KR102260037B1 - Compositions for preventing or treating bone disease comprising extracts of Trigonotis nakaii - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, a health functional food, a quasi-drug composition, and a feed composition for preventing or treating bone disease resulting from bone disease or oral disease, comprising an extract of chrysanthemum leaf extract as an active ingredient. The extract of the present invention is derived from a natural product and has low toxicity, remarkably inhibits the formation or differentiation of osteoclasts, remarkably promotes the formation or differentiation of osteoblasts, and Porphyromonas gingivalis ( By significantly inhibiting the activity of Porphyromonas gingivalis ), it can be usefully used for the prevention or treatment of bone diseases.

Description

Compositions for preventing or treating bone disease comprising extracts of Trigonotis nakaii

The present invention relates to a composition for preventing or treating bone disease, which includes an extract of chrysanthemum as an active ingredient, and specifically, a pharmaceutical composition for preventing or treating bone disease, a health functional food, and a quasi-drug comprising the extract of chamomile extract as an active ingredient. It relates to a composition and a composition for feed.

In osteoclasts, RAW2647 monocytes from mice are differentiated into multinucleated osteoclasts by receptor activator of nuclear factor ĸB (RANK) ligand (RANKL). In this differentiation process, RANKL outside the cell binds to RANK and promotes the activity of mitogen-activated protein kinase (MAPK). This is because a transcription factor called NF-ĸB enters the nucleus and TRAP related to osteoclast differentiation. It is possible by increasing the expression of (tartrate-resistant acid phosphatase), MMP-9 (matrixmetalloproteinase-9), c-Src tyrosine kinase, etc. Multinuclear osteoclasts formed through this process absorb mineralized bone. plays a role In addition, when RANKL binds to RANK, it promotes the activity of tumor necrosis factor receptor-associated factor 6 (TRAF6), thereby promoting the activity of MARK, or transcription factors such as NF-ĸB, AP-1, and 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 the signaling pathway activated by RANKL is important for the treatment of osteoclast-related diseases. It is recognized as one of the therapeutic approaches.

Osteoclasts cause the destruction and resorption of abnormal bone tissue due to imbalance with osteoblasts in the bone. fibrous dysplasia, in which bone tissue is replaced with fibrous connective tissue and immature trabecular trabeculae, periodontal disease in which alveolar bone is lost, and rheumatoid arthritis that causes joint destruction and deformation it is known As such, there is a close relationship between osteoclast differentiation and periodontal disease and bone disease.

Currently, antibiotics and antibacterial agents such as Sangquinarine, Listerine, Peroxide, and Chlorhexidine are widely used as therapeutic agents to treat 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, so it can be used only as a therapeutic agent. In addition, raw guinarin has the disadvantage of being unclear in its effect on bacteria in the oral cavity, its therapeutic effect on periodontal disease, and its high price. Listerine contains alcohol as its main ingredient and has a slight bacteriostatic effect, but in actual oral It exhibits only a temporary effect, and there is a disadvantage that it may also have a harmful effect on the tissue when used for a long time. In addition, piroxide, which has been recently added for whitening effect, is toxic to bacteria, but at the same time, it is also toxic to human tissues, so there is a problem in safety and sometimes bacteria resistant to pyroxide appear in bacteria. In addition, chlorhexidine is known to be the best known among agents for the prevention and treatment of periodontal disease as well as the inhibition of plaque formation, but it causes tissue irritation, tissue coloration and degeneration, and has a particularly irritating taste and strong odor. There are disadvantages in that it cannot be used for a long period of time for the purpose of treatment or, in particular, prevention, such as, as well as the problems shown, can cause mycosis mycosis, and has carcinogenic properties, limiting its use in pregnant women. Therefore, there is a need to develop a drug capable of effectively inhibiting the growth of strains that cause oral diseases without fear of side effects as described above.

Currently, bisphosphonate-based therapeutic agents are widely used to treat bone damage caused by osteoclasts, such as osteoporosis. However, in recent years, cases of various side effects such as osteonecrosis, severe atrial fibrillation, bone or joint incapacity, and musculoskeletal pain in patients taking bisphosphonate drugs are increasing every year (Br J Cancer 98:1736). -1740 (2008)). Therefore, there is a need to develop a drug that can supplement the disadvantages of the existing bisphosphonate-based drugs, have low toxicity, and effectively inhibit the formation and differentiation of osteoclasts.

Under this background, the present inventors have made intensive research to find a compound that is derived from a natural product and has low toxicity, inhibits the activity of oral disease-causing bacteria, and can inhibit the formation or differentiation of osteoclasts. By remarkably inhibiting the activity of the disease-causing bacteria, Porphyromonas gingivalis, and remarkably inhibiting the formation or differentiation of osteoclasts, it was confirmed that it can be usefully used for the prevention or treatment of oral diseases or bone diseases, The present invention was completed.

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

Another object of the present invention is to provide a health functional food for the prevention or improvement of bone diseases comprising the extract of chrysanthemum as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for the prevention or improvement of bone disease comprising the extract of chrysanthemum as an active ingredient.

Another object of the present invention is to provide a composition for feed for preventing or improving bone disease, comprising an extract of chrysanthemum marijuana as an active ingredient.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease comprising an extract of chrysanthemum marijuana as an active ingredient.

Specifically, the pharmaceutical composition for the prevention or treatment of bone disease of the present invention may include an extract or a fraction thereof.

The term of the present invention, " Trigonotis radicans var. sericea " is a perennial plant belonging to the family Solanaceae. In oriental medicine, the leaves and stems of champignon are known to be used as medicines for the symptoms of frequent urination and red white dysentery in children, but their effectiveness in preventing, treating or improving oral diseases or bone diseases is not yet known.

The present inventors have conducted various studies on substances exhibiting excellent preventive or therapeutic effects for oral diseases or bone diseases from natural resources present in nature, Porphyromonas gingivalis ) By inhibiting the activity of and inhibiting the formation or differentiation of osteoclasts, and promoting the differentiation of osteoblasts, it was confirmed that it can be usefully used in the prevention or treatment of bone diseases or bone diseases arising from oral diseases. Through this, it was found that the extract of chamomile extract can be used as a pharmaceutical composition for the prevention or treatment of bone diseases resulting from bone diseases or oral diseases. A composition comprising an extract of chrysanthemum chrysanthemum, which has the effect of preventing or treating the bone disease, has not been known so far, and it can be said that it is very significant in that it was first developed by the present inventors.

For the purpose of the present invention, the type of chrysanthemum is not limited, and cultivated ones may be used, commercially purchased ones, or used without limitation on the source.

In the present invention, the term "extract" refers to an extraction treatment of the A. serrata, but is not limited thereto, an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and adjustments thereof It may include extracts of all formulations that can be formed using the extract itself and the extract, such as preparations, purified substances, or mixtures thereof. In addition, it can be extracted from various organs of natural, hybrid, and variegated plants of the chrysanthemum, for example, roots, rhizomes, radish, stems, leaves, and fruits.

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

The extract may be extracted with water, an alcohol having 1 (C ₁ ) to 4 (C ₄ ) carbon atoms, or a mixed solvent thereof, but is not limited thereto, and the degree of extraction of the active ingredient depends on the organic solvent to be extracted. and the degree of loss may be different, so an appropriate organic solvent can be selected and used. Specifically, the extraction may be performed 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 include filtering the extract to remove suspended solid particles. For example, it is possible to filter the particles using cotton, nylon, or the like, or use ultrafiltration, cryofiltration, centrifugation, etc., but is not limited thereto.

Concentration of the extract may be performed by methods such as concentration under reduced pressure, reverse osmosis concentration, and the like.

For the drying step after concentration, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying or infrared drying may be used. In some cases, it may further include a process of pulverizing the final dried extract.

As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific component group from a mixture including various components.

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

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

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 region, and the oral region refers to a space in the mouth connected from the front lip to the pharynx in the rear palate. In the present invention, the oral disease is a concept that includes all diseases occurring in the oral cavity, regardless of the pathology, and non-limiting examples of the oral disease include dental caries, periodontal disease (periodontitis or gingivitis), toothache, sore throat, bad breath. and the like.

As used herein, the term “periodontal disease” is often referred to as gingivitis, and is divided into gingivitis and periodontitis according to the severity of the disease. It is a relatively light and quick-recovery form of oral disease. The form limited to the gums, that is, soft tissue, is called gingivitis, and when such inflammation has progressed to the gingiva and alveolar bones, it is called periodontitis.

There is a V-shaped gap between the gingiva (gum) and the teeth. Bacteria attack the subgingival part of the gingival sulcus and damage the periodontal ligament and adjacent tissues. As inflammation progresses and more tissue is damaged, the groove develops into a periodontal pocket, and the more severe the periodontal disease, the deeper the periodontal pocket. As the periodontal pocket deepens, the periodontal ligament becomes inflamed, and osteoclast differentiation is promoted by lipopolysaccharide (LPS) secreted by Porphyromonas gingivalis, leading to loss of alveolar bone.

In the present invention, the term “halitosis” is an odor derived from the oral cavity and adjacent organs, and 85 to 90% of bad breath originates from the oral cavity, and in particular, from the back of the tongue. The main component of bad breath is volatile sulfur compounds, and 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, making it a place for bacteria to live continuously. Although the production of volatile sulfur compounds by anaerobic bacteria is the most important cause of bad breath, it is also caused by oral diseases such as dental caries, periodontitis, and dry mouth. Many types of anaerobic bacteria are involved in the occurrence of bad breath, and a non-limiting example of the bacteria causing bad breath may include Porphyromonas gingivalis, which secretes many types and large amounts of enzymes.

As used herein, the term "Porphyromonas gingivalis or P. gingivalis" is one of several representative causative bacteria that cause oral diseases. The Porphyromonas gingivalis is a kind of Bacteroid-like bacteria and is a Gram-negative bacteria and anaerobic bacteria. The Porphyromonas gingivalis occurs in the oral cavity where periodontal disease occurs, and is also found in the upper gastrointestinal tract, respiratory tract and colon. Accordingly, it is possible to prevent, improve or treat oral diseases through antibacterial activity against Porphyromonas gingivalis.

In one embodiment of the present invention, as a result of administering the extract of the present invention to Porphyromonas gingivalis, it was confirmed that the extract of the present invention exhibits antibacterial activity and antibacterial lasting effect (FIG. 1). Through this, it was confirmed that the extract of chrysanthemum marijuana was effective in preventing or treating oral diseases through antibacterial activity and antibacterial sustained activity against Porphyromonas gingivalis.

As used herein, the term "bone disease" is meant to include all diseases induced by bone loss or a decrease in bone density, which appear due to a loss of balance between the generation and differentiation of osteoblasts and osteoclasts. Specifically, osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, periodontal disease, osteolysis, It may include fibrous dysplasia, Paget's disease, osteogenic imperfect, hypercalcemia, and the like, and more specifically, osteoporosis due to osteoclast differentiation. However, it is not limited thereto.

As used herein, the term "osteoclast" is a cell derived from a macrophage precursor, and the osteoclast precursor cells are macrophage colony stimulating factor (M-CSF), NF-κB It is differentiated into osteoclasts by receptor activator ligand (RANKL), etc., and means to form multinucleated osteoclasts through fusion. Osteoclasts bind to bone through αvβ3 integrin, etc. and create an acidic environment, while secreting various collagenases and proteases to cause bone resorption. Osteoclasts do not proliferate into fully differentiated cells and cause apoptosis at the end of their life span of about 2 weeks.

In one embodiment of the present invention, after administration of the extract of the present invention to mouse mononuclear cells differentiated into osteoclasts, tarrate-resistant acid phosphate (TRAP), a marker for osteoclast differentiation, was stained. As a result, the concentration-dependent TRAP By significantly inhibiting the activity, it was confirmed that osteoclast formation was inhibited ( FIGS. 2 and 3 ). Through this, it could be confirmed that the champignon extract is effective in preventing or treating bone diseases, such as osteoporosis or oral diseases, through the activity of inhibiting osteoclast formation.

As used herein, the term "osteoblast" refers to a cell having the ability to calcify bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as Ca and Mg ions in the matrix.

In one embodiment of the present invention, after administration of the extract of the present invention to mouse mononuclear cells differentiated into osteoblasts, ALP (alkaline phosphatase) staining, which is an osteoblast differentiation marker, was performed, and the concentration-dependent ALP activity was significantly increased. By activating it, it was confirmed that it promotes the formation of osteoblasts ( FIGS. 4 and 5 ). Through this, it was confirmed that the extract of champignon is effective in preventing or treating bone diseases, such as osteoporosis or oral diseases, by promoting the formation of osteoblasts.

As used herein, the term "prevention" refers to any act of inhibiting or delaying the onset of bone disease by administration of the pharmaceutical composition according to the present invention, and "treatment" is suspected of bone disease by administration of the pharmaceutical composition. and all actions in which the symptoms of the affected individual are improved or are beneficially changed.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring 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 gum, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid Lactic Acid), poly-L-lactic acid, and mineral oil.

The pharmaceutical composition is an oral dosage form 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 diseases or bone diseases. It may be formulated and used in the form of a solution, and in particular, when the pharmaceutical composition of the present invention is for preventing or treating oral diseases, it may have a formulation for oral spray, oral ointment or oral varnish. The carrier may include various amorphous carriers, microspheres, nanofibers, rosin, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

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

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

The content of the oleracea extract or a fraction thereof included in the pharmaceutical composition of the present invention is not particularly limited thereto, but based on the total weight of the final composition, 001 to 100% by weight, 001 to 50% by weight, more specifically 001 to 20% by weight % may be included.

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

Since the pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect on oral disease or bone disease, the method of the present invention comprising the step of administering it to an individual can be usefully utilized for the prevention or treatment of oral disease or bone disease. have.

As used herein, the term "individual" refers to all animals, including humans, that have or may develop oral disease or bone disease, for example, monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, and pigs. , goats, birds, fish, and the like, and specific examples may be mammals including humans, but is not limited thereto.

In the present invention, the term "administration" means introducing the composition to an individual by any suitable method, and the administration route may be administered through various routes as long as it can reach the target tissue. Specifically, the pharmaceutical composition of the present invention may be administered orally, intravenously, subcutaneously, intradermally, intranasally, intraperitoneally, intramuscularly, transdermally, etc., and if necessary for local treatment, suitable including intralesional administration It may be administered by any method or route. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or cerebrovascular injection, but is not limited thereto.

In the treatment method of the present invention, a pharmaceutical composition comprising an extract of chrysanthemum marijuana may be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level depends on the severity of the disease, the activity of the drug. , the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the rate of excretion, the duration of treatment, including drugs used in combination with or concurrently with the composition of the present invention factors and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered alone or in combination with a known therapeutic agent for pterygium. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.

In addition, the dosage of the pharmaceutical composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or the type of material used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at an amount of 1 mg/kg to 200 mg/kg, specifically 1 mg/kg to 100 mg/kg, and more specifically 20 to 40 mg/kg per adult. , The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present invention in any way.

As another aspect, it provides a health functional food for the prevention or improvement of bone diseases comprising the extract of chrysanthemum marijuana as an active ingredient.

Specifically, the health functional food for the prevention or improvement of bone disease of the present invention may include an extract or a fraction thereof.

In this case, the descriptions of "C. chrysanthemum", "extract", "fraction", "oral disease", "bone disease", and "prevention" are the same as described above.

As used herein, the term "improvement" refers to any action in which the symptoms of a suspected bone disease and the subject of the invention that are prevented or treated by using a composition comprising an extract of chrysanthemum or a fraction thereof as an active ingredient are improved or beneficial.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, etc., as long as it can contain the extract of the present invention or a fraction thereof, and is not limited thereto. In addition, it may include the form of pills, powders, granules, needles, tablets, capsules or liquids.

As used herein, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. and regulating nutrients for function or obtaining useful effects for health applications such as physiological action. On the other hand, health food means food that has an active health maintenance or promotion effect compared to general food, and health supplement means food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement food can be mixed. The health functional food of the present invention can be prepared by a method commonly used in the art. It can be manufactured in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and it can be excellent in portability.

When manufacturing the health functional food, it can be prepared by adding raw materials and ingredients commonly added in the industry, and the type is not particularly limited. For example, it may include, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As another aspect, there is provided a quasi-drug composition for the prevention or improvement of bone diseases comprising the extract of chrysanthemum marijuana as an active ingredient.

Specifically, the quasi-drug composition for the prevention or improvement of bone disease of the present invention may include an extract or a fraction thereof.

In this case, the descriptions of the "seaweed", "extract", "fraction", "oral disease", "bone disease", "prevention" and "improvement" are the same as described above.

In the present invention, the term "quasi-drug" refers to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, have weak action on the human body, or do not directly act on the human body, Or non-machines and similar products, as an article corresponding to one of the preparations used for sterilization, insecticide and similar purposes for the prevention of infection type, for diagnosing diseases of humans or animals. Items used for the purpose of treatment, alleviation, treatment or prevention, which are not instruments, machines, or devices, and items used for the purpose of influencing the structure and function of humans or animals, which are not instruments, machines, or devices means goods. In addition, the quasi-drugs may include skin external preparations or personal hygiene products.

The external preparation for skin is not particularly limited thereto, and specifically, it may be prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or gel. Specifically, but not limited to, soap, cosmetics, wet tissue, tissue paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, cheek touch, mascara, eye shadow, sunscreen lotion, hair care products, It may be an air freshener gel or a cleaning gel. In addition, another example of the quasi-drug composition of the present invention is a disinfectant cleaner, shower foam, mouthwash, oral spray, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

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

As another aspect, there is provided a composition for feed for preventing or improving bone disease, comprising an extract of chrysanthemum marijuana as an active ingredient.

Specifically, the composition for feed for preventing or improving bone disease of the present invention may include an extract of chrysanthemum or a fraction thereof.

In this case, the descriptions of the "seaweed", "extract", "fraction", "oral disease", "bone disease", "prevention" and "improvement" are the same as described above.

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

The dental composition can be added to fluorine varnish and applied to dental products such as antibacterial fluorine varnish, and oral gargle solution, dental hypersensitivity treatment agent, antibacterial dental adhesive, dental filling material, dental restoration material, dental use. It can be applied to and used in dental materials such as root canal fillers, dental root canal sealers, fissure sealants, dental coatings, and dental cements.

In addition, the dental composition may be used in all articles used for oral health. Examples of the article include dental appliances such as dental floss, toothbrushes, interdental toothbrushes, electric toothbrushes, tongue cleaners, mouthwashes, toothbrush sterilizers, etc., dental devices such as braces and implants, or all dental tools and devices used in dental care. but is not limited thereto.

Specifically, the article is immersed in the dental composition, the dental composition is applied or sprayed on the surface of the article, the surface of the article is washed several times with the dental composition, or the dental composition is permeated. It can be used on the article by a method such as wiping the surface of the article with a towel or the like, and is not particularly limited as long as the article and the dental composition can be in contact for a predetermined period of 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 a person skilled in the art can appropriately select it.

The extract of the present invention is derived from natural products and has low toxicity, remarkably inhibits the formation or differentiation of osteoclasts, remarkably promotes the formation or differentiation of osteoblasts, and Porphyromonas gingivalis ( By significantly inhibiting the activity of Porphyromonas gingivalis ), it can be usefully used for the prevention or treatment of bone diseases resulting from bone diseases or oral diseases.

1 is a graph showing the number of TRAP-positive cells according to the treatment concentration of the extract of chrysanthemum. Significance test was performed through the student t-test, and significance * compared to the control group represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
2 is a photograph showing the results of TRAP staining of differentiated osteoclasts according to the treatment concentration of the extract of chrysanthemum.
Figure 3 is a graph showing the number of ALP-positive cells according to the treatment concentration of the extract of chrysanthemum. Significance test was performed through the student t-test, and significance * compared to the control group represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
4 is a photograph showing the results of ALP staining of differentiated osteoblasts according to the treatment concentration of A. K. extract.
Figure 5 is a graph showing the growth inhibitory effect of Porphyromonas gingivalis according to the treatment concentration of the extract of chrysanthemum. Significance test was performed through the student t-test, and significance * compared to the control group represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

Hereinafter, the present invention will be described in detail.

On the other hand, the embodiment of the present invention can be modified in various other forms, the scope of the present invention is not limited to the embodiments described below. In addition, the embodiment of the present invention is provided in order to more completely explain the present invention to those of ordinary skill in the art. Furthermore, "including" a certain element throughout the specification means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

Hereinafter, Examples and Experimental Examples of the present invention will be specifically illustrated and described below. However, the Examples and Experimental Examples to be described below are merely illustrative of a part of the present invention, and are not limited thereto.

Example 1. Preparation of extract

In cooperation with domestic botanical taxonomists, cultivation experts, and herbal medicine differentiation experts, the wild flowers were collected in Korea, dried, put in 99.9% methanol, and extracted at 45℃ for 3 days. After 15 minutes of sonication during extraction, a 2-hour stop process was performed 10 times/day. Thereafter, filtration was performed using a non-fluorescent cotton filter, and concentration was performed using a rotary evaporator (N-1000SWD; EYELA) at 45° C., and 24 using a module spin 40 (Biotron co.). dried for an hour. Then, the extract was stored in a low temperature room at -4 ℃ after removing the solvent in the form of 20 mg/tube. The above procedure was performed at the Korea Plant Extract Bank, and a sample of 20 mg/tube was dissolved in DMSO to a concentration of 50 mg/ml for use in cell experiments, and this was diluted with a cell culture medium and used.

Example 2. Inhibitory effect on osteoclast differentiation of chammoth extract

Purchase a 5-week-old male ICR mouse, take the fermur and tibia, wash it with α-MEM containing antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) to obtain 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; 10 ng/mL) for 1 day. Non-adherent bone marrow cells were dispensed in a Petri dish and cultured for 3 days in the presence of M-CSF (30 ng/mL). After washing the non-adherent cells, the adherent cells were used as bone marrow-derived macrophages (BMM). In order to differentiate BMM cells into osteoclasts, BMM cells (1 × 104 cells/well) were applied to a 96-well plate in the presence of RANKL (10 ng/mL) and M-CSF (30 ng/mL), and Example The extract prepared in 1 was treated with different concentrations (0, 3, 10 and 30 μg/mL), and the final concentrations were 125, 250 and 500 μg/ml, and cultured for 4 days. As a negative control, cells treated with only RANKL were used. After culturing 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 tartrate-resistant acid phosphatase (TRAP), a differentiation marker of osteoclasts, 100 μl of TRAP buffer (100 mM sodium citrate pH 5.0, 50 mM sodium tartrate, 3 mM p-nitrophenyl phosphate) was added and 37 After reacting at ℃ for 5 minutes, 100 μl of 0.1 N NaOH was added to terminate the reaction. Then, by measuring the absorbance at 405 nm, the TRAP activity was measured.

As a result, as shown in FIGS. 1 and 2 , it was confirmed that the number of red-stained osteoclasts was significantly reduced while suppressing the TRAP activity in a concentration-dependent manner when RANKL-induced osteoclasts were treated with the extract. .

Through the above results, it can be seen that the extract of the present invention can be effectively used for the treatment of bone diseases because it exhibits an excellent inhibitory effect on osteoclast differentiation generation.

Example 3. Osteoblast differentiation-promoting effect of chamomile extract

The degree of promotion of osteoblast differentiation was observed by measuring the ALP activity of the expression level of Alkaline Phosphatase (ALP), an early biomarker of osteoblast differentiation. C2C12 cells, a myoblast cell line purchased from American Type Culture Collection (VA, USA), were grown with Dulbecco's Modified containing growth medium (GM; 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin). Eagle's Medium (DMEM)). Cells were seeded, and after 1 day, the medium was replaced with a differentiation medium (DM; DMEM containing 5% FBS and 50 ng/ml rhBMP-2) to differentiate the cells. At this time, the osteoblast differentiation promoting efficacy of the extract was evaluated using a differentiation medium with or without the extract. After 3 days, the cells were washed twice with phosphate-buffered saline (PBS), and 100 μl of 1-Step PNPP (p-nitrophenyl phosphate) substrate solution (Thermo Fisher Scientific, Mass., USA) was added. Thereafter, the cells were incubated at room temperature for 15 minutes, and the reaction was stopped by adding 50 μl of 2N NaOH. Then, the absorbance was measured at 405 nm using a microplate reader (Wallac EnVision, PerkinElmer, MA, USA). In addition, the osteoblast differentiation promoting efficacy of the extract was evaluated through ALP staining. Cells were prepared in the same manner as above, and after treatment with the extract, 3 days later, the cells were washed twice with PBS, fixed with 10% formalin in PBS for 30 seconds, and rinsed with deionized water, Staining was performed using an Alkaline Phosphatase Kit (Sigma, MO, USA). The degree of promotion of osteoblast differentiation can be confirmed through the degree of strong ALP staining.

As a result, as shown in FIGS. 3 and 4 , it was confirmed that the treatment of Osteoblasts extracts significantly increased the number of osteoblasts while promoting ALP activity in a concentration-dependent manner.

Through the above results, it can be seen that the extract of chrysanthemum of the present invention can be effectively used for the treatment of bone diseases because it exhibits an excellent osteoblast differentiation-promoting effect.

Example 4. Antibacterial effect of extract

In order to evaluate the oral antibacterial activity of the extract, P. gingivalis (Prophyromonas gingivalis; P. gingivalis, ATCC33277) was used. Porphyromonas gingivalis was activated by using BHI broth containing hemin and menadione as a medium, and _{cultured for 72 hours in an incubator at 37° C., CO 2} , each well of a 96-well plate 90 μL of bacteria were added to the (well), and 10 μL of the extract prepared in Example 1 was added. As a control group, 100 μL of bacteria alone was used, and 100 μL of BHI broth was additionally added to each well so that the final concentrations of the extracts of chrysanthemum extract were 250 μg/mL and 500 μg/mL. Then, the sample was 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 FIG. 5 , it was confirmed that the concentration-dependent inhibition of the growth of Porphyromonas gingivalis was confirmed when the extract was treated. Specifically, it was confirmed that the growth of Porphinomonas gingivalis was inhibited by about 50% when treated with 500 μg/mL of champignon extract.

Through the above results, it can be seen that the extract of champignon of the present invention exhibits excellent antibacterial and antibacterial lasting effects against the strains causing oral diseases, so it can be effectively used for the treatment of oral diseases and bone diseases resulting from oral diseases. there was.

As mentioned above, although the present invention has been described in detail through preferred preparation examples, examples and experimental examples, the scope of the present invention is not limited to the characteristic examples, and should be interpreted by the appended claims. In addition, those who have acquired ordinary knowledge in this technical field should understand that many modifications and variations are possible without departing from the scope of the present invention.

Claims (11)

Osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, osteolysis, and fiber containing the extract of chrysanthemum as an active ingredient A pharmaceutical composition for the prevention or treatment of any one or more bone diseases selected from the group consisting of fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
Any one selected from the group consisting of periodontal disease, periodontitis, and alveolar bone disease caused by Porphyromonas gingivalis containing an extract of chrysanthemum as an active ingredient A pharmaceutical composition for the prevention or treatment of abnormal bone disease.
delete delete delete The pharmaceutical composition of claim 1, wherein the prevention or treatment of bone disease is achieved through inhibition of osteoclast differentiation.
The pharmaceutical composition according to claim 1, wherein the prevention or treatment of bone disease is achieved through promotion of osteoblast differentiation.
Osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, osteolysis, and fiber containing the extract of chrysanthemum as an active ingredient A health functional food for the prevention or improvement of any one or more bone diseases selected from the group consisting of fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia.
Osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, osteolysis, and fiber containing the extract of chrysanthemum as an active ingredient A quasi-drug composition for the prevention or improvement of any one or more bone diseases selected from the group consisting of fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia.
Osteoporosis, osteomalacia, osteopenia, bone atrophy, osteoarthritis, rheumatoid arthritis, osteolysis, and fiber containing the extract of chrysanthemum as an active ingredient A feed composition for preventing or improving any one or more bone diseases selected from the group consisting of fibrous dysplasia, Paget's disease, osteogenic imperfect, and hypercalcemia.

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