KR101625887B1 - Antimicrobial composition comprising extract or fraction of Papenfussiella kuromo as active ingredient - Google Patents

Abstract

The present invention relates to an antimicrobial composition, and more particularly, to an antimicrobial composition comprising an extract or fraction of Papenfussiella kuromo , one of the marine plants, as an active ingredient, To an antimicrobial composition excellent in antimicrobial activity and excellent in prevention, improvement or therapeutic effect of an infectious disease caused by a bacterium.

Description

The present invention relates to an antimicrobial composition comprising extracts or fractions of pseudomonas sp.

The present invention relates to an antimicrobial composition comprising, as an active ingredient, an extract or fraction of Papenfussiella kuromo , one of the marine plants.

Staphylococcus aureus is a facultatively anaerobic gram-positive staphylococcus that is highly infectious, and is the most common cause of Staphylococcus infection. Staphylococci form colonies in the skin of healthy people and mucous membranes of the nasal passages and cause very common infections in the human body due to their high mortality. Infections caused by staphylococci can lead to various diseases ranging from skin infections such as acne, impetigo, and wounds to severe life-threatening diseases such as pneumonia, sepsis, and endocarditis.

Among the Staphylococcus aureus, methicillin-resistant Staphylococcus aureus ( MRSA), which is resistant to the penicillin antibiotic methicillin , is known as MRSA, and the MRSA is a β -lactam containing cephalosporin ) System antibiotics and other antibiotics.

MRSA was first reported in the United Kingdom in 1961 and has been reported in various parts of the world. Since then, MRSA has increased by 1-2% in Europe over the past 20 years . It accounts for more than 75% of aureus . In addition, most antibiotics are highly resistant to antibiotics that can be used during treatment, and the rate of post mortem infection is becoming a serious problem.

The main reason for the increased MRSA segregation rate is the use of indiscriminate antibiotics as well as intra-hospital infections. In other words, due to the abuse of existing antibiotics and side effects, the incidence of multidrug-resistant bacteria such as MRSA is increasing, and the infection is directly related to the mortality rate of the patients.

Therefore, it is necessary to develop antibiotics that are selective for MRSA and have a high success rate of treatment.

 Kim C, Milheirico C, Gardete S, Holmes MA, Holden MT, de Lencastre H, Tomasz A. Properties of a novel PBP2A protein homologue from Staphylococcus aureus strain LGA251 and its contribution to the β-lactam resistant phenotype. J Biol Chem. 2012.  Mine Y, Watanabe Y, Sakamoto H, Hatano K, Kuno K, Kamimura T, Tawara S, Matsumoto Y, Matsumoto F, Kuwahara S. Excellent activity of FK037, a novel parenteral broad-spectrum cephalosporin against methicillin-resistant staphylococci. J Antibiot (Tokyo). 1993; 46 (1): 99-119.

In order to solve the problems of the prior art as described above, it is an object of the present invention to provide an antimicrobial composition having an antimicrobial activity by containing an extract or fraction of Papanfussiella kuromo as an active ingredient.

In addition, the present invention has a synergistic antimicrobial activity due to the use of an extract of green beans or fractions thereof and a conventional antibiotic, and thus has excellent antimicrobial activity against Staphylococcus aureus which is highly infectious to skin, can reduce the amount of antibiotic used, And to provide an antimicrobial composition capable of alleviating the expression.

It is another object of the present invention to provide an antimicrobial composition which has a synergistic antimicrobial activity due to the use of a synthetic fungus extract or a fraction and a conventional antibiotic, and which is excellent in the prevention, improvement or therapeutic effect of an infectious disease caused by bacteria.

In order to accomplish the above object, the present invention provides an antimicrobial composition comprising as an active ingredient an extract or fraction of a foxtail extract.

The < RTI ID = 0.0 >

(a) extracting a foxtail extract with a solvent selected from water, an alcohol having 1 to 4 carbon atoms and a mixed solvent thereof to obtain a foxtail extract; And

 (b) adding distilled water to the extract, and successively fractionating an organic solvent, in particular, hexane, methylene chloride, ethyl acetate and butanol, to obtain respective solvent fractions;

.

Particularly, it is preferable that the extract or fractions of the flaxseed extract are hexane fraction of hexane.

It is preferable that the foliar fraction is contained in an amount of 20 to 70% by weight based on the total weight of the composition.

The antimicrobial composition of the present invention may further comprise an antibiotic substance.

The antibiotic may be selected from the group consisting of ampicillin, oxacillin, vancomycin, gentamicin, ciprofloxacin, norfloxacin, enrofloxacin, oxytetracycline oxytetracycline, streptomycin, cephalosporin, colistin, and the like may be used.

The antibiotic may be included in an amount of 20 to 70% by weight based on the total weight of the antimicrobial composition.

The antimicrobial composition may be selected from the group consisting of Staphylococcus aureus , Bacillus cereus , Clostridium perfringens , Escherichia coli , Salmonella enteritidis , ( Campylobacter jejuni ) and the like. In particular, the antimicrobial composition of the present invention has antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) or methicillin sensitive Staphylococcus aureus (MSSA).

The present invention also provides a pharmaceutical composition for preventing or treating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

The antimicrobial composition is preferably contained in an amount of 10 to 40% by weight based on the total weight of the pharmaceutical composition.

The present invention also provides a cosmetic composition for preventing or improving a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an effective ingredient.

The present invention also provides a food composition for preventing or ameliorating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

The present invention also provides an animal feed composition for preventing or ameliorating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

According to the present invention, it is possible to provide an antimicrobial composition excellent in antimicrobial activity by containing an extract or fraction of Papanfussiella kuromo as an active ingredient. In particular, the present invention relates to a method for preventing or ameliorating an infectious disease caused by a bacterium, which is excellent in antimicrobial activity against Staphylococcus aureus having a high infectivity to skin due to its synergistic antimicrobial activity by using a mixture of a green tea hair extract or a fraction with a conventional antibiotic substance, And is suitable for use as a pharmaceutical, cosmetic, food or animal feed composition. In addition, it is possible to provide an antimicrobial composition capable of reducing the amount of antibiotic used and alleviating the expression of antibiotic resistance.

Hereinafter, the present invention will be described in detail.

The antimicrobial composition of the present invention is characterized by comprising an extract of green beans or a fraction thereof as an active ingredient.

The Papenfussiella kuromo extract or fraction can be obtained by extracting, isolating and fractionating the natural extract using a method of extracting, isolating and fractionating, which are well known in the art. The 'extract' as defined in the present invention is extracted from the horseshoe horses using an appropriate solvent and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract of a horse chestnut.

As an appropriate solvent for extracting the extract from the horseshoe hair, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but the present invention is not limited thereto. For example, the solvent may be an alcohol having 1 to 4 carbon atoms such as purified water, methanol, ethanol, propanol, isopropanol, butanol, etc., Can be mixed and used. Particularly, as the solvent, methanol or ethanol (alcohol) is preferably used, and ethanol (alcohol) is particularly preferably used.

The extraction temperature is preferably 50 to 100 ° C. As the extraction method, methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used and more preferred is the use of hot water extraction method or reflux cooling extraction method Do.

In addition, the fractions of the foliar foliage can be obtained by fractionating the extract of the foliar extract, which has been extracted as described above, from the non-polar to the polar organic solvent.

Suitable fractions for fractionation of the foliar fraction include but are not limited to hexane, methylene chloride, ethyl acetate, butanol or mixtures thereof. Particularly, fractions obtained by fractionation with hexane, methylene chloride, ethyl acetate and butanol It is good. Particularly, since the hexane fraction has remarkably excellent antimicrobial activity, especially the antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), as compared with other fractions, the fraction of the chestnut flesh of the present invention is more preferably the hexane fraction Do.

After the extract is obtained using water or an organic solvent as described above, a liquid material can be obtained by freezing, heating and filtering at room temperature by a conventional method known in the art, or a liquid material can be obtained by further evaporating, spray drying or freeze- You may.

In addition, the extracts or fractions extracted and fractionated as described above may be prepared in powder form by an additional process such as vacuum distillation, freeze-drying, or spray drying, . The extract or fraction may be further purified by further chromatography using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like Can be obtained.

Therefore, the extracts and fractions of the artificial fungus used in the present invention include all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, and diluted solutions, concentrates or dried products thereof.

Preferably, the extract is at 20-70 wt%, more preferably at 50-70 wt%, based on the total weight of the antimicrobial composition. If the content is less than 20% by weight, the effect of the antimicrobial composition may be insufficient. If the content exceeds 70% by weight, adverse effects may occur.

The antimicrobial composition of the present invention comprising the above extract or fraction of the fimbrial follicle as an active ingredient may further comprise an antibiotic substance.

The antibiotic refers to conventional antibiotics. Specifically, the antibiotic includes ampicillin, oxacillin, vancomycin, gentamicin, ciprofloxacin, norfloxacin, norfloxacin, enrofloxacin, oxytetracycline, streptomycin, cephalosporin, colistin, and the like can be used. Particularly, in the present invention, oxacillin, norfloxacin, or a mixture thereof is preferably used as the antibiotic.

The antibiotic material is preferably included in an amount of 20 to 70% by weight, more preferably 50 to 70% by weight based on the total weight of the antimicrobial composition. If the content is less than 20% by weight, the effect of the antimicrobial composition may be insufficient. If the content exceeds 70% by weight, adverse effects may occur.

When the green foliar extract or fraction is mixed with an antibiotic, the blending ratio may be appropriately adjusted according to the kind of the antibiotic, and preferably 50 to 100 parts by weight of antibiotic is added to 100 parts by weight of the green foliar extract or the fraction To use. When the blending ratio is included in the above-mentioned range, the antibacterial activity is better.

In addition, the antimicrobial composition of the present invention comprising the extract or fraction and the antibiotic substance of the present invention may further contain an auxiliary component in an amount not affecting the antimicrobial activity of the antifungal extract or fraction and the antimicrobial activity Can be added. The auxiliary component may be sodium benzoate, allantoin, sodium propionate, or the like.

The sodium benzoate has a preservative effect and acts to increase the antimicrobial activity of the composition. The allantoin has a wound healing promoting action for reviving the cells of the wound tissue and acts to synergize with the extract of the hair follicle or the fraction to further promote the repair of the inflammation. In addition, the sodium propionate is an antifungal substance and can impart antifungal activity to the composition. The auxiliary components may be appropriately used within a range that does not affect the antimicrobial activity of the extract or fraction and the antibiotic substance.

The antimicrobial composition of the present invention as described above can be produced by mixing antioxidant extracts or fractions with conventional antibiotics to produce a bacterial, specifically Staphylococcus aureus , Bacillus cereus , When a component having antimicrobial activity or an existing antibiotic substance alone is used for Clostridium perfringens , Escherichia coli , Salmonella enteritidis , Campylobacter jejuni , etc., Lt; RTI ID = 0.0 > antimicrobial < / RTI > activity. In particular, the antimicrobial composition of the present invention exhibits a more excellent antimicrobial activity against Staphylococcus aureus , which is highly infectious to skin, and is useful as a medicillin resistant Staphylococcus aureus (MRSA) or a methicillin-susceptible Staphylococcus aureus (methicillin-sensitive Staphylococcus aureus , MSSA). Therefore, the antimicrobial composition of the present invention can be used for various purposes and applications requiring antimicrobial activity, and specifically for the purpose of treatment, prevention or improvement of bacterial infectious diseases. The bacterial infectious disease may be abscess, dermatitis, osteoarthritis, bacteremia, pneumonia, toxic shock syndrome, food poisoning, high fever, sepsis, acute breast cancer, edema, mastitis, intestinal colicosis and the like.

The present invention also provides a pharmaceutical composition for preventing or treating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

The antimicrobial composition is preferably contained in an amount of 20 to 70% by weight, more preferably 50 to 70% by weight based on the total weight of the pharmaceutical composition. If the content is less than 20% by weight, the effect may be insufficient. If the content is more than 70% by weight, side effects may occur.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, 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 are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The present invention relates to pharmaceutical compositions comprising an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, RTI ID = 0.0 > effective < / RTI > amount. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 0.001 to 100 mg / kg / day, and may be administered once a day, or divided into several doses.

The pharmaceutical compositions of the present invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The present invention also provides a cosmetic composition for preventing or improving a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an effective ingredient.

The antimicrobial composition is preferably contained in an amount of 10 to 40% by weight, more preferably 20 to 30% by weight based on the total weight of the cosmetic composition. If the content is less than 10% by weight, the effect may be insufficient. If the content is more than 40% by weight, side effects may occur.

The cosmetic composition of the present invention may further contain conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and the like which are conventionally used in cosmetic compositions, in addition to the above-mentioned effective ingredients. For example, the cosmetic composition may further contain an auxiliary component such as glycerin, butylene glycol, polyoxyethylene hardened castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, allantoin and the like .

Since the cosmetic composition of the present invention is basically applied to the skin, it can be prepared into any formulation conventionally produced by referring to the cosmetic composition of the related art. For example, they may be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritive cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a mask pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, the carrier component may include an animal oil, vegetable oil, wax, paraffin, starch, tracer, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, have.

When the formulation of the present invention is a powder or a spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like. Particularly in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane, dimethyl ether, and the like.

When the formulation of the present invention is a solution or an emulsion, the carrier component may include a solvent, a solubilizing agent, and an emulsifying agent. Specific examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.

When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol, propylene glycol or the like as a carrier component; Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, trachant, and the like.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Aliphatic alcohols, fatty acid glycerides, fatty acid diethanol amides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and the like.

The present invention also provides a food composition for preventing or ameliorating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

When the antimicrobial composition of the present invention is used as a food additive, the antimicrobial composition may be added as it is, mixed with other food or food ingredients, and used appropriately according to a conventional method.

The antimicrobial composition may be appropriately modified according to the intended use (prevention, health or therapeutic treatment), and the antimicrobial composition may be contained in an amount of 10 to 40% by weight based on the total weight of the food composition. By weight, more preferably from 20 to 30% by weight. If the content is less than 10% by weight, the effect may be insufficient. If the content is more than 40% by weight, side effects may occur.

As a specific example, the antimicrobial composition of the present invention is added in an amount of not more than 40% by weight, preferably not more than 30% by weight based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of foods to which the antimicrobial composition of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, , A drink, an alcoholic beverage, a vitamin complex, and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

The present invention also provides an animal feed composition for preventing or ameliorating a bacterial infectious disease comprising the above-mentioned antimicrobial composition as an active ingredient.

The antimicrobial composition is preferably contained in an amount of 10 to 40 wt%, more preferably 20 to 30 wt%, based on the total weight of the animal feed composition. If the content is less than 10% by weight, the effect may be insufficient. If the content is more than 40% by weight, side effects may occur.

The animal feed composition of the present invention comprising the above-described antimicrobial composition as an active ingredient can prevent or ameliorate an infectious disease caused by pathogenic bacteria in an animal by feeding to livestock, and can reduce methane production.

The animal to which the animal feed composition is administered may preferably be a ruminant, and the species may be a cattle, a camel, a deer, a giraffe, or the like. In addition, in the feed additive composition for livestock of the present invention, the livestock may be preferably a ruminant, which may be, but is not limited to, cattle, camel, deer or giraffe.

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

In addition, the animal feed composition may further contain auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterials, antioxidants, antifungal enzymes, living microbial preparations and the like. Specifically, the auxiliary component may be selected from the group consisting of cereals (for example, ground or crushed wheat, oats, barley, corn, rice, etc.), vegetable protein feeds (for example, (For example, animal fat, vegetable fat, etc.), animal protein feeds (e.g. blood, meats, bone meal, fish meal etc.), sugar and dairy products (e.g., dry ingredients comprising various powdered milk or whey powders) ), Nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents, etc.

The animal feed composition may be administered alone to an animal or may be administered in admixture with other feed additives in an edible carrier. In addition, the animal feed composition may be mixed with the top dressing of the feed, or may be easily administered in combination with a feed or a separate additive or a separate oral formulation. The dosage of the animal feed composition can be suitably adjusted by using a single daily intake or a divided daily intake administered in the art.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example 1. Preparation of extracts and fractions of green beans

After drying in air, 500 g was taken, and the mixture was cooled in 1,500 ml of 70% ethanol for 7 days, concentrated under reduced pressure, and lyophilized to obtain 6.78% of ethanol extract of green beans. The thus obtained ethanol extract of chondrule was dissolved in water, and then the process of separating the hexane layer was repeated. The thus-obtained hexane layer was concentrated under reduced pressure to obtain a powder of a hexane fraction of fumaric acid. Methylene chloride was added to the remaining water layer, followed by fractionation in the same manner as above, and concentration under reduced pressure, followed by lyophilization to obtain powdery methyl cellulose fraction in powder form. Then, ethylene acetate and butanol were sequentially added to the remaining water layer, followed by fractionation in the same manner as above. The filtrate was concentrated under reduced pressure and then lyophilized to obtain powdery ethyl acetate fraction and butanol fraction.

Example 2 Preparation of antimicrobial composition containing extract of green beans or fractions

The chondroitin hexane fraction, methylene chloride fraction, ethylene acetate fraction and butanol fraction isolated in Example 1 were dissolved in dimethylsulfoxide (DMSO) as a solvent at a minimum concentration of 0.97 μg / ml to a maximum concentration of 20 mg / ml And diluted to prepare respective antimicrobial compositions.

Example 3: Preparation of an antimicrobial composition containing oxacillin and extracts or fractions of green beans

Oxalcine (purchased from Sigma-Aldrich) was diluted stepwise in sterile distilled water to a concentration of 0.97 g / ml at the lowest concentration and to a concentration of 20 mg / ml at the highest concentration. An antimicrobial composition was prepared by mixing the antimicrobial composition of Example 3 with a diluted oxacillin solution at a weight ratio of 1: 1.

Example 4. Preparation of antibacterial composition containing northern foxtail extract or fraction and Norfloxacin

Norfloxacin (purchased from Sigma-Aldrich) was diluted stepwise in sterile distilled water to a minimum concentration of 0.97 g / ml to a maximum concentration of 20 mg / ml. An antimicrobial composition was prepared by mixing a diluent of Norfloxacin at a weight ratio of 1: 1 to the antimicrobial composition of Example 3 above.

Experimental Example

The methicillin resistant Staphylococcus aureus (MRSA) ATCC 33591 and the methicillin susceptible Staphylococcus aureus (MSSA) ATCC 25923 standard strains used in the experimental examples were purchased from the Korean Microorganism Conservation Center MRSA strains CCARM 3090, 3091, 3095 and 3102 were purchased from the culture collection of antimicrobial resistance microbes (CCARM). In addition, two strains of S. aureus MRSA (DPS-1 and DPS-2) were obtained from the nasal mucosa of MRSA carriers of plastic surgery in Wonkwang University and used as a clinical strain (Staphylococcal strains from the Department of Plastic Surgery Respectively.

Each of the above strains was incubated at -70 ° C? The cells were stored in deep freeze, incubated in a 37 ° C incubator for 24 hours, and then used in experiments with 0.5 McFarland standard turbidity (1 × 10 ⁸ CFU / ml).

Experimental Example 1. Antimicrobial Susceptibility Measurement

The disc diffusion method is widely used because it can measure the susceptibility of bacteria with rapid growth and is easy to test and has a low cost. Thus, the disk diffusion method was used to compare the antibacterial activity of the foliar fractions produced in Example 2 above.

MSSA ATCC 25923, MRSA ATCC 33591, two clinically isolated MRSA strains (DPS-1 and DPS-2), and MRSA strains were applied to the surface of high pressure sterilized agar using muller-hinton agar as the primary medium. CCARM 3091 were each inoculated in a total volume of 100 μl, and the mixture was uniformly coated with a cotton swab. Then, a sterilized paper disk (6 mm) was placed thereon, and the fraction 20 μl of the antimicrobial composition containing the butanol fraction was absorbed and absorbed and cultured in a 37 ° C. incubator for 24 hours. After the incubation, the diameter of the inhibitory ring formed around the disc was measured, and the experimental results of the fraction of hexane flesh and the fraction of methylene chloride are shown in Table 1 below. At this time, the inhibitory rings vary in size depending on the degree of sensitivity of the bacteria to antibiotics.

As shown in the above Table 1, it was confirmed that the chestnut flesh fraction as an active ingredient of the present invention exhibited excellent antimicrobial susceptibility to all the strains used in the experiment. Particularly, it was found that the sensory properties of the methylene chloride fractions of the flaxseed fraction of hexane fraction were better than those of methylene chloride fraction.

Experimental Example 2. Measurement of minimum inhibitory concentration

To determine the minimum inhibitory concentration (MIC) of the fractionated hexane fractions obtained in Example 1, a liquid medium dilution method was used according to the criteria of CLSI (Clinical and Laboratory Standards Institute).

First, 10 μl of each fraction was added to each well of a 96-well plate at a concentration ranging from 0.97 μg / ml to the maximum concentration of 20 mg / ml at the lowest concentration of DMSO (hexane fraction, oxacillin and norfloxacin) Respectively. Subsequently, MRSA ATCC 33591, two clinically isolated MRSA strains (DPS-1 and DPS-2) and MRSA CCARM 3090, 3091, 3095 and 3102 were diluted to 10 μl per well with a turbidity of 0.5 McFarland 1 × 10 ⁸ CFU / Each well was inoculated to a final concentration of 1.5 x 10 < ⁵ > CFU / spot. Then, when the culture was incubated in a 37 ° C incubator for 24 hours and visually observed, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of the hexane fraction in which the microorganisms did not proliferate. The results are shown in Table 2 Respectively.

As shown in Table 2, it was confirmed that the fractions of the artificial horsetail hexane as an active ingredient of the present invention showed an antimicrobial activity equal to or higher than that of oxacillin and norfloxacin, which are conventional antibiotics, in each experimental strain.

Experimental Example 3. Measurement of synergistic effect of hexane fraction and oxacillin

In order to measure the synergistic effect of the active ingredient of the present invention, the fraction of foliar hexane and the conventional antibiotic substance, a checkerboard dilution method was used. Oxacillin, a betalactam antibiotic, was used as an antibiotic, and the checkerboard experiment was conducted based on the above mentioned minimum inhibitory concentration experiment.

First, in the same manner as in the experiment for the minimum inhibitory concentration in Experimental Example 2, the fractionated pectin hexane fraction (PKH) was sequentially diluted in a horizontal direction at a high concentration and a low concentration in a 96-well plate, Cilin (OX) was injected into each well in the same amount, 10 쨉 l per well, over the flesh hair flesh hexane fraction.

Subsequently, MRSA ATCC 33591, two clinically isolated MRSA strains (DPS-1 and DPS-2) and MRSA CCARM 3090, 3091, 3095 and 3102 were diluted to 10 μl per well with a turbidity of 0.5 McFarland 1 × 10 ⁸ CFU / Each well was inoculated to a final concentration of 1.5 x 10 < ⁵ > CFU / spot. Then, the cells were cultured in a 37 ° C incubator for 18 hours.

The mixing effect was checked using a checkerboard dilution method and evaluated using a fractional inhibitory concentration (FIC) index. The FIC index was calculated by substituting the result of the compound concentration which appeared most efficiently into the following equations (1) and (2). The minimum inhibitory concentration (FICI) was calculated as the fractional inhibitory concentration index (FICI) when combined with hexane fraction and oxacillin. The FIC index was 0.5: synergy, and the FIC index was 0.5 to 0.75 : Partial synergy, FIC index = 0.75 to 1: additive effect, FIC index = 1 to 4: indifference, FIC index = 4 <antagonism And the results are shown in Table 3 below.

[Mathematical Expression]

FIC index (FICI) = FIC _A + FIC _B = [A] / MIC _A + [B] / MIC _B

In the above equation, FIC _A is [A] / MIC _A , FIC _B is [B] / MIC _B , MIC _A and MIC _B are MICs of a single drug A and B, It is the MIC of an A and B drug.

&Quot; (2) &quot;

(FICI) = (minimum inhibitory concentration of A drug / minimum inhibitory concentration of combined A drug) + (minimum inhibitory concentration of B drug / minimum inhibitory concentration of B drug after combination)

As shown in Table 3, ATCC 33591, DPS-1, DPS-2, CCARM 3090 and CCARM among the seven MRSA strains used in the experiment when the hexane fraction of the artificial hair of the present invention was combined with oxacillin, 3091 showed the synergistic effect and partial synergy effect.

Experimental Example 4. Measurement of synergistic effect of hexane fraction and norfloxacin

In order to measure the synergistic effect of the active ingredient of the present invention, hexane fraction of chondrites and norfloxacin (NR), which is a conventional antibiotic, the same procedure as in Experimental Example 3 was carried out, and the results are shown in Table 4 below.

As shown in Table 3, the ATCC 33591, DPS-1, DPS-2, CCARM 3090, CCARM 3091, and CCARM used in the experiment in combination with the active ingredient antifungal hexane fraction and norfloxacin, 3095 and CCARM 3102, respectively, showing synergistic effect and partial synergy effect.

Formulation Example 1. Preparation of pharmaceutical preparations

Acid production

20 mg of each of the antimicrobial compositions of Examples 2 to 4, 100 mg of lactose and 10 mg of talt were mixed and packed in airtight bags to prepare respective powders by a conventional method.

Tablet manufacture

Each of the tablets was prepared by mixing 10 mg of each of the antimicrobial compositions of Examples 2 to 4, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate and then tableting them by a conventional method.

Capsule preparation

10 mg of each of the antimicrobial compositions of Examples 2 to 4, 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate were mixed and filled in gelatin capsules by a conventional method to prepare respective capsules.

Injection manufacturing

Each injection (2 mL) per ampoule was prepared by the usual method using 10 mg of each of the antimicrobial compositions of Examples 2 to 4, 180 mg of mannitol, 2,974 mg of sterile distilled water for injection, and 26 mg of Na ₂ HPO ₄ .2H ₂ O .

Liquid preparation

To each of the purified water was added 20 mg of each of the antimicrobial compositions of Examples 2 to 4, 10 g of isomerized sugar and 5 g of mannitol and dissolved, and the lemon flavor was added in an appropriate amount. Then, purified water was further added thereto, adjusted to a total volume of 100 mL, filled in a brown bottle, sterilized, and each liquid preparation was prepared by a conventional method.

Formulation Example 2. Preparation of cosmetic preparation

Flexible longevity manufacturing

0.1% by weight of each of the antibacterial compositions of Examples 2 to 4, 5.2% by weight of 1,3-butylene glycol, 1.5% by weight of oleyl alcohol, 3.2% by weight of ethanol, 3.2% by weight of polysorbate 20, By weight, 1.0% by weight of a carboxyl vinyl polymer, 3.5% by weight of glycerin, a small amount of fragrance, a small amount of preservative and a remaining amount of purified water were mixed.

Milk lotion manufacturing

The antimicrobial compositions of Examples 2 to 4, 0.1 wt% of glycerin, 4.2 wt% of propylene glycol, 3.0 wt% of tocopheryl acetate, 4.6 wt% of liquid paraffin, 1.0 wt% of triethanolamine, 3.1 wt% of squalane, A mixture of 2.5% by weight of oil, 1.6% by weight of polysorbate 60, 1.6% by weight of sorbitan sesquioleate, 0.6% by weight of propylparaben, 1.5% by weight of carboxyl vinyl polymer, a small amount of fragrance, a small amount of preservative, Each milk lotion was prepared in a conventional manner.

Nutrition cream manufacturing

Antimicrobial compositions of Examples 2 to 4, 0.5% by weight of glycerin, 3.5% by weight of petroleum jelly, 2.1% by weight of triethanolamine, 5.3% by weight of liquid paraffin, 3.0% by weight squalane, 2.6% by weight of beeswax, 5.4% by weight of tocopheryl acetate , 3.2% by weight of polysorbate 60, 1.0% by weight of carboxyl vinyl polymer, 3.1% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative and a balance of purified water, .

Massage cream manufacturing

Antimicrobial compositions of Examples 2 to 4, antimicrobial compositions each containing 0.5% by weight of glycerin, 3.5% by weight of petroleum jelly, 3.5% by weight of triethanolamine, 24.0% by weight of liquid paraffin, 3.0% by weight squalane, 2.1% by weight wax, 0.1% by weight of tocopheryl acetate , 2.4% by weight of polysorbate 60, 1.0% by weight of a carboxyl vinyl polymer, 2.3% by weight of sorbitan sesquioleate, a small amount of fragrance, a small amount of preservative and a remaining amount of purified water were mixed, .

Formulation Example 3. Preparation of food preparation

Health food manufacturing

100 mg of the antimicrobial composition of each of Examples 2 to 4, a proper amount of vitamin A, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 g of biotin, 1.7 mg of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic 90 mg of potassium citrate, 100 mg of calcium carbonate and 24.8 mg of magnesium chloride were mixed and granules were prepared and each health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

Health drink manufacturing

100 mg of each of the antimicrobial compositions of Examples 2 to 4, 15 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A , 0.25 g of vitamin B1, 0.3 g of vitamin B2 and a predetermined amount of water were mixed and heated at 85 DEG C for about 1 hour with stirring. The resulting solution was filtered, sterilized in a sterilized 2 L container, Of healthy beverages were prepared. At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

Formulation Example 4. Production of animal feed

Feed additive manufacturing

100 g each of the antimicrobial compositions of Examples 2 to 4 and an appropriate amount of excipients were mixed and prepared according to a conventional method for preparing feed additives.

Feed manufacturing

The antimicrobial composition of each of Examples 2 to 4, 50 g of mushroom medium, 200 g of wheat bran, 30 g of beet pulp, 220 g of rice beer mug, 200 g of corn flake, 40 g of battery soybean, 100 g of starch powder, 200 g of corn silage, , Ryegrass (323 g), Geolite (14 g), and Tapioca (40 g) were mixed and prepared according to a conventional feed production method.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (16)

An antimicrobial composition comprising, as an active ingredient, an extract or fraction of Papenfussiella kuromo . The method according to claim 1,
Wherein the extract is obtained by extracting the artificial hair with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof. The method according to claim 1,
Wherein the seaweed flesh fraction is obtained by adding distilled water to a seaweed extract obtained by extracting seaweed follicles with at least one solvent selected from water, an alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, and sequentially fractionating an organic solvent Antimicrobial composition. The method of claim 3,
Wherein the organic solvent is at least one selected from the group consisting of hexane, methylene chloride, ethyl acetate and butanol. The method according to claim 1,
Wherein the extract or fractions of the flaxseed extract is a flaxseed fraction of hexane. The method according to claim 1,
Wherein the foliar fraction is contained in an amount of 20 to 70% by weight based on the total weight of the composition. The method according to claim 1,
Wherein the antimicrobial composition is selected from the group consisting of ampicillin, oxacillin, vancomycin, gentamicin, ciprofloxacin, norfloxacin, enrofloxacin, oxytetracycline wherein the antibiotic composition further comprises at least one antibiotic selected from the group consisting of oxytetracycline, streptomycin, cephalosporin, and colistin. delete 8. The method of claim 7,
Wherein the antibiotic substance is contained in an amount of 20 to 70% by weight based on the total weight of the antimicrobial composition. The method according to claim 1,
The antimicrobial composition may be selected from the group consisting of Staphylococcus aureus , Bacillus cereus , Clostridium perfringens , Escherichia coli , Salmonella enteritidis and Campylobacter jeju ( Campylobacter jejuni ). &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method according to claim 1,
Wherein the antimicrobial composition has antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA). A pharmaceutical composition for preventing or treating a bacterial infectious disease comprising the antimicrobial composition of claim 1 as an active ingredient. 13. The method of claim 12,
Wherein the bacterial infectious disease is any one or more selected from the group consisting of abscesses, dermatitis, osteoarthritis, bacteremia, pneumonia, toxic shock syndrome, food poisoning, high fever, sepsis, acute breast cancer, edema, mastitis and intestinal colicosis . A cosmetic composition for improving bacterial infectious diseases comprising the antimicrobial composition according to claim 1 as an active ingredient. A food composition for improving bacterial infectious diseases, comprising the antimicrobial composition of claim 1 as an active ingredient. An animal feed composition for improving a bacterial infectious disease comprising the antimicrobial composition of claim 1 as an active ingredient.