KR20110058009A - The composition for the treatment of cancers or inhibition of metastasis containing the extracts or fractions of asparagus cochinchinensis as active ingredient - Google Patents

Abstract

PURPOSE: A composition containing Asparagus cochinchinensis is provided to ensure cytotoxicity to cancer cells and to suppress cancer metastasis. CONSTITUTION: A composition for suppressing cancer metastasis contains Asparagus cochinchinensis extract as an active ingredient. The Asparagus cochinchinensis extract is isolated using water, low alcohol of C1-C4, or mixture solvent thereof. The low alcohol is methanol or ethanol. The composition additionally contains organic solvent fraction of Asparagus cochinchinensis extract as an active ingredient. The organic solvent is n-hexane, methylene chloride, ethyl acetate, or n-butanol. A health food for preventing and treating cancer contains Asparagus cochinchinensis extract or fraction thereof as an active ingredient.

Description

The composition for the treatment of cancers or inhibition of metastasis containing the extracts or fractions of Asparagus cochinchinensis as active ingredient}

The present invention relates to a composition for treating cancer or inhibiting cancer metastasis, containing the astronomical extract or a fraction thereof as an active ingredient.

Cancer refers to a population of abnormal cells caused by continuous division and proliferation due to the disruption of the balance between cell division and death by various causes, also called tumors or neoplasms. It usually affects more than 100 different parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops severe symptoms through infiltration into surrounding tissues and metastases to other organs (WHO, 2006). The causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, ionizing radiation and internal factors such as congenital gene mutations (Klaunig & Kamendulis, Annu). Rev Pharmacol Toxicol . , 44: 239-267, 2004). Recently, the association between chronic inflammation and cancer occurrence has emerged, and a lot of data supporting this has been reported. Infections and chronic inflammation account for 25% of all cancers and are known to be at much higher risk for cancer in patients with chronic inflammation and reactive oxygen species. Various mediators that control the inflammatory response, such as cytokines, free radicals, and growth factors, induce genetic and epigenetic changes such as mutations in tumor suppressor genes, DNA methylation, and post-transcriptional modifications, resulting in normal cell homeostasis. It is believed to alter the pathways necessary to maintain the risk and to develop and advance cancer (Hussain & Harris, Int J Cancer ., 121 (11): 2373-2380, 2007).

Cancer is the number one cause of death in Korea, accounting for 27%. Among them, liver cancer is the second most common cancer after lung cancer and is a malignant tumor with high mortality. Liver cancer is one of the leading causes of death among men in their 40s and 50s with high social activity and impact. In addition, liver disease, the seventh leading cause of death, is broadly included in the early stages of the development of liver cancer (National Statistical Office, 2006). The main cause of liver cancer is hepatitis B, which occupies an overwhelming proportion of 70%. Hepatitis carriers are known to be 100 times more likely to develop liver cancer than non-carriers. Therefore, studies on liver cancer have been conducted as a model of cancer development in chronic inflammation. However, to date, very few studies have been conducted on the mechanism of liver cancer, and since no clear identification has been made, there are many difficulties in treatment or diagnosis, and research is needed to effectively prevent and treat liver cancer.

In the early stages of cancer, there are treatments such as surgery, radiation therapy, and chemotherapy, but the side effects are also becoming a big problem. In the case of terminal cancer or metastatic cancer, life ends in a limited-time life without special treatment. Accordingly, as a new approach for the treatment of cancer, research is being conducted to develop carcinogenic inhibitors and cancer therapeutic agents having low side effects and excellent efficacy from relatively low toxicity natural products. According to various basic and clinical studies, herbal prescriptions have anti-cancer effects such as enhanced immune function and tumor growth inhibition, and side effects such as hematopoiesis and immune function inhibition that are commonly observed in chemotherapy and radiation therapy have been greatly reduced.

Asparagi Tuber is a perennial herbaceous plant belonging to the genus Liliaceae Asparagus. cochinchinensis (Lour.) Merr. Is a myocardium, which is mainly used for respiratory diseases and skin diseases due to its effects such as consonant, rhythm, green lung, and jinjin. (Cooperation teaching materials compilation committee, 2005), and it can be used as an ingredient in folk sake. It is native to China and is distributed on the coastal area of the southern part of the Korean peninsula. It is estimated that the seeds of native Chinese species are crowded by the ocean currents and form colonies on the coast of Korea, and are indirectly verified by molecular biological experiments. About the weakness of Astronomical dong in oriental medicine, it is known that it is sweet and bitter and its nature is very cold. Dongbogam describes that astronomical dong helps pulmonary function and is used for various diseases such as cough, asthma, stroke and diuresis (Chinese medicine dictionary, 1986; Woonkok Herbology, 2004; Chinese Pharmacopoeia, 2005). However, the anticancer and cancer metastasis inhibitory effects on the astronomical extract are not known.

Thus, the present inventors while studying to develop a cancer-derived drug or a drug for inhibiting cancer metastasis derived from natural products without toxicity and side effects, the fraction obtained from the astronomical extract shows significant cytotoxicity to cancer cells, the astronomical extract or its Since the fraction has the effect of inhibiting cancer metastasis by inhibiting cell migration and cell infiltration, the present invention was completed by confirming that the astronomical extract of the present invention or a fraction thereof can be usefully used as a composition for inhibiting cancer and cancer metastasis.

Disclosure of Invention An object of the present invention is to provide a composition for treating cancer or inhibiting cancer metastasis, which contains an astronomical extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a composition for suppressing cancer metastasis containing cheonmundong (Asparagus cochinchinensis) extract as an active ingredient.

In another aspect, the present invention provides a composition for treating cancer or inhibiting cancer metastasis, which contains an organic solvent fraction prepared by extracting the astronomical extract with an organic solvent as an active ingredient.

In addition, it provides a health functional food for the prevention and improvement of cancer or cancer metastasis containing cheonmundong extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a functional feed additive for preventing or improving cancer or cancer metastasis, which contains the astronomical extract or fractions thereof as an active ingredient.

Asparagus of the present invention cochinchinensis ) extracts or fractions thereof have remarkable cytotoxicity against hepatocellular carcinoma cells and are excellent in inhibiting cancer metastasis by inhibiting cell migration and cell infiltration, and have an excellent anti-cancer effect. It can be usefully used as.

Hereinafter, the term used by this invention is demonstrated.

As used herein, the term "prevention" means any action that inhibits cancer or delays progression by administration of a composition of the present invention.

As used herein, the terms "treatment" and "improvement" refer to any action by which administration of the composition of the invention improves or beneficially alters cancer.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

As used herein, the term "individual" means any animal, such as a human, monkey, dog, goat, pig or rat, having a disease in which cancer can be improved by administering the composition of the present invention.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which means the type of disease, the severity, the activity of the drug, the drug Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention is the Asparagus Cochinchinensis ) provides a composition for inhibiting cancer metastasis containing the extract as an active ingredient.

The present invention also provides a composition for treating cancer or inhibiting cancer metastasis, comprising a fraction of the astronomical extract as an active ingredient.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer, and more preferably liver cancer. One is not limited to this.

The astronomical extract is preferably prepared by a manufacturing method comprising the following steps, but not always limited thereto:

1) extracting by adding an extraction solvent to Asparagus cochinchinensis ;

2) cooling the extract of step 1) and then filtering; And

3) drying the filtered extract of step 2) under reduced pressure.

In the above method, the astronomical dong of step 1) can be used without limitation, such as cultivated or commercially available. The astronomical dong is preferably used, but not limited to dried roots.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, it is preferable to use C ₁ to C ₄ lower alcohols, ethanol or methanol is preferably used as the lower alcohol, more preferably 70% ethanol, but is not limited thereto. As the extraction method, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used, and the extraction may be preferably performed one to five times by the hot water extraction method, and more preferably three times of extraction. . The extraction solvent is preferably extracted by adding 2 to 10 times the weight of the dried astronomical copper root. The extraction temperature is preferably 50 to 100 ° C, more preferably 50 to 60 ° C, but is not limited thereto. In addition, the extraction time is preferably 1 to 4 hours, more preferably 2 hours is not limited thereto.

In the above method, the decompression concentration in step 3) preferably uses a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In a specific embodiment of the present invention, astronomical roots were washed with water and then dried for 7 days at shade and room temperature. The dried Cheonmundong root was crushed and placed in an extraction container, and extracted with an extraction solvent at 50 ~ 60 ℃ for 2 hours. After obtaining the cheonmundong extract, the solids were removed using a filter paper and the suspension was centrifuged to filter the supernatant under reduced pressure. The extract was concentrated under reduced pressure and lyophilized to prepare an astronomical extract.

The fraction of the astronomical dong extract is preferably prepared by a manufacturing method comprising the step of preparing an organic solvent fraction by adding an organic solvent to the astronomical extract, but is not limited thereto.

In the above method, the organic solvent is preferably n-hexane, methylene chloride, ethyl acetate or n-butanol, but is not limited thereto. The fraction may be obtained by repeating the fractionation process from 1 to 5 times, preferably 3 times, from the astronomical extract, preferably concentrated under reduced pressure after the fraction, but is not limited thereto.

In a specific embodiment of the present invention, the astronomical copper extract is suspended in distilled water, and then systematically fractionated in the order of n-hexane, methylene chloride, ethyl acetate, n-butanol and water, and each solvent fraction is concentrated under reduced pressure, and fractions are extracted from the astronomical copper extract. Was prepared.

In order to confirm the cytotoxicity of the astronomical extract or fractions thereof, the present inventors treated the 70% ethanol extract or fractions thereof in human liver cancer cell lines at different concentrations (0, 25, 50, 100, 200 or 400 µg / ml). Absorbance was measured to determine cell viability. As a result, ethyl acetate fraction (IC ₅₀ = 72.22 ± 0.34), methylene chloride fraction (IC ₅₀ = 112.68 ± 9.10) or n-butanol fraction (IC ₅₀ = 21.01 ± 1.37) showed a significant cytotoxic effect, in particular, The cytotoxic activity of n-butanol was found to be the best. In comparison, the astronomical dong 70% ethanol extract or its water fraction did not have an effect on apoptosis, and it was confirmed that induction of apoptosis in the astronomical dong extract at a concentration of 400 μg / ml or more (see FIG. 1).

The present inventors treated 70% ethanol extract or fraction (100 or 200 ㎍ / ml) of astronomical dong to human hepatocellular carcinoma cell line with increased cell migration by PMA to confirm the cell migration inhibitory effect of astronomical dong extract or fractions thereof. The cell migration was examined through nuclear staining. As a result, it was shown that significantly increased cell migration by PMA treatment was inhibited by the astronomical dong extract or fraction (see FIG. 2). In addition, by treating the astronomical extract of 0, 12.5, 25, 50, 100, 200 or 400 ㎍ / ㎖ concentration to confirm the effect of inhibiting cell migration by concentration, the cell migration significantly increased by PMA treatment of the extract It was inhibited concentration dependently at a concentration of 25-400 μg / ml (see FIG. 3).

In order to confirm the cell penetrating inhibitory effect of the astronomical extract and its fraction showing the cell migration inhibitory effect, the present inventors identified 70% ethanol extract or its water fraction (0, 25, 50, 100, 200 or 400 ㎍) in human liver cancer cell line. / Ml), matrix metalloproteinase (MMP) -2 and MMP-9, proteolytic enzymes that break down the main component type IV collagen of the basement membrane during cancer infiltration and metastasis. The activity of was measured by gelatin zymography. As a result, the activity of both MMP-2 and MMP-9 was increased by PMA treatment, but activity was suppressed in a concentration-dependent manner by the astronomical extract or water fraction (see FIG. 4).

Therefore, the astronomical dong extract of the present invention inhibits cell migration and cell infiltration, and thus may be usefully used as an active ingredient for the prevention and treatment of cancer, and the fraction of the extract has a cytotoxic effect through cancer cell death and cell migration. And since it inhibits cell infiltration, it can be usefully used as an active ingredient of cancer treatment or cancer metastasis inhibiting composition.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

Solid form preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and the like. 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 powders, granules, tablets, capsules, sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. It may be used in the form of a formulation, and preferably, an external skin pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasma agent may be prepared and used. It is not limited to this. Non-aqueous solvents and suspending agents may be used as vegetable oils such as propylene glycol pharmaceutical polyethylene glycol pharmaceutical olive oil, injectable esters such as ethyl oleate, etc. As a base of suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention may further include conventionally used carriers, excipients, disintegrants, sweeteners, lubricants, flavoring agents and diluents. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, and polyacryline Potassium and the like.

In addition, the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, Aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the pharmaceutical composition.

The pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the above components. The composition of the present invention may comprise from 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the fraction of the present invention based on the total weight of the composition.

In addition, the pharmaceutical composition of the present invention may be administered orally or parenterally, and when parenteral administration is selected for external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. It is desirable to.

 The dosage of the composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease of the patient, the daily dosage is the amount of the extract of astronomical dong 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and may be administered 1 to 6 times per day.

The composition of the present invention can be used alone or in combination with methods for using cancer, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the treatment of cancer or inhibition of cancer metastasis.

The present invention also provides a method for preventing and treating cancer or cancer metastasis, comprising administering to a subject a pharmaceutically effective amount of said extract or fraction thereof.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The method of administration may be administered orally or parenterally, intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, intrauterine dural, intracerebroventricular or intrathoracic It can be administered by injection.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, but is not limited thereto. Dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of a particular patient.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer, and more preferably liver cancer. One is not limited to this.

Asparagus of the present invention cochinchinensis ) extract or fractions thereof exhibits significant cytotoxicity against liver cancer cell lines, inhibits cell migration and reduces the activity of MMP-2 and MMP-9, key enzymes involved in cell infiltration, thereby preventing cancer or cancer metastasis. And useful for the treatment.

In addition, it provides a health functional food for the prevention and improvement of cancer or cancer metastasis containing cheonmundong extract or a fraction thereof as an active ingredient.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer, but more preferably liver cancer. It is not limited to this.

The astronomical copper extract or fractions thereof of the present invention can be added as it is or used with other foods or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of food. Examples of the food to which the cheonmundong extract or a fraction thereof may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, Tea, drink, alcoholic beverages and vitamin complexes, etc., and includes all of the health food in the usual sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the astronomical extract of the present invention or fractions thereof are various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, Glycerol, alcohols, carbonation agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Asparagus of the present invention cochinchinensis ) extract or fractions thereof exhibits significant cytotoxicity against liver cancer cell lines, inhibits cell migration and reduces the activity of MMP-2 and MMP-9, key enzymes involved in cell infiltration, thereby preventing cancer or cancer metastasis. And it can be usefully used for health functional food for improvement.

In addition, the present invention provides a functional feed additive for preventing or improving cancer or cancer metastasis, which contains the astronomical extract or fractions thereof as an active ingredient.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer, but more preferably liver cancer. It is not limited to this.

 The feed additive can prevent cancer or cancer metastasis by constantly ingesting poultry, livestock, and the like, and can treat cancer or cancer metastasis that has already occurred.

The feed additive of the present invention contains 0.1 ~ 20% by weight of the cheonmundong extract or fractions thereof, 0.001 to 0.01% by weight of lipase, 1 to 20% by weight of tricalcium phosphate, 0.01% of vitamin E ~ 0.1% by weight, enzyme powder 1 ~ 10% by weight, lactic acid bacteria 0.1 ~ 10% by weight, Bacillus (Bacillus) culture medium is composed of 0.01 to 10% by weight and glucose 20 to 90% by weight Preferred but not limited thereto, and if the fraction of the astronomical extract is added in an effective amount, it can be used as a feed additive of the present invention.

The effective amount means an amount capable of preventing cancer or cancer metastasis or treating cancer or cancer metastasis that has already occurred by allowing poultry, livestock, etc. to be continuously ingested. Moreover, it is preferable that the quantity which does not produce the bad influence beyond the benefit by addition is preferable.

In addition, the feed additive may additionally contain a carrier that is acceptable to poultry and livestock. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like, and various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added as necessary. The shape may be in a suitable state such as powder, granules, pellets, suspension, or the like. When supplying the feed additive of the present invention can be supplied alone or mixed in the feed for poultry and livestock.

Asparagus of the present invention cochinchinensis ) extract or fractions thereof exhibits significant cytotoxicity against liver cancer cell lines, inhibits cell migration and reduces the activity of MMP-2 and MMP-9, key enzymes involved in cell infiltration, thereby preventing cancer or cancer metastasis. And it can be usefully used in the functional feed additive for improvement.

Hereinafter, the present invention will be described in detail by examples and production examples.

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

< Example  1> Astronomical building  Preparation of Extract

Asparagus collected from Gyehwa-do, Jeollabuk-do cochinchinensis ) roots were dried and ground. 100 g of astronomical dong prepared by the above method was immersed in 1 L of 70% ethanol aqueous solution (70% EtOH), 100% ethanol (100% EtOH), 100% methanol (100% MeOH) or water (H ₂ O) and stirred. Then, the mixture was heated to reflux for 2 hours at an extraction temperature maintained at 50 to 60 ° C. and filtered through a filter paper. The extract was concentrated under reduced pressure using a concentrator at 50 to 55 ° C., and then freeze-dried to prepare 70% ethanol, 100% ethanol, 100% methanol, and water extracts of astronomical dong.

As a result, astronomical copper solvent extracts of 70% ethanol (15 g), 100% ethanol (15 g), 100% methanol (14 g) and water (17 g) were obtained, respectively.

< Example  2> Astronomical building  From extract Fraction  Produce

<2-1> n- Hexane Fraction

In order to prepare a fraction from the astronomical dong extract, 1760 kg of 70% ethanol extract was obtained from 11.4 kg of dried astronomical dong root according to the method of <Example 1>. After 1,760 g of the extract was suspended in 5 L of water (H ₂ O), the suspension and the same amount of n-hexane (n-hexane, Hx) were added thereto, and the mixture was left to separate into an aqueous layer and an Hx layer. After chemical equilibrium, the Hx layer was separated, and again, the same amount of Hx was poured and allowed to separate into the water layer and the Hx layer. The Hx layer obtained by repeating the layer separation process three times was concentrated under reduced pressure to obtain an Hx fraction (0.9 g).

<2-2> system Fraction

Separation of Hx fraction in the same manner as in Example <2-1> and methylene chloride (methylene chloride, dichloromethane, MC), ethyl acetate (EA), n-butanol (n) according to the polarity of the solvent in the remaining water layer -Bonolol, BuOH), water (H ₂ O) in order to extract the fractions, the astronomical copper methylene chloride, ethyl acetate, n-butanol and water fractions were prepared.

As a result, MC (3 g), EA (3 g), BuOH (29 g) and H ₂ O (680 g) astronomical copper fractions were obtained, respectively.

< Example  3> Cytotoxicity Experiment

Human liver cancer cell line HepG2 was purchased from American Type Culture Collection (ATCC HB-8065, USA) and added 100 units / ml penicillin, 100 μg / ml streptomycin with 10% fetal bovine serum (FBS) inactivated by heat. Incubated in DMEM medium at 37 ° C., 5% CO ₂ . Cell Counting Kit-8 (CCK-8) test was performed to confirm the cytotoxicity of the sample. HepG2 cells cultured in 96 well plates for 24 hours were treated with astronomical dong extract or fractions thereof by concentration (0, 25, 50, 100, 200, 400 μg / ml) and incubated for 24 hours. After incubation, the medium was removed, fresh medium to which 10% CCK-8 was added was added to each well, and further reacted at 37 ° C for 2 hours, followed by 450 nm using SpectaMax 340 reader (Molecular Devices, USA). Absorbance at was measured and cell viability was calculated by comparison with the control.

As a result, the cells in the EA (IC ₅₀ = 72.22 ± 0.34 µg / ml), MC (IC ₅₀ = 112.68 ± 9.10 µg / ml) and BuOH (IC ₅₀ = 21.01 ± 1.37 µg / ml) fractions as shown in FIG. It showed toxic effects, especially the cytotoxicity of the BuOH fraction was the best. The ethanol extract of cheonmundong showed cytotoxicity at the highest concentration of 400 ㎍ / ml, and the Hx fraction and the H ₂ O fraction showed little cytotoxicity (FIG. 1).

< Example  4> Confirmation of cell migration through nuclear staining

In order to confirm the cell migration inhibitory effect of the astronomical extract and fractions thereof, nuclear staining was performed. The lower plate of the 48-well microchemotaxis chamber (Neuro Probe, MD) was treated with 200 nM of PMA and samples at concentrations of 12.5, 25, 50, 100, 200 or 400 μg / ml and coated with 0.1% gelatin Put the cells in. After 8 hours, the lower surface of the filter membrane located in the middle of the upper and lower plates was stained with Diff-Quick solution (Dade Behring, DE) and observed under a microscope.

As a result, as shown in Figure 2, significantly increased cell migration by treatment with phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Inc., MO) It was inhibited by 100 or 200 μg / ml Astronomical Copper extract or fractions thereof (FIG. 2). In addition, as a result of confirming the cell migration inhibitory effect of the astronomical ethanol extract showing the cell migration inhibitory effect, as shown in Figure 3, significantly increased cell migration by the PMA treatment concentration (25 ~ 400 Μg / ml) dependently (FIG. 3).

< Example  5> Analysis of activity of cell penetrating enzymes

In order to confirm the cell penetrating inhibitory effect of ethanol extract and water fraction of astronomical dong, which showed cell migration inhibitory activity, gelatin zymography showed the activity of matrix metalloproteinase (MMP) -2 and MMP-9, which are proteolytic enzymes. It was measured using (gelatin zymography). HepG2 cells cultured in 24 well plates for 24 hours were treated with 200 nM of PMA and samples at concentrations of 12.5, 25, 50, 100, 200 or 400 μg / ml and incubated for 24 hours, after which the medium was obtained to give a centricon The sample was concentrated to (centricon). Proteins from concentrated samples were quantified using a Bio-Rad Protein Assay Kit (Hercules, CA) to determine non reducing sample buffer {0.5 M Tris-HCl, pH 6.8, 5% SDS, 20% glycerol, 1% bromophenol blue blue} and then loaded onto a 10% SDS-PAGE gel containing 1.5 mg / ml gelatin prepared beforehand.After electrophoresis, 2.5% (v / v) Triton The SDS of the gel was removed by X-100 solution, and added to zymogram development solution (50 mM Tris-HCl, pH 7.6, 5 mM CaCl ₂ ) and reacted at 37 ° C. for 18 hours.0.1% Coomassie brilliant blue G250 (in 45 The gel was stained with% (v / v) methanol / 10% (v / v) acetic acid) and then decolorized with 10% (v / v) acetic acid / 20% (v / v) methanol. The area was observed as a white band on a blue background, and the intensity of the band was measured by Multi Gauge software version 3.0 (Fuji Photo Film Co., LTd, Japan).

As a result, as shown in Figure 4, both MMP-2 and MMP-9 activity was increased by PMA treatment, the activity was inhibited in a concentration-dependent manner by the astronomical ethanol extract or water fraction.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Powder  Produce

2 g of ethyl acetate fraction of Example <2-2> of the present invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of ethyl acetate fraction of Example <2-2> of the present invention

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

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

<1-3> Preparation of Capsule

100 mg of butanol fraction of Example <2-2> of the present invention

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of the ring

1 g of butanol fraction of Example <2-2> of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

<1-5> Preparation of Granules

150 mg of butanol fraction of Example <2-2> of the present invention

Soybean Extract 50mg

Glucose 200 mg

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Manufacturing example  2> Manufacture of food

Food containing the cheonmundong extract of the present invention was prepared as follows.

<2-1> Production of flour food

0.5-5.0 parts by weight of 70% ethanol extract of <Example 1> of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1% to 5.0 parts by weight of 70% ethanol extract of <Example 1> of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles, and broths.

<2-3> ground Beef  Produce

10 parts by weight of 70% ethanol extract of <Example 1> of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> dairy products ( dairy products Manufacturing

5 to 10 parts by weight of 100% ethanol extract of <Example 1> of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

100% ethanol extract of <Example 1> of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with spray and hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds, and 100% ethanol extracts of <Example 1> prepared above were prepared by combining the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

100% ethanol extract (3 parts by weight) of <Example 1> of the present invention,

(0.5 part by weight),

Foxglove (0.5 part by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Substances such as liquid fructose (0.5%), oligosaccharides (2%), sugars (2%), salts (0.5%), water (75%) and 5 g of ethyl acetate fraction of Example 2 of the present invention. After homogeneous mixing and sterilization, it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of Vegetable Juice

5 g of the ethyl acetate fraction of Example <2-2> of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Preparation of Fruit Juice

1 g of the ethyl acetate fraction of Example <2-2> of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

< Manufacturing example  4> Preparation of feed additive

Using the butanol fraction of Example <2-2> as an active ingredient, the present inventors manufactured a feed additive with the following composition.

<The composition of the feed additive>

Butanol fraction of Example <2-2> of the present invention: 0.1 to 20% by weight,

Lipase: 0.001 to 0.01% by weight,

Tertiary calcium phosphate: 1-20% by weight,

Vitamin E: 0.01-0.1% by weight,

Enzyme powder: 1 to 10% by weight,

Lactic acid bacteria: 0.1 to 10% by weight,

Bacillus culture medium: 0.01 to 10% by weight, and

Glucose: 20 to 90% by weight.

As described above, the astronomical dong extract of the present invention or fractions thereof have an excellent effect on anti-cancer or cancer metastasis inhibition, the development of drugs related to cancer treatment or inhibition of cancer metastasis, or functional health food and functional feed additives for cancer or cancer metastasis improvement and It can be usefully used for production.

1 is a graph comparing the cytotoxicity of astronomical extract or fractions thereof:

EtOH-E: 70% ethanol extract;

Hx-E: n-hexane fraction;

MC-E: methylene chloride fraction;

EA-E: ethyl acetate fraction;

BuOH-E: n-butanol fraction; And

H ₂ OE: water fraction.

2 is a diagram showing the cell migration inhibitory effect of the astronomical extract or fractions thereof through nuclear staining.

Figure 3 is a diagram showing the effect of inhibiting cell migration by concentration of ethanol extract cheonmundong through nuclear staining:

PMA: phorbol 12-myristate 13-acetate; And

EtOH-E: 70% Ethanol Extract.

Figure 4 is a diagram showing the effect of inhibiting cell infiltration by concentration of ethanol extract or water fraction thereof.

Claims (9)

Asparagus cochinchinensis ) A composition for inhibiting cancer metastasis containing the extract as an active ingredient. The method of claim 1, wherein the astronomical extract is water, C ₁ A composition for inhibiting cancer metastasis, which is extracted with a lower alcohol of C ₄ or a mixed solvent thereof. 3. The composition for inhibiting cancer metastasis according to claim 2, wherein the lower alcohol is ethanol or methanol. The method of claim 1, wherein the cancer is any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer. A composition for inhibiting cancer metastasis. Cancer treatment or cancer metastasis inhibiting composition containing the organic solvent fraction prepared by extracting the cheonmundong extract with an organic solvent as an active ingredient. The composition for treating cancer or inhibiting cancer metastasis according to claim 5, wherein the organic solvent is n-hexane, methylene chloride, ethyl acetate or n-butanol. The method of claim 5, wherein the cancer is any one selected from the group consisting of liver cancer, colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer. Cancer treatment or composition for inhibiting cancer metastasis. Health functional food for the prevention and improvement of cancer or cancer metastasis containing cheonmundong extract or fractions thereof as an active ingredient. Functional feed additive for the prevention and improvement of cancer or cancer metastasis containing cheonmundong extract or a fraction thereof as an active ingredient.

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