KR102264167B1 - Cell wall extract from deinococcus radiodurans and anti-allergic composition comprising the same - Google Patents

Abstract

The present invention relates to a cell wall extract derived from Diinococcus radiodurans and an anti-allergic composition comprising the same, and more particularly, to a cell wall extract derived from Deinococcus radiodurans; Deinococcus radiodurans (Deinococcus radiodurans) step of disrupting the strain, separating and removing the precipitate containing the undisrupted strain by the first centrifugation and obtaining the supernatant, the cytoplasmic fraction by the second centrifugation removing the supernatant containing the supernatant and obtaining a precipitate, resuspending the precipitate in a buffer containing SDS, removing the supernatant containing the cell membrane fraction by third centrifugation, and obtaining a precipitate containing the cell wall fraction A method for producing a cell wall extract comprising the steps and removing nucleic acids and proteins from the precipitate; And Deinococcus radiodurans (Deinococcus radiodurans) It relates to an anti-allergic composition comprising a cell wall extract.

Description

Diinococcus radiodurans-derived cell wall extract and anti-allergic composition comprising the same {CELL WALL EXTRACT FROM DEINOCOCCUS RADIODURANS AND ANTI-ALLERGIC COMPOSITION COMPRISING THE SAME}

The present invention relates to a Dinococcus radiodurans-derived cell wall extract and an anti-allergic composition comprising the same, and more particularly, to a Diinococcus radiodurans-derived hydrophilic cell wall extract and an antiallergic composition comprising the same.

Allergy or allergy is a compound word created by combining the Greek words 'allos' meaning 'change' and 'ergo' meaning 'action', and has the meaning of an abnormal reaction. As can be inferred from the etymology, an allergen, a substance that does not show any reaction to ordinary people, causes hypersensitivity reactions such as hives, itchiness, runny nose, and cough in certain people due to abnormalities in innate or acquired immune function. say Typical allergens include pollen, drugs, vegetable fibers, bacteria, mold, house dust mites, food (eg, egg albumin, milk protein, peanuts), hair dye, and chemicals. Due to an increase in substances, environmental pollution, a decrease in immune function due to stress, and a change in diet, these allergic diseases are increasing worldwide.

Allergic reactions can be classified into five types: type I anaphylaxis, type II cytolytic type, type III immune complex type, type IV cell mediated type, and type V stimulatory hypersensitivity type, most of which belong to type I. , In general, when it comes to allergic reactions, it is used in the same sense as type I. Representative allergic diseases include atopic dermatitis, bronchial asthma, allergic rhinitis, allergic keratitis, skin urticaria, etc. In these allergic diseases, mast cells are known as important mediator cells.

Allergic reactions are induced through activation of intracellular signaling pathways when an allergen binds to IgE, which is bound to the IgE high affinity receptor (FcεRI) on the surface of these mast cells. Specifically, when an allergen invades a living body, T-helper cells and B cells are sequentially activated to generate IgE. Allergens that invade the living body are recognized by antigen-presenting cells, which differentiate Th0 (T helper cell type 0) into Th2 cells by presenting the allergen. These differentiated Th2 cells secrete cytokines such as IL-4, IL-5 and IL-13, which promote the development of eosinophils in the bone marrow and induce eosinophils to the inflamed tissue, as well as act on B cells. to induce the production of IgE. The generated IgE binds to the high-affinity IgE receptor (FcRIα) of the mast cell membrane, and when allergens such as mites and pollen bind to it and cross-link it, the receptor (FcRIα) aggregates. By crosslinking and aggregation of the receptor (FcRIα), mast cells are activated, and the ^{concentration of calcium ions (Ca ++} ) in the cytoplasm, etc. rises. The rise in calcium ion concentration acts on several types of intracellular regulators such as cholesterol in the cell membrane and the cytoskeletal system, and moves the granules to the cell membrane, and at the same time, degranulates through vacuolization of granules, fusion of granules, and fusion of granules and cell membrane causes When degranulation occurs, histamine, prostaglandin, leukotriene, and serotonin are secreted to cause vasodilation, vascular permeability enhancement, and contraction of bronchial smooth muscle. Inflammatory cytokines such as 1β, IL-4, IL-5, IL-6, and TNF-α are secreted, and neutrophils, eosinophils, macrophages, Th2 cells, basophils, etc. infiltrate into the tissue to induce an allergic inflammatory response.

Therefore, substances that inhibit the activation of mast cells, inhibit degranulation, or inhibit the production of inflammatory cytokines, etc. can be effective therapeutic agents for allergies. Currently, as anti-allergic drugs, antihistamines (alkylamines, desloratadine, etc.), mast cell stabilizers (cromoglycate, etc.), leukotriene blockers (montelukast, etc.), anti-IgE antibodies (Omalizumab), etc. are being used. However, in most cases, the effect of these drugs is temporary, and continuous treatment at a strong concentration induces side effects, thus limiting their use in the treatment of allergic diseases, which are chronic immune diseases.

Therefore, it can be said that it is still necessary to develop a new substance that has a preventive and ameliorating effect on allergic diseases while having few side effects and a continuous effect. In particular, natural products have the advantage of relatively low toxicity. In this regard, Korean Patent Application No. 2003-0038640 discloses a Diinococcus radiodurans cell membrane extract and a pharmaceutical composition for skin containing the same, but discloses only a description of a hydrophobic substance using an organic solvent. Accordingly, there is still a need to develop an effective eco-friendly material that can effectively control allergic diseases without side effects.

Accordingly, one aspect of the present invention is to provide a cell wall extract derived from Deinococcus radiodurans.

Another aspect of the present invention is to provide a method for obtaining a cell wall extract derived from Deinococcus radiodurans.

Another aspect of the present invention is to provide an anti-allergic composition comprising a cell wall extract derived from Deinococcus radiodurans.

According to one aspect of the present invention, Deinococcus radiodurans (Deinococcus radiodurans) cell wall extract is provided.

According to another aspect of the present invention, the step of disrupting the deinococcus radiodurans (Deinococcus radiodurans) strain; Separating and removing the precipitate containing the undisrupted strain by primary centrifugation to obtain a supernatant; removing the supernatant containing the cytoplasmic fraction by secondary centrifugation and obtaining a precipitate; After resuspending the precipitate in a buffer containing SDS, removing the supernatant containing the cell membrane fraction by third centrifugation and obtaining a precipitate containing the cell wall fraction; And there is provided a method for producing a cell wall extract comprising the step of removing the nucleic acid and protein from the precipitate.

According to another aspect of the present invention, there is provided an anti-allergic composition comprising the cell wall extract obtained by the method for producing the cell wall extract of the present invention.

According to another aspect of the present invention, there is provided an anti-allergic composition comprising a deinococcus radiodurans cell wall extract.

According to the present invention, there is provided a cell wall extract derived from Deinococcus radiodurans, which can effectively control allergic diseases, and inhibits the production of IL-4, IL-5 and IFN-γ cytokines to prevent inflammation and anti-allergy. Because it exhibits an effect, it can be usefully used as a pharmaceutical composition for the prevention and treatment of inflammation and allergic diseases, a cosmetic composition capable of alleviating inflammation and allergy-related symptoms, a health functional food, and the like.

1 schematically shows a method for preparing a cell wall extract of the present invention.
2 is a graph showing the results of Experimental Example 1.
3 is a graph showing the results of Comparative Experimental Example 1.
4 is a graph showing the results of Experimental Example 2.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, an eco-friendly material that can effectively control allergic diseases without side effects is provided, and in more detail, according to the present invention, a cell wall extract of Deinococcus radiodurans is provided.

In the present specification, "Dinococcus radiodurans cell wall extract" refers to a cell wall component obtained from Diinococcus radiodurans to be extracted, and "cell wall extract" may be used interchangeably with "cell wall fraction" . In particular, the cell wall extract contains a hydrophilic component.

The strain of Diinococcus radiodurans of the present invention is not particularly limited, and is obtained from nature or commercially available, for example, Diinococcus radiodurans R1, KCTC ( Diinococcus radiodurans BRD50 (used interchangeably with "#50" herein) deposited with the deposit number KCTC13953BP on August 17, 2019 in the Korean Collection for Type Cultures, as of August 17, 2019 Diinococcus radiodurans BRD54 deposited with accession number KCTC13954BP (used interchangeably with "#54" herein) may be used.

On the other hand, the method for preparing the Dinococcus radiodurans cell wall extract of the present invention is not particularly limited, and for example, it can be obtained by centrifugation after crushing the Dinococcus radiodurans strain. At this time, physiological saline may be used as a buffer during centrifugation, for example, phosphate buffered saline (PBS) may be used, and NaCl may be additionally included.

In more detail, the method for producing a cell wall extract of the present invention comprises the steps of disrupting a deinococcus radiodurans strain; Separating and removing the precipitate containing the undisrupted strain by primary centrifugation to obtain a supernatant; removing the supernatant containing the cytoplasmic fraction by secondary centrifugation and obtaining a precipitate; After resuspending the precipitate in a buffer containing SDS, removing the supernatant containing the cell membrane fraction by third centrifugation and obtaining a precipitate containing the cell wall fraction; and removing nucleic acids and proteins from the precipitate.

The step of crushing the Deinococcus radiodurans strain may be performed by a method selected from the group consisting of ultrasonic grinding, a compactor, a high-speed bead grinder and a ball mill, but is not limited thereto, and preferably ultrasonic It is carried out by crushing.

After disrupting the cells, the precipitate containing the undisrupted strain is separated and removed by primary centrifugation to obtain a supernatant. Bacteria (strain) not disrupted by the first centrifugation step and agglomerates of bacterial fractions not completely degraded are precipitated and removed.

To this end, the first centrifugation may be performed at 1,000 to 2,000 x g, for example, at 1,000 to 1,800 x g, preferably 1,200 to 1500 x g. When the centrifugation is performed at less than 1,000 x g, the separation may not be performed smoothly, and when it exceeds 2,000 x g, loss of some cell wall fraction components may occur.

A supernatant was obtained as a result of the first centrifugation, and further, the supernatant containing the cytosolic protein, which occupies the largest cytoplasmic fraction by the second centrifugation, other disrupted nucleic acids, and some disrupted lipid molecules removed and a precipitate is obtained.

After obtaining a precipitate as a result of the second centrifugation and resuspending the precipitate, the supernatant containing the cell membrane fraction is removed by the third centrifugation. For resuspension, a buffer containing SDS may be used, for example, PBS containing 0.5% SDS may be used, and incubation may be performed before centrifugation. When performing the incubation, for example, it may be carried out at a temperature of about 37 ℃ room temperature to 70 ℃ for about 10 minutes to 60 minutes. As a result of the third centrifugation, a precipitate containing a cell wall fraction can be obtained.

Subsequently, the step of removing nucleic acids and proteins from the precipitate is performed, and the order of the steps of removing nucleic acids and removing proteins is not particularly limited. For example, the nucleic acid removal step and the protein removal step are sequentially performed.

The nucleic acid removal step may be performed using DNA and RNA degrading enzymes such as DNAse I and RNAse A by resuspending in 1M Tris-HCl, and the type of DNA and RNA degrading enzymes is not particularly limited. Thereafter, the nucleic acid fraction contained in the supernatant is removed by fourth centrifugation, and a precipitate containing the cell wall is obtained.

The second to fourth centrifugation may be independently carried out at 13,000 to 25,000 x g, for example, at more than 15,000 to 21,000 x g, preferably at 17,000 to 20,000 x g. When the centrifugation is performed at less than 13,000 x g, the separation may not be performed smoothly, and when it exceeds 21,000 x g, some cell wall fraction components may be lost.

In order to remove proteins from the precipitate obtained after the fourth centrifugation _{, incubation may be subsequently performed in the presence of CaCl 2} , trypsin, etc., but the method for removing proteins is not particularly limited, for example, the incubation is about It can be carried out for 12 hours to 48 hours at a temperature of 37 ℃.

Meanwhile, the execution time of each centrifugation is not particularly limited, and for example, may be performed for 5 minutes to 60 minutes, preferably 10 minutes to 30 minutes, for example, 10 minutes to 20 minutes.

Furthermore, a drying step may be performed, and the drying method is not particularly limited, and, for example, freeze drying, vacuum drying, hot air drying, spray drying, etc. may be performed.

The meaning of the extract or fraction of the present invention is to include a dry solid extract from which the buffer has been removed.

According to the present invention, there is provided an anti-allergic composition comprising a Deinococcus radiodurans cell wall extract, for example, the Deinococcus radiodurans cell wall extract is obtained by the process of the present invention described above. it could be

As used herein, "anti-allergy" is meant to include alleviation, alleviation, treatment, prevention of allergic diseases or symptoms, for example, suppression or delay of onset.

The anti-allergic composition may be a pharmaceutical composition for improving, treating, or preventing allergic diseases.

As used herein, the term "active ingredient" alone exhibits a desired activity, or in the specification, "anti-allergy" means improvement (relief of symptoms), treatment, prevention (inhibition or delay of onset) of allergic diseases as defined below. ) is included.

As used herein, "allergic disease" means a pathological symptom caused by an allergic reaction mediated by the activation of mast cells, such as degranulation of mast cells, and the allergic disease includes atopic dermatitis, selected from the group consisting of asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, and allergic keratitis. There may be at least one.

The anti-allergic composition of the present invention uses the Deinococcus radiodurans cell wall extract as an active ingredient in any effective amount as long as it can exhibit the amelioration activity of the allergic disease intended for treatment, etc. according to the use, formulation, purpose of formulation, etc. can be included as As used herein, the term "effective amount" refers to the intended medical and pharmacological effects, such as the improvement of allergic diseases, when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied during the administration period as suggested by a medical professional. It refers to the amount of active ingredient included in the composition of the present invention. Such effective amounts can be determined empirically within the ordinary ability of one of ordinary skill in the art.

In addition to the active ingredient, the anti-allergic composition of the present invention may further include any compound or natural extract known to have anti-allergic activity and safety has already been verified to increase/reinforce anti-allergic activity.

The pharmaceutical composition of the present invention may be prepared as an oral dosage form or parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of the combination of two or more routes is a case in which two or more formulations of drugs according to the route of administration are combined. For example, one drug is first administered by an intravenous route and the other drug is secondarily administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopoeia of each country including the "Korea Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc. according to methods known in the art together with a suitable carrier It can be prepared in the form of Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol etc. are mentioned. In the case of formulation activity, an appropriate binder, lubricant, disintegrant, colorant, diluent, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrist, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as lubricant. sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, polyethylene glycol, and the like. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like.

When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or isotonic solution such as 5% dextrose may be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta agents, liniment agents, air rolls, and the like. In the case of a nasal inhalant, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like can be used.

The specific formulation of pharmaceutical compositions is known in the art.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.2 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, patient's severity, and administration route. kg range. Administration may be performed once or divided into several times a day. Such dosages should not be construed as limiting the scope of the invention in any respect.

Meanwhile, the anti-allergic composition may be a food composition. When the composition of the present invention is a food composition, its use may be understood to inhibit allergic skin hypersensitivity reaction.

The food composition of the present invention may be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, confectionery, noodles, etc. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and health functional food preparations such as bars can be prepared.

In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it conforms to the enforcement laws at the time of manufacture and distribution. For example, it is a health functional food according to Korea's "Health Functional Food Act", or confectionery, beans, tea, and beverages according to each food type in the Food Code of Korea (Food Sanitation Act) (Ministry of Food and Drug Safety Notification "Food Standards and Specifications") , food for special use, and the like.

The food composition of the present invention may contain food additives in addition to the active ingredients thereof. Food additives can be generally understood as substances that are added to and mixed with or infiltrated into food in manufacturing, processing or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be ensured. Food additives with guaranteed safety are limited in terms of ingredients or functions in the Food Additives Ordinance in accordance with the laws of each country that regulates the manufacture and distribution of food (“Food Sanitation Act” in Korea). In the Korean Food Additives Code (“Food Additive Standards and Specifications” notified by the Ministry of Food and Drug Safety), food additives are classified into chemically synthetic products, natural additives, and mixed preparations in terms of ingredients. It is classified into an agent, a preservative, an emulsifier, an acidulant, and a thickener.

The sweetener is used to impart appropriate sweetness to food, and both natural and synthetic ones may be used in the composition of the present invention. Preferably, a natural sweetener is used. Examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents may be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, it is a case where a natural thing is used. In the case of using a natural one, the purpose of nutritional enhancement in addition to flavor may be concurrently used. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, or the like, or obtained from green tea leaves, dandelion leaves, bamboo leaves, cinnamon, chrysanthemum leaves, and jasmine. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. The natural flavoring agent may be a liquid concentrate or a solid extract. Optionally, a synthetic flavoring agent may be used, and the synthetic flavoring agent may be an ester, an alcohol, an aldehyde, a terpene, or the like.

As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin etc. are mentioned, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. can be used as an acidulant. Acidulant may be added so that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing the taste.

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a bulking agent, and the like can be used.

The food composition of the present invention may contain, in addition to the food additives as described above, physiologically active substances or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutrition and guaranteed stability as food additives.

In the food composition of the present invention, the food additives as described above may be included in an appropriate amount to achieve the purpose of the addition according to the type of product. In relation to other food additives that may be included in the food composition of the present invention, reference may be made to the national food regulations or food additives regulations.

In addition, the anti-allergic composition may be a cosmetic composition. When the composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for suppressing allergic skin troubles, alleviating allergic skin irritation, and the like.

Even when the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified into any product in terms of its use or by law, and specifically functional cosmetics, non-functional products having uses such as improvement of skin troubles and improvement of atopic dermatitis It may be general cosmetics and the like. The product form may also take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax It can take the form of products such as foundation and spray. In the specific product form, it may be in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

The cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the active ingredient, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and fragrances, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.

The cosmetic composition of the present invention can be prepared according to the method for preparing a cosmetic composition conventionally performed in the art, except that it contains the active ingredient exhibiting anti-allergic activity.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

One. Dinocaucus Radio Durance ( Deinococcus radiodurans ) Preparation of cell wall extracts

(1) Acquisition of Diinococcus radiodurans strain

Diinococcus radiodurans R1 was obtained from ATCC, USA, and Diinococcus radiodurans #50 and #54 strains were obtained from Baengnokdam, Hallasan, respectively. In the following examples, for convenience, each strain is referred to as R1, #50 and #54 monarchs.

Diinococcus radiodurans #50 and #54 strains were obtained by the following process.

After obtaining permission to collect nationally-designated cultural assets from Jeju Special Self-Governing Province, 100 ml of fresh water, 100 g of soil, and 100 g of viscosity were collected from two places (Table 1) of Mt. Gamma rays were irradiated. After 30 ml of R2A liquid medium was added, the irradiated soil was cultured at 30° C. for 2 hours (enrichment), and then spread about 0.5 ml on R2A solid medium, and then cultured in an incubator at 30° C. for 3 days. Colonies appearing in R2A solid medium were selected and cultured for two days in R2A liquid medium. Some of the cultured liquid medium was made into a storage strain, and the rest were plated on R2A solid medium and cultured for two days in an incubator at 30°C. Through 16s rRNA analysis, 58 types of soil-derived, 49 types of clay-derived, and 3 types of freshwater-derived radiation-resistant microorganisms were isolated and identified. Finally, 110 radiation-resistant microorganisms including Diinococcus radiodurans #50 and #54 were isolated and identified. separated.

Soil harvesting coordinates location Sampling position 1 33°, 21’, 44.7“N 126°, 31’ 58.9”E Sampling position 2 33°, 21′, 44.6“N 126°, 32’ 01.3”E

To identify radiodurans (D. radiodurans) from the isolated 110 radiation-resistant microorganisms, genomic DNA was isolated and 16s mRNA was analyzed. 16s rRNA analysis was performed using 27F (5'-AGAGTTTGATCMTGGCTCAG-3) and 1492R primer (5'-TACGGYTACCTTGTTACGACTT-3') to BLAST the DNA sequencing results of the PCR product of about 1350 bp or more to select strains with high homology.

Among the 110 strains, 36 species of radiodurans (D. radiodurans) were identified, and two of them were named Diinococcus radiodurans #50 and #54, respectively, and # in KCTC (Korean Collection for Type Cultures). 50 was deposited with deposit number KCTC13953BP on August 17, 2019, and #54 was deposited with deposit number KCTC13954BP on August 17, 2019.

(2) Preparation of cell wall extract

As schematically shown in Figure 1, each strain pellet was suspended in 1M NaCl aqueous solution and disrupted by sonication (a). The first centrifugation was performed at 1,000xg for 15 minutes to remove undisrupted cells and precipitates including heavy cell lysates (b). The supernatant was again centrifuged at 18,800xg for 15 minutes to remove the supernatant containing cytosolic protein and light cell lysate, and a precipitate pellet was obtained (c). The pellet thus obtained was resuspended in PBS of 0.5% SDS and incubated at 60° C. for 30 minutes (d). After 3rd centrifugation at 18,800xg for 15 minutes, the pellet was resuspended in 1M Tris-HCl and treated with 10 μg/ml DNAse I and 50 μg/ml RNAse A at 37° C. for 2 hours. The pellet obtained by fourth centrifugation at 18,800 x g for 15 minutes _{was incubated in 10 mM CaCl 2} at 37° C. for 18 hours.

Subsequently, freeze-drying was performed to obtain a dried cell wall extract, weighed, and suspended in pyrogen-free water at concentrations of 0.1 mg/ml and 1 mg/ml.

2. Mouse allergy Establishment of an inflammatory response model

To induce an allergic inflammatory response in mice, 20 μg OVA (ovalbumin) antigen and 1 mg alum excipient were mixed and immunized twice a week with Balb/C mice at 7 weeks of age. After the spleen was isolated, the expression of allergens was induced by restimulation with 50 μg/ml OVA antigen in ^{5 x 10 5 cells/ml of splenocytes for 4 days.}

The induction of the allergic inflammatory response was confirmed by examining the expression of IL-4 and IL-5, which are allergen mediators, and the induction of the anti-allergic response was confirmed by examining the expression of IFN-γ, the anti-allergy mediator. IL4, IL-5, and IFN-γ expression was confirmed by the ELISA9 enzyme-linked immunosorbent assay method.

3. allergy Confirmation of Effect on Inflammatory Response

Experimental example 1: Confirmation of the effect of cell wall extract

D. radiocurans according to the process described in 1. R1, #50 and To investigate the ability of cell wall extracts isolated in #54 to modulate allergic inflammatory response, in the allergic inflammatory response model established in 2. above, each cell wall extract was administered at 10 μg/ml and 30 μg/ml upon restimulation of OVA antigen to splenocytes. was simultaneously treated with and 4 days later, the production of IL-4 and IL-5, which are mediators of allergic inflammatory response, in the supernatant was confirmed by the ELSIA method.

As a result, it was confirmed that each cell wall extract derived from Diinococcus radiodurans R1, #50, and #54 very strongly inhibited the production of IL-4 and IL-5, which are mediators of the allergic inflammatory response induced by the OVA antigen, The results are shown in FIG. 2 .

As can be seen in FIG. 2 , it was confirmed that IL-4 and IL-5 were inhibited by about 80% or more even at 10 μg/ml of the cell wall extract.

Comparative Experimental Example 1: Check the effect of cell membrane components

(1) Preparation of cell membrane components

Diinococcus radiodurans #50 strain pellet was suspended in 0.1M sodium citrate (pH 4.7) aqueous solution and crushed by sonication. The bacterial eluate was mixed with n-butanol in a 1:1 ratio, stirred at room temperature for 30 minutes, and then centrifuged at 18,800xg for 15 minutes to obtain an aqueous layer. The aqueous layer obtained by repeating the n-butanol extraction method three times is dialyzed in water for injection to remove salt and freeze-dried. Concentrations of 0.1 mg/ml and 1 mg/ml in pyrogen-free water. was suspended with

(2) Check the effect

In the allergic inflammatory response model established in 2. in order to investigate the ability of the cell membrane component of each strain obtained in (1) above to modulate the allergic inflammatory response, each cell wall extract was added at 10 μg/ml to the splenocytes upon restimulation of the OVA antigen. and 30 μg/ml were simultaneously treated, and the production of IL-4, an allergic inflammatory response mediator, in the supernatant after 4 days was confirmed through the ELSIA method.

As a result, the cell membrane component of the Diinococcus radiodurans #50 strain did not significantly inhibit IL-4 production as can be seen in FIG. 3 .

3. allergy Effect on Response

Experimental example 2: Confirmation of the effect of cell wall extract

D. radiocurans according to the process described in 1. R1, #50 and In order to investigate the ability of cell wall extracts isolated in #54 to control the allergic response, in the allergic inflammatory response model established in 2., splenocytes were simultaneously treated with 30 μg/ml of each cell wall extract at the time of restimulation with OVA antigen. 4 days The generation of IFN-γ, which is an anti-allergic reaction mediator, was confirmed in the supernatant after the ELSIA method, and the results are shown in FIG. 4 .

As a result, it was confirmed that each cell wall extract derived from Diinococcus radiodurans R1, #50, #54 increased the production of IFN-γ, an anti-allergic response mediator. As can be seen in FIG. 4 , it was found that the production of IFN-γ was increased by about 3 to 5 times at 30 μg/ml of the cell wall extract.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13953BP

Deposit date: 20190917

Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13954BP

Deposit date: 20190917

Claims (13)

delete delete delete delete delete delete delete delete Deinococcus radiodurans (Deinococcus radiodurans) Anti-allergic composition comprising a cell wall extract.
The anti-allergic composition according to claim 9, wherein the anti-allergic composition is a pharmaceutical composition for improving, treating, or preventing allergic diseases.
11. The method of claim 10, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis (allergic conjunctivitis), allergic dermatitis (allergic dermatitis), allergic contact At least one selected from the group consisting of allergic dermatitis (allergic contact dermatitis), and allergic keratitis, an anti-allergic composition.
10. The method of claim 9, wherein the anti-allergic composition is a food composition, anti-allergic composition.
10. The method of claim 9, wherein the anti-allergic composition is a cosmetic composition, anti-allergic composition.

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