WO2019050063A1 - Composition for preventing or treating periodontal diseases comprising as active ingredient scutellaria baicalensis extract treated with enzyme isolated from aspergillus niger - Google Patents

Abstract

The present invention relates to a composition for preventing or treating periodontal diseases, the composition comprising as an active ingredient an extract of Scutellaria baicalensis treated with an enzyme derived from Aspergillus niger . The extract of Scutellaria baicalensis treated with the enzyme derived from Aspergillus niger according to the present invention promotes the mineralization of osteoblasts, stimulates the proliferation of cells of the periodontal ligament and presents an excellent activity of prevention or treatment of periodontal diseases by the inhibition of the expression of interleukin 1β, TNF-α (tumor necrosis factor alpha) and MMP-1 (matrix metalloproteinase 1), and can therefore be used effectively as a pharmaceutical composition, functional health food or the like.

Description

Composition for preventing or treating periodontal disease comprising aspirin and its extractive gold-treated extract as active ingredients

The present invention relates to a composition for preventing or treating periodontal disease, and more particularly, to a composition for preventing or treating periodontal disease, which contains, as an active ingredient, a gold extract produced by treating an enzyme isolated from Aspergillus oryzae.

Periodontal disease refers to periodontal ligament that surrounds the teeth and symptoms related to inflammatory diseases that destroy hard tissues such as soft tissues such as gingiva and alveolar bone. As age increases, periodontal disease, periodontal inflammation, gingivitis, and alveolar bone osteodystrophy are the most common periodontal diseases when congenital and acquired alveolar bone deteriorates. 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. Gingivitis is a condition in which the gingivitis is not treated and the inflammation progresses to the gums and around the pubis. When the chronic inflammatory reaction stimulated by the bacterial toxin develops, the periodontal pocket is formed between the gums and the teeth, The deeper the deeper the depth of the periodontal ligament, the end result is inflammation of the periodontal ligament and bone loss occurs. In alveolar bone, osteoblast-induced bone formation and osteoclast-induced bone resorption are metabolized. When the bone resorption exceeds the osteogenesis due to various factors and the bone mass is decreased below the limit, alveolar bone formation disorders such as osteoporosis Occurs.

Among these periodontal diseases in the adult population globally, periodontitis is not only one of the most common oral diseases among the diseases that can occur in the mouth, but is also a major public health concern requiring significant cost to the health care system.

Periodontal disease is the most important issue for the prevention and prevention of oral hygiene in patients. In clinical practice, the treatment of periodontal diseases such as nonsurgical or surgical dental decompression, root resorption, gingival curettage, Of the total. However, these surgical treatments are cumbersome and difficult to treat effectively. In addition, the disease is limited only to the treatment which is not carried out when the disease progresses to some degree, and it is most likely to progress to chronic disease when the treatment is not performed. In addition, antibiotics acting on the whole body and antibiotics acting on the local region are used as additional treatments, but the drug is excessively delivered to areas other than the area where the periodontal disease occurs, resulting in side effects, Have been reported to cause serious problems in periodontal disease bacteria, which are resistant to antibiotics.

In addition, there is an increase in the expression of inflammatory mediators such as NO, IL-1β, MMP-1 and TNF-α in the progression of periodontal disease. These factors are factors that act strongly to induce bone resorption , And studies on periodontal diseases to date have been mostly concerned with the regulation of inflammatory mediators.

In addition, the periodontal disease is a complex feature caused by the action of periodontal disease bacteria such as loss of alveolar bone, inflammation, jinjy valis, etc. unlike general inflammation and arthritis, so that antibacterial and antiinflammation, bone formation of alveolar bone is promoted A combination of research and treatment methods such as exhibiting an action to inhibit bone resorption is required, but a drug study or treatment method for such treatment has not yet been developed.

Under such circumstances, it is necessary to develop a drug or a therapeutic agent capable of effectively preventing and treating periodontal disease. Recently, there have been attracted attention to improved natural products that have fewer side effects and have a variety of periodontal There is a growing need to develop a composition for prevention and treatment of diseases. Therefore, the present inventors observed that when the gold extract was treated with an aspergillus or its derivative, the inhibitory effect on the inherent inflammation of gold was drastically increased, and the alveolar bone formation effect was remarkably increased. As a result, The present invention can be used effectively for the prevention and treatment of periodontal disease.

[Prior Art Literature]

(Patent Document 0001) KR 10-1641213, 2016.07.14

(Patent Document 0002) KR 10-1704589, 2017.02.02

Accordingly, a main object of the present invention is to provide a composition for preventing or treating periodontal disease, which comprises an enzyme-treated gold extract as an active ingredient.

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

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease comprising an enzyme-treated golden extract derived from Aspergillus niger as an active ingredient.

The present inventors have searched for various natural substances in order to find a substance having an excellent effect for the prevention or treatment of periodontal disease and have made efforts to improve the activity of the discovered natural substances. As a result, it has been found that the enzyme isolated from Aspergillus niger The present invention has been accomplished based on the discovery that a golden extract has an antibacterial and antiinflammatory effect and an effect of promoting bone formation of alveolar bone and inhibiting bone resorption.

The composition of the present invention is a composition for prevention and treatment of periodontal disease using a gold extract, which is a natural material-derived material, and is safe without side effects even when applied to human body.

&Quot; Scutellaria radix " of the present invention means a root of Scutellaria baicalensis Georgi, which is a perennial herb with lepidoptera, which means that the root of the root is removed. The gold is conical, twisted and curved, 8 to 25 cm long, and 1 to 3 cm in diameter. The outer surface of the gold is yellowish brown or dark yellow, and there are sparse spots of lumpy spots. The old age is decayed or empty in the middle of the roots, dark brown is called now, and reddish brown is sometimes called high gold.

There are many phenolic compounds such as baicalin, baicalein, and wogonin in the gold, among which baicalin is the most abundant.

The term " extract " used in the present invention refers to an extract obtained by extracting a plant, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a fraction of the extract, And mixtures thereof, extracts of all the formulations which can be formed using the extract itself and the extract.

As the solvent for obtaining the gold extract in the present invention, any solvent conventionally used in the art can be used. For example, the extraction solvent may be selected from the group consisting of (a) an anhydrous or a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol, (D) ethylacetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethyl ether, (i) ) Butyl acetate or (j) water. Preferably, the extraction solvent is a polar solvent, more preferably a polar solvent selected from the group consisting of methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol, to be.

The gold extract may be extracted at room temperature or warmed under conditions in which the active ingredient is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the extract may differ. Therefore, an appropriate organic solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction. The gold extract of the present invention can be extracted from natural, hybrid, or variant plants of gold, and can be extracted from plant tissue cultures.

The term "fraction" as used herein means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents.

The golden fraction of the present invention means a fraction produced by fractionating the golden extract using a solvent having a different polarity in the fractionation column. Specifically, the golden fraction of the present invention can be obtained by suspending a gold extract with distilled water and then fractionating it with a solvent such as water, butanol, ethyl acetate, dichloromethane, or n-hexane. More specifically, the gold extract is suspended in distilled water, and a solvent such as water, butanol, ethyl acetate, dichloromethane, n-hexane or the like is added to the suspension in an amount of about 1 to 100 times, preferably about 1 to 5 times, And the polar and non-polar solvent soluble layers can be extracted and separated by repeating the above-mentioned steps for from 10 times to 10 times, preferably from 2 times to 5 times. Further, a conventional fractionation process may be performed (Harborne J. B. Plant Pathology, 1998, 3rd Ed. P6-7). More specifically, after suspending the gold extract in distilled water, the same amount of ethyl acetate as a solvent is continuously extracted to obtain a golden ethyl acetate soluble fraction.

The Aspergillus niger of the present invention can be isolated from the yeast, which is an edible strain.

The strain is harmless to the human body because it is an edible strain. Therefore, in performing the biotransformation process for converting the golden extract into the enzyme possessed by the strain, it is also possible to use only the crude extract or concentrate or extract the enzyme of the strain, Can be used in the form of using enzymes that separate the enzyme.

The Aspergillus niger may also be an Aspergillus oryzae strain distributed from KACC (Korean Agricultural Microbiology Resource Center). The Aspergillus niger may be selected from the group consisting of Aspergillus niger (microorganism deposit number KACC 41018), Aspergillus niger (microorganism deposit number KACC 41858), Aspergillus niger (microorganism deposit number KACC 42589), Aspergillus niger (Microorganism deposit number KACC 43547) and Aspergillus niger (microorganism deposit number KACC 44333).

Preferably, the Aspergillus niger may be Aspergillus niger (microorganism deposit number KACC 41018) or Aspergillus niger (microorganism deposit number KACC 41858).

Golden powder or gold extract may be added to the culture medium of Aspergillus niger . By adding a golden powder or a golden extract, it is possible to further improve the preventive or therapeutic effect of periodontal disease by biotransformation of the components in the golden extract. In the preparation examples of the present invention, crude extracts obtained from Aspergillus oryzae were treated with gold extracts, and Baicalin and Wogonoside, which are present in large quantities in the gold extract, were treated with Baicalein ) And Wogonin (see Table 1 and FIG. 1).

The method for preparing an enzyme having the activity of converting the golden extract into an active ingredient comprises the steps of: culturing Aspergillus niger in a culture medium; Centrifuging the culture solution to obtain an unpurified enzyme solution; Treating the enzyme solution with an organic solvent to precipitate an enzyme protein; Dissolving the precipitate in a buffer solution to dialyze and removing foreign matter to obtain an unpurified enzyme concentrate; And loading the unpurified enzyme concentrate into an ion exchange resin (ion exchange resin and cation exchange resin) and eluting it with a buffer solution to obtain a fraction of the enzyme. The organic solvent may be ethanol, isopropanol, acetone, or the like. The buffer solution to be treated with the precipitate may be a phosphoric acid buffer (posphate buffer), an acetate buffer, a tris buffer solution, or the like. The buffer solution used for the elution may be KCl buffer solution or NaCl buffer solution.

In the present invention, the enzyme-treated gold extract or its fraction itself is characterized by exhibiting an activity of preventing or treating periodontal disease.

In the present invention, the periodontal disease is exemplified by gingivitis, periodontal inflammation, alveolar bone breakage, and alveolar bone osteodystrophy. The alveolar bone disorder is osteoporosis but are not limited to, alveolar bone osteoporosis, alveolar bone osteomalacia, or alveolar bone osteopenia.

In the present invention, the prevention or treatment of periodontal disease of the enzyme-treated golden extract derived from Aspergillus niger promotes mineralization of osteoblasts, promotes the proliferation of periodontal ligament cells, 1 (interleukin-1?), TNF-alpha (tumor necrosis factor-alpha), and MMP-1 (matrix metalloproteinase-1).

In one embodiment of the present invention, the enzyme-treated gold extract according to the present invention promotes the proliferation of periodontal ligament cells (see FIG. 2), inhibits NO production as an inflammatory mediator (see FIG. 3) To promote the activation of alkaline phosphatase in osteoblasts (see FIG. 4), to promote mineralization (see FIG. 5), to prevent the alveolar bone from being actually absorbed in the animal model, (See Figs. 6 and 7).

The pharmaceutical composition for preventing or treating periodontal disease of the present invention may be in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated in the form of an oral preparation such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like and a sterile injectable solution according to a conventional method and may be formulated into powders, tablets, capsules, And a liquid agent are more preferable. Such formulation can be carried out by a method commonly used in the field of pharmacy, and can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA) .

The pharmaceutically acceptable carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

Solid form preparations for oral use include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like, and may include a lubricant such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like.

The composition for oral administration of the present invention may be formulated as a film, a toothpaste, a mouthwash solution, an ointment, a spray, a dressing solution, a coating agent, a dental floss or a periodontal tissue in addition to the formulations such as tablets and pills.

Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions and freeze-dried preparations, and non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, And the like.

The pharmaceutical composition of the present invention may contain 0.01 to 99.9% by weight, preferably 0.1 to 99% by weight, of the enzyme-treated gold extract in relation to the total weight of the composition, and the use of the composition for preventing or treating periodontal disease And the content of the active ingredient can be appropriately controlled depending on the purpose of use.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, The range varies depending on the condition, diet, administration time, administration method, excretion rate, and disease severity.

The daily dose of the enzyme-treated gold extract of the present invention is about 0.1-500 mg / kg, preferably 50-200 mg / kg.

The pharmaceutical composition of the present invention can be used alone or in combination with methods for the prevention and treatment of periodontal disease or using surgery and medication.

According to another aspect of the invention, the invention provides a dietary supplement for the prevention and treatment of periodontal disease containing the Aspergillus and this (Aspergillus niger) Gold extract derived enzyme treatment as an active ingredient.

The term " health functional food " in the present invention means a food prepared and processed by using a raw material or ingredient having a useful function in the human body according to Law No. 6727 on health functional foods, and " It means that the structure and function of the human body is ingested for the purpose of obtaining nutritional effect or useful effect for health use such as physiological action.

In the health functional food of the present invention, the enzyme treatment, the extraction solvent, the extraction method and the like are as described in the pharmaceutical composition. The health functional food of the present invention may contain 0.01 to 99.9% by weight of an enzyme-treated golden extract derived from Aspergillus niger , based on the total weight of the composition.

The health functional foods of the present invention may be formulations selected from powders, tablets, capsules, injections, liquids and dairy products, and the formulations thereof may be the same as those for preparing pharmaceutical compositions, .

There is no particular limitation on the kind of the food or health functional food of the present invention. Examples of foods to which the above-mentioned Aspergillus niger- derived enzyme-treated golden extract can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, ice cream , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

In addition to the above, the enzyme-treated gold extract of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the Aspergillus niger- derived enzyme-treated gold extract of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

The health functional food of the present invention includes a food added with a food additive for producing an enzyme-treated golden extract derived from Aspergillus niger of the present invention as a health functional food.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating periodontal disease, comprising as an active ingredient an enzyme-treated golden extract derived from Aspergillus niger , The present invention provides a method for preventing or treating periodontal disease in an animal other than a human, comprising the step of administering to a subject to prevent or treat periodontal disease.

The method of treatment according to the present invention does not mean that a method of treating an animal other than a human, but such a treatment method is ineffective in humans. Also, in the case of humans, it can be sufficiently used in the treatment of humans in consideration of having periodontal disease in which symptoms can be improved by administration of a pharmaceutical composition for the prevention or treatment of periodontal disease according to the present invention.

The term " animal excluding human being " in the present invention refers to an animal such as a horse, a sheep, a pig, a goat, a herb, a poultry, Camel, nutrition, and dog. By administering the pharmaceutical composition for preventing or treating periodontal disease according to the present invention to an animal other than a human, periodontal disease can be effectively prevented and treated.

The term " administering " in the present invention means introducing a predetermined substance into an animal by any appropriate method, and the administration route of the therapeutic composition according to the present invention may be administered orally, May be administered parenterally. In addition, the pharmaceutical composition for preventing or treating periodontal disease according to the present invention may be administered by any device capable of moving the active ingredient into the target cell.

The preferable dosage of the therapeutic composition according to the present invention varies depending on the condition and body weight of the animal to be treated, the degree of disease, the type of drug, route of administration and period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention may be administered in a dose of 0.1 to 500 mg / kg, preferably 50 to 200 mg / kg, once to three times a day.

Since the composition of the present invention is effective for the prevention and treatment of periodontal disease itself, it can be used directly as a composition for the prevention and treatment of periodontal disease and can be used by mixing with other ingredients containing natural extracts effective for periodontal disease It is expected that it will be able to improve the preventive and therapeutic effect of the existing periodontal disease.

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

(1) The present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease comprising an enzyme-treated golden extract derived from Aspergillus niger as an active ingredient.

(2) The Aspergillus niger- derived enzyme-treated gold extract of the present invention promotes mineralization of osteoblasts, promotes the proliferation of periodontal ligament cells, and promotes interleukin-1? ), TNF-alpha (tumor necrosis factor-alpha), and MMP-1 (matrix metalloproteinase-1) expression.

FIG. 1 is a graph showing the changes in the components of the gold extract and the enzyme-treated gold extract using HPLC (standards (A), before (B) and after (C). Peak 1, baicalin 2, wogonoside 3, baicalein; peak 4, wogonin).

FIG. 2 is a graph showing the effect of the enzyme-treated gold extract on cellular proliferation of periodontal ligament cells (HPDL) (in FIG. 2, *** means p-value <0.001).

FIG. 3 is a graph showing the effect of the enzyme-treated gold extract on inhibition of NO production, which is an inflammatory mediator (*** indicates p-value <0.001 as compared with normal group, p-value < 0.001).

FIG. 4 is a graph showing the effect of the enzyme-treated gold extract on the activity of Alkaline phosphatase in osteoblasts and periodontal ligament cells (*** means p-value <0.001).

FIG. 5 is a graph showing the effect of the enzyme-treated gold extract on the mineralization activity in osteoblasts and periodontal ligament cells (*** means p-value <0.001).

FIG. 6 is a 2D and 3D diagram showing an effect of improving periodontal disease in an animal model of an enzyme-treated gold extract. FIG.

FIG. 7 is a graph showing the CEJ-ABC distance between the first molar and the second molar end calculated from the photograph of FIG. 6, the CEJ-ABC distance between the second molar and the third molar, and the furcation involvement (** indicates p-value < 0.01 and *** indicates p-value < 0.001).

FIG. 8 is a graph showing expression levels of interleukin (IL) -1β, MMP-1 and tumor necrosis factor-alpha (inflammatory mediators) (*** indicates p-value <0.001 , ## indicates p-value <0.01 compared to LPS alone treatment group, and ### means p-value <0.001).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited to the embodiments disclosed below but may be embodied in various other forms, And to fully disclose the scope of the invention to a person having ordinary skill in the art to which the present invention belongs, and the present invention is only defined by the scope of the claims. In addition, the significance between the control group, the comparative group and the experimental group according to the experimental results described below was verified by One-Way ANOVA.

Example  1. Enzyme extraction

1-1. Strain isolation

The yeast was suspended in 10 ml of sterile distilled water. Thereafter, it was rotated at 200 rpm for 10 minutes. Sterile distilled water was prepared from the stock in sterile conditions at 10 ^-4 to 10 ^-6 dilutions. Successively diluted samples were inoculated on malt extract agar (Difco).

The Petri plates were then incubated at 30 +/- 1 DEG C for 3-4 days. After cultivation, colonies (black spores), which are presumed to be Aspergillus niger , were picked up by gross observation, transferred to potato dextrose agar (PDA, Difco) and cultured several times to obtain pure colonies.

1-2. Identification of strains (ITS Molecular marker  Sequencing)

Extraction of genomic DNA was performed using a commercial genomic DNA extraction kit (Solgent, Korea). For amplification, two previously known oligonucleotide fungal primers were used. ITS site primers (ITS 1, 5'-TCC GTA GGT GAA CCT GCG G-3 '; ITS 4, 5'-TCC TCC GCT TAT TGA for amplifying 5.8S gene, ITS 1 and ITS 2 non- TAT G-3 ') was constructed based on conserved regions of 18S (ITS 1) and 28S (ITS 4) rRNA genes. The purified PCR products were subjected to sequencing by Solgent Co. Ltd. ITS rRNA gene sequences of related taxa were obtained from the GenBank database and the query-based BioloMICS database (www.fungalbarcoding.org). The isolated strain exhibited about 99% similarity to the Aspergillus niger ITS sequence on the database.

1-3. Acquisition of enzyme

The isolated Aspergillus niger strain was cultured by liquid culture. For liquid culture, 1.5 g / L Beef Extract (Becton, Dickinson and Company), 2.5 g / L Peptone (BD), 0.05 g / L Magnesium Sulfate (Sigma) The culture was continued for 3 to 5 days at a temperature of 30-37 DEG C and 180 rpm in a medium containing 0.05 g / L of Calcium Chloride (Sigma) at 50-70%. Rice bran was added to the culture, and the culture was centrifuged at 7,000 rpm for 30 minutes to obtain an unpurified enzyme solution (crude enzyme solution). An acetone, which is an organic solvent (ethanol, isopropanol, acetone, etc. may be used), was added to the unpurified enzyme solution, and the enzyme protein was precipitated at 4 캜 for 4 hours. The precipitate was dissolved in a phosphate buffer, dialyzed, and then deionized to obtain a crude enzyme concentrate. Was loaded onto an ion exchange resin [anion exchange resin QAE-sephadex A50 (sigma) and cation exchange resin Sp-sephadex C50 (sigma)] and then eluted with NaCl buffer solution (Sigma) crude enzyme.

1-4. Enzymatic extraction from pre-marketed strains

Aspergillus niger (Microorganism Accession No. KACC 41018) and Aspergillus niger (Microorganism Accession No. KACC 41858), which were distributed at the Korea Institute of Bioscience and Biotechnology, were cultured, The coenzyme was almost completely isolated.

Example  2. Preparation of enzyme-treated gold extract

Domestic gold purchased from Kyungdong market was washed in running water to remove impurities. To 600 g of gold, 6 L of 70% ethanol was added, followed by stirring at room temperature for 3 days. The above procedure proceeded to 3-batch. The resulting extract was collected and concentrated under reduced pressure at 40 DEG C or lower to obtain an extract-concentrated extract. The concentrated gold extract was dissolved in 10 ml of 70% ethanol at a ratio of 1 mg, and then 3% (w / w) of the enzyme derived from Aspergillus niger extracted in Production Examples 1-3 and 1-4 was inoculated And cultured in a 55 ° C incubator for 24 hours. The culture was sterilized at 121 ° C for 15 minutes to inactivate the enzyme and centrifuged to collect only the supernatant. The enzyme-treated gold extract was concentrated under reduced pressure at 55 ° C using a rotary evaporator (N-1000, EYELA, Japan) and lyophilized to prepare a powder.

In order to compare the content of the indicator components of the above extracts, there was used an enzyme-untreated golden extract (Comparative Example), an enzyme-treated golden extract directly extracted from the koji of Example 1-3 (Example 2-1) (Examples 2-2 and 2-3) were analyzed for their components. For high performance liquid chromatography analysis, Thermo UHPLC U3000 was used. The HPLC analysis conditions are as follows. The column was a Sunfire C18 column (Waters, 250 x 4.6 mm, 5 m) and the column temperature was 25 캜. The UV wavelength was measured at 270 nm. The mobile phase was water and 0.1% formic acid in water and acetonitrile (gradientmode). The gold and enzyme-treated gold extracts were dissolved in mobile phase solvent at a concentration of 5 mg / ml and then injected with 10 uL and measured at a flow rate of 1 ml / min. The index component content of the extract is shown in Table 1 below.

Referring to Table 1 and FIG. 1, the gold extract of Example 2-1 contains Baicalin and Wogonoside, which are abundant in gold, and Baikalein and Wogonin, In the second half of the year. Baicalein and Wogonin are known to be effective against inflammation, and it is known that the enzyme-treated gold extract can be effective for periodontal diseases such as periodontitis. On the other hand, it was confirmed that the enzyme-treated gold extract obtained from the pre-sale strain of Examples 2-2 and 2-3 showed a similar pattern to the HPLC pattern of the enzyme-treated gold extract of Example 2-1.

Experimental Example  1. Enzyme-Treated Golden Extract Periodontal ligament  Evaluation of cell proliferation activity

The effect of the enzyme-treated gold extract (enzyme-treated gold extract of Examples 2-1, 2-2 and 2-3) obtained in Example 2 on the proliferation of human periodontal ligament progenitor cells (PDLC) was measured . In other words, periodontal ligament cells cultured in DMEM (Dulbecco's Modified Eagle's medium, Cambrex, USA) containing 10% fetal bovine serum (FBS) were inoculated in a 96-well plate at a concentration of 0.5 × 10 ⁴ cells / , And then cultured in a carbon dioxide incubator (5% carbon dioxide, 95% relative humidity, 37 ° C) for 24 hours. The culture solution was treated with the enzyme-treated gold extract at a concentration of 10 and 100 ug / ml and cultured for 24 hours. Then, 5 mg / ml of MTT (3- (4,5-dimethylthiazol-2-yl) diphenyl tetrazolium bromide) reagent was added to the culture solution and cultured at 37 ° C under 5% CO ₂ wet condition for 4 hours. In culture, purple crystals form in living cells. After 4 hours of incubation, the medium was removed, and 100 μl of DMSO was added. The crystal was dissolved in a shaking incubator for 10 minutes, and the absorbance was measured at 540 nm to measure the degree of cell growth. The test was repeated three times and the results obtained are shown in FIG.

2, the enzyme-treated gold extracts (ESBs) were superior to the corn-deficient extracts (ISD 100 ug / ml) or the untreated gold extracts (SB) Promoting activity.

In addition, the enzyme-treated gold extract (ESB 1) treated with enzymes directly extracted from yeast and the enzyme-treated golden extracts (ESB-2 and ESB-3) treated with the yeast extract enzyme from the depository organism promoted similar periodontal ligament cell proliferation Activity.

As a result, it was found that the composition of the present invention containing the enzyme-treated gold extract as an active ingredient promotes and activates the proliferation of periodontal ligament cells and is effective for the prevention and treatment of periodontal diseases such as periodontitis.

Experimental Example  2. Evaluation of NO inhibitory activity of enzyme-treated gold extracts

The effect of the enzyme-treated gold extract (the enzyme-treated gold extract of Examples 2-1, 2-2 and 2-3) obtained in Example 2 on the NO production involved in the inflammatory reaction was measured. Experiments were performed according to the method of Sherman et al. [Sherman et al., Biochem. Biophys. Res. Commun. RAW 264.7 (hereinafter referred to as RAW 274.7 cells, US cell line bank ATCC # TIB-71 (hereinafter referred to as &quot; RAW 264.7 cells &quot;), which was obtained by modifying Lipopolysaccharide ), The effect of the enzyme-treated gold extract on the production of nitric acid ions (NO ₃ ^- ), which is a stable oxidation product of NO, was measured as follows.

RAW 264.7 cells cultured in DMEM (Dulbecco's Modified Eagle's medium, Cambrex, USA) containing 1% fetal bovine serum (FBS) were used. RAW 264.7 cells were inoculated into a 48-well plate at a concentration of 2.5 × 10 ⁵ cells / ml. LPS (1 μg / ml) was added to induce activation and incubated in a carbon dioxide incubator (5% CO 2, 95% 37 &lt; 0 &gt; C) for 24 hours. To each 50 μl of each culture was added a grease reagent (Griess reagent: 37.5 mM sulphanilic acid, 12.5 mM N- (1-naphthyl) ethylenediamine dihydride, N- (1-naphthyl) ethylenediamine dihydride) Hydrochloric acid], reacted at room temperature for 10 minutes, and absorbance was measured at 550 nm using a spectrophotometer. At this time, by using a nitrate produced by each concentration of 0~50uM NO ₃ ^-, create an absorbance coefficient, the NO ₃ generated by the RAW 264.7 ^{cell-concentration} was calculated concentration. The enzyme-treated gold extracts prepared in Examples 2-1, 2-2 and 2-3 were dissolved in DMSO, respectively, and then added to RAW 264.7 cells at a final concentration of 10 and 100 ug / ml simultaneously with LPS to obtain NO ₃ ^- And the results are shown in Fig.

3, the enzyme-treated gold extracts (ESB-1, 2, 3) showed significantly improved NO production (100 ug / ml) compared to the corn-free extract Lt; / RTI &gt; Enzyme-treated gold extracts (ESB-2, KACC 41018) and enzyme-treated golden extracts (ESB 1), enzymes directly extracted from yeast, and enzyme-treated gold extracts ESB-3, and KACC 41858) showed similar NO production inhibitory activity. Thus, it was confirmed that the golden extract treated with the enzyme isolated from the koji regardless of the type of koji had an excellent activity to inhibit NO production.

Although high levels of NO inhibit the proliferation and differentiation of osteoblasts, low levels of NO stimulate osteoblast function [Raltson SH et al. Endocrinol. 135, p330-336, 1994; Riancho JA et al. J. Bone Miner Res. 10, p439-446, 1995], it was confirmed from the above experiment results that the golden extract treated with the enzyme from yeast has an excellent preventive and therapeutic activity against periodontitis by inhibiting the inflammatory reaction.

Experimental Example  3. Periodontal ligament  Identification of alkaline phosphatase activity in cells and osteoblasts

Osteoblast cells contain alkaline phosphatase (ALP) in the cell membrane, and alkaline phosphatase is an enzyme involved in calcium and phosphorus metabolism, and is found in high concentrations in the matrix vesicles of the extracellular and calcified tissues. The alkaline phosphatase enzyme exhibits optimal activity at basic pH 8-10. Alkaline phosphatase hydrolyzes organic phosphate and calcification is induced locally in the region where phosphate ion concentration is increased and calcium phosphate is deposited on the extracellular matrix. Therefore, the activity of preventing or treating periodontal disease of the enzyme-treated gold extract of the present invention was measured by confirming the alkaline phosphatase activity.

Alkaline phosphatase activity assay was performed by using alkaline phosphatase as a catalyst for the hydrolysis reaction of p-nitrophenyl phosphate (pNPP, Sigma, St. Louis, Mo., USA) The concentration of alkaline phosphatase is indirectly calculated by measuring the amount of nitrophenol. Human osteoblast-like MG63 cells (ATCC, Manisaas, USA) and periodontal ligament cells were treated with 10% fetal bovine serum (FBS), 100 IU / ml penicillin and 100 ug / ml streptomycin DMEM medium and cultured in a cell incubator maintained at 37 ° C in 5% CO ₂ .

The cultured cells were adjusted to 1 × 10 ⁴ cells / well and dispensed into 96 well plates. After 24 hours, the cells were cultured in differentiation induction medium [50 μg / ml Vit. C and 10 mM β-glycerophosphate (β-GP). This was incubated for 24 hours.

Phase; The enzyme-treated gold extracts obtained in Example 2 (enzyme-treated gold extracts of Examples 2-1, 2-2 and 2-3) were treated at a concentration of 10 and 100 μg / ml in the cultured medium, and after 3 days, , And then 20 쨉 l of 0.1% Triton X-100 was added thereto, and the cells were lysed at 37 째 C for 30 minutes. After dissolution, 20 μl of 0.1 M glycine-NaOH buffer (pH 10.4) and 20 μl of p-nitrophenyl phosphate (p-NPP) were added and reacted at 37 ° C for 30 minutes. After the reaction, the reaction was stopped with 100 μl of 0.1N NaOH and absorbance was measured at 405 nm.

The alkaline phosphatase activity was determined by measuring the p-nitrophenol (p-NP) produced from p-NPP and preparing a standard curve for p-nitrophenol, and then comparing the activity with that of the control group.

4, the treatment with the enzyme-treated golden extract (Example 2-1: ESB-1; Example 2-1: ESB-2; Example 2-3: ESB-3) It was confirmed that the enzyme-treated golden extract of the present invention has an effect of increasing the activity of alkaline phosphatase, and the activity of the enzyme-treated gold extract of the present invention Induction of periodontal disease, and the prevention and treatment of periodontal disease.

Enzyme-treated gold extract (ESB 1) treated with enzymes directly extracted from yeast, enzyme-treated golden extracts (ESB-2, KACC 41018) treated with enzymes directly extracted from yeast, and enzyme-treated gold extract (ESB-3, KACC 41858) showed similar effects of increasing alkaline phosphatase activity, and it was confirmed that the enzyme-treated gold extract had an effect of increasing the activity of alkaline phosphatase regardless of the type of yeast I could.

Experimental Example  4. Periodontal ligament  In cells and osteoblasts Mineralization  Verify Active

Mineralization activity was confirmed in order to confirm whether osteoblast and periodontal ligament cells differentiated by enzyme - treated gold extract directly formed bone.

Osteoblast and periodontal ligament cells were attached to a polystyrene cell culture dish and cultured in DMEM supplemented with 1% antibacterial-antifungal solution (PAA) containing penicillin and streptomycin and 10% FBS (PAA) The cells were incubated at 37 ℃ in CO ₂ and 95% humidity.

Each cell was treated with 3 × 10 ⁴ cells / well in a 24-well plate, and these cells were monolayered and cultured in differentiation induction medium [50 μg / ml Vit. C and 10 mM β-glycerophosphate (β-GP)]. Cells were cultured for one day and then ascorbic acid and beta-glycerophosphate, which plays a role of osteoblastizing osteoblasts and periodontal ligament cells in 24 well plates, were treated with 50 ug / ml and 10 mM To activate osteoblasts and periodontal ligament cells. The activated cells were divided into two groups. No substance was added to the first group (negative control group), and the second group was further divided into three groups to obtain a corn-non-inoculated extract, a non-enzyme-treated golden extract, (The enzyme-treated gold extracts of Examples 2-1, 2-2 and 2-3) were added to the culture medium at a concentration of 10 and 100 μg / ml, respectively.

The cells were cultured for 24 days at 3-day intervals while exchanging the medium of each test group. After incubation, the cells of each test group were fixed with 10% neutral formaldehyde solution, treated with Alizarin red-S staining solution for 5 minutes, washed, Of the calcified nodules were observed. After confirming the formation of nodules, a solution of 10 mM sodium phosphate (10% cetylpyridinium chloride, pH 7.0) was added at 1 ml / well and absorbance was measured at 550 nm using an ELISA reader. The results are shown in FIG.

As shown in FIG. 5, the results of mineralization of osteoblasts and periodontal ligament cells were compared with those of the enzyme-treated gold extract (Example 2-1: ESB-1, Example 2-2: ESB-2 , And ESB-1 (Example 2-3)) were significantly improved compared to the test groups treated with the corn-deficient extract and the enzyme-treated gold extract used as the positive control in the test groups . In other words, it was confirmed that the enzyme-treated golden extract promotes mineralization and thus has a direct effect on the prevention and treatment of periodontal disease.

Experimental Example  5. Prevention of alveolar bone absorption by using white paper model and confirmation of alveolar bone regeneration effect

Sprague-Dawley (Korea BioLink, Negro, Korea) Ligation with a silk suture was performed on the second molar of the rat mandible to induce periodontitis. To the rats in which the periodontal inflammation was confirmed, the enzyme-treated gold extract of Example 2-1 (100, 200 mg / kg) for 6 weeks, and then imaged with micro CT (Micro Computed Tomography, Micro CT, Inveon ™ Simens Medical Solutions USA, Inc.) to prevent alveolar bone absorption and alveolar bone regeneration. As a positive control group, a test group treated with corn sterilized extract (200 mg / kg) and untreated gold extract (200 mg / kg) was used.

FIG. 6 is a graph showing the results of the oral administration (negative control) of the vehicle (purified water), the oral administration group of the corn-depleted extract, the oral administration group of the gold extract without enzyme treatment, And the oral administration group of the enzyme-treated gold extract were respectively photographed with micro CT apparatus to confirm alveolar bone absorption prevention and alveolar bone regeneration effect.

Figure 7 also shows the CEJ-ABC (cemantoenamel junction-alveolar bone crest) distance between the first molar and the second molar in the test groups, the CEJ-ABC distance between the second molar and the third molar, and the Furcation involvement of the patient.

As shown in FIG. 7, the CEJ-ABC distance and the distance of the root root portion were reduced in all test groups as compared with the negative control group of periodontitis-induced negative control. In addition, the distance between the CEJ-ABC distance and the root apex was significantly reduced in the test group treated with the enzyme-treated gold extract than the group treated with the corn-unproblematic extract and the group treated with the golden extract.

Based on the results of FIGS. 6 and 7, it can be clinically confirmed that the enzyme-treated gold extract is more effective in preventing alveolar bone resorption and alveolar bone regeneration than the corn-deficient extract and gold extract used as a positive control.

Experimental Example  6. Inflammatory cytokines ( Cytokine ) Identification of expression level

The expression levels of interleukin (IL) -1β, MMP-1, and tumor necrosis factor-alpha (TNF-α), which are cytokines related to inflammatory diseases, were confirmed and the expression reduction effect was confirmed.

LPS (Lipopolysaccharide, Sigma-Aldrich, St. Louis, USA), which induces an inflammatory response, and Murine macrophage Raw 264.7 cell line (ATCC, Manisaas, USA) The enzyme-treated gold extracts isolated from the nuruk prepared in 2-1 were treated with Raw 264.7 cell lines at concentrations of 10 and 100 μg / ml, respectively. The expression levels of inflammatory cytokines IL-1β, MMP-1, and TNF-α were measured by enzyme-linked immuno sorbent assay.

1 μL of protein (1/50 dilution) and IL-1β, MMP-1, and TNF-α platinum ELISA kit (eBioscience, San Die Sea, USA) isolated from the Raw 264.7 cell line treated with the enzyme- (Sample Diluent, eBioscience, San Diego, USA) were mixed and dispensed into each well of a 96-well plate. (Biotin-Conjugate Antibody, eBioscience, San Diego, USA) was added to each well. The cells were washed twice with a buffer solution (Wash Buffer, eBioscience, San Diego, USA) And reacted for 2 hours. After washing twice with Wash Buffer (eBioscience, San Diego, USA), 100 μL of a conjugated antibody (Streptavidin-HRP conjugated antibody, eBioscience, San Diego, USA) . (Molecular Devices, Orleans, USA) after treatment with 100 μL of stop solution (Stop Solution, eBioscience, San Diego, USA) Were used to measure the absorbance at 450 nm for IL-1 [beta], MMP-1 and TNF- [alpha].

As shown in FIG. 8, in the gold extract-treated group treated with the enzyme of Example 2-1 isolated from the koji, the inflammatory cytokine IL-1β , MMP-1 and TNF-a were remarkably decreased.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention.

Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A pharmaceutical composition for the prevention or treatment of periodontal disease, comprising as an active ingredient an enzyme-treated golden extract derived from Aspergillus niger . The method according to claim 1, Wherein the gold extract is obtained by treating gold with at least one solvent selected from the group consisting of methanol, ethanol, propanol, butanol, n-propanol, iso-propanol and n-butanol. Gt; The method according to claim 1, Wherein said Aspergillus niger is a strain of Aspergillus niger (microorganism deposit number KACC 41018) or Aspergillus niger (microorganism deposit number KACC 41858), for the prevention or treatment of periodontal disease Red composition The method according to claim 1, Wherein said periodontal disease is at least one disease selected from the group consisting of gingivitis, periodontal inflammation, alveolar bone breakage, and alveolar bone osteodystrophy. A pharmaceutical composition for therapeutic use. The method according to claim 1, The prevention or treatment of periodontal disease of the enzyme-treated golden extract derived from Aspergillus niger may be used for promoting mineralization of osteoblasts, for promoting the proliferation of periodontal ligament cells, and for inhibiting interleukin-1 beta, A tumor necrosis factor-alpha (TNF-alpha), and a matrix metalloproteinase-1 (MMP-1) gene. The method according to claim 1, Wherein the composition is an oral formulation selected from the group consisting of powders, tablets, capsules, injections, and liquid preparations. A health functional food for the prevention or treatment of periodontal disease, which comprises an enzyme-treated golden extract derived from Aspergillus niger as an active ingredient. A pharmaceutical composition for the prevention or treatment of periodontal disease according to any one of claims 1 to 6, which comprises a step of administering to a subject in need of treatment a preventive or therapeutic periodontal disease, &Lt; / RTI &gt;

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