Inhibitor of ages generation in sweat

Abstract

To provide an AGEs formation inhibitor in sweat SOLUTION: The agent for suppressing the formation of AGEs in sweat contains an extract of Pashanbae and / or an extract of Chinese milk vetch as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for an inhibitor of AGE production in sweat. [Background technology]

[0002] AGEs (Advanced Glycation End Products) are advanced glycation end products produced by the protein glycation reaction (Maillard reaction), a non-enzymatic reaction between the amino groups of amino acids, peptides, and proteins and ketones or aldehydes (especially reducing sugars) (Patent Document 1). AGEs are known to accumulate in the body, and it has been reported that accumulated AGEs cause various skin-related symptoms such as age spots and wrinkles (Non-Patent Document 1).

[0003] As a prior art, Patent Document 2 discloses a method for screening drugs that have the effect of preventing or ameliorating disorders caused by AGEs, using holometabolous insect larvae.

[0004] Furthermore, Patent Document 3 discloses the use of a combination of celery extract and rutin to inhibit the production of AGEs. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-008460 [Patent Document 2] Japanese Patent Application Laid-Open No. 2011-095194 [Patent Document 3] Patent Publication No. 2021-175715 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of the Japanese Society of Cosmetic Chemists, Vol. 53, No. 2, 2019, pp. 83-90 Summary of the Invention [Problem to be solved by the invention]

[0007] In spite of the above-mentioned prior art, the present inventors have found that AGEs are produced by glycation of dermcidin. Based on the above findings, the present inventors discovered a screening technique for components that inhibit AGE production in sweat (AGE production inhibitors in sweat), and completed the present invention.

[0008] That is, an object of the present invention is to provide a novel method for screening for an agent that inhibits the production of AGEs in sweat. In particular, an objective of the present invention is to provide a novel screening method that uses the amount of AGEs produced by the glycation of dermcidin as an indicator. [Means for solving the problem]

[0009] The screening method of the present invention that solves the above problems comprises: This is a method for screening inhibitors of AGE formation in sweat, characterized in that the amount of AGEs formed by the glycation of dermcidin is used as an indicator to screen for inhibitors of AGE formation in sweat.

[0010] A preferred embodiment of the screening method of the present invention is characterized in that a candidate substance that produces a smaller amount of AGEs formed by glycation of dermcidin is judged to be a better inhibitor of AGE formation in sweat.

[0011] In a preferred embodiment of the screening method of the present invention, the index is based on the amount of AGEs produced in a sample containing dermcidin and glucose under UV irradiation and high temperature conditions.

[0012] Furthermore, a preferred embodiment of the screening method of the present invention includes the steps of: applying a candidate substance to a sample containing dermcidin and glucose; a measuring step of measuring the amount of AGEs produced by glycation of dermcidin in the sample after the applying step; The present invention is characterized by having the following.

[0013] Furthermore, a preferred embodiment of the screening method of the present invention is an application step of applying two or more candidate substances to two or more samples containing dermcidin and glucose, respectively; a measuring step of measuring the amount of AGEs produced by glycation of dermcidin in each of the samples after the applying step; a determining step for determining a candidate substance having a more excellent inhibitory effect on the production of AGEs in sweat based on the measurement results of the measuring step; The present invention is characterized by having the following.

[0014] The present invention also relates to a method for screening a topical skin composition for improving skin condition, which comprises using the AGE concentration of a sample containing dermcidin and glucose as an indicator to screen for active ingredients of the topical skin composition for improving one or more skin conditions selected from abnormal epidermal keratinization, collagen degradation, decreased function of the arrector pili muscles, inflammation, skin sagging, wrinkles, abnormal keratinization at the opening of pores, and abnormalities in the dermal collagen structure around pores.

[0015] The present invention also provides a method for measuring the degree of glycation of sweat using the amount of AGEs produced in sweat as an indicator.

[0016] The present invention also relates to an agent for inhibiting the production of AGEs in sweat, which contains as an active ingredient a Pashambe extract and / or a Rengeso extract.

[0017] In a preferred embodiment of the inhibitor of AGE production in sweat of the present invention, the inhibitor of AGE production in sweat of the present invention is intended to prevent and / or improve one or more skin conditions selected from abnormal epidermal keratinization, collagen degradation, and impaired function of the arrector pili muscles caused by contact of AGEs in sweat with the epidermis.

[0018] In a preferred embodiment of the inhibitor of AGE production in sweat of the present invention, the inhibitor of AGE production in sweat of the present invention is intended for preventing and / or improving one or more skin conditions selected from inflammation, sagging skin, wrinkles, and prominent pores, which are symptoms caused by factors produced in the epidermis by AGEs in sweat.

[0019] In a preferred embodiment of the agent for inhibiting the production of AGEs in sweat of the present invention, the agent for inhibiting the production of AGEs in sweat of the present invention is intended to prevent and / or improve deterioration of the dermal collagen structure around pores and / or abnormal keratinization at the opening of pores.

[0020] In a preferred embodiment of the agent for inhibiting the formation of AGEs in sweat of the present invention, the agent for inhibiting the formation of AGEs in sweat is intended to inhibit the glycation of dermcidin in sweat.

[0021] In a preferred embodiment of the agent for inhibiting the formation of AGEs in sweat of the present invention, the agent for inhibiting the formation of AGEs in sweat of the present invention is a composition for external application to the skin.

[0022] The present invention also relates to a skin condition-improving external application composition for skin, which comprises the aforementioned agent for inhibiting the production of AGEs in sweat and an anti-inflammatory and / or abnormal keratinization inhibitor.

[0023] In a preferred embodiment of the skin external composition for improving skin condition of the present invention, the anti-inflammatory and / or abnormal keratinization inhibitor has an inhibitory effect on the production of TNF-α and / or KRT16.

[0024] In a preferred embodiment of the skin condition-improving external composition of the present invention, the anti-inflammatory and / or abnormal keratinization inhibitor is a ripe bitter melon extract and / or an artemisia princeps extract.

[0025] The present invention also relates to an anti-inflammatory and / or abnormal keratinization inhibitor containing a ripe bitter melon extract and / or an artemisia extract as an active ingredient.

[0026] In a preferred embodiment of the anti-inflammatory and / or abnormal keratinization inhibitor of the present invention, the anti-inflammatory and / or abnormal keratinization inhibitor has an inhibitory effect on the production of TNF-α and / or KRT16. [Effects of the Invention]

[0027] According to the present invention, a novel method for screening for an inhibitor of AGE production in sweat can be provided. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows the test results of Test Example 1, for the amount of AGEs produced after 8 hours under UV irradiation and high temperature conditions. [Figure 2] FIG. 1 is a graph showing the inhibitory effects of Pashanbe extract and Astragalus extract on the production of AGEs in Test Example 2. [Figure 3] 1 shows test results of Test Example 3 showing the influence of AGEs on the expression level of the KRT16 gene. [Figure 4] 1 shows test results of Test Example 3 showing the influence of AGEs on the amount of TNF-α gene expression. [Figure 5] 1 shows the test results of Test Example 4-1 showing the effect of ripe bitter melon extract on the expression level of the KRT16 gene. [Figure 6] 4 shows the test results of Test Example 4-1 showing the effect of ripe bitter melon extract on the expression level of TNF-α gene. [Figure 7] 1 shows the test results of Test Example 4-2 showing the effect of a combination of ripe bitter melon extract and mugwort extract on the expression level of the KRT16 gene. [Figure 8] 1 shows test results showing the effect of a combination of ripe bitter melon extract and mugwort extract on the expression level of the TNF-α gene in Test Example 4-2. [Figure 9] 1 shows test results of Test Example 5 showing the effect of TNF-α on collagen gel contractility. DETAILED DESCRIPTION OF THE INVENTION

[0029] Preferred embodiments of the present invention will be described below, although it goes without saying that the technical scope of the present invention is not limited to the following embodiments.

[0030] <1> Screening Method The screening method of the present invention uses the amount of AGEs produced by glycation of dermcidin as an index.

[0031] Here, dermcidin and glucose are components contained in sweat. Therefore, the screening method of the present invention makes it possible to screen for inhibitors of AGE production in sweat that inhibit the glycation of sweat.

[0032] In this specification, "inhibition of the production of AGEs in sweat" refers to inhibition of the glycation of components in sweat.

[0033] In the screening method of the present invention, it is preferable that a candidate substance that produces a smaller amount of AGEs formed by glycation of dermcidin is judged to be a superior inhibitor of AGE formation in sweat.

[0034] The screening method of the present invention preferably comprises adding a candidate substance to a sample containing dermcidin and glucose, and measuring the amount of AGEs produced by glycation of dermcidin in the sample.

[0035] Specifically, it is preferable that the candidate substance be determined to be a candidate inhibitor of AGE production in sweat when the amount of AGEs produced in a sample to which the candidate substance has been added is statistically significantly less than the amount of AGEs produced in a comparison group to which the candidate substance has not been added.

[0036] Alternatively, if the amount of AGEs produced in a sample to which a candidate substance has been added is less than one-fold the amount of AGEs produced in a comparison group to which the candidate substance has not been added, the candidate substance may be determined to be a candidate inhibitor of AGE production in sweat.

[0037] Here, the sample containing dermcidin and glucose is not particularly limited as long as it allows measurement of the amount of AGEs produced.

[0038] In a sample containing dermcidin and glucose, the dermcidin content can be set as a guideline of 50 μg / mL or more, more preferably 80 μg / mL or more, and even more preferably 90 μg / mL or more.

[0039] In a sample containing dermcidin and glucose, the dermcidin content can be set as a guideline of 200 μg / mL or less, more preferably 150 μg / mL or less, and even more preferably 110 μg / mL or less.

[0040] The glucose content in the sample containing dermcidin and glucose was 1×10 -5 mol / L or more, more preferably 1×10 -4 mol / L or more, more preferably 1×10 -3 mol / L or more, more preferably 1×10 -2 mol / L or more, more preferably 1×10 -1 mol / L or more, and more preferably 1 mol / L or more can be used as a guideline.

[0041] In a sample containing dermcidin and glucose, the dermcidin content is preferably 1.1 μg / mL, more preferably 5 μg / mL or more, and even more preferably 10 μg / mL or more.

[0042] As a sample containing dermcidin and glucose, for example, a liquid sample containing dermcidin and glucose can be used. Sweat can also be used as a sample containing dermcidin and glucose.

[0043] Preferred methods for measuring the amount of AGEs produced by glycation of dermcidin in a sample include ELISA and HPLC.

[0044] More specifically, the present invention relates to a method for producing a pharmaceutical composition comprising: an application step of applying a candidate substance to a sample containing dermcidin and glucose; a measuring step of measuring the amount of AGEs produced by glycation of dermcidin in the sample after the applying step; It is also possible to have a form having the following.

[0045] The present invention also provides an application step of applying two or more candidate substances to two or more samples containing dermcidin and glucose, respectively; a measuring step of measuring the amount of AGEs produced by glycation of dermcidin in each of the samples after the applying step; a determining step for determining a candidate substance having a more excellent inhibitory effect on the production of AGEs in sweat based on the measurement results of the measuring step; It is also possible to have a form having the following.

[0046] Here, the applying step preferably includes a treatment in which the candidate substance is added to a sample containing dermcidin and glucose, and then the sample is allowed to stand under UV irradiation conditions.

[0047] For UV irradiation in the application step, UVB (280 to 315 nm) can be used.

[0048] Furthermore, the applying step preferably includes a treatment in which the candidate substance is added to a sample containing dermcidin and glucose, and then the sample is allowed to stand under high temperature conditions.

[0049] Here, the temperature condition in the application step is preferably 35°C or higher, more preferably 36°C or higher, and even more preferably 37°C or higher. By using the above-mentioned form, it is possible to more reliably screen for an AGE production inhibitor in sweat.

[0050] Furthermore, the temperature conditions in the screening method of the present invention are preferably 50°C or lower, more preferably 45°C or lower, and even more preferably 40°C or lower. By using the above-mentioned form, it is possible to more reliably screen for an AGE production inhibitor in sweat.

[0051] Here, in the application step, the standing time after adding the candidate substance to the sample containing dermcidin and glucose is preferably 4 hours or more, more preferably 7 hours or more, more preferably 20 hours or more, more preferably 36 hours or more, more preferably 50 hours or more, and more preferably 100 hours or more. By using the above-mentioned form, it is possible to more reliably screen for an AGE production inhibitor in sweat.

[0052] Furthermore, in the applying step, the time for leaving the sample containing dermcidin and glucose after adding the candidate substance is preferably less than 10 hours, more preferably less than 8 hours. By using the above-mentioned form, it is possible to more efficiently screen for an inhibitor of AGE production in sweat.

[0053] In the screening method of the present invention, the AGE production inhibitors in sweat to be screened are not particularly limited, and examples of candidate substances include commercially available compounds (including peptides), known compounds (including peptides), compounds obtained by combinatorial chemistry techniques, natural components derived from plants or marine organisms, and animal tissue extracts.

[0054] The term "animal or plant-derived extract" refers to not only the extract itself derived from an animal or plant, but also a fraction of the extract, a purified fraction, and a solvent-removed product of the extract, fraction, or purified product. Examples of plant-derived extracts include extracts from wild or cultivated plants, extracts sold as raw materials for herbal medicines, and commercially available extracts.

[0055] As will be shown in the Examples below, inhibiting the glycation of dermcidin contributes to the prevention and / or improvement of skin conditions caused by AGEs in sweat coming into contact with the epidermis.

[0056] That is, the present invention can also be used as a method for screening active ingredients of an external skin composition for improving skin condition, which prevents and / or improves skin conditions caused by contact of AGEs in sweat with the epidermis, using the degree of glycation of a sample containing dermcidin and glucose as an indicator.

[0057] Here, skin conditions caused by contact of AGEs in sweat with the epidermis include symptoms caused by factors (e.g., TNF-α and / or KRT16) produced by the epidermis in response to AGEs in sweat, such as abnormal epidermal keratinization, collagen degradation, decreased function of the arrector pili muscles, inflammation, sagging skin, wrinkles, and prominent pores.

[0058] Therefore, the present invention can also be embodied as a method for screening for an active ingredient of an external skin composition for improving skin condition, which uses the degree of glycation of a sample containing dermcidin and glucose as an indicator to prevent and / or improve one or more symptoms caused by factors produced from the epidermis in response to AGEs in sweat, selected from abnormal epidermal keratinization, collagen degradation, decreased function of arrector pili muscles, inflammation, sagging skin, and wrinkles.

[0059] Furthermore, by inhibiting the glycation of dermcidin, it is possible to prevent and / or improve abnormal keratinization at the opening of pores and deterioration of the dermal collagen structure around pores. That is, the present invention can also be embodied as a method for screening active ingredients of an external skin composition for improving skin condition, which uses the degree of glycation of a sample containing dermcidin and glucose as an indicator to prevent and / or improve abnormal keratinization at the opening of pores and deterioration of the dermal collagen structure around pores.

[0060] The present invention can also be used as a method for measuring the degree of glycation of sweat using the amount of AGEs produced in sweat as an indicator. The above-described contents can be applied to a preferred embodiment of the method of the present invention for measuring the degree of glycation of sweat using the amount of AGEs produced in sweat as an index.

[0061] <2> Inhibitor of AGE production in sweat and topical skin composition for improving skin condition <2-1> Inhibitor of AGE production in sweat The agent for inhibiting the production of AGEs in sweat of the present invention comprises a Pashanbe extract and / or a Rengeso extract.

[0062] Pashambe extract is a general term not only for the extract derived from Bergenia ligulata itself, but also for fractions of these extracts, purified fractions, and solvent-removed extracts, fractions, and purified products. Examples of extracts derived from Bergenia ligulata include extracts using wild-grown or cultivated plants, extracts using plants sold as raw materials for herbal medicines, and commercially available extracts.

[0063] When extracting the Pashambe extract, it is preferable to process the roots of Bergenia ligulata in advance by crushing or cutting them into small pieces to improve the extraction efficiency.

[0064] The extract can be obtained by the following method. 1 to 30 parts by weight of a solvent is added to 1 part by weight of Bergenia ligulata root or its dried product, and the mixture is immersed for several days at room temperature or for several hours at temperatures near the boiling point. After immersion, the mixture is cooled to room temperature, and insoluble matter is removed if desired. The solvent is then removed by, for example, vacuum concentration. The desired extract can then be fractionated and purified using column chromatography packed with silica gel or ion exchange resin.

[0065] The extraction solvent is preferably a polar solvent, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, 1,3-butylene glycol is a preferred extractant.

[0066] The term "Astragalus extract" refers not only to the extract derived from Astragalus itself, but also to fractions of these extracts, purified fractions, and products obtained by removing the solvent from the extracts, fractions, or purified products. Examples of extracts derived from Astragalus include extracts using wild-grown or cultivated plants, extracts using plants sold as raw materials for herbal medicines, and commercially available extracts. In addition, the astragalus extract can be prepared by extracting the whole plant or a dried product thereof from astragalus grown wild or cultivated in Japan.

[0067] In addition, when extracting the astragalus extract, it is preferable to process the whole plant of astragalus or a dried product thereof in advance by crushing or cutting into small pieces so as to improve the extraction efficiency.

[0068] The extract can be obtained by the following method. 1 to 30 parts by mass of solvent is added to 1 mass of whole or dried astragalus plant, and the mixture is immersed for several days at room temperature or for several hours at temperatures near the boiling point. After immersion, the mixture is cooled to room temperature, and insoluble matter is removed if desired. The solvent is then removed by, for example, vacuum concentration. The mixture is then fractionated and purified using column chromatography packed with silica gel or ion exchange resin to obtain the desired extract.

[0069] The extraction solvent is preferably a polar solvent, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, ethanol and 1,3-butylene glycol are preferred as extraction solvents.

[0070] As shown in the Examples below, Pashambe extract and Astragalus extract have the effect of inhibiting the production of AGEs from dermcidin and glucose. It has also been confirmed that AGEs from dermcidin and glucose significantly increase KRT16 gene expression. KRT16 is a gene associated with abnormal keratinization due to hyperproliferation of epidermal cells. That is, the active ingredient of the present invention is preferably applied to prevent and / or improve the deterioration of skin condition caused by AGEs in sweat coming into contact with the epidermis. More specifically, the active ingredient of the present invention can be used to prevent and / or improve abnormal epidermal keratinization and abnormal keratinization of pore openings caused by contact of AGEs in sweat with the epidermis.

[0071] Furthermore, as shown in the Examples below, it has been confirmed that AGEs derived from dermcidin and glucose significantly increase TNF-α gene expression. Here, TNF-α is known to cause a decrease in type I collagen production and an increase in collagen-degrading factor (MMP-1) in fibroblasts. Furthermore, TNF-α expression is low under normal conditions, but it has been reported as a representative inflammatory mediator in epidermal cells whose production increases in response to external stimuli such as contact dermatitis. That is, the active ingredient of the present invention can be used to prevent and / or improve one or more skin conditions selected from collagen degradation, impaired function and inflammation of arrector pili muscles, sagging skin, and wrinkles, which are symptoms caused by factors produced from the epidermis by AGEs in sweat.

[0072] The active ingredient of the present invention can also be used to prevent and / or improve abnormal keratinization at the opening of pores and deterioration of the dermal collagen structure around pores.

[0073] The present invention can also provide a method for inhibiting the glycation of sweat, which comprises applying a Pashanbe extract and / or a Rengeso extract to the skin.

[0074] Here, the method of the present invention for inhibiting glycation of sweat on the skin is a non-therapeutic method, preferably a cosmetic method, more preferably a cosmetic method for the skin.

[0075] The above-described contents can be applied to the preferred embodiment of the method for inhibiting glycation of sweat on the skin of the present invention.

[0076] Here, the agent for inhibiting the production of AGEs in sweat of the present invention is preferably in the form of a composition for external use on the skin.

[0077] Suitable examples of external skin compositions include cosmetics and pharmaceuticals.

[0078] Among these, it is preferable to use the cosmetic in the form of a cosmetic that can be used continuously, such as a lotion, emulsion, serum, cream, gel, sun care product, etc.

[0079] The content of Pashanbe extract and / or Astragalus extract in the composition for external use on skin is 0.05% by mass or more, preferably 0.08% by mass or more, and more preferably 0.1% by mass or more. The content of Pashambe extract and / or Astragalus extract in the composition for external use on skin is preferably 15% by mass or less, preferably 12% by mass or less, and more preferably 10% by mass or less.

[0080] More preferred forms of the external skin composition will be described below.

[0081] <2-2> Skin topical composition for improving skin condition The present invention is preferably in the form of an external skin composition for improving skin conditions, which contains the aforementioned agent for inhibiting the production of AGEs in sweat and an anti-inflammatory and / or abnormal keratinization inhibitor. By using the above-mentioned inhibitor of AGE production in sweat in combination with an anti-inflammatory and / or abnormal keratinization inhibitor, a skin condition-improving external composition for skin that exhibits even more excellent effects can be obtained.

[0082] Here, the anti-inflammatory and / or abnormal keratinization inhibitor to be combined with the aforementioned inhibitor of sweat AGE production is preferably one that has an inhibitory effect on the production of TNF-α and / or KRT16.

[0083] As shown in the examples below, ripe bitter melon extract and / or mugwort extract can be used for anti-inflammatory and / or abnormal keratinization suppression purposes by inhibiting the production of TNF-α and / or KRT16. Therefore, preferred examples of the anti-inflammatory and / or abnormal keratinization inhibitor to be combined with the aforementioned inhibitor of AGE production in sweat include ripe bitter melon extract and / or mugwort extract.

[0084] The term "ripe bitter melon extract" refers to not only the extract itself derived from ripe bitter melon, but also a collective term for fractions of these extracts, purified fractions, and solvent-removed extracts, fractions, and purified products. Examples of extracts derived from ripe bitter melon include extracts using wild-grown or cultivated plants, extracts sold as raw materials for herbal medicines, and commercially available extracts. In addition, the ripe bitter melon extract can be produced by extracting the fruit or dried product of ripe bitter melon that grows wild or is grown in Japan.

[0085] Furthermore, when extracting a ripe bitter melon extract, it is preferable to process the ripe bitter melon fruit or a dried product thereof in advance by crushing or shredding it so as to improve the extraction efficiency.

[0086] The extract can be obtained by the following method. 1 to 30 parts by mass of solvent is added to 1 mass of ripe bitter melon fruit or its dried product, and the mixture is immersed for several days at room temperature or for several hours at a temperature near the boiling point. After immersion, the mixture is cooled to room temperature, and insoluble matter is removed if desired, followed by removal of the solvent by vacuum concentration or the like. The mixture is then fractionated and purified using column chromatography packed with silica gel or ion exchange resin to obtain the desired extract.

[0087] The extraction solvent is preferably a polar solvent, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, 1,3-butylene glycol is a preferred extractant.

[0088] Artemisia extract is a general term that refers not only to the extract derived from Artemisia per se, but also to fractions of these extracts, purified fractions, and products from which the solvent has been removed from the extracts, fractions, and purified products. Examples of extracts derived from Artemisia include extracts using wild-grown or cultivated plants, extracts using plants sold as raw materials for herbal medicines, and commercially available extracts. In addition, the mugwort extract can be prepared by extracting the whole plant of mugwort that grows wild or is cultivated in Japan, or a dried product thereof.

[0089] In addition, when extracting an artemisia extract, it is preferable to process the whole plant of artemisia or its dried product in advance by crushing or cutting into small pieces so as to improve the extraction efficiency.

[0090] The extract can be obtained by the following method. 1 to 30 parts by mass of solvent is added to 1 mass of the whole plant of Artemisia or its dried material, and the mixture is immersed for several days at room temperature or for several hours at a temperature near the boiling point. After immersion, the mixture is cooled to room temperature, and insoluble matter is removed if desired, followed by removal of the solvent by vacuum concentration or the like. The mixture is then fractionated and purified using column chromatography packed with silica gel or ion exchange resin to obtain the desired extract.

[0091] The extraction solvent is preferably a polar solvent, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol, polypropylene glycol, and 1,3-butylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. Among these, ethanol and 1,3-butylene glycol are preferred as extraction solvents.

[0092] When provided in the form of a skin external composition, the composition is not particularly limited, and it may contain any commonly used optional ingredients as long as it does not impair the effects of the present invention. Such optional ingredients include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japan wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, ivotaro wax, lanolin, reduced lanolin, hard lanolin, and jojoba wax; liquid paraffin, squalane, pristane, ozokerite, paraffin, ceresin, petrolatum, mica, citric acid ... hydrocarbons such as crystalline wax; higher fatty acids such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, and undecylenic acid; higher alcohols such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol; cetyl isooctanoate, isopropyl myristate, hexyldecyl isostearate, diisopropyl adipate, sebacillus acidophilus, and the like; Synthetic ester oils such as di-2-ethylhexyl phosphate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, glycerin di-2-heptylundecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane erythritol tetra-2-ethylhexanoate, and other oils; fatty acid soaps (sodium laurate, sodium palmitate, etc.) anionic surfactants such as potassium lauryl sulfate and alkyl sulfate triethanolamine ether; cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride and laurylamine oxide; amphoteric surfactants such as imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine-based surfactants (alkyl betaine, amido betaine, sulfobetaine, etc.), and acyl methyl taurine;Sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic (registered trademark) types, Preferred examples of such surfactants include nonionic surfactants such as POE·POP alkyl ethers (POE·POP 2-decyltetradecyl ether, etc.), Tetronics, POE castor oil / hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, erythritol, sorbitol, maltitol, propylene glycol, and 2,4-hexanediol; moisturizing ingredients such as sodium pyrrolidonecarboxylate, lactic acid, and sodium lactate; para-aminobenzoic acid-based UV absorbers; anthranilic acid-based UV absorbers; salicylic acid-based UV absorbers; cinnamic acid-based UV absorbers; benzophenone-based UV absorbers; sugar-based UV absorbers; and UV absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane.

[0093] <2-3> Oral composition The present invention can also be in the form of an oral composition. When an oral composition is prepared, it is preferable to prepare a food composition containing the active ingredient of the present invention. Specifically, it can be in the form of a supplement having the dosage form of a general food, tablet, granule, drink, or the like.

[0094] Here, the contents and content ratios of Pashambe extract and / or Astragalus extract in the oral composition can be determined mutatis mutandis from the description of the preferred embodiment of the topical skin composition described above.

[0095] When the composition is prepared as an oral composition, optional ingredients may be appropriately blended within the range that does not impair the effects of the present invention. [Example]

[0096] The following shows various test results that support the findings that form the basis of the present invention.

[0097] <Test Example 1> Verification of AGE production by glycation reaction of peptide (Dermcidin)

[0098] (1) Method First, 1 M glucose / PBS was added to a 24-well dish. Next, 1 mg / mL dermcidin (Bachem Ag 4061590) was added in an amount 1 / 10 of the amount of glucose / PBS (final concentration: 100 μg / mL dermcidin).

[0099] The prepared samples were exposed to UV light (equivalent to 8 hours in summer) and then left to stand at 37°C for 8 hours, at which point the AGE concentration was measured (Figure 1: High temperature with UV). In addition, a sample that was left standing for 8 hours at 4°C without UV irradiation was used as a comparative example (low temperature / no UV in Figure 1). The results are shown in Figure 1.

[0100] (2) Results and considerations As shown in Figure 2, it was found that even after 8 hours, the peptide (dermcidin) and sugar (glucose) reacted to produce AGEs.

[0101] <Test Example 2> Selection of an extract that inhibits sweat glycation We investigated the components that inhibit the glycation of a peptide (dermcidin) contained in sweat.

[0102] (1) Method First, 1M glucose / PBS was added to a 24-well dish. Next, 1 mg / mL dermcidin (Bachem Ag 4061590) was added in an amount 1 / 10 of the amount of glucose / PBS (final concentration: 100 μg / mL dermcidin). In addition, Pashanbe extract and Astragalus extract were added in an amount 1 / 10 of the amount of glucose / PBS as candidate substances for inhibitors of AGE production in sweat (Example, final concentration: 10% Pashanbe extract, 10% Astragalus extract).

[0103] In addition, a comparative example was prepared without adding a candidate substance for an inhibitor of AGE production in sweat (no extract in Figure 2).

[0104] The prepared sample was irradiated with UV light (equivalent to one week in summer) and then left to stand at 37°C for one week. AGE concentrations were measured by ELISA. The results are shown in Figure 2.

[0105] (2) Results and considerations The results of this test confirmed that the combination of Pashambe extract and Astragalus extract had the effect of inhibiting the production of AGEs by Dermcidin. That is, it was found that the above method can be used to screen for sweat glycation inhibitors.

[0106] Furthermore, the results of this study showed that Pashanbe extract and Astragalus extract are inhibitors of AGE production in sweat, which have the effect of inhibiting the glycation of sweat.

[0107] <Test Example 3> Verification of the effect of AGEs in sweat on the expression of keratinizing factors and inflammatory factors in epidermal cells

[0108] (1) Method First, NHEK (normal human epidermal keratinocytes: Kurabo KK-4009, age: newborn, race: Caucasian, sex: male) were seeded (25,000 cells / well) into a 24-well dish. The seeded cells were cultured at 37°C in a 5% CO2 environment for 24 hours. The medium was replaced, and the cells were again cultured at 37°C in a 5% CO2 environment for 24 hours.

[0109] The medium was removed, and a medium containing 10% AGEs was added, followed by culturing for 24 hours at 37°C in a 5% CO2 environment (Example; AGEs-Dermcidin in the figure). Here, a medium containing no AGEs was used as a comparative example (Control in the figure).

[0110] After washing with PBS(-), mRNA was extracted according to standard methods, and cDNA was synthesized. Thereafter, quantitative RT-PCR was performed using QuantiTect Primer Assay (QIAGEN) to measure the gene expression levels of KRT16 and TNF-α. Here, the gene expression level was calculated using the comparative CT method, with β-actin as the endogenous control.

[0111] (2) Results As shown in Figure 3, it was confirmed that AGEs formed from dermcidin and glucose (AGEs-dermcidin; AGEs in sweat) significantly increased the production of KRT16.

[0112] Here, KRT16 is known to be associated with abnormal keratinization due to hyperproliferation of epidermal cells.

[0113] In other words, it was found that when AGEs in sweat come into contact with epidermal cells containing pores, abnormal keratinization occurs at the opening of the pores.

[0114] Furthermore, as shown in Figure 4, it was confirmed that AGEs formed from dermcidin and glucose (AGEs-dermcidin; AGEs in sweat) significantly increased the production of TNF-α, an inflammatory factor.

[0115] TNF-α is known to decrease type I collagen production and increase collagen-degrading factor (MMP-1) in fibroblasts. TNF-α is reported to be a major inflammatory mediator in epidermal cells, whose expression is low under normal conditions but whose production increases in response to external stimuli such as contact dermatitis.

[0116] In other words, it was found that when AGEs made from dermcidin and glucose (AGEs-dermcidin; AGEs in sweat) come into contact with epidermal cells, including pores, inflammation occurs in the epidermis. It was also found that when dermcidin and glucose come into contact with epidermal cells or penetrate into the pores made up of epidermal cells, collagen production decreases and collagen degradation occurs in the dermis.

[0117] <Test Example 4-1> Verification of the inhibitory effect of ripe bitter melon extract on the expression of keratinizing factors and inflammatory factors in epidermal cells

[0118] (1) Method First, NHEK (normal human epidermal keratinocytes: Kurabo KK-4009, age: newborn, race: Caucasian, sex: male) were seeded (25,000 cells / well) into a 24-well dish. The seeded cells were cultured at 37°C in a 5% CO2 environment for 24 hours. The medium was replaced and a medium containing ripe bitter melon extract was added to prepare the example (ripe bitter melon extract in the figure) (final concentration of ripe bitter melon extract: 0.25%). Here, a medium containing no ripe bitter melon extract was used as a comparative example (AGEs Control in the figure). Thereafter, the cells were again cultured at 37°C in a 5% CO2 environment for 24 hours.

[0119] The medium was removed, and medium containing ripe bitter melon extract and 10% AGEs was added, followed by incubation at 37°C in a 5% CO2 environment for 24 hours.

[0120] After washing with PBS(-), mRNA was extracted according to standard methods, and cDNA was synthesized. Thereafter, quantitative RT-PCR was performed using QuantiTect Primer Assay (QIAGEN) to measure the gene expression levels of KRT16 and TNF-α. Here, the gene expression level was calculated using the comparative CT method, with the comparative example set as 1 (standard). The results are shown in Figures 5 and 6.

[0121] (2) Results As shown in Figures 5 and 6, the ripe bitter melon extract was found to have an inhibitory effect on the expression levels of KRT16 and TNF-α genes.

[0122] <Test 4-2> Verification of the effect of combining ripe bitter melon extract with mugwort extract (1) Method First, NHEK (normal human epidermal keratinocytes: Kurabo KK-4009 Lot. 06445, age: newborn, race: Caucasian, sex: male) were seeded (25,000 cells / well) into a 24-well dish. The seeded cells were cultured at 37°C in a 5% CO2 environment for 24 hours. The medium was replaced with a medium containing ripe bitter melon extract and mugwort extract, and this was used as the example (ripe bitter melon extract + mugwort extract in the figure) (final concentrations of ripe bitter melon extract: 0.25%, mugwort extract: 0.0125%). Here, a medium containing neither fully ripe bitter melon extract nor mugwort extract was used as a comparative example (AGEs control in the figure). Thereafter, the cells were again cultured at 37°C in a 5% CO2 environment for 24 hours.

[0123] The medium was removed, and ripe bitter melon extract, mugwort extract, and medium containing 10% AGEs were added, followed by incubation at 37°C in a 5% CO2 environment for 24 hours.

[0124] After washing with PBS(-), mRNA was extracted according to standard methods, and cDNA was synthesized. Thereafter, quantitative RT-PCR was performed using QuantiTect Primer Assay (QIAGEN) to measure the gene expression levels of KRT16 and TNF-α. Here, the gene expression level was calculated using the comparative CT method, with the comparative example set as 1 (standard). The results are shown in Figures 7 and 8.

[0125] (2) Results As shown in Figures 9 and 10, it was found that the combination of ripe bitter melon extract and mugwort extract had the effect of suppressing the expression levels of KRT16 and TNF-α genes.

[0126] <Test Example 5> Verification that AGEs in sweat cause a decrease in the function of the arrector pili muscles (1) Method First, a collagen solution having the following composition was prepared.

[0127] [Table 1]

[0128] The prepared collagen solution was added to a 24-well plate at 250 μL / well and left to stand at 37°C in a 5% CO environment for 15 minutes. The prepared collagen solution was solidified to prepare a collagen gel.

[0129] The cell suspension was then diluted to 2.5 x 10 5 The diluted cell suspension was diluted 10-fold with collagen solution. The diluted cell suspension was added to the collagen gel at 1000 μL / well and allowed to stand at 37°C in a 5% CO 2 environment for 4 hours.

[0130] After standing, the gel was peeled off from the inner wall of the well using a spatula. To the experimental group (TNF-α addition group), 750 μL / well of TNF-α-containing FBS-containing D-MEM medium (final concentration: 50 ng / mL) was added. In addition, 750 μL / well of D-MEM medium containing 10% FBS was added to the comparative example (control group).

[0131] The TNF-α concentration was adjusted to a final concentration of 50 ng / mL in 2 mL (1.25 mL gel + 0.75 mL medium) on the day of seeding, and after the day of seeding, it was added to a final concentration of 50 ng / mL in 0.75 mL medium.

[0132] The cells were left standing for one week in a 37°C, 5% CO2 environment, with the medium being changed every two days. After standing, the surface areas of the gels were compared by taking photographs from above the medium under the same conditions, binarizing the images, and calculating the areas for both the comparative and example gels.

[0133] (2) Results When the Comparative Example (control group) was compared with the Example (TNF-α added group), the area of ​​the collagen gel seeded with smooth muscle cells was significantly larger in the Example (TNF-α added group). The above results indicate that collagen gel contraction was inhibited by TNF-α. In other words, it was found that the addition of TNF-α reduced the contractile ability of collagen gel.

[0134] In other words, it was found that the suppression of TNF-α production by the above-mentioned active ingredients makes it possible to prevent or improve the decline in the function of the arrector pili muscles. [Industrial Applicability]

[0135] The present invention can be applied to a method for screening for inhibitors of AGE production in sweat.

Claims

1. An inhibitor of AGEs production in sweat, containing Pashambe extract and / or Astragalus extract as active ingredients.

2. The AGE production inhibitor in sweat according to claim 1, for preventing and / or improving one or more skin conditions selected from abnormal epidermal keratinization, collagen degradation, and decreased function of the arrector pili muscles caused by contact of AGEs in sweat with the epidermis.

3. 3. An inhibitor of AGEs production in sweat according to claim 1 or 2, for preventing and / or improving one or more skin conditions selected from inflammation, sagging skin, and wrinkles, which are symptoms caused by factors produced from the epidermis by AGEs in sweat.

4. An inhibitor of AGEs production in sweat according to any one of claims 1 to 3 for preventing and / or improving abnormal keratinization at the opening of pores and / or deterioration of the dermal collagen structure around pores.

5. The agent for inhibiting the production of AGEs in sweat according to any one of claims 1 to 4, for inhibiting the glycation of dermcidin in sweat.

6. The AGEs production inhibitor in sweat according to any one of claims 1 to 5, which is a composition for external use on the skin.

7. The sweat AGEs production inhibitor according to any one of claims 1 to 6, an anti-inflammatory and / or abnormal keratinization inhibitor; A topical skin composition for improving skin condition, comprising:

8. The skin condition-improving external composition for skin application according to claim 7, wherein the anti-inflammatory and / or abnormal keratinization inhibitor has an inhibitory effect on the production of TNF-α and / or KRT16.

9. The skin condition-improving external composition for skin application according to claim 8, wherein the anti-inflammatory and / or abnormal keratinization inhibitor is a ripe bitter melon extract and / or an artemisia extract.

10. An anti-inflammatory and / or abnormal keratinization inhibitor containing a ripe bitter melon extract and / or a mugwort extract as active ingredients.

11. The anti-inflammatory and / or abnormal keratinization inhibitor according to claim 10, which has an inhibitory effect on the production of TNF-α and / or KRT16.

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