KR20090091477A - Composition comprising the extract of litsea japonica jussieu for preventing and treating cancer disease - Google Patents

Abstract

A composition containing a Litsea japonica Jussieu extract for preventing and treating cancer is provided to suppress the proliferation of cancer and have anticancer effect. A pharmaceutical composition for preventing and treating cancer comprises 0.1-50 weight% of Litsea japonica Jussieu as an active ingredient. The Litsea japonica Jussieu extract is isolated with branch, bark, leaves, flower, fruits or root using water, low alcohol of 1-4 carbon atoms, or their mixture solvent. A health functional food is used in the form of powder, granule, tablet, capsule or beverage.

Description

Composition comprising the extract of Litsea japonica Jussieu for preventing and treating cancer disease}

The present invention relates to a pharmaceutical composition and health functional food for the prevention and treatment of cancer diseases containing the extract of the crow bark (Litsea japonica Jussieu) as an active ingredient.

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

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

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

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

4 Orrenius S et al., Mitochondrial oxidativestress: implications for cell death. Annu. Rev. Pharmacol. Toxicol , 47 : 19.1-19.41,2007

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

Document 6 Jiang X et al. Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway, Science, 299 , 223-226, 2003

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

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

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

Tanaka et al., Butanolides from Litsea japonica, Phytochemistry, 29 , 857859, 1990

[11] Lee et al., Flavonoids from the Leaves of Litsea japonica and their anticomplement activity, Phytotherapy research, 19 , 273276, 2005

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

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

Sherwood et al., Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells, Exp. Cell Res., 215 (2) , 373-379, 1994

15, Luo, Y et al., Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol. 66, 479, 1997

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

Cancer is one of the incurable diseases that humanity has to solve, and huge capital has been invested in the development to cure it all over the world.In Korea, it is the number one disease cause of death among Koreans since 1983. More than 100,000 have been diagnosed, and more than 60,000 have died. Carcinogens, which cause cancer, are smoking, ultraviolet rays, chemicals, foods, and other environmental factors. However, various causes are not only difficult to develop a therapeutic agent, but also effective effects vary depending on the site of occurrence.

Currently used anticancer agents include biological preparations such as enzyme preparations or vaccines, pure synthetic medicines, and medicines derived from natural products. Among them, anticancer drugs using genes, enzymes, vaccines, etc. are not in a practical stage and are used by chemotherapy. Developed anticancer drugs

It has considerable toxicity and has a side effect of destroying not only cancer cells but also normal cells due to the inability to selectively remove only cancer cells. Therefore, there is an urgent need to develop an effective anticancer agent that is less toxic for preventing cancer and develops cancer cells.

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

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

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

Litsea japonica Jussieu is an evergreen sub-tree of the dicotyledonous plant of the genus Amphitaceae , and is distributed in southern Korea (Jeju Island, Gyeongnam, Jeonnam) and Japan. It is characterized by growing vertically at the beach and at the foot of the mountain within 700m above sea level. The bark is brown, the twigs are thick and hairy, the leaves are opposite and the long oval is narrow and the ends are narrow and thickly curled or curled backward. Flowers bloom in July-October in axillary inflorescences. The color is yellowish white. The gun has brown hairs on the outside and the casing is deeply divided into 6 pieces. Fruits are nucleus, oval, ripening in light purple in October of next year. The chemical components of the ravenous tree are also known as essential oils, fatty acids, lactones, alkaloids and terpenoids (Tanaka et al ., Butanolides from Litsea japonica, Phytochemistry , 29, 857859 1990), tiliroside, a flavonoids component isolated from leaves, is known to have an inhibitory effect on the complement system (Lee et al ., Flavonoids from the Leaves of Litsea japonica and their anticomplement activity, Phytotherapy research, 19, 273276, 2005).

However, none of the above documents teaches or discloses that the extract of the ravenous tree of the present invention can be used as a composition for preventing and treating cancer diseases by inducing a proliferation inhibitory effect of cancer cells.

Accordingly, the present inventors, through the study to investigate the anti-cancer effect of the ravenous tree, the ravenous tree extract induces apoptosis of cancer cells, specifically human promyelocytic leukemia and adriamycin-resistant human promyelocytic leukemia cells The present invention was completed by confirming an excellent anticancer effect of inhibiting the proliferation of cancer cells.

In order to carry out the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the extract of the crow bark (Litsea japonica Jussieu) as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of cancer diseases containing the extract of the crow bark (Litsea japonica Jussieu) as an active ingredient.

“Extract” as defined herein is characterized by one or more extracts selected from the group consisting of the branches, bark, leaves, flowers, berries or roots of the Litsea japonica Jussieu, preferably the leaf extracts of the crow bark do.

 "Extract" as defined herein is at least one solvent selected from the group consisting of water including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably a mixture of water and ethanol, more preferably 50 to Means an extract available in 100% ethanol.

     "Cancer disease" as defined herein includes common cancer diseases, preferably gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin or ocular melanoma, Uterine cancer, ovarian cancer, rectal cancer, anal muscle cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer Renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, promyelocytic leukemia, monocytic leukemia, lymphoid leukemia, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system ( Central nervous system (CNS) one or more diseases selected from the group consisting of tumors, primary CNS lymphomas, spinal cord tumors, brainstem glioma or pituitary adenoma, preferably chronic or acute leukemia, promyeloid Sex leukemia, monocytic leukemia, lymphocytic leukemia, uterine cancer, ovarian cancer, carpal carcinoma, endometrial carcinoma or cervical carcinoma.

“Litsea japonica Jussieu”, as defined herein, is a plant that is distributed in the seashores and foothills within 700 meters vertically of Jeju Island, Gyeongnam or Jeonnam, preferably Jeju Island, and more preferably Jeju Island, Japan, Korea. It features.

Hereinafter, the present invention will be described in detail.

      Crow tree extract of the present invention can be prepared as follows.

      The extract of the present invention is dried after crushing and crushing the leaves of the ravenous tree, the water of about 1 to 30 times the weight of the dried sample, preferably about 3 to 10 times the volume of water, a low number of carbon atoms 1 to 4 One or more solvents selected from the group consisting of alcohols or mixed solvents thereof, preferably a mixed solvent of water and ethanol, more preferably 50-100% ethanol, 0-120 ° C. temperature, preferably 4 ° C.-70 ° C. Extraction methods such as stirring extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for about 0.5 to 20 hours, preferably 1 to 5 hours at temperature, preferably 1 to 10 times by ultrasonic extraction, preferably The extract obtained after repeated extraction 2 to 6 times can be obtained by concentrating under reduced pressure.

The present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases containing the extract obtained by the production method as an active ingredient.

The composition for preventing and treating cancer diseases of the present invention comprises the extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

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

Compositions comprising extracts according to the invention are formulated 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. Carriers, excipients and diluents that may be included in the composition comprising the extract of the present invention 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 have. 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 may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate 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 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The composition of the present invention can be administered to mammals such as rats, 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 the prevention and improvement of cancer diseases containing the extract as an active ingredient.

      The composition comprising the extract of the present invention may be used in various ways, such as drugs, foods and drinks for the prevention and improvement of cancer diseases. Foods to which the extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

Since the extract itself of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

       The extract of the present invention may be added to food or beverage for the purpose of preventing and improving cancer diseases. At this time, the amount of the extract in the food or beverage is generally the health food composition of the present invention can be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml It can be added at a ratio of 0.3 to 1 g.

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

      In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The extract of the ravenous bark of the present invention may be useful as a pharmaceutical composition for preventing and treating cancer diseases by exhibiting an excellent anticancer effect of inhibiting the proliferation of cancer cells by inducing apoptosis of cancer cells.

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

Example 1. Crow Extract

The leaves (185 g) of Litsea japonica Jussieu, native to Jeju Island, were dried in the shade. The dry sample was crushed with a crusher and then immersed in 1L of 80% ethanol and extracted using ultrasonic waves at 20 ° C. for 1 hour. The extraction process was repeated three times. The supernatant was recovered and concentrated under reduced pressure. % Ethanol extract (43 g) was received from Jeju Biodiversity Research Institute (Schema 1; JBR-079). The ravenous 80% ethanol extract was dissolved in PBS (phosphate-buffered saline) or ethanol, and then diluted with the medium of Reference Example 1 according to the desired concentration, and used as a sample of the following Experimental Example. Business card)

Reference Example 1. Cell Culture

HL-60 (human promyelocytic leukemia) cells, HCT-15 (human colon cancer) cells, SK-OV-3 (human ovary cancer) cells, A- 549 (human lung cancer) cells and HEL-299 (normal cell line) cells were obtained from Korea Cell Line Bank, HL-60 / ADR (Adriamycin resistant human promyelocytic). leukemia) cells at the Research Center for Resistant Cells, and HL-60 / MX2 (mitoxantrone resistant human promyelocytic leukemia cell line) cells at the American Type Culture Collection. ) Was used for sale. Each cell contained 100 units / ml penicillin-streptomycin (GIBCO Inc, NY, USA) and 10% heat-inactivated fetal bovine serum (GIBCO Inc, NY, USA) in a T-25 flask. Cultures were incubated in a 37 ° C., 5% CO ₂ humidified incubator using RPMI 1640 medium, with subcultures allowing each cell to fill approximately 70-80% of the bottom of the T-25 flask. Once every 3-4 days.

Reference Example 2. Statistics Processing

The results shown are three or more independent test results, and the data are expressed as mean ± standard error values. Statistical significance test between groups was assessed using Student's t-test.

Experimental Example 1. Measurement of LJ cell proliferation inhibitory effect

In order to measure the effect of inhibiting the proliferation of cancer cells by LJ of Example 1, the experiment was carried out as follows using an MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] assay. was performed (Carmichael et al, Evaluation of a tetrazolium-based semiautomated colorimetric assay:.. assessment of chemisensitivity testing Cancer Research, 47, 944-946, 1987).

MTT assay is based on mitochondrial dehydrogenase activity in living cells. The water-soluble tetrazolium salt MTT (yellow) is reduced to formazan crystals (purple). (formazan crystal) production is increased, the absorbance is measured high.

Each cancer cell (2.5 × 10 ⁵ cells / ml) cultured by the method of Reference Example 1 was placed in a 96 well plate, and the LJ of Example 1 was treated at concentrations of 25, 50, 75 and 100 μg / ml. After incubating for 4 days, 50 μl of MTT (2 mg / ml, Sigma, MO, USA; After M2128) was added and reacted for 4 hours, the plate was centrifuged at 1000 rpm for 10 minutes to remove the supernatant. 150 μl of DMSO (Dimethylsulfoxide, Sigma, MO, USA; D2650) was added to dissolve the precipitate, and then the absorbance was measured at 540 nm using a microplate reader (Amersham Pharmacia Biotech, NY, USA). Compared with the average absorbance value of the control group showed the degree of inhibition of cell proliferation in each experimental group.

As a result, as shown in FIG. 1, the cell proliferation inhibitory effect of LJ (100 μg / ml) on cancer cells was about 83% for HL-60 cells, 64.5% for HL-60 / ADR cells, and HL-, respectively. 76.4% for 60 / MX2 cells, 7.2% for HCT-15 cells, 16.9% for SK-OV-3, 3.4% for A-549 cells, and for normal cells (HEL-299). The inhibitory effect of LJ on cell proliferation was 2.3%. Through this, LJ is not toxic to normal cell line, the cell growth inhibition effect of LJ was found to be the most excellent in HL-60 and HL-60 / ADR cells of cancer cells of Reference Example 1. Accordingly, in the following experimental examples, the effect of LJ was investigated using HL-60 and HL-60 / ADR cells as cancer cell lines (see FIG. 1).

Experimental Example 2. Measurement of apoptosis-inducing effects of HL-60 and HL-60 / ADR by LJ

Apoptosis is an active cell death process regulated by genes. Once a cell is signaled to die, chromatin in the nucleus is concentrated and lined up along the nuclear membrane, resulting in condensation, DNA fragmentation, and nuclear fragmentation. The surface of the cell changes prominently. This protruding part is separated into the apoptotic body surrounded by the cell membrane and undergoes a series of processes where it is separated and taken up by neighboring macrophages to disintegrate and remove. Therefore, in order to determine whether the proliferation inhibitory effect of HL-60 and HL-60 / ADR by LJ shown in Experimental Example 1 was caused by induction of apoptosis, the following experiments were performed.

2-1. DNA fragmentation analysis

In order to investigate whether LJ induces apoptosis of HL-60, whether or not DNA fragmentation, which is one of the characteristics of apoptosis process, was induced using agarose gel electrophoresis was conducted as follows (Oberhammer et. al ., Apoptotic death in epithelial cells: cleavage of DNA to 300 and / or 50 kb fragments prior to or in the absence of internucleosomal fragmentation, The EMBO Journal , 12 (9), 3679-3684, 1993).

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

LJ was treated to HL-60 cells (3.5 × 10 ⁵ cells / ml) at the concentration of 100 μg / ml, which was the most effective in Experimental Example 1, and then incubated for 3, 6 and 9 hours. After collecting the cells, DNA was isolated using the Promega Wizard Genomic DNA Purification Kit (Promega, WI, USA; # A1120). The isolated DNA was subjected to electrophoresis for 40 minutes (100 V) on a 1.2% agarose gel, then stained with EtBr (ethidium bromide; Sigma, MO, USA; E8751) and UV transilluminator (Spectronics Corporation Westbury, NY, USA) DNA fragmentation was observed under.

As a result, as shown in Figure 2, it was possible to observe the DNA fragmentation in the group treated with HL-60 cells LJ (100 ㎍ / ㎖) for 6 hours and 9 hours, through which LJ HL-60 cells It can be seen that the induction of apoptosis (see Fig. 2).

2-2. Cell cycle analysis

In order to quantitatively determine the degree of apoptosis of HL-60 and HL-60 / ADR by LJ, cells corresponding to the sub-G1 phase of the cell cycle, ie, HL-60 and HL-60 / ADR The experiment was carried out as follows using a flow cytometer through PI (propidium iodide; Sigma, MO, USA; P4170) staining (Sherwood et al ., Induction of apoptosis by the anti-tubulin drug colcemid: relationship of mitotic checkpoint control to the induction of apoptosis in HeLa S3 cells, Exp. Cell Res. , 215 (2), 373-379, 1994).

PI is a substance that fluoresces specifically by binding to DNA fragments in the nucleus of cells undergoing apoptosis.

HL-60 and HL-60 / ADR (2.5 × 10 ⁵ cells / ml) cells were treated with LJ at concentrations of 25, 50 and 100 μg / ml, respectively, and incubated for 48 hours, and then the cells were collected and collected in PBS, respectively. (Sigma, Mo, USA; P7994). After fixation with 70% ethanol at -20 ° C for 30 minutes, and after PBS washing, RNase A (1 mg / ml; Promega, WI, USA; # A1120) was treated with 4 ug / ml at 4 ° C for 30 minutes. . Then, PI was treated at 2 ug / ml concentration in the dark, stained for 30 minutes at 4 ℃ and analyzed the cell cycle using a COULTEREPICSXL ^TM flow cytometer (Coulter, Miami, FL, USA).

As a result, as shown in Figures 3 and 4, in the group treated with HL-60 cells at concentrations of 25, 50 and 100 μg / ml, the number of sub-G1 cells increased to 47, 61 and 84%, respectively. (See FIG. 3), in HL-60 / ADR cells, sub-G1 cells increased to 19, 22 and 31%, respectively, according to the treatment concentration of LJ (25, 50 and 100 μg / ml). (See FIG. 4). Through this, it could be confirmed that LJ causes apoptosis of HL-60 and HL-60 / ADR.

2-3. Morphological changes in the nucleus

To determine whether LJ induces apoptosis of HL-60 and HL-60 / ADR, H33342 fluorescein staining method that specifically binds DNA to morphological changes of the nucleus, one of the features of apoptosis The experiment was conducted as follows (Luo, Y et al., Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol . 66, 479, 1997).

.

Incubate HL-60 and HL-60 / ADR (2.5 × 10 ⁵ cells / ml) cells at concentrations of 25, 50 and 100 μg / ml for 3, 6, 9, 12, 24 and 48 hours, respectively. It was. Thereafter, a solution of Hoechst 33342 (H33342; Sigma, MO, USA; B2261), a fluorescent material that specifically binds to DNA, was treated at 2 ug / ml and stained at 37 ° C. for 10 minutes. Photographs were taken after fluorescence microscopy (IX-71, Olympus, Japan) at 400-fold magnification to observe changes in shape and shape with time.

As a result, as shown in Figure 5, in the LJ (100 ㎍ / ㎖) treatment group, the size of the HL-60 cells were reduced in time dependently compared to the control group, the shape of the nucleus was irregular and showed a partial nucleus agglomeration phenomenon. (See Figure 5).

6 and 7, in the LJ treatment group, the size of HL-60 and HL-60 / ADR cells was reduced in a concentration-dependent manner compared to the control group, and the nucleus was irregular in shape and partial nucleation of the nucleus. (See FIGS. 6 and 7).

This suggests that LJ induces apoptosis of HL-60 and HL-60 / ADR in a time and concentration dependent manner.

2-4. Changes in Bcl-2 / Bax Expression Patterns

Bcl among genes important for apoptosis-inducing regulation in order to determine the mechanism involved in inducing apoptosis of HL-60 cells and HL-60 / ADR by LJ identified through the results of Experimental Examples 2-1 to 2-3 The expression changes of the -2 and Bax genes were tested using a special protein detection test (western blot).

Although Bcl-2 is a cancer gene protein, unlike other oncogene proteins, Bcl-2 does not participate in cell proliferation and has a function of inhibiting cell survival, ie, apoptosis. Bax is a protein belonging to the Bcl-2 family and promotes cell death. There is a function to let.

HL-60 and HL-60 / ADR (3.5 × 10 ⁵ cells / mL) cells were treated with LJ (100 μg / mL), respectively, and incubated for 3, 6 and 9 hours, and then the respective cells were collected and PBS. (phosphate buffered) was washed 2-3 times. 500 μl of lysis buffer (50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO _3, 10 mM NaF, 1 mM Dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 [mu] g / ml aprotinin, 25 [mu] g / ml leupeptin) was added and reacted at 65 DEG C for 1 hour, followed by centrifugation at 12,000 rpm for 15 minutes to obtain each supernatant. The protein concentration of the supernatant of each cell was measured using a protein assay kit (BIO-RAD, USA; # 161-0158) using BSA (bovine serum albumin; Amresco; 0332) as a standard. Quantified accordingly. Each protein (20 ~ 30㎍) was denatured by 12% mini gel sodium dodecyl sulphate (PAGE) -Polyacrylamide Gel Electrophoresis (PAGE), and 200mA on PVDF membrane (BIO-RAD, USA; 162-0177). Transition for 2 hours. It was then immersed in TTBS (0.1% Tween 20 in TBS) solution containing 5% skim milk and reacted overnight at 4 ° C. to block the nonspecific proteins and 3 with TTBS. Washed twice. In this case, as an antibody for determining the expression level of Bcl-2 of each cell, mouse monoclonal anti-human Bcl-2 Ab (Santa-Cruz, USA; # SC-509), an antibody for determining the expression level of Bax, Rabbit polyclonal anti-human Bax Ab (Santa-Cruz, USA; # SC-493) was diluted 1: 1000 in TTBS solution and the reaction proceeded at room temperature for 2 hours. As a secondary antibody, antirabbit Ig G (Cell Signaling, USA; # 7074) combined with HRP (Horse Radish Peroxidase) and anti-mouse IgG (American Pharmacia Biotech, USA; # 94010) were diluted 1: 5000. The reaction proceeded for 30 minutes at room temperature. After washing 3 times with TTBS again, the reaction was reacted with ECL substrate (American Pharmacia Biotech, USA; 16021) for 1 ~ 3 minutes, and then exposed to X-ray film to analyze the expression changes of Bcl-2 and Bax proteins in each cell. It was.

As a result, as shown in Figures 8 and 10, the expression of Bcl-2 in HL-60 and HL-60 / ADR gradually decreased in time-dependent expression after 9 hours in the LJ treatment group, while Bax expression Was found to increase (see FIGS. 8 and 10). Through this, it was found that apoptosis of HL-60 and HL-60 / ADR by LJ is induced by Bcl-2 and Bax proteins.

2-5. Activation of Caspase-9 and Caspase-3

In order to determine the mechanism involved in inducing apoptosis of HL-60 and HL-60 / ADR by LJ identified through the results of Experimental Examples 2-1 to 2-3, Changes in the expression of the Caspase-9 and Caspase-3 genes were tested using the Western blot method (Sambrook J., Fritsch EF, Maniatis T. , "Molecular Cloning Laboratory Manual," 2nd ed., Pp 18.60-18.71, Cold Spring Harbor Laboratory Press, New York, 1989).

Caspase-9 and Caspase-3 are enzymes that degrade PARP, which are activated when apoptosis occurs and trigger apoptosis.

HL-60 and HL-60 / ADR (3.5 × 10 ⁵ cells / ml) cells were treated with LJ (100 μg / ml), respectively, and incubated for 3, 6 and 9 hours, and then each cell was collected and treated with PBS. Washed 3 times. 500 μl of lysis buffer (50 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO _3, 10 mM NaF, 1 mM Dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg / ml aprotinin, 25 μg / ml leupeptin) was added and reacted at 65 ° C. for 1 hour, followed by centrifugation at 12,000 rpm for 15 minutes to obtain supernatant. The protein concentration of the supernatant of each cell was quantified according to the protein assay kit (BIO-RAD, USA; # 161-0158) and its method using BSA (bovine serum albumin; Amresco; 0332) as a standard. The protein (20-30 μg) of each cell was denatured with 12% mini gel SDS (sodium dodecyl sulphate) -PAGE (Polyacrylamide Gel Electrophoresis) to denature and separate PVDF membrane (BIO-RAD, USA; 162-0177). ) Was transferred to 200 mA for 2 hours. It was then immersed in TTBS (0.1% Tween 20 in TBS) solution containing 5% skim milk and reacted overnight at 4 ° C. to block the nonspecific proteins and 3 with TTBS. Washed twice. At this time, the expression level of the rabbit polyclonal anti-human PARP Ab (Santa-Cruz, USA; SC-7150), Caspase-3 and (Caspase) -9 as an antibody to determine the expression level of each cell PARP As an antibody, rabbit polyclonal anti-human caspase-3 Ab (Cell Signaling, USA; # 9662) and Ab (Cell Signaling, USA; # 9502) of caspase-9 were TTBS solution. Diluted 1: 1000 at and used for 2 hours at room temperature. As a secondary antibody, antirabbit Ig G (Cell Signaling, USA; # 7074) combined with HRP (Horse Radish Peroxidase) and anti-mouse IgG (American Pharmacia Biotech, USA; # 94010) were diluted 1: 5000. The reaction proceeded for 30 minutes at room temperature. Then, washed three times with TTBS again, reacted with ECL substrate (American Pharmacia Biotech, USA; 16021) for 1 to 3 minutes, and then exposed to X-ray film to caspase-9 and kespase of each cell. The expression changes of caspase) -3 protein were analyzed.

As a result, as shown in Figures 9 and 11, the activity of caspase-9 and caspase-3 in HL-60 and HL-60 / ADR was 6 hours and After 9 hours, it gradually increased in time. In addition, PARP (116 kDa), one of the caspase-3 substrates, was degraded in time-dependent manner in proportion to the activity of caspase-9 and caspase-3, resulting in degradation of PARP from 9 hours. Protein (85 kDa) could be observed (see Figures 9 and 11). Through this, it was found that apoptosis of HL-60 and HL-60 / ADR by LJ is induced by the caspase-9 and the caspase-3 protein.

As such, it could be confirmed that LJ of the present invention specifically promotes apoptosis of cancer cells.

Examples of the formulation of the composition comprising the extract of the present invention will be described below, but the present invention is not intended to be limited thereto but merely to be described in detail.

Formulation Example 1 Preparation of Powder

LJ (Example 1) 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

LJ (Example 1) 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

LJ (Example 1) 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

LJ (Example 1) 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

LJ (Example 1) 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

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

Formulation Example 6 Preparation of Healthy Food

LJ (Example 1) 1000 mg

Vitamin mixture proper amount

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

50 μg folic acid

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

LJ (Example 1) 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 production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

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

      1 is a diagram showing the effect of inhibiting the proliferation of cancer cells by LJ,

Figure 2 is a diagram showing the DNA fragmentation of HL-60 cells by LJ using electrophoresis,

3 is a diagram showing the effect of increasing the number of sub-G1 phase cells by LJ in HL-60 cells using a flow cytometer,

Figure 4 is a diagram showing the effect of increasing the number of sub-G1 phase cells by LJ in HL-60 / ADR cells using a flow cytometer,

       Figure 5 is a diagram showing the morphological changes of HL-60 cells with LJ treatment time,

       6 is a diagram showing the morphological changes of HL-60 cells according to the concentration of LJ treatment,

Figure 7 is a diagram showing the morphological changes of HL-60 / ADR cells according to the concentration of LJ treatment,

8 is a diagram showing the effect of reducing the expression of Bcl-2 and Bax expression in HL-60 cells by LJ,

9 is a diagram showing the effect of increasing the activity of caspase-9 and caspase-3 in HL-60 cells by LJ,

10 is a view showing the effect of reducing the expression of Bcl-2 and Bax expression in HL-60 / ADR cells by LJ,

11 is a diagram showing the effect of increasing the activity of caspase-9 and caspase-3 in HL-60 / ADR cells by LJ.

Claims (8)

A pharmaceutical composition for the prevention and treatment of cancer diseases containing the extract of the crow bark (Litsea japonica Jussieu) as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is one or more extracts selected from the group consisting of branches, bark, leaves, flowers, fruits or roots of Litsea japonica Jussieu. The pharmaceutical composition of claim 1, wherein the extract is an extract available in at least one solvent selected from the group consisting of water including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof. The method of claim 1, wherein the cancer disease is gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin or ocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal muscle Cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis Cancer, prostate cancer, chronic or acute leukemia, promyelocytic leukemia, monocytic leukemia, lymphoid leukemia, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, 1 A pharmaceutical composition that is at least one disease selected from the group consisting of primary CNS lymphoma, spinal cord tumor, brain stem glioma or pituitary adenoma. The pharmaceutical composition according to claim 1, wherein the crow bark (Litsea japonica Jussieu) is a plant distributed in Jeju Island, Gyeongnam or Jeonnam in Japan, Korea. The pharmaceutical composition of claim 1, wherein the extract comprises 0.1 to 50% by weight of the total weight of the composition. A dietary supplement for the prevention and improvement of cancer diseases, which contains the extract of Licesea japonica Jussieu as an active ingredient. A dietary supplement according to claim 7 which is a powder, granule, tablet, capsule or beverage.

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