KR101484709B1 - A composition comprising the extract of Cnidium monieri (L) having potent immuo-stimulating activity and treating or preventing cancer disease - Google Patents

Abstract

More particularly, the present invention relates to a composition comprising an extract of Bee Casualties as an active ingredient. More specifically, the bee casualty extract has an effect of increasing the secretory activity and enhancing the phagocytosis of NO, TNF-α, which are immunological enhancers in macrophage Raw 264.7 cells In addition to exhibiting anticancer activity in colon cancer such as CT-26 cells mediated by immune cells, in animal model experiments of cancer xenografts, when co-administered with existing anticancer drugs such as 5-FU, TNF-a, and is useful as a component of foods, medicines and feedstuffs having excellent immunity enhancement and anticancer efficacy.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for treating or preventing an immune-enhancing and cancerous disease, which contains an extract of Bee Casualties as an active ingredient,

The present invention provides a composition for the treatment or prevention of immune enhancement and cancer diseases, which contains an extract of Bee Casualini as an active ingredient.

[1] Kim GY, Choi GS, Lee SH and Park YM. Acidic polysaccharide isolated from Phellinus linteus enhances through 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] 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: 3495-3499, 2012.

[7] 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: 2935-2943 2012.

[8] Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR and Delbanco TL. Unconventional medicine in the United States. Prevalence, costs, and patterns of use. N Engl J Med. 28: 328 (4): 246-252,1993.

[Literature 9] Korean Society of Pharmacopoeial Professors, Herbal Medicine, Korean Pharmaceutical Society, pp.889-892, 1995

Literature A, Fitting C, David B, Hamberger C and Cavaillon JM. Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards. J Immunol Methods. 26; 186 (2): 171-9. 1995.

[Document 11] Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 15: 47 (4): 936-942. 1987.

[12] Azizah MR, Kuak SH, Ainol SS, Rahim MN, Normaznah Y and Norella K. Association of the tumor necrosis factor alpha gene polymorphism with susceptibility and clinical-immunological findings of systemic lupus erythematosus.Asian Pac J Allergy Immunol. 22 (2-3): 159-163. 2004.

[Literature 13] Mills CD. Macrophage arginine metabolism to ornithine / urea or nitric oxide / citrulline: a life or death issue. Crit Rev Immunol. 21 (5): 399-425. 2001.

[14] Cheng AW, Wan FC, Wang JQ, Jin ZY, Xu XM, Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis Fish. International Immunopharmacology 8, 43-50. 2008.

[15] 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, 2006.

[Document 16] Holland EC. Mouse models of human cancer as tools in drug development .. Cancer Cell. 6 (3): 197-198. 2004.

[Literature 17] Lee B, Lee DY, Yoo KH, Baek NI, Park JH, Chung IS. Calenduloside E 6'-methyl ester induces apoptosis in CT-26 mouse colon carcinoma cells and inhibits tumor growth in a CT-26 xenograft animal model. Oncol Lett. 4 (1): 22-28. 2012.

[Literature 18] 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.

The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (first line of defense) is an anatomical physiological element that blocks all invaders, including microorganisms, regardless of their type. Nonspecific immunity (second line of defense) is a phagocyte that breaks through natural resistance and removes intruders. A specific immune system is a highly developed immune system with the ability to distinguish between memory and non-magnetic (Kim et al. Kim et al., 2008; Leiro et al., 2007; Han et al., 2009).

The leukocytes constitute a secondary or tertiary defense line, which breaks the primary line and takes charge of the foreign body. In the case of bacterial, viral infection or inflammatory reaction, the control of macrophage and lymphocyte activity is decided on the therapeutic effect of the drug It plays a pivotal role. Macrophage is a multifunctional cell that plays an important role in the immune system by producing various cytokines and NO in the context of oxidative stress. In particular, iNOS expressed by stimuli such as lipopolysaccharide (LPS), cytokine, and TNF-α in macrophages is known to produce large amounts of NO over a long period of time to promote NFκB activity. Superoxide anion by the activated macrophages (superoxide anion, O2 ^-), hydrogen peroxide _{(hydrogen peroxide, H 2 O 2} ) and active oxygen species such as; Production of (reactive oxygenspecies ROS) and nitrogen monoxide (nitric oxide, NO) is It is an important cytotoxic and cytostatic mechanism in nonspecific immunity. A number of studies have been conducted on which natural compounds affect the production of ROS and NO by macrophages. Macrophages play a central role in cell-mediated or humoral immunity as antigen-presenting, tumoricidal, or microbicidal cells. (Dawson TM, et al., 1996). These results indicate that NO produced by activated macrophages is a nonspecific host defense mechanism, and that it inhibits proliferation of bacteria and cancer cells.

Macrophages contain many lysosomes, which contain acid hydrolytic enzymes and peroxidase. It is also strongly attached to the glass surface and plastic surface and actively digests microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-y. They produce cytokines such as complement components, interferons, interleukin-1 and tumor necrosis factor, and can be enhanced by several cytokines produced from T-cells (Kim et al., 2004; Kim et al. 2008; Leiro et al., 2007; Han et al., 2009).

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- ). (Wettergren et al., 2012), which can lead to side effects such as myelosuppression, gastrointestinal reaction, leukopenia, and thrombocytopenia.

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.

The punishment casualty is the mature fruit of the annual herbivorous casualty ( Cnidium monieri (L). Cuss ) belonging to the Unbelliferae . Bee casualties are close to white, and when carefully observing the surface of the medicinal material, there are three ridges in the medicinal material, which are angular, and have lots of hairs on the skin. The taste of bee casualties is spicy, and the quality is warm and innocuous (Korean Society of Clinical Pharmacology, Herbal Medicine, 1995). It works on new, lung, and nonspecific. In one medicine, this medicine warms the kidneys and strengthens the stamina. It is applied to the pathologies of infertility. It has been used for men's erectile dysfunctions such as poisoning, hyperhidrosis, skin eczema, burning, improvement, etc. It is effective to warm the kidneys, to help the sexual function, to remove the wind, to dry and dry the humid. It is known that it is used for the treatment of men's ankle, scrotum warts, female pussy and pussy pruritus, infertility caused by cold of uterus, None of the above documents, however, discloses or teaches any description of the efficacy of the bee casualty extract as an immune enhancing, antibacterial and anticancer agent.

Therefore, the inventors of the present invention found that the extracts of bee casualties of the present invention showed an increase in the secretory activity of NO, TNF-α, which is an immunological enhancer in macrophage Raw 264.7 cells, In addition to exhibiting anticancer activity in colon cancer such as CT-26 cells mediated by immune cells, in combination with an existing anticancer agent such as 5-FU in an animal xenograft model, And TNF-α, which are necrotic factors, and thus can be effectively used as components of foods, medicines and feeds having excellent immunity enhancement and anticancer efficacy. The present invention has been completed based on this finding.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treatment or prevention of immune enhancement and cancer diseases, which comprises an extract of Bee Casualini as an active ingredient.

The present invention also provides a health functional food for the treatment or prevention of immune enhancement and cancer diseases, which contains an extract of Bee Casualties as an active ingredient.

The bee casualty extract defined in the present application is preferably a solvent selected from the group consisting of water containing 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 water and ethanol mixed solvent, Is an extract which is soluble in water or 60 to 100% ethanol.

In addition, the present invention provides an immunostimulant containing an insect casualty extract as an active ingredient.

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, colon cancer, and most preferably colon cancer.

The present invention also provides a pharmaceutical composition for preventing and treating cancer diseases, which comprises a combination of an insect casualty extract and an existing anticancer therapeutic agent as an active ingredient.

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

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.

The present invention also provides an anticancer adjuvant comprising an extract of Bee Casualini 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 5-fluorouracil (Cytarabine) and the anticancer activity can be maximized. For example, the weight ratio of the conventional anticancer agent and the bee-killer extract is 1: 0.1 to 10 w / ) ≪ / RTI >

Therefore, the present invention relates to antimetabolites such as the above-mentioned bee casualty extract and methotrexate, 6-mercaptopurine, 6-thioguanine, and 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 mediated by an immunological activity as an active ingredient.

The bee casualty extract of the present invention can be produced by the following production process.

Dry bees casualties can be prepared according to conventional extraction methods and can be prepared from C1 to C4 lower alcohols such as water, methanol or the like, having a volume of 1 to 20 times, preferably 5 to 15 times the dry weight of the dried bee casualties, And then the mixture is subjected to cold extraction, hot water extraction, and water extraction by repeating the reaction with a mixed solvent, preferably water, for 0.5 to 10 hours, preferably 2 to 5 hours, at 70 to 100 ° C for 1 to 10 times, preferably 2 to 5 times, The extraction method such as ultrasonic extraction and reflux cooling extraction is preferably performed by hot water extraction, the extract is filtered under reduced pressure through a filter paper, and the filtrate is concentrated and lyophilized to obtain the bee scarlet extract of the present invention.

The present inventors have found that the bee-scarlet extract obtained by the above-described method exhibits an effect of increasing the secretory activity and an increase in the phagocytosis of NO, TNF-α which are immunological enhancers in macrophage Raw 264.7 cells, , As well as in animal model experiments of cancer xenografts, it was confirmed that when co-administered with conventional anticancer drugs such as 5-FU, the growth of tumor tissue growth and the production of TNF-a, , And can be effectively used as a component of foods, medicines and feeds having excellent immunity enhancement and anticancer efficacy.

Accordingly, the present invention provides a pharmaceutical composition for the treatment or prevention of immune enhancement and cancer diseases, which comprises the bee scarlet extract obtained by the above production method as an active ingredient.

Also, the bee casualty is a medicinal substance that has been used for a long time as an edible or herb medicine, and the bee casualty extract of the present invention is also free from 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 administration 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 compound of the present invention may be formulated in an amount of 0.0001 to 50% by weight based on the total weight of the entire 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.

The present invention also provides a health functional food for the treatment or prevention of immune enhancement and cancer diseases, which comprises the bee scarlet extract obtained by the above production method as an 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.

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 an immunostimulating and anticancer efficacy containing the bee casualty extract as an active ingredient and a feed comprising the same.

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 bee casualty extract of the present invention exhibits an effect of increasing the secretory activity and enhancing the phagocytosis of immunological enhancers NO, TNF-a in macrophage Raw 264.7 cells, In addition to exhibiting anticancer activity in cancer cells such as colon cancer, it has been confirmed that in animal xenograft model experiments, co-administration with existing anticancer drugs such as 5-FU increases the inhibition of tumor growth and the production of tumor necrosis factor and TNF-a , And is useful as a component of foods, medicines and feeds having excellent immunity enhancement and anticancer efficacy.

FIG. 1 shows tumor necrosis factor, TNF-alpha (Tumor necrosis Factor) in RAW 264.7 cells according to extraction solvent;
FIG. 2 is a graph showing the immunoenhancing activity of a sample in RAW 264.7 cells. {(A) Cell viability test result; (B) NO production, (C) TNF-α, and (D) phagocytic activity in macrophages;
Figure 3 shows the results of the viability test of CT-26 cells through the immunological activity of the sample in RAW 264.7 cells.
FIG. 4 shows that the mixed administration group of 5-FU and pemocyte extract showed the most reduced cancer size;
FIG. 5 shows changes in blood TNF-alpha (Tumor Necrosis Factor) levels in the tumor control and 5FU treatment groups in the mixed administration group of 5-FU and bee casualty extract.

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  One. Punishment casualty  Preparation of extract

1-1. Punishment casualty  Preparation 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 casualties and heated at 100 ° C for 3 hours. The filtrate was concentrated under reduced pressure (EYELA Co., Ltd., N-1000, Japan) to obtain 126 g (hereinafter referred to as CMW) of bee casualty extract and used as a sample in 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 dried shreds were added to each of 500 ml of 70% ethanol and 100% ethanol, and the resulting mixture was heated at 100 ° C for 2 hours. This procedure was repeated three times, followed by vacuum filtration to obtain a filtrate. (Hereinafter referred to as CM70E) and 7.23 g (hereinafter, referred to as CM100E) of a 70% ethanol extract and a 100% ethanol extract of bee casualty, respectively, were concentrated under reduced pressure (EYELA, Respectively.

Reference Example  1. Cell culture, reagents and reagents

The mouse macrophage cell line (ATCC The Global Bioresource Center, USA) used in this experiment was purchased from DMEM containing 10% FBS and 1% penicillin / streptomycin, and 5% CO ₂ , And cultured under the condition of 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) and the kit (enzyme-linked immunosorbent assay (ELISA) kit for NO, TNF- (Fluoruuracil, 5-FU) was purchased from Ildong Pharmaceutical Co., Ltd., Seocho-Gu, Seoul, Korea and used in R & D Systems, Minneapolis,

Reference Example  2. Statistical 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. Tumor necrosis factor activity by extraction solvent

In order to confirm the immunoenhancing activity of the sample of the above example, experiments were conducted as follows (Ledur et al., 1995)

RAW 264.7 cells were dispensed in a 96-well microplate at a rate of 1 × 10 ⁵ per well, treated with the compound of the present invention at a concentration of 10, 30 and 100 μM, cultured at 37 ° C. in 5% CO ₂ for 24 hours , 50 μl of the cell culture supernatant was subjected to ELISA test for TNF-α (tumor necrosis factor-α) (R & D Systems Quantikine Mouse TNF-α kit). After that, an infinite 200 spectrophotometer The absorbance was measured at 540 nm using a spectrophotometer (Sigma, Switzerland). Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control.

As a result, as shown in Table 1 and FIG. 1, the compounds of the present invention were found to significantly increase the activity of tumor necrosis factor in all extract fractions compared to the normal control, and the activity of CMW tumor necrosis factor was the highest Respectively.

TNF-a (ng / ml) 10 μg / ml 30 μg / ml 100 μg / ml UN 0.03 0.03 LPS 2.26 + - 0.42 CMW 0.59 + - 0.12 1.02 + - 0.46 1.62 + 0.18 CM70E 0.29 + 0.02 0.80 + 0.02 0.93 + - 0.10 CM100E 0.03 0.03 0.45 + 0.07 0.98 + - 0.10

Experimental Example  2. Cells On survival  Effect on

In order to confirm the effect of the sample of the present invention on the cell viability, the following MTT assays were performed (Carmichael et al., 1987)

RAW 264.7 cells were treated with 3, 10, 30 and 100 mM concentrations for 24 hours and cultured. After completion of the incubation, the supernatant was discarded, 200 μl of MTT Assay reagent was added, and the mixture was incubated at 37 ° C. for 3 hours. Then, dmso was added and then the absorbance was measured at 590 nm using an Infinite 200 spectrophotometer (Tecan, Switzerland).

As a result of the present experiment, it was confirmed that the compounds of the present invention did not show significant cytotoxicity as compared with the negative control group (the compound not treated group) as shown in Table 2 and FIG. 2A.

Group Cell viability (%) of immune cells Control group 100.2 + 7.94 The positive control (LPS) 109.0 + - 6.85 Bee casualty extract 3μg / ml 100.1 ± 6.10 Bee casualty extract 10 μg / ml 99.4 ± 5.11 Bee Casualty Extract 30μg / ml 104.7 ± 2.44 Bee casualty extract 100 μg / ml 100.8 ± 5.29

Experimental Example  3. Immune enhancing activity

Experiments were carried out as follows (Ledur et al., 1995; Azizah et al., 2004; Mills 2001) by applying the method described in the literature to confirm the immunostimulatory activity of the above-

RAW 264.7 cells (ATCC, USA) and then by 1 × 10 per well in a 96-well microplate ⁵ frequency divider, and the process of the invention compounds at 10, 30 and a concentration of 100 mM, 37 ℃, in 5% CO ₂ conditions After incubation for 24 hours, the supernatant was taken and 50 μl of the cell culture supernatant was mixed with an equal amount of a grease reagent (Griess reagent, 0.1% (w / v) N- (1-naphthyl) ethylenediamine dihydrochloride + 1% ) sulfanilamide in 5% (v / v) phosphoric acid) was added and mixed at room temperature for 10 minutes. Then, the absorbance was measured at 540 nm using an Infinite 200 spectrophotometer (Tecan, Switzerland). Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control.

In addition, 50 μl of the cell culture supernatant was taken under the same conditions, and ELISA of tumor necrosis factor (TNF-α) was performed (R & D Systems Quantikine Mouse TNF-α kit).

As a result, the concentration of nitrite ion (NO2-) in the treated group of RAW 264.7 cells at 30 and 100 mg / ml of bee venom extract was 2.54 and 5.26 times higher than that of the control group, as shown in Tables 3 to 4 and FIG. 2-B . In the RAW 264.7 cells, tumor necrosis factor (TNF-α) was significantly higher than that of the control group (Fig. 2C). The results are shown in Fig. 2C. Fig. 2C shows the increase of TNF-α and NO secretion in the LPS-treated group.

Group NO  (μM) generation Control group 4.50 ± 1.48 The positive control (LPS) 37.72 + 1.89 Bee casualty extract 10 μg / ml 3.91 ± 1.12 Bee Casualty Extract 30μg / ml 11.43 ± 2.23 Bee casualty extract 100 μg / ml 23.71 + - 2.51

Group TNF-alpha (ng / ml) secretory ability Control group 0.03 0.02 The positive control (LPS) 2.26 ± 0.43 Bee casualty extract 10 μg / ml 0.59 + - 0.12 Bee Casualty Extract 30μg / ml 1.02 + - 0.47 Bee casualty extract 100 μg / ml 1.63 ± 0.19

Experimental Example  4. Immunocyte Pickling  activation

In order to confirm the phagocytosis activity of RAW 264.7 of the above-mentioned sample, the following experiment was carried out by applying the Neutral Red Uptake method described in the literature (Cheng, WX et al., 2008).

RAW 264.7 cells (ATCC, USA) for every 1 × 10 per well in a 96-well microplate ⁵ frequency divider, and 3, 10, 30 and the present invention extract at a concentration of 100 μg / ml in 37 ℃, 5% CO ₂ conditions After culturing for 24 hours, 100 μl of 0.075% Neutral Red was added and incubated at 37 ° C. for 1 hour. 200 μl of a solution containing 0.01% acetic acid and 100% ethanol in an equal volume was treated at room temperature for 2 hours, Absorbance was measured at 570 nm using a photometer (Tecan, Switzerland). Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control

As a result, as shown in Table 5 and FIG. 2D, the spermatogenic activity of RAW 264.7 cells according to the bee casualty extract treatment was increased up to 1.86 times in the 100 μM treatment group compared to the control group. This confirms that the bee casualty extract improves the phagocytosis of macrophages (see FIG. 2D).

Group Pickling  activation (%) Control group 100.0 ± 35.5 The positive control (LPS) 136.0 + - 25.2 Bee casualty extract 10 μg / ml 114.2 ± 49.7 Bee Casualty Extract 30μg / ml 148.3 ± 51.2 Bee casualty extract 100 μg / ml 186.8 ± 68.3

Experimental Example  5. 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 this embodiment, a mouse macrophage to examine the anticancer immune activity of the bee extract casualties RAW264.7cell 1 × 10 ⁵ cells / ml and the colon carcinoma cells of 1x10 CT-26cell ⁴ cells / ml (ratio 10 of effector / target cells : 1) were cultured together. Then, the bee casualty extract was treated for 24 hours at a concentration of 10, 30, and 100 μg / ml, and the cellular immune response of the immune cells to the cancer cells was evaluated through MTT assay. 1.

As shown in FIG. 3 and Table 6, similar to the positive control, lipopolysaccharide (LPS), stimulates macrophages to increase phagocytosis, which inhibits the growth of target cancer cells .

Group Anticancer activity of immune cells (%) Control group 214.4 ± 8.07 The positive control (LPS) 372.4 ± 23.7 Bee casualty extract 10 μg / ml 408.6 ± 20.8 Bee Casualty Extract 30μg / ml 308.8 ± 27.6 Bee casualty extract 100 μg / ml 390.0 占 10.1

Experimental Example  6. 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 flank 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.

Each group was treated as follows: (a) normal (normal; no treatment and no cancer); (b) control (control; saline and cancer induced) (f) 5-FU and pupae extract-treated group (50 mg / kg; dose: 100 mg / kg / dose) (5 mg / kg / dose; 100 mg / kg / dose; bee casualty extract) (g) 5-FU and pupae extract administration group (50 mg / kg; 5-FU, 300 mg / The tumor size was treated for 5 days after the onset and the bee casualty extract was treated daily for 14 days. Tumor volume was measured 7 times at 2-day intervals with a caliper 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 sacrificed overnight and the mice were sacrificed by ether anesthesia, and the blood was centrifuged to measure immune modulating substances in the blood.

The results showed that the treatment of tumor necrosis factor extract in CT-26 tumor-xenografted mice was significantly smaller than that of the control or 5FU treatment group. In particular, the combined administration group of 5-FU and necrotic extracts (Tumor Necrosis Factor) (TNF-a), which is the immunomodulator, was also significantly increased compared to the tumor control and 5FU treatment groups in the mixed treatment group of 5-FU and bee killer extract . (Fig. 5)

The pharmaceutical composition containing the extract or the compound of the present invention will be described by way of example, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

CM70E ----------------------------------------- 300 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

CM100E ---------------------------------------- 300 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

CMW ------------------------------------------ 300 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

CM70E ---------------------------------------- 300 mg

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

Sterile sterile distilled water for injection 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

CM100E --------------------------------------- 300 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

CMW ----------------------------------------- 1000 mg

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

Vitamin A Acetate --------------------------- 70 [mu] g

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

Vitamin B1 ----------------------------------- 0.13 mg

Vitamin B2 - 0.15 mg

Vitamin B6 ------------------------------------ 0.5 mg

Vitamin B12 ----------------------------------- 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 < RTI ID = 0.0 >

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

CM70E --------------------------------------- 300 mg

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

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

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

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

Nicotinic acid amide 3.5 g

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

Vitamin B1 ----------------------------------- 0.25 g

Vitamin B2 ------------------------------------ 0.3 g

Water -------------------------------------------- Quantitative

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 solution was filtered to obtain a sterilized 2-liter container, which was sealed and refrigerated It is used in the production of the health beverage composition of the present 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 (10)

A pharmaceutical composition for immunomodulation comprising an extract which is soluble in 60 to 100% ethanol (w / w) of fruit (casualty) Cnidium monieri (L) . delete delete delete delete delete Cnidium monieri (L). A health functional food for immunity enhancement containing extracts soluble in 60 to 100% ethanol (w / w) of fruit (casualty) as an active ingredient. delete Cnidium monieri (L). An animal feed additive containing an extract of 60% to 100% ethanol (w / w) of fruit (casualty) as an active ingredient. A feed comprising the animal feed additive of claim 9.

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