JP7718855B2 - Hyaluronidase activity inhibitor and method for enhancing the inhibitory effect of hyaluronidase activity - Google Patents

Description

The present invention relates to a hyaluronidase activity inhibitor and a method for enhancing the inhibitory effect of hyaluronidase activity.

Oral compositions contain a wide variety of ingredients to impart functions according to various applications. For example, with the aim of preventing periodontal diseases such as gingivitis and periodontitis, attempts have been made to incorporate active ingredients into oral compositions, such as bactericides that kill the causative bacteria (causative bacteria) and anti-inflammatory agents that suppress inflammation that occurs in periodontal tissues.

For example, Patent Documents 1 and 2 propose oral compositions containing benzalkonium chloride or chlorhexidine salts as disinfectants. Furthermore, Patent Document 3 proposes a topical composition containing one or more anti-inflammatory agents selected from the group consisting of glycyrrhetinic acid and its derivatives.

Japanese Patent Application Laid-Open No. 2017-7992 Japanese Patent Application Publication No. 3-200716 JP 2012-144490 A

On the other hand, it is known that inflammation in periodontal tissues increases the activity of proteolytic enzymes such as hyaluronidase, leading to further damage to periodontal tissues. However, it cannot be said that sufficient research has been done into ingredients that inhibit the activity of these enzymes. Furthermore, particularly when intended for application to periodontal tissues, there is a risk that the active ingredients will be diluted by saliva and not be sufficiently effective.

The present invention therefore aims to provide an active ingredient for inhibiting hyaluronidase activity, and an objective of the present invention is to provide a hyaluronidase activity inhibitor that can sufficiently inhibit hyaluronidase activity even when the concentration of the active ingredient is reduced by saliva, etc., and a method for enhancing the inhibitory effect of hyaluronidase activity.

In light of the above-mentioned problems, the inventors investigated various components that inhibit hyaluronidase activity, and found that the combined use of hinokitiol and a specific plant extract, which are both ineffective when used alone, synergistically increases the inhibitory effect of hyaluronidase activity. Furthermore, they found that a similar combination can achieve an inhibitory effect on hyaluronidase activity even at low concentrations where either component is completely ineffective when used alone.

That is, the present invention is characterized by the following (1) to (5).
(1) A hyaluronidase activity inhibitor characterized by comprising (A) hinokitiol and (B) at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and witch hazel.
(2) The hyaluronidase activity inhibitor according to (1), characterized in that the mass ratio (A)/(B) of the content of the component (A) to the content of the component (B) converted to solids is 0.1 to 2000.
(3) A method for enhancing the inhibitory effect of hyaluronidase activity, characterized by adding (A) hinokitiol and (B) at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and witch hazel.
(4) An oral composition characterized by containing the hyaluronidase activity inhibitor described in (1) or (2).
(5) An oral composition as described in (4), characterized in that the content of the (A) component is 0.00002 to 2 mass % and the content of the (B) component, converted to solid content, is 0.0000015 to 0.15 mass %.

According to the present invention, a highly effective hyaluronidase activity inhibitor can be obtained by combining hinokitiol with at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and hamamelis. Therefore, since the inhibitory effect of hyaluronidase activity can be exerted even when diluted to a low concentration, it can be effectively used in oral compositions.

Embodiments of the present invention are described in more detail below.

<Hyaluronidase activity inhibitor>
The hyaluronidase activity inhibitor of the present invention comprises (A) hinokitiol (hereinafter sometimes referred to as component (A)) and (B) at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and hamamelis virginiana (hereinafter sometimes referred to as component (B)).

[Component (A)]
Hinokitiol is an aromatic compound that is naturally extracted from the essential oils of Aomori Hiba and Taiwanese Cypress. Hinokitiol may be a natural extract or a chemically synthesized product.

[(B) component]
The hyaluronidase activity inhibitor of the present invention uses component (A) and at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and hamamelis.

Equisetum arvense extract (hereinafter also referred to as equisetum arvense extract), hawthorn extract (hereinafter also referred to as hawthorn extract), and hamamelis virginiana extract (hereinafter also referred to as hamamelis virginiana extract) can be commercially available products, or products extracted from the above three plants by the methods described below can be used. The method for extracting the plant extracts is not particularly limited, and any conventionally known method can be used. For example, extracts can be obtained by squeezing, solvent extraction, water distillation, steam distillation, or the like from any part of the above plants, either as is or after cutting or crushing. Solvent extraction methods known in the art can be used, and conventionally known extraction methods such as water (including warm water and hot water) extraction, alcohol extraction, supercritical extraction, microwave extraction, and compression extraction can be used.

When solvent extraction is performed, examples of the solvent include water; alcohols (whether anhydrous or hydrous) such as methanol, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, and 1,3-butylene glycol; ketones such as acetone; ethers such as diethyl ether and dioxane; nitriles such as acetonitrile; esters such as ethyl acetate; hexane, xylene, benzene, and chloroform.
The solvent for the solvent extraction is preferably water, alcohols, ketones, hexane, etc., more preferably water, alcohols, and ketones. These solvents may be used alone or in combination of two or more.

The resulting extract may be used as is, or may be dried before use. If necessary, the resulting extract may be purified and concentrated. Examples of purification include filtration or adsorption, decolorization, and separation using ion exchange resins or activated carbon columns. Examples of concentration include conventional methods such as evaporators. These extracts may then be further dried, such as by freeze-drying, or powdered using conventional methods. The extract thus obtained may be dissolved in water, alcohol, or the like, as needed.

Commercially available products include horsetail extract (Horsetail Extract), such as "Horsetail Extract" (trade name) and "Horsetail Extract BG" (trade name) manufactured by Maruzen Pharmaceutical Co., Ltd.; hawthorn extract (Hawthorn Extract), such as "Falcorex Hawthorn B" (trade name) and "Falcorex Hawthorn E" (trade name) manufactured by Ichimaru Pharcos Co., Ltd.; and witch hazel extract (Witch hazel extract), such as "Falcorex Hamamelis B" (trade name) manufactured by Ichimaru Pharcos Co., Ltd. and "Witch hazel Extract BG-J" (trade name) manufactured by Maruzen Pharmaceutical Co., Ltd.

In the hyaluronidase activity inhibitor of the present invention, the ratio (content ratio) of component (A) to component (B) is preferably such that the mass ratio ((A)/(B)) of the content of component (A) to the content of component (B) converted to solids is 0.1 to 2000. When the ratio of component (A) to component (B) is within this range, the inhibitory effects of component (A) and component (B) on hyaluronidase activity are synergistically improved, and the composition containing the hyaluronidase activity inhibitor of the present invention provides an excellent feel when used as a formulation and also improves formulation stability. The mass ratio (A)/(B) is more preferably 0.2 or more, even more preferably 2 or more, particularly preferably 10 or more, more preferably 1000 or less, even more preferably 200 or less, particularly preferably 40 or less, and most preferably 20 or less.

The details of the mechanism by which the combined use of components (A) and (B) enhances the inhibitory effect on hyaluronidase activity are not clear, but it is presumed that components (A) and (B) destabilize hyaluronidase.

The at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and hamamelis (witch hazel), which is component (B), is a plant extract and is therefore dissolved in an extract solution. The extract solution may contain other ingredients as long as they do not interfere with the effects of the present invention, such as the solvents, anti-inflammatory agents, surfactants, pH adjusters, preservatives, and herbal medicines described below.

<Oral composition>
The hyaluronidase activity inhibitor of the present invention is preferably incorporated into an oral composition for use. The shape and dosage form of the oral composition are not particularly limited. Oral compositions can be prepared in, for example, liquid, gel, paste, solid, etc. In addition, dosage forms of oral compositions include, for example, toothpastes such as toothpastes and liquid toothpastes, mouthwashes, mouth fresheners, mouthwashes (gargles), oral liniments, lozenges, chewing gum, etc. Among these, liquid oral compositions such as liquid toothpastes, mouthwashes, and mouthwashes are preferred.

The content of component (A) in the oral composition is preferably 0.00002 to 2% by mass. If the content of component (A) in the oral composition is too low, it will be difficult to achieve the inhibitory effect on hyaluronidase activity when used in combination with component (B), while if the content is too high, the feel of use will be reduced and formulation will be difficult, so it is preferable to contain it within the above range. The content of component (A) in the oral composition is more preferably 0.00005% by mass or more, even more preferably 0.0001% by mass or more, and more preferably 1% by mass or less, even more preferably 0.5% by mass or less, and particularly preferably 0.3% by mass or less.

The content of component (B) in the oral composition is preferably 0.0000015 to 0.15% by mass, calculated as solids content. If the content of component (B) in the oral composition is too low, it will be difficult to achieve the inhibitory effect on hyaluronidase activity when used in combination with component (A), while if the content is too high, the usability will be reduced and formulation will be difficult, so it is preferable to contain it within the above range. The content of component (B) in the oral composition is more preferably 0.000003% by mass or more, even more preferably 0.00001% by mass or more, and more preferably 0.1% by mass or less, even more preferably 0.05% by mass or less, and particularly preferably 0.02% by mass or less.

The ratio (content ratio) of component (A) to component (B) in the oral composition is the same as above, and the mass ratio ((A)/(B)) of the content of component (A) to the content of component (B) converted to solids is preferably 0.1 to 2000. When the ratio of component (A) to component (B) is within this range, the synergistic effect of components (A) and (B) improves the inhibitory effect on hyaluronidase activity, allowing hyaluronidase activity to be sufficiently suppressed. Furthermore, the formulation has an excellent feel when used and is also highly stable. The mass ratio (A)/(B) is more preferably 0.2 or greater, even more preferably 2 or greater, and particularly preferably 10 or greater, and more preferably 1000 or less, even more preferably 200 or less, particularly preferably 40 or less, and most preferably 20 or less.

In addition to the hyaluronidase activity inhibitor of the present invention, oral compositions may optionally contain various components necessary for the composition. Optional components include, for example, solvents, sweeteners, bactericides, surfactants, anti-inflammatory agents, anti-tartar agents, pH adjusters, enzymes, ion sources, preservatives, flavorings, herbal medicines, and colorants.

Examples of solvents include water such as purified water and ionized water, lower monohydric alcohols such as ethanol, and polyhydric alcohols such as glycerin, propylene glycol, and polyethylene glycol.

Sweeteners include, for example, stevioside, xylitol, erythritol, maltitol, sorbitol, sodium saccharin, sucralose, trehalose, reduced palatinose, aspartame, etc.

Examples of disinfectants include cationic disinfectants such as benzethonium chloride, cetylpyridinium chloride, benzalkonium chloride, chlorhexidine hydrochloride, and chlorhexidine gluconate, as well as triclosan, isopropylmethylphenol, thymol, and alkyldiaminoethylglycine hydrochloride solution.

Surfactants include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Specific examples of nonionic surfactants include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty acid diethanolamides, fatty acid monoglycerides, polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oil, alkyl glucosides, and polyoxyethylene polyoxypropylene block copolymers. Cationic surfactants include alkyltrimethylammonium chloride and dialkyldimethylammonium chloride. Anionic surfactants include sulfate ester salts, α-olefin sulfonates, sulfosuccinates, N-acylamino acid salts, and acylated methyl taurine salts. Amphoteric surfactants include betaine acetate amphoteric surfactants and imidazoline amphoteric surfactants.

Examples of anti-inflammatory agents include glycyrrhizinic acid and its derivatives, glycyrrhetinic acid and its derivatives, tranexamic acid, aminocaproic acid, allantoin and its derivatives, ascorbic acid and its salts, azulene, azulene sulfonate, and vitamin E (tocopherol).

Examples of anti-tartar agents include phosphates, polyphosphates, methoxyethylene maleic anhydride copolymers, zinc chloride, and zinc organic acids.

Examples of pH adjusters include monosodium phosphate, disodium phosphate, sodium citrate, citric acid, glucono-δ-lactone, sodium gluconate, sodium acetate, sodium carbonate, sodium bicarbonate, sodium hydroxide, and hydrates of these.

Enzymes include, for example, protease, dextranase, amylase, mutanase, lysozyme, etc.

Examples of ion sources include fluoride ion sources such as sodium fluoride, monofluorophosphate, and stannous fluoride, and calcium ion sources such as calcium phosphate, calcium gluconate, and hydroxyapatite.

Examples of preservatives include parahydroxybenzoic acid esters, sodium benzoate, potassium sorbate, and phenoxyethanol.

Fragrances include, for example, natural essential oils such as peppermint oil, spearmint oil, peppermint oil, eucalyptus oil, clove oil, thyme oil, rosemary oil, lemon oil, ginger oil, lime oil, cassia oil, cardamom oil, lavender oil, and cinnamon oil; fragrance ingredients such as l-menthol, l-carvone, carvacrol, eugenol, anethole, 1,8-cineole, vanillin, pinene, and 3-l-menthoxypropane-1,2-diol; mixtures of these; natural fragrances; blended fragrances; and synthetic fragrances.

Examples of herbal medicines include plant extracts other than horsetail extract, hawthorn extract, and witch hazel extract, which are components of the hyaluronidase activity inhibitor of the present invention, such as rosemary extract, Scutellaria root extract, Phellodendron bark extract, Angelica acutiloba extract, carrot extract, and fennel extract.

Examples of pigments include Blue No. 1, Blue No. 2, Blue No. 201, Yellow No. 4, Yellow No. 5, Yellow No. 202(1), Yellow No. 203, Red No. 3, Red No. 102, Red No. 104, Red No. 105, Green No. 3, and Green No. 201.

The oral composition can be prepared by heating the hyaluronidase activity inhibitor of the present invention and any optional ingredients, if necessary, and stirring the ingredients until homogeneous.

The hyaluronidase activity inhibitor of the present invention contained in the oral composition can suppress hyaluronidase activity in periodontal tissues, and the synergistic effect of components (A) and (B) enhances the effect. Therefore, even if the concentration of the hyaluronidase activity inhibitor is reduced by saliva, etc., sufficient hyaluronidase activity inhibitory effect can be exerted.

The present invention also provides a method for enhancing the inhibitory effect of hyaluronidase activity, which involves adding component (A) and component (B). As described above, by using component (A) and component (B) in combination, the inhibitory effect of hyaluronidase activity can be enhanced even at low concentrations where either component alone is ineffective. Furthermore, the effect can be enhanced by using component (A) in combination with other components that have hyaluronidase inhibitory effects.

Below are examples of formulations of oral compositions containing the hyaluronidase activity inhibitor of the present invention, where component (A) is hinokitiol and component (B) is at least one extract selected from the group consisting of extracts of horsetail, hawthorn, and hamamelis virginiana.
<Liquid oral composition: Formulation example>
(A) Component 0.00002 to 2% by mass
(B) Component (solid equivalent) 0.0000015 to 0.15% by mass
Ethanol 0 to 20% by mass
Propylene glycol 0 to 10% by mass
Glycerin 0 to 20% by mass
Xylitol 0-2% by mass
Polyoxyethylene hydrogenated castor oil 0-1% by mass
Fragrance 0-1% by mass
Methyl parahydroxybenzoate 0 to 0.5% by mass
Monosodium phosphate 0 to 0.2% by mass
Disodium phosphate 0 to 0.2% by mass
Purified water balance total 100% by mass

<Products>
The present invention also finds that hinokitiol, the component (A), has an excellent effect of inhibiting the growth of mold on specific resins.
Conventionally, the proliferation of microorganisms such as bacteria and mold on the surface of plastic containers for cosmetics and other products has been a problem. Products that are used for a certain period of time after opening are designed to have antiseptic properties in the formulation, taking into consideration the risk of contamination with microorganisms from the environment during use. However, antiseptic properties are usually not exerted in areas that do not come into contact with the formulation, and microorganisms may grow. For example, there have been reported cases of mold growing on the inner wall of the cap of containers containing oral compositions, and a means of suppressing the proliferation of microorganisms on the container surface has been desired.

The inventors have discovered that hinokitiol, a compound known to have antibacterial and antiseptic properties, has a high adsorption capacity for certain resins, specifically polyethylene (PE) and polypropylene (PP), and is therefore effective in inhibiting mold growth on these specific resins.

The product of the present invention preferably comprises a composition containing hinokitiol and a container containing a member formed from a resin comprising at least one of polyethylene and polypropylene. By filling a container containing a member formed from a resin comprising polyethylene and/or polypropylene with a composition containing hinokitiol, the growth of microorganisms on the surface of the member, specifically the inner surface of the container, can be inhibited even if the member formed from the resin comprising polyethylene and/or polypropylene is not in contact with the composition. Because hinokitiol is a naturally derived ingredient, it is possible to give the product an impression of safety and also ensure product stability.

Compositions containing hinokitiol may consist solely of hinokitiol, or may contain other ingredients as long as they do not interfere with the effects of the present invention. Examples of other ingredients include solvents, humectants, flavoring agents, surfactants, disinfectants, anti-inflammatory agents, pH adjusters, enzymes, preservatives, fragrances, colorants, thickeners, stabilizers, coating agents, and detergents.

The composition preferably contains 0.00002% or more by mass of hinokitiol, more preferably 0.001% or more by mass, and even more preferably 0.01% or more by mass. There is no particular upper limit, but it may be 100% by mass. The hinokitiol content in various compositions can be adjusted appropriately depending on the application. For example, when used in an oral composition, it is preferably in the range of 0.00002 to 2% by mass, with a lower limit of 0.00005% or more by mass, more preferably 0.0001% or more by mass, and an upper limit of 1% or less by mass, more preferably 0.5% or less by mass.

The container used in the present invention is not particularly limited as long as it includes at least a part formed from a resin comprising at least one of polyethylene and polypropylene, and can be used for any product, such as quasi-drugs, cosmetics, foods, beverages, detergents, deodorants, insecticides, and agricultural chemicals.
In particular, by molding the cap of the container from polyethylene resin or polypropylene resin, hinokitiol volatilizes and is adsorbed onto the inner surface of the cap, thereby providing an effect of inhibiting mold growth on the inner wall surface of the cap.

It is preferable that the headspace in the product be 1% or more of the container volume. Having a headspace of 1% or more of the container volume allows hinokitiol to easily volatilize from the composition and allows a sufficient amount to be adsorbed onto the container surface (inner surface of the container). If the amount of composition filled is too small, it may lead to product defects, so the upper limit of the headspace is preferably 10% or less.

The cap of the container preferably has a bottom area of 2 to 20 cm ^2. When the bottom area of the cap is within this range, the evaporated hinokitiol is evenly adsorbed to the entire bottom of the cap, thereby achieving the effect of inhibiting mold growth.

Furthermore, the surface area of the liquid surface of the composition when filled into a container is preferably 2 to 100 cm ^2. If the surface area of the liquid surface is less than 2 cm ² , hinokitiol will not volatilize sufficiently, and if it is more than 100 cm ² , the amount of volatilization will be too great, which may lead to a defective product.

The present invention also provides a method for imparting mold growth inhibitory effects to resins, which involves contacting vaporized hinokitiol with resins containing at least one of polyethylene and polypropylene. Because hinokitiol is highly volatile and highly adsorbent to polyethylene and polypropylene, vaporized hinokitiol adsorbs to the resin, safely inhibiting mold growth on the inner surface of containers while ensuring product stability.

Further explanation will be given below using the following test examples, but the present invention is not limited to these examples.

<Test Example 1>
1. Preparation of Samples Samples 1 to 9 were prepared according to the formulations shown in Table 1. Samples 1, 2, 4, 5, 8 and 9 are comparative examples, and samples 3, 6 and 7 are examples.
The horsetail extract used was "Horsetail Extract BG" manufactured by Maruzen Pharmaceutical Co., Ltd., and was formulated to have a solid content of 0.00015% by mass for samples 2 and 3, 0.000015% by mass for samples 5 and 6, and 0.015% by mass for sample 7.
The peony extract used was "Peony Extract BG-JC" manufactured by Maruzen Pharmaceutical Co., Ltd., and was blended in samples 8 and 9 so that the solid content was 0.00015 mass %.
The 0.1 mol/L acetate buffer solution was prepared by mixing a 0.1 mol/L acetic acid solution with a 0.1 mol/L sodium acetate solution and adjusting the pH to 3.5.

2. Hyaluronidase Activity Inhibition Test The inhibition rate of hyaluronidase activity was calculated according to the following procedure.
The preparation methods of the reagents used in the following procedures are as follows:
Hyaluronidase Type IV-S solution: Hyaluronidase Type IV-S (Sigma-Aldrich "Hyaluronidase from bovine tests") was added to 0.1 mol/L acetate buffer and dissolved, and the concentration was adjusted to 4000 units/mL.
Calcium chloride solution: Calcium chloride was added to and dissolved in a 0.1 mol/L acetate buffer solution to adjust the concentration to 2.5 mmol/L.
Sodium hyaluronate solution: Sodium hyaluronate ("Sodium hyaluronate for biochemistry, derived from rooster comb", manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to and dissolved in 0.1 mol/L acetate buffer solution, and the concentration was adjusted to 0.8 mg/mL.
Boric acid solution: 4.95 g of boric acid was dissolved in 50 mL of purified water, and the solution was adjusted to pH 9.1 with 1 M sodium hydroxide, and then purified water was added to make up to 100 mL.
p-DABA solution: Acetic acid was added to 12.5 mL of 10 M hydrochloric acid to bring the total volume to 100 mL, and 10 g of p-dimethylaminobenzaldehyde (p-DABA) was added and dissolved. The solution was diluted 10 times with acetic acid just before use.

(Test Method)
(1) 0.1 mL of hyaluronidase Type IV-S solution was added to 0.2 mL of a sample, and the mixture was allowed to react at 37°C for 20 minutes.
(2) 0.2 mL of calcium chloride solution was added, and the mixture was allowed to react at 37°C for 20 minutes.
(3) 0.5 mL of sodium hyaluronate solution was added, and the mixture was allowed to react at 37°C for 40 minutes.
(4) 0.2 mL of a 0.4 mol/L aqueous sodium hydroxide solution was added to stop the reaction, and after cooling, 0.2 mL of a boric acid solution was added to each reaction solution, and the mixture was boiled for 3 minutes.
(5) After cooling on ice, 6 mL of p-DABA solution was added and reacted at 37°C for 20 minutes.
(6) The absorbance of the resulting reaction solution at 585 nm was measured.
(7) As a control, 0.1 mol/L acetate buffer was used instead of the sample in (1), and the above steps (1) to (6) were carried out, followed by measuring the absorbance.
(8) As a control blank, 0.3 mL of 0.1 mol/L acetate buffer was used, and the above steps (2) to (6) were carried out to measure the absorbance.
(9) As a reaction solution blank, 0.1 mol/L acetate buffer was used instead of the hyaluronidase Type IV-S solution, and the above steps (1) to (6) were carried out, followed by measuring the absorbance.
(10) The hyaluronidase activity inhibition rate was calculated according to the following formula (1). The results are shown in Table 1.
Inhibition rate (%) = {(A - B) - (C - D)} / (A - B) × 100 ... (1)
In formula (1),
A: Absorbance of control B: Absorbance of control blank C: Absorbance of reaction solution D: Absorbance of reaction solution blank.

Comparing samples 1 and 2 with sample 3, samples 1 and 2, which contained component (A) and component (B) alone, had hyaluronidase activity inhibition rates of 8.2% and 8.9%, respectively, while sample 3, which contained a combination of component (A) and component (B), had a hyaluronidase activity inhibition rate of 34.0%.
Furthermore, samples 4 to 6 were obtained by diluting samples 1 to 3 by 10 times. Samples 4 and 5 were barely able to inhibit hyaluronidase activity, whereas sample 6 had a hyaluronidase activity inhibition rate of 5.7%.
In addition, sample 7 had increased contents of components (A) and (B) compared to sample 3, and the increased amounts resulted in a higher hyaluronidase activity inhibition rate.
Samples 8 and 9 contain peony extract as a plant extract, with sample 8 containing peony extract alone and sample 9 containing peony extract in combination with hinokitiol. Sample 8 had a hyaluronidase activity inhibition rate of 19.0%, while sample 9 had a hyaluronidase activity inhibition rate of 3.9%, indicating that peony extract alone exhibited an inhibitory effect on hyaluronidase activity, but that this effect was negated by combining it with hinokitiol.
These results demonstrate that the combined use of component (A) and component (B) significantly improves the inhibitory effect on hyaluronidase activity.

<Test Example 2>
1. Preparation of Samples Samples 10 to 14 were prepared according to the formulations shown in Table 2. Samples 10 to 12 are examples, and samples 13 and 14 are comparative examples.

2. Mold Growth Inhibition Test The mold growth inhibition effect was evaluated according to the following procedure.
In the following test methods, polypropylene flat plates (PP, thickness 0.75 mm), polyethylene flat plates (PE, thickness 0.8 mm), and polyethylene terephthalate flat plates (PET, thickness 1 mm) were used as test pieces, cut into 1 cm x 1 cm pieces.

(Test Method)
(1) 5 mL of saline containing 0.05% Tween 80 was added to a slant culture medium containing Cladosporium cladosporioides, a type of black mold, collected from a household bathroom. Spores were scraped off with a platinum loop, filtered through sterile gauze, and 0.1 mL of SCD liquid medium was added to obtain a spore solution.
(2) 1080 mL of the sample was filled into a 1120 mL white PET bottle and capped with a PE cap to which a test piece had been previously attached.
(3) The obtained test specimen was placed in a thermostatic bath at 60°C, and then removed after 7 days and allowed to stand until it reached room temperature (25±3°C).
(4) After leaving it to stand, the cap was removed, 5 μL of spore liquid was dropped onto the test piece at the bottom of the cap, the cap was closed again, and the test piece was left to stand in an incubator at 25°C. After 3 days, the growth of mold was visually confirmed.

(Evaluation criteria)
The growth of mold was confirmed by visual inspection, and samples with no mold growth were marked with "◯" and samples with mold growth were marked with "X." The results are shown in Table 3.

The results in Table 3 show that samples 10 to 12 were all able to inhibit mold growth on PP resin and PE resin. However, no mold growth inhibitory effect was obtained on PET resin. Furthermore, samples 13 and 14 were not able to inhibit mold growth on any of the PP resin, PE resin, or PET resin.
These results demonstrate that hinokitiol specifically adsorbs to PP resin and PE resin and exerts an inhibitory effect on mold growth.

Claims (5)

A hyaluronidase activity inhibitor characterized by comprising (A) hinokitiol and (B) horsetail extract . The hyaluronidase activity inhibitor according to claim 1, wherein the mass ratio (A)/(B) of the content of the component (A) to the content of the component (B) converted to solids is 0.1 to 2000. A method for enhancing the inhibitory effect of hyaluronidase activity, characterized by adding (A) hinokitiol and (B) horsetail extract . An oral composition containing the hyaluronidase activity inhibitor described in claim 1 or 2. The oral composition according to claim 4, characterized in that the content of component (A) is 0.00002 to 2% by mass, and the content of component (B) in terms of solids is 0.0000015 to 0.15% by mass.