KR20230007608A - Pharmaceutical composition for preventing or treating oral inflammatory disease comprising nucleic acid of Pediococcus acidilactici - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases. More specifically, the present invention includes gDNA of Pediococcus acidilactici to have an inhibitory effect on inflammatory mediators, thereby helping prevent, treat, or alleviate inflammatory diseases.

Description

Pharmaceutical composition for preventing or treating oral inflammatory disease comprising nucleic acid of Pediococcus acidilactici

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases in the oral cavity.

The present invention relates to paraprobiotics preparations.

Due to outbreaks of diseases caused by infection with pathogenic microorganisms worldwide, interest in the immune functional food market is continuously expanding. Due to current trends, probiotics are in the limelight in the related market due to various effects such as improving immune function and anti-inflammatory effect. In addition, it is reported that interest in probiotics will continue steadily according to experts' predictions of prolonged COVID-19. Accordingly, probiotics-related products are leading the expansion of the health functional food market in Korea. According to the statistics of the Korea Health Functional Food Association, probiotics in the functional food market in 2020 increased by nearly 20% from the previous year (741.5 billion won) to 885.6 billion won, showing a high share in the market. In addition, probiotics-related products such as prebiotics, postbiotics, and paraprobiotics are being released and related research is increasing.

Representative probiotics include Lactobacillus sp., Bifidobacterium sp., Bacillus sp., and Pediococcus sp. These probiotics must not be harmful to the host as live cells, and must have acid resistance, bile resistance, and excellent adhesion in the intestine to survive in the stomach and intestines. To this end, probiotics must be equipped with a complex coating process such as encapsulation, and low-temperature distribution is required to maintain a viable cell state. In order to maintain such a high concentration of probiotics, cost issues have emerged in the production and distribution process, and since they are taken as probiotics, there is a limitation that they cannot be used in various formulations. In addition, reports of severe adverse events such as neonatal necrotizing enterocolitis, sepsis, abdominal pain, diarrhea, hives, abdominal distension, and immune imbalance continue to be reported when taken by people with weak immunity, infants, or the elderly. In addition to this, some probiotics have antibiotic resistance genes, so there is a problem that they are recognized as potentially dangerous strains. In order to compensate for this, research on paraprobiotics, which can produce probiotic effects using active substances of lactic acid bacteria rather than live bacteria, is increasing.

Paraprobiotics are collectively referred to as paraprobiotics, substances having biological activity isolated from probiotic strains. Research is being conducted on this as a material that can overcome the limitations of probiotics, a health functional food material, and secure efficacy and safety. Representative paraprobiotics include cell wall peptidoglycan, cytoplasmic proteins and peptides, and nucleic acids, and according to current studies, it has been reported that their efficacy is similar or even more excellent when compared to probiotics. Currently, various domestic companies are researching and developing paraprobiotics as an alternative to probiotics with the advantage that there are no problems with probiotics such as maintenance of live bacteria, cost in the manufacturing and distribution process, and interaction with the host. Until now, it is still incomplete.

Korean Registered Patent No. 2207872

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

An object of the present invention is to provide a quasi-drug composition for preventing or improving inflammatory diseases.

An object of the present invention is to provide a food composition for preventing or improving inflammatory diseases.

An object of the present invention is to provide a paraprobiotics preparation.

1. A pharmaceutical composition for preventing or treating inflammatory diseases containing gDNA (genomic DNA) of Pediococcus acidilactici .

2. The prevention of inflammatory diseases according to the above 1, wherein the Pediococcus acidilactitis is at least one of Pediococcus acidilactis K10 (Accession No. KCTC14575BP) and Pediococcus acidilactish HW01 (Accession No. KCTC14576BP) or a pharmaceutical composition for treatment.

3. The pharmaceutical composition for preventing or treating inflammatory diseases according to 1 above, wherein the inflammation is oral inflammation.

4. A quasi-drug composition for preventing or improving inflammatory diseases containing gDNA (genomic DNA) of Pediococcus acidilactici .

5. A food composition for preventing or improving inflammatory diseases containing gDNA (genomic DNA) of Pediococcus acidilactici .

6. A paraprobiotic preparation containing gDNA (genomic DNA) of Pediococcus acidilactici .

The pharmaceutical composition, quasi-drug composition, food composition, and paraprobiotics preparation of the present invention can help prevent, treat, or improve inflammatory diseases because of their inhibitory effect on inflammatory mediators.

1 is a result of confirming that the material isolated from Pediococcus acidilactici is DNA.
Figure 2 is a result of confirming the optimal processing time conditions for the P. acidilactici K10 gDNA and the P. acidilactici HW01 gDNA for the Porphyromonas gingivalis induced inflammatory response inhibitory effect.
3 shows the results of confirming the inhibitory effect of Escherichia coli -induced inflammatory response of P. acidilactici K10 gDNA and P. acidilactici HW01 gDNA.
4 is a result of confirming the optimal treatment concentration conditions for the P. acidilactici K10 gDNA and the P. acidilactici HW01 gDNA for the effect of inhibiting the Porphyromonas gingivalis-induced inflammatory response.
Figure 5 shows the effect of P. acidilactici K10 gDNA and P. acidilactici HW01 gDNA on Porphyromonas gingivalis-induced inflammatory response inhibition compared to the inflammatory response inhibition effect of P. acidilactici K10 and P. acidilactici HW01 whole bacteria. It is the result of confirming that it is excellent.
6 is a result of confirming the intracellular signal transduction mechanism of the inflammatory response inhibitory activity of P. acidilactici K10 gDNA and P. acidilactici HW01 gDNA.

Hereinafter, the present invention will be described.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising gDNA (genomic DNA) of Pediococcus acidilactici .

Pediococcus ecdylactis Pediococcus ecdylactis at least one of K10 and Pediococcus aesidiractici HW01.

Pediococcus ecdylactish K10 was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC, Korean Collection for Type Cultures) and registered as accession number KCTC14575BP.

Pediococcus esidilactici HW01 was deposited at the Korea Research Institute of Bioscience and Biotechnology (KCTC, Korean Collection for Type Cultures) and registered under the accession number KCTC14576BP.

The gDNA of Pediococcus escidiractici can be extracted by the following method.

(a) adding lysozyme to the culture medium of the strain;

(b) adding a nuclear membrane dissolving solution to the material obtained in step (a) and heating;

(c) adding RNase to the material obtained in step (b);

(d) adding a protein precipitating solution to the material obtained in step (c) and obtaining a supernatant; and

(e) obtaining a DNA fraction in the supernatant with an alcohol solvent.

In step (e), the alcohol solvent may be isopropanol or ethanol.

The step (e) may be mixing the supernatant with isopropanol and centrifuging to obtain a DNA precipitate, washing the DNA precipitate with ethanol, centrifuging, and evaporating the ethanol to obtain a DNA fraction.

The method may further include (f) hydrating and concentrating the obtained DNA fraction.

In step (a), the culture solution of the strain may be obtained by culturing the Pediococcus escidilactici strain in a medium and then centrifuging to remove the supernatant.

According to one embodiment, Pediococcus aesidiractici K10 or HW01 may exhibit an IL-1β inhibitory effect induced by lipopolysaccharide (LPS) of Porphyromonas gingivalis .

According to one embodiment, Pediococcus aesidiractici The gDNA of K10 or HW01 may exhibit an inhibitory effect on IL-1βIL-6 and MCP-1 induced by LPS of Porphyromonas gingivalis or Escherichia coli .

The pharmaceutical composition of the present invention has an excellent preventive or therapeutic effect on inflammatory diseases by inhibiting inflammatory mediators.

Inflammation mediators are messengers that act on blood vessels or cells to promote sequential inflammatory responses, for example, Porphyromonas gingivalis or Escherichia coli virulence factor LPS (lipopolysaccharide) may be induced by

Inflammation mediating factors may be specifically cytokines, chemokines, and the like, and more specifically, IL-1βIL-6 and MCP-1.

gDNA refers to genomic DNA as the total DNA in a cell. The Pediococcus escidilactici gDNA included in the pharmaceutical composition of the present invention has a remarkably superior anti-inflammatory effect compared to the case of using the Pediococcus escidilactici itself. In addition, in the case of using Pediococcus escidilactici gDNA, it is possible to overcome the cost problem in the case of using live bacteria or the limitation that it cannot be used in various formulations.

The inflammatory disease may be induced by lipopolysaccharide (LPS), a virulence factor of Porphyromonas gingivalis or Escherichia coli .

The inflammatory disease may be selected from the group consisting of oral inflammation, arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, and degenerative neuroinflammation. there is.

Inflammation in the oral cavity may be inflammation induced by Porphyromonas gingivalis . Since the LPS of Porphyromonas gingivalis affects periodontitis or gingivitis, more specifically, the inflammatory disease may be periodontitis or gingivitis.

The term “prevention” refers to prophylactic measures that result in any degree of reduction in the likelihood of developing a condition to be prevented or a recurrence or recurrent condition, including minor, substantial or major reduction in the likelihood of developing or recurring the condition, as well as total prevention. Refers to a measure, and the degree of likelihood reduction is at least a minor reduction.

The term "treatment" refers to treatment that results in a beneficial effect on a subject or patient suffering from the condition being treated, including not only cure but also relief of any degree, including minor, substantial, major relief, the degree of relief being At least a slight relief.

The pharmaceutical composition of the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. It may, but is not limited thereto.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc., such solid preparations may contain at least one excipient such as starch or calcium carbonate in the compound. It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

In the pharmaceutical composition of the present invention, the effective amount may vary depending on the patient's age, sex, and body weight, and is generally 1 to 6000 mg per kg of body weight, preferably 60 to 600 mg may be administered once or divided into three times. there is. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

The present invention provides a quasi-drug composition for preventing or ameliorating inflammatory diseases containing gDNA (genomic DNA) of Pediococcus acidilactici .

The quasi-drug composition of the present invention has an excellent effect of preventing or improving inflammatory diseases by inhibiting inflammatory mediators.

Pediococcus esidilactitis, gDNA, inflammation, inflammatory mediators, and the term "prevention" may be within the foregoing scope, but are not limited thereto.

The term "improvement" means any action that at least reduces the severity of a parameter associated with the condition being treated, eg a symptom.

The quasi-drug composition may be a quasi-drug for oral use. Ingredients included in the quasi-drug composition are active ingredients, and may include ingredients commonly used in oral quasi-drug compositions in addition to the above active ingredients, such as abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, medicinal ingredients, flavoring agents, Colorants, solvents, brighteners, solubilizers or pH adjusters may be included.

The quasi-drug composition for oral use may be prepared in any formulation commonly manufactured in the art, for example, toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, mouthwash. And it may have a formulation such as gum massage cream, but is not limited thereto.

As an example, when the oral quasi-drug composition is a toothpaste formulation, it may include a wetting agent, an abrasive, a binder, a foaming agent, a flavoring agent, a sweetening agent, a coloring agent, a preservative, a medicinal component, a solvent, a pH adjusting agent, and the like.

The humectant is to make the powder of toothpaste ingredients paste and to prevent the toothpaste from hardening in the air, and glycerin, sorbitol, propylene glycol, polyethylene glycol, etc. alone or in combination of two or more, 1 to 60% by weight of the total weight of the composition , Specifically, 10 to 50% by weight may be used.

The foaming agent diffuses toothpaste into the oral cavity to increase the cleaning effect and acts as a surfactant to clean oral contamination. 0.5 to 10% by weight, specifically 0.5 to 5% by weight of the total weight of the composition may be used by mixing two or more kinds.

The binder prevents separation between the powder and liquid components in the toothpaste, and is composed of cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylcellulose, and sodium alginate, carrageenan, and xanthan gum alone or in combination of two or more. 0.1 to 5% by weight, specifically 0.3 to 2% by weight of the total weight may be used.

Abrasives remove the attachments on the tooth surface without damaging the tooth surface and restore _the original _{gloss of} the _{tooth .} H ₂ O), aluminum hydroxide (Al(OH) ₃ ), potassium pyrophosphate, magnesium carbonate, etc. alone or in combination of two or more, 1 to 60% by weight, specifically 10 to 50% by weight of the total weight of the composition can

The flavoring agent is to enhance the feeling of use by imparting a refreshing feeling and smell to the toothpaste, and is 0.01 to 60% by weight, specifically 0.01 to 5, of the total weight of the composition by mixing peppermint oil, spearmint oil, menthol, etc. alone or in combination of two or more. Weight percent can be used.

The sweetener is to remove the unpleasant taste caused by the toothpaste raw material and improve the refreshing feeling, and 0.01 to 60% by weight of the total weight of the composition, specifically 0.01 ~ 5% by weight can be used.

The active ingredients are for the prevention of dental caries, periodontal disease, toothache prevention, cold tooth prevention, bad breath removal, etc. Triclosan, tocopherol acetate, sodium monofluorophosphate, etc. may be used alone or in combination of two or more. In the present invention, the compound may be used as an additional active ingredient.

The quasi-drug composition may be used alone or overlapping, or overlapping with other quasi-drug compositions other than the present invention.

The present invention provides a food composition for preventing or alleviating inflammatory diseases containing gDNA (genomic DNA) of Pediococcus acidilactici .

The food composition of the present invention has an excellent effect of preventing or improving inflammatory diseases by inhibiting inflammatory mediators.

Pediococcus ecdylactitis, gDNA, inflammation, inflammatory mediators, the term "prevention" and the term "improvement" may be within the foregoing scope, but are not limited thereto.

The food composition may be prepared and processed in the form of an orally disintegrating film, tablet, capsule, powder, granule, liquid, pill or the like for the purpose of preventing or improving inflammatory diseases.

A food composition may contain conventional food additives, and the suitability as a food additive is determined according to the standards and standards for the item in accordance with the General Rules of the Code of Conduct for Food Additives and General Test Methods approved by the Korea Food and Drug Administration, unless otherwise specified. judged by

Items listed in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, alkali additives for noodles, preservative preparations, and tar color preparations.

For example, a food composition in the form of an oral disintegrating film may be prepared in the form of a film by mixing the composition with a film-forming base, and if necessary, a thickener, an emulsifier, a plasticizer, a sweetener, a flavoring agent, or an antifoaming agent may be further included as additives. can be manufactured

A food composition in tablet form may be granulated by a conventional method of mixing the composition with an excipient, a binder, a disintegrant, and other additives, and then compression-molded by adding a lubricant or the like, or the mixture may be directly compression-molded. . In addition, the food composition in the form of a tablet may contain a flavoring agent and the like, if necessary. Food compositions in the form of tablets may include chewable tablets, orally disintegrating tablets, uncoated tablets, film-coated tablets, coated tablets, multi-layered tablets, press-coated tablets, inner core tablets, effervescent tablets, dispersed tablets, dissolving tablets, and the like.

Among food compositions in the form of capsules, hard capsules can be prepared by filling a mixture obtained by mixing the composition with additives such as excipients in a conventional hard capsule, and soft capsules are a mixture obtained by mixing the composition with additives such as excipients. It can be prepared by filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like, if necessary.

The food composition in the form of a ring may be prepared by molding a mixture obtained by mixing the composition with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and, if necessary, may be coated with sucrose or other coating agent, or starch, The surface can also be coated with a material such as talc.

A food composition in the form of a granule may be prepared by a conventionally known method of a mixture of the composition and an excipient, a binder, a disintegrant, etc., and may contain a flavoring agent, a flavoring agent, and the like, if necessary.

Food compositions include processed sugar products, jellies, soft candies, candies, beverages, meat, chocolate, foods, and confectionery. It may be pizza, ramen, other noodles, chewing gum, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

The food composition may be applied orally for use as a nutrient, and the application form is not particularly limited. For example, when administered orally, the daily intake is preferably 5000 mg or less, more preferably 2000 mg or less, and most preferably 500 to 1500 mg or 650 mg daily. When formulated into capsules or tablets, one tablet can be administered with water once a day.

The present invention provides a paraprobiotics preparation containing gDNA (genomic DNA) of Pediococcus acidilactici .

The paraprobiotics preparation of the present invention inhibits inflammatory mediators and has an excellent effect of preventing or improving inflammatory diseases.

Pediococcus escidilactici, gDNA, inflammation and inflammation mediators may be within the above ranges, but are not limited thereto.

Paraprobiotics refers to substances with biological activity isolated from probiotic strains.

As described above, Pediococcus escidilactici gDNA has a remarkably superior anti-inflammatory effect compared to the case of using Pediococcus escidilactici itself. In addition, in the case of using Pediococcus escidilactici gDNA, it is possible to overcome the cost problem in the case of using live bacteria or the limitation that it cannot be used in various formulations.

Paraprobiotics preparations can be prepared and administered in various formulations and methods according to methods known in the art. For example, the gDNA of Pediococcus escidilactisi included in the paraprobiotics preparation of the present invention is mixed with carriers commonly used in the pharmaceutical field to form powders, liquids and solutions, and tablets. ), capsules, syrups, suspensions, or granules. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant and a flavoring agent, but is not limited thereto. In addition, the administration dose can be appropriately selected according to the degree of absorption of the active ingredient in the body, inactivity rate, excretion rate, age, sex, animal species, condition and severity of the disease of the recipient to be administered.

Paraprobiotics preparations can be used as food additives. In this case, the paraprobiotics may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to its purpose of use (prevention, health or therapeutic treatment). In general, the paraprobiotics of the present invention are added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on raw materials during production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or health control, the mixed amount may be less than the above range, and since there is no problem in terms of safety, the mixed amount may be used in an amount greater than the above range. There is no particular restriction on the type of food to which paraprobiotics preparations can be added, and examples include bread, candy, snacks, confectionery, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks and vitamin complexes, etc. There is, and includes all health foods in the usual sense.

Hereinafter, examples will be described in detail to explain the present invention in detail.

Example

Materials and Methods

Nucleic acid extracts of Pediococcus acidilactici K10 and Pediococcus acidilactici HW01 were obtained through the following steps.

One. Pediococcus acidilactici K10 and Pediococcus acidilactici Steps for culturing HW01

In order to culture and prepare appropriate concentrations of lactic acid bacteria for the DNA fraction of domestic lactic acid bacteria, Pediococcus acidilactici K10 and Pediococcus acidilactici HW01, native strains were cultured in MRS liquid medium to 2 × 10 CFU/mL.

2. Step of concentrating and obtaining cultured bacteria

The bacteria cultured to 2 × 10 CFU/mL were centrifuged at 14000 x g for 2 minutes using a centrifuge, and the supernatant was removed to concentrate the bacteria.

3. Fractionation of active substance, gDNA, from bacteria

EDTA and Lysozyme were added to the concentrated bacteria and allowed to stand at 37°C for 1 hour. Thereafter, the supernatant was removed by centrifugation at 14,000 x g for 2 minutes using a centrifuge, heated at 80 ° C for 5 minutes using a nuclear membrane lysis solution, and cooled at room temperature. After mixing with RNase, the mixture was left at 37°C for 1 hour and then cooled at 4°C. Then, after adding the protein precipitation solution, centrifugation was performed at 14000 x g for 3 minutes, and only the supernatant was taken. The supernatant was mixed with isopropanol and centrifuged to obtain a DNA precipitate. The DNA precipitate was treated with 70% ethanol to wash impurities mixed with DNA. DNA was extracted by evaporating ethanol after centrifuging the DNA precipitate treated with ethanol.

4. Concentrating the fractionated gDNA

The obtained DNA fraction was rehydrated and concentrated to obtain pure gDNA.

result

1. Lactobacillus Active Substance Separation

1-1. Electrophoresis results

Using 10 μL of Dnase I (1 μg/mL, containing MgCl2 5 mM) to 10 μL of gDNA (300 μg/mL) isolated from Pediococcus acidilactici K10 and Pediococcus acidilactici HW01, the gDNA was electrophoresed on a 1% agarose gel. It was confirmed and the results are shown in Figure 1.

As shown in FIG. 1A, it can be confirmed that the band completely disappeared in the group treated with DNase, indicating that the band confirmed by electrophoresis is pure DNA.

1-2. gDNA purity measurement result

The purity of DNA is determined by the absorbance at 260 nm, and the purity is confirmed by the ratio to the absorbance at 280 nm. If the value of 260nm / 280nm is between 1.8 and 1.9, it is pure DNA. In this experiment, as shown in Table 1 below, it was confirmed that it was pure DNA with K10 1.88 and HW01 1.86, and this was used in all experiments.

Sample ID Nucleic Acid Conc.
(nucleic acid concentration) 260nm/280nm K10 347.3 1.88 HW01 301 1.86

2. Confirmation of the inflammatory response modulating effect in macrophages of mice

2-1. Establish optimal processing time conditions

After pre-treatment with Pediococcus acidilactici K10 and Pediococcus acidilactici HW01 gDNA extracts isolated from macrophages (RAW 264.7, mouse macrophage cell line) at different times (3, 9, 15 hours), P. gingivalis LPS (1 μg/mL) was applied for 3 hours. processed. Then, total RNA was isolated, cDNA was synthesized, and expression changes of various inflammation-related cytokines/chemokines (IL-1β, IL-6, MCP-1) were confirmed using qRT-PCR to establish optimal processing time conditions. .

As a result of confirming various inflammatory response mediators as shown in the results of FIG. 2, it was confirmed that the highest inflammatory response control effect was obtained under the 15-hour pretreatment condition. Based on this result, the efficacy evaluation experimental condition was established as a 15-hour pretreatment.

2-2. P. acidilactici K10 and P. acidilactici HW01 gDNA E. coli Inhibiting effect of LPS-induced inflammatory response

Mouse macrophages were pretreated with P. acidilactici K10 and P. acidilactici HW01 gDNA extracts for 15 hours and E. coli LPS (1 μg/mL) for 3 hours, and total RNA was isolated to synthesize cDNA. Expression changes of various inflammation-related cytokines/chemokines (IL-1β, IL-6, MCP-1) induced by LPS of E. coli, a representative pathogen, were confirmed by qRT-PCR, and P. acidilactici K10, P. acidilactici HW01 gDNA was evaluated for its inflammatory response modulating effect.

As shown in FIG. 3, as a result of confirming the inflammatory response mediators, the regulatory efficacy of P. acidilactici K10 and P. acidilactici HW01 gDNA on the inflammatory response induced by LPS of E. coli under 15-hour pretreatment conditions was confirmed, This suggests the regulatory effect of P. acidilactici K10 and P. acidilactici HW01 gDNA on the inflammatory response induced by various pathogenic bacteria.

2-3. Establish optimal treatment concentration conditions

P. acidilactici K10 and HW01 gDNA extracts isolated from macrophages (RAW 264.7, mouse macrophage cell line) were pretreated at different concentrations (0.1, 1, and 10 μg/mL), and then P. gingivalis LPS (1 μg/mL) was added. It was treated for 3 hours. Then, total RNA was isolated, cDNA was synthesized, and expression changes of various inflammation-related cytokines/chemokines (IL-1β, IL-6, MCP-1) were confirmed using qRT-PCR to establish optimal treatment concentration conditions. .

As a result of confirming various inflammatory response mediators as shown in the results of FIG. 4, it was confirmed that the highest inflammatory response regulating effect was obtained under the 1 μg/mL concentration condition. Based on this result, the concentration condition of the gDNA extract of P. acidilactici K10, HW01 was established as 1 µg/mL.

2-4. P. acidilactici K10 and P. acidilactici HW01 gDNA P. gingivalis Inhibiting effect of LPS-induced inflammatory response

The efficacy of P. acidilactici K10 and P. acidilactici HW01 gDNA in regulating the inflammatory response induced by P. gingivalis LPS was evaluated. Rat macrophages were pretreated with 1 μg/mL of gDNA extract of K10 and HW01 for 15 hours, pretreated with K10 and HW01 whole bacteria 10 ⁷ CFU/mL for 15 hours, and then treated with 1 μg/mL of P. gingivalis LPS for 24 hours, and the supernatant was After obtaining, changes in the expression of inflammatory cytokines and chemokines (IL-1β, IL-6, MCP-1) were confirmed using ELISA.

2-4. P. acidilactici K10 and P. acidilactici HW01 gDNA P. gingivalis Inhibiting effect of LPS-induced inflammatory response

The efficacy of P. acidilactici K10 and P. acidilactici HW01 gDNA in regulating the inflammatory response induced by P. gingivalis LPS was evaluated. Rat macrophages were pretreated with 1 μg/mL of gDNA extract of K10 and HW01 for 15 hours, pretreated with K10 and HW01 whole bacteria 10 ⁷ CFU/mL for 15 hours, and then treated with 1 μg/mL of P. gingivalis LPS for 24 hours, and the supernatant was After obtaining, changes in the expression of inflammatory cytokines and chemokines (IL-1β, IL-6, MCP-1) were confirmed using ELISA.

2-5. P. acidilactici K10 and P. acidilactici Intracellular signal transduction mechanism of inflammation modulating effect by HW01 gDNA

Cell signal transduction mechanisms of P. acidilactici K10 and P. acidilactici HW01 gDNA on the inflammatory response induced by P. gingivalis LPS were identified. Rat macrophages were pre-treated with 1 μg/mL of gDNA extracts of K10 and HW01 for 15 hours, and then treated with 1 μg/mL of P. gingivalis LPS for 24 hours to induce MAPKs (Mitgen-activated protein kinases) such as ERK, p38, and JNK, Iκbα was confirmed.

As shown in Figure 6, the intracellular mechanism of the gDNA extracts of P. acidilactici K10 and P. acidilactici HW01 suppresses the inflammatory response through MAPKs (Mitgen-activated protein kinases) such as MAPKs, ERK, p38, and JNK, and NF-κB It can be confirmed that

Korea Research Institute of Bioscience and Biotechnology KCTC14575BP 20210520 Korea Research Institute of Bioscience and Biotechnology KCTC14576BP 20210520

Claims (6)

A pharmaceutical composition for preventing or treating inflammatory diseases comprising gDNA (genomic DNA) of Pediococcus acidilactici .
The method according to claim 1, wherein the Pediococcus acidilactic acid is at least one of Pediococcus acidilactic acid K10 (Accession No. KCTC14575BP) and Pediococcus acidic acid HW01 (Accession No. KCTC14576BP) Prevention or treatment of inflammatory diseases pharmaceutical composition for use.
The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1, wherein the inflammation is oral inflammation.
A quasi-drug composition for preventing or improving inflammatory diseases comprising gDNA (genomic DNA) of Pediococcus acidilactici .
A food composition for preventing or improving inflammatory diseases comprising gDNA (genomic DNA) of Pediococcus acidilactici .
A paraprobiotics preparation containing gDNA (genomic DNA) of Pediococcus acidilactici .

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