KR101484710B1 - A composition comprising the extract of Cnidium monieri (L) for treating or preventing pathological microbe infection - Google Patents

Abstract

The present invention relates to a composition comprising an extract of bee casualties as an active ingredient. More particularly, the bee casualty extract not only protects against infectious pathogenic microorganisms by improving the immunity of Raw 264.7 cells, And it is useful as a component of foods, medicines and feeds having an immunosuppressive effect against a pathogenic microbial infection.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating and preventing a pathogenic microbial infectious disease containing an extract of bee-killer as an active ingredient,

The present invention provides a composition for the treatment and prevention of pathogenic microbial infectious diseases which contains an extract of bee scabbard 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] Korean Society of Pharmacopoeial Professors, Herbal Medicine, Korean Pharmaceutical Society, pp.889-892, 1995

[Literature 7] Ledur 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.

[8] 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.

[Literature 9] Kim Jong Man et al., Journal of Agricultural Science, 39, pp.1-7, 1997

[Literature 10] Naknukool S, Horinouchi I, Hatta H. Stimulating macrophage activity in mice and humans by oral administration of quillaja saponin.Biosci Biotechnol Biochem. 75 (10): 1889-1893, 2011.

[Literature 11] Ishida-Fujii K, Sato R, Goto S, Yang XP, Kuboki H, Hirano S, Sato M. Prevention of pathogenic Escherichia coli infection in mice and stimulation of macrophage activation in rats by an oral administration of probiotic Lactobacillus casei I-5. Biosci Biotechnol Biochem. 71 (4): 866-873, 2007.

[Literature 12] Korea Food and Drug Administration, Toxicological Testing Standards for Drugs, etc., Korea Food and Drug Administration Notice 2005-60, 2005

[Document 13] OECD Guideline for the Testing Chemicals revised draft guideline 420, Organization for Economic Cooperation and Development (OECD), 2001

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 (secondary line) 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).

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). Medicines derived from medicinal herbs have been focused on the identification of active ingredients for the prevention and treatment of diseases. Although the efficacy of medicinal herbs has been mentioned in past oriental medicine books such as Dongbok-bong, 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 components of the celestial water exerts a direct effect. Cheonryu water, which is known to have an immune enhancing effect in Cheonryo water contained in Dongyoungbok and Chinese Medicine Dictionary, needs to be tested for its immunostimulatory effect 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 casualties is spicy, and the quality is warm and innocuous (Korean Pharmacology Professor Council, Herbal Medicine, Korean Pharmaceutical Association, 1995). In one room, the medication warms the kidneys and strengthens the stamina, which is applied to the menstrual syndrome and the pathology of female 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, disclose or teach any description of efficacy as a therapeutic agent for pathogenic microbial infections through the immune enhancement of bee casualty extract.

Accordingly, the inventors of the present invention confirmed that the extract of bee casualty of the present invention has an effect of protecting the infectious pathogenic microorganism by enhancing the immunity of Raw 264.7 cells while confirming the substance having immunity enhancing effect from the extract of natural products, The present invention has been completed upon confirming that it can be effectively used as a component of foods, medicines and feeds having an immunosuppressive effect against a pathogenic microbial infection.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of pathogenic microbial infectious diseases, which comprises an extract of bee scarlet as an active ingredient.

The present invention also provides a health functional food for the treatment and prevention of pathogenic microbial infectious 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.

Pathogenic microbial infections as defined herein include pathogenic bacteria such as, for example, Escherichia coli, Staphylococci and Pneumococcus; Protozoa such as malaria and amoeba; Yeast such as Crepotococus; Fungi which cause skin diseases such as sporotrichosis and chromomycosis; Intestinal parasites such as hookworm, tapeworm, and roundworm; Or an infectious disease caused by infection with other parasitic infections such as colon amoeba and dysentery, preferably food poisoning, upper or lower infection, or skin infections of bacteria.

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 include C1 to C4 lower alcohols, such as water, methanol, etc., at a volume of 1 to 20 times, preferably 5 to 15 times the dry weight of the dried bee casualties, , 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, , Ultrasonic extraction, and reflux cooling extraction. Preferably, the extract is subjected to hot-water extraction, the extract is filtered under reduced pressure through a filter paper, the filtrate is concentrated and lyophilized, and then the bee scarlet extract of the present invention can be obtained.

The inventors of the present invention found that the bee scarlet extract obtained by the above method exhibits an immunological defense effect against a pathogenic microorganism, a survival protection effect of an animal model inducing an Escherichia coli infection, and an immunological enhancement effect of an animal model inducing an Escherichia coli infection , And it can be used effectively as a component of foods, medicines, and feeds that have an effect on pathogenic microbial infection.

Accordingly, the present invention provides a pharmaceutical composition for the treatment and prevention of pathogenic microbial infectious diseases, which comprises the bee scarlet extract obtained by the above-described 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 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.

The present invention also provides a health functional food for treating and preventing pathogenic microbial infectious diseases, which contains 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.

The present invention also provides an animal feed additive for the treatment and prevention of pathogenic microbial infectious diseases containing the bee casualty extract as an active ingredient and a feed comprising the animal feed additive.

The composition for animal feed additive may be in the form of a 20% to 90% high concentration concentrate or in the form of a 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 mentioned above, the bee casualty extract of the present invention not only showed an effect of protecting the infectious pathogenic microorganisms by improving the immunity of Raw 264.7 cells, but also confirmed the survival rate and the immunity enhancement effect of the animal model of Escherichia coli infection , And is useful as a component of foods, medicines and feedstuffs having immunosuppressive efficacy against an excellent pathogenic microbial infection.

Brief Description of the Drawings Fig. 1 is a graph showing the immunostimulation index NO of extracts of bee scabbard according to extraction solvents in RAW 264.7 cells. Fig.
FIG. 2 is a graph showing the ability of RAW 264.7 cells to colonize E. coli. FIG.
FIG. 3 is a graph showing a survival rate defense effect of an animal model that induced Escherichia coli infection.
FIG. 3 shows the results of an experiment in which the number of Escherichia coli in the blood of an animal model that induced Escherichia coli infection was measured.
FIG. 4 is a graph showing the expression of cytokines {(A) IL-12; (B) IFN-γ}.

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 and cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin at 5% CO ₂ , 37 ° C Lt; / RTI > 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). LPS and DMSO were purchased from Sigma, St. Louis, MO, USA, and fluorescein-5-isothiocyanate FITC-labeled E. coli was purchased from Molecular Probe, Immunosorbent assays (ELISA) kit for IL-12 and IFN-γ were purchased from R & D Systems, Minneapolis, MN, USA.

Escherichia coli (E. coli; Escherichia coli ) were purchased from ATCC The Global Bioresource Center with K18 strain (ATCC 23518). The experimental animals were purchased from Koatech (Pyungtaek, Korea) with 26 male ICR mice at 5 weeks of age.

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  One. Punishment casualty  Immune enhancing activity by extraction solvent

In order to confirm the immunoenhancing activity of the sample of the above example, the following experiment was conducted by applying the method described in the literature (Ledur et al., 1995, Mills 2001)

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.

As a result, as shown in Table 1 and FIG. 1, the compounds of the present invention were found to significantly increase the immunostimulating factor NO in all the extract fractions compared to the normal control, and it was confirmed that CMW immunity enhancing activity was the highest Respectively.

NO (μM) 10 μg / ml 30 μg / ml 100 μg / ml UN 4.50 ± 1.47 LPS 37.7 ± 1.8 CMW 5.68 ± 2.01 10.8 ± 2.26 26.5 ± 1.75 CM70E 3.91 ± 1.11 11.4 ± 2.22 23.7 ± 2.50 CM100E 3.62 ± 1.04 4.71 ± 0.64 17.86 ± 1.98

Experimental Example  2. Punishment casualty  Extract On pathogenic microorganisms  About Immune  Defense effect

In order to confirm the immunosuppressive effect against the pathogenic microorganism of the bee scarlet extract of the above example, the following experiment was conducted using E. coli labeled with fluorescence (FITC) described in the literature (Han et al., 2009).

RAW 264.7 cells were dispensed in a 96-well microplate at a rate of 1 × 10 ⁵ per well, and the extract of the present invention was cultured at 37 ° C. and 5% CO ₂ for 24 hours at a concentration of 3, 10, 30 and 100 μM, (FITC-labeled E. coli, Molecular Probe, Eugene, OR, USA) was incubated at 37 ° C for 2 hours and then treated with 0.25% trypan blue for 1 hour at room temperature. Cells were lysed with lysis buffer . Absorbance was measured at 480-520 nm using an Infinite 200 spectrophotometer (Tecan, Switzerland). Lipopolysaccharide (LPS, 100 ng / ml) was used as a positive control

As a result of the experiment, as shown in Table 2 and FIG. 2, E. coli robustness of RAW 264.7 cells treated with bee scarlet extract was increased by 1.26, 1.37, and 1.46 times as compared with the control group. It was confirmed that the bee casualty extract enhances the pathogenic microorganism pickling ability of macrophages (see FIG. 2B).

Group Coliform bacteria (%) Control group 100.0 ± 18.2 The positive control (LPS) 207.5 ± 33.6 Bee casualty extract 10mg / ml 126.9 ± 18.4 Punishment casualty  Extract 30 mg / ml 137.8 ± 23.2 Bee Casualty Extract 100mg / ml 146.6 ± 24.9

Experimental Example  3. In an animal model that induced E. coli infection Survival rate  Defense effect

In order to confirm the survival rate defense effect of an animal model that induced Escherichia coli infection of the bee casualty extract of the above-mentioned sample, experiments were conducted as follows (Kim Jong Man et al., 1997).

The test was used in three groups: untreated control group, E. coli inoculation positive control group, and E. coli inoculated group. After extracts were dissolved in water, the mice were orally administered to group 2 and group 3 mice at a dose of 300 mg / kg once at an interval of 5 times. After 24 hours, each mouse received 5 × 10 ⁷ (CFU / mouse) ) Concentration in the peritoneal cavity. The number of dead plants was recorded for 3 days after the inoculation, to investigate the pathogenic bacterial defense rate of the bee casualty extract.

As a result, as shown in FIG. 3, the animals of the positive control group (Ⅰ) of E. coli inoculation showed mortality rate of 30% on the first day after the inoculation of E. coli, but after the administration of the bee casualty extract, The inoculation group showed 70% of E. coli inoculation group after 3 days before the onset of all the deaths in the vaccination effect test against pathogenic Escherichia coli, resulting in 2 out of 2 days and 1 day after 1 day of E. coli administration, The survival rate is considered to be a protective effect of bacteria.

Experimental Example  4. Immune enhancement effect of an animal model inducing E. coli infection

In order to confirm the immunological enhancement effect of the animal model that induced Escherichia coli infection of the bee casualty extract of the sample of the above example, an experiment was conducted as described below by applying the method described in the literature (Naknukool et al., 2011; Ishida-Fujii et al., 2007).

The test was used in three groups: untreated control group, E. coli inoculation positive control group, and E. coli inoculated group. The extracts were dissolved in water and administered orally to the mice of groups 2 and 3 once at a dose of 300 mg / kg five times at an interval of 1 day. After 24 hours, intraperitoneal administration of 5 × 10 ⁴ (CFU / mouse) . Four days after the inoculation, the experiment was terminated and the number of bacteria in the blood was measured by sacrificing the experimental animals. IL-12 and IFN-γ, which are immunologically active cytokines in serum, were measured.

As a result, it was confirmed that the number of Escherichia coli in blood was reduced by 5.51 times compared with the group administered only Escherichia coli in the group treated with bee casualty extract (Fig. 4 and Table 3). The cytokine capable of protecting Escherichia coli from the serum was IL-12 (Fig. 5, Tables 4 and 5). These results suggest that the bee casualty extract is effective in protecting pathogenic microorganisms through immunological activity.

Group The number of E. coli in blood (CFU / g of blood) Escherichia coli 5825.0 + - 1441.3 Bee Casualty Extract + Escherichia coli 1057.1 ± 388.60

Group IL -12 ( pg / ml ) Control group 75.2 + - 12.3 Escherichia coli infection group 366.9 ± 20.0 Bee casualty extract + Escherichia coli infection group 689.4 ± 91.2

Group IFN-y (pg / ml) Control group 46.7 ± 13.1 Escherichia coli infection group 205.9 ± 55.9 Bee casualty extract + Escherichia coli infection group 216.8 ± 29.6

Experimental Example  5. Oral toxicity test

In this experiment, mice were fasted for 12 hours before the injection of bee-casualties using 7-week-old ICR mice, and the body weight was measured. The toxicity test standard of the Korea Food and Drug Administration (Korea Food and Drug Administration, ), 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 the bee casualty extract, no deaths were observed at the highest dose of 2,000 mg / kg administered in the test animals and conditions used in this study, Toxicity by administration of the bee casualty extract was not induced in any of the observed indicators according to the Toxicological Test Standard. In conclusion, the bee casualty extract was found to be safe without toxicity 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

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 < RTI ID = 0.0 >

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

Na2HPO412H2O -------------------------------- 26 mg

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

Formulation example  5. Liquid  Produce

CM100E ------------------------------------- 300 mg

Isolation 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 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 g

Nicotinic acid amide 1.7 mg

Folic acid ---------------------------------------- 50 μg

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

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

Ferrous sulfate - 1.75 mg

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 ------------------------------------------- Quantitation

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 (7)

Pharmacy for the treatment and prevention of food poisoning caused by infectious diseases caused by Escherichia coli containing extracts soluble in 60 to 100% ethanol (v / v) of fruit (casualty) of Cnidium monieri (L. Cuss ) Composition. delete delete Health for the prevention and improvement of food poisoning caused by infectious diseases caused by Escherichia coli containing extracts soluble in 60 to 100% ethanol (v / v) of fruit (casualty) of Cnidium monieri (L) Functional foods. delete Animals for the treatment and prevention of food poisoning caused by infectious diseases caused by Escherichia coli containing extracts soluble in 60 to 100% ethanol (v / v) of fruit (casualty), Cnidium monieri (L) Feed additives. A feed comprising the animal feed additive of claim 6.

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