Skin irritation inhibitor

Abstract

To provide a material and a formulation effective for reducing rash caused by menstrual blood.SOLUTION: A rash reducing agent for menstrual blood comprises, as an active ingredient, one or more plants selected from ginger, horse chestnut, and bladderwrack, or an extract thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a swelling inhibitor that suppresses the onset of swelling caused by menstrual blood.

Background Art

[0002] In women, the genitals (delicate area) often become swollen due to menstruation, vaginal discharge, sweating, rubbing against underwear, or menstrual pads. Such swelling in the delicate area is considered to be non-allergic contact dermatitis caused by irritation from excretions such as urine and blood, or menstrual products. Menstrual blood is the blood discharged from the vagina together with the endometrium during menstruation. It has been reported that such menstrual blood has pro-inflammatory properties (Non-Patent Document 1), causing strong itching and rashes during or immediately after menstruation, and in severe cases, swelling or blisters may occur.

[0003] For such swelling caused by menstrual blood, in order to suppress the developed inflammation, conventionally, drugs such as steroids, antihistamines, urea, and moisturizers have been used. However, their effects are not always sufficient, and various side effects are often observed with steroids, and there are safety problems for long-term continuous use. Therefore, it is desired to identify the inflammatory substances and suppress the onset of swelling.

[0004] On the other hand, it has been reported that ginger rhizome, Zingiber officinale, has an action of suppressing the activity of human fibroblast-derived elastase (Patent Document 1) and an action of inhibiting the activity of human fibroblast-derived neutral endopeptidase (Patent Document 2). Also, Malus domestica has an action of promoting the expression of antibacterial peptides derived from human skin (Patent Document 3) and an action of improving wrinkles (Patent Document 4), and Actaea rubra has an action of strengthening blood vessels (Patent Document 5) and an action of inhibiting the activity of pancreas-derived elastase (Patent Document 6), etc. However, it has not been known so far that Zingiber officinale, Malus domestica, and Actaea rubra have an action of inhibiting neutrophil elastase activity or an action of suppressing swelling.

Prior Art Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2000-119189 [Patent Document 2] Japanese Patent Publication No. 2001-335495 [Patent Document 3] Japanese Patent Publication No. 2020-7258 [Patent Document 4] Japanese Patent Publication No. 2005-8571 [Patent Document 5] Japanese Patent Publication No. 2006-036673 [Patent Document 6] Japanese Patent Publication No. 2001-181167 [Non-patent literature]

[0006] [Non-Patent Document 1] Cutan and Ocul Toxicol 24:243-246 2005 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention relates to providing materials and formulations that are effective in suppressing rashes caused by menstrual blood. [Means for solving the problem]

[0008] The inventors comprehensively analyzed the protein components in menstrual blood and investigated their relationship with skin irritation. They discovered that neutrophil elastase expression is elevated in individuals with skin irritation, and that menstrual blood with a higher neutrophil elastase content has a greater ability to induce IL-1a production in epidermal cells. They then found that extracts of ginger, horse chestnut, and bladderwrack have neutrophil elastase activity inhibitory effects, and that these are useful for suppressing skin irritation caused by menstrual blood.

[0009] In other words, the present invention relates to the following 1) to 2). 1) A rash inhibitor for menstrual blood, containing one or more plants selected from ginger, horse chestnut, and bladderwrack, or their extracts, as active ingredients. 2) A neutrophil elastase activity inhibitor containing one or more plants selected from ginger, horse chestnut, and bladderwrack, or their extracts, as an active ingredient. [Effects of the Invention]

[0010] The present invention's menstrual blood-induced rash inhibitor or neutrophil elastase activity inhibitor is useful for suppressing rashes caused by contact with menstrual blood, such as rashes in delicate areas. [Brief explanation of the drawing]

[0011] [Figure 1] Intergroup comparison of neutrophil elastase content in menstrual blood. [Figure 2] Relationship between neutrophil elastase content in menstrual blood and IL-1α production induction ability. [Figure 3] The inhibitory effect of plant extracts on neutrophil elastase activity. [Modes for carrying out the invention]

[0012] In this invention, "shokyo" refers to the rhizome of ginger (Zingiber officinale Roscoe) of the family Zingiberaceae. "Maronie" refers to the European horse chestnut tree (Aesculus hippocastanum), which belongs to the family Hippocastanaceae. "Hibamata" refers to Fucus distichus ssp. evanescens or Fucus evanescens, a species of bladderwrack belonging to the genus Fucus in the family Fucusaceae.

[0013] The parts of Zingiber officinale, Celtis sinensis, and Petasites japonicus (hereinafter also referred to as "the plants of the present invention") used are the rhizomes for Zingiber officinale, and for Celtis sinensis and Petasites japonicus, any of their parts, such as flowers, flower buds, flower spikes, fruit skins, fruits, stems, leaves, branches, branch leaves, trunks, tree barks, rhizomes, roots, seeds, whole herbs, or combinations thereof can be used. For Celtis sinensis, it is preferable to use its tree bark or seeds, and seeds are more preferable. For Petasites japonicus, it is preferable to use whole algae or petioles, and whole algae are more preferable.

[0014] Such plants can be used as they are, or as juice obtained by squeezing them, dried products obtained by drying the plant bodies themselves or their pulverized products, or extracts extracted from these, but it is preferable to use them as extracts.

[0015] The extracts of the plants of the present invention include various solvent extracts, dilutions thereof, concentrated solutions thereof, or dried powders thereof obtained by known extraction methods, such as extracting these at normal temperature or under heating, or using an extraction instrument such as a Soxhlet extractor for extraction. Examples of known extraction methods include immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, and microwave extraction.

[0016] The extraction solvent used to obtain the extract can be either a polar solvent or a non-polar solvent. Examples include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform, and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; and pyridines. These can be used individually or in mixtures. Of these, water, alcohol-based (alcohols and / or polyhydric alcohols) solvents, and water-alcohol mixed solvents are preferred, with water-alcohol mixed solvents being preferred. Furthermore, methanol, ethanol, propanol, and butanol are more preferred among the alcohols, with ethanol being even more preferred. Also, butylene glycol is more preferred among the polyhydric alcohols. More suitable water-alcohol mixed solvents include aqueous ethanol solutions with a concentration of 10% (v / v) or higher, and 95% (v / v) or lower, preferably 60% (v / v) or lower. For example, 10-95% (v / v) aqueous ethanol solutions, preferably 10-60% (v / v) aqueous ethanol solutions, are available. More suitable water-polyhydric alcohol mixed solvents include aqueous solutions of butylene glycol with a concentration of 5% (v / v) or more and 95% (v / v) or less, preferably 60% (v / v) or less. For example, 5-95% (v / v) aqueous solutions of butylene glycol, preferably 5-60% (v / v) aqueous solutions of butylene glycol, are available.

[0017] For obtaining the ginger extract, it is preferable to use a water-polyhydric alcohol mixed solvent from the above-mentioned extraction solvents, more preferably an aqueous butylene glycol solution, and even more preferably a 40-60% (v / v) aqueous butylene glycol solution. For obtaining the horse chestnut extract, it is preferable to use a water-alcohol mixed solvent from the above-mentioned extraction solvents, more preferably an aqueous ethanol solution, and even more preferably a 40-60% (v / v) aqueous ethanol solution. For obtaining the extract of Fucus vesiculosus, it is preferable to use a water-polyhydric alcohol mixed solvent from among the above-mentioned extraction solvents, more preferably an aqueous butylene glycol solution, and even more preferably a 5-15% (v / v) aqueous butylene glycol solution.

[0018] The extract of the present invention can be obtained, for example, by using 1 to 50 parts by mass of the extraction solvent per 1 part by mass of the plant of the present invention and extracting at 4 to 100°C for 0.5 hours to 30 days. More specifically, when water is used as the extraction solvent, 5 to 30 parts by mass per 1 part by mass of plant is preferred, and extraction is performed at 40 to 100°C for 1 hour to 1 day. When a water-ethanol mixed solvent is used as the extraction solvent, 5 to 30 parts by mass per 1 part by mass of plant is preferred, and extraction is performed at room temperature under reflux for 1 hour to 20 days. These operations may also be repeated.

[0019] The above extract can be used as is, or it can be diluted, concentrated, or freeze-dried and then prepared in powder or paste form before use. In addition to the extracted product, commercially available products may also be used.

[0020] Furthermore, it is preferable to remove inert impurities from the extract using known techniques such as liquid-liquid partitioning, solid-liquid partitioning, filtration membranes, activated carbon, adsorption resins, and ion exchange resins. The solvent used at this time may be one of the examples of extraction solvents described above. These may also be treated with deodorization, decolorization, or other known methods as necessary before use. Furthermore, when further purifying the extract, the known techniques and methods may be used.

[0021] As shown in the reference examples below, when we comprehensively analyzed the protein components in menstrual blood of individuals with rashes in the delicate area, divided them into a non-rash group and a rash group, we found that the rash group had higher expression levels of neutrophil elastase compared to the non-rash group (Figure 1). Furthermore, we found that menstrual blood with a higher neutrophil elastase content had a higher ability to induce IL-1α production in epidermal cells (Figure 2). Therefore, it is thought that neutrophil elastase inhibitors can suppress rashes caused by menstrual blood.

[0022] As shown in the examples below, the extracts of ginger, horse chestnut, and bladderwrack each have neutrophil elastase activity inhibitory effects. Therefore, the plants or their extracts of the present invention can serve as neutrophil elastase activity inhibitors or inhibitors of menstrual blood-induced skin irritation, and can also be used to produce neutrophil elastase activity inhibitors or inhibitors of menstrual blood-induced skin irritation. In other words, the plants or their extracts of the present invention can be used to inhibit neutrophil elastase activity or to suppress skin irritation in humans who may come into contact with menstrual blood. Here, use on humans may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical procedures, that is, methods of performing surgery, treatment, or diagnosis on humans, and more specifically, methods of performing surgery, treatment, or diagnosis on humans by a physician or a person under the direction of a physician.

[0023] The neutrophil elastase activity inhibitor or the anti-rash agent caused by menstrual blood may be in the form of using the plant or its extract alone, or in the form of a composition containing it (for example, a pharmaceutical or quasi-drug, a sanitary product, etc.). That is, the neutrophil elastase activity inhibitor or the anti-rash agent caused by menstrual blood of the present invention may be a pharmaceutical, quasi-drug, or sanitary product that exhibits a neutrophil elastase activity inhibitory effect or an anti-rash effect caused by menstrual blood, or it may be a material or formulation used in combination with such pharmaceutical, quasi-drug, sanitary product, etc.

[0024] In this invention, "neutrophil elastase" refers to a protease with a molecular weight of approximately 30,000 present in neutrophils in the blood, which has degrading activity for elastin, collagen, etc., and is registered as EC3.4.21.37. The base sequence of the neutrophil elastase gene is publicly known, for example, in humans and mice, and the human neutrophil elastase gene and protein are registered as NCBI accession numbers NM_1972.4 and NP_001963. Proteases that have degrading activity for elastin, collagen, etc. are collectively called elastases, and in addition to the neutrophil elastase of this invention, there are several other elastases in humans, such as fibroblast-derived elastase and pancreatic elastase, but each has its own unique EC number, expression site, active site type, and substrate specificity, and is a completely different enzyme. Neutrophil elastase activity inhibition means reducing or eliminating the activity of neutrophil elastase. The mode of inhibition may be substrate inhibition, competitive inhibition, non-competitive inhibition, non-competitive inhibition, or a combination of two or more of these inhibitions (mixed inhibition). Neutrophil elastase activity can be measured by the method described in the examples below.

[0025] In this invention, "rash caused by menstrual blood" refers to contact dermatitis caused by contact between menstrual blood and the skin, and more specifically, to irritant contact dermatitis (primary irritant contact dermatitis). The symptoms include erythema, papules, edema, vesicles, and other lesions that appear at the site of contact with the menstrual blood, the causative substance, and are accompanied by severe itching. For example, typical examples include rashes in the delicate area, urinary area, anal area, buttocks, groin, and lower back caused by absorbent items such as sanitary napkins and tampons that contain menstrual blood absorbent materials. Here, "delicate area" refers to the female genital area (groin), and in this specification, "groin area" or "delicate groin area" refers to the part of the delicate area between the groin and the labia. Furthermore, in this invention, "suppression of rash" refers to suppressing the onset of a rash.

[0026] The pharmaceuticals, quasi-drugs, or sanitary products containing the plant or its extract of the present invention may be in the form of external preparations or internal preparations, but preferably include external skin preparations (e.g., ointments, creams, emulsions, lotions, gels, aerosols, etc.), body cleansers, bath additives, etc. Such formulations can be obtained by general manufacturing methods, either directly or by mixing and dispersing them with formulation-acceptable carriers, such as various oils, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohols, water, chelating agents, thickeners, UV absorbers, emulsifying stabilizers, pH adjusters, dyes, fragrances, etc., and then processing them into the desired form. Furthermore, these pharmaceuticals, quasi-drugs, or sanitary products may be appropriately formulated with other plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, cooling agents, anti-seborrheic agents, etc., as appropriate, within limits that do not interfere with the effects of the present invention, depending on the respective formulation.

[0027] Pharmaceuticals, quasi-drugs, or sanitary products are applied directly or indirectly to the skin that comes into contact with menstrual blood, and this includes applications by incorporating these compositions into topical preparations, absorbent articles, or wipes. Here, "absorbent article" means any article capable of receiving and / or absorbing and / or containing and / or retaining bodily fluids or exudates, including menstrual blood, and preferably includes sanitary napkins, panty liners, tampons, etc. The composition can be incorporated into any component of the absorbent article by spraying, immersion, transfer, soaking, etc.

[0028] In embodiments of the present invention, the content of the active ingredient in a composition using the menstrual blood-induced rash inhibitor or neutrophil elastase activity inhibitor of the present invention can be appropriately determined depending on the form of the composition. The content of the plant or its extract of the present invention in the pharmaceutical, quasi-drug or sanitary article is generally 0.00001% by mass, preferably 0.001% by mass or more, and 10% by mass or less, preferably 1% by mass or less, as a solid content concentration. Furthermore, it is preferably 0.00001 to 10% by mass, and more preferably 0.001 to 1% by mass.

[0029] The dosage of the above-mentioned pharmaceuticals, quasi-drugs, or sanitary products is not particularly limited as long as an effective amount is obtained, and may vary depending on the subject's condition, weight, sex, age, or other factors. However, for an adult (60 kg) person, the daily dose of each extract (on a dry basis) is preferably 0.5 mg or more, and 1000 mg or less, and preferably 500 mg or less. It is also preferable that the dose be between 0.5 and 1000 mg, and more preferably between 0.5 and 500 mg. Furthermore, the preparation may be taken and administered according to any intake and administration plan, but it is preferable to administer it once or several times a day for several weeks to several months.

[0030] Furthermore, the target population for use of antiperspirants for menstrual blood-induced rashes or neutrophil elastase activity inhibitors is not particularly limited as long as it is necessary, but examples include people who come into contact with or may come into contact with menstrual blood, such as women during or immediately after menstruation. [Examples]

[0031] The present invention will be described in more detail below with reference to examples. Example 1: Proteomic analysis of menstrual blood proteins (1) Selection of subjects Under the Helsinki Declaration, a study was conducted on women aged 20 to 49 with stable menstrual cycles. Physicians assessed rash symptoms in the genital area using a 5-point scoring system (0: none, 1: mild, 2: mild, 3: moderate, 4: severe) for erythema, papules, maceration, and desquamation. Nine subjects with an erythema score of 0 in the groin and labia during menstruation were selected as the non-rash group, while eight subjects with an erythema score of 1 or higher in either the groin or labia were selected as the rash group.

[0032] (2) Collection of menstrual blood On the second or third day after the start of menstruation, menstrual blood was collected using a sanitary napkin with a collection dish placed on top. The collected menstrual blood was transferred to a centrifuge tube, centrifuged at 860 × g for 10 minutes, and the supernatant was collected.

[0033] (3) Solubilization and fragmentation of proteins 50 μL of menstrual supernatant was diluted 10-fold with PTS buffer (0.1 M Tris-HCl pH 9.0, 12 mM sodium deoxycholate, 12 mM sodium N-dodecanoylsarcosinate). After removing insoluble matter from the menstrual supernatant by centrifugation (21500 × g, 30 min, 4°C), hemoglobin was adsorbed and removed by mixing with an equal volume of nickel column beads (Ni-NTA) by inversion at room temperature for 1 hour. To the supernatant from which hemoglobin had been removed from menstrual blood supernatant, DTT (dithiothreitol) and iodoacetamide were added to final concentrations of 5 mM and 15 mM, respectively, and the mixture was shaken at room temperature for 1 hour to perform reductive alkylation. After dilution fivefold with 50 mM ammonium bicarbonate aqueous solution, the protein concentration was quantified using the EZQ Protein Quantitation Kit®, and 1 μg of trypsin (derived from porcine pancreas, mass spectrometry grade, 202-15951, Fujifilm Wako Pure Chemical Industries, Ltd.) per 100 μg was added, and the mixture was reacted at 37°C for 24 hours to perform peptide fragmentation.

[0034] Trifluoroacetic acid was added to inactivate the enzyme to a final concentration of 0.5% (v / v), and then the mixture was centrifuged at 15,000 rpm for 10 minutes to remove insoluble matter. The supernatant was mixed with an equal volume of ethyl acetate and stirred with a test tube mixer for 2 minutes to transfer sodium deoxycholate and sodium N-dodecanoyl sarcosinate to the organic layer. The mixture was then centrifuged at 21,500 x g for 2 minutes to separate the organic layer (ethyl acetate layer) from the aqueous layer. The aqueous layer was collected and dried under reduced pressure using a centrifugal evaporator (50°C, overnight). The obtained dry material was dissolved in 300 μL of Buffer A (0.1% (v / v) trifluoroacetic acid, 5% (v / v) acetonitrile aqueous solution), and applied to a Stage-tip column (solid-layer extraction column for proteome analysis) (BUNSEKI KAGAKU 57(12), 1011-1018, 2008) equilibrated with the same Buffer A. The peptide was adsorbed onto the column by centrifugation (500 × g, room temperature, 4 min). After washing the column with 1 mL of Buffer A, the peptide was eluted with 0.1 mL of Buffer B (0.1% trifluoroacetic acid, 80% acetonitrile aqueous solution) (500 × g, room temperature, 3 min). The eluate was dried under reduced pressure at 35°C, and the resulting precipitate was suspended in 100 μL of 0.1% formic acid, 2% acetonitrile to obtain the peptide solution.

[0035] (4)LC-MS / MS analysis The peptide concentration of the peptide solution obtained in (3) was quantified by nanoLC-UV, the concentration was adjusted to 0.2 μg / μL, and LC-MS / MS analysis was performed. The detailed conditions for each are shown in Tables 1 and 2.

[0036] [Table 1]

[0037] [Table 2]

[0038] Proteome Discoverer ver.2.2 (ThermoFisher Scientific) was used to analyze the spectral data obtained by MS / MS analysis. For protein identification, Swiss Prot was set as the reference database, and Mascot database search (Matrix Science) was used. Proteins satisfying a false discovery rate (FDR) p<0.01 were included in the analysis. The analysis was performed using the normalized abundance value of each protein.

[0039] (5) Results A differential expression analysis was performed between the rash group and the non-rash group, and when the menstrual supernatant components correlated with the presence or absence of rash were analyzed, as shown in Figure 1, neutrophil elastase in the menstrual supernatant tended to be higher in the rash group compared to the non-rash group.

[0040] Reference Example 2: Relationship between neutrophil elastase content in menstrual blood and IL-1α production induction ability (1) Method Epidermal cells derived from neonatal foreskin were used. The culture medium used was EpiLife® Medium, with 60 μM calcium (Thermo Fisher Cat. No. MEPI500CA) supplemented with HuMedia-KG growth additive set (Kurabo Cat. No. KK-6150). Epidermal cells were cultured in a 9.0 × 10⁶ size. 4 Cells were seeded in 24-well plates at a concentration of cells / well, and menstrual blood was added to the culture medium at a concentration of 0.1-10% (v / v) while the cells were subconfluent. When adding menstrual blood, the culture medium used was Epilife-KG2 medium minus medium, which was de-enhanced by removing growth factors (EGF and BPE). After 24 hours of exposure culture, the amount of the inflammatory cytokine IL-1α secreted into the collected medium was quantified using ELISA (R&D systems) according to the attached protocol and used as an indicator of inflammatoryity.

[0041] (2) Results When we analyzed the correlation between the inflammatory properties of menstrual blood (IL-1α secretion) and the neutrophil elastase content in the menstrual supernatant, which tended to be higher in the rash group in Reference Example 1, we found that the inflammatory properties of menstrual blood (IL-1α secretion) were positively correlated with the amount of neutrophil elastase in the menstrual supernatant (Figure 2).

[0042] Example 1: Evaluation of neutrophil elastase activity inhibitors of various plant extracts (1) Material Purified enzyme: Neutrophil elastase (Enzo Life Sciences, Inc., BML-SE284-9090) Materials used for evaluation: The following three types of plant extracts were used. • Ginger extract; Ginger extract (K) (Koei Kogyo), 50% (v / v) butylene glycol aqueous solution extract of ginger rhizome, evaporation residue 6.6% by mass • Horse chestnut extract; Horse chestnut extract (Maruzen Pharmaceutical), 50% (v / v) ethanol aqueous solution extract of horse chestnut seeds, evaporation residue 1.43% by mass • Fucus vesiculosus extract; Falcorex Fucus vesiculosus EX (Ichimaru Falcos), 10% (v / v) butylene glycol aqueous solution extract of Fucus vesiculosus (whole plant), evaporation residue 1.6% by mass

[0043] (2) Measurement of neutrophil elastase activity Enzyme activity was measured using the Neutrophil elastase colormetric drug discovery kit (BML-AL497). As shown in Table 3 below, the reaction solutions were prepared and incubated at 37°C for 1 to 20 hours. The absorbance at 405 nm was measured at 1, 2, 4, and 20 hours. Elastatinal, an elastase inhibitor, was used as an indicator of the inhibitory effect at final concentrations of 1, 10, and 100 μM. To eliminate the influence of the evaluation material on fluorescence intensity, wells containing only the evaluation material (without adding cell extract) were prepared and their fluorescence intensity measured in the same way. These wells were used as blanks, while wells without the evaluation material were used as controls.

[0044] [Table 3]

[0045] (3) Results As shown in Figure 3, extracts of ginger, horse chestnut, and bladderwrack showed neutrophil elastase activity inhibitory effects, indicating that these plant extracts can be used as inhibitors of menstrual blood-induced rashes.

Claims

1. A neutrophil elastase activity inhibitor comprising ginger or an extract thereof as an active ingredient, wherein the extraction solvent for the extract is water, an alcohol-based solvent, or a water-alcohol-based mixed solvent.

2. The neutrophil elastase activity inhibitor according to claim 1, wherein the extraction solvent for the extract is an aqueous solution of butylene glycol.

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