KR20040009983A - Extracts and fractions of Betula platyphylla var. japonica with antioxidant and/or anticancer activities - Google Patents

Abstract

PURPOSE: Provided are extracts and fractions of Betula platyphylla var. japonica having antioxidant and/or anticancer activities. They alleviate and/or treat cell damage by reactive oxygen species, remove radicals to inhibit the formation of lipid peroxide and have anticancer activity. CONSTITUTION: Betula platyphylla var. japonica is extracted with a polar solvent, such as 70% methanol. Therefore, the extract has antioxidation effects, such as inhibition of lipid peroxide formation and cell apoptosis, radical removal, activation of superoxide dismutase, catalase or glutathione peroxylase, and the like. A fraction of the Betula platyphylla var. japonica extract is fractioned with ethylacetate, butanol, hexane, dichloromethane or water.

Description

Extracts and fractions of Antioxidant and / or Anticancer Activity Extracts and fractions of Betula platyphylla var. japonica with antioxidant and / or anticancer activities}

The present invention relates to extracts and fractions having antioxidant and anticancer activity, and more particularly, to extracts and fractions extracted from Betula platyphylla var. Japonica.

Living bodies that breathe aerobics, such as the human body, produce oxygen by reducing oxygen. O ₂ is indispensable to occur by-product in the process _{^{-, OH -, H 2 O}} 2 and the like reactive so large that small amounts as, due to which causes oxidative damage to react with various substances in the living body of the cell functions are inhibited It causes apoptosis. These are called reactive oxygen pecies, and oxidative damage of DNA, proteins, lipids and small cell molecules caused by reactive oxygen species has been associated with neurological disorders such as heart disease (Witztum, 1993) and Alzheimer's disease (Richardson, 1993), many diseases such as mutations and cancer are known to cause aging (Finkel and Holbrook, 2000). Hydrogen peroxide, one of the free radicals, has been known to cause lipid peroxidation and DNA damage in cells (S. Ahmad, 1995). Hydrogen peroxide (H ₂ O ₂ ) is also found in various copper concentrations at concentrations above 50 μM. Toxic to plant and bacterial cells (B. Halliwell et al., 2000).

Oxidative damage by reactive oxygen species results from an imbalance between the rate of radical production and the rate of elimination. There are enzymes in the cell that can remove radicals on their own. These enzymes that act as antioxidants convert superoxide dismutase (SOD) or hydrogen peroxide, which breaks down superoxide, into water and oxygen. Catalase (CAT), an enzyme that contains selenium in its active site, glutathione peroxidase (GPX) that destroys hydrogen peroxide as well as other peroxides (Blake et al., 1987). In addition, other intramolecular substances that can react with reactive oxygen species include glutathione (GSH), flavonoids, ubiquinol-10, glucose, and albumin. . Other substances that can be taken externally for antioxidant activity are vitamins C, E, A and pro-vitamin A, small amounts of selenium and zinc (Halliwell and Gutteridge, 1998). ).

A lot of research is being conducted worldwide to make safe and strong antioxidants by separating the components with antioxidant activity from natural products. Research and reporting on various natural antioxidants such as soybeans, oilseed seeds, herbs, tea, berries, ginkgo biloba, ginseng extracts, fruits and vegetables. Resveratrol, a large amount of grape skin, inhibits DNA synthesis and cytotoxicity in HL-60 cells, a human promyelocytic leukemia cell line. It has been reported to have antioxidant activity by reducing the death of rat pheochromocytoma (PC12) cells induced by the virus (MV Clement et al., 1998). Epigallocatechin-3-galate from green tea leaves also inhibited liver damage in rats induced by D-galactosamine (GaIN) and formed intracellular reactive oxygen species in DU145 cells, a human prostate cancer cell line. Apoptosis is induced by increasing the activity (LY Chung et al., 2001). Ginsenosides isolated from Panax ginseng also abolished superoxide anion (O ₂ ⁻ ) produced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in HL-60 cells and were inhibited by TPA in the skin of normal mice. It is known to inhibit the production of induced tumor necrosis factor (SJ Lee et al., 2000; YS Keum et al., 2000). Among many natural products, it has been studied that phenolic substances have a potential antitumor effect (YJ Shur, 1998). Curcumin, present in Ginger ( Curcuma longa Linn), has a diarylheptanoid, which is known to have anticancer and antimutagenic effects in cells cultured with various experimental animals (KK Soudamini et al., 1989; CV Rao et. al., 1995). Programmed cell death, or apoptosis, is considered an important topic for many researchers in terms of various human diseases and pathological mechanisms, including cancer (CBThompson, 1995). Apoptosis is an active death mechanism in which enzymes in cells are actively involved in cell degradation. Formally, DNA fragmentation and apoptosis are reached at the final stage, with reduced cell weight, cell membrane destruction, and chromosome condensation. Cheera forms cysts. Apoptosis is also caused by oxidative stress, increased intracellular calcium levels, changes in mitochondrial membrane potential, and various stimulators that stimulate cell membrane receptors, and in fact many cytotoxic substances induce apoptosis in malignant tumor cells in vitro . (SM Planchon et al., 1995; I. Muller et al., 1997).

Antioxidants are substances with a wide range of biochemical activities. They inhibit the formation of reactive oxygen species, directly or indirectly eliminate radicals generated in cells, and change the oxidation and reduction power in cells (C.K. Sen, 1998). Some antioxidants have been used as inhibitors of cell death. This is because cells are killed through a process called apoptosis as a result of damage caused by oxidative stress (D.M. Hockenhery et al., 1993). Antioxidants, including sulfur, such as N-acetylcysteine and dimercaptopropanol, are known to cause apoptosis through p53-tumor suppressor genes in transformed cell lines (M. Liu et al., 1998). Many kinds of natural medicines have been studied as antioxidants. Skin peel of the subject of the present invention has been used as a traditional Chinese medicine to treat pneumonia, nephritis, chronic bronchitis. However, studies on the antioxidant and anticancer activity of hides, particularly at the molecular level, have been insufficient.

An object of the present invention is to inhibit the radicals and lipid peroxides, which are the main causes of cell damage, and to increase the activity of antioxidant enzymes in the cells, thereby removing the active oxygen species, which can be used in the beneficial and effective drug for the human body to prevent cell damage. To provide an extract or fraction.

It is another object of the present invention to provide an extract having anticancer activity by inducing cell nuclear segmentation, inducing chromosomal DNA segmentation, and inducing cell death in cancer cells. Those skilled in the art can readily recognize other objects and advantages of the present invention from the accompanying drawings, the detailed description of the invention, and the contents of the claims.

1 is a diagram showing that the skin extract has an effect of preventing cell death induced by hydrogen peroxide, A is a cell death curve by hydrogen peroxide, B is a skin extract to protect the cell death induced by hydrogen peroxide The curve shows the effect.

2 is a diagram showing that the bark extract and fraction has DPPH radical scavenging activity. ●-Methanol extract, ○-Hexane, ▲-Dichloromethane, △-Ethyl acetate, ■-Butanol, □-Water layer

3 is a diagram showing that the skin extract has activity to protect the nuclear segmentation of cells induced by hydrogen peroxide, A is a normal control group, B is a nuclear segmentation of cells induced by hydrogen peroxide, C is a skin The extract has a protective effect on the nuclear segmentation of cells induced by hydrogen peroxide.

Fig. 4 is a diagram showing that the bark extract and the acetylacetate fraction have an activity of preventing peroxidation of cell membrane phospholipids induced by hydrogen peroxide. ●-Methanol extract, △-Ethyl acetate fraction

5 is a diagram showing that the extract extract has an action of increasing the activity of superoxide dismutase, catalase and Glutathione peroxidased, enzymes showing antioxidant enzyme activity, A is superoxide dismutase, B is catalse C is increased activity of glutathione peroxidase Indicates.

FIG. 6 is a diagram showing that the extracts have cytotoxic effects on epithelial cell cancer cell line (KB) (A) and blood cancer cell line (HL-60) (B).

7 is a fluorescence micrograph showing that the skin extract has an effect of inducing nucleus fragments in the hematological cancer cell line (HL-60), where A represents a normal control group and B represents a nucleus segment induction effect of the extract.

8 is a diagram showing that the skin extract has an effect of inducing chromosomal DNA fragmentation in the blood cancer cell line (HL-60).

9 is a diagram showing the results of flow cytometry analysis that the skin extract has an effect of inducing apoptosis in the hematologic cancer cell line (HL-60).

10 is a diagram showing that skin extracts affect the expression and activity regulation of proteins that induce apoptosis in hematological cancer cell line (HL-60). Bcl-2 is unchanged and Bax is expressed. Is shown to increase caspase-3 activity and PARP cleavage.

Table 1 is a table showing that the bark extract has an activity that inhibits the production of subdiploid cells by hydrogen peroxide.

Table 2 shows that skin extracts have the effect of inducing the production of subdiploid cells in the hematological cancer cell line (HL-60) and thus causing a change in cell cycle distribution of cells.

In order to achieve the above object, the extract of the present invention exhibits antioxidant and anticancer activity, and is a bark extract extracted with a polar solvent.

In addition, the fraction of this invention is a skin fraction which shows antioxidant activity. The bark extract of the present invention is preferably a 70% methanol extract and the bark fraction is a fraction extracted with nucleic acid, dichloromethane, ethyl acetate, butanol, or water. More preferably, the extract or fraction according to the present invention is characterized in that the lipid peroxide inhibitory activity, radical scavenging activity, the activity of inhibiting the formation of apoptosis by hydrogen peroxide, G ₂ / M cell cycle arrest phenomenon of cells by hydrogen peroxide It is characterized by promoting the activity that does not occur, superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPX). Also preferably, the bark extract according to the present invention, the activity of inhibiting the nuclear segmentation of cells induced by reactive oxygen species, the activity of inhibiting the formation of apoptotic cells, the antioxidant activity in the cells treated with free radicals, free radicals Anticancer activity in cancer cells treated with the species, induction of cell nuclear fragments and induction of chromosomal DNA fragments in cancer cells, the extract is to increase the expression of Bax in cancer cells, or the extract is the activity of caspase-3 in cancer cells It is characterized by showing the activity to promote.

The present invention will be described in more detail with reference to the following examples. However, embodiments of the present invention are intended to illustrate the invention, not to limit the scope of the invention.

1. Extract Preparation Method

1) Preparation of Methanol Extracts and Fractions of Hwapi Hwapi was purchased from Gyeongdong Market and prepared as follows. After pulverizing the purchased skin finely put about 100 g and 70% methanol in a round flask, and heated to 80 ℃ reflux and extracted for more than 3 hours. The temperature was lowered to room temperature and the remaining sample was filtered again using the filter paper, and the remaining sample was repeatedly extracted in the same manner as described above, and the filtered solutions were concentrated under reduced pressure. It was dissolved in distilled water and freeze-dried to make a powder and stored at -70 ℃. The fractions were extracted by adding hexane, dichloromethane, ethyl acetate and butanol in stages, and the solvent was evaporated with a reduced pressure concentrator (Eyela, Japan) and dissolved in dimethyl sulfonic acid (dimethylsulfoxide, DMSO) to 100 μg / ml and stored at -70 ° C. . In this study, they were diluted to various concentrations in cell culture medium or phosphate-buffered saline.

2) Cell Culture

The cell lines used in this study were V79-4 (chinese hamster lung cell line; ATCC CCL-93), KB (human oral epidermoid carcinoma cell line; ATCC CCL-17), and HL-60 (human promyelocytic leukemia cell line). V79-4 cells were treated with 5% fetal bovine serum (FBS; Bio-whittaker, USA), 100 μg / ml streptomycin (GibcoBRL, USA), 100 unit / ml penicillin (GibcoBRL, USA) in DMEM (GibcoBRL, USA) medium. 2 mM L-glutamine (Bio-whittaker, USA) was added and incubated at 37 ° C. under 5% CO ₂ /95% air. KB and HL-60 cell lines were also cultured under the same conditions, except KB cells were cultured in DMEM mixed with 10% FBS and HL-60 cells in RPMI-1640 (GibcoBRL, USA) medium mixed with 10% FBS. .

2. Experimental method

Experiment 1. Cell survival test

Cell survival test was performed using the MTT assay. V79-4 cells were placed in 96-well microplates and treated with 0, 4, 20 and 100 μg / ml skin extract. After 1 hour of incubation, 100 μM hydrogen peroxide was added to the medium and incubated for another 24 hours. 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide (MTT; Sigma, USA) was dissolved in physiological saline at 5 mg / ml, filtered through a 0.2 μm filter, and then dispensed at -20 ° C. It was stored and used when dissolved. After 20 μl of MTT was added and incubated for 4 hours, the culture solution of each well was removed, 100 μl of isopropanol containing 0.04 N HCl was added to the formed formazan salt, and gently shaken to completely dissolve the ELISA reader (Bio-Rad, USA). Absorbance was measured at 540 nm wavelength (reference 655 nm). MTT assay was also used for the cytotoxicity test of the bark extract. KB and HL-60 cells were cultured in 96-well microplates and then treated with 0, 20, 100 and 500 μg / ml skin extracts. For 48 hours and 72 hours in the case of KB was measured for cytotoxicity, 48 hours in the case of HL-60 cells were cultured to determine the toxicity. The absorbance was measured at 570 nm (reference 655 nm).

Experiment 2. DPPH radical scavenging measurement experiment

600 μl of 1.5 × 10 ^-4 M 1,1-diphenyl-2-picrylhydrazyl (DPPH; Sigma) was added to the skin extract concentrations of 0.8, 4, 20 and 100 μg / ml, respectively. Made to 3 ml. After shaking well for 10 seconds, the absorbance was measured at 520 nm, and the scavenging activity of the DPPH radical was calculated by the following equation.

Radical scavenging activity (%) = {(ODcontrol-ODsample) / ODcontrol} × 100

The antioxidant activity of each sample was expressed as IC ₅₀ (the concentration of sample required to inhibit DPPH radical formation by 50%).

Experiment 3. Measurement experiment of lipid peroxide inhibitory activity

Phospholipid unsaturated fatty acids, which are components of the cell membrane, cause peroxidation by radicals, and the lipid peroxide produced at this time is known to be a major cause of cell membrane damage. Therefore, inhibition of lipid peroxidation reaction has been used as an important indicator for antioxidant activity assay. In this study, as the causative agents causing oxidative damage was the the H ₂ O ₂ generating species, radicals hydroxyl radical, by varying the cell incubation time after adding the concentration of H ₂ O ₂ and H ₂ O ₂ to form lipid peroxidation The optimum conditions were 1 mM H ₂ O ₂ and 1 hour incubation time. In order to measure the lipid peroxidation inhibitory activity, the concentration of each herbal extract was added to the cultured V79-4 cells at 100, 20 and 4 μg / ml, and then cultured for 1 hour. Next, in order to cause oxidative stress, the concentration of hydrogen peroxide was added to 1 mM and incubated for another 1 hour. The treated cells were washed well with physiological saline, and then, 500 μl of 1.15% KCl was added to harvest the cells, transferred to a microfuge tube, and homogenized while maintaining 4 ° C. To 100 μl of this ground homogenate , 200 μl of 8.1% SDS and 1.5 ml of 20% acetic acid with pH adjusted to 3.5 and 1.5 ml of 0.8% TBA were added according to the method of Ohkawa et al. (Ohkawa et. Al., 1979) . With a final volume of 4 ml. After mixing the solution well, the mixture was heated and reacted at 95 ° C. for 2 hours, cooled to room temperature, and the resulting red colored material was extracted with a mixture of n-butanol: pyridine (15: 1, v / v) and 10 at 1800 rpm. Centrifuged for a minute. The supernatant was taken, the absorbance was measured at 532 nm, and lipid peroxide inhibitory activity was obtained.

Experiment 4. Superoxide dismutase (SOD) activity measurement experiment

SOD activity was determined by the NBT (nitroblue tetrazolium) method of Beauchamp and Fridovich (1971). 0.05 M sodium carbonate buffer (pH 10.2) was added to the treated cells and homogenized. 50 μl of this grinding homogenate was shaken with 3 mM xanthine, 0.75 mM NBT, 3 mM EDTA (ethylenediaminetetraacetic acid), 1.5 mg / ml BSA (bovine serum albumin), and 50 μl of 0.1 mg / ml xanthine oxidase was added to room temperature. It was left for 30 minutes at. The reaction was stopped by adding 6 mM CuCl ₂ and then centrifuged at 1500 rpm for 10 minutes. The supernatant was taken, the absorbance was measured at 560 nm, and the SOD activity was determined.

Experiment 5. Catalase (CAT) Activity Measurement Experiment

To 100 μl of the treated ground homogenate, 12 μl of 3% hydrogen peroxide was added and the final volume was filled with 1 ml of 50 mM phosphate buffer solution (pH 7.0). After leaving this solution at 37 ° C. for 2 minutes, the absorbance change of hydrogen peroxide was measured at 240 nm for 5 minutes (Carrillo et al., 1991).

Experiment 6. Glutathione peroxidase (GPX) activity measurement experiment

To 100 μl of the ground homogenate treated with the sample according to the method of Paglia and Valentine (1967), 1 mM EDTA, 10 mM GSH, 1 mM NaN ₃ , 1 unit glutathione reductase and 1.5 mM NADPH were added for 10 minutes at 37 ° C. It was left. Hydrogen peroxide was added to the solution to 1 mM, and the absorbance change of NADPH was measured at 340 nm.

Experiment 7: Nuclear Segment Protection or Induction Confirmation Experiment

V79-4 cells were cultured in sterile cover glass and treated with 100 μg / ml of bark extract, followed by incubation for 24 hours. The cover glass to which cells were attached was carefully taken out, washed with physiological saline, and dried in air. The treated V79-4 cells were soaked in 100% ethanol for at least 5 hours to completely fix the cells. HL-60 cells were added to 500 ㎍ / ml skin extract in a 60 mm dish and harvested after 24 hours. Physiological saline was added to a cell concentration of 5 × 10 ⁵ cells / ml to prepare a cell suspension, and 50 μl of these were taken and cells were attached to the slides using a cell spin (Hanil, Korea) at 500-1000 rpm for 10 minutes. The slides with the cells attached were dried in air. The treated HL-60 cells were soaked in 100% ethanol for at least 5 hours to completely fix the cells. The fixed cells were washed with physiological saline and stained with propidium iodide (PI), a DNA-specific fluorescent dye, for 10 minutes. Stained cells were observed with a fluorescence microscope (Nikon eclipse E800, Japan).

Experiment 8. Chromosome DNA Segmentation Induction Confirmation Experiment

HL-60 cells at 1 × 10 ⁶ cells / ml concentration were added with 0, 4, 20, 100 and 500 μg / ml of bark extract and incubated for 24 hours. In another condition, 500 μg / ml of bark extract was added and incubated for 0, 3, 7, 16, 24 and 48 hours. After incubation, each cell was harvested and centrifuged at 750 × g for 5 minutes and the precipitate washed with ice-cold saline. After centrifugation once again under the same conditions as above, 1 × TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA) was added to the cell precipitate to a concentration of 1 × 10 ⁶ cells / ml. Five times lysis buffer (0.5% SDS, 25 mM Tris, 5 mM EDTA, pH 7.5) was then added to lyse the cell precipitate. The final concentration of Proteinase K was added to the sample to 1 mg / ml and the sample was left at 50 ℃ for 3 hours or ON. The phenol: chloroform (1: 1) extraction was repeated 2-3 times to remove the protein from the sample and chlroform was added to completely remove the phenol remaining in the sample. DNA was precipitated by adding 1/10 times 3 M sodium acetate and 2 and 1/2 times ice-cold ethanol to ON at -20 ° C or 1 hour at -70 ° C. Centrifuge at 15000 x g for 20 minutes and remove supernatant. 70% ethanol was added to wash the precipitate and the DNA was dried under vacuum (speed vacuum; Savant, Holbrook, NY), and the dried DNA was dissolved in 100 μl of 1 × TE. DNase-free RNase was added to a final concentration of 200 μg / ml and left at 37 ° C. for 1 hour and at 65 ° C. for 5 minutes. The isolated DNA was electrophoresed on 1.5% agarose gel, stained with ethidium bromide (0.5 ㎍ / ml), and visualized by UV irradiation.

Experiment 9: Flow cytometry analysis

V79-4 cells were treated with 100 μM hydrogen peroxide and incubated for 7 hours and 24 hours. In addition, 100 ㎍ / ml of the bark extract was treated in advance, hydrogen peroxide was added and incubated for 7 hours and 24 hours as above. HL-60 cells were treated with 500 μg / ml skin extract on HL-60 cells at a concentration of 1 × 10 ⁶ cells / ml and incubated for 0, 3, 7, 16, 24 and 48 hours. The cultured V79-4 cells and HL-60 cells were centrifuged at 750 × g for 5 minutes each. The cell precipitate was washed with physiological saline and then centrifuged again under the same conditions. 1 ml of a 1: 1 mixed solution of physiological saline and Mcllvain's buffer (0.2 M Na ₂ HPO ₂ , 0.1 M citric acid, pH 7.5) was added, followed by 2x cold-ethanol. After storage for 1 hour at 4 ℃ centrifuged for 10 minutes at 750 × g. After washing twice with physiological saline and 2 ml PI staining solution (10 ㎍ / ml propidium iodide, 100 ㎍ / ml DNase-free RNase in PBS) and incubated for 2 hours at 37 ℃ dark room. Samples were analyzed by flow cytometer (FACS-caliber, Becton Dickinson).

Experiment 10. Western blot analysis

HL-60 cells were treated with bark extract at different concentrations and times. Cells were harvested and centrifuged at 750 × g for 5 minutes. After washing the cell precipitate with physiological saline, centrifugation once again and 100 μl of ice-cold lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 1% Triton X-100, 1.5 mM MgCl ₂ , 1 mM NaVO _3). , 100 mM NaF, 10% glycerol, 1 mM EGTA, 10 mM Na pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, pH 7.5) were added to lyse the cells for 30 minutes. The dissolved samples were centrifuged at 15000 × g for 15 minutes and protein concentration was measured by Lowry assay (Bio-rad, USA). 30-50 μg of protein was electrophoresed on 7-15% SDS-polyacrylamide gel. After electrophoresis, SDS-polyacrylamide gel was transferred to nitrocellulose membrane (Schleicher & Schuell, Germany) for 2 hours (30 mA, Pharmacia biotech) and transferred to TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween-). 20, pH 7.8) was added to skim milk made of 5% and incubated for at least 2 hours. Then washed three times for 10 minutes with TBST buffer. Primary antibody (anti-Bcl-2; Oncogene, Mouse monoclonal antibody, 1: 1000 in TBST buffer, anti-Bax; Santa Cruz Biotechnology, rabbit polyclonal antibody, 1: 500 in TBST buffer, anti-caspase; Transduction on nitrocellulose membrane) , Laboratories, mouse monoclonal antibody, 1: 500 in TBST buffer, and anti-PARP; Santa Cruz Biotechnology, rabbit polyclonal antibody, 1: 2000 in TBST buffer) were added and allowed to stand at room temperature for 1 hour and in TBST buffer for 10 minutes. Wash three times. The horseradish-conjugated secondary antibody diluted 1: 1000 was added thereto, followed by incubation for 40 minutes, and washed with TBST as above. Proteins transferred to membrane were examined for protein expression using an enhanced chemiluminescence (ECL) detection kit (Amersham Life Sciences, Arlington Heights, IL).

3. Experimental Results

1] Antioxidant Effect of Skin Extract

Antioxidant activity of skin extracts and fractions was measured by viability of cells (Experiment 1), DPPH radical scavenging activity (Experiment 2), lipid peroxide inhibitory activity (Experiment 3) and the like.

1) Cytotoxic Protective Effects of Skin Extracts by Hydrogen Peroxide

The results of the cell viability measurement experiment (Experiment 1) are shown in FIGS. 1A and 1B. It can be seen from Figures 1A and 1B that the bark extract has an effect of preventing cell death induced by hydrogen peroxide. FIG. 1A is a cell death curve by hydrogen peroxide, and FIG. 1B is a curve showing the effect of protecting the apoptosis extract from cell death induced by hydrogen peroxide. Referring to FIG. 1A, when only normal cells V79-4 were treated with hydrogen peroxide, the survival rate of the cells was clearly reduced. After each treatment the 1, 12.5, 25, 50, 100, and hydrogen peroxide in 1000 μM in cells, were a survival rate of the cells decreased as the concentration of hydrogen peroxide increases this time, the concentration of hydrogen peroxide to reduce the survival rate of cells _50% IC 50 The 50% inhibitory concentration was about 100 μM. Referring to Figure 1B, it can be seen that the addition of 4, 20 and 100 ㎍ / ml of bark extract to the cells and then treated with 100 μM hydrogen peroxide inhibits cell death by hydrogen peroxide. Perianth represents a respective 18, 27 and ERA 84% depending on the concentration of the extract, the concentration ED _{50 (50%} effective dose) of the mortality of cells for a 100 μM hydrogen peroxide perianth extract defending 50% is 37.7 ㎍ / ml .

2) DPPH Radical Scavenging Activity of Skin Extract and Fraction

DPPH radical scavenging activity experiment (Experiment 2) results of the skin extract and fractions are shown in FIG. Referring to FIG. 2, the concentration dependence of the radical scavenging activity was increased with increasing concentrations in all samples. The methanol extract of the hide showed a high scavenging activity of IC ₅₀ 2.4 μg / ml. In the fraction samples, the IC ₅₀ values of ethyl acetate, butanol and water fractions were 5.9, 8.1, and 11 μg / ml, respectively. The IC ₅₀ values of the hexane and dichloromethane fractions were both> 20 μg / ml, indicating no significant results.

3) Inhibitory Activity of Lipid Peroxide on Methanol Extracts and Fractionated Samples

The results of the experiment for measuring the lipid peroxide inhibitory activity of the methane of the bark extract and the fraction sample (Experiment 3) are shown in FIG. 4. Referring to Figure 4, the lipid peroxide inhibitory activity of the cells treated with methanol extract showed a very high value of IC ₅₀ <4.0 μg / ml. In the case of ethyl acetate fraction, IC ₅₀ 6.5 μg / ml showed slightly lower lipid peroxide inhibitory activity than methanol extract.

4) SOD activity

SOD, CAT, and GPX activities in V79-4 cells treated with extracts of methane were measured to investigate the changes in the antioxidant enzyme activity due to the antioxidant activity of the hides. The results of the SOD activity measurement experiment (Experiment 4) are shown in FIG. 5A. SOD activity was increased by 21, 31, and 36% by increasing the concentration of methanol extract to 4, 20, 100 μg / ml. SOD activity in control cells without medicinal herbs was 24.9 ± 1.5 U / mg protein.

5) CAT active

The results of the CAT activity measurement experiment (Experiment 5) are shown in FIG. 5B. CAT activity showed a slightly lower increase than SOD activity. When methanol extracts were added at concentrations of 4, 20 and 100 μg / ml, CAT activity was increased to 12, 20 and 24%, respectively (Figure 5B). All samples showed a concentration-dependent increase in CAT activity in the concentration range of 4 to 100 μg / ml, and the CAT activity of control cells was 14.9 ± 1.9 U / mg protein.

6) GPX active

The results of the GPX activity measurement experiment (Experiment 6) are shown in Figure 5C. The activity of GPX showed a very high increase compared to the previous two cases. GPX activity increased 51, 62, and 67%, respectively, when treated with 4, 20, and 100 μg / ml skin extracts (FIG. 5C). Like other antioxidant enzyme activities, GPX activity was observed to increase its concentration with increasing sample concentration. GPX activity of control cells without sample was 11.7 ± 1.3 U / mg protein.

7) Inhibitory effect of nuclear segmentation on cells induced by hydrogen peroxide

3A and 3B show the results of the nuclear segmentation defense effect confirmation experiment (Experiment 7). Morphological changes of intracellular nuclei were observed using propidium iodide (PI), a DNA-specific fluorescent dye (magnification × 200). In the control group, no change in the morphology of the nucleus was observed, indicating the state of normal cells (FIG. 3A). As a result of observing the cells after treatment with 100 μM hydrogen peroxide, the size of the nucleus in the cell was observed to be smaller than that of the control group, indicating that the nuclear segmentation, a morphological change, occurred (FIG. 3B). However, when 100 μg / ml of bark extract was treated beforehand, hydrogen peroxide was added, and unlike when only hydrogen peroxide was treated, no significant change occurred in the nucleus and there was no difference in comparison with the control group (FIG. 3C). Therefore, it can be seen that the bark extract protects nuclear fragmentation generated by hydrogen peroxide treatment.

7) Inhibitory Activity of Apoptosis Formation by Hydrogen Peroxide

Flow cytometry analysis (Experiment 9) to investigate the effect of skin extract on apoptotic formation and cell cycle (Experiment 9) shows that skin extract recovers G2 / Marrest induced by oxidative stress. there was.

After treatment with 100 μM hydrogen peroxide only in V79-4 cells and pre-treatment with 100 μg / ml skin extracts incubated for 7 hours and 24 hours, respectively, the experimental groups were cultured for 7 hours and 24 hours with hydrogen peroxide stress. The following results were observed. For the cells treated with hydrogen peroxide only for 7 hours, 42.7% of the cells in the G2 / M phase were pretreated with the bark extract, but the amount of cells decreased to 35.6%. In addition, the cells treated with hydrogen peroxide alone were 24% in the G2 / M phase, but the amount of cells in the G2 / M phase was 33.4%.

Table 1. Table showing that bark extract has activity to inhibit the production of cell death body by hydrogen peroxide

2] Anticancer Effect of Skin Extract

1) Cytotoxic Effects of Skin Extracts on Cancer Cell Lines

To determine the effect of skin extracts on the survival of epithelial cancer cells KB and HL-60 hematological cancer cells, the survival rate of cells according to the concentration of skin extracts was measured. (Experiment 1). Experiment 1 results are shown in Figs. 6A and 6B. Referring to Figure 6 A, B, it can be seen that as a result of treatment of KB, HL-60 cells with 0, 4, 20, 100, and 500 μg / ml of bark extract, respectively, the survival rate of the cells decreases in a concentration-dependent manner. The IC ₅₀ value of the bark extract, a concentration that reduces the survival rate of cells by 50%, was 304.0 μg / ml for KB cells (FIG. 6A) and 159.0 μg / ml for HL-60 cells (FIG. The above results indicate that HL-60 cells are more sensitive to skin extract.

2) Induction of Nuclear Segmentation by Skin Extracts in HL-60 Cells

The results of experiments confirming the nuclear nuclear fragment induction effect of the bark extract in HL-60 cells are shown in Figures 7A and B. In this experiment, intracellular nuclear fragmentation was observed using propidium iodide (PI), a DNA-specific fluorescent dye (magnification × 200) (Experiment 7). As a control, HL-60 cells treated with 0.1% DMSO showed normal morphology in the shape of the nucleus (FIG. 7A). In contrast, HL-60 cells were cultured for 24 hours in a medium containing 500 μg / ml skin extract, and nuclear fragmentation was observed (FIG. 7B).

3) Effect of Skin Extract on Chromosomal DNA Segmentation in HL-60 Cells

One of several mechanisms that induce apoptosis is to increase apoptosis through apoptosis. Therefore, agarose gel electrophoresis, one of the biochemical characteristics of apoptosis, confirmed the mechanism of induction of apoptosis by identifying ladder-type DNA fragments (Experiment 8). The results of Experiment 8 are shown in Figs. 8A and 8B. In this experiment, depending on the concentration, 0, 4, 20, 100 and 500 μg / ml of the bark extract were incubated for 24 hours or 500 μg / ml of the bark extract for 0, 3, 7, 16, 24 and 48 hours. Incubated. After DNA extraction, electrophoresis on 1.5% agarose gel and observed by ethidium bromide staining revealed DNA fragmentation with time and concentration. As can be seen in FIG. 8A, DNA fragmentation was observed at 100 μg / ml at 24 μg treatment of different concentrations of skin extract for 24 hours, and 500 μg / ml of skin extract was treated in a concentration-dependent manner. Segmentation was also observed in. In FIG. 8B, DNA fragmentation began to appear after treatment of 500 ㎍ / ml skin extract for about 3 hours, and segmentation was observed even after the treatment for 48 hours.

4) Induction of Apoptosis Formation in HL-60 Cells

Flow cytometry analysis was carried out to determine whether the extracts induce the apoptosis in HL-60 cells (Experiment 9). The results of this experiment are shown in Figures 9A and B. As shown in Table 2, an increase in the number of subdiploid cells was observed in a time- and concentration-dependent manner. Treatment of 500 μg / ml of bark extracts on HL-60 cells for 3, 7, 16, 24, and 48 hours, respectively, showed time-dependent patterns of subdiploid cells with 25.5, 46.3, 58.2, 72.9, and 87.7%. The number increased (Figure 9A). In addition, 4, 20, 100 and 500 ㎍ / ml of the bark extract was treated for 24 hours, as shown in Figure 9A the number of subdiploid cells increased and the number was increased to 0, 5.7, 12.2, and 64% ( Figure 9B). Table 2 shows that skin extracts induce the production of subdiploid cells in the hematological cancer cell line (HL-60) and thus have the effect of altering cell cycle distribution of cells.

Table 2. Table showing the effect of skin extracts on the production of apoptotic bodies in hematologic cancer cell lines, thus leading to changes in cell cycle distribution

5) Induction of Apoptosis Formation in HL-60 Cells at the Molecular Level

To understand the molecular mechanism of apoptosis induced by skin extracts, we analyzed the expression changes of apoptosis-related proteins (Experiment 10). The result of experiment 13 is shown in FIG. In this experiment, we first confirmed whether apoptosis was related to caspase-3 activation in HL-60 cells, and subsequently, the expression levels of Bcl-2, Bax, and Poly (ADP-ribosyl) polymerase (PARP) proteins. saw. First of all, we observed whether apoptosis occurred through expression of anti-apoptotic Bcl-2 protein and pro-apoptotic Bax in mitochondria.Bcl-2 did not change protein amount over time, but Bax did not change over time. The amount of protein increased. Therefore, the ratio of Bcl-2 and Bax was found to decrease relatively. Caspase-3 was activated over time to observe the cleavage of the 32 kDa precursor. PARP, a substrate of caspase-3, was also cleaved over time, reducing the precursor 116 kDa and increasing the protein of 85 kDa (Fig. 8).

3. Consideration of Experimental Results

In this experiment, the protective effect of bark extract against oxidative stress was verified by confirming cell viability, DPPH radical scavenging and sensitization of apoptosis by fluorescence staining. Treatment with only hydrogen peroxide to normal cells, V79-4, reduced the cell viability in a concentration-dependent manner, and in the presence of 100 μM hydrogen peroxide, about 50% of the cells were killed. However, the pre-treatment and stress treatment of the bark extract increased the survival rate of the cells. Also, at a low concentration of 2.4 μg / ml, skin extracts reduced DPPH radicals by 50%. In addition, it can be seen that the fraction samples prepared using five organic solvents having different polarities exhibited DPPH radical scavenging activity. Hwapi extract and ethyl acetate fractions showed the activity of inhibiting the formation of phospholipid peroxide of the excellent cell membrane. In addition, the bark extract increased the activity of the antioxidant enzymes SOD, CAT, and GPX in cells. This deficiency suggests that the mechanism of action of antioxidant extract is due to the increased activity of antioxidant enzymes. Microscopic observation through fluorescence staining observed that cells grown in 100 μM hydrogen peroxide-treated medium formed apoptosis, a product of apoptosis, but little cell death was observed in cells treated with skin extracts. These results suggest that the bark extract has a significant protective effect against cellular damage caused by hydrogen peroxide. In addition, the flow cytometry results showed an interesting fact. Comparing cells treated with hydrogen peroxide only and cells treated with bark extracts, we observed that G2 / M arrest in V79-4 cells treated with hydrogen peroxide only decreased due to the bark extract treatment.

In addition, we demonstrated by various methods that bark extracts cause death in HL-60 cells. By treating skin extracts with KB and HL-60 cells, it has been demonstrated that this substance inhibits the growth of cancer cells. Agarose gel electrophoresis showed that apoptosis progressed actively in HL-60 cells treated with skin extracts by DNA fragmentation in the nucleus, and the results through flow cytomerty showed that the cell apoptosis increased with time. As a result, it was further confirmed that skin extracts induce apoptosis in cancer cells.

There are two major genes involved in apoptosis, one of which is the Bcl-2 family of genes that play a role in inhibiting or increasing cell death, and the other is the caspase gene family. killer gene (T. Okura et al., 1998). The Bcl-2 family is a gene found in B-cell lymphoma and consists of more than 12 proteins. They are divided into three functional groups, among them Bcl-2 (26 kDa) with anti-apoptotic activity and Bax (21 kDa) with pro-apoptotic activity, which act by forming homo- or heterodimers with each other. do. However, their exact mechanism of action is not yet known (M.L. Kuo et al., 1996; D.M. Finucane etal., 1999).

The pro- and anti-apoptotic Bcl-2 family meet on the surface of the mitochondria and compete with each other to regulate the release of cytochrome c. If the pro-apoptotic group is increased, cytochrome c is released and apoptosis is activated in mitochondria (S.O. Klaus et al, 1998). Therefore, the protein level of anti-apoptotic Bcl-2 was unchanged, but the amount of pro-apoptotic Bax increased, resulting in the reduction of the protein ratio of Bcl-2 / Bax. I could conclude that.

Caspase is known to activate one type of caspase and another type of caspase based on the substrate specificity of cleavage following aspartic acid in the substrate. This mechanism leads to the mutual activation of caspase enzymes. These enzymes also cleave several substrates, such as PARP, Lamin, cytoskeletal proteins, and PAK2, thus losing their function, resulting in various structural and biochemical properties of apoptosis in stressed cells. PARP, an enzyme present in the nucleus, has been known to be involved in repairing DNA damage and to inhibit the activity of DNA hydrolase in cells. The catalytic domain of PARP is cleaved and separated from the DNA binding site by caspase-3 including caspase-3 in various cells undergoing apoptosis. Thus, truncated PARP is thought to act as a signal of apoptosis in many higher cells (S. Nigata, 2000; P.J. Duriez et al., 1997; X. Li et al., 2000)

In conclusion, it can be concluded that the bark extract has antioxidant effects in normal cells subjected to oxidative stress by reactive oxygen species in vitro , as well as cancer cell death in KB, and HL-60 cells.

According to the present invention, the bark extract and fractions inhibit radicals and lipid peroxides, which are the main causes of cell damage, at high concentrations, and increase the activity of antioxidant enzymes in the cells to prevent cell damage by removing reactive oxygen species. Therefore, the extract of the present invention can be used as an antioxidant drug to prevent cell damage.

In addition, the bark extract according to the present invention, by inducing the segmentation of the cell nucleus and chromosomal DNA segmentation in cancer cells, and has anticancer activity by regulating protein expression inducing cell death. Therefore, it can be used as an anticancer drug.

[references]

B. Halliwell, MV Clement and LH Long (2000), Hydrogen peroxide in the human body, FEBS Lett , 486, 10-13

B. Halliwell, JMC. Gutteridge, Free Radicals in Biology and Medicine. 3 ^rd ED., 1998, Oxford University

KK Soudamini, and R. Kuttan (1989) Inhibition of chemical carcinogenesis by curcumin, J. Ethnopharmacol ., 27, 227-233

CB Thompson (1995) Apoptosis in the pathogenesis and treatment of doisease, Science , 267, 1456-1462

CK Sen (1998) Redox signaling and the emerging therapeutic potential of thiol antioxidants, Biochem. Pharm ., 55, 1747-1758

CV Rao, A. Rivenson, B. Simi, and BS Raddy (1995) Chemoprev ention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound, Cancer Res ., 55, 259-266

DM Finucane, BW Ella, NJ Waterhouse, TG Cotter, and DR Green (1999) Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL, JBC , 274 (4), 2225-2233

D.M. Hockenhery, Z.N. Oltavai, and S.J. Korsmeyer (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis, Cell, 75, 241-251

I. Muller, A. Jenner, G. Bruchelt, D. Niethanmmer, and B. Halliwell (1997) Effect of concentration on the cytotoxic mechanism of doxorubicin-apoptosis and oxidative DNA damage, Biochem, Biophys. Res. Commun ., 230, 254-257

LY Chung, YC Cheung, SK Kong, KP Fung, and TT Kwork (2001) Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells, Life Sciences , 68, 1207-1214

ML Kuo, TS Huang, and JK Lin (1996) Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells, Biochem. Biophys. Acta ., 1317, 95-100

M. Liu, JC Pelling, J. Ju, E. Chu, and DE Brash (1998) Antioxidant action via p53-mediated Apoptosis, Cancer research , 58, 1723-1729

MV Clement, JL Hirpara, S. Chawdhury, and S. Pervaiz (1998) Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells, Blood , 92 (3), 996-1002

PJ Duriez and GM Shah (1997) Cleavage of poly (ADP-ribose) polymerase: a sensitive parameter to study cell death, Biochem. Cell. Biol , 75, 337-349

S. Ahmad (1995), Oxidative stress and antioxidant defenses in biology, Chapman & Hall press , 1-61 and 210-237

SJ Lee, WG Ko, JH Kim, JH Sung, SJ Lee, CK Moon, and BH Lee (2000) Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c -mediated activation of caspase-3 protease, Biochem. Pharm ., 60, 677-685

SM Planchon, S. Wuerzberger, B. Frydman, P. Huston, DR Church, G. Wilding, and DA Boothman (1995) β-Lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent response, Can. Res ., 55, 3706-3711

S. Nigata (2000) Apoptotic DNA fragmentation, Exp. Cell. Res , 256, 12-18

SO Klaus, F. Davide, L. Marek, W. Sebastian, and EP Marcus (1998) Apoptosis signaling by death receptors, Eur . J. Biochem ., 254, 439-459

T. Finkel, NJ. Holbrook, Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408; 239-47

T. Okura, M. Igase, Y. Kitami, T. Fukuoka, M. Maguchi, K. Kohara, and K. Hiwada (1998) Platelet-derived growth factor induces apoptosis in vascular smooth muscle cells, Biochem. Biophys. Acta ., 1403, 245-253

X. Li, and Z. Darzynkiewicz (2000) Cleavage of PARP measured in Situ in individual cells: relationship to DNA fragmentarion and cell cycle position during apoptosis, Exp. Cell. Res , 255, 125-132

YJ Shur (1998) Cancer chemoprevention by dietary phytochemicals: a mechanistic viewpoint, Cancer J. , 11, 6-10

YS Keum, KK Park, JM Lee, KS Chun, and YJ Shur (2000) Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng, Cancer letters , 150, 41-48

ZL. Witztum, The role of oxidized lox density lipoproteins in atherogenesis. Br. Heart J 1993; 69; 12-14

Claims (21)

Skin extract extracted with a polar solvent showing antioxidant and anticancer activity The extract of claim 1, wherein the polar solvent is 70% methanol. 2. The extract of claim 1, wherein the extract is characterized by an antioxidant activity that inhibits the formation of lipid peroxide. The extract of claim 1, wherein the extract of the bark is characterized in that it exhibits an antioxidant activity of eliminating radicals. The extract of claim 1, wherein the extract is an antioxidant, characterized in that it exhibits an antioxidant activity that inhibits the formation of cell death bodies by reactive oxygen species The extract of claim 1, wherein the extract is characterized by an antioxidant activity promoting the activity of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPX). The extract of claim 1, wherein the extract is characterized by an antioxidant activity that inhibits nuclear fragmentation of cells induced by reactive oxygen species. The extract of claim 1, wherein the extract is characterized in that the antioxidant extract exhibits antioxidant activity to prevent G ₂ / M cell cycle arrest of the cells by reactive oxygen species. 2. The extract of claim 1, wherein the extract is characterized by an anticancer activity that induces cell nuclear fragments in cancer cells. 2. The extract of claim 1, wherein the extract is characterized by an anticancer activity that induces chromosomal DNA fragmentation in cancer cells. 2. The extract of claim 1, wherein the extract is characterized by an anticancer activity that increases the expression of Bax in cancer cells. The extract of claim 1, wherein the extract is an anticancer extract, characterized in that it exhibits anticancer activity promoting the activity of caspase-3 in cancer cells. 13. The extract of claim 1, wherein the cancer cells are hematologic cancer cells or epithelial cancer cells. Skin fractions showing antioxidant activity 15. The skin fraction according to claim 14, wherein the skin fraction is extracted from ethyl acetate or butanol. 15. The skin fraction of claim 14 wherein the skin fraction is a skin fraction extracted with nucleic acid, dichloromethane, or water. 17. The skin fraction according to claim 15 or 16, wherein the skin fraction has an antioxidant activity that inhibits the formation of lipid peroxide. 17. The extract of claim 15 or 16, wherein the skin fraction has an antioxidant activity that prevents G2 / M cell cycle arrest of cells caused by reactive oxygen species. 17. The skin fraction according to claim 15 or 16, wherein the skin fraction extracted by ethyl acetate, butanol, or water exhibits antioxidant activity to eliminate radicals. 17. The skin fraction according to claim 15 or 16, wherein the skin fraction has an antioxidant activity that inhibits the formation of cell death bodies by reactive oxygen species. 17. The skin fraction according to claim 15 or 16, wherein the skin fraction exhibits an antioxidant activity that promotes the activity of super oxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPX).