JPH11276184A - Antimicrobially active substance and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject a new antimicrobally active substance produced by Acinetobacter calcoaceticus adhered to sponge algae and useful as a medicine for humans, animals or fishes, and as agrochemical, food preservative or the like. SOLUTION: This new substance is obtained by the following procedure: sponge algae (Scientific name: Ceratodictyon spongiosum Zanardini) is washed with seawater and a phosphate buffer solution followed by addition of a phosphate buffer solution and then crushing the sponge algae; the resulting mixed liquid is centrifuged, the Acinetobacter calcoaceticus strain is isolated from the resulting supernatant and then cultured; the resulting supernatant afforded by centrifuging the culture solution is subjected to extraction with ethyl acetate, and the resulting extract is purified by column chromatography to obtain the objective new substance as high-purity extract which has high activity to suppress growth of Gram-positive bacteria (e.g. staphylococcus, streptococcus), also having an activity against Gram-negative bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel antibacterial active substance, particularly to an antibacterial active substance obtained by separating from a marine microorganism and a method for producing the same.

[0002]

2. Description of the Related Art In recent years, with advances in medicine and the development of the environment, infectious diseases have been controlled and infectious diseases can be controlled in Japan and developed countries. Therefore, the disease caused by the conventional pathogenic microorganisms has been considerably overcome by applying antibiotics and antibacterial agents. However, the evolution of pathogenic microorganisms is unexpectedly rapid, and there are cases where unexpected infectious diseases pose a great social threat. This is due to changes in the causative bacteria and the emergence of resistant bacteria, fulminant streptococci,
Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, enterohemorrhagic Escherichia coli O157: H7, and the like have become major problems in Japan. Among these pathogens, there are also indigenous human bacteria, and patients and medical workers who are carriers are often present in hospitals, making infection prevention measures difficult. In addition, cases in which resistant bacteria are the causative bacteria are often intractable, which is a clinical problem. In addition, antibacterial agents and disinfectants generally have a problem of resistant bacteria caused by using the same agent for a long period of time. Therefore, providing different kinds of antibacterial agents and bactericides having high antibacterial activity and bactericidal activity is significant for the treatment of infectious diseases for humans or animals, and there is a strong demand for the development of new antibacterial substances.

At present, extraction and screening of substances produced by animals, plants and microorganisms inhabiting on land and in water to find useful activity as pharmaceutical preparations are being vigorously performed. In particular, in recent years, the target has shifted from land to freshwater systems and the ocean, and the activities of substances extracted from fish and shellfishes and algae that inhabit there, the structural analysis, and the search for total synthetic routes have been performed. So far, cytotoxic pigment compounds have been extracted from sponge algae, and their antitumor and antibacterial activities have been reported (JP-A-1-
No. 500993, Tetrahedron 44, 1727-1734 (198
8), J. Natural Products 58, 1596-1599 (1995)). These substances are compounds obtained by subjecting sponge algae to an organic extraction solvent.

[0004] In addition, several species of substances produced by strains of the genus Acinetobacter sp., Which are microorganisms that adhere to and inhabit the surface of sponge alga, have been extracted,
As for the pharmacological activity, only an antitumor effect has been reported (JP-A-54-20196), and no antibacterial activity has been reported yet. Further, the substances described in the publication have no antibacterial activity.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an antibacterial and bactericidal activity which is different in origin from existing natural extracts used as antibacterial agents or bactericides for humans or animals. To provide a very high natural extract.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have conducted extensive extraction and screening of seaweeds inhabiting the seas near Japan and the waters of Micronesia, and as a result, Acinetobacter calcoaceticus ( Acineto
It has been found that a substance produced by B. bacter calcoaceticus has an extremely excellent inhibitory action on the growth of Gram-positive bacteria (Staphylococci) and Gram-negative bacteria, and thus completed the present invention.

That is, the present invention provides an Acinetobacter
Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), an antibacterial active substance produced by Acinetobacter calcoaceticus, is cultured.
Acinetobacter calcoaceticus, an antibacterial active substance obtained by collecting from the culture
a method for producing an antibacterial active substance, comprising culturing netobacter calcoaceticus) and collecting from the culture supernatant.
An antibacterial agent and a bactericide containing the antibacterial active substance, or an antibacterial composition and a bactericide composition containing the antibacterial active substance and one or more pharmaceutically acceptable carriers.

Hereinafter, the present invention will be described in detail. Bacteria belonging to the genus Acinetobacter are Neisseriac (Neisseriac)
eae) is a Gram-negative aerobic bacterium. However, Acinetobacter is exceptionally an oxidase-negative bacterium among Neisseriaceae. Among the species of the genus Acinetobacter, Acinetobacter calcoaceticus (Acinetoba
cter calcoaceticus), which lacks spores and flagella and has a rod-like shape when cultured. It is a fungus that originally exists in water and soil, but is a typical opportunistic pathogen and known as a causative organism for hospital-acquired infection. This Acinetobacter calcoaceticus (Acinetobacter calcoaceticus)
Can be easily obtained. For example, culture and isolation can be performed by developing a supernatant obtained by centrifuging fresh water, seawater, soil, or the like on an agar medium.

The antibacterial active substance of the present invention can be obtained by isolating and cultivating Acinetobacter attached and inhabiting sponge algae, and extracting a substance produced by the microorganism.
Sponge algae (scientific name: Ceratodictyon spongiosum Zanardini)
Means southern Japan, Southwest Islands, Malay Archipelago, Polynesia,
Distributed in Australia, the Indian Ocean, and the Atlantic Ocean, its body is spongy, diverging irregularly, with only a fruit-tip tip protruding hairy, and inhabiting intertidal rocks. The antibacterial active substance of the present invention is a known substance extracted from sponge algae (Japanese Patent Laid-Open No. 1-5).
00993, Tetrahenron, 44, 1727-1734, (1988), J. Na
Tural Products 58, 1596-1599 (1955)) are completely different substances, differing in the extraction method and the habitat of the sponge itself. In addition, Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) inhabiting soil and water
Is a completely new marine microbial product having a different microbial habitat than that of a known substance (Japanese Patent Application Laid-Open No. 54-20196).

The method for separating the antibacterial active substance of the present invention is not particularly limited as long as it is a method usually used for extracting a substance from seaweed. A preferred method in the present invention will be described.

(Extraction method) (1) An extract obtained from a sponge algae by first washing the sponge with artificial seawater and a phosphate buffer, further adding a phosphate buffer, and crushing the sponge. It also releases microorganisms attached to sponge algae. By the way, conventionally, when trying to obtain an extract from seaweed, the collected seaweed is firstly used for lower alcohols such as methanol,
Alternatively, the method is generally applied to other organic solvents, and most of the extracts are alkaloid compounds. (2) The obtained mixture of the phosphate buffer and the crushed sponge is centrifuged, and the Acinetobacter calcoaceticus strain is isolated from the supernatant.

(3) The obtained Acinetobacter calcoaceticus strain was cultured in a liquid medium for 4 days, and the supernatant obtained by centrifugation was extracted with ethyl acetate, and the obtained extract was subjected to column chromatography. Purify by chromatography. The column chromatography is preferably a silica gel column chromatography. In particular, a high purity extract can be obtained by performing the silica gel column chromatography shown in FIG. 1 in two steps.

The solvent eluted in the first step is not particularly limited as long as it is a water-immiscible organic solvent such as hexane, ethyl acetate, ether, chloroform, diethyl ether, dichloromethane, benzene, etc., and particularly preferably, n-hexane , Diethyl ether and ethyl acetate (2: 3: 5). The active fraction having the most significant antibacterial action (PEAK I in FIG. 1) among the eluted samples is subjected to the second stage silica gel column chromatography. The elution solvent at this time is also not particularly limited, but it is preferable to use a mixture of benzene and chloroform (1:10), and to obtain an active fraction (PEAK A in FIG. 1) containing an extract without impurities. be able to. Also,
In order to obtain a highly pure extract from the active fraction, after the above operation, the active fraction is further purified by silica gel thin layer chromatography and / or high performance liquid chromatography to obtain a completely purified extract. Can be.

The fractions containing the extract (antibacterial active substance) of the present invention can be pooled and evaporated to dryness, and the residue can be crystallized with methanol, ethyl acetate, acetone, acetonitrile or the like. The extract obtained by this method is about 10 mg to 100 mg per 10 L of the culture solution. The extract thus obtained has an extremely strong activity for inhibiting the growth of gram-positive bacteria such as staphylococci and streptococci, and also has an antibacterial activity for gram-negative bacteria. As a preservative for agricultural chemicals or foods. Less than,
This extract is called the antibacterial active substance of the present invention.

When the antibacterial active substance of the present invention is used as a medicament for humans, the dosage depends on the purpose of administration (treatment or prevention), the severity of the symptoms, sex, weight, age,
Although it varies depending on the kind of the pathogenic bacterium infected, etc., it is generally 50 mg to 1 g, particularly 100 mg to 3 g per adult per day.
It is preferably in the range of 00 mg. In addition, when the antibacterial active substance of the present invention is used as a pharmaceutical for animals, the dosage varies depending on the purpose of administration, the type and size of the animal to be administered, the degree of the infected pathogen, and the like. It is preferable that the dose be in the range of 1 mg to 200 mg / kg of body weight, especially 5 mg to 100 mg / kg of body weight per day. In addition, in the case of a medicine for humans and a medicine for animals, these daily doses are once a day or 2 times a day.
It is preferable to administer in 4 to 4 divided doses. Further, the daily dose may exceed the above-mentioned amount if necessary. The antibacterial active substance of the present invention has activity against a wide range of microorganisms that cause various infectious diseases, and is used for treating, preventing, or reducing symptoms caused by these pathogens. Can be.

The bacteria or bacteria-like microorganisms for which the antibacterial active substance of the present invention is effective include Staphylococcus, Streptococcus, Enterococcus, Peptostreptococcus, Neisseria, Escherichia, Salmonella, Vibrio, Examples include Citrobacter, Shigella, Klebsiella, Enterobacter, Serratia, Brotheus, Pseudomonaz, Haemophilus influenzae, Acinetobacter, Helicobacter, Campylobacter, Trachomach chlamydia, and the like.

Diseases caused by these pathogens include folliculitis, cough, rash, erysipelas, cellulitis, lymphangitis, glanders, subcutaneous abscess, sweat glanditis, acne conglomerate, infectious nodules, perianal area. Abscess, mastitis, superficial secondary infections such as trauma / burn / surgical wound, pharyngolaryngitis, acute / chronic bronchitis, tonsillitis, bronchiectasis, diffuse panbronchiolitis, chronic respiratory disease Secondary infection, pneumonia, pyelonephritis, cystitis, prostatitis, epididymitis, gonococcal urethritis, nongonococcal urethritis, cholecystitis, cholangitis, bacterial dysentery, enteritis, uterine appendixitis, intrauterine infection, Bartholin's glanditis, blepharitis, stye, lacrimal cystitis, blepharitis, corneal ulcer, otitis media, sinusitis, periodontitis, pericoronitis, jaw inflammation, endocarditis, sepsis, meningitis, Skin infections and the like can be exemplified.

Further, the antibacterial active substance of the present invention includes various microorganisms that cause infectious diseases of animals, for example, Escherichia, Salmonella, Pasteurella, Haemophilus, Bordetella, Bacillus, Staphylococcus, Mycoplasma. It is effective for genera. Specific disease names caused by these pathogens include, in birds, colibacillosis, chick dysentery, chicken paratyphoidosis, poultry cholera, infectious coryza, staphylococci, mycoplasmosis, etc., in pigs, colibacillosis, salmonellosis, Pasteurellosis, hemophilus infection, atrophic rhinitis, exudative epidermitis, mycoplasma infection, etc.E. coli in cattle, salmonellosis, hemorrhagic sepsis, mycoplasma infection, bovine lung disease, anthrax, mastitis, etc., E. coli in dogs Examples include sexual sepsis, salmonella infection, hemorrhagic sepsis, pyometra, cystitis, etc., and in cats, exudative pleurisy, cystitis, chronic rhinitis, hemophilus infection, kitten diarrhea, mycoplasma infection and the like.

The antibacterial active substance of the present invention can be used as a pharmaceutical preparation by blending with one or more pharmaceutically acceptable carriers, and can be in any known form. Examples of pharmaceutically acceptable carriers include fillers, bulking agents, binders, humectants, disintegrants, surfactants, lubricants and the like. Representative examples of pharmaceutical preparation forms include tablets, pills, suppositories, injections, pastes, ointments, creams, gels, gel creams,
Lotion, poultice, plaster, liniment, liquid,
Aerosols, powders, granules, capsules, syrups, oral preparations, eye drops, nasal drops and the like can be mentioned. Usually, they can be stably administered systemically or locally, orally or parenterally. .

Hereinafter, preparation examples of typical pharmaceutical preparations will be described. (Example of tablet formulation) A tablet can be prepared by a known method using a known carrier. Such carriers include lactose, sucrose, sodium chloride, glucose, urea, excipients such as starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution. , Gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, lauryl Disintegrants such as sodium sulfate, stearic acid monoglyceride, starch, lactose, sucrose, stearin,
Disintegration inhibitors such as cocoa butter and hydrogenated oil, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin, pentonite, colloidal silicic acid, etc. Lubricants such as adsorbents, purified talc, stearates, boric acid powder, and polyethylene glycol can be exemplified. Furthermore, tablets may be coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated enteric coated tablets,
It can be a film-coated tablet, a double tablet, or a multilayer tablet.

(Pills) Pills are prepared by using known carriers.
It can be prepared by a known method. Such carriers include excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, kaolin, talc, binders such as gum arabic, powdered tragacanth, gelatin, and ethanol;
Disintegrants such as laminaran agar can be exemplified.

(Suppositories) Suppositories can be prepared by known methods using known suppository bases. Such suppository bases include lipophilic bases, water-soluble bases, emulsion bases and the like, and can be appropriately selected and used.
Examples of such suppository bases include cocoa butter, hydrogenated peanut oil, hydrogenated coconut oil, polyethylene glycols, monolen, turure, and pluronic. Suppositories may contain, for example, 0.01 to 10% by weight of the antibacterial active substance of the present invention and a UV absorber 0.1% in such a base.
0.01 to 5% by weight, and if necessary 0.01% of an antioxidant
~ 5% by weight can be prepared. In addition, local anesthetics, antihistamines, local astringents, sulfa drugs, antibiotics, wound treatments, surfactants,
Vitamins, herbal extracts, bile acids, preservatives, excipients,
Additives such as absorption promoters and amino acids can be added as necessary.

(Injection) An injection can be prepared by a known method using a known diluent or the like. As an injection, a sterile solution, emulsion, or suspension is used, and is preferably made isotonic with blood. Examples of the diluent used in the form of solutions, emulsions and suspensions include water, aqueous lactic acid, ethyl alcohol, propylene glycol, polyoxyethylene sorbitan fatty acid esters and the like, and other additives. In addition, salt, glucose or glycerin, a solubilizer, a buffer, a soothing agent, etc., as well as a coloring agent, a preservative, a flavor, a flavor, a sweetener, etc. in an amount sufficient to prepare an isotonic solution. Can be used.

(Ointment) An ointment can be prepared by a known method using a known ointment base. Examples of ointment bases include higher fatty acids or esters thereof (eg, adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipic ester, myristic ester, palmitic ester, diethyl sebacate, lauric acid) Hexyl acid, cetyl isooctanoate, etc.), waxes (eg, spermaceti, beeswax, ceresin, etc.),
Surfactants (eg, polyoxyethylene alkyl ether phosphate), higher alcohols (eg, cetanol, stearyl alcohol, cetostearyl alcohol, etc.), silicone oils (eg, dimethyl polysiloxane, methyl phenyl polysiloxane, glycol methyl poly) Siloxane, silicone glycol copolymer, etc.), hydrocarbons (eg, hydrophilic petrolatum, white petrolatum, purified lanolin, liquid paraffin, etc.), water, absorption enhancers (eg, propylene carbonate, diisopropyl adipate, crotamiton,
Examples thereof include azone, pyrothiodecane, etc.), humectants (eg, glycerin, propylene glycol, butylene glycol, sorbitol, etc.), anti-rash agents and other additives, and are appropriately selected from these and used.

A specific example of the preparation of an ointment is as follows: 5 to 15% by weight of a higher fatty acid ester, 1 to 10% by weight of a surfactant and 0.01 to 10% by weight of the antibacterial active substance of the present invention at room temperature Mix under heating, add 4 to 10% by weight of waxes and 50 to 90% by weight of hydrocarbon, and heat or heat and melt,
Keep at 50-100 ° C. After all the components become a transparent solution, the mixture is uniformly mixed with a homomixer, and then the temperature is lowered to room temperature with stirring to obtain the ointment of the present invention. In addition, this manufacturing example is only an example,
It goes without saying that it can be produced by known methods and formulations similar thereto. In addition, the order of compounding each compound is not particularly limited. Hereinafter, formulation examples of various formulations,
Production examples will be described, and the same applies to them.

(Gel) The gel can be prepared by a known method using a known gel base. Examples of gel bases include lower alcohols (eg, ethanol, isopropyl alcohol, etc.), water, gelling agents (eg, carboxyvinyl polymer, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, propylene glycol alginate). Esters), neutralizing agents (eg, triethanolamine, diisopropanolamine, sodium hydroxide, etc.), surfactants (eg, sorbitan sesquioleate, sorbitan trioleate, sorbitan monooleate, sorbitan monostearate, Sorbitan monolaurate, polyethylene glycol monostearate, polyoxyethylene nonylphenyl ether, polyoxyethylene cetyl ether, polyoxyethylene lauryl ether Ether, etc.), absorption promoters (e.g., propylene carbonate, diethyl sebacate, diisopropyl adipate,
Crotamiton, azone, propylene glycol, pyrothiodecane, etc.), rash preventives, and other additives.

Specific examples of the production of gels are shown in (A)
0.5 to 5% by weight of a gelling agent is added to 55% by weight or less of water to swell, while (B) 0.01 to 50% by weight of the antibacterial active substance of the invention
10% by weight is dissolved or suspended in a dissolving agent.
The gel agent of the present invention can be obtained by dissolving in a mixture of not more than 5% by weight, then adding (B) to (A), adding a neutralizing agent, and adjusting the pH to 4 to 7. .

(Cream) A cream can be prepared by a known method using a known cream base. Examples of cream base include higher fatty acid esters (eg, myristic acid ester, palmitic acid ester, diethyl sebacate, hexyl laurate, cetyl isooctanoate), lower alcohols (eg, ethanol, isopropanol, etc.), hydrocarbons (Eg, liquid paraffin, squalane, etc.), polyhydric alcohols (eg, propylene glycol, 1,3-butylene glycol, etc.), higher alcohols (eg, 2-hexyldecanol, cetanol, 2-octyldodecanol, etc.), emulsifiers ( Examples: polyoxyethylene alkyl ethers, fatty acid esters, polyethylene glycol fatty acid esters, etc.), preservatives (eg, paraoxybenzoate), absorption promoters (eg, propylene carbonate, diethyl sebacate, diisopropyl adipate, crotamiton) Azone, pirotiodecane, etc.), an irritation inhibitor, and other additives and the like, appropriately selected and used from among these.

(Gel-like cream) A gel-like cream having properties intermediate between a cream and a gel is obtained by adding a gelling agent (eg, carboxyvinyl polymer, hydroxyethylcellulose, hydroxypropylcellulose, ethylcellulose) to the above cream. , Carboxymethylcellulose, etc.) and a neutralizing agent (eg, diisopropanolamine, triethanolamine, sodium hydroxide, etc.).
It can be obtained by adjusting the H value to 4 to 9, preferably 5 to 7.

Specific examples of the preparation of the gel cream are as follows: (A) 0.01 to 10% by weight of the antibacterial active substance of the present invention.
In a mixture of 25% by weight or less of higher fatty acid ester and 40% by weight or less of lower alcohol,
% By weight and 5% by weight or less of emulsifier. on the other hand,
(B) 0.5-5% by weight of a gelling agent is added to water to swell, then (B) is added to (A) and homogenized uniformly with a homomixer. By adjusting the value to 4 to 9, the gel cream of the present invention can be obtained.

(Poultice) The poultice can be prepared by a known method using a known poultice base. Examples of compress bases include thickeners (eg, sodium polyacrylate,
Synthetic water-soluble polymers such as polyacrylic acid, poval, polyvinylpyrrolidone, polyethylene oxide and polyvinyl methacrylate, natural products such as gum arabic, starch and gelatin, methylcellulose, hydroxypropylcellulose, alginic acid, sodium alginate, ammonium alginate, carboxymethylcellulose Sodium), wetting agents (eg, urea, glycerin, propylene glycol, butylene glycol, sorbitol, etc.), fillers (eg, kaolin, zinc oxide, talc, titanium, benite, epoxy resins, organic acids (citric acid,
Tartaric acid, maleic acid, maleic anhydride, succinic acid, etc.),
Calcium, magnesium, aluminum, etc.), water, dissolution aids (eg, propylene carbonate, crotamiton, diisopropyl adipate, etc.), tackifiers (eg, rosin, ester gum, polybutene, polyacrylate, etc.), anti-rash agents ( Examples: diphenhydramine hydrochloride, chlorpheniramine maleate, glycyrrhizic acid, dexamethasone, betamethasone, fluocinolone acetonide, and other additives (eg, salicylic acid, methyl salicylate, glycol salicylate, l-menthol, camphor, vanillyl amide nonylate) Thymol, capsicum extract, peppermint oil, azone, pyrothiodecane, etc.) and the like.

A specific example of the production of a poultice is shown in (A)
0.01 to 10% by weight of the antibacterial active substance of the present invention is mixed and dissolved with 0.5 to 8% by weight of a solubilizing agent to make it uniform.
Next, (B) 5 to 20% by weight of a thickener, preferably 10 to 1%
5% by weight of wetting agent 5-40% by weight, water 10-80% by weight
And 20% by weight or less of filler is added to obtain a uniform kneaded product. Next, (A) is added to (B) and mixed to obtain a uniform kneaded product. The spread of the kneaded product is spread on a support by a usual method, and then a release coating is stuck thereon, whereby the poultice of the present invention can be obtained. The support is appropriately selected from stretchable or non-stretchable fabric, nonwoven fabric, non-woven paper, etc., and the release coating is appropriately selected from polyethylene, polypropylene, polyvinyl chloride, polyester, polyvinylidene chloride, siliconized paper, etc. Used as

(Paster) A plaster can be prepared by a known method using a known base for plaster. Examples of bases for plasters include known polymer bases (eg, acrylic compositions which are copolymers with vinyl monomers such as methacrylates, acrylonitrile, vinyl acetate, vinyl propionate, silicone resins, Polyisoprene rubber, polyisobutylene rubber, natural rubber, acrylic rubber, styrene-butadiene-styrene block copolymer, styrene-isoprene-styrene block copolymer, etc.), fats or higher fatty acids (eg almond oil, olive oil, camellia oil) , Versic oil, peanut oil, oleic oil, liquid paraffin, polybutene, etc.), tackifiers (eg, rosin, rosin-modified maleic acid, hydrogenated rosin ester, etc.), anti-rash agents, and other additives (eg,
dl-camphor, 1-menthol, thymol, nonylate vanillylamide, pepper tincture, peppermint oil, crotamiton, peppermint oil, azone, pyrothiodecane, etc.), which are appropriately selected and used.

A mixture of these plaster bases and the antibacterial active substance of the present invention is blended with a stretchable or non-stretchable support (eg, polypropylene, polyester, polyvinylidene chloride, polyacryl, polyurethane, rayon, cotton). ,
After spreading and applying the composition to an ethylene-vinyl acetate copolymer, cloth, nonwoven fabric, non-woven paper, etc.) and then applying a release coating thereon, the plaster of the present invention can be obtained. As an example of the compounding composition, in a known cool or warm plaster, the medicinal ingredient is replaced with the antimicrobial active substance of the present invention by 0.01 to 10% by weight, and further, the ultraviolet absorber is 0.01 to 5% by weight.
Alternatively, the plaster of the present invention can be obtained by adding 0.01 to 5% by weight of an antioxidant as needed.

(Liniment) The liniment can be prepared by a known method using a known liniment base. Specific examples of preparation are as follows: alcohols (eg, monohydric alcohols such as ethanol, propanol and isopropanol, polyhydric alcohols such as polyethylene glycol, propylene glycol and butylene glycol) 10 to 70% by weight, water 55% by weight The fatty acid ester (eg, various esters of adipic acid, sebacic acid, myristic acid, etc.) is 60% by weight or less and the surfactant (eg, polyoxyethylene alkyl ether) is 10% by weight or less. Antibacterial active substance 0.01-1
The liniment of the present invention can be prepared by adding 0% by weight and further adding 0.01 to 5% by weight of an ultraviolet absorber and, if necessary, 0.01 to 5% by weight of an antioxidant.

The liniment of the present invention can be obtained by substituting the active ingredient in the known liniment with the antibacterial active substance of the present invention and further blending an ultraviolet absorber. In the liniment of the present invention,
If necessary, a neutralizing agent for pH adjustment, a viscosity imparting agent such as methylcellulose, carboxyvinyl polymer, or hydroxypropylcellulose, an anti-rash agent or other additives (eg, salicylic acid, methyl salicylate, glycol salicylate, l-menthol, Camphor, mint oil,
Capsicum extract, vanillyl amide nonylate, thymol, crotamiton, Aison, propylene carbonate, diisopropyl adipate, pyrothiodecane, etc.) can also be blended.

(Liquid) A liquid can be prepared by a known method using a known liquid base. The liquid preparation of the present invention can be used as a disinfectant / disinfectant. The liquid preparation of the present invention contains 0.01 to 10% by weight of the antibacterial active substance of the present invention.
It is preferable to mix them to a certain degree. As the liquid base, a lower monoalcohol, a nonionic surfactant, a water-soluble polymer compound, a humectant, water and the like can be used. As lower monoalcohols, primary, secondary and primary carbon atoms having 1 to 4 carbon atoms,
Tertiary monoalcohols are preferred and include, for example, ethanol, ethanol for disinfection, absolute ethanol, isopropanol, propanol, butanol and the like. The mixing amount of these lower monoalcohols is 6
It is preferably from 0 to 90% by weight. If the amount of the lower monoalcohol is less than 60% by weight, a sufficient antibacterial or bactericidal effect cannot be obtained. If the amount is more than 90% by weight, the retention time of the foam is short and may not be practical.

Examples of the nonionic surfactant to be added to the liquid preparation of the present invention include polyoxyethylene sorbitan acyl ester polysorbate 80, polysorbate 60, polysorbate 20, and dimethylsiloxane methyl (PO) of silicone polyether copolymer.
E) siloxane copolymers and the like; polyoxyethylene acyl ester-based polyoxyl 40 and polyoxyethylene lauryl ether; and the like; polyoxyethylene alcohol ether-based lauromacrogol and the like; stearic acid glycerin ester-based glyceryl monostearate; Decaglycerin monolaurate and the like; sorbitan fatty acid ester span60 monostearate and the like; sorbitan acyl ester sorbitan sesquioleate and the like; polyoxy hydrogenated castor oil HCO-60, HCO-
50 and the like; Pluronic F68 of polyoxyethylene propylene glycol monofatty acid ester and the like can be mentioned. These nonionic surfactants can be used alone or
Species or more may be used in combination at an appropriate ratio. The HLB of these nonionic surfactants is preferably 10 or more. If the HLB is less than 10, it may be difficult to obtain a foam having appropriate stability. The amount of the nonionic activator is preferably 0.5 to 3% by weight, particularly preferably 1 to 3% by weight, based on the whole liquid preparation. If the compounding amount is out of this range, it may be difficult to obtain a foam having appropriate stability as described above.

Examples of the water-soluble polymer compound to be added to the liquid preparation of the present invention include natural or synthetic polymer compounds. Examples of the natural polymer compound include soluble polysaccharides such as acacia gum, xanthan gum, pectin, carrageenan, sodium alginate, etc., soluble polypeptides such as gelatin, chitins such as chitin and chitosan. Examples of the synthetic high molecular compound include partially chemically processed natural high molecular compounds, such as sodium carboxymethylcellulose, which is a soluble polysaccharide, and hydroxypropylcellulose. Examples thereof include polyvinylpyrrolidone and its derivatives, and polyvinylpyrrolidone-based compounds such as polyvinylpyrrolidone and its derivatives. Among these, xanthan gum is preferred from the viewpoint of foam stability. The compounding amount of these water-soluble polymer compounds is preferably 0.01 to 3% by weight based on the whole liquid preparation. If the compounding amount is out of this range, it may be difficult to obtain a foam having appropriate stability as described above.

Examples of the humectant incorporated in the liquid preparation of the present invention include glycerin, propylene glycol, sorbitol, 1,3-butylene glycol, macrogol 400, and hyaluronic acid.
Glycerin is preferred from the viewpoint of defoaming properties and moisture retention. One or more of these humectants can be blended in any ratio. Among the above, the humectants other than hyaluronic acid are 1 to 10% by weight based on the whole liquid, and the hyaluronic acid is the whole liquid. It is preferable to mix 0.01 to 1% by weight with respect to the weight.

The liquid preparation of the present invention may be mixed with water. In this case, the mixing amount is 3 to 35% by weight of the whole liquid preparation.
It is preferable that Water acts on a surfactant to improve foaming and acts on a water-soluble polymer compound to stabilize foam. It has been conventionally known that when alcohols such as ethanol, which are useful as a liquid base, are mixed with a foam at a high concentration, the foam becomes unstable and the foam disappears at an early stage. Therefore, by combining a high-concentration lower monoalcohol with a predetermined nonionic surfactant, the foam retention time can be adjusted appropriately. Usually, when about 0.5 to 2 g of a foam liquid is applied to the palm and the hands are rubbed and used, the range in which the user perceives that the liquid spreads sufficiently and that the remaining foam is not too long depends on the experience. Typically 5 to 30 seconds. In order to prepare an effective solution that is easy to handle, it is necessary to use a high-concentration alcohol and keep the foam holding time within this range. Further, the liquid preparation of the present invention can maintain the state of the foam clearly and stably by appropriately mixing the water-soluble polymer compound.

The liquid preparation of the present invention can be usually mixed with a propellant to form an aerosol composition. Examples of the propellant include dimethyl ether, liquefied petroleum gas, nitrogen gas, nitrous oxide gas, carbon dioxide gas, and chlorofluorocarbon gas, which are commonly used in aerosols. Also, compressed air can be used without using a propellant. Further, a mixture of these may be used. The amount of the propellant is not particularly limited, but is preferably 0.1 to 80% by weight based on the total amount of the liquid agent of the present invention. In addition, in addition to the above, if necessary, a component usually blended in a fragrance or an external preparation, for example, 1-
Menthol, tocopherol acetate, and vegetable oils such as castor oil and squalene and oils and fats such as animal oils may be used in appropriate amounts.

[0043]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the present invention. (Example 1) A sponge algae collected off the coast of Amami Oshima, Kagoshima Prefecture (scientific name: Ceratodictyon spongi)
osum Zanardini) 10 kg was washed with artificial seawater (manufactured by Jamalin) and phosphate buffer solution (PBS, pH 7.5), immersed in 4 times PBS (w / v) for 24 hours, and crushed with a crusher. , Centrifugation (3000 rpm, 20 minutes, 4
° C) to obtain a supernatant, and a solution obtained by filtering the supernatant with a 0.45 µm filter is separated and cultured to obtain Acinetobacter calcoaceticus.
The strain was isolated.

This strain was added to a liquid medium (22.5 L of artificial seawater, 50 g of polypeptone, 10 parts of yeast extract in 30 L of medium).
g, pH 7.6), and cultured at 37 ° C. for 4 days in a 5% CO ₂ atmosphere, followed by centrifugation (4000 rpm).
pm, 20 minutes, 20 ° C), and the obtained supernatant was extracted with ethyl acetate. After the extract was concentrated under reduced pressure, it was separated by silica gel column chromatography (silica gel 60, 35 × 300 mm) using n-hexane / diethyl ether / ethyl acetate (2: 3: 5) as a solvent. The obtained eluted portion was subjected to a second stage silica gel column chromatography using a mixed solution of benzene and chloroform (1:10) as a solvent. Further, n-hexane / ethanol (4: 1) was used as a developing solution.
Impurities were removed twice by silica gel thin layer chromatography using v / v), and the range of Rf = 0.2 was recovered. Further, purification was performed by high performance liquid chromatography. The mobile phase in this high performance liquid chromatography was chloroform containing 5% methanol, and the column stationary phase was a silica gel column (Inetsil, 20 × 250 m).
m, manufactured by GL Science), and the flow rate of the mobile phase is 5
ml / min, wavelength of ultraviolet spectrum measuring instrument is 230n
m. Extract 2 completely purified in this way
0 mg was obtained.

FIG. 1 shows a flow chart of the purification of the extract of the present invention. Also, the high performance liquid chromatogram is shown in FIG.
The mass spectrum data of the obtained extract is shown in FIG. 3, and the visible ultraviolet absorption spectrum is shown in FIG. The physicochemical properties of the obtained extract are as shown below. (1) Property: pale yellow solid (powder) (2) MS (TOF-MS) (m / z): 447.08 [M + Na] (3) UV (λmax; nm): 210, 270

(Test Example 1) The test strains used in this test were:
Gram positive bacteria Staphylococcus aureus (IFO 1503
5), methicillin-resistant Staphylococcus aureus (GIFU123
61) (MRSA), Bacillus subtilis (IFO14419) and Gram-negative bacterium Vibrio parahaemolyticus (IFO12711)
It is. The culture of the test strain is Staphylococcus aureus
(IFO 15035), methicillin-resistant Staphylococcus aureus
(GIFU12361) (MRSA) and Bacillussubtilis (IFO
14419) includes a TSA medium (Difco Laboratories)
Using Vibrio parahaemolyticus (IFO12711),
Marine Agar medium (Difco Laboratories) was used. In the liquid culture of each test strain, a medium obtained by removing agar components from each agar medium was used.

The antibacterial activity test was performed as follows. One platinum loop of each test strain was contacted with each liquid medium, and
After shaking culture at 3 ° C. for 3 days, 0.4 ml of the obtained culture solution was added to 20 ml of soft agar medium (containing 0.8% agar) and transferred to a square petri dish to prepare a solid medium. A sterilized paper disk (diameter 8 mm, manufactured by Toyo Roshi Kaisha, Ltd.) was arranged on a solid medium, and an aqueous solution of the extract of the present invention was impregnated with 50 μl per disk. After culturing this medium at 25 ° C. for 1 day, the growth inhibition circle formed around the paper disk is visually observed, and the minimum growth inhibition concentration (MIC: the minimum drug concentration required to completely inhibit the growth of the strain) ) Was recorded. Table 1 shows the results.

[0048]

Table 1 Test strain Minimum growth inhibitory concentration (MIC, μg / μg / disk) −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Staphylococcus aureus (IFO15035) 0.75 Staphylococcus aureus (GIFU12361) ) 0.75 Bacillus subtilis (IFO14419) 1.5 Vibrio parahaemolyticus (IFO12711) 6.0 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

As shown in Table 1, the extract of the present invention was confirmed to have a clear growth inhibitory activity at low concentrations against the Gram-positive bacteria and Gram-negative bacteria used in the test.

[0050]

According to the present invention, a novel antibacterial active substance can be obtained from a microorganism attached to a marine sponge. The extract of the present invention has a growth inhibitory effect on Gram-positive bacteria and Gram-negative bacteria, and can be used as an antibacterial agent for humans and animals, a bactericide, a pesticide, a preservative for foods, and the like.

[Brief description of the drawings]

FIG. 1 is a flowchart showing a procedure for extracting an antibacterial active substance of the present invention from a microorganism attached to a sponge.

FIG. 2 is a view showing a liquid chromatogram of the extract of the present invention.

FIG. 3 is a view showing mass spectrum data of the extract of the present invention.

FIG. 4 is a view showing a visible ultraviolet absorption spectrum of the extract of the present invention.

Claims (8)

[Claims] 1. An antibacterial substance produced by Acinetobacter calcoaceticus. 2. An antibacterial active substance obtained by culturing Acinetobacter calcoaceticus and collecting from the culture. 3. A method for producing an antibacterial active substance, comprising culturing Acinetobacter calcoaceticus and collecting from the culture supernatant. 4. After washing a sponge algae with seawater and a phosphate buffer, immersed in a phosphate buffer, and isolated from the immersion solution, Acinetobacter calcoaceticus (Acinetobact.
4. The method for producing an antibacterial active substance according to claim 3, wherein the bacterium is cultured. 5. An antibacterial agent containing the antibacterial active substance according to claim 1. 6. A fungicide containing the antibacterial active substance according to claim 1. 7. An antimicrobial composition comprising the antimicrobial active substance according to claim 1 or 2 and one or more pharmaceutically acceptable carriers. 8. A fungicidal composition containing the antibacterial active substance according to claim 1 or 2 and one or more pharmaceutically acceptable carriers.