KR102234310B1 - COMPOSITION COMPRISING Machilus thunbergii FOR PREVENTING, IMPROVING OR TREATING ORAL DISEASE - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of oral diseases comprising Hubak tree as an active ingredient, and provides a pharmaceutical composition for the prevention or treatment of oral diseases, including the extract of Hubak tree (Trichosanthes kirilowii) as an active ingredient. do.
In addition, the present invention provides an oral care composition for the prevention or amelioration of oral disease including magnolia (Trichosanthes kirilowii) extract as an active ingredient.
Accordingly, since the present invention exhibits a growth inhibitory activity against oral disease-causing bacteria, a drug containing this as an active ingredient has an effect of preventing and treating oral diseases such as dental caries and periodontal disease. In particular, when the natural extract according to the present invention is used as a toothpaste, mouthwash, etc., there is an effect of effectively preventing the formation of plaque, which is a bacterial precipitate, by inhibiting the growth and activity of oral pathogens. In addition, the natural extract according to the present invention has the advantage of having very few side effects since it uses a natural product with proven safety as an active ingredient.

Description

Composition for the prevention, improvement or treatment of oral diseases that contain the red oak as an active ingredient {COMPOSITION COMPRISING Machilus thunbergii FOR PREVENTING, IMPROVING OR TREATING ORAL DISEASE}

The present invention relates to a composition for the prevention, improvement, or treatment of oral diseases comprising a red oak as an active ingredient, and a pharmaceutical composition and oral hygiene composition capable of preventing, improving or treating various oral diseases using a red oak extract About.

Oral disease is an important infectious disease that can be prevented as a major health problem of dental caries and periodontal disease. Oral health affects your overall quality of life, and neglect of oral health can lead to chronic or systemic disease. As the biofilm in the oral cavity develops, microorganisms such as Lactobacillus casei , Streptococcus sanguis , Streptococcus mutans , Streptococcus mitis, and Streptococcus sobrinus may occur, which causes damage to tooth enamel and gum tissue. Since oral diseases are caused by collapsing the equilibrium of the oral ecosystem according to the production of such biofilms, mechanical removal of biofilms plays an important role in preventing tooth decay and periodontal disease.

The development of caries is closely related to gram-positive acidogenic or acidoduric bacteria, mainly mutans streptococci ( referring to S. mutans and S. sobrinus ), lactobacilli and actinomycetes. Corresponds to this. These bacteria transform sucrose into organic acids such as lactic acid through metabolic processes, and the acid dissolves calcium phosphate in teeth, causing tooth decay.

As described above, the biofilm, which is formed into a thin film in the form of a film by continuously accumulating insoluble glucan, which plays a key role in plaque formation, prevents bacteria from adhering to teeth or coagulation (coagulated mass) between bacteria. It is a great help to form plaque as a result, and it greatly affects the induction of oral diseases such as dental caries and periodontal disease. In addition, the biofilm formed in the form of a bacterial clot is known to be transferred to the entire body along the esophagus, etc. in addition to tooth attachment, causing cardiovascular disease and inflammatory diseases in the body.

Accordingly, it is necessary to suppress streptococci in the oral cavity in order to prevent or treat dental caries and periodontal diseases. In order to safely treat oral diseases without side effects, research is being conducted based on substances extracted from natural products. One study found that mutastein isolated from the culture of Aspergillus terreus inhibited the glucosyltransferase activity of Streptococcus mutans, thereby inhibiting the synthesis and adhesion of insoluble glucans. There is a report.

'Patent Document 1'relates to a composition for preventing or treating periodontal diseases containing a mixed extract, and provides a composition for preventing or treating periodontal diseases containing a mixed extract of saengjihwang, hyeonsam, and corncobs. Another technical document,'Patent Document 2', relates to a natural oral composition using sea salt, cheonnyeoncho, mint, green tea, and laurel leaves.

However, conventional natural extracts that have an effect on dental caries and periodontal diseases can effectively prevent or treat oral diseases because they function only for each of them, such as exhibiting only antibacterial activity or only exhibiting the activity of inhibiting the formation of enzymes that produce biofilms. There is no problem.

KR 10-2019-0000955 A1 (2019.01.04.) KR 10-2015-0027352 A2 (2015.03.12.)

The present invention has been conceived to solve the above problems, and the problem to be solved in the present invention is to have antimicrobial properties and at the same time use a natural extract capable of suppressing the production of biofilms, Machilus thunbergii , as an active ingredient. It is to provide a composition for preventing, improving or treating oral diseases, including.

The present invention for solving the above problems is a technical feature to provide a composition for preventing, improving or treating oral diseases comprising an extract of Machilus thunbergii as an active ingredient.

In addition, the present invention for solving the above problems is a technical feature to provide an oral hygiene composition for the prevention or improvement of oral diseases comprising the extract as an active ingredient.

Since the present invention exhibits a growth inhibitory activity against an oral disease-causing strain, a pharmaceutical product containing it as an active ingredient has the effect of preventing and treating oral diseases such as dental caries and periodontal disease.

In particular, when the natural extract according to the present invention is used as a toothpaste, mouthwash, etc., there is an effect of effectively preventing the formation of plaque, which is a bacterial precipitate, by inhibiting the growth and activity of oral pathogens.

In addition, the natural extract according to the present invention has the advantage of having very few side effects since it uses a natural product with proven safety as an active ingredient.

1 to 3 is a graph showing the results of time-kill and growth inhibition curves of the Ethanol extract and Ampicillin or Ethanol Extract and Gentamicin

Terms and words used in the present specification and claims conform to the technical idea of the present invention based on the principle that "the inventor can appropriately define the concept of terms in order to describe his or her invention in the best way." It should be construed as a meaning and concept, but not limited to a conventional or dictionary meaning.

Therefore, the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all the technical spirit of the present invention, and thus various equivalents that can replace them at the time of application It should be understood that there may be water and variations.

Machilus thunbergii (Machilus thunbergii) grows up to 20m in height. The bark is brown with bark eyes and the young branches are green. The bark of the old tree falls like a piece of scale. Leaves that run alternately appear to be turned at the ends of the branches, obovate or oblong, and have a flat edge. The surface is green and the quality is thick, and there are no hairs on both sides. The petiole is thick, about 2~3cm long. The flowers are bisexual, and yellow-green flowers bloom on the conical inflorescences from the axils from May to June. Fruits are berry, round, and ripen in black purple in July-September of the following year. Hubak tree is an evergreen broad-leaved arboreous tree that is native to Korea and grows at the foot of a mountain in the southern part of Ulleungdo. It is weak against cold but has strong composition resistance and grows well in fertile coastal areas.

Since ancient times, the cortex has been used as a medicinal for leg swelling, abdominal distension, and pain. Isoquinoline alkaloids are present in the root of the black oak tree, lignin, catechin, and polysaccharides are present in the heartwood, and essential oil (essential oil) is present in the leaves. oils), volatile components in fruits, and lignans and neoignans in the cortex are known.

The present invention relates to a pharmaceutical composition for the prevention and treatment of oral diseases having antibacterial activity against oral pathogens containing Machilus thunbergii ethanol extract (MTEE) as a main component. The ethanol extract of the present invention can be used alone, but when used together with an antibiotic, it exhibits more excellent antibacterial activity and anti-biofilm activity against oral pathogens.

Experiments to be described below were performed three times for each experimental example, and statistical analysis was performed by parameter tests (two-way analysis of variance [ANOVA], parametric tests) using commercial software (SPSS 22.0), and the results were averaged ± Expressed as standard deviation, but considered significant at the p <0.05 level.

Example. Preparation of Ethanol Extract of Blackberry

200 g of dried black oak trunks were pulverized, ethanol of 80% concentration was added in a volume of 10 times and reacted at a temperature of 120° C. for 24 hours to recover and extract only the liquid layer. The extract was again filtered under reduced pressure with only water paper filter paper, and the remaining solids were removed, and the remaining liquid was concentrated under reduced pressure in a concentrator equipped with a cooling condenser and freeze-dried to prepare an extract having a concentration of 80 brix.

Experimental Example 1. Minimum inhibitory concentration / minimum bactericidal concentration analysis (Minimum inhibitory concentrations/minimum bactericidal concentrations assay, MIC/MBC assay)

1-1. Preparation for experiment

The dental caries-inducing bacteria (cariogenic bacteria) used in this study is the Streptococcus mutans ATCC 25175, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 27607, Streptococcus ratti KCTC 3294, Streptococcus criceti KCTC 3292, Streptococcus anginosus ATCC 31412, Streptococcus gordonii ATCC 10558.

In addition, periodontopathogenic bacteria used in this experiment were Aggregatibacter actinomycetemcomitans ATCC 43717, Fusobacterium nucleatum ATCC 51190, Prevotella intermedia ATCC 49046 and Porphylomonas gingivalis ATCC 33277.

Here, KCTC stands for Korean collection for type culture, and ATCC stands for American type culture collection.

Excluding two oral pathogens such as Prevotella intermedia ATCC 49046 and Porphylomonas gingivalis ATCC 33277, 1% yeast extract (Difco) was added to Brain-Heart Infusion (Difco Laboratories, Detroit, MI) medium, and Prevotella intermedia ATCC 49046. And Porphylomonas gingivalis ATCC 33277 were used in BHI medium to which 1 μg/ml of hemin (Sigma, St. Louis, MO, USA) and 1 μg/ml of menadione were added.

1-2. Experimental process

The minimum inhibitory concentration was measured by the broth dilution method of the ethanol extract of Hubak tree prepared through the examples, and this was repeated three times.

11 oral pathogens cultured in the process of Experimental Example 1-1 were cultured at 37° C. under anaerobic conditions. Facultative anaerobic bacteria were cultured for 18 hours, microaerophilic bacteria for 24 hours, and obligate anaerobic bacteria for 1-2 days.

The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the test sample that completely inhibited visible growth in the medium. The concentrations at which 50% and 90% of oral bacteria were inhibited were expressed as MIC ₅₀ and MIC ₉₀ , respectively. The minimum sterilization concentration (MBC) was determined as the minimum concentration of the ethanol extract of Humpback when 99.9% of the test bacteria were killed.

Ampicillin and gentamicin (Sigma) were used as standard antibiotics to compare the sensitivity of the ethanol extract of H. mulberry to oral bacteria.

1-3. Experiment result

Table 1 shows the results showing the minimum inhibitory concentration (MIC) and the minimum sterilization concentration (MBC) of the ethanol extract of Hubak tree against oral pathogens.

Referring to Table 1, the results of antibacterial activity and ethanol extract (MTEE) from Mumbai were caries-causing bacteria (MICs, 12.5 to 50 μg/mL; MBSs, 50 to 200 μg/mL), periodontal pathogenic bacteria (MICs, 25 to 50). It showed antimicrobial activity against μg/ml; MBSs, 50 to 200 μg/ml). Ampicillin showed activity at 0.0625/8-0.0125/32 µg/ml, while gentamicin showed activity at 4/8-128/512 µg/ml. The ranges of MIC ₅₀ and MIC ₉₀ showed activity in the ranges of 3.13 to 12.5 µg/ml and 12.5 to 50 µg/ml, respectively.

Ethanol extract of H. mulberry showed strong antimicrobial activity against S. gordonii ATCC 10558 (MIC/MBC, 12.5/50 μg/ml) than other oral pathogens (MIC/MBC, 25/50-50/200 μg/ml). . That is, MIC ₅₀ and MIC ₉₀ showed the lowest concentrations of 3.13 µg/ml and 12.5 µg/ml, respectively.

Experimental Example 2. Checker-board dilution test

2-1. Preparation for experiment

It was carried out in the same procedure as in Experimental Example 1-1.

2-2. Experimental process

Synergistic effect is a phenomenon in which the drug effect when two different drugs are administered simultaneously within a certain dose is stronger than that when each drug is administered alone. (additive effect) is a phenomenon that is the same as or similar to the drug effect when two different drugs are administered simultaneously within a certain amount of the drug and the drug effect when each drug is administered alone. In the present invention, ampicillin or gentamicin was used together to confirm the synergistic effect of antimicrobial activity.

The fractional inhibitory concentration index (FICI) is the sum of the fractional inhibitory concentration (FIC) values in the most effective combination, and is most commonly used when interpreting the results of studies of antibiotic combination effects.

Equation 1 is a calculation method of FIC.

(A) means the MIC when a drug to be used in combination with drug A is used in combination, and MIC _A refers to the MIC when drug A is used alone.

Equation 2 is a calculation method of FICI.

(A) refers to the MIC when a drug to be used in combination with drug A is used in combination, MIC _A refers to the MIC when drug A is used alone, and (B) is used in combination with drug B. It means the MIC when the drug to be used is used in combination, and MIC _B means the MIC when the drug B is used alone.

In addition, the fractional bactericidal concentration (FBC) is the same as the calculation method of FIC, and the fractional bactericidal concentration index (FBCI) is the same as the calculation method of FICB. Calculate. FBCI is the sum of the values of FBC in the most effective combination.

If the FICI and FBCI values are less than 0.5, it is considered that there is a synergistic effect according to the combination of the ethanol extract and antibiotics of H. It was judged to show only the concentration-dependent effect, that is, additive action, and marked as'additional value'. If it is more than 1.0 and less than 2.0, it is considered to be indiffernt, and if it is more than 2.0, it is considered to be antagonistic.

Through the checkerboard dilution test, the antibacterial effect was confirmed by the combination of the ethanol extract and antibiotics. Serial dilutions of two different antibiotics (Ampicillin and Gentamicin) were mixed in a cation-supplemented Mueller-Hinton medium. Then, after incubating 11 oral pathogens at 37°C for 24-48 hours, the minimum concentration at which visible growth stops was determined as the minimum inhibitory concentration (MIC), and the minimum concentration at which 99.9% of the test bacteria are killed is the minimum. It was determined by sterilization concentration (MBC). The results are shown in Table 2 (Ampicillin) and Table 3 (Gentamicin).

In Tables 2 and 3, “alone” means using a reagent alone, and “combination” means using a combination with antibiotics. FIC and FBC mean "(amount after combination)/(amount before combination)".

2-3. Experiment result

Table 2 shows the results of a checkerboard dilution test of ethanol extract and ampicillin from Humbak tree against oral pathogens.

It can be seen that the MIC is reduced by more than 4 times when the ethanol extract of H. mulberry and ampicillin are combined.

In addition, FICI was confirmed to be less than 0.5 in the other oral pathogens except S. gordonii, and it can be seen that synergy occurs accordingly. However, in the case of S. gordonii , it can be seen that additive action occurs with FICI less than 0.75.

In addition, when ampicillin was combined, FBCI was measured to be 0.5 or less in all oral pathogens except S. ratti, S. criceti, and P. gingivalis, indicating that a synergistic effect occurs.

Table 3 shows the results of a checkerboard dilution test of ethanol extract and gentamicin of H. oak tree against oral pathogens.

When gentamicin is combined, it can be seen that MIC is reduced by 4 to 8 times or more in all oral pathogens except S. criceti and P. gingivalis, and FICI is less than 0.5, which is synergistic. And in all other oral pathogens except S. sanguinis, S. ratti, S. anginosus, and F. nucleatum , it can be seen that FBCI is less than 0.5 and synergistically.

Eventually, as confirmed in Tables 2 and 3, it was confirmed that the combination of ethanol extract and antibiotics of H. oak tree for most oral pathogens had a synergistic effect according to the mixing compared to that used alone.

Experimental Example 3. Time-kill and growth inhibition curves assay

3-1. Preparation for experiment

It was carried out in the same procedure as in Experimental Example 1-1.

3-2. Experimental process

The oral pathogen strain suspension was inoculated into a tube containing Mueller-Hinton's medium and _{an antibiotic of MIC 50} ^{concentration, and the final number of bacteria in the final test tube was maintained at 5 ~ 7 × 10 6} CFU/mL.

Then, after culturing in an anaerobic chamber at 37°C, the number of viable cells was calculated according to the passage of time every 0, 0.5, 1, 2, 3, 4, 5, 6, 12 and 24 hours. The test tube was centrifuged in an anaerobic chamber at 37° C. for 48 hours, washed and sterilized, and then successively diluted 10 times in phosphate saline (pH 7.3) to perform experiments and observations.

The time-bacterial killing curve was measured by dividing the ethanol extract of Humbak tree as a mixture of the ethanol extract of Humbak tree and the mixture of ampicillin and gentamicin in order to confirm the antimicrobial activity effect of the mixture of the ethanol extract and antibiotics. .

3-3. Experiment result

Time-killing and growth inhibition curve analysis confirmed that when used together with the ethanol extract of H. chinensis and ampicillin or gentamicin, there was an inhibitory effect on oral bacteria.

Specifically, as shown in Figs. 1 to 3, it can be seen that when the ethanol extract of Hubak tree is used alone, it dies little by little over time. However, it can be seen that, when the ethanol extract of Humbak tree is used together with ampicillin or gentamicin, the killing time is much shorter, and the killing rate is also very fast.

Eventually, this indicates that the ethanol extract of the present invention has antibacterial activity against oral pathogens. In addition, it can be seen that the synergistic effect of antimicrobial activity against most oral pathogens is excellent when used in combination with antibiotics such as ampicillin and gentamicin compared to the case of using the ethanol extract of Humbak tree alone.

On the other hand, the ethanol extract of the present invention can be used as a pharmaceutical without being limited to a specific formulation, and in particular, can be used as an active ingredient for oral disease prevention and treatment.

Specifically, solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid formulations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .

Forms for parenteral administration include toothpaste, mouthwash, topical administration (creams, ointments, dressing solutions, sprays, and other coatings). An example of the formulation of the topical administration agent may be a carrier such as gauze made of natural fibers or synthetic fibers impregnated with a composition for preventing or treating oral diseases, or extracts of red oak as an active ingredient.

Claims (10)

delete delete delete delete delete As an oral hygiene composition for preventing or improving oral diseases comprising an extract of Machilus thunbergii as an active ingredient,
The antibiotic ampicillin or gentamicin is added to the composition,
In the case of ampicillin,
The causative agent of the oral disease is at least one of Streptococcus mutans ATCC 25175, Streptococcus sobrinus ATCC 27607, Aggregatibacter actinomycetemcomitans ATCC 43717 and Prevotella intermedia ATCC 49046,
In the case of gentamicin,
The causative agent of the oral disease is any one or more of Streptococcus mutans ATCC 25175, Streptococcus sobrinus ATCC 27607 and Prevotella intermedia ATCC 49046,
The oral disease is a dental caries disease, characterized in that, oral hygiene composition for preventing or improving oral disease.
delete delete delete delete

Images