KR100519477B1 - Periodontal disease prevention and treatment composition containing trichromic acid, urodessoxycholine acid derivative and milky skin extract - Google Patents

Abstract

The present invention relates to a composition for preventing and treating periodontal disease containing triclosan, ursodesoxycholic acid derivatives, and ulmus extract. The periodontal disease prevention and treatment composition of the present invention not only has excellent bactericidal effect and periodontal disease prevention effect, but also can treat periodontal disease, thereby promoting oral hygiene.

Description

Periodontal disease prevention and treatment composition containing trichromic acid, urodessoxycholine acid derivative and milky skin extract

The present invention relates to a composition for preventing and treating periodontal disease containing triclosan, ursodesoxycholic acid derivatives, and ulmus extract. The periodontal disease prevention and treatment composition of the present invention not only has excellent bactericidal effect and periodontal disease prevention effect, but also can treat periodontal disease, thereby promoting oral hygiene.

Periodontal disease refers to the loss of teeth due to gingival inflammation and bleeding, periodontal pocket formation, and destruction of alveolar bone. This periodontal disease progresses to the process of colonization of bacteria, invasion of periodontal tissues of bacteria, and destruction of periodontal tissues. First, saliva proteins in the oral saliva are adsorbed to dentin and chalky surfaces to form a film and mainly on the surface of the film. As the bacteria, such as Streptococcus and Actinomyces grow, form plaques and, as time passes, the plaques move in the direction of the root of the tooth and at the same time, Porphyromonas and Actinobacillus Anaerobic gram-negative bacteria such as) grows and these bacteria, bacterial components and bacterial products penetrate into the gingival connective tissue through the gingival epithelial epithelium, forming periodontal pockets, and metabolism of these bacteria results in poisonous hydrogen sulfide, ammonia and toxic amines. While secreting toxins from cells such as Direct oxygen is destroyed by endotoxins such as polysaccharides or prostaglandins secreted outside the cell by various actions of humoral and cellular immune systems stimulated by the biological immune system, Prostaglandins, Inflammation of gums is caused by several cytokines such as leukotriens, histamine, and interleuckins, and Tumor Necrosis Factro α . Then, collagen, which is a substrate of periodontal tissue, is degraded by enzymes such as collagenase secreted from bacteria and leukocytes, gum retraction occurs, and if left unattended, it progresses to periodontal disease.

In an effort to prevent the occurrence of periodontal disease, antimicrobial agents such as Chlorhexidine gluconate, Cetylpyridium chloride and Sanguinarine have been developed to kill periodontal disease bacteria in a short time. Although it has been applied to oral products such as toothpaste and toothpaste, the occurrence of periodontal disease has not been fundamentally prevented yet.

Therefore, in recent years, efforts have been actively made to suppress periodontal disease using herbal extracts such as myrrh, baekbaekpi, horse riding, green tea, licorice, gold, pogongyoung, and gold and silver coins, but prostaglandins play a decisive role in causing periodontal disease. Plant extracts that inhibit the production of collagenase, which inhibits production or degrades periodontal tissue, have not been identified, and in most cases, inhibit the formation of plaque, or inhibit antibacterial, anti-inflammatory, astringent, hemostasis, and blood circulation. It is to prevent the occurrence of periodontal disease through the action of herbal medicine.

Until now, the most widely used method to treat periodontal disease is to use an agent that inhibits the activity of collagenase enzymes that break down the periodontal tissue or inhibits the production of cytokines or prostaglandins, which are inflammation-causing agents. .

European Patent No. 528468 A1 describes a method for treating periodontal disease by developing a toothpaste and a mouthwash containing triclosan to inhibit the production of periodontal disease by inhibiting the production of prostaglandins, the cause of periodontal disease. In USP 5356615, oral products using triclosan were suggested to have an excellent effect on suppressing plaque formation. USP 5571501 also suggested the efficacy of triclosan on the bactericidal effect of oral microorganisms. In addition, clinical preventive dentistry (Clinical Preventive Dentistry vol. 14 (16), 1992), etc., tried to provide the effect of inhibiting plaque formation and preventing periodontal disease using triclosan. U.S. Patent No. 5,2308,895 A develops a system in which a drug containing tetracycline is continuously delivered to treat periodontal disease by inhibiting the enzyme activity of collagenase, which breaks down periodontal tissue. In Korean Patent Application Nos. 96-61927 and 96-69522, milk extracts inhibit the enzymatic activity of collagenase, which degrades periodontal tissue, and promote collagen synthesis. It has been shown to have an excellent effect on regeneration. In addition, Korean patent applications Nos. 96-30337 and 96-30449 have shown the efficacy of urodesoxycholine acid as a medicament that inhibits the production of cytokines or prostaglandins, which are inflammation-causing agents in periodontal disease. However, the above-mentioned medicinal agents not only inhibit the production of periodontal disease-causing substances in the treatment of periodontal disease, but also inhibit the activity of periodontal tissue degrading enzymes and at the same time inhibit the production of cytokines and prostaglandins that cause gum inflammation. It does not have three effects and has a limit to completely treat periodontal disease. In particular, tetracycline is highly susceptible to side effects when used for a long period of time. Side effects such as vomiting, pain in the gastrointestinal tract, diarrhea and tooth pigmentation occur very often. Can also be a problem.

Accordingly, the present inventors effectively inhibit the production of cytokine or prostaglandin, which are mediators of effective sterilization and gum inflammation of oral harmful microorganisms for the prevention and treatment of oral hygiene and periodontal disease. In addition, the present study was conducted to find a composition that inhibits the activity of periodontal tissue degrading enzymes and promotes the regeneration of periodontal tissue. Therefore, the present inventors confirmed that it has a strong bactericidal effect on oral harmful microorganisms through experiments, and selected tricroic acid, which is recognized for medical and toxicological safety, as an effective fungicide of the present invention. In addition, the present inventors established the method of measuring collagenase activity and collagen synthesis quantifying the degradation of the periodontal tissue, and searched for herbal medicines and plant extracts that are safe for humans to inhibit collagenase activity and inhibit collagen activity. Milky skin extract, which has an excellent effect on promoting synthesis, was selected as an effective drug for preventing periodontal tissue destruction and for tissue regeneration. And the present inventors have used scientific methods such as quantification of the messenger RNA molecule of Tumer-Nicrosis Factor Alpha and Interleukins, which are cytokines that cause periodontal disease, quantification of prostaglandins (PGE ₂ ), and superoxide production assays. Ursodesoxycholic acid, chenodesoxycholic acid, the active ingredients of wood and ungdam showed excellent efficacy in suppressing the production of cytokines and prostaglandins (PGE ₂ ) causing periodontal disease using At least one of (Chenodesoxycholic acid) and its derivative Taurodesoxycholic acid was selected as a preventive and therapeutic agent for gingivitis.

Therefore, the present inventors confirmed that the application of these three pharmacological agents at the same time can maximize the efficacy and effect in promoting oral hygiene and preventing periodontal disease prevention than using these medicinal agents alone or in combination of the two, As a result of clinical trials targeting patients with periodontal disease, the present invention was completed by confirming that the efficacy is maximized when these three active agents are used simultaneously, rather than using these active ingredients alone or two drugs at the same time.

The composition of the present invention contains 0.001 to 1% by weight of tricroic acid, 0.001 to 1% by weight of urethanesoxycholine acid derivatives and 0.001 to 3% by weight of milky skin extract.

Trichromic acid is composed of 2,4,4'-trichloro-2'-hydroxydiphenyl ether and is known to have an antimicrobial activity in the bacterial cell membrane and excellent tooth retention effect.

Yubaekpi is dried and dried in the sun by extracting the root of the stem or root of Ulmus pumila, an elmaceae plant distributed in the north central Korea and China. It is known that there is, the main component is composed of beta sitosterol ( β- Sitosterol), phytosterol, and tannin. Milky skin extract contains both polar and non-polar extracts of the elm tree's root bark, and elm trees include Ulmus pumila, Ulmus parvifolia Jacq., Ulmus macrocarpa Hance, and large-leaf elm. (Ulmus macrocarpa var. Amcrophylla Nak.) And Ulmus davidiana Planch are all common names.

Fut tauri (gallbladder of Bos taurus demesticus Gemlin) and ungdam (gallbladder of Fel ursi, Selenarctos thibetanus G. Cuvi, etc.) are safe for humans and are used as herbal medicines such as edema, sedation, dysentery, chronic hepatitis, sore throat, Efficacy is known in life support, anti-inflammatory, and the like, and main active ingredients include urodessoxycholine acid, chinodesoxycholine acid, and its derivatives, taurusodesoxycholine acid.

Detailed description of the invention is as follows.

It is preferable to use the trichromic acid used in the present invention at a predetermined ratio within the content of 0.001 to 1% by weight based on the total composition.

In the method of manufacturing herbal extracts of milky skin, dried herbal medicines are dried in small shades and powdered, and 50 ml of powder is added to 500 ml of 95% ethanol for 3 days, followed by immersion extraction for 3 days, and the extract is filtered using No. 1 Whatman No. 1. After concentration under reduced pressure, it is used in the present invention. Milky skin herb extract used in the present invention is preferably used in the oral product composition by mixing 0.001 to 3% by weight, preferably 0.01 to 1% by weight. At the concentrations of the herbal extracts of less than 0.001% by weight, the efficacy and effect on periodontal disease cannot be expected. If the content exceeds 3% by weight, the synergistic effect of the three medicinal agents cannot be expected. And the content of another active ingredient used in the present invention urodes oxycholine acid, chinodes oxycholine acid and its derivatives taurusodesoxycholine acid, respectively, or suitable for use in oral product compositions Do. 0.001 to 1% by weight, preferably 0.01 to 0.1% by weight, based on the total composition. When the content of the urethanesoxycholine acid derivatives is less than 0.001% by weight, the efficacy and effect on periodontal inflammation cannot be expected, and when it exceeds 1% by weight, the synergistic effect of the three medicinal agents cannot be expected.

The present invention removes the causative agent of the development of gingivitis in the usual ingredients such as toothpaste, mouthwash, oral ointment, oral products, and at the same time, trichromic acid, milk white skin extract, and urethanesoxycholine for the treatment and prevention of immunological gingivitis The present invention relates to a composition for promoting oral hygiene and preventing and treating periodontal disease simultaneously using one or more of these acids as an active ingredient among acids, chinodesoxycholine acid, and derivatives thereof, taulorsodecoxycholine acid. As another component of this invention, it mix | blends using the appropriate amount of the component normally used according to the kind of composition for oral cavity, and a use purpose. For example, the toothpaste composition of the present invention can be prepared by the following method.

Abrasive components include calcium monohydrogen phosphate, precipitated silica, silica gel, sodium bicarbonate, calcium carbonate, hydrous alumina, insoluble sodium phosphate, sodium pyrophosphate, etc. As a fluorine compound which is an agent that promotes remineralization of the tooth and strengthens the tissue of the tooth, sodium fluoride and sodium monofluorophosphate may be mixed or used alone, and its content is suitably 0.01 to 2.0% by weight. It is not suitable as an oral preparation because the content is ineffective at a concentration of 0.01% by weight or less and exceeds 2.0% by weight because it may affect the safety of the human body.

Wetting agents are used to maintain the state of the toothpaste composition and to prevent drying. Such wetting agents include glycerin, sorbitol, polyethylene glycol and propylene glycol, and are used alone or in combination of two or more. In addition, the toothpaste component is used to combine the liquid and solid components to maintain the form of the toothpaste and ensure the stability of natural or synthetic polymer materials such as gum, acacia gum and the amount is used 0.1 to 5% by weight. The foaming agent complements the cleaning action of the abrasive and not only penetrates the drug to the areas where the toothbrush is hard to reach, but also generates bubbles to increase the brushing feeling, helps the cleaning action, speeds up the dispersion and penetration of the drug, It reduces the interfacial tension, so foreign substances in the oral cavity easily fall. Commonly used foaming agents are anionic surfactants such as sodium lauryl sulfate and sodium alkyl sulfate, and secondary nonionic surfactants are polyoxyethylene polyoxypropylene copolymers, polyoxyethylene sorbitan fatty acids, alkanolamide fatty acid esters and sucrose fatty acid esters. , Polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters and polyoxyethylene castor hardened oil derivatives are used. The amount of foaming agent is preferably 0.5 to 5% by weight of anionic or nonionic surfactants alone or in combination of two or more.

In addition, flavors and sweeteners are used to control the hot or somewhat bitter taste. The flavor is mainly used in the natural flavors such as peppermint and spearmint oil is used 0.1 to 1% by weight in toothpaste. As the sweetener, synthetic or natural non-fermentable sugars are mainly used. Examples of the sweeteners include sodium saccharin, aspartame, lactose, maltose, and xyitol, and a suitable sweetener is preferably 0.05 to 1% by weight of saccharin sweetener. Do. In addition, as a buffer to adjust the pH of the toothpaste composition, alkali metal salts of regular phosphoric acid, especially sodium phosphate, dibasic sodium phosphate, trisodium phosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate Can be used. In order to prevent contamination of microorganisms that may occur during the manufacture and use of toothpaste compositions, 0.1 to 0.5, alone or mixed with methyl paraoxyansiate, benzoic acid, sodium benzoate, salicylic acid, etc. Use by weight.

The composition for improving oral hygiene and prevention and treatment of periodontal disease of the present invention is usually used as a medicament of the ingredients used in the product, and as a medicament, triclosan and milky skin extract, urodessoxycholine acid, chinodesoxycholine It is characterized by using at least one of these three substances simultaneously as an active ingredient in an acid and its derivatives, tauruthoradexoxycholine acid.

In the following experimental example, using the experimental group containing triclosan, milky skin extract and urodesoxycholine acid in the composition shown in the following Table 1 was tested for the bactericidal effect.

TABLE 1

Experimental Example 1. The bactericidal effect of the oral microorganisms of the experimental groups 1 to 15>

Experiments included Actinomyces viscosus ATCC 15987, Candida albicans ATCC 10231, Streptococcus mutans ATCC 10449, and Actinobacillus actinobacillus actinomycecicom 84. ) And five strains of Porphyromonas gingivalis ATCC 381 were used.

Actinomyces viscosus ATCC 15987 and Candida albicans ATCC 10231 were cultured in YM medium (glucose 10g / L, yeast extract 3g / L, malt extract 3g / L, bactopeptone 5g / L), and Streptococcus mutans ATCC 10449 was incubated in TH medium (Todd Hewitt broth 30g / L, sucrose 50g / L), Actinobacillus actinomycetum comitans ATCC 33384 and Porphyromonas gingivalis ATCC 381 was incubated in Thioglycollate Medium. The culture temperature was the Actinomyces viscosus ATCC 15987 cultured in an anaerobic incubator in 26 ℃, Candida albicans ATCC 10231 and Streptococcus mutans ATCC 10449 is a 37 ℃, Actinobacillus actinomycetumcomitans ATCC 33384 and Porphyromonas gingivalis ATCC 381 were cultured in an anaerobic incubator for 37 ℃.

The bactericidal test was carried out after subculture of these strains, three times before 24 hours, and inoculated with 1 × 10 ⁶ strains after adding 0.1 ml of the experimental group agonist to 9.9 ml of each suitable group medium. After incubation for 18 hours, strains were measured by colony count using agar dilution method.

<Experiment Result>

In the experimental results, the growth of microorganisms was not observed in the experimental groups 2, 5, 7, 8, 9, 12, 14, 15, 16, 19, 21, 22, which showed that tricroic acid has an excellent bactericidal effect against oral microorganisms. Indicates.

Experimental Example 2 Superoxide Production Inhibition Effect of Experimental Groups 1 to 22

Intact venous blood collected using citric acid as an anticoagulant from healthy adults without systemic disease was centrifuged at 1200 rpm for 10 minutes, and then the leukocyte concentrates were recovered first, and then diluted 1: 1 with RPMI 1640 medium. After 12 ml of Ficoll-Paque was added to a 50 ml centrifuge tube, 30 ml of diluted blood was carefully added to form a middle layer, followed by centrifugation at 1600 rpm for 30 minutes, and then the upper layer containing serum was removed. After careful collection of the cell-containing thickening, add three times RPMI 1640 medium and centrifuge at 800 rpm for 10 minutes, discard the supernatant, add 10 ml of RPMI 1640 medium and gently pipet at 800 rpm After centrifugation for 10 minutes, the supernatant was discarded and pipetted with HBSS (HANKS 'BALANCED SALT SOLUTION) buffer solution, followed by 24-well human mononuclear leukocytes. 10 ⁶ to ^late After dispensing 0.45 ml into cells / well and incubating for 2 hours aseptically under 95% air, 5% CO ₂ , 100% humidity conditions, FMLP ((N-Formyl-Met-Leu-Phe) was added to 10 ^-6 M Treated with 0.05 ml and cultured at 37 ° C. for 14 minutes to stimulate the cells, followed by 80 μM of 0.1 ml Cytochrome C, 30 μg of 0.1 ml Superoxide dismutase, and the composition of the experimental group. 0.1 ml of HBSS was added to make the remaining total reaction solution 0.9 ml, and then warmed at 37 ° C. for 10 minutes, and the final concentration of the phylogenized Zymosan A was 1.3. After adding 0.1 ml to mg / ml and warming for 90 minutes at 37 ℃ while shaking, the reaction was stopped for 10 minutes at 4 ℃, centrifuged at 4 ℃, 1500 rpm, 10 minutes and the supernatant at 550 nm The optical density was measured and the amount of superoxide anion produced was calculated by the following equation. .

In the above test results, as shown in the experimental groups 8, 15 and 22, the superoxide production inhibitory effect was better when trichromic acid, baekbaekpi and ursodeoxycholic acid were prescribed at the same time. From this, it can be seen that the application of tricroic acid, baekbaekpi and urodessoxycholine acid at the same time increases the superoxide production inhibitory effect.

Experimental Example 3. Inhibitory Effect on Collagenase, an Periodontal Tissue Degrading Enzyme of Experimental Groups 1 to 22

Experimental results of carrying out the inhibitory effect on the collagenase, a periodontal tissue degrading enzyme with the composition of the present invention are as follows.

This test method is a collagenase enzyme contained in the saliva and gingival fluid of periodontal patients secreted from periodontal pathogens such as Pyromonas ginjivalis, polymorphonucle leukocytes and neutrophils. It is an experimental method that mimics the bio-oral environment in which gum degeneration occurs due to the decomposition of collagen, which is a matrix of periodontal tissue by the action of. Each 100 ml of Azocoll, a 2% red collagen substrate, was added to 1.5 ml Eppendorf, and one eppendorf tube was used as a blank and three tubes were purchased from Sigma. Standard enzyme solution of collagenase type 1 (collagen degrading activity: 315 units / mg) was added to 10, 100, 200 ppm and Separyl S from the saliva and gingival sulcus of patients with periodontal disease was added to each tube of the experimental group. 100 μl of the purified collagenase enzyme was added through -200 chromatography, and 10 μl of the inventive composition (Experimental Group 1 to 22) was added to each of the tubes, followed by buffer solution (0.05 M Tris-HCl, 1 nM CaCl ₂ , 7.8) was added to 500 μl of total reaction solution, reacted for 18 hours at 37 ° C incubator, centrifuged at 10000 g for 5 minutes to precipitate undigested collagen, and the supernatant containing the degraded collagen was taken. W by measuring the absorbance at 540 nm to write and, in terms of the activity concentration of the enzyme evaluation from the standard curve for enzyme activity by standard activity curve. The results were as follows;

In the above test results, as shown in experimental groups 8, 15, and 22, when trichromic acid, milk extract, and ursodesoxycholine acid were simultaneously prescribed, the inhibitory effect on collagenase activity was higher than that when they were prescribed alone. Appeared excellent. This suggests that the treatment of collagenase activity is elevated when tricroic acid, baekbaekpi and urodesoxycholine acid are simultaneously administered.

Experimental Example 4. Inhibitory Effect on the Production of Tumer Nicrosensor Alpha of Mononuclear Leukocytes of Experimental Groups 1 to 22>

Blood mononuclear leukocytes isolated from blood were added to the 24-well plate in 0.8 ml to give 10 ⁶ Cells were well-dispensed and the wells to which 200 µl of RPMI 1640 medium were added were incubated for 24 hours. To a well of a 96-well plate to which a Tummer Nicosfector alpha antibody is attached, 50 μl of standard solution (0, 10.24, 25.6, 64, 160, 400 pg / well) is added, and the cell culture solution 50 is added to the experimental group well. Add μl, add 50 μl Biotinylated Antibody Reagent to all wells, hold at 25 ° C. for 2 hours, wash three times with wash buffer solution, Streptavidin-H conjugate Conjugate was added to all wells and maintained again at 25 ° C. for 30 minutes, then washed three times with wash buffer solution and 100 μl of enzyme substrate was added immediately, and the plate was opened at 25 ° C. in the dark. After maintaining for 30 minutes and adding 100 μl of 0.18 M sulfuric acid, the absorbance at 450 nm was measured with a microplate reader to prepare a standard curve using the absorbance value of the standard solution. Determined.

Trichoic acid, milky skin and ursodesoxycholine acid were tested as shown in experimental groups 8, 15 and 22. At the same time, the effects of suppressing the production of tomericosis factor alpha were better than those prescribed by them alone. From this, it can be seen that when the tricroic acid, milk white skin and urodesoxycholine acid are simultaneously prescribed, the effect of suppressing the production of tumer nicrosis factor alpha is increased.

Experimental Example 5. Inhibitory Effect on the Production of Interleukin (IL-1 β ) in Mononuclear Leukocytes of Experimental Groups 1 to 22>

A well in which blood mononuclear leukocytes isolated from blood was added to a 24-well plate was added to a control group, 100 μl of E. coli LPS (250 ppm) and 100 μl of LPS (250 ppm) and 100 μl of the composition of the present invention. One well is used as the experimental group and incubated for 18 hours. To a well of a 96-well plate to which an antibody of interleukin (IL-1 β ) is attached, 50 μl of standard solution (0, 10.24, 25.6, 64, 160, 400 pg / well) is added, and then the cells are added to the experimental group wells. Add 50 [mu] l of culture, add 50 [mu] l of biotinylated antibody reagent to all wells, maintain at 25 [deg.] C. for 3 hours, wash three times with wash buffer, and streptavidin-HRP conjugate to all wells. After addition, the mixture was maintained at 25 ° C. for 30 minutes and washed again with washing buffer solution three times. 100 μl of enzyme substrate was added immediately, and the mixture was kept at 25 ° C. in the dark for 30 minutes with the lid of the plate open and 0.18 M sulfuric acid. After adding 100 μl, absorbance at 450 nm was measured with a microplate reader, and a standard curve was prepared using the absorbance values of the standard solution.

As a result of testing the efficacy and effect of IL-1 β production in human mononuclear leukocytes stimulated with E. coli LPS, Triclosan, Milky Peel and Ursodesoxycholine acid simultaneously When prescribed, the effect of inhibiting the production of IL-1 β was better than that of these alone. From this, it can be seen that increasing the inhibitory effect of IL-1 β production when trichromic acid, baekbaekpi and urodesoxycholine acid were simultaneously prescribed.

Experimental Example 6. Inhibitory Effect on Prostaglandin (PGE ₂ ) Production of Mononuclear Leukemia in Experimental Groups 1 to 22>

Experimental results of the composition and the inhibitory effect on the production of prostaglandins causing periodontal disease are as follows. This study was performed to evaluate the production of prostaglandins (PGE ₂ ) induced by stimulating human mononuclear leukocytes with lipopolysaccharide, a cell wall component of the periodontal disease causing bacterium Popiromonas jinjivalis during the course of periodontal disease. A specific test method was a 0.1 M phosphate buffer solution containing 50 μl buffer (0.9% NaCl, 0.1% bovine serum albumin, 0.5% Kathon) in a blank well of 96-well plate to which anti-mouse lgG of goats was attached. Add 50 μl of the standard solution to a standard (0, 2.5, 5, 10, 20, 40, 80, 160, 320 pg) well, and then add 50 μl of the inventive composition to the well set as the experimental group. Add 50 μl of antibody against PGE ₂ to all wells except the blank wells and then add 50 μl of PGE ₂ conjugated peroxidase to all wells except the blank and cover 96 well-plate and 25 ° C. After maintaining for 1 hour in a washing buffer (phosphate buffer containing 0.05% Tween 20: pH 7.5), washed four times and at room temperature 150 μl of enzyme substrate (3,3 dissolved in 20% dimethylformamide) ', 5,5'-tetramethylbenzidine / hydrogen peroxide) was added immediately and at 25 ° C. for 30 minutes After maintaining and adding 100 μl of 1 M sulfuric acid, absorbance was measured at 450 nm with a microplate reader, and a standard curve was prepared using the absorbance values of the standard solution to calculate the amount of prostaglandin produced in the experimental group.

Efficacy and efficacy of PGE ₂ production in human mononuclear leukocytes stimulated with E. coli LPS were tested, and as shown in experimental groups 8, 15, and 22, tricroic acid, milky skin, and ursodesoxycholine acid may be prescribed simultaneously. In the case of PGE ₂ production inhibitory effect was better than when they were prescribed alone. This suggests that the treatment of trigemic acid, baekbaekpi and urodesoxycholine acid at the same time increases the inhibitory effect of PGE ₂ production.

Experimental Example 7. Effects on the Production of Polygenic Proteins of Gingival Fibroblasts of Experimental Groups 1 to 22

Primary cultured gingival fibroblasts were dispensed in 24-well plates at 10 ⁶ cell / well, incubated in DMEM medium containing 10% FBS for 1 day, and then replaced with fresh medium 24 After incubation for a period of time, the cell layer attached to the bottom was washed with HBSS buffer solution, and then 0.8 ml of MEM medium containing no serum and proline was added, and then 100 μl of ¹⁴ C-proline (10 μCi) was treated. Cells were incubated with the culture medium containing the cells, and total protein and collagen protein were measured after 24 hours. First, in order to measure the amount of extracellular total protein, the culture medium of each well was placed in a sealed dialysis tube at one end and sealed at the other end of the cold buffer solution (Tris-HCl 0.05 mol / L, NaCl 0.2 mol / L, CaCl). ₂ Complete the dialysis with 0.05 mol / L and 0.3 mN of phenylmethylsulfonyl fluoride for 24 hours, and then take 100 μl of each of them into a counting vial and add 10 ml of scintillation cocktail (Ccintillation coctail) Radioactivity was measured for 1 minute with (Liquid Scintillation Counter (LSC)). To measure the total amount of intracellular total protein, 0.1 N NaOH and 0.3 mN of phenylmethylsulfonyl fluoride were added to each well from which the cell culture medium was removed, and then maintained at 60 ° C. for 3 minutes to break down the cell membrane. In one end sealed dialysis tube and the other end sealed cold buffer (Tris-HCl 0.05 mol / L, NaCl 0.2 mol / L, CaCl ₂ 0.05 mol / L, phenylmethylsulfonyl fluoride 0.3 mN) After completion of dialysis for 24 hours, 100 μl of each was put in a counting vial, 10 ml of scintillation cocktail was added, and radioactivity was measured for 1 minute with a liquid scintillation counter. To measure the amount of intracellular and extracellular collagen protein, 100 μl of dialysis-produced cell culture solution and cell homogenate were added to 1.5 ml microtubes, and the collagenase buffer solution (0.5 M Tris-HCl, 1 mN CaCl ₂ , 0.3 mN phenylmethylsulfonyl fluoride) and 100 μl of collagenase enzyme (100 ppm) were added and then maintained at 37 ° C. for 3 hours to completely break down collagen and then remove 50% of undigested protein. 500 μl of a solution containing trichloroacetic acid and 1% tannic acid was added, and then precipitated at 4 ° C. for 30 minutes, followed by centrifugation at 1000 × g for 5 minutes, followed by separation of the supernatant, and then 100 μl of the counting vial. 10 ml of scintillation cocktails were put in and the radioactivity was measured for 1 minute by LSC.

In order to study the efficacy and effect on the collagen synthesis of human fibroblasts, the present invention was treated with a cell culture medium containing ¹⁴ C-proline, and as shown in experimental groups 8, 15 and 22, triclosan, milk milk and urine When Sodesoxycholine acid was prescribed at the same time, collagen synthesis promoting effect was better than that when they were prescribed alone. From this, it can be seen that increasing the collagen synthesis promoting effect when tricarboxylic acid, baekbaekpi and urodesoxycholine acid at the same time.

In addition to inhibiting the activity of collagenase enzymes that break down collagen protein, which is a substrate of periodontal tissues, for the prevention of periodontal disease, triclosan, which has excellent bactericidal and periodontal disease effects, promotes oral hygiene and prevents and treats periodontal disease. Milky skin extract, which promotes collagen protein synthesis, and another component, desoxy, which treats periodontal disease by inhibiting the production of superoxide, prostaglandin (PEG ₂ ), tumer nicrosis factor alpha, and interleukin-1 β When one or more of the choline acids were used as an active ingredient, these substances were simultaneously applied, and as shown in the above experimental items, significant synergistic effects on oral hygiene and periodontal disease were confirmed.

<Examples 1 to 9 and Comparative Examples 1 to 3>

The following is an example of a toothpaste composition as an Example and a comparative example for demonstrating this invention in detail. Plaque formation inhibitory effect and gingival inflammation inhibitory effect experiments were performed using toothpaste to which the experimental group of the experimental example was added. The manufacturing procedure of these toothpaste compositions is as follows. Powder components such as carboxymethyl cellulose sodium, saccharin, and preservatives were dispersed in the wet sorbitol solution, diluted with purified water, mixed firstly in a mixer, and then charged with an abrasive and a medicament agent such as calcium impression hydrogen dihydrogen. . Finally, a toothpaste composition was prepared by adding a foaming agent sodium sulfate, a safety agent and a perfume component and mixing in a vacuum.

The experimental method first selects the subjects and then explains the oral examination. After dividing the subjects randomly by 10 persons into each of the Examples and Comparative Examples, and teaching the correct tooth brushing method, the oral examination was conducted and the dental index (Quigley-Hein Index) modified by Turesky) and Gingival Bleeding Index (Loe-Silness Index). After the scaling of all the teeth in the experimental group, the control toothpaste was used for 2 weeks, followed by an oral examination. At this time, the plaque index and the gingivitis index were initial values. Example Toothpaste and Comparative Example Toothpaste was distributed to each subject and brushing was performed three times a day for six months in the same manner as usual, and oral examination was performed to score plaque index and gingivitis index. The experimental results were compared with the initial values of the experimental group and the score after 6 months, and then statistically tested by T-test.

<Experiment Result>

<Discussion>

1. Using Examples 1 to 9 and Comparative Examples 1 to 3 after measuring the plaque index for 6 months and statistically significant test results were used in Examples 1 to 9 and Comparative Examples 1 to 3 in the effect of inhibiting plaque formation It has been found to have significant effects before and after use, and when pretreated with urosoceoxycholine acid, milk extract and triclosan at the same time, the effect of inhibiting plaque formation is excellent. Appeared.

2. As a result of statistically significant test of gingivitis index after using Examples 1 to 9 and Comparative Examples 1 to 3 for 6 months, the results before and after use in Examples 1 to 9 and Comparative Examples 1 to 3 in gingivitis relief effect It was found that there was a significant effect after use, and when urosodes oxycholine acid, milk extract, and triclosan were prescribed at the same time, the gingivitis relieving effect was excellent. .

Example 10 Preparation of Gum Ointment

Gum ointment was prepared with the following composition.

Example 11. Preparation of Oral Cleaner

An oral cleanser was prepared with the following composition.

In Examples 10 to 11, the results were excellent in inhibiting plaque formation and reducing gingival inflammation.

The periodontal disease prevention and treatment composition of the present invention is excellent in inhibiting plaque formation and gingival inflammation, and excellent bactericidal effect and periodontal disease prevention effect, as well as treatment of periodontal disease to promote oral hygiene. do.

Claims (3)

It contains 0.001 to 1% by weight of trichromic acid, 0.001 to 1% by weight of urethane desoxycholine acid derivatives and 0.001 to 3% by weight of milky skin extract. A periodontal disease prophylactic and therapeutic agent composition comprising chinodes oxine choline acid, taurusoledes oxic acid, or a mixture thereof. 2. The prevention of periodontal disease according to claim 1, wherein the milky skin extract comprises ethanol extract of the root bark of the elm tree, wherein the elm tree is selected from among the birch tree, the elm tree, the elm tree, the large leaf elm tree and the elm tree. Therapeutic composition. The composition for preventing and treating periodontal disease according to claim 1 or 2, wherein the formulation of the composition is a fest, ointment, liquid, gel or spray.