Oral flora improver

Abstract

To provide an intraoral flora improver.SOLUTION: An intraoral flora improver contains kaempferide and bethretol. The mass ratio of the bethretol to the kaempferide is 1.5 or more and 4 or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oral flora-improving agent. [Background technology]

[0002] Various bacteria coexist in the human oral cavity, forming a bacterial community called the bacterial flora. The bacterial flora in the oral cavity (hereinafter referred to as the "oral bacterial flora") contains bacteria that cause periodontal disease and dental caries (hereinafter also referred to as "bad bacteria"), but also contains bacteria that have antibacterial properties against bad bacteria (hereinafter also referred to as "good bacteria"), suppressing the onset of periodontal disease and dental caries. However, when there is a change in physical condition due to a change in diet, stress, aging, etc., or when plaque accumulates on the teeth or periodontium, the proliferation of bad bacteria cannot be suppressed. As a result, periodontal disease and dental caries develop.

[0003] Various techniques have been proposed to prevent periodontal disease and dental caries. For example, Patent Document 1 proposes a technique of using a composition containing (A) ε-polylysine and / or a salt thereof and (B) an antibacterial component derived from a natural product for preventing dental caries and periodontal disease.

[0004] In addition, in Patent Document 1, the antibacterial components (B) derived from natural products include lysozyme, chitosan, snowbell extract, hiba oil, hinokitiol, pectin hydrolysate, forsythia extract, artemisia capillaris extract, perilla leaf extract, mustard extract, wasabi extract, moso bamboo extract, chili pepper extract, knotweed extract, udo extract, garlic extract, pimenta extract, fig extract, mulberry extract, grape seed extract, pepper extract, madake extract, oregano extract, rice husk extract, clove extract, ginger extract, sage extract, acetophenone extract, grape skin extract, Japanese laurel extract, yucca foam extract, and rosemary extract. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2004-123630 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an oral bacterial flora-improving agent. [Means for solving the problem]

[0007] The present inventors have discovered that a composition containing kaempferide and betuletol in a specified ratio exhibits antibacterial activity against harmful bacteria in the oral flora, while exhibiting little antibacterial activity against beneficial bacteria in the oral flora, and have thus completed the present invention.

[0008] The gist of the present invention is as follows. [1] An oral flora improving agent comprising kaempferide and betuletol, wherein the mass ratio of the betuletol to the kaempferide is 1.5 or more and 4 or less. [2] The oral flora improving agent according to [1], wherein the mass ratio of the betuletol to the kaempferide is 2 or more and 3 or less. [3] The oral flora improving agent according to [1] or [2], characterized in that the total content of the kaempferide and the betuletol is 1 μg to 200 μg per 1 ml of the oral flora improving agent. [4] A periodontal disease preventive agent comprising kaempferide and betuletol, wherein the mass ratio of the betuletol to the kaempferide is 1.5 or more and 4 or less. [5] The periodontal disease preventive agent according to [4], characterized in that the mass ratio of the betuleitol to the kaempferide is 2 or more and 3 or less. [6] The periodontal disease preventive agent according to [4] or [5], characterized in that the total content of the kaempferide and the betuleitol is 1 μg to 200 μg per 1 ml of the periodontal disease preventive agent. [7] A caries prevention agent comprising kaempferide and betuletol, wherein the mass ratio of the betuletol to the kaempferide is 1.5 or more and 4 or less. [8] A caries prevention agent according to [7], characterized in that the mass ratio of the betulethol to the kaempferide is 2 or more and 3 or less. [9] The caries prevention agent according to [7] or [8], characterized in that the total content of the kaempferide and the betulethol is 1 μg to 200 μg per 1 ml of the caries prevention agent. Effect of the Invention

[0009] According to the present invention, an oral bacterial flora-improving agent can be provided. [Brief description of the drawings]

[0010] [Figure 1a] FIG. 1a is a graph showing the optical density (OD value at 595 nm) (Test 1a). [Figure 1b] FIG. 1b is a graph showing the optical density (OD value at 595 nm) (Test 1b). [Figure 2a] FIG. 2a is a graph showing the optical density (OD value at 595 nm) (Test 2a). [Figure 2b] FIG. 2b is a graph showing the optical density (OD value at 595 nm) (Test 2b). [Diagram 3] FIG. 3 is a graph showing the optical density (OD value at 595 nm) (Test 3). [Figure 4] FIG. 4 is a graph showing the optical density (OD value at 595 nm) (Test 4). [Figure 5a] FIG. 5a is a graph showing the optical density (OD value at 595 nm) (Test 5a). [Figure 5b] FIG. 5b is a graph showing the optical density (OD value at 595 nm) (Test 5b). [Figure 6a] FIG. 6a is a graph showing the optical density (OD value at 595 nm) (Test 6a). [Figure 6b]FIG. 6b is a graph showing the optical density (OD value at 595 nm) (Test 6b). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, one embodiment of the present invention will be described.

[0012] The present embodiment relates to an oral microflora improving agent.

[0013] As used herein, "improving oral flora" refers to improving the ratio of beneficial bacteria to harmful bacteria in the oral flora, and more specifically, to increasing the ratio of beneficial bacteria to harmful bacteria in the oral flora compared to before improvement.

[0014] In this specification, "bad bacteria" refers to bacteria that cause periodontal disease or dental caries, and "good bacteria" refers to bacteria that have an antibacterial effect against bad bacteria. In this specification, the antibacterial effect is a concept that includes both bacteriostasis, which prevents bacteria from multiplying, and sterilization, which kills bacteria.

[0015] Here, examples of bacteria (bad bacteria) that cause dental caries include Streptococcus mutans and Streptococcus sobrinus.

[0016] Furthermore, examples of bacteria that cause periodontal disease (bad bacteria) include Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans serotype b, and Prevotella intermedia.

[0017] On the other hand, examples of bacteria (good bacteria) that exhibit antibacterial effects against these bad bacteria include Streptococcus salivarius K12 (Streptococcus salivarius subsp. salivarius Andrewes and Horder (ATCC BAA-1024 (registered trademark))) and Streptococcus salivarius strein M18 (Streptococcus salivarius Andrewes and Horder (ATCC BAA-2593 (registered trademark))).

[0018] The oral flora improving agent of the present embodiment contains kaempferide and betuletol.

[0019] Kaempferide contained in the oral bacterial flora-improving agent of this embodiment is a compound represented by the following formula (1) (3,5,7-trihydroxy-3-methoxy-flavone), which is a type of flavone.

[0020] [ka]

[0021] Betuletol contained in the oral bacterial flora improving agent of this embodiment is a compound represented by the following formula (2) (3,5,7-trihydroxy-6,3-dimethoxy-flavone), which is a type of flavone.

[0022] [ka]

[0023] Kaempferide and betuletol share the same basic structure (3,5,7-trihydroxy-3-methoxy-flavone), but differ in the substituent at position 6. Specifically, the substituent at position 6 in kaempferide is hydrogen, while the substituent at position 6 in betuletol is a methoxy group.

[0024] In the oral flora improving agent of this embodiment, the mass ratio of betuletol to kaempferide is 1.5 or more and 4 or less. From the viewpoint of easily exhibiting an antibacterial effect against harmful bacteria regardless of the type, the mass ratio of betuletol to kaempferide is preferably 2 or more and 3 or less.

[0025] The ratio of kaempferide and betuletol contained in the oral flora improving agent can be determined, for example, by extracting the oral flora improving agent with about 70% to 80% ethanol, concentrating the extract to obtain a crude extract, and then quantifying the respective concentrations by UPLC (ultra-performance liquid chromatography) with reference to known literature. If the oral flora improving agent contains components other than kaempferide and betuletol that dissolve in about 70% to 80% ethanol, a removal process may be performed to remove such components from the ethanol extract. The method of removal may be set according to the components to be removed, and known methods may be used.

[0026] In the oral flora improving agent of this embodiment, the contents of kaempferide and betuletol are not particularly limited, but the total content of kaempferide and betuletol can be, for example, 0.1 μg to 400 μg per 1 ml of oral flora improving agent, and from the viewpoint of further improving the ratio of bad bacteria to good bacteria, it is preferably 1 μg to 200 μg, and more preferably 5 μg to 200 μg.

[0027] Kaempferide and betuletol can be obtained from natural materials containing polyphenols (hereinafter also referred to as "polyphenol-containing materials"). Examples of polyphenol-containing materials containing kaempferide and betuletol include propolis, red grape extract, red grape skin extract, red grape leaves, white grape leaves, red grape fruits, white grape fruits, white grape skins, grape seeds, ginkgo leaves, French west coast pine, young barley leaves, young wheat leaves, young rice leaves, young soybean leaves, blueberry fruits, skins, seeds, perilla leaves, rose leaves, rose flowers, yacon, Jerusalem artichoke, purple sweet potato and their leaves, Asteraceae plants, or herbs such as echinacea, lemon balm, turmeric, guava, lemongrass, and lavender, as well as green tea, black tea, and oolong tea.

[0028] Here, a method for adjusting the ratio of kaempferide to betuletol will be described. As described above, kaempferide and betuletol can be obtained from polyphenol-containing materials, but although it depends on the extraction method, it is usually difficult to adjust the ratio of kaempferide to betuletol to the above-mentioned ratio (1.5 to 4) simply by obtaining an extract containing betuletol and kaempferide from a polyphenol-containing material.

[0029] Therefore, as a method for adjusting kaempferide and betuletol to the above-mentioned ratio (1.5 or more and 4 or less), one example is to separately separate (isolate) betuletol and kaempferide from a polyphenol-containing material and then mix them in the above-mentioned ratio (1.5 or more and 4 or less).

[0030] The method for separating kaempferide and betuletol from a polyphenol-containing material is not particularly limited, and a known method can be used. In addition, for example, the following method may be used. First, the polyphenol-containing material is extracted with about 70 to 80% (for example, 70 to 80%) ethanol, and the extract is concentrated to obtain a crude extract. Next, the ethanol extract of the polyphenol-containing material is applied to a column filled with a carrier (Sephadex LH-20, manufactured by GE Healthcare Japan Co., Ltd.), and separated using 100% by volume ethanol as a separation solvent. The separation solvent is allowed to fall naturally, and a portion that emits fluorescence in response to excitation light in the ultraviolet region is separated using an ultraviolet irradiation device and concentrated at 39°C. This concentrated liquid is added to a silica gel column and separated. A mixed solution of chloroform and methanol (volume ratio 95:5) is used as the separation solvent, and the flow rate for separation can be 1.0 ml / min. The betuletol fraction and the kaempferide fraction are each separated using a photodiode array. The separated solutions are each concentrated in a vacuum concentrator, and then dried at 40°C using a vacuum dryer, allowing betuletol and kaempferide to be separated.

[0031] In the above-mentioned method, the extraction solvent used for extracting the polyphenol-containing material may be, in addition to ethanol, hydrophilic solvents such as water, methanol, propanol, acetone, ethyl acetate, benzene, chloroform, etc. In addition to ethanol, the separation solvent used in the above-mentioned method may be, in addition to ethanol, water, methanol, propanol, butanol, etc.

[0032] The method for adjusting the mass ratio of betuletol to kaempferide to the above-mentioned range (1.5 to 4) is not limited to the above-mentioned method, and kaempferide and betuletol do not have to be separated separately. In this case, for example, two or more extracts containing kaempferide and betuletol at different content ratios are obtained from a polyphenol-containing material, and these extracts are mixed together to adjust the ratio of kaempferide to betuletol to the above-mentioned range (1.5 to 4).

[0033] Furthermore, as described above, it is difficult to adjust kaempferide and betuletol to the above-mentioned ratio (1.5 or more and 4 or less) simply by obtaining an extract containing betuletol and kaempferide from a polyphenol-containing material; however, if the extract of a polyphenol-containing material is an extract in which the mass ratio of betuletol to kaempferide is within the above range, that extract may be used in the oral flora improvement agent of this embodiment.

[0034] The oral flora improving agent of this embodiment may be composed of only kaempferide and betuletol, or may contain other components other than kaempferide and betuletol. Examples of other components include abrasives, preservatives, anti-inflammatory agents, glucosyltransferase (GTase) inhibitors, plaque inhibitors, hypersensitivity inhibitors, tartar prevention agents, adhesives, thickening agents, excipients, lubricants, flavors, sweeteners, cooling agents, colorants, deodorants, surfactants, and pH adjusters.

[0035] The form of the oral bacterial flora improving agent of this embodiment is not particularly limited, and may be any of liquid, solid, semi-solid (gel, ointment, paste), and the like.

[0036] The method of applying the oral flora improving agent of this embodiment to the oral cavity is not particularly limited, so long as it can be retained in the oral cavity for a predetermined period of time.

[0037] The oral flora improving agent of the present embodiment may be edible or inedible as long as it can be retained in the oral cavity for a predetermined period of time. For example, the oral flora improving agent of the present embodiment may be in the form of a food (including functional foods, foods for specified health uses, foods for the sick, etc.), or in the form of a medicine (including quasi-drugs and designated quasi-drugs) or oral cosmetics.

[0038] The amount of the oral flora improving agent of the present embodiment to be applied to the oral cavity is not particularly limited, but may be, for example, an amount that allows 5 to 20 μg of kaempferide and betuletol to remain in the oral cavity in total per application. The frequency of application of the oral flora improving agent of the present embodiment is not particularly limited, but may be, for example, 1 to 3 times a day.

[0039] The formulation form of the oral flora improving agent of this embodiment is not particularly limited as long as it can be retained in the oral cavity for a predetermined period of time, and examples include edible oral preparations such as troches, edible films, chewing gums, candies, gummy candies, tablets, granules, fine granules, powders, and capsules, and non-edible formulation forms such as liquid dentifrices, toothpastes, lubricants, toothpaste powders, mouthwashes, mouth rinses, gargles, and mouth fresheners (mouth sprays, etc.).

[0040] The method for producing the oral bacterial flora improving agent of the present embodiment is not particularly limited, and any known method can be used depending on the formulation form.

[0041] The oral flora improving agent of the present embodiment described above exhibits antibacterial activity against bad bacteria that cause periodontal disease and dental caries, but is less likely to exhibit antibacterial activity against good bacteria. Therefore, the oral flora improving agent of the present embodiment can improve the ratio of bad bacteria to good bacteria in the oral flora.

[0042] As described above, the oral flora improving agent of the present embodiment exhibits antibacterial activity against bad bacteria that cause periodontal disease and dental caries, but is less likely to exhibit antibacterial activity against good bacteria. Therefore, the oral flora improving agent of the present embodiment can also be used as a periodontal disease preventive agent that prevents the occurrence of periodontal disease, or a caries preventive agent that prevents the occurrence of dental caries. EXAMPLES

[0043] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0044] [Acquisition of Kaempferide and Betuletor] 50g of raw propolis from Brazil was added with 120g of 77% ethanol and extracted at 60℃ and 200 rpm for 1-2 hours. The obtained extract was suction filtered with No. 2 filter paper manufactured by Advantec Toyo Co., Ltd., and 103g of the obtained filtrate was cooled to -15℃ or lower. It was further naturally filtered with No. 2 filter paper, and the obtained filtrate was used as propolis extract (yield: 83g, solid content: 20%).

[0045] Next, the ethanol extract of propolis was applied to a column packed with a carrier (Sephadex LH-20, GE Healthcare Japan Co., Ltd.) and separated using 100% ethanol by volume as the separation solvent. The separation solvent was allowed to fall naturally, and the part that emitted fluorescence in response to excitation light in the ultraviolet region was separated using an ultraviolet irradiation device. A fraction separated with 100% ethanol by volume, 10 times the column volume, was obtained, and this was applied to a vacuum concentrator and concentrated at 39°C.

[0046] This concentrated solution was added to a silica gel column for separation. A mixed solution of chloroform and methanol (volume ratio 95:5) was used as the separation solvent, and the flow rate for separation was 1.0 ml / min. The betuletol fraction and the kaempferide fraction were each separated using a photodiode array. The separated solutions were each subjected to a reduced pressure concentrator and concentrated. They were then dried at 40°C using a vacuum dryer, and betuletol and kaempferide were separated. It was confirmed by HPLC analysis that the obtained substances were betuletol and kaempferide.

[0047] [Acquisition of test subject] Using the fractionated betuletol and kaempferide, test substances A to E shown in Table 1-1 below were obtained. Also, using the fractionated betuletol and kaempferide, test substances F to G shown in Table 1-2 below were obtained. Note that test substances B to D and test substances F to G were obtained by mixing the fractionated kaempferide and betuletol. [Table 1-1] [Table 1-2]

[0048] [Test 1(a) (Antibacterial effect against bad bacteria (bacteria that cause periodontal disease)] To evaluate the antibacterial effect against harmful bacteria, the following tests were carried out.

[0049] Fusobacterium nucleatum ATCC31647 (obtained from Sumitomo Pharma International Co., Ltd.) was prepared as the harmful bacteria that causes periodontal disease.

[0050] Antibacterial activity was measured by the broth microdilution method according to the standard method of the Japanese Society of Chemotherapy. First, the bad bacteria that cause periodontal disease (Fusobacterium nucleatum ATCC31647) were cultured anaerobically at 37°C in a mixture of nitrogen (83%), hydrogen (7%) and carbon dioxide (10%) gases using Gifu Anaerobic Medium (GAM) containing 5 μg / ml hemin and 1 μg / ml menadione. The bad bacteria that cause dental caries (Streptococcus mutans ATCC25175) in Test 3 described below were cultured aerobically at 37°C using Brain Heart Infusion (BHI) medium. The cultured bad bacteria were cultured at 2 × 10 6 100μL of bacterial culture solution prepared to cfu / ml was added to each well of a 96-well microplate. 100μl of 0.8% to 0.01% DMSO aqueous solution mixed with test substances A to E was added to the wells to which the bacterial culture solution was added, so that the concentration of each test substance in the solution in the well was 5μg / ml. As a control, 100μl of 0.8% to 0.01% DMSO aqueous solution was added to the wells to which the bacterial culture solution was added.

[0051] Then, for Fusobacterium nucleatum ATCC31647, the bacterial culture was cultured at 37°C for 96 hours, and the optical density (OD value at 595 nm) of the solution in the well was measured using a microplate reader. The results are shown in Figure 1a.

[0052] As shown in Figure 1a, when test substances C and D were added, the OD value was lower than the control, and was almost 0. The OD value being lower than the control indicates that the bacteria had died. From this result, it was understood that an antibacterial effect against Fusobacterium nucleatum (bad bacteria) was observed when the mass ratio of betulethol to kaempferide was within the range of 1.5 to 4.

[0053] On the other hand, as shown in Figure 1a, the OD values ​​of test substances A, B, and E were comparable to that of the control. These results indicate that when only one of kaempferide and betuletol is contained, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 to 4, it is difficult to show antibacterial activity against Fusobacterium nucleatum (bad bacteria).

[0054] [Test 1(b) (Effect of concentration)] In this test, the test substance added to the wells was at a concentration different from that in test 1(a) to evaluate the effect of the test substance concentration on the antibacterial activity.

[0055] In this test, test substances C and D, which were confirmed to have antibacterial effects against Fusobacterium nucleatum (bad bacteria) in Evaluation 1(a), were used as test substances. In this test, the concentration of the test substance in the solution in the well was set to 10-60μg / ml. Other than these conditions, Fusobacterium nucleatum ATCC31647 was cultured in the same manner as in Test 1(a), and the optical density (OD value at 595nm) of the solution in the well was measured. The results are shown in Figure 1b.

[0056] As shown in Figure 1b, even when the test substances were added at concentrations of 10-60μg / ml, the OD value was lower than that of the control when test substances C and D were added, and was nearly 0. From this result, it was understood that as long as the mass ratio of betulethol to kaempferide is within the range of 1.5 to 4, it will have an antibacterial effect against Fusobacterium nucleatum (bad bacteria) regardless of the concentration.

[0057] [Test 2(a) (Antibacterial effect against other bad bacteria (causing periodontal disease)] In this study, harmful bacteria different from those used in Study 1 were used to evaluate the antibacterial effect.

[0058] In this test, the bad bacteria used was Aggregatibacter actinomycetemcomitans strain Y4 (obtained from the Microorganism Materials Development Laboratory (JCM) of the RIKEN BioResource Research Center (RIKEN BRC), National Research and Development Agency), which causes periodontal disease. In this test, the concentration of the test substance in the solution in the well was 40 to 200 μg / ml. Other than these conditions, the culture was carried out for 40 hours in the same manner as in test 1 (a), and the optical density of the solution in the well (OD value at 595 nm) was measured. The results are shown in Figure 2a.

[0059] As shown in Figure 2a, when test substances C and D were added, the OD value was lower than that of the control, and was less than 1 / 6 of the OD value of the control, regardless of the concentration of the test substances in the range of 40 to 200 μg / ml. From this result, it was understood that when the mass ratio of betulethol to kaempferide is in the range of 1.5 to 4, it also shows antibacterial activity against Aggregatibacter actinomycetemcomitans.

[0060] On the other hand, as shown in Figure 2a, the OD values ​​of test substances A, B and E were lower than that of the control, but were more than three times higher than those of test substances C and D. From these results, it was revealed that when only one of kaempferide and betuletol is contained, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 to 4, antibacterial activity against Aggregatibacter actinomycetemcomitans (bad bacteria) is exhibited, but the antibacterial activity is significantly weaker than when the mass ratio of betuletol to kaempferide is within the range of 1.5 to 4.

[0061] [Test 2(b) (Antibacterial activity of test substances F and G)] In this test, the test substances F to G were used instead of the test substances A to E used in test 2(a), and the test substance concentrations in the solution in the wells were set to 60 to 200 μg / ml. Except for this, the culture was carried out for 40 hours in the same manner as in test 2(a), and the optical density (OD value at 595 nm) of the solution in the wells was measured. The results are shown in Figure 2b.

[0062] As shown in Figure 2b, when test substances F to G were added, the OD value was almost 0, lower than the control, regardless of the concentration of the test substances within the range of 60 to 200 μg / ml. From this result, it was understood that when the mass ratio of betuletol to kaempferide was 1.5 or when the mass ratio of betuletol to kaempferide was 4, antibacterial activity was shown against Aggregatibacter actinomycetemcomitans.

[0063] [Test 3 (Antibacterial effect against other bad bacteria (causing dental caries)] In this study, harmful bacteria different from those used in Study 1 were used to evaluate the antibacterial effect.

[0064] In this test, Streptococcus mutans ATCC25175 (obtained from the Joint Microbial Research and Development Laboratory (JCM), RIKEN BioResource Research Center (RIKEN BRC)) which causes dental caries was used as the bad bacteria. In addition, in this test, the concentration of the test substance in the solution in the well was set to 200μg / ml. Other than these conditions, the same method as in Test 1(a) was used for culturing for 40 hours, and the optical density of the solution in the well (OD value at 595nm) was measured. The results are shown in Figure 3.

[0065] As shown in Figure 3, when test substances C and D were added, the OD value was lower than that of the control, and was about half that of the control. From this result, it was understood that when the mass ratio of betuletol to kaempferide is within the range of 1.5 to 4, it also shows antibacterial activity against Streptococcus mutans.

[0066] On the other hand, as shown in Figure 3, test substances A and E had OD values ​​comparable to that of the control. Furthermore, even though test substance B had a lower OD value than the control, it still had an OD value about 1.5 times higher than test substances C and D. These results demonstrate that when test substances contain only one of kaempferide or betuletol, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 to 4, it is difficult to demonstrate antibacterial activity against Streptococcus mutans (bad bacteria).

[0067] [Test 4 (Antibacterial effect against other bad bacteria (causing periodontal disease)] In this study, harmful bacteria different from those used in Study 1 were used to evaluate the antibacterial effect.

[0068] In this test, Prevotella intermedia ATCC25611 (obtained from the Joint Microbial Research and Development Laboratory (JCM), RIKEN BioResource Research Center (RIKEN BRC), National Research and Development Agency), which causes periodontal disease, was used as the bad bacteria. In addition, in this test, the concentration of the test substance in the solution in the well was set to 100 μg / ml. Other than these conditions, the culture was performed for 48 hours in the same manner as in Test 1, and the optical density (OD value at 595 nm) of the solution in the well was measured. The results are shown in Figure 4.

[0069] As shown in Figure 4, when test substances C and D were added, the OD value was lower than the control, being 1 / 6 or less of the OD value of the control. From this result, it was understood that an antibacterial effect against Prevotella intermedia (bad bacteria) was observed when the mass ratio of betulethol to kaempferide was within the range of 1.5 to 4.

[0070] On the other hand, as shown in Figure 4, the OD values ​​of test substances A and E were comparable to that of the control. Furthermore, even though test substance B had a lower OD value than the control, it still had an OD value about four times higher than that of test substances C and D. These results demonstrate that when only one of kaempferide or betuletol is contained, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 to 4, it is difficult to exhibit antibacterial activity against Prevotella intermedia (bad bacteria).

[0071] [Test 5(a) (Antibacterial effect against beneficial bacteria)] In this test, good bacteria were used instead of the bad bacteria used in test 1, and the antibacterial effect was evaluated.

[0072] In this test, instead of the bad bacteria used in Test 1, the good bacteria Streptococcus salivarius strein M18 (obtained from Sumitomo Pharma International Co., Ltd.) was used. In addition, in this test, the concentration of the test substance in the solution in the well was set to 40 to 200 μg / ml. Other than these conditions, the culture was carried out for 40 hours in the same manner as in Test 1, and the optical density (OD value at 595 nm) of the solution in the well was measured. The results are shown in Figure 5a.

[0073] As shown in Figure 5a, the OD values ​​of test substances C and D were comparable to that of the control at any concentration within the range of 40-200 μg / ml. This result indicates that when the mass ratio of betulethol to kaempferide is within the range of 1.5 to 4, it is difficult to show antibacterial activity against beneficial bacteria (Streptococcus salivarius M18).

[0074] [Test 5(b) (Antibacterial activity of test substances F and G)] In this test, the culture was carried out for 40 hours in the same manner as in Test 5(a), except that Tests F to G were used instead of Tests A to E, and the optical density (OD value at 595 nm) of the solution in the well was measured. The results are shown in Figure 5b.

[0075] As shown in Figure 5b, the OD values ​​of test substances F to G were comparable to that of the control at any concentration within the range of 40 to 200 μg / ml. This result revealed that when the mass ratio of betuletol to kaempferide was 1.5 or when the mass ratio of betuletol to kaempferide was 4, the antibacterial effect against the good bacteria (Streptococcus salivarius M18) was poor.

[0076] [Test 6(a) (Antibacterial effect against other bad bacteria (causing periodontal disease)] In this study, harmful bacteria different from those used in Study 1 were used to evaluate the antibacterial effect.

[0077] In this test, the bad bacteria used were Porphyromonas gingivalis strain 381 (obtained from the Microorganisms Research Division (JCM) of the RIKEN BioResource Research Center (RIKEN BRC)) and Porphyromonas gingivalis ATCC33277 (obtained from the Microorganisms Research Division (JCM) of the RIKEN BioResource Research Center (RIKEN BRC)). In this test, test substances C and D were used as the test substances at concentrations of 10 to 60 μg / ml in the solution in the well. The other conditions were the same as in Test 1(a), except that Porphyromonas gingivalis strain 381 was cultured for 72 hours and Porphyromonas gingivalis ATCC33277 was cultured for 48 hours, after which the optical density (OD value at 595 nm) of the solution in the well was measured. The results are shown in Figure 6a.

[0078] As shown in Figure 6a, in both tests using P. gingivalis strain 381 and P. gingivalis ATCC33277, the OD values ​​of test substances C and D were lower than those of the control at any concentration within the range of 10-60 μg / ml, and some of the test substances had an OD value of almost 0. From these results, it was revealed that when the mass ratio of betulethol to kaempferide was within the range of 1.5 to 4, it showed antibacterial activity against P. gingivalis (bad bacteria).

[0079] [Test 6(b) (Antibacterial activity of test substances F and G)] In Test 6(a) using Porphyromonas gingivalis ATCC33277 as the bad bacteria, the same method as Test 6(a) was used except that Tests F and G, the concentrations of which in the solution in the wells were 20-80μg / ml, were used instead of Tests C and D. The culture was carried out for 48 hours, and the optical density (OD value at 595nm) of the solution in the wells was measured. The results are shown in Figure 6b.

[0080] As shown in Figure 6b, the OD values ​​of test substances F and G were lower than that of the control at any concentration within the range of 20 to 80 μg / ml, and were almost 0. From these results, it was revealed that when the mass ratio of betuletol to kaempferide was 1.5 or when the mass ratio of betuletol to kaempferide was 4, antibacterial activity was exhibited against Porphyromonas gingivalis (bad bacteria).

[0081] From the results of the above-mentioned tests 1 to 6, it was understood that when the mass ratio of betuletol to kaempferide is 1.5 or more and 4 or less, it shows an antibacterial effect against any bad bacteria, but is difficult to show an antibacterial effect against good bacteria. In other words, when the mass ratio of betuletol to kaempferide is 1.5 or more and 4 or less, it shows a stronger antibacterial effect against bad bacteria than good bacteria, and it was understood that it is possible to improve the ratio of bad bacteria to good bacteria in the oral bacterial flora.

[0082] In addition, from the results of the above-mentioned tests 1 to 6, it was understood that when the mass ratio of betuletol to kaempferide is 1.5 or more and 4 or less, the antibacterial effect is more likely to be exhibited regardless of the type of bad bacteria, and in addition, the antibacterial effect against bad bacteria is stronger, compared to when only one of kaempferide and betuletol is contained, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 or more and 4 or less. In other words, it was also made clear that when the mass ratio of betuletol to kaempferide is 1.5 or more and 4 or less, the ratio of bad bacteria to good bacteria in the oral bacterial flora can be improved more than when only one of kaempferide and betuletol is contained, or when the mass ratio of betuletol to kaempferide is outside the range of 1.5 or more and 4 or less.

Claims

1. 1. An oral flora improving agent comprising kaempferide and betuletol, wherein the mass ratio of said betuletol to said kaempferide is 1.5 or more and 4 or less.

2. The oral flora improving agent according to claim 1, characterized in that the mass ratio of said betuletol to said kaempferide is 2 or more and 3 or less.

3. 3. The oral flora improving agent according to claim 1, wherein the total content of said kaempferide and said betuletol is 1 μg to 200 μg per 1 ml of said oral flora improving agent.

4. A periodontal disease preventive agent comprising kaempferide and betuletol, the mass ratio of said betuletol to said kaempferide being 1.5 or more and 4 or less.

5. The periodontal disease preventive agent according to claim 4, characterized in that the mass ratio of said betuletol to said kaempferide is 2 or more and 3 or less.

6. 6. The periodontal disease preventive agent according to claim 4, wherein the total content of said kaempferide and said betuletol is 1 μg to 200 μg per 1 ml of said periodontal disease preventive agent.

7. A caries prevention agent comprising kaempferide and betuletol, the mass ratio of said betuletol to said kaempferide being 1.5 or more and 4 or less.

8. 8. The caries prevention agent according to claim 7, characterized in that the mass ratio of said betuletol to said kaempferide is 2 or more and 3 or less.

9. 9. The caries prevention agent according to claim 7, wherein the total content of said kaempferide and said betulethol is 1 μg to 200 μg per 1 ml of said caries prevention agent.

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