Fungicide for Porphyromonas gingivalis and method for producing ingredient having antibacterial activity against Porphyromonas gingivalis

Abstract

To provide a fungicide that has excellent antifungal action against Porphyromonas gingivalis.SOLUTION: The problem can be solved by a fungicide for Porphyromonas gingivalis of the present invention containing, as an active ingredient, at least a component exhibiting an Rf value in the range of 0.04 to 0.25 in thin-layer chromatography using a solvent in which hexane:ethyl acetate are mixed at 20:1 as a mobile phase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a fungicide for Porphyromonas gingivalis and a method for producing a component having antibacterial activity against Porphyromonas gingivalis. [Background technology]

[0002] It has become clear that periodontal disease is caused by oral bacteria, and Porphyromonas gingivalis is known to be one of the causative bacteria of periodontal disease. It has been disclosed that cis-octadecenoic acid has an antibacterial effect against Porphyromonas gingivalis (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 6-25000 [Non-patent literature]

[0004] [Non-Patent Document 1] Oral Microbiology and Immunology, 1996 (Denmark), Vol. 5, p. 350-255 [Non-patent document 2] Journal of Essential Oil Bearing Plants, 2018 (India), Vol. 1, pp. 40-51 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the antibacterial activity of cis-octadecenoic acid against Porphyromonas gingivalis was insufficient. Therefore, an object of the present invention is to provide a bactericide having excellent antibacterial activity against Porphyromonas gingivalis. [Means for solving the problem]

[0006] The present inventors have conducted extensive research into antibacterial agents that have excellent antibacterial activity against Porphyromonas gingivalis, and have surprisingly found that a specific component extracted from fennel exhibits excellent antibacterial activity against Porphyromonas gingivalis. The present invention is based on this finding. Therefore, the present invention provides [1] A fungicide for Porphyromonas gingivalis, containing as an active ingredient a component that exhibits an Rf value in the range of at least 0.04 to 0.25 in thin-layer chromatography using a solvent mixture of hexane and ethyl acetate at a ratio of 20:1 as a mobile phase; [2] The fungicide for Porphyromonas gingivalis according to [1], wherein the ratio of petroselinic acid in the components showing an Rf value in the range of 0.04 to 0.25 is 66% or less. [3] (1) A method for producing an ingredient having antibacterial activity against Porphyromonas gingivalis, comprising a step of immersing fennel fruits and / or seeds in n-hexane; [4] (2) The method for producing a component having antibacterial activity against Porphyromonas gingivalis according to [3], further comprising a step of fractionating the hexane extract obtained in the step (1) using a normal phase column. [5] (3) A method for producing a component having an antibacterial activity against Porphyromonas gingivalis according to [4], further comprising a step of fractionating the fraction obtained in the step (2) using a reverse-phase column. [6] An antibacterial food composition against Porphyromonas gingivalis, comprising the bactericide according to [1] or [2]. [7] A food composition for preventing periodontal disease, comprising the bactericide according to [1] or [2]. [8] An antibacterial pharmaceutical composition against Porphyromonas gingivalis, comprising the bactericide according to [1] or [2]; and [9] A pharmaceutical composition for preventing or treating periodontal disease, comprising the bactericide according to [1] or [2]. Regarding. It has been reported that petroselinic acid (cis-octadecenoic acid) is contained in fennel (Non-Patent Document 2), and it has been disclosed that fennel contains a substance that inhibits the protease of Porphyromonas gingivalis (Patent Document 1). However, it is surprising that a specific component extracted from fennel exhibits excellent antibacterial activity against Porphyromonas gingivalis. [Effects of the Invention]

[0007] The bactericide of the present invention exhibits excellent antibacterial activity against Porphyromonas gingivalis. The production method of the present invention makes it possible to simply produce a component having excellent antibacterial activity against Porphyromonas gingivalis. [Brief explanation of the drawings]

[0008] [Figure 1] This diagram shows the purification process of the antibacterial component against Porphyromonas gingivalis from fennel. [Figure 2] FIG. 1 is a diagram showing a schematic diagram of calculation of Rf values by thin-layer chromatography. [Figure 3] Photographs showing the active ingredients in the hexane extract and fraction 3 separated by thin layer chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0009] [1] Fungicide The disinfectant of the present invention contains, as an active ingredient, a component that exhibits an Rf value in the range of at least 0.04 to 0.25 in thin layer chromatography using a 20:1 mixture of hexane and ethyl acetate as the mobile phase. The components exhibiting an Rf value of 0.04 to 0.25 include peteroselic acid, as well as other active ingredients.

[0010] Thin-layer chromatography The Rf value is measured using thin-layer chromatography (TLC). Thin-layer chromatography is a type of liquid chromatography that uses a fine powder thinly coated on a support as the stationary phase and an appropriate solvent as the mobile phase. Materials such as glass, aluminum, and plastic are used as supports. Various fine powders are used as the stationary phase, and separation methods based on various mechanisms such as adsorption, ion exchange, partitioning, and molecular sieving can be selected depending on their properties. When a sample moves with the mobile phase (solvent), substances with a high affinity for the stationary phase move slowly because they interact with each other. Conversely, substances with a low affinity for the stationary phase move quickly. This mechanism allows the separation of substances in the sample.

[0011] The conditions for thin layer chromatography to determine the Rf value of the present invention are as follows. Stationary phase: TLC aluminum sheet silica gel 60F 254 (Merck) Mobile phase: Hexane and EtOAc mixed at a ratio of 20:1 (1) The sample is dissolved in DMSO (dimethyl sulfoxide) and spotted onto a thin-layer plate (stationary phase) using a capillary tube (Figure 2A). (2) Pour the mobile phase solvent into a glass developing tank to a height of several centimeters and leave it to stand for 1-2 hours to allow the solvent vapor in the tank to reach equilibrium. (3) Place the plate (1) into the development tank (2), close the lid, and leave it until the solvent reaches about 1 cm from the top of the plate (Figure 2B, C). (4) Remove the plate and let it dry. (5) The components of the plate (4) are detected by the sulfuric acid method. Specifically, the plate (4) is sprayed or dipped in a 10% aqueous solution of sulfuric acid, and then heated on a hot plate or similar device. The organic spots are carbonized by dehydration, and the black staining is confirmed.

[0012] The Rf value is calculated as shown in Figure 2C. Specifically, the distance traveled by the substance from the sample spot position is defined as A, and the distance traveled by the solvent is defined as B. Then, A / B is the R f The retention factor value is R. f The value is specific to the substance in TLC under the thin layer chromatography conditions.

[0013] As described above, the Rf value of the active ingredient in the present invention is at least in the range of 0.04 to 0.25. Strictly speaking, as shown in fraction 3 of Figure 3(B), the lower limit of the Rf value of the active ingredient is 0, where the sample has hardly moved from the spot position. The upper limit of the Rf value is 0.27. Therefore, the Rf value of the active ingredient in the present invention is 0 to 0.27. However, taking into consideration even slight variations due to variations in TLC conditions, the lower limit of the Rf value is 0 or more, and in some embodiments, 0.01 or more, in some embodiments, 0.02 or more, in some embodiments, 0.03 or more, and in some embodiments, 0.04 or more. In some embodiments, the upper limit is 0.29 or less, in some embodiments, 0.28 or less, in some embodiments, 0.27 or less, in some embodiments, 0.26 or less, and in some embodiments, 0.25 or less. The lower limit and upper limit can be combined as appropriate. That is, slight variations in the upper and lower limits of the Rf value due to experimental procedures in the thin layer chromatography are included in the range of the Rf value of the active ingredient of the present invention.

[0014] The components exhibiting an Rf value in the range of at least 0.04 to 0.25 include peteroselic acid, but also include an active ingredient other than peteroselic acid (hereinafter, sometimes referred to as active ingredient A). This active ingredient A may be a supplementary ingredient that enhances the antibacterial activity of peteroselic acid against Porphyromonas gingivalis. It may also be a component that exhibits direct antibacterial activity against Porphyromonas gingivalis. However, the present invention is not limited by any of these possibilities. The ratio of peteroselic acid in the components exhibiting an Rf value in the range of 0.04 to 0.25 is preferably 66% or less, in one embodiment 50% by weight or less, in one embodiment 46.8% or less, and in one embodiment 40% or less. The ratio (%) is calculated from the peak area of peteroselic acid by HPLC.

[0015] [2] Manufacturing method of antibacterial ingredients The method for producing the antibacterial component of the present invention includes (1) a step of soaking fennel seeds in n-hexane (hereinafter, sometimes referred to as soaking step (1)). This step is a step of extracting a component having antibacterial activity against Porphyromonas gingivalis from fennel.

[0016] Fennel Fennel (Foeniculum vulgare) is a perennial plant in the Apiaceae family, but is usually cultivated as an annual. Native to Europe, Egypt, India, and China, fennel grows to a height of 1.5 meters, is cold-hardy, and easy to cultivate. When the seeds begin to ripen and turn yellow, the stalks are harvested, dried, and threshed to extract the seeds. The leaves are trimmed to 1-2 cm from the base in June, when they reach a height of over 20 cm. Both the seeds and leaves are widely used in fish dishes, including sauces for fish, raw fish, and salted fish. They are also used as a flavoring in sweets, soups, meat dishes, liqueurs, and vermouth, and as a main ingredient in curry powder. In India, the seeds are chewed after meals to aid digestion and eliminate bad breath. They are also commonly used in herbal teas.

[0017] 《Soaking process (1)》 In the soaking step (1), fennel seeds are soaked in n-hexane. The fennel fruit (seeds) used in the production of the fennel extract of the present invention may be used as is or may be dried. Furthermore, the fennel may be processed into a crushed or powdered form to improve extraction efficiency. The ratio of fennel to n-hexane is not particularly limited as long as the active ingredient can be efficiently extracted, but it is 1 to 100 parts by weight, preferably 5 to 20 parts by weight, of n-hexane per 1 part by weight of fennel.

[0018] The soaking (extraction) temperature is not particularly limited, but is, for example, 4 to 60° C., and preferably 10 to 30° C. The soaking (extraction) time is not particularly limited, but is, for example, 5 minutes to 48 hours, and preferably 30 minutes to 2 hours.

[0019] First fractionation step (2) In the first fractionation step (2), the hexane extract obtained in the step (1) is fractionated using a normal phase column. Examples of normal-phase columns include, but are not limited to, silica gel columns, aminopropyl silica gel (NH silica gel), cyanopropyl silica gel (CN silica gel), and 1,2-dihydroxy-3-propoxypropyl silica gel (diol silica gel). Examples of developing solvents used for fractionation include, but are not limited to, hexane, ethyl acetate, chloroform, methanol, cyclohexane, toluene, benzene, heptane, dichloromethane, acetone, acetonitrile, tetrahydrofuran, petroleum ether, diethyl ether, ethanol, propanol, butanol, water, or a combination of two or more of these. Acetic acid, formic acid, and trifluoroacetic acid can be used as additives.

[0020] Second fractionation step (3) In the second fractionation step (3), the fraction obtained in the step (2) is fractionated using a reverse phase column. The reversed-phase column is not particularly limited, but examples thereof include a silica gel column to which a functional group is attached. Examples of functional groups include octadecyl groups (C18), but other examples include octyl groups (C8), butyl groups (C4), and trimethyl groups (C3). The developing solvent used for fractionation is not particularly limited, but examples include hexane, ethyl acetate, chloroform, methanol, acetonitrile, tetrahydrofuran, or a combination of two or more of these.

[0021] 《Effect》 The mechanism by which the bactericide of the present invention exhibits excellent antibacterial activity against Porphyromonas gingivalis has not been clearly analyzed, but can be assumed as follows: However, the present invention is not limited to this assumption. Fennel contains peteroselic acid, an antibacterial substance against Porphyromonas gingivalis. It is presumed that fennel also contains a component A other than peteroselic acid that exhibits antibacterial activity against Porphyromonas gingivalis. This component A is presumed to be a supplementary component that enhances the antibacterial activity of peteroselic acid against Porphyromonas gingivalis, or a component that exhibits direct antibacterial activity against Porphyromonas gingivalis. It is believed that component A can be extracted from fennel with n-hexane together with peteroselic acid. Therefore, it is believed that the fungicide of the present invention exhibits excellent antibacterial activity against Porphyromonas gingivalis due to the synergistic effect of peteroselic acid and component A.

[0022] [3] Food composition The food composition of the present invention contains the disinfectant of the present invention in a conventional food. Specific examples of the disinfectant-containing food include freshly prepared foods such as salads; cooked foods such as steaks, pizzas, and hamburgers; stir-fried foods such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and processed vegetable products; confectioneries such as cookies, bread, biscuits, hardtack, cakes, rice crackers, yokan, puddings, jellies, ice creams, chewing gum, crackers, chips, chocolates, and candies; noodles such as udon, pasta, and soba; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; condiments such as miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, and roux; soybean foods such as tofu; processed agricultural and marine products such as furikake, tsukudani, and cereals; and konjac.

[0023] Examples of beverages include coffee beverages; cocoa beverages; vegetable juices obtained from the above-mentioned vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, green tea, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, sparkling wine, etc.), sake, plum wine, happoshu, whiskey, brandy, shochu, rum, gin, and liqueurs; dairy beverages; soy milk beverages; liquid diets; and sports drinks.

[0024] The food or beverage includes feed and beverage for animals, such as primates including humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, and mice.

[0025] These foods or beverages may contain, as desired, food additives and food ingredients, such as antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meat, seafood, etc. The amounts of these food ingredients and food additives to be added can be determined appropriately within the range that does not impair the object of the present invention.

[0026] These foods or beverages can be subjected to common sterilization processes, such as heat-pressure sterilization using retort and autoclave, batch sterilization, plate sterilization, electric heating sterilization, microwave heating sterilization, and steam sterilization such as injection and infusion.

[0027] Foods and beverages include functional foods (drinks) and health foods (drinks). As used herein, "health foods (drinks)" refers to foods or beverages that have or are expected to have some effect on health, and "functional foods (drinks)" refers to foods or beverages among the aforementioned "health foods (drinks)" that are designed and processed to fully exhibit bioregulatory functions (i.e., the function of preventing the onset of allergic symptoms or alleviating or treating allergic symptoms). In this specification, supplements (drinks) are included in functional foods (drinks). Functional foods and health foods can be in granular, solid, liquid, capsule, gel, or tablet form.

[0028] The food composition of the present invention exhibits excellent antibacterial activity against Porphyromonas gingivalis, a bacterium that causes periodontal disease. Therefore, the food composition of the present invention can be used as a food composition for preventing periodontal disease. By ingesting the food composition of the present invention, Porphyromonas gingivalis in the oral cavity can be reduced, and periodontal disease can be prevented.

[0029] [4] Pharmaceutical composition The pharmaceutical composition of the present invention contains the disinfectant of the present invention as an active ingredient. The pharmaceutical composition of the present invention may contain the active ingredient in an amount of, but not limited to, 0.01 to 99% by weight, preferably 0.1 to 80% by weight. The dosage of the pharmaceutical composition of the present invention can be determined appropriately depending on, for example, age, sex, weight, severity of symptoms, or administration method, and can be administered orally. The administration method, dosage, administration period, and administration interval of the pharmaceutical composition to humans are preferably determined through controlled clinical trials.

[0030] Solid compositions for oral administration include tablets, powders, granules, etc. In such solid compositions, one or more active ingredients are mixed with at least one inert excipient, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, and / or magnesium aluminum metasilicate. The compositions may contain inert additives, such as lubricants such as magnesium stearate, disintegrants such as sodium carboxymethyl starch, stabilizers, and solubilizers, according to conventional methods. Tablets or pills may be coated with a sugar coating or a film of a gastric or enteric substance, if necessary. Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., and contain commonly used inert diluents, such as purified water or ethanol. In addition to the inert diluents, the liquid compositions may contain adjuvants such as solubilizing agents, wetting agents, and suspending agents, as well as sweeteners, flavors, aromatics, and preservatives.

[0031] The pharmaceutical composition of the present invention exhibits excellent antibacterial activity against Porphyromonas gingivalis, a bacterium that causes periodontal disease. Therefore, the pharmaceutical composition of the present invention can be used as a pharmaceutical composition for treating or preventing periodontal disease. Administration of the pharmaceutical composition of the present invention reduces Porphyromonas gingivalis in the oral cavity, thereby enabling the treatment or prevention of periodontal disease. [Example]

[0032] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0033] Example 1 In this example, an extract was obtained from fennel seeds using n-hexane. Fennel seed powder (including fruit and seed powder) was weighed into a centrifuge tube, n-hexane was added, and the tube was shaken. The centrifuge tube was centrifuged, and the supernatant was collected and concentrated under reduced pressure using an evaporator to obtain an extract.

[0034] The antibacterial activity of the obtained hexane extract against Porphyromonas gingivalis was measured by measuring the minimum bactericidal concentration. The bacterium used was Porphyromonas gingivalis ATCC 33277. The liquid medium was Bacto TM The liquid medium used was Brain Heart Infusion (DIFCO LABORATORIES) supplemented with 5 μg / mL hemin and 1 μg / mL menadione. Blood agar was prepared by adding defibrinated sheep blood and agar to the liquid medium, filling plates, and solidifying them. 2 μL of each diluted sample was dispensed into each well of a 96-well plate (1). 8 200 μL of bacterial solution adjusted to CFU / mL was added to each well and mixed. Culture was performed under anaerobic conditions, and the turbidity (OD) (595) was measured periodically for 48 hours to confirm bacterial growth. 160 μL of liquid medium without bacteria was added to another 96-well plate (2). After 48 hours, 40 μL of the bacterial solution from each well of the cultured 96-well plate (1) was removed and added to each well of the 96-well plate (2), and cultured under anaerobic conditions. After 72 hours, the turbidity (OD) (595) was measured, and the sample concentration at which no bacterial growth occurred was determined to be the MBC (Minimal Bactericidal Concentration). The resulting hexane extract demonstrated antibacterial activity against Porphyromonas gingivalis.

[0035] Example 2 In this example, the hexane extract obtained in Example 1 was fractionated using a silica gel column. The extract was loaded onto a silica gel column (Φ20 × 240 mm) and separated into four fractions. The developing solvents used were hexane, hexane:EtOAc = 20:1, 10:1, 1:1, CHCl3:MeOH = 100:1, and CHCl3:MeOH:H2O = 9:1:0.1, in that order. The antibacterial activity of the obtained fractions 1 to 4 against Porphyromonas gingivalis was measured in the same manner as in Example 1. Fraction 3 showed a strong antibacterial activity.

[0036] Thin-layer chromatography The hexane extract of Example 1 and fractions 1 to 4 obtained in Example 2 were analyzed by thin layer chromatography. The mobile phase solvent was poured into a glass developing tank to a height of several centimeters, and the tank was left to stand for 1-2 hours to allow the solvent vapor to equilibrate. TLC Aluminum Sheet Silica Gel 60F 254 The hexane extract and fractions 1 to 4 dissolved in DMSO were spotted onto a plate (Merck), which was then placed in a glass development tank and capped. The plate was left to stand until the solvent reached approximately 1 cm from the top of the plate. The plate was then removed, dried, and stained using the sulfuric acid method. As shown in FIG. 3, in the TLC of the hexane extract of Example 1 and Fraction 3 of Example 2, in which antibacterial activity was observed, common components were observed with Rf values in the range of 0 to 0.27.

[0037] Example 3 In this example, Fraction 3 obtained in Example 2 was fractionated into five fractions by adsorption onto an HPLC reverse-phase column μ-Bonda pak C18 (Φ25 × 100 × 2). The developing solvents used were 70%, 80%, 85%, 90%, and 100% MeOH. The antibacterial activity of the obtained fractions 3-1 to 3-5 against Porphyromonas gingivalis was measured in the same manner as in Example 1. Fraction 3-3 showed a strong antibacterial activity.

[0038] NMR analysis of fraction 3-3 confirmed that it contained petroselinic acid. Comparison of the HPLC peak area with the calibration curve for petroselinic acid revealed that 66% of the components in fraction 3-3 were petroselinic acid. Furthermore, 46.8% of the components in fraction 3 were petroselinic acid.

[0039] The hexane extract obtained in Example 1, Fraction 3 obtained in Example 2, and Fraction 3-3 obtained in Example 3 were diluted to contain the same amount of peteroselic acid. For example, as shown in Table 1, the concentrations of the hexane extract containing 2.7 μg / mL of peteroselic acid, Fraction 3, and Fraction 3-3 were 8 μg / mL, 6 μg / mL, and 4.2 μg / mL, respectively. The hexane extract, Fraction 3, and Fraction 3-3 were prepared to contain 2.7 μg / mL, 3.4 μg / mL, 4.1 μg / mL, 4.8 μg / mL, and 5.5 μg / mL of peteroselic acid. The minimum bactericidal concentration (MBC) of each diluted sample was measured. As shown in Table 1, the minimum bactericidal concentration of peteroselic acid was 5.5 μg / mL, while the minimum bactericidal concentration of fraction 3-3 was 5.2 μg / mL, which corresponds to 3.4 μg / mL of peteroselic acid, and the minimum bactericidal concentrations of the hexane extract and fraction 3 were 12 μg / mL and 9 μg / mL, which corresponds to 4.1 μg / mL of peteroselic acid.

[0040] [Table 1]

[0041] These results indicate that the hexane extract, fraction 3, and fraction 3-3 contain components other than peteroselic acid that exhibit antibacterial activity against Porphyromonas gingivalis, and that they exhibit excellent antibacterial activity against Porphyromonas gingivalis due to a synergistic effect with peteroselic acid. [Industrial Applicability]

[0042] The disinfectant of the present invention can be used for the prevention or treatment of periodontal disease.

Claims

1. A fungicide for Porphyromonas gingivalis, comprising as an active ingredient a component contained in an extract of fennel fruit and / or seeds, which component exhibits an Rf value in the range of at least 0.04 to 0.25 in thin layer chromatography using a solvent mixture of hexane and ethyl acetate in a ratio of 20:1 as the mobile phase.

2. 2. The fungicide for Porphyromonas gingivalis according to claim 1, wherein the ratio of peteroselic acid in the components exhibiting an Rf value in the range of 0.04 to 0.25 is 66% or less.

3. (1) A method for producing an ingredient having antibacterial activity against Porphyromonas gingivalis, comprising the step of immersing fennel fruits and / or seeds in n-hexane.

4. The method for producing a component having antibacterial activity against Porphyromonas gingivalis according to claim 3, further comprising: (2) a step of fractionating the hexane extract obtained in step (1) using a normal phase column.

5. The method for producing a component having antibacterial activity against Porphyromonas gingivalis according to claim 4, further comprising: (3) a step of fractionating the fraction obtained in step (2) using a reverse-phase column.

6. An antibacterial food composition against Porphyromonas gingivalis, comprising the bactericide according to claim 1 or 2.

7. A food composition for preventing periodontal disease, comprising the bactericide according to claim 1 or 2.

8. 3. A pharmaceutical composition for use in treating Porphyromonas gingivalis, comprising the bactericide according to claim 1.

9. A pharmaceutical composition for preventing or treating periodontal disease, comprising the bactericide according to claim 1 or 2.

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