KR102451860B1 - A probiotic microbial agent for preventing or improving the loss of alveolar bone - Google Patents

Abstract

The present invention relates to a probiotic microbial preparation for preventing or improving alveolar bone loss, wherein the oral lactic acid bacteria Weissella cibaria CMU (oraCMU) contains 10 ⁸ to 10 ¹⁰ CFU (colony forming unit) per 1 g. The microbial probiotic preparation for preventing or improving alveolar bone loss of the present invention, characterized in that it is contained in a vegetable oil base in an amount and prepared as a concentrate, is applied to a place that induces periodontitis and acts locally, thereby pro-inflammatory cytokines in the inflamed tissue It has the effect of inhibiting alveolar bone loss by inhibiting phosphorus TNF-α and IL-1ßIL-6 and reducing the number of total bacteria. In particular, because live lactic acid bacteria are used as oral care probiotics (OCPs), it is possible to control even harmful bacteria that live in blind spots where toothbrushes or gargles do not reach while living in the oral cavity when ingested. The microbial probiotic of the present invention is a natural-friendly next-generation oral care solution that has no side effects that can fundamentally prevent and improve periodontal disease by selectively controlling harmful bacteria and restoring the balance of the broken microflora in the oral cavity.

Description

Probiotic microbial agent for preventing or improving the loss of alveolar bone {A probiotic microbial agent for preventing or improving the loss of alveolar bone}

The present invention relates to a probiotic microbial probiotic preparation for preventing or improving alveolar bone loss, and more specifically, a microorganism for preventing or improving alveolar bone loss, including a novel oral lactic acid bacterium Weissella cibaria CMU (oraCMU). It relates to probiotics.

Periodontal disease refers to symptoms related to inflammatory diseases that destroy soft tissues such as periodontal ligaments and gingiva surrounding teeth and hard tissues such as alveolar bone.

Periodontal disease occurs when congenital or acquired alveolar bone deteriorates with increasing age, and typical periodontal diseases include periodontal inflammation, gingivitis, and alveolar bone osteodystrophy. Gingivitis is an early periodontal disease in which the soft tissues of the gums are inflamed, and the symptoms are relatively mild and the recovery is reversible. Periodontitis is a case in which gingivitis has not been treated and the inflammation has progressed to the gums and pubic area. When a chronic inflammatory reaction stimulated by bacterial toxins progresses, an infected periodontal pocket is formed and separated between the gums and teeth. The more severe it is, the deeper the periodontal pocket becomes, and eventually, the periodontal ligament becomes inflamed and bone loss occurs. In alveolar bone, when the metabolism of bone formation by osteoblasts and bone resorption by osteoclasts in alveolar bone exceeds bone formation and the bone mass decreases below the limit due to various factors, alveolar bone formation disorders such as alveolar osteoporosis occur.

Among these periodontal diseases, periodontitis is not only one of the most common oral diseases in the adult population worldwide among the diseases that can occur in the oral cavity, but also a major public health concern that imposes a significant cost on the medical management system.

For periodontal disease, prevention through the establishment of improved oral hygiene is the most important, and in clinical practice, surgical treatment such as non-surgical or surgical calculus removal, root planing, gingivectomy, and periodontal tissue regeneration using renal attachment is the most important. is predominantly However, these surgical treatment methods are cumbersome, difficult to treat effectively, and are limited to treatment performed when the disease has progressed to some extent rather than prevention of the disease. In addition, although systemic antibiotics and topical sustained-release preparations are used as additional treatments, excessive drug delivery to unnecessary areas is causing side effects. reported to cause

In addition, in the progression of periodontal disease, an increase in inflammatory mediators such as NO, IL-1β, MMP-1, and TNF-α has been observed. As it turned out, studies related to periodontal disease to date are mostly related to the regulation of the inflammatory mediators. In addition, periodontal disease has a complex characteristic caused by the action of periodontal disease bacteria such as loss of alveolar bone, inflammation, gingivalis, etc., unlike general inflammation and arthritis, etc. A complex study and treatment method, such as exhibiting an action to inhibit bone resorption, is required, but drug research or treatment for such treatment has not yet been developed.

Under this background, there is a need for the development of drugs or therapeutic agents that can effectively prevent and treat periodontal disease. And the need for the development of therapeutic compositions is gradually increasing.

Therefore, the present inventors prevent periodontal tissue loss due to periodontitis, and while researching a substance that can promote periodontal tissue regeneration, oral lactic acid bacteria Weissella cibaria CMU (oraCMU) induces periodontitis in a mouse model. It was confirmed that TNF-alpha, IL-1beta, and IL-6, which are pro-inflammatory cytokines, were inhibited in the inflamed tissue by applying locally to the affected area, and the effect of suppressing alveolar bone loss by reducing the total number of bacteria By confirming that this exists, the present invention was completed.

The present invention is to provide a microbial probiotic preparation for preventing or improving alveolar bone loss.

In order to solve the above technical problem, in the present invention, oral lactic acid bacteria Weissella cibaria CMU; oraCMU) is included in the vegetable oil base in an amount of 10 ⁸ to 10 ¹⁰ CFU (colony forming unit) per 1 g. It provides a microbial probiotic preparation for preventing or improving alveolar bone loss, characterized in that it is prepared as a concentrate.

According to one embodiment of the present invention, the vegetable oil base extends the survival period of lactic acid bacteria and is any one of sunflower seed oil, coconut oil, olive oil, peanut oil, sesame oil, medium-chain neutral fatty acid, D-alpha-tocopherol, or It is characterized in that it is a mixture of these.

According to one embodiment of the present invention, the concentrate is characterized in that it is prepared in the form of a capsule.

According to one embodiment of the present invention, the concentrate is characterized in that it is prepared in the form of gauze so that it can seep into the periodontal disease site.

According to one embodiment of the present invention, the concentrate is characterized in that it is prepared in the form of a patch that can be attached to the periodontal disease site.

According to one embodiment of the present invention, the concentrate is prepared in the form of liquid drops.

According to one embodiment of the present invention, it is characterized in that it is prepared in the form of a gum including the concentrate mixed with the vegetable extract in the vegetable oil base.

According to one embodiment of the present invention, oral lactic acid bacteria Weissella cibaria CMU (oraCMU) is a lactic acid bacterium, characterized in that it binds to anaerobic bacteria causing bad breath to prevent tooth support bone (alveolar bone) loss. In addition, the present invention is characterized in that it relates to the utilization of Weissella cibaria CMU (oraCMU) KCTC 10650BP, a lactic acid bacterium of the Weissella genus that binds to anaerobic bacteria that cause bad breath and generates hydrogen peroxide in aerobic and anaerobic conditions.

According to a specific embodiment of the present invention, a cell (RAW 264.7 cell) and animal (ICR mouse) evaluation model was established for evaluating the periodontal disease improvement effect of oral lactic acid bacteria oraCMU, and oraCMU in an in vitro model using RAW 264.7 cells. did not have cytotoxicity, inhibited NO production following the induction of LPS, a periodontitis-inducing substance, and confirmed the reduction of TNF-α and IL-1ßIL-6, the inflammation-related cytokines of RAW 264.7 cells, in both live and dead cells.

According to a specific embodiment of the present invention, in an in vivo model using ICR mouse, oraCMU reduced alveolar bone loss by 36.3% compared to the control group, and P. gingivalis , a periodontitis-causing bacterium, was not detected at all in the teeth of the oraCMU intake group, In addition, it was confirmed that total bacteria in the gingival tissue and teeth were also reduced, and the expression of TNF-α, IL-1β, and IL-6, which are factors related to periodontal inflammation, was significantly decreased in the oraCMU ingested group.

In the present invention, the periodontal disease may include gingivitis, periodontal inflammation, alveolar bone breakage, alveolar bone osteodystrophy, and the like, and the alveolar osteodystrophy is alveolar osteoporosis ( alveolar bone osteoporosis), alveolar bone osteomalacia, or alveolar bone osteopenia.

The probiotic preparation for preventing or treating periodontal disease of the present invention may be in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injection solutions according to conventional methods, and powders, tablets, capsules, injections and It is more preferable that it is a liquid preparation. Such formulation may be carried out by a method conventionally performed in the pharmaceutical field, and is preferably formulated according to each disease or added ingredient using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA). can do.

The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like.

Solid preparations for oral use include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose. ), gelatin, and the like, and lubricants such as magnesium stearate and talc. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and may include water and diluents such as liquid paraffin, wetting agents, sweetening agents, fragrances, and preservatives.

In the oral preparation of the present invention, in addition to the above formulations such as powders, tablets, capsules, etc., it may be formulated as, for example, a film, toothpaste, mouthwash, ointment, spray, dressing solution, coating agent, dental floss or periodontal sack.

Parenteral preparations include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, and freeze-dried preparations, and non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, ethylol injectable esters such as lactate; and the like.

The microbial probiotic preparation of the present invention can be orally applied or administered according to a desired method, and the dosage depends on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease. The range varies accordingly.

The microbial probiotic preparation of the present invention can be used alone or in combination with methods using surgery and drug treatment for the prevention and treatment of periodontal disease.

The microbial probiotic preparation containing Weissella cibaria CMU (oraCMU) of the present invention is applied to a place that induces periodontitis and acts locally, thereby pro-inflammatory cytokines TNF-α, IL in the inflamed tissue. It has the effect of inhibiting alveolar bone loss by inhibiting -1ß IL-6 and reducing the number of total bacteria. In particular, because live lactic acid bacteria are used as oral care probiotics (OCPs), it is possible to control even harmful bacteria that live in blind spots where toothbrushes or gargles do not reach while living in the oral cavity when ingested. The microbial probiotic of the present invention is a natural-friendly next-generation oral care solution that has no side effects that can fundamentally prevent and improve periodontal disease by selectively controlling harmful bacteria and restoring the balance of the broken microflora in the oral cavity.

1 is a result of confirming the safety of cytotoxicity in RAW 264.7 cells of Weissella cibaria CMU (oraCMU).
2 is a graph showing the efficacy of inhibiting LPS-induced NO production according to oraCMU treatment in RAW 264.7 cells.
Figure 3 shows the evaluation of the efficacy of oraCMU treatment in the in vivo periodontal disease model.
4 is a graph showing the distance from the maxillary crest boundary to the alveolar ridge (CEJ-ABC) of the distal surface of the maxillary second molar for each experimental group.
5 is a graph showing the distance from the maxillary crest boundary to the alveolar ridge (CEJ-ABC) of the mesial surface of the maxillary second molar for each experimental group.
6 is a graph comparing the quantitative comparison of P. gingivalis and total bacteria between the experimental groups in the gingival tissue.
7 is a graph comparing the quantitative comparison of P. gingivalis and total bacteria between experimental groups in teeth.
8 is a graph showing the cytokine production inhibitory effect in LPS-induced RAW 264.7 cells by treatment by oraCMU concentration.
9 is It is a graph showing the cytokine expression change by oraCMU treatment in the ICR mouse periodontal disease model gingiva (A, C, E, G) and serum (B, D, F, H).

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe a preferred embodiment of the present invention, and the scope of the present invention is not limited and interpreted by the matters described in the following examples.

<Example 1> in vitro (cells) and in vivo (Animal) periodontal disease evaluation model development

(1) In vitro model: Induction of inflammation by LPS treatment on RAW 264.7 cells

After the addition of LPS to the cultured RAW 264.7 cells and cultured to induce inflammation, the degree of induction of the inflammatory response was measured through NO (nitrc oxide) assay.

(2) In vivo model: ICR mice induce periodontal disease through ligature model in maxillary second molars

Induced periodontal disease through ligature model in maxillary second molars in ICR mice. Using a 6-0 silk suture, the left second molar was ligated to prevent loosening during the experiment. After 14 days, the amount of bone loss was confirmed through micro-CT to confirm whether or not periodontal disease was caused.

<Test Example 1> in vitro Model building and safety evaluation

To confirm the safety of cytotoxicity in RAW 264.7 cells of Weissella cibaria CMU (oraCMU), cytotoxicity was observed after culturing RAW 264.7 cells and oraCMU 1 x 10 ⁹ CFU together.

1 is a result of confirming the safety of cytotoxicity in RAW 264.7 cells of Weissella cibaria CMU (oraCMU). As shown here, cell death by lactic acid bacteria was not observed.

<Test Example 2> in vitro Efficacy evaluation of oral care probiotics in a periodontal disease model

After LPS treatment on RAW 264.7 cells, the efficacy of oraCMU was confirmed by analyzing NO production changes by Weissella cibaria CMU (oraCMU).

In cultured RAW 264.7 cells, oraCMU was treated with LPS at doses of 1 x 10 ⁷ CFU (low), 1 x 10 ⁸ CFU (medium), and 1 x 10 ⁹ CFU (high), respectively, and inhibition of NO production was observed.

2 is a graph showing the efficacy of inhibiting LPS-induced NO production according to oraCMU treatment in RAW 264.7 cells. As shown here, the treatment of oraCMU suppressed all NO production in RAW 264.7 cells by LPS treatment. The inhibitory effect on live cells was superior to that of dead cells, and the high concentration of oraCMU treatment showed better NO production inhibition than the low concentration oraCMU treatment.

<Test Example 3> in vivo Efficacy evaluation of oral care probiotics in a periodontal disease model

(1) Organization of the experimental group

Negative control (group without treatment) (Ctrl)

Positive control group (group without any treatment after induction of periodontitis by ligation) (LIP/Ctrl)

Group treated with PBS after inducing periodontal disease by ligation (LIP/PBS)

Group treated with 1 x 10 ⁷ CFU of oraCMU after induction of periodontal disease by ligation (LIP/WC-L)

Group treated with 1 x 10 ⁸ CFU of oraCMU after induction of periodontal disease by ligation (LIP/WC-M)

Group treated with 1 x 10 ⁹ CFU of oraCMU after inducing periodontal disease by ligation (LIP/WC-H)

(2) Test method

Micro-CT image acquisition of sampling tissue (maxillary molar area): 14 days after periodontitis caused by ligation, it is collected as a block including alveolar bone and dental tissue from experimental animals, fixed in formaldehyde, and requested for Micro-CT imaging by an external institution did.

Evaluating the effect of inducing and inhibiting periodontal disease by measuring the distance between the cementoenamel junction-alveolar bone crest (CEJ-ABC): Measure the distance between the cementoenamel junction and the alveolar crest in a cross-section using the captured image and periodontal disease inhibitory effect by comparing each experimental group

Sampling tissue (maxillary molar gingiva) P. gingivalis - specific 16s rRNA quantitation to confirm the effect on infection: After isolating genomic DNA from gingival tissue and teeth, real- Efficacy was confirmed by comparing between experimental groups by performing time PCR and performing relative quantification using the Delta delta CT method for the amount of P. gingivalis and total bacteria in the gingival tissue.

(3) Primer base sequence used in the experiment

(4) Real-time PCR analysis conditions using PowerUp SYBR Green Master Mix

(5) Mechanism of action analysis

Among P. gingivalis virulence factors, LPS is known to act on TLR-2 and -4, and cytokines (TNF-α, IL-1β, IL-6, IL-10, etc.) secreted by this signal were measured.

In the in vitro efficacy evaluation, the cell culture supernatant was analyzed, and in the in vivo efficacy evaluation, the inflammation inhibition mechanism was analyzed by ELISA analysis of serum or periodontal tissue.

Figure 3 shows the evaluation of the efficacy of oraCMU treatment in the in vivo periodontal disease model. Here, A) negative control (group without treatment) (Ctrl), B) positive control (group without treatment after induction of periodontitis by ligation) (LIP/Ctrl). C) is a group treated with PBS after inducing periodontitis by ligation (LIP/PBS), D) is a group treated with 1 x 10 ⁷ CFU of oraCMU after induction of periodontitis by ligation (LIP/WC-L), E) After inducing periodontitis by ligation This is the group treated with oraCMU 1 x 10 ⁸ CFU (LIP/WC-M), and F) the group treated with 1 x 10 ⁹ CFU of oraCMU after induction of periodontitis by ligation (LIP/WC-H).

As shown in FIG. 3 , after sacrificing the experimental animal, the specimen was photographed with micro-CT, and the CEJ-ABC distance of the maxillary left second molar was measured at the same angle to confirm the degree of alveolar bone loss. The negative control group (4A) showed the shortest CEJ-ABC distance compared to other experimental groups, whereas the positive control group (4B) had the most alveolar bone loss, confirming that periodontal disease was normally induced.

After inducing periodontitis, the PBS-treated group (C) and the oraCMU 1 x ^{10 7} CFU treatment group (4D) did not show a significant difference from the positive control group. It decreased compared to the control group, but there was no statistical significance. The least alveolar bone loss was observed in the oraCMU 1 x 10 ⁹ CFU-treated group compared to the positive control group.

Then, the alveolar bone loss of the distal surface of the maxillary left second molar was quantitatively analyzed through micro-CT image analysis. 4 is a graph showing the distance from the maxillary crest boundary to the alveolar ridge (CEJ-ABC) of the distal surface of the maxillary second molar for each experimental group. As shown here, it was confirmed that the oraCMU 1 x 10 ⁹ CFU treatment group after ligation had a significant 36.6% inhibitory effect on alveolar bone destruction compared to the positive control group.

Also, alveolar bone loss on the mesial surface of the maxillary left second molar was quantitatively analyzed through micro-CT image analysis. 5 is a graph showing the distance from the maxillary crest boundary to the alveolar bone ridge (CEJ-ABC) of the mesial surface of the maxillary second molar for each experimental group. As shown here, the amount of alveolar bone loss was slightly decreased in the oraCMU 1 x 10 ⁹ CFU treatment group after ligation compared to the positive control group, but no statistical significance was observed.

<Test Example 4> In an animal model Phorphyromonas gingivalis detection test

Quantitative comparison of P. gingivalis and total bacteria was compared between experimental groups in gingival tissue and teeth. 6 is a graph comparing the quantitative comparison of P. gingivalis and total bacteria between the experimental groups in the gingival tissue.

In the gingival tissue, P. gingivalis was not detected at all in the gingival tissue of the periodontitis-inducing group, perhaps because periodontitis was induced by binding with a ligation thread without inducing periodontitis by P. gingivalis . It was confirmed that a small number was observed.

7 is a graph comparing the quantitative comparison of P. gingivalis and total bacteria between experimental groups in teeth.

In the teeth, P. gingivalis was detected in 3 animals in the periodontitis-inducing group, but no P. gingivalis was detected in the oral lactobacillus group, and in total bacteria, similar to the gingival tissue results, less total bacteria were observed in the oral lactobacillus group.

Considering that the total number of bacteria in the gingival tissue and teeth of the oral lactobacillus group is relatively lower than that of the control group, it can be said that the effect of the oral lactic acid bacteria oraCMU.

In particular, in the teeth, P. gingivalis was detected in three control mice, but no P. gingivalis was detected in the oral lactobacillus group. Therefore, the effect of the oral lactic acid bacteria oraCMU on reducing alveolar bone loss and oral bacteria was confirmed.

<Test Example 5> in vitro cytokine assay in the model

Among P. gingivalis virulence factors, LPS is known to act on TLR-2 and -4, and the cell culture supernatant for pro-inflammatory cytokines (TNF-α, IL-1ßIL-6, etc.) Inflammation inhibition mechanism was analyzed by serum or periodontal tissue ELISA analysis.

8 is a graph showing the cytokine production inhibitory effect in LPS-induced RAW 264.7 cells by treatment by oraCMU concentration. As shown here, in the case of oraCMU treatment, both live and dead cells inhibited the cytokine expression of RAW 264.7 cells. The cytokine secretion inhibitory effect was higher in live cells than in dead cells, and cytokine secretion inhibition was higher in the oraCMU 1 x 10 ⁹ CFU treatment group.

<Test Example 6> in vivo cytokine assay in the model

After induction of periodontal disease, the change of inflammatory cytokines in gingival tissue and serum according to oraCMU treatment was confirmed.

9 is It is a graph showing the cytokine expression change by oraCMU treatment in the ICR mouse periodontal disease model gingiva (A, C, E, G) and serum (B, D, F, H). As shown here, in the case of the gingival tissue, the expression of all cytokines measured in the positive control group that induced periodontal disease was significantly increased, indicating an inflammatory state. However, in the case of the oraCMU 1 x 10 ⁹ CFU-treated group, the expression of TNF-α, IL-1β, IL-6 and IL-10 was significantly decreased compared to the positive control group, indicating that inflammation was relieved.

Claims (7)

A composition for preventing and treating periodontal disease, characterized in that Weissella cibaria CMU (oraCMU) is included in a vegetable oil base in an amount of 10 ⁹ CFU (colony forming unit) per 1 g and prepared as a concentrate . The method of claim 1,
The vegetable oil base is sunflower seed oil, coconut oil, olive oil, peanut oil, sesame oil, medium-chain neutral fatty acid, D-alpha-tocopherol any one or a mixture thereof, periodontal disease prevention and treatment composition, characterized in that. The method of claim 1,
The concentrate is a composition for preventing and treating periodontal disease, characterized in that it is prepared in the form of a capsule. The method of claim 1,
A composition for preventing and treating periodontal disease, characterized in that it is prepared in the form of gauze so that the concentrate can seep into the periodontal disease site. The method of claim 1,
The concentrate is a composition for preventing and treating periodontal disease, characterized in that it is prepared in the form of a patch that can be attached to a periodontal disease site. The method of claim 1,
The concentrate is a composition for preventing and treating periodontal disease, characterized in that it is prepared in the form of a liquid drop. 7. The method of claim 6,
A composition for preventing and treating periodontal disease, characterized in that it is prepared in the form of a gum including a concentrate mixed with a vegetable extract in the vegetable oil base.

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