KR20160118851A - Compositions for preventing or treating oral diseases comprising silibinin - Google Patents

Abstract

The present invention relates to a composition for preventing and treating an oral disease containing silibinin as an active ingredient. The composition for preventing and treating the oral disease containing silibinin or salt of the silibinin as the active ingredient expresses an excellent antibacterial effect regarding Streptococcus mutans and Porphyromonas gingivalis which are causative bacteria of cavities or periodontitis, suppresses the production of proinflammatory cytokines which brings about the periodontitis, and has an effect capable of suppressing the formation of a bio film on the surface of teeth by the Streptococcus mutans. Therefore, the composition can be usefully used as the composition for preventing and treating the cavities, a bad breath, gingivitis, or the periodontitis. Moreover, the silibinin or the salt of the silibinin has no side effects for a human body as a chemical substance which is derived from plants and thus is safe. Therefore, the composition can be safely applied to a pharmacological or mouthwash composition.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating an oral disease comprising silibinin as an active ingredient,

The present invention relates to a composition for preventing or treating oral diseases, which comprises siibinin as an active ingredient. More specifically, the present invention relates to a composition for inhibiting the growth of a cavity causing periodontitis, To a composition for preventing or treating oral diseases such as tooth decay, halitosis, gingivitis, or periodontitis, which contains, as an active ingredient, silibinin or a salt thereof having an effect of inhibiting biofilm formation on tooth surfaces.

It is known that 200-300 kinds of microorganisms usually reside in the oral cavity of a person between the tooth surface, between the teeth and gums of the root portion, and on the surface of the tongue. The presence of these microorganisms may be a normal phenomenon, but when proper oral hygiene activities are not carried out, biofilms formed on the surface of the teeth by pathogenic microorganisms, ie dental plaques, are formed and dental caries, , Gingivitis, and periodontal disease, which can lead to the loss of teeth, and to the health and the aesthetics of the tooth.

In addition, as the food industry develops, the incidence of oral diseases such as tooth decay increases, and the age of occurrence is also lowered, thereby causing a great deal of treatment costs for teeth.

Oral bacteria that play a crucial role in the oral disease by participating in the formation of the dental flora are Streptococcus mutans , Sterptococcus salivarius , Streptococcus gordonii , Lactobacillus acidophilus , Lactobacillus casei and Enterococcus faecalis exist (Kolenbrander, PE, et al. , 2002, Microbiol. Mol., Biol. , 66: 486-505 .; Marsh, PD, 1992, J. Den Res., 71: 1431-1438.).

Among the oral bacteria, Streptococcus gordonii is known to act as an early adhesion bacteria that reacts with the glycoprotein membrane formed on the tooth surface among various bacteria in the formation of a dental plaque (plaque) composed of various species of bacteria . In addition, Streptococcus mutans decomposes carbohydrates, especially glucose and fructose, contained in foods that reside in the dental plaque membrane in the oral cavity, and releases lactic acid, which is a byproduct of the metabolism, mainly lactic acid Thereby delaminating the tooth mass. In addition, glucosyltransferase (GTFase) produced from Streptococcus mutans catalyzes the synthesis of a viscous, non-water-soluble polysaccharide glucan using sucrose as a substrate (Gibbons, RJ and J. Van Houte., 1975, Anuu Rev. Microbiol., 29: 19-44 .; Hamada, S. and HD Slade., 1980, Microbiol. Rev., 44: 331-384 .; Jacquelin, LF et al. , 1995, Patho. Biol., 43: 371-379). The glucan attaches Streptococcus mutans to the tooth surface to attract other microorganisms, and enables adhesion and proliferation at the tooth surface. In addition, since the presence of glucan in the dentine bacterial membrane is dense and water-insoluble, saliva, which has acid diffusion or buffering action from the dentine bacterial membrane, becomes a barrier against penetration into the cell membrane, To continue the demineralization action to cause dental caries. To prevent dental caries, individuals should thoroughly manage the oral environment and remove the bacteria immediately when they form on the tooth surface. However, it is not easy to thoroughly remove the bacterial membrane. Therefore, many studies have been conducted to inhibit microorganisms such as Streptococcus mutans present in the dentine bacterial membrane.

Antibacterial agents such as chlorohexidine gluconate, cetylpyridinium chloride, and triclosan have been developed and used in oral products such as toothpaste and toothpaste to solve oral diseases caused by these bacteria come.

US Patent No. 5356615 suggests that plaque formation inhibition effect can be obtained by oral products using triclosan, and US Patent No. 5571501 (14) (16), 1992), the use of triclosan to inhibit plaque formation and to prevent periodontal disease has been demonstrated in the Clinical Preventive Dentistry Vol. In addition, EP 528468 A1 suggests that toothpastes and mouthwashes containing triclosan can inhibit the production of prostaglandins, a periodontal disease inducing substance. However, in the case of triclosan, the cytotoxicity is high, so there is a high possibility of side effects in long-term use. In the case of a mature biofilm such as a plaque, the bacteria existing in the biofilm have antimicrobial resistance, Problems are being raised.

In order to minimize the side effects of artificially synthesized materials, functional materials effective for inhibiting adhesion of pathogenic microorganisms in the oral cavity to oral surfaces or inhibiting dental plaque formation from natural materials have been developed. As representative examples, there may be mentioned, for example, Korean white pine bark (Korean Patent Laid-Open Publication No. 2014-0141754), turmeric and milky water (Korean Patent No. 10-1445177), omija (Korean Patent Publication No. 2014-00312512) 2014-0020131). However, in the case of a substance having antimicrobial activity against oral bacteria extracted from such a natural substance, the effective concentration of the substance showing efficacy is low, and cultivation must be carried out in agricultural land.

Accordingly, the inventors of the present invention have continued research on a plant-derived compound in order to develop a substance having an improving effect on oral diseases such as cavity, bad breath, gingivitis or periodontitis. Among the plant-derived compounds, And not only inhibits the growth of periodontal bacteria, but also has excellent safety and stability, thereby completing the present invention.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a composition comprising, as an active ingredient, silibinin or a salt thereof having an improving effect on oral diseases such as tooth decay, bad breath, gingivitis or periodontitis.

In order to solve the above technical problems, the present invention provides a composition for preventing or treating oral diseases comprising silibinin or a salt thereof as an active ingredient.

Preferably, the composition for preventing or treating oral diseases according to the present invention is a composition for preventing or treating cavities, bad breath, gingivitis or periodontitis.

The active ingredient of the composition of the present invention, silibinin, is one of the polyphenolic flavonoid materials, and can be represented by silybin or silymarin. The IUPAC name is (2R, 3R) - 3- (4-hydroxy-3-methoxyphenyl) -2- (hydroxymethyl) -2,3-dihydro-1 2 - [(2R, 3R) -3,5-dihydro-2H- 3R) -3- (4-hydroxy-3-methoxyphenyl) -2- (hydroxymethyl) -2,3-dihydro-1,4-benzodioxin-6-yl] -3,4- dihydro- 4-one}, which has a structure represented by the following formula (1).

The silibinin has been known to exhibit the efficacy of hepatitis treatment, treatment and prevention of osteoporosis, neuroprotective effect against inflammatory reaction, and anti-cancer (Korean Patent No. 10-1435242; Korean Patent No. 10-1254847; Yeo H ..... et al, 2007, Korean J. Orient Int Med, 28 (1): 166-175 .; gilwon No. 2012, Sungkyunkwan University doctoral thesis, National Library, call number 617-12-44). . However, the therapeutic efficacy of silibinin in caries, bad breath, gingivitis or periodontitis is still unknown.

The silibinin may be extracted from a flower or seed of milk thistle ( Silybum marianum ) or chemically synthesized.

(B) an anhydrous or a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol as an extraction solvent. Preferably, extraction is carried out using an aqueous methanol solution, an aqueous ethanol solution or an aqueous butanol solution, more preferably an aqueous butanol solution. It is apparent to those skilled in the art that the extract of the present invention having substantially the same effect can be obtained by using not only the above-mentioned extraction solvent but also other extraction solvent.

In addition, the above-mentioned silibinin can be obtained through a conventional purification process in addition to the extraction method using the above-mentioned extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, Silibinin can also be obtained through fractionation.

Salivin salts of the present invention may be in the form of pharmaceutically acceptable salts. Such salts include acid addition salts formed by pharmaceutically acceptable free acids or metal salts formed by the base. For example, inorganic acid and organic acid can be used as the free acid. As the inorganic acid, hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid or phosphoric acid can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid and the like can be used. As the metal salt, an alkali metal salt, an alkaline earth metal salt, a sodium salt, a potassium salt or a calcium salt can be used. But is not limited thereto.

The composition comprising the silibinin or a salt thereof as an active ingredient of the present invention inhibits the growth of Streptococcus mutans , a causative organism of tooth decay, and Porphyromonas gingivalis , a causative organism of periodontitis, Inhibits the production of IL-6 and TNF- [alpha], an inflammatory factor induced by lipopolysaccharide (LPS) secreted from Porphyromonas gingivalis in Streptococcus mutans , The effect of inhibiting the formation of biofilm on the surface of the teeth caused by the tooth surface.

Accordingly, the present invention provides a composition for the prevention or treatment of oral diseases containing silibinin or a salt thereof as an active ingredient, which comprises a pharmaceutical composition for preventing or treating cavities, bad breath, gingivitis or periodontitis, or a composition for preventing or treating cavities, An oral cleanser composition for preventing periodontitis is provided.

The composition for preventing or treating oral diseases of the present invention may contain 0.0001 to 15% by weight, preferably 0.0001 to 10% by weight, more preferably 0.0001 to 10% by weight, based on the total weight of the composition, of the active ingredient, 5% by weight.

The composition for preventing or treating oral diseases according to the present invention may further comprise a silivinin derivative obtained through various addition or substitution reaction of substituents known in the art with the nucleus of the silivinin of the formula 1 as an active ingredient As shown in FIG. For example, the silivin derivative may be one having an acetyl group, a hydroxyl group, a halo group, a nitro group or an alkyl group having 1 to 3 carbon atoms bonded to the silivinine of the above formula (1) Similar efficacy can be demonstrated.

In the present specification, the terms "inhibiting cariogenic bacteria" and "inhibiting periodontitis causing bacteria"

As used herein, the term "prophylactic " means inhibiting the occurrence of a disease or disorder in an individual who has never been diagnosed as having a disease or condition, but is likely to be susceptible to such disease or disease. As used herein, the term "treatment" refers to (a) inhibiting the development of a disease or disease, (b) relieving the disease or disease, and (c) As used herein, the term "individual" means a mammal, including a human, having a disease whose symptoms may be ameliorated by administration of the composition of the present invention, such as a monkey, cow, horse, pig, sheep, dog, cat, rat, Means a mammal.

A pharmaceutical composition according to one embodiment of the present invention may comprise a pharmaceutically acceptable carrier in addition to the silivinin or a salt thereof.

The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one which is usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polybinpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. When the composition is an external preparation, it is preferable to apply the composition in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day for one month or longer. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulating it using a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by a person having ordinary skill in the art, Can be manufactured by dipping. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets, patches, emulsions or capsules, and may additionally contain dispersing or stabilizing agents .

In addition, the composition for oral care for the prevention of oral diseases can be formulated without particular limitation as long as it is applicable to the oral cavity, and more specifically, it can be formulated into paste, solid, liquid or chewing gum. For example, toothpaste, dentifrice, jelly, caramel, candy, mouthwash, chewing gum, mouth rinse, gum massage cream, and the like, but is not limited thereto.

The silivinin or a salt thereof of the present invention is a plant-derived compound harmless to the human body and has no toxicity and side effects, so that it can be safely used for long-term use.

The composition comprising the silibinin or a salt thereof of the present invention exhibits excellent antibacterial activity against Streptococcus mutans and Porphyromonas gingivalis which are causative bacteria of tooth decay and periodontitis, Since the effect of suppressing the production of cytokine and inhibiting the formation of biofilm on the tooth surface by Streptococcus mutans is excellent, it is useful as a composition capable of preventing or treating cavities, bad breath, gingivitis or periodontitis Can be used. In addition, the silibinin or its salt is a chemical substance derived from a plant and is safe because it has little side effects on the human body, so that it can be safely applied to a pharmaceutical or mouthwash composition.

FIG. 1 shows results of disc diffusion experiments of inhibiting the growth of Streptococcus mutans and Porphyromonas gingivalis of the active ingredient of the present invention, silibinin.
FIG. 2 is a graph showing inhibitory effects of Streptococcus mutans and Porphyromonas gingivalis , which are the active ingredients of the present invention, on the proliferation of silibinin.
FIG. 3 is a graph showing the effect of inhibiting biofilm formed by Streptococcus mutans in the treatment of silibinin, an active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for explaining the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention.

Example 1: Evaluation of antimicrobial activity of silibinin

The antimicrobial activity of Streptococcus mutans , known as the causative organism of the worms, and Porphyromonas gingivalis , the causative organism of periodontitis, was measured using the agar diffusion method for the silibinin Respectively.

1-1. Culture of target strain

First, Streptococcus mutans (ATCC 25275) and Porphyromonas gingivalis ( ATCC 10556) were distributed from ATCC (American Type Culture Collection, Rockville, MD, USA). Then, the two strains were each plated on a plate medium and incubated for 24 hours at 37 ° C under anaerobic conditions. The anaerobic conditions for cultivation were maintained at 90% N ₂ , 5% H ₂ and 5% CO ₂ , A medium of the following composition was used.

(Final) menadione (H5533, Sigma Chemical Co. USA) solution +1.0 mg / L (final). The composition of the plate medium was as follows: Tryptic Soy Agar (TSA, Difco Laboratories, USA) with 5% defibrinated Sheep Blood +5.0 mg / (M5625, Sigma Chemical Co. USA) solution.

1-2. Measurement of antimicrobial activity using a paper disk

The antimicrobial activity against the causative strains of oral diseases was measured by using a paper disc containing the antimicrobial substance, silibinin, and the medium diffusion method and the minimum inhibitory concentration.

First, a paper disk containing silibinin was treated with a solution of silymarinin (Sigma-aldrich, USA) at a concentration of 1,000 mg / ml using 10 mg / ml (10%) DMSO (dimethyl sulfoxide, Sigma, USA) (8 cm in diameter, 1.5 cm in thickness; paper, Advantec Filter Paper, manufactured by Toyo Roshi Kaisha Co., Ltd.) Ltd., Japan) and dried.

Streptococcus mutans (ATCC 25275) and Porphyromonas gingivalis (ATCC 10556) were mixed with BHI (brain heart infusion broth, DIFCO laboratories, Ditroit, MI , USA; 5 mg hemin and 0.5 mg vitamin K / ml content) and then using the culture solution 1X10 ⁶ adjusted to CFU / ml and spread on plate medium per 100 ㎕, anaerobic conditions _{(90% N 2, 5%} H 2 And 5% CO ₂ ) at 37 ° C for 24 hours to measure the degree of growth of causative microorganisms in oral diseases. The measurement of the degree of growth of the causative microorganism of the oral disease was performed by measuring the size (mm unit) of the circular growth inhibition ring formed around the paper disk and measuring the oral disease caused by sodium fluoride (NaF; positive control) And to compare the degree of growth of the causative microorganisms. All experiments were performed independently 3 times and the mean values of the results were used. The results are shown in Fig.

As shown in Fig. 1, silibinin inhibits the growth of Streptococcus mutans (ATCC 25275), known as a causative organism causative of cavities, and Porphyromonas gingivalis (ATCC 10556), a causative organism of periodontitis , in a concentration-dependent manner Therefore, it was confirmed that there was an antimicrobial activity.

1-3. Determination of the minimum inhibitory concentration (MIC) of a solution containing silibinin

Streptococcus mutans (ATCC 25275) and Porphyromonas gingivalis (ATCC 10556) described above 1-1 were mixed with BHI (brain heart infusion broth, DIFCO laboratories, Ditroit, MI, USA; and were each 100 ㎕ added to each well of a 0.5 mg vitamin K / ml content) by using the culture broth 1X10 ⁶ CFU / ml and then adjusted to 96- well plates (96-well plate, Falcon, USA). One row of the wells was used as a control group and 100 쨉 l of the culture solution was added instead of the culture solution. Then, the plate to which the sample was added was incubated at 37 ° C. for 24 hours under anaerobic conditions (90% N ₂ , 5% H ₂ and 5% CO ₂ ) The concentration not to proliferate was determined as the minimum inhibitory concentration (MIC). The results are shown in Fig.

As shown in Fig. 2, the silibinin inhibits the growth of Streptococcus mutans (ATCC 25275), which is known as a causative organism causing cavities, and Porphyromonas gingivalis (ATCC 10556), a causative organism of periodontitis , in a concentration-dependent manner Therefore, it was confirmed that there was an antimicrobial activity. In particular, it has been confirmed that at a concentration of 3 mg / ml or more, the silibinin inhibits the growth of Streptococcus mutans (ATCC 25275) and Porphyromonas gingivalis (ATCC 10556).

Example 2: Measurement of inflammation-inhibiting activity of periodontal cells in a solution containing silibinin

(IL-6) and tumor necrosis factor (TNF-α) expressed by lipopolysaccharide (LPS) produced by Porphyromonas gingivalis ( ATCC 10556) ) On the inhibitory effect of silibinin on periodontitis.

2-1. Lipopolysaccharide (LPS) extraction from Porphyromonas gingivalis ( ATCC 10556)

First, Porphyromonas gingivalis ( ATCC 10556) was cultured in a culture medium containing BHI (brain heart infusion broth, DIFCO laboratories, Ditroit, MI, USA; 5 mg hemin and 0.5 mg vitamin K / ml) 90% N ₂ , 5% H ₂ and 5% CO ₂ ) at 37 ° C for 24 hours. Then, the cultured strain was suspended in distilled water, and 90% phenol equivalent to that of distilled water was added at 60 DEG C and stirred for 20 minutes. Then, the aqueous solution layer was separated by centrifugation at 7,000 rpm for 15 minutes using a centrifuge maintained at 4 ° C, and dialyzed at 4 ° C for 3 days using non-ionized water. The mixture was then centrifuged at 40,000 rpm for 4 hours using a centrifuge maintained at 4 ° C to obtain a precipitate. The precipitate was purified by dialysis against lipopolysaccharide (LPS), vacuum dried, and stored at 4 ° C.

2-2. Inhibitory effect of silibinin-containing solution on the expression of inflammatory factors in the oral cavity

Mouse-derived macrophages (RAW 264.7) were inoculated into DMEM medium containing 4.5 g / l glucose, 10% serum and 1% antibiotic, and cultured at 37 ° C for 24 hours. Then, the macrophages were subcultured to a concentration of ⁵ × 10 ⁵ UFC / mL in a 96-well plate, and 0.1 ppm of the lipopolysaccharide (LPS) prepared in the above Example 2-1 and 0.1 mg / The vinin solution was treated at a concentration of 1 mg / ml, 2 mg / ml and 3 mg / ml for 3 days at 37 ° C. After the incubation, the medium was removed and the cells were recovered by treating the cells with lysis buffer (0.1 M potassium phosphate buffer, pH 7.8, 1% Triton X-100/1 mM DTT / 2 mM EDTA) 6 (IL-6) and tumor necrosis factor (TNF-α) were measured by enzyme-linked innumosorbent assay (ELISA). As a control group, macrophages without treatment with silibinin were used. The results are shown in Table 1 below.

sample Treatment concentration
(mg / mL) TNF-a production
% Inhibition IL-6 production
% Inhibition Control (LPS 0.1 ppm) - 100 100 Silibinin One 25 18 2 32 29 3 54 41

As shown in Table 1, it was confirmed that secretion levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in macrophages treated with silibinin of the present invention were inhibited in a concentration-dependent manner .

Example 3: Measurement of inhibitory effect on biofilm formation

We observed that Streptococcus mutans (ATCC 25275) inhibited the production of biofilm biofilms from artificial teeth for the silibinin samples.

Specifically, Streptococcus mutans (ATCC 25275) was cultured in a BHI medium (BHI, DIFCO laboratories, Diroit, MI, USA) for 24 hours at 37 ° C under anaerobic conditions, sucrose was added to promote adhesion. Next, the artificial teeth were treated with the silylinine-free culture medium (Control.), The 1 mg / mL silibinin treatment culture medium and the 0.1% sodium fluoride (NaF; positive control) cultured with Streptococcus mutans (ATCC 25275) Were reacted for 48 hours each. The biofilm formed on the artificial teeth by Streptococcus mutans (ATCC 25275) was then stained with 0.1% sapranin solution. The results are shown in Fig.

As shown in FIG. 3, it was found that the silibinin inhibited the biofilm produced by Streptococcus mutans (ATCC 25275) to a level similar to that of the positive control sodium fluoride.

Claims (4)

A composition for preventing or treating oral diseases comprising silibinin or a salt thereof as an active ingredient.
The composition according to claim 1, wherein the oral disease is cavities, bad breath, gingivitis or periodontitis.
The composition of claim 1, wherein the silibinin or salt thereof is included in an amount of 0.0001 to 15% by weight based on the total weight of the composition.
The composition of claim 1, wherein said composition is a pharmaceutical composition or oral cleanser composition for the prevention or treatment of oral diseases.

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