KR101733436B1 - Composition for preventing or treating periodontal disease - Google Patents

Abstract

The present invention relates to a method for preventing or treating periodontal disease using an extract of a superfine plant having an activity of inhibiting the growth of periodontal disease-causing bacteria, inhibiting the expression of MMPs destroying the periodontal tissue extracellular matrix and promoting osteoclast bone regeneration ≪ / RTI >

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating periodontal disease,

The present invention relates to a method for producing zanthoxylum The present invention relates to a composition for preventing or treating periodontal disease.

As life expectancy is extended due to improvement of people's standard of living and medical technology development, importance of oral health care for increasing the quality of life of the elderly population is increasing. Dental caries and periodontal diseases are typical dental diseases that can be caused by oral health problems. In particular, periodontal diseases show various clinical symptoms such as gingival bleeding and swelling, formation of periodontal ligament, loss of attached gingiva, alveolar bone destruction and bad breath , And is a major cause of tooth loss (Ali RW et al., J. Clin. Periodontol. 1997 24: 830-835; Socransky SS et al., J. Clin. Periodontol. 1998 15: 440-444). Periodontal disease can be divided into gingivitis and periodontitis depending on the severity of the disease. It is a relatively light and quick-recovery type of periodontal disease called gingivitis, which is limited to soft tissues such as gingival bleeding, And gingival bone (alveolar bone) around the alveolar bone and the absorption of the alveolar bone occurs (petersen and Ogawa, J Periodontol. 2005 76; 2187-2193).

It is known that periodontal disease is one of the most common chronic diseases that cause infectious disease, but it is known to be present in 10 to 60% of adults (Xiong X et al., BJOG 2006; 135-143; Papapanou PN. Ann Periodontol 1996 1: 1-36, Albandar JM and Rams TE, Periodontol 2002; 29, 7-10). Periodontal disease is a specific inflammatory disease caused by microorganisms that coexist in biofilm called oral plaque and microbial secretions that cause inflammation (Feng Z and Weinberg A, Periodontol 2006, 40; 50-76. ) Is triggered by insufficient oral care, smoking, and aging (Burt, J Periodontol. 2005 76; 1406-1419). Recent studies suggest that periodontal disease is associated with systemic diseases such as arteriosclerosis, myocardial infarction, stroke, diabetes, and pregnancy complications (Desvarieux, M. et al., Circulation, 2005 111; 576-582; Offenbacher S et al., J. Periodontol. 1996 67: 1103-1113; Garcia RI et al., Periodontol. 2001 25: 21-36; Champagne CM. Et al., J. Int. Acad. Thus, the importance of oral health management to prevent periodontal disease as well as treatment of periodontal diseases that have already occurred is further increased. It is getting bigger.

There are more than 350 microorganisms in the oral cavity. Bacteria involved in the progression of periodontal disease are usually less than 5%. The oral cavity, which contains fewer oxygen contacts and contains a large amount of dead epithelial cells, saliva proteins and food waste, provides an environment in which specific microorganisms can live, and these microorganisms cause diseases. Among the microorganisms present in the subgingival plaque in the lower part of the gingiva as a major cause of periodontal disease, Porphyromonas belonging to the so-called "red complex" gingivalis , Tannerella forsythia , Treponema denticola and Prevotella intermedia, etc. are known. Fusobacterium , which plays an important role in the process of development of late plaques composed of various microorganisms that can cause early disease, nucleatum, etc. are known (Loomer PM et al., Periodontol 2000. 2004; 34: 49-56). These microorganisms release toxins such as lipopolysaccharide, nuclease, leukotoxin, and hydrogen sulfide, which are metabolites or cell wall components, during the process of adhering or infiltrating the host tissues and cells, causing an inflammatory reaction to the surrounding periodontal tissues. At this time, cytokines produced by host cells and immune substances produced in response to cytokines further destroy periodontal tissue (Noda D et al. J Periodontal Res 2007; 42 (6): 566-571; Dong Chen et al., 2008 Pharmacology 82 (4): 264-269). When inflammatory responses are induced in periodontal cells, matrix metalloproteinases (MMPs), including inactive collagenases, are activated or their secretion is stimulated. Metrix mallotropeptidease is a calcium and zinc-dependent peptidase secreted from a variety of cells such as polymorphonuclear leukocytes, macrophages, gingival fibroblasts, bone cells, and acts at neutral pH and utilizes a variety of extracellular substrates as substrates (Jeng AY et al., Bioorg Med Chem Lett 1998: 8: 10-15, 1998). In this study, we investigated the effects of matrix metalloproteinase on collagen degradation, 897 .; Emingil G et al., J Clin Periodontol 2008 35 (3): 221-229). With this progression, attachment loss of the connective tissue and alveolar bone loss lead to tooth loss.

Periodontal disease is characterized by several reactions, including the action of certain bacteria, host inflammation, MMP, and bone loss. For the treatment of periodontal disease, continuous administration of general antiinflammatory or antimicrobial agents may have various side effects such as digestive disorders, hypersensitivity reactions, renal toxicity, oral toxicity, tooth discoloration and so on. Studies are being actively conducted to search for natural substances.

It is an object of the present invention to provide a composition for the prevention or treatment of periodontal disease using a plant extract.

Other objects and specific objects of the present invention will be described below.

The present invention relates to a composition for the prevention or treatment of periodontal disease, which comprises an extract of P. falciparum as an active ingredient, as confirmed in the following examples and experimental examples. The present inventors carried out an experiment to confirm the antimicrobial effect of the extract of P. falciparum on the periodontal disease-causing bacteria and the dental caries bacteria, and it was confirmed that the extract of the present invention has a specific antimicrobial activity against periodontal disease-causing bacteria. As periodontal disease progresses, inflammation reaction occurs by periodontal disease causing bacteria, and the expression or activity of MMP among various proteins induced by inflammatory reaction increases, destroying the extracellular matrix (ECM) The extract of the present invention reduced the expression of MMP in HGF-1 cells, which are gingival fibroblasts, and the expression of TIMP1, which regulates the activity of MMP, was increased. In addition, the extracts of Bombyx mori showed bone regeneration activity by regulating osteogenic gene expression. Therefore, the composition of the present invention has antimicrobial effect against a specific microorganism causing periodontal disease, inhibits the expression of MMP affecting periodontal tissue and has an osteoprotegerase activity, and thus can be provided as a composition for preventing or treating periodontal disease have.

In view of the above, the present invention can be grasped as a composition for preventing or treating periodontal disease, which comprises an extract of P. falciparum as an active ingredient. In another aspect, the present invention relates to a method for the treatment of periodontal disease, ( Porphyromonas gingivalis , or Fusobacterium nucleatum , which is an antimicrobial composition.

Zanthoxylum piperitum ) is a rhododendron and deciduous shrub with the leaves are alternate phyllotaxis and bloom mainly in May to June. It is distributed in Korea, Japan and China. Young leaves are used for food, fruit for medicinal use or spice, and the skin of fruit is used for spice. Since ancient times, pine berry fruit has helped Yang, helped digestion, prevented diarrhea, stopped diarrhea, and was widely used for fever, detoxification, antiparasitic, analgesic and anti-inflammatory drugs. Fruit peels are known to be effective against headaches and insomnia. Leaves were used as an antidote to food during cooking. Studies on antioxidant, antioxidant, anti-thrombotic, and anti-inflammatory effects have been known mainly in the oil field of chestnut tree (Aihara T. Yakugaku Zasshi 1950 70: 409-411; Kim KK et al., J Life Sci 2007 17: 1121-1128; Jang MJ et al., J Korean Soc Food Sci Nutr 35: 21-27).

The primrose may be any of roots, stems, leaves, flowers, fruits and / or seeds, preferably leaf parts.

As used herein, the term "extract" refers to any substance that can be extracted regardless of the extraction method, including water, anhydrous or lower alcohol having 1-4 carbon atoms (methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol ), Extracts obtained by extracting with acetone, ethyl acetate, methylene chloride, chloroform, 1,3-butylenecyl alcohol, hexane, diethyl ether or a mixed solvent thereof and extracts obtained by fractionation with the above- . As long as it is extracted through the step of immersing the object to be extracted in the extraction solvent, it is understood that any of extraction methods such as cold beating, refluxing, heating, and ultrasonic wave can be applied. Nevertheless, the extract is preferably obtained by extracting the extraction object with water, ethanol or a mixed solvent thereof, and includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed. And more preferably an extract obtained by using a mixed solvent of water and ethanol (80% ethanol) as an extraction solvent.

As used herein, the term "active ingredient" alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.

In the meantime, the "composition for the prevention or treatment of periodontal disease" of the present invention may contain any amount (effective amount) as long as it can exhibit the periodontal disease prevention or therapeutic activity, Will be determined within the range of 0.001 wt% to 99.900 wt% based on the total weight. The term "effective amount" as used herein refers to the amount of active ingredient capable of preventing, ameliorating, treating, or delaying the onset of symptoms of hangover. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

In the present specification, the term " periodontal disease "is also referred to as a style, and can be understood as a disease occurring in periodontal tissues including gingivitis and periodontal gingiva (gums), periodontal ligament, and bone tissue.

Terms that are not specifically defined in this specification are intended to have a Korean national meaning or a meaning commonly used in the art.

The composition for preventing or treating periodontal disease and antimicrobial composition (hereinafter, referred to as "composition of the present invention ") of the present invention is, in a specific embodiment, a toothpaste, a liquid oral mouthwash, a dietary film, And the like, and each composition can be prepared by a conventional method.

When the composition for preventing or treating periodontal disease of the present invention is formulated into a toothpaste, the toothpaste of the present invention contains an extract of a superfine tree as an essential ingredient, and further contains a wetting agent, a thickening agent, a foaming agent, a preservative, a sweetening agent, May be further contained.

The thickening agent of the toothpaste is a component that prevents phase separation depending on time by combining an inorganic powder component insoluble in water and a liquid component in the composition and imparts a viscosity to the composition. Examples of the component include carboxymethyl cellulose (CMC), carrageenan, Gum arabic, xanthan gum, other gums, and the like may be mixed and used.

As other additives, an abrasive such as calcium phosphate which gives a tooth surface gloss, a brightener such as a fluorine agent or a titanium oxide having a cavity inhibiting function, or an edible pigment such as blue No. 1 can be used.

In addition, when the composition of the present invention is formulated into a toothpaste form, it may further include another ingredient for improving the functionality. Examples of the ingredient for preventing gingivitis or periodontitis include aminocaproic acid, allantoin, allantoin chlorohydroxyaluminum, Tranexamic acid, pyridoxine hydrochloride, sodium chloride, tocopherol acetate, and the like. Further, sodium pyrophosphate may be further mixed and used as a component for preventing dental deposition. As toothpaste components having a plaque removal effect, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, precipitated calcium carbonate, calcium carbonate, tricalcium phosphate, colloidal silicon dioxide, silicon dioxide, calcium phosphate anhydride, hydrated silicon dioxide, May be further mixed and used.

In addition, when the composition for preventing or treating periodontal disease of the present invention is formulated into an oral cleanser, the oral cleanser of the present invention contains an extract of Chrysanthemum morifolium extract as an essential ingredient and further contains a wetting agent, a foaming agent, a preservative, a sweetener, And the like.

The wetting agent may be a mixture of at least one member selected from the group consisting of glycerin, sorbitol solution, amorphous sorbitol solution, polyethylene glycol, and polyhydric alcohol such as propylene glycol as a component for suppressing the evaporation of water in the mouth rinse composition.

The foaming agent has an effect of emulsifying or solubilizing a water-soluble component and an oil-soluble component in an oral cleanser composition, and has a cleansing effect in the oral cavity. Examples thereof include sodium lauryl sulfate, sodium N-lauroylsalicylate, At least one selected from the group consisting of anionic and nonionic surfactants such as salts, sucrose fatty acid esters, polyoxyethylene hardened castor oil, sorbitan fatty acid esters and polyoxyethylene polyoxypropylene copolymers can be used in combination.

The preservative is a component for prolonging the preservation period of the oral cleanser, and methyl paraoxybenzoate, benzoic acid, sodium benzoate and the like can be used.

The sweetener and / or fragrance may be saccharin sodium, aspartame, stevioside, licorice acid, and the like. Examples of the sweetener include peppermint, spearmint oil, Menthol, carbo- nons, anethole, and augenol.

As other additives, stabilizers such as sodium citrate for pH control, fluorinating agents having a cavity inhibiting function, astringent agents such as zinc chloride, and food colors such as blue No. 1 can be used.

Ethanol may be used as a solvent for the composition of the oral cleanser, and the remaining components are made of purified water.

In another specific embodiment, the composition of the present invention can be identified as a food composition.

When the composition of the present invention is identified as a food composition, it can be used as a food composition such as beverage such as juice, carbonated beverage, ionic drink, processed oil such as milk and request route, gum, rice cake, Korean food, bread, , Capsules, and the like.

The food composition of the present invention can be manufactured as a health functional food, a health supplement food, a special nutrition supplement food, a functional beverage and the like.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

As the mineral, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium and zinc can be used.

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

Examples of the preservative which can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate and EDTA (ethylenediaminetetraacetic acid).

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

The composition of the present invention may be identified as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be formulated into a wide variety of dosage forms, including pharmaceutically acceptable carriers, excipients and the like, in addition to the active ingredient, and may be formulated into a wide variety of dosage forms such as a localized preparation such as cream, lotion, ointment (semi-solid external drug), micro loo emulsion, (TTS) (e.g., patches, bandages, etc.), spray, dog goggles, and the like.

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin and the like), alginic acid, and the like. Depending on the formulation of the pharmaceutical composition of the present invention, one or more suitable ones may be selected and used. Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences (19th ed., 1995). The pharmaceutical composition of the present invention may further comprise an emulsifier (e.g., TWEENS), a wetting agent (e.g., sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, a preservative, water, saline, a phosphate buffer solution and the like.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

As described above, according to the present invention, it is possible to provide a composition for preventing or treating periodontal disease, which comprises an effective component of a plant extract.

The composition for the prevention or treatment of periodontal disease of the present invention may be manufactured into a toothpaste, a mouthwash, a dietary film, a mouse spray or chewing gum, or may be used as a pharmaceutical composition or a food composition.

Figure 1 shows the results of confirming the inhibitory effect on the growth of the periodontal disease strain of P. ficus-indica extract. The left side was treated with 0.2 mg and the right side was treated with 2.0 mg. [A, P. gingivalis ; B, F. nucleatum ; C, S. mutans ]
FIG. 2 shows the results of expression of TIMPs and MMPs mRNA in HGF-1 cells by the extract of Ficus spp. In HGF-1 cells.
FIG. 3 shows the results of expression of osteogenic markers by MG-63 cells.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Manufacture of Secondary Pinus densiflora Extract

Zanthoxylum purchased from Jeju area piperitum ) leaves were shaded after shading. Three pieces of the first dried pine tree were immersed in 80% ethanol for 48 hours and repeatedly extracted three times by ultrasonic disruption. The extract was filtered, concentrated under reduced pressure, and the solvent was removed, followed by lyophilization to obtain a powdery extract. The extraction yield was 21.3 wt% with respect to the dry sample.

< Experimental Example  1> Identification of periodontal disease improving activity of extracts

<Experimental Example 1-1> Inhibitory effect of periodontal disease-inducing bacteria

In order to confirm the effect of the supernatant extract of the present invention on the growth of periodontal disease-causing bacteria, a disk diffusion assay was performed.

Porphyromonas gingivalis (KCTC 5352), Fusobacterium nucleatum (KCTC 2640), Streptococcus Mutans (KCTC 3065) strain was used in the experiment. P. gingivalis is known to be the most common chronic periodontitis strain in periodontal disease of adults and F. nucleatum is known to play an important role in the development of late periodontal disease. S. mutans is known to be a representative organism.

&Lt; Culture of strain &

(A) 1 g of P. gingivalis , 1 g of Trypticase soy broth, 1 g of yeast extract, 1 ml of 5 ㎎ / ㎖ Hemin solution (Hemin 62.5 mg / 0.125 ml 1N NaOH + DW 12.5 ml), 1 mg / ml Menadione solution (menadione 20 mg / 20 ml EtOH), and 5% sheep blood and 1.5% agar were added to the agar culture medium.

(B) In order to culture F. nucleatum , the cells were cultured in a medium supplemented with 37 g of Brain heart infusion, 5 g of yeast extract, 1 ㎖ of 5 ㎎ / ㎖ hemin solution and 10 ㎖ of 1 mg / ml Menadione solution, When cultured in medium, 5% sheep blood and 1.5% agar were added.

(C) S. mutans was cultured in a medium supplemented with 37 g of brain heart infusion per liter, and 1.5% agar was added when cultured in agar medium.

Each of the microorganisms was cultured in an anaerobic tank in each liquid medium. The gas pack was changed every 2 days. When the OD600 value was 0.8 to 1, subculture was performed.

<Disc diffusion method>

To confirm the antimicrobial activity against periodontal disease, the disk diffusion method was used. After each strain was cultured in the medium, when the OD600 value reached 0.8 or more, an appropriate amount was taken using a sterilized cotton swab and the resultant was smeared.

The extracts were prepared at 10 mg / ml and 100 mg / ml, and then 20 μl of each extract was added to 8 mm paper disks (ADVANTEC, JAPAN) to prepare paper disks at a concentration of 0.2 and 2 mg . Each of the paper disks was placed on a microorganism-free medium and cultured for 48 hours in an anaerobic incubator (OXOID, USA) at 37 ° C. The diameters of inhibitory rings were measured and the results are shown in Table 1 and FIG. 1, Respectively. As a positive control, 10 μg of erythromycin was used.

<Result>

Inhibitory effect of extracts of P. ficus-indica on the growth of periodontal bacteria (unit: cm) Positive control group Herb extract 0.2mg Mulberry extract 2.0mg P. gingivalis 4.5 2.6 4.7 F. nucleatum 3.6 2.6 4.2 S. mutans 5.3 - -

As shown in [Table 1] and [Fig. 1], growth inhibitory effect on the periodontal disease-inducing bacteria and dental caries-inducing bacteria was not observed in S. mutans , a dental caries inducing strain, , And P. gingivalis and F. nucleatum , which are pathogenic bacteria of periodontal disease. Especially, treatment with high concentration (2 mg) showed similar or superior growth inhibitory effect to erythromycin, which is a positive control group. From these results, it can be expected that the bark extract of the present invention will have a specific effect on periodontal disease.

<Experimental Example 1-2> Inhibitory effect of MMP expression on periodontal tissue cells

Gingivitis and periodontitis are caused by gingival associations and the activity of plaque bacteria in the plaque accumulated on the tooth surface adjacent to the gingival sulcus. Lipolysaccharide (LPS) and endotoxin, which are major antigens of gingival bacillus, cause inflammation reaction in gingival tissue. In order to eliminate this inflammation reaction, immune response by immune cells occurs inside the gingival tissue. It is known to regulate the expression of MMP genes in relation to the progression of periodontal disease among various actions caused by inflammatory factors such as IL-1, TNF-α, TGF-α, β and IFN-γ in periodontal tissues. (MMP) has been reported to modulate the degradation of cell interstitials by blocking activity by TIMP (tissue matrix metalloproteinases inhibitor). In this study, we investigated the effect of the extracts of Mulberry (Pinus densiflora) extracts on HGF-1 cells, which are human gingival fibroblasts, on MMPs And TIMPs expression.

<Cytotoxicity confirmation experiment>

HGF-1 (human gingival fibroblast) cells purchased from ATCC were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37 ° C and maintained at 5% CO 2 Lt; / RTI &gt; The cultured cells were seeded at a density of 1 × 10 4 cells / well in a 96-well plate. After 24 h, the extracts were treated with MTT solution (0-100 μg / ml) nm.

< MMP  Control confirmation experiment>

HGF-1 cells were seeded at a density of 1 × 10 ^ 6 cells / well in a 6-well plate and treated with LPS (10 μg / ml) and samples (25, 50 and 100 μg / ml). Then, total RNA was extracted from each cell using trizol and quantified using Nanodrop. The final quantified total RNA was synthesized by cDNA synthesis kit (TaKaRa). The synthesized cDNA was prepared by reacting each primer and SYBR green at 95 ° C for 10 minutes, followed by 95 ° C for 30 seconds, 57 ° C for 1 minute, and 72 ° C 30 seconds was repeated for 40 cycles and then relative quantified. The primers used for confirming the amount of MMP expression induced by induction of inflammation are shown in [Table 2] and the results are shown in [Fig. 2].

Primer sequence Gene name Primer sequences (5'-3 ') GAPDH 5-GCTTAAGAGACAGCCGCATCT-3 (sense)
5-CGACCTTCACCATTTTGTCTACA-3 (antisense) MMP-2 5-TCCCGAGATCTGCAAGCAAG-3 (sense)
5-AGAATGTGGCCACCAGCAAG-3 (antisense) MMP-3 5-TGATGGGCCTGGAATGGTC-3 (sense)
5-TTCATGAGCAGCAACCAGGAATAG-3 (antisense) MMP-7 5-GACATTGCAGGCATCCAGAAGTTA-3 (sense)
5-AGGGCGTTTGCTCATTCCAG-3 (antisense) MMP-9 5-AGCCGGGAACGTATCTGGA-3 (sense)
5-TGGAAACTCACACGCCAGAAG-3 (antisense) MMP-13 5-CCCTGGAATTGGCGACAAAG-3 (sense)
5-GCATGACTCTCACAATGCGATTAC-3 (antisense) TIMP-1 5-CATCTCTGGCCTCTGGCATC-3 (sense)
5-CATAACGCTGGTATAAGGTGGTCTC-3 (antisense) TIMP-2 5-GACACGCTTAGCATCACCCAGA-3 (sense)
5-CTGTGACCCAGTCCATCCAGAG-3 (antisense)

<Result>

As a result of the cytotoxicity test, the extract of P. ficus did not show any toxicity to HGF-1 cells (IC ₅₀ ≥100) at all concentrations in the gingival fibroblast. Thus, inflammatory response was induced by treatment with LPS in gingival fibroblasts, and MMP expression was confirmed by the extract of P. ficus.

The expression of MMPs and TIMPs mRNA in Hipp-1 cells was measured and the expression of TIMP1 was increased in a concentration-dependent manner and the expression of MMP1, 2 and 9 was decreased. However, expression of TIMP2 showed a slight decrease, and no change was observed for MMP3 and 7. MMPs that play a major role in human oral health and periodontitis are known as MMP1, 2, and 9 (Makela et al., J Dent Res. 1994 73: 1397-1406 .; Seguier S et al., J Periodontol 2001 72: 1398-1406 .; Ejeil AL et al., J Periodontol 2003: 74: 188-95). Thus, the extract of the present invention is effective for the prevention of periodontal disease by inhibiting inflammation and extracellular matrix breakage Able to know.

< Experimental Example  2> Cherrywood extract Bone regeneration  Check the effect

Periodontal disease can cause alveolar bone uptake and tooth loss can occur if it continues. Therefore, we carried out an experiment to confirm bone regeneration activity to inhibit the progression of periodontal disease and to prevent tooth loss.

RANKL is a TNF-α ligand family that is present in osteoblasts and induces osteoclast formation and activity from progenitor cells (Lossdorfer et al., 2002; 71 (1): 45-52). These RANKLs are inhibited by OPG, and the relative ratios of RNAKL and OPG affect osteoclast formation and regulate osteogenesis (Simonet et al., Cell. 1997 Apr 18; 89 (2): 309- 19.). For this reason, in this study, the expression pattern of osteogenic markers OPG and RNAKL of the extracts of P. falciparum on MG-63 osteoblast was measured and the results are shown in FIG.

< Bone formation  Confirmation experiment>

To determine whether osteoblast-induced bone formation was induced in osteoblast-like MG-63 cells, mRNA levels of osteogenic biomarkers, OPG and RANKL, were measured. MG-63 cells were seeded in a 6-well plate at 1 × 10 6 cells / well and treated with the samples (25 μg, 50 μg, 100 μg / ml) after 24 hours. Then, total RNA was extracted from each cell using trizol and quantified using Nanodrop. The final quantified total RNA was synthesized by cDNA synthesis kit (TaKaRa). The synthesized cDNA was prepared by reacting each primer and SYBR green at 95 ° C for 10 minutes, followed by 95 ° C for 30 seconds, 57 ° C for 1 minute, and 72 ° C 30 seconds was repeated for 40 cycles and then relative quantified. The primers used to confirm the expression levels of OPG and RANKL are shown in Table 3 and the results are shown in FIG.

Primer sequence Gene name Primer sequences (5'-3 ') OPG 5-CAATTTGCCTGGCACCAAAG-3 (sense)
5-AGGTGAGGTTAGCATGTCCA-3 (antisense) RANKL 5-TGGATGCCTTGAATAATAAG-3 (sense)
5-AATTTGCGGCACTTGTGGAA-3 (antisense)

<Result>

MG-63 osteoblast cells were examined for osteogenic effect. OPG and RNAKL were used as bone formation markers and the results are shown in FIG. OPG and RANKL showed a tendency to decrease in concentration-dependent manner when they were treated with the extract of P. ficus-indica, and the expression ratio of OPG was higher than that of RANKL. The bone - forming activity of the extracts of P. japonica can be used not only to inhibit the progression of periodontal disease but also to restore periodontal tissue and teeth that have already been lost to periodontal disease. In addition, the bone regeneration activity of the extract of P. ficus-like extract prepared according to the embodiment of the present invention can be used for treatment of various bone diseases including osteoporosis as well as periodontal disease.

Claims (11)

( Porphyromonas gingivalis ) which comprises an extract of a plant as an active ingredient.
The method according to claim 1,
Wherein the extract is obtained by using water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
The method according to claim 1,
Wherein the supernatant is an 80% ethanol extract.
4. The method according to any one of claims 1 to 3,
Wherein the composition of the composition is a toothpaste, a mouthwash, a dietary film, a mouse spray, or a chewing gum.
4. The method according to any one of claims 1 to 3,
Wherein the composition is a pharmaceutical composition.
4. The method according to any one of claims 1 to 3,
Wherein the composition is a food composition.
delete ( Fusobacterium nucleatum ) comprising an extract of Liliaceae barkifolia as an active ingredient.
9. The method of claim 8,
Wherein the composition of the composition is a toothpaste, a mouthwash, a dietary film, a mouse spray, or a chewing gum.
9. The method of claim 8,
Wherein the composition is a pharmaceutical composition.
9. The method of claim 8,
Wherein the composition is a food composition.

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