KR102061981B1 - Compositions for preventing or treating oral disease or bone disease comprising extracts of Brachypodium sylvaticum - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a food composition, a quasi-drug composition, and a dental composition for preventing or treating oral diseases or bone diseases containing a Brachypodium sylvaticum extract as an active component. Specifically, the Brachypodium sylvaticum extract of the present invention is derived from natural products, thereby having less toxicity, significantly inhibiting the activity of Porphyromonas gingivalis, which is an oral disease causing bacterium, and significantly inhibiting the formation or differentiation of osteoclasts. Therefore, the Brachypodium sylvaticum extract can be usefully used for preventing or treating oral diseases or bone diseases.

Description

Composition for preventing or treating oral disease or bone disease comprising extract of forest wheat wheat as an active ingredient {Compositions for preventing or treating oral disease or bone disease comprising extracts of Brachypodium sylvaticum}

The present invention relates to a composition for the prevention or treatment of oral diseases or bone diseases, including forest wheat extract, specifically, the pharmaceutical composition, food for the prevention or treatment of oral diseases or bone diseases comprising forest wheat extract as an active ingredient A composition, a quasi-drug composition, and a dental composition.

Osteoclasts differentiate RAW264.7 monocytes from mice into multinucleated osteoclasts by RANKL (receptor activator of nuclear factor ĸB (RANK) ligand). This differentiation process promotes the activation of mitogen-activated protein kinase (MAPK) by RANKL binding to RANK, which is associated with osteoclast differentiation by the transcription factor NF-ĸB entering the nucleus. It is possible by increasing the expression of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, and c-Src tyrosine kinase, and multinucleated osteoclasts formed by this process produce mineralized bone. Absorbs. In addition, binding of RANKL to RANK promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6), thereby promoting the activity of transcription factors such as MARK or NF-ĸB, AP-1, NFATc1 (LEE ZH, KIM). HH.Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts.Biochem Biophys Res Commun. 2003 May 30, 305, 211-4). Therefore, blocking of signaling pathways activated by RANKL is recognized as one of the therapeutic approaches in the treatment of diseases related to osteoclast differentiation.

Osteoclasts cause abnormal bone tissue destruction and absorption due to imbalances with osteoblasts in the bone, thereby causing osteoporosis, which reduces bone mass and bone density, osteomalacia, in which bone is depleted from bone, and normal Fibrous dysplasia, in which bone tissue is replaced by fibrous connective tissue and immature bone ossein, periodontal disease in which the alveolar bone is lost, and rheumatoid arthritis, which causes joint destruction and deformation. It is known. As such, there is a close relationship between osteoclast differentiation, periodontal disease, and bone disease.

In the case of porphyromonas gingivalis, it is known to reduce osteogenicity by inhibiting osteoblast differentiation by host mediated inflammatory reaction through lipopolysaccharide (LPS) or cilia (Wilson, 1998; Kadono et al. LPS is a well known stimulating factor for the synthesis of inflammatory cytokines or eicosanoids as a pathogenic component present in the bacterial envelope, and stimulates osteoblasts to cause osteolytic factors such as IL-1, IL-6, PGE2, and oxidation. It is known as a major structure that destroys periodontal tissue by releasing nitrogen. In addition, differentiation of osteoclasts is directly promoted and activated by LPS itself, which can ultimately induce bone loss (Koide et al., 2000; Taubman et al., 2005; Islam et al., 2007; Hou et al., 2013; Teramachi et al. 2017). As such, there are many data on the effects of osteoblasts or osteoclasts on the differentiation of periodontal disease-inducing strains, and there is a close relationship between periodontal disease and bone disease, such as therapies for treating osteoarthritis and periodontal disease. This exists.

Currently, antibiotics and antibacterial agents such as Sangquinarine, Listerine, Peroxide, Chlorhexidine, and the like are widely used as treatments for treating oral diseases including periodontal disease. However, since antibiotics can cause the appearance of resistant bacteria in the oral cavity and superinfection along with systemic side effects on our body, long-term use is difficult and can be used only as a therapeutic agent. In addition, raw guarinin has a disadvantage that the effect on bacteria in the oral cavity is unclear, and the treatment effect on periodontal disease is less clear, and the price is expensive. In addition to a temporary effect, there is a drawback that may cause harmful effects on the tissue in the long-term use. In addition, pyroxide, which has been recently added for the whitening effect, is toxic to bacteria, but at the same time toxic to human tissues, there is a problem in safety as well as sometimes resistant bacteria to pyroxide appear in bacteria. In addition, chlorhexidine is known to be the best among the formulations known to date to prevent plaque formation and to prevent and treat periodontal disease. However, chlorhexidine causes irritation to tissue, pigmentation and degeneration of tissues, and particularly has a strong irritating taste and odor. Not only there is a problem that can be shown, myocerosis can be caused, there is a carcinogenic disadvantage that can not be used for a long time for the purpose of treatment or prevention, such as limited use in pregnant women. Therefore, there is a need to develop a drug that can effectively inhibit the growth of strains causing oral diseases without concern for such side effects.

Currently, bisphosphonate-based therapeutics are widely used to treat bone damage caused by osteoclasts such as osteoporosis. However, the number of adverse events such as osteonecrosis, severe atrial fibrillation, incapacitation of bones or joints, and musculoskeletal pain has been increasing yearly in patients taking bisphosphonate drugs (Br J Cancer 98: 1736). -1740 (2008). Therefore, there is a need to develop a drug that can compensate for the shortcomings of existing bisphosphonate-based drugs, less toxic and effectively suppress the formation and differentiation of osteoclasts.

Under these backgrounds, the present inventors conducted a research effort to find a compound that is derived from natural products and is less toxic, inhibits the activity of oral disease-causing bacteria and inhibits the formation or differentiation of osteoclasts. By inhibiting the activity of Porphyromonas gingivalis , a disease-causing bacterium, and significantly inhibiting the formation or differentiation of osteoclasts, it can be usefully used for the prevention or treatment of oral or bone diseases. The present invention has been completed.

One object of the present invention to provide a pharmaceutical composition for the prevention or treatment of oral diseases or bone diseases comprising the forest wheat wheat extract as an active ingredient.

Another object of the present invention is to provide a food composition for the prevention or improvement of oral diseases or bone diseases comprising forest wheat wheat extract as an active ingredient.

Another object of the present invention to provide a quasi-drug composition for the prevention or improvement of oral diseases or bone diseases comprising the forest wheat wheat extract as an active ingredient.

Yet another object of the present invention is to provide a dental composition for the prevention or improvement of oral diseases, including forest wheat wheat extract as an active ingredient.

In order to achieve the above object, one embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of oral diseases or bone diseases comprising forest wheat extract as an active ingredient.

Specifically, the pharmaceutical composition for preventing or treating oral or bone diseases of the present invention may include forest wheat wheat extract or fractions thereof.

The term "brachypodium sylvaticum " of the present invention is a perennial plant belonging to Gramineae and Brachypodium , and inhabits the southern part of Korea, particularly coastal islands.

Forest wheat is known to be used as a feed for livestock, but various pharmacological properties and its prevention, treatment or improvement of oral or bone diseases are not known yet.

The inventors of the present invention have conducted various studies on substances exhibiting a prophylactic or therapeutic effect of natural oral or bone diseases from natural resources. As a result, forest wheat extract is Porphyromonas gingivalis , which is an oral disease-inducing bacterium. By inhibiting the activity of and inhibiting the formation or differentiation of osteoclasts, it was confirmed that it can be useful for the prevention or treatment of oral diseases or bone diseases. Through this, it was found that forest wheat wheat extract can be used as a pharmaceutical composition for preventing or treating oral diseases or bone diseases. The composition comprising forest wheat extract having a prophylactic or therapeutic effect of the oral disease or bone disease is not known until now, it can be said that the significance is very significant in that it was first developed by the present inventors.

For the purposes of the present invention, forest wheat is not limited to the type thereof, and may be used without limitation to its source or to those used in cultivation or commercially available.

In the present invention, the term "extract" refers to extracting the forest wheat wheat, but is not limited thereto, the extract obtained by the extraction process, the dilution or concentrate of the extract, the dried product obtained by drying the extract, these adjustments Extracts of all formulations that can be formed using the extract itself and the extract, such as offerings, purified products or mixtures thereof. In addition, it can be extracted from various organs of the natural, hybrid, varieties of the forest wheat wheat, for example roots, roots, ground, stems, leaves, berries and the like.

Forest wheatgrass extract of the present invention can be prepared using common extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but methods such as dipping extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction may be used.

The forest wheatgrass extract may be extracted with water, alcohol having 1 (C ₁ ) to 4 carbon atoms (C ₄ ) or a mixed solvent thereof, but is not limited thereto, and the degree of extraction of the active ingredient according to the organic solvent to be extracted and Since the degree of loss may vary, an appropriate organic solvent may be selected and used. Specifically, it can be extracted using water, an organic solvent or a mixed solvent thereof. More specifically, ethanol may be used, but is not limited thereto.

In addition, the solvent extract may further comprise the step of filtering the extract to remove the suspended solid particles. As an example, the particles may be filtered using cotton, nylon, or the like, or ultrafiltration, freezing, centrifugation, or the like may be used, but is not limited thereto.

Concentration of the extract may be used, such as concentrated under reduced pressure, reverse osmosis concentration.

The drying step after concentration may be a freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying or infrared drying. In some cases, the method may further include grinding the final dried extract.

As used herein, the term "fraction" refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture comprising several different components.

The fractionation method of obtaining the fraction in the present invention is not particularly limited as long as it can exhibit a prophylactic or therapeutic effect of oral disease or bone disease, but may be performed according to a method commonly used in the art. For example, solvent fractionation is performed by treating various solvents, ultrafiltration fractionation is carried out through an ultrafiltration membrane having a constant molecular weight cut-off value, and separation according to various chromatography (size, charge, hydrophobicity or affinity) is performed. Chromatography), and combinations thereof.

The kind of the fractionation solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water, distilled water and alcohols; Nonpolar solvents, such as hexane, ethyl acetate, chloroform, dichloromethane, etc. are mentioned. These may be used alone or in combination of two or more thereof. In the case of using the alcohol in the fractionation solvent, it is preferable to use an alcohol having 1 (C ₁ ) to 4 (C ₄ ) carbon atoms.

In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

In the present invention, "oral disease" refers to various diseases occurring in the oral cavity, and the oral cavity refers to a space in the mouth connected to the pharynx from the anterior lip to the posterior bulb. In the present invention, if the oral disease is a disease occurring in the oral cavity is a concept including all irrespective of the condition, non-limiting examples of the oral disease is dental caries, periodontal disease (periodonitis or gingivitis), toothache, ache, bad breath Etc. can be mentioned.

As used herein, the term "periodontal disease" is often referred to as flavor, and is divided into gingivitis and periodontitis according to the degree of the disease. It is a relatively mild and fast recovery type of oral disease called gingivitis, which is limited to the gums, or soft tissues, and it is called periodontitis when the inflammation progresses around the gingiva and alveolar bone.

There is a V-shaped gap between the gingiva (gingiva) and the teeth, which is a periodontal disease in which bacteria attack the lower gingiva of the gingival sulcus, damaging the periodontal ligament and adjacent tissues. As the inflammation progresses and more tissue is damaged, the groove develops into a periodontal pocket, and the more severe the periodontitis, the deeper the periodontal sac becomes. As the periodontal sac becomes deeper, the periodontal ligaments become inflamed, and lipopolysaccharide (LPS) secreted by Porphyromonas gingivalis promotes osteoclast differentiation, resulting in alveolar bone loss.

In the present invention, the term "bad breath" is an odor derived from the oral cavity and adjacent organs, and 85 to 90% of the bad breath is derived from the oral cavity, particularly from the back of the tongue. The main components of bad breath are volatile sulfur compounds, of which 90% of the total amount of volatile sulfur compounds is hydrogen sulfide made from cysteine and methyl mercaptan and dimethyl sulfide made from methionine. These components are mainly produced by protein enzymes secreted by anaerobic bacteria, and the back of the tongue is the most important habitat. This area is not well cleaned by saliva, and there are many small depressions, which is a place where bacteria continue to live. The generation of volatile sulfur compounds by anaerobic bacteria is the most important cause of bad breath, but is also caused by oral diseases such as dental caries, periodontitis, dry mouth. Many types of anaerobic bacteria are involved in the development of bad breath, and non-limiting examples of the cause of bad breath include Porphyromonas gingivalis , which secrete many types and large amounts of enzymes.

In the present invention, the term " Porphyromonas gingivalis or P. gingivalis " is one of several representative causes of oral disease. Porphyromonas gingivalis is a type of bacteroid flexible bacteria, gram-negative bacteria and anaerobic bacteria. Porphyromonas gingivavalis occurs in the oral cavity in which periodontal disease occurs, and is also found in the upper gastrointestinal tract, respiratory tract and colon. Accordingly, it is possible to prevent, ameliorate or treat oral diseases through the antibacterial activity against Porphyromonas gingivalis.

In one embodiment of the present invention, the result of administering the forest wheatgrass extract of the present invention to Porphyromonas jinjibali, it was confirmed that exhibits the antibacterial activity against porphyromonas jinjivalis (Fig. 4), the present invention It was confirmed that the forest wheat extract of the antibacterial lasting effect (Fig. 5). Through this, it was confirmed that the forest wheat extract is effective for the prevention or treatment of oral diseases through the antimicrobial activity and antimicrobial sustaining activity against porphyromonas jinjibali.

In the present invention, the term "bone disease" is meant to include all diseases induced by bone loss or bone mineral density, which appear due to the loss of balance between the production and differentiation of osteoblasts and osteoclasts. Specifically, osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, periodontal disease, osteolysis, osteolysis, Fibrous dysplasia, Paget's disease, osteogenic imperfect, hypercalcemia, and more specifically, osteoporosis due to differentiation of osteoclasts. May be, but is not limited thereto.

In the present invention, the term "osteoclast" is a cell derived from a macrophage precursor (macrophage precursor), the osteoclast progenitor cells are a macrophage colony stimulating factor (M-CSF), NF-κB Differentiation into osteoclasts by receptor activator ligands (RANKL) and the like means the formation of multinucleated osteoclasts through fusion. Osteoclasts bind to bone through αvβ3 integrin, etc., create an acidic environment and secrete various collagenases and proteases to cause bone resorption. Osteoclasts do not proliferate into fully differentiated cells and cause apoptosis at the end of their lifespan for about two weeks.

In one embodiment of the present invention, after administering the forest wheatgrass extract of the present invention to the mouse monocytes differentiated into osteoclasts, the result was a concentration-dependent TRAP activity as a result of staining the osteoclast differentiation marker TRAP (tarrate-resistant acid phosphate) By inhibiting significantly, it was confirmed that the inhibition of osteoclast formation (Fig. 6). Through this, it was confirmed that the forest wheat extract is effective for the prevention or treatment of bone diseases, such as osteoporosis or oral disease through the osteoclast formation inhibitory activity.

As used herein, the term "prevention" refers to any action that inhibits or delays the development of a bone disease by administration of a pharmaceutical composition according to the present invention, and "treatment" is suspected of bone disease by administration of the pharmaceutical composition. And any action in which the symptoms of the affected individual improve or beneficially change.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent, which may comprise a non-naturally occuring carrier. Specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid, poly-L-lactic acid, Mineral oil and the like.

The pharmaceutical composition is an oral formulation such as pills, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injections according to conventional methods for preventing and treating oral or bone diseases. It may be used in the form of a solution, and in particular, when the pharmaceutical composition of the present invention is for the prevention or treatment of oral diseases, it may specifically have a formulation of oral sprays, oral ointments or varnishes for oral cavity. The carrier may include various amorphous carriers, microspheres, nanofibers, rosin, and the like.

When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used.

Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.

The content of forest wheatgrass extract or fractions thereof contained in the pharmaceutical composition of the present invention is not particularly limited, but is 0.01 to 100% by weight, 0.01 to 50% by weight, more specifically 0.01 to 20% by weight based on the total weight of the final composition It may be included in the amount of%.

The present invention provides a method for preventing or treating oral or bone diseases, comprising administering the pharmaceutical composition to a subject.

Since the pharmaceutical composition of the present invention exhibits a prophylactic or therapeutic effect of oral or bone diseases, the method of the present invention comprising administering the same to an individual can be usefully used for the prevention or treatment of oral or bone diseases. have.

As used herein, the term "individual" means any animal, including humans that may or may develop oral or bone diseases, such as monkeys, dogs, cats, rabbits, morphotes, rats, mice, cattle, sheep, pigs. , Goat, bird, fish, etc., and may be, for example, a mammal including a human, but is not limited thereto.

As used herein, the term "administration" means introducing the composition into a subject in any suitable manner, and the route of administration may be administered through a variety of routes as long as the target tissue can be reached. In particular, the pharmaceutical compositions of the present invention may be administered orally, intravenously, subcutaneously, intradermal, nasal, intraperitoneal, intramuscular, transdermal, and the like, including suitable intravenous administration if necessary for local treatment. Administration by methods and routes. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections, but is not limited thereto.

The therapeutic method of the present invention may be administered in a pharmaceutically effective amount of a pharmaceutical composition comprising forest wheat extract.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dose level refers to the severity of the disease, the activity of the drug, and the like. Including the age, body weight, health, sex of the patient, sensitivity to the drug of the patient, time of administration of the composition of the present invention used, route of administration and duration of release, treatment with the composition of the invention used or co-administered Factors, and factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered alone or in combination with known pterygium therapeutics. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.

In addition, the dosage of the pharmaceutical composition may be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. For example, the pharmaceutical composition of the present invention can be administered at 1 mg / kg to 200 mg / kg, specifically 1 mg / kg to 100 mg / kg, more specifically 20 to 40 mg / kg per adult However, the frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or multiple times in divided doses. The dosage does not limit the scope of the invention in any aspect.

As another aspect, it provides a food composition for the prevention or improvement of oral disease or bone disease comprising forest wheat extract as an active ingredient.

Specifically, the food composition for preventing or improving oral disease or bone disease of the present invention may include forest wheat wheat extract or fractions thereof.

At this time, the description of the "forest wheat", "extract", "fraction", "oral disease", "bone disease", and "prevention" are as described above.

As used herein, the term "improvement" refers to all the actions of improving or benefiting the suspicion and benefit of suspicion and onset of an oral disease or bone disease that is prevented or treated using a composition containing forest wheat extract or fractions thereof as an active ingredient. .

In the present invention, the term "food" is meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products, including ice cream, various soups, beverages, tea, drinks, alcoholic beverages It includes all foods in the usual sense, such as vitamin complexes, health functional foods, and the like, as long as it may include the forest wheat wheat extract or fractions thereof. It may also include the form of pills, powders, granules, acupuncture, tablets, capsules or solutions.

In the present invention, the term "health functional food" refers to a food prepared and processed using raw materials or ingredients having a useful function to the human body according to Act No. 6767 of the Health Functional Food Act, "functional" is the structure of the human body And it means to obtain a useful effect for health use, such as regulating nutrients or physiological action for function. On the other hand, health food refers to foods that have active health maintenance or promotion effect compared to general foods, and health supplement food means foods for the purpose of health supplement, in some cases, the term functional health food, health food, health supplement food Can be used interchangeably. Health functional food of the present invention can be prepared by a method commonly used in the art. It can be prepared in various forms of formulation, unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material and can be excellent in portability.

When preparing the food composition may be prepared by adding the raw materials and components commonly added in the art, the kind is not particularly limited. For example, various herbal extracts, food acceptable food additives, or natural carbohydrates may be included as additional ingredients, such as conventional foods, but are not limited thereto.

As another aspect, the present invention provides a quasi-drug composition for the prevention or improvement of oral diseases or bone diseases comprising the forest wheat wheat extract as an active ingredient.

Specifically, the quasi-drug composition for preventing or improving oral disease or bone disease of the present invention may include forest wheatgrass extract or a fraction thereof.

At this time, the description of the "forest wheat", "extract", "fraction", "oral disease", "bone disease", "prevention" and "improvement" are as described above.

As used herein, the term "quasi drug" refers to a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or animal, has a weak action on the human body or does not directly act on the human body, Or non-machinery and the like, as well as preparations for the use of disinfection, insecticides and similar applications for the prevention of infectious diseases, for the diagnosis of human or animal diseases. Other than articles used for the purpose of treatment, alleviation, treatment, or prevention, other than instruments, machines or devices, and not goods, machines or devices used for the purpose of pharmacologically affecting the structure and function of humans or animals Means the goods. In addition, the quasi-drug may include external skin preparations or personal hygiene products.

The external skin preparations are not particularly limited thereto, and in particular, may be prepared in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or gels, and in particular for the personal hygiene products Specific examples include, but are not limited to, soaps, cosmetics, wipes, tissue paper, shampoos, skin creams, face creams, toothpastes, lipsticks, perfumes, makeup, foundations, ball touch, mascara, eye shadows, sunscreen lotions, hair care products, Air freshener gel or cleaning gel. In addition, another example of the quasi-drug composition of the present invention is a disinfectant cleaner, shower foam, mouthwash solution, oral spray, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

When the forest wheat wheat extract or fractions thereof of the present invention is used as an quasi-drug additive, the extract or fraction may be added as it is or used together with other quasi-drugs or quasi-drug components, may be appropriately used according to a conventional method, and the amount of the active ingredient may be used. It may be appropriately determined depending on the purpose.

As another aspect, it provides a dental composition for the prevention or improvement of oral diseases, including forest wheat wheat extract as an active ingredient.

Specifically, the dental composition for preventing or improving oral diseases of the present invention may include forest wheatgrass extract or a fraction thereof.

At this time, the description of the "forest wheat", "extract", "fraction", "oral disease", "prevention" and "improvement" are as described above.

In the present invention, the term "dental" may mean a use for preventing or treating diseases or damage in the teeth and their supporting tissues and oral cavity.

The dental composition can be applied to a dental product such as antibacterial fluorine varnish by addition to the fluorine varnish, oral tooth care solution, dental perceptual hypersensitivity agent, antibacterial dental adhesive, dental filler, dental restoration, dental It can be applied to a dental material such as root canal filling material, dental root canal sealer (sealer), subfoveal sealant, dental coating and dental cement.

In addition, the dental composition can be used in all articles used for the health of the oral cavity. Examples of such articles include dental devices such as dental floss, toothbrushes, interdental toothbrushes, powered toothbrushes, tongue cleaners, mouthwashes, toothbrush sterilizers, dental devices such as braces, implants, etc., or any dental tools and devices used in dental practice. But it is not limited thereto.

Specifically, the article is immersed in the dental composition, the dental composition is applied or sprayed onto the surface of the article, the surface of the article is washed several times with the dental composition, or the dental composition soaks. Any towel may be used for the article by wiping the surface of the article, and the like, and the article and the dental composition are not particularly limited as long as the article can be contacted for a predetermined time. In addition, the time for performing the method is not limited as long as the dental composition can sufficiently react with the surface of the article, and those skilled in the art can appropriately select it.

Forest wheat wheat extract of the present invention is derived from natural products, while less toxic, oral disease by significantly inhibiting the activity of Porphyromonas gingivalis ( porphyromonas gingivalis ), which causes oral disease, and significantly inhibits the formation or differentiation of osteoclasts. Or it can be usefully used for the prevention or treatment of bone diseases.

1 is a graph showing the cytotoxicity (cell viability) according to the treatment concentration of forest wheat wheat extract.
2 is a graph showing the effect of inhibiting the production of nitric oxide according to the treatment concentration of forest wheat wheat extract. Significance test was performed by the student t-test, the significance # of the LPS treated group compared to the control group is p <0.001. Significance of forest wheat extract treatment group * compared to LPS treatment group * p <0.05, ** indicates p <0.01, *** indicates p <0.001.
Figure 3 is a graph showing the inhibitory effect of inflammatory cytokines, (a) IL-6 and (b) TNF-α production according to the treatment concentration of forest wheat wheat extract. Significance test was performed by the student t-test, the significance # of the LPS treated group compared to the control group is p <0.001. Significance of forest wheat extract treatment group * compared to LPS treatment group * p <0.05, ** indicates p <0.01, *** indicates p <0.001.
Figure 4 is a graph showing the growth inhibitory effect of porphyromonas ginjivalis according to the treatment concentration of forest wheatgrass extract. The significance test was performed through the student t-test, and the significance * was p <0.05, ** was p <0.01, and *** was p <0.001.
5 is a graph showing the sustained effect of growth inhibition of porphyromonas ginjivalis according to the control group, fluorine varnish treated group, fluorine varnish + forest wheat wheat treated group. Significance test was carried out through the student t-test, the significance # # # of the fluoride varnish treated group compared to the control group is p <0.001. Significance *** of fluorine varnish + forest wheat wheat treated group compared to fluorine varnish treated group indicates p <0.001.
6 is a graph showing (a) the number of TRAP positive cells and (b) TRAP staining of differentiated osteoclasts according to the treatment concentration of forest wheatgrass extract. The significance test was performed through the student t-test, and the significance * was p <0.05, ** was p <0.01, and *** was p <0.001.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Forest Wheat Extract

Through the cooperation of domestic plant taxonomy, cultivation specialists and herbalist discrimination specialists, native forest wheat was collected and dried, and the outpost was dried in 99.9% methanol for 3 days. After 15 minutes of sonication, the extraction was followed by a 2-hour stoppage of 10 cycles / day. Subsequently, the resultant was filtered using a non-fluorescent cotton filter, concentrated using a rotary evaporator (N-1000SWD; EYELA) at 45 ° C, and using a module spin 40 (Biotron co.). Dried for hours. Thereafter, the extract was removed in 20 mg / tube solvent and stored in a -4 ℃ low temperature room. The procedure was carried out in the Korea Plant Extract Bank, a sample of 20 mg / tube was dissolved in DMSO to make a concentration of 50 mg / ml for use in cell experiments, it was used diluted to the cell culture medium.

Example 2. Anti-inflammatory Effect of Forest Wheatgrass Extract

RAW264.7 cells were purchased from ATCC (Rockville, MD), 10% fetal bovine serum (FBS, HyClone Logaa, UT), streptomycin (100 μg / mL), and penicillin (100 U / mL) The added growth medium (CM, DMEM) was incubated in 37 ℃, 5% CO ₂ humid air.

Example 2-1. Cytotoxicity Assessment

4 × 10 ³ RAW264.7 cells were plated in 96-well plates of GM. After 24 hours of incubation, the forest wheat extract prepared in Example 1 was treated by concentration (0, 3, 10 and 30 μg / mL) for 1 day in the presence of lipopolysaccharide (LPS, 1 μg / mL) in the cells. It was. Cell viability (%) was measured three times using Cell Counting Kit-8 (CCK-8, Dojindo, Rockville, ML, USA) according to the manufacturer's manual. Absorbance was measured with a microplate reader (VERSA max ^™ , Molecular Devices, Sunnyvale, Calif.) And converted to cell number using a standard curve.

As a result, as shown in Figure 1, when treated with forest wheat wheat extract, it showed that the cell viability of about 100% at all concentrations showed no cytotoxicity. These results show that the antimicrobial effect or osteoclast generation inhibitory effect to be confirmed later is not due to the toxic effect on the cells, but the antibacterial effect or the osteoclast generation inhibitory effect by the forest wheat extract.

Example 2-2. Nitric oxide production inhibitory activity

Cells were plated with 1 × 10 ⁶ cells in a 60 mm dish, and then treated with the concentration of the forest wheat extract prepared in Example 1 (0, 3, 10 and 30 μg / mL) for 2 hours. Then LPS (1 μg / mL) was added. After 24 hours of incubation, nitric oxide (NO) levels were analyzed using a Greries reaction. Cell culture medium (100 μL) was mixed with grease reagent and incubated for 10 minutes at room temperature. Absorbance was measured with a microplate reader at 540 nm. As blank in all experiments, fresh culture medium was used. Nitrite (NO ₂ ) was measured with a standard curve using sodium nitrite (NaNO ₂ ).

As a result, as shown in Figure 2, the addition of LPS significantly increased the nitric oxide level compared to the control, but the addition of LPS after the treatment of forest wheat extract significantly reduced the nitric oxide level than the addition of only LPS Confirmed. These results show that forest wheat extract exhibits anti-inflammatory effect by inhibiting the production of nitric oxide, a mediator of inflammation in cells.

Example 2-3. Cytokine production inhibitory activity

The cells were plated into 1 × 10 ⁶ cells in a 60 mm dish, and then treated with the concentration of the forest wheat extract prepared in Example 1 (0, 3, 10 and 30 μg / mL) for 2 hours. Then LPS (1 μg / mL) was added. Cells were incubated for 24 hours. The cytokine amount of the supernatant was measured using an ELISA kit (R & D, Minneapolis, MN) measuring IL-6 and TNF-α according to the manufacturer's manual.

As a result, as shown in Figure 3, the addition of LPS significantly increased IL-6 and TNF-α levels compared to the control group, IL-6 than LPS only added LPS after forest wheat wheat extract treatment And it was confirmed that the TNF-α level is significantly reduced. These results show that forest wheat extract exhibits anti-inflammatory effect by inhibiting the production of inflammatory cytokines IL-6 and TNF-α.

Example 3. Antibacterial Effect of Forest Wheat Extract

Example 3-1. Antimicrobial Activity of Forest Wheat Extract

In order to evaluate the oral antimicrobial activity of forest wheatgrass extract, P. physiobacilli ( Prophyromonas gingivalis ; P. gingivalis , ATCC33277 ) was used. BHI broth containing hemin and menadione was used as a medium to activate Porphyromonas jinjivalis bacteria, followed by incubation for 72 hours at 37 ° C. in a CO ₂ incubator, and then each well of a 96-well plate. 90 μL of the bacteria was added to the wells, and 10 μL of the forest wheat extract prepared in Example 1 was added thereto. As a control group, only 100 μL of bacteria were used, and 100 μL of BHI broth was added to each well so that the final concentration of the forest wheatgrass extract was 125 μg / mL, 250 μg / mL, and 500 μg / mL. Thereafter, the samples were incubated for 72 hours. Absorbance was measured at 600 nm with an ELISA kit (Spectra MAX250, Molecular Devices Co.) to determine the concentration at which the growth of bacteria was inhibited.

As a result, as shown in Figure 4, when processing the forest wheat wheat extract was confirmed to inhibit the growth of porphyromonas ginjivalis in a concentration-dependent manner. Specifically, when treated with 125 μg / ml and 250 μg / ml of forest wheat extract, about 20% growth of porphyromonas gingivalis is inhibited, and when treated with 500 μg / ml of forest wheat extract, porphyromonas It was confirmed that the growth of ginjivalis about 40%.

Example 3-2. Antimicrobial Sustaining Activity of Forest Wheat Extract

Evaluation of the antimicrobial sustained activity of Porphyromonas jinjivalis of forest wheatgrass extract was performed as follows. 5 mm diameter disk-shaped film (OHP film) was placed in a sterile wrapping paper and sterilized with EO gas, and then the samples were prepared as shown in Table 1, and evenly applied to the entire surface of the film evenly and evenly to the same thickness. At this time, three films were made per sample, placed in a petri dish having a diameter of 35 mm, and dried in a clean bench with UV on for 30 minutes. After putting 2000 μL of sterilized distilled water into a Petri dish with a film to which the sample was applied, the coated surface was faced downward so that the sample could be eluted with water. After storage at 37 ° C. for 5 days at 80 rpm in a shaking water bath, the film was removed from the Petri dish and the antibacterial activity was evaluated using an agar diffusion test.

Sample 1
(Control) Film itself Sample 2 Fluorine varnish KR610 Sample 3 Forest wheat flour 50 mg / ml (50 μL) + Fluorine varnish KR610 (50 μL)

The agar diffusion method was performed as follows. Porphyromonas gingivalis was incubated in BHI broth medium for 72 hours at 37 ° C. in a CO ₂ incubator, and then inoculated onto agar medium coated with base agar.

After the top agar solidified, the film stored at 80 rpm in a shaking bath at 37 ° C. for 5 days was placed on agar plate. At this time, the surface of the film to which the sample was applied was allowed to be bonded to the agar medium downward. Thereafter, after culturing in a CO ₂ incubator at 37 ° C. for 72 hours, the diameter of the porphyromonas gingivalis inhibitor band formed around the OHP film was measured in a right angle direction, and the average was determined as an inhibition zone (mm).

As a result, as shown in Figure 5, the fluorine varnish treatment group showed a significant antimicrobial lasting effect as compared to the control group, the fluorine varnish + forest wheat wheat treated group showed a significant antibacterial lasting effect compared to the fluorine varnish treatment group. .

Above, through the above results it can be seen that the forest wheat wheat extract of the present invention can be effectively used in the treatment of oral diseases because it exhibits excellent antimicrobial effect and antimicrobial lasting effect on the strain causing oral disease.

Example 4. Inhibitory Effect of Forest Wheat Extract on Osteoclast Differentiation

Buy 5 week old male ICR mice to take the femur and tibia and wash them with α-MEM containing antibiotics (100 units / mL penicillin and 100 μg / mL streptomycin) to bone marrow cells ) Was obtained. The bone marrow cells were cultured in α-MEM containing 10% fetal bovine serum (FBS) and macrophage colony-stimulating factor (M-CSF) for 10 days. Non-adherent myeloid cells were dispensed into Petri dishes and incubated for 3 days in the presence of M-CSF (30 ng / mL). After washing the non-adherent cells, adherent cells were used as bone marrow-derived macrophage (BMM). To differentiate BMM cells into osteoclasts, BMM cells (1 × 10 4 cells / well) were applied to 96-well plates in the presence of RANKL (10 ng / mL) and M-CSF (30 ng / mL), Example Forest wheatgrass extract prepared in 1 was treated by concentration (0, 3, 10 and 30 μg / mL), the final concentration was 125, 250 and 500 μg / ml and incubated for 4 days. The negative control group used cells treated with RANKL only. After incubation for 4 days, multinucleated osteoclasts were fixed with 3.7% formalin for 10 minutes and treated with 0.1% Triton X-100 for 10 minutes to ensure permeability. The osteoclasts were stained red by treatment with TRAP solution (Sigma Aldrich, USA). Then, to measure the activity of osteoclast differentiation marker TRAP (tartrate-resistant acid phosphatase), 100 μl of TRAP buffer (100 mM sodium citrate pH 5.0, 50 mM sodium tartrate, 3 mM p-nitrophenyl phosphate) was added 37 After reacting for 5 minutes at ° C, 100 μl of 0.1 N NaOH was added to terminate the reaction. Then, the absorbance at 405 nm was measured to measure the TRAP activity.

As a result, as shown in Figure 6, when processing the forest wheat wheat extract to RANKL-induced osteoclasts, it was confirmed that the number of red stained osteoclasts significantly reduced while inhibiting TRAP activity in a concentration-dependent manner.

Above, through the above results it can be seen that the forest wheat wheat extract of the present invention can be effectively used for the treatment of oral diseases and bone diseases because the osteoclasts exhibit an inhibitory effect on the differentiation production.

From the description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (11)

Pharmaceutical composition for the prevention or treatment of oral diseases or bone diseases comprising forest wheat extract as an active ingredient.
The pharmaceutical composition of claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms or mixed solvents thereof.
According to claim 1, wherein the oral disease is periodontal disease or bad breath, pharmaceutical composition.
The pharmaceutical composition of claim 3, wherein the periodontal disease is periodontitis, gingivitis, or a combination thereof.
According to claim 1, wherein the bone disease (osteoporosis), osteomalacia (osteomalacia), osteopenia (osteopenia), bone atrophy (bone atrophy), osteoarthritis (rheumatoid arthritis), periodontal disease (periodontal disease) Pharmaceutical composition, which is at least one selected from the group consisting of osteolysis, fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia. .
According to claim 1, wherein the bone disease is osteoporosis (osteoporosis), pharmaceutical composition.
According to claim 1, wherein the prevention or treatment of oral disease is achieved through the inhibition of Porphyromonas gingivalis activity, pharmaceutical composition.
According to claim 1, wherein the prevention or treatment of oral disease or bone disease is achieved through the inhibition of osteoclast differentiation, pharmaceutical composition.
Food composition for the prevention or improvement of oral disease or bone disease comprising forest wheat extract as an active ingredient.
A quasi-drug composition for the prevention or improvement of oral diseases or bone diseases comprising forest wheat wheat extract as an active ingredient.
Dental composition for the prevention or improvement of oral diseases comprising forest wheat extract as an active ingredient.

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