KR101426238B1 - Herbal Extract For Preventing and Treating Disease of Oral Cavity - Google Patents

Abstract

The present invention relates to an extract having activity for the prevention and treatment of oral diseases, which is isolated from a natural material, and has a growth inhibitory activity of 50% or more and activity of 50% or more against Streptococcus mutans Wherein the extracts exhibit oxygen species inhibitory activity at the same time.

Description

[0001] The present invention relates to a natural extract for preventing and treating oral diseases,

The present invention relates to a extract with the release oral disease preventive and therapeutic activity for from natural materials, and more particularly, Streptococcus mutans (Streptococcus mutans) or more than 50% growth inhibition and 50% of active oxygen for Lt; RTI ID = 0.0 > inhibitory < / RTI > activity.

In the human mouth (oral cavity), various kinds of bacteria form a cluster. There are various proteins in oral saliva and secretions. For example, in the mouth, a needle containing an enzyme called lysozyme is continuously secreted to control microorganisms. However, the nutrient-rich oral environment is most suitable for microbial growth It does not induce complete killing because it can be a condition. In particular, oral tongue and dental plaque (dental plaque) is used as a refuge of pathogenic microorganisms.

The number of microorganisms suspected to survive in a human oral cavity is known to be about 600 to 800 or more, and most of them are bacteria, but archaea and fungi and yeast It is known to contain many.

Most of these are harmless general bacteria or microorganisms that are in symbiotic relationship with them. However, some microorganisms are opportunistic pathogens, which are diseases such as dental cavities and periodontal disease .

Dental caries is caused by microorganisms forming a plaque (dental plaque) through fermentation metabolism using nutrients in the mouth to make various organic acids such as lactic acid and to remove calcium from the tooth surface. In the plaque, the protein in the saliva forms a thin film on the tooth, and Streptococcus sobrinus strain and Streptococcus mutans , which are kind of streptococci, It is made by forming a film (biofilm) and thickened by Fusobacterium.

Once the tooth surface enamel layer is destroyed by organic acids, the underlying dental layer is also destroyed by the proteolytic enzyme secreted by the microorganism, resulting in increased dental caries.

Periodontal disease is a disease that causes inflammation of the periodontal tissue and loss of structure, also called taste. Specifically, periodontal disease is an inflammation of the surrounding tissues of the teeth that hold the teeth. It is clinically caused by gingival bleeding and swelling, formation of the periodontal pocket, and destruction of the alveolar bone. Resulting in loss of teeth.

The major cause of periodontal disease is plaques. When these plaques are mechanically accumulated in the periodontal pockets, they become habitats of bacteria existing around them. These forms gradually migrate into aerobic, breathable, gram-positive bacteria, . At this time, the toxin and all products of the anaerobic Gram-negative bacteria that are multiplied together directly destroy the tissues or stimulate the immune system and cause irritation along with periodontal tissue destruction due to various actions from the stimulated immune system.

Recent studies have shown that periodontal disease is an inflammatory disease that causes tissue damage as a result of intricate interactions between bacterial and host immune responses and is associated with a pathogenesis of bacterial and host- oxygen species, ROS) are also reported to be involved.

Host cells and germs are defending against damage by reactive oxygen species and have an antioxidant defense system to eliminate them.

The major extracellular antioxidants located in saliva, gingival crevicular fruid or plasma include ascorbic acid (Vitamin C), uric acid, α-tocopherol (Vitamin E) Carotenoids (Vitamin A), bilirubin, reduced glutathione, transferrin, lactoferrin, haptoglobin, and the like.

Recently, studies on herbal medicine extracts that can be safely used without side effects and effective removal of bacteria in oral cavity have been actively carried out in order to prevent dental caries. This is because the herbal medicine preparations have already been used for many purposes in civilian for a long time, Because of its proven stability.

Herbal extracts that exhibit antimicrobial activity against Streptococcus mutans , a major causative agent of dental caries, include Pagoja, Grapefruit, Cucumber, Angelica, Cinnamon, Propolis, Seaweed, Cocoa polyphenols, Flavonoids, Cacti, etc. are known. In addition, many polyphenols have been isolated from tea extracts such as green tea, black tea, and oolong tea, which are known to partially inhibit the production of biofilm-forming materials that are released by caries.

In addition, herbal medicine extracts for preventing or treating periodontal disease include myrrha, fresh mulberry extract, sanguinaria extract, corn extract, succulent extract, ginkgo biloba extract, jujube extract, milky white extract, And extracts of non-toxic extracts.

However, the conventional natural extract has only antibacterial activity or exhibits only antioxidative activity, and thus has a problem that it is limited to the prevention and treatment of some diseases of oral diseases.

Therefore, there is a high need for a natural material having an inhibitory activity against oral disease bacteria and periodontal disease-inducing active oxygen species.

The invention of streptococcus mutans (streptococcus mutans), oral disease causing strains in order to solve the technical problem, which has been requested from the problems, and of the prior art It is an object of the present invention to provide a natural extract having both a growth inhibitory activity and an active oxygen species inhibitory activity as a periodontal disease inducing factor.

Thus, as an extract with a natural extract, isolated from natural materials oral disease prevention and therapeutic activity in accordance with the present invention, streptococcus (Strepococcus mutans ) at a concentration of 50% or more and an active oxygen species-suppressing activity of 50% or more at the same time.

The 50% growth inhibitory activity and the 50% active oxygen species inhibitory activity are the minimum concentrations for judging the effectiveness of antimicrobial substances for the prevention and treatment of oral diseases, that is, antimicrobial activity. In the case of plant extracts, It is judged to be antimicrobial when the bacterial growth inhibitory power of 50% or more is confirmed.

The natural extract may be isolated from a natural material selected from the group consisting of a deciduous tree, a black gruel, and a rhizome.

The cervical spine ( Thuja orientalis L.) is an evergreen needlework tree belonging to the Quercus mongolica family, 25 m high and 1 m in diameter, but belongs to the shrubs. Small branches grow vertically. Leaves and berries are used as medicinal materials in leaves. Leaves are weak and pleasant, and they act as hemostatic, blood, convergence, and diuretic, so they can be used as blood, blood, colitis, dysentery, hypertension Is used for the symptoms of. Fruit is sweet and flat and has the efficacy of nourishment, calm, and lump. It is a treatment for nervous breakdown, insomnia, heart palpitations, physical weakness and constipation. It is used.

Further, Sparganium erectum (Sparganium stoloniferum ) is a multi-year-old herb with black ginseng, which grows in the pond and ditch, with rootstock extending sideways and stems emerging from some places. Roots and stems are used as extinction, tongkuk economy, and yujeju. The Korean herb medicine, which is dried in the roots of black ginseng roots, is a scentless and tender flavor, but it tastes slightly fried and chewed. It has a function to protect the flag and strengthen the flow of the flame and to remove the eosinophilia. It is used for postpartum hemorrhagic abdominal pain, menstrual relief, dysmenorrhea, menstrual occlusion, dysentery, ingestion, etc., . The efficacy of pharmacological effects on ectopic pregnancy, the effect on primary malignant tumors, gastric cancer, liver cancer, esophageal cancer, cervical cancer, breast cancer, rectal cancer and lung cancer, hemorrhage due to cerebral thrombosis, It has been known that the effect of coronary artery disease was also improved around the appendiceal process.

In addition, the ginseng tree is a deciduous shrub belonging to the camphor tree which grows on the sunny side of the mountain. It is also referred to as cedarwood or white mugwort. According to the picture book of Korea, the coral reef (mountain pepper) is the fruit of the gabbia tree (Lindera glauca Blume). The taste is spicy, the quality is warm, and the efficacy is not in the sense of a paralysis, and it is used for the cold barrel of the thong. Symptoms that can not be said suddenly by paralysis, take in water and dalyeoseo to relieve pain caused by the abdomen. . Therefore, in the private sector, it is used in the treatment of lowering, paralysis, arthralgia and myalgia caused by customs, treatment of chest cold pain, trauma bleeding, cold, headache,

Thus, although various pharmacological activities have been reported for natural materials such as leaves, fruits, and the like, the antimicrobial activity against the oral disease-causing strains and the antioxidative effect against the active oxygen species, At the same time, there have been no reports or studies on the pharmacological activity.

The natural material may be a leaf, fruit or stem of a spruce tree, may be an outpost of a black gruel, or may be a stem or a leaf of a gentian tree.

The content of leaves, fruits or stem extracts of Cervus spp. To have a growth inhibitory activity over 50% against Streptococcus mutans is at least 500 μg / ml or more.

In addition, the content of the black ginseng seedling extract to have a growth inhibitory activity of 50% or more against Streptococcus mutans is at least 100 / / ml.

In addition, the content of the stem or leaf extract of Rhizoma leaves to have a growth inhibitory activity of 50% or more against Streptococcus mutans is at least 100 μg / ml or more.

In addition, the polyphenol contents of the leaves, fruits or stems of the cabbage trees showing 50% or more active oxygen species inhibitory activity were measured by using a calibration curve by gallic acid, and the mg content per 100 g of dry sample (mg GAE / 100 g dw (Mg RE / 100 g dw) per 100 g of the dry sample using a calibration curve by rutin, the flavonoid content was 250.14 ± 18.26 mg (mg / 100 g dw), calculated as 395.27 ± 24.39 mg GAE / The content of leaves, fruit or stem extracts of the cabbage trees exhibiting 50% DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) radical scavenging activity is above 33.10 ± 3.67 ㎍ / ㎖ at rutin / The content of leaves, fruit or stem extracts of the cabbage trees showing the radical scavenging activity of ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) in the% is 62.96 ± 5.23 ㎍ / ㎖ or more.

At this time, 1 mg / ml of the extract of Quercus serrata, Fruit and Stem shows nitrite scavenging activity produced by 90.44 ± 1.18% periodontal disease inflammation.

The polyphenol content of the black ginseng outpost showing 50% or more active oxygen species inhibitory activity was calculated as mg content (mg GAE / 100 g dw) per 100 g of dry sample using a calibration curve with gallic acid, ± 13.57 mg GAE / 100 g dw and the flavonoid content was 61.01 ± 3.62 mg rutin / 100 g dw or more calculated as mg content (mg RE / 100 g dw) per 100 g of dry sample using a calibration curve by rutin , The content of the black ginseng seedling extract showing 50% DPPH radical scavenging activity was 166.30 ± 5.94 ㎍ / ㎖ or more and the content of black ginseng seedling extract showing 50% of ABTS radical scavenging activity was 155.76 ± 9.05 ㎍ / ㎖ or more.

At this time, 1 mg / ml of black ginseng seed extract showed nitrite scavenging activity produced by 60.29 ± 4.12% periodontal disease inflammation.

In addition, the polyphenol content of the stem or leaf of Rhizopus japonicus, which exhibits more than 50% of active oxygen species inhibitory activity, is determined by using a calibration curve by gallic acid, and the mg content (mg GAE / 100 g dw) per 100 g of dry sample Calculated as 189.70 ± 4.33 mg GAE / 100 g and the flavonoid content was calculated as mg content (mg RE / 100 g dw) per 100 g of dry sample using a calibration curve by rutin, 67.50 ± 1.45 mg RE / 100 g dw , The content of extracts of the stem or leaf extracts of P. vivax showed 50% of DPTS radical scavenging activity of 28.45 ± 3.96 ㎍ / ㎖ and 50% of ABTS radical scavenging activity, 34.52 占 .34 占 퐂 / ml or more.

At this time, 1 mg / ml of extracts of Rhizopus japonicus and leaves showed nitrite scavenging activity produced by 67.64 ± 7.06% periodontal disease inflammation.

The natural extract according to the present invention can be prepared by adding the above natural material to water or alcohol and heating to obtain an extract, and concentrating the extract.

For example, about 150 g of the natural material of the dried bamboo tree is put into an extraction bottle, and distilled water is poured thereon to extract it at a temperature of 80 to 121 ° C for 5 hours three times, or 150 g of a dried natural material is dissolved in 95 to 99% Or methanol in a volume of 1 to 15 times, and the extract was heated at 70 to 80 ° C for 12 hours in an extractor equipped with a cooling condenser. The extracted liquid was again filtered under reduced pressure, and then the solid content was removed. The filtrate was concentrated with a condenser , Preferably 50 to 90 brix, followed by lyophilization.

The above-mentioned natural extracts can be used as medicines regardless of the formulations as an active ingredient of the oral disease prevention and treatment agent, and include, for example, liquid toothpaste, mouthwash, oral spray, Can be used.

As described above, the natural extract according to the present invention simultaneously exhibits a growth inhibitory activity of 50% or more and an active oxygen species inhibitory activity of 50% or more against Streptococcus mutans, which is an oral disease-causing strain, at a predetermined concentration , And the medicines containing it as an active ingredient have an advantage of being able to simultaneously prevent and treat dental caries and periodontal disease.

In particular, when the natural extract according to the present invention is used as a toothpaste, an oral cleaning agent, etc., the growth of Streptococcus mutans is inhibited by 50% or more, and the advantage of being able to efficiently remove plaque as a bacterial deposit have.

In addition, since the above-mentioned medicines and the like use natural extracts having proven safety as active ingredients, there is an advantage that side effects are extremely small.

FIG. 1 is a graph showing the determination of the 50% growth inhibitory concentration of Streptococcus mutans in the extracts of Cryptomeria japonica, Black Ginseng, and Ganoderma lucidum extracted according to the embodiment of the present invention.

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

≪ Example 1 >

150 g of natural material of dried cedar wood, black gruel, and ganoderma lucidum were placed in an extraction bottle, distilled water was poured, and the mixture was extracted three times at 90 ° C for 5 hours. The extract solution was filtered again with a filter paper made of water and paper, and the solid content was removed. The remaining solution was concentrated under reduced pressure in a condenser equipped with a cooling condenser and lyophilized (90 brix).

<Experimental Example 1>

Using the natural extracts prepared in Example 1, experiments were carried out to determine the growth inhibition activity of the bacteria causing dental caries disease and the growth inhibitory activity concentration of the dental disease-causing bacteria. The efficacy was determined as the minimum concentration indicating a growth inhibitory effect of 50% or more. This is the minimum inhibitory activity concentration of bacteria to be used for preventive and therapeutic purposes.

Streptococcus mutans , an oral disease-causing strain, was used as an experimental bacterium. Streptococcus mutans was purchased from Streptococcus mutans KCTC3065 was used.

Streptococcus mutans were inoculated into brain heart infusion medium (Sheep brain and heart extract 37%) and cultured at 37 ° C under aerobic conditions for 20 to 24 hours. Natural extracts were added to the liquid medium to determine the 50% growth inhibitory activity concentration (쨉 g / ml) of the respective natural products against oral disease-causing bacteria, and 0 to 500 ㎍ / ml And then sterilized. Each of the bacteria pre-cultured in the medium supplemented with the concentration of each natural extract was inoculated at a density of 2 × 10 ⁶ cells / ml at a OD _{600 nm} of a spectrophotometer and cultured under the growth conditions of each bacterium. After the incubation, the absorbance was measured at OD _{600 nm} at the time of inoculation and at the end of the culture using a spectrophotometer, and the 50% growth inhibitory activity concentration value (쨉 g / ml) was measured. The results are shown in Table 1 below.

[Table 1] Minimum inhibitory concentration for Streptococcus mutans

상기 표 1에서 보는 바와 같이, 본 발명에 따른 각각의 추출물의 100 ~ 500 ㎍/㎖의 농도를 첨가하였을 경우, 구강질환 유발 세균인 스트렙토코쿠스 뮤탄스(Streptococcus mutans)의 생장이 억제됨을 알 수 있다. 실험에 사용한 시료가 완전히 정제되지 않은 추출물로 낮은 성장 저해 활성을 나타내었지만 정제 할 경우 이보다 낮은 농도에서 성장 저해 활성을 나타낼 것으로 예측된다. 이는 구강 위생 제품 생산 시, 낮은 농도에서도 구강질환 유발 세균의 생장을 억제함으로 적은 양을 첨가하더라도 효과적으로 구강질환 예방 및 치료 효과를 발휘할 수 있음을 의미한다.

As shown in Table 1, when the concentration of each extract according to the present invention was 100 to 500 μg / ml , the growth of Streptococcus mutans , an oral disease-causing bacterium, was inhibited have. Although the sample used in the experiment was not completely purified, it showed low growth inhibitory activity but it would be expected to exhibit growth inhibitory activity at a lower concentration when purified. This means that the oral hygiene product can inhibit the growth of the oral disease-causing bacteria even at a low concentration, which means that the addition of a small amount can effectively prevent or treat oral diseases.

The results of the experiment were verified after 3 repeated measurements. However, the extract of each natural material obtained as described above may vary in efficacy depending on the growth environment of the plant, such as the time of collection of the plant and the habitat.

&Lt; Experimental Example 2 > Measurement of total polyphenol content and total flavonoid content

The total polyphenol content and total flavonoid content were measured using the natural extract prepared in Example 1.

Polyphenol has been known as a major substance for applications in foods and medical care because of its antioxidant effect. 100 쨉 l of 10% Na ₂ CO _{3 was} added to 1 ㎎ / ml of the natural extract according to the present invention, and the mixture was allowed to stand at room temperature for 5 minutes.

The extract mixture and 50% Folin-Ciocalteu 100 μl of the reagent (Folin-ciocalteau phenol reagent) was mixed and reacted. After storing in a dark room at room temperature for 60 minutes, the absorbance was measured at 725 nm using a spectrophotometer. Gallic acid was prepared at a concentration of 0 ~ 1,000 mg / ml and the total phenol content of the sample extract was calculated from the standard calibration curve obtained by analyzing in the same manner as the sample.

100 쨉 l of 10% aluminum chloride was added to 1 ㎎ / ml of the natural extract according to the Moreno method (Ref. Nieva Moreno 2000), and the mixture was allowed to stand at room temperature for 5 minutes. M potassium acetate were mixed and reacted. After storage at room temperature for 40 minutes, the absorbance was measured at 420 nm using a spectrophotometer. The total flavonoid content of the extracts was calculated from standard calibration curves obtained at concentrations ranging from 0 to 1,000 mg / ㎖ using Rutin as a reference material.

The content of each ingredient was expressed as mg content per 100 g of dry sample using a calibration curve prepared from standard samples gallic acid and rutin according to the recommendation of Food and Drug Administration. The results are shown in Table 2 below.

[Table 2] Total polyphenol and total flavonoid content

1. mg content per 100 g dry sample using calibration curve with gallic acid

2. mg content per 100 g of dry sample using calibration curve by rutin

As shown in Table 2, the content of total polyphenols and total flavonoids of the extract according to the present invention was found to be the largest in the cabbage trees. As the antioxidant material, it was found to be more desirable than the extracts of black ginseng and ginseng extract.

The test results were expressed as mean ± SD after 3 repetitions. The significance of each sample was verified and analyzed by Duncan's multiple range test at p <0.05. However, the extract of each natural material obtained as described above may vary in efficacy depending on the growth environment of the plant, such as the time of collection of the plant and the habitat.

<Experimental Example 3> Measurement of total antioxidant capacity using DPPH radical scavenging ability and ABTS radical

DPPH antioxidant activity test and ABTS antioxidant activity test were performed for measuring the free radical scavenging activity for confirming the activity inhibition activity of the active oxygen species using the natural extract prepared in Example 1.

The DPPH radical scavenging activity was measured by mixing 60 μl of 0.2 mM DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) and 60 μl of the prepared sample in a dark room at room temperature for 15 to 30 minutes. Then, using a spectrophotometer, The absorbance was measured.

The total antioxidant activity of ABTS radicals was determined by measuring the antioxidant activity of ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) free radicals produced by the reaction with potassium persulfate To discolor the cyan color of the radical-specific index.

Equal amounts of 7.4 mM ABTS and 2.6 mM potassium persulfate are mixed and reacted in the dark for 15-24 hours. Immediately before use, dilute with distilled water to 73 (± 0.03) absorbance at 734 nm. 50 μl of sample was added to 950 μl of the diluted solution, and the mixture was reacted at room temperature for 10 minutes, and the absorbance was measured at 734 nm.

The degree of antioxidant activity in the DPPH test is degree of antioxidant activity of a display and, ABTS test the 50% DPPH radical scavenging activity concentration of SC ₅₀ values are expressed as 50% ABTS radical scavenging activity concentration of ₅₀ SC value. The DPPH SC ₅₀ value of gallic acid used as a control was 9.07 ± 0.2 ㎍ / ㎖, and the ABTS SC ₅₀ value was 4.96 ± 0.28 ㎍ / ㎖.

In addition, nitrite scavenging ability test of natural extracts showing possibility of using as antioxidant material was conducted to confirm the therapeutic effect of periodontal disease inflammation.

The nitrite scavenging activity was performed by the method of Kato et al. (Ref. Kato H, Lee IE 1987). Add 40 μl of natural product extract to 40 μl of 1 mM NaNO ₂ . After adjusting to pH 1.2 with 0.1 N HCl, react at 37 ° C for 1 hour. 1 ml of 2% acetic acid and 100 μl of Griess reagent were added, reacted at room temperature for 15 minutes, and absorbance was measured at 520 nm. The results are shown in Table 3 below. Vitamin C (ascorbic acid) 1mg / ㎖ was used as a control for the nitrite scavenging activity produced by periodontal disease inflammation and the scavenging activity of vitamin C as a control group was found to be 96.64 ± 0.26%.

[Table 3] Antioxidant efficacy evaluation

As shown in Table 3, the antioxidant activity of DPPH and ABTS according to the present invention was excellent in antioxidant activity in the Quercus serrata and Quercus serrata, and the nitrite scavenging activity of Quercus serrata was superior.

As shown in Tables 2 and 3, the extracts selected from the group consisting of the natural wood materials of the present invention, which are selected from the group consisting of Quercus serrata, Black ginseng and Rhodophyta, have a 50% growth inhibitory activity against Streptococcus mutans And 50% of the active oxygen species inhibitory activity, respectively.

Experimental results were expressed as mean eee standard deviation after 3 repetitions. The significance of each sample was tested and analyzed by Duncan's multiple range test at p <0.05.

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (6)

A pharmaceutical composition for the prevention and treatment of oral diseases containing at least 50% of a growth inhibitory activity and at least 50% of active oxygen species inhibitory activity against Streptococcus mutans and containing natural extracts isolated from Rhizoma trees as an active ingredient Composition. The pharmaceutical composition for preventing or treating oral diseases according to claim 1, wherein the natural extract is prepared by adding a stem or a leaf of Phalaciput in water or alcohol to obtain an extract, and concentrating the extract. The pharmaceutical composition for preventing or treating oral diseases according to claim 1, wherein the natural extract has a content of extract of 100 占 퐂 / ml or more for inhibiting the growth inhibition of Streptococcus mutans by 50% or more Composition. The method according to claim 1, wherein the natural extract is calculated as mg content (mg GAE / 100 g dw) per 100 g of dry sample using a calibration curve by gallic acid, 100 mg / g, and the flavonoid content was 67.50 ± 1.45 mg RE / 100 g dw, calculated as mg content (mg RE / 100 g dw) per 100 g of dry sample using a calibration curve by rutin , The content of the extract showing 50% DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) radical scavenging activity was 28.45 ± 3.96 ㎍ / ㎖ or more and the content of extract having 50% of ABTS radical scavenging activity was 34.52 ± 0.34 占 퐂 / ml or more. [Claim 2] The method according to claim 1, wherein the material of the natural extract is a stem or a leaf of Phalaenopsis, and the content of the extract for obtaining 50% or more of a growth inhibitory activity and 50% of antioxidative activity against Streptococcus mutans 100 쨉 g / ml or more. The pharmaceutical composition for preventing and treating oral diseases according to claim 1, wherein the formulation of the pharmaceutical composition is liquid toothpaste, mouthwash, oral spray, oral ointment or oral varnish.

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