution provided by Zinochet Co., Ltd. and used to fix 24 Stealth Micro spotting pins using a pixsys 5500 arrayer (Cartesian Technologies, CA). ) Was deposited on CMT-GAPS II silane slide glass (Corning, NY). Dropped slides were rehydrated with 1 X SSC solution for 1 minute, the DNA was linked to the slides with a UV crosslinker (Stratagene, Calif.) And then succinic anhydride / sodium borate solution with moderate agitation. Soak for 15 minutes and leave for 2 minutes in a 95 ℃ water bath (water bath). After that, the slides were quickly transferred to 95% ethanol, left for 1 minute, and then centrifuged at 3,000 rpm for 20 seconds to dry. Extraction of total RNA for DNA chip analysis was carried out using HeLa cells treated with EECP for 48 hours and control HeLa cells untreated using a tree reagent (TRI REAGENT; MRC, OH) according to the manufacturer's recommendations. Fluorescent label complementary DNA (cDNA) probes were constructed using 40 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen, NY). Reverse transcripts consisted of 400 U SuperScript RNase H-reverse transcriptase (Invitrogen), 0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP and 0.1 mM Cy3 or Cy5 labeled dCTP (NEN Life Science Product Inc.). After reverse transcription, sample RNA was added with 5 µl of stop solution (0.5M NaOH / 50m EDTA) and reacted at 65 ° C. for 10 minutes to stop reverse transcription. The labeled cDNA mixture is concentrated by ethanol precipitation. Re-dissolve in 20 μl hybridization solution (Ginosek, Korea) with concentrated Cy3 and Cy5 labeled cDNAs. The two cDNAs were mixed and denatured at 95 ° C. for 2 minutes, and then reacted in a 45 ° C. water chamber for 20 minutes, and then the cDNA mixture was placed on a spotted slide and covered with a cover slip (22 mm × 22 mm). . The slides are hybridized in a hybridization chamber at 62 ° C. for 12 hours. The hybridized slides were washed for 2 minutes with 2 X SSC and 0.1% SDS solution, and then washed for 3 minutes with 1 X SSC solution and 2 minutes with 0.2 X SSC solution at room temperature. The slides are then centrifuged at 3000 rpm for 20 seconds to dry. Hybridization slides are scanned with an Exxon Instruments GenePix 4000B scanner to scan the scanned images with GenePix Pro 5.1 (Axon, CA) and GeneSpring 6.1 (Sillicongenetics, CA), R. Analysis was done using the package program. The analysis of the DNA chip results was carried out using global normalization and intensity dependent normalization using the signal intensity data obtained through scanning. In addition, block-wise standardization was performed to minimize the difference between each block of the chip. The distribution of all genes obtained as a result of the experiment was displayed as a histogram and compared with the distribution of the standardized data to confirm the accuracy of the experiment. Significance between the experimental group and the control group was classified as a gene representing a difference in expression. As a result of hybridization experiment, the gene expression variation showed expression variation of more than 2 times that a total of 115 genes were significant level in the microarray experiment. A total of 69 genes, including the interleukin 1 alpha gene (IL1A gene), overexpressed more than twofold (fold), whereas 79 genes, including the AKAP12 gene, overexpressed more than twofold.

Among genes related to DNA metabolism, self-killing, cell cycle, transcription, cell proliferation, and signaling among 115 genes, important genes whose expression is increased or decreased by EECP are summarized in Tables 2 to 7 below. As shown in Table 2 below, TOP2A, a gene involved in DNA metabolism, was reduced by two or more times by EECP treatment. As shown in Table 3, IGF1R, a gene related to cell death, was reduced by two or more times, and PPP1R15A, PHLDA2, IL1A, and the like increased by two or more times. As shown in Table 4, as a gene related to the cell cycle, GAS1, FOSB, CENPF, MAP2K6, KNTC2, IGF1R, and the like decreased by two or more times. As shown in Table 5, as a gene related to cell proliferation, SOC3, SKI, DAB2, and the like decreased more than two times, FGF18 and GPNMB more than two times increased. As shown in Table 6 below, as genes related to transcription, FOS, INSM1, etc. were reduced more than two times and JUN increased more than two times. As shown in Table 7, as a gene related to signal transduction, AKAP12, TGFBR1, VTN, LIFR, etc. decreased more than two times and PPP2R5B increased more than two times. As a result, EECP has the effect of inhibiting the proliferation of cells and inducing cell death through the inhibition of the cell cycle and the transcriptional inhibition by acting on HeLa cells, and confirmed that it can be useful as a composition for preventing and treating uterine cancer. .

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_001067 0.46 Topoisomerase (DN A) Ⅱ alpha 170kDa TOP2A

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_000875 0.50 Insulin-like growth factor 1 receptor IGF1R NM_014330 2.15 Protein phosphatase 1, regulatory (inhibitor) subunit 15A PPP1R15A NM_003311 2.29 Pleckstrin homology-like domain, family A, member 2 PHLDA2 NM_000575 7.24 Interleukin 1, alpha IL1A

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_002048 0.32 Growth arrest-specific 1 GAS1 NM_006732 0.35 FBJ murine osteosarcoma viral oncogene homolog B FOSB NM_016343 0.36 Centromere protein F, 350 / 400ka (mitosin) CENPF NM_002758 0.39 Mitogen-activated protein kinase kinase 6 MAP2K6 NM_006101 0.50 Kinetochore associated 2 KNTC2 NM_000875 0.50 Insulin-like growth factor 1 receptor IGF1R

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_003955 0.30 Suppressor of cytokine signaling 3 SOCS3 NM_003036 0.35 KIAA0508 protein SKI NM_001343 0.39 Disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAB2 NM_000875 0.50 Insulin-like growth factor 1 receptor IGF1R NM_003862 2.05 Fibroblast growth factor 18 FGF18 NM_002510 2.78 Glycoprotein (transmembrane) nmb GPNMB

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_005252 0.33 V-fos FBJ murine osteosarcoma viral oncogene homolog FOS NM_006732 0.35 FBJ murine osteosarcoma viral oncogene homolog B FOSB NM_002196 0.40 Insulinoma-associated 1 INSM1 NM_002228 3.46 V-jun sarcoma virus 17 oncogene homolog (avian) JUN

Gene Bank No. Expression Fold Description Gene Symbol DOWN UP NM_005100 0.28 A kinase (PRKA) anchor protein (gravin) 12 AKAP12 NM_004612 0.37 Transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53kDa) TGFBR1 NM_000638 0.41 Vitronectin (serum spreading factor, somatomedin B, complement S-protein) VTN NM_002310 0.43 Leukemia inhibitory factor receptor LIFR NM_006244 2.48 Protein phosphatase 2, regulatory subunit B (B56), beta isoform PPP2R5B

Experimental Example  7. Small Bead Coral  In mouse models of extracts Celiac cancer  Cell line inhibitory effect test

The following experiment was performed to investigate the inhibitory effect of celiac cancer extract in small animal models. As an animal model, C3H / HeNTacSam-MTV male mice (Samtaco, Osan, Gyeonggi-do) weighing 20 to 24 g and 6 to 8 weeks old were used. After one week of adaptation, the subcultured sarcoma cells were diluted in buffer to 1 × 10 ⁶ cells and injected through the hind limbs to induce cancer. Experiments were performed in which mice showing signs of cancer, such as abdominal distension and swelling of hair, were selected over 10 to 14 days after cancer cell injection, and composed of four groups of five animals per group. The negative control group was untreated group, 1 ml of physiological saline, the positive control group was orally administered 3 mg / kg of adriamycin, an anticancer agent, and 40 mg / kg of EECP, respectively. At 15 days after drug administration, mice were sacrificed and dissected to extract cancerous tissues from the abdominal cavity, and the weight and volume of the cancerous tissues were measured. The volume was calculated by substituting Equation 1 below and the anticancer efficiency in Equation 2 below, and all experimental values were statistically treated by Student's t test. Experimental results As shown in Figure 6 and Table 8, compared with the control (A) of Figure 6, the group treated with adriamycin with a positive control (B) showed a 61.1% cancer inhibitory effect, EECP 40 mg / kg The treated experimental group (C) was confirmed to show a cancer suppression effect of 72.1%.

Volume = (width × depth × length) / 2

Anticancer Efficiency = [1- (Tumor Volume Average of EECP Treatment / Tumor Volume Average of Control)] × 100

Control group (n = 5) Adriamycin (n = 5) EECP (n = 5) Body weight (g) 31.2 ± 4.8 385 ± 56 27.6 ± 0.32 Cancer tissue volume (mm ³ ) 445.5 ± 88.4 173.5 ± 81.4 ^a 121.8 ± 13.9 ^a Cancer Tissue Weight (mg) 385 ± 56 158 ± 67 ^a 154 ± 23 ^a Anticancer efficiency (%) 0 61.1 72.1 ^a comparison to the control, p <0.05

Hereinafter, an example of the preparation of a pharmaceutical composition containing an extract of the present invention will be described, but the present invention is not intended to limit the present invention but is only intended to be described in detail.

Formulation example  One. Powder  Produce

300 mg of EECP of Example 1

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

300 mg of EECP of Example 1

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

300 mg of EECP of Example 1

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

300 mg of EECP of Example 1

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

300 mg of EECP of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, the lemon flavor is appropriately added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

EECP 1000 mg of Example 1

Vitamin Mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

Formulation example  7. Manufacture of health drinks

EECP 1000 mg of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed, sterilized and refrigerated It is used to prepare a healthy beverage composition of the present invention. 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.

Small beaded horsetail extract of the present invention induces cancer cell suppression effect and self-killing and exhibits in vivo anti-cancer activity in a mouse model, which can be usefully used in pharmaceutical compositions and health functional foods for preventing and treating cancer diseases. Can be.

Claims (6)

Small-Beaded Coral ( Corallina) pilulifera ) Pharmaceutical composition for the prevention and treatment of cancer diseases containing the extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the cancer disease is liver cancer, thyroid cancer, ovarian cancer, gallbladder cancer, colon cancer, leukemia, esophageal cancer, gastric cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, lung cancer, and laryngeal cancer. The pharmaceutical composition according to claim 1, wherein the extract is an extract soluble in a solvent selected from water, methanol, ethanol or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the extract is contained in an amount of 0.02 to 50% by weight based on the total weight of the composition. Health functional food for cancer disease prevention and improvement including small bead coral extract and food acceptable food supplement. A dietary supplement according to claim 5 which is a powder, granule, tablet, capsule or beverage.

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