JP7204846B1 - Hinokitiol-containing composition - Google Patents

Abstract

An object of the present invention is to provide a method for suppressing periodontal disease bacteria that have invaded oral cells. A composition containing hinokitiol as an active ingredient for enhancing digestive activity in oral cells. [Selection figure] None

Description

TECHNICAL FIELD The present disclosure relates to compositions containing hinokitiol, uses thereof, and the like. It should be noted that the contents of all documents mentioned herein are hereby incorporated by reference.

Periodontal disease is an inflammatory disease caused by infection with bacteria (periodontal disease bacteria). Therefore, one of the important factors for the prevention or treatment of periodontal disease is to suppress the activity of periodontal disease bacteria. are mentioned. For this reason, many oral compositions have been researched and developed for the purpose of sterilizing or suppressing periodontal disease bacteria (for example, Patent Document 1).

JP 2012-111732 A JP 2020-019724 A JP 2020-019725 A

J Periodontal Res. 2021 Jul 13. doi: 10.1111/jre.12915.

P. gingivalis, a periodontal pathogen, is known to invade cells, especially gingival epithelial cells. Invading P. gingivalis exerts potent cytotoxicity. Some of the invading P. gingivalis is degraded in lysosomes, and autophagy is induced in the cell to avoid this degradation pathway. As a result, P. gingivalis is incorporated into autophagosomes, where it escapes intracellular degradation reactions, proliferates, and exerts cytotoxicity (Non-Patent Document 1). Furthermore, nearly half of the bacteria that have invaded the cell exit the cell via the recycling pathway from early endosomes and re-invade the surrounding cells. For this reason, P. gingivalis can move between cells, survive, proliferate, and continue to infect (for example, see the following web page: http://web.dent.osaka-u.ac.jp/~ prevent/research01.html).

The invasion of periodontal bacteria into the gingival epithelial cells leads to escape from the disinfectant, in addition to the above-mentioned harmful effects. That is, even if an oral composition containing an active ingredient that exhibits a bactericidal or inhibitory effect is applied in the oral cavity, it is difficult for the active ingredient to reach the periodontal bacteria that have invaded the cells. It becomes difficult to sterilize or suppress pericobacteria. Therefore, means for suppressing the invasion of periodontal bacteria into cells have been investigated (Patent Documents 2 and 3).

This time, the present inventors have investigated a method for suppressing (preferably sterilizing) periodontal bacteria that have invaded gingival epithelial cells.

The present inventors found that hinokitiol has the effect of enhancing digestive activity in oral cells, and that this effect can suppress periodontal bacteria that have invaded oral cells (especially gingival epithelial cells). Made improvements.

The disclosure includes, for example, subject matter described in the following sections.
Section 1.
A composition for enhancing digestive activity in oral cells, containing hinokitiol as an active ingredient.
Section 2.
A composition for activating the intralysosomal degradation reaction of oral cells, containing hinokitiol as an active ingredient.
Item 3.
A composition containing hinokitiol as an active ingredient for enhancing the resistance of oral cells or tissue composed thereof to periodontal bacteria.
Section 4.
A composition containing hinokitiol as an active ingredient for enhancing the defense ability of oral cells or tissue composed thereof against periodontal disease bacteria.
Item 5.
A composition for eliminating periodontal bacteria in oral cells or tissue composed thereof, containing hinokitiol as an active ingredient.
Item 6.
Item 6. The composition according to Items 1 to 5, wherein the concentration of hinokitiol is 50 to 2000 ppm.
Item 7.
Item 7. The composition according to any one of Items 1 to 6, which is an oral composition.
Item 8.
Item 8. The composition according to any one of Items 1 to 7, for a subject having Porphyromonas gingivalis present in oral cells.
Item 9.
Item 9. The composition according to any one of Items 1 to 8, wherein the oral cells are gingival epithelial cells.

It is possible to suppress and eliminate periodontal bacteria that have invaded oral cells and purify the cells. In addition, it is possible to provide oral cells with resistance and defense against invaded periodontal disease.

P. invaded gingival epithelial cells. The results of examining whether various materials can suppress g bacteria are shown. P. invaded gingival epithelial cells. The results of examining whether each concentration of hinokitiol can suppress bacteria g are shown. P. The results of examining whether each concentration of hinokitiol can sterilize bacteria g are shown. The results of examination of the toxicity of each concentration of hinokitiol to gingival epithelial cells are shown in terms of cell viability. P. Gingival epithelial cells invaded by bacteria were examined for lysosomal enzyme activity using a fluorescence kit (fluorescence detection image) without hinokitiol (left) and with hinokitiol (right). show. Based on the image in FIG. 5a, the amount of intracellular degrading enzymes per cell (area of lysosomal active fraction/number of cells) is shown as a relative value with the case where hinokitiol was not added as 100%.

Each embodiment included in the present disclosure will be described in further detail below. The present disclosure preferably includes, but is not limited to, hinokitiol-containing compositions and uses thereof, and includes everything disclosed herein and recognized by a person skilled in the art.

A hinokitiol-containing composition included in the present disclosure is a composition for enhancing digestive activity in oral cells. Such compositions encompassed by the present disclosure are sometimes referred to as "compositions of the present disclosure."

Hinokitiol has the following formula:

It is a compound represented by Hinokitiol used in the composition of the present disclosure may be a synthetic product or may be extracted from a natural product (for example, Hiba).

The content of hinokitiol in the composition of the present disclosure is not particularly limited as long as it is within the range where the effect is exhibited. For example, the composition of the present disclosure preferably contains about 50 to 2000 ppm of hinokitiol. The upper or lower limit of the range is, for example, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, or 1950 ppm. For example, the range may be 60-1500 ppm. Although not particularly limited, the hinokitiol content is particularly preferably 300 ppm from the viewpoint that the effect of enhancing the digestive activity in oral cells is exhibited more strongly. Although not particularly limited, the hinokitiol content is particularly preferably less than 1000 ppm, and more preferably 800, 750, or 700 ppm or less, from the viewpoint of avoiding severe damage to oral cells by hinokitiol.

Hinokitiol exerts an effect of enhancing digestive activity in oral cells by being applied to oral cells. As a result, periodontal bacteria that have invaded into oral cells can be efficiently digested and suppressed. In particular, when hinokitiol is applied to oral cells, it activates the degradation reaction in lysosomes, thereby enhancing the digestive activity in oral cells and efficiently digesting and suppressing periodontal bacteria that have invaded oral cells. can do.

Therefore, the composition of the present disclosure containing hinokitiol is preferably used as an oral composition. In addition, it is particularly preferably used for the prevention or treatment of periodontal disease. That is, one particularly preferred form of the composition encompassed by the present invention is an anti-periodontal oral composition.

In addition, the composition of the present disclosure can also be preferably used as a composition for activating an intralysosomal degradation reaction. The composition for activating the intralysosomal degradation reaction can be preferably used particularly for oral cells invaded by periodontal bacteria (that is, periodontal bacteria exist inside).

In addition, the composition of the present disclosure is used to strengthen the resistance to periodontal bacteria in oral cells and tissues composed of oral cells (especially periodontal tissue), or to improve the protective power against periodontal bacteria. For strengthening, it can be preferably used for.

The oral cells are not particularly limited, but gingival epithelial cells are particularly preferred. This is because gingival epithelial cells are susceptible to invasion and damage by periodontal disease bacteria.

Periodontal disease bacteria for which the composition of the present disclosure is particularly effective are not particularly limited as long as the effects of the present invention are exhibited, but a preferred example is Porphyromonas gingivalis (P. gingivalis).

In addition, the subject to which the composition of the present disclosure is applied is preferably a subject in which periodontal bacteria have invaded oral cavity cells (that is, subjects in which periodontal disease bacteria are present in oral cells). In addition, it can be preferably used for mammals including humans (e.g., dogs, cats, mice, rats, sheep, horses, cows, monkeys, etc.), with humans being particularly preferred.

Compositions of the present disclosure can be, for example, solid compositions, liquid compositions, and the like. In addition, the composition of the present disclosure (particularly oral compositions) can be prepared according to conventional methods, such as ointments, pastes, pastes, gels, liquids, sprays, mouthwashes, and liquid dentifrices. It can be in the form (dosage form) of an agent, toothpaste, gum, or the like. Among them, mouthwashes, liquid dentifrices, toothpastes, ointments, pastes, liquids and gels are preferred.

The composition of the present disclosure may further contain, for example, one or two or more optional components that can be blended in an oral composition, as long as the effects are not impaired.

For example, nonionic surfactants, anionic surfactants or amphoteric surfactants can be blended as surfactants. Specifically, for example, nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid esters, maltose fatty acid esters, and lactose fatty acid esters; fatty acid alkanolamides; glycerin fatty acid esters; sorbitan fatty acid esters; fatty acid monoglycerides; Polyoxyethylene alkyl ether having an addition coefficient of 8 to 10 and an alkyl group having 13 to 15 carbon atoms; Polyoxyethylene alkylphenyl ether having a polyoxyethylene addition coefficient of 10 to 18 and an alkyl group having 9 carbon atoms; diethyl sebacate; polyoxyethylene hydrogenated castor oil; fatty acid polyoxyethylene sorbitan; Examples of anionic surfactants include sulfuric acid ester salts such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; sodium cocoyl sarcosinate and lauroyl methylalanine. acyl amino acid salts such as sodium; cocoyl methyl taurate sodium; Zwitterionic surfactants include betaine acetate type surfactants such as betaine lauryldimethylaminoacetate and betaine coconut oil fatty acid amidopropyldimethylaminoacetate; imidazoline type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine sodium. Active agent: amino acid-type active agent such as N-lauryldiaminoethylglycine, and the like. These surfactants can be blended singly or in combination of two or more. The blending amount thereof is usually 0.1 to 5% by mass based on the total amount of the composition.

Flavoring agents such as menthol, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronol, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineol, Linalool, ethyl linalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, Perfumes such as d-borneol, fennel oil, cinnamon oil, cinnamaldehyde, peppermint oil and vanillin can be used. These may be blended singly or in combination of two or more in an amount of, for example, 0.001 to 1.5% by mass based on the total amount of the composition.

Sweeteners that can be used include, for example, saccharin sodium, acesulfame potassium, stevioside, neohesperidyl dihydrochalcone, perillartine, thaumatin, aspartylphenylalanylmethyl ester, p-methoxycinnamic aldehyde, and the like. These can be blended, for example, in an amount of 0.01 to 1% by mass relative to the total amount of the composition.

Furthermore, as wetting agents, sorbit, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylit, maltit, lactit, polyoxyethylene glycol, etc. may be blended alone or in combination of two or more. can.

Binders include sodium carboxymethyl cellulose, carboxymethyl ethyl cellulose salt, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, crystalline cellulose, cellulose derivatives such as crystalline cellulose and carmellose sodium, and microbial productivity such as xanthan gum. Natural polymers or natural gums such as molecules, tragacanth gum, karaya gum, gum arabic, carrageenan, dextrin, agar, pectin, pullulan, gellan gum, locust bean gum, sodium alginate, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyvinyl methyl ether, Synthetic polymers such as sodium polyacrylate, thickening silica, inorganic binders such as Veegum, and cationic binders such as O-[2-hydroxy-3-(trimethylammonio)propyl]hydroxyethylcellulose chloride. be done. These caking agents can be used individually or in combination of 2 or more types.

As preservatives, parabens such as methylparaben, ethylparaben, propylparaben and butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethylglycine hydrochloride, and the like can be added.

As a coloring agent, legal dyes such as Blue No. 1, Yellow No. 4, Red No. 202 and Green No. 3, mineral dyes such as ultramarine blue, enhanced ultramarine blue and Prussian blue, titanium oxide, and the like may be blended.

As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or chemically possible salts thereof, sodium hydroxide, or the like may be added. These may be blended singly or in combination of two or more so that the pH of the composition is in the range of 4-8, preferably 5-7. The blending amount of the pH adjuster may be, for example, 0.01 to 2% by weight.

The oral cavity composition of the present disclosure further includes vitamin E compounds such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate as active ingredients, amphoteric fungicides such as dodecyldiaminoethylglycine, triclosan, Nonionic fungicides such as isopropylmethylphenol, anionic fungicides such as sodium lauroyl sarcosinate, cationic fungicides such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, dextranase, amylase, protease, Enzymes such as mutanase, lysozyme, lytic enzyme (retech enzyme), alkali metal monofluorophosphates such as sodium monofluorophosphate and potassium monofluorophosphate, fluorides such as sodium fluoride and stannous fluoride, tranexamic acid or epsilon aminocaproic acid, aluminum chlorohydroxyallantoin, dihydrocholesterol, glycyrrhetinic acid, glycyrrhizic acid, sodium copper chlorophyllin, glycerophosphate, chlorophyll, sodium chloride, calopeptide, allantoin, carbazochrome, potassium nitrate, palatinit, etc., either alone or in combination of two or more. They can be formulated in combination.

As a base, alcohols, silicon, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastibase, etc. can be added.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Also, the present disclosure encompasses any and all combinations of the constituent elements described herein.

Also, the various characteristics (property, structure, function, etc.) described for each of the embodiments of the disclosure described above may be combined in any way to identify subject matter encompassed by the disclosure. That is, the present disclosure encompasses all subject matter consisting of any and all possible combinations of the features described herein.

Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples. Unless otherwise specified, % other than % indicating CO ₂ concentration indicates % by mass.

<P. Culture of gingivalis>
10 ⁶ cells/mL of Porphyromonas gingivalis strain OMZ314 (hereinafter Pg) were inoculated into a test tube containing 10 mL modified GAM medium. After that, the cells were cultured under 37° C. anaerobic conditions for one day (preculture).

1 mL of the bacterial solution obtained in the pre-culture was transferred to a test tube containing 10 mL of new modified GAM medium, and cultured under 37° C. anaerobic conditions for 1 day (main culture).

P. spp. 10% FBS-containing MEM medium. D600 was adjusted to 0.1 and P.D. It was used for the test as a g bacterial solution.

<Culture of gingival epithelial cells>
Gingival epithelial cells (Ca9-22 strain) were suspended in 10% FBS-containing MEM medium and adjusted to 2.0×10 ⁵ cells/mL, then 1 mL was dispensed into each well of a 12-well plate and placed at 37° C. It was cultured for 1 day under 5% _CO2 conditions and used for the test.

<Preparation of test material solution>
Hinokitiol, sodium copper chlorophyllin (copper chromate), tocopherol acetate (VEA), tocopherol nicotinate (VEN), and pyridoxine hydrochloride (VB6) were used as test materials. For each material, a solution of 1% ethanol dissolved in 10% FBS-containing MEM medium was used as a solvent to prepare a solution having a predetermined concentration. Hereinafter, the solutions of each material indicate those dissolved in the solvent.

Examination of P. gingivalis intracellular degradation (Experiment 1)
After culturing the gingival epithelial cells, the medium was changed to P. The strain was replaced with P.g bacterial solution and co-cultivated at 37° C., 5% CO ₂ for 2 hours. g induced invasion of the bacteria into gingival epithelial cells. After 2 hours of culture, non-invaded P. spp. After washing the bacteria, 1 mL of each material solution adjusted to a concentration of 100 ppm was added to the gingival epithelial cells and cultured for 2 hours under conditions of 37° C. and 5% CO ₂ . After 2 hours of culture, the cells were washed with PBS, replaced with fresh 10% FBS-containing MEM medium, and cultured for 20 hours under conditions of 37° C. and 5% CO ₂ . After 20 hours of culture, the cells were washed with PBS, treated with 200 μL of 0.25% Trypsin-EDTA solution at 37° C. for 5 minutes to detach the cells, and then the reaction was stopped with 1.8 mL of MEM medium containing 10% FBS. , the solution was recovered. The recovered solution was inoculated on a blood agar medium, cultured at 37° C. under anaerobic conditions for 5 days, and colonies formed on the agar medium were counted to detect intracellular P. The decomposition activity of g bacteria was evaluated.

The results were obtained by comparing the number of colonies in the control (similar examination using only the solvent without using the material) to the intracellular P.C. Fig. 1 shows the relative value when the number of g bacteria is 100%. Hinokitiol inhibited P. cerevisiae that invaded gingival epithelial cells. It was found that the digestion of bacterium g was efficiently promoted.

P. Examination of intracellular decomposition of gingivalis (Experiment 2)
Solutions containing different concentrations of hinokitiol were prepared, and in the same manner as described above (Experiment 1), depending on the amount of hinokitiol (solution concentration), P. It was examined whether the digestive enhancement effect of g bacteria was changed. The results are shown in FIG. (Similar to Fig. 1, the number of colonies in the control (study conducted in the same manner using only the solvent without using hinokitiol) is shown as a relative value when the number of intracellular P.g bacteria is 100%.) Hinokitiol When the concentration of P. is above about 40 ppm, P. It was found that the effect of promoting digestion of g bacteria was improved. In addition, when the concentration of hinokitiol exceeds about 300 ppm, P. spp. It was found that the effect of enhancing digestion of g bacteria was further improved.

P. gingivalis sterilization study (experiment 3)
Solutions containing different concentrations of hinokitiol were prepared, whereas 1/10 amount of P. g bacterial solution was added and cultured at 37° C. under anaerobic conditions for 2 hours. Then, 10 volumes of fungicide-inactivated medium (modified GAM medium supplemented with 0.07% lecithin and 0.5% Tween 80) was added, which was inoculated onto blood agar medium and anaerobicly treated at 37°C. conditions for 5 days. After that, by counting the colonies on the agar medium, P. hinokitiol was detected. g Bactericidal effect was evaluated. The results were compared with the number of colonies in the control (similar examination using only the solvent without using hinokitiol). Fig. 3 shows the relative value when the number of g bacteria is 100%. Hinokitiol itself includes P. It was found that there was almost no effect of sterilizing g bacteria.

Moreover, from the above results, hinokitiol is P. g that invaded gingival epithelial cells rather than directly suppressing the bacteria. By enhancing the digestion of P. g bacteria, P. g It was thought that the effect of suppressing g bacteria could be exhibited.

Cytotoxicity evaluation test (Experiment 4)
After carrying out the steps up to culturing the gingival epithelial cells for 20 hours in the same manner as in the above (Experiment 1), after washing with PBS, WST-1 solution was added, and the cells were placed under conditions of 37°C and 5% _CO2 . for 30 minutes. The absorbance of the reaction solution at a wavelength of 450 nm was measured using an absorption plate reader. At this time, the absorbance at a wavelength of 600 nm was taken as a blank value. The viability of gingival epithelial cells was calculated from the measured absorbance. The results are shown in FIG. 4 as relative values when the absorbance in the control (similar examination using only a solvent without using hinokitiol) is defined as 100% survival rate. It was suggested that when the hinokitiol concentration is 1000 ppm or more, damage to gingival epithelial cells may progress.

Examination of activation of intracellular degradation (Experiment 5)
Using Lysolive ^™ EsterGreen ^™ (Marker Gene Technologies, Inc), a kit capable of monitoring the activity of lysosomal enzymes, the digestive activity in gingival epithelial cells (intralysosomal degrading enzyme activity) was examined.

In the same manner as in Experiment 1, the following operations were carried out up to the step of treating the material solution for 2 hours, and after washing with PBS, Lysolive ^TM EsterGreen ^TM adjusted with 0.1% DMSO-containing MEM medium according to the instructions attached to the kit was added. It was added to gingival epithelial cells and cultured for 1 hour at 37° C. and 5% CO ₂ while shielding from light. After culturing, the cells were washed with PBS and replaced with fresh 10% FBS-containing MEM medium, and fluorescence detected by lysosomal activity (excitation light: 490 nm, emission light: 525 nm) was observed under a fluorescence microscope (Fig. 5a). In addition, the number of cells in the field of view and the area of the fluorescently-stained lysosomal active fraction present in the cells were counted, and the amount of intracellular degrading enzymes (area of lysosomal active fraction/number of cells) was calculated. The results are shown in Figure 5b. HT100 ppm in FIGS. 5a and 5b indicates that a hinokitiol concentration of 100 ppm solution was used. From the results, it was found that hinokitiol improves lysosomal enzyme activity. Therefore, this result also suggests that hinokitiol inhibits P. cerevisiae that has invaded gingival epithelial cells. By enhancing the digestion of P. g bacteria, P. g It was thought that the effect of suppressing g bacteria could be exhibited.

Claims (7)

An oral composition containing 500 to 2000 ppm of hinokitiol as an active ingredient for eliminating periodontal bacteria in oral cells or tissue composed thereof. The composition according to claim 1 , wherein the concentration of hinokitiol is 550-2000 ppm. 3. The composition of claim 1 or 2 for subjects having Porphyromonas gingivalis in oral cells. The composition according to any one of claims 1 to 3 , wherein the oral cells are gingival epithelial cells. The composition according to any one of claims 1 to 4, wherein the periodontal bacteria is Porphyromonas gingivalis. The composition according to any one of claims 1 to 5, which is applied to the oral cavity such that digestive activity in oral cells is enhanced. The composition according to any one of claims 1 to 5, which is applied to the oral cavity such that the intralysosomal degradation reaction of oral cells is activated.

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