KR20110088978A - Oral composition containing herbal extract - Google Patents

Abstract

PURPOSE: A composition containing herb extract is provided to prevent dental caries and prevent and treat periodontitis. CONSTITUTION: A composition for oral cavity contains extract of Asiasari Radix, Caryophylli Flos, and Paeoniae Radix. The extract is a hot water extract or 70-95% ethanol extract. The composition is formed in a toothpaste, oral cleaner, chewing gum, or gingival massage cream.

Description

Oral composition containing herbal extracts

The present invention relates to a composition for oral cavity having the prevention and treatment of periodontitis with the prevention of caries by inhibiting plaque formation, more specifically sessin extract having an inhibitory effect on the causative bacteria, inhibitory effect and anti-inflammatory The present invention relates to an oral composition containing all of the potent clove extract and the peony extract having the inhibitory activity of glucosyltransferase.

In general, important diseases that occur in the teeth and surrounding tissues are caries and periodontal disease. The cause of tooth decay is due to various plaques (or plaques) composed of bacteria and metabolites that reside in the oral cavity. The cause of periodontal disease is also due to the metabolic habitat and metabolic activity of certain bacteria in the teeth and gums. It is understood.

There are several known microorganisms that form plaques, but Streptococcus mutans is a major factor. Streptococcus mutans secrete an enzyme called glucosyltransferase (GTase) in extracellular or cell surface to produce glucose and fructose by using sucrose in food as a substrate. Form glucan, a water insoluble polymer. These glucans attach various microorganisms that live in the oral cavity to the tooth surface to form a bacterial membrane, called plaque (or plaque).

When the lactic acid bacteria, including Streptococcus mutans, grow in the plaque, lactic acid is produced using fructose as a carbon source, and when the lactic acid is collected in adherent glucan, it is concentrated to a high concentration of acid to dissolve calcium ions in the tooth and consequently enamel of the tooth ( enamel) causes caries ( J. Periodontol . 63: 322-331, 1992).

Periodontal disease is often referred to as taste, which is divided into gingivitis and periodontitis depending on the severity of the disease. Periodontal disease is a relatively light and fast-recovering form of periodontal disease called gingivitis, which is limited to gums, or soft tissues. Periodontitis is an inflammatory lesion of peri-dental tissue caused by certain bacteria or groups of bacteria in the periodontology. Periodontal pockets, gingival contractions and periodontal ligaments and alveolar bone fractures, resulting in normal tooth loss. ”(National Institute of Periodontology and Dental Sciences. Periodontal Science. Seoul: Gunja Publishing House. 2004: 116).

More specifically, periodontal disease is a mixed infection by several bacterial groups present in plaque caused by the progression of inflammation in response to tissues around the teeth. Among these bacteria, Porphyromonas gingivalis, which belongs to the red complex, is known as the most important causative agent of periodontal disease.

Porphyromonas gingivavalis gram negative anaerobic bacteria increases in the number of lesions of periodontal disease, and as the periodontal disease progresses, the phytopymonas gingivalis is often found. Pyphyromonas gingivalis is a cytokine produced by these cells that react with P. pylorimonas gingivalis, although it may directly metabolize or infiltrate host tissues and cells to produce metabolites or toxins. In addition, the periodontal tissue is further destroyed by substances produced by host cells that respond to these cytokines. In other words, periodontal disease is caused by Pyromonas ginalivalis and is accompanied by inflammation and bleeding. In severe cases, it is necessary to use denture after extraction.

In order to prevent tooth decay and gum disease, it is necessary to inhibit or sterilize the growth of Streptococcus mutans bacteria and Popyromonas gingivalis. Examples of such a substance include penicillin, erythromycin, tetracycline, and spiramycin. There are antibiotics and triclosan, a nonionic fungicide, is the most representative. Korean Patent 1999-0079220 proposes a composition for oral cavity using triclosan, and Korean Patent 10-2006-0089890 proposes a method of increasing the residual effect of triclosan in the oral cavity.

However, antibiotics have side effects such as resistant bacteria expression, hypersensitivity reactions and gastrointestinal disorders. Especially, antimicrobial agents such as chlorohexidine have the effect of inhibiting tooth decay, but they are known to have side effects such as destruction of oral ecosystems and tooth coloring. have.

Therefore, as an agent capable of minimizing or supplementing the side effects exhibited by these chemicals, research on antimicrobial and fungicides using natural products that have been used for a long time is being actively conducted.

As a potent substance for inhibiting the growth of Streptococcus mutans, Pogongyoung extract in Korean Patent 1994-025562, and Yarrow and Horse Chestnut extract in Korean Patent 1994-008668 have been presented.

In addition, the Republic of Korea Patent 2000-0000342 proposed a method for extracting the antimicrobial material from the curcuma nagging, the Republic of Korea Patent 2003-0080471 presented a composition for oral cavity containing nagging sol.

In the case of gum disease, when certain inflammatory reactions are induced by certain bacteria, reactions such as activating inactive collagenase or stimulating the release of collagenase and other matrix metalloproteinases Happens.

The above-mentioned matrix metalloproteinases are calcium and zinc dependent peptidases secreted from various cells such as polymorphic neutrophils, macrophages, gingival fibroblasts, osteoblasts, and act at neutral pH. Various extracellular matrices are used. The mechanism of production of these enzymes is that tissues are directly destroyed by endotoxins such as lipopolysaccharide, a cell wall component of anaerobic Gram-negative bacteria such as bacteroids and Actinobacillus, or the biological immune system. Inflammation of gums is caused by various kinds of cytokines such as free radicals, prostaglandins, and interleukins secreted outside the cell by various actions of the immune system, and the stimulation of these inflammatory mediators By collagenase secreted by the collagenase and bacteria secreted by the collagen (collagen), which is a substrate of the periodontal tissue is decomposed, gum retraction occurs, if left untreated, it progresses to periodontal disease.

It is known to contribute to the destruction and destruction of periodontal tissue as well as the regeneration and development of everyday periodontal tissues. Representative enzymes that play such a role are matrix metalloproteinase-1 (MMP-1) and MMP-8. In gum disease, extracellular matrix degradation, ie collagen in periodontal tissues, weakens. MMP-1 regulates the regeneration of connective tissue and is present at particularly high concentrations in areas with inflammatory bowel disease. Inhibiting MMPs to prevent collagen breakdown of these sites is critical for preventing gum disease. Korean Patent 1998-0063510 proposes a method for treating gum disease using collagenase inhibitory effect of hyssop extract and milky skin extract.

As described above, the technology for preventing tooth decay and periodontal disease using the disclosed natural products has the advantage of using natural antibacterial and anti-inflammatory materials which have little side effects to the human body, but Streptococcus, the main causative agent of caries and periodontitis, is prevented. Mutans or periodontitis-inducing bacterium Popiromonas gingivalis has a problem of inhibiting the specific one of the species and there is a disadvantage that can not simultaneously provide a function to alleviate or treat after the periodontitis.

The development of caries and periodontitis is caused or accelerated by various causes as well as harmful bacteria in the oral cavity. Therefore, there is a need for development of a composition for oral cavity having the effect of preventing the formation of plaque and inhibiting inflammation causing agents more effectively by inhibiting the activity of glucosyltransferase as well as antibacterial against harmful bacteria in the oral cavity.

Accordingly, the present inventors are studying natural ingredients for the preparation of oral compositions excellent in the prevention and treatment of tooth decay and periodontal disease, the composition of the extract of sessin, cloves and peony suppresses the cavities and periodontitis causative bacteria In addition, the present invention was found to have the effect of inhibiting the activity of glucosyltransferase and inhibiting the production of inflammatory substances, and completed the present invention.

The present invention was devised to solve the above-mentioned problems, an object of the present invention is to remove the causative bacteria and periodontitis causative bacteria using the extract of sessin, cloves, peony and to suppress the activity of glucosyltransferase to prevent caries and periodontitis At the same time to provide a composition for oral care that can be prevented and treated.

In addition, another object of the present invention is to provide a composition for oral cavity containing extracts of sessin, cloves, peony, toothpaste, toothpaste, oral massage cream, chewing gum and the like which can prevent and treat tooth decay and periodontitis at the same time.

The present invention for achieving the above object is to remove the causative and periodontitis causative bacteria using the extract of sessin, cloves, peony and inhibit the activity of glucosyltransferase for oral cavity that can simultaneously prevent and treat caries and periodontitis. To provide a composition.

The present invention also provides a composition for oral cavity containing extracts of sessin, cloves, peony, toothpaste, dentifrice, oral massage cream, chewing gum and the like which can prevent and treat tooth decay and periodontitis at the same time.

The following describes the features of the main components contained in the composition for oral cavity of the present invention.

The plant extract used in the present invention is obtained from the following plants.

◎ slender (細辛, Asiasarum Root and Rhizome) are the roots and rhizomes of North slender (Asiasarum heterotropoides F. Maekaw var. Mandshuricum F. Maekawa) or Seoul jokdori Pool (Asiasarum sieboldii Miquel var. Seoulense Nakai ).

Clove is a bud of Clove ( Symzygium aromaticum Merrill et Perry).

Peony Root is the root of Peony ( Paeonia lactiflora Pallas ) or other related plant (Peaonyaceae Paeoniaceae).

Extract of the present invention is prepared using hot water or 70% to 95% ethanol, in particular, it is preferable to extract using 70% to 95% ethanol. The hot water extracts of sessin, cloves and peony are prepared by washing and cutting these vegetable medicines, adding several times the amount of distilled water to each medicine or mixed medicine and refluxing and filtering at 90 ° C. to 100 ° C. for at least 1 hour. In the case of ethanol extraction, washed or shredded vegetable herbs in 70% to 95% ethanol solution, respectively or mixed, extracted by stirring for at least 24 hours at room temperature, and filtered and purified.

The extract of sessin, cloves and peony, which is an active ingredient according to the present invention, may be contained in 0.001% by weight to 5.0% by weight, preferably 0.01 to 1.0% by weight of the total composition. When the concentration is less than the above-mentioned range, the efficacy is weak, and when combined with more than 5.0% by weight, the feeling is not good due to the inherent taste and aroma, and there is a problem that the cytotoxicity and manufacturing cost increase.

The present invention is applied to an appropriate amount of the components other than the extract of sessin, cloves, peony, commonly used according to the type and purpose of the composition for oral use. In the case of the toothpaste which is one embodiment of the present invention, a fluorine compound, an abrasive, a humectant, a sweetener, a flavoring agent, etc. may be applied in addition to the above components.

The fluorine compound serves to prevent tooth decay by increasing the acid resistance of the tooth, and may use at least one of sodium fluoride, sodium monofluorophosphate, tin fluoride, and the like, and the content thereof is preferably 0.01 to 2.0% by weight.

The abrasive may be used alone or in combination of two or more kinds of precipitated silica, calcium carbonate, hydrous alumina, calcium dihydrogen phosphate, sodium bicarbonate, and the content thereof is preferably 10 to 60% by weight.

As the humectant, an amorphous sorbitol solution, glycerin, propylene glycol, polyethylene glycol, or the like may be used alone or in combination of two or more thereof, and the content thereof is preferably 3 to 60% by weight.

The sweetening agent uses sodium saccharin, xylitol, erythritol, stevia, aspartame and the like, and may be used alone or in combination of two or more thereof, and the content thereof is preferably 0.01 to 2% by weight.

The foaming agent may be used alone or in combination of two or more kinds of sodium lauryl sulfate, sodium lauryl sarcosinate, cocaidopropyl betaine, polyoxyethylene hardened castor oil, polyoxyethylene polyoxypropylene-based condensation polymer, and the like. The content is preferably 0.5 to 5% by weight.

As the binder, carrageenan, xanthan gum, sodium carboxymethylcellulose, polyvinylpyrrolidone, or the like may be used alone or in combination of two or more thereof, and the content thereof is preferably 0.1 to 5% by weight.

The flavoring agent may be used by mixing a proper amount of peppermint oil, spearmint oil, menthol and the like. As a preservative, methyl paraben, propyl paraben, etc. can be mixed and used suitably. As the pH adjusting agent, sodium triphosphate, monobasic sodium phosphate, citric acid, sodium citrate, phosphoric acid, tartaric acid, sodium tartarate and the like can be used, and the preferred pH is 5-8. As a dye, blue 1, yellow 203, titanium oxide, etc. can be mixed and used in an appropriate amount.

Other compositions for oral cavity of the present invention can also be prepared by a conventional method with the plant extract of the present invention by selecting the appropriate components according to the type and purpose.

The composition for oral cavity according to the present invention contains the extract of sessin, cloves and peony, removes the causative agent of caries and periodontitis, inhibits the activity of glucosyltransferase, and inhibits the induction of anti-inflammatory substances, thereby simultaneously preventing caries and periodontitis. And therapeutic effects.

Hereinafter, the present invention will be specifically described as Examples and Experimental Examples. However, these examples are merely to aid the understanding of the present invention, but the scope of the present invention is not limited to these examples. In the following Experimental Examples and Examples, unless otherwise specified,% represents% by weight.

Experimental Example 1

Comparison of Glucosyltransferase Inhibitory Activity of Plant Extracts

1) Subject

95% ethanol extract of the following plant herbal medicines: fruit, guchu, rhubarb, bark skin, country of origin, pesticide, upper limb, peony, breeding, pageokcheon

2) Experiment Method

In order to measure the glucosyltransferase inhibitory activity of the various plant extracts, each plant herbal was washed with water and washed, and then extracted by stirring for 24 hours at room temperature by adding 10 times (w / v) of 95% ethanol to each sample. And filtered to prepare each extract.

Streptococcus mutans was inoculated into 2L of BHI (brain heart infusion) medium and anaerobically incubated for 48 hours at 37 ° C, followed by centrifugation (4 ° C, 12000g, 20 minutes) to remove the supernatant. Ultrafiltration with (Millipore, Billerica.Mass., USA), concentrated to 200ml and then dialyzed in buffer solution (potassium phosphate, 50mM, pH6.9) overnight at 4 ℃ to prepare glucosyltransferase coenzyme 1.4unit Dilute to / mL.

150 μl of each plant extract prepared above was mixed with 600 μl of the enzyme solution prepared above, pre-incubated at 37 ° C., and 150 μl of 8.4% sucrose was reacted for 1 hour, followed by methanol in the reaction solution. 900 μl is added to stop the reaction and centrifuged (10000 rpm, 10 minutes) to obtain a precipitate (insoluble glucan). The precipitate was washed sequentially with 1 mL of distilled water and 1 mL of 70% ethanol, and then 200 μl of distilled water and 800 μl of anthrone color developing reagent (anthrone 200mg / H ₂ SO ₄ 100mL) were added and heated by boiling water for 10 minutes. After cooling for 3 minutes, the absorbance at 550 nm was measured with a spectrophotometer.

If the absorbance decreases at the wavelength, glucosyltransferase (GTase) inhibitory activity means that glucan production is inhibited. Inhibitory activity of glucosyltransferase was calculated as shown in Equation 1 below, and the inhibitory activity of 95% ethanol extract of each plant was shown in Table 1.

[Equation 1]

GTase inhibitory activity (%) = 1- (absorbance of sample solution / absorbance of control solution) × 100

However, the absorbance of the control solution is the absorbance of the reaction solution except the plant extract.

3) results

GTase Inhibitory Activity of 95% Ethanol Extracts from Various Plants Botanical Extract (95% Ethanol Extract) Inhibition rate (%) Fruiting -43.3 Gucchi -53.3 rhubarb 33.3 Neck skin 25.0 Country -26.7 Medicine -6.7 Upper limb 55.0 Peony 75.0 Breeding 46.7 Pagokcheon 20.0

According to Table 1, it can be seen that the inhibitory activity against glucosyltransferase of ethanol extracts such as peony, breeding, etc. is relatively high, and it can be seen that the inhibitory activity of peony extract is particularly excellent.

Experimental Example 2

Comparison of Antibacterial Efficacy of Oral Bacteria by Measuring Growth Inhibition of Clove, Sessin and Peony Extracts

1) Test subject

95% ethanol, 70% ethanol, hydrothermal extract of the following plant herbal medicines: cloves, sessin, peony (excluding peony hot water extract)

2) Experiment Method

① As shown in Table 2 below, two microorganisms were inoculated in 5 mL of liquid culture medium and incubated in a 35 ° C. incubator for 2 to 7 days under each culture condition.

② The bacterial cultures obtained by liquid culture were measured by 595 nm OD (Optical density value) by ELISA (Enzyme-linked immunosorbant assay) to dilute the microorganism to 1 × 10 ⁶ cfu / mL.

③ Dispense and dilute the bacterial dilution solution into the culture plate medium by injection plate method. 8 mm disks were loaded at regular intervals on the culture plate medium.

(4) 50 μl / mL of each sample was dispensed into the disc and incubated for 2 to 7 days in a 35 ° C. incubator.

⑤ After that, the growth of the microorganisms and growth inhibition ring was measured.

Experimental strains and culture conditions Test strain Culture medium Oxygen demand Incubation temperature S.mutans
(KCTC 3065) Brain Heart Infusion (BHI) Aerobic 35 ℃ P.gingivalis
(KCTC 5352) TSA HEMIN MENADIONE MEDIUM Anaerobic 35 ℃

3) Experiment result

As shown in Table 3 below, the growth inhibitory results of two oral harmful microorganisms using eight samples of each extraction solvent for three herbal medicines were as follows. It was confirmed to have. However, peony hot water extract was excluded from the experiment due to re-dissolution problem.

Growth inhibitory test results (growth inhibitory ring unit: mm) Test Sample Extraction solvent Concentration (mg / mL) S.mutans
(KCTC 3065) P.gingivalis (KCTC 5352) cloves 95% ethanol 100 15 25 70% ethanol 100 18 25 Hydrothermal 100 16 13 Sesin 95% ethanol 100 15 13 70% ethanol 100 13 9.5 Hydrothermal 100 × × Peony 95% ethanol 100 12 10.5 70% ethanol 100 13 10

[Experimental Example 3]

Comparison of antimicrobial efficacy of oral harmful bacteria by measuring minimum inhibitory concentration (MIC 90) on clove, sessin and peony extract

1) Subject

95% ethanol, 70% ethanol, hydrothermal extract of the following plant herbal medicines: cloves, sessin, peony (excluding peony hot water extract)

2) Experiment Method

① 2 microorganisms were inoculated into 5 mL of liquid culture medium as shown in Table 2 above, and cultured in a 35 ° C. incubator for 2 to 7 days under each culture condition.

② The bacterial cultures obtained by liquid culture were measured by 595 nm OD (Optical density value) by ELISA (Enzyme-linked immunosorbant assay) to dilute the microorganisms to 2 × 10 ⁵ cfu / mL.

③ Dilute 100 μl of liquid culture medium and 8 test samples to a maximum concentration of 10 mg / mL in a 96-well plate, and divide 100 μl into two-fold steps. 10% DMSO was also performed.

(4) 100 µl of the 96-well plate liquid culture medium or 100 µl of the diluted solution was incubated for 2 to 7 days in a 35 ° C incubator.

⑤ After 2-7 days, 595nm OD value of the ELISA reader was measured to determine the growth of bacteria and the minimum inhibitory concentration (MIC90).

 3) Experiment result

As shown in Table 4 and Table 5, MIC90 was measured for 8 samples of each extraction solvent for three herbal medicines, and the antimicrobial activity was measured. The lowest inhibitory concentration value was shown in the ethanol extract. In the antimicrobial test against Popiomonas gingivalis, the causative agent of periodontitis, 95% ethanol extract of clove showed the lowest minimum inhibitory concentration.

Minimum inhibitory concentration measurement results for S.mutans KCTC3065 Test Sample Extraction solvent Concentration (mg / mL) MIC 90 (μg / mL) cloves 95% ethanol 10 1,134 70% ethanol 10 1,160 Hydrothermal 10 2,189 Sesin 95% ethanol 10 562 70% ethanol 10 1,121 Hydrothermal 10 Over 5,000 Peony 95% ethanol 10 4,460 70% ethanol 10 3,654

Minimum Inhibitory Concentration Measurement Results for P.gingivalis KCTC5352 Test Sample Extraction solvent Concentration (mg / mL) MIC 90 (μg / mL) cloves 95% ethanol 10 444 70% ethanol 10 1,841 Hydrothermal 10 3,048 Sesin 95% ethanol 10 Over 5,000 70% ethanol 10 Over 5,000 Hydrothermal 10 Over 5,000 Peony 95% ethanol 10 4,983 70% ethanol 10 Over 5,000

[Experimental Example 4]

Determination of Nitric Oxide (NO) Production of Clove, Sessin and Peony Extracts

1) Subject

95% ethanol, 70% ethanol, hydrothermal extract of the following plant herbal medicines: cloves, sessin, peony (excluding peony hot water extract)

2) Experiment Method

① Inoculate rat-derived macrophage (Raw 264.7) to the bottom of the culture dish and contain penicillin (100U / mL), streptomycin (100µg / mL), 10% FBS (fetal bovine serum), 25mM / L HEPES Put Dulbecco's Modified Eagle's Medium (DMEM) medium and incubate at 37 ° C and 5% CO ₂ .

② Raw 264.7 cells were inoculated in a 35 pie plate at a concentration of 1 × 10 ⁶ / mL and incubated for 24 hours.

③ After incubation, all of the medium was removed and the sample was prepared to the final concentration by using a medium containing no serum containing 100ng / mL LPS, and then 1mL was added to each well.

④ After culturing for 24 hours, the cells grown in 35 pies were collected by centrifugation, and the medium supernatant was taken.

⑤ NO standard (NaNO ₂ ) was prepared: Stock solution (Stock sol .; 80mM in DW) concentration was 0, 10, 20, 40, 80μM (Raw 264.7 cell Serum Free Medium)

⑥ Into a 96-well plate, 100 μl of NO standard and the supernatant taken in No. 5 were added, and 100 μl of the same amount of Greases reagent was added.

⑦ The absorbance value was measured at 540 nm using an ELISA reader.

3) Experiment result

As shown in Table 6 below, eight samples of each extractant for three herbal medicines were tested for inhibition of the production of nitric oxide, which is closely related to the inflammatory response. It was confirmed that the NO production inhibitory effect in the% ethanol extract.

Measurement result of NO production inhibitory activity of each extract Sample Name Concentration (㎍ / mL) NO production inhibition (%) control 0 100 LPS 0.1 0 Clove 95% Ethanol Extract One 27.9 10 44.4 20 55.3 Clove 70% Ethanol Extract One 31.7 10 37.5 20 38.0 Clove Hydrothermal Extract One 31.2 10 28.7 100 74.9 Sessin 95% Ethanol Extract One 26.4 10 31.1 100 37.7 Sesine 70% Ethanol Extract One 18.4 100 38.1 200 52.3 Sessin Hot Water Extract One 22.9 10 28.9 20 26.8 Peony 95% Ethanol Extract One -11.3 10 3.1 20 17.2 Peony 70% Ethanol Extract One -3.0 10 6.4 20 9.5

[Experimental Example 5]

Determination of Interleukin-1 Production Inhibition of Clove, Sessin and Peony Extracts

1) Subject

95% ethanol, 70% ethanol, hydrothermal extract of the following plant herbals: cloves, sessin, peony

2) Experiment Method

① Cell culture and medium recovery

i. Raw 264.7 cells were seeded in 35 mm plates at a concentration of 1 × 10 ⁶ / well.

ii. After 24 hours of incubation, a serum free medium treated with LPS 100ng / mL was used to make the final concentration of the sample determined by MTT assay.

iii. After 24 hours, the medium was recovered and centrifuged at 12,000 rpm and 5 min to recover the supernatant and used for the experiment.

② Mouse IL-1α / IL-1 F1 (R & D Systems DY400)

I. Plate Preparation

i. Empty 96-well plates are prepared and prepared by diluting the capture antibody in the kit with a working solution.

ii. 100 μl of the capture antibody (in PBS) diluted and prepared at a concentration of 2.0 μg / mL was added to each well, and then sealed and incubated overnight at room temperature.

iii. The capture antibody was removed as much as possible, 400 μl of wash buffer was added to each well and allowed to stand for 1 minute, and then shaken on a paper towel to completely remove the solution. (3 times in total)

iv. 300 μl of reagent diluent was added to each well of the washed plate and blocked for at least 1 hour at room temperature.

v. Step iii was carried out to completely remove the reaction solution.

II. Essay Procedure

i. In each well, 100 μl of standard (in the reagent diluent) and the sample were added and covered with an adhesive strip, followed by reaction at room temperature for 2 hours.

ii. Steps I-iii were carried out to completely remove the reaction solution.

iii. In each well, 9 μg / mL detection antibody was diluted to 50ng / mL using a reagent diluent, and put into 100 μl, and then covered with an adhesive cover.

iv. Steps I-iii were carried out to completely remove the reaction solution.

v. Each well was diluted with 100 μl of streptavidin-HRP (diluted with a reagent diluent at a concentration of 1: 200), covered with an adhesive cover, and reacted at room temperature for 20 minutes (incubation in a cow). .

vi. Steps I-iii were carried out to completely remove the reaction solution.

vii. 100 μl of standard solution was added to each well and covered using an adhesive cover, followed by reaction at room temperature for 20 minutes (incubation in a cow).

viii. 50 μl of stop solution was added to each well to terminate the reaction, and then the plates were mixed by tapping.

ix. Absorbance was measured at 450 nm using a microplate reader (wavelength correction: 540 nm or 570 nm).

3) Experiment result

As shown in Table 7 below, the inflammatory response was confirmed by the production of IL-1, which is a kind of pre-inflammatory cytokine, expressed in the early stages of the inflammatory response to eight samples of the extractant for three herbal drugs. It was indirectly confirmed whether the effect of inhibiting. In this experiment, 95% ethanol extract, 70% ethanol extract, and hydrothermal extract of clove were found to be effective in inhibiting the production of IL-1 induced by LPS.

Measurement result of NO production inhibitory activity of each extract Sample Name Concentration (㎍ / mL) NO production inhibition (%) control 0 0 LPS 100ng / mL 625.04 ± 17.58 Clove 95% Ethanol Extract One 597.55 ± 29.93 10 544.61 ± 15.15 20 393.07 ± 1.07 Clove 70% Ethanol Extract One 588.72 ± 19.46 10 541.66 ± 2.25 20 547.27 ± 9.43 Clove Hydrothermal Extract One 630.35 ± 17.11 10 870.43 ± 13.31 100 570.51 ± 8.46 Sessin 95% Ethanol Extract One 640.33 ± 18.01 10 637.42 ± 28.92 100 912.83 ± 15.36 Sesine 70% Ethanol Extract One 640.33 ± 18.01 100 1137.69 ± 17.50 200 1297.99 ± 32.12 Sessin Hot Water Extract One 870.57 ± 40.22 10 879.60 ± 19.16 20 784.09 ± 19.61 Peony 95% Ethanol Extract One 1220.95 ± 21.30 10 1190.40 ± 10.69 100 1332.59 ± 20.25 Peony 70% Ethanol Extract One 1197.52 ± 2.35 10 1224.91 ± 6.38 100 1386.21 ± 6.18

Experimental Example 6

For the toothpaste compositions of Examples 1 to 5 and Comparative Examples 1 to 4 of Table 8, the plaque formation inhibitory effect and the gingivitis inhibitory effect were tested as follows.

Toothpaste Composition (Unit: wt%) ingredient Example 1 Example
2 Example
3 Example
4 Example
5 compare
Example
One compare
Example
2 compare
Example
3 compare
Example
4 Silicon dioxide 19 19 19 19 19 19 19 19 19 Sorbitol Liquid (70%) 50 50 50 50 50 50 50 50 50 Polyethylene glycol 5 5 5 5 5 5 5 5 5 Sodium Saccharin Hydrate 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 0.13 Sodium Lauryl Sulfate 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Carboxymethyl Cellulose Sodium One One One One One One One One One Clove 95% Ethanol Extract - - - - - - 0.1 - - Sessin 95% Ethanol Extract - - - - - - - 0.1 - Peony 95% Ethanol Extract - - - - - - - - 0.1 Clove, Sessin, Peony 95% Ethanol Extract 0.01 0.1 One - - - - - - Clove, Sessin, Peony 70% Ethanol Extract - - - 0.1 - - - - - Clove, Sessin, Peony Hot Water Extract - - - - 0.1 - - - - Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100

1) Experimental method

Using the double-blind method, the researchers and the subjects did not know what kind of effect the toothpaste had, and all the subjects examined baseline such as gingivitis index and plaque index before the experiment. After scaling, the two-week residual effect was removed. The summary of the measurement rules for each oral environmental index is as follows. In addition, the target tooth for measuring these indices was selected by the narrowing and the tongue of the six teeth, such as 16, 11, 26, 36, 31, 46 teeth, respectively, and the method commonly used in the world.

2) Experiment and result of plag inhibition effect

For testing, 20 ml of water was mixed with 10 drops of staining solution (disclosing solution), held in the mouth for 1 minute, spited out and lightly brushed with water. The coloration was performed by a dental orthodontic examination using a probe and alveolar, and the criteria for the score of the dental bacterium were applied by Quigley & Hein's "Turesky modification" criteria. ) And the sum of the indices was averaged by the number of teeth. At this time, the third molar was excluded from the examination regardless of eruption.

Bacterial Membrane Rating Standard

0 = when the dental plaque is not attached at all

1 = when there is a bactericidal bacterium on the gingival edge

2 = toothed part annularly with 1mm or less toothpaste

3 = Tooth surface less than 1/3 of the cervical side when there is an annular plaque greater than 1mm

When tooth surface less than 2/3 tooth side cervical bacterial membrane

5 = When there is a toothpaste bacterium on the tooth surface more than 2/3 of the cervical side

Bacterial Membrane Index = Total Score / Score

During the two-week elimination period, control toothpaste was used three times a day after each meal. After homogenizing the oral environment of the subjects for two weeks, each toothpaste was distributed to the subjects who were cross-distributed based on the baseline, and used three times a day for two months thereafter. The plaque index was measured. The statistical significance was tested by t-test by comparing the initial values of the experimental group and the scores after 2 months.

In the two-month experiment, changes in the plaque index for each group were shown in Table 9. As a result of measuring the plaque index according to the use of each toothpaste composition, the statistically significant test resulted in statistically significant plaque suppression effect over time compared to the initial value in all experimental groups before and after each toothpaste and comparative example. there was.

In addition, in the measurement of plaque index, the experimental group of Comparative Example 4 using the peony extract as an active ingredient and the Examples 1 to 5 experimental groups containing both clove, sessin and peony extract were found to have excellent plaque suppression effect. However, there was no significant difference in the improvement index of Comparative Example 1, Comparative Example 2 and Comparative Example 1 and Comparative Example 3.

Plaque index measurement results Experimental group Initial value 1 month later 2 months later Improvement Index Example 1 1.52 ± 0.33 1.31 ± 0.32 1.08 ± 0.35 0.44 ± 0.30 Example 2 1.54 ± 0.27 1.12 ± 0.28 0.94 ± 0.25 0.60 ± 0.27 Example 3 1.49 ± 0.32 1.01 ± 0.24 0.75 ± 0.23 0.74 ± 0.28 Example 4 1.51 ± 0.31 1.14 ± 0.32 1.02 ± 0.29 0.49 ± 0.29 Example 5 1.52 ± 0.29 1.19 ± 0.30 1.05 ± 0.24 0.47 ± 0.25 Comparative Example 1 1.53 ± 0.26 1.45 ± 0.33 1.36 ± 0.29 0.17 ± 0.29 Comparative Example 2 1.53 ± 0.25 1.39 ± 0.26 1.25 ± 0.22 0.28 ± 0.24 Comparative Example 3 1.52 ± 0.32 1.35 ± 0.31 1.26 ± 0.28 0.26 ± 0.30 Comparative Example 4 1.51 ± 0.33 1.13 ± 0.26 1.03 ± 0.23 0.48 ± 0.29

3) Experiment and result of measuring gingivitis inhibitory effect

The gingiva per tooth are divided into lingual and lingual surfaces, and then calculated as the average of the calculated indexes. Each gingivitis is evaluated from 0 to 3 points according to the Loe & Silness score rating, and the gingivitis index of each individual is calculated by average grade.

Gingivitis Rating Criteria

0 = normal value

1 = minor inflammation, color tone change, mild edema but no periodontal bleeding

2 = bleeding due to severe inflammation, redness, edema, gingival change and mild irritation to the periodontal probe

3 = Severe inflammation, redness, edema, ulcers and natural bleeding

Gingivitis index = total score / score

Changes in the gingivitis index, which is evaluated as an item for assessing the degree of inflammation in the gingiva, showed a decrease in the gingivitis index over time in both the control group and the experimental group, as shown in Table 10, but in Comparative Examples 1, 3, and 4, the initial value and 2 There was no statistically significant difference in the gingivitis index after months, and all of the examples showed statistically significant differences. In Comparative Example 2 using the clove extract also showed a significant difference in the gingivitis index change. Therefore, it can be seen that the use of clove extract is effective in preventing gingivitis and is more effective when the extract of cloves, sessin and peony is used as an active ingredient.

Gingivitis index measurement result Experimental group Initial value 1 month later 2 months later Improvement Index Example 1 1.57 ± 0.41 1.37 ± 0.37 1.21 ± 0.38 0.36 ± 0.39 Example 2 1.55 ± 0.38 1.24 ± 0.28 1.07 ± 0.29 0.48 ± 0.33 Example 3 1.52 ± 0.36 0.92 ± 0.28 0.74 ± 0.28 0.78 ± 0.30 Example 4 1.58 ± 0.39 1.30 ± 0.32 1.15 ± 0.25 0.43 ± 0.33 Example 5 1.55 ± 0.40 1.35 ± 0.39 1.17 ± 0.35 0.38 ± 0.38 Comparative Example 1 1.56 ± 0.42 1.50 ± 0.38 1.48 ± 0.39 0.8 ± 0.40 Comparative Example 2 1.58 ± 0.42 1.34 ± 0.40 1.18 ± 0.39 0.30 ± 0.39 Comparative Example 3 1.54 ± 0.37 1.48 ± 0.33 1.47 ± 0.34 0.07 ± 0.35 Comparative Example 4 1.53 ± 0.35 1.50 ± 0.39 1.48 ± 0.35 0.05 ± 0.37

The composition for oral cavity mixing all the extracts of sessin, cloves and peony according to the present invention can inhibit the glucosyltransferase activity involved in plaque formation and inhibit the growth of caries and periodontitis, thereby preventing caries, plaque and periodontitis. It inhibits the production of inflammation-causing substances and provides the effect of simultaneously relieving and treating periodontitis.

Claims (6)

Oral composition containing all the extracts of sessin, cloves and peony. The composition for oral cavity according to claim 1, which contains 0.001% by weight to 5.0% by weight of extract of sessin, cloves and peony based on the whole composition. The oral composition according to claim 1, wherein the extract of sessin, cloves and peony is contained in an amount of 0.01% by weight to 1.0% by weight based on the whole composition. The oral composition according to claim 1, wherein the extracts of sessin, cloves and peony are hot water extracts or 70-95% ethanol extracts. The composition for oral cavity according to claim 4, wherein the extract of sessin, cloves and peony is 95% ethanol extract. The oral composition according to claim 1, wherein the formulation of the composition for oral cavity is toothpaste, mouthwash, chewing gum or gum massage cream.