KR20090087709A - Composition for treating of gingival disorders comprising saponin as an active ingredient - Google Patents

Abstract

A composition for preventing or treating periodontal diseases, which contains a saponin from ginseng or red ginseng is provided to have anti-inflammatory effect and antioxidative effect. A composition for preventing or treating periodontal diseases comprises a saponin as an active ingredient. The saponin is contained in a ginseng or red ginseng extract and is a ginsenoside]-Rg1, -Re, -Rf, -Rh1, -Rg2, -Rb1, -Rc, -Rb2, -Rd, -Rg3 or -Rh2. The saponin is an enriched protopanaxadiol saponin. The composition has treating effect of periodontal diseases and a composition for toothpaste, or oral cavity protection.

Description

Composition for Treating of Gingival Disorders Comprising Saponin as an Active Ingredient}

The present invention relates to a composition for preventing or treating periodontal disease comprising saponin as an active ingredient. More specifically, saponins, preferably ginseng or red ginseng saponins, including the active ingredient, there is no cytotoxicity and skin side effects, and relates to a composition for preventing or treating periodontal disease having excellent anti-inflammatory, antioxidant and antimicrobial effects.

Periodontal disease is an inflammatory disease in the peri-dental tissue that begins with the excessive proliferation of certain anaerobic Gram-negative bacillus and leads to the destruction of connective tissue of the gums and irreversible bone absorption. The progression of the disease is caused by the interaction of bacteria and host (human) defense reactions, and various types of cytokines are continuously produced in cells during host defense reactions against harmful substances from pathogens and pathogens. It is secreted in large quantities, and the periodontal tissue destruction occurs. Such cytokines include interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-α) and prastaglandin E ₂ (PGE _2). ).

Lipopolysaccharide (LPS), secreted from Gram-negative bacteria, acts strongly on proinflammatory agents and causes a number of host-induced inflammatory processes. In particular, Aggregatibacter ( Aggregatibacter) LPS of actinomycetemcomitans (AA) is an important cause of focal acute periodontitis and is known to participate in connective tissue destruction and bone resorption. LPS acts as a stimulant to the periodontal ligament cells and gingival fibroblasts of the periodontal tissues and is involved in inflammation of the periodontal tissues.

Periodontal ligament is a dense connective tissue located between the calcified tissue (alveolar bone) and the cementum of the tooth in the oral cavity and plays an important role in maintaining the teeth and maintaining the structural properties of the hard tissue. It is also known to be involved in repair and regeneration of adjacent bone and cementum in vivo (McCulloch CAG, Periodontology 2000 1: 16-25 (1993)).

Periodontal ligament cells are not composed of homogeneous cells but consist of several subpopulation cells with different phenotypes and functions. And progenitor cells of cells that form bone and cementum, such as the formation of calcified nodules (Cho MI et al., Calcif Tissue Int 50: 459-467 (1992); Goseki et al., Mech Ageing Dev 91: 171-183 (1996); Kawase et al., Bull Kanagawa Dent Coll 18: 135-141 (1990); McCulloch & Bordin, J Periodontal Res 26: 144-154 (1991).

Gingival fibroblasts are constituent cells of the gingival tissue that responds first to the external environment, and can regenerate and repair tissues without external stimulation. The regeneration process is different from the regeneration of skin muscles, which is more like a developmental regeneration.

Recent trends in the prevention and treatment of periodontal disease have been the most effective recognition of the mechanical removal of biofilms that have been traditionally done, but in addition, reports have been reported to have a good effect in combination with various drug treatments. Inflammatory response control of fibroblasts has been introduced as a way to prevent and control the progression of periodontitis, and research on the effects of various drugs on these cells is ongoing.

The scientific name of ginseng is Panax ginseng C.A. Meyer has long been used to promote health and treat diseases in many countries in East Asia. In general, ginsenoside (ginsenoside) and its metabolites are thought to have the main pharmacological effect of ginseng extract. The effects of ginseng revealed so far have been proved to be effective in various fields, such as enhancing immunity, anti-inflammatory effect, analgesic effect, hemostatic action or cardiovascular improvement. In particular, for the anti-inflammatory effect, anti-inflammatory and anti-allergic function by ginsenoside-Rh1, reduction of TNF-α by ginsenoside-Rb1, enhancement of immune function by ginsenoside-Rg1, and phospho-phosphate by ginseng extract Degradation of lipase 2 function and a decrease in the number of neutrophils have been reported.

According to a recent report by Rhule et al., When stimulating macrophages with LPS and treating Chinese ginseng (Panax notoginseng) extracts by concentration, the secretion of TNF-α and IL-6 is significantly reduced depending on the concentration. Expression of COX-2 and IL-1β mRNA involved in PGE ₂ production also decreased with ginseng treatment. In the case of TNF-α production, ginseng treatment before LPS stimulation was effective 24 hours before or at the same time, but ginseng treatment after 8 hours after LPS stimulation was ineffective. In addition, when treated with ginsenoside-Rb1 and Rg1 for the administration of components, the decrease in TNF-α production occurred, but the degree was weak compared to the extract.

In the present invention, the effect of LPS-stimulated periodontal ligament cells and gingival fibroblasts treated with ginseng or red ginseng saponin on the secretion of inflammatory substances IL-6, IL-8 and COX-2.

Throughout this specification many patent documents are referenced and their citations are indicated. The disclosures of the cited patent documents are incorporated by reference herein in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop a substance capable of preventing or treating periodontal disease, which is an inflammatory disease caused by destruction of the periodontal tissue by a large amount of cytokines secreted during the host's defense against the pathogen and pathogens. As a result, saponins (especially ginseng or red ginseng saponins) are expressed in inflammatory substances interleukin-6, -8 and COX-2 in periodontal tissues (periodontal ligament cells and gingival fibroblasts) stimulated with inflammation-inducing substances that cause periodontal disease. By confirming the inhibition, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for preventing or treating periodontal disease.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating periodontal disease comprising saponin as an active ingredient.

The present inventors have tried to develop a substance capable of preventing or treating periodontal disease, which is an inflammatory disease caused by destruction of the periodontal tissue by a large amount of cytokines secreted during the host's defense against the pathogen and pathogens. As a result, saponins (especially ginseng or red ginseng saponins) are expressed in inflammatory substances interleukin-6, -8 and COX-2 in periodontal tissues (periodontal ligament cells and gingival fibroblasts) stimulated with inflammation-inducing substances that cause periodontal disease. It was confirmed that it inhibits.

Saponin used as an active ingredient in the present invention may be a saponin contained in soybeans, green onions, deodeok, bellflower, buttercups, garlic, onions, ganoderma lucidum, ginkgo (plants), 칡, ginseng or red ginseng, preferably ginseng Or use saponin contained in the red ginseng extract.

As used herein, the term “ginseng” refers to Korean ginseng ( Panax ginseng), three sessions (P. quiquefolius), jeonchilsam (P. notoginseng), jukjeol three (P. japonicus), three yeopsam (P. trifolium), Himalayan cedar (P. pseudoginseng), Vietnam, three (P. vietnamensis) and American hemp ( Panax quinquefolium ), but is not limited thereto. Preferably, the ginseng used in the present invention is Korean ginseng ( Panax ginseng ).

As used herein, the term “red ginseng” means prepared by heating ginseng through steam or sun-drying, preferably steam.

Ginseng or red ginseng extract containing saponin as an active ingredient of the present invention includes extracts obtained by conventional extraction procedures known in the art. For example, (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol with water, (d) acetone The extract obtained by using (e) ethyl acetate, (f) chloroform, (g) butyl acetate or (h) 1,3-butylene glycol as an extraction solvent is included in ginseng or red ginseng extract. Preferably, the ginseng or red ginseng extract is obtained by using water, ethanol or a mixed solvent of ethanol and water as an extraction solvent, and most preferably, water is used as an extraction solvent.

On the other hand, it will be apparent to those skilled in the art that the extract of the present invention can be obtained ginseng or red ginseng extract having substantially the same effect using not only the aforementioned extraction solvent but also other extraction solvents. In addition, the extract of the present invention includes not only the extract by the above-described extraction solvent, but also the extract that has undergone a conventional purification process. Obtained by various additional purification methods, such as separation using ultrafiltration membranes having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). The fraction is also included in the ginseng or red ginseng extract of the present invention.

Ginseng or red ginseng extract containing saponin of the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

"Saponin" used as an active ingredient in the present invention has the same meaning as ginsenosides, that is, ginseng saponin unless otherwise specified. "Saponin" used in the present invention preferably means ginsenosides unique to ginseng or red ginseng. More preferably, saponins used in the present invention are (a) ginsenosides of the protopanaxadiol type, ie ginsenosides Ra1-3, Rb1-3, Rc, Rd, Rg3, Rh2, Ri, Rs1 and Rs2 , Malonyl-ginsenosides Rb1-2, Rc and Rd; And (b) ginsenosides of the protopananac triol type, ie ginsenosides Re, Rf, Rg1-2 and Rh1.

The saponins used in the present invention may be used in the form of isolated and purified saponins, but are preferably ginsenosides contained in the powder or extract of ginseng or red ginseng. That is, the powder or extract of ginseng or red ginseng containing saponin can be used in the present invention.

As used herein, the terms "periodontal disease" or "gum disease" are used interchangeably.

According to a preferred embodiment of the present invention, the saponin used as the active ingredient of the present invention is ginsenoside -Rg1, -Re, -Rf, -Rh1, -Rg2, -Rb1, -Rc, -Rb2, -Rd,- At least one ginsenoside selected from the group consisting of Rg3 and -Rh2, more preferably ginsenoside-Rg1, -Re, -Rf, -Rh1, -Rg2, -Rb1, -Rc, -Rb2, It is a mixture of -Rd, -Rg3 and -Rh2.

According to another preferred embodiment of the present invention, the saponin used in the present invention is enriched with Protopanaxadiol (PD) saponin, and more preferably, At least one ginsenoside selected from the group consisting of senoside-Rb1, -Rc, -Rb2, -Rd, -Rg3 and -Rh2.

According to another preferred embodiment of the present invention, the content ratio of protopanaxatriol (PT) to protopanaxatriol (PT) -based saponins of saponins enriched with protopanaxadiol-based saponins ( PD / PT) is 1.6-4.0.

The term PD used while referring to the content and ratio of the ProtoPanaxadiol (PD) -based saponins and Protopanaxatriol (PT) -based saponins means Rb1, Rc, Rb2, Rd, Rg3 and Rh2. , PT means Rg1, Re, Rf, Rh1 and Rg2.

According to another preferred embodiment of the present invention, the protopanaxadiol-based saponin is 60-80% by weight based on the total weight of saponin.

According to a preferred embodiment of the present invention, the content of ginsenosides -Rb1, -Rg1 and -Rh1 included in the mixture is 20-30% by weight based on the ginsenoside total weight.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the active ingredient of the present invention described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the above-described efficacy or activity of the active ingredient of the present invention.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Preferably, the dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg body weight.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

According to another preferred embodiment of the invention, the composition of the present invention has a therapeutic effect of periodontal disease, the composition is a toothpaste (or toothpaste), oral cleansing composition or composition for oral care. As used herein, the term “composition for preventing or treating periodontal disease” may be replaced with “composition for tooth and mouth care” or “composition for tooth and mouth cleaning”.

Saponin of the present invention can treat or prevent periodontal disease by promoting the physiological activity of the fibroblasts of gum tissue and regenerating damaged gum tissue through rapid healing of gum wounds.

According to a preferred embodiment of the present invention, the periodontal disease treated or prevented by the composition of the present invention is gum inflammation. In detail, it effectively inhibits inflammation-related cytokines (especially IL-6 and IL-8) and COX-2, an enzyme involved in the production of PGE ₂ , and has excellent anti-inflammatory effects and excellent antioxidants that inhibit cellular oxidative damage. The effect and excellent antimicrobial effect can effectively treat periodontal disease (gum disease).

According to a preferred embodiment of the present invention, saponin used as the active ingredient of the present invention is 0.01-70.0% by weight, more preferably 0.1-50.0% by weight, most preferably 5-20.0% by weight based on the total composition to be. When the weight of saponin as an active ingredient is less than 0.01% by weight, the effect is less likely to appear. When the weight of the saponin exceeds 70.0% by weight, the effect of increasing the content is very weak, and there is a problem in that formulation stability is not secured. .

The features and advantages of the present invention are summarized as follows:

(Iii) The composition of the present invention contains saponin, preferably saponin contained in ginseng or red ginseng extract as an active ingredient.

(Ii) Saponin used as an active ingredient of the present invention has an effect of preventing or treating periodontal disease, and in detail, is an enzyme involved in the production of inflammation-related cytokines (especially IL-6 and IL-8) and PGE ₂ . It can effectively treat periodontal disease (gum disease) by effectively inhibiting COX-2, excellent anti-inflammatory effect, excellent antioxidant effect to inhibit oxidative damage in cells and excellent antibacterial effect.

(Iii) The composition of the present invention can be safely applied to pharmaceutical compositions, toothpaste, oral treatment and oral protection composition because there is no cytotoxicity and skin side effects.

As described above in detail, the present invention provides a composition for preventing or treating periodontal disease, including saponin, preferably saponin contained in ginseng or red ginseng extract as an active ingredient. Saponin is used as an active ingredient of the present invention has a preventive or therapeutic efficacy of periodontal diseases, particularly inflammatory cytokines (in particular, IL-6 and IL-8) and the COX-2 enzyme which is involved in PGE ₂ produced It can effectively treat periodontal disease (gum disease) by effectively inhibiting the excellent anti-inflammatory effect, excellent antioxidant effect to inhibit the oxidative damage in the cell and excellent antibacterial effect. In addition, the composition of the present invention can be safely applied to pharmaceutical compositions, toothpaste, oral treatment and oral protection composition because there is no cytotoxic and skin side effects.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experimental Materials and Experimental Methods

sample

Ginseng extract gold base or red ginseng extract (KGC provided) containing saponin was used. The red ginseng extract fraction saponin used in the present invention is named saponin (saponin, SA), ginseng extract gold base RG 1 and red ginseng extract RG 2 respectively. The content of ginsenosides contained in the ginseng or red ginseng extract of the present invention is as described in Tables 1 to 3 below:

SA (Saponin) Water content Ginsenoside content (mg / crude saponin 100 mg) 2.50% Rg1 Re Rf Rh1 Rg2 Rb1 Rc Rb2 Rd Rg3 Rh2 Sum 0.90 1.66 2.48 1.89 7.60 6.91 5.74 3.49 2.46 9.17 0.21 42.51

RG1 (Ginseng Extract Gold Base) Water content Ginsenoside content (mg / wet Ext g) 35.20% Rg1 Re Rf Rh1 Rg2 Rb1 Rc Rb2 Rd Rg3 Rh2 Sum 1.32 2.56 1.28 0.65 2.34 7.90 5.89 5.03 2.41 3.16 3.09 35.63

RG2 (red ginseng extract) Water content Ginsenoside content (mg / wet Ext g) 32.20% Rg1 Re Rf Rh1 Rg2 Rb1 Rc Rb2 Rd Rg3 Rh2 Sum 0.43 0.72 1.06 1.04 4.96 3.55 1.81 1.75 1.70 5.40 0.10 22.52

Total ginsenosides were quantified using HPLC / UV (203 nm).

Cell culture

in media containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in α-MEM (alpha-Minimum Essential Medium, GIBCO) under 95% air, 5% CO ₂ and 37 ° C Human periodontal ligament cells (Seoul National University Cell Line Bank) were cultured aseptically. In addition, gingival fibroblasts (Seoul National University Cell Line Bank) were treated in DMEM (Dulbecco's Modified Eagle Medium) at 95% air, 5% CO ₂ , and 37 ° C in medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Incubated. Then, change the medium every 2-3 days and when the cells become semiconfluent, the cells are suspended in a culture dish using 0.125% trypsin-EDTA, and then the cell suspension is collected for 10 minutes at 1200 rpm. After precipitation by centrifugation, cell suspension was subcultured by making a cell suspension with medium. Experiments were performed by selecting cells of 6-8 passages. 1 × 10 ⁵ cells were dispensed into 6 well plates for RNA extraction, and 3.3 × 10 ³ cells were dispensed into 96 well plates for MTS experiments. The cells used in each experiment were incubated for two days to allow the cells to stably adhere and proliferate after dispensing, and the cell culture was exchanged with a medium containing 1% fetal bovine serum, followed by each experiment.

Eggrigatibacter  Actinomycetem Comitans Aggregatibacter actinomycetemcomitans , A.A.) lipopolysaccharide ( LPS ) extraction

AA (Seoul National University Strain Bank) was used in a Todd-Hewitt broth containing 1% yeast extract at 37 ° C. and anaerobic (80% N ₂ , 10% H _2). And 10% CO ₂ ). LPS was extracted using the LPS extraction kit and degrading the protein through proteolytic enzyme K and solubilization precipitation. The extracted LPS was freeze-dried, quantified, dissolved in deionized water, and stored at -20 ° C.

A.A. LPS of Life  Confirm

Normally, when LPS is treated to cells, intercellular adhesion molecules-1 and ICAM-1 are expressed. The extracted LPS of AA was confirmed to be bioactive through FACS (fluorescence activated cell sorter) analysis. To measure bioactivity, each cell was treated with 100 ng / ml AA LPS and 10 ng / ml E. coli LPS for 18 hours. Treated cells were detached with 20 mM trypsin and centrifuged to obtain only cell pellets and then suspended in PBS. Anti-ICAM-1 rabbit polyclonal primary antibody was attached to each suspension and reacted at 4 ° C for 30 minutes. After washing with PBS, anti-rabbit-FITC-conjugated secondary antibody was added and reacted at 4 ° C. for 30 minutes, then washed with PBS again and analyzed by FACScan flow cytometer (BD FACSCant II).

Sodium Nitroprusside ( SNP ) And A.A. LPS Treatment of

SNPs (Sigma) and A.A. Two hours before the LPS treatment, the red ginseng saponin-based extracts SA, RG 1 and RG 2 of the present invention were treated at each concentration. Nitrogen oxides (NO) were treated with SNPs at 4.0 mM and A.A. LPS was added at 0 μg / ml, 1 μg / ml and 5 μg / ml to react for 18 hours.

MTS  Assessment of Cell Proliferation Using Assay

MTS (3- (4,5-dimethylthiazole-2) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H- by mitochondrial dehydrogenase expressed in metabolic cells Tetrazolium, intramolecular salt, and Sigma) were converted to formazan, a blue water-soluble product, to determine the proliferation of the cells.

Periodontal ligament cells and gingival fibroblasts were incubated in 96-well plates and reacted with each sample. After 4 days of incubation, MTS solution was added to the medium for 3 hours at 37 ° C, followed by microplate reader (Bio-). Absorbance (OD) value was measured using Tek).

RNA  Differentiation gene expression at level

Using RNA extraction kits, each sample and A.A. After extracting RNA of periodontal ligament cells treated with LPS and quantifying the extracted RNA, cDNA was synthesized using reverse transcriptase. A.A. Primers of cyclooxygenase 2 (COX 2), interleukin-6 (IL-6), interleukin-8 (IL-8) and GAPDH expressed by LPS treatment were prepared to perform RT-PCR of the synthesized cDNA. . PCR products were then electrophoresed on 1.5% agarose gel and stained with EtBr.

division Omnidirectional Primer Reverse primer COX2 5'-TTCAAATGAGATTGTGGGAAAATTG-3 ' 5'-AGATCATCTCTGCCTGAGTATCTT-3 ' IL-6 5'-ATGAACTCCTTCTCCACAAGCGC-3 ' 5'-GAAGAGCCCTCAGGCTGGACTG-3 ' IL-8 5'-ATGACTTCCAAGCTGGCCGTGGCT-3 ' 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3 ' GAPDH 5'-CACTGACACGTTGGCAGTGG-3 ' 5'-CATGGAGAAGGCTGGGGCTC-3 '

Experiment result

Periodontal Ligament Cell  Check survival rate

First, the reaction of samples of periodontal ligament cells was examined to determine the concentration of ginseng or red ginseng-related reagent. As can be seen in Figure 1, by treating the three ginseng or red ginseng extracts (SA, RG 1 and RG 2) by concentration using the MTS analysis, the survival rate of the periodontal ligament cells drastically dropped at more than 100 ㎍ / ㎖ It can be seen that RG 1 affects the periodontal ligament cell viability at 1000 μg / ml and RG 2 at 750 μg / ml or more.

SNP  For stimulation Periodontal Ligament Cell  Check survival rate

In addition, the concentration of reagents that protect the periodontal ligament cells against stimulation of external harmful substances was examined. Sodium nitroprusside (SNP) is a nitric oxide (NO) generator, resulting in lower cell survival rates when applied in excess. As can be seen in Figure 2, the periodontal ligament cell survival rate is lowered to 40% or less after 4 mM treatment to the periodontal ligament cells. Meanwhile, various concentrations of SA, RG 1 and RG 2 were pretreated to the periodontal ligament cells and 4 mM SNP. As a result, cell viability was 75 μg / ml for SA, 500 μg / ml for RG 1 and 750 for RG 2. It was found that the best results were obtained at μg / ml.

Based on the experimental results, the concentration of reagents to be used in the periodontal ligament cell experiment was determined to be 60 μg / ml for SA, 600 μg / ml for RG 1 and 700 μg / ml for RG 2. In addition, it was confirmed that the ginseng or red ginseng extract containing saponin of the present invention alone protects the periodontal ligament cells and increases the survival rate.

LPS  Against noxious stimuli Life  Confirm

Periodontal ligament cell protective function of ginseng extracts against LPS harmful stimuli was first performed using the most widely used LPS of E. coli . Although the results were not shown, when the ginseng or red ginseng extract of the determined concentration was pre-treated and treated with LPS of E. coli , the cell survival rate was significantly higher than that of the control group without the ginseng extract. However, grasping the function of ginseng extract against AA LPS, which is an important cause of periodontitis, is the main purpose of the present invention, so AA LPS was produced with the help of Seoul National University strain bank. To verify the effectiveness of the produced AA LPS, we compared E. coli LPS and bioactivity.

As can be seen in Figure 3, confirming the secretion of ICAM-1 was found to appear to be valid. In addition, as can be seen in Figure 4, after treatment of periodontal ligament cells with the concentrations of 1 μg / ml and 5 μg / ml, which are commonly used AA LPS, the cell survival rate was significantly different from that of the control group. It can be seen that it is not visible. This means less toxicity than E. coli LPS, and the number of cells in the amount of cytokine secretion is irrelevant.

COX Of -2 and cytokines (interleukin-6 and 8) Expression  Confirm

The concentration of each sample was determined through the results of the proliferation experiment of the cells treated with SNP and the proliferation experiment of the cells of ginseng extract, and A.A. After treatment with LPS 1 μg / ml and 5 μg / ml, the expression of enzymes and cytokines was confirmed by RT-PCR (FIG. 5).

As can be seen in Figure 6, COX-2 is an enzyme that mediates the production of PGE ₂ cells in the periodontal ligament cells increased the amount produced according to the concentration during LPS treatment. However, in the group pre-treated with ginseng or red ginseng extract 2 hours before LPS treatment, the amount produced was reduced compared to the control without LPS treatment. On the other hand, the results were different depending on the type of ginseng or red ginseng extract. In the case of SA and RG 1, the production of COX-2 was greatly reduced when the concentration of LPS was 1 ㎍ / ml, but not significantly at 5 ㎍ / ml. I could not see. RG 2, on the other hand, reduced COX-2 production in both LPS concentrations of 1 μg / ml and 5 μg / ml.

As can be seen in Figure 7, the production of periodontal ligament cells of IL-6 for LPS stimulation also increased compared to the untreated control group, but it can be seen that the concentration is not affected. However, in the group treated with ginseng or red ginseng extract 2 hours before LPS treatment, the amount of IL-6 produced was significantly reduced when the concentration of LPS was 1 ㎍ / ml. In addition, it was confirmed that the amount of IL-6 produced also decreased when the concentration of LPS is 5 μg / ml. In conclusion, with respect to the expression of IL-6, it can be seen that the difference between the three types of reagents is not large.

As can be seen in Figure 8, the production of periodontal ligament cells of IL-8 for LPS stimulation showed similar results as in the case of IL-6. However, in the group treated with ginseng or red ginseng extract 2 hours before LPS treatment, the amount of IL-8 produced was 5 μg / ml when the concentration of LPS was 1 μg / ml, unlike IL-6. The amount was greatly reduced.

SNP  And LPS  For stimulation Gingival fibroblast  Check survival rate

The response of gingival fibroblasts to ginseng or red ginseng extract was examined. As a result of treatment of three ginseng or red ginseng extracts (SA, RG 1 and RG 2) by concentration using MTS analysis, as shown in FIG. 9, SA and RG 1 have a cell viability at the concentration applied to the periodontal ligament cells. It was confirmed to decrease. However, no significant change was found for RG 2. On the other hand, when treated with SNP also increased the cell survival rate was found that ginseng or red ginseng extract has a cell protective function for gingival fibroblasts.

As shown in FIG. 10, the results of LPS stimulation were similar, indicating that cell viability decreased when SA and RG 1 were treated, and that there was no significant difference in cell viability when RG 2 was treated. have.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a graph showing the results of confirming the toxicity of the reagent on the cells through the treatment of human periodontal ligament cells, the active ingredients of the present invention SA, RG 1 and RG 2 after MTS analysis. SA is a saponin fraction of red ginseng extract, RG 1 is a ginseng extract containing saponin and RG 2 represents a red ginseng extract containing saponin.

Figure 2 is treated with each of the concentrations SA, RG 1 and RG 2 of the active ingredients of the present invention to human periodontal ligament cells after reacting SNP (4 mM) to generate nitric oxide (NO) cells after 18 hours This graph shows the result of measuring toxicity. The SNP stands for sodium nitroprusside.

3 is a graph showing the result of confirming the bioactivity by FACS analysis of the extracted AA LPS, E. coli LPS. The AA represents Aggregatibacter actinomycetem comitans (AA).

4 is A.A. in periodontal ligament cells. LPS treatment and incubation for 4 days and then the graph showing the results of checking the cell proliferation.

Figure 5 is A.A. in periodontal ligament cells using RT-PCR. 18 hours after LPS treatment, the photo shows the expression of enzyme COX-2 and cytokine.

Figure 6 is A.A. in periodontal ligament cells using RT-PCR. Photos and graphs showing the expression level of COX-2 18 hours after LPS treatment.

7 is A.A. in periodontal ligament cells. Photos and graphs showing the results of confirming the expression of IL-6 using RT-PCR 18 hours after LPS treatment.

8 is A.A. in periodontal ligament cells. After 18 hours of treatment with LPS, the expression level of IL-8 was confirmed using RT-PCR.

9 is a cell after treatment with gingival fibroblasts, SNP (4 mM) reagent that generates the intracellular nitric oxide (NO) after 18 hours of treatment with each of the active ingredients of the present invention SA, RG 1 and RG 2 at different concentrations This graph shows the result of measuring toxicity.

10 is A.A. in gingival fibroblasts. It is a graph showing the results of confirming the proliferation of cells after treatment with LPS and treatment for 4 days at each concentration of the active ingredients SA, RG 1 and RG 2 of the present invention.

Claims (11)

Periodontal disease prevention or treatment composition comprising saponin (saponin) as an active ingredient. The composition of claim 1, wherein the saponin is contained in ginseng or red ginseng extract. The method of claim 1, wherein the saponin is selected from the group consisting of ginsenosides -Rg1, -Re, -Rf, -Rh1, -Rg2, -Rb1, -Rc, -Rb2, -Rd, -Rg3 and -Rh2 At least one ginsenoside. The method of claim 3, wherein the saponin is a mixture of ginsenosides -Rg1, -Re, -Rf, -Rh1, -Rg2, -Rb1, -Rc, -Rb2, -Rd, -Rg3 and -Rh2 Composition. The composition of claim 1, wherein the saponin is enriched with Protopanaxadiol (PD) saponin. 6. The method according to claim 5, wherein the protopanaxadiol-based saponin is at least one ginsenoside selected from the group consisting of ginsenoside-Rb1, -Rc, -Rb2, -Rd, -Rg3, and -Rh2. Composition. The content ratio (PD / PT) of protopanaxatriol (PT) to saponin of protopanaxatriol (PT) based on saponin enriched with the protopanaxadiol-based saponin Is 1.6-4.0. 6. The composition according to claim 5, wherein the protopanaxadiol-based saponins are 60-80 wt% based on the total weight of saponins. The composition of claim 4, wherein the content of ginsenosides-Rb1, -Rg1 and -Rh1 included in the mixture is 20-30% by weight based on the total weight of ginsenosides. The method of claim 1, wherein the composition comprises (a) a pharmaceutically effective amount of saponin; And (b) a pharmaceutically acceptable carrier. The composition of claim 1, wherein the composition has a therapeutic effect of periodontal disease, and the composition is a toothpaste, a mouthwash composition, or a composition for oral care.

Images