Composition for inhibiting pseudomonas syringae, and skin disinfectant comprising the composition

Abstract

To provide a Pseudomonas syringae-inhibiting composition which contains alcohol but still inhibits the growth of Pseudomonas syringae while inhibiting the production of IL-1α caused by the alcohol.SOLUTION: It was found that a composition which contains a monovalent alcohol having 1-3 carbon atoms and also contains a copolymer (P) having a specific structure can inhibit the growth of Pseudomonas syringae while inhibiting the production of IL-1α. The findings have led to the completion of the present invention.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a Pseudomonas syringae suppression composition that is a true bacterium and a skin disinfectant containing the composition.

Background Art

[0002] Pseudomonas syringae ( P. syringae ) is a true bacterium that infects plants such as Japanese larch, broad bean, and kiwifruit. It lives on the surface of plant leaves and branches, and may also live in the human living environment or adhere to the human body surface. As an action on host plants of Pseudomonas syringae, it is known to cause symptoms such as forming spots on the leaf surface, deforming leaves, and necrotizing flowers. As a method for suppressing the growth of Pseudomonas syringae, the method described in Patent Document 1 is known. In this method, the activity of Pseudomonas syringae is inhibited by using an N-ethyl-N-methyl-formamidine-based compound having a specific substituent and a strobilurin-based compound.

[0003] Since some strobilurin-based compounds exhibit thyroid toxicity and the like, it is difficult to apply them to the human body from a safety perspective. In view of the fact that Pseudomonas syringae adheres to the human body surface as described above, it is preferable that a growth inhibitor of Pseudomonas syringae can be applied to human skin without problems. For example, cationic surfactant-based compounds such as benzalkonium chloride and benzethonium chloride, biguanide compounds such as chlorhexidine salts typified by chlorhexidine gluconate, phenolic compounds such as cresol, and iodine compounds such as iodoform can be applied to human skin and are effective bactericidal components against many microbial species including Pseudomonas syringae. However, these components are affected by pH and coexisting organic substances. That is, depending on the formulation and the usage environment, the efficacy may change, and there may be cases where a sufficient growth inhibitory effect against Pseudomonas syringae cannot be exhibited.

[0004] Examples of methods for suppressing IL-1α production include the following techniques. Patent Document 2 discloses that the extract of Primula sieboldii E. Morren has the effect of suppressing IL-1α production. Patent Document 3 also discloses that the extract of Arnica montana L. has the effect of suppressing IL-1α production. In Patent Document 2, human epidermal cells are exposed to a 50 μg / mL LPS (lipopolysaccharide) medium to intentionally promote the production of IL-1α, and the presence or absence of the IL-1α inhibitory effect of the extract is evaluated. In Patent Document 3, mouse-derived macrophages are exposed to a 100 ng / ml LPS medium to intentionally promote the production of IL-1α, and the presence or absence of the IL-1α inhibitory effect of the extract is evaluated. However, the extracts described in any of the documents have a different composition from the Pseudomonas syringae suppression composition of the present invention, and furthermore, it is not shown whether they have the effect of suppressing the production amount of IL-1α that occurs when alcohol is exposed to human skin or the like.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Alcohols such as ethanol and isopropanol are not easily affected by changes in pH and coexisting organic substances, and are excellent in quick-drying property and immediate effect. Therefore, it is preferable to use alcohol (treatment by spraying, rubbing, etc.) for suppressing the growth of Pseudomonas syringae. Alcohol has no thyroid toxicity like strobilurin compounds, and there are almost no safety problems when usually applied to the skin. However, in some alcohol disinfectant users, for example, people with sensitive skin who show an excessive reaction to alcohol, frequent contact of alcohol with the skin may promote the production of cytokine IL-1α and affect the skin such as the formation of wrinkles (Non-Patent Document 1). Therefore, when frequently contacting the skin of people with sensitive skin with alcohol, it is important to suppress the production of IL-1α by alcohol from the viewpoint of preventing wrinkle formation. More specifically, the problem of the present invention is to provide a Pseudomonas syringae inhibitory composition that can suppress the growth of Pseudomonas syringae while suppressing the production of IL-1α by alcohol while containing alcohol.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that a composition containing a monohydric alcohol having 1 to 3 carbon atoms and a copolymer (P) having a specific structure described below can suppress the production of IL-1α while suppressing the growth of Pseudomonas syringae, and thus completed the present invention.

[0009] That is, the present invention is as follows. [1] (a) 35 to 75 w / v% of a monohydric alcohol having 1 to 3 carbon atoms, and (b) having a weight average molecular weight of 5,000 to 5,000,000, comprising a structural unit based on 2-methacryloyloxyethyl phosphorylcholine represented by the following formula (1) and a structural unit based on butyl methacrylate represented by the following formula (2), and the ratio of the content of the structural unit (1) to the structural unit (2) is 10:90 to 90:10 in molar ratio, 0.001 to 1.0 w / v% of the copolymer (P) A Pseudomonas syringae inhibitory composition comprising

Chemical formula

Chemical formula

Advantages of the Invention

[0010] The Pseudomonas syringae inhibitory composition of the present invention can suppress the growth of Pseudomonas syringae while suppressing the production of IL-1α by alcohol, even though the composition contains alcohol.

Modes for Carrying Out the Invention

[0011] Hereinafter, the present invention will be described in more detail. The Pseudomonas syringae inhibitory composition of the present invention comprises the following. (a) 35 to 75 w / v% of a monohydric alcohol having 1 to 3 carbon atoms, and (b) having a weight average molecular weight of 5,000 to 5,000,000, comprising a structural unit based on 2-methacryloyloxyethyl phosphorylcholine represented by the following formula (1) and a structural unit based on butyl methacrylate represented by the following formula (2), and the ratio of the content of the structural unit (1) to the structural unit (2) is 10:90 to 90:10 in molar ratio, 0.001 to 1.0 w / v% of the copolymer (P) In addition, in this specification, when preferred numerical ranges (for example, ranges of content or weight-average molecular weight) are described stepwise, each lower limit value and upper limit value can be combined independently. For example, in the description "preferably 10 to 100, more preferably 20 to 90", "preferred lower limit value: 10" and "more preferred upper limit value: 90" can be combined to "10 to 90".

[0012] The "Pseudomonas syringae suppression" of the present invention includes the sterilization, disinfection, antibacterial, and growth inhibition of Pseudomonas syringae. The "suppression of IL-1α production" means that when the control (for example, the production amount of IL-1α in disinfectant ethanol) is set to 100, it is less than 90%, preferably less than 75%, and more preferably less than 50%.

[0013] <(a) Monohydric alcohol having 1 to 3 carbon atoms> The composition or inhibitor of the present invention contains (a) a monohydric alcohol having 1 to 3 carbon atoms. The monohydric alcohol having 1 to 3 carbon atoms contained as the component (a) of the present invention is not particularly limited, and examples thereof include methanol, ethanol, normal propanol (1-propanol), isopropanol (2-propanol), etc. From the viewpoints of the impact on the environment and the safety to the body, ethanol and isopropanol are preferred. The monohydric alcohol having 1 to 3 carbon atoms as the component (a) of the present invention may be used alone or in combination of two or more. When two or more kinds of the component (a) are used in combination, the content thereof means the total amount thereof. In addition, as the above monohydric alcohol as the component (a), commercially available products for pharmaceuticals and cosmetics can be used. The content of component (a) of the present invention is 35 to 75 w / v%, preferably 45 to 70 w / v%, and more preferably 55 to 65 w / v% from the viewpoints of the inhibitory effect on the growth of Pseudomonas syringae and quick-drying property. When the content is less than 35 w / v%, the inhibitory effect on the growth of Pseudomonas syringae or the quick-drying property may not be exhibited. On the other hand, when it is more than 75 w / v%, almost no improvement in the inhibitory effect on the growth of Pseudomonas syringae or the quick-drying property is observed, which is not preferable from the viewpoint of cost-effectiveness.

[0014] <Copolymer (P)> The inhibitory composition or inhibitor of the present invention contains a copolymer (P) composed of a structural unit based on 2-methacryloyloxyethyl phosphorylcholine (MPC: the following formula 1) and a structural unit based on butyl methacrylate (the following formula 2).

Chemical formula

Chemical formula

[0015] The copolymer (P) is a copolymer having a weight average molecular weight of 5,000 to 5,000,000 obtained by copolymerizing 2-methacryloyloxyethyl phosphorylcholine and butyl methacrylate by a copolymerization method known per se. The weight average molecular weight can be measured using gel permeation chromatography (GPC).

[0016] In the copolymer (P), the ratio of the content of the structural unit based on MPC to the content of the structural unit based on butyl methacrylate ([structural unit based on MPC]:[structural unit based on butyl methacrylate]) is not particularly limited, but is 10:90 to 90:10 in terms of molar ratio, preferably 20:80 to 80:20, and more preferably 25:75 to 75:25. Examples of the copolymer (P) can include the following. · 2-Methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [Copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 600,000] · 2-Methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [Copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 100,000] · 2-Methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [Copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 1,000,000] · 2-Methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [Copolymer ratio (molar ratio) 30 / 70, weight average molecular weight: 100,000]

[0017] The copolymer (P) of the present invention (2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer) may be used alone or in combination of two or more. The content in the composition or agent of the copolymer (P) of the present invention means the total amount when two or more are used in combination. In addition, as the copolymer (P) (2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer), commercially available products for pharmaceuticals and cosmetics can be used. The weight average molecular weight of the copolymer (P) of the present invention is 5,000 to 5,000,000, and in view of ease of stretching etc. when applied to the skin, it is preferably 10,000 to 2,000,000, more preferably 10,000 to 1,000,000, and even more preferably 10,000 to 200,000.

[0018] The content of the copolymer (P) of the present invention in the composition or agent of the present invention is 0.001 to 1.0 w / v%. When the content is less than 0.001 w / v%, the Pseudomonas syringeae inhibitory effect or the IL-1α production inhibitory effect may not be exhibited. When it is more than 1.0 w / v%, since the solid content increases, the quick-drying property may be reduced depending on the usage method. Further, from the balance between the Pseudomonas syringeae growth inhibitory effect and the quick-drying property, it is preferably 0.0025 to 0.5 w / v%, and more preferably 0.005 to 0.25 w / v%.

[0019] The polymerization form of the copolymer (P) is not particularly limited, and it may be a random copolymer or a block copolymer, but a random copolymer is preferred.

[0020] The copolymer (P) can be produced by a known production method. For example, a monomer mixture containing MPC and butyl methacrylate can be polymerized by a known method such as solution polymerization in the presence of a radical polymerization initiator under an inert gas atmosphere such as nitrogen. The content ratio of each monomer may be a ratio corresponding to the content ratio of each structural unit in the copolymer (P). The copolymer (P) can be produced and used by the above-described polymerization method, but a commercially available product as a solution or dispersion of water or a polyhydric alcohol can also be used. The copolymer (P) of the present invention can also be used as an active ingredient of an IL-1α production inhibitor caused by alcohol disinfection of the skin. That is, it has a weight average molecular weight of 5,000 to 5,000,000 and is composed of a structural unit based on 2-methacryloyloxyethyl phosphorylcholine represented by the formula (1) and a structural unit based on butyl methacrylate represented by the formula (2), and the content ratio of the structural unit (1) to the structural unit (2) is 10:90 to 90:10 in molar ratio. It is an IL-1α production inhibitor containing 0.001 to 1.0 w / v of the copolymer (P).

[0021] The inhibitory composition or inhibitor of the present invention may contain, in addition to the components described above, a humectant, an emollient, a pH adjuster, and / or a fragrance, etc. that can be used in general external preparations, etc., as long as the effects of the present invention are not inhibited. Examples of the humectant include glycerin, propylene glycol, butylene glycol, sorbitol, sodium lactate, hyaluronic acid and its salts, sorbitol, pyrrolidone carboxylic acid and its salts, N-cocoyl-L-arginine ethyl ester DL-pyrrolidone carboxylate, and urea, etc. One or more of these can be included. Examples of the emollient include fatty acid esters such as isopropyl myristate, isopropyl palmitate, isopropyl stearate, isobutyl oleate, and isobutyl maleate, etc. One or more of these can be included. Examples of the pH adjuster include hydrochloric acid, gluconic acid, primary amines, secondary amines, sodium hydroxide, and potassium hydroxide, etc. One or more of these can be included. Examples of the fragrance include menthol, peppermint oil, methyl salicylate, vanillin, benzyl succinate, methyl eugenol, anethole, limonene, ocimene, citronellyl acetate, cineole, ethyl linalool, spearmint oil, peppermint oil, and eucalyptus oil, etc. One or more of these can be included.

[0022] The inhibitory composition or inhibitor of the present invention can be produced according to a conventional method. For example, it can be produced by adding water, a wetting agent, and other general additives as necessary to the above-described MPC copolymer and the monohydric alcohol having 1 to 3 carbon atoms, mixing appropriately, and making it uniform. In addition, the inhibitory composition or inhibitor of the present invention can be used, for example, as pharmaceuticals, quasi-drugs, or their components.

[0023] The inhibitory composition or inhibitor of the present invention can be provided in the form of a liquid, a semi-solid form such as a paste, etc. From the viewpoints of ease of use and safety during use, etc., the suppression composition or suppressant of the present invention is preferably in a liquid form. The suppression composition or suppressant of the present invention can be housed in a pump container or a spray container. Specifically, it is preferably made into products such as a liquid discharge type filled in a container with a dispenser pump, a spray type filled in a container with a spray pump, a foam discharge type filled in a container with a former pump or an aerosol container, an impregnation type impregnated in a cleaning wipe, etc. Considering the ease of use during use, the liquid discharge type is particularly preferable. As an example of using the suppression composition or suppressant of the present invention, when used on the skin of fingers, an appropriate amount (about 1 mL to 3 mL) of the suppression composition or suppressant of the present invention can be taken and applied while rubbing it into the hand. When impregnating the suppression composition or suppressant of the present invention into a cleaning wipe, the suppression composition or suppressant of the present invention is impregnated in an amount of about 10 parts by mass to 400 parts by mass with respect to 100 parts by mass of the cleaning wipe, and the skin can be wiped with the wipe for use.

Examples

[0024] Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

[0025] Each component used in the Examples and Comparative Examples is as follows. <Component (a)> Ethanol: "Anhydrous Ethanol" manufactured by Fujifilm Wako Pure Chemical Corporation <Component (b)> Polymer A: 2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 600,000] Polymer B: 2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 100,000] Polymer C: 2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [copolymer ratio (molar ratio) 80 / 20, weight average molecular weight: 1,000,000] Polymer D: 2-methacryloyloxyethyl phosphorylcholine·butyl methacrylate copolymer [copolymer ratio (molar ratio) 30 / 70, weight average molecular weight: 100,000] <Component (b)'> Aminoalkyl methacrylate copolymer E: "Oidrad 100" manufactured by Evonik Japan Co., Ltd. Hydrophobized hydroxypropyl methylcellulose: "Sanjelose 60L" manufactured by Daido Kasei Kogyo Co., Ltd. Hydroxyethyl cellulose: "Natrosol 250 HHR CS" manufactured by Ashland Japan Co., Ltd.

[0026] <Weight average molecular weight measurement> The weight average molecular weight was measured under the following conditions using gel permeation chromatography (GPC). ·GPC system: EcoSEC system (manufactured by Tosoh Corporation) ·Columns: Shodex OHpak SB-802.5HQ (manufactured by Showa Denko K.K.) and SB-806HQ (manufactured by Showa Denko K.K.) connected in series ·Developing solvent: 20 mM sodium phosphate buffer (pH 7.4) ·Detector: Differential refractive index detector ·Molecular weight standard: EasiVial PEG / PEO (manufactured by Agilent Technologies) ·Flow rate: 0.5 mL / min ·Column temperature: 40 °C ·Sample: The obtained Polymers A - D were diluted with the developing solvent to a final concentration of 0.1 wt%. ·Injection volume: 100 μL

[0027] (Example 1) 80 mL of ethanol (Component (a)) was weighed, and Polymer A (Component (b)) was added while stirring. Then, an appropriate amount of purified water was added to make the total volume 100 mL, and the composition of Example 1 was obtained. The blending amounts of each component are as shown in Table 1 below. (Examples 2 to 9 and Comparative Examples 1 to 3) The compositions of Examples 2 to 9 and the compositions of Comparative Examples 1 to 3 were produced by the same method as the production method of the composition of Example 1.

[0028] Regarding the compositions of the examples and comparative examples, the following Pseudomonas syringeae growth inhibition evaluation and IL-1α production rate evaluation were performed.

[0029] Pseudomonas syringeae growth inhibition evaluation (1) Test microorganism: Pseudomonas syringeae ( Pseudomonas syringae (NBRC 14084)) (2) Preparation of test bacterial solution For each test bacterium, using sterilized physiological saline, a test bacterial solution was prepared to be 1×10 8 Colony Forming Unit (CFU) / mL. CFU means a unit indicating the number of viable (capable of forming colonies) microbial cells contained in the seeded microorganism. (3) Preparation of medium 1.3 g of rehydration solution 702 "Digo" (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to 100 ml of purified water, shaken well, dissolved by heating, dispensed into containers, and autoclaved at 121°C for 15 to 20 minutes. (4) Measurement of minimum inhibitory concentration (MIC) Using each composition of the examples and comparative examples as a sample, 100 μL of each sample was serially diluted two-fold with the medium to prepare a dilution series with a final concentration of the sample of 0.5 μg / mL. After adding 50 μL of each test bacterial solution to the prepared dilution series, it was cultured at 37°C for 24 hours. After the culture was completed, the turbidity of the medium was visually determined, and the minimum concentration (μg / mL) at which the growth of the test bacterium was not observed was taken as the MIC. (5) Evaluation of growth inhibition effect Based on the obtained MIC values, the inhibitory effect on the growth of Pseudomonas syringae was evaluated according to the following criteria. (6) Evaluation criteria When the MIC was 7.8 μg / mL or less, it was designated as "A"; when it was greater than 7.8 μg / mL and 15.6 μg / mL or less, it was designated as "B"; when it was greater than 15.6 μg / mL and 31.3 μg / mL or less, it was designated as "C"; and when it was greater than 31.3 μg / mL, it was designated as "D". Those classified as A, B, and C were determined to have an inhibitory effect on the growth of Pseudomonas syringae.

[0030] IL-1α production rate evaluation <Handling of LabCyte EPI-MODEL> In this example, evaluation was performed using a human three-dimensional cultured epidermis LabCyte EPI-MODEL 24 plate (manufacturer: Japan Tissue Engineering Co., Ltd., product number: 401151). For the culture method of LabCyte EPI-MODEL, OECD TG439 adopted on July 26, 2013 was referred to. Specifically, it is as follows. (1) 25 μL of the composition of each example and comparative example or ethanol for disinfection (manufacturer: Ken-ei Pharmaceutical Co., Ltd.) was brought into contact with the epidermal side of the pre-cultured LabCyte EPI-MODEL and exposed for 5 minutes. The number of repetitions in this test was set to 4. (2) LabCyte EPI-MODEL was washed with DPBS, cultured in a new assay medium for 48 hours, and then 150 μL of the assay medium was sampled (hereinafter referred to as the recovered medium). It was stored at -20°C until the start of IL-1α quantification. <IL-1α quantification> The test method described in the manual of the human IL-1α measurement ELISA kit (manufacturer: Proteintech Group, Inc., product number: KE00123) (Common manual for human IL1-alpha measurement ELISA kit, https: / / www.cosmobio.co.jp / product / detail / human-il1-alpha-elisa-kit-pgi.asp?entry_id=35058) was followed. (1) The recovered medium and various reagents used were left at room temperature and thawed. (2) 100 μL each of the recovered medium and the Standard dilution series were added to the wells of a dedicated plate, and incubated for 120 minutes with the cover on. (3) The inner liquid was discarded into a washbasin or the like, and the remaining inner liquid was removed by tapping the plate. (4) It was washed 4 times with 350 μL of Wash Buffer. After the 4th wash, the plate was tapped. (5) 100 μL of 1× Detection Antibody was added to the wells of a dedicated plate, and incubated for 60 minutes with the cover on. (6) The washing steps shown in (3) and (4) above were carried out in the same manner. (7) 100 μL of 1× HRP-conjugated Antibody was added to the wells of a dedicated plate, and incubated for 40 minutes with the cover on. (8) The washing steps shown in (3) and (4) above were carried out in the same manner. (9) 100 μL of TMB Substrate was added to the wells of a dedicated plate, and incubated for 20 minutes. (10) Subsequently, 100 μL of Stop Solution was added to the wells of a dedicated plate. (11) The absorbance at 450 nm and 630 nm was measured using a plate reader. (12) A calibration curve (Four-parameter logistic) was created from the Standard dilution series, and the amount of IL-1α (pg / μL) in each recovered medium was quantified. The average value of 4 repetitions of each recovered medium was taken as the amount of IL-1α in each composition. <IL-1α production rate evaluation> The value obtained by dividing the amount of IL-1α (pg / μL) in the recovered medium of each composition by the amount of IL-1α (pg / μL) in ethanol for disinfection was defined as the IL-1α production rate. Regarding the IL-1α production rate, when it is less than 50%, it is designated as "A"; when it is 50% or more and less than 75%, it is designated as "B"; when it is 75% or more and less than 90%, it is designated as "C"; when it is 90% or more, it is designated as "D". Those classified as A, B, and C were determined to have the effect of suppressing the IL-1α production amount.

[0031]

Table 1

[0032] As shown in Table 1 above, all the compositions of the examples were evaluated to have the effect of suppressing the growth of Pseudomonas syringae and the effect of suppressing IL-1α production. In particular, the compositions of Examples 8 and 9 containing Polymer D were evaluated to have an excellent effect of suppressing the growth of Pseudomonas syringae and an excellent effect of suppressing IL-1α production. On the other hand, the compositions of the comparative examples containing component (b)' instead of component (b) were evaluated to have no effect of suppressing the growth of Pseudomonas syringae or the effect of suppressing IL-1α production. In addition, since the composition of the present invention contains a monohydric alcohol having 1 to 3 carbon atoms (particularly ethanol), it has quick-drying property and immediate effect, and also has the effect of reducing wrinkle formation due to the ability to suppress IL-1α production.

Industrial Applicability

[0033] According to the present invention, it is possible to provide a composition that can suppress the growth of Pseudomonas syringae while suppressing the production of IL-1α in the skin by alcohol, while containing alcohol in the composition.

Claims

1. (a) 35 to 75 w / v% of a monohydric alcohol having 1 to 3 carbon atoms, and (b) a copolymer (P) having a weight average molecular weight of 5,000 to 5,000,000, comprising a structural unit based on 2-methacryloyloxyethyl phosphorylcholine represented by the following formula (1) and a structural unit based on butyl methacrylate represented by the following formula (2), wherein the ratio of the content of the structural unit (1) to the content of the structural unit (2) is 10:90 to 90:10 in molar ratio, 0.001 to 1.0 w / v% and A Pseudomonas syringeae inhibitory composition comprising. 【Chemical Formula 1】 【Chemical Formula 2】

2. A skin disinfectant comprising the composition according to Claim 1.

Images