KR20160030921A - A composition comprising the extract of Morus alba L. for treating or preventing cancer disease - Google Patents

Abstract

The present invention relates to a composition for treating and preventing cancer diseases, comprising a Morus alba L. extract as an active ingredient. The Morus alba L. extract of the present invention shows anti-cancer activity in cells of a macrophage Raw 264.7 and colorectal cancer such as CT-26 cells, and inhibition activity of tumor tissue growth, when co-administering a conventional anticancer agent such as 5-FU in a cancer xenograft animal model experiment, thereby being able to be helpfully used as foods, drugs, and components of feeds having an excellent anti-cancer effect.

Description

[0001] The present invention relates to a composition for treating or preventing a cancerous disease,

The present invention provides a composition for treating or preventing a cancerous disease, which comprises a topical extract as an active ingredient.

[1] Kim GY, Choi GS, Lee SH and Park YM. Acidic polysaccharide

from Phellinus linteus enhances through the up-regulation of Nitric oxide and Tumor necrosis factor-α from peritoneal macrophages. Journal of Ethnopharmacology, 95, 69-76. 2004.

[2] Kim GS, Kim DH, Lim JJ, Lee JJ, Han DY, Lee WM, Jung WC, Min WG, Won CG, Rhee MH, Lee HJ and Kim S. Biological and antibacterial activities of the natural herb Houttuynia cordata Water extract against the inteacellular bacterial pathogen Salmonella within the RAW 264.7 macrophage. Biological and Pharmaceutical Bulletin, 31 (11), 2012-2017. 2008.

[3] Leiro JM, Castro R, Arranz JA and Lamas J. Immunomodulating activities of acidic sulphated polysaccharides obtained from the seaweed Ulva rigida C. Agardh. International Immunopharmacology, 7, 879-888. 2007.

[4] Han EH, Choi JH, Hwang YP, Park HJ, Choi CY, Chung YC, Seo JK, Jeong HG. Immunostimulatory activity of aqueous extracts from Prunella vulgaris.Food Chem Toxicol. 47 (1): 62-69. 2009.

[Literature 5] Dawson TM and Dawson VL: Nitric oxide synthase: role as a transmitter / mediator in the brain and endocrine system. Annu Rev Med. 47: 219-227. 1996.

[Literature 6] Boring CC, Squires TS, Tong T: Cancer statistics, CA Cancer J Clin. 43 (1): 7-26. 1993.

[7] Kim NH, Kim SN, Oh JS, Lee S, Kim YK. Anti-mitotic potential of 7-diethylamino-3 (2'-benzoxazolyl) -coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620 / 5-FU. Biochem Biophys Res Commun, 2012.

[Reference 8] Kanetaka K, Enjoji A, Furui J, Nagata Y, Fujioka H, Shiogama T, et al. Effects of Intermittent 5-Fluorouracil and Low-dose Cisplatin Therapy on Advanced and Recurrent Gastric Cancer. Anticancer Res 32: 3495-9, 2012.

[9] Wettergren Y, Carlsson G, Odin E, Gustavsson B. Pretherapeutic uracil and dihydrouracil levels of colorectal cancer are associated with sex and toxic side effects during adjuvant 5-fluorouracil-based chemotherapy. Cancer; 118: 2935-43, 2012.

[Literature 10] Eisenberg et al., N. Eng. J. Med., 328, pp. 246-252 1993.

[Document 11] Gang, Byeong-soo, etc., Herbology, Younglim, Seoul, Korea 2000.

[Document 12] Namba, T., The Encyclopedia of Wakan-Yaku with Color Pictures Vol. 1 Hoikusha, Osaka, Japan, 1993.

[13] Jensen MM, Jørgensen JT, Binderup T and Kjaer A: Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper. BMC Med Imaging. 16: 8-16, 2008.

[14] Alessandria G, Filippeschi S, Sinibaldi P, Mornet F, Passera P, Spreafico F, Cappa PM and Gullino PM: Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells. Cancer Res 47: 4243-4247, 1987.

[Literature 15] Wang, M., Guilbert, L. J., Li, J., Wu, Y., Pang, P., Basu, T.K. &Amp; Shan, J.J. Int Immunopharmacol, 4, pp 311-315. 2004.

The present invention provides a composition for treating or preventing a cancerous disease, which comprises a topical extract as an active ingredient.

Malignant tumors (cancer) are the second leading cause of death following heart disease (Boring et al., 1993)

Cancer may eventually lead to an increase in the number of abnormal or neoplastic cells derived from normal tissue, an increase in the number of neoplastic tumor cells entering adjacent tissues, and a local lymph node and remote site through the blood or lymph system, It is characterized by the formation (metastasis) of spreading malignant cells. In the cancer stage, cells proliferate under conditions that normal cells do not grow. Cancer appears in a variety of forms characterized by different degrees of invasiveness and invasiveness.

Cancer is widely classified into blood cancer and solid cancer, and it occurs in almost all parts of the body such as lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer and skin cancer. Of the methods used to treat malignant tumors, chemotherapeutic agents other than surgery or radiation therapy are collectively referred to as anticancer drugs, and most of them exhibit anticancer activity by inhibiting the synthesis of nucleic acids. 5-fluorouracil (5-FU) is a water-soluble fluorinated pyrimidine derivative, which is used as an antitumor agent. (Kim et al., 2012). (Kanetaka et al., 2012), which is used alone or in combination with leucovorin (LV) for solid tumors such as liver cancer, stomach cancer, colon cancer, pancreatic cancer and breast cancer and has a plasma half- ), Bone marrow weakness, gastrointestinal reactions, leukopenia, and thrombocytopenia (Wettergren et al., 2012).

At present, many natural products are being used to improve health through immunity enhancement around the world. In the United States, natural products are recognized not only as medicines but also as health-functional foods. It is known that not only in Asia but also one third of the US population is taking natural products at least once in the form of alternative medicines (Eisenberg et al., 1993). Drugs derived from natural products have been focused on the identification of active ingredients for the prevention and treatment of disease. While traditional medicine books such as Dongbok-bong have mentioned the efficacy of natural products, the information in the literature is not associated with the current specific diseases or symptoms. In addition, it has not been revealed which of the ingredients of the natural product has a direct effect. Natural products, which are known to have immunostimulatory effects among the natural products listed in Dong-Bo-bo-gyung and the Chinese Medicine Dictionary, require verification of immunity enhancement efficacy by modern scientific research.

In Oriental medicine, the mulberry leaves are called mulberry leaves, the roots are full mulberry leaves, the root husks are mulberry leaves, the branches are mulberry leaves, the berries are mulberry leaves, The milk from the leaves is called 桑 葉 汁 juice, the milk taken from the shell is called the uvuli juice (mulberry juice), and the material bearing the wood is called 桑 柴 灰. Medicinal materials related to mulberry are generally not toxic and can be safely used. It is effective in eliminating the bad heat accumulated in the body, and is applied to hypertension and diabetes (Kang, Byeong-soo, 2000).

When the fruit of Morus alba L., which is a deciduous tree belonging to Moraceae, is found in magenta, it is used as a medicinal herb after drying, and it is used in the treatment of dizziness, tinnitus, (Namba et al., 2000). In Japan, it is known to control the efficacy and fever of bloody blood and apply it to the treatment of tinnitus, pain medicine, insomnia, tinnitus, dizziness, back pain and constipation ). In Dong-bo-gyung, it is said that ruling the noise, benefiting the five days, and gathering the mulberry trees.

Therefore, the inventors of the present invention have obtained a patent for a substance having an immunopotentiating effect from a natural-product-derived extract, confirming that the extract has an effect of enhancing the immune system (Patent Registration No. 10-839096).

However, none of the above references discloses or teaches any description of the anticancer efficacy of the elixir extract.

As a follow-up study of the present invention, the inventors of the present invention have found that the extract of the present invention exhibits anticancer activity in colon cancer such as CT-26 cells in macrophage Raw 264.7 cells, as well as in 5-FU The present invention has been accomplished by confirming that the compound of the present invention can be effectively used as a component of foods, medicines and feeds having excellent anticancer efficacy by confirming the inhibitory activity against cancerous tissue growth upon co-administration with existing anticancer drugs.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for treating or preventing a cancerous disease, which comprises a topical extract as an active ingredient.

In addition, the present invention provides a health functional food for preventing or ameliorating cancer diseases, which comprises a topical extract as an active ingredient.

The sickle cell extract as defined herein is a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably water or a mixed solvent of water and ethanol, Is an extract which is soluble in water or 60 to 100% ethanol.

As used herein, the term "cancer disease" as used herein refers to any disease or condition that is suspected to be a cancer, such as a cancer selected from the group consisting of colon cancer, prostate cancer, breast cancer, cervical cancer, colon cancer, leukemia, small bowel cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, gastric cancer, Skin cancer, brain tumor, bladder cancer, ovarian cancer, or gallbladder cancer, preferably colon cancer, prostate cancer, breast cancer, cervical cancer, or colorectal cancer, most preferably colon cancer.

The present invention also provides a pharmaceutical composition for the prevention and treatment of cancer diseases, which comprises, as an active ingredient, a combination of a topical extract and an existing anticancer therapeutic agent.

The present invention also provides a health functional food for prevention and improvement of cancer diseases, which comprises a combination of a topical extract and an existing anticancer therapeutic agent as an active ingredient.

Existing chemotherapeutic agents as defined herein include 5-FU, Fluorouracil, LV (leucovorin), adriamycin, cyclophosphamide, amsacrine, daunomycin, (taxol), preferably 5-FU.

The pharmaceutical dosage forms of the extract of the present invention may be administered alone or in combination with other therapeutically active compounds, such as 5-FU (Fluorouracil), LV (leucovorin), adriamycin, (5-fluorouracil), such as cyclophosphamide, amsacrine, daunomycin, taxol and the like, preferably 5-FU (Fluorouracil) Can be used as anticancer adjuvants for inhibiting the growth of cancer cells having multi-drug resistance (MDR) against anticancer drugs.

In addition, the present invention provides an anti-cancer adjuvant containing an extract of Suksikji as an active ingredient.

Accordingly, the present invention provides a method for the metabolism of an anticancer agent, preferably a methotrexate, a 6-mercaptopurine, a 6-thioguanine, a 5-fluorouracil or a cytarabine, Antimetabolites; Alkylating agents such as chlorambucil, cyclophosphamide, ethylene imine compound, thiotepa, alkyl sulfone, carmustine, and dacarbazine, ); Antitumor agents such as anticancer agents such as actinomycin D, doxorubicin, bleomycin and mitomycin, plant alkaloids such as vincristine and vinblastine, and mitotic inhibitors such as the mitotic inhibitor taxol, drugs); Hormones such as adrenocortical hormone or progesterone; Platinum-containing anticancer agents such as cisplatin; More preferably, at least one anticancer agent selected from adriamycin, cyclophosphamide, 5-FU, amsacrine, taxol, preferably methotrexate, Antimetabolites such as 6-mercaptopurine, 6-thioguanine, 5-fluorouracil and Cytarabine; More preferably, the combination ratio of the pyrimidine derivative (5-fluorouracil, Cytarabine) and the anticancer activity is maximized, for example, the weight ratio of the conventional anticancer agent and the heder extract is 1: 0.01 to 100 w / ), More preferably from 1: 0.1 to 10 (w / w).

Accordingly, the present invention relates to antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, 5-fluorouracil and Cytarabine; Alkylating agents such as chlorambucil, cyclophosphamide, ethylene imine compound, thiotepa, alkyl sulfone, carmustine, and dacarbazine, ); Antitumor agents such as anticancer agents such as actinomycin D, doxorubicin, bleomycin and mitomycin, plant alkaloids such as vincristine and vinblastine, and mitotic inhibitors such as the mitotic inhibitor taxol, drugs); Hormones such as adrenocortical hormone or progesterone; A combination of at least one anticancer agent selected from platinum-containing anticancer agents such as cisplatin, more preferably adriamycin, cyclophosphamide, 5-FU, amsacrine, and taxol, An anticancer agent comprising the active ingredient is provided.

The sickle cell extract of the present invention can be produced by the following production process.

Dried bulkheads may be prepared according to conventional extraction methods and may be prepared from C1 to C4 lower alcohols such as water, methanol, etc., or water, methanol or the like, in an amount of from 1 to 20 times, preferably from 5 to 15 times, Preferably at a temperature of 70 ° C to 100 ° C, for 0.5 to 10 hours, preferably 2 to 5 hours, in a mixed solvent, preferably water or a mixed solvent of water and ethanol, more preferably water or 60 to 100% Preferably 10 to 10 times, preferably 2 to 5 times. The extract is preferably subjected to extraction by hot water extraction, such as cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction. The extract is filtered under reduced pressure through a filter paper, Followed by lyophilization, and then the extract of the present invention can be obtained.

The inventors of the present invention found that the extract obtained from the above-described method does not only exhibit anticancer activity in colon cancer such as CT-26 cells but also exhibit cancer tissue growth upon co-administration with an existing anticancer agent such as 5-FU And thus it can be used as a component of foods, medicines and feeds having excellent anticancer efficacy.

Accordingly, the present invention provides a pharmaceutical composition for treating or preventing a cancerous disease, a health functional food, a health food, and a feed, which contains the extract of Shinjunggukja obtained by the above-mentioned method as an active ingredient.

In addition, the healed person is a medicinal substance which has been used for a long time as a food or a herbal medicine, and thus the extract of the present invention has no toxicity and side effects.

The pharmaceutical composition of the present invention contains 0.1 to 50% by weight of the above extract relative to the total weight of the composition.

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

Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing the extract of the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one excipient such as starch, calcium carbonate, sucrose, ), Lactose, gelatin and the like.  

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol and vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, and glycerol gelatin can be used.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, once or several times per day. In the composition, the extract of the present invention may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine and intracerebroventricular injections.

In addition, the present invention provides a health functional food for improving or preventing a cancerous disease, which contains the extract as a active ingredient.

The extract of the present invention can be used variously for medicines, foods and beverages for the prevention and treatment of a target disease. Examples of foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, and health supplement foods, and they can be used in the form of powders, granules, tablets, have.

In addition, the present invention provides a health supplement for improving or preventing cancer diseases, which comprises a topical extract as an active ingredient.

The extract of the present invention can be added to food or beverage for the purpose of prevention and treatment of a target disease. At this time, the amount of the extract in food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 30 g of 100% Can be added at a ratio of 0.3 to 10 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like , Xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

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

In addition, the present invention provides an animal feed additive having anticancer efficacy and an animal feed comprising the same as an effective ingredient.

The composition for animal feed additive may be 20 to 90% high concentrate or may be in the form of powder or granules.

The composition for animal feed additive of the present invention is a composition containing an organic acid such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, a phosphate such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin ), Natural antioxidants such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid and phytic acid.

Animal feed additives containing the extract of the present invention and feeds containing the extract of the present invention can be used as auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, living microbial agents, For example, crushed or shredded wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; It is preferable to mix all of the dry ingredients comprising the sugar and the milk product such as the various powdered milk and the whey powder and the dry additive and then mix the liquid ingredient with the ingredient to be liquid after heating, In addition to the main components such as fat and vegetable fat, can be used together with materials such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The composition for animal feed additives can be administered to animals alone or in combination with other feed additives in edible carriers.

In addition, the composition for animal feed additives can be easily administered as top dressing or they can be mixed directly with animal feed, or separately from feed, in separate oral formulations, by injection or percutaneously, or in combination with other ingredients.

Typically, a single daily dose or a divided daily dose can be used as is well known in the art.

When the composition for animal feed additives is administered separately from an animal feed, the dosage form of the composition, as is well known in the art, may be prepared in an immediate release or sustained release formulation in combination with their non-toxic pharmaceutically acceptable excipient . Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, it may be in the form of a soft gelatin capsule, or a syrup or liquid suspension, emulsion or solution. In addition, the dosage form may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoting agents and the like.

In addition, the feed comprising the animal feed additive of the present invention may be any protein-containing organic fructose commonly used to meet dietary needs of an animal. These protein-containing flours usually consist mainly of corn, soy flour or corn / soy flour mix.

The animal feed additive may be added to the animal feed by immersion, spraying or mixing.

The present invention is applicable to a number of animal diets including mammals, poultry, and fish. More specifically, the diets may be used in commercial mammals, such as pigs, cows, sheep, goats, laboratory animals (e.g., rats, mice, hamsters and gervilus rats), fur- , And zoo animals (e.g., monkeys and tailless monkeys), as well as livestock (eg, cats and dogs). Common commercially important poultry include chicken, turkey, duck, goose, pheasant and quail. Commercial breeding fish such as trout can also be included.

In the animal feed compounding method including the animal feed additive according to the present invention, the animal feed additive is incorporated into the animal feed in an amount of about 1 g to 100 g per 1 kg of the feed on a dry weight basis.

Also, after the feed mixture is thoroughly mixed, lightly viscous granulation or granulation material is obtained depending on the degree of crushing of the components. It is supplied as a mesh or is formed into a desired discrete shape for further processing and packaging. At this time, in order to prevent separation during storage, it is preferable to add water to the animal feed, followed by a conventional pelleting, expansion, or extrusion process. Excess water can be dried off.

As described above, the extract of the present invention exhibits anticancer activity in colon cancer, such as CT-26 cells, in macrophage Raw 264.7 cells, , It is useful as a component of foods, medicines and feeds having excellent anticancer efficacy.

Figure 1 shows the results of the viability test of CT-26 cells in AW 264.7 cells; Each data represents mean (mean ± SD) and significance is considered to be the difference from the non-treatment group (nor) (* p <0.05, ** p <0.01);
FIG. 2 is a chart showing the most reduced arm size and weight in the mixed administration group of 5-FU and MB.
FIG. 3 is a graph showing changes in spleen immunocyte activity levels in a mixed administration group of 5-FU and MB extract. FIG.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

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

Example 1: Preparation of sickle leaf extract

1-1. Manufacture of water extract

Cnidium monieri (L.Cuss ) purchased from the University of Iksan City, Jeollabuk- do , was naturally dried, finely chopped, and 10 kg of distilled water was added to 1 kg of dried cured chilled water and heated at 100 ° C for 3 hours. The filtrate was concentrated under reduced pressure (EYELA, N-1000, Japan) to obtain 126 g (hereinafter referred to as MFW) of the extract of the fresh heart. The filtrate was used as a sample of the following experimental example.

1-2. Preparation of 70% and 100% ethanol extracts

In the same manner as in the above-mentioned 1-1, 50 g of the dried bulkhead was added with 500 ml of 70% ethanol and 100% ethanol, respectively, and the resulting mixture was heated at 100 ° C for 2 hours. This operation was repeated three times, followed by vacuum filtration to obtain a filtrate (Hereinafter, referred to as MF70E) and 7.23 g (hereinafter referred to as MF100E) of a 70% ethanol extract and a 100% ethanol extract of a fresh syrup were respectively concentrated under reduced pressure (EYELA, N-1000, ), Respectively.

Reference Example 1. Cell culture, reagents and reagents

The mouse macrophage cell line and mouse macrophage cell line (ATCC The Global Bioresource Center, USA) used in this experiment were purchased from the respective companies and cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 5% CO ₂ , 37 ° C. Cell culture media and reagents were purchased from the company (Thermo Scientific Hyclone, Waltham, Mass., USA) and DMEM and FBS were purchased from the company (GibcoBRL, Grand Island, NY, USA). Lipopolysaccharide (LPS) and dimethylsulfoxide (DMSO) were purchased from Sigma, St. Louis, MO, USA. Fluoruuracil and 5-FU were purchased from Ildong Pharmaceutical Co., Ltd., Seocho-Gu, Seoul , Korea).

Reference Example 2. Statistics Processing

The results were accepted as significant a p value less than .05 by the black to the average value (Mean ± SD) and statistical significance is shown Student assay (Students t -test) a.

Experimental Example 1. Anticancer Activity through Immune Activity

In order to confirm the anticancer activity of the sample of the above example, the following experiment was conducted by applying the method described in the literature (Jensen et al., 2008)

In order to investigate the anticancer activity of the extract obtained from the present example, 1 × 10 ⁵ cells / ml of mouse macrophages RAW 264.7 cells and CT-26cell of colon cancer cells were cultured at a ratio of 1 × 10 ⁴ cells / ml 10: 1) were cultured together. Then, the extracts were incubated at 10, 30, and 100 μg / ml for 24 hours. Cellular immune responses of the cells were evaluated by MTT assays. (1).

As a result of the experiment, it was confirmed that the samples of the present invention inhibited the growth of the target cancer cells, as shown in the following Fig. 1 and Table 1. [

Group Anticancer activity (%) Control group 70.3 ± 5.32 The positive control (LPS) 0.1 μg / ml 72.8 ± 4.25 1 μg / ml 158.9 ± 1.53 Sickle water extract 10 μg / ml 143.2 ± 26.3 30 μg / ml 165.3 ± 14.9 100 μg / ml 152.3 + - 12.3

Experimental Example  2. Cancer heterogeneity  Anti-cancer experiments in animal models

(Holland EC, 2004; Lee B et al., 2012), in order to confirm the anticancer effect in the animal xenograft model experiment of the sample of the above example.

(22 ± 1 ° C), relative humidity (55 ± 1%) and 12-hour day and night control period (Standardized pellet) under the conditions of anesthesia, and animal protection regulations and standards (institutional, Wonkwang University Animal Care and Use Committee). To establish an allograft colon carcinoma animal model, cells (5 × 10 ⁵ CT-26c ell) in 200 μL PBS were injected into the right lobe of the mouse (BALB / cmice). The tumors were cultured for 7 days until they were visible, and were divided into 7 groups of 9 animals each.

(B) 5-FU treated group (50 mg / kg), (C) 5-FU treated group, and 50-mg subcutaneous extract group treated with 50 mg (50 mg / kg, 5-FU, 300 mg / kg / dose, 5 mg / kg body weight) 5-FU was treated for 5 days after the onset of tumor size, and the sickle leaf extract was treated daily for 14 days. Tumor volume was measured caliper 21 times at daily intervals and calculated according to the following equation (2).

Here, the maximum tumor diameter and width were calculated according to the minimum tumor diameter according to the literature (Alessandria G. et al., 1987.).

At the end of the experiment, the mice were fasted overnight and the animals were sacrificed after ether anesthesia. Tumor tissues were extracted and weighed, and the blood was centrifuged to measure immune modulating substances in the blood.

Group Tumor weight (g) Control group 6.8 ± 0.4 5 FU 50 mg / kg 2.8 ± 0.3 5 FU 50 mg / kg + Sickle water extract 100mg / kg 2.7 ± 0.6 + Heartburn water extract 300mg / kg 2.7 ± 0.5

The results of this study showed that the treatment of tumor cell extracts in CT-26 tumor-xenografted mice was significantly reduced compared to the control or 5FU treatment group. In particular, the mixed treatment group of 5-FU and MB (Fig. 2). Tumor necrosis Factor (TNF-a), which is an immunomodulator, was also significantly increased in the mixed treatment group of 5-FU and SMB extracts compared to the tumor control and 5FU treatment groups. (Fig. 3)

Experimental Example  3. Cancer heterogeneity  Immunocytochemical activity in an animal model experiment

CT-26 tumor-to release the spleen are not sensible by taking the spleen cells from the mouse xenograft the following, 1 x 10 ⁶ spleen cells in 96-well microplates gae Pipette. To evaluate the cytotoxic T cell activity, melanoma cell line B16 melanoma cancer cells were cultured with 1 × 10 ⁴ cells / ml (ratio of effector / target cells 10: 1), followed by measurement of immune cell immune response against cancer cells In order to evaluate the activity of natural killer cells, the cells were incubated with 1 × 10 ⁴ cells / ml of erythroleukemia K562 cells (ratio of effector / target cells: 10: 1) Respectively. The activity of cytotoxic T cells and natural killer cells was calculated as shown in Equation (1).

In addition, the proliferation of mouse spleen cells was measured according to the concentration of Con A through the MTS method (CellTiter96 ™ AQueous Assay) (Wang, M. et al., 2004).

As a result of the above experiment, as shown in FIG. 3A, the extract of Suksikjang increased cell proliferation as compared to 5-FU and increased the activity of cytotoxic T cell and natural killer cell as shown in FIG. 3B and C, respectively. Therefore, it was found that sickle cell extract is involved not only in innate immunity but also immunity of T cell as a central cell in acquired immunity.

Experimental Example  4. Oral dose toxicity test

In this experiment, mice were fasted for 12 hours before ICR mice were administered and ICR mice were weighed at 7 weeks of age. After the body weight was measured, toxicological test standards (Korea Food & Drug Administration, Korea Food and Drug Administration, 2005, 2005 ), And the test substance was diluted with purified distilled water to prepare test substances of 5 mg / kg, 50 mg / kg and 500 mg / kg, respectively. The administration was made by oral administration, which is the main route of application in the clinic. The method of administration was as follows: The animal was fixed by the adriamycin fixation method and then injected directly into the stomach using a metal oral administration unit. 10 ml per kg of body weight was administered once a day on the basis of body weight on the day of feeding. Feed was restricted for 2 hours after administration of the test substance to the test animal, and then feed and water were continuously supplied during the test period.

In accordance with the Toxicological Testing Standards of the Food and Drug Administration and the guidelines of the International Organization for Economic Cooperation and Development (OECD Guidelines for the Testing Chemicals revised draft guideline 420, 2001) (S) of the animal's skin, hair, eye, mucous membrane, respiratory system, circulatory system, autonomic nervous system, central nervous system, physical activity, behavioral form and test animal tremors, convulsions, salivation, diarrhea, The presence of lethargy, sleep, coma, etc. were observed. The general clinical symptoms were observed once a day for 14 days after the administration and the individual body weight of the test animals was measured once a day before and after administration of the test substance and during the test period.

ICR mice were anesthetized with ethyl ether on the 14th day after the injection, and all the organs were removed by bled lethal injection to observe gross lesions of individual test animals and weigh the organs.

As a result of the acute toxicity test by single oral administration of sickle leaf extract, no mortality was observed at the highest dose of 2,000 mg / kg administered in the test animals and conditions used in this study, In all the observed parameters according to the toxicity test criteria, no toxicity was induced by the administration of the extract of the fresh heart. In conclusion, sickle leaf extract was safe without toxicity even at the maximum recommended dose of 2,000 mg / kg of acute toxicity test.

The preparation examples of the pharmaceutical composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.

Formulation example  One. Sanje  Produce

MFW Extract ------------------------------------ 20 mg

Lactose ----------------------------------------- 100 mg

Talc ------------------------------------------ 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

MF70E Extract ---------------------------------- 10 mg

Corn starch ----------------------------------- 100 mg

Lactose ----------------------------------------- 100 mg

Magnesium stearate ---------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

MF100E Extract --------------------------------- 10 mg

Crystalline cellulose - 3 mg

Lactose ------------------------------------ 14.8 mg

Magnesium stearate - 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

MF100E Extract --------------------------------- 10 mg

Mannitol --------------------------------------- 180 mg

Sterile sterilized distilled water used 2974 mg

Na ₂ HPO ₄ .12H ₂ O --------------------------------- 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

MF70E Extract ---------------------------------- 20 mg

Ising Party --------------------------------------- 10 g

Mannitol ------------------------------------------ 5 g

Purified water -----------------------------------------

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

MFW Extract ----------------------------------- 1000 mg

Vitamin mixture -----------------------------------

Vitamin A Acetate ---------------------------- 70 g

Vitamin E -------------------------------------- 1.0 mg

Vitamin B ₁ ------------------------------------ 0.13 mg

Vitamin B ₂ ------------------------------------ 0.15 mg

Vitamin B ₆ ------------------------------------- 0.5 mg

Vitamin B ₁₂ ------------------------------------ 0.2 g

Vitamin C -------------------------------------- 10 mg

Biotin ---------------------------------------- 10 μg

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------ 50 g

Calcium pantothenate -------------------------------- 0.5 mg

Inorganic mixture ----------------------------------------------------------------------------------------------------------------------------------

Ferrous sulfate - 1.75 mg &lt; RTI ID = 0.0 &gt;

Zinc oxide - 0.82 mg

Magnesium carbonate -------------------------------- 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary 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 mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

MF70E Extract ---------------------------------- 100 mg

Vitamin C ---------------------------------------- 15 g

Vitamin E (powder) --------------------------------- 100 g

Lactic Acid Iron --------------------------------------- 19.75 g

Zinc oxide --------------------------------------- 3.5 g

Nicotinic amide ------------------------------------- 3.5 g

Vitamin A --------------------------------------- 0.2 g

Vitamin B ₁ ------------------------------------- 0.25 g

Vitamin B ₂ --------------------------------------- 0.3g

Water ---------------------------------------------- QUANTITY

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (3)

After 5 to 15 times as much water as the dry weight of the dried Morus alba L. was added and heated for 2 to 5 hours at 70 ° C to 100 ° C for 2 to 5 times, the extract was subjected to filtration under reduced pressure Then, the filtrate is concentrated and lyophilized to obtain a supernatant water extract; And
Mixing 5-fuluoruracil (5-FU) and the bulkhead water extract at a weight ratio of 1: 2;
To produce a pharmaceutical composition for the treatment or prevention of cancer diseases having an anticancer activity and cancer tissue growth inhibitory effect on colon cancer containing 5-fluorouracil (5-FU) as an active ingredient, Way. After 5 to 15 times as much water as the dry weight of the dried Morus alba L. was added and heated for 2 to 5 hours at 70 ° C to 100 ° C for 2 to 5 times, the extract was subjected to filtration under reduced pressure Then, the filtrate is concentrated and lyophilized to obtain a supernatant water extract; And
Mixing 5-fuluoruracil (5-FU) and the bulkhead water extract at a weight ratio of 1: 2;
(5-FU) as an active ingredient, and a health functional food for preventing and ameliorating cancer diseases having an inhibitory effect on cancer tissue growth How to. After 5 to 15 times as much water as the dry weight of the dried Morus alba L. was added and heated for 2 to 5 hours at 70 ° C to 100 ° C for 2 to 5 times, the extract was subjected to filtration under reduced pressure Then, the filtrate is concentrated and lyophilized to obtain a supernatant water extract; And
Mixing 5-fuluoruracil (5-FU) and the bulkhead water extract at a weight ratio of 1: 2;
(5-FU) as an active ingredient, and a method for producing an animal feed additive having anticancer activity and cancer tissue growth inhibitory activity against colon cancer, which comprises 5-fluorouracil (5-FU) as an active ingredient.

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