Anti-inflammatory drugs

Abstract

The objective is to provide an anti-inflammatory agent that can reduce oxidative damage and allergic symptoms of the scalp by using a substance that preferentially removes more damaging peroxides without hindering the progression of the oxidation reaction in hair dyeing. [Solution] An anti-inflammatory agent that reduces scalp inflammation, injury, and allergic inflammatory reactions that occur when dyeing hair with hair dyes that involve oxidation, and includes an antioxidant that removes peroxides produced by the reaction between the oxidative dyes contained in the hair dye and hydrogen peroxide without inhibiting the oxidation reaction during hair dyeing.

Description

[Technical Field]

[0001] This invention relates to an anti-inflammatory agent that reduces scalp inflammation, injury, and allergic inflammatory reactions that occur during hair dyeing using oxidation reactions. [Background technology]

[0002] Traditionally, most hair dye products use hair dyes containing oxidative dyes such as paraphenylenediamine (PPD), an aromatic amine compound. Some consumers are seeking hair dyes with natural ingredients and low irritation, and this demand is steadily increasing, leading to a growing market for hair dyes that do not contain oxidative dyes. However, hair dyes containing oxidative dyes are generally inexpensive and widely available, and compared to other compounds, they produce vivid and long-lasting colors, making them the dominant choice for many beauticians and consumers, and they currently dominate the hair dye market. According to 2010 statistics from the International Agency for Research on Cancer (IARC), it is estimated that 50-80% of women worldwide have used hair dye at least once in their lives. Furthermore, past surveys have shown that in Europe, 50.9% of people have experienced hair coloring at least once in their lifetime, and 81.4% of those were women. A past survey in South Korea reported that over 50% of people develop gray hair in their early 40s, and 60% of those continue to dye their hair regularly despite side effects. Furthermore, hair dyes containing PPD are said to account for more than 70% of the market share in Europe, the United States, and East Asia.

[0003] Hair dyes containing this oxidative dye produce pigment when oxidized with hydrogen peroxide (H2O2) to color the hair. However, as a side reaction, peroxides and Bandlovsky bases are produced, causing allergic symptoms and skin inflammation. When an allergy develops to diamine-based oxidative dyes such as PPD, mild cases result in irritant contact dermatitis such as burning or stinging during dyeing, while moderate cases result in allergic contact dermatitis such as itching, redness, and bumps that persist into the next day or later. After an allergic reaction appears, it worsens with each use of hair dye, and in severe cases, systemic symptoms may occur. In addition, although there are individual differences, even people without allergies may experience scalp inflammation due to oxides produced during the oxidation reaction of oxidative dyes, resulting in symptoms such as itching and redness. It is presumed that these allergic symptoms and inflammatory reactions can be improved if the above oxidation reaction is suppressed and the production of peroxides is inhibited. However, when reducing agents are used, even if oxidative damage is suppressed, the dyeing reaction, which is also an oxidation reaction, is also suppressed, so they could not be used. Furthermore, while applying protective creams to the scalp can reduce skin inflammation and oxidative damage, if these creams get on the hair, they can inhibit the dyeing process, resulting in uneven coloring. Therefore, the creams must be applied only to the scalp, avoiding the hair, which is time-consuming and impractical. On the other hand, for example, Patent Document 1 discloses a staining composition that suppresses skin inflammation by containing PPD and orthoaminophenol in predetermined proportions. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Unexamined Patent Publication No. 56-15205 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] According to Patent Document 1, it is possible to suppress skin inflammation by including orthoaminophenol, which prevents the formation of bandlovsky bases. However, at present, no anti-inflammatory agent has been realized that can effectively suppress skin inflammation without interfering with hair dyeing.

[0006] This invention has been made in view of the above circumstances, and aims to provide an anti-inflammatory agent that can reduce oxidative damage and allergic symptoms of the scalp by using a substance that does not hinder the progress of the oxidation reaction in hair dyeing and preferentially removes more damaging peroxides. [Means for solving the problem]

[0007] An anti-inflammatory agent according to the present invention, which serves the above purpose, is an anti-inflammatory agent that reduces scalp inflammation, injury and allergic inflammatory reactions that occur when dyeing hair with hair dyes that involve oxidation, and includes an antioxidant that removes peroxides produced by the reaction between the oxidative dyes contained in the hair dye and hydrogen peroxide without inhibiting the oxidation reaction during hair dyeing. Here, the antioxidant should be capable of removing the peroxide produced by the reaction between the oxidative dye and hydrogen peroxide. However, it must be an antioxidant that eliminates radicals without becoming a radical itself, and does not inhibit the reaction between hydrogen peroxide and the oxidative dye. In all hair dyes that use oxidative dyes and oxidizing agents, radicals are generated as an intermediate. Therefore, the anti-inflammatory agent according to the present invention can reduce damage and decomposition caused by radicals, and this agent is effective when dyeing hair using hair dyes that involve oxidation. Specifically, the oxidation reaction when dyeing hair with hair dyes (hair dyes) containing oxidative dyes such as paraphenylenediamine, toluene-2,5-diamine, para-aminophenol, aminophenol, 2,4-diaminophenol, and bis-aminophenol, which involve the oxidation of amine groups; dyes that involve the oxidation of carbonyl groups such as 2,2'-dichloroindigo, alizarin, carminic acid, chromium orange, naphthol, and hematoxylinic acid; dyes that involve the oxidation of imine groups such as rhodamine B, chlorineol, metaallergyline, alizarin red, and ligron, or dyes that involve the oxidation of azo groups such as azobenzene, azodyred, azoblue, azopurple, azoorange, and azogreen, is an oxidation reaction that involves the generation of radicals similar to those described above, and therefore this agent can be used.

[0008] In the anti-inflammatory agent according to the present invention, the antioxidant is preferably contained in a soil-derived extract obtained from soil collected from a geological layer formed more than 300,000 years ago when marine plants and marine animals accumulated under anaerobic conditions. Here, the soil collected from strata formed by the deposition of marine plants and marine animals under anaerobic conditions refers, for example, to strata formed when a part of the sea, isolated by land uplift more than 300,000 years ago, was covered by debris flows and / or volcanic ash, and the marine plants and marine animals in this covered sea repeatedly fermented and decomposed. Specifically, soil collected from strata 10 to 80 meters underground in the Isahaya region of Nagasaki Prefecture (commonly called siamarin) is preferably used, but it is not limited to this. Furthermore, the soil may be collected from strata dating back, for example, 100 million years ago, as long as it was deposited under anaerobic conditions and the ashification of organic matter has not progressed significantly.

[0009] In the anti-inflammatory agent according to the present invention, the soil-derived extract is preferably obtained by extracting water-soluble substances contained in the soil. Here, as the extraction solvent, aqueous solvents such as water, aqueous ethanol, and ethanol can be used. Also, the obtained soil-derived extract may be in a liquid state or may be a dried powder obtained by freeze-drying or the like. Note that one type of soil-derived extract (soil-derived extract = cialmarin extract (cialmarin is a registered trademark)) is a sugar chain nutrient extract, and as a specific example, Noguchi Catalyzer 21 (Catalyzer and Noguchi Catalyzer 21 are registered trademarks) manufactured and sold by the applicant of this application can be mentioned. The reaction rate constant between hydroxy radicals and antioxidants is as high as at least 10 9 M -1 ·S -1 On the other hand, for hydrogen peroxide, it is 10 1 ~10 4 M -1 ·S -1 and is about 10 5 ~10 8 There is a difference in the order of the reaction rate. Therefore, radical scavenging is possible as long as it is within a concentration range that does not inhibit the dyeing oxidation reaction by hydrogen peroxide. In the dyes being used, the concentration of hydrogen peroxide is about 10 -1 ~10 1 M. So, if the concentration is such that it does not inhibit the oxidation reaction between hydrogen peroxide and the dye, that is, a concentration of 1 / 100 or more with respect to the concentration of hydrogen peroxide used, it hardly inhibits dyeing. In theory, the lower the reaction rate constant between the antioxidant and hydrogen peroxide, the higher the additive concentration can be increased. However, in practice, due to the influence on pH and other effects other than the oxidation reaction caused by the addition of the antioxidant, it is desirable to add the minimum amount possible. Specifically, those with a high reaction rate constant with hydrogen peroxide are 10 1 mM or less, and those with a low reaction rate constant are 10 2 mM or less. If it is below this concentration, scalp oxidation damage can be suppressed without inhibiting the reaction of the dye. The soil-derived extract has a relatively mild reaction with hydrogen peroxide at about 10 1 M -1 ·S -1 and contains an antioxidant at a concentration of about 1 mM, so it can be added up to the maximum as long as it does not affect the viscosity of the hair dye. Therefore, there is no problem in adding such soil itself containing an antioxidant to the hair dye.

[0010] The anti-inflammatory agent according to the present invention may be added to the hair dye and used. Here, the anti-inflammatory agent can be added to the hair dye at the time of hair dyeing within a range that does not affect the viscosity of the hair dye, and the addition ratio is preferably at most 50% or less. In addition, the anti-inflammatory agent may be added to the hair dye in the state of a soil-derived extract (soil-derived extract solution), or may be added in the state of soil (the soil itself).

[0011] The anti-inflammatory agent according to the present invention may be applied to the scalp before the hair dyeing operation with the hair dye. Here, the anti-inflammatory agent may be applied to the scalp in the state of a soil-derived extract (soil-derived extract solution), or may be applied to the scalp in the state of soil (the soil itself).

Effects of the Invention

[0012] The anti-inflammatory agent according to the present invention contains an antioxidant that removes peroxides generated by the reaction of an oxidation dye and hydrogen peroxide contained in the hair dye without inhibiting the oxidation reaction during hair dyeing, thereby effectively suppressing only the oxidative damage of the scalp while maintaining a good dyeing reaction with the hair dye.

[0013] In the anti-inflammatory agent according to the present invention, when the antioxidant is contained in a soil-derived extract extracted from soil collected from a stratum formed by the deposition of marine plants and marine animals under anaerobic conditions more than 300,000 years ago, it is excellent in safety.

[0014] In the anti-inflammatory agent according to the present invention, when the soil-derived extract is obtained by extracting water-soluble substances contained in the soil, it is excellent in safety and mass productivity.

[0015] When the anti-inflammatory agent according to the present invention is added to the hair dye and used, there is no need to perform application of the anti-inflammatory agent etc. separately from the hair dyeing operation, and the workability is excellent.

[0016] When the anti-inflammatory agent according to the present invention is applied to the scalp before hair dyeing work with a hair dye, it can exhibit a high effect in suppressing scalp inflammation.

Brief Description of Drawings

[0017] [Figure 1] It is an explanatory diagram showing the reaction pathway of PPD and hydrogen peroxide during hair dyeing. [Figure 2] It is a diagram explaining the damage and inflammatory reaction to the living body that occur when a hair dye containing PPD reacts with an oxidizing agent. [Figure 3] (A) to (E) are images showing the results of the anti-inflammatory evaluation test using the anti-inflammatory agent according to the present invention. [Figure 4] It is a graph measuring the cell viability from the images of FIGS. 3(A) to (E). [Figure 5] (A) to (C) are graphs showing the results of the evaluation test of the ability of the anti-inflammatory agent according to the present invention to scavenge hydroxyl radicals. [Figure 6] It is a graph showing the peak of hydroxyl radicals in FIGS. 5(A) to (C). [Figure 7] It is an image confirming the influence of the anti-inflammatory agent according to the present invention on the dyeing ability of PPD. [Figure 8] It is a graph measuring the influence of the anti-inflammatory agent according to the present invention on the dyeing ability of PPD by absorbance. [Figure 9] It is an image confirming the influence of the anti-inflammatory agent according to the present invention on hair dyeing with PPD. [Figure 10] (A) to (C) are images confirming the influence of the anti-inflammatory agent according to the present invention on the cuticle of hair. [Figure 11] (A) to (C) are images confirming the influence of the concentration of the anti-inflammatory agent according to the present invention on hair dyeing, and (D) is a graph showing the result of calculating the dyeing rate from the images of (A) to (C). [Figure 12] It is an image confirming the influence of various antioxidants on hair dyeing. [Figure 13] It is a graph showing the result of calculating the dyeing rate from the image of FIG. 12. [Figure 14] This graph shows the results of an evaluation test to determine the inhibition of inflammatory substance production and release by the anti-inflammatory agent according to the present invention. [Modes for carrying out the invention]

[0018] Next, with reference to the attached drawings, embodiments of the present invention will be described to facilitate understanding of the invention. As shown in Figures 1 and 2, hair dyes (hair colorants) containing paraphenylenediamine (PPD), a representative example of an oxidative dye, are oxidized with hydrogen peroxide (H2O2). This reaction between PPD and H2O2 produces QDI (quinone diimine), which then reacts further to undergo oxidation, polymerization, and combination with other pigments to form the final pigment compound that colors the hair. However, it has been pointed out that PPD itself can induce allergic reactions associated with an increase in intracellular ROS, and it has also become clear that PPD oxidized by hydrogen peroxide generates mutagenic compounds called bandlovsky bases and hydroxyl radicals through oxidation reactions. In other words, as shown in Figure 2, the formation of (2) peroxides, (3) bandlovsky bases and peroxides associated with their oxidation leads to the progression of scalp damage and inflammation through processes (4), (5), and (6). Conversely, if the formation of (2) and (3) can be suppressed without inhibiting the formation of QDI, the associated damage and inflammation can be suppressed or prevented.

[0019] An anti-inflammatory agent according to one embodiment of the present invention reduces scalp inflammation, injury, and allergic inflammatory reactions that occur when dyeing hair using hair dyes containing oxidative dyes such as PPD. This anti-inflammatory agent should contain antioxidants that can remove peroxides produced by the reaction of oxidative dyes with hydrogen peroxide. Specifically, it should have a high reaction rate constant with hydrogen peroxide. 1 Below mM, with the lowest being 10 2Antioxidants containing less than mM of a substance can suppress oxidative damage to the scalp without inhibiting the reaction of hair dyes. Such antioxidants are found, for example, in soil-derived extracts taken from soil collected from geological layers (3-80m underground) formed more than 300,000 years ago when marine plants and marine animals (fish, shellfish, seaweed, moss, etc.) accumulated under anaerobic conditions.

[0020] This soil-derived extract may be a dried powder obtained by, for example, freeze-drying the soil, but it is preferable that it be in liquid form. The liquid soil-derived extract (soil-derived extract solution) is obtained by extracting water-soluble substances contained in the soil. Specifically, it is a solution obtained by extracting water-soluble substances from crushed or micronized soil and removing impurities. This soil-derived extract solution contains humic acid (humic acid and fulvic acid) as well as natural sugar chain nutrients. It is preferable that the soil-derived extract solution is sterilized. Water, aqueous ethanol, ethanol, and other aqueous solvents are preferably used as the extraction solvent, but are not limited to these. By adding this soil-derived extract (soil-derived extract solution) to hair dye as an anti-inflammatory agent, it is possible to suppress oxidative damage to the scalp without inhibiting the reaction of the hair dye. Alternatively, instead of adding this soil-derived extract (soil-derived extract solution) to hair dye, it can also be applied to the scalp as an anti-inflammatory agent before dyeing hair with a common (commercial) hair dye containing PPD. Furthermore, soil itself can be used as an anti-inflammatory agent instead of the soil-derived extract (soil-derived extract solution). [Examples]

[0021] Next, we will describe experiments conducted to confirm the effects of the present invention, but the following examples do not limit the technical scope of the present invention in any way.

[0022] <Evaluation study of inflammation (cytotoxicity) suppression using keratinocyte cells> The anti-inflammatory effect of the present invention on hair dyes containing PPD as an oxidative dye was verified using keratinocyte cells.

[0023] (Comparative Example 1) Human epidermal keratinocyte line HaCaT was subcultured in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, West Bio). 5 × 10 4 Cells were seeded at cells / ml, MQ water (Millipore) was added after 24 hours, and the cells were cultured for another 24 hours before being photographed using a microscope (TE2000-E, Nikon Instec). The HaCaT cells used (provided by the Department of Cell Control Engineering, Faculty of Agriculture, Kyushu University) are a type of human keratinocyte, a non-cancerous human keratinocyte cell line, and are widely used in fields such as skin research and cosmetic safety testing.

[0024] (Comparative Example 2) The evaluation was carried out in the same manner as in Comparative Example 1, except that 80 μg / ml of PPD and 2 mM hydrogen peroxide (H2O2) were added instead of MQ water (Millipore).

[0025] (Example 1) The evaluation was carried out in the same manner as in Comparative Example 2, except that 1% of Noguchi Catalyzer 21 (manufactured by Noguchi Research Institute Co., Ltd., a glycan nutrient extract), which is a type of soil-derived extract and acts as an anti-inflammatory agent according to the present invention, was added to PPD and H2O2.

[0026] (Example 2) The evaluation was carried out in the same manner as in Example 1, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 5%.

[0027] (Example 3) The evaluation was carried out in the same manner as in Example 1, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 10%.

[0028] When keratinocyte cells from Comparative Example 1 (untreated) were photographed, it was confirmed that cells were present across the entire image, as shown in Figure 3(A). In contrast, as shown in Figure 3(B), in Comparative Example 2, where keratinocyte cells were treated only with PPD and H2O2, it was found that the cells had detached, resulting in a patchy appearance. Furthermore, in Examples 1 to 3, where the anti-inflammatory agent according to the present invention was added to PPD and H2O2, it was confirmed that cell detachment decreased as the concentration of the added anti-inflammatory agent increased, as shown in Figures 3(C) to (E). In addition, the graph in Figure 4, which measures the number of cells in Figures 3(A) to (E), shows that cell survival is dependent on the concentration of the anti-inflammatory agent. From these results, it can be said that the cell damage and inflammation caused by PPD and H2O2 are suppressed by the anti-inflammatory agent. All tests were performed independently three times, and the graphs show the average values. Error bars indicate the standard deviation unless otherwise specified (the same applies to the following tests).

[0029] <Evaluation test of hydroxyl radical scavenging ability using magnetic resonance (ESR) spectrometer> The ability of the present invention's anti-inflammatory agent to scavenge hydroxyl radicals against hair dyes containing PPD as an oxidizing dye was verified using magnetic resonance imaging (ESR). Hydroxyl radicals generated by PPD and H2O2 can be trapped using DMPO, a radical spin trapping agent, and detected by ESR.

[0030] (Comparative Example 3) The DMPO-OH adduct strength was measured for a reaction solution (200 μL) consisting of 2 mg / ml PPD, 178 mM DMPO, and 2% H2O2. The reaction solution was prepared in a tube containing all reactants except the H2O2 solution. ESR measurements were performed using a free radical monitor (JES-FR30, JEOL Ltd.). The data acquisition parameters were as follows: magnetic field: 336 ± 10 mT, microwave power: 10 mW, modulation frequency: 100 kHz, modulation amplitude: 0.1 mT, microwave frequency: 9.4271 GHz, sweep time: 1 minute, time constant: 0.03 seconds.

[0031] (Example 4) The evaluation was carried out in the same manner as in Comparative Example 3, except that 1% of Noguchi Catalyzer 21 (manufactured by Noguchi Research Institute Co., Ltd., a glycan nutrient extract), which is a type of soil-derived extract and acts as an anti-inflammatory agent according to the present invention, was added to the reaction solution.

[0032] (Example 5) The evaluation was carried out in the same manner as in Example 4, except that the amount of anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 10%.

[0033] The evaluation results for Comparative Example 3 and Examples 4 and 5 are shown in Figures 5 and 6. As shown in Figures 5(A) and 6, in Comparative Example 3, where only PPD and 2% H2O2 were added and the anti-inflammatory agent according to the present invention was not added, radicals were trapped by DMPO and detected as a typical hydroxyl radical peak (DMPO-OH adduct). In Examples 4 and 5, where the anti-inflammatory agent according to the present invention was added to PPD and H2O2, as shown in Figures 5(B), (C) and 6, it was confirmed that the DMPO-OH adduct decreased as the concentration of the added anti-inflammatory agent increased. From these results, it is considered that radicals generated by PPD and H2O2 are reduced by the anti-inflammatory agent, and that the anti-inflammatory agent of the present invention functions effectively as a radical scavenger and can suppress various types of damage caused by radical (peroxide) generation.

[0034] <Confirmation of the effect of anti-inflammatory drugs on pigment production> The effect of the anti-inflammatory agent according to the present invention on the staining ability of PPD, an example of an oxidative dye, was investigated.

[0035] (Comparative Example 4) The coloration of a liquid to which only 2% H2O2 was added was photographed.

[0036] (Comparative Example 5) The coloring process of a reaction solution prepared by adding 2% H2O2 to 2% PPD was filmed. The absorbance of the reaction solution after 30 minutes was also measured to confirm the amount of pigment produced.

[0037] (Example 6) The evaluation was carried out in the same manner as in Comparative Example 5, except that 0.1% of Noguchi Catalyzer 21 (manufactured by Noguchi Research Institute Co., Ltd., a glycan nutrient extract), which is a type of soil-derived extract, was added to the reaction solution as an anti-inflammatory agent according to the present invention.

[0038] (Example 7) The evaluation was carried out in the same manner as in Example 6, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 1%.

[0039] (Example 8) The evaluation was carried out in the same manner as in Example 6, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 2%.

[0040] (Example 9) The evaluation was carried out in the same manner as in Example 6, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 5%.

[0041] (Example 10) The evaluation was carried out in the same manner as in Example 6, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 10%.

[0042] (Comparative Example 6) The evaluation was carried out in the same manner as in Comparative Example 5, except that the PPD concentration was set to 0.2%.

[0043] (Example 11) The evaluation was carried out in the same manner as in Example 6, except that the PPD concentration was set to 0.2%.

[0044] (Example 12) The evaluation was carried out in the same manner as in Example 7, except that the PPD concentration was set to 0.2%.

[0045] (Example 13) The evaluation was carried out in the same manner as in Example 8, except that the PPD concentration was set to 0.2%.

[0046] (Example 14) The evaluation was carried out in the same manner as in Example 9, except that the PPD concentration was set to 0.2%.

[0047] (Example 15) The evaluation was carried out in the same manner as in Example 10, except that the PPD concentration was set to 0.2%.

[0048] As shown in Figure 7, when H2O2 is added to PPD, coloring progresses over time. However, comparisons of Examples 6-10 and Examples 11-15 confirm that coloring is accelerated as the concentration of the added anti-inflammatory agent increases. This is also evident from the absorbance measurement results shown in Figure 8, indicating that the anti-inflammatory agent of the present invention does not suppress, but rather accelerates, the dyeing of hair by hair dye containing PPD.

[0049] <Confirmation of the effects of anti-inflammatory agents on hair dyeing> The effect of the anti-inflammatory agent according to the present invention on hair dyeing using PPD, an example of an oxidative dye, was investigated.

[0050] Figure 9 shows images of the dyed state after 30 minutes when human gray hair was added to the reaction solutions similar to those used in Comparative Example 5 and Examples 6-10, and Comparative Example 6 and Examples 11-15. Purchased human gray hair was used for the tests, and for the reaction solutions in Examples 6-10 and 11-15, the gray hair was added immediately after the anti-inflammatory agent was added.

[0051] As seen in the image in Figure 9, no visible changes in staining were observed under any of the conditions (regardless of the concentration of the anti-inflammatory agent, including 0%). Furthermore, as shown in Figures 10(A) to (C), the surfaces of undyed hair (gray hair), hair dyed under the same conditions as Comparative Example 5, and hair dyed under the same conditions as Example 10 were photographed, and the effect on the cuticle was examined. No change was observed whether or not an anti-inflammatory agent was added. Based on these results, it can be said that even in actual hair dyeing using hair dyes containing PPD, the anti-inflammatory agent of the present invention does not have any visible effect on the dyeing reaction and does not damage the hair.

[0052] Next, the effect of the anti-inflammatory agent according to the present invention on hair dyeing was investigated using actual commercially available hair dyes. (method) When mixing commercially available dye solutions 1 and 2, anti-inflammatory agents at various concentrations (0, 10, and 30%) adjusted with MQ water were added to make up 10% of the total volume. This mixture was then applied to human gray hair and dyed. Figures 11(A) to (C) are photographs of gray hair after application and subsequent time intervals (2.5 minutes, 5 minutes, 7.5 minutes, and 10 minutes), at which point the dyeing reaction was stopped by washing. Figure 11(D) is a graph showing the dyeing rate of Figures 11(A) to (C) calculated using image analysis software (ImageJ). As the dyeing time increased (5 minutes, 7.5 minutes, and 10 minutes), the dyeing-promoting effect of the anti-inflammatory agent concentration could not be confirmed. However, at the initial stage (2.5 minutes), it was confirmed that the dyeing-promoting effect was observed depending on the concentration of the anti-inflammatory agent. Based on these results, it can be said that the anti-inflammatory agent of the present invention does not actually inhibit dyeing, but rather can promote it, and at the very least, does not inhibit dyeing.

[0053] Next, we investigated the effects of various antioxidants on hair dyeing using commercially available hair dyes. Figure 12 shows images (photographs) of hair dyed for 10 minutes, starting from left to right, in the case of no additives and in the case of mixing the anti-inflammatory agent of the present invention, 10 mM ascorbic acid, 10 mM reduced glutathione, 10 mM cysteine, 10 mM gallic acid, and 10 mM hydroquinone, respectively. Figure 13 is a graph showing the dyeing rate of the hair dyed under each condition, obtained by image analysis of each image in Figure 12.

[0054] When each antioxidant was mixed at a concentration of 10 mM, ascorbic acid showed almost no change compared to the control (no additive), but reduced glutathione showed a slight effect compared to the control (no additive), with a lower staining rate, and cysteine, gallic acid, and hydroquinone also showed an effect on staining. Ascorbic acid has a low reaction rate constant with hydrogen peroxide (approximately 10 mM). 2 M -1 ·S -1), and therefore it is thought that it did not inhibit staining. Alternatively, it is possible that it became a monodehydroascorbic acid radical through its own oxidation, and as a result, promoted the oxidation reaction. The higher inhibition rate of cysteine ​​compared to glutathione can be attributed to the fact that its reaction rate constant with hydrogen peroxide is approximately 10^2 higher than that of reduced glutathione. Furthermore, the high inhibition rates of gallic acid and hydroquinone are presumed to be due to their three reducing groups, resulting in a quantitatively higher rate of reduction reaction. While ascorbic acid appears suitable as an anti-inflammatory agent, its own oxidation triggers a chain reaction, making it a radical generator. Therefore, it is considered unsuitable as an anti-inflammatory agent according to the present invention. Both reduced glutathione and cysteine ​​have thiols as reducing groups, and upon oxidation, they become oxidized glutathione and cystine, respectively, and hardly become radicals themselves. Antioxidants that do not generate such radicals are suitable as anti-inflammatory agents, but considering the reaction rate with hydrogen peroxide, it is necessary to add them at concentrations below those that do not affect staining. These results suggest that suitable antioxidants as anti-inflammatory agents are those that do not inhibit the oxidation reaction in staining and do not generate radicals after the reaction. In such cases, it is important to use them at concentrations below those that do not inhibit the reaction between hydrogen peroxide and PPD. Alternatively, a method that uses multiple antioxidants to suppress the generation of antioxidant radicals may also be used.

[0055] <Evaluation test of inhibition of inflammatory substance production and release by anti-inflammatory agents> In allergic reactions, mast cells scattered throughout the skin come into contact with allergens, releasing inflammatory substances such as histamine, which triggers an inflammatory response (allergic symptoms). More specifically, when high-affinity IgE receptors (FcεRI) on mast cells are stimulated by IgE antibodies that react with allergens, the mast cells become activated, releasing inflammatory mediators such as histamine contained in granules, causing inflammation. Using the widely used rat basophil-like cell line RBL-2H3, we investigated the inhibitory effect of the present invention's anti-inflammatory agent on the production and release capabilities of inflammatory substances.

[0056] (Comparative Example 7) RBL-2H3 cells were seeded in a 96-well culture plate and sensitized with anti-dinitrophenyl (DNP) IgE antibody for 2 hours. After removing the culture medium, the cells were washed with Tyrode's buffer (MT buffer) and cultured in MT buffer for 10 minutes. Subsequently, the cells were stimulated with DNP-conjugated human serum albumin (DNP-HSA) for 3 hours, and the supernatant was collected on ice. The cells were then lysed and collected in Tyrode's buffer containing 0.1% Triton X-100, and protein quantification was performed using the cell lysates. 4-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside, a chromogenic substrate for β-hexosaminidase, was added to the collected culture supernatant and cell lysates. After stopping the enzymatic reaction, the absorbance at 405 nm was measured using an absorbance microplate reader to determine the enzymatic activity of β-hexosaminidase. Based on these measurement results, the rate of β-hexosaminidase release per unit of protein from cells (amount of inflammatory substance release) was calculated.

[0057] (Comparative Example 8) The evaluation was performed in the same manner as in Comparative Example 7, except that 100 nM wartmannin was added to the MT buffer as a degranulation inhibitor during cell culture for 10 minutes.

[0058] (Example 16) The evaluation was carried out in the same manner as in Comparative Example 7, except that when culturing cells for 10 minutes, Noguchi Catalyzer 21 (manufactured by Noguchi Research Institute Co., Ltd., a glycan nutrient extract), a type of soil-derived extract that acts as an anti-inflammatory agent according to the present invention, was added to the MT buffer to a final concentration of 0.1%.

[0059] (Example 17) The evaluation was carried out in the same manner as in Example 16, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 1%.

[0060] (Example 18) The evaluation was carried out in the same manner as in Example 16, except that the amount of anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 5%.

[0061] (Example 19) The evaluation was carried out in the same manner as in Example 16, except that the amount of the anti-inflammatory agent (Noguchi Catalyzer 21) added was set to 10%.

[0062] As shown in Figure 14, the amount of inflammatory substances released was highest in Comparative Example 7, where neither a degranulation inhibitor nor an anti-inflammatory agent was added. In Comparative Example 8, where wartmannin was added as a degranulation inhibitor, the amount of inflammatory substances released was suppressed compared to Comparative Example 7. Furthermore, in Examples 16 to 19, where an anti-inflammatory agent was added, the amount of inflammatory substances released was suppressed more than in Comparative Example 8. In particular, in Example 16, where 0.1% of the anti-inflammatory agent was added, and in Example 17, where 1% was added, concentration-dependent suppression of inflammatory substances was confirmed, with Example 17 showing the lowest amount of inflammatory substances released. Significant suppression of inflammatory substances was also confirmed in Example 19, where 10% of the anti-inflammatory agent was added. From these results, it is presumed that the anti-inflammatory agent of the present invention has the effect of suppressing degranulation by mast cells in allergic patients, and a similar suppressive effect on allergic symptoms can be expected in people with PPD allergies.

[0063] The anti-inflammatory agent of the present invention (Noguchi Catalyzer 21) is a solution containing stable antioxidant capacity. If the oxidation reaction were suppressed by the anti-inflammatory agent, the staining itself would not proceed. However, the results of the above test show that staining is not actually inhibited. This means that the oxidation reaction of hydrogen peroxide (H2O2), which is essential for hair dye components, has little to no effect on the staining reaction. Therefore, if the target of the anti-inflammatory agent is a peroxide such as a hydroxyl radical, a scheme can be established to suppress cell damage without inhibiting the staining reaction. Since hydroxyl radicals are highly reactive and their reaction rate constant is about 10^6 larger than that of hydrogen peroxide, there is a good possibility that the anti-inflammatory agent selectively eliminates hydroxyl radicals without eliminating hydrogen peroxide.

[0064] Furthermore, even people without allergies can expect to benefit from the anti-inflammatory effect of this agent in preventing inflammation. This is because when the skin barrier is damaged due to the generation of hydroxyl radicals and peroxides, these harmful substances can easily penetrate the skin, resulting in inflammation. However, with the anti-inflammatory agent of this invention, the skin barrier is protected by the removal of peroxides, and as a result, inflammation is prevented by inhibiting the penetration of harmful substances and the entry of allergens. It can also be expected that the inflammatory response will be suppressed by inhibiting the degranulation reaction by mast cells when allergens enter the skin. In the above example, PPD was used as the oxidation dye. However, as explained earlier, in hair dyeing with hair dyes containing oxidation dyes that involve the oxidation of amine groups, carbonyl groups, imine groups, or azo groups, oxidation reactions involving the generation of radicals occur. Therefore, the anti-inflammatory agent according to the present invention can be widely applied to hair dyeing using hair dyes that involve oxidation, and can reduce scalp inflammation, damage, and allergic inflammatory reactions by eliminating (removing) radicals (peroxides).

[0065] Although embodiments of the present invention have been described above, the present invention is not limited to the above-described forms, and includes other embodiments and modifications that can be conceivable within the scope of the matters described in the claims, and any changes to conditions that do not depart from the gist of the invention are all within the scope of application of the present invention.

Claims

1. An anti-inflammatory agent that reduces scalp inflammation, injury, and allergic inflammatory reactions that occur when dyeing hair with hair dyes that involve oxidation, An anti-inflammatory agent characterized by containing an antioxidant that removes peroxides produced by the reaction between the oxidative dye contained in the hair dye and hydrogen peroxide, without inhibiting the oxidation reaction during hair dyeing.

2. The anti-inflammatory agent according to claim 1, characterized in that the antioxidant is contained in a soil-derived extract obtained from soil collected from a geological layer formed more than 300,000 years ago by the accumulation of marine plants and marine animals under anaerobic conditions.

3. The anti-inflammatory agent according to claim 2, characterized in that the soil-derived extract is obtained by extracting water-soluble substances contained in the soil.

4. The anti-inflammatory agent according to claim 1, characterized in that it is used by being added to the aforementioned hair dye.

5. The anti-inflammatory agent according to claim 1, characterized in that it is applied to the scalp before the hair dyeing process using the aforementioned hair dye.

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