KR100605116B1 - Composition comprising Notoginseng Radix extract for preventing and treating of periodentitis as an effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of periodontal disease, which contains the extract of Schisandra chinensis as an active ingredient, specifically, inhibits the release of tumor necrosis factor-alpha (TNF-α) and selectively kills only activated T cells. Periodontal extract, which promotes the proliferation and differentiation of osteoblasts, reduces the formation and activity of osteoclasts, inhibits the destruction of alveolar bone, and increases the proliferation and cellular activity of periodontal ligament cells. It relates to a composition for preventing and treating diseases. The composition for preventing and treating periodontal disease of the present invention not only inhibits inflammation but also prevents destruction of alveolar bone and is effective in proliferation of periodontal ligament cells, and thus may be useful for medicines or health foods for the prevention and treatment of periodontal disease. have.

Description

Composition comprising Notoginseng Radix extract for preventing and treating of periodentitis as an effective component             

Figure 1 is a schematic diagram showing the extraction and separation method of the three muscles extract of the present invention, Figure 2 is a graph showing the effect of the three muscles extract of the present invention on the inhibition of tumor necrosis factor-alpha (TNF-α),

Figure 3 is a graph measuring the selective cell death of the three muscles of the present invention activated T cells,

4 is a graph showing the change in osteoblast proliferation according to the concentration of Samchil root extract,

FIG. 5 is a graph showing the effect of Samchil-Geun extract on collagen, basic phosphatase, osteopontin and osteocalcin RNA expression in RCC cells;

COL: collagen, ALP: basic phosphatase,

OPN: Osteopontin, OC: Osteocalcin,

R-F1M: Ethyl alcohol extract of Shamil root, R-F1W: Hot water extract of Shamil root

Figure 6 is an electrophoresis picture showing the effect of Samchil-Geun extract on OPG expression of osteoblasts,

Figure 7 is a graph showing the expression of the transcription factor (Runx2) for differentiation when the three-root extract was treated on C2C12 cells inserted with the 6xOSE2-Luc vector, compared with the control group,

8 is a graph showing the effect of Samchil-Geun extract on phosphatase activity of HOS cells,

9 is a graph showing the effect of Samchil-Geun extract on the formation of osteoclasts,

10 is a graph showing the effect of Samchil-Geun extract on osteoclast activity,

11 is a graph showing the effect of the three-stage root extract of the present invention on the cell activity of human periodontal ligament cells,

Figure 12 is a graph showing that the three-root muscle extract of the present invention is active in the proliferation of human periodontal ligament cells.

The present invention relates to a composition for preventing and treating periodontal disease, which contains the extract of Schisandra chinensis as an active ingredient, and more specifically, inhibits the release of tumor necrosis factor-alpha (TNF-α) and selectively kills only activated T cells. It inhibits inflammation, promotes the proliferation and differentiation of osteoblasts, reduces the formation and activity of osteoclasts, inhibits the destruction of alveolar bone, and contains Samchil-geun extract which increases the proliferation and cellular activity of periodontal ligament cells as an active ingredient. It relates to a composition for preventing and treating periodontal disease.

Periodontal tissue is composed of alveolar bone, gingiva (gum) and periodontal ligament. The gum is part of the supportive tissue that we see in our mouth, where the disease (gingivitis) begins, and when the disease grows deeper and spreads deeper into the supportive tissue, the periodontal ligament is coupled to the root of the tooth and the bone tissue surrounding the tooth. Is destroyed, and the alveolar bone surrounding the teeth is also destroyed and periodontal disease occurs.

Periodontal disease, including gingivitis and periodontal disease, is an inflammatory state of tooth support tissue caused by bacteria, and is a disease that causes loss of teeth due to bleeding, formation of periodontal pockets, and destruction of alveolar bone. The periodontal disease proceeds to the process of colonization of bacteria, periodontal penetration of bacteria and periodontal tissue destruction. Specifically, due to poor oral hygiene, oral bacteria can form when the bacterial membrane is inflamed, sometimes bleeding and bad breath from the gums, and further progresses in this condition deepens the gap between the teeth and gums, resulting in periodontal sac Periodontal disease is caused by the growth of bacteria that cause periodontal disease. As periodontitis progresses, even weak stimuli such as brushing may bleed and swell the gums, often turning into acute inflammation, causing pain. This inflammation decreases the ability to make bones, increases the ability to absorb bones, and the alveolar bones are lowered and destroyed, resulting in loss of teeth.

There are many causes of periodontal disease, but as a local factor, when plaque accumulates in the periodontal sac, it becomes a habitat for anaerobic gram-negative bacteria in the periphery and proliferates deep into the periodontal sac. At this time, the toxins and all products of the proliferated anaerobic Gram-negative bacteria directly destroy tissues or stimulate the immune system, and cause inflammation along with periodontal tissue destruction by various actions from the stimulated immune system. As a defense mechanism, the function and immune response of polymorphonuclear leukocytes act as systemic factors.

In other words, the metabolism of anaerobic gram-negative bacteria, the toxins of cells such as hydrogen sulfide, ammonia and toxic amines are secreted into the periodontal tissues, and the tissues are directly organized by endotoxins such as lipopolysaccharide, which is a cell wall component. Free radicals secreted extracellularly by various actions of the humoral and cellular immune system stimulated by stimulating the biological immune system, prostaglandins, Leukotriens, histamine Gum inflammation is caused by several types of cytokines such as interleuckins. In addition, collagen, which is a substrate of periodontal tissue, is degraded by enzymes such as collagenase secreted from bacteria and leukocytes, gum retraction occurs, and if left unattended, it progresses to periodontal disease.

Therefore, antibacterial and bacteriostatic action against anaerobic gram-negative bacteria, which are the underlying factors, and methods of removing toxic products of these bacteria or restoring lost periodontal tissues are known to be important for prevention and treatment of periodontal disease. have.

The treatment of periodontal disease has been used for the establishment of improved oral hygiene, non-surgical or surgical calculus removal, root graft, gingival sopa and regeneration of periodontal tissue. However, these surgical methods are difficult to treat effectively due to the hassle of going to the dentist for treatment, and they are limited to the treatments performed when the disease progresses rather than preventing the disease. In most cases. As an additional treatment, systemic antibiotics and topical sustained-release drugs have been used, but cases of periodontal disease bacteria have been reported that result in too much drug delivery to unnecessary areas resulting in side effects and resistance to recently used antibiotics. There is a serious problem.

In order to supplement the limitations of the surgical treatment and the use of antibiotics, and to express the effect of preventing and treating periodontal disease, the appearance of an agent having the activity of restoring damaged and lost periodontal tissues was required.

The inventors have found that CD4 ⁺ allows the secretion of various types of inflammatory cytokines (TNF-α, IFN-γ, GM-CSF, IL-2, IL-6) secreted from a biological immune system stimulated by bacterial toxins. On the basis of the fact that T cells are activated, they express osteoprotegerin ligand on the surface to promote osteoclastogenesis and play a decisive role in cartilage destruction. It was deemed necessary to develop a drug that inhibits release and selectively kills only activated T cells, thereby inhibiting inflammation and preventing alveolar bone destruction (Kong YY et al., Nature 402, 304-309, 1999). .

Accordingly, the present inventors inhibit the release of cytokines in herbal extracts, selectively kill only activated T cells to inhibit inflammation, promote osteoblast proliferation and differentiation, and reduce osteoclast formation and activity of alveolar bone. Samchil-geun extract that can prevent the destruction by inhibiting the inflammation of the periodontal disease by the above action and confirmed that it is effective in the proliferation of periodontal ligament cells and completed the present invention.

An object of the present invention is to inhibit the inflammation caused by periodontal disease as well as to promote the proliferation and differentiation of osteoblasts and to reduce the formation and activity of osteoclasts to prevent the destruction of alveolar bone, and is effective in the proliferation of periodontal ligament cells It is to provide a composition for preventing and treating periodontal disease containing Samchil root extract as an active ingredient.

The present invention provides a composition for preventing and treating periodontal disease, containing the extract of Samchil-geun as an active ingredient.

The composition of the present invention includes a pharmaceutical composition and health food composition for preventing and treating periodontal disease.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing and treating periodontal disease, containing the extract of Samchil-geun as an active ingredient.

The present invention provides a extract of Samchil-Keun having extract and treatment activity of periodontal disease by extracting and concentrating Samchil-Geun with ethyl alcohol.

Panax notoginseng (Burk.) FH Chen is a perennial herb that is smaller than ginseng, has 7 leaflets, and is rooted in the shape of small North Koreans. The plant has three leaves and seven leaves attached to the samchil, or its shape is similar to Chosun ginseng, also called samchil ginseng. The root contains 3 to 8% of saponins, the main constituents of which are ginsenosides Rb1, Rg1, Re and notoginsenosides R1, R2, Fa, Fc, and a small amount of ginsenosides ( ginsenosides) R2, b2, d, e, c, with or without R0. Essential oils are less than ginseng and contain oleanolic acid. Roots have hemostatic and cardiac effects, and animal studies have shown that they increase the blood flow in the coronary arteries, reduce the oxygen consumption of myocardium, and reduce the lipid and cholesterol content in the blood. In addition, it is effective for many inflammatory diseases including hepatitis due to its excellent anti-inflammatory, analgesic and hemostatic effects. It is also effective for bleeding and internal bleeding caused by trauma or cuts, and it is known to be effective even when applied directly to a wound or eaten. have.

Samchil-geun extract of the present invention can be extracted with water, alcohol or a mixed solvent of water and alcohol, wherein the alcohol is preferably ethanol. At this time, the ethanol extraction is preferably made for 3 to 5 hours at 70 to 80 ℃, the concentration is preferably made of vacuum decompression at 20 to 40 ℃.

The extraction method may be a conventional extraction method such as cold needle, warm needle, heating using the solvent. In order to further isolate the active ingredient of the extract, although not particularly limited to the separation method that can be used, such as organic solvent extraction, chromatography, it is preferable to extract with ethylacetate (ethylacetate), as in the embodiment of the present invention, More preferred is extraction with n-butanol. In addition, the extraction with ethyl alcohol is preferably repeated three or more times.

Samchil-geun extract of the present invention by inhibiting the release of tumor necrosis factor-alpha (TNF-α), it can be used as a medicine or health food useful for the prevention and treatment of periodontal disease.

In order to examine the effect of the extract of the present invention on the tumor necrosis factor-alpha (TNF-α), lipopolysaccharide (lipopolysaccharide, human mononuclear cell line) using THP-1 cell line LPS) and the three-root muscle extract of the present invention, the free amount of tumor necrosis factor-alpha (TNF-α) in the cell culture was measured by the enzyme immunoassay (ELISA). As a result, it was confirmed that the Samchil-Geun extract of the present invention significantly inhibited the free amount of tumor necrosis factor-alpha (TNF-α) when treated at a concentration of 10 μl / ml ( see FIG. 2 ).

In addition, the Samchil root extract of the present invention can be used as a medicine or health food useful for the prevention and treatment of periodontal disease by selectively killing only activated T cells.

In order to confirm whether the trichophyte extract of the present invention selectively kills activated T cells, lymph nodes of 5 week-old female rats were extracted and crushed to extract cells, and then activated only T cells in culture, and activation of activated T-lymphocytes. Cell death was measured. As a result, the three-stage muscle extract of the present invention does not affect the killing of inactivated T cells when treated at a high concentration of 5 μl / ml or more, and selectively kills only activated T cells ( see FIG. 3 ).

In addition, the three-stage muscle extract of the present invention promotes the proliferation and differentiation of osteoblasts, reduces the formation and activity of osteoclasts, and can be used as a medicine or health food useful for the prevention and treatment of periodontal disease.

Samchil root extract of the present invention can prevent the destruction of alveolar bone by affecting the proliferation of osteoblasts and osteoclasts, which are the main cells of bone. Specifically, the proliferation of osteoblasts, the mRNA expression level for the characteristic genes of osteoblasts, the expression of OPG (osteoprotegerin) inhibiting the production of osteoclasts by osteoblasts, the activity of the expressed proteins of the gene and osteoblast differentiation. In the expression of transcription factors, etc., the three-stage muscle extract improves the function of osteoblasts (see FIGS. 4, 5, 6, 7 and 8 ).

In addition, Samchil-geun extract of the present invention strongly inhibits osteoclast function by greatly reducing the formation of TRAP (+) multinucleated cells and osteoclast activity, which are considered as osteoclasts, and contribute more to the reduction of bone tissue destruction ( FIG. 9). And 10 ). Therefore, the three-stage muscle extract of the present invention promotes the proliferation and differentiation of osteoblasts, and can inhibit the destruction of the alveolar bone by reducing the formation and activity of osteoclasts, which is effective for the prevention and treatment of periodontal disease.

In addition, in order to find out whether the Samgyuk-geun extract of the present invention can effectively treat periodontal disease by the above-described action, a human periodontal ligament cell proliferation experiment was performed. It was confirmed that it can effectively promote periodontal cell proliferation of ( see FIGS. 11 and 12 ).

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the three-stage muscle extract.

The composition of the present invention may contain one or more active ingredients exhibiting a different function in addition to the three-stage muscle extract.

In addition to the above-described active ingredient for administration of the pharmaceutical composition for the prevention and treatment of periodontal disease, containing the three-root muscle extract of the present invention as an active ingredient may be prepared by including one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on diet, administration time, administration method, excretion rate and severity of disease. The daily dosage is 0.1 to 10 mg / kg, preferably 0.1 to 3 mg / kg, and more preferably divided once to several times a day when the oral administration of the three-stage muscle extract of the present invention.

As a result of the toxicity experiment when orally administering the three-stage root extract of the present invention to mice, the 50% lethal dose (LD ₅₀ ) by the oral toxicity test was found to be at least 5 g / kg or more proved to be a safe substance.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of periodontal disease.

The composition of the present invention is effective in periodontal diseases such as gingivitis and periodontitis, and not only reduce the inflammation of the gums, but also to strengthen the gums to be resistant to bacterial infection because it is not antibiotics, preventing and treating periodontal disease For oral compositions can be prepared.

The composition for oral cavity containing the three-stage muscle extract provided by the present invention is not particularly limited in the formulation and may have a conventional formulation, and specifically, may have a formulation such as toothpaste, mouthwash or mouthwash. The composition for oral cavity provided by the present invention may contain various bases and additives necessary for formulation according to the formulation, and the kind and amount of these components can be easily selected by those skilled in the art. For example, when the formulation of the composition for oral cavity is a toothpaste, it can be prepared by adding an abrasive, a wetting agent, a foaming agent, a binder, a sweetening agent, a pH adjusting agent, a preservative, an active ingredient, a fragrance, a brightener, a pigment, a solvent and the like.

The composition of the present invention can be added to health food for the purpose of improving periodontal disease. When the three roots of the present invention is used as a food additive, the three roots of the extract may be added as it is, or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the three-quarters root extract of the present invention is added in an amount of 100% by weight or less, preferably 50% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.1-20 g, preferably about 1-10 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.05 to 50 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example 1 Preparation of Samchil-Geun Extract

Samchil-keun was used for commercial production in commercial building.

<1-1> Samchil-geun crude extract

1) Schizophrenic alcohol extract

The samchil muscle was sectioned to a size of 1 to 2 cm and then washed in running water to remove impurities. The cut ginseng root was ground to a powder, 200 g of powder was placed in a 3 L flask, and heat extraction was repeated three times for 4 hours while reflux stirring at 78.5 ° C. using 2,000 ml of ethanol. The extract was filtered and concentrated under vacuum at 40 ° C. or lower using a vacuum rotary evaporator to obtain crude root extracts containing 2.7 g of the three muscles powder (RF1M) (into the three muscles powder). Yield: 1.35%).

2) Tricuspid Muscle Extract

The extraction was performed in the same manner as in 1) except that water was used instead of ethanol as the extraction solvent.

3) Crude extract of Samgye-geun mixed solvent

Extracted in the same manner as the extraction method of 1), except that a mixed solvent of water 25% and ethanol 75% instead of ethanol as the extraction solvent.

<1-2> Isolation of Trichophyta japonica Extract

The crude extract (RF1M) obtained in 1) of <Example 1> was additionally fractionated with 500 ml of normal butanol (n-butanol) at room temperature using a separatory funnel three times to obtain a fraction (RF1MB).

This RF1MB fraction was separated using column chromatography to separate the RF1MB4 fraction. Further column chromatography was performed on the RF1MB4 fraction to obtain a final fraction of the three-stage muscle extract (RF1MB4b).

Extraction of thirty-seven root extract according to the present invention, isolation method is shown in Figure 1;

In the following experimental examples, the final fraction (RF1MB4b) of Samchil-Geun extract was concentrated and lyophilized, and then diluted in water to perform in vitro and animal experiments.

Experimental Example 1 Free Inhibition Experiment of Tumor Necrosis Factor-alpha (TNF-α) by Samchil-Geun Extract of the Present Invention

In order to examine the effect of the three-stage muscle extract of the present invention on the free inhibition of cytokine tumor necrosis factor-alpha (TNF-α) released from THP-1 cells, which are human monocytic cell lines, The experiment was conducted.

<1-1> Cell Selection and Cell Culture

THP-1 cells (ATCC No. TIB-202), a human-derived cell line, were purchased from ATCC (Rockville, USA) and added 10% FBS (fetal bovine serum) to RPMI 1640 (Gibco, BRL, USA). After culturing at and used in the experiment, the effect of the three-stage muscle extract prepared in the above Example on the tumor necrosis factor-alpha (TNF-a) of the human was evaluated.

<1-2> Free amount measurement of tumor necrosis factor-alpha (TNF-α)

In order to determine the effect of the extract of the trichophyte root (RF1MB4b) on the tumor necrosis factor-alpha (TNF-α) release, tumor necrosis factor-alpha (TNF) using the cells prepared in Experimental Example 1-1 The free amount of -α) was measured by enzyme immunoassay (ELISA).

Cells were aliquoted to ⁵ × 10 ⁵ per ml in a 96-well plate and lipopolysaccharide (LPS) was added to activate the cells to release tumor necrosis factor-alpha (TNF-α).

Simultaneously with LPS, the experimental group was treated with 2 or 10 μl / ml concentration of Samchil-geun extract (RF1MB4b) prepared in Example, and the amount of tumor necrosis factor-alpha in the cell culture was measured by enzyme-linked immunoassay (ELISA). It was.

As a result, as shown in Figure 2 , when the three-root muscle extract (RF1MB4b) of the present invention was treated at a low concentration (2 μl / ㎖) compared to the control group treated with the three-muscle extract extract tumor necrosis factor-alpha ( Although the free amount of TNF-α) was not significantly inhibited, the free amount of tumor necrosis factor-alpha (TNF-α) was significantly inhibited when the three-root muscle extract (RF1MB4b) of the present invention was treated at a concentration of 10 μl / ml. It was confirmed.

Therefore, it can be seen that the three-stage muscle extract of the present invention effectively inhibits the release of tumor necrosis factor-alpha (TNF-α).

Experimental Example 2 Confirmation of Selective Apoptosis of Activated T Cells by the Trichophyta grandis Extract of the Present Invention

In order to confirm whether the trichophyte extract of the present invention can selectively kill only activated T cells, the following experiment was performed.

<2-1> Isolation and Activation of T Cells

Lymph nodes of 5 week-old female rats were extracted and crushed cells were extracted with a sterile syringe tip. Single cells that had passed through a cell filter (Falcon, NJ USA) were washed with PBS and then added to the culture at 2 × 10 ⁶ cells / ml. The culture solution was used by adding 10% FBS (fetal bovine serum) to RPMI 1640 (Gibco, BRL, USA).

In order to activate only T cells in culture, 5 μg / ml of concanavalin A was added and cultured for 48 hours. After 48 hours of incubation, 10 mg / ml of methyl-alpha-D-Mannopyranoside (Sigma, Germany) was added thereto, followed by incubation for 30 minutes. 48 hours after washing 3 times with PBS and keeping the cell number at 2 × 10 ⁶ cells / ml in the culture medium containing 100 units / ml of human Interleukine-2 (hIL-2, R & D, MN, USA) Cultured (Lenardo MJ et al. Nature 353 (6347): 858-61. 1991).

<2-2> Selective Cell Death Measurement of Activated T Cells

Activated T cells were adjusted to 1 × 10 ⁶ cells / ml and put into 200 μl in 96 well plates (Falcon, USA). At this time, 100 units / ml of hIL-2 were added together.

The control group was used without the extract of Samchil-Geun extract, and the experimental group was prepared by the final fraction (RF1MB4b) of the Samchil-Geun extract prepared in the above example by concentration (5 μg / ml, 10 μg / ml, 20 μg / ml). Incubated for 24 hours.

Inactivated cells as a control was prepared as follows.

Single cells extracted from the spleen were adjusted to 2x10 ⁶ cells / ml, and 200 µl was dispensed into 96-well plates, and the same extract as the activated cells was prepared by incorporating the Samchil-Geun extract prepared in the above example for 24 hours. After 24 hours, the cells were transferred to a flow tube, followed by propidium iodide (PI), and the living cell number was measured for 20 seconds using the CellQuest program of a flow cytometer (FACSCaliver, Becton Dickinson, France).

Cell death was calculated as (1-F extract treated cells / untreated cells) x100. In this method, an effective drug was selected through a screening process of candidate drugs and a low cell death rate of inactivated naive T cells was selected (Sabapathy K, et al., Curr Biol). 11; 9 (3), 116-25. 1999).

As a result, as shown in Figure 3 , when treated with a high concentration of the three muscles extract of the present invention 5 μl / ㎖, did not affect the killing of inactivated T cells, and selectively killed only activated T cells It was confirmed.

Therefore, it can be seen that the three-stage muscle extract of the present invention has an effect of selectively killing activated T cells according to the concentration.

Experimental Example 3 Osteoblast Proliferation by Samchil-Geun Extract

In order to determine the effect of the three-stage muscle extract fraction of the present invention isolated from <Example 1> on the proliferation of osteoblasts and the increase of cell activity, the following experiments were performed.

<3-1> Osteoblast Screening and Cell Culture

Using the following three types of cells was confirmed the effect of the three-stage muscle extract of the present invention on the proliferation of osteoblasts. Human cells include MG-63 cells (ATCC No. CRL-1427), a cell line derived from human osteosarcoma, and mouse muscle cells, C2C12 cells (ATCC No. CRL-1772), ATCC (Rockville, USA). ) Was used in the experiment after incubation in a medium in which 10% FBS (fetal bovine serum) was added to DMEM (Gibco, BRL, USA).

In addition, as the primary cultured cells, osteoblast group (RCC) obtained from the cranial canal of Sprague-Dawley rat was used. That is, on the 19th day of birth, the frontal and parietal bones were extracted from the cranial canal of the Sprague-Dawley rats, and then, each at 10 ° C. at 37 ° C. with an enzyme solution containing 0.1% collagenase, 0.05% trypsin, and 0.5 mM EDTA. Treatment was performed continuously for 10 minutes, 10 minutes, 20 minutes, and 20 minutes to separate cells of groups I to V, and to mix cells of groups IV and V, in which cells with osteoblastic characteristics were mainly present. After primary culture in DMEM containing 10% FBS, cells were collected with trypsin-EDTA solution and the number of cells was used for each experiment.

<3-2> Measurement of Osteoblast Proliferation

In order to examine the effects of Samchil-Geun extract on the proliferation of osteoblasts, ³ H-thymidine introduced into the DNA of cells during cell proliferation using the three types of osteoblasts prepared in Example <3-1> thymidine) was measured. Specifically, osteoblasts were dispensed to be 2 × 10 ^{4 per} well of a 24-well plate, and replaced with DMEM containing 1% FBS the next day. Samchil-geun extract prepared by diluting continuously at concentrations of 10 ⁻⁴ , 10 ⁻³ , 10 ⁻² , 10 ⁻¹ , 1 or 10 μl / ml in the replaced culture medium was added by concentration and incubated for 48 hours. After incubation with 3 μCi / well of ³ H-thymidine for the last 4 hours, the cells were washed with phosphate buffered saline (PBS) and treated with ice-cold 5% trichloroacetic acid (TCA) insoluble fraction. Was separated and dissolved in 0.1 M NaOH. ³ H-thymidine influx was measured by taking a double fraction and measuring radioactivity on a liquid scintillation counter.

The level of osteoblastic cell proliferation, according to various thirty-seven muscle extract concentration by the process shown in FIG. From this, the low concentrations of Schizophyllus koji extracts were not significantly different from the control without treatment of Schizophyllus chinensis extract, but proliferation was more pronounced than that of control osteoblasts from 1 μl / ml to 10 μl / ml in proportion to the treatment concentration. It was confirmed to increase ( FIG. 4 ).

<3-3> mRNA expression for characteristic genes of osteoblasts (type 1 collagen, basic phosphatase, osteopontin and osteocalcin)

In order to evaluate the effect of Samchil-Geun extract on osteoblast differentiation, after preparing and culturing cranial canal cells of Sprague-Dawley rat by the method described in Example <3-1> , the primary Passaged cells were collected and aliquoted into 60 mm tissue culture dishes at a density of 63 × 10 ⁴ / dish. Osteoblasts were incubated in α-minimum essential medium containing 10% FBS, and 50 ㎍ / ml of ascorbic acid and β-glycerophosphate from the third day of culture. 10 mM was added to induce differentiation of osteoblasts. From the day after the start of the culture, it was added to the culture dish of osteoblasts at a concentration of 1 μl / ml, and the calcified nodules were formed in the osteoblasts by adding the same concentration of Samchil-Gil extract every two days. Incubated for days up to 15 days. In order to examine the effects of the three-stage muscle extract, after 3 days, 7 days of culture and 14 days of incubation, total RNA was isolated using an Easy Blue ^TM total RNA isolation kit. . Of these, 25 μg of total RNA was subjected to electrophoresis on a 1.2% agarose / formaldehyde denaturing gel and transferred to a nylon filter (Hybond-N). Prehybridization reactions were performed in an ExpressHyb ^™ hybridization solution. Pro-α1 (I) -collagen, basic phosphatase, osteopontin, or osteocalcin probes labeled with ³² P-dCTP were then isolated using a megaprime DNA labeling kit. Hybridization was carried out in the hybridization solution contained for 1 hour, washed twice with Wash Solution 1 (2X SSC, 0.05% SDS) and once with Wash Solution 2 (0.1X SSC, 0.1% SDS), followed by autoradiography. Comparison of the expression level of the protein was performed using a Fuji Bas 1500 image analyzer.

As a result of measuring the type 1 collagen, basic phosphatase, osteopontin, and osteocalcin mRNA expression of the osteoblasts examined by the above procedure, the extract of Samchil-geun was added to the type 1 collagen, basic phosphatase, It was confirmed to promote the expression of pontin ( FIG. 5 ). At this time, basic phosphatase is an enzyme bound to the cell membrane of osteoblasts and is used as a marker for osteoblasts and is thought to be involved in various stages of bone tissue formation (Farley, JR and Baylink, D., Metabolism , 35). : 563-571, 1986). Collagen type 1, basic phosphatase, osteopontin and osteocalcin are known as characteristic genes for osteoblasts (Gerstenfeld, LC et al ., Develop. Biol ., 122: 49-60, 1987; Yoon, K. et al ., Biochem. Biophys. Res. Commun. , 148: 1129-1136, 1987). Expression of the characteristic protein of the osteoblasts seems to have reduced the expression of the gene rather than the control after 8 days since the osteoblast differentiation has already achieved sufficient bone formation.

<3-4> Expression of Osteoblastic OPG (Osteoprotegerin) That Inhibits Osteoclast Production

The MG63 cell line was cultured confluent in a 60 mm tissue culture dish, followed by addition of 2 ml of serum-free DMEM containing Samchil root extract at concentrations of 1, 4, 20 and 100 μg / ml for 16 hours. After incubation, the supernatant was obtained. The culture supernatant was concentrated with 20-25% trichloroacetic acid (TCA), dissolved in SDS-PAGE sample buffer and electrophoresed on 10% acrylamide SDS-PAGE gel. Samples were transferred from gels to PVDF membranes by standing at 250 kPa for 1 hour after electrophoresis. The membrane was transferred to a blocking buffer (5% skim milk) for 1 hour, and then the anti-OPG antibody (R & D system), a primary antibody, was diluted 1:25 in a blocking buffer and placed in a solution at room temperature. It was left for about 2 hours. After washing the membrane twice for 15 minutes with TBST solution (100 mM Tris-Cl, 0.9% NaCl, 0.1% tween 20, pH 7.5), the secondary antibody (also known as Promega) with alkaline phosphate was added. The membrane was left for 1 hour in a solution diluted in blocking buffer at: 7000. After washing the membrane three times for 20 minutes with the TBST solution, the substrate was reacted for 15 minutes and then analyzed using RAS (Fujifilm).

As a result of measuring the expression of OPG, a substance that inhibits the production of osteoclasts in osteoblasts, the expression of OPG in osteoblasts was promoted in proportion to the concentration up to 20 ㎍ / ml as the concentration of the extract of Schizophyllum was increased. The tendency to ( Fig. 6 ). In Figure 6 , the number at the bottom represents the density (density) of each band, which means that the wider the expression amount is higher.

<3-5> Expression of major transcription factor (Runx2) in osteoblast differentiation

Transient transfection of C2C12 cells (ATCC No. CRL-1772) with a 6xOSE2-Luc vector inserted into the pGL3 promoter vector, a series of six OSE2 DNA sequences bound by Runx2, a transcription factor essential for bone formation The method of determining the expression level of Runx2 as luciferase activity was administered by administering the test fraction.

In order to promote bone formation, osteoblast differentiation must be preceded. In particular, in order to promote osteoblast differentiation, expression of Runx2, which is a transcription factor for differentiation, is essential. This increases the expression of several genes (osteocalcin, osteopontin, type 1 collagen, bone sialoprotein) that osteoblasts require. The promoter of these genes contains the response element of Runx2 (also called osteoblast specific factor binding element), and its central consensus sequence is PuACCPuCA (where P is the purine base). And u represents the pyrimidine base). This increased osteoblastic gene expression in osteoblasts has been shown to be involved in the promotion of bone formation (Lee, KS., Et al ., Mol. Cell. Biol ., 2000 Dec; 20 (23): 8783-92 Park, MH, et al ., J. Bone Miner Res ., 2001 May; 16 (5): 885-92). The detailed experimental method is as follows.

Day 0

Each C2C12 cell is seeded at 1 × 10 ⁵ density per well of a 6 well plate and then incubated in a 5% CO ₂ incubator for 24 hours.

2. Day 1

(1) p6xOSE2-Luc: lipopeptamine complexes were prepared using Lipofectamine. Specifically, Solution I was prepared by mixing 0.5 μg of p6xOSE2-Luc, 3 μl of PLUS reagent, and 100 μl of serum-free DMEM medium per 6 well plate at room temperature for 15 minutes.

Next, Solution II was prepared, including DMEM without 3 μl of lipofectamine and 100 μl of serum, and then the solution I and solution II were mixed and reacted at room temperature for 15 minutes to allow p6xOSE2-Luc: lipo. Pectamine complexes were prepared.

(2) transformation

1) After removing the culture medium of the cells for 15 minutes, the cells were washed with DMEM without serum. DMEM without 800 μL of serum was added per well of a 6 well plate.

2) 207 μl of p6xOSE2-Luc: lipopeptamine complex was added to the wells, followed by incubation in a 5% CO ₂ incubator for 3 hours.

3) After 3 hours of incubation, 1 ml of DMEM containing 30% FBS was added per well, followed by incubation in a 5% CO ₂ incubator for 24 hours.

3. Day 2

After removing the culture medium of the cells, the cells were washed with DMEM without serum. For 24 hours after treatment with serum-free DMEM with 2 ml of bFGF added at 10.0 ng / ml per well of 6-well plate, the control group treated with serum-free DMEM without bFGF. Incubated in a 5% CO ₂ incubator.

Day 3

After 24 hours of incubation, the cells were washed with PBS solution and treated with 500 μl of bright glo lysis buffer (Promega, WI, USA) and then bright glo substrate (Promega, WI, USA). After reacting for 5 minutes by adding the same amount, Luciferase activity was measured using a luminometer (Junior, EG & G BERTHOLD, Australia).

As a result of measuring the effect of the three muscles extract on p6xOSE2-Luc-temporally transduced C2C12 cells, it was confirmed that the expression of Runx2 gene is increased compared to the control ( Fig. 7 ).

<3-6> Basic Phosphatase Activity of Osteoblast Marker

To evaluate the effect of Schizophyllum paniculata extract on osteoblast activity, HOS osteoblasts (ATCC No. CRL-1543) were purchased from the American Type Culture Collection, Rockville, USA (ATCC) and stored in 96-well plates for each well. Aliquot to 5x10 ³ cells and multiply into monolayers, and then add Samchil-Geun extract to the culture at concentrations of 10 ^-4 , 10 ^-3 , 10 ^-2 , 10 ^-1 , 1 μl / ml, containing 2% FBS After 48 hours of incubation in 96-well plates with DMEM, cells were treated with 0.1% Triton X-100 / saline for 30 minutes at 37 ° C. A certain amount of the cell treatment solution was reacted with 0.1 N glycine-NaOH buffer (pH 10.4) for 30 minutes in the presence of 100 mM PNPP as a substrate for 30 minutes at 37 ° C. to determine the amount of PNP released from the substrate PNPP at 405 nm. Colorimetric determination was made to investigate the effect on the activity of basic phosphatase.

As a result of measuring the activity of the basic phosphatase of osteoblasts by the above process, Samchil-Geun extract had little effect on the activity of the enzyme ( FIG. 8 ).

Experimental Example 4 Inhibition of Osteoclast Proliferation by Samchil-Geun Extract

In order to confirm the inhibition of the production of osteoclasts and the decrease of cell activity by the three-stage muscle extract, the present inventors conducted the following experiments.

<4-1> Pure Isolation of Osteoclast Progenitor Cells and Induction of Differentiation into Mature Osteoclasts and Treatment of Schizophrenic Extract

In order to separate the mouse bone marrow cells, 7 to 9 weeks old male mice were sacrificed with cervical torsion, followed by sterile extraction of the femur and tibia, removal of soft tissues, and the cutting of both ends of the long bone. Inject 1 ml of enzyme solution containing 0.1% collagenase (Gibco), 0.05% trypsin, and 0.5 mM EDTA (Gibco) to remove bone marrow and stir for 30 minutes to collect bone marrow cells. Uncultivated cells were obtained after incubation in the included α-Minimum Medium (α-MEM) for 24 hours. Unattached cells, which become progenitor cells of osteoclasts, were cultured by aliquoting 2 x 10 ⁵ cells per well. Samchil prepared in <Example 1> in α-MEM containing 20 ng / ml macrophage-colony stimulating factor (Peprotech, USA) and 30 ng / ml RANKL (Peprotech, USA) during 8 days of culture The root extract was treated and incubated. After completion of the culture, in order to examine the production of osteoclasts, the cells were fixed and subjected to TRAP staining. In addition, in order to examine the activity of osteoclasts, the cells were detached and observed at the site of absorption.

<4-2> TRAP (+) Inhibition of Multinucleated Cells

After cell culture, adherent cells were washed with PBS and then fixed with citrate-acetate-formaldehyde for 5 minutes, naphthol AS-BI phosphate, fast garnet GBC. TRAP staining was performed by incubating the solution and 37 mM acetate buffer (pH 5.0) containing 7 mM tartrate buffer (pH 5) for 1 hour. At this time, TRAP (+) multinucleated cells having three or more nuclei were considered as osteoclasts.

Osteoclasts are derived from cells of the monocyte / macrophage lineage in the bone marrow, which are circulated into the blood and are known to proliferate and proliferate in the endothelial layer to form multinucleated cells (Scheven, BAA et al. , Nature , 321: 79-81, 1986), osteoclasts have a tartrate-resistant acid phosphatase (TRAP), an acid phosphatase that is characteristically resistant to tartrate, which is different from other bone tissue cells. It is known to be used as a distinguishable cytochemical marker of osteoclasts (Minkin, C., Calcif. Tissue Int. , 34: 285-290, 1982).

In the present invention, in order to induce the differentiation of osteoclasts, we used a known bone marrow that the precursor cells of osteoclasts, a positive (+) in the TRAP considering the multi-core of cells to osteoclasts, and culturing the osteoclast precursor cells <carried After treatment with Samchil-Geun extract prepared in Example 1> and cultured for 8 days, the change in the number of TRAP-positive multinucleated cells was measured. As a result of measuring TRAP activity, which is a marker enzyme of osteoclasts, the group treated with Samchil-Geun extract significantly reduced the number of TRAP (+) multinucleated cells from 100% to less than 10% compared to the control group. From the above results, it was confirmed that the three-stage muscle extract of the present invention has an inhibitory effect on the production of osteoclasts ( FIG. 9 ).

<4-3> Inhibition of osteoclast activity

In order to confirm the growth and activity of osteoclasts, osteoclast progenitor cells were cultured on a plate coated with hydroxide apatite (OAAS, OCT Inc.), and the TRAP activity and resorption pit, markers of osteoclasts, were observed. . Specifically, the culture medium was removed after cell culture. After cell culture, in order to remove the adhered cells, wash the OAAS plate with distilled water, add 5% sodium hypochlorite solution, leave for 5 minutes, wash it again with distilled water, dry it and image the absorbed area. Observations were made using Image pro plus. In other words, to examine the activity of osteoclasts, which are mainly responsible for bone resorption, bone extracts were extracted while culturing the precursor cells of osteoclasts using calcium and phosphate-coated plates made similar to the minerals of bone tissues. Upon treatment, it was observed whether there was a change in absorption area.

As a result, the osteoclast progenitor cells were cultured on the plate coated with calcium-phosphate when the three-muscle root extract was added, and it was confirmed that the activity and absorption of the multinucleated cells showing TRAP (+) were significantly reduced ( FIG. 10 ).

Experimental Example 5 Confirmation of Human Periodontal Ligament Cell Proliferation by Samchil-Geun Extract Fraction

In order to determine the effect of the Samchil-Geun extract fraction isolated in < Example 1> on the proliferation and cell activity of human periodontal ligament cells, the following experiments were performed.

<5-1> Screening and Cell Culture of Human Periodontal Ligament Cells

The periodontal ligament of the premolar extracted for healthy orthodontic treatment was aseptically scraped using a curette and placed on a culture dish, and 10% FBS (fetal bovine serum) was added to DMEM (Gibco, BRL, USA). A small amount of the added culture was added and cultured. At this time, microscopic observation while culturing confirms that the cells emerge from the tissue into the implant. After the cells were grown in clusters, the periodontal ligament tissue specimens were removed, and the cells were grown in monolayers, which were then passaged and used for experiments.

<5-2> Cell Viability of Human Periodontal Ligament Cells

In a 96-well plate, human periodontal ligament cells per well were put into 5000 cell numbers, incubated for 48 hours in a 5% CO ₂ incubator, and then replaced with a culture solution containing three muscle root extracts. It was further cultured. After treatment with 20ul MTT (5 mg / ml), the culture was removed, and isopropanol was added, followed by absorbance measurement (562, 650 nm). As a result of the experiment, it was confirmed that there was no toxicity to the cells, but rather increased the cell activity ( FIG. 11 ).

<5-3> Proliferation of human periodontal ligament cells

Human periodontal ligament cells were incubated in a 24-well plate containing 20000 cells per well for 48 hours in a 5% CO ₂ incubator, then replaced with a culture solution containing three concentrations of extracts from each concentration for another 48 hours. It was. After removing the culture, trypsin-EDTA was added, and the cell number was counted by a hemacytometer. Experimental results confirm that the growth of the cells up to 16% depending on the concentration ( Fig. 12 ).

In conclusion, it was confirmed that the Samchil-Geun extract fraction of the present invention not only increases the cell activity of human periodontal ligament cells, but also can effectively proliferate human periodontal ligament cells.

Experimental Example 6 Acute Toxicity Test by Samchil-Geun Extract

Samchil-geun used in the present invention was widely used for edible, so it was determined that there is no problem in stability, but the oral administration and intraperitoneal toxicity experiments were carried out to confirm this.

Acute toxicity test was performed using SPF SD rats at 6 weeks of age. Two animals per group were suspended orally at a dose of 5 g / kg in each of the R-F1M and R-F1W of the present invention suspended in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all three extracts showed no change in toxicity in rats up to 5 g / kg and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of more than 5 g / kg.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example 1  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

Samchiol-Geun Extract 2g

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

Samchil root extract 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

Samchil root extract 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Formulation Example 2  : Manufacture of mouthwash goggles

Samchil root extract 0.01 ~ 1.0g

Xyitol 5 ~ 10g

Ethyl alcohol 5 ~ 15g

Sorbitol 5 ~ 15g

Saccharin Sodium 10-100mg

Sodium Monofluorophosphate 500 ~ 1000mg

Sodium Lauryl Sulfate 100-200mg

Polysorbate 20 100 ~ 1000mg

Peppermint Flavor 10-100mg

Sodium benzoate 10-100 g

Purified water

color quantity

100 g in total

According to the above ingredients was prepared according to the oral cleaning agent goggle gargles composition for oral cavity having a periodontal prevention and treatment effect.

Formulation Example 3  : Manufacture of food

Food containing the three-stage root extract of the present invention was prepared as follows.

1. Preparation of Cooking Seasonings

Samchil-geun extract of the present invention 20 to 95% by weight to prepare a cooking seasoning for health promotion.

2. Preparation of Tomato Ketchup and Sauce

Health-promoting tomato ketchup or sauce was prepared by adding 0.2 to 1.0% by weight of Samchil-Geun extract of the present invention to tomato ketchup or sauce.

3. Manufacturing of Flour Foods

0.5 to 5.0% by weight of the three-stage root extract of the present invention was added to wheat flour, and bread, cake, cookies, crackers, and noodles were prepared using the mixture to prepare foods for promoting health.

4. Preparation of soups and gravy

Samchil-geun extract of the present invention was added to 0.1 ~ 5.0% by weight to soup and gravy to prepare a health-promoting meat products, soup of noodles and gravy.

5. Preparation of Ground Beef

10% by weight of the extract of Samchil-geun of the present invention was added to ground beef to prepare ground beef.

6. Manufacture of Dairy Products

5 to 10% by weight of the extract of Samchil root of the present invention was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, yulmu was alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The extract of the present invention was concentrated under reduced pressure and concentrated in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

It was prepared by combining the dry powder of the grains, seeds and samchil root extract prepared above in the following ratio.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder of samchil-geun extract (3% by weight),

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 4  : Preparation of Beverages

1. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing at 20 ℃ for 180 seconds to mix with a cooling water 1: 1: ratio of carbon dioxide gas was injected 0.5 ~ 0.82% to prepare a carbonated beverage containing the samchil root extract of the present invention.

2. Manufacture of health drinks

Instant sterilization by homogeneously mixing subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%) , Packed in a small packaging container such as plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

5 g of the three-stage root extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

1 g of the extract of Samchil-geun of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

The three-arm muscle composition for preventing and treating periodontal disease of the present invention has the effect of inhibiting the release of tumor necrosis factor-alpha (TNF-α), selectively killing only activated T cells, and inhibiting inflammation, It prevents destruction and is effective in the proliferation of periodontal ligament cells, and can be usefully used in medicine or health food for the prevention and treatment of periodontal disease.

Claims (6)

Pharmaceutical composition for the prevention and treatment of periodontal disease containing Samchil root extract as an active ingredient. Oral composition for the prevention and treatment of periodontal disease containing Samchil root extract as an active ingredient. 3. The composition of claim 2, wherein the composition for oral cavity is a toothpaste, mouthwash or mouthwash. Health food composition for the prevention and treatment of periodontal disease containing Samchil root extract as an active ingredient. The composition according to claim 1, wherein the three-quarters root extract is extracted with water, alcohol or a mixed solvent of water and alcohol. 6. The composition for preventing and treating periodontal disease according to claim 5, wherein the alcohol is ethanol.

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