JPH10167945A - Orally applicable composition containing extract of achyranthes japonica or bark of ulmus macrocapra - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an orally applicable composition suppressing generation of periodontal disease inducer and activity of periodontium, decomposing enzyme and accelerating collagen synthesis by containing an extract of Achyranthes japonica and/or an extract of bark of Ulmus macrocapra. SOLUTION: This orally applicable composition is obtained by mixing respectively 0.001-5wt.% of (A) an extract of Achyranthes japonica and/or (B) an extract of bark of Ulmus macrocapra, a polisher (e.g. sodium monohydrogenphosphate), as necessary, a medicinally active component such as a fluorine compound (e.g. sodium fluoride), a wetting agent, a binder, a foaming agent, a flavor, a sweetener, a buffer, an antiseptic agent, a surfactant, a solubilizer and a medicinally active component transferring agent, etc. The extract component A and the component B as solid extracts are respectively obtained by drying dried Achyranthes japonica and bark of Ulmus macrocapara in the shade, thinly cutting, pulverizing, extracting with water or alcohol, filtering and concentrating the resultant filtered solution under a reduced pressure. The composition can be made to a dentifrice composition or an ointment for curing periodontal disease by selecting the mixing component, as necessary.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an oral composition containing an extract of beef knee or Japanese white peel. More specifically,
The present invention relates to an oral composition containing at least one active ingredient from a bovine knee or yuba skin extract, which is extracted from bovine knee or yuba skin and has excellent therapeutic effects on periodontal disease. Examples of the composition of the present invention include a dentifrice composition and an ointment composition.

[0002]

BACKGROUND OF THE INVENTION Periodontal disease is clinically gingival inflammation and bleeding,
It refers to loss of teeth due to formation of periodontal sac and destruction of alveolar bone. Such periodontal disease progresses through a series of processes consisting of bacterial colonization, bacterial infiltration of periodontal tissue, and destruction of periodontal tissue.First, salivary proteins in oral saliva are transferred to dentin and chalky surfaces. A film is formed while being adsorbed, and Streptococcus (St.
Bacteria such as reptococcus) and Actinomyces form plaques as they grow, and as time passes, such plaques move toward the apical roots, while at the same time porphyromonas and Actinobacillus. The anaerobic gram-negative bacteria such as grow, such bacteria, bacterial components, and bacterial products penetrate into the gingival connective tissue through the gingival epithelium (pocket) to form a periodontal sac and the metabolic results of such bacteria It secretes toxic cell toxins such as toxic hydrogen sulfide, ammonia, and amines into periodontal tissues, and at the same time destroys tissues directly by endotoxins such as lipopolysaccharide, which is a cell wall component, or Stimulating, stimulated reactive oxygen species secreted extracellularly by various actions of the humoral and cellular immune system, prostaglandins, leukotrienes, Gingival inflammation is induced by various cytokines such as stamin and interleukin, and collagen, which is a substrate of periodontal tissue, is degraded by enzymes such as collagenase secreted from bacteria and leukocytes, and gingival regression occurs. Then progress to periodontal disease.

In an effort to prevent the occurrence of such periodontal diseases, chlorohexidine gluconate, cetylpyridum chloride, and sanguinarine capable of exposing and killing periodontal disease bacteria for a short period of time have been proposed. And triclosan
) And triamcinolone acetonide (t
Anti-inflammatory agents such as riamcinolone acetonido have been developed and applied to oral products such as toothpastes, dentifrices and ointments, but the occurrence of periodontal disease is still fundamentally unpreventable.

[0004] Recently, myrrh has been used in Korea.
Mulberry white bark, masuma, green tea, licorice, yellow rice, kamikohide, gold and silver flowers,
Efforts are being made to control periodontal disease using crude drug extracts such as corn, but it is important to suppress the production of prostaglandin, which plays a crucial role in inducing periodontal disease, Plant extract that suppresses the activity of collagenase that degrades periodontal tissue is a fact that cannot be confirmed,
In most cases, it only prevents plaque formation or prevents periodontal disease by the action of Chinese medicine such as antibacterial, anti-inflammatory, astringent, hemostatic, and promoting blood circulation. To date, the most widely used treatments for periodontal disease are drugs that inhibit the activity of collagenase enzymes that degrade periodontal tissue or suppress the production of prostaglandin, a periodontal disease-inducing substance This is a method using a physically active ingredient.

An example of such a method is disclosed in US Pat.
European Patent No. 2308955 describes that a periodontal disease can be treated by suppressing the enzymatic activity of collagenase, which degrades periodontal tissue, by developing a system for continuously delivering an active substance containing tetracycline. Patent Publication No. 528468 A1 discloses a treatment for periodontal disease by suppressing the production of prostaglandin, a periodontal disease-inducing substance, by using a dentifrice and a mouthwash containing triclosan to suppress periodontal disease. Possible ways are presented.

However, the above-mentioned pharmacologically active ingredients have two functions, namely, the action of suppressing the production of periodontal disease-inducing substances during the treatment of periodontal disease and the action of suppressing the activity of periodontal tissue degrading enzyme. Since it has only one action in the action,
Not only is there a limit to the complete treatment of periodontal disease, but there is a risk of various side effects when it is used for a long time because it is a synthetic substance. Especially, tetracycline is usually 250mg
(1 tablet) four times a day for a total of 1000 mg per day for 5-7 days. In general, it has been reported that the administration of tetracycline has improved clinically and bacteriologically to a steady state. The result has come out. According to a study report on long-term administration of low concentrations, administration of tetracycline 1000 mg a day for 2 weeks and then 250 mg once a day for 48 weeks, clinically reduces gingivitis, It was reported that motile strains in subliminal plaque disappeared, indicating a decrease in periodontal capsule depth and loss of bacterial adherence.
However, long-term use of low concentrations of tetracycline often leads to the development of resistance to Gram-negative bacteria,
Careful attention must be paid to the emergence of such resistant strains, as they often occur even when antibiotic administration is discontinued, and as side effects, vomiting, gastrointestinal pain complaints, diarrhea, tooth coloring, etc. often occur, There are problems such as the need to ban pregnant women and children.

[0007]

SUMMARY OF THE INVENTION Accordingly, the present inventors
A method for measuring the activity of collagenase that degrades periodontal tissue using various Chinese herbs and plant extracts in order to find substances that have excellent side-effect and periodontal disease treatment effects even with long-term use. Of collagenase using scientific methods such as interleukins-1β and prostaglandin (PGE ₂ ) quantification method, superoxide production measurement method, and collagen synthesis quantification method which induces and periodontal disease Chinese herbs and plant extracts were shown to show excellent efficacy in inhibiting the activity of or of producing interleukins-1β and prostaglandin and promoting collagen synthesis. As a result, as described below,
It has been confirmed that the specific extract of beef knee or yu white bark has the effect of suppressing the production of periodontal disease-inducing substance, and at the same time, suppressing the activity of periodontal tissue degrading enzyme and promoting collagen synthesis. It was completed.

That is, the present invention is as follows. (1) An oral composition containing a cow knee extract, a Japanese white skin extract or a mixture thereof. (2) The extraction solvent is water, methanol, ethanol, propanol or butanol (1).
A composition according to claim 1. (3) The composition according to (1), wherein the oral composition is a dentifrice composition. (4) Each of the cow knee extract and the Japanese white peel extract was separately used in 0.1 ml.
The composition according to (3), which contains 001 to 5% by weight. (5) The cow knee extract and the Japanese white peel extract were each added to 0.1% each.
The composition according to (3), which is contained in an amount of from 0.1 to 3% by weight. (6) The composition according to (3), wherein the cow knee extract and the Yumaku bark extract are mixed together and contained in an amount of 0.002 to 10% by weight. (7) The composition according to (3), further comprising 20 to 60% by weight of calcium hydrogen phosphate, precipitated silica or calcium carbonate as an abrasive. (8) Further, sodium fluoride and / or
Alternatively, the composition according to (3), further comprising 0.1 to 2% by weight of each of the first sodium fluorophosphates. (9) The composition according to (1), wherein the oral composition is an ointment for treating periodontal disease. (10) 0.1% of cow knee extract based on the total weight of the composition.
The composition according to (9), which is contained in a ratio of 001 to 5% by weight. (11) The composition according to (9), which contains the extract of Yuzuki bark at a ratio of 0.001 to 5% by weight based on the total weight of the composition. (12) The composition according to (9), wherein the composition contains a mixture of the cow knee extract and Yuzuki bark in a ratio of 0.002 to 10% by weight based on the total weight of the composition. (13) One or more of a surfactant, a solubilizer, a wetting agent, a drug transmitting agent, a buffer, a preservative, a sweetener and a flavor are further compounded (9) to (11). )). (14) The composition according to (13), wherein a polyoxyethylene-polyoxypropylene copolymer is blended as a surfactant at a ratio of 5 to 30% by weight based on the total weight of the composition. (15) The composition according to (13), wherein a lower alcohol solvent is blended as a solubilizer in a ratio of 1 to 20% by weight based on the total weight of the composition. (16) One or more components selected from glycerin, sorbitol solution, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600 and polyethylene glycol 1000, propylene glycol, porosamer and monoglyceride as a wetting agent are added to the total composition. 5-40 based on weight
The composition according to (13), wherein the composition is blended in a ratio of% by weight. (17) Gelatin or pectin or a mixture thereof as a drug delivery agent, based on the total weight of the composition, from 1 to 3
The composition according to (13), wherein the composition is compounded at a ratio of 0% by weight. (18) The buffer is selected from monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide and sodium pyrophosphate and pyrophosphate. A composition in which one or more components are blended and the pH is adjusted to 5.0 to 8.0.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an oral composition containing a cow knee extract, a Japanese white peel extract or a mixture thereof.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION A crude drug extract used as an active ingredient in the oral composition of the present invention is extracted from cow's knees or yuzuki bark. One of the active herbs of the present invention is cattle knee (Achyranthes japonica, Achyranthe) which is a plant distributed in all regions of Korea, Japan and the sub-Yellow region of China.
s fauriei, Achyranthes bidentata), dug out after removing the whiskers and dried in gray, yellow-yellow or tan rods, or slightly bent lengths of 15-9
There are many vertical wrinkles and traces of beards in some places as a circumference of 0 cm and a diameter of 3 to 7 cm. The folded surface is grayish or light brown and flat, the central xylem is yellowish white, and the quality is hard and brittle. It is almost odorless, tastes slightly sweet and mucous. When the cross section is observed with a microscope, the bark and xylem can be distinguished as distinct cambium. At the center of the xylem is a small native xylem, which contains calcium hydroxide crystals in parenchyma cells,
Starch grains are not visible. It is known to have a pharmacological effect on diuretic effect, menstruation, arthritis, etc. The main components are saponin and oleanolic glycan
oside), an insect metamorphosis hormone [inokososterone (inokos
terone), ecdysterone], betaine hydrate and the like.

Further, the active herbal medicine used in the composition of the present invention, Yumaku, is Ulmus macrocapra, Ulmus macrocapra, which is a plant belonging to the family Elaceae distributed in central and northern Korea, China and Japan.
The root bark of pumila or Ulmus davidiana is harvested and sun-dried.It is known to have pharmacological effects of water irrigation, dissemination, tsunogo, edema, and erysipelas. Β-sitosterol (β-si
tosterol), plant sterols and tannins. The term "yu white bark" is also referred to as "yu root bark" or "tree bark" according to the country and region, but "yu white bark" is often used.

[0012] In the present invention, the cow knee extract and the Japanese white peel extract use water or alcohol extracts, respectively. The cow knee extract and Yu white skin extract used in the present invention are obtained by shaving dried cow knee and Japanese white skin, shaving into small pieces, powdering, extracting with water or alcohol, filtering, and filtering the filtrate. Concentrate under reduced pressure to obtain a solid extract.

[0013] Examples of the alcohol extraction solvent that can be used for extraction of beef knee and Yuzhi bark include lower alcohol solvents having 1 to 4 carbon atoms such as methanol, ethanol, propanol and butanol, and ethanol is preferably used. .

The crude drug extract of the present invention is contained in a dentifrice composition or an ointment composition, and has an activity of treating periodontal disease. In each of the compositions according to the present invention, the extract of beef knee and yu white skin was 0.001 to 5% by weight based on the total weight of the composition,
Preferably, it is used in a ratio of 0.01 to 3% by weight. In the composition of the present invention, it is preferable that the extracts of beef knee and Yuzhi bark are also mixed and blended in the composition of the present invention so that each of the extracts is present in the above-described blending ratio. However, when two crude drug extracts are combined, it may be 0.002 to 10% by weight based on the total weight of the composition. If the content of each crude drug extract is less than 0.001% by weight, the intended efficacy and effect on periodontal disease cannot be expected, and if it exceeds 5% by weight, the stability of the product may decrease. Not desirable. The weight percent of the above extract is the weight percent of the solid extract of each of the beef knee and the Japanese white peel.

<Dentifrice composition> The dentifrice composition according to the present invention can be specifically produced by the following method.

As abrasive components, potassium monohydrogen phosphate, precipitated silica, silica gel, sodium bicarbonate, calcium carbonate,
One or more selected from hydrated alumina, insoluble sodium metaphosphate and sodium pyrophosphate are used. Preferred abrasive components are calcium monohydrogen phosphate, hydrous silica and calcium carbonate. The abrasive component is usually used in an amount of 1 to 90% by weight in the entire dentifrice composition,
Preferably it is 20 to 60% by weight.

As a medicinal ingredient for promoting remineralization of a tooth and strengthening a tissue of a tooth, a fluorine compound is exemplified.
Specifically, sodium fluoride or sodium monofluorophosphate is used alone or as a mixture of both. Its content is suitably from 0.01 to 2.0% by weight in the whole dentifrice composition. If the content of the pharmaceutically active ingredient is less than 0.01% by weight, the effect is not sufficient, and if it exceeds 2.0% by weight, the safety of the human body may be affected, so that it is not suitable for use as an oral preparation.

The humectant is used to maintain the condition of the dentifrice composition and prevent drying. Examples of the humectant include glycerin, sorbitol solution, polyethylene glycol and propylene glycol, and these may be used alone or in combination of two or more. The above mixture is used in an amount of 20 to 60% by weight in the total dentifrice composition.

In the dentifrice component, a binder is used to bind the liquid and solid components to maintain the dentifrice form and to ensure safety. Such binders are mainly sodium or calcium alginate, carboxylate. Natural or synthetic high-molecular substances such as sodium methylcellulose, xanthan gum, acacia gum, carrageenan and the like are used, and the content thereof is 0.1 to 5% by weight in the whole dentifrice composition.

The foaming agent complements the cleaning action of the abrasive, and not only allows the medicinal ingredient to penetrate into the area hardly reachable by the toothbrush, but also serves to generate air bubbles and increase the tooth brushing feeling, thereby assisting the cleaning action and improving the medicinal effect. Since the dispersion and penetration of the components are accelerated and the interfacial tension is reduced, foreign substances in the oral cavity can be easily removed. As the foaming agent mainly used, sodium lauryl sulfate and sodium alkyl sulfate which are anionic surfactants are used, and polyoxyethylene, polyoxypropylene and copolymer which are auxiliary nonionic surfactants are used. Polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid, alkanolamide fatty acid ester, sucrose fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester and polyoxyethylene hydrogenated castor oil derivative are used. The content of the foaming agent is 0.5 to 5 by using an anionic or nonionic surfactant alone or by mixing two or more kinds.
It is preferred to use% by weight.

In addition to the above additives, flavors and sweeteners may be used to give a refreshing sensation and to control the bitter taste.
As the fragrance, peppermint and spearmint oil, which are natural fragrances, are often used. The amount of the fragrance used is 0.1 to 1% by weight in the whole dentifrice composition. As the sweetener, synthetic or natural non-fermentable sugars and the like are mainly used, and typical ones are saccharin sodium, aspartame,
Lactose, maltose, xylitol and the like,
Preferred is saccharin sodium and the like. The amount of the sweetener used is 0.05 to 1% by weight in the whole dentifrice composition.

Examples of the buffer for adjusting the pH of the dentifrice composition include, for example, alkali metal salts of orthophosphoric acid, particularly sodium monophosphate, sodium phosphate dibasic, sodium phosphate tribasic, citric acid and citric acid. Examples thereof include sodium acid, phosphoric acid, hydrochloric acid, sodium hydroxide, sodium pyrophosphate and pyrophosphate, and are preferably sodium monophosphate, sodium phosphate dibasic, and sodium phosphate tribasic. One or more components selected from the above buffering agents are blended to adjust the pH to 5.0 to 8.0.
Adjust to

Preservatives are used to prevent microbial contamination that can occur during the manufacture and use of the dentifrice composition. As the preservative, preservatives generally used for foods and pharmaceuticals can be used, and specific examples include methyl parahydroxybenzoate, benzoic acid, sodium benzoate, and salicylic acid. These preservatives can be used alone or in combination of two or more. The amount of the preservative used is 0.01 to 0.5% by weight in the whole dentifrice composition.

In the case of using a mixture of beef knee and Japanese white peel extract in the dentifrice composition of the present invention, the use amount of the extract is 0.002 to 10% by weight in the whole dentifrice composition. The mixing ratio of the two is 1: 5000 to 5000: 1 for beef knee: yu white skin.
Degree, preferably 1: 0.1 to 10.0.

<Ointment Composition> The ointment composition of the present invention contains additives usually used in the field of ointment production, such as surfactants and solubilizers for stabilizing the state of the ointment. Ingredients such as a humectant, a drug delivery agent for oral soft tissues, a buffer, a preservative, a sweetener and a flavor can be further added.

In the ointment composition of the present invention, a polyoxyethylene-polyoxypropylene copolymer is used as a surfactant for the state stability of the ointment.
For example, Pluronic F-127 or Pluronic F-
Pluronic derivatives such as 108 can be used. In the ointment composition of the present invention, such a surfactant can be used in a ratio of 5 to 30% by weight based on the total weight of the composition.

In the ointment composition of the present invention, a lower alcohol solvent can be used as a solubilizing agent for the extract of beef knee and Japanese white peel. Lower alcohol solvents used for such purposes include ethanol, isopropyl alcohol, and the like,
A mixture of two or more alcohol solvents can be used. The lower alcohol solvent in the composition of the present invention,
It is preferable to mix them in a ratio of 1 to 20% based on the total weight of the ointment composition.

In the ointment composition of the present invention, a humectant is further used for maintaining the state of the ointment and preventing drying. Examples of the humectant include glycerin, sorbitol solution, polyethylene glycol 200 and polyethylene glycol 4.
Polyethylene glycol such as 00, polyethylene glycol 600 and polyethylene glycol 1000, propylene glycol, Poloxamer 40
One component selected from 7) and monoglyceride (Myverol 18-99) can be used alone or as a mixture of two or more components. The amount of wetting agent used is preferably from 5 to 40% by weight, based on the total weight of the composition.

On the other hand, as a drug delivery agent which adheres to the oral soft tissue so that the drug efficacy of the active ingredient can be transferred, gelatin and pectin are used alone or in combination with each other in an amount of 1 to 30% by weight based on the total weight of the composition. % Can be used.

As the buffer for adjusting the pH of the ointment composition, alkali metal salts of orthophosphoric acid (particularly sodium monophosphate, sodium phosphate dibasic, sodium phosphate tribasic), citric acid and sodium citrate , Phosphoric acid, hydrochloric acid, sodium hydroxide, pyrophosphoric acid and pyrophosphate (mainly sodium salt), and the like.
Sodium phosphate. 1 selected from the above buffers
The pH of the mixture is adjusted to 5.0 to 5.0
Adjust to 8.0.

In addition to the above-mentioned additives, in view of the fact that the composition of the present invention is particularly applied to the oral cavity, it is possible to use a flavor and a sweetener to give a refreshing feeling and to control a slightly bitter taste. Good. As the fragrance, peppermint and spearmint oil, which are natural fragrances, are often used. The amount of fragrance used
It is 0.1 to 1% by weight in the total dentifrice composition. As the sweetener, synthetic or natural non-fermentable sugars and the like are mainly used,
Representative examples include saccharin sodium, aspartame, lactose, maltose, xylitol and the like, and preferably saccharin sodium and the like. The amount of the sweetener used is 0.05 to 1% by weight in the entire dentifrice composition.
It is.

Preservatives are used to prevent microbial contamination that can occur during the manufacture and use of the dentifrice composition. As the preservative, preservatives generally used for foods and pharmaceuticals can be used, and specific examples include methyl parahydroxybenzoate, benzoic acid, sodium benzoate, and salicylic acid. These preservatives can be used alone or in combination of two or more. The amount of the preservative used is 0.01 to 0.5% by weight in the whole dentifrice composition.

When the ointment composition of the present invention is used clinically, the composition formulated in the above ratio is applied to an affected area with periodontal disease in an amount of 50 to 500 mg, preferably 1 to 50 per day.
It is suitable to use the drug in an amount of 100 to 300 mg, but the dose may be adjusted appropriately in consideration of factors such as the severity of periodontal disease and the size of the affected part.

[0034]

The present invention will be described in more detail with reference to the following Examples and Comparative Examples, which should not be construed as limiting the present invention.

Production Example 1 50 g of powder obtained by pulverizing dried cow knees, 500 ml of 95% ethanol was added thereto, and the mixture was subjected to sedimentation extraction for 3 days, followed by using No. 1 Watman (Whatman No. 1). After filtration, the filtrate was concentrated under reduced pressure to obtain 15 g of a dried cow knee extract.

Production Example 2 50 g of powder obtained by pulverizing dried Yuzuki skin was added to 500 ml of 95% ethanol, and the resulting mixture was subjected to sedimentation extraction for 3 days, followed by using No. 1 Watman (Whatman No. 1). , Filtered and concentrated under reduced pressure, dried 20 g of dried white peel extract
I got

Example 1-18 and Comparative Example 1-18 Toothpaste compositions of Examples and Comparative Examples were produced according to the composition ratios shown in Tables 1 to 9 below.

[0038]

[Table 1]

[0039]

[Table 2]

[0040]

[Table 3]

[0041]

[Table 4]

[0042]

[Table 5]

[0043]

[Table 6]

[0044]

[Table 7]

[0045]

[Table 8]

[0046]

[Table 9]

Experimental Example 1-1: Toothpaste of the present invention (Example 1)
1-1-1) Inhibitory effect on superoxide formation
The venous blood collected from healthy adults without systemic disease using citric acid as an anticoagulant was centrifuged at 1200 rpm for 10 minutes, and then the leukocyte concentrate in the middle layer was collected. RPMI1640 medium was added to the leukocyte concentrate of this middle layer at 1:
It was added and diluted to a ratio of 1. After 12 ml of Ficoll-Paque was added to a 50 ml centrifuge tube, 30 ml of the diluted blood was carefully added so as to form a middle layer, and centrifuged at 1600 rpm for 30 minutes. The upper layer containing serum was removed, and the middle layer containing mononuclear cells was diluted by carefully adding 3 times RPMI1640 medium, followed by centrifugation at 80 rpm for 10 minutes. The supernatant was discarded, and 10 ml of RPMI1640 medium was added, followed by gentle pipetting, followed by centrifugation at 800 rpm for 10 minutes. Discard the supernatant and remove HBSS (hanks'balanced sa
After adding a buffer solution and pipetting, 0.45 ml of the obtained human mononuclear leukocytes was dispensed into a 24-well plate at 10 ⁶ cells / well, and 95% air and 5% The cells were cultured aseptically under CO ₂ and 100% humidity for 2 hours. This is followed by FMLP (N-Formyl-Met-Le
u-Phe) was added to a concentration of 10 ⁻⁶ M and 0.05 ml.
After culturing at 7 ° C. for 15 minutes to stimulate the cells, 80 μM
0.1 ml of Cytochrome C
C), 0.1 ml of superoxide dismutase (SOD) was added to a concentration of 30 μg, and the dentifrice composition diluted 3 times in Examples and Comparative Examples excluding the cytotoxic surfactant was added. HBSS (hanks'balanced) so that the total amount of the remaining reaction solution is 0.9 ml.
After adding a salt solution), the mixture was kept at 37 ° C. for 10 minutes. Thereafter, 0.1 ml of zymosan A, which was phagocytosed as a stimulant, was added to a final concentration of 1.3 mg / ml, and the mixture was incubated at 37 ° C. for 90 minutes with shaking.
The reaction was stopped at 4 ° C. for 0 minute and stopped at 4 ° C. for 150 minutes.
After centrifugation at 0 rpm for 10 minutes, the supernatant was collected. The absorbance at 550 nm of the supernatant was measured,
The production amount of superoxide anion was determined by the following equation.

[0048]

(Equation 1)

The measurement results are shown in Table 10 below.

[0050]

[Table 10]

From the above experimental results, it was found that the cow knee extract
In Examples 1-1, 5, 7, 9, and 11 containing not less than 01%, the superoxide production inhibitory effect was shown to increase as the concentration of the cow knee extract increased, while Examples 1-2, 4 containing the extract
Nos. 6, 8, 10 and 12 showed no superoxide formation inhibitory effect. Examples 1-13, 14, 1 containing both the cow knee extract and the Yuzuki skin extract
5, 16, 17 and 18 also showed that the inhibitory effect on superoxide production increased with the concentration of cow knee extract.

Experimental Example 1-2: Toothpaste of the present invention (Example 1-
Collagenase which is a periodontal tissue degrading enzyme of 1-1-18)
The effect of inhibiting the enzyme activity on the enzyme activity of collagenase, which is a periodontal tissue-degrading enzyme, is as follows. In this test method, periodontal disease-causing bacteria Porphyromonas gingivalis and Polymorphonuclear
Collagen, a substrate of periodontal tissue, is degraded by the action of collagenase oxygen contained in saliva and gingival fluid secreted from leukocytes and neutral leukocytes (Neutrophil) in patients with periodontal disease. This is an experimental method that imitates the oral environment of a living body in which the phenomenon occurs.

To 25 1.5 ml Eppendorf tubes, 100 μl of an azocol solution (Azocoll solution) as a 2% red collagen substrate was added. One eppendorf tube was used as a blank and three tubes contained collagenase type I purchased from Sigma.
Standard enzyme solution (collagen decomposition activity: 315un
its / mg) was added to give 10, 100, and 200 ppm. To each of the remaining tubes was added 100 μl of collagenase oxygen purely separated from the saliva and gingival oral fluid of periodontal disease patients by Sephacryl S-200 chromatography, one more tube was used as a control group, In each of the tubes, the experimental group dentifrice (Examples 1-1 to 1-18) and the comparative group dentifrices (Comparative Examples 1-1 to 1-18) excluding the cytotoxic surfactant were added to distilled water: The mixture was mixed at a ratio of 2 and completely homogenized.
Centrifuged for 0 minutes. Thereafter, the supernatant was treated with 10 μl, and a buffer solution (0.05 M Tris-HCl, InM CaCl ₂ , 7.8) was added so that the total reaction solution became 500 μl.
After reacting for a period of time, these Eppendorf tubes are centrifuged at 10,000 g for 5 minutes to precipitate undegraded collagen, and the supernatant containing the degraded collagen is taken and the absorbance is measured at 540 nm to prepare a standard activity curve. did. The activity concentration of the enzyme was converted from the standard activity curve to compare and evaluate the enzyme activities of the experimental group and the target group, and the following results were obtained (see Tables 11 and 12).

[0054]

[Table 11]

[0055]

[Table 12]

From the above experimental results, it was found that the cow knee extract was
Examples 1-1, 5, 7, 9 and 1 containing 1% or more
1 showed that the inhibitory effect on collagenase activity increased as the concentration of cow knee extract increased. Examples 1-2, 4, containing a white peel extract
6, 8, 10 and 12 also showed that the inhibitory effect on collagenase activity increased as the concentration of white peeled skin increased. Example 1-1 in which both the cow knee extract and the Yu-bark extract have excellent inhibitory effects on collagenase activity, and contain both the cow knee extract and the Yu-bark extract
3, 14, 15, 16, 17 and 18 were shown to have an increasing effect.

Experimental Example 1-3: Toothpaste of the present invention (Example 1-
1-1-18) mononuclear leukocyte interleukin (IL
1 beta) were dispensed in 0.8ml added to a 10 ⁶ cell / well of blood mononuclear leukocytes isolated from suppression blood in 24-well plates for generating. E. coli lipopolysaccharide (LPS) (250 ppm) 1 with well control group supplemented with 200 μl of RPMI 1640 medium
Well containing 100 μl and LPS (250 ppm) 100 μl
Example (1-) in which a surfactant having cytotoxicity was excluded.
1 to 18) and Comparative Examples (1-1 to 1-18) were cultured for 24 hours using wells to which 100 μl of the three-fold diluted dentifrice composition was added, and then 50 μl of arachidonic acid was added thereto for an additional 30 minutes. Incubate for minutes. Interleukin (IL
(1β) was added to the wells of the 96-well plate to which the antibody of (1β) was attached, and 50 μl of the biotinylated antibody reagent was added to all wells.
After being kept at 5 ° C. for 30 minutes, and washed three times with a washing buffer solution, a streptavidin-horseradish peroxidase (HRP) conjugate (Streptavidin-HRP Conjugat) was added.
e) was added to all wells, kept at 25 ° C. for 30 minutes, and washed again three times with a washing buffer solution. to this,
Immediately add 100 μl of enzyme substrate, keep the plate open at 25 ° C. in a dark room for 30 minutes, and add 0.18 M
After adding 100 μl of sulfuric acid, the absorbance at 450 nm was measured with a microplate reader, and a standard curve was created as the absorbance value of the standard solution to calculate the amount of interleukin produced in the experimental group. The measurement results are shown in Table 13 below.

[0058]

[Table 13]

I. of human mononuclear leukocytes stimulated with E. coli LPS
As a result of testing the efficacy and effect on the production of L-1β,
1- containing a cow knee extract at a concentration of 0.01% or more
The inhibitory effects in 3, 5, 7, 9 and 11 were shown to increase as the concentration of the cow knee extract increased, while Example 1-2 containing the extract of Yuhaku bark. ,
Nos. 4, 6, 8, 10 and 12 showed no IL-1β production inhibitory effect. Examples 1-13, 14, 1 containing both the cow knee extract and the Yuzuki skin extract
5, 16, 17 and 18 also showed that the inhibitory effect on superoxide production increased with the concentration of cow knee extract.

Experimental Example 1-4: Toothpaste of the present invention (Example 1-
1-1-18) mononuclear leukocyte prostaglandin (PG
E ₂ ) Inhibitory effect on production The tooth dentifrice of the present invention was used to inhibit the production of prostaglandin, a periodontal disease-inducing substance. The experimental results are as follows. This test method generates periodontal in disease progression periodontal disease causes a bacteria Porphyromonoas gingivalis is a cell wall component lipopolysaccharide by induced by stimulating mononuclear leukocytes humans prostaglandins (PGE ₂₎ By using an antigen-antibody immunodiagnosis method to remove the cytotoxic surfactant from the experimental group (Examples 1-1 to 1-18)
And a control group dentifrice (Comparative Examples 1-1 to 1-18) were mixed with distilled water at a ratio of 1: 2, completely homogenized, and centrifuged at 5000 g for 10 minutes to treat the supernatant. Comparative evaluation was performed. A specific test method is a goat anti-mouse I
Buffer solution (0.9% NaCl, 0.1% bovine serum albumin, 0.5%
50 µl of Kathon-containing 0.1 M phosphate buffer solution)
Add standard (0, 2.5, 5, 10, 20, 40, 80, 160, 320p
g) 50 μl of standard solution of appropriate concentration was added to the wells. In addition, the experimental group dentifrice excluding the cytotoxic surfactant produced as described above (Examples 1-1 to 1-1)
8) and 50 μl of the comparative dentifrice (Comparative Examples 1-1 to 1-18)
Was added to the wells set in the Examples and Comparative Examples, and 50
The μl antibody to PGE ₂ in was added to all wells excluding the blank wells, the PGE ₂ binding peroxidase 50μl is added to all wells excluding the blank wells, covered with a 96-well plate for 1 hour at 25 ° C. After holding, the plate was washed four times with a washing buffer solution (phosphate buffer solution containing 0.05 Tween 20: pH 7.5), and then washed 150 times at room temperature.
μl of enzyme substrate (3,3 ′, 5,5′-tetramethylbenzidine / hydrogen peroxide dissolved in 20% dimethylformamide) was immediately added and reacted at 25 ° C. for 30 minutes. After adding 100 μl of 1 M sulfuric acid thereto, the absorbance at 450 nm is measured with a microplate reader, and a standard curve is created with the absorbance value of the standard solution to calculate the amount of prostaglandin produced in the experimental group. The measurement results are shown in Tables 14 and 15 below.

[0061]

[Table 14]

[0062]

[Table 15]

Human mononuclear leukocytes stimulated with E. coli LPS
PGE_TwoEffect on the production, as a result of implementing the effect, cow knee
1-3, 5, 7, 9, 9 and 11 containing the extract
The inhibitory effect of cattle knee extract increased as the concentration increased
While it was shown to rise, it contained Yu-bark extract
In Examples 1-2, 4, 6, 8, 10, and 12
Is PGE_TwoIt was shown that there was no production suppression effect. Cow knee extraction
Example 1-1 containing both exudates and Yuzhi bark extract
3, 14, 15, 16, 17 and 18 are also PGE _TwoGeneration suppression
Control effect increases with cow knee extract concentration
Was.

Experimental Example 1-5: Toothpaste of the present invention (Example 1-
1-1-18) Collagen protein production of gingival fibrous cells
Effect on adult gingival fibroblast cells were cultured in a 24-well plate.
After dispensing to obtain ⁶ cells / well, 10% FB
After culturing for one day in a DMEM medium containing S, the existing medium was replaced with a fresh medium the next day, and the cells were further cultured for 24 hours. After this, the washing layer on the bottom was washed with HBSS buffer solution, and then serum-free and proline-free ME were removed.
Experimental group toothpaste (Examples 1-1 to 1-18) and Comparative group toothpaste (Comparative examples 1-1 to 1-18) excluding the cytotoxic surfactant by adding 0.8 ml of M medium were distilled. 1: 2 with water
The mixture was added at a ratio of
After centrifuging at 0 g for 10 minutes and treating the supernatant, ¹⁴ C-
The cells were cultured in a culture solution containing 100 μl of proline (10 Ci). After 24 hours, the total protein and collagen protein were measured. First, in order to measure the amount of extracellular protein synthesized, the culture solution in each well was placed in a dialysis tube with one sealed, and the other was sealed with a cold buffer (Tris-HCl 0.05m2).
ol / L, NaCl 0.2 mol / L, CaCl ₂ 0.05 mol / L, phenylmethylsulfonyl fluoride 0.3 mN) after completion of dialysis for 24 hours, take 100 μl of each, put in a count vial and put 10 ml of scintillation cocktail. Radioactivity was measured for 1 minute in a liquid scintillation counter (LSC). 0.1 N NaOH and 0.3 mN of phenylmethylsulfonyl fluoride were added to each well from which the cell culture solution had been removed in order to measure the amount of total intracellular protein synthesis, and then maintained at 60 ° C. for 30 minutes to break the cell membrane. After that, the cell homogenate was put into a dialysis tube sealed on one side, and the other was sealed on a cold buffer (Tris-HCl 0.05 mol / L, NaCl
After dialysis with 0.2 mol / L, CaCl ₂ 0.05 mol / L, phenylmethylsulfonyl fluoride 0.3 mN) for 24 hours, take 100 μl of each and put in a counting vial.
The scintillation cocktail of ml was put, and radioactivity was measured by LSC for 1 minute. In order to measure the amount of intracellular and extracellular collagen protein synthesis, 100 μl of each of the dialyzed cell culture solution and cell homogenous solution was taken and placed in a 1.5 ml microtube, and a collagenase reaction buffer (0.05 M Tris-H
Cl, lmM CaCl ₂ , 0.03 mM phenylmethylsulfonyl fluoride) and 100 μl of collagenase enzyme (100 ppm) are added and reacted at 37 ° C. for 3 hours to completely degrade collagen.
After adding 500 μl of a solution containing 1% trichloroacetic acid and tannic acid, the mixture was precipitated at 4 ° C. for 30 minutes, and then centrifuged at 1000 × g for 5 minutes.
0 μl was taken, placed in a counting vial, filled with 10 ml of scintillation cocktail, and radioactivity was measured by LSC for 1 minute. The measurement results are shown in Table 16 below.

[0065]

[Table 16]

In order to study the efficacy and effect of human fibrous cells on collagen synthesis, the dentifrice composition of the present invention was treated with a cell culture solution containing ¹⁴ C-proline as described above. 1-2, 4, 6, containing
8, 10 and 12, the collagen synthesis promoting effect was shown to increase as the concentration of the white peel on the skin increased, while Examples 1-3, 5, and 5 containing the cow knee extract
7, 9 and 11 showed no collagen synthesis promoting effect. Examples 1-13, 14, 15, 16, and 17 containing both cow knee extract and Yuzuki skin extract
And 18 also showed that the collagen synthesis-promoting effect increased with the concentration of the extract of Japanese white peel.

Example 1-6: Toothpaste of the present invention (Example 1-
Clinical experiments on efficacy and effects of 1-1-18) The results of clinical experiments on periodontal disease treatment with the dentifrice of the present invention are as follows. The subjects were periodontal disease patients with complete dentition and no missing teeth. The subjects were age-specific 30- to 50-year-olds. After selecting the group, the experimental group toothpaste was divided into 60 groups each (Examples 1-1 to 1).
-18) and a comparative group toothpaste (Comparative Examples 1-1 to 1-18) were subjected to clinical experiments on the treatment effect on periodontal disease by the following method. First, the clinical experiment method of the dentifrice of the present invention was performed by the following method. After having divided the experimental group into the experimental group and the comparative group, after having been trained in oral health education and the correct brushing method,
The same control toothbrush was supplied to all experimental groups, and the initial gingivitis index was scored by performing toothbrushing, and the experimental group and comparative group toothpaste were supplied and used for one week, one month, After 3 months and 6 months, oral examination is conducted to check the gingivitis index. The method of measuring the gingivitis index is to insert the gingival probe into the gingival row and continue around each tooth without applying force. After 30 seconds, the bleeding state was measured, and the score was recorded by the following method to obtain a result. Points Description 0 points: no bleeding state 1 point: point bleeding state 2 points: linear bleeding state 3 points: interdental site triangular bleeding state 4 points: whole gum bleeding state The measurement results are shown in Table 17 below.

[0068]

[Table 17]

From the above experimental results, a cow knee extract capable of suppressing the enzyme activity of collagenase, which suppresses the production of IL-1 and prostaglandin which induce periodontal disease and suppresses superoxide and degrades periodontal tissue. Alternatively, the dentifrice of the present invention (Examples 1-1 to 1-12) containing the extract of Yuzuki skin which can promote collagen synthesis and suppress the enzyme activity of collagenase has a therapeutic effect on periodontal disease of 1
From the week to the lapse of 6 months, the efficacy was shown to be superior to the comparative examples (1-1 to 1-18) which did not contain the cow knee extract or the extract of Yuzuki skin, and the gingivitis index of the comparative example Is continuously increased over time after one month, while the use of the dentifrice developed according to the present invention suppresses the occurrence of periodontal disease and the gingivitis index decreases significantly over time Examples 1-13, 14, 15, containing both the cow knee extract and the Japanese white peel extract
16, 17, and 18 were shown to have an increasing effect on periodontal disease suppression.

Examples 2-1 to 2-18 and Comparative Example 2-1
～2-18 each component in accordance with normal ointment manufacturing method by component compounding ratio shown in Table 18-26 below were dissolved and gelled buffer, ointment compositions according to the present invention (Examples 2-1 ~
2-18) and comparative compositions (Comparative Examples 2-1 to 2-18).

[0071]

[Table 18]

[0072]

[Table 19]

[0073]

[Table 20]

[0074]

[Table 21]

[0075]

[Table 22]

[0076]

[Table 23]

[0077]

[Table 24]

[0078]

[Table 25]

[0079]

[Table 26]

Experimental Example 2-1 Supermarket of the ointment of the present invention
Oxide Production Inhibiting Effect The inhibitory effect of the ointment composition of the present invention on the production of superoxide, a substance that induces periodontal disease, was measured by the following method. 1200 r of venous blood collected from healthy adults without systemic disease using citric acid as an anticoagulant
After centrifugation at pm for 10 minutes, the leukocyte concentrate in the middle layer was recovered. The PRMI164 was added to the leukocyte concentrate in the middle layer.
It was added and diluted to a ratio of 1: 1 with medium 0. 50
After adding 12 ml of Ficoll-Paque to the centrifuge tube of 30 ml, 30 ml of the diluted blood was carefully added so as to form a middle layer, and centrifuged at 1600 rpm for 30 minutes.
The upper layer containing serum was removed, and the middle layer containing mononuclear cells was carefully removed with a sterile pipette into a new centrifuge tube. Then, three times the volume of PRMI1640 medium was added, and the mixture was centrifuged at 800 rpm for 10 minutes. separated. The supernatant was discarded, and 10 ml of a PRMI1640 medium was added, followed by gentle pipetting, followed by centrifugation at 800 rpm for 10 minutes. Discard the supernatant and remove HBSS (hanks' balanced salt s).
olution) After adding buffer solution and pipetting, human mononuclear leukocytes were placed in a 24-well plate at 10 ⁶ cells.
Dispense 0.45 ml at a time to give 95%
Air, 5% CO ₂ After culturing aseptically for 2 hours under 100% humidity, FMLP (N-Formyl-Met-Leu-Phe) 0.
The cells were treated with 05 ml (10 ⁻⁶ M) and cultured at 37 ° C. for 15 minutes to stimulate the cells. 0.1 ml of cytochrome C
(80 μM), 0.1 ml of SOD (30 μM), and 0.1 ml of each of the ointment compositions of Examples and Comparative Examples diluted three-fold with HBSS, and then added HBSS so that the total reaction solution was 0.9 ml. The mixture was incubated at 37 ° C. for 10 minutes, and 0.1 ml of stimulant phagocytosed Zymosan A was added to a final concentration of 1.3 mg / ml. The reaction mixture was incubated at 37 ° C. for 90 minutes with shaking, then placed in a 4 ° C. refrigerator for 10 minutes to stop the reaction, and then centrifuged at 4 ° C. for 10 minutes at 1500 rpm. 55
The absorbance of the supernatant was measured at 0 nm, and the amount of generated superoxide anion was calculated by the following equation.

[0081]

(Equation 2)

The measurement results are shown in Table 27 below.

[0083]

[Table 27]

From the results shown in Table 27 above, Examples 2-1 to 5, 7, and 7 containing 0.001% or more of cow knees.
In Examples 9 and 11, the superoxide production inhibitory effect was shown to increase as the concentration of cow knees was increased, while Examples 2-2, 4, 6, 8,
In Nos. 10 and 12, it was shown that there was no superoxide generation inhibitory effect. Examples 2-13, 14, 15, 16, 17, 17 and 18 containing both cow knees and Yuzhi bark
It was also shown that the inhibitory effect on superoxide production increased with cow knee concentration.

Experimental Example 2-2: Collagena, an ointment of the present invention
Inhibitory Effect on the Activity of the Enzyme The inhibitory effect of the ointment composition of the present invention on collagenase which is a periodontal tissue degrading enzyme was tested by the following method. In this test method, Porphyromonas gingivalis and Polymorphonu, the causative bacteria of periodontal disease
Living oral environment in which collagen, which is a substrate of periodontal tissue, is degraded by the action of collagenase enzyme contained in saliva and gingival oral fluid of patients with periodontal disease secreted from clear leukocytes and neutral leukocytes, and gingival regression occurs This is an experimental method that mimics the above. To each of the 25 1.5 ml Eppendorf tubes, 100 μl of 2% red collagen substrate azocol solution was added. One eppendorf tube was used as a blank, and three tubes contained one collagenase type 1 standard enzyme solution (collagen decomposition activity: 315 units / mg) purchased from Sigma.
It was added so as to be 0, 100, and 200 ppm. To each of the remaining tubes, 100 μl of collagenase enzyme purely separated from the saliva and gingival oral fluid of periodontal disease patients by Sephacryl S-200 chromatography was added, one of which was used as a control group, Each of the remaining tubes contains an experimental group ointment according to the present invention (Example 2-1).
-2-18) and Comparative Group Ointment (Comparative Examples 2-1 to 2-18)
Was mixed with distilled water in a ratio of 1: 2 to completely homogenize 5
10 μl of the supernatant centrifuged at 000 g for 10 minutes was added with a micropipette, and the buffer solution (0.05 M Tri) was added.
s-HCl, lnM CaCl ₂ 7.8) was added to make the total reaction solution 500 μl. Each eppendorf tube was placed in a 37 ° C. incubator and reacted for 18 hours, and then centrifuged at 10,000 g for 5 minutes to precipitate undegraded collagen. The supernatant containing the degraded collagen was collected and the absorbance was measured at 540 nm. A standard activity curve was created from the results obtained from the tubes to which the standard enzyme solution was added, and the enzyme activity in the remaining tubes was converted from the standard curves to compare and evaluate the enzyme activities of the experimental group and the control group. did. The measurement results are shown in Table 28 below.

[0086]

(Equation 3)

[0087]

[Table 28]

From the results shown in Table 28 above, Examples 2-1 to 5, 7, and 7 containing 0.001% or more of cow knees.
In Examples 9 and 11, the inhibitory effect on collagenase activity was shown to increase as the concentration of cow knee increased, and Examples 2-2 and 4,
6, 8, 10 and 12 also show that the inhibitory effect on collagenase activity increases as the concentration of Yu-bark increases, and that both the cow knee extract and the Yu-bark extract have excellent inhibitory effects on collagenase activity. Example 2-1 containing both a cow knee extract and a Japanese white peel extract
3, 14, 15, 16, 17 and 18 were shown to have an increasing effect.

Experimental Example 2-3: Mononuclear leukemia of the ointment of the present invention
Inhibitory effect on sphere interleukin (IL-1β) production
The effect of the ointment composition according to the present invention on the production of interleukin (IL-1β), a periodontal disease-inducing substance, was measured by the following method. 0.8 ml of blood mononuclear leukocytes separated from blood is added to a 24-well plate and 10 ⁶ ce
ll / well, and dispensed to RPMI1640.
The control group, E. coli LPS
(250 ppm) well and LPS (250 ppm) to which 100 μl was added
A well to which 100 µl and 100 µl of an ointment diluted three-fold with RPMI1640 medium were added was used as an experimental group, and cultured for 24 hours. Then, 50 µl of arachidonic acid was added, and the cells were further cultured for 30 minutes. 96 with IL-1β antibody attached
-A standard solution of IL-1β (0, 10.24, 25.6, 64, 160, 400 pg) was added to the wells of the well plate.
After adding 50 μl / well, 50 μl of the above cell culture solution was added to the wells of the experimental group, and 50 μl of the biotinylated antibody reagent was added to all wells, and then 25 ° C.
For 30 minutes, followed by washing buffer solution (Tween 20).
It was washed three times with a 0.01 M phosphate buffer solution containing 0.05% by weight, pH 7.5). 100 .mu.l of the outreptoavidin-HRP conjugate was added to all wells and maintained at 25.degree. C. for 30 minutes, followed by washing three times again with the washing buffer solution, and 100 .mu.l of the enzyme substrate was immediately added.
After keeping the plate open for 30 minutes in a dark room at 0 ° C. and adding 100 μl of 0.18 M sulfuric acid, the absorbance was measured at 450 nm using a microplate reader to create a standard curve from the absorbance values of the standard solution. The interleukin production of the experimental group was calculated based on the above. The measurement results are shown in Table 29 below.

[0090]

[Table 29]

From the results shown in Table 29 above, E. coli L
As a result of examining the inhibitory effect of the ointment of the present invention on the production of IL-1β in human mononuclear leukocytes stimulated with PS, 0.01%
Example 2-3 containing a cow knee extract at a concentration of at least
5, 7, 9 and 11, the inhibitory effect was shown to increase as the concentration of the cow knee extract was increased, while Examples 2-2, 4 containing the Yuzuki bark extract were shown. ,
Nos. 6, 8, 10 and 12 were shown to have no IL-1β production inhibitory effect. Examples 2-13, 14, 15, 16, 17, and 18, which contain both cow knees and Yuzuki skin, also showed that the inhibitory effect on IL-1β production increased with cow knee concentrations.

Experimental Example 2-4: Mononuclear leukemia of the ointment of the present invention
Inhibition of sphere prostaglandin (PGE ₂ ) production
The effect of the ointment composition of the present invention on the production of prostaglandin, a periodontal disease-inducing substance, was tested by the following method. This test method antigens the production of PGE ₂ which lipopolysaccharide is a cell wall component of porphyromonasgingivalis a periodontal disease-causing bacteria in periodontal disease progression was induced by stimulating mononuclear leukocytes of a human - antibody immune diagnosis The experimental group ointment (Examples 2-1 to 2-18) and the comparative group toothpaste and ointment (Comparative Examples 2-1 to 2-18) were mixed with distilled water at 1:
Mix at a ratio of 2 to completely microbialize, and
After centrifugation for minutes and treating the supernatant, the inhibitory effect was compared and evaluated. The specific experimental method is as follows. 50 μl buffer solution (0.9% NaCl, 0. 0, 0, 1) was placed in blank wells of a 96-well plate to which goat anti-mouse IgG had been attached.
0.1% bovine serum albumin containing 0.5% Kathon.
1M phosphate buffer solution) and add 50μl to the standard wells.
l appropriate concentration (0, 2.5, 5, 10, 20, 40, 8
0,160,320 pg) of the standard solution of PGE ₂ and then the experimental group ointment prepared as described above (Example 2).
-1 to 2-18) and the comparative group ointment (Comparative Examples 2-1 to 2-)
18) was added to 50μl of the set well Comparative Example group and examples, all that the PGE ₂ binding peroxidase 50μl after adding the antibody 50μl against PGE ₂ to all wells excluding the blank wells except blank wells And cover with 96-well plate 2
After maintaining at 5 ° C. for 1 hour, washing buffer solution (0.05 T
Wash four times with phosphate buffer solution containing ween 20: pH 7.4, and at room temperature enzyme substrate (3,3 ', 5,5'-tetramethylbenzine / hydrogen peroxide dissolved in 20% dimethylformamide) ) Add 150 μl immediately and add at 25 ° C
And then add 100 μl of 1M sulfuric acid, measure the absorbance at 450 nm with a microplate reader, create a standard curve from the absorbance values of the standard solution, and determine the amount of prostaglandin produced in the experimental group from the standard curve. (Unit pg) was calculated. The measurement results are shown in Table 30 below.

[0093]

[Table 30]

From the results shown in Table 30 above, E. coli L
Results of testing the inhibitory effect of the ointment composition of the invention on PGE ₂ production of stimulated human mononuclear leukocytes by PS, the in 2-3,5,7,9 and 11 contains a Achyranthes extract While the inhibitory effect was shown to increase as the concentration of the cow knee extract increased, PGEs were obtained in Examples 2-2, 4, 6, 8, 10 and 12 containing the extract of Yuzuki bark. ₂ It was shown that there was no production suppression effect. Examples 2-13, 14, 1 containing both cow knees and Yuzhi bark
5, 16, 17 and 18 also showed that the PGE ₂ production inhibitory effect increased with the concentration of cow knees.

Experimental Example 2-5: Gingival fiber of ointment of the present invention
Effect of Tooth Cells on Collagen Protein Production The effect of the ointment composition of the present invention on the production of collagen proteins in gingival fibroblasts was measured by the following method.
Pre-cultured gingival fibroblast cells in 24-well plate
0 ⁶ cell / day after well become as 10% FBS was DMEM medium 1ml being contained and cultured for one day dispensed into each well, after 24 hours of incubation with alternate existing medium with fresh medium After washing the bottom wash layer with HBSS buffer, serum and proline-free M
Immediately after adding 0.8 ml of the EM medium, the experimental group ointment (Examples 2-1 to 2-18) and the comparative group ointment (Comparative Example 2)
-1 to 2-18) were diluted with DMEM medium at a ratio of 1: 2, completely homogenized, and 100 μl of the supernatant obtained by centrifugation at 5000 g for 10 minutes was treated. Immediately thereafter, ¹⁴ C-proline (10 μCi) was used. Cells were cultured in a culture solution containing 100 μl. After 24 hours, the collagen protein was measured. First, in order to measure the total amount of extracellular protein synthesized, the culture solution in each well was placed in a dialysis tube sealed on one side, and the other was sealed on a cold buffer (Tris-HCL 0.05 mol / L, NaCl
After dialysis with 0.2 mol / L, CaCl ₂ 0.05 mol / L, phenylmethylsulfonyl fluoride 0.3 mN) for 24 hours, take 100 μl of each, place in a counting vial, add 10 ml of scintillation cocktail, and use LSC. 1
The radioactivity was measured for a minute. 0.1N was added to each well from which the cell culture solution was removed to measure the amount of total intracellular protein synthesis.
NaOH and phenylmethylsulfonyl fluoride
After adding 3 mN each and maintaining at 60 ° C. for 30 minutes to break the cell membrane, the cell bacterial solution was placed in a dialysis tube, one of which was sealed, the other was sealed, and a cold buffer solution (Tris-HCL 0.0
5 mol / L, NaCl 0.2 mol / L, CaCl ₂ 0.05 mol / L,
24 with phenylmethylsulfonyl fluoride (0.3mN)
After dialysis for 100 hours, 100 μl of each was placed in a counting vial, 10 ml of scintillation cocktail was added, and radioactivity was measured by LSC for 1 minute. To measure the total amount of intracellular and extracellular total collagen protein synthesis, 100 µl of each of the dialyzed cell culture solution and the cell homogenous solution was taken and placed in a 1.5 ml microtube, and a collagenase reaction buffer (0.05 M Tris-HCl) was added. , 1 mM CaCl ₂ , 0.03 mN
After adding 100 μl of phenylmethylsulfonyl fluoride) and collagenase enzyme (100 ppm), the mixture was maintained at 37 ° C. for 3 hours to completely degrade collagen, and then 50% trichloroacetic acid and 1% to remove undegraded proteins. After adding 500 μl of a solution containing tannic acid and precipitating at 4 ° C. for 30 minutes, 1000 × g
After centrifugation for 5 minutes at 100 ° C., take 100 μl of the supernatant, place in a counting vial, and add scintillation cocktail 1
0 ml was added and the radioactivity was measured by LSC for 1 minute. The measurement results are shown in Table 31 below.

[0096]

[Table 31]

From the results shown in Table 31 above, as a result of treating the ointment composition of the present invention with a cell culture solution containing ¹⁴ C-proline, it was found that Example 2-2 containing an extract of white peel was obtained. ,
In 4, 6, 8, 10 and 12, the effect of promoting collagen synthesis was shown to increase as the concentration of Japanese white peel increased, while Example 2 containing cow knee extract was used.
3, 5, 7, 9 and 11 showed no collagen synthesis promoting effect. Examples 2-13, 14, 15, 16, 17, and 18 containing both cow knees and Yuhzhi also showed that the collagen synthesis promoting effect increased with the concentration of Yuhzhi.

Experimental Example 2-6: Periodontal disease of the ointment of the present invention
Therapeutic effect (clinical experiment) The therapeutic effect of the ointment of the present invention on periodontal disease treatment was confirmed by the following clinical experiment method. After the test subjects were divided into the experimental group and the comparative group, the initial gingivitis index was measured by performing tooth polishing, and the experimental group and the comparative group ointment were supplied and 200 mg at a time, one day. Was used three times after eating, and one week and one month later, oral examination was performed to examine the gingivitis index. Here, the gingivitis index in each case was determined by inserting a gingival probe into the gingival row and continuously probing around each tooth in a state where no force was applied. Scores were recorded based on the criteria shown in Table 32, and the results were obtained. The measurement results are shown in Table 33 below.

[0099]

[Table 32]

[0100]

[Table 33]

From the results shown in Table 33 above, it is possible to suppress the production of IL-1β and prostaglandin which induce periodontal disease and the enzyme activity of collagenase which suppresses superoxide and degrades periodontal tissue. The treatment effect on periodontal disease of the ointment of the present invention (Examples 2-1 to 2-12) containing a cabbage extract or a skin extract of Yuhi, which can promote collagen synthesis and suppress the enzyme activity of collagenase, From one week to one month, the efficacy was shown to be superior to that of the comparative examples (2-11 to 2-18) containing no beef knee or Japanese white peel, and the gingivitis index of the comparative example was one month. On the other hand, it gradually increases with time, but the use of the ointment developed according to the present invention suppresses the occurrence of periodontal disease and shows that the gingivitis index decreases significantly with time. Cage, in Examples 2-13,14,15,16,17 and 18 contain both Achyranthes and Nireshiro skin has been shown as having the increased effect on periodontal disease suppression.

[0102]

The dentifrice composition and the ointment composition according to the present invention contain superoxide, prostaglandin (PGE ₂ ) and interleukin-1β which are periodontal disease-inducing substances.
In addition to suppressing the production of collagen, it is possible to effectively treat periodontal disease by suppressing the activity of collagenase enzyme, which degrades collagen protein, which is a substrate of periodontal tissue, and at the same time, promoting collagen protein synthesis. it can.

Continuing on the front page (72) Inventor Soo Seok Kyung, Republic of Korea, Dejong, Dedokgu, Yongchukdong, 102, Dugong apartment, 107-403 1505

Claims (18)

[Claims] 1. An oral composition comprising a cow knee extract, a Japanese white peel extract or a mixture thereof. 2. The composition according to claim 1, wherein the extraction solvent is water, methanol, ethanol, propanol or butanol. 3. The composition according to claim 1, wherein the oral composition is a dentifrice composition. 4. The composition according to claim 3, comprising 0.001 to 5% by weight of each of the cow knee extract and the Japanese white peel extract. 5. The composition according to claim 3, comprising 0.01 to 3% by weight of each of the cow knee extract and the Japanese white peel extract. 6. The composition according to claim 3, wherein the cow knee extract and the Yu-bare extract are mixed together and contain 0.002 to 10% by weight. 7. A polishing agent containing calcium monohydrogen phosphate, precipitated silica or calcium carbonate in an amount of 20 to 60, respectively.
The composition according to claim 3, wherein the composition contains 0.1% by weight. 8. Further, sodium fluoride and / or sodium monofluorophosphate are each used as a fluorine compound in an amount of 0.1 to 0.1%.
The composition according to claim 3, wherein the composition contains 1 to 2% by weight. 9. The composition according to claim 1, wherein the oral composition is an ointment for treating periodontal disease. 10. The composition according to claim 9, wherein the cow knee extract is contained in a ratio of 0.001 to 5% by weight based on the total weight of the composition. 11. The composition according to claim 9, comprising the extract of Yuzuki skin in a ratio of 0.001 to 5% by weight based on the total weight of the composition. 12. The composition according to claim 9, wherein the composition comprises 0.002 to 10% by weight of a mixture of beef knee extract and Yuzuki bark based on the total weight of the composition. . 13. A surfactant, a solubilizer, a wetting agent,
10. A method according to claim 9, wherein one or more of a drug transmitting agent, a buffer, a preservative, a sweetener and a flavor are blended.
The composition according to any one of claims 11 to 11. 14. The composition according to claim 13, wherein a polyoxyethylene-polyoxypropylene copolymer is blended as a surfactant in a ratio of 5 to 30% by weight based on the total weight of the composition. 15. The composition according to claim 13, wherein a lower alcohol solvent is incorporated as a solubilizer in a ratio of 1 to 20% by weight based on the total weight of the composition. 16. A humectant selected from glycerin, sorbitol solution, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600 and polyethylene glycol 1000, propylene glycol, porosamer and monoglyceride.
14. The composition according to claim 13, wherein one or more components are incorporated in a ratio of 5 to 40% by weight based on the total weight of the composition. 17. The composition according to claim 13, wherein gelatin or pectin or a mixture thereof is incorporated as a drug delivery agent in a ratio of 1 to 30% by weight based on the total weight of the composition. 18. A monobasic sodium phosphate as a buffer,
One or more components selected from dibasic sodium phosphate, tribasic sodium phosphate, citric acid and sodium citrate, phosphoric acid, hydrochloric acid, sodium hydroxide, sodium pyrophosphate and pyrophosphate are blended. A composition whose pH has been adjusted to 5.0 to 8.0.