JP2020083783A - Function-activating agent of fibroblast - Google Patents

Abstract

To provide a function-activating agent of fibroblast which is safe even when taking the agent for a long period, and can be used as food and drink.SOLUTION: There are provided a function-activating agent of fibroblast including a processed product of barley young leaves as an active ingredient, food and drink containing the function-activating agent, and a cosmetic containing the function-activating agent. Because such a function-activating agent, food and drink, and cosmetic have function-activating effect of fibroblast, effects such as anti-aging of skin, that is, reduction of resilience or elasticity of skin, prevention or improvement of aging of skin such as wrinkle and sag, restoration of scar or inflammation, furthermore prevention or improvement (treatment) of a disease or a state (symptom) caused by reduction of fibroblast function (activity) are expected.SELECTED DRAWING: None

Description

The present invention relates to a function-utilizing agent for fibroblasts, and more specifically, a function-utilizing agent for fibroblasts containing a certain processed product of young barley leaves as an active ingredient, a fiber comprising the function-utilizing agent. The present invention relates to a food or drink for the prevention or amelioration (treatment) of a disease or condition (symptoms) caused by a decline in the function of blast cells, and a cosmetic containing the agent for promoting function.

Fibroblasts are one of the cells that form connective tissue and produce dermal components (hereinafter sometimes referred to as "extracellular matrix components") such as collagen, elastin (elastic fiber), and hyaluronic acid. A decrease in extracellular matrix components of the dermis, particularly collagen and hyaluronic acid, is involved in aging phenomena such as reduced elasticity of skin and wrinkle formation. Therefore, it is considered that skin aging can be prevented or improved by activating the function of fibroblasts and increasing the production of extracellular matrix components. In addition, fibroblasts are activated in the process of repairing wounds and inflammation and play a role of early repairing wounds.

Here, it is known that naturally-derived substances (polyphenols) such as catechin derived from green tea and lathveratrol contained in red wine have a fibroblast activation effect. Further, in Patent Document 1, for example, a substance obtained by subjecting a crushed part of Fuyu persimmon young leaves to cellulase treatment with supercritical extraction with liquefied carbon dioxide or a substance obtained by crushing cypress family juniperus genus Mus musculus seeds and then extracting with hexane (certain polyphenol Is disclosed to have a activating effect on fibroblasts. Furthermore, Non-Patent Document 1 discloses that a plurality of proteins having a molecular weight of 10 KDa or more, whose activity is lost by heat treatment of barley young leaf extract, are involved in promoting the production of type I collagen by fibroblasts.

On the other hand, young leaves of barley, especially young barley leaves, have been used for many years as functional foods and drinks that are highly safe and have no side effects even when taken for a long period of time. Barley young leaves have been used so far for anti-ulcer action, anti-cholesterol action, anti-inflammatory action, hypoglycemic action, anti-mutagenic action, anti-thrombotic action, vascular protective action, adipocyte differentiation inhibitory action, melanin synthesis inhibitory action, osteoporosis prevention It has been reported to have various physiological actions such as action and active oxygen scavenging enzyme (SOD) gene expression promoting action (see, for example, Non-Patent Document 2 and Patent Documents 2 to 5). In addition, various proposals have been made regarding the manufacturing method, and it has been reported that stable mass production is possible (see, for example, Patent Documents 6 to 8). However, as far as the present inventors are aware, no report has been made regarding the fact that the processed barley leaf product of the present invention has a function-activating effect on fibroblasts.

JP, 2006-298800, A JP, 2008-56580, A JP, 2011-51948, A JP, 2013-63940, A JP, 2015-140338, A JP, 2009-148213, A JP, 2003-33151, A JP, 2002-65204, A

2007 Pharmaceutical Society of Japan 127th Meeting (Toyama) 28P1-am091 "Study of green juice component of young barley leaf (52nd report) Human collagen production promoting action of young barley leaf" Yoshihide Hagiwara, et al. Journal of Japan Food Chemistry Engineering Vol. 48, No. 10, 712-725 2001

As described above, if the function of fibroblasts can be activated, it is considered to be effective for preventing or improving skin aging and repairing wounds and inflammation. However, conventional fibroblast activators of natural origin have problems such as difficulty in absorption, mild action, instability, and difficulty in acquisition.

The object of the present invention is highly safe and easy to obtain, has no side effects even after long-term administration, and has an excellent function activating (activating) action of fibroblasts, It is intended to provide a food or drink for the prevention or amelioration (treatment) of a disease or condition (symptoms) caused by a functional decline of fibroblasts containing a function enhancer, and a cosmetic containing the function enhancer. ..

As a result of diligent studies on the above problems, the present inventor has found that a certain processed product of young barley leaf, which is safe even after long-term administration, has an excellent function-activating effect on fibroblasts. The present invention has been accomplished based on such findings.

That is, the present invention is as specified by the following matters.
(1) A function-utilizing agent for fibroblasts comprising a heat-treated barley leaf or an organic solvent extract thereof, or an organic solvent extract of barley leaf as an active ingredient.
(2) The functionalized agent according to (1), wherein the heat-treated product is squeezed powder or dried powder of young barley leaves.
(3) The functional activation is at least one action selected from the group consisting of fibroblast proliferation promoting action, type I collagen production promoting action and hyaluronic acid production promoting action, (1) or The function stimulating agent according to (2).
(4) The functional agent according to any one of (1) to (3) above, wherein the organic solvent is a protic polar solvent or an aprotic polar solvent.
(5) A food or drink for preventing or improving (treating) a disease or condition (symptoms) caused by a decline in the function of fibroblasts, which comprises the function enhancer according to any one of (1) to (4) above. .
(6) A cosmetic comprising the function-utilizing agent according to any one of (1) to (4) above.

Further, as another embodiment of the present invention, heating of the young barley leaf to a subject in need of prevention or amelioration (treatment) of a disease or condition (symptoms) caused by a decrease in function (activity) of fibroblasts The function (activity) of fibroblasts, which comprises a step of administering a treated product or an organic solvent extract thereof or an organic solvent extract of young barley leaves (hereinafter, these may be abbreviated as “treated barley leaves”). ) Use as a method for preventing or ameliorating (treating) a disease or condition (symptoms) caused by a decrease, or as a preventive or ameliorating (treatment) agent for a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts The treated barley leaf for treatment, or the treated barley leaf for use in the prevention or amelioration (treatment) of a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts, or a fiber Examples thereof include the use of the treated barley leaf product for producing a preventive or ameliorating (treating) agent for a disease or condition (symptoms) caused by a decrease in blast cell function (activity). In the present invention, the function activating agent (or function activating agent) means an agent having a limited use of function activating. Therefore, the present invention is a use invention in which the use of function utilization of fibroblasts is specified.

According to the present invention, the treated barley leaf as referred to in the present invention has a function activating (activating) action of fibroblasts, so that anti-aging of the skin, that is, reduction of skin tension and elasticity, wrinkles and sagging, etc. The effects of preventing or improving skin aging, repairing wounds and inflammation, etc. can be expected. In addition, for a subject in need of prevention or improvement (treatment) of a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts, prevention or improvement of such disease or condition without side effects. (Treatment) is expected.

Treated barley young leaves used in the present invention, there is no problem in safety at all, since it can be freely prepared in the form of cosmetics, foods and drinks or pharmaceuticals, not only healthy people, elderly people, sick people, A person after illness can use or ingest for a long period of time.

Hereinafter, embodiments of the present invention will be described in detail, but the following description is an example of the embodiments of the present invention, and the present invention is not limited to the following description. In this specification, when the expression "to" is used, it is used as an expression including numerical values or physical properties before and after the expression.

In a preferred embodiment of the present invention, the function-utilizing agent for fibroblasts is a treated product of young barley leaves, that is, a heat-treated product of young barley leaves or an organic solvent extract thereof or an organic solvent extract of young barley leaves as an active ingredient. .. As the treated barley leaf, a treated barley leaf (stem leaf) harvested from the beginning of splitting to the beginning of heading (plant height of about 20 to 40 cm) is preferably used.

Here, as barley, for example, two-row barley, four-row barley, six-row barley, as long as it belongs to Hordeum vulgare species such as bare barley, any species, any variety can be used, but two-row barley, six-row barley Barley and bare barley are preferable, and six-row barley is particularly preferable.

More specifically, examples of the varieties of Nijo barley (beer barley) include, for example, Aominori, Agrimochi, Asaka Gold, Kinuyutaka, Omi Yutaka, Kirinijou, Haruna Nijo, Amagi Nijo, Fuji Nijo, Ishuku Shiraz, Misato Golden, Kawahonami, Kawamizu, Kinuka Nijo, Komaki Nijo, Sakitama Nijo, Tone Nijo, Sachiho Golden, Satsukibare, Shunrei, Mihal Gold, Sky Golden, Takachiho Golden, Tsuyu Shizuri, Tone Nijo, Nasu Nijo, Nishino Gold , Nishino Chikara, Nishino Hoshi, Haruka Nijo, Nirasaki Nijo, Nira Nijo, Harushizuku, Satsuki Nijo, Hoshiun, Mikamo Golden, Miho Golden, Myogi Nijo, Yashio Golden, Yachiho Golden, Kitaiku No. 41, Sai Stars, Ryofu, etc.

Examples of the varieties of six-row barley include asamamugi, kashimagol, mino-ryugi, cuttlefish, musashinomugi, drillmugi, sanadamugi, hagami, pod, cold, shunrai, silky snow, shinjuboshi, setsugenmochi, natriomugi, hayamayutaka, hayamayutaka, hayamayutaka. Examples include barley, fiber snow, double harvest, and Miyuki barley.

Examples of naked barley varieties include Ichibanboshi, Senbonhadaka, Sanukihadaka, Kikaihadaka, Sanshu, Nanpuuhadaka, Shiratamahadaka, Yunagihadaka, Daishimochi, Toyonokaze, Hayatehadaka, Ichimoku, Bimanihadaka, Beiwarohadaka, Beiwarohadaka, Beihirohadaka, Etc.

These varieties are cultivated by various agricultural test stations, agricultural research centers, private companies, etc., and fruits and young leaves are used for food (including beverages). From these varieties, it is preferable to select and grow varieties suitable for the cultivation area and to harvest young leaves (stems and leaves).

As described above, the barley young leaves in the present invention mean young leaves (stems and leaves) that have grown to a height of about 20 to 40 cm from the beginning of division to the beginning of heading.

As a processed product of young barley leaves, a form that can be ingested by a subject in need of prevention or improvement (treatment) of a disease or condition (symptoms) caused by a decrease in function (activity) of fibroblasts, or a form that can be used for cosmetics Is a heat-treated product of young barley leaves or an organic solvent extract. Specifically, for example, squeezed processed product of barley young leaves, crushed processed product, shredded processed product, dried processed product, roasted processed product, extracted processed product, powdered processed product, frozen processed product, and squeezed juice.・Dry powdered products, dried/pulverized products, pulverized/filtered products (pure, juice), shredded/hot water extraction processed products, and the like, or processed products thereof in combination, or The thing which carried out heat processing and/or organic solvent extraction after that is mentioned.

Of these, the commonly used forms include powdered processed products of squeezed young leaves (stems and leaves) (squeezed juice) (hereinafter sometimes referred to as “squeezed powder”) and dried young leaves (stems and leaves). A processed product (hereinafter sometimes referred to as "dry powder") is suitable.

Here, the squeezed powder is, for example, according to the method described in Patent Documents 6 to 8 or the like, after the young leaves of barley are harvested (cut), a liquid (squeezed liquid) squeezed out by squeezing is used as necessary. Can be prepared by mixing with a suitable excipient and spray drying. This squeezed powder can be suitably used as a heat-treated product or a solvent extract as it is, or by adding other excipients, etc., as a function activating agent for the fibroblasts of the present invention.

Further, the dry powder is obtained by, for example, harvesting (cutting) young leaves of barley according to the method described in Patent Documents 7 and 8 or the like, followed by shredding, drying treatment and pulverization treatment, and blanching as necessary. It can be prepared by combining treatments. This dry powder can be suitably used as a heat-treated product or a solvent extract, as it is, or by adding other excipients, etc., as a function activating agent for the fibroblasts of the present invention.

Here, the heat treatment in the present invention may be a treatment under the condition that the protein of young barley leaves is inactivated (denatured), and has the same meaning as a normal sterilization treatment. The heat treatment may be carried out under the conditions used in ordinary sterilization treatment, which are an appropriate combination of temperature, time, and pressure. Specifically, for example, there may be mentioned conditions in which temperature: 60 to 140° C. and time: 3 seconds to 60 minutes are combined. Further, it may be a high-pressure treatment used in a method of sterilizing food.

In the present invention, extraction with an organic solvent may be carried out by a commonly used method known per se. As the organic solvent, for example, an aprotic polar solvent and a protic polar solvent are preferable. Examples of the aprotic polar solvent include N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate and the like. Examples of the protic polar solvent include methanol, ethanol, n-propanol, isopropanol, n-butanol, nitromethane, acetic acid and the like. Among these, dimethylformamide is preferable as the aprotic polar solvent, and ethanol is preferable as the protic polar solvent. Preferably, the extraction with the organic solvent in the present invention is performed under the condition that the protein of young barley leaf is inactivated (denatured). In addition, as a matter of course, the protein is not extracted by the organic solvent.

In the present invention, the treated barley leaf, as specifically shown in Examples described later, has a function activating (activating) action of fibroblasts, so the function stimulant of fibroblasts of the present invention is: Anti-aging of the skin, that is, effects such as reduction of skin tension and elasticity, prevention or improvement of skin aging such as wrinkles and sagging, repair of wounds and inflammation can be expected. In addition, for a subject in need of prevention or improvement (treatment) of a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts, prevention or improvement of such disease or condition without side effects. (Treatment) is expected.

Here, the function activation (activation) of fibroblasts is at least one action selected from the group consisting of a dermal fibroblast proliferation promoting action, a type I collagen production promoting action and a hyaluronic acid production promoting action. Means that.

The agent for stimulating the function of fibroblasts of the present invention is a treatment of young barley leaves for a subject in need of prevention or amelioration (treatment) of a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts. Method for preventing or ameliorating (treating) a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts, which comprises a step of administering a substance, and due to a decrease in the function (activity) of fibroblasts Treated barley leaf for use as a preventive or ameliorating (treating) agent for a disease or condition (symptoms), and preventing or ameliorating (treating) a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts. ), and a treated barley leaf for producing a preventive or ameliorating (therapeutic) agent for a disease or condition (symptoms) caused by a decrease in the function (activity) of fibroblasts. Expected to be used.

In the present specification, the term “prevention or amelioration” of a disease or condition is used to mean including the regulation, delay of progression, alleviation, onset prevention, recurrence prevention, suppression, etc. of a disease or condition.

The processed barley leaf of the present invention has a high affinity with water, can be dissolved or suspended, and has excellent miscibility with components that can be usually added to foods and drinks, pharmaceuticals or cosmetics. In addition, young barley leaf is a component that has already been used as a food and drink for many years. Therefore, the function-utilizing agent for fibroblasts containing the processed product of young barley leaves as an active ingredient can be used or ingested without any worry, by appropriately incorporating it into daily skin care (makeup) and diet. ..

The function-utilizing agent for fibroblasts of the present invention is used for “promoting function activation (activation) of fibroblasts” (in the present specification, “function-utilizing fibroblasts”). Is a limited product), which is an agent containing a processed product of young barley leaves as an active ingredient for activating (activating) fibroblasts, and can be used alone as a food or drink, a drug (formulation), or a cosmetic. You can Further, it can be contained in various compositions such as foods and drinks, pharmaceuticals and cosmetics as an active ingredient for activating (activating) the function of fibroblasts. Thereby, a food/beverage composition, a pharmaceutical composition, or a cosmetic composition can be obtained in which the function of fibroblasts is utilized. Use of the obtained food/drink composition, pharmaceutical composition or cosmetic composition for functional utilization of fibroblasts is effectively used for the diseases or conditions (symptoms) resulting from the above-mentioned decrease in function (activity) of fibroblasts. You can

The agent for stimulating the function of fibroblasts in the present invention is not particularly limited as long as it is a form that can be orally ingested or applied to the skin, such as a solution, a suspension, an emulsion, a powder, and a solid molded product. Specific examples include solid agents, powder agents, capsules, tablets, granules and powders, as well as forms used in ordinary foods such as beverages, foods and cosmetics, pharmaceuticals and cosmetics. In addition, as the form of the preparation, powders, capsules, tablets, granules, drinks and the like, which can easily control the intake, can be preferably exemplified.

In the present invention, the treated barley leaf may be used as it is, or may be used by producing a desired preparation by a conventionally known ordinary method. For example, it can be mixed with various kinds of additives permitted in the production of the preparation and molded into a composition. As the additive, any additive may be used as long as it does not impair the effects of the present invention. For example, in addition to crude drugs, vitamins, minerals, etc., excipients, surfactants, coating agents, oils and fats, waxes. Kinds, sweeteners, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, colorants, fragrances, buffers, antioxidants, pH adjusting agents, citric acid, malic acid, gluconic acid and other acidulants, Etc.

Examples of the crude drug include ginseng, american ginseng, seven ginseng ginseng, ganoderma lucidum, propolis, agarix, blueberries, ginkgo biloba and extracts thereof. Examples of the above vitamins include oil-soluble vitamins such as vitamins D and K, and water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid, and biotin.

Examples of the excipient include lactose, starch, cellulose, maltitol, dextrin and the like. Examples of the surfactant include glycerin fatty acid ester and sucrose fatty acid ester. Examples of the coating agent include gelatin, pullulan, shellac, zein and the like. Examples of the oils and fats include wheat germ oil, rice germ oil, and safflower oil. Examples of the waxes include beeswax, rice bran wax, carnauba wax and the like. Examples of the sweetener include sucrose, glucose, fructose, stevia, saccharin, sucralose and the like.

Further, according to the present invention, there is provided a food or drink, a pharmaceutical product, or a cosmetic product, which comprises a function stimulating agent for fibroblasts, which contains a processed barley leaf as an active ingredient. The term “comprising” as used herein may include a physiologically acceptable carrier depending on the desired product form, and also means that it contains other auxiliary components that can be used in combination.

In the present invention, the food or drink is something other than a medicine and is not particularly limited as long as it is in a form that can be orally ingested as described above. Specifically, for example, instant noodles, retort foods, canned foods, microwave foods, instant soups and miso soups, freeze-dried foods and other instant foods; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea Beverages such as beverages, powdered beverages, concentrated beverages, alcoholic beverages; bread, pasta, noodles, cake mix, fried flour, bread crumbs and other flour products; candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, Confectionery such as snacks, crackers, Japanese confectionery, dessert confectionery; sauces, tomato processing seasonings, flavor seasonings, cooking mixes, sauces, dressings, sauces, seasonings such as curry and stew bases; processed oils and fats, Fats and oils such as butter, margarine, mayonnaise; dairy products such as milk drinks, yogurts, lactic acid bacteria drinks, ice creams, creams; processed egg products, fish meat hams and sausages, seafood products such as fish paste; livestock ham and Processed livestock products such as sausages; canned agricultural products, jams and marmalades, pickles, boiled beans, cereals and other processed agricultural products; frozen foods and the like.

Food and drink include health foods (for example, functional foods, dietary supplements, health supplements, nutrition-enhanced foods, nutrition-adjusted foods, supplements, etc.), health foods (for example, foods for specified health uses, foods with nutritional function, Foods with functional claims, etc.), special-purpose foods (eg, foods for sick people, infant formula, milk powder for pregnant or lactating women, etc.), as well as increased lipid absorption and decreased fibroblast function (activity) Also included are foods and drinks that are labeled with a risk reduction, prevention, or improvement of the disease or condition (symptoms) caused by them.

According to another aspect of the present invention, a food or drink containing a function stimulating agent for fibroblasts, the prevention of diseases or conditions that can be prevented or ameliorated by activating (activating) the functions of fibroblasts, Alternatively, food and drink may be provided with the improved function displayed.

In the present invention, the term "medicament" refers to a preparation prepared as an oral preparation or a parenteral preparation in accordance with a conventional method in combination with an additive acceptable for formulation. In the case of an oral preparation, as described above, it is not particularly limited as long as it is a form that can be orally ingested. Further, in the case of a parenteral preparation, it can be in the form of an injection or a suppository. From the viewpoint of simplicity, an oral preparation is preferable. As the additive acceptable for formulation, the same ones as described above can be mentioned.

In the present invention, the content of the active ingredient (treated barley leaf) in the function-utilizing agent for fibroblasts is difficult to uniformly define depending on the type of preparation, the form, the purpose of prevention or improvement, etc. , Adult (body weight 60 kg) per day, as a squeezed powder of barley young leaves (heat-treated product), usually about 15 mg to 15 g, preferably about 50 mg to 9 g, and as dry powder of young barley leaves, usually about 50 mg to 15 g. , Preferably about 100 mg to 9 g. Further, the content in the preparation may be appropriately set in consideration of the daily intake so that the necessary daily intake of the active ingredient can be taken (taken).

In the present invention, the cosmetics are, for example, those prepared as cosmetics for application to the skin for use and hair cosmetics for application on the scalp and hair. The cosmetics include quasi-drugs in addition to the cosmetics prescribed by the Pharmaceutical Affairs Law.

When the agent for promoting the function of the fibroblast of the present invention is provided as a cosmetic, the dosage form that can be taken is not particularly limited as long as it can be applied to the skin, scalp, and hair. Specifically, solid, liquid, emulsion, gel, cream, ointment, foam, mist, aerosol, etc. dosage forms, soap, lotion, emulsion, cream, gel, jelly, essence, It can be provided as a lip cream, a pack, a mask or the like.

Other optional ingredients that these cosmetics may contain are not particularly limited, and additives and the like that can be blended in ordinary cosmetics can be used. As such additives, for example, water, oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, astringents, bactericidal/antibacterial agents, whitening agents, ultraviolet absorbers, Moisturizers, cell activators, anti-inflammatory/anti-allergic agents, antioxidants, vitamins, natural extracts and the like can be mentioned. The content of these other optional components is not particularly limited and can be appropriately selected according to the desired dosage form and the like.

The content of the active ingredient (treated barley leaf) in cosmetics varies depending on the type and form of the preparation, the purpose of use, the frequency of use, etc. and is not particularly limited, and it is difficult to uniformly define fiber The content is preferably such that the blast cell activation action (effect) is achieved. Specifically, for example, the lower limit is usually 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and the upper limit is usually 3% by mass or less, preferably 1% by mass. % Or less, more preferably 0.3% by mass or less.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the technical scope of the present invention is not limited to these exemplifications. In the following examples, the test substance concentration (%) is the “barley young leaf extract powder” concentration (mass %) unless otherwise specified.

[Example 1] Preparation of barley young leaf juice powder Prepared as follows according to the method described in JP-A-2009-148213.
10 kg of cut barley young leaves (stems and leaves) were pressed, and 8 kg of the obtained juice was sterilized by heating and then dried to obtain 0.5 kg of juice powder (hereinafter sometimes referred to as “young barley extract powder”). .

Example 2 Cell proliferation effect 1 Materials and test methods 1-1 Cells Human neonatal-derived dermal fibroblast cell line NB1RGB cells (RIKEN BRC, Japan) and, CO ₂ incubator (CO ₂ concentration% (v / v), This test was carried out by culturing at 37°C.

1-2 medium 10.0% (v/v) Fetal Bovine Serum (FBS, Cat No. SH30071.03, Hyclone, UK) and 1.0% (v/v) antifungal agent (Antibiotic-Antimycotic 100X, Cat) No. 15240-062, Invitrogen, USA) Eagle's Minimal Essential Medium (EMEM, Cat No. 051-07615, Wako, Japan) was used.

1-3 Test substance To the barley green leaf extract powder prepared in Example 1, a 50% ethanol solution (CAS No. 64-17-5, Japan Alcohol, Japan) in an amount three times that amount was added and stirred, and ultrasonic waves were applied for 10 minutes. Processed. The sonicated barley leaf extract powder solution was centrifuged at 12,000 xg for 5 minutes at room temperature, and the supernatant was recovered and used as a test substance. This test substance was diluted with a 50% ethanol solution to prepare a total of 2 concentrations (10%, 30%) before use. Furthermore, the prepared test substance was diluted with 30 μML(+)-ascorbic acid (CAS No. 50-81-7, Kanto Chemical, Japan) and 0.1% FBS-containing EMEM to give a total of 2 concentrations (0.3%, 1%) was prepared at the time of use and used for the test (final concentration: 0.1%, 0.3%).

1-4 Test configuration In the cell proliferation test, 3 wells of a 96-well plate (Cat No. 3595, Corning) were used for each treatment group. Further, the test was carried out by providing one test substance group and one control group per plate. Operations related to the test including preparation of the test substance were performed at room temperature unless otherwise specified.

1-5 Test Operation (1) Cell Culture 2.0×10 ⁴ cells/200 μL of NB1RGB cells were seeded per well in a 96-well plate, and cultured in a CO ₂ incubator for 24 hours. Further, in order to prevent drying during culture, 200 μL of Phosphate buffer saline (PBS (-), Cat No. 198601, Nissui, Japan) was added to the wells not used for the test.
After 24 hours, 100 μL of each of the test substance and the control was added to a 96-well plate and cultured in a CO ₂ incubator for 48 hours (final concentrations of the test substance were 0.1% and 0.3%). As a control, EMEM containing 30 μML(+)-ascorbic acid and 0.1% FBS was used.
After 48 hours, the number of cells in the 96-well plate from which the culture supernatant had been removed was evaluated by the following method to evaluate the cell proliferating effect of the test substance.

(2) Evaluation of Cell Proliferation Action Each well of the 96-well plate from which the medium was removed was gently washed once with 200 μL of PBS(−) heated to 37° C.
200 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-ul)-2,5-diphenyltetrazolium bromide (MTT, CAS No. 298-93-1, Sigma-Aldrich, USA) solution per well In addition, the cells were cultured for 2 hours in a CO ₂ incubator.

After the culture was completed, the MTT solution was removed, and the wells were washed with 200 μL of PBS(−). In order to solubilize the produced insoluble formazan, 2-propanol (CAS No. 67-63-0, Kanto Chemical, Japan) containing 0.04N hydrochloric acid (CAS No. 7647-01-0, Kanto Chemical, Japan) was used. ) Was added and the mixture was allowed to stand at room temperature for 1 hour under light shielding.

The 96-well plate was shaken at 270 rpm for 10 seconds to uniformly disperse the dye in the well, and then the absorbance at 570 nm (OD ₅₇₀ ) was measured using a microplate reader (SPARK ^™ 10M, TECAN, Switzerland). Test substance administered group of _{OD 570} of the control group _{OD 570} as 100%, i.e. to calculate the effect of cell proliferation as a cell growth rate of the test substance (%).

2 Test Results The cell growth rate ± standard deviation of the test substance 0.1% and 0.3% addition groups when the cell growth amount (OD ₅₇₀ ) of the control group was 100% was , 117.7 ± 2.8% and 149.7 ± 19.7%. A significant cell proliferation effect was observed in the barley young leaf extract powder-added group as compared with the control group.

[Example 3] Collagen and hyaluronic acid production promoting action 1 Materials and test method 1-1 cells The same cells as 1-1 of Example 2 were cultured under the same conditions to carry out this test.

1-2 Medium A medium (EMEM) having the same composition as 1-2 in Example 1 was used.

1-3 Test substance Dimethyl sulfoxide (DMSO, CAS N0. 67-68-51, Wako, Japan) was added to the barley young leaf extract powder prepared in Example 1 to prepare a 30% concentration. This was centrifuged at 12,000 xg for 5 minutes at room temperature to collect the supernatant. The collected supernatant was diluted with DMSO to prepare a concentration of 3% before use. Furthermore, the prepared test substance was diluted 100-fold with EMEM containing 30 μML(+)-ascorbic acid (CAS No. 50-81-7, Kanto Chemical, Japan) and 0.1% FBS to use it at a concentration of 0.03%. It was prepared at the time (0.01% final concentration).

1-4 Test configuration To calculate the number of cells, collagen production and hyaluronic acid production, a 96-well plate per one treatment group (cell activation: Cat No. 3595, Corning, USA, collagen production and hyaluronic acid production: Cat No. 3855) , Thermo scientific, USA). Further, the test was carried out by providing one test substance group and one control group per plate. Operations related to the test including preparation of the test substance were performed at room temperature unless otherwise specified.

1-5 Test Operation (1) Cell Culture The cells were cultured for 48 hours in the same manner as in 1-5(1) of Example 2.
After 48 hours, the culture supernatant was dispensed into a new 96-well plate and stored frozen (-80°C). The amount of collagen and the amount of hyaluronic acid in this culture supernatant were measured by the following method using Enzyme-Linked ImmunoSorbent Assay (ELISA). Further, the number of cells in the 96-well plate from which the culture supernatant was removed was measured by the same method as 1-5(2) of Example 2.

(2) Collagen production promoting action The effect of the test substance on the production amount of type I collagen, which is said to give elasticity and elasticity to the skin, was directly evaluated by ELISA as follows.

100 μL of collagen standard solution per well and the culture supernatant cryopreserved in the above (1) “Cell culture” were added to a high-adsorption type 96-well plate, and allowed to stand at 4° C. overnight.
The microplate was washed twice with 200 μL of 0.05% Tween20 in PBS(-) (Wash buffer, Tween20: Cas No. 9005-64-5, Sigma-Aldrich, USA), and 200 μL of 1% Bovine Serum Albumin (BSA , Cat No. PRL68700-50G, Proliant, USA) in PBS was added and left at room temperature for 1 hour.

After 1 hour, the cells were washed twice with 200 μL of Wash buffer, 100 μL of Biotin-conjugated anti-collagen type I antibody (Cat No. 600-406-103, ROCKLAND, USA) solution was added, and the mixture was allowed to stand at room temperature for 1 hour. After 1 hour, the plate was washed 4 times with 200 μL of Wash buffer, 100 μL of Avidin-horseradish peroxidase (Cat No. CJ30H, Prozyme, USA) solution was added, and the mixture was allowed to stand at room temperature for 30 minutes.

After 30 minutes, it was washed 4 times with 200 μL of Wash buffer and once with 200 μL of ultrapure water (CAS No. 7732-18-5, Wako, Japan) to obtain 2,2′-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS, Cat No. 5110-0010, KPL, USA) was added in an amount of 100 μL per well, and the mixture was allowed to stand at room temperature for 5 minutes.

The 96-well plate was shaken at 270 rpm for 30 seconds to homogenize the dye in the well, and then the absorbance at 405 nm (OD ₄₀₅ ) was measured using a microplate reader.

A value obtained by dividing the OD ₄₀₅ of the control substance and the test substance by the OD ₅₇₀ (the number of cells) measured by the method (1) above was calculated as the collagen production amount per cell. Furthermore, the collagen production rate (%) per cell of the test substance was calculated by setting the collagen production rate per cell of the control group as 100%.

(3) Hyaluronic Acid Production Promoting Effect The effect of the test substance on the production amount of hyaluronic acid, which is said to hold water between collagen and elastin, was evaluated by sandwich ELISA as follows.

A 100 μL solution of 5 μg/mL Hyaluronan binding protein (HABP, Cat No. BC40, Hokudo, Japan) was added to a high-adsorption type 96-well plate and left standing at 4° C. for 24 hours. The HABP solution was removed, and the mixture was washed 3 times with 200 μL of 0.05% Tween 20 in 1.5M NaCl solution (Wash buffer, USA NaCl: Cas No. 7647-14-5, Wako, Japan).

100 μL of 5% BSA solution was added, and the mixture was left standing at 4° C. overnight. Next, the BSA solution was removed, the plate was dried after washing twice with 200 μL of Wash buffer.

100 μL of culture supernatant diluted 100-fold with PBS (−) containing 0.5 M NaCl, 0.02% Tween 20 and 1% BSA was added to each well, and the mixture was allowed to stand at room temperature for 2 hours.

After 2 hours, the plate was washed 4 times with 200 μL of Wash buffer, 100 μL of biotin-labeled HABP (Cat No. BC41, Hokudo, Japan) solution was added, and the mixture was allowed to stand at room temperature for 30 minutes.

After 30 minutes, the plate was washed 4 times with 200 μL of Wash buffer and once with 200 μL of ultrapure water, 100 μL of streptavidin-labeled HRP solution was added, and the mixture was allowed to stand at room temperature for 30 minutes. After 30 minutes, the plate was washed 4 times with 200 μL of Wash buffer and once with 200 μL of ultrapure water, 100 μL of ABTS was added to each well, and the mixture was allowed to stand at room temperature for 10 minutes.

The 96-well plate was shaken at 270 rpm for 30 seconds to homogenize the dye in the well, and then the absorbance at 405 nm (OD ₄₀₅ ) was measured using a microplate reader.

A value obtained by dividing the OD ₄₀₅ of the control substance and the test substance by the OD ₅₇₀ (the number of cells) measured by the method of (1) above was calculated as the hyaluronic acid production rate per cell. Furthermore, the hyaluronic acid production rate (%) per cell of the test substance was calculated by setting the hyaluronic acid production rate per cell of the control group as 100%.

3. Test Results When the amount of collagen produced per cell in the control group was 100%, the collagen production rate (%) of the test substance (young barley leaf extract powder) 0.01% addition group was 118.5 ± 4.7 (% )Met. The barley young leaf extract powder-added group showed a significant collagen production promoting action as compared with the control group.

In addition, the hyaluronic acid production rate (%) of the test substance (young barley leaf extract powder) 0.01% addition group was 115.5±17.4, when the production amount of hyaluronic acid per cell in the control group was 100%. (%)Met. Compared with the control group, the barley green leaf extract powder-added group was found to have a hyaluronic acid production promoting action.

Claims (6)

A function-utilizing agent for fibroblasts comprising a heat-treated barley leaf or an organic solvent extract thereof, or an organic solvent extract of barley leaf as an active ingredient. The function-utilizing agent according to claim 1, wherein the heat-treated product is squeezed powder or dried powder of young barley leaves. The function activation is at least one action selected from the group consisting of a fibroblast proliferation promoting action, a type I collagen production promoting action and a hyaluronic acid production promoting action. Function enhancer. The organic solvent is a protic polar solvent or an aprotic polar solvent, The functional stimulant according to any one of claims 1 to 3, wherein the organic solvent is a protic polar solvent or an aprotic polar solvent. A food or drink for preventing or ameliorating (treating) a disease or a condition (symptoms) caused by the functional decline of fibroblasts, which comprises the function stimulating agent according to any one of claims 1 to 4. A cosmetic comprising the function stimulating agent according to any one of claims 1 to 4.