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    • 专利摘要:
    The present invention relates to pharmaceutical compositions. The pharmaceutical composition comprises at least one matrix metalloproteinase inhibitor, quercetin, rutin, taxoxyline, camphorol, myricetin, curcumin, resveratrol, arecoline, apigenin, ugo selected from the group consisting of milk white skin and green tea One or more nitric oxides and superoxide generation inhibitors selected from the group consisting of nin, luteolin and tetogenegenin and one or more prostaglandin production inhibitors selected from the group consisting of oils and fats, sessile and gastric glands. This pharmaceutical composition is excellent in periodontal disease, anti-inflammatory treatment effect. In addition, the pharmaceutical composition of the present invention is excellent in inhibiting collagenase, nitric oxide, superoxide, prostaglandin, interleukin-1β and tumor necrosis factor production. Thus, it prevents diseases such as periodontal disease, wounds, cancer metastasis, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer, trauma, wrinkles, acne, AIDS, burns, atherosclerosis, fractures associated with these efficacy And useful for the treatment.
    公开号:KR20000041190A
    申请号:KR1019980056996
    申请日:1998-12-22
    公开日:2000-07-15
    发明作者:김문무；석재균；김상년；김정훈；박상기；이학모
    申请人:성재갑；주식회사 엘지화학；
    IPC主号:
    专利说明:

    Pharmaceutical composition
    [Industrial use]
    The present invention relates to pharmaceutical compositions, and in particular, to pharmaceutical compositions useful for preventing and treating diseases such as periodontal disease, inflammation, rheumatoid arthritis and cancer metastasis.
    [Prior art]
    Matrix metalloproteinases are calcium and zinc dependent endopeptides secreted from cells such as polymorphonuclear neutrophils, macrophage, gingival fibroblasts, bone cells It operates at zero neutral pH and uses various extracellular substrates as substrates.
    These proteases are not only numerous physiological processes such as embryonic development, tissue formation, salivary gland formation, tooth development, but also wounds, cancer metastasis, periodontal disease, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer It is known to be involved in various diseases such as trauma, wrinkles, acne, AIDS, burns, arteriosclerosis, fractures.
    Matrix metalloproteinases are divided into 12 groups and are known to secrete enzymes produced by the expression of genes of these enzymes by growth factors and cytokines. In addition, cytokines such as growth factor and interleukin-1β, tumor necrosis factor-α, platelet-derived growth factor, and epidermal growth factor in mesenchymal and keratinocytes of mesoderm tissues generated by blood vessels, connective tissue and lymphatic vessels may While facilitating gene expression, interfetongamma and gluticocorticoids and transforming growth factor-β inhibit the gene expression of the enzyme. And parathyroid hormone and endotoxins and prostaglandins are known to increase the synthesis of enzymes in connection with bone resorption.
    These genetic regulatory mechanisms include fibroblasts in which the activation factor protein-1 for the gene is modified by transcription factors such as c-fos and c-jun, and the synergistic action of the PEA3 and NIP protein binding sites. Induces maximal transcriptional activity in the collagenase and stromelysin promoters. The regulation of neutrophil metalloproteinases is primarily regulated by granules of lysosomes rather than at the level of transcription.
    The molecular structure of matrix metalloproteinases is generally characterized by five regions, such as signal peptides, propeptide, catalytic sites, hinge sites, and pepsin. Gelatinase, matrix metalloproteinase-2, and matrix metalloproteinase-9 have the same collagen site as one fibronectin, and matrix metalloproteinase-9 contains one additional type V collagen site. Have. These enzymes are secreted into inactivated proenzymes and Zymogens, which are not stored in cells and find activity as signal peptides are lost. The function of the propeptide is involved in maintaining the inactivation of the enzyme and is cleaved at several stages. Inducing the activity of these enzymes involves detergents, oxidizing agents, organometallic substances and enzymes such as trypsin and plasmin, which release cysteine from the zinc at the enzyme catalytic site, causing zinc to interact with water to hydrolyze the substrate. . Substrate metalloproteinases of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidative processes, which are first generated by intracellular hydrogen peroxide and hypochlorous acid by myeloperoxidase in the presence of chlorine ions. And this inactivates the substrate metalloproteinase. Matrix metalloproteinases in fibroblasts are activated by proteolytic enzymes such as trypsin and prasmin.
    Thus, the activated enzyme only needs to be secreted and activated in a small amount to perform a predetermined physiological function and causes various diseases mentioned above. The substrate specificity of the enzyme is determined by the specificity of the hemopexin or vitronectin site and basically collagenase shows specificity for fibrous collagen, while gelatinase degrades type IV collagen of denatured collagen and basement membrane.
    Specifically, substrate metalloproteinase-2 belongs to 72-KD gelatinase (A) and the substrates are gelatin, collagen type IV, V, VIII, VIII, elastin and fibronectin. Substrate metalloproteinase-3 belongs to stromelysin-1 and substrates include proteoglycans, fibronectin, laminin, procollagease, collagen types IV, V, VIII, VIII and elastin. Matrix metalloproteinase-7 belongs to Matrilysin and its substrates include lamin, fibronectin, collagen type IV, gelatin, procollagenase, proteoglycans and urokinase. Matrix metalloproteinase-8 belongs to collagenase of polymorphonuclear leukocytes and the substrate is the same as matrix metalloproteinase-1. The substrate metalloproteinase-10 belongs to stromelysin-2 and the substrate is the same as the substrate metalloproteinase-3. The substrate metalloproteinase-11 belongs to stromelin-3 and the substrate is not determined. Matrix metalloproteinase-12 belongs to the metalloestalases of macrophages and the substrate is elastin.
    In particular, 72-KD 92-KD-11 collagenase, a type IV collagenase among matrix metalloproteinases, breaks down type IV collagen, a major structural component of the basement membrane, the first barrier to cancer metastasis. The enzyme is said to be the most important enzyme in cancer cell infiltration and metastasis. Therefore, in order to develop drugs useful for the treatment of rheumatoid, periodontal disease, and corneal ulcer caused by cancer invasion and metastasis and degradation of collagen connective tissue, studies on inhibitors of type IV collagenase are being conducted.
    Among the diseases described above, periodontal disease is clinically referred to bring about tooth loss due to gingival inflammation and bleeding, periodontal pocket formation and destruction of the alveolar bone. Such periodontal disease proceeds to the process of colonization of bacteria, periodontal tissue penetration of bacteria, and periodontal tissue destruction, which will be described in more detail as follows.
    First, the saliva protein in the oral saliva is adsorbed on the dentin and chalky surfaces to form a film. On the surface of the film, bacteria such as Streptococcus and Actinomyces form plaques. Thereafter, as the time passes, the formed plaque moves toward the root portion and anaerobic Gram-negative bacteria such as Porphyromonas and Actinobacillus grow.
    The bacteria, bacterial components, and bacterial products thus formed penetrate into the gingival connective tissue through the gingival spherical epithelium to form periodontal pockets. As a result of the metabolism of these bacteria, the periodontal tissue secretes toxins of cells such as toxic hydrogen sulfide, ammonia and toxic amines. At the same time, tissue is directly destroyed by endotoxins such as lipopolysaccaride, a cell wall component. Alternatively, nitric oxide, superoxide, prostaglandins, and rucottriens secreted outside the cell by various actions of the humoral and cellular immune system stimulated by the biological immune system. Inflammation of the gums is caused by several types of cytokines such as (Luekotriens), histamine (Histamine), Interluekins, and Tumor necrosis factor α. In addition, 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.
    To prevent the development of this periodontal disease, antibacterial agents such as chlorohexidine gluconate, Cetylpyridium chloride, Sanguinarine and Triclosan have been developed to It has been applied to oral products such as toothpaste. However, such an antimicrobial agent does not fundamentally prevent the occurrence of periodontal disease.
    Recently, efforts have been actively made to suppress periodontal disease by using herbal extracts such as myrrh, baekbaekpi, horse riding, green tea, licorice, golden, pogongyoung, gold and silver coins. However, plant extracts that inhibit the production of prostaglandins or inhibit the activity of collagenase that degrades periodontal tissue have been identified. In most cases, it prevents the occurrence of periodontal disease through the action of herbal medicines such as inhibiting the formation of plaque or antibacterial, anti-inflammatory, astringent, hemostasis, blood circulation.
    To date, the most widely used method for treating periodontal disease uses an agent that inhibits the activity of collagenase enzymes that break down the periodontal tissue or inhibits the production of prostaglandins, which is a periodontal disease-causing substance.
    U.S. Pat.No. 5,230,895 confirmed that periodontal disease can be treated by developing a system in which a drug containing tetracycline is continuously delivered to inhibit the enzyme activity of collagenase, which breaks down periodontal tissue. In addition, European Patent Publication No. 528468 A1 has developed a toothpaste and a mouthwash containing Triclosan to confirm that periodontal disease can be suppressed by inhibiting the production of prostaglandin, a periodontal disease-causing substance. It has been suggested that it can be treated.
    However, the pharmacological agent introduced above suppresses the production of periodontal disease-causing substances in the treatment of periodontal disease and at the same time does not have two effects of inhibiting the activity of periodontal tissue degrading enzymes. Not only does it have limitations, but because it is a synthetic material, there are many side effects of long-term use.
    In addition, the US patent for administering tetracycline describes a method of administering tetracycline, usually 250 mg (one tablet) four times a day, all 1000 mg for one day, for 5 to 7 days. In general, it has been reported that the tetracycline administration is clinically and bacteriologically improved, and the tetracycline daily treatment after the removal of tartar has a good effect. Research reports on long-term low-dose administration showed that periodontal sac was reduced and gingival sac was eliminated and gingival sac was eliminated in the periodontal subgingiva with 48 mg of 250 mg once a day for 2 weeks. The loss of depth and adhesion was also reduced. However, long-term use of low concentrations of tetracycline often results in resistance expression of Gram-negative bacteria. Expression of these resistant strains is very frequent even when the antibiotics are stopped. Side effects include vomiting, pain complaints in the gastrointestinal tract, diarrhea, and tooth engraftment, and there are limitations that pregnant women and young children should refrain from.
    The present invention is to solve the above problems, an object of the present invention is to provide a pharmaceutical composition capable of inhibiting collagenase activity and the production of prostaglandins to prevent and treat periodontal disease.
    Another object of the present invention is to provide a pharmaceutical composition capable of inhibiting the activity of the matrix metalloproteinase, thereby preventing and treating the disease.
    [Means for solving the problem]
    In order to achieve the above object, the present invention is one or more substrate metal protease activity inhibitors selected from the group consisting of milk white skin and green tea; Quercetin, Rutin, Taxifolin, Campifol, Kaempferol, Myricetin, Curcumin, Resveratrol, Arecolin, Apigenin ), One or more nitric oxide and superoxide inhibitors selected from the group consisting of Ugonin, Luteolin, and Tectorigenin; And one or more prostaglandin production inhibitors selected from the group consisting of oils and fats, sessile and gastric glands.
    Hereinafter, the present invention will be described in more detail.
    The present invention prevents and treats diseases such as wounds, cancer metastasis, periodontal disease, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcers, osteoporosis, gastric ulcers, trauma, wrinkles, acne, AIDS, burns, atherosclerosis, fractures Extensive research has been conducted on substances known to be important drugs. In this study, we measured the activity of collagenase that degrades connective tissues that cause inflammation, quantitative determination of nitric oxide and superoxide production, and quantification of prostaglandins to inhibit collagenase activity in herbal medicines and plant extracts. Herbal and plant extracts were screened for or inhibiting the production of nitric oxide and superoxide and prostaglandin production and the inhibition of tumor necrosis factor and interleukin-1β production.
    As a result of these experiments, it was found that the synergistic effect of the substances exhibiting the efficacy is excellent, and to develop a composition that can suppress periodontal disease, inflammation, rheumatoid arthritis and cancer metastasis to complete the present invention.
    The pharmaceutical composition comprises at least one matrix metalloproteinase inhibitor, quercetin, rutin, taxoxyline, camphorol, myricetin, curcumin, resveratrol, arecoline, apigenin, ugo selected from the group consisting of milk white skin and green tea One or more nitric oxides and superoxide generation inhibitors selected from the group consisting of nin, luteolin and tetogenegenin and one or more prostaglandin production inhibitors selected from the group consisting of oils and fats, sessile and gastric glands.
    The composition of the present invention comprises a matrix metal protease activity inhibitor that degrades collagen protein, which is the substrate of connective tissue, in an amount of 0.0001 to 5% by weight relative to the total weight of the pharmaceutical composition. If the content of the matrix metalloproteinase activity inhibitor is less than 0.0001% by weight, the effect is insignificant, and if the content of the substrate metal protease activity exceeds 5% by weight, problems occur in the formulation and affect color. One or more of milky skin or green tea may be used as the substrate metal protease inhibitor.
    Yubaekpi was extracted from the stems or roots of the elm, Ulmus macrocarpa, Ulmus pumila, and Ulmus davidiana, which are distributed in North Korea, China, and Japan. Use what is sun dried. The pharmacological action of baekbaekpi is known to have diuretic, small seedlings, canned, species, and sole action, the main components are composed of beta sitosterol (β-Sitosterol), plant rolls and tannins.
    For green tea, pick the leaves of Camellia sinensis O. Ktze. The pharmacological action of green tea is effective in antibacterial, hepatitis, diseases of the heart circulation and atherosclerosis, and has been reported to have anticancer activity. Main components include catechin, epicatechin, epicatechin gallate, and epigallocatechin gallate.
    The pharmaceutical composition of the present invention includes inhibitors of production of nitric oxide and superoxide in an amount of 0.0001 to 5% by weight based on the total weight of the pharmaceutical composition. When the content of the inhibitors of the production of nitric oxide and superoxide is less than 0.0001% by weight, the effect is insignificant, and when it exceeds 5% by weight, problems occur in the formulation and affect color. Such production inhibitors may be used alone or in combination with quercetin, rutin, taxoxyline, camperol, myricetin, curcumin, resveratrol, arecoline, apigenin, ugonin, luteolin or tectorizenin.
    The pharmaceutical composition of the present invention also contains a prostaglandin production inhibitor in an amount of 0.000 to 5% by weight based on the total weight of the pharmaceutical composition, thereby inhibiting the production of prostaglandins, particularly prostaglandin E2, thereby preventing pain and inflammation. When the content of the prostaglandin production inhibitor is less than 0.0001% by weight, the effect is insignificant, and when the content of the prostaglandin production inhibitor is higher than 5% by weight, problems occur in the formulation and affect color. Such prostaglandin-producing inhibitors may be used at least one of oils, sesame or gastric glands. Oily Fatigue uses stem bark of Salix babylonica Linnaeus, a salicaceae plant, and is dried in the sun. Its pharmacological action is known for its efficacy in Isu, Sojong, Jitong, Geopung, Sole, Toothache. The main active ingredients of oils and fats include salicycin (Salicin), galallotannin, indole-3-acetic acid, populin and Juncein.
    Fructus is harvested from the mature fruit of the Rudaceae plant, Evodia officinalis, and dried in the sun. It is known that the pharmacological action of sewage milk is effective in analgesic action, disinfection action, and gastritis. The main components of sewage oil are Evodiamine, Evodene, Evodol, Rutaecarpine.
    For the lieutenant, the roots of Clematis mandshurica, a plant of the Ranunculaceae, are harvested and dried in the sun. It is known that the pharmacological action of the gastrointestinal tract is effective in analgesic action, antibacterial action, osteomalacia, etc. The main active ingredients include anemonin, annemol, and saponin.
    The manufacturing method of the pharmaceutical composition of this invention containing such a component is as follows. Dry herbal preparations used as substrate metal protease inhibitors, nitric oxide and superoxide inhibitors and prostaglandin inhibitors are shaded and powdered. Alcohol, such as ethanol, is added to the obtained powder, and extraction is carried out for about 3 days. The extract is filtered using filter paper and concentrated under reduced pressure to obtain a herbal extract.
    The pharmaceutical composition of the present invention comprising the above-described components can inhibit matrix metalloproteinase activity inhibition, nitric oxide and superoxide inhibition, and prostaglandin E 2 production, as well as the production of interleukin-1β and tumor necrosis factor. . Therefore, it can be used as a major medicament for preventing and treating diseases such as periodontal disease, wound, cancer metastasis, trauma, wrinkles, acne, AIDS, burns, arteriosclerosis, fractures associated with such efficacy.
    Such a pharmaceutical composition of the present invention may be prepared in any form of a drug as a complex preparation for inhibiting the production of nitric oxide and superoxide, inhibiting activity of substrate metalloproteinases such as collagenase, and inhibiting prostaglandin production. . Such forms may also be in tablets, capsules, powders, ointments, solutions, gels, pastes, patching agents, granules, but in the present invention, the application of these ingredients simultaneously as an active ingredient in toothpastes and anti-inflammatory ointments, which are oral products, An example will be described.
    1.Method of Preparing Toothpaste Composition
    Toothpaste compositions are prepared by mixing pharmaceutical compositions, abrasives, wetting agents, binders and foaming agents comprising a matrix metalloproteinase inhibitor, a nitrile oxide and a superoxide inhibitor and a prostaglandin inhibitor.
    The toothpaste composition of the present invention comprises 0.0001 to 5% by weight, preferably 0.01 to 3% by weight of the substrate metal protease activity inhibitor, the nitric oxide and superoxide generation inhibitor, and the prostaglandin production inhibitor, respectively, based on the total toothpaste composition weight. It includes a pharmaceutical composition. When the content of the inhibitors is less than 0.0001% by weight, respectively, the efficacy and effect on periodontal disease cannot be expected. When the content of the inhibitors exceeds 5% by weight, the safety of the product is unsuitable.
    An abrasive is mainly used as an inorganic compound powder as a component that removes deposits on the tooth surface and gives the teeth its original shine without damaging the tooth surface. Representative examples of abrasives include calcium monohydrogen phosphate, precipitated silica, silica gel, sodium bicarbonate, calcium carbonate, hydrous alumina, insoluble sodium metaphosphate, sodium pyrophosphate, hydrous silica, and the like. , Calcium monohydrogen phosphate, hydrous silica, calcium carbonate is mainly used. The content of the abrasive component may be 1-90% by weight, preferably 20-60% by weight, based on the total toothpaste composition weight.
    Wetting agents are ingredients used to maintain the state of the dentifrice composition and to prevent drying. Such humectants include glycerin, sorbitol solution, polyethylene glycol, propylene glycol, and the like, and are used alone or in combination of two or more of 20-60 wt%.
    The binder included in the toothpaste composition of the present invention is used to maintain the form of the toothpaste and ensure safety by combining the liquid and solid components of the toothpaste component. As the binder, natural or synthetic high molecular materials such as sodium carginate or calcium salt, sodium carboxymethyl cellulose, xanthan gum, and acacia gum are used, and the amount thereof is used in an amount of 0.1-5% by weight.
    The foaming agent complements the cleaning action of the abrasive and not only penetrates the medicinal agent to the areas where the toothbrush is hard to reach, but also generates bubbles to increase the sensation of teeth, helps the cleaning action, speeds up the dispersion and penetration of the medicinal agent, By reducing the interfacial tension, foreign matter in the oral cavity easily falls. Commonly used foaming agents include sodium lauryl sulfate, sodium alkyl sulfate, and anionic surfactants such as polyoxyethylene, polyoxypropylene, copolymers, polyoxyethylene hardened castor oil, polyoxyethylene sorbitan fatty acid, Alkanol amide fatty acid esters, 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.
    The toothpaste composition of the present invention may further include a medicament that promotes remineralization of the tooth to strengthen the tissue of the tooth. Agonists having such a function include sodium fluoride and sodium monofluorophosphate mixed or used alone as a fluorine compound, and its content is suitably 0.01 to 2.0% by weight. If the content of the fluorine compound is less than 0.01% by weight is not effective, if it exceeds 2.0% by weight is not suitable for oral preparations because it may affect the safety of the human body.
    In addition, flavors and sweeteners are used to control the bitter or somewhat bitter taste. The flavoring is mainly used in the natural flavors peppermint and spearmint oil, 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, buffers for adjusting the pH of the toothpaste composition include alkali metal salts of phosphate, particularly sodium phosphate dibasic, sodium diphosphate dibasic, sodium triphosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate. It may be used, in particular, mainly used by appropriately mixing two kinds of sodium phosphate, sodium phosphate, sodium phosphate. The pH of the toothpaste composition is adjusted to 5 to 8.0.
    In order to prevent contamination of microorganisms that may occur during the manufacture and use of dentifrice compositions, 0.01 to 0. alone or in combination with paraoxymethyl benzoate, benzoic acid, sodium benzoate, salicylic acid, etc., which are generally approved for use in foods and pharmaceuticals. 5% by weight is used.
    2. Preparation of Ointment Composition
    The pharmaceutical compositions, lower alcohols, state stabilizers, wetting agents and drug delivery systems of the present invention are mixed. This mixture is dissolved in a buffer solution and gelled to prepare an ointment composition.
    The ointment composition of the present invention comprises a pharmaceutical composition comprising a substrate metal protease activity inhibitor, a nitric oxide and a superoxide production inhibitor, and a prostaglandin production inhibitor, each 0.0001 to 5% by weight relative to the total ointment composition weight. If the amount of the inhibitor is less than 0.0001% by weight, respectively, the effect is insignificant. If the amount of the inhibitor exceeds 5% by weight, problems occur in the formulation, and color is affected.
    As the lower alcohol used in the present invention, 0 to 20% of ethanol and isopropyl alcohol can be used, respectively or in combination. State stabilizers are used to stabilize the state of the ointment composition, and fluoric derivatives may be used. As the fluoric derivative, poloxamer 407 or poloxamer 348 may be used, and the amount thereof may be used in an amount of 5 to 30 wt%.
    Wetting agents are used to maintain the state of the ointment composition and to prevent drying. Such wetting agents include glycerin, sorbitol solution, polyethylene glycol and propylene glycol, poloxamer sold under the tradename Poloxamer 407, monoglycerides sold under the trade name Miverol 18-99, alone or Two or more kinds can be mixed and used. The amount of wetting agent used is from 1 to 20% by weight based on the total weight of the ointment composition.
    Buffers to adjust the pH of the ointment composition include alkali metal salts of phosphate, in particular sodium phosphate dibasic, sodium phosphate dibasic, sodium triphosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate. It can be used, and it is mainly used by mixing suitably two kinds of sodium phosphate dibasic, sodium diphosphate dibasic, and sodium triphosphate suitably. The pH of the ointment composition is adjusted to 5 to 8.0.
    In addition, flavors and sweeteners are used to control the bitter or somewhat bitter taste. The flavor is mainly used in the natural flavors peppermint and spearmint oil is used 0.1 to 1% by weight in the ointment composition. 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 order to prevent contamination of microorganisms that may occur during the manufacture and use of ointment compositions alone or mixed with methyl paraoxybenzoate, propyl paraoxybenzoate, benzoic acid, sodium benzoate, salicylic acid, etc., which are generally approved for use in foods and pharmaceuticals. 0.01 to 0.5% by weight can be used.
    EXAMPLE
    Hereinafter, preferred examples and comparative examples of the present invention are described.
    1.Pharmaceutical composition
    (Examples 1 to 5)
    Milk dry skin, oil skin, green tea, and curcumin were dried and dried in a dry herbal medicine. 500 g of 95% ethanol was added to 50 g of powdered powder, which was then immersed and extracted for 3 days. The extract was filtered using No. 1 Whatman No. 1 and concentrated under reduced pressure to obtain a herbal extract. This herbal extract was mixed in the amounts shown in Table 1 below.
    (Unit: weight%)Milky skinMaintenancegreen teaCurcumin Example 10.0010.0010.0010.001 Example 20.010.010.010.01 Example 30.10.10.10.1 Example 4OneOneOneOne Example 51.51.51.51.5
    (Examples 6 to 10)
    The same procedure as in Example 1 was carried out except that quercetin was used in the amount shown in Table 2 instead of curcumin.
    (Unit: weight%)Milky skinMaintenancegreen teaQuercetin Example 60.0010.0010.0010.001 Example 70.010.010.010.01 Example 80.10.10.10.1 Example 9OneOneOneOne Example 101.51.51.51.5
    (Examples 11 to 15)
    It was carried out in the same manner as in Example 1, except that taxoxyline was used instead of curcumin in the amount shown in Table 3 below.
    (Unit: weight%)Milky skinMaintenancegreen teaTaxoxyline Example 110.0010.0010.0010.001 Example 120.010.010.010.01 Example 130.10.10.10.1 Example 14OneOneOneOne Example 151.51.51.51.5
    (Examples 16 to 20)
    The procedure was carried out in the same manner as in Example 1, except that the routine was used in the amount shown in Table 4 instead of curcumin.
    (Unit: weight%)Milky skinMaintenancegreen teaRoutine Example 160.0010.0010.0010.001 Example 170.010.010.010.01 Example 180.10.10.10.1 Example 19OneOneOneOne Example 201.51.51.51.5
    (Examples 21 to 25)
    Resveratrol instead of curcumin was carried out in the same manner as in Example 1 except for using the amount shown in Table 5.
    (Unit: weight%)Milky skinMaintenancegreen teaResveratrol Example 210.0010.0010.0010.001 Example 220.010.010.010.01 Example 230.10.10.10.1 Example 24OneOneOneOne Example 251.51.51.51.5
    2. Toothpaste Composition
    (Examples 26 to 27 and Comparative Examples 1 and 2)
    Toothpaste compositions were prepared by mixing the ingredients shown in Table 6 below.
    (Unit: weight%) Example 26Example 27Comparative Example 1Comparative Example 2 Humectantglycerin200200 Amorphous Sorbitol Liquid023023 BinderCarrageenan0.800.80 Carboxymethyl Cellulose0One0One SurfactantsSodium Lauryl SulfateOne2One2 Sorbitan monooleate0.20.40.20.4 abrasiveCalcium dihydrogen phosphate040040 Precipitated silica250250 Calcium carbonate0000 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skin0.010.050.010 Maintenance0.010.0500.05 green tea0.010.0500 Quercetin0.010.0500 Sodium fluoride0.2200.220 Sodium fluorophosphate00.7600.76 Spices 0.750.60.750.6 Food coloring 0.0002500.000250 Purified water RemainderRemainderRemainderRemainder
    (Examples 28 to 29 and Comparative Examples 3 to 4)
    Toothpaste compositions were prepared by mixing the ingredients shown in Table 7 below.
    (Unit: weight%) Example 28Example 29Comparative Example 3Comparative Example 4 Humectantglycerin200200 Amorphous Sorbitol Liquid023023 BinderCarrageenan0.800.80 Carboxymethyl Cellulose0One0One SurfactantsSodium Lauryl SulfateOne2One2 Sorbitan monooleate0.20.40.20.4 abrasiveCalcium dihydrogen phosphate040040 Precipitated silica250250 Calcium carbonate0000 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skin0.10.500 Maintenance0.10.500 green tea0.10.50.10 Curcumin0.10.500.5 Sodium fluoride00.2200.22 Sodium fluorophosphate0.7600.760 Spices 0.750.60.750.6 Food coloring 0.0002500.000250 Purified water RemainderRemainderRemainderRemainder
    (Examples 30 to 31 and Comparative Examples 5 to 6)
    Toothpaste compositions were prepared by mixing the ingredients shown in Table 8 below.
    (Unit: weight%) Example 30Example 31Comparative Example 5Comparative Example 6 Humectantglycerin200200 Amorphous Sorbitol Liquid023023 BinderCarrageenan0.800.80 Carboxymethyl Cellulose0One0One SurfactantsSodium Lauryl SulfateOne2One2 Sorbitan monooleate0.20.40.20.4 abrasiveCalcium dihydrogen phosphate400400 Precipitated silica025025 Calcium carbonate0000 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skinOne1.500 MaintenanceOneOne00 green teaOne1.501.5 TaxoxylineOneOneOne0 Sodium fluoride00.2200.22 Sodium fluorophosphate0.7600.760 Spices 0.750.60.750.6 Food coloring 0.0002500.000250 Purified water RemainderRemainderRemainderRemainder
    3. Ointment Composition
    (Examples 32 to 33 and Comparative Examples 7 to 8)
    Cetylpyridium chloride, pluronic, lower alcohol, glycerin, gelatin, pectin, carboxymethylcellulose, poloxamer 407, monoglyceride (Myverol 18-99), propylene glycol or polyethylene glycol, menthol and preservatives Mix in the amounts shown in Table 9. This mixture was prepared by dissolving and gelling in buffer.
    (Unit: weight%) Example 32Example 33Comparative Example 7Comparative Example 8 Humectantglycerin5050 Polyethylene glycol0505 Poloxamer 4070.100.10 Monoglycerides (Myverol 18-99)10151015 Drug Delivery Systemgelatin5555 pectin5555 SurfactantsSodium Lauryl Sulfate200200 Sorbitan monooleate020020 Low alcoholethanol5050 Isopropyl Alcohol0505 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skin0.010.050.010 Maintenance0.010.0500.05 green tea0.010.0500 Quercetin0.010.0500 Spices 0.750.60.750.6 Food coloring 0.000200.00020 Purified water RemainderRemainderRemainderRemainder
    (Examples 34 to 35 and Comparative Examples 9 to 10)
    Except for using the components shown in Table 10 was carried out in the same manner as in Example 34.
    (Unit: weight%) Example 34Example 35Comparative Example 9Comparative Example 10 Humectantglycerin100100 Polyethylene glycol0505 Carboxymethylcellulose0.50.10.50.1 Poloxamer 40710010Monoglycerides (Myverol 18-99)020 20 Drug Delivery Systemgelatin3232 pectin3232 SurfactantsSodium Lauryl Sulfate150150 Sorbitan monooleate015015 Low alcoholethanol5050 Isopropyl Alcohol0505 antisepticParaoxymethyl Benzoate0.10.10.10.1 Paraoxy Benzoate0.050.050.050.05 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skin0.10.500 Maintenance0.10.500 green tea0.10.50.10 Quercetin0.10.500.5 Spices 0.750.60.750.6 Food coloring 0.000200.00020 Purified water RemainderRemainderRemainderRemainder
    (Examples 36 to 37 and Comparative Examples 11 to 12)
    It carried out similarly to Example 34 except having used the component of Table 11 below.
    (Unit: weight%) Example 36Example 37Comparative Example 11Comparative Example 12 Humectantglycerin150150 Polyethylene glycol0505 Carboxymethylcellulose0.50.10.50.1 Poloxamer 4075One5One Monoglycerides (Myverol 18-99)0505 Drug Delivery SystemgelatinOne10One10 pectinOne5One5 SurfactantsSodium Lauryl Sulfate100100 Sorbitan monooleate010010 Low alcoholethanol5050 Isopropyl Alcohol0505 antisepticParaoxymethyl Benzoate0.10.10.10.1 Paraoxy Benzoate0.050.050.050.05 SweetenerZaccarin Sodium0.10.20.10.2 BufferSodium monophosphate0.10.20.10.2 Trisodium Phosphate0.050.050.050.05 Active ingredientMilky skin1.001.5000 Maintenance1.001.0001.5 green tea1.001.50One0 Taxoxyline1.001.010.050.1 Spices 0.750.60.750.6 Food coloring 0.000200.00020 Purified water RemainderRemainderRemainderRemainder
    A. Inhibition of collagenase enzyme activity, type IV collagenase activity, superoxide production inhibition, prostaglandin E2 production inhibition, interleukin-1β production inhibition and tumor necrosis factor production inhibition The experiment was carried out as follows.
    Experimental Example 1: Inhibitory Effect on Collagenase, a Periodontal Tissue Degrading Enzyme, of the Pharmaceutical Compositions of the Present Invention on Enzyme Activity
    Experimental results of carrying out the inhibitory effect on collagenase, a periodontal tissue degrading enzyme with the present invention drug system is as follows.
    This test method was performed in the salivary and gingival sulcus of patients with periodontal disease, which were secreted from porphyromonas gingivalis and polymorphonuclear leukocytes and neutral leukocytes (Neutrophil). It is an experimental method that mimics the bio-oral environment in which gum retraction is caused by the degradation of collagen RPS, which is the substrate of periodontal tissues by the action of collagenase enzyme.
    To each of 25 1.5 ml Eppendorf tubes 100 μl of Azocoll solution, 2% red collagen substrate, were added. One eppendorf tube was used as a blank and three tubes were added with 10, 100 and 200 ppm of standard enzyme solution (collagen degradation activity: 315 units / mg) of collagenase type I purchased from Sigma. G. To each of the remaining tubes was added 100 [mu] l of collagenase enzyme purely separated from the saliva and gingival fissures of periodontal disease patients via Sephacryl S-200 chromatography.
    One tube was used as a control, and each of the remaining tubes was treated with 10 μl of the experimental group at an appropriate concentration, and then buffer solution (0.05 M, Tris-HClm 1 nM CaCl 2 , pH 7.8) was added so that the total reaction solution was 500 μl.
    Subsequently, the obtained eppendol tube was reacted for 18 hours in a 37 ° C thermostat, and the eppendorf tube was centrifuged at 10000 g for 5 minutes. Undigested collagen was precipitated by centrifugation and the supernatant containing the degraded collagen was taken to measure absorbance at 540 nm to create a standard activity curve. The enzyme activity was converted from the standard curve to evaluate the enzyme activity of the experimental group and the control group. The results are shown in Table 12 below. In addition, the results obtained using the pharmaceutical compositions of Examples 1 to 25 are shown in Tables 13 to 15 below.
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectMilky skinMilky skinMilky skinMilky skingreen teagreen teagreen teagreen tea Test concentration [%]0.0010.010.1One0.0010.010.1One %5073One70252One
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 1Example 2Example 3Example 4Example 5Example 6Example 7Example 8 %753OneOne842
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 9Example 10Example 11Example 12Example 13Example 14Example 15Example 16 %OneOne9542211
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 17Example 18Example 19Example 20Example 21Example 22Example 23Example 24Example 25 %74OneOne1052OneOne
    As shown in Table 12, when the milky skin and green tea were used, the inhibitory effect on collagenase activity was found to be excellent when used at a concentration of 0.01% or more. However, as shown in Tables 13 to 15, the composition of Examples 1-25 containing all three components contains collagen only even if it contains only 0.001% of each component (Examples 1, 6, 11, 16, 21). It can be seen that the inhibitory effect on the activating activity increases. The inhibitory effect on the collagenase activity of the Example was found to be 89% to 93% compared to the control, and at the concentration of 0.01% or more, the inhibitory effect on the collagenase activity of 95% or more.
    Experimental Example 2: Inhibitory Effect on the Type IV Collagenase Activity of the Compositions of the Present Invention (Examples 1 to 25)
    In preparing the acrylamide gel, 34 mg of Eppendorf tubes were prepared by making a separation gel to which 1 mg / ml of gelatin as a substrate was added. One Eppendorf tube was used as a blank, and nine tubes were added with 10, 100 and 200 ppm of Type IV collagenase purchased from Calbiocam, and 100 μl of enzyme and test group were added to each of the remaining tubes. 10 μl each was treated to a concentration. Sodium dodecyl sulfate (SDS) sample buffer solution (4% SDS, 125 mM Tris-Cl, pH 6.8, 10% glycerol) was added to all tubes, mixed with 500 μl of total reaction solution, followed by electrophoresis. It was.
    After electrophoresis, the gel was washed twice with 50 mM Tris-HCl (pH 7.5) buffer containing 2.5% Triton X-100 for 30 minutes to remove SDS, followed by reaction buffer (50 mM Tris-HCl, pH). 7.5, 10 mM CaCl 2 , 0.01% NaN 3 ) was reacted for 5 hours at 37 ℃. Thereafter, the gel was stained with 0.1% Cooasie brilliant blue, and then decolorized to prepare a standard activity curve using a densitometer of the gelatin resolution. To compare the enzyme activity of the results are shown in Table 16 below. In addition, the results obtained using the pharmaceutical compositions of Examples 1 to 25 are shown in Tables 17 to 19 below.
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectMilky skinMilky skinMilky skinMilky skingreen teagreen teagreen teagreen tea Test concentration [%]0.0010.010.1One0.0010.010.1One %70252One83527One
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 1Example 2Example 3Example 4Example 5Example 6Example 7Example 8 %2373OneOne2582
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 9Example 10Example 11Example 12Example 13Example 14Example 15Example 16 %OneOne21842220
    (Unit:% = enzyme activity of experimental group × 100 ÷ enzyme activity of control group) SubjectExample 17Example 18Example 19Example 20Example 21Example 22Example 23Example 24Example 25 %74OneOne2282OneOne
    As shown in Table 16, the inhibitory effect on the type IV collagenase enzyme activity of milky skin and green tea showed 75% and 80% inhibitory effects at concentrations of 0.001%, respectively. In addition, as shown in Tables 17 to 19, when all three components are included, collagenase activity is also affected even when only 0.001% of each component is contained (Examples 1, 6, 11, 16, and 21). The inhibitory effect was found to be synergistic, with values ranging from 89% to 93%. In addition, the concentration of 0.01% or more was shown to have an inhibitory effect on type IV collagenase enzyme activity of 95% or more.
    Experimental Example 3: Inhibitory effect on the production of superoxide of the composition of the present invention
    Collected venous blood was centrifuged at 1200 rpm for 10 minutes using citric acid as an anticoagulant from healthy adults without systemic disease. After centrifugation, the leukocyte concentrate in the middle layer was recovered, and diluted with the concentrate and the volume ratio of 1: 1 with RPMI 1640 medium. Subsequently, 12 ml of Ficoll-Plaque was added to a 50 ml centrifuge tube, followed by careful addition of 30 ml of diluted blood to the middle layer. After centrifugation of the centrifuge tube at 1600 rpm for 30 minutes, the upper layer containing serum was removed and the middle layer containing monocytes was carefully diluted.
    Three times RPMI 1640 medium was added to the diluent, centrifuged at 800 rpm for 10 minutes, the supernatant was discarded, and 10 ml of RPMI 1640 medium was added. After the sample was collected by gently pipetting the obtained product, the sample was centrifuged at 800 rpm for 10 minutes. The supernatant was discarded from the resultant obtained by centrifugation, HBSS (Hanks' Balanced Salt Solution) buffer solution was added, and the sample was collected by pipetting. This sample was dispensed into a 24-well plate at 0.45 ml of human mononuclear leukocytes at 10 6 cell / well.
    After incubating the sample and the plate containing leukocytes aseptically for 2 hours under 95% air, 5% CO 2 , and 100% humidity conditions, FMLP (N-Formul-Met-Leu-Phe) was added to 10 -6 M. Cells were stimulated by 0.05 mL treatment and incubated at 37 ° C. for 15 minutes. 0.1 ml of Cytochrome C to 80 µM, 0.1 ml of Superoxide dismutase to 30 µg, 0.1 ml of the test subjects were added to the culture medium, and the total reaction solution was 0.9 ml. HBSS was added.
    The mixture was kept at 37 ° C. for 10 minutes, and 0.1 ml of the stimulating substance, phylated Zymosan A, was added at a final concentration of 1.3 mg / ml and kept at 37 ° C. for 90 minutes with shaking, followed by 4 ° C. After the reaction was stopped for 10 minutes and centrifuged at 4 ° C., 1500 rpm for 10 minutes, the supernatant was measured at 550 nm to measure the optical density of Sample B in Table 20 below.
    The optical density of Samples A, C and D in Table 20 was measured in the same manner except that the amount of use was changed as shown in Table 20 below. Using the optical density, the amount of superoxide anion produced was calculated according to Equation 1 below, and the results are shown in Tables 21 and 22.
    [Equation 1]
    O- 2 = (Δ OD / 21.0) × 10 3 (nmmoles / 10 6 cell.min)
    Δ O. D. = (B-D)-(A-C) = (B + C)-(D + A)
    Sample ASample BSample CSample D Mononuclear leukocytes [ml]0.50.450.50.45 Cytochrome C [ml]0.10.10.10.1 SOD [ml]000.10.1 FMLP [ml]00.0500.05 Zymoic acid [mL]00.100.1 Test subject group [mL]00.100 Reaction liquid [mL]0.40.20.30.2 Total [ml]1.001.001.001.00
    (Unit:% = excess hydrogen oxide generation amount in the experimental group × 100 ÷ excess hydrogen oxide generation amount in the control group) Test subjectTest subject concentration [%]% Curcumin0.00116 0.017 0.12 OneOne Routine0.00112 0.014 0.1One OneOne Quercetin0.00111 0.013 0.12 OneOne Taxoxyline0.00112 0.012 0.1One OneOne Camperol0.00145 0.0111 0.12 OneOne Apigenin0.00132 0.017 0.1One OneOne
    (Unit:% = amount of excess hydrogen oxide produced in the experimental group × 100 ÷ amount of excess hydrogen oxide produced in the control group) Test subjectTest subject concentration [%]% Ugonin0.00142 0.0111 0.12 OneOne Luteolin0.00130 0.017 0.1One OneOne Mycetin0.00145 0.0111 0.12 OneOne Resveratrol0.00113 0.0111 0.12 OneOne Arecoline0.00127 0.017 0.1One OneOne Ttoritorinin0.00132 0.017 0.1One OneOne
    The same results using the pharmaceutical compositions of Examples 1 to 25 are shown in Tables 23 to 25 below.
    (Unit:% = amount of excess hydrogen oxide produced in the experimental group × 100 ÷ amount of excess hydrogen oxide produced in the control group) SubjectExample 1Example 2Example 3Example 4Example 5Example 6Example 7Example 8 %853OneOne1082
    (Unit:% = amount of excess hydrogen oxide produced in the experimental group × 100 ÷ amount of excess hydrogen oxide produced in the control group) SubjectExample 9Example 10Example 11Example 12Example 13Example 14Example 15Example 16 %OneOne12842214
    (Unit:% = amount of excess hydrogen oxide produced in the experimental group × 100 ÷ amount of excess hydrogen oxide produced in the control group) SubjectExample 17Example 18Example 19Example 20Example 21Example 22Example 23Example 24Example 25 %74OneOne852OneOne
    In the above experimental results, curcumin, quercetin, taxoxyline, rutin, and restoratrol showed superior effect of inhibiting superoxide formation, and the same effects were obtained in Examples 1 to 25 each containing these medicinal agents. Showed not to be inhibited by blood and green tea.
    Experimental Example 4: Inhibitory effect on the production of nitric oxide (NO) of mononuclear leukocytes of the present compositions (Experimental Examples 1 to 22)
    Incubate RAW 264.7 cells in DMEM (dulbecco's modified eagle medium) containing 10% fetal bovine serum. When the cells were filled in a cell culture plate of 10 cm in diameter, 1 mL was dispensed into 10 6 cells / well in a 24-well plate. Plates containing RAW cells were incubated for 24 hours aseptically under 95% air, 5% CO 2 , 100% humidity conditions.
    The cultured groups were treated for 1 hour to a concentration of 100 ppm. Only cultures without addition of the test subjects were used as controls. Subsequently, E. coli lipopolysaccharide and interferon gamma were treated at 1 ppm and 100 U, respectively, to induce the production of nitric oxide, and then incubated for 24 hours. Five minutes after mixing one grease reagent at 1: 1, the optical density was measured at 540 nm, and a standard curve was prepared using the absorbance values of the standard solution to calculate the amount of nitric oxide in the experimental group. The results are shown in Tables 26 to 27 below.
    (Unit:% = amount of superoxide produced by experimental group × 100 ÷ amount of superoxide produced by control group) Test subjectTest subject concentration [%]% Curcumin0.00115 0.012 0.12 OneOne Routine0.00124 0.014 0.1One OneOne Quercetin0.00117 0.015 0.12 OneOne Taxoxyline0.00118 0.013 0.1One OneOne Camperol0.00123 0.0111 0.12 OneOne Apigenin0.00132 0.0124 0.1One OneOne
    (Unit:% = amount of superoxide produced by experimental group × 100 ÷ amount of superoxide produced by control group) Test subjectTest subject concentration [%]% Ugonin0.00125 0.0115 0.12 OneOne Luteolin0.00131 0.0122 0.1One OneOne Mycetin0.00132 0.0121 0.12 OneOne Resveratrol0.00113 0.017 0.12 OneOne Arecoline0.00127 0.0112 0.1One OneOne Ttoritorinin0.00132 0.0121 0.1One OneOne
    The same experiment was carried out using the compositions of Examples 1 to 25 and the results are shown in Tables 28 to 30 below.
    (Unit:% = amount of superoxide produced by experimental group × 100 ÷ amount of superoxide produced by control group) SubjectExample 1Example 2Example 3Example 4Example 5Example 6Example 7Example 8 %753OneOne962
    (Unit:% = amount of superoxide produced in experimental group × 100 ÷ amount of superoxide produced in control group) SubjectExample 9Example 10Example 11Example 12Example 13Example 14Example 15Example 16 %2One973OneOne14
    (Unit:% = amount of superoxide produced in experimental group × 100 ÷ amount of superoxide produced in control group) SubjectExample 17Example 18Example 19Example 20Example 21Example 22Example 23Example 24Example 25 %84OneOne1232OneOne
    In the above experimental results, as in the superoxide production inhibitory effect, curcumin, quercetin, taxoxyline, rutin, and restoratrol were excellent in inhibiting the production of nitric oxide, each containing 0.001% of these drugs. Examples 1, 6, 11, 16, 21 showed a synergistic effect in inhibiting the production of nitric oxide, and even at a concentration of 0.01% or more, the effect is not inhibited by milk white skin, oil skin and green tea and is excellent Is showing.
    Experimental Example 5: Inhibitory effect on the production of prostaglandins (PGE 2 ) of mononuclear leukocytes of the composition of the present invention (Experimental Group 1 to 25)
    Experimental results of carrying out the inhibitory effect on the production of the prostaglandin as a periodontal disease-causing substance with the composition of the present invention are as follows. In this test method, Lipopolysaccharide (LPS), a cell wall component of E. coli, was used to stimulate the production of prostaglandins (PGE2) induced by stimulating human mononuclear leukocytes. It was.
    In a specific test method, 0.8 ml of blood mononuclear leukocytes isolated from blood was added to a 24-well plate and aliquoted to 10 6 cell / well. A well to which 200 μl of RPMI 1640 medium was added to a well plate to which blood mononuclear leukocytes was added was used as a control, and 100 μl of wells and LPS (250 ppm) to which the well plate was added 100 μl of E. coli LPS (250 ppm). And the wells to which 100 μl of the test subjects were added at appropriate concentrations were incubated for 24 hours.
    In a blank well of a 96-well plate to which 1 gG of goat anti-mouse was attached, containing 50 μl buffer solution (0.9% NaCl, 0.1% bovine serum albumin, 0.5% Kathon). 0.1 M phosphate buffer solution) was added. To the standard well was added 50 μl of appropriate concentrations of prostaglandin standard solution (40, 80, 160, 320 pg).
    50 μl of the culture supernatant treated with the test subject group was added to the wells selected as the test group. In addition, 50 μl of antitease for PGE 2 was added to all wells except the blank wells followed by 50 μl of PGE 2 Conjugate Peroxidase to all wells except the blank wells and 96-well The plate was covered and kept at 25 ° C. for 1 hour. Wash all well plates four times with wash buffer (phosphate buffer solution containing 0.05% Tween 20: pH 7.5) and at room temperature 150 μl of enzyme substrate (3,3 ′, 5 dissolved in 20% dimethylformamide). , 5'-tetramethylbenzidine / hydrogen peroxide) was added immediately, held at 25 ° C. for 30 minutes, and 100 μl of 1M sulfuric acid was added followed by measurement of absorbance at 450 nm with a microplate reader. A standard curve was prepared from the absorbance values of the standard solution to calculate the amount of prostaglandin produced in the experimental group. Oily skin, sessile and gastric glands, each of which inhibits prostaglandin production, were used as experimental groups, and the experimental results of the control group and the LPS-treated group are shown in the following table. In addition, the results obtained using the pharmaceutical compositions of Examples 1 to 25 as the test subject groups are shown in Tables 31 and 32.
    SubjectPGE 2 (pg / ml)SubjectPGE 2 (pg / ml) Control12Sewage 0.001126 LPS treatment group312Sewage 0.0120 Oil Blood 0.001124Osu 0.17 Yuji Blood 0.00570Osu 1.07 Yuji 0.0124Maternity Line 0.001130 Yuji Blood 0.18Maternity Line 0.0130 Yuji Blood 1.07Maternity Line 0.18 Yuji Blood 1.57Maternity Line 1.08
    ExamplePGE 2 (pg / ml)ExamplePGE 2 (pg / ml) One1328 3847 58617 77810 98107 11211215 13121410 1591617 178187 198207 21152210 238247 257
    Efficacy and effects of PGE 2 production in human mononuclear leukocytes stimulated with E. coli LPS have been shown to increase with increasing concentrations, especially at concentrations above 0.005%. Increased. In addition, Examples 1 to 25, each containing such an pharmacological agent, also Example 1, wherein the inhibitory effect on the production of PGE 2 was not inhibited by the milky skin, the oily skin and the green tea, but rather contained 0.01% of its concentration, respectively. , 6, 11, 16, and 21 showed synergistic effects.
    Experimental Example 6: Inhibitory effect on the production of interleukin-1beta (IL-1β) of protein leukocytes of (Examples 1 to 25) of the composition of the present invention
    Blood mononuclear leukocytes isolated from blood were added to 0.8 wells in 24-well plates and aliquoted to 10 6 cell / well. A well to which 200 μl of RPMI 1640 medium was added to this plate was used as a control, and 100 μl of well and LPS (250 ppm) and 100 μl of E. coli LPS (250 ppm) were added to the plate, and Examples 1 to 25 were used. The well to which 100 μl was added at the proper concentration was used as an experimental group and incubated for 24 hours.
    In a well of a 96-well plate to which an antibody of interleukin (IL-1β) was attached, a standard solution of interleukin-1β (0, 10, 24, 25.6, 64, 160, 400 pg / well) was added. 50 µl was added, 50 µl of the cell culture solution was added to the experimental wells, and 50 µl of the biotinylated antibody reagent was added to all the wells.
    All wells were kept at 25 ° C. for 3 hours and then washed three times with wash buffer solution, streptavidin-HRP conjugate was added to all wells and held again at 25 ° C. for 30 minutes. Again washed three times with wash buffer solution and 100 μl of enzyme substrate was added immediately. The wells are then kept at 25 ° C. in the dark, with the lid of the plate open for 30 minutes, and 100 μl of 0.18 M sulfuric acid is added and the absorbance at 450 nm is measured with a microplate reader to measure the standard curve to the absorbance value of the standard solution. The amount of interleukin produced in the experimental group was calculated by. The results are shown in Table 33 below.
    ExampleIL-1β (pg) Control624 LPS treatment group1908 One872 2687 3524 472 524 6723 7638 873 962 1025 11824 12724 13523 1438 1534 16825 17725 18562 1961 2024 21871 22723 23632 2454 2524
    As a result of testing the efficacy and effect on IL-1β production of human mononuclear leukocytes stimulated with E. coli LPS, Examples 1 to 25 showed that the efficacy increased with increasing concentrations. Its potency increased dramatically, indicating that IL-1β content was reduced compared to the control group that did not stimulate LPS.
    Experimental Example 7: Inhibitory effect on the production of tumor necrosis factor (TNF-α) of mononuclear leukocytes of (Inventive Examples 1 to 25) of the composition of the present invention
    Blood mononuclear leukocytes isolated from blood were added to 0.8 wells in 24-well plates and aliquoted to 10 6 Cells / well. A well to which 200 μl of RPMI 1640 medium was added to this well-plate was used as a control, and 100 μl of well and LPS (250 ppm) to which 100 μl of E. coli LPS (250 ppm) was added, and Examples 1 to 25 Composition 100 The well to which the μl was added to the proper concentration was used as the experimental group and incubated for 24 hours.
    To a well of a 96-well plate to which an antibody of TNF α is attached, 50 µl of a standard solution of tumor necrosis factor (0, 10.24, 25.6, 64, 160, 400 pg / well) was added, and the experimental group 50 μl of the above cell culture was added to the wells.
    In addition, 50 μl of biotinylated antibody reagent was added to all wells, and then maintained at 25 ° C. for 3 hours, followed by washing the wash buffer solution three times, and streptoavidin-HRP conjugate was added to all wells again. Hold at 30 ° C. for 30 minutes. After all wells were washed again three times with wash buffer solution, 100 μl of enzyme substrate was added immediately and held at 25 ° C. in the dark with the lid of the plate open for 30 minutes and 100 μl of 0.18 M sulfuric acid was added, The 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 TNF α generated in the experimental group. The results are shown in Table 34 below.
    ExampleTNF α (pg) Control743 LPS treatment group2408 One1052 2887 3524 4272 5421 61054 7638 8373 9262 10125 111024 12724 13523 14238 15134 161305 17725 18632 19461 20224 211310 22923 23825 24454 25324
    As a result of testing the efficacy and effect on the TNF α production of human mononuclear leukocytes stimulated with E. coli LPS, Examples 1 to 25 showed that the efficacy increased as the concentration was increased. As in the test results of interleukin-1 beta, the effect was rapidly increased at a concentration of 0.1% or more, indicating that the content of TNF α was decreased compared to the control group that did not stimulate LPS.
    Experimental Example 8: Clinical Trial of Efficacy and Effect of the Toothpaste of the Present Invention (Examples 26 to 31)
    Results of conducting a clinical trial for the treatment of periodontal disease with the toothpaste of the present invention are as follows.
    Subjects were selected from 120 oral subjects with 30 oral examinations of 30 or 50 patients at age intervals from 30 to 50 years of age. The clinical trials on the treatment effect of periodontal disease of the experimental group toothpaste (Examples 26 to 31) and the comparative group toothpaste (Comparative Examples 1 to 6) were performed by the following method.
    First, the clinical experiment method of the toothpaste of the present invention was performed by the following method.
    The experimental group was divided into the experimental group and the comparative group, and the oral health education and the correct brushing method were taught. The same control toothbrush was supplied to all test subjects and the toothpaste was performed to score the initial gingivitis index, and the test toothpaste and the control toothpaste were supplied to the test subjects for 1 week, 1 month, 3 months, and 6 months. The gingivitis index was measured, and the gingivitis index was measured by inserting a periodental probe into the gingival bulb and continuously probing around each tooth without applying force. Table 36 shows the results obtained by recording the scores by the method shown in Table 35 below.
    scoreContents 0 pointsBleeding 1 pointDot bleeding 2 pointsGlandular bleeding 3 pointsInterdental triangle bleeding 4 pointsGingival bleeding
    Example or Comparative Example NumberEarly1 week1 month3 months6 months ExampleComparative exampleExampleComparative exampleExampleComparative exampleExampleComparative exampleExampleComparative example 261.041.051.261.431.642.451.653.241.703.56 271.051.021.201.351.532.371.553.111.563.41 281.041.031.201.441.252.421.453.341.503.54 291.051.011.071.371.072.391.073.221.103.45 301.041.031.041.431.042.451.043.251.073.64 311.051.041.051.381.052.421.053.311.073.48
    These results suggest that milk curd, which can inhibit the enzyme activity of collagenase, which degrades periodontal tissues, curcumin, quercetin, taxoxyline, rutin or resveratrol, and periodontal disease that inhibit the production of superoxide and superoxide. Treatment effect of the periodontal disease of the toothpaste of the present invention (Examples 26 to 31) containing oil-derived inhibiting the production of prostaglandin induced by Comparative Examples 1 to 6 containing these drugs alone from 1 week to 6 months It can be seen that the efficacy is more excellent. Therefore, the case of including all the drugs showed that the effect is increased.
    While the gingivitis index of the comparative example is continuously increased as the period elapses after one month, the use of the toothpaste developed according to the present invention is suppressed in the occurrence of periodontal disease, and the gingivitis index is significantly decreased over time.
    Experimental Example 9: Animal Experiments on Anti-Inflammatory Efficacy and Effects of the Invention Ointment Compositions (Examples 32 to 37)
    Clinical trial method of the ointment composition of the present invention was carried out by the following method.
    Wistar male rats weighing 200-250 g were divided into experimental and comparative groups, and then lightly anesthetized with ether. A rat was injected into the right rear sole of the foot, and the group was injected with Complete Freynd's adjuvant, and the group injected with 0.1 ml of liquid paraffin was used as the control group. From 5 days to 19 days after the injection of the experimental ointment composition (Examples 32 to 37) and Comparative Example ointment composition (Comparative Examples 7 to 12) at the same time on the right hind paw of the rats at the same time, the initial stage, Foot thickness measured on day 10 and 20 was expressed in% with the control group and the results are shown in Table 37 below.
    Experimental groupEarly10 days20 days ExampleComparative exampleExampleComparative exampleExampleComparative example Control1.05 1.47 2.53321.031.041.211.451.302.52 331.061.061.181.371.192.42 341.041.061.171.381.182.48 351.051.051.051.361.152.49 361.061.071.061.411.102.43 371.041.061.041.401.042.44
    In the above results, curcumin, quercetin, taxoxyline, rutin and resveratrol, periodontal disease, which inhibit milk protein and green tea nitric oxide and superoxide production, which can inhibit the enzyme activity of collagenase, which degrades periodontal tissue, The inhibitory effect of the occurrence of foot edema of the ointment composition of the present invention (Examples 32 to 37) containing fats and oils that inhibit the production of prostaglandins to be induced is superior to Comparative Examples 7 to 10 from 1 week to 1 month. appear.
    The foot edema of the comparative example is continuously increased as time passes after 5 days, while using the ointment composition developed according to the present invention, the occurrence of foot edema is suppressed, and it is shown that the time is significantly reduced. It was shown that ointment compositions containing three inhibitors at the same time have an excellent synergistic effect against anti-inflammatory as compared to the ointment compositions mixed alone.
    As described above, the pharmaceutical composition of the present invention is excellent in periodontal disease, anti-inflammatory treatment effect. In addition, the pharmaceutical composition of the present invention is excellent in the effect of inhibiting collagenase, nitric oxide, superoxide, prostaglandin, interleukin-1β and tumor necrosis factor production. Thus, it prevents diseases such as periodontal disease, wounds, cancer metastasis, rheumatoid arthritis, inflammation, parathyroidism, diabetes, corneal ulcer, osteoporosis, gastric ulcer, trauma, wrinkles, acne, AIDS, burns, atherosclerosis, fractures associated with these efficacy And useful for the treatment.
    权利要求:
    Claims (2)
    [1" claim-type="Currently amended] At least one matrix metalloproteinase activity inhibitor selected from the group consisting of milky skin and green tea;
    Produces one or more nitric oxides and superoxides selected from the group consisting of quercetin, rutin, taxoxyline, camperol, myricetin, curcumin, resveratrol, arecoline, apigenin, ugonin, luteolin and tectorizenin Inhibitors; And
    One or more prostaglandin inhibitors selected from the group consisting of oils and fats, sessile and gastric glands
    Pharmaceutical composition comprising a.
    [2" claim-type="Currently amended] The method of claim 1,
    Wherein the amounts of the matrix metalloproteinase inhibitor, the nitric oxide and superoxide inhibitor, and the prostaglandin inhibitor are 0.0001 to 5% by weight, respectively, based on the total weight of the composition.
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    同族专利:
    公开号 | 公开日
    KR100599934B1|2006-11-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-12-22|Application filed by 성재갑, 주식회사 엘지화학
    1998-12-22|Priority to KR1019980056996A
    2000-07-15|Publication of KR20000041190A
    2006-11-30|Application granted
    2006-11-30|Publication of KR100599934B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR1019980056996A|KR100599934B1|1998-12-22|1998-12-22|Composition for preventing or treating periodontal disease|
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