WO2014081124A1 - Composition for treating or alleviating dental pulp diseases, containing butein as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating dental pulp diseases, containing butein as an active ingredient, and a food composition for preventing or alleviating dental pulp diseases, containing butein as an active ingredient. The dental pulp disease can be any one selected from the group consisting of pulp hyperemia, pulpitis, pulp degeneration, pulpal necrosis, pulp gangrene and a combination thereof. In addition, the food composition for preventing or alleviating dental pulp diseases can be a dietary supplement. The butein shows remarkable cytoprotective effects in dental pulp cells and excellent inhibition of reactive oxygen species, and thus can be useful in a pharmaceutical composition or a food composition for treating, preventing or alleviating diseases associated with the dental pulp.

Description

Treatment for or improvement of pulp disease comprising butane as an active ingredient

The present invention relates to a composition for treating or improving pulp disease comprising butein as an active ingredient.

The dental pulp is a soft connective tissue that fills the pulp cavity inside the tooth and is abundantly distributed in nerves and blood vessels. The nerves of the pulp are located in the ivory cells adjacent to the dentin inner surface of the tooth at the outermost layer of the pulp. The branch reaches the surface of dentin.

Heme Oxygenase-1 (HO-1) is responsible for inducing the oxidation of ham to break down into biliverdin, free iron and carbon monoxide. As an enzyme, chlorophyll is converted to bilirubin by biliverdin reductase, which acts as an ROS scavenger and shows antioxidant effects. In addition, carbon monoxide has an anti-inflammatory effect, free iron is involved in ferritin synthesis and exhibits a cytoprotective effect.

Thus, the pathway of hematin oxidase-1 has a great effect on both the mechanism of repairing damaged cells in human periodontal ligament cells and the mechanisms involved in adapting the cells to stress situations.

The pulp tissue is easily inflammatory response or tissue damage due to the surrounding environment, it is easy to cause damage to the surrounding tissue such as dentinogenesis, and treatment is required, and prevention is required before the tissue damage. Publication No. 2011-0017599).

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of pulp disease comprising butane as an active ingredient.

Another object of the present invention to provide a food composition for preventing or improving pulp disease comprising butane as an active ingredient and a health functional food comprising the food composition as an active ingredient.

In order to achieve the above object, the pharmaceutical composition for preventing or treating pulp disease according to an embodiment of the present invention includes butane as an active ingredient. The pulp disease may be any one selected from the group consisting of pulp red blood, pulp salt, pulp degeneration, pulp necrosis, pulp necrosis, and a combination thereof.

A food composition for preventing or improving pulp disease according to another embodiment of the present invention includes butane as an active ingredient.

Functional health food for preventing or improving pulp disease according to another embodiment of the present invention includes the food composition as an active ingredient.

In this aspect, the present invention provides a pharmaceutical composition for preventing or treating pulp disease comprising butane as an active ingredient. The pulp disease may be any one selected from the group consisting of pulp red blood, pulp salt, pulp degeneration, pulp necrosis, pulp necrosis, periodontitis and gingivitis.

In addition, the present invention provides a food composition for preventing or improving pulp disease comprising butane as an active ingredient.

In addition, the present invention may be a health functional food for preventing or improving pulp disease comprising a food composition as an active ingredient.

The present invention also relates to a method for separating butane excellent in preventing or improving pulp disease from lacquer trees.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention completed the present invention by confirming that there is an excellent pulp cell protection effect on the butane isolated from the lacquer tree extract while researching a natural derivative having a therapeutic or prophylactic effect.

The pharmaceutical composition for preventing or treating pulp disease according to an embodiment of the present invention includes butane as an active ingredient.

The butane includes a compound having a structure of tetrahydroxychalcone as a flavonoid-based material, and the tetrahydrochalcone refers to a form in which four hydroxy groups are bonded to the chalcone structure.

The tetrahydroxychalcone is 2 ', 3,4,4'-tetrahydroxychalcone (2', 3,4,4'-Tetrahydroxychalcone), 2 ', 4', 3,4-tetrahydroxychalcone (2 ', 4', 3,4-Tetrahydroxychalcone), 3,4,2 ', 4'-tetrahydroxychalcone (3,4,2', 4'-Tetrahydroxychalcone), 2 ', 4', 3,4- It may be any one selected from the group consisting of tetrahydroxychalcone (2 ', 4', 3, 4-Tetrahydroxychalcone) and combinations thereof.

The butane may be isolated from the sumac extract.

The sumac ( Rhus verniciflua ) belongs to the sumacaceae , and the flower is herbaceous , light greenish yellow flowers bloom in May, and the octopus-like nucleus is deleted in October. The lacquer contains a substance called urushiol (urushiol) to induce lacquer, lacquer is used as a herbal medicine, such as schizophrenia, menopause or heart pain.

The sumac extract may be extracted by a conventional extraction method in the field to which the present invention belongs, and the butane may be separated by a conventional separation method in the field to which the present invention belongs.

The pulp disease includes all lesions occurring in the pulp tissue of the tooth, and specifically, the pulp disease may be any one selected from the group consisting of pulp congestion, pulpitis, pulp degeneration, pulp necrosis, pulp necrosis, periodontitis and gingivitis. .

The pulp can be caused by the expansion of the blood vessels of the pulp when a physical, chemical or bacterial stimulus is applied to the pulp, and the pulp inflammation means that the pulp is inflamed by the stimulus applied to the pulp.

The pulp necrosis refers to a disease in which pulp tissue necrosis when a circulatory disorder occurs due to inflammation, etc., and a state in which the necrotic pulp tissue decays or decomposes is called pulp necrosis.

The dimensional change means that degenerative or atrophic changes occur due to aging even if the difference is not changed or stimulated. The pulp is enclosed by hard dentin so that the circulatory disorder is easily caused when inflammation or the like occurs, and the disease may be more easily worsened. Therefore, it is further required to prevent or initially treat the pulp disease.

The butane may treat or ameliorate pulp disease by inhibiting cellular damage and reactive oxygen species in pulp.

Specifically, when the expression of hematine oxidase-1 (HO-1) is increased by the butane, the oxidation of hematin (heme) is induced by the hematin oxidase-1, thereby biliverdin ( It was experimentally confirmed that the cleavage into biliverdin, free iron and carbon nomoxide inhibits the damage of pulp cells to external stimuli by the degradation products.

Heme Oxygenase-1 (HO-1) induces the oxidation of ham (heme) involved in cleavage into biliverdin (biliverdin), free iron (carbon) and carbon monoxide (carbon monoxide) As an enzyme, chlorophyll is converted into bilirubin by biliverdin reductase, which acts as an ROS scavenger and shows an antioxidant effect. In addition, carbon monoxide has an anti-inflammatory effect, free iron is involved in ferritin synthesis, and has a cytoprotective effect.

The pulp disease prevention or treatment composition comprising the butane as an active ingredient may include 0.001% to 99.9% or 0.1% to 99% or 1% to 20% by weight of the butane relative to the total weight However, the present invention is not limited thereto, and the amount of the butane may be appropriately adjusted to a pharmaceutically effective amount or a preferred amount according to the use mode and method of use of the composition.

The composition for preventing or treating pulp disease of the present invention may include the above-described butane as an active ingredient, and may further include a pharmaceutically acceptable carrier, excipient, or diluent according to the formulation, method of use, and purpose of use. Can be.

The pulp disease prevention or treatment composition may be directly applied to animals including humans.

The animal is a biological group corresponding to a plant, and the organic material is mainly consumed as nutrients, and the digestive organ, the excretory organ, and the respiratory organ are differentiated. Preferably, the animal may be a mammal, more preferably a human.

The butane may be used alone as an active ingredient in a composition for preventing or treating pulp disease, and may further include other pharmacologically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients.

In detail, when the butane is used as a medicine or for medicinal or pharmaceutical use, the butane may be mixed with a pharmaceutically acceptable carrier or excipient or diluted with a diluent according to a conventional method. More specifically, in addition to the active ingredient, nutrients, vitamins, electrolytes, flavoring agents, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal deposits, pH adjusting agents, stabilizers, preservatives , Glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.

In the present invention, the expression `` pharmaceutically acceptable '' means physiologically acceptable and when administered to an animal, preferably a human, usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or the like. do. The pharmaceutically effective amount may be appropriately changed depending on the disease and its severity, the patient's age, weight, health condition, sex, route of administration and duration of treatment.

Examples of such pharmaceutically acceptable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, One or more selected from the group consisting of dextrin, calcium carbonate, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline, but is not limited thereto, and any conventional carrier, excipient or diluent may be used.

The components may be added independently or in combination with the butane, the active ingredient.

In addition, the composition for preventing or treating pulp disease is a conventional fillers, extenders, binders, disintegrating agents, anticoagulants, lubricants, wetting agents, pH adjusting agents, nutrients, vitamins, electrolytes, alginic acid and salts thereof, pectic acid and salts thereof, protection Sex colloids, glycerin, fragrances, emulsifiers or preservatives; The components may be added independently or in combination to the composition for preventing or treating pulp disease.

In addition, the composition of the present invention may further include a substance used for preventing or treating pulp disease in addition to the active ingredient. The substance used for the prevention or treatment of the known pulp disease is preferably added in the range of 0.1 to 20 parts by weight or 100 parts by weight of the plant extract, which is the active ingredient, per 100 parts by weight of the pulp disease prevention or treatment composition. It may be added in the range of 5 parts by weight to 200 parts by weight, respectively, but is not limited thereto.

When the composition for preventing or treating pulp disease is used as a medicine, the method of administration may be oral or parenteral, for example, oral, transdermal, subcutaneous, intravenous or intramuscular.

In addition, the formulation of the composition for preventing or treating pulp diseases may vary depending on the method of use, and is well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated using the method. Examples of such formulations include granules, powders, syrups, solutions, suspensions, tablets, injections, spirits, cataplasma, capsals, soft gelatin capsules or hard gelatin capsules.

For the formulation, excipients, for example, conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, sweeteners, fragrances or preservatives may be further included.

Generally, solid preparations for oral administration include tablets, pills, soft or hard capsules (CAPSULES), pills (PILLS), powders (POWDERS) and granules (GRANULES), one such preparation. The above excipients may be prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used.

In addition, liquid preparations for oral administration include suspensions (SUSTESIONS), liquid solutions, emulsions (EMULSIONS) and syrups (SYRUPS), and various excipients, in addition to commonly used simple diluents such as water or liquid paraffin, Humectants, sweeteners, fragrances, preservatives and the like.

Furthermore, the compositions of the present invention may be formulated using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). Can be.

The dosage of the composition can be appropriately determined by those skilled in the art in consideration of the method of administration, the age, sex and weight of the recipient, the severity of the disease, the condition, the absorption of the active ingredient in the body, the inactivation rate and the drug used in combination. For example, based on the daily active ingredient of the composition, the plant extract is 0.0001 mg / kg (body weight) to 500 mg / kg (body weight), 0.01 mg / kg (body weight) to 400 mg / kg (body weight) Or 1 mg / kg (body weight) to 300 mg / kg body weight, and may be administered once or several times a day. However, the above dosage does not limit the scope of the present invention in any aspect.

Composition according to another embodiment of the present invention is a composition for preventing or improving pulp disease comprising butane as an active ingredient.

The pulp disease prevention or improvement composition is preferably a food composition for preventing or improving pulp disease, the food may be a health functional food for preventing or improving pulp disease.

The active ingredient and the pulp disease is the same as described in the pharmaceutical composition.

The food means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain process. It is meant to include all beverages, food additives and beverage additives.

The food may be, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, and the like. In addition, the food in the present invention includes special nutritional products (e.g., crude oil, young, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasonings ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruit and vegetable beverages, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup).

The food, health functional food, beverages, food additives and beverage additives may be prepared by conventional manufacturing methods.

In the present invention, a health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition which has added value to the food by using physical, biochemical, biotechnological techniques, etc. It means the food processed and designed to fully express the function of the gymnastics for recovery.

The dietary supplement may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of dietary supplements.

In the present invention, the drink refers to a generic term of drinking to quench thirst or enjoy the taste, it is intended to include a health functional beverage.

The beverage is not particularly limited to other ingredients other than including the butane as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .

Examples of the natural carbohydrates may be monosaccharides such as glucose or fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin, cyclodextrin, or sugar alcohols such as xylitol, sorbitol, and erythritol. . The flavoring agent may be a natural flavoring agent such as stevia extract such as taumartin or rebaudioside A or glycyrgin, or a synthetic flavoring agent such as saccharin, aspartame or the like.

The amount of the natural carbohydrate added may generally be about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention may further contain a pulp for the production of natural fruit juices, fruit juice drinks, vegetable drinks.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of the butane of the present invention.

The health functional beverage is a biological defense rhythm control, disease prevention and recovery of a beverage group or beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. It refers to a beverage that is designed and processed to fully express the gymnastics function in relation to the living body.

The health functional beverage is not particularly limited to other ingredients except the butane of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

Examples of the natural carbohydrates may be monosaccharides such as glucose or fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin, cyclodextrin, or sugar alcohols such as xylitol, sorbitol, and erythritol. . The flavoring agent may be a natural flavoring agent such as stevia extract such as taumartin or rebaudioside A or glycyrgin, or a synthetic flavoring agent such as saccharin, aspartame or the like.

The amount of the natural carbohydrate added may generally be about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the food composition of the present invention.

In addition, the composition of the present invention may further contain a pulp for the production of natural fruit juices, fruit juice drinks, vegetable drinks.

In addition, in the food composition for the purpose of preventing or improving pulp disease, the amount of butane may be included as 0.01 to 15% by weight of the total food weight, the beverage composition is 0.02 g to 100 ml based on 100 ml 5 g, preferably 0.3 g to 1 g.

The butane was confirmed through in vitro experiments and animal experiments, it was confirmed that the effect of inhibiting pulp cell damage by increasing the expression and activity of hematine oxidase-1.

In the food composition for preventing or improving pulp disease comprising the butane as an active ingredient, the amount of the butane may be 0.01 wt% to 15 wt% of the total food weight. In addition, based on 100 ml of the butane beverage composition may be included in a ratio of 0.02 g to 5 g or 0.3 g to 1 g.

The present invention also relates to a method for separating butane excellent in preventing or improving pulp disease from lacquer trees.

According to another aspect, the present invention relates to a method for preparing a pulp disease prevention or improvement composition comprising butane as an active ingredient.

The method of separating the butane may include an extraction step, a fractionation step, and a compound separation step.

Specifically, the method of separating the butane is the lacquer cutting step of washing, drying and cutting the lacquer; Preparing a lacquer methanol extract by adding and extracting methanol to the shredded lacquer; N-hexane, ethyl acetate, and water are sequentially added to the lacquer methanol extract, and fractionated by solvent fractionation to prepare an ethyl acetate fraction of the lacquer methanol extract; Preparing a silica gel column chromatography fraction by fractionating the ethyl acetate fraction of the lacquer methanol extract using a mixed solvent of hexane and acetone mixed at a ratio of 3: 1 (hexane: acetone) and silica gel column chromatography; And performing reverse phase medium pressure liquid chromatography on the silica gel column chromatography fraction using a 50% aqueous methanol solution to prepare a reverse phase medium pressure liquid chromatography fraction.

The method of separating the butane may be carried out by washing and drying the lacquer, and then cutting it.

The preparing of the lacquer methanol extract may be performed by adding methanol to the shredded lacquer and extracting it while heating to 50 ° C. to 100 ° C., preferably 70 ° C. to 90 ° C.

The step of preparing the ethyl acetate fraction of the methanol extract of the lacquer tree may be carried out by fractionation by solvent fractionation while sequentially adding n-hexane, ethyl acetate and water to the lacquer methanol extract.

The preparing of the silica gel column chromatography fractions is performed by performing silica gel column chromatography using a mixture of hexane and acetone mixed with ethyl acetate fraction of the lacquer methanol extract in a volume ratio of 3: 1 (hexane: acetone). You can proceed in this way.

The preparing of the reverse phase medium pressure liquid chromatography fraction may be performed by performing reverse phase medium pressure liquid chromatography using the obtained silica gel column chromatography fraction as a solvent in a 50% aqueous methanol solution.

Butane prepared by the method of the present invention or the fraction containing the butane is excellent in the effect of inhibiting pulp cell damage by increasing the expression and activity of hematine oxidase-1, pharmaceutical for the treatment or prevention of pulp disease It can be used as an active ingredient of the composition or food composition for improving or preventing pulp disease.

Butane obtained by the production method of the present invention increases the expression and activity of hematine oxidase-1 and has an effect of protecting pulp cells from external stimuli through degradation products by the enzymes, thereby preventing inflammation or tissue damage. It can be used to prevent or treat pulp disease by preventing damage to pulp cells that can easily occur and cause tissue damage such as dysentery, which is the surrounding tissue, and, unlike artificially synthesized compounds, side effects are not a problem and safety Since it is also recognized, it can be widely used not only in pharmaceutical compositions but also in food compositions, including dietary supplements.

1 is a flow chart illustrating a method for separating butane from lacquer tree according to an embodiment of the present invention.

2A and 2B are data relating to a material separated from the lacquer tree according to an embodiment of the present invention, and FIG. 2A shows NMR data of a material separated from the lacquer tree, and FIG. 2B shows a chemical of the material separated from the lacquer tree. It represents the structure.

Figure 3 is a graph showing the cell survival rate for confirming the cytotoxicity of butane in human dental pulp cells (HDP, human dental pulp cells) according to an embodiment of the present invention, the vertical axis of the graph is a control of the survival rate of the cell ( Relative to-), the abscissa represents the throughput of butane and-represents the control without any treatment.

4A to 4D are graphs showing the human-derived pulp cell protection effect of butane according to an embodiment of the present invention, and FIGS. 4A and 4C are human-derived pulp cells treated with hydrogen peroxide (H ₂ O ₂ ). 4b and 4d are graphs showing the effect of ROS scavenging, and the vertical axis of the graphs of FIGS. 4a and 4c represents the survival rate of the pulp cells, and the vertical axis of the 4b and 4d graphs is the fluorescence intensity. It is shown as a relative value to the control, the horizontal axis of each graph indicates whether or not the treatment of a particular substance.

Figures 5a to 5d shows the activity of hematin oxidase-1 (HO-1) in human-derived pulp cells (HDP) according to an embodiment of the present invention, Figures 5a and 5c is hematin oxidase- 1 is a photograph confirming the expression, Figure 5b and 5d is a graph showing the enzyme activity of hematin oxidase-1, the vertical axis of Figure 5b and 5d shows the activity of hematin oxidase-1, The horizontal axis of FIG. 5B represents the concentration of the butanes treated, and the horizontal axis of FIG. 5D represents the treatment time of the butanes.

6a to 6c are diagrams showing the relationship between butane and mitogen activated protein kinase (MAPK) activity according to an embodiment of the present invention, Figure 6a is mitogen activity according to the butane treatment time Figure 6b is a picture showing the active pathway of protein kinase, Figure 6b is a picture showing the activation of hematin oxidase-1 according to the treatment of butane and the presence of mitogen-activated protein kinase expression inhibitor, Figure 6c is a treatment of butane And it is a graph showing the survival rate of human-derived pulp cells in the presence or absence of mitogen-activated protein kinase expression inhibitor, the vertical axis of Figure 6c represents the survival rate of the pulp cells, the horizontal axis represents the treatment of each component, + Means the case of processing and-means the case of not processing.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

Preparation Example: Preparation of Plant Extract and Separation of Butane

After washing, drying and cutting 1 kg of Rhus verniciflua Stokes purchased from Kyungdong Market, 1 L of methanol was added thereto, and the resultant was extracted by heating at 80 ° C. for 3 hours, and the solvent was removed to obtain 37.78 g of Methanol extract of Rhus verniciflua Stokes. After dissolving the methanol extract in water, n-hexane, ethyl acetate and water were added to prepare each fraction by a solution-solution partition, and 8.71 g of ethyl acetate fraction was obtained in the prepared fraction.

A mixture of nucleic acid and acetone obtained by mixing the ethyl acetate fraction obtained in the ratio of 3: 1 (hexane: acetone) on the basis of the volume ratio and silica gel column chromatography (silicagel CC, 6.5 × 60 cm, 200 to 300 mesh, Merck, USA) to obtain four fractions. Each fraction was divided by TLC (Thin Layer Chromatography) analysis. As a result, a spot having an Rf value similar to butane was found in the second fraction, and the experiment was conducted with the second fraction. Of the four fractions, the second fraction (660 mg) was subjected to reverse phase medium pressure liquid chromatography (MPLC, ODS-S-50 B, 120 kPa, 50 µm) using a 50% methanol aqueous solution solvent using a gradient gradient elution method. Thus, three fractions were obtained. Three fractions were divided by TLC (Thin Layer Chromatography) analysis. In order to identify the substance of the first fraction (28.8 mg) of the three fractions, ¹ H-NMR was performed, the results are shown in Figure 2a. Results summarized from the graph identified in FIG. 2A are as follows.

¹ H NMR (500 MHz, Acetone-d6) δ: 8.14 (1H, d, J = 8.25 Hz, H-6 ′), 7.79 (1H, J = 15.1 Hz, H-β), 7.71 (d, 1H, J = 15.5 Hz, H-α), 7.38 (1 H, brs, H-2), 7.23 (1 H, d, J = 7.8 Hz, H-6), 6.92 (1 H, d, J = 8.25 Hz, H- 5), 6.49 (1H, d, J = 8.25 Hz, H-5 '), 6.38 (1H, brs, H-3').

The structure of the material identified from the results summarized by the graph is shown in FIG. 2B. The structure shown in Fig. 2b is compared with Chen's YP et al , Journal of Chinese Pharmaceutical Sciences 17, p82-p86 (2008), and the material obtained by the fraction is identical to that of the butane. It was confirmed that the material was butane.

Experimental Example 1. Measurement of cytotoxicity in human-derived pulp cells

Confirmation of cell viability for measuring cytotoxicity of the cell line of butane obtained from the lacquer tree in the preparation example was performed by applying the method disclosed in the literature (Mosmann T, Journal of Immunological Methods 65, p55-p63). (1983)). Specifically, the experimental method is as follows.

First, 10% heat-inactivated FBS and human-derived pulp cells (HDP, sold at Wonkwang University Dental College, 2 × 10 ⁴ cells / well) were used to measure the cytotoxicity of the butanes obtained in the above preparation. Dispensed in DMEM medium containing penicillin G (100 IU / ml, Gibco co, USA) and streptomycin (100 μg / ml, Gibco co, USA) and using a 5% CO ₂ incubator (Sanyo, MCO175) 37 Incubated for 24 hours at ℃. Thereafter, the butanes were treated by 1, 2, 5, 10, 20 and 30 μM for each concentration, and further incubated for 24 hours in a 5% CO ₂ incubator. After the incubation, cell viability was measured using the MTT method. After each experiment was repeated three times, the average value of the measurement results is shown in Figure 3 as a relative value for the control group did not perform any treatment.

As shown in FIG. 3, when the concentration of butane is 30 μM, the cell viability is decreased. Hereinafter, an experiment for confirming the effect of the pulp cell protection of butane on the concentration of butane is less than 30 μM, Specifically, it was performed at a concentration of 20 μM or less.

Experimental Example 2. Confirmation of the effect of butane on pulp cell protection

In order to confirm the protective effect of butane obtained from the preparation example on human-derived pulp cells (HDP), 2.5, 5, 10 and 20 μM of the butane obtained in the preparation example was added to the human-derived pulp cells of Experimental Example 1. After each pretreatment with 1 mM hydrogen peroxide (hydrogen peroxide) treated to induce toxicity, and incubated in a 5% CO ₂ incubator for 12 hours. After the culture, the cell protective effect on human-derived pulp cells was confirmed by MTT method. In the experiment, 20 μM of CoPP, a hematine oxidase-1 (Heme oxygenase-1, HO-1) expression inducing agent, was used as a positive control group (FIGS. 4A and 4B). As a comparative example, 100 μM of SnPP (Tin protophorphyrin, porphyrin products) was used (FIGS. 4C and 4D).

In relation to the cellular protective effect on the human-derived pulp cells, the results of measuring cell viability using the MTT method are shown in FIGS. 4A and 4C, and the degree of inhibition of reactive oxygen species (ROS) production was measured using fluorescence assay. One result is shown in FIGS. 4B and 4D.

Cell viability measurement method using the MTT method was performed by the following method. First, human-derived pulp cells were incubated for 24 hours at a concentration of 2 × 10 ⁴ cells / well in a 96 well plate, and treated with 2.5, 5, 10, and 20 μM of butane, followed by 1 mM hydrogen peroxide (H ₂ O ₂ ). Was treated for 12 hours to induce cytotoxicity. After inducing the cytotoxicity, MTT (Sigma, USA) reagent was added at a concentration of 0.5 mg / ml, and further incubated for 4 hours in a 5% CO ₂ incubator. After the incubation, the supernatant was discarded and 150 μl of DMSO (Junsei, 35535-0350) was added and shaken for 30 minutes, and then the absorbance was measured at 570 nm using an ELISA reader (Bio-Rad, Microplate reader model 680). Was performed. The experiment was performed three times each repeated experiment, the average value of the experimental results are shown in Figure 4a and 4c as a cell protection rate for the control group.

The measuring method of the reactive oxygen species (ROS) was performed by the following method. First, the measurement of the generation of reactive oxygen species was carried out by washing the cultured human-derived pulp cells with PBS, using a Hank's balanced salt solution containing 10 μM of 2 ', 7'-dichlofluorescein diacetate (DCFDA, Fluka, Netherlands). Added and reacted in the dark for 30 minutes. After the reaction, the fluorescence of the cells (Spectramax Gemini XS, Molecular Devices, Sunnyvale, U.S.A.) was measured under the conditions of excitation wavelengh of 490 nm and emission wavelengh of 525 nm. The experiment was repeated three times each, the average value of the experimental results are shown in Figure 4b and 4d compared with the control group without any sample.

As shown in FIG. 4A and FIG. 4C, it was confirmed that the viability of the cells was increased when the butane was treated, and in particular, when the 20 μM was treated, the cell survival was increased to about the same level as the positive control group. In addition, as shown in Figures 4b and 4d, the rate of inhibition of reactive oxygen species generation or the removal of reactive oxygen species was also confirmed to increase when treated with butane, especially when treated with butane at 20 μM to about the same degree as the control group It has been shown to inhibit the production of reactive oxygen species. On the other hand, when the treatment with SnPP, the inhibitor of hematine oxidase-1 alone, did not affect the cells, the treatment with hematine oxidase-1 alone did not affect the treatment with hematine oxidase-1 alone. In addition, the cell viability and inhibition of reactive oxygen species production were reduced, and the increase in cell survival rate and reactive oxygen species production inhibition by the butane treatment was predicted to be due to hematin oxidase-1.

In order to confirm the effectiveness of the human-derived pulp cell cytoprotective effect and reactive oxygen species removal effect of butane identified from the above measurement results, the inventors of the present invention confirmed a similar effect, specifically, isolated from the extract of Caesalpinia sappan Efficacy was compared with one sapanchalcone (C ₁₆ H ₁₄ O ₅ ).

First, the EC ₅₀ value for the cellular protective effect on the human-derived pulp cells was measured based on the measured results, and the results were separated from Sapanchalcon (C ₁₆ H ₁₄ O ₅ ) isolated from the extract of Caesalpinia sappan . ) Was measured by comparing the EC ₅₀ value for the cell protective effect on human-derived pulp cells through the same experiment, and the results are shown in Table 1 below.

Table 1

	EC 50 (μM)
Butane	7.89 ± 2.34
Sappanchalcone	19.32 ± 2.71

As shown in Table 1, butane was confirmed to have the same effect only with a content of about 40% compared to Sapanchalcone, and confirmed to have a cell protection effect on human-derived pulp cells significantly superior to Sapanchalcone. It became.

In addition, based on the measured results, the IC ₅₀ value for the inhibition of reactive oxygen species production or the removal rate of reactive oxygen species in the human-derived pulp cells was measured. In addition, by performing the same experiment as described above using Sapanchalcon (C ₁₆ H ₁₄ O ₅ ) isolated from the extract of Caesalpinia sappan , in the rate of inhibition of the generation of reactive oxygen species or removal of reactive oxygen species in the human-derived pulp cells for IC ₅₀ values were determined, to the IC ₅₀ value for the inhibition of reactive oxygen species generation or active oxygen removal rate and the measured butane derived from human of the swash plate chalkon cell dimensions are given in Table 2.

TABLE 2

	IC 50 (μM)
Butane	9.76 ± 1.08
Sappanchalcone	22.32 ± 3.42

As shown in Table 2, butane was confirmed to have the same effect with only about 43% of the content of sapanchalcone, and significantly inhibited the generation of reactive oxygen species in human-derived pulp cells compared to sapanchalcone or It was found to have an active oxygen free removal effect.

Experimental Example 3. Confirmation of the effect on the expression and activity of butane hematin oxidase-1 (HO-1)

In order to confirm the protective effect of the butane obtained from the preparation example in human-derived pulp cells, the expression level of hematine oxidase-1 (Heme oxygenase-1, HO-1) having anti-oxidative activity and cytoprotective activity was measured. To this end, experiments were carried out using Western blot analysis described in the literature (Li B et al , European Journal of Pharmacology 614, p58-p65 (2009)).

First, human-derived pulp cells were treated with butane at a concentration of 2.5, 5, 10, and 20 μM for 12 hours, and cobalt protophorphyrin (CoPP), which promotes the expression of hematine oxidase-1, was used as a positive control group. (FIG. 5A). Thereafter, the human-derived pulp cells were treated with butane at a concentration of 20 μM for 3, 6, 12, 18 and 24 hours (FIG. 5C).

Specifically, in order to confirm the effect of promoting the expression of hemate oxidase-1 according to the content of butane and the treatment time, an experimental method of performing a western blot analysis is as follows.

First, the human-derived pulp cells were incubated for 24 hours at a concentration of 3 × 10 ⁶ cells / ml in a 60 mm dish, followed by concentration of 20 μM of cobalt protophorphyrin (CoPP) or butane, a hematin oxidase-1 inducer. Treated and incubated for 12 hours. After the incubation, RIPA (89900, Thermo) buffer was added and centrifuged for 25 minutes at 4 ℃ and 14,000 X g, the supernatant was separated. Protein quantification in the isolated supernatant was performed using a BSA protein experiment kit (Pierce Biotechnology).

Specifically, the supernatant was electrophoresed for 1 hour using 12.5% SDS-polyacrylamide gel, and then transferred from the polyacrylamide gel using a nitrocellulose membrane (NC membrane). It was. The transcribed nitrocellulose membrane was stopped for 1 hour in a fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline) containing 5% nonfat milk. After stopping the reaction, the membrane was washed three times every 10 minutes with PBST (PBS, 0.1% Tween 20), and then the antibody (Ab) of hematin oxidase-1 (374087, Calbiochem) was 1: 1000. Diluted and the reaction was carried out for 1 hour. After the reaction, the plate was washed three times with PBST once every 10 minutes, and the secondary antibody (Anti-mouse IgG, NA934, Amersham Pharmacia Biotech) was diluted 1: 1000 and reacted for 1 hour.

After the reaction, the solution was washed three times with PBST once every 10 minutes. Then, ECL (Amersham Pharmacia Biotech) solution was mixed well in a 1: 1 ratio and poured onto nitrocellulose membrane (NC membrane) to emit light. It developed after photosensitive to a film. Actin content was measured in the same manner using an antibody against Actin (SC1616, Santacruz biotechnology, USA).

In addition, in order to confirm the protective effect of the butane obtained in the preparation in human-derived pulp cells, the activity of heme oxygenase-1 (Heme oxygenase-1, HO-1) having antioxidant and cytoprotective activity To determine, hematine oxidase-1 enzyme activity was measured by the amount of biliverdin increased due to the hematin oxidase-1 expression as described in the literature (Tenhunen R et al , The Journal of laboratory and clinical medicine, 75, p 410-421 (1970).

First, in order to confirm the activity of hematin oxidase-1 enzyme, an enzyme showing antioxidant and cytoprotective effects, butane was treated for 12 hours at concentrations of 2.5, 5, 10 and 20 μM on human-derived pulp cells. Cobalt protophorphyrin (CoPP), which promotes expression of hematine oxidase-1, was used as a positive control (FIG. 5b). Thereafter, the human-derived pulp cells were treated with butane at a concentration of 20 μM for 3, 6, 12, 18 and 24 hours (FIG. 5D).

A reaction comprising nicotinamide adenine dinucleotide phosphate and biliverdin reductase obtained from rat liver cytosol to a microsome obtained from the treated human-derived pulp cells Solution, specifically 100 mM PBS, 2 mM magnesium chloride (MgCl ₂ ), 3 mg rat liver cytosol, 0.8 mM nicotinamide adenine dinucleotide phosphoric acid (NADPH), 2 mM glucose-6- Phosphate (glucose-6-phosphate), 0.2 U of glucose-6-phosphate dehydrogenase were mixed, 20 μM of hemin was treated with a substrate, and then 1 at 37 ° C. After reacting in the dark for 1 hour, 1 ml of chloroform was added to terminate the reaction.

After the reaction was completed, the absorbance (SpectramaxGemini XS, Molecular Devices, Sunnyvale, CA, USA) was measured at 450 nm, and the absorbance value was substituted for the enzymatic activity of hematin oxidase-1 by substituting the formula of Equation 1 below. Was measured, and the examples of the values derived by the following formula are shown in Table 3. The enzyme activity measured according to the butane treatment content and treatment time is shown in FIGS. 5A-5D.

[Equation 1]

OD _{450 nm} (y) = A + B × HO concentration (x) + C × {HO concentration (x)} ²

A = 0.0585, B = 0.373, C = 0.00188

TABLE 3

HO concentration (ng / ml)	0.0	0.125	0.250	0.5	1.0	2.0	4.0	8.0
OD 450	0.091	0.134	0.143	0.214	0.421	0.786	1.325	3.021

As shown in FIG. 5a and FIG. 5b, the expression and activity of hematin oxidase-1 were increased when the butane was treated, especially when the butane was treated with a 10 μm content. It was confirmed that the expression level and activity of 1 was increased, and cobalt protoporphyrin (CoPP), a positive control group, was treated with butane at a content of 20 Μm.

In addition, as shown in Figures 5c and 5d, the longer the treatment time of the butane was confirmed that the overall activity of the enzyme in the pulp cells, but the enzyme activity was reduced rather than 18 hours in 24 hours Bar, it was confirmed that the treatment with 18 hours can exhibit the highest expression level and the highest activity of hematine oxidase-1.

Experimental Example 4. Confirmation of the effect on the expression of butane MAPK

In order to confirm the protective effect of the butane obtained in the preparation in human-derived pulp cells, the effect on the expression of mitogen activated protein kinase (MAPK) of the butane obtained in the preparation was The effects on p-ERK, p-p38 and p-JNK expression belonging to mitogen active protein kinases were measured.

Specifically, the expression of p-ERK, p-p38 and p-JNK is treated with 0 μm, 15 minutes, 30 minutes, 45 minutes, and 60 minutes of butane at 20 μM concentration in human-derived pulp cells, respectively. Experiments were performed using Western blot analysis disclosed in the literature described in Experimental Example 3 (Li B et al , European Journal of Pharmacology 614, p58-65 (2009)). The results are shown in Figures 6a to 6c.

6A to 6C, it was confirmed that the expression of p-JNK in human-derived pulp cells gradually increased as the treatment time of butane was increased (FIG. 6A), but it was treated with butane only or PD98059. (P-ERK inhibitor) or SB203580 (p-p38 inhibitor), together with hematine oxidase-1 and butane, after treatment with SP600125, a inhibitor of p-JNK, was treated with hematine oxidase- It was confirmed that the expression of 1 is suppressed (FIG. 6B). In addition, this treatment showed the same tendency in confirming the cell viability (Fig. 6c). Through the above results, the protection effect of human derived pulp cells by the treatment of butane, specifically hematin oxidase-1 expression and the increase of enzyme efficiency, is closely related to the p-JNK pathway. This was confirmed.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims (10)

1. Pharmaceutical composition for preventing or treating pulp disease comprising butane as an active ingredient.
2. The method according to claim 1,

   The butane is a flavonoid-based compound having a structure of tetrahydroxychalcone.
3. The method according to claim 2,

   The tetrahydroxychalcone is 2 ', 3,4,4'-tetrahydroxykal, 2', 4 ', 3,4-tetrahydroxychalcone, 3,4,2', 4'-tetrahydroxychalcone And 2 ', 4', 3,4-tetrahydroxychalcone.
4. The method according to claim 1,

   The pulp disease is selected from the group consisting of pulp red blood, pulp salt, pulp degeneration, pulp necrosis, pulp necrosis, periodontitis and gingivitis.
5. Food composition for preventing or improving pulp disease comprising butane as an active ingredient.
6. The method according to claim 5,

   The butane is a food composition of the flavonoid series having a structure of tetrahydroxychalcone.
7. The method according to claim 6,

   The tetrahydroxychalcone is 2 ', 3,4,4'-tetrahydroxykal, 2', 4 ', 3,4-tetrahydroxychalcone, 3,4,2', 4'-tetrahydroxychalcone And 2 ', 4', 3,4-tetrahydroxychalcone.
8. The method according to claim 5,

   The pulp disease is selected from the group consisting of pulp red blood, pulp salt, pulp degeneration, pulp necrosis, pulp necrosis, periodontitis and gingivitis.
9. Health functional food for preventing or improving pulp disease comprising the food composition of any one of claims 5 to 8.
10. The method according to claim 9,

    The pulp disease is a health functional food selected from the group consisting of pulp red blood, pulp salt, pulp degeneration, pulp necrosis, pulp necrosis, periodontitis and gingivitis.

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