JP6288759B2 - Transglutaminase activator - Google Patents

Description

  The present invention relates to a transglutaminase activator, a ceramide production promoter, and an involucrin expression promoter.

  The stratum corneum of the epidermis has a barrier function to prevent transpiration of moisture in the body, irritation from the outside world, and entry of foreign substances. The stratum corneum is composed of corneocytes and intercellular lipids, and the corneocytes are encapsulated in a cell membrane-like structure called a cornified envelope. The cornified envelope contributes to the construction of a stable keratinocyte structure and is an important structure for maintaining the barrier function of the skin. The cornified keratinocytes in the cornified basal layer are keratinized, and involucrin (also referred to as “IVL” in this specification), loricrin, and the like are synthesized in the cornified envelope. Proteins are formed by cross-linking by activation of transglutaminase. The expression and synthesis of involucrin and the activity of transglutaminase are important for the normal formation of the cornified envelope, the normal keratinization of the epidermis, and the maintenance and improvement of the skin moisturizing function. In this regard, it has been reported that involucrin expression promotion and transglutaminase activation lead to normal skin keratinization, barrier function improvement, and rough skin improvement (for example, Non-Patent Documents 1 and 2 and Patents). Reference 1 and 2).

In addition, ceramide, which is one of the sphingolipids, is a lipid that exists only in a trace amount in the entire living body. However, in the stratum corneum, the outermost layer of the skin, ceramide occupies about half of the lipid and plays an important role in the skin moisturizing and barrier mechanisms. This ceramide functions by constructing a lamellar structure between cells in the stratum corneum after being produced and secreted in epidermal cells. However, in skin diseases such as dry skin, rough skin, atopic dermatitis, senile psoriasis and psoriasis, the healthy metabolism of ceramide is hindered, the amount of ceramide in the stratum corneum is reduced, and the skin's moisture retention ability is reduced. It has been reported many that it causes a decrease, keratinization failure of the epidermis, a decrease in barrier ability, etc. (see Non-Patent Document 3). Substances that promote ceramide production can be expected to have effects such as suppression of animal cell proliferation, induction of differentiation, and induction of apoptosis. As a result, diseases caused by abnormal cell proliferation or differentiation such as inflammatory diseases and malignant tumors. It is thought that the therapeutic effect with respect to can be expected (refer nonpatent literature 4). Furthermore, ceramide has a bone resorption inhibitory action, a bone strengthening action, and an alveolar bone loss inhibitory action, and is useful for the prevention and improvement of osteoarthritis such as osteoporosis, fracture, low back pain, rheumatism (see Patent Document 3). It has also been reported that ceramide has an effect in preventing periodontal disease (see Patent Document 4), and that ceramide has an effect of imparting firmness and stiffness to the hair and improving touch (see Patent Document 5).
Thus, ceramide can be expected to have various effects, and there is a demand for a substance that can promote the production of ceramide.

  In addition, keratin-related proteins such as involucrin are known to be involved in hair formation. For example, as a result of gene expression analysis in hair roots, the expression level of the involucrin gene is significantly increased in those with comb hair, and a substance that increases the expression level of the involucrin gene is a promoter or wave of comb hair or curly hair. It has been reported that it can be a chemical accelerator (see Patent Document 6).

As for the physiological activity of the extract of Peristrophe japonica , one of the plants belonging to the genus Peristrophe belonging to the family Acanthaceae, it has an inhibitory effect on melanin production and an inhibitory effect on dopa oxidase activity, etc. Is known (see, for example, Patent Document 7). However, the extract of gypsophila activates transglutaminase, promotes the production of ceramide and the expression of involucrin, maintains or improves the barrier function and moisturizing function of the skin, prevents or improves rough skin, and makes the hair become bruised and shrunk. It has not been known so far to be useful for hair and wave formation.

JP 2004-91376 A Japanese Patent Laid-Open No. 2007-1914 JP 2001-158736 A JP 2001-158735 A Japanese Patent Laid-Open No. 10-152421 International Publication 2011/043330 Pamphlet International Publication 2012/099247 Pamphlet

Kalinin, A., et al., Journal of Cell Science, 2001, vol. 114, p. 3069-3070 Rawlings, A. V., et al., Journal of Investigative Dermatology, 2005, vol. 124, p. 1099-1110 Ishikawa, J., et al., Journal of Cosmetic Dermatology, 2013, vol. 12, p. 3-11 David E. Modrak, et al., Molecular Cancer Therapeutics, 2006, vol. 5 (2), p. 200-208

An object of the present invention is to provide a transglutaminase activator that activates transglutaminase and is useful for maintaining or improving the skin barrier function and moisturizing function, and preventing or improving rough skin.
Another object of the present invention is to provide a ceramide production promoter that promotes the production of ceramide and is useful for maintaining or improving the skin barrier function and moisturizing function, preventing or improving rough skin, and the like.
Furthermore, the present invention is an involucrin expression promoter that promotes the expression of involucrin, and is useful for maintaining or improving the skin barrier function and moisturizing function, preventing or improving rough skin, as well as hair combing, curling, and waving. The issue is to provide

  As a result of intensive studies in view of the above-mentioned problems, the present inventors have found that an extract of Agrobacterium activates transglutaminase, promotes ceramide production, and promotes involucrin expression. The present invention has been completed based on these findings.

TECHNICAL FIELD The present invention relates to a transglutaminase activator comprising an extract of agglomerated as an active ingredient.
Moreover, this invention relates to the ceramide production promoter which uses the extract of a sunflower as an active ingredient.
Moreover, this invention relates to the involucrin expression promoter which uses the extract of a sunflower as an active ingredient.
In addition, the present invention relates to an epidermis keratinization improving agent comprising an extract of Amaranthus as an active ingredient.
Moreover, this invention relates to the skin moisturizing function improving agent which uses the extract of a sunflower as an active ingredient.
Moreover, this invention relates to the skin barrier function improving agent which uses the extract of a sunflower as an active ingredient.
Moreover, this invention relates to the rough skin prevention or improvement agent which uses the extract of a sunflower as an active ingredient.
Furthermore, the present invention relates to a combing hair agent containing an extract of Amaranthus as an active ingredient.

The transglutaminase activator of the present invention activates transglutaminase and is useful for maintaining or improving the skin barrier function and moisturizing function, and preventing or improving rough skin.
The ceramide production promoter of the present invention promotes the production of ceramide and is useful for maintaining or improving the barrier function and moisturizing function of skin, preventing or improving rough skin, and the like.
The involucrin expression promoter of the present invention promotes the expression of involucrin, and is useful for maintaining or improving the barrier function of skin, preventing or improving rough skin, and combing hair, curly hair, and making waves.

In the present specification, “improvement” refers to improvement of a disease, symptom or condition, prevention or delay of deterioration of the disease, symptom or condition, or reversal, prevention or delay of progression of the disease, symptom or condition.
Further, in the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by therapy.
In the present specification, “prevention” means prevention or delay of the onset of a disease or symptom in an individual, or reduction of the risk of onset of a disease or symptom in an individual.
Furthermore, in this specification, “rough skin” means that the moisture retention capacity of the skin is reduced and the moisture of the skin is taken away, and the skin surface is crushed or cracked, or the skin surface roughness is increased. State. Skin in such a state is also referred to as “that skin” or “dry skin”.

The transglutaminase activator, the ceramide production promoter, and the involucrin expression promoter of the present invention contain an extract of sunflower as an active ingredient. As demonstrated in the examples described later, the extract of sunflower has a transglutaminase activation effect, a ceramide production promoting effect, and an involucrin expression promoting effect.
In addition, the epidermis keratinization improving agent, skin moisturizing function improving agent, skin barrier function improving agent, and rough skin preventing or improving agent of the present invention also have an extract of hazelnut as an active ingredient. As mentioned above, activation of transglutaminase, promotion of ceramide production, and promotion of involucrin expression are very effective in improving keratinization of the epidermis and skin moisturizing function, maintaining the barrier function of skin, and preventing or improving rough skin. Is important to. Therefore, an extract of angiosperm having transglutaminase activation effect, ceramide production promotion effect, and involucrin expression promotion effect is used as an epidermis keratinization improving agent, skin moisturizing function improving agent, skin barrier function improving agent, and rough skin prevention or improvement. It can be an active ingredient of an agent.
As described above, ceramide is involved in the control of cell proliferation, differentiation, apoptosis, and the like. For this reason, the extract of Agrobacterium that promotes ceramide production prevents diseases caused by abnormal cell growth or differentiation, such as inflammatory diseases and malignant tumors, by suppressing the growth of animal cells, inducing differentiation, and inducing apoptosis. Or it is useful for the pharmaceutical for treatment, a quasi-drug, etc. Moreover, the extract of the sunflower can be used for prevention or improvement of osteoarthritis such as osteoporosis, bone fracture, low back pain, rheumatism, pharmaceuticals for preventing or improving periodontal disease, quasi drugs, and the like. Furthermore, the extract of gypsophila is also useful for applications such as quasi-drugs and cosmetics for imparting firmness and stiffness to the hair and improving the feel of the hair.
Furthermore, as described above, a substance that increases the expression level of the involucrin gene can be a promoter for comb hair and curly hair or a promoter for wave formation. Therefore, the extract of the sunflower which has the effect of promoting the expression of involucrin can be used as an active ingredient of the hair-cutting agent. In the present specification, “combination” includes the promotion of comb hair and curly hair and the promotion of wave formation.

In the present specification, the term “Annelina” refers to a plant belonging to the genus Amaranthaceae.
Any part of the plant can be used for the production of the plant extract, including whole grass, root, tuberous root, rhizome, trunk, branch, stem, leaf (leaf blade, petiole, etc.), bark, sap, resin, flower (Petals, ovaries, etc.), fruits (mature fruits, immature fruits, etc.), seeds and the like can be used. A combination of these parts may also be used. Especially, it is preferable that the extract of the sunflower used for this invention is the extract of the whole plant of a sunflower.

The extract of the sunflower used in the present invention can be obtained by an ordinary extraction method used for plant extraction or the like. The extraction method can be appropriately set, and is preferably obtained by extracting the plant at room temperature or under heating or using an extraction tool such as a Soxhlet extractor.
For the preparation of the extract of the sunflower, the sunflower can be used as it is or after being dried and ground. Moreover, a steam-distilled product or a pressed product of the sunflower can be used, and these can be purified from essential oils or the like, or commercially available products can also be used. You may use the sunflower, or its steam distillate or compressed product, alone or in combination of two or more.

  The extraction solvent used for the preparation of the extract of the sunflower can be selected as appropriate, and those usually used for the extraction of plant components, such as water; alcohols such as methanol, ethanol, propanol, butanol; ethylene glycol, propylene glycol, 1 Polyhydric alcohols such as 1,2-butylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate Chain and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether Benzene, torr Aromatic hydrocarbons such as ene; pyridines; supercritical carbon dioxide; fats and oils, waxes, and other oils. These may be used alone or in combination of two or more. Among these, water, ethanol, and an aqueous ethanol solution are preferable, an aqueous ethanol solution is more preferable, an aqueous ethanol solution having an alcohol content of 30% by volume or more is more preferable, and an aqueous ethanol solution having an alcohol content of 40% by volume or more is particularly preferable. Further, acid or alkali may be added during extraction to adjust the pH of the extraction solvent.

  As extraction conditions, normal conditions can be applied. For example, the sunflower can be immersed or heated to reflux at 0 ° C. to 100 ° C. for 0.5 hours to 30 days. In order to increase the extraction efficiency, stirring may be performed together or homogenization treatment may be performed in a solvent. The amount of the extraction solvent to be used is not less than 1 time (preferably not less than 5 times) and not more than 100 times (preferably not more than 50 times, more preferably not more than 40 times) the weight of the sunflower.

In the present invention, the extract of sunflower can be used as it is, or a fraction having high activity can be fractionated and used by an appropriate separation means such as gel filtration, chromatography, precision distillation or the like. Moreover, after diluting, concentrating, or freeze-drying the extract of the obtained sunflower, it can also be prepared and used for powder or paste form. In addition, the extract obtained by the above method can be used by being dissolved in a solvent different from the extraction solvent.
In the present invention, the extract includes various solvent extracts obtained by the extraction method as described above, diluted solutions thereof, concentrated solutions thereof, purified fractions thereof, dried powders thereof or transferred solutions thereof.

  The forms of transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, skin roughness inhibitor or improver, and hair growth agent of the present invention are: For example, it can be a pharmaceutical composition, a cosmetic composition or a food composition, or can be contained in these.

  When preparing a pharmaceutical composition, it is usually prepared as a preparation containing the active ingredient and preferably a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier generally refers to an inert, non-toxic, solid or liquid, bulking agent, diluent or encapsulating material that does not react with the active ingredient, eg, water. , Ethanol, polyols (for example, propylene glycol, butylene glycol, glycerin, and polyethylene glycol), suitable mixtures thereof, solvents or dispersion media such as vegetable oils, and the like.

  The pharmaceutical composition is orally, parenterally, e.g., in the oral cavity, in the skin, subcutaneously, in the mucosa, intravenously, in the artery, in the muscle, in the abdominal cavity, in the vagina, in the lungs. Administered intracerebrally, ocularly and intranasally. Examples of the preparation for oral administration include tablets, granules, fine granules, powders, capsules, chewables, pellets, syrups, solutions, suspensions and inhalants. As parenteral preparations, suppositories, retention enemas, drops, eye drops, nasal drops, pessaries, injections, mouth washes, ointments, creams, gels, controlled release patches and patches Skin external preparations, and the like. The pharmaceutical compositions may be administered parenterally in the form of sustained release subcutaneous implants or in the form of targeted delivery systems (eg, monoclonal antibodies, vector delivery, ion implantation, polymer matrices, liposomes and microspheres). .

  The pharmaceutical composition may further contain additives conventionally used in the pharmaceutical field. Such additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents, and the like, and can be used as necessary. In order to achieve sustained release so that it can act for a long time, it can also be coated with a known retarder or the like. Examples of excipients include sodium carboxymethylcellulose, agar, light anhydrous silicic acid, gelatin, crystalline cellulose, sorbitol, talc, dextrin, starch, lactose, sucrose, glucose, magnesium metasilicate magnesium phosphate, calcium hydrogen phosphate, etc. Can be used. Examples of the binder include gum arabic, sodium alginate, ethyl cellulose, sodium caseinate, sodium carboxymethyl cellulose, agar, purified water, gelatin, starch, tragacanth, and lactose. Examples of the disintegrant include carboxymethyl cellulose, carboxymethyl cellulose sodium, carboxymethyl cellulose calcium, crystalline cellulose, starch, hydroxypropyl starch and the like. Examples of the lubricant include stearic acid, calcium stearate, magnesium stearate, talc, hydrogenated oil, sucrose fatty acid ester, waxes and the like. Examples of the antioxidant include tocopherol, gallic acid ester, dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), ascorbic acid and the like. Other additives and drugs as required, such as antacids (sodium bicarbonate, magnesium carbonate, precipitated calcium carbonate, synthetic hydrotalcite, etc.), gastric mucosa protective agents (synthetic aluminum silicate, sucralfate, copper chlorophyllin sodium, etc.) ) May be added.

  When preparing a cosmetic composition, the form thereof can be selected as appropriate, such as solution, emulsion, powder, water-oil two-layer system, water-oil-powder three-layer system, gel, tablet and other solids, aerosol, It can be in any form such as mist, capsule, and sheet. In addition, the product form of the cosmetic composition is also arbitrary. For example, skin care cosmetics such as face wash, makeup remover, lotion, cosmetic liquid, pack, milky lotion, cream and sunscreen, foundation, makeup base, lipstick, eye Makeup cosmetics such as shadow, eyeliner, mascara, eyebrow, blusher and nail enamel, hair cosmetics such as hair shampoo, hair rinse, hair styling, hair dye and hair restorer, soap, body soap, deodorant cosmetic and bath preparation And other body cleansing agents, oral cosmetics such as dentifrices and mouthwashes, and aromatic cosmetics such as perfumes. Moreover, this cosmetic may belong to either cosmetics or quasi-drugs in accordance with Japanese Pharmaceutical Affairs Law.

The cosmetic composition includes other components commonly used in cosmetics, quasi drugs and pharmaceuticals, such as powder components, liquid fats and oils, solid fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, silicones, anions. Surfactant, cationic surfactant, amphoteric surfactant, nonionic surfactant, humectant, water-soluble polymer, thickener, film agent, UV absorber, sequestering agent, lower alcohol, polyhydric alcohol , Sugar, amino acids, organic amines, polymer emulsions, pH adjusters, skin nutrients, vitamins, antioxidants, antioxidant auxiliaries, fragrances, water, etc. may be blended as necessary and manufactured by conventional methods. it can.
Examples of other components that can be incorporated into the cosmetic composition include preservatives (ethyl paraben, butyl paraben, etc.), anti-inflammatory agents (eg, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin, etc. ), Whitening agents (for example, ascorbic acid and its derivatives, placenta extract, saxifrage extract, arbutin, etc.), various extracts (for example, buckwheat, auren, shikon, peonies, assembly, birch, sage, loquat, carrot, aloe , Mallow, iris, grape, yokuinin, loofah, lily, saffron, senkyu, ginger, hypericum, onionis, garlic, capsicum, chimpi, red snapper, seaweed, etc.), activator (eg royal jelly, photosensitizer, cholesterol derivative, etc.) , Blood circulation promoter For example, nonyl acid valenylamide, nicotinic acid benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, gingerone, cantalis tincture, ictamol, tannic acid, α-borneol, nicotinic acid tocopherol, inositol hexanicotinate, cyclandrate, cinnarizine , Trazoline, acetylcholine, verapamil, cephalanthin, γ-oryzanol, etc.), antiseborrheic agents (eg, sulfur, thianthol, etc.), anti-inflammatory agents (eg, tranexamic acid, thiotaurine, hypotaurine, etc.) and fungicides (eg, triclosan, Cetylpyridinium chloride, thymols, benzalkonium chloride, etc.).

The pharmaceutical composition and cosmetic composition can be applied in the form of an oral composition, an external composition, an internal use composition, etc., and is preferably used in the form of a skin external composition.
When used in the form of a composition for external use on the skin, in addition to the extract of gypsophila, components used in normal composition for external use on the skin, such as surfactants, oily substances, polymer compounds, preservatives, various medicinal ingredients, powders UV absorbers, dyes, fragrances, emulsion stabilizers, pH adjusters, and the like can be appropriately blended. As a medicinal component, in the case of an epidermis keratinization improving agent or a skin moisturizing function improving agent, for example, vitamin D3, a sphingosine derivative, oleanolic acid, clofibric acid, oleethanolamide may be mentioned.

  When preparing a food composition, the form can be selected as appropriate, and beverages are also included. In addition to general foods, treatment, prevention, or improvement can be achieved by either keratinization of the epidermis or improvement / maintenance of skin moisturizing and barrier functions, activation of transglutaminase, promotion of ceramide production, and promotion of involucrin Also included are foods and drinks that indicate the concept of treatment, prevention or improvement of possible diseases or conditions, that is, health foods, functional foods, foods for the sick, foods for specified health use, and the like. Health foods, functional foods, foods for patients and foods for specified health use are specifically formulated in various forms such as fine granules, tablets, granules, powders, capsules, syrups, liquids, and liquid foods. Can be used for these formulations. A food composition in the form of a preparation can be produced in the same manner as a pharmaceutical preparation. The active ingredient and a carrier acceptable as a food, for example, an appropriate excipient (eg, starch, processed starch, lactose, glucose, water, etc.) ) And the like, and then can be produced using conventional means. In addition, food compositions include soups, juices, milk beverages, tea beverages, coffee beverages, cocoa beverages, jelly-like beverages and other liquid food compositions, pudding, yogurt and other semi-solid food compositions, breads, udon Such as noodles such as cookies, chocolate, candy, gum, rice crackers and the like, spreads such as sprinkles, butter, jam and the like. The food also includes feed.

  Food compositions include various food additives such as antioxidants, fragrances, various esters, organic acids, organic acid salts, inorganic acids, inorganic acid salts, inorganic salts, pigments, emulsifiers, preservatives, seasonings Additives such as additives, sweeteners, acidulants, fruit juice extracts, vegetable extracts, nectar extracts, pH adjusters, and quality stabilizers may be used alone or in combination.

Administration target of transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, skin roughening preventive or improver, or hair growth agent of the present invention Is preferably a warm-blooded vertebrate, more preferably a mammal. As used herein, mammals include, for example, humans and non-human mammals such as monkeys, mice, rats, rabbits, dogs, cats, cows, horses, and pigs. Transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, rough skin preventive or improver, or hair growth agent of the present invention is human. Suitable for administration to primates such as monkeys, especially humans.
The extract used in the present invention, and the transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermal keratinization improver, skin moisturizing function improver, skin barrier function improver, rough skin prevention or improvement of the present invention The agent or combing agent can be applied to a subject who wants to prevent or treat keratinization failure of the epidermis, moisturize the skin, improve the skin barrier function, prevent or improve rough skin, or make the hair comb. . The extract or agent is preferably applied under necessary conditions (preferably under low humidity and dry conditions). The extract or agent is preferably applied to the skin, scalp or hair.

  The above-mentioned effects in the transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, rough skin preventive or ameliorate agent, or combing agent of the invention The dosage of the components can be appropriately determined according to the individual's condition, body weight, sex, age, material activity, administration or intake route, administration or intake schedule, formulation form, or other factors. For example, based on the mass of the active ingredient, it is preferably 0.001 mg or more, 1 g or less, or preferably 0.001 to 1 mg per kg of body weight per day. The active ingredient can be taken or administered once a day to several times a day, or at an arbitrary period and interval.

  The above-mentioned effects in the transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, rough skin preventive or ameliorate agent, or combing agent of the invention The content of the components can be appropriately determined so as to achieve the above dose. For example, in the transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improving agent, skin moisturizing function improving agent, skin barrier function improving agent, skin roughening preventing or improving agent, or hair growth agent of the present invention. The content of the active ingredient is preferably 0.00001% by mass or more, more preferably 0.0001% by mass or more, further preferably 0.0005% by mass or more, preferably 20% by mass or less, more preferably 10% by mass or less, and 5% by mass. The following is more preferable, 0.00001 to 20% by mass is preferable, 0.0001 to 10% by mass is more preferable, and 0.0005 to 5% by mass is further preferable.

  In relation to the above-described embodiment, the present invention further includes the following transglutaminase activator, ceramide production promoter, involucrin expression promoter, epidermis keratinization improving agent, skin moisturizing function improving agent, skin barrier function improving agent, rough skin prevention or improvement. Disclosed are agents, combing agents, manufacturing methods, methods and uses.

<1> A transglutaminase activator comprising an extract of agglomerates as an active ingredient.
<2> A ceramide production promoter comprising an extract of agglomerates as an active ingredient.
<3> An involucrin expression promoter, comprising an extract of Amaranthus as an active ingredient.
<4> An epidermis keratinization improving agent comprising an extract of agglomerates as an active ingredient.
<5> A skin moisturizing function-improving agent comprising an extract of agglomerates as an active ingredient.
<6> A skin barrier function-improving agent comprising an extract of agglomerates as an active ingredient.
<7> An agent for preventing or improving rough skin, comprising an extract of agglomerates as an active ingredient.
<8> A combing hair agent, comprising an extract of agglomerates as an active ingredient.

<9> The transglutaminase activator, the ceramide production promoter, or the involucrin expression promoter according to any one of <1> to <8>, wherein the extract of the sunflower is an extract of whole plant of sunflower , An epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, a rough skin preventing or improving agent, or a combing hair agent.
<10> The extract of the sunflower is obtained by extracting the sunflower using an aqueous ethanol solution (preferably an ethanol aqueous solution having an alcohol content of 30% by volume or more (more preferably 40% by volume or more)) as an extraction solvent. Transglutaminase activator according to any one of <1> to <9>, ceramide production promoter, involucrin expression promoter, epidermis keratinization improver, skin moisturizing function improver, skin barrier function improver, An agent for preventing or improving rough skin, or a combing hair agent.
<11> The content of the active ingredient is 0.00001% by mass or more (preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more), 20% by mass or less (preferably 10% by mass or less, more preferably 5% by mass or less). The transglutaminase activator according to any one of <1> to <10>, a ceramide production promoter, an involucrin expression promoter, an epidermal keratinization improving agent, a skin moisturizing function improving agent, and a skin barrier A function improving agent, a rough skin preventing or improving agent, or a wrinkling agent.

<12> Use of an extract of Agrobacterium as a transglutaminase activator, a ceramide production promoter or an involucrin expression promoter.
<13> Use of an extract of sunflower for the production of a transglutaminase activator, a ceramide production promoter or an involucrin expression promoter.
<14> A method of using an extract of Amaranthus as a transglutaminase activator, a ceramide production promoter or an involucrin expression promoter.
<15> A method for activating transglutaminase, a method for promoting ceramide production, or a method for promoting involucrin expression, which uses an extract of Amaranthus.
<16> The above extract is applied to a subject who desires prevention or treatment of keratinization failure of the epidermis, skin moisturization, improvement of skin barrier function, prevention or improvement of rough skin, or hair combing, <15> The method described in the item>.
<17> The method according to <15> or <16>, wherein the extract is applied under conditions that require application of the extract (preferably under dry conditions with low humidity).
<18> The method according to any one of <15> to <17>, wherein the extract is applied to skin, scalp or hair.
<19> The use or method according to any one of <12> to <18>, wherein the extract of the sunflower is an extract of the whole plant of sunflower.
<20> The extract of the sunflower is obtained by extracting the sunflower using an ethanol aqueous solution (preferably an ethanol aqueous solution having an alcohol content of 30% by volume or more (more preferably 40% by volume or more)) as an extraction solvent. The use or method according to any one of <12> to <19>.
<21> Transglutaminase activator, ceramide production promoter or involucrin expression promoter, the content of the extract of trichomes is 0.00001 mass% or more (preferably 0.0001 mass% or more, more preferably 0.0005 mass% or more) 20 mass % Or less (preferably 10% by mass or less, more preferably 5% by mass or less). The use or method according to any one of <12> to <20>.

<22> Use of an extract of agglomerated as a skin keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, a rough skin preventing or improving agent, or a wrinkling agent.
<23> Use of an extract of agglomerated for production of an epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, a rough skin preventing or improving agent, or a hair-cutting agent.
<24> A method of using an extract of agglomerated as an epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, a rough skin preventing or improving agent, or a wrinkling agent.
<25> A method for improving skin keratinization, a method for improving skin moisturizing function, a method for improving skin barrier function, a method for preventing or improving rough skin, or a method for combing hair, which comprises applying an extract of Amaranthus.
<26> Applying the extract to a subject who desires prevention or treatment of keratinization failure of the epidermis, skin moisturization, improvement of skin barrier function, prevention or improvement of rough skin, or hair combing, <24 > Or <25>.
<27> The method according to any one of <24> to <26>, wherein the extract is applied under conditions that require application of the extract (preferably under dry conditions with low humidity).
<28> The method according to any one of <24> to <27>, wherein the extract is applied to skin, scalp or hair.
<29> An extract of Amaranthus used for a method for preventing or treating skin epikeratinization failure, a method for improving skin moisturizing function, a method for improving skin barrier function, a method for preventing or improving rough skin, or a method for combing hair.
<30> Use of an extract of Amaranthus for the manufacture of a prophylactic or therapeutic agent for skin keratinization failure, a skin moisturizing function improving agent, a skin barrier function improving agent, a rough skin preventing or improving agent, or a combing hair fixative.
<31> Use of an extract of sunflower that is used for a non-therapeutic treatment method for skin keratinization failure, skin moisturizing function, skin barrier function, rough skin, or comb hair.
<32> The use according to <31> above, wherein the extract of Amaranthus is applied in the form of a pharmaceutical composition or a cosmetic composition.
<33> The use according to <32> above, wherein the extract of sunflower is applied in the form of a composition for external use.
<34> The use according to <31> above, wherein the extract of sunflower is applied in the form of food or beverage.
<35> The use or method according to any one of <22> to <34>, wherein the extract of the sunflower is an extract of the whole plant of sunflower.
<36> The extract of the sunflower is obtained by extracting the sunflower using an ethanol aqueous solution (preferably an ethanol aqueous solution having an alcohol content of 30% by volume or more (more preferably 40% by volume or more)) as an extraction solvent. The use or method according to any one of <22> to <35>.
<37> The content of the extract of the sunflower in the epidermis keratinization improving agent, the skin moisturizing function improving agent, the skin barrier function improving agent, the rough skin preventing or improving agent, or the shading agent is 0.00001% by mass or more (preferably Any one of the above <22> to <36>, wherein 0.0001% by mass or more, more preferably 0.0005% by mass or more) is 20% by mass or less (preferably 10% by mass or less, more preferably 5% by mass or less). Use or method as described.

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

Preparation Example 1
10 g of whole plant of sunflower (manufactured by Shinwa Bussan Co., Ltd.) was shredded, 100 mL of a 30% by volume aqueous ethanol solution was added, and extraction was carried out for 7 days under room temperature and standing conditions. Thereafter, filtration was performed to obtain 61 mL of an extract of nematode. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 1.10% (w / v). This was diluted with a 30% by volume ethanol aqueous solution to prepare a 1.0% (w / v) (extract solid content) extract.
<Calculation of evaporation residue>
When 1 mL of the extract of sunflower was dried at 105 ° C. for 6 hours using a dryer (DRY Thermo Unit DTU-1C (trade name, manufactured by TAITEC CORPORATION)), 11.0 mg of a dried product was obtained. The evaporation residue of this extract was calculated as 11.0 / 1000 × 100 = 1.10% (w / v). In the following preparation examples, the evaporation residue of each extract is calculated in the same manner.

Preparation Example 2
Extraction was performed using a 50% by volume ethanol aqueous solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 58 mL of an extract of nematode. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.92% (w / v). After concentrating this, 1.0% (w / v) (extract solid content) extract was prepared with 50 volume% ethanol aqueous solution.

Preparation Example 3
Extraction was performed using a 60% by volume aqueous ethanol solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 58 mL of an extract of nematode. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.79% (w / v). After concentration, 1.0% (w / v) (extract solid content) extract was prepared with 60% by volume ethanol aqueous solution.

Preparation Example 4
Extraction was performed using a 70 vol% aqueous ethanol solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 60 mL of an extract of nematode. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.70% (w / v). After concentrating this, 1.0% (w / v) (extract solid content) extract was prepared with 70 volume% ethanol aqueous solution.

Preparation Example 5
Extraction was performed using an 80% by volume aqueous ethanol solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 55 mL of an extract of nebula. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.60% (w / v). After concentrating this, 1.0% (w / v) (extract solid content) extract was prepared with 80 volume% ethanol aqueous solution.

Preparation Example 6
Extraction was performed using a 90 volume% aqueous ethanol solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 53 mL of the extract of nebula. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.36% (w / v). This was concentrated to dryness, and then 1.0% (w / v) (extract solid content) extract was prepared with 90% by volume ethanol aqueous solution.

Preparation Example 7
Extraction was performed using a 99.5% by volume aqueous ethanol solution under the same conditions as in Preparation Example 1. Thereafter, filtration was performed to obtain 52 mL of an extract of nebula. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.17% (w / v). This was concentrated to dryness, and then an extract of 1.0% (w / v) (extract solid content) was prepared with a 99.5% by volume ethanol aqueous solution.

Preparation Example 8
600 g of whole plant of sunflower (manufactured by Affinity Products Co., Ltd.) was chopped, 6000 mL of a 99.5% by volume ethanol aqueous solution was added, and extraction was performed for 7 days under room temperature and standing conditions. Thereafter, filtration was performed to obtain 4504 mL of the extract of nebula. After 2,000 mL of hexane was added to 4000 mL of the resulting extract and stirred, 6000 mL of water was further added. After liquid-liquid partitioning, 8367 mL of the lower layer was extracted. 1000 mL of 1,3-butylene glycol was added to 2500 mL of the obtained extract and concentrated with an evaporator to remove ethanol and water. After adding 1500 mL of water to this, it was starched at 5 ° C. for 4 days. After removing insolubles by filtration, 40 volume% 1,3-butylene glycol aqueous solution was added to 2403 L of the obtained filtrate to prepare 3266 mL of an extract. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 0.02% (w / v).

Test Example 1 Measurement of transglutaminase activity Human epidermal keratinocyte cell line HEKn (manufactured by KURABO) was seeded at 4 × 10 ⁴ cells / well in a 12-well plate and cultured. As the medium, commercially available EpiLife-KG2 manufactured by Kurabo Industries was used. After culturing at 37 ° C. and 5% CO ₂ for one day, the medium was replaced with a medium not containing growth factors (BPE, EGF), and the final concentration of the extract of the sunflower extract prepared in Production Examples 1 to 8 was shown in Table 1. Each was added so that Further, instead of the extract of sunflower, 50% by volume ethanol as an extraction solvent was added at a final concentration of 0.1% (v / v) as a control, and CaCl ₂ was added as a positive control at a final concentration of 1.5 mM. . CaCl ₂ is known to promote keratinization and was used as a positive control. All of these were further cultured at 37 ° C. for 3 days.
After completion of the culture, the culture solution was removed, washed twice with PBS (−), and 150 μL of extraction buffer (10 mM Tris-HCl buffer, pH 7.4 containing 0.5 mM EDTA, 1% TritonX-100, and protease inhibitors). The cells were collected using a cell scraper to obtain a cell disruption solution by ultrasonic treatment. The supernatant obtained by centrifugation (15,000 rpm, 10 minutes) was used for evaluation as a lysate. Enzyme activity was measured using Transglutaminase Colorimetric Microassay Kit (trade name) according to the manufacturer's instructions. The protein concentration was quantified using BCA Protein Assay Kit (trade name, manufactured by Thermo Scientific) according to the manufacturer's instructions.

  The evaluation results are shown in Table 1. The transglutaminase activity of each extract sample was shown as a relative value when the transglutaminase activity of the control to which 50% by volume of ethanol was added was 1.

As is apparent from Table 1, in the system to which the extract of sunflower was added, the transglutaminase activity was greatly increased to the same level or more than that of the positive control.
Furthermore, transglutaminase activity is related to maintenance of skin barrier function, maintenance or improvement of moisturizing function, and prevention or improvement of rough skin, and activation of transglutaminase prevents keratinization failure of the epidermis and moisturizing function of skin. Improvement and prevention or improvement of rough skin are possible. Therefore, the extract of the sunflower which has a transglutaminase activation effect can be used as an active ingredient of an epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, and a rough skin prevention or improving agent.

Test Example 2 Verification of ceramide production promotion effect Normal human epidermal keratinocytes (trade name: NHEK (F), manufactured by KURABO) in a culture solution (trade name: EpiLife-KG2, manufactured by KURABO) using a culture plate ) At 37 ° C. and 5% CO ₂ .
Thereafter, the culture solution was changed to EpiLife-KG2 from which growth factors such as epidermal growth factor were removed, and the extract of the sunflower prepared in Preparation Example 2 was adjusted so that the concentration was 1 w / v% in terms of solid content. 0.1% amount was added. Also, instead of an extract of sunflower, Eucalyptus globulus extract prepared as shown below as a positive control with 50% by volume of ethanol as a control at a final concentration of 0.1% v / v. The final concentrations shown in Table 2 were added respectively. Eucalyptus extract is known to have an effect of promoting ceramide production and was used as a positive control.
After culturing for 3 days, each cell was collected together with 1 well.

The organic phase from which the lipid was extracted from the collected cells by the Bligh and Dyer method was transferred to a glass tube, solidified with nitrogen, and then redissolved with chloroform and methanol to obtain a lipid sample.
In addition, 0.1N NaOH, 1% SDS aqueous solution is added to the cells after lipid extraction, and the protein is solubilized by heating at 60 ° C. for 2 hours, cooled to room temperature, and then neutralized by adding 2N HCl. The amount of protein was quantified by the BCA method.

The prepared lipid sample was horizontally developed by thin film chromatography (TLC) twice with chloroform: methanol: acetic acid = 190: 9: 1. The copper sulfate solution was sprayed and sprayed with a hot plate to detect ceramide, and the amount was determined. The amount of ceramide was expressed as a relative value, assuming that the amount of ceramide when ethanol of 50% by volume was added was 1.
The results are shown in Table 2.

(Reference example)
40 g of Eucalyptus globules Labillardiere (manufactured by Shinwa Bussan) was cut into small pieces , 400 mL of 50% by volume ethanol was added, and extraction was performed for 7 days under room temperature and standing conditions. Then, it filtered and 291 mL of eucalyptus extracts were obtained. When the evaporation residue was calculated for the obtained extract, the evaporation residue was 3.16% (w / v). This was diluted with a 50% by volume aqueous ethanol solution to prepare a 1.0% (w / v) extract.

As is clear from Table 2, the ceramide output was increased in the system to which the extract of sunflower was added compared to the control system. Therefore, it turns out that the ceramide production promoter of this invention which uses an extract of hagrose as an active ingredient can promote ceramide production.
Furthermore, ceramide is related to maintaining or improving the skin barrier function, moisturizing function, and preventing or improving rough skin, and promotes the production of ceramide, thereby preventing keratinization failure of the epidermis and improving skin moisturizing function. , And prevention or improvement of rough skin are possible. Therefore, an extract of trichomes having an effect of promoting production of ceramide can be used as an active ingredient of an epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, and a rough skin preventing or improving agent.

Test Example 3 Evaluation of Involucrin Expression Level (1) Cell Culture The human epidermal keratinocyte cell line HaCaT was prepared by inactivating 10% fetal bovine serum, 1% penicillin-streptomycin (manufactured by Gibco) into DMEM (manufactured by Gibco). The cultured medium was cultured under the conditions of 37 ° C. and 5% CO ₂ .

(2) Evaluation of IVL expression For the primary screening, Involucrin ELISA Assay Kit BT-650 (trade name, manufactured by BTI) was used with a partly modified protocol. The detailed protocol is shown below.
In a 24-well plate, HaCaT cells were seeded at 5 × 10 ⁴ cells / well and cultured. On the next day, the extract of the sunflower extract that was 1.0% (w / v) in terms of solid content prepared in Preparation Example 2 was replaced with a medium containing the final concentration shown in Table 3, and further cultured for 24 hours. In addition, instead of the extract of sunflower, a culture medium in which 50% ethanol as an extraction solvent was replaced with a medium containing a final concentration of 0.5% v / v and cultured for 24 hours was used as a control. Twenty-four hours after the addition of the extract, the cells were washed twice with PBS (−), and Complete Mini Protease Inhibitor Cocktail (trade name, manufactured by Roche) was added to 20 mM Tris-HCl (pH: 7.5) · 2 mM EDTA. 400 μL of the extraction buffer containing was added to the cells. One Protease Inhibitor Cocktail was added to 10 mL of extraction buffer. Cells were detached and collected with a cell scraper in the presence of this extraction buffer, and then sonicated with a sonicator. The supernatant collected by centrifugation of this solution was used as an lysate for evaluation.
The total protein concentration of the lysate was quantified using BCA Protein Assay Kit (trade name, manufactured by Thermo Scientific) according to the manufacturer's instructions. Dilute lysate with buffer B (2 mM EDTA, 5 g / L Tween 20, 2.5 g / L Gelatin in PBS) to a total well of 3 μg per well of a 96-well plate and add to 96-well plate Then, an anti-IVL antibody (kit accessory, dilution ratio 1/300) was added, and a primary antibody reaction was performed overnight at 4 ° C. Also, human IVL protein (kit accessory) was added to each 96-well plate (Nalgenunk, 469078) by 1 ng / well, and EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, a condensing agent, Kanto The antigen was adsorbed overnight at 4 ° C. together with 10 μg / well manufactured by Chemical Co., Ltd. On the next day, the solution of the plate on which the antigen was adsorbed was removed by suction, 100 μL of 0.1 M NH ₄ Cl was added, and the mixture was reacted at room temperature for 30 minutes. After the reaction, it was washed four times with distilled H ₂ O (manufactured by Gibco) and once with buffer B, and 100 μL of the primary antibody reaction solution of the previous day was added and allowed to react at room temperature for 30 minutes. After this reaction, it was washed 5 times with buffer B, anti-rabbit-AP Conjugate antibody (dilution ratio 1/2000) attached to the kit was added, and allowed to react at room temperature for 1 hour. After the reaction, it was washed once with buffer B and once with buffer D (1 mM MgCl ₂ .6H ₂ O in 0.05M carbonate / bicarbonate (manufactured by Sigma, product name: C3041-50CAP), and p-nitrophenylphosphate (1 mg / mg). Color development reaction was performed by adding 100 μL of a solution in mL in buffer D (manufactured by Sigma) After confirming the color development, the absorbance at 405 nm was measured to quantify the amount of IVL.
The evaluation results are shown in Table 3. The IVL amount of the extract sample was shown as a relative value when the IVL amount of the control to which 50% ethanol was added was 1.

As is apparent from Table 3, the expression level of involucrin was increased in the system to which the extract of sunflower was added.
Furthermore, the expression of involucrin is related to the maintenance and improvement of the skin barrier function, the moisturizing function, and the prevention or improvement of rough skin, and by promoting the expression of involucrin, the prevention of epikeratosis and the moisturizing function of the skin. Improvement and prevention or improvement of rough skin are possible. In addition, a substance that increases the expression level of the involucrin gene can be a comb hair or curly hair promoter or a wave promoter. Therefore, the extract of the sunflower which has an involucrin expression promoting effect can be used as an active ingredient of an epidermis keratinization improving agent, a skin moisturizing function improving agent, a skin barrier function improving agent, and a rough skin prevention or improving agent and a hair growth agent.

Claims (4)

A transglutaminase activator comprising as an active ingredient an extract of perilla ( Peristrophe japonica ). A ceramide production promoter comprising an extract of perilla ( Peristrophe japonica ) as an active ingredient. An involucrin expression promoter containing an extract of perilla ( Peristrophe japonica ) as an active ingredient. A wrinkling agent containing an extract of perilla ( Peristrophe japonica ) as an active ingredient.