KR101520821B1 - Composition for preventing and alleviating periodontal diseases and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a functional composition for the prevention and improvement of periodontal disease and a method for producing the same. The functional composition for preventing and improving periodontal disease according to the present invention is characterized by containing a plant composition in which a topical extract, a topical extract and quercetin are mixed.

Description

Technical Field [0001] The present invention relates to a functional composition for preventing and improving periodontal disease,

The present invention relates to a functional composition for the prevention and improvement of periodontal disease and a method for preparing the same, and more particularly to a functional composition for prevention and improvement of periodontal disease, which comprises an extract of an active ingredient extracted from the upper and lower leaves and quercetin And a manufacturing method thereof.

Periodontal disease is divided into gingivitis and periodontitis, which are chronic bacterial infections affecting the teeth and gingiva supporting the teeth. Gingivitis is an early periodontal disease that causes inflammation of the soft tissues of the gingiva, and its symptoms are relatively light and the recovery is reversible. Periodontitis is a condition in which gingivitis is not treated and the inflammation progresses to the gums and around the pubic bone. When the chronic inflammatory reaction stimulated by the bacterial toxin develops, the periodontal pocket is formed between the gums and the teeth and the periodontal pocket is formed. Deepening of the depth of the periodontal ligament results in inflammation of the periodontal ligament and bone loss.

Accordingly, there is a need to develop a functional composition capable of preventing and improving periodontal disease.

The present invention has been made in view of the above problems, and it is an object of the present invention to inhibit the production of gingival and periodontitis related substances (Nitric oxide, PGEM, TNF-α, IL-1β) (COX-2, LOs, iNOS) and inhibiting the activity of the periodontal disease-inducing enzyme (COX-2, LOs, iNOS) and preventing and improving periodontal disease, and a method for producing the functional composition.

It is another object of the present invention to provide a functional composition for preventing and ameliorating inflammation, pain and hemorrhage caused by periodontal disease, so that there is no concern about side effects compared with other conventional synthetic drugs, The present invention provides a functional composition having an excellent effect for improving and preventing fundamental symptoms, and a method for producing the functional composition.

This object is achieved by a functional composition for the prevention and / or ameliorating periodontal disease, which comprises a plant composition in which a root extract is mixed with a topical extract and quercetin.

It is preferable that the mixed plant composition is mixed at a ratio of 20-40 g of the leaf extract and 20-40 g of quercetin to 100 g of the root extract.

The formulations to which the functional composition is administered are preferably capsules, rings, granules, powders, candies, gums, tablets, beverages, and syrups.

According to another aspect of the present invention, there is provided a method for preparing a functional composition for preparing a topical extract of an extract contained in a functional composition of the present invention, To 99% ethanol aqueous solution at 30 to 80 ° C for 2 to 8 hours, 1 to 4 times, followed by cooling and filtration; A second step of concentrating the filtrate obtained in the first step under reduced pressure at 60 to 100 占 폚; And a third step of lyophilizing the extract obtained in the second step to produce a powdery extract, wherein the indicator substance Rutin extracted through the above process is 0.01 to 2% by weight. And a method for producing a functional composition for prevention and improvement.

The above object can also be accomplished by a method for preparing a functional composition for preparing a topical extract of an extract contained in a functional composition of the present invention, comprising the steps of: mixing the top leaves with a 4- to 10-fold weight ratio aqueous alcoholic solution at 0 to 80% 2 to 8 hours, 1 to 4 times, followed by cooling and filtration; A second step of concentrating the filtrate obtained in the first step under reduced pressure at 60 占 폚; And a third step of lyophilizing the extract obtained in the second step to produce a powdery extract, wherein chlorogenic acid, which is an index substance extracted from the process, is 0.01 to 2% by weight And a method for producing a functional composition for preventing and ameliorating periodontal disease.

According to the present invention, the antioxidative effect (DPPH radical scavenging activity, SOD-like activity), the inhibition of the production of Nitric oxide and PGEM (Prostaglandin E metabolite), the periodontal disease inducing substance, and the COX- Inhibition of IL-2 (Cycloxygenase-2) and LOs (Lipoxygenases) activity, inhibition of inflammatory cytokines TNF-α (Tumor Necrosis Factor-α) and IL-1β (Interleukin- Improvement of bad breath and gingivitis index (plaque index, gingival index, gingival depth, gingival recession, clinical attachment level, bleeding index) in a simple clinical trial is excellent in inhibiting the expression of iNOS (inducible nitric oxide synthase) By producing a functional plant composition having an excellent effect, it exhibits a useful effect as a preventive agent for periodontal disease and as a symptom improving agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings.

The present invention relates to a method for inhibiting the production of Nitric oxide and PGEM (Prostaglandin E metabolite), which are superior to the respective extracts, by optimizing the extract to a predetermined amount, (Tumor Necrosis Factor-α) and IL-1β (Interleukin-1β), which are inflammatory cytokines, inhibit the activity of COX-2 (cyclooxygenase-2) and LOs (lipoxygenases) (Gingival index, gingival index, depth of gingival recession, gingival recession, and clinical attachment level) in a simple clinical study. Bleeding index) of the present invention. In addition, the present invention relates to a multifunctional composition useful for prevention of periodontal disease and improvement of symptoms by having harmful active oxygen radical scavenging effect and antioxidative effect, which are potential inducers of periodontal disease and are intensified factors of the disease.

Here, a brief description of the left and right minders is as follows.

It saddened chairs (Audi) According to classical literature, such as mulberry (Morus alba L.) fruit of a Compendium of Materia Medica, blind, Jean-term, Article mouthfeel properties Garage, dalmyeo taste, no poison, relieve and comfort sogaljeung the five viscera , To help with diabetes and joints, to pass through the blood, and to forget the hunger if you eat for a long time. The components of the newborn are Albafuran A, Albafuran B, Albafuran C, Albanol A, Albanol B, Alboctalol, alpha-Pinene (+, -), Artecanin, Astragalin, Bergapten, Hydroxystigmast-5-en-7-one, Beta-Resorcylaldehyde, Beta-Sitosterol, Betulinic acid Mulberrofuran T, Omega-Hydroxymoracin N, Oxydihydroresveratrol, Oxyresveratrol (E-form), Quercetin, Resorcinol, Resveratrol, Sanggenon C, Sanggenon D, Sanggenon E, Sanggenon P, Scopoletin, Stigmasterol, Umbelliferone, and Zeatin (Source: Food and Drug Administration).

The leaves of Morus alba L. are the leaves of Morus alba L. It is known that it removes the fever in the main stem of the river, lowers the hemorrhage, stops the bleeding and heals the eye disease. In addition, many anti-diabetic pharmacological actions have been reported. Among the essential oils, there are acetic acid, propionic acid, valeric acid, and capronic acid (Source: Korea Food & Drug Administration). Food raw material search).

In such a composite composition, the following method was used for preparing the composition by extracting the extract of Sambilha mushroom extract, the mulberry extract and quercetin, respectively, with water or an alcoholic aqueous solution.

First, a method for preparing a composition by extracting a bulkhead is as follows.

The bulkhead was pulverized at 500 to 1000 rpm using a food pulverizer and then extracted with a 4- to 10-fold volume of 0 to 99% ethanol aqueous solution at 30 to 80 ° C for 2 to 8 hours for 1 to 4 times, mesh) stainless steel sieve. The filtrate obtained in the first step is subjected to a second step of concentrating the filtrate under reduced pressure at 20 to 100 占 폚 to 20 byrix. A third step of lyophilizing the extract obtained in the second step and producing a powdery extract is carried out.

Rutin is usually contained in 0.01 to 2% by weight in the fresh extract powder obtained by the above method.

Next, the method for preparing the composition by extracting the top leaves is as follows.

The upper leaves were pulverized at 500 to 700 rpm for 10 to 20 minutes using a food pulverizer and then extracted and cooled 1 to 4 times at 60 to 100 ° C for 10 to 100% Perform the first step of filtration with a stainless steel sieve of standard mm (80 mesh). The filtrate obtained in the first step is subjected to a second step of concentrating the filtrate under reduced pressure at 60 DEG C to 20brix. A third step of lyophilizing the extract obtained in the second step and producing a powdery extract is carried out.

Particularly, the bulk extract obtained by the above method contains 0.01 to 2% by weight of chlorogenic acid.

Through the above process, the mixture of 20 g to 40 g of the leaf extract and 20 to 40 g of quercetin is contained in 0.01 g to 2 wt% of Rutin.

The plant extracts may be used as such, but may be formulated with acceptable moisture absorbents, forming agents, dilutes, carriers, etc., which can be edible for making into powders, tablets, granules or gels. It can be mixed and used.

In addition, the present invention relates to a functional composition for preventing and improving periodontal disease, comprising a combination selected from the group consisting of extracts of the extracts obtained from the above-mentioned methods, the extracts of the leaf extracts and quercetin as main components, and the selected combination of vitamins and the like as additional components As well as other edible ingredients.

The composition includes beverages, special nutritional compositions, health supplements, functional foods and other foods.

On the other hand, the form of the composition includes powders, granules, tablets, capsules, liquid or drink forms.

In addition, the present invention relates to a method for producing a food, comprising the step of mixing the composition with a flavoring agent, a coloring agent and an aging agent (such as cheese, chocolate, etc.), such as a bactericide, spice, seasoning agent, various nutrients, vitamins, minerals ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, And a method of using the food additive. In this case, the food additive may be added to the food by immersing, spraying or mixing the food. The ratio of such additive is not so important, but is generally selected in the range of 0 to 20 parts by weight per 100 parts by weight of the functional composition of the present invention to be.

It can also be added to foods or beverages for the purpose of preventing periodontal disease and improving symptoms. In this case, the amount of the composition in the food or beverage may generally be 10 to 90% by weight, preferably 20 to 70% by weight, based on the total weight of the functional composition of the present invention, In a proportion of 0.1 to 30 g, preferably 1 to 30 g.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by the following Examples.

REFERENCE EXAMPLE 1: Preparation of sickle leaf extract

The bulkhead was pulverized with a food pulverizer at 700 rpm for 10 minutes and then extracted twice with 4 times 30% ethanol aqueous solution at 60 ° C for 3 hours, cooled, and then filtered using a stainless steel sieve of 0.125 mm (80 mesh) Filtered. The obtained filtrate was concentrated under reduced pressure to 60 ° C at 20 ° C. The obtained extract was lyophilized to prepare a powdery extract.

Reference Example 2: Preparation of leaf extracts

The upper leaves were pulverized using a food pulverizer at 700 rpm for 10 minutes, then extracted twice with 8 times of 30% aqueous ethanol solution at 60 ° C for 3 hours, cooled and filtered with a stainless steel sieve of 0.125 mm (80 mesh) size. The obtained filtrate was concentrated under reduced pressure to 60 ° C at 20 ° C. The obtained extract was lyophilized to prepare a powdery extract.

Reference Example 3: Quercetin < RTI ID = 0.0 >

Products containing more than 90% quercetin and 85% polyphenol were purchased and used.

Example 1: Preparation of a mixture of sickle leaf extract, mulberry leaf extract, quercetin

It is preferable that the plant composition in which the topical extract of the present invention is mixed with the topical extract and the quercetin is mixed at a ratio of 20 to 40 g of the topical extract and 20 to 40 g of quercetin to 100 g of the topical extract.

Hereinafter, as an embodiment of the present invention, 100 g of the extract according to the present invention is weighed with an electronic balance at a weight of 20 g of the leaf extract and 20 g of quercetin, and placed in a double cone mixer and mixed at a rotational speed of 40 hz for 30 minutes.

Experimental Example 1: DPPH radical scavenging experiment

DPPH (2,2-Diphenyl-1-picrylhydrazyl, Sigma, MO, USA), which is a very stable free radical, is converted from purple to yellow diphenylpicrylhydrazine by hydrogen proton-radical scavenger. Respectively. The results are shown in the following [Graph 1] and [Table 1].

[1] A graph is a mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention showing the SC ₅₀ value of the DPPH radical scavenging 50% at a concentration of ppm.

[Graph 1]

[Experimental Method]

After dissolving each sample by concentration, 75 μl of each sample is mixed with 750 μl of DPPH (0.2 mM in ethanol) and 675 μl of tertiary purified water. After reacting at room temperature for 30 minutes, 200 μl of the reaction solution was transferred to a 96-well plate, and the absorbance was measured at 520 nm using an ELISA reader (Molecular Devices, CA, USA). The DPPH radical scavenging activity (%) was calculated from the formula (1-absorbance of sample 1-added sample / absorbance of sample-free sample) X 100. The SC ₅₀ value is the minimum concentration required to eradicate 50% of the generated radical in ppm.

DPPH radical scavenging ability (SC ₅₀ , ppm) Reference Example 1 187.41 + - 21.51 Reference Example 2 107.66 ± 25.65 Reference Example 3 38.56 + - 22.54 Example 1 88.49 + - 11.35

As shown in [Table 1] and [Graph 1], the DPPH radical of Reference Example 3 prepared by the present invention had the best scavenging ability, and Example 1 also showed a remarkably excellent effect.

Experimental Example 2: SOD-like activity test

In order to measure the scavenging activity of the superoxide radical of each plant extract composition, the scavenging activity of oxygen radical liberated from xanthine by xanthine oxidase was compared. As shown in the following [Graph 2] and [Table 2].

[2] a graph is a graph showing the SC ₅₀ value represents the reference examples 1 to 3 and Example 1 of mixed extract the SOD-like activity produced by the present invention at a concentration of ppm.

[Graph 2]

[Experimental Method]

(0.4 mM) and NBT solution (0.24 mM, nitro blue tetrazolium) in a solution of xanthine oxidase (0.045 U / ml) dissolved in a buffer solution (0.1 M phosphate buffer, pH 8.0) The reaction was stopped by addition of SDS (70 mM) solution. The degree of discoloration due to oxidation of cytochrome c was measured at 560 nm.

SOD-like activity (SC ₅₀ , ppm) Reference Example 1 212.55 ± 31.56 Reference Example 2 134.94 + 21.41 Reference Example 3 64.84 ± 14.25 Example 1 94.51 + - 19.54

As shown in [Graph 2] and [Table 2], SOD-like activity of Reference Example 3 prepared by the present invention was the most excellent, and Example 1 also showed a remarkably excellent effect.

Experimental Example 3: Preparation of H ₂ O ₂  Scavenging experiment

In order to measure the antioxidative activity of hydrogen peroxide, which is a product generated after decomposition of superoxide dismutase (SOD), which is a harmful active oxygen radical generated in vivo, into safer water and oxygen, it is produced by Fenton reaction And the degree of absorption at 520 nm of MDA (malondialdehyde) generated by oxidizing 2-deoxyribose by hydroxyl radicals was used. The results are shown in [Graph 3] and [Table 3].

[3] a graph is a graph, see examples 1-3 and carrying out the mixed extract prepared by the example 1 of the present invention showing the SC ₅₀ value of clearing the H ₂ O ₂ 50% at a concentration of ppm.

[Graph 3]

[Experimental Method]

To 10 μl of FeSO ₄ 7H ₂ O (50 mM) -EDTA (50 mM) solution, 10 μl of 2-deoxyribose (50 mM) solution was added and 20 μl of the sample dissolved in each concentration was added. phosphate buffer, pH 7.0), 10 μl of H ₂ O ₂ (50 mM) and 20 μl of distilled water were added and reacted at 37 ° C for 4 hours. 100 μl of 2.8% trichloroacetic acid and 1% 2- thiobarbituric acid was added and heated at 100 ° C for 15 minutes. After rapid cooling, the absorbance was measured at 530 nm using a spectrophotometer. Hydroxy radical scavenging activity (%) was calculated by the formula (1-absorbance of sample added sample / absorbance of sample not added sample) x 100.

H ₂ O ₂ scavenging ability (SC ₅₀ , ppm) Reference Example 1 203.51 ± 32.66 Reference Example 2 176.81 + - 23.58 Reference Example 3 45.28 + - 39.54 Example 1 85.26 ± 17.22

As shown in [Graph 3] and [Table 3], the H ₂ O ₂ scavenging ability of Reference Example 3 produced by the present invention was the most excellent, and Example 1 also showed remarkably excellent effects.

Experimental Example 4: Inhibition of LPS induced NO production

The effect of each sample in inhibiting the production of NO (nitric oxide), a periodontal disease inducing substance, was determined by measuring the nitrite concentration in the culture liquid produced by treating LPS (lipopolysaccharide) in mouse macrophages using the Griess reaction, Graph 4] and [Table 4].

[Graph 4] shows that the mixed extracts prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibit the production of nitric oxide induced by LPS in mouse macrophages.

[Graph 4]

[Experimental Method]

NO concentration was measured by Griess reaction. First, RAW 264.7 cells derived from mouse macrophages at 1 × 10 ⁴ cells / well were cultured in DMEM medium containing FBS and antibiotics in a 96-well culture plate, and cultured for 24 hours at 37 ° C. in a 5% CO 2 incubator. After 24 hours, the medium used for the previous culture was removed and a new DMEM medium containing no FBS and antibiotics was dispensed, and then the plant composition prepared according to Example 1 was treated by concentration. After 1 hour, 1 μg / mL of LPS was treated and cultured for 24 hours. The amount of NO produced during the culture was measured using the Griess reagent and the total NO ₂ ^- concentration in the cell culture. 50 μl of the cell culture supernatant and 50 μl of Griess reagent were mixed and incubated in 96-well plates for 10 min. Absorbance was measured at 540 nm using a reader. The percent elimination of nitric oxide (%) was calculated by using the equation (x1) (absorbance of the reaction group to which 1 sample was added / absorbance of the control group to which no sample was added).

Nitric Oxide Production (μM) Negative control group 4.51 ± 0.51 Positive control group 29.84 ± 1.66 Reference Example 1 16.58 ± 2.33 Reference Example 2 19.56 ± 0.52 Reference Example 3 28.25 + 1.52 Example 1 19.51 + - 3.21

As shown in [Graphs 4] and [Table 4], when the plant extract composition prepared according to the present invention was used, production of NO, which is one of the periodontal disease-causing substances induced by LPS, in mouse macrophages was remarkably suppressed . In particular, the inhibition of the formation of NO was remarkably excellent in the case where Reference Example 1 and Example 1 were treated.

Experimental Example 5: Inhibition of COX-2 activity

In order to examine the effect of each sample on the activity of COX-2 (Cyclooxygenase-2), the periodontal disease inducing enzyme, arachidonic acid (arachidonic acid) , And the results are shown in [Graph 5] and [Table 5].

[Graph 5] shows the IC ₅₀ value in ppm of the COX-2 enzyme inhibited by the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.

[Graph 5]

[Experimental Method]

First, 10 μl of COX-2 enzyme (30 units / mL), 10 μl of heme solution (150 μM / mL), and 10 μl of a reaction buffer (10 μl) were added to a 1.5 ml tube to cause the enzymatic reaction of COX-2 producing prostaglandin using arachidonic acid as a substrate (0.1 M Tris-HCl, 5 mM EDTA, 2 mM phenol, pH 8.0) was added thereto, and 20 μl of the diluted sample was added thereto at a sequential concentration, followed by stabilization at 37 ° C for 5 minutes. 10 아 of arachidonic acid was added thereto, followed by reaction at 37 째 C for 2 minutes, followed by addition of 50 쨉 l of 1M HCl, and the reaction was stopped. 100 μl of SnCl ₂ solution (50 mg / ml) was added, mixed well and allowed to react at room temperature for 5 minutes.

In order to perform a competitive ELISA with the prostaglandin produced in the above reaction, the reaction solution was diluted 2000-fold and 4000-fold with EIA buffer, and 50 μl was added to a 96-well plate coated with prostaglandin antibody. Then, 50 μl of PGs screening AChE tracer, Was added and reacted at room temperature for 18 hours. The 96 wells were washed 5 times with buffer, 200 쨉 l of Ellman's reagent was added, and developed for 60 minutes, and the absorbance was measured at a wavelength of 410 nm.

The degree of inhibition of COX-2 activity was indicated by IC ₅₀ (ppm), which is a 50% inhibitory concentration according to the manufacturer's method. Experimental results were expressed as mean ± standard deviation by repeating twice.

Inhibition of COX-2 activity (IC ₅₀ , ppm) Reference Example 1 135.24 ± 30.59 Reference Example 2 194.25 ± 25.36 Reference Example 3 59.88 ± 59.56 Example 1 104.64 ± 45.55

As shown in [Graph 5] and [Table 5], when the plant extract composition prepared according to the present invention was used, the activity of COX-2, which is a periodontal disease inducing enzyme, was remarkably suppressed. And the inhibition of the activity of COX-2 in the case of Example 1 was excellent.

Experimental Example 6: Inhibition of LOs (Lipoxygenases)

To measure the effect of inhibiting the periodontal disease-inducing enzyme LOs, the principle of measuring the amount of hydrogen peroxide, which is the product of the lipoxygenation reaction by LOs, was used using the Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical Com, MI, USA) , And the results are shown in [Graph 6] and [Table 6].

[Graph 6] shows the inhibition rate of the activity of Lipoxygenase enzyme by the mixed extract prepared in Reference Examples 1 to 3 and Example 1 of the present invention as compared with the untreated group of the sample.

[Graph 6]

[Experimental Method]

After adding 90 μl of 5-LO (220 units / ml) and 10 μl of 1mM linoleic acid to 10 μl of the sample, the mixture was reacted at room temperature for 5 minutes. 100 μl of chromogen was added thereto and reacted at room temperature for 5 minutes. Absorbance was measured at a wavelength of 490 nm by a device. The results were expressed as the 50% inhibitory concentration (IC ₅₀ ) value and the 5-LO inhibition rate (%) was expressed as the IC ₅₀ value using the equation [(absorbance of control group - absorbance of experimental group) / absorbance of control group] × 100.

Lipoxygenases Inhibition (%) Reference Example 1 54.50 + - 2.53 Reference Example 2 2.52 + - 6.56 Reference Example 3 25.81 + - 3.94 Example 1 9.81 ± 6.52

As shown in [Graphs 6] and [Table 6], when the plant extract composition prepared according to the present invention was used, the activity of the periodontal disease-related enzyme LO was inhibited. In particular, 1 inhibition of LO activity was found to be excellent.

Experimental Example 7: Inhibition of TNF-α production

In order to examine the effect of each sample in reducing the in vivo production of TNF-α, an inflammation inducer, MIA-induced chronic inflammatory animal models were measured using a TNF-α sandwich ELISA kit (eBiosience, Vienna, Austria) The results are shown in [Graph 7] and [Table 7].

[Graph 7] shows the amount of TNF-α produced in the edema of the chronic inflammatory animal model by MIA by the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.

[Graph 7]

[Experimental Method]

Each sample was sacrificed by oral administration of MIA-induced chronic inflammatory animal model once daily for the following period and dose, and blood plasma and plasma separation were performed. Then, the concentration of TNF-α in the plasma was measured by the method proposed by the manufacturer of the ELISA kit .

First, TNF-α in plasma was bound to an anti-rat TNF-α antibody coated on a 96-well plate. Each well was washed five times, and biotin-conjugated anti-rat TNF-α Antibody was reacted, each well was washed 5 times, treated with streptavidin-HRP, finally developed with tetramethyl-benzidine, and absorbance was measured at 450 nm using an ELISA reader. The amount of TNF- [alpha] was calculated in an arithmetic proportion using the standard curve of the standard recombinant TNF- [alpha] contained in the ELISA kit.

TNF-α production inhibition (pg / mL) Negative control group 358.66 ± 112.50 Positive control group 3265.26 + 265.33 Reference Example 1 906.51 + - 305.11 Reference Example 2 2261.46 ± 205.61 Reference Example 3 1681.64 ± 100.59 Example 1 1308.54 ± 115.69

As shown in [Graphs 7] and [Table 7], when the plant extract composition prepared according to the present invention was used in a chronic inflammatory animal model by MIA, the in vivo production of TNF- In particular, the in vivo production inhibition of TNF-a was very excellent in the case of the treatment of Reference Example 1 and Example 1. [

Experimental Example 8: Inhibition of IL-1β production

In order to examine the effect of each sample in reducing the in vivo expression of IL-1β, an inflammation inducer, IL-1β sandwich ELISA kit (eBiosience, Vienna, Austria) was administered to carrageenan- ), And the results are shown in [Graph 8] and [Table 8].

[Graph 8] shows the amount of IL-1? Produced in the edema of chronic inflammatory animal model by MIA by the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.

[Graph 8]

[Experimental Method]

Each sample was sacrificed by oral administration of MIA-induced chronic inflammatory animal model once daily for the following period and dose, and blood plasma and plasma separation were performed. The concentration of IL-1β present in the plasma was measured by the method proposed by the manufacturer of the ELISA kit .

First, IL-1β was bound to the anti-rat IL-1β antibody coated on a 96-well plate, and each well was washed five times with biotin-conjugated anti-rat IL-1β antibody. Was washed 5 times with streptavidin-HRP, finally developed with tetramethyl-benzidine, and absorbance was measured at 450 nm using an ELISA reader. The amount of IL-1β was calculated in an arithmetic proportion using the standard curve of the standard recombinant IL-1β contained in the ELISA kit.

IL-1β production inhibition (pg / mL) Negative control group 64.51 + - 14.51 Positive control group 775.26 + - 42.60 Reference Example 1 215.90 ± 64.58 Reference Example 2 488.54 ± 70.36 Reference Example 3 594.56 ± 64.82 Example 1 350.84 ± 45.99

As shown in [Graphs 8] and [Table 8], when the plant extract composition prepared by the present invention is used in a chronic inflammatory animal model by MIA, the in vivo production of IL-1β, a periodic disease-related factor, is suppressed In particular, inhibition of in vivo production of IL-1β was shown to be excellent in the case where Reference Example 1 and Example 1 were treated.

Experimental Example 9: Inhibition test of PGs (Prostaglandins)

To determine the efficacy of each sample to inhibit the production of periodontal disease-causing factor Prostaglandins (PGs) in a chronic inflammatory animal model using MIA, PGs and Tracer were measured using a Ptostaglandin Screening EIA Kit (Cayman Chemical Co., MI, USA) (PGs artificially bound to acetylcholinesterase) competitively bind to anti-PGs-antibody, and the results are shown in [Graph 9] and [Table 9].

[Graph 9] shows that the mixed extracts prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibit the production of prostaglandins in edema of chronic inflammatory animal models by MIA.

[Graph 9]

[Experimental Method]

Prostaglandin AChE Tracer and antiserum were placed in a 96-well plate coated with anti-PGS antibody, and 50 μl of the standard prostaglandin or each sample-treated medium was added thereto. The reaction was carried out for 18 hours at room temperature After washing each well 5 times, 200 ㎕ of Ellman's Reagent is added, and developed for 70 minutes. Absorbance is measured at 420 nm wavelength using an ELISA reader. The amount of PGs was calculated in an arithmetic proportion using a standard curve for the reaction of standard PGs contained in the ELISA kit.

Prostaglandins production inhibition (pg / mL) Negative control group 11.42 + - 8.62 Positive control group 355.99 ± 25.40 Reference Example 1 94.98 + - 6.53 Reference Example 2 224.66 ± 31.51 Reference Example 3 168.84 ± 9.54 Example 1 130.67 + - 8.50

As shown in [Graphs 9] and [Table 9], when the plant extract composition prepared according to the present invention was used, the production of PGs, which is one of the periodontal disease inducers induced by LPS, in mouse macrophages was inhibited In particular, the inhibition of PGs formation in Reference Example 1 and Example 1 was found to be excellent.

EXPERIMENTAL EXAMPLE 10 Inhibition of In vivo Expression of iNOS, COX-2 and 5-LO

In order to measure the inhibitory effect of iNOS, COX-2 and 5-LO on the in vivo expression of periodontal disease inducing enzymes in each sample treated with the concentration of [Table 10] in the inflammatory cell model using LPS, immunocytochemical staining Immunocytochemistry) was performed, and the results are shown in [Photo 1], [Photo 2] and [Photo 3].

[Picture 1], [Picture 2] and [Picture 3] show that the mixed extracts prepared according to Reference Examples 1 to 3 and Example 1 of the present invention exhibited the iNOS expression in the inflammatory response of mouse macrophages induced by LPS, COX-2, 5-LO.

[Picture 1]

[Picture 2]

[Picture 3]

[Experimental Method]

Mouse macrophage-derived Raw264.7 cells were cultured in a 24-well culture medium containing glass coverslips for 12 mm round optical microscope specially coated for cell proliferation using DMEM medium containing 10% FBS and antibiotics 2 × 10 ⁴ cells / well were plated on a well culture plate and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. After 24 hours, the medium used for the previous culture was removed and a new DMEM medium containing no FBS and antibiotics was dispensed and each sample was treated by concentration. After 1 hour, the cells were treated with LPS at a concentration of 1 μg / mL and cultured for 24 hours. After 24 hours, the cells were fixed with acetone, treated with 0.3% H ₂ O ₂ for 10 minutes for inactivation of endogenous peroxidase, washed three times with PBS, treated with chlorine serum to prevent non-specific binding of the primary antibody, The primary antibody was reacted at 4 ° C for 24 hours. After 24 hours, the cells were washed three times with PBS, and the signal was amplified using a VECTASTAIN elite ABC kit (VECTOR laboratories, CA, USA) and stained with DAB (Sigma, MO, USA). Cell-mounted cover slides were removed from 24-well culture plates using a forceps, dehydrated and attached to a slide glass using Canada balsam (Sigma, MO, USA). The slide thus prepared was observed with an optical microscope at a magnification of 1000 times. The dilution concentrations of iNOS (Santa Cruz Biotechnology, CA, USA), COX-2 (Cell Signaling Technology, MA, USA) and 5-LO (Santa Cruz Biotechnology, CA, USA) 1.

division Treatment concentration (24 hours) LPS not processed Negative control group Purified water Treatment with LPS (1 ug / ml) Positive control group Purified water Reference Example 1 Sickleberry extract 200ppm Reference Example 2 Mulberry extract 200ppm Reference Example 3 Quercetin 200ppm Example 1 Composition 200 ppm

Inhibition of iNOS, COX-2 and 5-LO in vivo expression of the inflammatory enzymes of Reference Example 1 and Example 1 prepared according to the present invention, as shown in [Picture 1] to [Picture 3] iNOS, COX-2, and 5-LO were highly expressed and the number of colored cells was decreased compared to the positive control).

Experimental Example 11: Measurement of cell (Raw 264.7) toxicity

MTT assay was performed to measure the toxicity of Example 1 against mouse macrophage Raw264.7 cells, and the results are shown in [Graph 10].

[Experimental Method]

The toxicity of macrophages was determined by measuring MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] (Sigma, MO, USA). Raw264.7 cells were plated at 1 × 10 ⁴ cells / mL per well in 96 wells and cultured at 37 ° C and 5% CO ₂ for 24 hours. 24 hours in the previous removing the medium used for the culture and to the back of the sample was dissolved in DMSO the final concentration of DMSO is diluted with medium to a 1% by concentration of the extract, respectively, 37 ℃ in (μg / mL), 5% CO 2 5 hours and 24 hours, respectively. After treatment, 20 μl of 0.2% MTT solution was added to each well, followed by reaction at 37 ° C in a 5% CO ₂ incubator for 3 hours. After the reaction, all of the supernatant was removed, 150 μl of DMSO was added, the formazan produced at room temperature was completely dissolved for 10 minutes, and the absorbance at 570 nm was measured using an ELISA reader. To investigate the effects on DMSO, experiments were performed by dividing into a control group and a control group for 1% DMSO. Cell viability (%) was expressed by the following equation. Cell survival rate (%) = (absorbance of sample treated group / absorbance of control group) x100

As shown in [Graph 10], it can be seen that the cytotoxicity of Example 1 produced by the present invention is not toxic even at a high concentration of 2500 ppm.

[Graph 10]

Experimental Example  12: ICR  Animals using a mouse Single toxicity  Experiment

To determine the toxicity of Example 1 to mice, an animal monotoxicity experiment was conducted and the results are shown in [Graph 11], [Graph 12], and [Graph 13].

[Experimental Method]

Sprague Dawley (SD) rats (Orient Bio, Korea) were used as experimental animals. Control group and control group were 5 male and 5 female. These animals were not observed for general symptoms during acquisition and during the adaptation period. The feeding conditions were set at 22 ± 2 ℃, 55 ± 10% of relative humidity and 12 hours of illumination time (6:00 am to 6:00 pm). The animals were fed with solid feed (Samtaco, Korea) (Tap water) was freely consumed.

In the single toxicity test of Example 1, the experimental group was administered with dissolving the distilled water in Example 1, the control group was treated with distilled water, the rat was fasted 12 hours before the administration of the sample and used as a single dose oral dose of the normal health functional food Kg body weight and 2 g / kg body weight.

Clinical symptoms were observed according to the Korea Food & Drug Administration 's toxicity test standard and OECD test guideline 420 (TG 420). That is, all the animals were observed every hour for 6 hours after the administration on the day of administration, and the change of the general state of the animal, the expression of the toxic symptoms and the deaths were observed once a day from the next day to the 14th day. The body weight change on the day of the test substance administration and on the 14th day was also measured. After the end of the test, the animals were anesthetized and sacrificed, and visual appearance of the external appearance and internal organs was visually observed.

[Graph 11]

[Graph 12]

[Graph 13]

There were no deaths in the male and female according to the administration of Example 1. There were no specific behavioral changes in the NSAIF treated group compared with the control group after 4 times of observation on the same day and 1 time of daily observation for 14 days from the next day. In addition, there was no significant change in the weight change after 14 days of sample administration and the change of organ weight after autopsy compared with the control group. Thus, it can be seen that Example 1 has no animal monotoxicity.

Hereinafter, examples of formulations of foods or health functional foods containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Preparation Example 1: Preparation of powder

300 mg of the functional composition of Example 1, 100 mg of lactose, 10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

Production Example 2: Preparation of tablets

50 mg of the functional composition of Example 1, 100 mg of corn starch, 100 mg of lactose, 2 mg of magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Preparation Example 3: Preparation of capsule

50 mg of the functional composition of Example 1, 100 mg of corn starch, 100 mg of lactose, 2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Production Example 4: Preparation of liquid agent

100 mg of the functional composition of Example 1, 10 g of isomerized sugar, 5 g of mannitol,

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and then purified water was added thereto. The whole was then adjusted to 100 ml with purified water, Filled and sterilized to prepare a liquid preparation.

Production Example 5: Preparation of Health Functional Foods

1000 mg of the functional composition of Example 1, a proper amount of vitamin A, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 13 mg of vitamin B10, 15 mg of vitamin B20, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of niacinamide, 1.7 mg of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, a suitable amount of inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic Mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Preparation Example 6: Preparation of health drinks

1,000 mg of the functional composition of Example 1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of a plum concentrate, 1 g of taurine and purified water were added to adjust the total amount to 900 ml.

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

On the other hand, in Example 1 described above, when the composition obtained by mixing 20 g of the mulberry leaf extract and 20 g of quercetin with 100 g of the extract of the fresh mint extractively evaluated the results of each of Experimental Examples 1 to 12, It is judged to be a good composition.

The reason for the highest composition ratio of the extracts was that the extracts of Sangsik extract had the most excellent effects in inhibiting nitric oxide and prostaglandins formation, inhibiting TNF-α and IL-1β production as described above, The extracts and quercetin show excellent effects even in the most effective items. In addition, the extracts of mulberry leaf and quercetin have a bitter taste, so they are not suitable for product development in various food forms if they are contained in large quantities in foods.

As described above, according to the present invention, it is possible to prevent the periodontal disease-inducing substances NITRICO and PGEM (Prostaglandin E metabolite) from being produced, and the periodontal disease inducing enzyme (TNF-α) and IL-1β (Interleukin-1β), which are inflammatory cytokines, and inhibit the inhibition of COX-2 and LOs Inhibition, and inhibition of the expression of iNOS (inducible nitric oxide synthase), which is a periodontal disease inducing enzyme.

In this study, we investigated the effects of iNOS (inducible nitric oxide synthase), iNOS (iNOS), and TNF-α and IL-1β on periodontitis, and inhibit the formation of periodontitis, Nitric oxide and Prostaglandins, , COX-2 (cyclooxygenase-2) and 5-LO (5-lipoxygenase) in vivo and quercetin provides antioxidative effects (DPPH scavenging activity, SOD-like activity, H ₂ O ₂ scavenging ability) COX-2 and Lipoxygenases.

Claims (5)

A composition for preventing and / or improving periodontal disease, which comprises a mixture of a topical extract, a topical extract and quercetin. The method according to claim 1,
Wherein the mixture is a mixture of 20 to 40 g of the leaf extracts and 20 to 40 g of quercetin per 100 g of the extract of the topical extract. 3. The method according to claim 1 or 2,
The composition for preventing and improving periodontal disease is characterized in that the dosage form of the composition for preventing or improving periodontal disease is any one of capsules, rings, granules, powders, candies, gums, tablets, beverages and syrups. delete delete