KR100691525B1 - A composition comprising the extract of Ptercarpus santalinus for the prevention and treatment of cancer - Google Patents

Abstract

The present invention relates to a composition containing the sweet potato extract as an active ingredient, specifically, the sweet potato extract of the present invention has various cancer cell inhibitory effect and induction effect, to the pharmaceutical composition and health functional food for cancer disease prevention and treatment It can be usefully used.

Rosewood fragrance, cytotoxicity, anticancer activity, cancer, pharmaceutical composition, health functional food

Description

A composition comprising the extract of Ptercarpus santalinus for the prevention and treatment of cancer}

1 is a graph showing the survival rate of HeLa cells according to the concentration of rosewood extract,

2 is a graph showing the inhibition rate of HeLa cell proliferation according to the concentration of rosewood extract,

3 is a cell cycle analysis of HeLa cells,

Figure 4 shows the morphological changes of HeLa cells treated with Rosewood extract for 48 hours through DAPI staining,

Figure 5 shows the DNA fragmentation of DMSO treatment (A), alveolar extract (B), DNase I treatment (C) HeLa cells through tunel staining,

6 is a result of Western blot analysis of HeLa cells treated with Rosewood extract for 48 hours,

Figure 7 shows the effect of the rosewood extract on mouse liver toxicity.

Apoptosis is the most important pathway leading to cell death and plays a very important role in the maintenance of eukaryotic homeostasis and tissue growth control (Green and JC Reed, et al., Science , 281, pp 1308-1312 , 1998; MO Hengartner, Nature, 407, pp 770-776, 2000). Cell death and cell proliferation are necessary processes to maintain intracellular balance, and in order to perform this process normally, cells need to be in a steady state. However, this disruption of intracellular equilibrium causes humans to cause a variety of diseases, including cancer (CB Thompson et al., Science , 267 , pp 1456-1462, 1995). Apoptosis occurs through the extracellular suicide receptor dependent pathway and the intracellular mitochondrial dependent pathway. This self-killing is also induced by treatment with chemotherapeutic agents (SH Kaufmann and WC Earnshaw, Experimental Cell Research, 256, pp 42-49, 2000; PR Walker, C. Smith et al., Cancer Research, 51 , pp 1078-). 1085, 1991). Caspases, a species of cysteine proteases in the cytoplasm, play an important role in the practice of self-killing. Procaspase-8, the first practitioner, is transformed into caspase-8 by its own self-processing for stimuli that lead to cell death. Procaspase-9, on the other hand, is activated by the mitochondrial pathway (G. Denecker, D. Vercammen et al., Cell. Mol . Life Sci ., 58, pp356-370, 2001). It also plays an important role in inducing cells into cell death by signals such as deficiency of growth factors, ultraviolet light, cyclophosphamide (leukemia treatment), and anticancer agents such as etoposide (SE Wilson). , Exp.Eye Res., 69 , pp 255-266, 1999). The mitochondrial pathway occurs with the mediation of Bcl-2 termination proteins. Bcl-2 species include Bcl-2 and Bax proteins, which regulate the transport of cytochrome- C in mitochondria (Kelekar and CB Thompson, Trends Cell Biol ., 8, pp.324-330 , 1998). Self-killing occurs in cells that undergo various auto-killing induction signals, in which cytochrome- C is released into the cytoplasm from mitochondria (DD Newmeyer and S. Ferguson-Miller, Cell, 112, pp481-490, 2003). Released Cytochrome- C interacts with dATP, apoptosis protease activating factor (APaf1) and procaspase-9 in the cytoplasmic apoptosome complex and consequently acts as an active caspase for procaspase-9 Modified with spase-9 (J. Chandra, A. Samali et al., Free Radic . Biol . Med ., 29, pp. 323-333, 2000). Activated caspase-9 activates performers such as caspase-3, -6, -7 (GS Salvesen and VM Dixit, Cell, 91, pp443-446, 1997). Performer caspases cleave inhibitors of poly ADP-ribose polymerase (PARP), cellinas (laminas) and casad activated DNase (ICAD). Thus, these cleaved proteins are used as target proteins for self-killing. These phenomena result in morphological characteristics such as condensation of chromosomes, fragmentation of DNA, and formation of apoptosis bodies (M. Germain, EB Affar, et al., J. Biol. Chem. 274, pp 28379-28384, 1999).

Plants can be used as a valuable resource for natural bioactive substances and have been used for a long time in medicine, including Korea. Currently, the scientific approach to oriental medicine is increasing for the development of new drugs including anticancer drugs (W. Hu and JJ Kavanagh, Lancet Oncology, 4, pp721-729, 2003; SM Lee, ML Li, et al., Life Science, 71, pp 2267-2277, 2002). In Korea Plants known as rosewood ( Ptercarpus santalinus ) have been widely used as dyes in Southeast Asia and India (D. Palevitch, Z. Yaniv, Oryx Press, Arizona, 1986) . Known as a medicinal plant in Korea, it has been used as a folk remedy for the treatment of inflammation, mental disorders and ulcers.

However, no data have been published that show the exact evidence of rosewood in vitro and in vivo. Therefore, based on this information, this study investigated the cytotoxic effects of various cells using extracts of rosewood and its mechanism in HeLa cells.

       It is an object of the present invention to provide a composition for preventing and treating cancer diseases, including a rosewood extract having anticancer activity by inducing cell growth inhibition and self-killing.

In accordance with the above object, the present invention provides a composition for the prevention and treatment of cancer diseases, using the extract of the algae ( Ptercarpus santalinus ) as an active ingredient.

The extract means an extract selected from water, methanol, lower alcohols such as ethanol or a mixed solvent thereof, preferably 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.

       The sweet potato extract of the present invention can be obtained as follows.

The sweet almond extract of the present invention, after washing the altar flavor clean, about 1 to 10 times, preferably about 2 to 8 times water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof of the weight (kg) Cold extraction, hot water extraction, ultrasonic extraction, reflux for about 10 hours to 40 hours, preferably 20 hours to 30 hours at an extraction temperature of 0 to 100 ℃, preferably 10 to 50 ℃ with a solvent selected from Extraction is repeated several times by an extraction method such as cold extraction, preferably cold leaching, and additionally about 1 hour to 24 hours, preferably 10 hours to 15 hours at an extraction temperature of 0 to 100 ° C, preferably 50 to 70 ° C. Extraction method such as cold sediment extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction for several times, preferably by reflux cooling extraction several times and then filtered to obtain the sweet potato extract of the present invention Can be.

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 cell growth inhibition and self-apoptosis-inducing activity in various cancer cells of the extract of the sweet potato extract of the present invention obtained by the above method, it was confirmed that it can be usefully used as a composition for preventing and treating cancer diseases.

In addition, the rosewood fragrance has been used as a herbal medicine for a long time, the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects.

It provides a pharmaceutical composition for preventing and treating cancer diseases containing the sweet almond 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.

Pharmaceutical dosage forms of the extracts of the present 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 any 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, it provides a health functional food for cancer disease prevention and improvement containing the sweet almond 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.

The health functional food for preventing and improving cancer diseases of the present invention comprises the extract in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on 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 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 only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1. Preparation of Rosewood Extract

After cutting 10Kg of the rosewood fragrance stem from Hung-Hwan Co., Seoul, washed with purified water, 30L of ethanol was added and repeated cold extraction three times at room temperature for 24 hours and additionally extracted under reflux condition for 12 hours at 70 ℃. Was repeated twice, and the resultant was filtered with a filter paper (Whatman, USA), concentrated under reduced pressure with a vacuum condenser (EYELA, N-1000, Japan), and then lyophilized to extract an extract of sweet potato (produced amount: 635.46 g). ) Was obtained. This extract was dissolved in DMSO (Sigma, St Louis, MO), stored at 4 ° C. and used as a sample of the present invention (hereinafter referred to as EEPS).

Reference Example 1. Cell Lines and Culture Media

1-1. HeLa cell culture

HeLa cells, a cervical cancer cell line, were purchased from the American Type Culture Collection, Rockville. HeLa cells were cultured at 37 ° C. and 5% CO ₂ with DEME (Dulbecco's modified Eagle's medium) added 10% (v / v) Fetal bovine serum (FBS) and 0.1% gentamycine. It was.

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 cultured at 37 ° C. and 5% CO ₂ with RPMI 1640 using 10% (v / v) FBS (Fetal bovine serum) and 0.1% gentamycine.

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 cultured at 37 ° C. and 5% CO ₂ , using Dulbecco's modified Eagle's medium (DEME) added 10% (v / v) Fetal bovine serum (FBS) and 0.1% gentamycine. It was.

1-4. A549 cell culture

Human lung cancer cell line A549 cells were purchased from the American Type Culture Collection, Rockville (ATCC). A549 cells were cultured under RPMC 1640 with 10% (v / v) FBS (Fetal bovine serum) and 0.1% gentamycine added at 37 ° C and 5% CO ₂ .

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 cultured under RPMI 1640 with 10% (v / v) FBS (Fetal bovine serum) and 0.1% gentamycine added at 37 ° C and 5% CO ₂ .

1-6. Jurket E6-1 Cell Culture

Jurket E6-1 cells, a human blood cancer cell line, were purchased from the American Type Culture Collection, Rockville. Jurket E6-1 cells were cultured under conditions of 37 ° C. and 5% CO ₂ using RPMI 1640 as a medium containing 10% (v / v) FBS (Fetal bovine serum) and 0.1% gentamycine.

Experimental Example 1. Measurement of cytotoxic activity and cytostatic activity

1-1. Cytotoxicity Measurement

The cytotoxicity of EEPS against each cancer cell was measured. Cytotoxicity was determined by MTT assay (T. Mosmann, Journal of immunological methods, 65 , pp55-63, 1983), and cultured by dispensing an initial cell number of 1 × 10 ⁴ into a microtiter (Elisa). EEPS was treated at different concentrations (5-75 μg / ml) and the control was treated with 0.1% DMSO. After treatment for 48 hours at 37 ℃, MTT solution was added and incubated for 4 hours at 37 ℃. Optical concentration was measured at 550 nm using an ELISA reader. As shown in Table 1, the cytotoxic activity of EEPS on each cell line was confirmed that the cancer cell inhibition rate was excellent even at low concentrations. In addition, the cytotoxicity of HeLa cells of EEPS showed a higher activity as shown in Figure 1, the higher the activity was confirmed in a concentration-dependent.

Cell line IC ₅₀ (ug / ml) HT29 19.5 HepG2 25.1 A549 30.3 T24 33.3 HeLa 40 Jurket E6-1 34.5

1-2. Cell proliferation inhibitory measure

To examine the effect of EEPS on the inhibition of proliferation of cancer cells, trypan blue exclusion assay (KH Jones and JA Senft, The journal of histochemistry and cytochemistry, 33 , pp77-79, 1985) was used.

HeLa cells were dispensed in a 35 mm culture dish, treated with different concentrations of EEPS (5, 25, 50, 75 μg / ml) and incubated for 7 days, and trypan blue staining (Trypan) was performed at 2-day intervals to measure live cells. blue exclusion) was used. The cells were trypsinized and washed with PBS (phosphate-buffered saline), and the cells were suspended by the addition of trypan blue staining solution. Live cells were measured using a hemocytometer. Cells treated with EEPS compared to control cells showed a significant difference from day 3, and when treated with 25 ㎍ / ㎖ showed a 59.1% survival rate compared to 100% of the control. In addition, the concentration of EEPS was gradually increased to 5, 25, 50, 75 µg / ml and cultured for 7 days. As shown in FIG. 2, 17.6 ± 10.0%, 41.1 ± 5.1%, 90.2 ± 2.6%, It was confirmed that the survival rate was concentration-dependently lowered to 97.8 ± 1.0%.

Experimental Example 2. Flow 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 and treated with 5, 12.5, 25 μg / ml EEPS, and 0.1% DMSO for 48 hours. The cells were removed from the dish with trypsin, washed once with PBS, and the cells were collected and resuspended in PBS. DNA staining was done using the CycleTEST ^™ PLUS kit (Becton Dickinson, Heidelberg). Nuclear fractions stained with propidium iodide (PI) were obtained by following the experimental procedure of the kit. Fluorescence intensity was determined using a FAC Scan flow cytometer, and analysis was performed using CellQuest software (Becton Dickinson). As a result, as shown in FIG. 3, the sub-G1 population showing self-killing by EEPS was significantly increased compared to the control 2.5% (5 μg / ml: 5.5%, 12.5 μg / ml: 10.0). %, 25 μg / ml: 39.9%). In the non-self killing population, low concentrations of EEPS increased G1 cell population (control: 50.2%, 5µg / ml: 66.5%, 12.5µg / ml: 74.1%). Cell group (control: 29.7%, 5 µg / ml: 20.8%, 12.5 µg / ml: 14.1%) and G ₂ / M group (control: 20.1%, 5 µg / ml: 12.8%, 12.5 µg / ml: 11.8%) was gradually decreased. When EEPS was treated at a high concentration (50 µg / ml), it was confirmed that the G1 group slightly decreased than the lower concentration (67.6%). This decrease in cell number was accompanied by an increase in cells in the sub-G1 phase. As a result of cell cycle analysis, it was confirmed that EEPS stops the cell cycle of HeLa cells in G1 phase in a concentration-dependent manner (see the arrow in FIG. 3) and induces autokilling.

Experimental Example 3. Induction of self-killing by EEPS

In order to confirm that the cell proliferation inhibitory effect of EEPS is a phenomenon caused by self-killing, HeLa cells treated with EEPS for 48 hours were subjected to nuclear staining with DAPI. HeLa cells were washed once with PBS and the cells were fixed for 10 minutes at room temperature using 3.7% paraformaldehyde. Fixed cells were washed with PBS and treated with DAPI (4, 6-diamidino-2-phenylindole; Sigma) solution and stained for 10 minutes at room temperature. Stained cells were washed twice with PBS and analyzed by fluorescence microscopy. As shown in FIG. 4, chromatin was condensed in the nucleus of HeLa cells treated with 5 μg / ml EEPS (B) than 0.1% DMSO-treated control group (A). B; see the arrows), which is typical of the autologous killing. This phenomenon increases the number of autologous cells as the concentration of EEPS increases.

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

Self-killing was determined to confirm DNA fragmentation because DNA fragmentation is another characteristic of self-killing (ID Bowen, SM Bowen et al., Chapman and Hall, London, 1998). 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. Next the cells were washed twice with PBS. After fixing for 25 minutes at room temperature using 4% paraformaldehyde solution and washed twice with PBS. The cells were soaked in 0.2% Triton X-100 solution for 5 minutes and washed with PBS. Negative controls and positive controls were prepared by treatment with DMSO (A) and DNase I (C), respectively. After the above procedure, the cells were equilibrated with buffer for 5 minutes at room temperature. Next, a TdT enzyme reaction mixture and a biotinylated nucleotide 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 PBS, treated with 0.3% (v / v) hydrogen peroxide solution for 5 minutes, rinsed with PBS and treated with DAB until the area was light brown in color. The stained cells were observed under an optical microscope. It was. As shown in FIG. 5, the nucleus (B) of the cells treated with EEPS was stained dark brown, and only a few cells of the control cells were identified.

Experimental Example 5 Up-down Regulatory Effects of Self-killing Associated Proteins by Treatment of EEPS

Self-killing requires the activation of caspase species. Procaspases-8 and -9 are early implementers and are activated in their own processes and make downstream procaspase-3 an active dimer, caspase-3, the execution factor for self-killing. To verify that EEPS induces the activation of these caspases, HeLa cells were exposed to EEPS followed by Western blot analysis using procaspase-3, -8, -9 and cytochrome- C antibodies. HeLa cells were treated with 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) in 0.1% Triton X-100, 1 mM ATP and protease inhibitors. 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 and actin was used as a control. SDS-PAGE was performed. Caspase-3, -8, -9, 15% of cytochrome- C and actin and 8% of PARP were used as separation gels. After protein electrophoresis, the protein in the gel was transferred to PVDF membrane and blocked for 1 hour at room temperature using a blocking solution (Blockace ^™ , Dai-Nippon). The primary antibody was reacted at 37 ° C. for 1 hour and the membrane was washed with 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). The reaction was performed at 37 ° C. for 1 hour using a peroxidase-conjugated secondary antibody (Pierce), and the immunoreactive protein was detected by a chemiluminescence system (SuperSignal West Femto Maximum sensitivity Substrate, Pierce). . The degree of reaction was quantified using Fluorchem ^™ 5500 (Alpha Innotech). Actin, PARP, Caspase-8, -9 Antibodies are listed in Santa Cruz Biotechnology Inc. Antibodies of caspase-3, cytochrome- C protein at were purchased from BD Biosciences Pharminogen.

As a result, as shown in Figure 6, Pro caspase-8 (53 kDa) was activated with caspase-8 (20 kDa), and procaspase-9 (43 kDa) was activated cas It was changed to spase-9 (37 kDa) and reduced the expression of procaspase-3. The release of cytochrome- C from the mitochondria into the cytoplasm suggests that self-killing is induced by the mitochondrial dependent self-killing pathway. When EEPS was treated at a concentration of 10-50 μg / ml, it was confirmed that the release of cytochrome- C into the cytoplasm increased in a concentration-dependent manner. Cleavage of PARP, one of the caspase-3 substrates, is also a feature found in self-killing. Activated caspase-3 cleaves 116 kDa PARP into 85 kDa fragments. After the EEPS treatment as shown in Figure 6 it can be seen that the PARP is cut to 85kDa. These results confirm that EEPS induces apoptosis of HeLa cells, which is a mitochondria dependent signal pathway.

Experimental Example  6. DNA chip analysis

In this experiment, we used the Platinum Biochip cancer 3.0K oligo chip manufactured by Genochem Co., Ltd. to confirm the difference in gene expression variation between HeLa cells treated with sweet potato ethanol extract and untreated controls. Total RNA samples were synthesized by cDNA with a specific primer through reverse transcription process, and the first fusion was performed on the Platinum oligo chip. And it was labeled during the second fusion process. The Genotype Platinum Biochip Human cancer 3.0K oligo chip used in this experiment is known gene (2959) and unknown EST gene (8159) and housekeeping of human cancer oligo set of Array_Ready Oligo set provided by Qiagen Operon. A total of 3096 spots containing Arabidopsis DNA, housekeeping gene and control gene, are composed of 23 columns and 23 rows in one block. have. Each oligonucleotide was dissolved in 15 μl of spotting solution provided by Ginosque, Inc., and used 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 × SSC solution for 1 minute and the DNA was linked to the slides with a UV crosslinker (Stratagene, Calif.). Next, the slides are soaked with succinic anhydride / sodium borate solution for 15 minutes with moderate stirring. Soak for 2 minutes in a water bath. The slides are then quickly transferred to 95% ethanol for 1 minute and dried by centrifugation at 3,000 rpm for 20 seconds. Total RNA extraction for DNA chip analysis was performed using TRI REAGENT (MRC, OH) and HeLa cells treated with EEPS for 48 hours, according to the manufacturer's recommendation. Fluorescent label cDNA probes were prepared by oligo (dT) ₁₈ -primary polymerization using 40 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen, NY). Reverse transcription reactions consisted of 400 U SuperScript RNase H- reverse transcriptase (Invitrogen), 0.5 mM dATP, dTTP and dGTP, dCTP labeled 0.2 mM dCTP and 0.1 mM Cy3 or Cy5 (NEN Life Science Product Inc.). After reverse transcription, sample RNA was digested by adding 5 μl of stop solution (0.5M NaOH / 50m EDTA) and incubating at 65 ° C. for 10 minutes. The labeled cDNA mixture is concentrated by ethanol precipitation. Re-dissolve in 20 μl hybridization solution (GenoCheck, KR) with concentrated Cy3 and Cy5 labeled cDNAs. The two cDNAs are mixed, denatured at 95 ° C. for 2 minutes, and reacted for 20 minutes in a 45 ° C. water chamber. The cDNA mixture is then placed on a spotted slide and covered with a coverslip (22 mm x 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 were scanned with an Axon 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 DNA chip results was performed using global intensity and intensity dependent normalization using the signal intensity numerical 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 more than two-fold expression variation in which a total of 148 genes were significant in the microarray experiment. A total of 69 genes, including the CDKN1A gene, a cyclin-dependent kinase inhibitor 1A (p21, Cip1), overexpressed more than two-fold, while down-expression decreased more than two-fold. The expression genes were 79 including lipoprotein lipase (LPL) genes. Among the genes related to DNA regeneration, self-killing, cell circulation, cell proliferation, and transcription among 48 genes, important genes whose expression is increased or decreased by EEPS are shown in Tables 1 to 5 below. By EEPS treatment, XRCC5 and NTHL decreased more than twofold among genes repairing DNA damage. The genes involved in cell death were more than two-fold decreased in CASP9, PRKCZ, PRKCE, MYBL2 and IGF1R, and more than two-fold increased in NFKB1A, TNFRSF25, TNFRSF10D and IL1A. The genes related to cell cycle were more than doubled in CDC7, MCM5 and CCND3, and more than doubled in CDC34. The genes related to cell proliferation were reduced by more than 2 times in MXD4 and UHRF1. The genes related to transcription decreased by 2 times in HOXB8, SMARCA3, ATF7 and BATF and MAFF more than 2 times. As a result, EEPS has the effect of inhibiting cell proliferation and inducing cell death through HeLa cells by inhibiting the cell cycle and transcription inhibition, it can be usefully used as a composition for preventing and treating uterine cancer. .

 Degree of gene expression repairing DNA damage in EEPS treatment Gene bank  No. Expression Fold Description Gene Symbol down up NM_021141 0.45 X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand-break rejoining; Ku autoantigen, 80kDa) XRCC5 NM_002528 0.49 Nth endonuclease III-like 1 (E. coli) NTHL1

 Degree of Gene Expression Associated with Apoptosis in EEPS Treatment Gene bank  No. Expression Fold Description Gene Symbol down up NM_001229 0.32 Caspase 9, apoptosis-related cysteine protease CASP9 NM_002744 0.37 Protein kinase C, zeta PRKCZ NM_005400 0.42 Protein kinase C, epsilon PRKCE NM_002466 0.44 V-myb myeloblastosis viral oncogene homolog (avian) -like 2 MYBL2 NM_000875 0.45 Insulin-like growth factor 1 receptor IGF1R NM_020529 2.00 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha NFKBIA NM_003790 2.94 Tumor necrosis factor receptor superfamily, member 25 TNFRSF25 NM_003840 3.30 Tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain TNFRSF10D NM_000575 5.67 Interleukin 1, alpha IL1A

Degree of Gene Expression Related to Cell Cycle in EEPS Treatment Gene bank  No. Expression Fold Description Gene Symbol down up NM_003503 0.38 CDC7 cell division cycle 7 (S. cerevisiae) CDC7 NM_006739 0.44 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae) MCM5 NM_002466 0.44 V-myb myeloblastosis viral oncogene homolog (avian) -like 2 MYBL2 NM_000875 0.45 Insulin-like growth factor 1 receptor IGF1R NM_001760 0.48 Cyclin D3 CCND3 NM_004359 2.18 Cell division cycle 34 CDC34

Gene Expression Related to Cell Proliferation in EEPS Treatment Gene bank  No. Expression Fold Description Gene Symbol down up NM_006454 0.38 MAX dimerization protein 4 MXD4 NM_003503 0.38 CDC7 cell division cycle 7 (S. cerevisiae) CDC7 NM_000875 0.45 Insulin-like growth factor 1 receptor IGF1R NM_013282 0.48 Ubiquitin-like, containing PHD and RING finger domains, 1 UHRF1

Gene expression related to transcription during EEPS treatment Gene bank No. Expression Fold Description Gene Symbol down up NM_006454 0.38 MAX dimerization protein 4 MXD4 NM_024016 0.41 Homeo box B8 HOXB8 NM_006739 0.44 MCM5 minichromosome maintenance deficient 5, cell division cycle 46 (S. cerevisiae) MCM5 NM_013282 0.48 Ubiquitin-like, containing PHD and RING finger domains, 1 UHRF1 NM_003071 0.48 SWI / SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 3 SMARCA3 NM_006856 0.48 Activating transcription factor 7 ATF7 NM_006399 2.27 Basic leucine zipper transcription factor, ATF-like BATF NM_012323 2.41 V-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) MAFF

Experimental Example 7. Toxicity Test of Rosewood Extract

Animal experiments were performed to confirm the toxicity of the almond extract. C57bl / 6 mice were divided into four groups of 10 males each of 20 to 25 g, and then the algae extract of the present invention (EEPS) was orally administered at doses of 125, 250, 500 and 1000 mg / kg, respectively. After oral administration, toxicity was observed for 2 weeks, and none of the 125 and 250 mg / kg groups died, and both the 500 and 1000 mg / kg groups died (see Table 6). The autopsy revealed that histologically no symptoms were found. As a result of extracting the livers of the deceased mice and measuring the hepatotoxicity, as shown in FIG. There was no difference and the algae extract was not toxic.

Dosage Survival rate (death / test subject) EEPS 1g / kg 5/5 EEPS 500mg / kg 5/5 EEPS 250mg / kg 0/5 EEPS 125mg / kg 0/5

Experimental Example 8. Inhibition effect of celiac cancer cell line in mouse model

In order to investigate the inhibitory effect of peritoneal extract in real animal model was performed as follows.

Animal models consisted of four groups of five rats of each group using three to four week old C57bl / 6 male mice (weight 13-17 g). After one week of adaptation, cancer-causing cancers were injected by injecting sarcoma cells that were passaged into cells at 5 × 10 ³ cells in the abdominal cavity. At the same time, the negative control group was treated as physiological saline 1ml, the positive control group was treated with 3 mg / kg of adriamycin, an anticancer drug, and the experimental group was treated with 100 mg / kg of algae crude extract, respectively. Intraperitoneal cancer, the peritoneal circumference and body weight were measured at intervals of 6 to 3 days from the day of significant occurrence, and the extract of the algae extract inhibited tumor growth. As a result, it was confirmed that adriamycin, an anticancer agent treated with a positive control, showed an inhibitory effect of 60%, and 100mg / kg of sweet potato extract exhibited a 50% ascites inhibitory effect.

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

Formulation Example 1 Preparation of Powder

Rosewood extract 300 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

Rosewood extract 300 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

Rosewood extract 300 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

Rosewood extract 300 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 12} H 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

Rosewood extract 300 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve, 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 Preparation of Healthy Food

Rosewood extract 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

Formulation Example 7 Preparation of Healthy Drink

Rosewood extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage 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.

The algae extract of the present invention exhibits various cancer cell inhibitory effects and anti-cancer activity inducing self-killing, and can be usefully used in pharmaceutical compositions and health functional foods for preventing and treating cancer diseases.

Claims (6)

A pharmaceutical composition for preventing and treating liver cancer, colorectal cancer, leukemia, uterine cancer, lung cancer or bladder cancer, containing ethanol extract of rosewood stem as an active ingredient. delete delete 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 preventing and improving liver cancer, colorectal cancer, leukemia, uterine cancer, lung cancer or bladder cancer, including ethanol extract of rosewood stem and food acceptable food supplement. delete

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