JP2019202987A - Sensory stimulation reducing agent - Google Patents

Abstract

To provide a sensory stimulation reducing agent that reduces sensory stimulation caused by paraben.SOLUTION: A sensory stimulation reducing agent contains sulforaphane, or a plant containing sulforaphane or a glycoside thereof, or extract thereof, as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a sensory stimulus reducing agent that relieves the feeling of irritation caused by parabens.

  In many cases, external preparations for skin, such as skin cosmetics, contain a preservative in the preparation in order to prevent contamination and propagation of bacteria. Among them, parabens are most often blended in topical skin preparations as antiseptics having high antibacterial activity and safety. However, people who are sensitive to ultraviolet rays, dryness, chemical substances, etc., so-called sensitive skin, may feel a temporary unpleasant irritation such as a tingling or tingling sensation due to parabens.

  On the other hand, conventionally, as a thing which suppresses irritation | stimulation with respect to skin, for example, the irritation | stimulation inhibitor (patent document 1) which consists of a hydrophobic group containing polysaccharide derivative which suppresses irritation | stimulation with respect to surfactant, irritation | stimulation with respect to hair dye A stimulus inhibitor for hair dyes that suppresses hair loss (Patent Document 2) has been reported. In addition, it has been reported that a TRPA1 inhibitor can be used as a paraben stimulation inhibitor (Patent Document 3).

On the other hand, germinated broccoli which is a germinant of broccoli (Brassica oleracea var. Italica) belonging to the Brassica family Brassica is known as a plant having a high content of sulforaphane, and its extract contains preventive agents such as whitening, wrinkles and tarmi. It has been reported that there is an improving action (Patent Document 4), a sebum production promoting action (Patent Document 5), and the like.
However, there are no reports of preventing or improving skin sensitive to germinated broccoli extracts or sulforaphane.

JP 2001-64185 A JP 2002-363053 A JP 2008-79528 A JP 2003-155221 A Japanese Patent Application No. 2009-114152

  The present invention relates to providing a sensory stimulation reducing agent that reduces sensory stimulation by parabens.

  The present inventors examined sensory stimulation induced by parabens. As a result, methylparaben was metabolized in vivo by the metabolic enzyme carboxylesterase 1 (CES1) and converted to p-hydroxybenzoic acid (PHBA). That there is almost no sensory stimulation, and that CES1 expression is reduced in people with sensitive skin compared to healthy subjects, and that CES1 expression promoting substances can be a sensory stimulation reducing agent by parabens. I found And the plant extract containing sulforaphane, such as sulforaphane and germination broccoli, has the expression promotion effect of CES1, and discovered that there was a sensory stimulus reduction effect by parabens.

That is, the present invention relates to the following 1) to 2).
1) A sensory irritation reducing agent comprising sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.
2) A CES1 expression promoter containing sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the parasites can be decompose | disassembled rapidly in the living body, and the sensory stimulus reduction agent of a new mechanism which reduces the sensory stimulus induced by parabens can be provided.

IL-1α production in the three-dimensional cultured epidermis. MP: methyl p-hydroxybenzoate added, PHBA: p-hydroxybenzoic acid added. Data are mean ± SD. Skin sensory stimulation score in sensitive skin persons. MP: methyl p-hydroxybenzoate application, PHBA: p-hydroxybenzoic acid application. CES1 expression level in healthy and sensitive skin. Data are mean ± SD. Metabolic activity of methyl p-hydroxybenzoate in healthy and sensitive skin. Data are mean ± SD. Sensory stimulus reducing effect of germinated broccoli extract by methyl p-hydroxybenzoate.

In the present invention, sulforaphane refers to 4- (methylsulfinyl) butyl isothiocyanate.
Sulforaphane can be obtained by isolating and purifying it from a plant containing sulforaphane or a glycoside thereof by a known method. In addition, the isolated and purified sulforaphane is commercially available from Sigma-Aldrich, etc., and in the present invention, the commercially available product can also be used.

As an active ingredient of the present invention, not only the isolated sulforaphane but also a plant containing the above sulforaphane or a glycoside thereof or an extract thereof can be used.
Examples of plants containing sulforaphane or glycosides thereof include Brassicaceae plants, preferably Brassicaceae plants. Specific examples include broccoli, cauliflower, cabbage and the like. Of these, broccoli (Brassica oleracea var. Italica) is preferable from the viewpoint of the content of sulforaphane, and germinated broccoli (also referred to as “broccoli sprout”) until germination after germination is more preferable.
Examples of sulforaphane glycosides include sulforaphane glucosinolate.

As the plant containing sulforaphane or a glycoside thereof, all or a part of the plant, a pulverized product, a lyophilized product, a juice, or the like can be used. For example, in germinating broccoli, dried pulverized product of germinated buds, cotyledons, seeds, part of roots or whole plants can be suitably used.
Examples of the plant extract include various solvent extracts obtained by extraction by a known extraction method, diluted solutions thereof, concentrated solutions thereof, and dried powder thereof. Examples of known extraction methods include immersion, decoction, leaching, solid-liquid extraction, reflux extraction, supercritical extraction, ultrasonic extraction, and microwave extraction. For example, the immersion may include immersion and leaching for 1 hour to 4 weeks at 0 ° C. to the solvent boiling point (preferably 15 to 40 ° C.), and the solid-liquid extraction may be performed at 0 ° C. to the solvent boiling point (preferably 15 to 40). And stirring or shaking at 30 to 1000 rpm for 30 minutes to 2 weeks. In order to prevent oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by passing an inert gas such as boiling degassing or nitrogen gas may be used in combination. Moreover, in the case of reflux extraction, it can be performed using an extraction tool such as a Soxhlet extractor.

  As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include, for example, water; monovalent, divalent or polyhydric alcohols; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; Cyclic ethers; Polyethers such as polyethylene glycol; Saturated or unsaturated hydrocarbons such as hexane; Aromatic hydrocarbons such as benzene and toluene; Halogenated carbonization such as dichloromethane, chloroform, dichloroethane, and carbon tetrachloride Examples include hydrogens; pyridines; dimethyl sulfoxide; acetonitrile; carbon dioxide, supercritical carbon dioxide; fats and oils, waxes, other oils; and mixtures thereof. Preferable examples include water, alcohols, and mixed liquids in terms of pharmacological activity and versatility.

  Although it does not specifically limit as said alcohol, For example, monohydric alcohols, such as methanol, ethanol, propanol, butanol, amyl alcohol, hexanol, heptanol, octanol; 1, 3- butylene glycol, ethylene glycol, propylene glycol, 1 , 4-butanediol, 1,5-pentanediol, 1,6-hexanediol and other dihydric alcohols; trivalent or higher alcohols such as glycerin, and the like. Among these, monohydric alcohols and dihydric alcohols are preferable in terms of pharmacological activity and operability. Preferably, the alcohol may be an alcohol having 1 to 10 carbon atoms, more preferably an alcohol having 1 to 4 carbon atoms.

  Preferable examples of the alcohols include methanol, ethanol, 1,3-butylene glycol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, t-butanol and the like.

  The concentration of alcohol in the aqueous solution of the alcohol is preferably 20% by mass or more, more preferably 30% by mass or more, more preferably 40% by mass or more, and preferably 80% by mass or less, more preferably 70% by mass. It is not more than mass%, more preferably not more than 60 mass%. Moreover, the concentration of the alcohol in the aqueous solution of the alcohol is preferably 20 to 80% by mass, more preferably 30 to 70% by mass, and still more preferably 40 to 60% by mass.

  Of the alcohols, ethanol is more preferable from the viewpoint of versatility. Therefore, more suitable solvents for preparing the plant extract include water, ethanol, and aqueous ethanol.

  As a usage-amount of the solvent in extraction, 1-1000 mL is preferable with respect to 1 g of plants (dry mass conversion). Here, in this specification, the volume means the volume at 25 ° C. Moreover, dry mass means the mass of the dry solid content which is the residue which dried the sample with the 105 degreeC electric constant temperature dryer for 3 hours, and removed the volatile substance. The extraction conditions are not particularly limited as long as sufficient extraction can be performed. For example, the extraction time is preferably 1 hour or longer, more preferably 3 days or longer, more preferably 1 week or longer, while preferably 2 months or shorter, more preferably 5 weeks or shorter, more preferably 2 weeks or shorter. . The extraction temperature is preferably 0 ° C. or higher, more preferably 5 ° C. or higher, on the other hand, the solvent boiling point or lower is preferable, and 90 ° C. or lower is more preferable. Usually, extraction is performed for a long time at a low temperature and for a short time at a high temperature. Examples of extraction conditions include 15 to 40 ° C. for 3 days to 5 weeks, preferably 1 to 2 weeks, 60 to 90 ° C. for 1 to 5 hours, and the like. Can be selected.

  In the present invention, the plant extract can be used as it is, but after the extract is diluted, concentrated or dried, it can also be prepared and used in the form of powder or paste. In addition, when lyophilized and used, a solvent usually used for extraction, such as water, ethanol, propylene glycol, 1,3-butylene glycol, water / ethanol mixture, water / propylene glycol mixture, water / 1,3-butylene glycol It can also be diluted with a solvent such as a mixed solution. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  The plant extract thus obtained may be subjected to a separation / purification step such as a treatment with a synthetic adsorbent, a filtration treatment, a concentration treatment, and the like, if necessary, to increase the content of sulforaphane. As a germinated broccoli extract with an increased content of sulforaphane, “broccoli sprout extract” (Oryza Oil & Chemical Co., Ltd.) is commercially available, and in the present invention, the commercially available product can also be used.

As shown in the Examples below, the germinated broccoli extract, which is sulforaphane and a plant containing sulforaphane, has an action of promoting the expression of CES1 and reducing sensory stimulation by parabens.
Therefore, sulforaphane or a plant containing sulforaphane or a glycoside thereof or an extract thereof can be a sensory stimulus reducing agent or a CES1 expression promoter, and can be used to promote the expression of CES1 in order to reduce sensory stimulus, It can be used to produce a sensory stimulus-reducing agent or CES1 expression promoter.
Here, “use” may be administration or ingestion to a human or non-human animal, and may be therapeutic use or non-therapeutic use. Note that “non-therapeutic” means a concept that does not include medical practice, that is, a concept that does not include a method for operating, treating, or diagnosing a person, more specifically, a doctor or a person who has received instructions from a doctor It is a concept that does not include a method of performing surgery, treatment or diagnosis.

In the present invention, “reduction of sensory stimulation” means to suppress or alleviate a temporary unpleasant irritation caused by parabens, such as a tingling sensation or a tingling sensation in the skin. Here, as the “parabens”, for example, methylparaben (methyl p-hydroxybenzoate), ethylparaben (ethyl p-hydroxybenzoate), propylparaben (propyl p-hydroxybenzoate), isopropylparaben (p-hydroxybenzoate) Acid isopropyl), butylparaben (butyl p-hydroxybenzoate), isobutylparaben (isobutyl p-hydroxybenzoate), benzylparaben (benzyl p-hydroxybenzoate) and the like, and sodium salts thereof.
Such a sensory stimulus reduction effect can be measured by sensory evaluation as shown in the examples described later.

In the present invention, “CES1” refers to carboxylesterase 1 and is also known as serine esterase 1 or SES1. This enzyme is a member of the family of mammalian liver carboxyesterases (EC 3.1.1.1) and hydrolyzes endogenous substrates with ester, thioester or amide functional groups.
As shown in the reference examples described later, after culturing with the addition of methyl p-hydroxybenzoate (MP) or p-hydroxybenzoic acid (PHBA) to a human three-dimensional cultured epidermis model, inflammatory cytokine (IL-1α in the medium) ) When the amount was measured, IL-1α production increased when MP was added, but almost no IL-1α production was seen when PHBA was added (FIG. 1). In addition, when MP or PHBA was applied to the skin of a person conscious of sensitive skin and the irritation was tested, some irritation was felt in the MP application, whereas irritation was almost felt in the application of PHBA. (Figure 2). Furthermore, when skin was collected from healthy subjects and sensitive skin subjects, and the metabolic activity of MP and the expression level of CES1 in the skin were measured, the skin activity of MPs in sensitive skin subjects was higher than that of healthy subjects. It was found that the CES1 expression level was low and much lower (FIGS. 3 and 4).
These results suggest that a substance that promotes the expression of CES1 is useful as an agent for reducing sensory stimulation by parabens.

In the present invention, “CES1 expression” includes expression of CES1 protein, activity of CES1 protein, expression of CES1 mRNA encoding the protein, and activity of promoter of CES1 gene, preferably CES1 mRNA expression.
In the present invention, the CES1 expression promoting effect is achieved by, for example, contacting a cell capable of expressing CES1 with the active ingredient of the present invention and measuring the expression of CES1 in the cell (for example, expression of CES1 mRNA, etc.). Can be evaluated.

The sensory stimulation reducing agent and CES1 expression promoter may be sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof alone, or an oil, pigment, fragrance, preservative, chelate Agent, pigment, antioxidant, vitamin, mineral, sweetener, seasoning, preservative, binder, extender, disintegrant, surfactant, lubricant, dispersant, buffer, coating agent, carrier, dilution It may be a composition combined with additives and excipients used in various preparations such as pharmaceuticals, cosmetics, quasi-drugs, and daily necessities. Moreover, those forms are not particularly limited, and can be prepared in any form such as a solution, emulsion, suspension, gel, solid, powder, granule, aerosol, and the like.
The amount of sulforaphane or the plant containing sulforaphane or glycoside thereof in the composition or the extract thereof is 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.001% by mass or more of the total mass of the formulation as sulforaphane. Is 0.01% by mass or more, more preferably 0.1% by mass or more, and 99% by mass or less, preferably 95% by mass or less, more preferably 90% by mass or less, and further preferably 80% by mass or less. is there. Moreover, 0.001-99 mass%, Preferably it is 0.01-95 mass%, More preferably, it is 0.1-90 mass%, More preferably, 1-80 mass% is mentioned. Further, when the composition contains a sulforaphane or a plant containing the glycoside thereof or an extract thereof, the content is 0.001% by mass or more, preferably 0, based on the total mass of the preparation as a dry solid content. 0.01% by mass or more, more preferably 0.1% by mass or more, further preferably 1% by mass or more, and 99% by mass or less, preferably 95% by mass or less, more preferably 90% by mass or less, further preferably Is 80 mass% or less. Moreover, 0.001-99 mass%, Preferably it is 0.01-95 mass%, More preferably, it is 0.1-90 mass%, More preferably, 1-80 mass% is mentioned.

  The sensory irritation reducing agent and CES1 expression promoting agent of the present invention include, for example, a composition containing the above-described parabens (skin cleaning agent, hair cleaning agent, makeup agent, bath agent, permanent wave agent, hair dye, Soaps, kitchen detergents, laundry detergents, cosmetics such as dentifrices, quasi-drugs, pharmaceuticals, daily necessities, etc.) or a composition that is separate from the composition containing parabens The irritation sensation caused by the parabens can be reduced by preparing as above and using it simultaneously with the composition or before and after using the composition. That is, the sensory stimulus-reducing agent or CES1 expression promoter of the present invention is preferably applied to a human who uses a composition containing parabens, and a human who seeks to reduce the irritation caused by parabens. Can be mentioned.

When the sensory stimulus reducing agent and CES1 expression promoter of the present invention are used together with a composition containing parabens, the amount of sensory stimulus reducing agent and CES1 expression promoter used is not particularly limited as long as the sensory stimulus is reduced. For example, with respect to 1 part by mass of parabens, the sulforaphane of the present invention, the plant containing sulforaphane or a glycoside thereof, or an extract thereof is preferably 10 ⁻⁷ parts by mass or more, more preferably 10 ⁻⁶ parts by mass as sulforaphane. Part or more, more preferably 10 ⁻⁵ parts by weight or more, and preferably 10 parts by weight or less, more preferably 1 part by weight or less, still more preferably 0.1 parts by weight or less. Moreover, Preferably it can be set as the ratio of 10 < ^-7 > -10 mass parts, More preferably, it is 10 ^<-6 > -1 mass part, More preferably, it is a ratio of 10 < ^-5 > -0.1 mass part.

Regarding the above-described embodiment, the following aspects are further disclosed in the present invention.
<1> A sensory irritation reducing agent comprising a sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.
<2> A CES1 expression promoter comprising sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.
<3> Use of sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof for producing a sensory stimulus reducing agent.
<4> Use of sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof for producing a CES1 expression promoter.
<5> Sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof for use in reducing sensory stimulation.
<6> A sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof for use in promoting the expression of CES1.
<7> Use of sulforaphane, or a plant containing sulforaphane or a glycoside thereof, or an extract thereof for reducing sensory stimulation (non-therapeutic).
<8> Use (non-therapeutic) of sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof to promote CES1 expression.
<9> A method of reducing sensory stimulation using sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof.
<10> A method for promoting CES1 expression using sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof.
<11> In the above item <1>, <3>, <5>, <7> or <9>, the sensory stimulation is a stimulation sensation caused by perabenes.
<12> In the above <1> to <11>, the plant containing sulforaphane or a glycoside thereof is a germinated broccoli.
<13> In the above <1> to <12>, sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof is used by blending with a composition containing parabens, or the composition The composition is prepared as a separate composition, and is used simultaneously with the composition or before and after use of the composition.
<14> In the above <13>, sulforaphane, a plant containing sulforaphane or a glycoside thereof, or a extract thereof is a plant containing sulforaphane, sulforaphane or a glycoside thereof, or 1 The extract, as sulforaphane, is preferably 10 ⁻⁷ parts by mass or more, more preferably 10 ⁻⁶ parts by mass or more, further preferably 10 ⁻⁵ parts by mass or more, and preferably 10 parts by mass or less, more preferably 1 Less than mass part, More preferably, it is a ratio of 0.1 mass part or less, Preferably it is 10 < ^-7 > -10 mass part, More preferably, it is 10 < ^-6 > -1 mass part, More preferably, it is 10 < ^-5 > -0.1 mass. It is used at the ratio of parts.
<15> In the above <1> and <3>, the amount of sulforaphane or the plant containing sulforaphane or a glycoside thereof in the composition is 0.0001% by mass of the total mass of the formulation as sulforaphane Or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, further preferably 0.1% by mass or more, and 99% by mass or less, preferably 95% by mass or less, more preferably 90% by mass or less, more preferably 80% by mass or less, or 0.001 to 99% by mass, preferably 0.01 to 95% by mass, more preferably 0.1 to 90% by mass, and further preferably 1 -80 mass%. <16> In the above items <1> and <3>, the amount of the sulforaphane or the plant containing the glycoside thereof in the composition is 0.001% by mass of the total mass of the preparation as a dry solid content Or more, preferably 0.01% by mass or more, more preferably 0.1% by mass or more, further preferably 1% by mass or more, and 99% by mass or less, preferably 95% by mass or less, more preferably 90% by mass. % Or less, more preferably 80% by mass or less, or 0.001 to 99% by mass, preferably 0.01 to 95% by mass, more preferably 0.1 to 90% by mass, and further preferably 1 to 80%. % By mass.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.

Reference Example 1 Comparison of sensory irritation induced by parabens (evaluation with three-dimensional cultured epidermis)
LabCyte EPI-MODEL24 (Japan Tissue Engineering Co., Ltd.) was used as a human three-dimensional cultured epidermis model. After pre-culturing overnight (5% CO ₂ , 37 ° C.) with 500 μL of dedicated assay medium, the culture cup was transferred to a 24-well plate with fresh assay medium. After adding 50 μL of 16.4 mM aqueous solution of methyl p-hydroxybenzoate (MP) or p-hydroxybenzoic acid (PHBA) to the epidermal tissue in the culture cup and culturing for 6 hours, the culture cup was filled with phosphate buffer. After 10 washes, the cups were transferred to 24-well plates with fresh assay medium and further incubated for 18 hours. After the supplemental culture, the amount of IL-1α in the medium was quantified using a Human IL-1 alpha / IL-1F1 Quantikine ELISA Kit (R & D Systems) according to the attached protocol. The results are shown in FIG.
From FIG. 1, the amount of IL-1α, which is known to be enhanced by sensory stimulants, is very small in PHBA compared to MP, and the sensory stimulation of PHBA was considered to be extremely weak.

Reference Example 2 Comparison of sensory irritation caused by parabens (human evaluation)
Those who met all of the following criteria were selected as subjects with sensitive skin.
1. Age: 20-55 years old Gender: Female 3. Those who are aware of sensitive skin A person who falls under one or more of the following a or b: a. Select and use products that appear to be sensitive skin sensitive products b. Within the past year, I have experienced a tingling and tingling sensation using facial cleansers, skin care products and makeup bases.

Stinging test A was performed on the selected subjects by the following method.
2cm x 5cm non-woven fabric impregnated with 600µL of 16.4mM aqueous solution of methyl p-hydroxybenzoate or p-hydroxybenzoic acid after cleansing with make-up remover and face wash, acclimatized for more than 5 minutes in an environment of 23 ° C and 50% humidity Touch the cheeks, and after 30 seconds, 1 minute, 2 minutes, 3 minutes, and 5 minutes after the start of contact, the sensory stimuli perceived by the subject (pain, tingling, tingling, tingling, warm, hot ) (0: No feeling, 0.5: Slight or slightly uncomfortable, 1.0: Slightly uncomfortable, 1.5: Slightly uncomfortable, 2.0: (Slight discomfort, 2.5: Somewhat considerable discomfort, 3.0: Unbearable or considerable discomfort) 30 seconds to 5 minutes later, the maximum value of all scores from 30 seconds to 5 minutes A value obtained by subtracting the maximum value of the score when the test was conducted was statistically analyzed by a Wilcoxon signed rank sum test as a sensory stimulus score. The results are shown in FIG.
From FIG. 2, it was considered that the sensory stimulation of PHBA was very weak and hardly contributed to the sensory stimulation.

Reference Example 3 Comparison of CES1 expression level and metabolic activity between healthy skin and sensitive skin (1) Sting test B
After washing with a make-up remover and face wash, acclimatize for 5 minutes or more in an environment of 23 ° C. and 50% humidity, and contact a 10 cm ² non-woven fabric impregnated with 600 μL of a 16.4 mM aqueous solution of methyl p-hydroxybenzoate on the right cheek. , 30 seconds after the start of contact, 1 minute, 2 minutes, 3 minutes later, the score reported for the sensory stimulus recognized by the subject (0: feel nothing, 0.5: feel slightly, 1.0: Feel a little, 2.0: Feel clearly, 3.0: Feel very strong, I want to remove the non-woven fabric to a strong stimulus) The maximum value of all scores from 30 seconds to 3 minutes later The stimulation score was used. Next, after wiping the right neck with 70% isopropyl alcohol, a 2 cm × 2.5 cm nonwoven fabric impregnated with 300 μL of a 16.4 mM aqueous solution of methyl p-hydroxybenzoate (MP) was brought into contact with the wiping site, and contact was started. Scores declared for sensory stimuli perceived by subjects after 30 seconds, 1 minute, 2 minutes, 3 minutes (0: feel nothing, 0.5: feel slightly, 1.0: feel slightly, 2.0: Feel clearly, 3.0: Feel very strong, I want to remove the non-woven fabric to a strong stimulus) The maximum value of all scores from 30 seconds to 3 minutes later is the sensory stimulus score of the neck did.

(2) Selection of subjects Three people were selected as healthy skin subjects from those who meet all of the following criteria.
・ Age: 20-55 years old ・ Gender: Female ・ Person who is aware that it is not sensitive skin ・ Students who have a sensory stimulation score of 0 (no feeling) in cheeks and neck in sting test B

Three people who selected all of the following criteria were selected as sensitive skinners.
・ Age: 20-55 years old ・ Gender: Female ・ Person who is aware of sensitive skin ・ Chest sensory stimulation score is 2 (feels clear) in sting test B and neck sensory stimulation score is 0.5 More than (feel slightly)

(3) Skin biopsy Three individuals each from healthy and sensitive skin were selected, and the skin was collected from the right neck (site where the sting test was performed) with a 4 mmφ biopsy trepan, and the collected skin was liquid. Immediately frozen with nitrogen and stored at −80 ° C. until analysis.

(4) Expression level and activity of carboxylesterase 1 in biopsy skin 4mmφ biopsy skin is halved with a scalpel, one piece for carboxylesterase 1 expression level analysis and the remaining piece for carboxylesterase 1 activity analysis It was.
Ripple biopsy skin tissue for expression level analysis with scissors and add protease inhibitor mix (Wako Pure Chemical Industries) into microtube containing biopsy skin tissue (tissue) Weight (mg) × 20) μL was added, homogenized with a homogenizer (Hiscotron, Microtec Nithion Co., Ltd.), and then allowed to stand on ice for 30 minutes. After 30 minutes, sonication was performed at 4 ° C. for 2 minutes, and centrifugation was performed at 10,000 rpm and 4 ° C. for 20 minutes to obtain the supernatant as a skin extract. Using Pierce BCA Protein Assay Kit (Thermo Scientific), the amount of protein in the skin extract was quantified using bovine serum albumin as a standard protein.

Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed using the skin extract as a sample as follows.
The skin extract was separated by SDS-PAGE and transferred to a PVDF membrane (BIO RAD). The transferred PVDF membrane is soaked in 5% skim milk solution, blocked, and then subjected to a primary antibody reaction using an anti-carboxyl esterase 1 antibody (R & D Systems, Cat # MAB4920) diluted 1: 200, and diluted 1: 2000. The secondary antibody reaction was performed using the horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody (R & D Systems, Cat # HAF007). Light was emitted by Clarity Western ECL Substrate (BIO RAD), and antibody reaction was detected using LAS-4000 (Fuji Film Co., Ltd.). Bands of CES1 and actin were separately selected from the photographed Western blot image using image analysis software CS Analyzer (Atto Co., Ltd.), and the band intensity was measured to calculate the intensity of CES1 per actin. In addition, it was verified by a similar experiment using an anti-β-actin antibody as the primary antibody whether the amount of the sample loaded onto the polyacrylamide gel was uniform.

  After biopsy skin tissue for activity analysis is cut into small pieces with scissors, a 50 mM HEPES / 150 mM KCl (pH 7.4) solution (tissue weight (mg) × 20) μL is added, and a homogenizer (Hiscotron, Microtech Nichion Co., Ltd.). And then centrifuged at 10,000 rpm and 4 ° C. for 20 minutes to obtain the supernatant as a skin extract. The amount of protein in the skin extract was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). The final protein concentration of each skin extract was adjusted to 50 μg / mL, methyl p-hydroxybenzoate, which is a substrate for carboxylesterase 1, was added to 200 μM, and the mixture was reacted at 37 ° C. for 5 hours. Thereafter, the same amount of cold acetonitrile as the reaction solution was added to stop the reaction, and the metabolite p-hydroxybenzoic acid was quantified by HPLC, and the amount of p-hydroxybenzoic acid produced per unit protein and unit time was determined as CES1. Metabolic activity. The equipment, columns, and measurement conditions used for HPLC are shown below.

Equipment: Alliance 2695 Separation Module, 2996 Photodiode Array Detector (Waters)
Column: L-column ODS (4.6 × 150 mm, 5 μm, Chemical Substance Evaluation Research Organization)
Column temperature: 40 ° C
Mobile phase: 45% methanol / 0.1% formic acid Flow rate: 1.0 mL / min
Sample injection volume: 50 μL
Detection wavelength: 254 nm

The results are shown in FIGS.
From FIG. 3 and FIG. 4, it was confirmed that the skin of sensitive skin had lower CES1 expression level and CES1 metabolic activity than healthy skin.

Example 1 CES1 expression promoting action Normal human epidermal keratinocytes (Life Technologies Japan) were seeded in 6-well plates so that each well had 1 × 10 ⁵ , cultured overnight in HuMedia-KG2 (Kurabo), and then sulforaphane ( Sprouting broccoli extract ("Broccoli sprout extract" (for Oriza Oil & Chemicals Co., Ltd., cosmetics)) so that the final concentration is 0.1, 1, 10 μM, the solid concentration is 0.0001 w / v%. Alternatively, the cells were exposed to 0.001 w / v% and cultured for 48 hours. The cultured cells were washed with a phosphate buffer (D-PBS (−)), and RNA was extracted using RNeasy Mini Kits (Qiagen) according to the attached manual. The RNA concentration was calculated using NanoDrop1000 (Thermo Fisher Scientific). After performing reverse transcription reaction of Total RNA extracted using High-Capacity RNA-to-cDNA Kit (Life Technologies Japan), real-time PCR was performed using 7500 Fast Real-Time PCR System (Life Technologies Japan). Carried out. Real-time PCR was performed using the reverse-transcribed cDNA and carboxylesterase 1 or Taqman primer of RPLP0 as an endogenous control (Life Technologies Japan, CES1: Hs00275627-m1, RPLP0: HS00420895_gH), Taqman Gene Expression Master Mix (Life Technologies Japan). ) Were used to compare gene expression levels by the ΔΔCT method. The results are shown in Tables 1 and 2.

  From Tables 1 and 2, an increase in the expression level of CES1 mRNA was observed with sulforaphane or germinated broccoli extract.

Example 2 Sensory Stimulation Reduction Action A male sprouting broccoli extract having a solid content concentration of 0.01% was selected as a test subject who was aware of sensitive skin and felt sensory stimulation with methyl p-hydroxybenzoate (MP). Applying 10% ethanol solution ("Broccoli sprout extract" (Oryza Oil Chemical Co., Ltd., for cosmetics)) twice a day, once in a 500 yen coin size to the entire face of the subject for 1 week. The sensory stimulation scores from the front and rear sting test C were compared. Sting test C was carried out by the following method.
After washing the face with make-up remover and face wash, acclimatize for 5 minutes or more in an environment of temperature 23 ° C. and humidity 50%, apply 10 cm ² of nonwoven fabric impregnated with 600 μL of 16.4 mM aqueous solution of methyl p-hydroxybenzoate to the cheeks, Scores reported for sensory stimuli perceived by subjects 30 seconds to 3 minutes after application (0: feel nothing, 0.5: feel slightly, 1.0: feel slightly, 2.0: feel clear, 3 0.0: I want to remove the nonwoven fabric from a strong stimulus that feels very strong) and subtract the maximum value of the score when a similar test is performed with distilled water, and use the Wilcoxon signed rank sum as a sensory stimulus score. Statistical analysis was performed by test. The results are shown in FIG.

  From FIG. 5, it was confirmed that the sensory stimulation of methyl p-hydroxybenzoate was reduced by the germinated broccoli extract.

Claims (4)

  A sensory irritation reducing agent comprising sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.   The sensory stimulation reducing agent according to claim 1, wherein the sensory stimulation is a stimulation sensation caused by perabenes.   A CES1 expression promoter comprising sulforaphane, a plant containing sulforaphane or a glycoside thereof, or an extract thereof as an active ingredient.   The agent of any one of Claims 1-3 whose plant containing sulforaphane or its glycoside is germination broccoli.