JP2011052011A - Evaluation system for onset of periodontal disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluation system of onset of periodontal disease which is simple and easy, is conducted at high speed and is full of objectivity, a screening method of a prophylactic-curative agent of the periodontal disease, a culture substance of a model organ of the periodontal disease, a screening set of the prophylactic-curative agent of the periodontal disease, and a model animal of the onset of the periodontal disease and the like. <P>SOLUTION: A lower jaw is extirpated from a mouse, a grinder and an incisor are pulled out under a stereoscopic microscope, alveolar bones with uniform size are gathered, an LPS or a TLR2 ligand is added to an organ culture system of the alveolar bones, the culture system is cultured for 6 days under existence or non-existence of a subject substance, bone absorbing activity is determined by quantitative analysis of calcium concentration in a culture supernatant after culturing. The LPS is administered to gums outside the lower jaw grinder together with the subject substance or independently under the stereoscopic microscope and a bone density of the alveolar bone is measured. It is found that polymethoxy flavonoids included in a flat lemon extract or the like are effective against prevention-cure of the periodontal disease by the above screening. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an evaluation system for periodontal disease onset comprising an alveolar bone of a non-human animal and TLR4 ligand and / or TLR2 ligand, and more specifically, a screening method for a prophylactic / therapeutic agent for periodontal disease, periodontal disease model The present invention relates to organ cultures, periodontal disease preventive / therapeutic agent screening sets, periodontal disease onset animals, and the like.

  Periodontal disease is a disease in which the gums and bones supporting the teeth are suppurated or destroyed by dirt (plaque) on the tooth surface, and is a national disease that affects 80% of the Japanese population. . Various treatment methods for periodontal diseases include surgical treatment methods, drug treatment methods using antibiotics such as periocrine and funkisone, osteoporosis, fractures and periodontal diseases consisting of thiazole compounds or pharmaceutically acceptable salts thereof. A therapeutic agent for bone diseases / disorders (for example, see Patent Document 1), a therapeutic agent for periodontal disease containing an anti-CD14 antibody as an active ingredient (for example, see Patent Document 2), vascular endothelial growth factor / vascular permeability A therapeutic agent for periodontal disease (for example, see Patent Document 3) characterized by comprising a preparation containing an antibody against a factor (VEGF / VPF) is known.

  In addition, as a method for evaluating substances useful for the prevention and treatment of periodontal disease, a thread is wound around an animal's tooth so that plaque easily adheres to it, and it is left for several months. A method using a spontaneous onset model (see, for example, Non-Patent Document 1) or a human periodontal disease bacteria that develops a disease, then extracts a periodontal tissue to prepare a section, and observes and evaluates morphologically A method of administering periodontal disease bacteria that develop by infecting animals by infecting them with live bacteria (for example, see Non-patent Document 2) has been reported.

  However, in the case of the evaluation method using the spontaneous development model, the evaluation is performed using a morphological technique by microscopic observation using a section, but the periodontal tissue has teeth with high calcification, It is necessary to decalcify, and the process of preparing the slice includes embedding and slicing a fixed tissue, which requires labor and time, and requires a skillful technique. In addition, in the case of the method of administering periodontal disease bacteria, it is necessary to keep the animals in a sterile environment and it is necessary to improve the implementation environment, so the screening system lacks versatility and the evaluation method in this case is also Due to the morphological technique, there is a problem that it takes time for infection of animals, and it takes several months when section preparation and morphological evaluation are combined.

  On the other hand, the Toll gene is involved in the determination of the dorsoventral axis during Drosophila embryogenesis (see, for example, Non-Patent Documents 3 and 4) and innate immunity to detect invading pathogens in adults (for example, In recent years, mammalian homologues of the Toll gene have been identified (see, for example, Non-Patent Documents 8 to 12), and the human Toll-like receptor (TLR) family includes TLR2 and TLR4. Ten species have been reported so far. The role of the TLR family is as a pattern recognition receptor that recognizes bacterial common structures, distinguishing different pathogen-associated molecular patterns, and similar intracellular signaling leading to translocation of the transcription factor NF-κB into the nucleus. Causes activation of the pathway. Such signal transduction pathways ultimately produce inflammatory cytokines and induce host defense responses, as well as host defense responses against acquired immunity. In recent years, many TLR ligands have been reported.

  Examples of TLR2 ligands include peptidoglycan (PGN) of Gram-positive bacteria, bacteria-derived triacylated lipoproteins, mycoplasma-derived diacylated lipoproteins, and other lipoproteins derived from various bacteria, tuberculosis lipoarabinomannans, yeast zymosan, and cruise. Various bacterial components such as the GPI anchor of Trypanosoma cruzi have been reported (for example, see Non-Patent Documents 13 to 17). As TLR4 ligand, lipopolysaccharide (LPS), a glycolipid specific to the cell wall of Gram-negative bacteria, is known, and LPS is also known to have a strong innate immunity-inducing activity against the host. (For example, see Non-Patent Documents 18 and 19). LPS is an amphiphile composed of a hydrophilic polysaccharide part composed of a core region and an O-antigen, and a hydrophobic glycolipid collectively referred to as lipid A. It has a repeating structure with specific oligosaccharides, and is used for serological classification of Gram-negative bacteria, and the core region is an oligosaccharide containing characteristic sugar components such as Kdo and heptose. Its structure is less diverse than O-antigen polysaccharides. Lipid A has a structure in which a plurality of acyl groups are bonded to phosphorylated glucosamine disaccharide, and is recognized as the main body of LPS immunity-inducing activity.

JP-A-11-209284 JP-A-10-114679 JP 2002-097157 A

J.Periodont Res.39: 107-110,2004 J.Clin.Invest.106: R59-R67,2000 Cell 52, 269-279, 1988 Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996 Nature 406, 782, 2000 Nat. Immunol. 2, 675, 2001 Annu. Rev. Immunol. 20, 197, 2002 Nature 388, 394-397, 1997 Proc. Natl. Acad. Sci. USA 95, 588-593, 1998 Blood 91, 4020-4027, 1998 Gene 231, 59-65, 1999 Science 285, 732, 1999 Science 285, 736, 1999 J. Biol. Chem. 274, 33419, 1999 J. Immunol. 164, 554, 2000 Nature 401, 811, 1999 J. Immunol. 167, 416, 2001 Trends Glycosci. Glycotechnol., 14, 53-68, 2002 Crit. Rev. Microbiol., 16, 477-523, 1989

  As described above, conventionally, methods have been used in which an animal's periodontal tissue is excised and sliced, and morphologically observed and evaluated. However, these methods are not simple and time until evaluation is used. It was an evaluation system that took several months. Therefore, it is difficult to screen a large number of candidate substances, and morphological evaluation is likely to vary depending on the preparation state of the slices prepared and the person evaluating them, and differences in experimental results between the practitioner and the facility are likely to occur. Therefore, it is not suitable as a screening system that requires objective evaluation. An object of the present invention is to provide a simple, rapid and objective evaluation system for the development of periodontal disease, a screening method for a prophylactic / therapeutic agent for periodontal disease, a periodontal disease model organ culture, and a prophylactic / therapeutic agent for periodontal disease It is to provide a screening set, a periodontal disease onset model animal, and the like.

  The present inventor removes the mandible from the mouse, extracts the molar teeth and incisors under a stereomicroscope, collects the alveolar bone of the same size, and adds LPS or TLR2 ligand to the alveolar bone organ culture system. When the bone resorption activity was measured by quantifying the calcium concentration in the culture supernatant after the culture, it was found that the addition of LPS or TLR2 ligand increased the bone resorption activity in a concentration-dependent manner. As a result of examining the bone resorption activity of alveolar bone by adding indomethacin in combination, it was found that bone resorption activity by LPS or TLR2 ligand is completely suppressed when indomethacin is added in combination. In addition, the present inventor found that the bone density of the alveolar bone is reduced by administering LPS to the gingiva outside the lower molars of the mouse under a stereomicroscope. It has been found that the bone density decrease of the alveolar bone is completely suppressed. The present invention has been completed based on the above findings.

  That is, the present invention relates to (1) an agent for preventing and treating periodontal disease comprising polymethoxyflavonoids as active ingredients.

  The present invention also provides (2) a periodontal disease prevention / treatment agent according to (1) above, wherein the polymethoxyflavonoids are citrus-derived polymethoxyflavonoids, and (3) the polymethoxyflavonoids are effective against tangeretin. The present invention relates to the preventive / therapeutic agent for periodontal disease according to the above (1) or (2), which is a polymethoxyflavonoid as a component.

  Further, the present invention provides (4) a food or food material for preventing or treating periodontal disease containing polymethoxyflavonoids as an active ingredient, and (5) the polymethoxyflavonoids are citrus-derived polymethoxyflavonoids. The food or food material for periodontal disease prevention / treatment as described in (4) above, or (6) the polymethoxyflavonoid is tangeretin for the prevention or treatment of periodontal disease as described in (4) or (5) above. It relates to food or food materials.

  Periodontal disease preventive / therapeutic agent screening method, periodontal disease model organ culture, periodontal disease preventive / therapeutic agent screening set, periodontal disease onset model animal, Candidate substances useful for prevention or treatment can be evaluated in a short time of about one week. The in vitro screening system using mouse alveolar bone organ culture system makes it possible to easily check the effectiveness of many candidate substances for periodontal disease and is particularly suitable as a primary screening system. . In addition, in an in vivo screening system using a periodontal disease onset model animal, it is possible to evaluate the effectiveness of a candidate substance for periodontal disease in a very short time using a living body. Therefore, it is particularly suitable as a material for determining the presence or absence of effectiveness in humans. These two evaluation systems are used as screening systems when examining the addition of active ingredients for oral care products such as dentifrices, foods and supplements such as gums and salmon, and preventive and therapeutic agents for periodontal diseases. Is also suitable.

In vitro screening system of the present invention (organ culture system of mouse alveolar bone: increase in bone resorption activity by LPS) and in vivo screening system (periodontal disease evaluation by bone density of mouse alveolar bone: bone density by LPS administration to mandible It is a figure which shows the result of (decrease). It is a figure which shows the result of the in vitro screening system (organ culture system of mouse alveolar bone: increase in bone resorption activity by LPS, TLR2 ligand and suppression of increase in bone resorption activity by combined use of indomethacin) of the present invention. It is a figure which shows the result of the in-vivo screening system (periodontal disease evaluation by the bone mineral density of a mouse | mouth alveolar bone: the bone density reduction by LPS administration to a lower jaw, and the suppression of the bone density reduction by indomethacin combined use) of this invention. It is a figure which shows the result of the bone resorption inhibitory effect of nobiletin and tangeretin in an alveolar bone organ culture system. It is a figure which shows the result of the administration effect of nobiletin and tangeretin on the bone density of the alveolar bone of a periodontal disease model mouse.

  The evaluation system for the onset of periodontal disease according to the present invention is characterized in that a TLR4 ligand and / or a TLR2 ligand is brought into contact with the alveolar bone of a non-human animal. Examples include a screening method for a prophylactic / therapeutic agent for periodontal disease using an evaluation system, a periodontal disease model organ culture, a screening set for a prophylactic / therapeutic agent for periodontal disease, and a periodontal disease onset model animal. In addition, examples of the non-human animal in the present invention include mice, rats, rabbits, dogs, cats, sheep, horses, cows, monkeys, etc., but it is possible to develop periodontal disease in the alveolar bone in a short period of time. A mouse is particularly preferable because it can be easily handled.

  The screening method for the preventive / therapeutic agent for periodontal disease of the present invention can be broadly classified into in vitro system and in vivo system. As an in vitro system, an alveolar bone of a non-human animal is organ-cultured and then used as a culture solution. The screening method is not particularly limited as long as it is a screening method in which TLR4 ligand and / or TLR2 ligand and a test substance are added and further cultured, and the calcium concentration in the culture supernatant is measured and evaluated. It is preferable to measure and compare the calcium concentration in the control without addition. When the non-human animal is a mouse, as an alveolar bone for organ culture in an in vitro system, those extracted from molars and incisors can be advantageously used. Here, organ culture refers to removing organ pieces from a living body. This culture means organic growth without losing the three-dimensional structure. The in vivo system is particularly a screening method in which TLR4 ligand and / or TLR2 ligand and a test substance are administered to the gingiva near the alveolar bone of a non-human animal, and the bone density of the alveolar bone is measured and evaluated. Although not limited, it is preferable to measure and compare the bone mineral density of the alveolar bone in the control to which the test substance is not administered, and in particular, either the left or right other than the test substance in combination in the same animal. It is preferable to measure and compare the bone density of the alveolar bone. When the non-human animal is a mouse, the gingiva on the outer side of the mandibular molar can be suitably exemplified as the gingiva near the alveolar bone in the in vivo system.

  As the TLR4 ligand, LPS can be preferably exemplified. Examples of TLR2 ligands include gram-positive bacteria PGN, bacteria-derived triacylated lipoproteins, mycoplasma-derived diacylated lipoproteins, and other bacterially derived lipoproteins, tuberculosis lipoarabinomannans, yeast zymosan, and cruising trypanosoma. The GPI anchor of (Trypanosoma cruzi) can be specifically mentioned. The concentration and amount used of these TLR4 ligands and TLR2 ligands can be appropriately changed depending on the type of ligand, animal species and their body weight, but the concentration and amount used are set so that periodontal disease develops in the alveolar bone in a short period of time. For example, in the case of a mouse using LPS, in an in vitro screening system, it may be added so that the concentration in the culture solution is 0.1 to 10 μg / ml, preferably 1 to 10 μg / ml. In an in vivo screening system, it may be administered at 825 to 1650 μg / kg. In the case of a mouse using a TLR2 ligand, the in vitro screening system may be added so that the concentration in the culture medium is 30 to 300 μg / ml, preferably 100 to 300 μg / ml, and in the in vivo screening system, 8. What is necessary is just to administer so that it may become 3-83 mg / kg.

  The test substance is not particularly limited, but examples include analgesic / anti-inflammatory / anti-inflammatory drugs (NSAID, steroids, etc.), antibiotics, extracts from animals and plants, herbal medicines, nucleic acids, sugars, sugar alcohols, polypeptides, etc. However, it can be applied to oral care products such as toothpastes such as extracts from animals and plants, herbal medicines, sugars, sugar alcohols and the like, and foods and supplements such as gums and candy.

  The analgesic / anti-inflammatory / anti-inflammatory drugs as candidate drugs include ibuprofen piconol, indomethacin, ufenamate, ketoprofen, glycyrrhetinic acid, diclofenac sodium, suprofen, piroxicam, felbinac, bufexamac, flurbiprofen, pendazac, diphenhydramine, lauryl sulfate Diphenhydramine, Amsinonide, Prednisolone valerate acetate, Diflucortron valerate, Dexamethasone valerate, Betamethasone valerate, Diflorazone acetate, Hydrocortisone acetate, Difluprednate, Betamethasone dipropionate, Dexamethasone, Triamcinolone acetonide, Halsinonide, Pivalate Mometasone furoate, fluocinonide, fluocinolone acetonide, flurdoxy Koruchido, prednisolone, alclometasone propionate, clobetasol propionate, can be given dexamethasone propionate, deprodone propionate, beclomethasone propionate, clobetasone butyrate, hydrocortisone butyrate, butyrate propionate, hydrocortisone, betamethasone butyrate propionate, acetate and the like prednisolone. Examples of the antibiotics as candidate drugs include oxytetracycline hydrochloride, tetracycline hydrochloride, gentamicin, fradiomycin, erythromycin, pimaricin, bleomycin sulfate, synthetic penicillin, synthetic cephalosporin, vancomycin and the like. Furthermore, examples of extracts from plants include citrus-derived polymethoxyflavonoids such as nobiletin, tangeretin, nariltin, and synencetin. In addition to these polymethoxyflavonoids, polymethoxyflavonoids such as seeker extract Extracts derived from citrus fruits including citrus can also be used as candidate drugs.

  The periodontal disease preventive / therapeutic agent of the present invention is not particularly limited as long as it is obtained by the above-described method for screening for the preventive / therapeutic agent of periodontal disease of the present invention. Specifically, indomethacin, nobiletin, Examples thereof include polymethoxyflavonoids such as tangeretin, nariltin, and synencetin. These may be used singly or in combination of two or more, or extracts derived from citrus fruits containing polymethoxyflavonoids such as seeker extract which is a mixture thereof. When these periodontal disease preventive / therapeutic agents are used as pharmaceutical therapeutic agents, pharmaceutically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegrating agents In addition, various blending ingredients such as a solubilizer, a solubilizer, and an isotonic agent can be added. These therapeutic agents can be administered orally or parenterally, for example, can be administered orally in a dosage form such as powder, granules, tablets, capsules, syrups, suspensions, etc., or For example, oral preparations such as toothpastes, oral ointments, gargle solutions, and preparations such as emulsions and suspensions can be administered parenterally as injections. Moreover, the periodontal disease preventive / therapeutic agent of this invention can also be added and mix | blended with a foodstuff or a foodstuff material, and it can also be set as the foodstuff or foodstuff material for a periodontal disease prevention / treatment. Examples of the food or food material include yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, tea, sencha, oolong tea, sports drinks, pudding, cookies, bread, cakes, jelly, rice crackers, etc. Japanese confectionery such as baked confectionery, sheep candy, frozen confectionery, bread and confectionery such as chewing gum, noodles such as udon and soba, fish paste products such as kamaboko, ham and fish sausage, miso, soy sauce, dressing, mayonnaise, sweeteners, etc. It can be used as a food by blending it with dairy products such as cheese, butter and other dairy products, tofu, konnyaku, other boiled rice, dumplings, croquettes and salads. The term “for periodontal disease prevention / treatment” refers to a case where it is indicated on the packaging container or the instructions that it is effective for the prevention or treatment of periodontal disease.

  The periodontal disease model organ culture of the present invention is particularly suitable if it is obtained by organ-culturing the alveolar bone of a non-human animal such as a mouse and then further adding TLR4 ligand and / or TLR2 ligand to the culture solution. The periodontal disease model organ culture is not limited, and can be advantageously used for in vitro screening of preventive / therapeutic agents for periodontal disease, as well as for elucidating the mechanism of periodontal disease onset. be able to. Organ culture of alveolar bone, the alveolar bone is taken out from a living body, without loss of three-dimensional construction of the original, there in any way, including the known methods as long as the culture to perform organic growth May be.

  As a screening set for a prophylactic / therapeutic agent for periodontal disease of the present invention, medium for culture of alveolar bone of non-human animals such as mice, TLR4 ligand and / or TLR2 ligand, and measurement of calcium concentration in the culture supernatant If it is a set which has a kit, it will not restrict | limit in particular, As a culture medium for an alveolar bone organ culture, BGJb containing 1 mg / ml BSA, Penicillin (P) (63.2 mg / l), Streptomycin (S) (100 mg / l) A liquid culture medium, an alpha MEM liquid culture medium, etc. can be mentioned.

  As the periodontal disease onset model animal of the present invention, a TLR4 ligand and / or a TLR2 ligand is added to the gingiva near the alveolar bone of a non-human animal such as a mouse, rat, rabbit, dog, cat, sheep, horse, cow, monkey or the like. It is not particularly limited as long as it is a non-human animal obtained by administration, and such a periodontal disease onset model animal can be advantageously used for screening of preventive / therapeutic agents for periodontal disease in vivo. It can be advantageously used to elucidate the mechanism of

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[In vitro screening system (mouse alveolar bone organ culture system: increase in bone resorption activity by LPS)]
The mandible was collected from a 6-week-old ddy mouse and the molar and incisor extracted under a stereomicroscope were used as alveolar bone. Using a 48-well plate, the cells were cultured at 37 ° C. for 24 hours in 500 μl of 1 mg / ml BSA / BGJb medium / Penicillin (P) (63.2 mg / l), Streptomycin (S) (100 mg / l) culture solution. Thereafter, the medium was changed to the same culture solution, ie, a medium in which LPS (1 μg / ml; manufactured by Sigma) was added to 200 μl of 1 mg / ml BSA / BGJb medium / PS, and further cultured for 6 days. After the culture, the calcium concentration in the culture supernatant was measured with calcium C test Wako (manufactured by Wako) (n = 4). The calcium concentration in the case of only the culture solution was subtracted, and the increase in the calcium concentration was judged to be calcium released from the alveolar bone, and was defined as bone resorption activity. The results are shown in FIG. 1 (left). In FIG. 1 (left), * represents p <0.001. As shown in FIG. 1 (left), an increase in bone resorption activity was observed with the addition of LPS.

[In vivo screening system (assessment of periodontal disease by bone mineral density of mouse alveolar bone: reduction of bone density by LPS administration to mandible)]
Under a stereomicroscope, LPS (0.5 mg / ml) was divided into two portions of 6-week-old ddy mice external to the left mandibular molar and 25 μl (total 50 μl) was administered 3 times every other day (LPS (25 μg / body / day)). PBS was similarly administered to the gingiva outside the right lower molar. Three days after the third administration, the alveolar bone was removed from the left and right lower jaws, and after extraction, the bone density (BMD) of the alveolar bone was measured by the DEXA (dual energy X-ray absorptiometry: Aloka) method (n = 4). . The results are shown in FIG. 1 (right). In FIG. 1 (right), * represents p <0.001. As shown in FIG. 1 (right), a decrease in bone density was observed with LPS administration.

[In vitro screening system (organ culture system of mouse alveolar bone: increase in bone resorption activity by LPS, TLR2 ligand and suppression of increase in bone resorption activity by combined use of indomethacin]]
As in Example 1, the mandible was collected from a 6-week-old ddy mouse, and the molar and incisor extracted under a stereomicroscope were used as alveolar bone. Using a 48-well plate, the cells were cultured at 37 ° C. for 24 hours in 500 μl of 1 mg / ml BSA / BGJb medium / PS culture solution. Thereafter, a medium supplemented with LPS (1 μg / ml; manufactured by Sigma) and a medium supplemented with LPS (1 μg / ml; manufactured by Sigma) and indomethacin (10-6M; manufactured by Sigma) were added to 200 μl of the same culture solution. The medium was supplemented with lipopeptide (300 μg / ml; manufactured by BACHEM), which is a TLR2 ligand, and the medium supplemented with lipopeptide (300 μg / ml; manufactured by BACHEM) and indomethacin (10-6M; manufactured by Sigma). The culture was further continued for 6 days. After the cultivation, the calcium concentration in the culture supernatant was measured with calcium C test Wako (manufactured by Wako) (n = 4 for each). The calcium concentration in the case of only the culture solution was subtracted, and the increase in the calcium concentration was judged to be calcium released from the alveolar bone, and was defined as bone resorption activity. The results are shown in FIG. In FIG. 2, * represents p <0.01, and ** represents p <0.001. As shown in FIG. 2, an increase in bone resorption activity was observed by the addition of LPS and TLR2 ligand, but the increase in bone resorption activity was suppressed by the combined use of indomethacin.

[In vivo screening system (periodontal disease assessment by bone mineral density of mouse alveolar bone: bone density reduction by LPS administration to the mandible and suppression of bone density reduction by combined use of indomethacin)]
In the same manner as in Example 2, under a stereomicroscope, 25 μl each of LPS (0.5 mg / ml) alone was divided into two portions on the gingiva outside the left mandibular molar teeth of 6-week-old ddy mice (total 50 μl), LPS (0.5 mg / ml) is divided into two parts, 25 μl each (total 50 μl), indomethacin (Sigma) 1 μg, LPS (0.5 mg / ml) is divided into two parts, 25 μl each (total 50 μl) 10 μg of indomethacin (Sigma) was administered. PBS was similarly administered to the gingiva outside the right mandibular molar. Seven days after administration, the alveolar bone was removed from the left and right lower jaws, and after extraction, the bone density (BMD) of the alveolar bone was measured by the DEXA (dual energy X-ray absorptiometry: Aloka) method (n = 4 each). The results are shown in FIG. In FIG. 3, * represents p <0.05 compared to the control, ** represents p <0.001, and ## represents p <0.001 compared to administration of LPS alone. As shown in FIG. 3, a decrease in bone density was observed by LPS administration, but the decrease in bone density was suppressed in a concentration-dependent manner by the combined use of indomethacin.

[Inhibition of bone resorption by nobiletin and tangeretin in alveolar bone organ culture system]
The mandible was collected from the mouse, and the molar and incisor extracted under the stereomicroscope were used as the alveolar bone. After culturing in 1 mg / ml BSA / BGJb medium / PS for 24 hours, LPS (10 μg / ml) and nobiletin (30 μM) represented by formula (1) or tangeretin (30 μM) represented by formula (2) were further treated. After culturing for 3 days, the Ca concentration in the culture supernatant was measured to determine bone resorption activity. The result is shown in FIG. On the third day after treatment, bone resorption activity was significantly increased by LPS treatment (* P <0.001 vs control), but significantly decreased by combined treatment with nobiletin or tangeretin (#### P <0.001) vs LPS).

[Effects of nobiletin and tangeretin on bone mineral density of alveolar bone in periodontal disease model mice]
Mice in which 6-week-old mice were administered with the solvent (DMSO / PEG300 1: 4) three times every other day on the gingiva of the lower mandibular molar group were mice in the control group and LPS (30 μg / body / day) was administered in the same schedule As an LPS group, periodontal disease model mice were prepared. Furthermore, LPS mixed with nobiletin (0.3 mg / body / day) or tangeretin (0.5 mg / body / day) was co-administered 3 times every other day. For group 4, the alveolar bone was removed 3 days after the third administration, and after tooth extraction, the bone density of the alveolar bone was measured by the Dual Energy X-ray Absorptiometry (DEXA) method. The result is shown in FIG. Alveolar bone density decreased by LPS administration was maintained at a control level by combined administration of nobiletin (0.3 mg / body / day) (LPS administration group: * P <0.001 vs control, nobiletin group: ## P <0.01 vs LPS). In addition, the bone density of the alveolar bone decreased by the LPS administration was significantly recovered by the combined administration of tangeretin (0.5 mg / body / day) (tangeretin group: #P <0.05 vs LPS).

Claims (6)

Periodontal disease preventive and therapeutic agent comprising polymethoxyflavonoids as active ingredients. The preventive / therapeutic agent for periodontal disease according to claim 1, wherein the polymethoxyflavonoids are citrus-derived polymethoxyflavonoids. The preventive / therapeutic agent for periodontal disease according to claim 1 or 2, wherein the polymethoxyflavonoids are polymethoxyflavonoids containing tangeretin as an active ingredient. A food or food material for preventing or treating periodontal disease comprising polymethoxyflavonoids as an active ingredient. The food or food material for periodontal disease prevention and treatment according to claim 4, wherein the polymethoxyflavonoids are citrus-derived polymethoxyflavonoids. The food or food material for preventing or treating periodontal disease according to claim 4 or 5, wherein the polymethoxyflavonoid is tangeretin.