JP2009274957A - Bactericidal agent against pathogenic bacterium of periodontal disease comprising red longhorn frass as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bactericidal agent against a pathogenic bacterium of a periodontal disease, which has (a) a potent bactericidal effect against an anaerobic gram-negative rod-shaped bacterium causing a periodontal disease, while having (b) a weak bactericidal effect against another streptococcus group of normal dental plaque bacterium living in an oral cavity; an oral cavity hygienic agent containing the bactericidal agent against the pathogenic bacterium of the periodontal disease; and a food containing the bactericidal agent against the pathogenic bacterium of the periodontal disease. <P>SOLUTION: (1) The bactericidal agent against the pathogenic bacterium of the periodontal disease containing a component derived from a frass of a red longhorn beetle (Purpuricenus temminckii); (2) the oral cavity hygienic agent containing the bactericidal agent mentioned in (1); and (3) the food containing the bacterididal agent described in (1) are disclosed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a periodontal pathogen fungicide.

Periodontal disease is a national disease that affects about 80% of people over the age of 40.
In recent years, a large-scale epidemiological study conducted in the United States found that patients with severe periodontal disease not only have a high incidence of arteriosclerosis and pneumonia, but also that periodontal disease is deeply related to diabetes and premature birth. did. It has been strongly pointed out that periodontal disease can cause and induce systemic diseases and cause various chronic diseases. As the number of elderly people increases, the number of periodontal disease patients is increasing and countermeasures are urgently needed.
About 700 species and 6 billion bacteria are permanently present in the oral cavity, and it is known that periodontal disease is caused by anaerobic gram-negative bacilli that live in the gingival crevice. Periodontal disease is not an infection caused by a single bacterium, but develops after a long period of incubation with a group of attenuated bacteria. Periodontopathic bacteria produce many pathogenic factors such as endotoxins that are toxic to many cells, leukocyte toxins that directly destroy immunocompetent cells, and proteolytic and collagenolytic enzymes that destroy gingival tissue.
Therefore, prevention by eliminating individual pathogenic factors is difficult, and it is necessary to prevent an increase in pathogenic bacteria.
However, these causative bacteria are considered to be a kind of resident bacteria, normal plaque (plaque) produced by normal bacteria in the oral cavity, that is, adheres to biofilms, and is a strong pathogenic bio that is dominated by periodontopathic bacteria Form a film.
The removal of this biofilm is extremely difficult and plaque control by professional tooth cleaning, ie scaling, is the best method but requires regular visits. Therefore, it is often difficult for the elderly and workers who are restricted in time, and there is an urgent need to easily develop a drug that suppresses the growth of periodontal pathogens.

  Currently, plaque control, which is performed for the purpose of preventing oral infections of caries and periodontal disease, is considered to be the most effective method, but this scaling requires specialized techniques. Therefore, daily oral infection prevention is centered on brushing with a dentifrice.

A bactericidal agent can be used as a preventive agent for oral infection together with a dentifrice. Moreover, what added the bactericidal agent to the mouthwash can be used. When this disinfectant is used, the following problems are pointed out, and its use is limited.
As a disinfectant that is a drug, there is chlorohexidine glucuronate. It has been pointed out that it adsorbs on the tooth surface and causes tooth coloring and calculus deposition due to long-term use, and cannot be said to be a good drug.
Popidone iodine has a strong bactericidal effect but a strong mucosal irritation effect. In some cases, dentifrices containing enzyme preparations are used, but strong effects cannot be expected.
In addition, antibiotics may be used, but because resistant bacteria appear after long-term use, it may cause opportunistic infections due to fungal substitution, and it is difficult to use for purposes other than short-term treatment. is necessary.

On the premise of the above, it is necessary to develop natural products with a strong bactericidal effect.
In the oral cavity, a streptococci group that occupies a large number as normal plaque bacteria and a bacterial group that normally exhibits a pathogenicity with an increase in the number of bacteria are present.
The former is useful for adhering to the tooth surface and mucous membrane surface and inhibiting the colonization of exogenous bacteria.
Includes Gram-positive streptococci such as Streptococcus mitis.
The latter includes anaerobic gram-negative bacteria such as periodontopathic bacteria Porhyromonas gingivaris (FDC381), Porhyromonas gingivaris (ATCC53978), Prevotella intermedia (ATCC25611).
Therefore, for the development of antibacterial agents, the above-mentioned antibacterial agents that act against all the bacteria cannot be effective, and the former beneficial resident bacteria group has as little influence as possible, while the latter periodontal pathogens It is necessary to develop a drug having the property of exerting a strong antibacterial action against bacteria.

The following examples are known for conventional anti-periodontal disease drugs.
Patent Document 1 is “Anti-periodontal disease agent containing ω-alkenyl isothiocyanate, ω-alkylthioalkyl isothiocyanate, ω-phenylalkyl isothiocyanate (wasabi component)”.
It describes that the bacteria targeted by periodontal pathogen fungicides are periodontal disease-causing bacteria such as Porhyromonas gingivaris, Actinobacillus atinomycetemcomitans, Fusobacterium nucleatum, and Prevotella nigrescens. This invention is considered to act equally on the action against oral streptococci. In terms of the action of the drug, it results in acting equally on periodontal disease-causing bacteria and oral streptococci.
Similarly, the same can be pointed out with respect to the invention (Patent Document 2) which is “a composition for preventing or treating periodontal disease comprising polyphenols and specific lignans”. In addition, polyphenols themselves have been used as anti-carious and anti-periodontal disease compositions (Patent Document 3, Patent Document 4, Patent Document 5).
“A composition for oral cavity containing an extract of at least one plant selected from pine bark, rose butts and lychee seeds as a protease inhibitor capable of inhibiting proteases released by pathogenic bacteria and immune cells” (Patent Literature) 6), “A novel dental caries and periodontal treatment pharmaceutical composition using lactoferrin and bovine colostrum and bovine colostrum immunoglobulin concentrate” (Patent Document 7), “Disease involving cysteine protease is osteoporosis, dental Cysteine protease inhibitor that is either peri-oral or bacterial / viral infection "(Patent Document 8)," At least one lactone selected from delta-lactones having 11 to 16 carbon atoms is used as an active ingredient Periodontal pathogen adhesion inhibitor "(Patent Document 9)," Dihydrofarnesol, Isocamphylcyclohexanol, etc. Antibacterial perfume for addition to the oral composition. ”(Patent Document 10),“ Antimicrobial oral composition containing a macrocyclic lactone having a specific structure and anti-proliferative ability against caries and periodontal bacteria. The above-mentioned antibacterial composition for oral cavity ”(Patent Document 11) is known.
Neither of them mentions that it acts selectively as a periodontal pathogen fungicide.

Patents using bamboo components include Patent Document 12.
This is a bamboo characterized by being a water-in-oil emulsion composition containing an oily base (A) and bamboo vinegar (B), and optionally water (C) and an emulsifier (D). It is a creamy composition of vinegar. The technical contents are different from the present invention.
Patent Document 13 uses a combination of Sosetsu bamboo extract and ε-polylysine. Also, the target bacteria are: “The cause of dental caries is mainly the growth of Streptococcus mutans, and the cause of periodontal disease is mainly Porphyromonas gingivaris and Actinobacillus actinos. It is called “Mectemcommitans” (Actinobacillus atinomycetemcomitans).

As is clear from the above prior art, medicines used in the oral cavity have been used for both oral streptococci and periodontopathic bacteria, and have a weak bactericidal action against oral streptococci and periodontopathic bacteria. Therefore, there has been a demand for the development of a medicine having a different action against both bacteria, such as a sufficient bactericidal action against bacterium.
JP 2006-131542 A JP 2005-162654 A JP-A-1-90124 JP-A-7-285876 JP-A-1-90124 JP 2005-179206 A Japanese Patent Laid-Open No. 2003-137809 JP 2005-139084 A JP 2003-300850 A JP 2002-265978 A JP 2001-163744 A Japanese Patent Laid-Open No. 2005-200393 JP 2004-123630 A

  The subject of the present invention is (a) a strong bactericidal power against anaerobic gram-negative bacilli causing periodontal disease, while (b) another normal plaque bacterium that lives in the oral cavity. It is to provide a periodontal pathogen bactericidal agent having a weak bactericidal power, an oral hygiene agent containing this periodontal pathogen bactericidal agent, and a food containing this periodontal pathogen bactericidal agent.

The present inventors diligently researched on the above problems, and found the following to complete the present invention. About Benicum kirifras extract that the present inventors have found to be effective as a drug, when used as a periodontal pathogen bactericidal agent for periodontal pathogenic bacteria, 12-15% growth inhibitory effect is recognized, Even at 0.06%, the growth inhibitory effect was 10%.
Actinomyces spp., Which adheres to many oral bacteria and plays an important role in plaque accumulation, also showed a bactericidal effect, and growth inhibition of about 40% was observed when 0.125% was added. On the other hand, the growth inhibitory effect against beneficial oral streptococci Streptococcus gordonii, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus mitis necessary for maintaining oral health is completely observed when 0.125% of the growth medium is added. I couldn't.

According to the present invention, the following inventions are provided.
(1) Periodontopathic fungicide containing Benica longiflas component.
(2) An oral hygiene agent comprising the periodontal pathogen bactericidal agent according to (1).
(3) A food comprising the periodontal pathogen fungicide according to (1) above.

ADVANTAGE OF THE INVENTION According to this invention, the periodontal pathogen bactericidal agent containing a Benica longiflas component, the oral hygiene agent containing a periodontal pathogen bactericidal agent, and the foodstuff containing a periodontal pathogen bactericidal agent are obtained.
In the case of containing about 1% by weight of the red beetle flax component, it acts on the gram-positive streptococci group and periodontopathic bacteria, and the growth of all these test bacteria has almost growth inhibitory activity. When it is about 0.25%, it acts on periodontopathic bacteria and has a remarkable growth inhibitory activity of 30 to 60%, and when it is about 0.1%, it has an inhibitory activity of about 10 to 15%. . For the gram-positive streptococci group, the inhibitory activity is about 0 to 40% at about 0.25%, and hardly affects at about 0.1%. From the above, the medicament of the present invention, the oral hygiene agent and food containing it are useful as periodontal pathogen bactericides and preventive agents.

  Benicamikiriflas and pharmaceuticals using the Benicumakifurras component have already been invented by the present inventors (Japanese Patent Laid-Open No. 2003-277276). In this invention, it discovered that it was useful as a foodstuff containing a periodontal pathogen fungicide, an oral hygiene agent containing a periodontal pathogen fungicide, and a periodontal pathogen fungicide.

Examples of the bamboo used as a raw material for obtaining Benicumakirisura and Benikamichifurasu ingredients that are the subject of the present invention include bamboo species such as Mosouchiku, Shibuchiku, Madake, Shudosasa, Sasamorpha, Narihiratake, and Tochiku. Used. Of these, the bamboo genus Machitake and Hachiku are preferably used.
When these bamboos are cut out and left unattended, the red beetle (Purpuricenustemminckii Guerin-Meneville 1844) lays eggs and the larvae eat the bamboo and grow. During the growth process, excrement remains in the bamboo. This is called Benikamikiriflas. By collecting the red beetle, the red bean and the red bean component are used as target substances such as the periodontal pathogen fungicide of the present invention.
In Japan, red beetle is distributed mainly in Honshu (Yabutsubaki belt-lower part of beech belt), Sado, Shikoku, Kyushu, Sanuki, Tsushima, etc., and appears from spring to summer.

  Benica longiflas obtained from bamboo is obtained as a moist granular light yellowish composition. Since the red-breasted beetle contains water, it can be dried to form a powdery body. In order to dry, it carries out on the conditions of 30-80 degreeC. Treatment at a high temperature exceeding 80 ° C. is not preferable because it involves decomposition. This powder can be used as a medicine by directly contacting the affected part using gauze or the like. Moreover, this red-breasted crab can also be used as a periodontal pathogen fungicide as it is without removing special moisture. Moreover, the red beetle fluff component extracted from this red beetle with water or an organic solvent can be used as a periodontal pathogen fungicide.

  When the water extract is taken out from the red beetle, it is brought into contact with an excessive amount of water (hot water) in a container. It is effective to use hot water as water. Usually, the amount of water is adjusted to a concentration of about 0.001 to 0.007% by weight. The extraction is heated under reflux conditions. The heating time is appropriately set. The heat treatment usually requires at least 15 minutes or more and about 1 hour. As a result, the extracted component of the red beetle can be transferred to the hydrothermal layer. It is effective to repeat this hot water treatment twice or more, usually about 3 times. By this first operation, a liquid material is generally obtained at a ratio of about 0.3 to 1.0%, usually about 0.7% by weight with respect to the weight of the red beetle. The liquid obtained in this way is concentrated under reflux conditions. Finally, the extract of Benicum kirifras is obtained as a brownish powder. The yield is about 1.0 to 2.0% by weight with respect to the weight of the red beetle. Hot water can also be used. It can also be performed under conditions under pressure. The powdery water extract thus obtained is appropriately dissolved in water before use and used as a medicine. As shown in a later Example, as a result of performing water extraction using 10 g of Benicum krillus, 176.2 mg of a frass extract was obtained.

  An extract obtained from an organic solvent can be used as a drug by treating the beetle chrysanthemum in contact with the organic solvent. Examples of the organic solvent include alcohols such as ethanol, methanol, and isopropanol, polyhydric alcohols such as glycerin, ethylene glycol, propylene glycol, and 1,3-butylene glycol or derivatives thereof, ketones such as acetone and methyl ethyl ketone, ethyl acetate, and acetic acid. Mention may be made of esters such as isopropyl and ethers such as ethyl ether and isopropyl ether. In addition, aliphatic hydrocarbons such as petroleum ether, n-hexane, and n-pentane can be used. Among these, ethanol, 1,3-butylene glycol, propylene glycol and the like are used. These can be used as a mixed solvent including water. In any case, after solvent extraction, it can be further brought into contact with another solvent to extract further specific components. However, ethanol or 1,3-butylene glycol solvent can be appropriately used without such operation. As a concentration, it can be used by applying directly to the affected area.

  In the organic solvent extraction of Benica longiflas, extraction with hexane or the like is performed, and the residue is separated into a hexane extract and a residue. The residue is extracted with methanol, and the methanol extract and the residue are separated. And the butanol-dissolved component, and various dissolved components can be taken out by extracting the residue with water.

In the case where Benica longiflas is used as an oral hygiene agent, it is common to use a water extract or an organic solvent extract having a concentration of about 0.1 to 1.10% by weight.
The periodontopathic fungicide containing the Benica longiflas component has (a) a strong bactericidal power against anaerobic gram-negative bacilli causing periodontal disease, while (b) inhabits the oral cavity. Since it is a medicine with weak bactericidal activity against another normal plaque bacterium Streptococcus group, the periodontal pathogen bactericidal agent that becomes a periodontal disease therapeutic agent and periodontal disease preventive agent, this periodontal pathogen bactericidal agent It can be used as a food containing the contained oral hygiene agent and periodontal pathogen fungicide. While it has a strong bactericidal power against anaerobic gram-negative bacilli that cause periodontal disease, (b) for streptococci, another normal plaque bacterium that lives in the oral cavity When used as a drug with weak sterilizing power, it is effective to adjust the concentration to 0.5% or less, preferably 0.25% or less, more preferably 0.1%. is necessary.

The periodontal pathogen bactericidal agent of the present invention can be applied by adding it to foods and drinks or oral hygiene agents.
The dosage form of the oral hygiene agent to be added may be any of a liquid agent, a solid agent, and a semi-solid agent, and is applicable to a dentifrice, a troche, a liquid or powdered mouthwash, a coating liquid, a chewing gum, and the like.
In addition, as foods and drinks to which the periodontal pathogen bactericidal agent of the present invention is blended, soft drinks such as juice and confectionery such as candy and chewing gum are suitable.
The periodontal pathogen bactericidal agent of the present invention is added to the final product so as to have a concentration of preferably 0.001 to 1% by mass, particularly preferably about 0.002 to 0.06% by mass. Used. If the addition amount is less than the lower limit mass, the periodontal disease inhibitory effect is not exhibited, and if the addition amount exceeds the upper limit mass, it may not be suitable as a periodontal pathogen bactericidal agent for blending in foods and beverages, oral hygiene agents, and the like.

The dentifrice composition is as follows.
(A) Benica longiflas component 0.01-0.5%
(B) 0.4 to 2.0% by mass of xanthan gum,
(C) 0.5 to 10.0% by mass of a nonionic surfactant and / or an amphoteric surfactant,
(D) Water 10-40 mass%

The composition of the toothpaste composition is as follows.
Product name Mass parts Calcium hydrogen phosphate 20
Silicic anhydride 8
Sorbit liquid 43
Glycerin 19
Thickener 0.6
Perfume 0.8
Water 8.595
Benica longiflas ingredient 0.005
Total 100

Chewing gum product name Mass gum base 22
Sugar 49.5
Glucose 10
Mizuame 17.7995
Fragrance 0.5
Benica longiflas ingredient 0.005
Total 100

Mouth cleaner product part by mass ethanol 10
Polyoxyethylene hydrogenated castor oil 1
Saccharin sodium 0.2
Sodium benzoate 0.1
Glycerin 6
Fragrance 0.2
Sodium citrate appropriate amount (pH adjusted)
Benica longiflas ingredient 0.005
Water balance 100

The contents of the present invention will be described below with reference to examples. As a method for producing the extract of Benica longiflas of the present invention, a method for producing an extract with an organic solvent and a method for producing an extract with water will be described. Regarding the effect of Benicum kirifras of the present invention as a drug, the growth inhibition rate and turbidity according to the concentration used in the periodontopathic bacteria Porhyromonas gingivaris (FDC381), Porhyromonas gingivaris (ATCC53978) and Prevotella intermedia (ATCC25611) As a result of measuring the degree of growth, the rate of growth inhibition and turbidity depending on the concentration used in Streptococcus gordonii (ATCC10558), Streptococcus sanguinis (ATCC10556), Streptococcus oralis (ATCC10557), and Streptococcus mitis (ATCC903) Was determined from the measurement results.
As a toxicity test for various skin diseases of the red beetle frass component, a “24-well multiwell screening method” using fibroblasts can be performed, and the presence or absence of the toxicity can be confirmed from the result.
The contents and experimental results will be described in detail below.

(Manufacture of extract of Benica longiflas with organic solvent)
591 g of red beetle flax obtained from bamboo is extracted with hexane, divided into 332 mg of hexane extract and the residue, this residue is extracted with methanol, and 14.6 g of methanol extract is divided into the residue. It can be divided into 3.685 g of the category dissolved in ethyl acetate and the category dissolved in water, and the segment dissolved in water can be divided into 1.931 g of butanol-soluble category and water category by treating with n-butanol solvent. The methanol extraction residue was subjected to water (hot water) extraction to obtain 12.132 g of a water extract.

(Manufacture of extract of Benica longiflas with organic solvent)
Extract 1037 g of red beetle from bamboo with hexane, divide it into 509.9 mg of hexane extract, extract this residue with methanol, divide it into methanol extract and residue, It can be divided into the ethyl acetate dissolution section 5.41g and the water dissolution section, and the section dissolved with water can be treated with n-butanol solvent and divided into 2.53g butanol dissolution section and 3.65g water section, The methanol extraction residue was subjected to water (hot water) extraction to obtain 27.132 g of a water extract.

(24-well multiwell screening method (toxicity test))
Toxicity test of the drug of Benicari liflas of the present invention was performed by a 24-well multiwell screening method (described in Eiko Takaoka, “Introduction to Tissue Culture” published by the Society Publishing Center, page 68).
(In this method, each ABCD line (row) and 1 to 6 columns of wells (total of 24 wells) are prepared, containing 0.1 ml of drug stock solution for A line (A1), and the concentration is diluted twice. ((A2 to A5), containing no drug (A6)) Next, a suspension of cells is prepared, and the number of cells is about 5 × 10 ⁴ / ml. Dispense 0.9 ml of the cell suspension in a horizontal position, shake gently to the left and right, and incubate in a carbon dioxide funnel, incubate for 3-7 days, rinse the medium, and gently inject methanol. And fixed for 10 minutes or more.After washing with running water, Giemsa staining is performed, and toxicity is determined by whether or not staining abnormality is observed.)
The contents of the toxicity test were as follows.
(1) Preparation of drug extract Benica longiflas water extract (Benica longiflas 3.9 g, 20 cc of pure water was added and stirred, heated from room temperature to just before 100 ° C. (just before boiling) and boiled at this temperature. The state was kept for 1 hour, cooled to room temperature, and centrifuged with a centrifuge (1200 rpm for 10 minutes), and the supernatant was sterilized by filtration to prepare a drug stock solution.
(2) The 24-well multiwell screening method was performed as a toxicity test.
(3) The following cells were used. NHDF-NEO CRYOPRESERVED (human skin fibroblast neonate). BIO WHITTAKER (manufactured by ACAMBREX COMPANY: May 10, 1999 Lot number: 9F0889 with warranty attached)
(4) The following culture media were used as culture media.
FBM (modified MCDB202 serum added)
(5) The addition conditions were as follows. The stock solution was added to the multiwell at 0.1 ml per well. For dilution, PBS (phosphate buffered saline) was used, and the final concentration was made into four stages of 0%, 12.5%, 25%, and 50%.
(6) Culture condition 0.9 ml of cell suspension suspended in the culture medium FBM was added to each 24 well. Warm for 4 days at 36 ° C.
(7) Appraisal method (a) The medium in the well was discarded (the cells were grounded in the well), and immediately fixed with methanol, followed by Giemsa staining. It observed with the microscope and recorded the photograph (FIG. 3).
(B) A 50% stock solution was added in a 25 cm ² plastic culture bottle and observed for 8 days. Moreover, Giemsa staining was performed after culture.
(8) Result of Appraisal Method (A) According to the control result obtained by the above operation by the 24-well multiwell screening method, the control result (0%) and the final concentration of 0%, 12.5%, Compared to the 25% and 50% results, the final concentrations of 0%, 12.5%, 25%, and 50% were found to have no change in growth and morphology and were not toxic.
(9) Appraisal result (b) As a result of adding 50% of the stock solution in a 25 cm ² plastic culture bottle and observing for 8 days, neither growth nor morphology was observed. According to the result of Giemsa staining after the culture, there was no abnormality.
(10) Conclusion It can be said that the water extract of Benicum kirifras does not affect human-derived fibroblasts and is safe when used as a drug.

Periodontal disease-causing bacteria Porhyromonas gingivaris (FDC381) that has been cryopreserved in advance,
As a result of measuring the growth inhibition rate and the degree of turbidity according to the concentration used in Porhyromonas gingivaris (ATCC53978) and Prevotella intermedia (ATCC25611), Streptococcus gordonii (ATCC10558), Streptococcus sanguinis (ATCC10556), Streptococcus For oralis (ATCC 10557) and Streptococcus mitis (ATCC 903), the results of measuring the growth inhibition rate and the degree of turbidity according to the concentration used were calculated.
The results are summarized in Table 1. The values on the left side of Table 1 indicate the degree of turbidity. Figures in parentheses indicate the inhibition rate.
The results are shown in FIG. 1 and FIG.

  In the case of containing about 0.75% by weight of the extract of Benica longiflas water, it acts on Gram-positive streptococci and periodontopathic bacteria, and the growth of all these test bacteria has almost growth inhibitory activity. ing. When it is about 0.25%, it acts on periodontopathic bacteria and has a remarkable growth inhibitory activity of 30 to 60%, and when it is about 0.1%, it has an inhibitory activity of about 10 to 15%. . For the gram-positive streptococci group, the inhibitory activity is about 0 to 40% at about 0.25%, and there is almost no effect at about 0.1%. From the above, it can be seen that the drug of the present invention is useful as a periodontal pathogen fungicide and a preventive agent.

It is a figure which shows the periodontal disease bacteria growth inhibitory effect. It is a figure which shows a plaque formation microbe growth inhibitory effect. The figure which shows the control result by a 24-well multiwell screening method.

Claims (3)

  Periodontopathic fungicide containing Benica longiflas component.   An oral hygiene agent comprising the periodontal pathogen bactericidal agent according to claim 1. A food comprising the periodontal pathogen fungicide according to claim 1.

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