JP2804232B2 - Anti-caries, periodontal agent and oral composition containing it - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for preventing and treating dental caries and periodontal disease derived from plants of the genus Eucalyptus, and an oral composition containing the same.

[0002]

2. Description of the Related Art Caries and periodontal diseases are two major diseases in the oral cavity, and it has been revealed that both are infections caused by specific bacteria.

[0003] Streptococcus mutans and Streptococcus sobrinus are found in human carious lesions and dental plaque, and play an important role in caries development. This strain produces the enzyme glucosyltransferase, synthesizes highly sticky high molecular weight polysaccharide glucan using sucrose as a substrate, and strongly establishes on the enamel surface layer of teeth to form plaque. Microbial communities inhabit the formed plaque, and the enamel of the teeth is demineralized and caries are induced by the acid production accompanying the sugar metabolism.

[0004] Periodontal disease, on the other hand, is a multifactorial disease consisting of factors, the environment and the host. Bacteria play the most etiological role among them, and recent studies have reported several pathogens and their virulence factors. Among them, Porphyromonas gingivalis is frequently isolated from periodontal pockets of patients with periodontitis, and has strong pathogenic factors such as collagenase, immunoglobulin degrading enzyme, fibroblast growth inhibitory activity, and volatile sulfide. Therefore, it is considered to be the most common pathogen of adult periodontitis among periodontal diseases.

[0005] Therefore, in order to prevent dental caries and periodontal disease, it is apparent that it is important to inhibit the growth of the fungus in the lesion and to suppress the pathogenic factor of the fungus. From the viewpoint of controlling bacteria, it is effective to apply an effective antibacterial substance to the fungus. Conventionally, antibiotics such as tetracycline and minocycline have been used. However, although they have a strong action, they cannot be said to be suitable for daily use due to the appearance of resistant bacteria and side effects.

In recent years, attempts have been made to use natural products, especially eucalyptus extracts, for the prevention and treatment of dental caries and periodontal disease. It is known that the essential oil component of eucalyptus (eucalyptus oil) and cineol (eucalyptol), which is a main component thereof, enhance the effect as a preservative and the action of an antibacterial active substance (Japanese Patent Laid-Open No. Sho 6)
2-289511, antibacterial oral composition). However, these products have a weak antibacterial activity against caries and periodontal disease-causing bacteria of eucalyptus oil and cineole itself, and are considerably limited in use and addition amount when used in the oral cavity due to its strong unique odor. There is.

Further, JP-A-5-306252 (new macrocarpals and a method for producing the same) discloses that macrocarpals collected from a solvent extract of Eucalyptus plants show aldose reductase inhibitory activity and antibacterial activity. Although it has been disclosed that its antibacterial activity only acts selectively against Gram-positive bacteria (see the same publication, page 11, column 19, below, lines 11 to 10), It is insufficient for the prevention and treatment of caries and periodontal disease, which need to be effective against both Gram-positive and negative bacteria.

On the other hand, Japanese Patent Application Laid-Open No. 58-39615 (toothpaste composition) discloses that a combination of a solvent extract of eucalyptus oil residue and solvent extraction residue with sodium alkylsulfate shows antibacterial activity against periodontal disease-causing bacteria. It is disclosed that. However, this eucalyptus solvent extract expresses antibacterial activity only when used in combination with sodium alkylsulfate, suggesting that the antibacterial activity itself does not exist or is very weak. In addition, no effect on caries has been observed in this composition.

Japanese Patent Application Laid-Open No. 3-44316 (plaque formation inhibitor composition) discloses a glucosyltransferase enzyme which causes decay by extracting a residue obtained by defatting and decolorizing eucalyptus with an organic solvent containing an acid. It discloses that it has an inhibitory effect. However, this one is disclosed in
Contrary to the composition according to JP-A-39615, there is an anti-cariogenic effect, but no effect on periodontal disease has been recognized.

As described above, various Eucalyptus extracts have been disclosed, but they are all insufficient for prevention and treatment of dental caries and periodontal disease and have low versatility.

[0011]

Accordingly, the present inventors have conducted intensive studies on extracting effective components for the prevention and treatment of both caries and periodontal disease from Eucalyptus plants. The extract obtained by extracting the residue from which the component (eucalyptus oil) has been removed with a polar solvent and the phloroglucinol derivative contained therein have a very strong antibacterial activity against caries and periodontal disease-causing bacteria. Have also found that they also have a collagenase inhibitory effect produced by Porphyromonas gingivalis which is a periodontal disease-causing bacterium, and have completed the present invention.

[0012] Phloroglucinol derivatives contained in eucalyptus plants are considered to have antiviral activity, HI
V-reverse transcriptase inhibitory activity and the like have been reported, but the anti-cariogenic and periodontal disease effects of the present invention are not known and have been clarified for the first time by the present invention.

[0013]

The present invention provides an extract obtained by treating a residue obtained by removing an essential oil component (eucalyptus oil) from a plant of the genus Eucalyptus with a polar solvent, and a phloroglucinol derivative obtained as an active substance from the extract. An anti-carious and periodontal agent as a component and an oral composition using these.

Hereinafter, the present invention will be described with reference to an example of extraction production of the preparation.

First, a plant of the genus Eucalyptus, for example, eucalyptus leaves, is pulverized by a suitable pulverizing means such as a pulverizer, and the essential oil component is removed preferably by using a steam distillation apparatus usually used for essential oil extraction. The removal of the essential oil component can be carried out at normal temperature using a low-polarity solvent such as n-hexane or petroleum ether.

The essential oil extraction residue thus obtained is subjected to extraction by applying at least one of polar solvents such as ethyl acetate, acetone, ethanol, methanol, water and the like, and the obtained extract is concentrated under reduced pressure. Thereby, the preparation of the present invention is obtained in the form of an extract.

The thus obtained preparation is further separated and purified using a suitable separation and purification means such as column chromatography to obtain a phloroglucinol derivative which is the preparation of the present invention in the form of a compound.

This phloroglucinol derivative has the general formula

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(Wherein R is C ₁₅ H ₂₃ , C ₁₅ H ₂₅ O, C ₁₅
H ₂₃ O ₂ , C ₁₅ H ₂₇ O _2, which may contain a double bond, a carbonyl group, a hydroxyl group, or may form a ring) and is white to slightly yellow non-crystalline Powder.

Specific examples of the phloroglucinol derivative used as the preparation of the present invention include those having the following structural formulas.

[0021]

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[0022]

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[0023]

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[0024]

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[0025]

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[0026]

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[0027]

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[0028]

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[0029]

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The column chromatography used for separation and purification of the phloroglucinol derivative includes adsorption chromatography, partition chromatography, high performance liquid chromatography, thin layer chromatography and the like.

Although there are various uses for the preparation of the present invention, it is excellent in fragrance and taste, and has high safety, so that it is blended in food and sanitary goods such as chewing gum, candy, tablet confectionary, mouthwash, toothpaste and the like. It can be used on a daily basis. When the preparation of the present invention is incorporated into food or sanitary goods, it is preferable to add the preparation so as to have a ratio of 0.001 to 10% by weight.

Hereinafter, the present invention will be described by way of examples, test examples, and application examples, but these examples do not limit the scope of the present invention.

[0033]

Example 1 After removing oil-forming components from 1 kg of eucalyptus leaf powder using a steam distillation apparatus, 5 liters of ethanol was added, and the mixture was refluxed under heating for 3 hours. The obtained filtrate was concentrated under reduced pressure to obtain 214 g of an extract which is the preparation of the present invention.

Example 2 After removing oil components from 1 kg of eucalyptus leaf powder using a steam distillation apparatus, 5 liters of a mixed solution of ethanol and water (1: 1) was added, and the mixture was refluxed for 3 hours. The obtained filtrate was concentrated under reduced pressure to obtain 234 g of an extract of the present invention.

Example 3 After 100 g of the extract obtained in Example 2 was dispersed by adding water, the mixture was partitioned with ethyl acetate, and the ethyl acetate-soluble fraction (2
9.5 g). The ethyl acetate-soluble fraction was adsorbed on a column packed with 1 kg of silica gel (Wakogel C-200, Wako Pure Chemical Industries, Ltd.).

The column was eluted with a dichloromethane-methanol (50: 1 to 2: 1) mixture to fractionate. When developed with a mixed solution of dichloromethane-methanol (3: 1) using a TLC plate (60F254, Merck), UV absorption was observed around an Rf value of 0.2 to 0.4,
Fractions showing purple, gray or brown spots were collected with 10% sulfuric acid. This fraction is separated into octadecylsilanol type (OD) for high performance liquid chromatography (HPLC).
S) Using a preparative column (senshu science), elute with methanol-water (90:10 to 100: 0) and elute with 285
Each peak was collected while monitoring the UV absorption of the nanometer.

The respective peaks were further subjected to dichloromethane-methanol (85:15 to 98: 2) using a silica gel preparative column for HPLC (senshu science), and further to methanol-water using an ODS preparative column for HPLC. (90:10 to 100: 0) and acetonitrile-water (90:10 to 100: 0) were repeatedly purified to obtain Compounds 1 to 9 of the preparation of the present invention.

The physicochemical properties of Compounds 5 to 9 are described below.

(Compound 5)

(Equation 1)

[0040]

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(Compound 6)

(Equation 2)

[0042]

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(Compound 7)

(Equation 3)

[0044]

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(Compound 8)

(Equation 4)

[0046]

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(Compound 9)

(Equation 5)

[0048]

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Test Example 1 The preparations of the present invention prepared in Examples 1 to 3 were tested for antibacterial activity against oral bacteria by the following method.

The antibacterial activity of the preparation of the present invention was compared with eucalyptus oil and thymol known as a bactericide.

1) Test bacterial strain, culture medium and culture conditions Test bacterial culture medium Culture conditions • Caries-causing bacteria (Gram-positive bacteria) 1. Streptococcus mutans Ingbritt strain A 37 ° C aerobic culture 2. Streptococcus mu Tance LA7 strain A 37 ℃ aerobic culture 3. Streptococcus sobrinus 6715 strain A 37 ℃ aerobic culture 4. Streptococcus sobrinus B13 strain A 37 ℃ aerobic culture ・ Periodontal disease-causing bacteria (gram-negative bacteria) 5. Porphyro Monas gingivalis ATCC33277 strain B 37 ° C anaerobic culture 6. Prevotella intel media ATCC25611 strain B 37 ° C anaerobic culture 7. Prevoteira melaninogenica ATCC25845 strain B 37 ° C anaerobic culture 8. Capnosite Faga Ochracea ATCC33596 strain B 37 ° C anaerobic Culture A: Brain heart infusion liquid medium B: Trymince soy liquid medium with hemin (5 (μg / ml) Menadione (0.5μg / ml)

2) Test method for antibacterial activity Four strains of cariogenic bacteria and six strains of periodontal disease were cultured for 24 to 48 hours in the respective media and culture conditions, and the bacteria were sufficiently grown. Using a 96-well flat bottom microplate, a 2-fold dilution series of the sample solution (100 μl) was prepared in each well. Each of the bacteria was inoculated into a medium prepared at a double concentration, and 100 μl thereof was added to each well.

After culturing for 24-48 hours, the turbidity (550 nm) of each well was measured using a microplate reader to determine the growth of the bacteria, and the minimum growth inhibitory concentration (M
IC).

Table 1 shows the test results. The preparation of the present invention showed antibacterial activity against cariogenic bacteria and periodontal causative bacteria, and it was found that the action was considerably stronger than that of thymol. The effect of eucalyptus oil was remarkably inferior to the antibacterial activity of the preparation of the present invention.

[0055]

[Table 1]

Test Example 2 Collagenase inhibitory activity of the preparations of the present invention prepared in Examples 1 to 3 against oral bacteria was tested by the following method.

The collagenase activity was measured using Collageno Kit CLN-100 (Cosmo Bio). Eucalyptus oil and tetracycline hydrochloride, for which a collagenase activity inhibitory effect was reported, were used as collagenase activity inhibitor samples, and the effects were compared.

1) Collagenase preparation method The collagenase solution used in this test was prepared by the following method.

Polyphyromonas gingivalis FDC3
81 strains of Trypticus soy liquid medium [Hemin (5μ
g / ml) and menadione (0.5 μg / ml)]
Contact with 1500 ml and anaerobically culture at 37 ° C for 3 days.
The culture is centrifuged, and the supernatant is treated with ammonium sulfate 8 at 4 ° C.
0% saturation, centrifugation, removal of supernatant, 0.05M Tris-HCl buffer containing 5 mM calcium chloride (pH
7.5) The precipitate was recovered in 300 ml. Perform sufficient dialysis using the same buffer, and filter the dialysate solution (0.22 μm).
m) was used as a collagenase solution.

2) Collagenase Inhibitory Activity Test Method Collageno Kit CLN-100 uses FITC-labeled collagen as a substrate, selectively transforms only the decomposed products produced after the reaction with collagenase at 35 ° C., and emits the fluorescence of the decomposed products extracted with ethanol. Is measured to determine the collagenase activity.

As an experimental system, a collagenase solution of 100 μm was used.
l, sample solution 100μl, substrate collagen solution 200μ
The total collagenase activity was measured by reacting at 35 ° C. for 2 hours in a total of 400 μl. The collagenase inhibitory activity of the sample was defined as the collagenase activity of the control (no sample added) as 100, and the degree of decrease in the collagenase activity when the sample was added was expressed in%.

Table 2 shows the test results. The formulation of the present invention has a stronger P.C. than tetracycline hydrochloride, which is known as a collagenase inhibitor. Zimbaris collagenase had an inhibitory effect. Eucalyptus oil did not show any collagenase inhibitory activity.

[0063]

[Table 2]

Application Example 1 Using the phloroglucinol derivative (compounds 1 to 5) prepared by the method of Example 3, chewing gum, candy, tablet confectionery, mouthwash and toothpaste were produced according to the following formulation.

[0065]

[0066]

[0067]

[0068]

[0069]

Application Example 2 A chewing gum was produced in the same manner as in Application Example 1, except that Compound 6 was replaced by 0.005% and sugar was 55.195%.

Application Example 3 A candy was produced in the same manner as in Application Example 1 except that Compound 7 was replaced with Compound 2 by 8.2% and sugar by 42%.

Application Example 4 Tablets were produced in the same manner as in Application Example 1 except that Compound 8 was used instead of Compound 3.

Application Example 5 An impregnating agent was produced in the same manner as in Application Example 1 except that Compound 9 was used instead of Compound 4.

Application Example 6 A toothpaste was produced in the same manner as in Application Example 1, except that the eucalyptus extract obtained by the method of Example 1 was used instead of compound 5.

Application Example 7 A chewing gum was produced in the same manner as in Application Example 1 except that the eucalyptus extract obtained by the method of Example 2 was used instead of Compound 1.

[0076]

EFFECTS OF THE INVENTION The preparation of the present invention highly inhibits the growth of cariogenic bacteria and periodontal disease-causing bacteria and has a high inhibitory effect on collagenase produced by the periodontal disease-causing bacteria Porphyromonas gingivalis. , Is a periodontal agent. Eucalyptus, which is a raw material of the preparation of the present invention, is used as a herbal tea or a fragrance component, and there is no problem in its safety, and there is no possibility of side effects or the like seen when an antibiotic is used.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ identification symbol FI // C07C 47/565 C07C 47/565 (58) Investigated field (Int. Cl. ⁶ , DB name) A61K 7/26 A61K 31 / 11 A61K 31/12 A61K 35/78 C07C 49/443 C07C 47/565 CA (STN) REGISTRY (STN) WPIDS (STN)

Claims (3)

(57) [Claims] 1. An essential oil component is removed from a plant of the genus Eucalyptus,
An anti-caries and periodontal agent, comprising an extract obtained by extracting the residue with a polar solvent as an active ingredient. 2. An essential oil component is removed from a plant of the genus Eucalyptus,
The residue was extracted with a polar solvent and the extract was further separated and purified
The general formula obtained by (Wherein R is C ₁₅ H ₂₃ , C ₁₅ H ₂₅ O, C ₁₅ H ₂₃ O ₂ , C
_A phloroglucinol derivative represented by ₁₅ H ₂₇ O _2, which may contain a double bond, a carbonyl group, a hydroxyl group, or may form a ring) as an active ingredient. Anti-caries, periodontal agent. 3. An oral composition comprising the anti-caries and periodontal agent according to claim 1 or 2.