KR101760178B1 - Composition comprising the extract of Hizikia Fusiforme for treating periodontal disease - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of periodontal disease, which comprises a root extract. The present invention has few side effects as a natural product, and has an excellent effect of alleviating inflammation and activating alveolar bone formation of periodontal ligament cells. Thus, periodontal disease can be effectively prevented, treated or improved.

Description

[0001] The present invention relates to a composition for treating periodontal disease,

The present invention relates to a composition for the prevention or treatment of periodontal disease, which comprises a root extract.

The tooth is structurally the hardest hard tissue in the human body and is supported by the alveolar bone. The alveolar bone is firmly attached to the base bone of the jaw, and the 2-3 mm portion immediately adjacent to the root may be referred to as a periodontal bone, but the alveolar bone generally refers to both the hard tissue including the periodontal bone. When the alveolar bone is lowered, the tooth root is exposed and the symptoms called the taste appear. Also, when teeth are lost, alveolar bone tends to gradually disappear.

The periodontal ligament is a layer with an average thickness of 0.2 mm, which attaches to the teeth and attaches and supports the teeth to the alveolar bone. In addition, periodontal ligaments disperse the force to the alveolar bone when the tooth chews the food to buffer and transmit the sense of the tooth to the alveolar bone. Because there are undifferentiated mesenchymal cells in the periodontal ligament, bone reconstruction can be continued without changing the overall structure and function. The undifferentiated mesenchymal cells in the periodontal ligament migrate to the alveolar bone and modify the alveolar bone, or move toward the teeth to produce the collagen fibers and firmly support the teeth.

The incidence of periodontal disease increases with age. If periodontal disease is the only gingivitis, it is called gingivitis. If the inflammation progresses to the vicinity of the gums and gums, it is called periodontitis. If periodontal disease progresses, it will damage the periodontal ligament and even the alveolar bone. An example of alveolar bone damage is alveolar bone osteodystrophy, which further includes alveolar bone osteoporosis, alveolar bone osteomalacia, and alveolar bone osteopenia. If the alveolar bone is damaged, the tooth is eventually lost.

Currently, periodontal disease is mainly treated by surgical methods such as nonsurgical or surgical dental decompression, root resorption, gingival curettage, and periodontal regeneration. However, these surgical treatments are not only painful and troublesome to the patient but also applied to a certain degree of disease rather than preventing the disease. In addition to surgical treatment methods, antibiotics that work systemically as a medication or that are released from a local site are used. However, in this case, too much of the drug is delivered to unnecessary sites, resulting in side effects and no effect on antibiotic resistant bacteria have.

In order to effectively treat periodontal disease, infection, inflammation, periodontal ligament and alveolar bone damage should be treated in a complex manner, but they have not been developed to date as drugs or treatment methods for such treatment.

Korean Patent No. 10-0605116 (Title of the Invention: Composition for preventing and treating periodontal disease containing extract as a active ingredient)

The present invention aims at providing a pharmaceutical composition and a food composition for preventing or treating periodontal disease, which comprises a root extract as an active ingredient.

The present invention provides a pharmaceutical composition for prevention or treatment of periodontal disease, which comprises a top extract as an active ingredient.

The present invention also provides a food composition for preventing or ameliorating periodontal disease, comprising an extract of Aspergillus as an active ingredient.

In the present invention, the fusiformis (Hizikia fusiforme ) is a perennial algae inhabiting the lower part of the intertidal zone about 30 cm above the hypocenter and present only in the metamorphic age. In Korea, it grows from south of Jeomjin to the west coast of Jangsan - gan, and it is widely distributed in south coast and Jeju area. They belong to the taxonomic Heterokontophyta, Phaeophyceae, Fucales, Sargassaceae and Hizikia. It is dark brown with 3 ~ 5 stalks in short roots and the branches are very short and the branches have many leaves and bubbles. The size of the plant is about 20 to 60 cm. The stem is shaped like a whip, and the leaves are in the shape of a spoon with an aphrodite or a mount, and the bubble is a little swollen in shape with a center, and there is no houseplant.

In the present invention, it is possible to use the tops of the tops.

In the present invention, the extraction method such as hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction can be used. The number of times of extraction is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto. It is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration, but the present invention is not limited thereto. By concentrating the extract, the extract is extracted in liquid or powder form. The extract can be obtained by extracting with an extraction solvent selected from the group consisting of water, an alcohol having 1 to 4 carbon atoms and an alcohol aqueous solution having 1 to 4 carbon atoms, and can be a semi-dried or dried powder of the extract thus obtained . Preferably, the extraction solvent may be an aqueous ethanol solution, more preferably a 40 to 90% aqueous ethanol solution. The extraction with the aqueous ethanol solution can be carried out at room temperature for 1 to 7 days, preferably for 3 days. It may also be carried out under shading and / or warming (e.g., warming) conditions, if desired. The amount of the above-mentioned extraction solvent is 2 to 15 times the weight of the dry weight. The top extract of the present invention has an amount of 0.1 to 95% by weight based on the total weight of the composition containing the top extract.

The pharmaceutical composition or food composition of the present invention can be used to prevent or treat periodontal disease. Periodontal disease refers to diseases of the gums and adjacent tissues. Periodontal disease starts from bacterial infection, and periodontal pocket is formed between the gum and tooth when the inflammation reaction is progressed by the toxin of the bacteria. As the disease deepens, the depth of the periodontal cavity deepens and the inflammation spreads as the periodontal disease progresses. Loss will occur. Or, if the balance between osteogenesis and bone resorption in the alveolar bone is broken and the bone resorption exceeds the osteogenesis and the bone mass falls below the limit, alveolar bone formation disorder, which is a type of periodontal disease, occurs.

Periodontal diseases that can be prevented, treated or ameliorated by the pharmaceutical composition or food composition of the present invention include gingivitis, periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy. . Alveolar bone formation disorders include alveolar bone osteoporosis, alveolar bone osteomalacia, and alveolar bone osteopenia.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier, which contains the extract of the present invention as an active ingredient, and may be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like Oral formulations, external preparations, and sterile injectable solutions. The pharmaceutically acceptable carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

In addition, the composition of the present invention includes a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient. Solid form preparations for oral use include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like, and may include a lubricant such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like. Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions and freeze-dried preparations, and non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, And the like.

The compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, Lt; / RTI > Preferably, the composition of the present invention can be administered orally or subcutaneously. However, the route of administration is not limited thereto.

In the compositions and methods of the present invention, the dosage of the active ingredient varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but may be suitably selected depending on the case. For example, the top extract may be administered at a dose of 0.0001 to 500 mg / kg per day, preferably 0.01 to 300 mg / kg per day, and the administration may be administered once a day or divided into several times.

The formulation of the food composition of the present invention may be prepared by a conventional method and may be formulated in the form of capsules or other tablets, granules, powders, beverages, porridge, etc. after drying together with the carrier. .

The food composition of the present invention can be used as a health functional food. "Health functional food" refers to foods prepared and processed using raw materials or ingredients having useful functions in the human body pursuant to Law No. 6727 on Health Functional Foods, and the term "functional" It is intended to take nutrients for the purpose of controlling nutrients and obtaining effects that are useful for health use such as physiological action.

The food composition of the present invention may contain conventional food additives and, unless otherwise specified, the suitability of the food additives as "food additives" is to be determined by the Food and Drug Administration in accordance with General Regulations and General Test Methods approved by the Food and Drug Administration. Shall be determined according to the relevant standards and standards.

Examples of the substances described in the above-mentioned "Food Additive Code" include natural compounds such as ketones, chemical compounds such as glycine, potassium citrate, nicotinic acid and cinnamic acid, persimmon extracts, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, a tar coloring agent and the like.

The health functional food composition of the present invention can be used for various foods and beverages, and can be used for various foods, beverages, gums, tea, vitamin complex, health functional supplement, food additive, , Capsule, ring or beverage. May be in any food form other than those described above.

The formulation of the food composition of the present invention may be prepared by a conventional method and may be formulated in the form of capsules or other tablets, granules, powders, beverages, porridge, etc. after drying together with the carrier. .

The present invention provides a method for preventing, ameliorating, or treating periodontal disease by administering a top extract to a patient.

The present invention has a small natural side effect and is effective in treating infections, alleviating inflammation, and activating alveolar bone formation of periodontal ligament cells. Thus, periodontal disease can be effectively prevented, treated or improved.

Fig. 1 is a graph showing the evaluation of NO production inhibitory activity.
FIG. 2 is a graph showing an increase in the activity of alkaline phosphatase in periodontal ligament cells.
3 is a graph showing the increase in mineralization activity of periodontal ligament cells.
Fig. 4 is a 3D diagram showing the effect of improving periodontitis in the white paper. Fig.
FIG. 5 is a graph showing CEJ-ABC values and Furcation Involvement values at the first molar and second molar end calculated from the view of FIG.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

Manufacturing example . Manufacture of extracts

The dried Totsu (Wando deckhouse, KR) was washed in running water to remove impurities. Water (20 L) was added to 1 kg of the washed tops and subjected to hot water extraction at 100 ° C. The above procedure was carried out in 3 batches. The obtained extracts were combined and concentrated under reduced pressure at 40 DEG C or lower to obtain 628 g (yield: 20.9%) of a semi-solid form of the extract.

Example  1. Evaluation of inhibitory activity of NO extract on extracts

It is reported that high concentration of NO inhibits the proliferation and differentiation of periodontal ligament cells by inducing an inflammatory reaction while low concentration of NO inhibits the inflammatory reaction and stimulates the function of periodontal ligament cells. Therefore, whether the extract of the present invention inhibited NO production was evaluated.

(Biochem. Biophys. Res. Commun. 191, p. 1301-1308, 1993).

RAW 264.7 cell line, a mouse macrophage line, was inoculated at 2.5 × 10 ⁵ cells / ml into a 48 well plate containing DMEM containing 10% fetal bovine serum (Dulbecco's Modified Eagle's medium, Cambrex, USA) In group 3, LPS (10 ng / ml) was added to the second group to induce macrophage activation. Group 3 was further divided into three groups, 1 μg / ml for group 3-1, 10 μg / ml for group 3-2 and 100 μg / ml for group 3-2 were added with LPS (10 ng / ml). Each was incubated in a carbon dioxide incubator (5% CO 2, 95% relative humidity, 37 ° C) for 24 hours.

Each of the above culture solutions was supplemented with a grease reagent (37.5 mM sulphanilic acid, 12.5 mM N- (1-naphthyl) ethylenediamine dihydride, 6.5 mM hydrochloric acid ]), And then the absorbance was measured at 550 nm using a spectrophotometer. The concentration of NO ₂ produced by RAW 264.7 cells in the culture was calculated based on the NO ₂ -concentration-absorbance correlation coefficient generated using nitrate of 0 to 50 μM concentration.

The results are shown in Fig. In FIG. 1, it can be seen that the extracts of the plant extract significantly inhibited the production of NO in a concentration-dependent manner (in FIG. 1, * indicates p-value <0.05 and ** indicates p-value <0.01).

From the above results, it can be seen that the extract of the present invention inhibits NO production, inhibits the inflammatory reaction, and stimulates the action of periodontal ligament cells.

Example  2. Alkaline phosphatase activation assay

Alkaline phosphatase (ALP) is known as an early indicator of periodontal ligament cell differentiation. Therefore, it was evaluated whether or not the extract of the present invention increases alkaline phosphatase activity.

Human periodontal ligament cells (Periodontal ligament progenitor cells (PDLC) 10% FBS (fetal bovine serum; FBS), 100 IU / ml penicillin and 100 ug / ml streptomycin into the DMEM medium is added 5% CO _2, And cultured in a cell incubator maintained at 37 ° C. The cultured cells were plated at 1 × 10 ⁵ cells / ml in a 24-well plate and cultured in the cell incubator for 24 hours.

The cultured cells were divided into two groups. No treatment was given to the first group, and the second group was further subdivided into three groups: 1 μg / ml for the 2-1 group, 10 μg / ml for the 2-2 group, 2 -3 group were added 100 μg / ml of the extract. After 72 hours, the medium was removed from the plate and washed three times with PBS. Then, 50 μl of 0.15% Triton X-100 was added to the plate, and the cells were lysed by incubating in a 37 ° C. incubator for 30 minutes . After centrifugation at 3,000 rpm for 5 minutes, 40 μl of supernatant, 40 μl of 0.1 N glycine, and 20 μl of 100 mM p-nitrophenylphosphate (p-NPP) were added and reacted for 30 minutes at 37 ° C. After the reaction, 50 μl of 0.1 N NaOH was added to stop the reaction, and the absorbance at 405 nm was measured to calculate the amount of p-NPP converted to p-nitrophenol (p-NP) by ALP enzyme.

The results are shown in Fig. In FIG. 2, it can be seen that the extracts from the roots increase ALP activity (* indicates p-value <0.05 and ** indicates p-value <0.01 in FIG. 2).

From the above results, it can be seen that the extract of the present invention promotes the differentiation of periodontal ligament cells by increasing ALP activity.

Example  3. Top extract Mineralization  Promotion evaluation

Periodontal ligament cells produce calcium and release it outside the cell. Therefore, mineralization occurs in the medium where the periodontal ligament cells are cultured and calcium accumulates around the cells. Therefore, it was evaluated whether or not the periodontal ligament cells of the present invention increase mineralization.

Human periodontal ligament cells (Periodontal ligament progenitor cells (PDLC) ) with 10% FBS (fetal bovine serum; FBS), 100 IU / ml penicillin and 100 ug / ml streptomycin into the DMEM medium is added 5% CO ₂ , And cultured in a cell incubator maintained at 37 ° C. The cultured cells were subcultured in a 24-well plate at 1 × 10 ⁵ cells / ml and cultured in the cell incubator for 24 hours.

Ascorbic acid and beta-glycerophosphate, which are known to activate periodontal ligament cells, were added to the medium at concentrations of 50 μg / ml and 10 mM, respectively, to activate periodontal ligament cells. The activated cells were divided into two groups. No substance was added to the first group (control group), and the second group was further divided into four groups. 1 μg / ml for the 2-1 group, 5 μg for the 2-2 group / ml, 10 μg / ml in group 2-3, and 50 μg / ml in group 2-4 were added to the culture medium. And the medium was replaced with a medium containing the extract of the present invention at intervals of 3 days and cultured for 24 days.

After each cell was fixed with 10% neutral formaldehyde solution, it was treated with Alizarin red-S staining solution for 5 minutes and washed to determine whether or not a red calcified nodule was formed on the naked eye. Respectively. After confirming nodule formation, 10 mM sodium phosphate (10% cetylpyridinium chloride, pH 7.0) was added at 1 ml / well and absorbance was measured at 550 nm with an ELISA reader.

The results are shown in FIG. In FIG. 3, the extracts of the primates showed 1.5 to 2 times increase in the phototoxic activity of periodontal ligament cells as compared with the control group (in FIG. 3, * indicates p-value <0.05 and ** indicates p-value < 0.01, and *** indicates p-value < 0.001).

From the above results, it can be confirmed that the extract of the present invention promotes mineralization of bone by increasing the mineralization activity of periodontal ligament cells.

Example  4. Check for periodontal disease improvement

As an animal model to check for periodontal disease improvement, there is a model that induces periodontal disease by using tooth ligation in the white paper, a model that induces calculus formation and alveolar bone damage, and a bacteria that causes periodontitis ( Entamoeba gingivalis, etc.). In the present invention, the effect of improving the periodontal disease on the extract of the top extract was confirmed by an experiment using an animal model of tooth ligation in a white paper.

The left lower second molar of a 7-week-old male rat was ligated with a 3-0 suture, and then ligated for 6 weeks to induce periodontitis. Doxycycline was given daily for 7 weeks in one group (positive control, control), but no dose was given in one group (negative control) after dividing into 5 groups. (HE 50, HE 100, and HE 200, respectively) were administered to the three groups at 50, 100, and 200 mg / kg, respectively, for 7 weeks. On the other hand, the rats that did not cause periodontitis were regarded as normal. After 7 weeks, the alveolar bone was sacrificed at the sacrifice of the white paper, and 3D images were obtained using Micro-CT.

Methods for verifying the improvement of periodontal disease in animal experiments include the method of measuring the distance between the alveolar bone and the cementum and enamel boundary of the tooth CEJ-ABC, the method of measuring the distance between the alveolar bone and the bottom of the tooth called furcation involvement, And pocket depth between the gums. In the present invention, CEJ-ABC and furcation involvement method were used to confirm. The results are shown in Fig. In FIG. 4, the red line A and C in the second upper right corner of FIG. 4 indicate the CEJ-ABC between the first molar and the second molar and between the second molar and the third molar, respectively, and the red line B Furcation involvement. If the alveolar bone of this part is broken and widening occurs, the tooth does not wipe well, resulting in a vicious cycle in which the widening becomes worse. In FIG. 4, in the case of the negative control group, the alveolar bone is lost and the teeth are exposed on the surface. However, in the case of the group treated with the extract, the higher the dose, the more the damage of the alveolar bone is improved. In HE200 group, It can be confirmed that there is not.

The CEJ-ABC value and the furcation involvement value were measured using the 3D photograph of FIG. 4, and the results are shown in FIG. In FIG. 5, CEJ-ABC and Furcation involvement index values were lower in the extract-treated group than in the negative control group, and the higher the dose of the extract, the better.

From the above results, it can be seen that the extracts of topotum prevent the damage of the alveolar bone in the animal experiment, help the alveolar bone regeneration, and improve the periodontal disease.

Claims (6)

A pharmaceutical composition for the prevention or treatment of periodontal disease comprising as an active ingredient an extract of Hizikia fusiforme obtained by using water as an extraction solvent. A food composition for preventing or ameliorating periodontal disease, which comprises, as an active ingredient, a safflower extract obtained by using water as an extraction solvent. The method according to claim 1, An extract, a semi-dried or dried powder of the extract. The composition of claim 1, wherein the periodontal disease is gingivitis, periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy. 5. The composition of claim 4, wherein the alveolar bone formation disorder is alveolar bone osteoporosis, alveolar bone osteomalacia, or alveolar bone osteopenia. The composition of claim 1, wherein the composition is for oral administration.

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